Abstract: Following infection, naïve CD8+ T cells bearing pathogen-specific T cell receptors (TCRs) differentiate into a mixed population of short-lived effector and long-lived memory T cells to mediate an adaptive immune response. How the TCR regulates memory T cell development has remained elusive. Using a mutant TCR transgenic model, we found that point mutations in the TCR beta transmembrane domain (betaTMD) impair the development and function of CD8+ memory T cells without affecting primary effector T cell responses. Mutant T cells are deficient in polarizing the TCR and in organizing the nuclear factor kappaB signal at the immunological synapse. Thus, effector and memory states of CD8+ T cells are separable fates, determined by differential TCR signaling.

Abstract: The affinity of the T-cell receptor (TCR) for self antigen is the basis for the selection of a useful (MHC-restricted) and safe (self-tolerant) T-cell repertoire. However, it has been difficult to understand how thymocytes measure ligand affinity and translate this signal into a cellular response. In this Opinion article, we propose a new model that describes how the TCR discriminates between low- and high-affinity ligands, which is based on the duration of TCR-ligand interactions and a 'zipper' mechanism that mediates the interaction of the TCR and co-receptor molecules to initiate negative-selection signalling.

Abstract: The ex vivo induction of alloantigen-specific hyporesponsiveness by costimulatory pathway blockade or exposure to immunoregulatory cytokines has been shown to inhibit proliferation, IL-2 production, and the graft-versus-host disease (GVHD) capacity of adoptively transferred T-cells. We hypothesized that inhibition of the intracellular NF-kappaB pathway in alloreactive T-cells, which is critical for T-cell activation events including IL-2 transcription, could lead to alloantigen hyporesponsiveness and loss of GVHD capacity. We demonstrate that treatment of mixed lymphocyte reaction (MLR) cultures with PS1145, a potent inhibitor of NF-kappaB activation, can induce T-cell hyporesponsiveness to alloantigen in primary and secondary responses while preserving in vitro responses to potent mitogenic stimulation. GVHD lethality in recipients of ex vivo PS1145-treated cells was profoundly inhibited. Parking of control or PS1145-treated MLR cells in syngeneic Rag(-/-) recipients resulted in intact contact hypersensitivity (CHS) responses. However, GVHD lethality capacity also was restored, suggesting that lymphopenic expansion uncoupled alloantigen hyporesponsiveness. These results indicate that the NF-kappaB pathway is a critical regulator of alloresponses and provide a novel small molecule inhibitor based approach that is effective in preventing early posttransplant GVHD lethality but that also permits donor T-cell responses to recover after a period of lymphopenic expansion.

Abstract: The use of appropriate fluorescent proteins has allowed the use of FRET microscopy for investigation of intermolecular interactions in living cells. This method has the advantage of both being dynamic and of working at the subcellular level, so that the time and place where proteins interact can be visualized. We have used FRET microscopy to analyze the interactions between the T cell antigen receptor and the coreceptors CD4 and CD8. This chapter reviews data on how these coreceptors are recruited to the immunological synapse, and how they interact when the T cell is stimulated by different ligands.

Abstract: TCR engagement by peptide-MHC class I (pMHC) ligands induces a conformational change (Deltac) in CD3 (CD3Deltac) that contributes to T cell signaling. We found that when this interaction took place between primary T lineage cells and APCs, the CD8 coreceptor was required to generate CD3Deltac. Interestingly, neither enhancement of Ag binding strength nor Src kinase signaling explained this coreceptor activity. Furthermore, Ag-induced CD3Deltac was developmentally attenuated by the increase in sialylation that accompanies T cell maturation and limits CD8 activity. Thus, both weak and strong ligands induced CD3Deltac in preselection thymocytes, but only strong ligands were effective in mature T cells. We propose that CD8 participation in the TCR/pMHC interaction can physically regulate CD3Deltac induction by "translating" productive Ag encounter from the TCR to the CD3 complex. This suggests one mechanism by which the developmentally regulated variation in CD8 sialylation may contribute to the developmental tuning of T cell sensitivity.

Abstract: The CD8 coreceptor contributes to the recognition of peptide-MHC (pMHC) ligands by stabilizing the TCR-pMHC interaction and enabling efficient signaling initiation. It is unclear though, which structural elements of the TCR ensure a productive association of the coreceptor. The alpha-chain connecting peptide motif (alpha-CPM) is a highly conserved sequence of eight amino acids in the membrane proximal region of the TCR alpha-chain. TCRs lacking the alpha-CPM respond poorly to low-affinity pMHC ligands and are unable to induce positive thymic selection. In this study we show that CD8 participation in ligand binding is compromised in T lineage cells expressing mutant alpha-CPM TCRs, leading to a slight reduction in apparent affinity; however, this by itself does not explain the thymic selection defect. By fluorescence resonance energy transfer microscopy, we found that TCR-CD8 association was compromised for TCRs lacking the alpha-CPM. Although high-affinity (negative-selecting) pMHC ligands showed reduced TCR-CD8 interaction, low-affinity (positive-selecting) ligands completely failed to induce molecular approximation of the TCR and its coreceptor. Therefore, the alpha-CPM of a TCR is an important element in mediating CD8 approximation and signal initiation.

Abstract: Most mechanisms of cell development, physiology, and signal transduction are controlled by protein-protein interactions. Immunoprecipitation of multiprotein complexes detected by flow cytometry (IP-FCM) is a means to quantitatively measure these interactions. The high sensitivity of this method makes it useful even when very little biomaterial is available for analysis, as in the case of rare primary cell subsets or patient samples. Detection of the T cell antigen receptor associated with the CD3 multiprotein complex from as few as 300 primary murine T cells is presented as an example. The method is compatible with quantitative flow cytometry techniques, making it possible to estimate the number of coimmunoprecipitated molecules. Both constitutive and inducible protein-protein interactions can be analyzed, as illustrated in related methodology using glutathione S-transferase-fusion protein pull-down experiments. IP-FCM represents a robust, quantitative, biochemical technique to assess native protein-protein interactions, without requiring genetic engineering or large sample sizes.

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Abstract: T cell tolerance depends on the T cell receptor's affinity for peptide/major histocompatibility complex (MHC) ligand; this critical parameter determines whether a thymocyte will be included (positive selection) or excluded (negative selection) from the T cell repertoire. A quantitative analysis of ligand binding was performed using an experimental system permitting receptor-coreceptor interactions on live cells under physiological conditions. Using three transgenic mouse strains expressing distinct class I MHC-restricted T cell receptors, we determined the affinity that defines the threshold for negative selection. The affinity threshold for self-tolerance appears to be a constant for cytotoxic T lymphocytes.

Abstract: Cyclic AMP (cAMP) is an important negative regulator of T cell activation, and an increased level of cAMP is associated with T cell hyporesponsiveness in vitro. We sought to determine whether elevating intracellular cAMP levels ex vivo in alloreactive T cells during primary mixed lymphocyte reactions (MLR) is sufficient to induce alloantigen-specific tolerance and prevent graft-versus-host disease (GVHD). Primary MLRs were treated with exogenous (8)Br-cAMP and IBMX, a compound that increases intracellular cAMP levels by inhibition of phosphodiesterases. T cell proliferation and IL-2 responsiveness in the treated primary MLR cultures were greatly reduced, and viable T cells recovered on day 8 also had impaired responses to restimulation with alloantigen compared to control-treated cells, but without an impairment to nonspecific mitogens. Labeling experiments showed that cAMP/IBMX inhibited alloreactive T cell proliferation by limiting the number of cell divisions, increasing susceptibility to apoptosis, and rendering nondeleted alloreactive T cells hyporesponsive to alloantigen restimulation. cAMP/IBMX-treated CD4(+) T cells had a markedly reduced capacity for GVHD lethality in major histocompatibility complex class II disparate recipients, but maintained the capacity to mediate other CD4(+) T cell responses in vivo. Thus, our results provide the first preclinical evidence of using cAMP-elevating pharmaceutical reagents to achieve long-term alloantigen-specific T cell tolerance that is sufficient to prevent GVHD.

Abstract: The immune system can be roughly divided into innate and adaptive compartments. The adaptive compartment includes the B and T lymphocytes, whose antigen receptors are generated by recombination of gene segments. The consequence is that the creation of self-reactive lymphocytes is unavoidable. For the host to remain viable, the immune system has evolved a strategy for removing autoimmune lymphocytes during development. This review discusses how T lymphocytes are generated, how they recognize antigens, and how their antigen receptor directs the removal of self-reactive T cells.

Abstract: A healthy individual can mount an immune response to exogenous pathogens while avoiding an autoimmune attack on normal tissues. The ability to distinguish between self and non-self is called 'immunological tolerance' and, for T lymphocytes, involves the generation of a diverse pool of functional T cells through positive selection and the removal of overtly self-reactive thymocytes by negative selection during T-cell ontogeny. To elucidate how thymocytes arrive at these cell fate decisions, here we have identified ligands that define an extremely narrow gap spanning the threshold that distinguishes positive from negative selection. We show that, at the selection threshold, a small increase in ligand affinity for the T-cell antigen receptor leads to a marked change in the activation and subcellular localization of Ras and mitogen-activated protein kinase (MAPK) signalling intermediates and the induction of negative selection. The ability to compartmentalize signalling molecules differentially in the cell endows the thymocyte with the ability to convert a small change in analogue input (affinity) into a digital output (positive versus negative selection) and provides the basis for establishing central tolerance.

Abstract: The src family kinases p56lck (Lck) and p59fyn (Fyn) are the most proximal signaling molecules to be activated downstream of the T-cell receptor. Using an inducible transgenic model, we can regulate the expression of Lck in primary T cells and ask how the signaling cascade and differentiation potential are affected by the absence or the presence of reduced levels of Lck. We show that in naïve T cells, Lck controls the threshold of activation by preferentially regulating multiple signaling pathways that result in the mobilization of Ca2+ through activation of phospholipase C-gamma and protein kinase C as well as activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway. Fyn is also able to stimulate the ERK/MAPK pathway in primary T cells but has little influence on the mobilization of Ca2+. Only Lck efficiently stimulates production of diacylglycerol and therefore RasGRP1 recruitment to the plasma membrane and phosphorylation of Shc, suggesting that Fyn activates ERK via a different upstream signaling route. Finally, we show that signals through Lck are essential for the development of T-cell-effector potential, particularly for effective cytokine transcription.

Abstract: The T cell receptor (TCR) can recognize a variety of cognate peptide/major histocompatibility complex (pMHC) ligands and translate their affinity into distinct cellular responses. To achieve this, the nonsignaling alphabeta heterodimer communicates ligand recognition to the CD3 signaling subunits by an unknown mechanism. In thymocytes, we found that both positive- and negative-selecting pMHC ligands expose a cryptic epitope in the CD3 complex upon TCR engagement. This conformational change is induced in vivo and requires the expression of cognate MHC. We conclude that TCR engagement with a cognate pMHC ligand induces a conformational change in the CD3 complex of thymocytes and propose that this marks an initial event during thymic selection that signals the recognition of self-antigen.

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Abstract: The CD3epsilon proline-rich sequence (PRS) binds to the cytosolic adaptor molecule Nck after TCR ligation. It has been proposed that this interaction is essential for immunological synapse formation and T cell activation. To assess the physiological importance of the CD3epsilon PRS, we have generated mice that lack this motif (CD3epsilon.PRS(M)). Pull-down experiments demonstrated the inability of Nck to bind to the CD3epsilon PRS in thymocytes from mutant mice after TCR ligation. Surprisingly, no differences were observed in the number and percentage of T cell subsets in the thymus and spleen, and there was no apparent defect in positive or negative selection. Furthermore, the proliferative response of CD3epsilon.PRS(M) T cells to staphylococcal enterotoxin B and anti-CD3 Ab was normal. TCR surface expression, constitutive internalization, and Ag-induced down-modulation were also normal. These data suggest that the interaction between the CD3epsilon PRS and Nck, or any other Src homology 3 domain-containing molecule, is not essential for T cell development and function.

Abstract: Naive T cells become programmed for clonal expansion and contraction during the early hours of antigenic signaling. Recent studies support an 'autopilot' model, wherein the commitment to proliferate and the magnitude of the proliferative response are simultaneously determined during a single, brief period of antigen exposure. Here, we have examined whether the proliferation of naive CD4+ T cells must occur on 'autopilot', or whether extended periods of antigenic signaling can impact primary proliferative responses to antigen-presenting macrophages (macrophage APC). We found that a single exposure to antigen (18 h) simultaneously committed T cells to (1) up-regulate surface TCR above the level expressed on naive T cells, (2) undergo minimal cell division, and (3) acquire susceptibility to TCR-dependent activation-induced cell death. However, continued antigenic signaling between 18 and 72 h was required to amplify the number of daughter cells derived from the already committed T cells. Thus, a discrete commitment time was followed by a 'tuning' period, where extended antigenic signaling determined the volume of the proliferative response. We conclude that T cell commitment to full clonal expansion versus TCR-dependent death susceptibility represent two separate programming events whose timing can be segregated by macrophage APC.

Abstract: We have studied the role of the T cell receptor (TCR) beta chain transmembrane and cytoplasmic domains (betaTM/Cyto) in T cell signaling. Upon antigen stimulation, T lymphocytes expressing a TCR with mutant and betaTM and Cyto domains accumulate in large numbers and are specifically defective in undergoing activation-induced cell death (AICD). The mutant TCR poorly recruits the protein adaptor Carma-1 and is subsequently impaired in activating NF-kappaB. This signaling defect leads to a reduced expression of Fas ligand (FasL) and to a reduction in AICD. These beta chain domains are involved in discriminating cell division and apoptosis.

Abstract: T lymphocytes are generated in the thymus, where developing thymocytes must accept one of two fates: They either differentiate or they die. These fates are chiefly determined by signals that originate from the T cell receptor (TCR), a single receptor complex with a remarkable capacity to decide between distinct cell fates. This review explores TCR signaling in thymocytes and focuses on the kinetic aspects of ligand binding, coreceptor involvement, protein phosphorylation, and mitogen-activated protein kinase (MAPK) activation. Understanding the logic of TCR signaling may eventually explain how thymocytes and T cells distinguish self from nonself, a phenomenon that has fascinated immunologists for 50 years.

Abstract: Recognition by CD8+ cytotoxic T lymphocytes (CTLs) of antigenic peptides bound to major histocompatibility class (MHC) I molecules on target cells leads to sustained calcium mobilization and CTL degranulation resulting in perforin-dependent killing. We report that beta1 and beta3 integrin-mediated adhesion to extracellular matrix proteins on target cells and/or surfaces dramatically promotes CTL degranulation. CTLs, when adhered to fibronectin but not CTL in suspension, efficiently degranulate upon exposure to soluble MHC.peptide complexes, even monomeric ones. This adhesion induces recruitment and activation of the focal adhesion kinase Pyk2, the cytoskeleton linker paxillin, and the Src kinases Lck and Fyn in the contact site. The T cell receptor, by association with Pyk2, becomes part of this adhesion-induced activation cluster, which greatly increases its signaling.

Abstract: T cells expressing T cell receptor (TCR) complexes that lack CD3 delta, either due to deletion of the CD3 delta gene, or by replacement of the connecting peptide of the TCR alpha chain, exhibit severely impaired positive selection and TCR-mediated activation of CD8 single-positive T cells. Because the same defects have been observed in mice expressing no CD8 beta or tailless CD8 beta, we examined whether CD3 delta serves to couple TCR.CD3 with CD8. To this end we used T cell hybridomas and transgenic mice expressing the T1 TCR, which recognizes a photoreactive derivative of the PbCS 252-260 peptide in the context of H-2K(d). We report that, in thymocytes and hybridomas expressing the T1 TCR.CD3 complex, CD8 alpha beta associates with the TCR. This association was not observed on T1 hybridomas expressing only CD8 alpha alpha or a CD3 delta(-) variant of the T1 TCR. CD3 delta was selectively co-immunoprecipitated with anti-CD8 antibodies, indicating an avid association of CD8 with CD3 delta. Because CD8 alpha beta is a raft constituent, due to this association a fraction of TCR.CD3 is raft-associated. Cross-linking of these TCR-CD8 adducts results in extensive TCR aggregate formation and intracellular calcium mobilization. Thus, CD3 delta couples TCR.CD3 with raft-associated CD8, which is required for effective activation and positive selection of CD8(+) T cells.

Abstract: The application of TCR transgenic mice to transplantation immunology is hampered by the limited lines available. Recently, we reported CD4+ T cell receptor (TCR) transgenic mice specific for I-Abm12 expressed on B6.C.H-2bm12 mice. Here, we characterized rejection of skin and vascularized cardiac allografts in these mice, which we term ABM (for anti-bm12). In vivo proliferative experiments reveal that all CD4 T cells in ABM mice react to bm12 antigens. Surprisingly, while ABM mice have accelerated (compared to B6 recipients) rejection of bm12 skin allografts, they, like B6 recipients, fail to acutely reject bm12 cardiac allografts. This is not due to lack of immunogenicity of bm12 hearts, as these grafts are acutely rejected by primed ABM recipients, although not by primed B6 recipients. Lastly, long-term surviving bm12 grafts in ABM recipients are relatively free from chronic rejection (compared with B6 recipients), which may be due to skewing of the CD4 repertoire towards direct alloreactivity, and consequent lack of CD4 mediated indirect allorecognition as evidenced by the lack of IgG deposition in those grafts. The results indicate that a complex interplay between responder frequency, priming and repertoire dictates the occurrence, or lack thereof, of acute and chronic rejection.

Abstract: We have developed an adoptive transfer model system to visualize the dynamics of alloantigen-specific CD4+ T-cell activation in vivo. Using TCR-transgenic (tg) mice reactive to I-Ab(m12), we studied the clonal expansion and differentiation of alloreactive T cells by tracking the fate of adoptively transferred TCR-tg CD4+ T cells in syngeneic mice transplanted with skin grafts expressing I-Ab(m12). Following transplantation, alloantigen-specific TCR-tg CD4+ T-cell expansion was observed initially in the draining lymph nodes followed by the spleen. TCR-tg CD4+ T cells up-regulated CD69 and CD25 expression, developed an effector/memory surface phenotype and produced IFN-gamma in response to alloantigen ex vivo. Furthermore, we validate the model system as a means for studying the effects of tolerogenic regimens on alloreactive CD4+ T cells, demonstrating that CTLA4Ig inhibits alloantigen-dependent clonal expansion and effector function of TCR-tg CD4+ T cells in vivo. We describe the first model for tracking alloreactive CD4+ T-cell activation in vivo. It provides a powerful tool for studying CD4+ T-cell mediated alloimmune responses and mechanisms of tolerance induction in vivo.

Abstract: Dead cells are a prominent feature of the thymic landscape as only 5% of developing thymocytes are exported as mature T cells. The remaining thymocytes die by one of two mechanisms; most thymocytes die because they are not positively selected and do not receive a survival signal, whereas a minority of thymocytes undergo T-cell receptor (TCR)-mediated apoptosis, a process known as negative selection. Negative selection is extremely important for establishing a functional immune system, as it provides an efficient mechanism for ridding the T-cell repertoire of self-reactive and potentially autoimmune lymphocytes. This review discusses several cellular and molecular aspects of negative selection.

Abstract: It is important to understand how T-cell antigen receptor (TCR) engagement and signaling are regulated throughout an immune response. This review examines the dynamics of surface TCR expression and signaling capacity during thymic and effector T-cell development. Although the TCR can undergo vast changes in surface expression, T cells remain capable of sustaining TCR engagement for long periods of time. This may be achieved by a combination of mechanisms that involve (a) controlling the quantity of surface TCR available for ligand interaction and (b) controlling the quality of surface TCR expression during T-cell activation. TCR signaling itself appears to be one of the main quantitative modulators of surface TCR expression, and it can cause both downregulation and upregulation at different times of T-cell activation. Recent studies indicate that the degree of upregulation is tunable by the strength of antigenic stimulation. There is evidence that qualitatively distinct forms of the TCR exist, and their potential role in sustained antigenic signaling is also discussed. A goal of future studies will be to better characterize these modulations in surface TCR expression and to clarify their impact on the regulation of immune responses.

Abstract: The origin and specificity of alphabeta TCR(+) T cells that express CD8alphaalpha have been controversial issues. Here we provide direct evidence that precursors of functional CD8alphaalpha T cells are positively selected in the thymus in the presence of agonist self-peptides. Like conventional positive selection, this agonist selection process requires functional TCR alpha-CPM, whereas it is independent of CD8beta expression. Furthermore, CD8alphaalpha expression on mature, agonist-selected T cells does not imply selection by MHC class I, and CD8alphaalpha(+) T cells can be either class I or class II restricted. Our data define a distinct agonist-dependent, positive selection process in the thymus, and they suggest a function for CD8alphaalpha distinct from the conventional TCR coreceptor function of CD8alphabeta or CD4.

Abstract: To generate peripheral T cells that are both self-MHC restricted and self-MHC tolerant, thymocytes are subjected to positive and negative selection. How the TCR discriminates between positive and negative selection ligands is not well understood, although there is substantial evidence that the CD4 and CD8 coreceptors play an important role in this cell fate decision. We have previously identified an evolutionarily conserved motif in the TCR, the alpha-chain connecting peptide motif (alpha-CPM), which allows the TCR to deliver positive selection signals. Thymocytes expressing alpha-CPM-deficient receptors do not undergo positive selection, whereas their negative selection is not impaired. In this work we studied the ligand binding and receptor function of alpha-CPM-deficient TCRs by generating T cell hybridomas expressing wild-type or alpha-CPM-deficient forms of the T1 TCR. This K(d)-restricted TCR is specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide(252-260) IASA-YIPSAEK(ABA)I and is therefore amenable to TCR photoaffinity labeling. The experiments presented in this work show that alpha-CPM-deficient TCRs fail to cooperate with CD8 to enhance ligand binding and functional responses.

Abstract: Signal transduction in T cells is a dynamic process involving a large number of membrane and cytosolic proteins. The TCR macromolecular complex (signalosome) is initiated by receptor occupancy and becomes more elaborate over time. This review describes how 'vertical' displacement mechanisms and lateral coalescence of lipid-raft-associated scaffold proteins combine to form distinct signalosomes, which control signal specificity.

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Abstract: Both the direct and indirect antigen presentation pathways are important mechanisms for T cell-mediated allograft rejection. Studies using knockout mice and monoclonal antibodies have demonstrated that CD4+ T cells are both necessary and sufficient for the rejection of allogeneic tissues, including skin, heart, and islet. Furthermore, combined blockade of the CD28/B7 and CD154/CD40 costimulatory pathways induces tolerance in multiple CD4+ T-cell dependent allograft models. In this study, we addressed the T-cell requirement for costimulation in direct antigen presentation. We demonstrated that class II-specific alloreactive T-cell receptor transgenic T cells were sufficient to mediate allograft rejection independent of costimulatory blockade. Analysis of the costimulatory capacity of different antigen presenting cell (APC) populations demonstrated that APCs resident within the donor skin, Langerhans cells, are potent stimulators not requiring CD28- or CD154-dependent costimulation for direct major histocompatibility complex (MHC) antigen presentation. These results complement previous work examining the role of costimulation on CD8+ T cells, supporting a model in which the effectiveness of costimulatory blockade in the setting of transplantation may be selective for the indirect pathway of MHC alloantigen presentation.

Abstract: Natural killer T (NKT) cells are a subset of mature alpha beta TCR(+) cells that co-express NK lineage markers. Whereas most NKT cells express a canonical Valpha14/Vbeta8.2 TCR and are selected by CD1d, a minority of NKT cells express a diverse TCR repertoire and develop independently of CD1d. Little is known about the selection requirements of CD1d-independent NKT cells. We show here that NKT cells develop in RAG-deficient mice expressing an MHC class II-restricted transgenic TCR (Valpha2/Vbeta8.1) but only under conditions that lead to negative selection of conventional T cells. Moreover development of NKT cells in these mice is absolutely dependent upon an intact TCR alpha-chain connecting peptide domain, which is required for positive selection of conventional T cells via recruitment of the ERK signaling pathway. Collectively our data demonstrate that NKT cells can develop as a result of high avidity TCR/MHC class II interactions and suggest that common signaling pathways are involved in the positive selection of CD1d-independent NKT cells and conventional T cells.

Abstract: Alloreactive T cell precursor frequency was measured in vivo using fluorescent dye labeling in combination with novel models based on lymphocyte activation and recovery. CFSE-labeled C57BL/6 (H-2(b)) spleen and lymph node cells were adoptively transferred to C57BL/6xDBA F(1) (H-2(b/d)) recipients, a parent-->F(1) MHC mismatch in which only donor cells respond. Recipients were sacrificed at serial time points to assess engraftment efficiency, and the extent of donor cell activation and proliferation. These data were used to calculate alloreactive T cell frequencies that varied 30-fold (0.71 +/- 0.31% to 21.05 +/- 3.62%), depending upon whether it was assumed that all donor cells injected became established and were capable of responding, or that only those present at later time points (24-72 h) were available to respond. By measuring the number of cells established in the recipient 24 h after transfer, before proliferation, we calculated an in vivo alloreactive frequency of approximately 7%. Using CD69 expression at 48 h to quantify activation, we found that 40-50% of the alloactivated CD4(+) donor T cells do not divide. Studies of cotransferred congenic and allogeneic cells demonstrated that bystander proliferation does not occur. We conclude that accurate calculations of alloreactive precursor frequency must account for both proliferation and cell engraftment. When this is done, a high percentage of alloreactive T cells exists across an MHC mismatch, but not all alloreactive cells proliferate in vivo. Bystander proliferation is negligible, revealing exquisite specificity to the alloresponse. These data provide a novel approach to quantify alloreactive T cell responses during specific immunomodulatory strategies in vivo.

Abstract: The widely accepted kinetic proofreading theory proposes that rapid TCR dissociation from a peptide/MHC ligand allows for stimulation of early but not late T cell activation events, explaining why low-affinity TCR ligands are poor agonists. We identified a low-affinity TCR ligand which stimulated late T cell responses but, contrary to predictions from kinetic proofreading, inefficiently induced early activation events. Furthermore, responses induced by this ligand were kinetically delayed compared to its high-affinity counterpart. Using peptide/MHC tetramers, we showed that activation characteristics could be dissociated from TCR occupancy by the peptide/MHC ligands. Our data argue that T cell responses are triggered by a cumulative signal which is reached at different time points for different TCR ligands.

Abstract: A key role in the communication between the alphabetaTCR and the CD3/zeta complex is played by a specific motif within the connecting peptide domain of the TCR alpha chain (alpha-CPM). T cell hybridomas expressing an alpha-CPM-mutated TCR show a dramatic impairment in antigen-driven interleukin-2 production. This defect can be complemented by a calcium ionophore, indicating that activation of the calcium pathway is impaired. Several lines of evidence implicate Fyn in the regulation of calcium mobilization, at least in part through the activation of phospholipase Cgamma. Here we have investigated the potential involvement of Fyn in the TCR alpha-CPM signaling defect. Using T cell hybridomas expressing either a wild-type TCR or an alpha-CPM mutant, we show that Fyn fails to be activated by the mutant receptor following SEB binding and fails to generate tyrosine-phosphorylated Pyk2, a member of the focal adhesion kinase family. This defect correlated with an impairment in phospholipase Cgamma phosphorylation. Production of interlukin-2 and activation of the transcription factor NF-AT in response to triggering of the TCR alpha-CPM mutant with SEB were fully restored in the presence of constitutively active Fyn. Hence the signaling defect generated by the TCR alpha-CPM mutation results at least in part from an impaired coupling of the TCR.CD3 complex to Fyn activation.

Abstract: T cell development is tightly controlled by thymic stromal cells. Alterations in stromal architecture affect T cell maturation and the development of self-tolerance. The monogenic autoimmune syndrome APECED (autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy) is characterized by the loss of self-tolerance to multiple organs. Although mutations in the autoimmune regulator (AIRE) gene are responsible for this disease, the function of AIRE is not known. Here we report on the spatial and temporal pattern of murine Aire expression during thymic ontogeny and T cell selection. Early during development, thymic Aire transcription is critically dependent on RelB and occurs in epithelial cells in response to lymphocyte-mediated signals. In adult tissue, Aire expression is confined to the medulla and the corticomedullary junction, where it is modulated by thymocytes undergoing negative selection. Aire may determine thymic stromal organization and with it the induction of self-tolerance.

Abstract: To investigate the molecular basis that makes heterodimeric CD8alphabeta a more efficient coreceptor than homodimeric CD8alphaalpha, we used various CD8 transfectants of T1.4 T cell hybridomas, which are specific for H-2Kd, and a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). We demonstrate that CD8 is palmitoylated at the cytoplasmic tail of CD8beta and that this allows partitioning of CD8alphabeta, but not of CD8alphaalpha, in lipid rafts. Localization of CD8 in rafts is crucial for its coreceptor function. First, association of CD8 with the src kinase p56lck takes place nearly exclusively in rafts, mainly due to increased concentration of both components in this compartment. Deletion of the cytoplasmic domain of CD8beta abrogated localization of CD8 in rafts and association with p56lck. Second, CD8-mediated cross-linking of p56lck by multimeric Kd-peptide complexes or by anti-CD8 Ab results in p56lck activation in rafts, from which the abundant phosphatase CD45 is excluded. Third, CD8-associated activated p56lck phosphorylates CD3zeta in rafts and hence induces TCR signaling and T cell activation. This study shows that palmitoylation of CD8beta is required for efficient CD8 coreceptor function, mainly because it dramatically increases CD8 association with p56lck and CD8-mediated activation of p56lck in lipid rafts.

Abstract: Positive selection allows thymocytes that recognize an individual's own major histocompatibility complex (self-MHC) molecules to survive and differentiate, whereas negative selection removes overtly self-reactive thymocytes. Although both forms of thymic selection are mediated by the alphabeta T-cell receptor (TCR) and require self-MHC recognition, an important question is whether they are controlled by distinct signalling cascades. We have shown that mutation of an essential motif within the TCR alpha-chain-connecting peptide domain (alpha-CPM) profoundly affects positive but not negative selection. Using transgenic mice expressing a mutant alpha-CPM TCR we examined the contribution of several mitogen-activated protein kinase (MAPK) cascades to thymic selection. Here we show that in thymocytes expressing a mutant alpha-CPM receptor, a positively selecting peptide failed to activate the extracellular signal-regulated kinase (ERK), although other MAPK cascades were induced normally. The defect in ERK activation was associated with impaired recruitment of the activated tyrosine kinases Lck and ZAP-70, phosphorylated forms of the TCR component CD3zeta and the adaptor protein LAT to detergent-insoluble glycolipid-enriched microdomains (DIGs). Therefore, an intact DIG-associated signalosome is essential for sustained ERK activation, which leads to positive selection.

Abstract: Antagonist peptides inhibit T cell responses by an unknown mechanism. By coexpressing two independent T cell receptors (TCRs) on a single T cell hybridoma, we addressed the question of whether antagonist ligands induce a dominant-negative signal that inhibits the function of a second, independent TCR. The two receptors, Valpha2Vbeta5 and Valpha2Vbeta10, restricted by H-2Kb and specific for the octameric peptides SIINFEKL and SSIEFARL, respectively, were coexpressed on the same cell. Agonist stimulation demonstrated that the two receptors behaved independently with regard to antigen-induced TCR downregulation and intracellular biochemical signaling. The exposure of one TCR (Valpha2Vbeta5) to antagonist peptides could not inhibit a second independent TCR (Valpha2Vbeta10) from responding to its antigen. Thus, our data clearly demonstrate that these antagonist ligands do not generate a dominant-negative signal which affects the responsiveness of the entire cell. In addition, a kinetic analysis showed that even 12 h after engagement with their cognate antigen and 10 h after reaching a steady-state of TCR internalization, T cells were fully inhibited by the addition of antagonist peptides. The window of susceptibility to antagonist ligands correlated exactly with the time required for the responding T cells to commit to interleukin 2 production. The data support a model where antagonist ligands can competitively inhibit antigenic peptides from productively engaging the TCR. This competitive inhibition is effective during the entire commitment period, where sustained TCR engagement is essential for full T cell activation.

Abstract: The zeta-chain homodimer is a key component in the TCR complex and exerts its function through its cytoplasmic immunoreceptor-tyrosine activation motif (1). The zeta-chain extracellular (EC) domain is highly conserved; however, its functional and structural contributions to the TCR signaling have not been elucidated. We show that the EC domain of the zeta homodimer is essential for TCR surface expression. To gain a more detailed structural and functional information about the zeta-chain EC domain, we applied a cysteine scanning mutagenesis to conserved amino acids of the short domain. The results showed that the interchain disulfide bridge can be displaced by seven or eight amino acids along the EC domain. The TCR signaling efficacy was dramatically reduced during peptide/MHC engagement in the zeta mutants containing the displaced disulfide bond. These signaling defective zeta mutants produced an unconventional early tyrosine phosphorylation pattern. While the tyrosine phosphorylated forms of zeta (p21 and p23) could be observed during Ag stimulation, downstream signaling events such as the generation of phospho-p36, higher m.w. forms of phospho-zeta, and phospho-zeta/ZAP-70 complexes were impaired. Together these results suggest an important function of the phylogenetically conserved zeta-EC domain.

Abstract: Hybrids of New Zealand Black (NZB) and New Zealand White (NZW) mice spontaneously develop a disease similar to human systemic lupus erythematosus. MHC and non-MHC genes contribute to disease susceptibility in this murine model. Multiple studies have shown that the NZW H2z locus is strongly associated with the development of lupus-like disease in these mice. The susceptibility gene(s) within H2z is not known, but different lines of evidence have pointed to class II MHC genes, either H2-E or H2-A (Ez or Az in NZW). Recent studies from our laboratory showed that Ez does not supplant H2z in the contribution to lupus-like disease. In the present work we generated C57BL/10 (B10) mice transgenic for Aaz and Abz genes (designated B10.Az mice) and used a (B10.Az x NZB)F1 x NZB backcross to assess the contributions of Az genes to disease. A subset of backcross mice produced high levels of IgG autoantibodies and developed severe nephritis. However, no autoimmune phenotype was linked to the Az transgenes. Surprisingly, in the same backcross mice, inheritance of H2b from the nonautoimmune B10 strain was strongly linked with both autoantibody production and nephritis. Taken together with our previous Ez studies, the present work calls into question the importance of class II MHC genes for lupus susceptibility in this model and provides new insight into the role of MHC in lupus-like autoimmunity.

Abstract: Unlike parental New Zealand Black (NZB) or New Zealand White (NZW) mice, (NZB x NZW)F1 mice exhibit a lupus-like disease characterized by IgG autoantibody production and severe immune complex-mediated nephritis. In studies of the genetic susceptibility to disease in this F1 model, the NZW MHC (H2z) has been strongly linked with the development of disease, and it was hypothesized that class II MHC genes, particularly Ez genes, may underlie this genetic contribution. In the present study, we bred transgenic B6 mice expressing I-Ez or congenic B6 mice carrying H2z with NZB mice and used a backcross analysis to test the hypothesis that Ea(z) and/or Eb(z) genes account for the effect of H2z on disease. The genetic analysis of different backcross combinations showed that unlike mice carrying H2z, mice inheriting Ez transgenes do not demonstrate increased IgG autoantibody production or increased incidence of nephritis. Surprisingly, in the same transgenic backcross mice, inheritance of the endogenous H2b from the B6 strain was strongly linked with the production of IgG autoantibodies, but not with disease. Additional experiments suggested that the level of IgG3 autoantibody production, which is controlled by H2, may be important in the pathogenesis of renal disease. Contributions to autoantibody production were also detected from an NZB locus on distal chromosome 1 (previously named Nba2). Together, these studies provide new insight into the role of MHC in lupus-like autoimmunity.

Abstract: The T cell receptor (TCR) is internalized following activation of protein kinase C (PKC) via a leucine (Leu)-based motif in CD3gamma. Some studies have indicated that the TCR is recycled back to the cell surface following PKC-mediated internalization. The functional state of recycled TCR and the mechanisms involved in the sorting events following PKC-induced internalization are not known. In this study, we demonstrated that following PKC-induced internalization, the TCR is recycled back to the cell surface in a functional state. TCR recycling was dependent on dephosphorylation of CD3gamma, probably mediated by the serine/threonine protein phosphatase-2A, but independent on microtubules or actin polymerization. Furthermore, in contrast to ligand-mediated TCR sorting, recycling of the TCR was independent of the tyrosine phosphatase CD45 and the Src tyrosine kinases p56(Lck) and p59(Fyn). Studies of mutated TCR and chimeric CD4-CD3gamma molecules demonstrated that CD3gamma did not contain a recycling signal in itself. In contrast, the only sorting information in CD3gamma was the Leu-based motif that mediated lysosomal sorting of chimeric CD4-CD3gamma molecules. Finally, we found a correlation between the phosphorylation state of CD3gamma and T cell responsiveness. Based on these observations a physiological role of CD3gamma and TCR cycling is proposed.

Abstract: The two lineages of T cells, alphabeta and gammadelta, differ in their developmental requirements: only alphabeta T cells require major histocompatibility complex recognition, a process known as positive selection. The alphabeta T cell receptor (TCR), but not its gammadelta counterpart, contains a motif within the alpha-chain connecting peptide domain (alpha-CPM) that has been conserved over the last 500 million years. In transgenic mice expressing an alphabeta TCR lacking the alpha-CPM, thymocytes were blocked in positive selection but could undergo negative selection. Thus, the alpha-CPM seems to participate in the generation of signals required for positive selection.

Abstract: Antigen stimulation through the T cell receptor (TCR) induces phosphorylation of the associated CD3 gamma delta epsilon- and zeta-chain cytoplasmic tails. These events lead to the induction of the intracellular signaling pathways with concomitant receptor down-regulation. The TCR is down-regulated from the cell surface by the activation of protein kinase, C (PKC) and subsequent serine phosphorylation of the CD3 gamma-chain. We report here that the TCR alpha-chain cytoplasmic tail is also necessary for PKC-mediated internalization of the TCR complex. The requirement for the TCR alpha-chain cytoplasmic tail is specific for internalization of the TCR complex, since down-regulation of CD4 is still intact in hybridoma cells expressing a tailless TCR alpha-chain. The absence of TCR internalization directly correlates with defective PKC-mediated phosphorylation of the CD3 gamma-chain. Despite deficient PKC-mediated TCR down-regulation, the tailless alpha beta TCR still transduces antigenic signals resulting in the production of interleukin-2. Although the TCR tails are not obviously required for signal transduction, the TCR alpha-tail may serve as a targeting domain for PKC-mediated down-regulation of the TCR complex.

Abstract: A single amino acid residue, Gln136, located within the connecting peptide domain of Cbeta controls the ability of the alpha/beta TCR to transmit a full signal. TCRs in which this Cbeta residue is mutated to Phe, the residue found in TCR-gamma, are unresponsive to antigenic ligands. Interestingly, this Cbeta residue is either polar or charged in every species studied thus far, including the trout and the skate. In contrast, the analogous residue in Cgamma is always hydrophobic. In spite of their compromised antigen responsiveness, the mutant TCR complex contains the CD3-gamma, -delta, -epsilon, and -zeta chains, and undergoes zeta chain phosphorylation and ZAP-70 recruitment. However, the biological response of the mutant TCR could be rescued with a calcium ionophore, implying that mutant TCRs are defective in generating a calcium-mediated signal. The implications of the differences between Cbeta and Cgamma are considered.

Abstract: The zeta chain is required in the TCR complex to guarantee its surface expression and function. However, an understanding of the interaction(s) between the zeta chain and the other proteins in the TCR/CD3 has not yet been achieved. In this report, we attempt to assign a functional role to the short extracellular (EC) domain of the zeta chain by studying its unique positive charge, a lysine at position 9, because of its interesting location to the interchain disulphide bond of the zeta chain homodimer. We show that amino acid exchanges of lysine 9 to glycine, serine, cysteine or asparagine generate TCR complexes which are clearly defective in antigenic signalling. Interestingly, the non-conservative point mutations were segregating TCR complex signalling pathways. However, lysine 9 is not critical for TCR complex surface expression unless the positively charged lysine is exchanged for the negatively charged amino acid aspartic acid. The zeta chain mutant bearing a lysine to cysteine exchange is the sole mutant to be inefficiently co-precipitated with the TCR/CD3 complex suggesting a loose interaction of the zeta chain within the TCR complex.

Abstract: Mutant alphabeta TCRs were generated by replacing domains of the alpha and beta chain constant regions with homologous domains from TCR delta and gamma chains, respectively. Chimeric TCRs in which the alpha chain contains TCR delta chain sequences within the connecting peptide domain are unresponsive to alloantigens and superantigens, and have defective interactions with the CD3/zeta complex. Although these antigen-unresponsive TCRs undergo zeta chain phosphorylation upon stimulation with superantigen, they do not generate a full signal capable of producing IL-2. Mutant TCRs acquire signaling activity with a combination of superantigen and calcium ionophore, indicating a defect in calcium-mediated signaling. Finally, a conserved motif, FETDxNLN, present in the alpha chain connecting peptide domain, is disrupted in all signaling-defective TCRs. This conserved alpha chain connecting peptide motif might mediate the transfer of signals from the alphabeta heterodimer to the CD3/zeta complex.

Abstract: F1 progeny of New Zealand Black (NZB) and New Zealand White (NZW) mice spontaneously develop an autoimmune process remarkably similar to human systemic lupus erythematosus. Previous studies have implicated major genetic contributions from the NZW MHC and from a dominant NZB gene on chromosome 4. To identify additional NZB contributions to lupus-like disease, (NZB x SM/J)F1 x NZW backcross mice were followed for the development of severe renal disease and were comprehensively genotyped. Despite a 50% incidence of disease, significant associations between the presence of the NZB genotype and disease were noted on chromosomes 1, 4, 7, 10, 13, and 19. The data indicated that multiple NZB genes, in different combinations, contribute to severe renal disease, and that no single gene is required. To further investigate this NZB contribution, NZB x SM/J (NXSM) recombinant inbred (RI) strains were crossed with NZW mice, and F1 progeny were analyzed for the presence of lupus-like renal disease. Interestingly, nearly all of the (RI x NZW)F1 cohorts studied expressed some level of disease. Five RI strains generated a high incidence of disease, similar to (NZB x NZW)F1 mice, and nearly one-half of the cohorts developed disease at intermediate levels. Only two cohorts demonstrated very little disease, supporting the conclusion that multiple genes are capable of disease induction. Experiments correlating the genotypes of these RI strains with their ability to generate disease revealed that none of the disease-associated loci defined by the backcross analysis were present in all five RI strains that generated disease at high levels. Overall, both the backcross data and RI analysis provide additional support for the genetic complexity of lupus nephritis and uphold the conclusion that heterogeneous combinations of contributing NZB genes seem to operate in a threshold manner to generate the disease phenotype.

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Abstract: Current models of T cell receptor (TCR) structure are generally based on the homology observed between the TCR and the immunoglobulins. Furthermore, these models have predicted the locations of framework and complementarity determining regions within the alpha- and beta-chain variable regions. In order to test the validity of these models, we have generated a series of mutations within the V beta domain of an allo-reactive TCR and determined their effect on antigen recognition.

Abstract: We have compared the transmembrane sequences of 72 vertebrate antigen receptor (mIg and TCR) polypeptides. This allowed us to identify a Conserved Antigen Receptor Transmembrane (CART) motif which is present in all antigen receptor transmembrane domains from species as far removed as cartilaginous fish. Most of the amino acids in the CART motif are polar or aromatic and may interact with other proteins in the lipid environment. In addition, modeling the antigen receptor transmembrane domain in an alpha helical conformation places the CART residues on one face of the alpha helix. Thus, the CART motif may encode a structural unit which plays a role in the assembly and/or the signaling properties of lymphocyte antigen receptors. We speculate on the potential role of the CART motifs in lymphocyte signaling.

Abstract: Lupus-like autoimmunity in (NZB x NZW)F1 mice is frequently marked by the development of a severe and fatal renal disease. Genes from both NZB and NZW parents are required for the full expression of disease. We applied a mapping technique based on polymorphism in simple sequence repeats to the analysis of (NZB x NZW)F1 x NZW backcross mice to determine the NZB genetic contribution to disease. The results show that a single NZB locus or tightly linked group of loci on the distal part of chromosome 4 provides the strongest association with renal disease and death. This locus, designated here as nba-1 (New Zealand Black autoimmunity), lies distal to the locus elp-1, 60-70 centimorgans from the centromere. It is of interest that a gene encoding a receptor for tumor necrosis factor maps to the vicinity of this disease-associated gene.

Abstract: The hapten/class I MHC-specific soluble immunoregulatory molecules produced by CD8+T cells from dinitrobenzene sulfonate-primed mice express the binding specificity and serologic determinants of alpha/beta TCR. To examine the genes used to encode these soluble immunoregulatory molecules, we utilized a surface TCR expressing Ts hybridoma, which constitutively produces a DNP/Kd-specific regulatory molecule. Northern and Southern analyses indicated that MTs 79.1 cells use a V beta 8 and a V alpha 4 gene to encode the variable regions of the surface alpha/beta TCR. A panel of TCR- variants was generated by subjecting MTs 79.1 cells to gamma-irradiation. Twelve of the TCR- variants were chosen for detailed characterization. Northern blot analyses indicated the absence of the MTs 79.1 V alpha 4 chain transcript in five of the variants and the absence of the parental V beta 8 chain transcript in the other seven. Southern blot analyses demonstrated the deletion of the parental gene encoding the alpha- or beta-chain from the genome of the respective mutant. None of the 12 TCR gene deletion mutants produced the parental suppressive activity. Expression of the parental TCR beta-chain gene in one of the beta-chain gene deletion mutants reconstituted the ability to produce this activity. As with the MTs 79.1 molecule, the regulatory molecule produced by the beta-chain gene transfectant was bound by and eluted from Sepharose columns coupled with either DNP or anti-V beta 8 antibodies. These results establish a strong linkage between the suppressor molecules produced by these Ts and TCR alpha- and beta-chain gene transcription.

Abstract: Self-tolerance is the process by which the T cell repertoire develops without expressing self-reactive specificities. The mechanisms which functionally eliminate self-reactive T cells are clonal deletion and clonal inactivation, and both of these phenomena have been studied in T cell populations reactive to endogenous superantigens that are encoded by endogenous mouse mammary tumor proviruses (Mtv). The studies described here demonstrate that the kinetics of Etc-1 (encoded by the Mtv-9 open reading frame gene)-mediated deletion are much slower than that seen for Mls 1a (encoded by the Mtv-7 open reading frame), and that Etc-1-reactive T cells are present in the periphery up to 2 wk after birth. The deletion of peripheral Etc-1-reactive T cells late in ontogeny indicates an efficient mechanism of peripheral clonal deletion in these animals. The clonal deletion of Etc-1-reactive cells is abrogated in B cell-suppressed animals; however, clonal elimination of peripheral V beta 5+ or V beta 11+ (Etc-1 reactive) T cells can be induced when these mice are allowed to recover their B cell population after cessation of anti-mu treatment. Finally, we establish that peripheral Etc-1-reactive V beta 11+/CD4+ T cells remaining in B cell-suppressed and recovering animals are markedly less responsive to stimulation through the TCR than are control T cells. These data support the idea that peripheral self-reactive T cells can be rendered tolerant by two mechanisms which may be temporally related. This model suggests that clonal hyporesponsiveness may be followed by clonal deletion.

Abstract: Mls-1 is an endogenous superantigen that leads to in vivo deletion and in vitro stimulation of T cell receptor (TCR) V beta 6-, 7-, 8.1-, and 9-expressing cells. The MA/MyJ mouse deletes the identical set of TCR from its mature T cell repertoire; however, it does not contain Mtv-7, the murine mammary tumor provirus (MMTV), whose sag gene encodes Mls-1. Interestingly, the superantigen activity of this mouse strain segregates with a new mammary tumor provirus, Mtv-43, not seen in other inbred strains. The predicted amino acid sequence of the sag gene of Mtv-43 was compared with that of Mtv-7. Strikingly, the COOH terminus of the two molecules is very similar, while all other MMTV-encoded superantigens differ 100% in this segment.

Abstract: A monoclonal antibody, B20.1, was generated by fusing spleen cells from a Lou rat immunized with a soluble alpha/beta T cell receptor (TcR; V alpha 2/V beta 2) to mouse myeloma cells. Analysis of a panel of V alpha 2 mRNA-expressing T cell lines, hybridomas and transfectants revealed that the B20.1 antibody was specific for murine TcR V alpha 2 chains. The V alpha 2+ T cell population was examined in various inbred strains by two-color immunofluorescence using B20.1 and CD4- and CD8-specific antibodies with the following results: (a) the B20.1 antibody detected most members of the TcR V alpha 2 subfamily in the four TcR V alpha haplotypes tested; (b) in most strains examined, TcR V alpha 2 expression was biased to the CD4 subset (7.4%-17.4% V alpha 2+ T cells) as compared to the CD8 compartment (3.8%-13.3%); (c) TcR V alpha 2 expression was not influenced by Mls gene products and (d) increased positive selection of V alpha 2+ CD8+ T cells by H-2k major histocompatibility complex molecules occurred in all murine strains tested of the TcR V alpha a, but not in those bearing the TcR V alpha b haplotype.

Abstract: Several murine superantigens in association with class II major histocompatibility complex proteins have been shown to cause the deletion of T cells based on the expression of particular beta-chain variable region (V beta) gene segments. We have previously shown that mice expressing the Etc-1 superantigen, encoded by an open reading frame within the 3' long terminal repeat of the endogenous mouse mammary tumor provirus (Mtv), Mtv-9, delete T cells expressing either V beta 5 or V beta 11 gene segments. Comparison of several Mtv 3' long terminal repeat open reading frame sequences has indicated that the carboxyl terminus likely encodes the V beta specificity of these proteins. Our analysis of C57BL/6 x DBA/2 recombinant inbred strains of mouse revealed three Mtv-9-negative strains that nevertheless have a low frequency of V beta 5-expressing T cells. Here we demonstrate that a second endogenous superantigen, responsible for the deletion of V beta 5-bearing T cells, is encoded by a gene mapping to Mtv-6 on chromosome 16. Surprisingly, the carboxyl-terminal sequences of the Mtv-6 and -9 superantigens are extremely divergent, in spite of the fact that they both mediate the deletion of V beta 5+ lymphocytes.

Abstract: We have characterized a panel of monoclonal antibodies (mAb) produced by immunizing rats with two distinct soluble mouse alpha/beta T cell receptor (TcR). Fifty mAb were found to react with the corresponding surface-bound TcR. Such observations suggest that the soluble TcR molecules used as immunogen are folded in a conformation similar to the native structure. Furthermore, the binding to T cells of four antibodies was found to correlate with the expression of the V alpha 8, V beta 2 or V beta 10 gene segments. Finally, staining of T lymphocytes from various mouse strains suggests that (a) the two anti-V alpha 8 antibodies recognize different epitopes, and each on only a fraction of V alpha 8+ cells; (b) the anti-V beta 10 mAb identifies a V beta 10 polymorphism among mouse strains, and (c) T cells expressing the V beta 2 or V beta 10 gene segments are not subject to major clonal deletion events induced by the major histocompatibility complex class II and Mls products which were tested.

Abstract: The identity of minor lymphocytes stimulating (Mls) antigens, endogenous superantigens that can activate, or induce the deletion of, large portions of the T-cell repertoire, has recently been revealed: they are encoded by mouse mammary tumor viruses (MMTV) that have integrated into the germ line as DNA proviruses. As Hans Acha-Orbea and Ed Palmer point out, Mls-mediated modulation may be only the tip of the retrovirus iceberg; already murine leukemia virus (MuLV), with similar superantigen properties, has been discovered.

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Abstract: H-2b class I-restricted, TNP-specific CTL clones were obtained by limiting dilution cloning of either short term polyclonal CTL lines or spleen cells of TNP-immunized mice directly ex vivo. Sequence analyses of mRNA coding for TCR alpha- and beta-chains of 11 clones derived from CTL lines from individual C57BL/6 mice revealed that all of them expressed unique but clearly nonrandom receptor structures. Five alpha-chains (45%) employed V alpha 10 gene elements, and four of those (36%) were associated with J beta 2.6-expressing beta-chains. The alpha-chains from these four TCR, moreover, contained an acidic amino acid in position 93 of their N or J region-determined sequences. Clones isolated directly from spleen cells carried these types of receptors at lower frequency, 27% V alpha 10 and 19% J beta 2.6, indicating that bulk in vitro cultivation on Ag leads to selection for these particular receptors. However, even in TNP-specific CTL cloned directly ex vivo, V alpha 10 usage was increased about fivefold over that in Ag-independently activated T cells in H-2b mice (4 to 5%). The selection for V alpha 10/J beta 2.6-expressing cells was obtained repeatedly in other TNP-specific CTL lines from C57BL/6 mice but not in FITC-specific CTL from the same strain or in TNP-specific CTL lines from B10.BR (H-2k) or B10.D2 (H-2d) mice. We conclude from this (a) that the selection for V alpha 10/J beta 2.6+ T cells is driven by the complementarity of these receptors to a combination of TNP and MHC epitopes and (b) that predominant receptor structures reflect the existence of a surprisingly limited number of "T cell-relevant" hapten determinants on the surface of covalently TNP-modified cells.

Abstract: The majority of endogenous superantigens in the mouse (including the Mls loci) is encoded by mouse mammary tumor proviruses (Mtv) carried in the germline. To understand the differences between the highly stimulatory viral superantigens such as Mls-1a (encoded by Mtv-7), which have biologic activity in vivo and in vitro, and the poorly stimulatory viral superantigens such as Etc-1 (encoded by Mtv-9), which are active only in vivo, the physiologic expression of each Ag was studied in the Mtv-7+ (Mls-1a+), Mtv-9+ (Etc-1+) C57BL/6 x DBA/2 F1 (BDF1) mouse. Using the T cell hybridomas, 1BVB11.40 (anti Etc-1) and 18bbm.19 (anti Mls-1a), we found that similar to Mls-1a, B cells from the spleen and from the thymus present the Etc-1 superantigen, whereas macrophages and dendritic cells do not. Small, resting B cells present the Mls-1a and Etc-1 superantigens poorly; however, the same cells treated with LPS or IL-4 are at least eightfold more efficient in the presentation of these gene products. Furthermore, the effects of LPS and IL-4 are synergistic, but this synergy is not fully explained by the enhancement of I-A and I-E expression. The depletion of IgM+ B cells from neonatal BDF1 mice prevents the clonal deletion of V beta 5+ and 11+ (Etc-1-reactive) cells but not the deletion of V beta 6+ and 8.1+ (Mls-1a reactive) T cells. Despite the persistence of Mls-1a-mediated clonal deletion in B cell-depleted BDF1 mice, these results taken together, argue that the highly stimulatory Mls-1a gene product and the weakly stimulatory Etc-1 gene product are expressed on similar cell types and that their presentation is regulated in a similar way by agents active with B lymphocytes. It is argued that the differences between the highly stimulatory and weakly stimulatory superantigens reflect differences in avidity between the relevant V beta domain and its class II MHC protein/superantigenic ligand.

Abstract: The mAb MR9-4 and MR9-8 react with T cells expressing the V beta 5.1 and -5.2 chains of the TCR. T cells expressing V beta 5.1 TCR were stained by both antibodies with similar surface fluorescence intensity. For the T cell clones and hybridomas expressing V beta 5.2 TCR, staining intensity with MR9-8 varied from negative to comparable to that stained with the anti-pan V beta 5 mAb MR9-4, whereas every V beta 5-positive T cell can be activated with either MR9-4 or -9-8 mAb, suggesting a differential binding affinity of MR9-8 mAb to V beta 5 TCR molecules. Analysis of J beta segment and V alpha chain usage in the V beta 5-positive T cell hybridomas revealed that a differential binding of MR9-8 mAb to the V beta 5.2 chain is not dependent on either the J beta segment usage or the associating V alpha chain alone. These results suggest that the differential binding of MR9-8 mAb to V beta 5.2 TCR is due to the conformational change of the V beta chain created by a combination of the V alpha (possibly J alpha) and D beta-J beta segment associating with the V beta 5.2 chain.

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Abstract: Superantigens are defined by their ability to stimulate T cells based predominantly on their V beta expression and ability to delete T cells in the thymus when expressed endogenously. We show here that the expression of one endogenous superantigen, Etc-1, is controlled by the expression of the open reading frame region of the 3' long terminal repeat of the mouse mammary tumor proviral gene, Mtv-9. We show that Mtv-8 controls a superantigen with similar specificity, and that both Mtv-8 and Mtv-9 stimulate some V beta 17+ T cells. A third provirus, Mtv-6, controls a superantigen with specificity for V beta 3. Data presented raise the possibility that endogenous superantigens may compete for class II molecules in a single B cell.

Abstract: A special class of self-antigens (endogenous superantigens) is capable of deleting many murine T cells on the basis of their expression of particular T-cell receptor V beta gene segments. In mice that endogenously express these antigens, tolerance is mediated in part by the clonal deletion of the relevant V beta-bearing T cells. The deletion of I-E-reactive V beta 5.2-bearing T cells is dependent on the coexpression of an I-E tolerogenic coligand (Etc)14 and the gene for one of these coligands, Etc-1, maps to chromosome 12, near the mouse mammary tumour viral integrant, Mtv-9. Here we report a perfect genetic linkage between Etc-1 and Mtv-9 and show that Etc-1 is also involved in the I-E-dependent deletion of T cells bearing V beta 5.1 and V beta 11 domains. We also demonstrate that Mtv-9 transcripts are present in B cells expressing Etc-1 and suggest that the coligand recognized by roughly 15% of all T lymphocytes is encoded by the Mtv-9 genome.

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Abstract: The effects of quantitative differences in class II cell surface expression have been difficult to address in intact animals. This study uses several lines of H-2s/s mice carrying an A beta k transgene that differ significantly in terms of class II cell surface expression. Due to inefficient chain pairing, mice carrying 60 to 65 copies of this transgene express only low levels of A alpha s/A beta k on the cell surface, and cell surface expression of the endogenous A alpha s/A beta s complex (and total Ia) is severely reduced (to 7-15% control levels). The significant decrease in class II cell surface expression in the thymic cortex of these mice did not affect the frequency of peripheral T cells expressing at least 10 distinct TCR V beta chains. However, T cell proliferative responses to the A alpha s/A beta s-restricted peptide MBP 89-101 were abrogated in high copy number A beta k mice. Experiments using bone marrow chimeras demonstrated that both inefficient Ag presentation and failure to positively select appropriate T cells contributed to this lack of response. Inefficient Ag presentation was clearly the dominant defect, and the density of class II cell surface expression required for positive selection appeared to be quite low.

Abstract: Clonal deletion plays a major part in the maintenance of natural self-tolerance in both normal and transgenic mice. Self antigens that are expressed in the thymus result in the physical elimination of autoreactive thymocytes at a particular stage in their development. For example, the majority V beta 6- and V beta 8.1-bearing T cells that recognize the minor lymphocyte-stimulating antigen, Mls-1a (ref. 10) , are clonally deleted in the thymuses of normal mice and transgenic mice expressing Mls-1a (refs 2, 3, 9). In contrast, a very different mechanism of tolerance involving the functional inactivation, but not elimination, of autoreactive cells, termed clonal inactivation or clonal anergy, has been implicated in some experimentally manipulated systems of tolerance. To test further the mechanisms involved in self-tolerance, we have generated transgenic mice expressing a V beta 8.1 beta chain on greater than 95% of peripheral T cells and have tested tolerance to Mls-1a in these mice. Surprisingly, a significant fraction of the CD4+ peripheral cells that survived deletion were non-responsive in vitro to any stimulus tested. Naturally occurring tolerance to a self antigen expressed in the thymus can thus be mediated by clonal anergy, as well as by clonal deletion.

Abstract: The TCR beta-chain locus of NZW mice carries an 8.8-kb deletion which encompasses the C beta 1, D beta 2, and all six J beta 2 gene segments. On a theoretical basis, the absence of D beta 2 and J beta 2 gene segments in this strain should result in a 70% reduction of the diversity of the TCR repertoire. To experimentally assess the effects of this deletion, we bred the NZW TCR beta-chain allele onto a BALB/c background and tested the ability of this new congenic strain to respond to a panel of 22 random Ag. T cells from BALB/c.beta NZW mice responded to all 22 Ag tested but the magnitude of the response to a large proportion of these Ag (11 of 22) was markedly reduced when compared with T cells from BALB/c mice. Responses to the remaining Ag were either comparable (9 of 22) or occasionally even enhanced (2 of 22) compared with BALB/c mice. In addition, we found that the frequency of V beta 6- and V beta 8.1-bearing T cells was increased by approximately 20% in BALB/c.beta NZW mice. These results suggest that D beta 2 and J beta 2 gene segments are required to maintain a diverse T cell repertoire and that their deletion from the genome may confer a significant selective disadvantage in the wild.

Abstract: We have previously shown that T cells bearing V beta 5+ T-cell receptors (TCRs) are frequent in B10 (H-2b) and B10.Q (H-2q) mouse strains but are rare in the congenic strain B10.BR (H-2k). Furthermore, we have found that V beta 5 bearing T cells appear to be excluded from the B10 alloresponse to I-Abm12 despite the participation of most other V beta bearing cells. To further study MHC effects on V beta 5 expression, we have generated two V beta 5 specific monoclonal antibodies and show here that V beta 5 expressing T cells are clonally deleted from strains expressing a class II, I-E molecule. Furthermore, I-E- strains generate few CD4+ V beta 5+ T cells despite significant numbers of V beta 5+ T cells in the CD8+ subset. Thus, V beta 5 bearing T cells are positively selected by class I MHC molecules, clonally deleted by class II I-E molecules, and poorly selected by class II I-A molecules.

Abstract: T cells that express the T cell receptor V beta 5.2 domain react with the class II major histocompatibility complex (MHC) molecule I-E, and V beta 5.2+ T cells are deleted in mouse strains that express I-E glycoproteins. By examination of genetically defined recombinant inbred (RI) mouse strains, it was found that the deletion was dependent on the expression of I-E and one of a limited number of non-MHC gene products (cotolerogens). The gene encoding one of these cotolerogens maps to chromosome 12 and is linked to the endogenous provirus Mtv-9. These observations suggest that the I-E-mediated and minor lymphocyte-stimulating antigen (Mls)-mediated deletions of alpha beta T cells from the repertoire are similar; both require the expression of a class II MHC glycoprotein and a second non-MHC gene product.

Abstract: We have generated a rat mAb, TR310, which recognizes a determinant encoded by the murine V beta 7 gene segment of the TCR. TR310 immunoprecipitates TCR from cell lysates, co-modulates with CD3, and can be used for immunofluorescence staining of T cells. By using this antibody, we found that the average percentage of V beta 7+ peripheral T cells in Mls-1b mice was 3.8%, but only 0.8% in Mls-1a mice. A similar difference was also observed in the mature TCRhi thymocyte subsets, suggesting that V beta 7+ T cells are deleted during intrathymic maturation in Mls-1a mice. TR310 should prove to be a valuable reagent in further studies of the TCR repertoire and the analysis of factors which alter it.

Abstract: We have generated an mAb, RR3-15, that recognizes murine TCRs containing the V beta 11 domain. Using this antibody to stain peripheral T cells, we have demonstrated that V beta 11-bearing T cells are largely absent from strains of mice that express the class II MHC molecule, I-E. Studies with F1 mice demonstrate that this effect is dominant, consistent with tolerance. The clonal deletion of V beta 11-bearing T cells appears to occur intrathymically, as immature but not mature V beta 11+ T cells are present in the thymus of I-E-bearing mice. Examination of B6 x DBA/2 recombinant inbred strains demonstrates that the expression of I-E molecules is necessary for the clonal deletion of V beta 11-bearing T cells, but that other non-MHC genes control the clonal deletion process, as well. Paradoxically, only a small fraction of V beta 11+ T cell hybridomas are I-E reactive.

Abstract: We have studied the genetic diversity of the TCR repertoire to the murine alloantigen I-Abm12 by generating a panel of 178 C57BL/10-derived I-Abm12-reactive T cell hybridomas. The expression of V alpha and V beta gene families was examined in this panel and the frequency of expression of V beta, but not ofV alpha, gene families differed significantly from that observed in a companion panel of random C57BL/10-derived hybridomas. The V beta 5 gene family was expressed significantly less frequently while the V beta 14, V beta 15, and V beta 16 genes were expressed significantly more frequently in the panel of I-Abm12-reactive than in the panel of random hybridomas. The junctional regions (VJ alpha and VDJ beta) of TCR V alpha and V beta genes from selected I-Abm12-specific hybridomas were amplified using the polymerase chain reaction, and directly sequenced. Surprisingly, no conserved J alpha, D beta, J beta, or N region-encoded sequences among these selected I-Abm12-reactive TCRs were identified. Thus, the T cell response to an I-A alloantigen that differs by only three amino acid residues from the I-A molecule of the responding strain is genetically complex but nonrandom. We have estimated that the repertoire to this alloantigen is comprised of at least 37 different TCRs.

Abstract: We have examined the frequency of expression of individual alpha and beta chain V gene families in a population of immature T cells that has not been selected or tolerized. To accomplish this, we generated 519 T cell hybridomas from freshly isolated thymocytes of newborn C57BL/10 mice and subjected RNA from these hybrids to hybridization analysis with 11 V alpha, 16 V beta, C gamma and C delta probes. Comparison of the expressed repertoire of V beta gene segments in this newborn thymocyte population with similar data previously generated from adult peripheral T cells (Bill et al., Proc. Natl. Acad. Sci. USA 1988. 85: 9184.) revealed two V beta genes, V beta 12 and V beta 15, whose expression is decreased in the periphery possibly due to the effects of tolerance. An additional V beta gene segment (V beta 10) and a V beta gene family (V beta 5.1, V beta 5.2) were expressed more frequently in the mature, peripheral population than in the newborn thymus. These findings may represent two instances of positive selection of T cells. Furthermore, unlike VH gene segments, D beta-proximal V beta genes are not overrepresented on this collection of immature thymocyte hybridomas. A similar analysis of V alpha gene family expression was hampered by the fact that V alpha gene segments were expressed in only 15% of newborn thymocyte hybridomas (compared to 58% of adult hybridomas). An unexpectedly large fraction (57%) of those newborn hybrids expressing a V alpha gene segment was also found to express C delta mRNA and further examination revealed that several V alpha gene probes were actually detecting delta chain mRNA. The most notable of these was the V alpha 7 gene family, which accounted for approximately one-third of the expressed V alpha genes but was expressed exclusively as part of a delta chain mRNA. We found no examples of hybridomas co-expressing both full-length alpha and delta chain mRNA, despite significant numbers of hybridomas co-expressing full-length beta and gamma chain transcripts. This observation suggests that a large number of mature alpha/beta T cells may have proceeded through development without having gone through a stage where a delta chain message is expressed, thereby precluding their development into gamma/delta cells.

Abstract: Unlike parental New Zealand Black (NZB) or New Zealand White (NZW) mice, (NZB x NZW)F1 mice exhibit a lupus-like disease characterized by high serum levels of IgG anti-nuclear antibodies and a fatal immune complex glomerulonephritis. Previous results from studying [(NZB x NZW)F1 x NZB] backcross mice indicated that the NZW major histocompatibility complex (MHC) or gene(s) closely linked to this locus provides the major dominant NZW genetic contribution to the F1 disease. A surprising feature of the results was the 12% frequency of discordance between the autoimmune phenotype and the presence of the NZW H-2z haplotype. In the current study, we attempted to precisely define the position of the NZW gene(s) required for lupus-like renal disease by mapping genes in individual backcross mice that are both centromeric and telomeric to the MHC and then correlating genotypes for each locus with disease. The data indicate that an adjacent NZW locus does not provide a more accurate correlation with the autoimmune phenotype compared with MHC genes themselves. Thus, the imperfect association of MHC haplotype with disease in this murine model is not explained by genetic recombination with linked genes. These data may provide insight into the mechanisms by which MHC antigens increase the probability of developing autoimmune disease and may help explain the difficulty of defining MHC relationships in human systemic lupus erythematosus.

Abstract: The I-Abm12 mutation has been used extensively to study the relationship between structure and function of murine class II major histocompatibility molecules. I-Abm12 differs from I-Ab by three amino acid replacements in the A beta chain, and the proposed structural model of the I-Abm12 molecule places these three amino acid substitutions along one of the alpha-helices where they may affect both antigen and TCR binding. Two of the substitutions, Ile----Phe67 and Thr----Lys71, are thought to point into the binding site, whereas the third substitution, Arg----Gln70, is thought to point up and hence, be available for binding to the TCR. These predicted orientations are consistent with serologic analysis of the bm12 molecule, which demonstrates that residue 70 is uniquely accessible to mAbs distinguishing I-Ab from I-Abm12. In this study we have determined the influence of each of these amino acid substitutions on the ability of the resulting molecules to stimulate a panel of I-Abm12 (allo) reactive T cell hybridomas. Our experiments indicate that reversion of the amino acid at position 70 from Gln (I-Abm12) to Arg (I-Ab) interferes with allorecognition by 33 of 35 I-Abm12-reactive hybridomas. On the other hand, many hybrids can tolerate amino acid substitutions at positions 67 or 71. Single amino acid substitutions at position 67, 70, or 71 are recognized by only a minority of I-Abm12-specific hybrids and usually the reactivity is greatly diminished. These data are most consistent with the idea that the amino acid at position 70 directly interacts with the TCR during allorecognition. The additional effects of residues 67 and 71 are consistent with a contribution by bound peptide to the allorecognition process.

Abstract: Three rat mAb, RR3-15, RR3-16, and RR3-18, were established by fusing spleen cells from a rat immunized with the male Ag-specific cytolytic T cell clone, OH6, to mouse myeloma cells. The mAb was identified by their capacity to focus the cytolytic activity of the OH6 CTL clone on nonspecific target cells via FcR-FcR interaction. That all three mAb recognized the OH6 TCR was confirmed by immunoprecipitation studies in which each antibody precipitated a 90 kDa disulfide-linked heterodimer characteristic of the TCR. Surface immunofluorescence staining of a panel of T cell lines and splenic T cell populations showed that RR3-16 reacted not only to the OH6 T cell clone but also to a minor fraction of normal T cells. This reactivity was found to be due to the expression of a gene in the V alpha 3 family. However, RR3-16 did not react with all T cell lines and clones known to express genes from the V alpha 3 family. cDNA sequences of three independent RR3-16+ T cell hybridomas analyzed by polymerase chain reaction were identical to the previously published V alpha 3 sequence of the CTL clone C9. Thus, the mAb RR3-16 is specific for a single member of the TCR V alpha 3 gene family. Analysis of the expression of RR3-16+ TCR in CD4+ and CD8+ subsets of peripheral T cells demonstrated preferential expression on CD8+ T cells, suggesting regulated expression of this particular TCR V alpha gene.

Abstract: A monoclonal antibody secreting hybridoma was established by fusing spleen cells from a rat immunized with a murine T cell clone, OI11, which has I-Ab restricted specificity for the male H-Y antigen and unrestricted specificity for the minor lymphocyte stimulating antigen, Mls-1a, to the mouse myeloma P3X63AG8.653 and screening for the capacity of the hybridoma supernatants to stimulate the OI11 T cell clone. An antibody (RR4-7) was found to be specific not only for the immunizing T cell clone but virtually for all T cells using the V beta 6 TCR gene product as part of their surface antigen receptor. When the expression of the V beta 6 gene in various strains of mice was analyzed, it was found that strains expressing the Mls-1a antigen contained few T cells expressing V beta 6-encoded TCRs. The majority of T cell hybridomas which expressed V beta 6-encoded TCRs were found to be reactive to the Mls-1a antigen. These data confirm the finding of H. R. MacDonald et al. (Nature (London) 332, 40, 1988) that most TCRs encoded by the V beta 6 gene have a biased specificity for the Mls-1a antigen.

Abstract: To investigate the possible function(s) of T cell receptor (TcR) gamma delta expressing lymphocytes, we generated a series of gamma delta TcR surface positive hybridomas. Spontaneous producers of IL-2 were quite common among these hybridomas, particularly those expressing a certain V delta gene or gene family (V delta M23). Several other experiments indicated that IL-2 production in these hybridomas is triggered via TcR gamma delta. Surprisingly, every spontaneously reactive gamma delta+ hybridoma was further stimulated by purified protein derivative (PPD) of Mycobacterium tuberculosis, perhaps due to crossreaction with a bacterial antigen homologous to certain eukaryotic heat shock proteins. The finding of an antigen recognized by a gamma delta TcR could aid in understanding the functional role of the gamma delta TcR+ lymphocytes.

Abstract: T lymphocytes differentiate in the thymus, where functionally immature, CD4+CD8+ (double positive) thymocytes develop into functionally mature CD4+ helper cells and CD8+ cytotoxic (single positive) T cells. The thymus is the site where self-reactive T cells are negatively selected (clonally deleted) and where T cells with the capacity to recognize foreign antigens in association with self-proteins encoded by the major histocompatibility complex (MHC) are positively selected. The net result of these developmental pathways is a T-cell repertoire that is both self-tolerant and self-restricted. One unresolved issue is the identity of the thymic stromal cells that mediate the negative and positive selection of the T-cell repertoire. Previous work has pointed to a bone-marrow-derived macrophage or dendritic cell as the inducer of tolerance, whereas a radiation-resistant, deoxyguanosine-resistant thymic cell seems to mediate the positive selection of self-MHC restricted T cells. Thymic stromal cells in the cortex interact with the T-cell antigen receptor on thymocytes. Using several strains of transgenic mice that express the class II MHC molecule I-E in specific regions of the thymus, we show directly that the positive selection of T cells is mediated by an I-E-bearing cell in the thymic cortex.

Abstract: Using a quantitative RNA hybridization method, we have identified a third murine T-cell receptor beta-chain variable (V) region gene, V beta 9, that engenders recognition of Mls-1a gene products. V beta 9-expressing T cells are clonally deleted from the periphery of mice that carry the Mls-1a allele, and the fine specificity of V beta 9+ hybridomas suggests that V beta 6-, V beta 8.1-, and V beta 9-encoded T-cell receptors may recognize a similar antigenic epitope. Comparison of these three protein sequences identifies two residues that may be important in determining this recognition specificity.

Abstract: The data presented here indicated that both membrane Ig and IL-4 receptors transduce signals across the plasma membrane of quiescent B cells, which results in the induction of c-fos and c-myc proto-oncogene mRNA expression. Monoclonal anti-Ig antibodies with specificity for mu, delta, or kappa chains, regardless of mitogenicity, induced increased c-fos and c-myc mRNA expression with kinetics and magnitude similar to that observed following stimulation of B cells with IL-4. Maximal levels of c-fos mRNA, approximately 30-fold over background, were observed 30 min after stimulation. Maximal levels of c-myc mRNA, approximately 10-fold over background, were observed 60 min after stimulation. Phorbol myristate acetate alone induced expression of these two oncogenes in a similar fashion, suggesting that protein kinase C may be involved in the regulation of their expression following anti-Ig crosslinking. Ionomycin induced only a small increase in c-myc and c-fos message (three- to four-fold), and did not synergize with phorbol myristate acetate, suggesting that the membrane Ig-mediated calcium mobilization may not play a major role in regulation of c-myc or c-fos expression in mouse B cells. In vitro nuclear run-on analyses indicate that c-myc expression is primarily regulated post-transcriptionally, whereas c-fos expression is regulated at the level of transcription. Anti-sense transcription was found to be constitutive for both the c-myc and C-fos loci and was further induced by anti-Ig and IL-4, suggesting an additional mechanism for regulation of these genes. The observation that both anti-Ig and IL-4 regulate the expression of c-fos and c-myc suggests that multiple second messenger generating systems regulate the expression of these oncogenes in normal B cells and that their expression may be necessary, but is not sufficient to drive quiescent B cells into cell cycle.

Abstract: The physiological role of lymphocytes bearing the gamma delta T-cell receptor (TCR) is still unclear. A function for a subset of these cells, however, is inferred from the finding that certain gamma delta chain-bearing lymphocytes are stimulated in a receptor-dependent fashion by mycobacterial antigens. We found that hybridomas derived from such cells in newborn murine thymus not only responded to mycobacterial purified protein derivative (PPD), but also exhibited an apparent autoreactivity. In neither response was haplotype-specific major histocompatibility (MHC) restriction demonstrable. To investigate the nature of antigen recognition by these gamma delta+ cells, we sequenced the gamma- and delta-chains from 28 PPD-reactive hybridomas, and found that a specific gamma-chain, together with one of a limited set of delta-chains, was needed to generate the PPD specificity. The reactive gamma delta pairs exhibited considerable junctional diversity, which may act to produce differences in the fine specificities of the responding cells.

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Abstract: Recent evidence suggests that the major histocompatibility complex class II molecules of B lymphocytes function as signal-transducing receptors during the generation of T lymphocyte-dependent humoral immune responses. By analogy with other receptors, we postulate that perturbation of the class II molecules is coupled to the generation of intracellular second messengers through interactions involving the transmembrane and/or cytoplasmic domains of the class II molecules. We report a series of experiments that assess which amino acids of the class II molecule I-Ak are required for coupling it to the signal-transduction pathway. We prepared a series of B-lymphocyte transfectants that express I-Ak molecules with COOH-terminal truncations of either the Ak alpha or Ak beta chain or both. The ability of each transfected class II molecule to transduce a signal after being bound by monoclonal antibody was found by monitoring the translocation of protein kinase C from the cytosol to the "nuclear compartment" of the transfected B lymphocyte. Results indicate that the Ak beta chain plays the dominant role in signal transduction and that the 6 cytoplasmic amino acids of Ak beta chain most proximal to the inner plasma membrane are of greatest importance in coupling I-Ak molecules to the molecules of the signaling cascade.

Abstract: Our understanding of the immune mechanisms that lead to systemic lupus erythematosus has been greatly advanced by the availability of murine models which display both serological and clinical features of the human disease. Studies have demonstrated that CD4+ T cells are required for the full expression of disease in these mice. (NZB X NZW)F1 mice exhibit a lupus-like disease (elevated levels of IgG antinuclear antibodies and a fatal glomerulonephritis) that is not characteristic of either parent. At least three gene loci have been identified in NZW mice that could potentially contribute to a T cell-dependent autoimmune disease, including the T cell receptor alpha- and beta-chain gene complexes and the major histocompatibility complex (MHC). The NZW T cell receptor beta-chain complex appeared to be particularly unusual in that the C beta 1, D beta 2, and J beta 2 gene segments have been deleted. However, an analysis of (NZB X NZW)F1 X NZB back-cross mice revealed no association of disease expression with the presence of this allele. There was also no correlation of disease incidence with the presence of the NZW T cell receptor alpha-chain allele. In contrast, nearly 90% of the backcross mice with the NZW MHC expressed severe autoimmune disease compared with 12% of the mice that did not carry this haplotype. Additional studies strongly suggested that the gene(s) within the NZW MHC is the only dominant NZW genetic contribution to F1 disease. We also determined if self-reactive T cells are able to escape thymic tolerance in autoimmune New Zealand and MRLlpr/lpr mice. In nonautoimmune mice expressing I-E, T cells utilizing V beta 17a and V beta 11 encoded domains have been shown to be clonally eliminated in the thymus. Similarly, V beta 8.1+ and V beta 6+ T cells are tolerized in nonautoimmune mice expressing Mls-1a. These T cell subsets were quantified in the lymph nodes and spleens of (NZB X NZW)F1, (NZB X SWR)F1, and MRL-lpr/lpr mice before and after the development of lupus-like disease. The results indicate that peripheral T cells in these mice, including the massive CD4-, CD8- T cell population in lpr mice, have been modified by normal mechanisms of tolerance such that potential self-reactive V beta specificities have been eliminated in the thymus.

Abstract: The TCR alpha/beta variable element V beta 17a is expressed in all strains of mice carrying the V beta a complex in which about half of the V beta elements have been lost due to a large deletion. The mAb KJ23a detects V beta 17a containing alpha/beta receptors in these mice. Mouse strains with the V beta b complex carry a full complement of V beta genes including a structural gene for V beta 17 (V beta 17b), but no T cells reactive with KJ23a are present in these mice. Among random peripheral T cell hybridomas prepared from V beta b mice, occasional V beta 17b transcripts are found. The sequence of one of these transcripts reveals a single base difference from V beta 17a which results in a termination codon within the coding region inactivating the gene.

Abstract: The T-cell-activating protein TAP is a murine phosphatidylinositol-anchored glycoprotein whose expression is controlled by the Ly-6 locus. Previous studies have suggested an important role for this protein in physiological T-cell activation. Using oligonucleotide probes, we have now isolated a cDNA clone whose predicted sequence would encode a protein with an NH2-terminal sequence identical to that of the TAP molecule. Further analysis of the predicted protein sequence revealed a cysteine-rich protein with a hydrophobic domain at the COOH terminus and without N-linked glycosylation sites--all features consistent with our previous analysis of the TAP protein. In Southern blot analysis, the Ly-6.2 cDNA clone detects a multigene family and a restriction fragment length polymorphism that maps precisely to the Ly-6 locus. Expression of the cDNA clone in COS cells demonstrates that it codes for TAP and clarifies the relationship between the epitopes recognized by various alpha Ly-6 monoclonal antibodies. Finally, we have studied the expression of Ly-6 mRNA in a variety of cell lineages. Ly-6 transcripts were detected in all organs examined, including spleen, kidney, lung, brain, and heart. This demonstrates that the Ly-6 locus is transcriptionally active in a wide range of organs and suggests that the role of TAP or TAP-like proteins might extend to other tissues.

Abstract: In contrast to parental New Zealand Black (NZB) or New Zealand White (NZW) mice, (NZB x NZW)F1 mice exhibit a lupus-like disease characterized by high levels of immunoglobulin G (IgG) antinuclear antibodies in their serum and a fatal immune-complex glomerulonephritis. At least three gene loci have been identified in NZW mice that could potentially contribute to a T cell-dependent autoimmune disease, including the T cell receptor alpha- and beta-chain gene complexes and the major histocompatibility complex (MHC). The NZW T cell receptor beta-chain complex appeared to be particularly unusual in that the C-beta-1, D-beta-2, and J-beta-2 gene segments have been deleted. Approximately one half of (NZB x NZW)F1 x NZB backcross mice developed severe renal disease and elevated levels of IgG antibodies to double-stranded deoxyribonucleic acid and histone, suggesting that only one dominant gene or closely linked group of genes accounts for the NZW genetic contribution to F1 disease. Despite the extremely unusual nature of the NZW T cell receptor beta-chain gene complex, we found no association of disease expression with the presence of this allele in the backcross mice. There was also no correlation of disease incidence with the presence of the NZW T cell receptor alpha-chain allele. In contrast, nearly 90 percent of the backcross mice with the NZW MHC expressed severe autoimmune disease compared with 12 percent of the mice that did not carry this haplotype. Thus, the NZW MHC or gene(s) linked to this locus appears to be the only dominant NZW genetic contribution to F1 disease. Recent preliminary studies mapping genes that are located centromeric and telomeric to the NZW MHC suggest that the disease-associated gene(s) lies within the MHC.

Abstract: Antigen receptors on most T-cells are heterodimeric glycoproteins, comprised of an alpha chain and a beta chain. These chains are encoded by discontiguous variable (V), diversity (D) and joining (J) gene segments that rearrange to produce a contiguous and functional alpha or beta chain gene. To investigate the size and diversity of the germline repertoire of alpha-chain gene segments, we have characterized and sequenced 20 alpha chain cDNAs. Among these cDNA clones, we have found 4 J alpha and 4 V alpha sequences that have not yet been described. The relationship of these "new" gene segments to those already characterized is discussed.

Abstract: We report the molecular cloning of a human gene MER-2 located on chromosome 11 that encodes a cell surface antigen which is polymorphic on red blood cells. An essential element of the cloning strategy was cotransfection-induced linkage of pSV2-neo, which encodes resistance to the antibiotic G418, to the human MER-2 gene. An important feature of the pSV2-neo construct is that the same gene (the transposon, Tn5) that encodes G418 resistance in eukaryotic cells confers neomycin resistance in bacteria. Chinese hamster ovary (CHO) cells were cotransfected with pSV2-neo and genomic DNA from a CHO X human cell hybrid containing a single human chromosome (chromosome 11). Transfectants expressing both the human MER-2 gene and G418 resistance were isolated by selection in the antibiotic G418, followed by indirect immunofluorescence using the monoclonal antibody 1D12, which recognizes the MER-2 antigen, manual enrichment, and single-cell cloning. Genomic DNA from a primary transfectant positive for MER-2 expression and G418 resistance was used to construct a cosmid library and cosmid clones able to grow in neomycin were isolated. Of 150,000 cosmid clones screened, 90 were resistant to neomycin and of these, 11 contained human repetitive sequences. Five neomycin-resistant cosmid clones containing human repetitive DNA were able to transfect CHO cells for G418 resistance and MER-2 expression.

Abstract: The receptors of two T cell hybridomas that recognize class I and class II major histocompatibility complex (MHC) molecules, respectively, have been compared. In both cases these receptors are hybrid molecules formed as a result of cellular fusion. The receptors contain the same alpha chain, contributed by the tumor cell fusion partner, and related beta chains, contributed by the normal T cell component. Thus, surprisingly, the same alpha chain can contribute to recognition of class I and class II MHC molecules. Moreover, the finding that in two independent examples hybrid receptor molecules created randomly by in vitro cell fusion recognize MHC supports the theory that the T cell repertoire has an intrinsic affinity for MHC.

Abstract: The genetic control of the cytotoxic T-cell response to the male histocompatibility antigen, H-Y, was analyzed in BALB/cKe(C) and SJL/J(J) which are both nonresponders. However, the (C X J)F1 hybrid is a responder. Therefore, two dominant complementing genes are involved. Analysis of a set of (C X J) recombinant inbred (RI) lines reveals that these two complementing gene products are a restricting element (R) encoded by the H-2 (MHC) locus on chromosome 17 and a subunit of the T-cell receptor (anti-R) encoded by the Tar alpha-locus on chromosome 14. The order and orientation of gene segments within the Tar alpha-locus has also been established relative to the chromosome 14 marker, Es-10. The existence of two RI strains which are recombinant at chromosome 14 has made it possible to determine that this order is Es-10--v alpha-1--v alpha-2--[C alpha--Np-2]--centromere. The implications of these data for the antigen-specific regulation of immune responsiveness are discussed in terms of the dual recognitive-single receptor model.

Abstract: Two allelic forms of the T cell antigen receptor alpha chain gene were discerned by restriction fragment length polymorphism (RFLP) employing the T cell antigen receptor alpha chain probe pGA5, and the restriction enzyme Bgl II. Analysis revealed that the polymorphic fragments are detected by a probe specific for the constant region exon of the T cell antigen receptor alpha chain gene. Furthermore, the polymorphic fragments were shown to segregate within families. The two allelic forms yield two homozygous states, 3.2/3.2 and 2.9/2.9, at a frequency of 76.5 and 2.9%, respectively, within the normal population. The heterozygous state was observed in 20.6% of the population. The discovery of allelic forms of both the alpha and beta chains of the T cell antigen receptor genes may provide a unique opportunity to study heritable markers of T cell function in several human diseases.

Abstract: To isolate DNA segments specific to chromosome band 14q11, which has been implicated in a number of human T-cell malignancies, a genomic DNA library was prepared from a variant cell subline of the human lymphoblastic KE37 cell line. This subline (KE37-R) bears a t(8;14) (q24;q11) translocation, and the breakpoint on the resulting chromosome 8q+ has been located at the 3' end of the third c-myc exon. Three molecular clones were isolated by screening the library with a c-myc exon 3 probe, and one of them (lambda K40) was analyzed in detail. It contains a 15-kb insert consisting of 4.5 kb of sequence from chromosome 8 (e.g., downstream of c-myc exon 3) and sequences from chromosome 14. The origin of these latter sequences was established by hybridizing DNA from chromosomes sorted by flow cytometry to a lambda K40 subclone containing only chromosome 14 presumptive sequences and by Southern blot analysis of rodent X human somatic hybrid cell DNA with the same probe. No cross-hybridization was found between the lambda K40 clone and a cDNA clone for the alpha chain T-cell receptor gene which is also located in 14q11. A preliminary survey of DNAs from human T-cell malignancies with a probe corresponding to chromosome 14 sequences of lambda K40 clone revealed for some of them restriction patterns different from those of the germ line DNA. The fact that the rearrangement observed in a leukemic patient was not found in DNA from lymphocytes obtained during remission excluded any polymorphism.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: The genes encoding the alpha chain of the human T-cell receptor have been mapped to chromosome 14, the chromosome on which the human immunoglobulin heavy chain locus resides. Thus, genes encoding two different classes of antigen receptor are present on the same chromosome. Furthermore, breaks involving chromosome 14 are frequently seen in tumors of T-cell origin. The potential relation of these chromosome abnormalities to alpha-chain genes is discussed.

Abstract: The kinetics and order of rearrangements in the gene complex encoding T-cell-receptor beta chains were studied by Southern blot hybridization in a collection of hybridomas derived from fetal thymocytes at various stages of ontogeny (day 14 to day 17). Our results show a steady increase in the frequency of rearranged beta complexes during this period and suggest that these rearrangements occur within the thymus. beta-chain diversity region (D beta) to beta-chain joining region (J beta) joining preceded other types of rearrangements. More complex hybridization patterns consistent with fully rearranged functional beta-chain genes did not begin to accumulate until day 16, 1 day prior to significant surface expression of the receptor protein.

Abstract: Three independent T cell hybridomas were isolated that have identical specificities for antigen and products of the major histocompatibility complex (MHC). All three react with the same clone-specific antireceptor antibody, and Southern blots show all three contain the same rearranged alpha and beta genes. Variants of one of these hybridomas, DO-11.10, were isolated that had lost the ability to respond to antigen plus MHC. These proved to have lost the DO-11.10-specific alpha or beta genes or both. Fusion of alpha-loss variants to beta-loss variants restored reactivity. These results indicate that the specific recognition of antigen plus MHC is determined solely by the alpha/beta-containing T cell receptor.

Abstract: We have investigated the structure of the murine Y chromosome by first developing a novel method for specifically cloning Y-encoded DNA and then generating a library enriched for Y-specific DNA sequences. Three randomly chosen Y DNA clones were studied and found to share several interesting properties: all three are members of small Y-specific multisequence families; all three are mouse-specific; and all three probes detect Y-encoded restriction fragments that are polymorphic. Examination of polymorphic Y chromosome restriction fragments in male DNA from nine different inbred strains suggests that only two polymorphic forms of Y chromosomal DNA exist among inbred strains of mice.

Abstract: The T-cell receptor has been studied intensely over the past 10 years in an effort to understand the molecular basis for major histocompatibility complex (MHC) restricted antigen recognition. The use of anti-receptor monoclonal antibodies to isolate and characterize the receptor from human and murine T-cell clones has shown that the protein consists of two disulphide-linked glycopeptides, alpha and beta, distinct from known immunoglobulin light and heavy chains. Like immunoglobulin light and heavy chains, however, both the alpha- and beta-chains are composed of variable and constant regions. Molecular cloning has revealed that the beta-chain is evolutionarily related to immunoglobulins, and is encoded in separate V (variable), D (diversity), J (joining) and C (constant) segments that are rearranged in T cells to produce a functional gene. We report here cDNA clones encoding the alpha-chain of the receptor of the human T-cell leukaemia line HPB-MLT. Using these cDNA probes, we find that expression of alpha-chain mRNA and rearrangement of an alpha-chain V-gene segment occur only in T cells. The protein sequence predicted by these cDNAs is homologous to T-cell receptor beta-chains and to immunoglobulin heavy and light chains, particularly in the V and J segments.

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Abstract: A total of 358 lys2 mutants of Saccharomyces cerevisiae have been characterized for suppressibility by the following suppressors: UAA and UAG suppressors that insert tyrosine, serine or leucine; a putative UGA suppressor; an omnipotent suppressor SUP46; and a frameshift suppressor SUF1-1. In addition, the lys2 mutants were examined for phenotypic suppression by the aminoglycoside antibiotic paromomycin, for osmotic remediability and for temperature sensitivity. The mutants exhibited over 50 different patterns of suppression and most of the nonsense mutants appeared similar to nonsense mutants previously described. A total of 24% were suppressible by one or more of the UAA suppressors, 4% were suppressible by one or more of the UAG suppressors, while only one was suppressible by the UGA suppressor and only one was weakly suppressible by the frameshift suppressor. One mutant responded to both UAA and UAG suppressors, indicating that UAA or UAG mutations at certain rare sites can be exceptions to the specific action of UAA and UAG suppressors. Some of the mutants appeared to require certain types of amino acid replacements at the mutant sites in order to produce a functional gene product, while others appeared to require suppressors that were expressed at high levels. Many of the mutants suppressible by SUP46 and paromomycin were not suppressible by any of the UAA, UAG or UGA suppressors, indicating that omnipotent suppression and phenotypic suppression need not be restricted to nonsense mutations. All of the mutants suppressible by SUP46 were also suppressible by paromomycin, suggesting a common mode of action of omnipotent suppression and phenotypic misreading.

Abstract: Previous work from our laboratory has demonstrated that a subclass of the aminoglycoside antibiotics, those containing the drug fragment paromamine, stimulates mistranslation in cell-free protein-synthesizing systems derived from eucaryotic cells. We report here experiments which show that the ciliate Tetrahymena thermophila (formerly T. pyriformis, syngen 1) is sensitive to the paromamine-containing aminoglycoside antibiotics. The drugs are active with respect to growth inhibition, inhibition of protein synthesis in the whole organism, inhibition of protein synthesis in vitro and the stimulation of mistranslation in cell-free protein-synthesizing systems. Because of their misreading properties, these drugs may be useful in isolating and propagating strains carrying mutations which can be translationally suppressed (that is, missense and nonsense mutations).

Abstract: R factor proteins are synthesized in R factor-containing Escherichia coli minicells. Half of this protein remained associated with the minicell membrane upon lysis of the minicells. Over 90% of the membrane-associated protein was extracted by sodium lauryl sarcosinate, suggesting a location of these proteins in the inner membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these membrane preparations demonstrated the presence of multiple peptides, including a prominent band with a molecular weight of 28,000 to 30,000. A polypeptide of similar size was seen in membrane preparations from minicells harboring R factors from five different compatibility types. This major R factor membrane peptide was seen with R factors repressed or derepressed for pilus synthesis, with and without antibiotic resistances. It was associated with R factor deoxyribonucleic acid in membrane-deoxyribonucleic acid complexes. Its possible role in R factor replication and/or transfer is being investigated.

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