Abstract: The nuclease ARTEMIS is an essential factor of V(D)J recombination during lymphocyte development and in the repair of DNA double-strand breaks (DSB) by the nonhomologous end joining (NHEJ) pathway. Patients with mutations in the DCLRE1C gene, which encodes ARTEMIS, suffer from radiosensitive B(-/low) T(-/low) severe combined immunodeficiency (SCID) or radiosensitive Omenn syndrome. To date, causative DCLRE1C mutations inherited as a recessive trait have been reported in 49 patients. In this study, molecular diagnoses of 29 novel patients presenting with the phenotype of B(-/low) SCID revealed mutations in the DCLRE1C gene. In total, 13 different mutated DCLRE1C alleles were detected, nine of which have not been described before. By far the most frequent mutations (59%) were gross deletions of exons 1-3 or exons 1-4 due to a homologous recombination of the wild-type DCLRE1C gene with a pseudo-DCLRE1C gene located 61.2 kb 5' to the DCLRE1C start codon. Fine mapping of the recombination intervals revealed private mutations in most cases. MEIG1, a gene encoding a protein that is essential for spermatogenesis in mice, is lost by the gross deletions. Functional analyses on patients' fibroblasts demonstrated that the corresponding alleles carry null mutations of the DCLRE1C gene.
Abstract: A large proportion of patients with mutations in the Wiskott-Aldrich syndrome (WAS) protein gene exhibit the milder phenotype termed X-linked thrombocytopenia (XLT). Whereas stem cell transplantation at an early age is the treatment of choice for WAS patients, therapeutic options for patients with XLT are controversial.In a retrospective multicenter study we defined the clinical phenotype of XLT and determined the probability of severe disease related complications in patients above the age of 2 years with documented WAS gene mutations and mild to moderate eczema or mild, infrequent infections.Enrolled were 173 patients (median age 11.5 years) from 12 countries spanning 2830 patient years. Serious bleeding episodes occurred in 13,9%, life threatening infections in 6.9%, autoimmunity in 12.1%, and malignancy in 5.2% of patients. Overall and event free survival probabilities were not significantly influenced by the type of mutation or IVIG or antibiotic prophylaxis. Splenectomy resulted in increased risk of severe infections.This analysis of the clinical outcome and molecular basis of XLT patients demonstrates excellent long term survival but also a high probability of severe disease related complications. These observations will allow better decision making when considering treatment options for individual XLT patients.
Abstract: Nijmegen breakage syndrome (NBS) is characterized by chromosomal instability, radiation hypersensitivity, characteristic facial appearance, immunodeficiency and strong predisposition to lymphoid malignancy. Traditionally, NBS patients have not undergone hematopoietic SCT (HSCT) owing to concerns about increased toxicity. We therefore report on the HSCT experience in NBS patients in Europe. Six patients were transplanted either for resistant or secondary malignancy (four patients) or severe immunodeficiency (two patients). Five patients received reduced-intensity conditioning regimens. After a median follow-up of 2.2 years, five patients are alive and well. One patient who received myeloablative conditioning died from sepsis before engraftment. Acute GVHD grades I-II occurred in three of five patients, mild chronic GVHD in one. All five surviving patients exhibit restored T-cell immunity. The experience in these six patients suggests that HSCT in NBS is feasible, can correct the immunodeficiency and effectively treat malignancy. Acute toxicity seems to be reasonable with reduced-intensity conditioning regimens.
Abstract: More than 20 North American academic centers account for the majority of hematopoietic stem cell transplantation (HCT) procedures for primary immunodeficiency diseases (PIDs), with smaller numbers performed at additional sites. Given the importance of a timely diagnosis of these rare diseases and the diversity of practice sites, there is a need for guidance as to best practices in management of patients with PIDs before, during, and in follow-up for definitive treatment. In this conference report of immune deficiency experts and HCT physicians who care for patients with PIDs, we present expert guidance for (1) PID diagnoses that are indications for HCT, including severe combined immunodeficiency disease (SCID), combined immunodeficiency disease, and other non-SCID diseases; (2) the critical importance of a high degree of suspicion of the primary care physician and timeliness of diagnosis for PIDs; (3) the need for rapid referral to an immune deficiency expert, center with experience in HCT, or both for patients with PIDs; (4) medical management of a child with suspicion of SCID/combined immunodeficiency disease while confirming the diagnosis, including infectious disease management and workup; (5) the posttransplantation follow-up visit schedule; (6) antimicrobial prophylaxis after transplantation, including gamma globulin administration; and (7) important indications for return to the transplantation center after discharge. Finally, we discuss the role of high-quality databases in treatment of PIDs and HCT as an element of the infrastructure that will be needed for productive multicenter clinical trials in these rare diseases.
Abstract: Effective techniques for the genetic modification of peripheral T-cells would facilitate functional gene studies and the development of gene therapeutic approaches. However many approaches to genetically modify T-cells are hampered by low transfection efficiency, direct cell toxicity and the need for specialized laboratory space. In this study we investigated the Amaxa Nucleofector platform, a non-viral technique to transfect primary human T-cells. A plasmid equipped with two different promoters enabled concomitant expression of a gene of interest and of a cell surface marker allowing for immunomagnetic cell enrichment. This resulted in highly purified populations of gene modified T-cells and after repeated enrichment steps provided stably and homogenously transfected, fully functional human T-cells. In summary this study provides proof of principle that human T-cells can be altered to homogenously and stably express a gene of interest with a non-viral technique. This should enable further studies on T-cell physiology and ultimately facilitate the translation of treatment approaches either for diseases that are caused by defective gene function in T-cells or for diseases that require genetically designed T-cell therapy.
Abstract: Background: Defects in the development or activation of T cells result in immunodeficiency associated with severe infections early in life. T-cell activation requires Ca2+ influx through Ca2+-release activated Ca2+ (CRAC) channels encoded by the gene ORAI1. Objective: Investigation of the genetic causes and the clinical phenotype of immunodeficiency in patients with impaired Ca2+ influx and CRAC channel function. Methods: DNA sequence analysis for mutations in the genes ORAI1, ORAI2, ORAI3, and stromal interaction molecule (STIM) 1 and 2, as well as mRNA and protein expression analysis of ORAI1 in immunodeficient patients. Immunohistochemical analysis of ORAI1 tissue distribution in healthy human donors. Results: We identified mutations in ORAI1 in patients from 2 unrelated families. One patient is homozygous for a frameshift nonsense mutation in ORAI1 (ORAI1-A88SfsX25), and a second patient is compound heterozygous for 2 missense mutations in ORAI1 (ORAI1-A103E/L194P). All 3 mutations abolish ORAI1 expression and impair Ca2+ influx and CRAC channel function. The clinical syndrome associated with ORAI1 deficiency is characterized by immunodeficiency with a defect in the function but not in the development of lymphocytes, congenital myopathy, and anhydrotic ectodermal dysplasia with a defect in dental enamel calcification. In contrast with the limited clinical phenotype, we found ORAI1 protein expression in a wide variety of cell types and organs. Conclusion: Ca2+ influx through ORAI1 is crucial for lymphocyte function in vivo. Despite almost ubiquitous ORAI1 expression, the channel has a nonredundant role in only a few cell types judging from the limited clinical phenotype in ORAI1-deficient patients. (J Allergy Clin Immunol 2009;124:1311-8.)
Abstract: Primary immunodeficiency diseases are not only characterized by an improper immune defence against infections but in addition can present with characteristic abnormalities of other organs. These findings can affect bone, teeth, hair, and skin and may direct diagnostic considerations leading to genetically defined disorders. This article summarizes some of these multisystem disorders with immunodeficiency including definition, diagnostic approach, and current therapy options.
Abstract: Adoptive transfer of naturally occurring CD4(+)CD25(+) regulatory T cells can tolerize transplantation alloresponses in animal models. However isolation of these cells in sufficient numbers from humans is cumbersome and prone to contamination with alloreactive CD25(+) T cells. Incubation of ethylenecarbodiimide-coupled antigen presenting cells (APC) with naive T cells and antigen has been shown to induce tolerance in various experimental models. We therefore investigated whether ECDI-coupled allogeneic APC were able to induce an expandable human CD4(+) Treg population. CD4(+) and CD4(+) CD25(-) cells cultured for 5 days with ECDI-treated human PBMC exhibited potent suppressive capacity in a mixed lymphocyte reaction. Induction of these ECDI-Tregs was associated with up-regulation of Foxp3 mRNA and protein expression and they maintained high expression of CD62L and CD27 as well as low CD127 expression. ECDI-treated APC displayed reduced expression of the co-stimulatory signaling molecules CD40 and CD80, and failed to stimulate proliferation and cytokine secretion in co-cultured CD4(+) T cells. Restimulation in the presence of rapamycin and hrIL-2 led to expansion of ECDI-Tregs with increasing Foxp3 levels and suppressive activity significantly higher than expanded naturally occurring CD4(+)CD25(+) Tregs. In summary these findings support the hypothesis that ECDI-coupled APC can convert naive CD4(+) T cells into functional Tregs with different phenotypic characteristics than naturally occurring CD4(+)CD25(+) Tregs. These inducible Tregs could provide a novel approach that might facilitate the translation of ex vivo generated and expanded Tregs into clinical settings.
Abstract: Engraftment syndrome (ES) has been recognized as an inflammatory condition during neutrophil recovery after hematopoietic stem cell transplantation (HSCT) characterized by noninfectious fever and skin rash. It has been reported to occur frequently after autologous HSCT in children and adults, and has been shown to be an independent risk factor for increased transplant-related mortality (TRM). However, virtually no data exist on its occurrence after allogeneic HSCT in children. To determine incidence, predisposing factors for, and complications of ES in a pediatric transplant cohort, we analyzed 61 consecutive recipients of a myeloablative allogeneic HSCT for the occurrence of ES. Diagnosis of ES was established when children presented with > or =2 of the following symptoms within 7 days before engraftment: (1) fever >38.0 degrees C, (2) skin rash, (3) weight gain and albumin drop, or (4) dyspnea, hypoxia, and pulmonary infiltrates. Incidence of ES in this cohort was 48% (29 of 61). In a univariate analysis, posttransplant granulocyte-colony stimulating factor (G-CSF) administration (P = .02), and high mononuclear cell count (MNC) (P = .002) were identified as significant risk factors predisposing for the development of ES. In a multiple logistic regression analysis, amphotericin B therapy (P = .009) and high MNC (P = .004) were significant explanatory variables for ES risk. There was a slight trend toward a higher rate of chronic GVHD (cGVHD) in patients with ES (P = .11). However, after a median follow-up of 9.5 years overall survival (OS) (P = .53) and TRM (P = .65) did not differ between the 2 groups. ES presenting with fever, rash, weight gain, and pulmonary symptoms should be recognized as a frequent complication of allogeneic HSCT after myeloablative conditioning in children. Treatment with G-CSF, amphotericin B, and a high nucleated cell count of the graft predisposed for the development of ES in this study. OS and TRM in this cohort were not affected by the occurrence of ES.
Abstract: Adenosine deaminase (ADA) deficiency is a systemic metabolic disease that causes an autosomal recessive variant of severe combined immunodeficiency (SCID) and less consistently other complications including neurologic abnormalities. Hematopoietic stem cell transplantation (HSCT) is able to correct the immunodeficiency, whereas control of nonimmunologic complications has not been extensively explored. We applied HSCT in 15 ADA-deficient patients consecutively treated at our institutions since 1982 and analyzed long-term outcome. Seven patients received transplants without conditioning from HLA-matched family donors (MFDs); the other 8 patients received conditioning and were given transplants either from HLA-mismatched family donors (MMFDs; n = 6) or from matched unrelated donors (MUDs; n = 2). At a mean follow-up period of 12 years (range, 4-22 years), 12 patients are alive with stable and complete immune reconstitution (7 of 7 after MFD, 4 of 6 after MMFD, and 1 of 2 after MUD transplantation). Six of 12 surviving patients show marked neurologic abnormalities, which include mental retardation, motor dysfunction, and sensorineural hearing deficit. We were unable to identify disease or transplantation-related factors correlating with this divergent neurologic outcome. The high rate of neurologic abnormalities observed in long-term surviving patients with ADA deficiency indicates that HSCT commonly fails to control CNS complications in this metabolic disease.
Notes: 0006-4971 (Print) xD;Journal Article xD;Multicenter Study
Abstract: Oral chronic graft vs. host disease (GVHD) frequently presents in patients with sclerotic features of skin GVHD and is often associated with considerable limitations of oral food intake and decreased quality of life. Systemic tacrolimus is efficacious for prophylaxis and treatment of acute and chronic GVHD and topical tacrolimus has shown activity in chronic GVHD skin lesions. We therefore initiated a pilot study to investigate the safety and efficacy of topical tacrolimus ointment in children with oral GVHD. Six patients suffering from oral GVHD (five chronic and one acute) were included in the study. Tacrolimus ointment 0.1% was applied twice daily using sterile gauze. The only side-effects observed were a slight burning discomfort after the first application in one patient and after food intake in another patient. Tacrolimus was absorbed systemically in four of six patients. Of six patients, we observed a complete response in two, a very good partial response (VGPR) in two, and a PR in two patients, respectively. We conclude that topical application of tacrolimus ointment holds promise as a safe and efficacious treatment for oral GVHD in children. The Food and Drug Administration has recently issued a health advisory about a potential cancer risk associated with topical tacrolimus treatment of the skin; therefore, its benefits should be weighed against its potential risks and diligent long-term follow-up should be carried out especially in children.
Abstract: "Hair-on-end" skull changes are typically seen in individuals suffering from thalassaemia. They are induced by widening of the diploic space due to marrow expansion that is a consequence of ineffective and excessive erythropoiesis. We present a child with severe congenital neutropenia who exhibited the typical hair-on-end sign on plain skull radiographs and MRI. In this patient the skull changes were very likely induced by the expansion of white blood cell precursors induced by long-term daily injections of recombinant human granulocyte colony stimulating factor (G-CSF) to treat his confounding disease. This case report is the first description of hair-on-end changes associated with the use of G-CSF.
Abstract: Intravenous immunoglobulin (IVIg) preparations are increasingly used for therapy of several neuroimmunologic diseases. IVIg therapy is considered safe, although serious side effects like aseptic meningitis, cerebral vasospasm, or ischemic encephalopathy have been reported. These side effects are frequently associated with neutrophilic pleocytosis in the cerebrospinal fluid (CSF), suggesting a neutrophil-mediated mechanism. To elucidate the potential role of neutrophil activation, we analyzed IVIg preparations from 5 different commercial sources for the presence of antineutrophil cytoplasmic antibody (ANCA)-like immunoglobulins against ethanol-fixed peripheral-blood neutrophils, purified human antigens, and a panel of human and nonhuman tissues. All IVIg batches tested (n = 13) contained atypical ANCAs (IgG titer up to 1:2048, IgA up to 1:512). Moreover, all preparations were capable of inducing hydrogen peroxide production in TNFalpha-primed human neutrophils, with a significant correlation (P < .005) between atypical ANCA titers in IVIg preparations and neutrophil activation. Fc-mediated binding and activation was ruled out by the use of IVIg-F(ab')(2) fragments. Our findings strongly suggest that in vivo activation of TNFalpha-primed neutrophils by atypical ANCAs of IVIg may contribute to the side effects of IVIg therapy and for the first time demonstrate that the activation of neutrophil granulocytes by IVIg occurs in an Fc receptor (FcR)-independent, hence antigen-dependent, way.
Abstract: Pathological angiogenesis is increasingly recognized to be an important feature of pathogenesis in solid tumors and also in leukemias. Specific blockers of angiogenesis are now being introduced into early clinical trials with encouraging results. Vascular endothelial growth factor (VEGF) seems to play a central role in tumor angiogenesis and is associated with a poor prognosis in both solid tumors and adult leukemias. In pediatric acute lymphocytic leukemia however, the expression of angiogenic molecules and its relation to prognosis and relapse are unknown. Therefore, we prospectively analyzed 46 pediatric patients with precursor B cell acute lymphocytic leukemia by semi-quantitative RT-PCR for expression of the angiogenic molecules VEGF, VEGF-C, iNOS and TGF-beta and correlated relapse and survival data with the expression of these factors. We found a high mRNA expression of TGF-beta and iNOS, a moderate expression of VEGF but no expression of bFGF and VEGF-C. A significantly higher expression of VEGF mRNA was found in patients with late relapses compared to patients without relapses (p=0.043). A significantly higher mRNA expression of iNOS was found in surviving patients compared with non-surviving patients (p=0.023). Angiogenic factors are expressed in the bone marrow of patients with pediatric B cell precursor ALL and VEGF is a potential candidate for therapeutic intervention as it is significantly higher expressed in children with late relapses. The mRNA expression of iNOS in the surviving children possibly reflects an increased activity of the immune system against the leukemia which leads to a superior survival.
Abstract: BACKGROUND: The [(3)H]thymidine incorporation assay and staining of living cells with fluorescent dyes like carboxyfluorescein diacetate, succinimidyl ester (CFSE) have evolved as valuable methods for studying T cell responses. To assess proliferation of cells already labeled by FITC, CFSE, GFP, or other "green" molecules or to simultaneously track two otherwise indistinguishable cell populations in mixed cell cultures, it would be desirable to have a dye with distinct fluorescent properties for this application. METHODS: We analyzed the dilution of the far red fluorescent dye SNARF-1 in proliferating cells by flow cytometric analysis. The results were compared with the CFSE dilution technique as well as the [(3)H]thymidine incorporation assay. RESULTS: Staining of primary human lymphocytes revealed that SNARF-1 labeling was equivalent to CFSE for estimating proportions of proliferating cells in stimulated cell cultures and yielded results comparable to [(3)H]thymidine incorporation. We showed that SNARF-1 offers the possibility to simultaneously analyze the proliferation of phenotypically indistinguishable subsets of hematopoietic cells and can also be used to track uniformly proliferating, non hematopoietic cells like HEK293. CONCLUSIONS: In summary, we have demonstrated that labeling of cells with SNARF-1 allows for estimating cell proliferation of cells of hematopoietic and non-hematopoietic origin.
Abstract: The Wiskott-Aldrich syndrome is an X-linked hereditary disorder associated with combined immunodeficiency, thrombocytopenia, small platelets, eczema, and increased susceptibility to autoimmune disorders and cancers. It is caused by mutations in the gene (WAS) for the Wiskott-Aldrich syndrome protein (WASP). We investigated family members of the patients originally described by Wiskott in 1937 and identified a new frame shift mutation in exon 1 of WAS. This mutation is likely to be the hypothesized genotype that caused the severe form of the Wiskott-Aldrich syndrome in the three brothers described by Wiskott.
Abstract: Many paediatric oncology centres apply parenteral nutrition (PN) in children with severe oral mucositis after chemotherapy. However, no convincing data exist to support this treatment strategy. The aim of our study was to elucidate a possible advantage of PN versus intravenous replacement fluid therapy (FT). In a prospective randomized study, 30 children with mucositis WHO grade IV were assigned to receive either PN or intravenous replacement FT. Weight, total body water, fat-free mass (measured by impedance analysis) and peripheral white blood cells were assessed daily. For aspects of quality of life and economics, the length of hospital stay, the incidence of infections, the days on intravenous antibiotics and delay of scheduled chemotherapy were examined. Children with PN gained body weight significantly compared to baseline and to FT due to an augmentation of fat mass while total body water and fat-free mass significantly decreased. In children with FT, body weight remained stable while total body water and fat-free mass significantly increased, thereby loosing fat mass. We observed no differences in recovery of peripheral white blood cells (WBC), incidence of infections, hospitalization time, days on intravenous antibiotics, days on opioid analgesics and delay of the next scheduled chemotherapy cycle. Although children with PN gained weight in form of fat mass, this did not translate into a clinical benefit for the patients such as earlier recovery of WBC counts, shorter hospitalization time, a decreased use of analgesics or less delay of the next scheduled chemotherapy cycle. Our findings therefore do not support the hypothesis that PN is superior to FT when used for less than 10 days for oral mucositis.
Abstract: Advances in immunosuppressive therapies have made tissue and organ transplantation a common procedure in clinical medicine. However, true donor and recipient tolerance is not regularly achieved and almost all transplant recipients continue to require immunosuppressants throughout life, which is associated with side effects of the drugs. The identification and characterisation of regulatory T cells (Tregs) has recently opened up exciting opportunities for new ways of adoptive immunotherapy in transplantation. CD4+CD25+ Tregs of thymic origin have been shown to be key regulators of unseasoned immune responses in mice and in humans, preventing graft-versus-host disease and organ graft rejection in the transplantation setting. Although these cells can be found in the peripheral blood of healthy individuals, their isolation to a satisfying degree of purity is time-consuming and ineffective. Therefore, a variety of different methods to expand or induce regulatory T cells ex vivo have been advocated. Antigen-specific activation of Tregs is a prerequisite for their optimal function, making the design of new strategies to create and expand antigen-specific Tregs highly desirable. This review will focus on recent advances achieved in the field of transplantation tolerance using naturally occurring Tregs (CD4+CD25+), as well as other Tregs, and will discuss future applications of these cells in immunotherapy.
Abstract: TCR affinity dictates T cell selection in the thymus and also has a high impact on the fate of peripheral T cells. Graft-vs-host disease (GVHD) is a pathological process initiated by activation of donor T cells after adoptive transfer into an allogeneic recipient. How TCR affinity affects the potential of alloreactive T cells to induce GVHD is unclear. Using alloreactive CD4+ and CD8+ TCR transgenic (Tg) T cells, GVHD models are presented that allow for the visualization of how CD8+ alloreactive T cells behave in response to alloantigens with different TCR affinity in the absence or presence of CD4 help. In a nonmyeloablative transplant model where GVHD lethality is due to marrow aplasia, alloreactive CD8+ TCR Tg T cells induced significantly more severe GVHD in the recipients that express an intermediate-affinity alloantigen than in the recipients that express a high-affinity alloantigen. In a myeloablative transplant model where GVHD lethality is due to epithelium injury, CD8+ TCR Tg cells were also more pathogenic in the recipients with an intermediate-affinity alloantigen than in those with a high-affinity alloantigen. The presence of alloreactive CD4+ TCR Tg cells enhanced the potential of CD8+ TCR Tg cells to cause GVHD in recipients with an intermediate-, but not with a high-, affinity alloantigen. These findings underscore that alloantigen affinity and CD4 help control the fate and pathogenicity of alloreactive CD8+ T cells in vivo.
Abstract: PFAPA syndrome is characterized by periodic episodes of high fever, aphthous stomatitis, pharyngitis, and/or cervical adenitis. It is of unknown etiology and manifests usually before 5 years of age. We determined serum and intracellular cytokine levels in six PFAPA patients (4 males, 2 females, mean age 8 years (+/- 1.2 SEM), range 4-13) during the symptom-free period as well as 6-12 hours and 18-24 hours after fever onset. Values were compared to age-matched, healthy controls. Febrile PFAPA attacks led to a significant increase in IL-6 and IFN-gamma serum concentrations compared to symptom-free periods and to controls, with IL-1beta, TNF-alpha and IL-12p70 levels being significantly higher than in controls. Lymphocytic IFN-gamma and CD8+ IL-2 production was consistently significantly elevated compared to healthy children. During the asymptomatic period, serum concentrations of IL-1beta, IL-6, TNF-alpha and IL-12p70 were significantly increased compared to controls. Intracellular TNF-alpha synthesis was not elevated at any time point. Soluble TNFRp55 levels were even lower in between febrile episodes, reaching values comparable to controls during attacks, whereas soluble TNFRp75 levels increased during attacks compared to healthy children. Anti-inflammatory IL-4 in serum was at all times lower in PFAPA patients compared to controls with no difference in levels of intracellular IL-4 and IL-10 or serum IL-10. The observed increase of pro-inflammatory mediators, even between febrile attacks, suggests a dysregulation of the immune response in PFAPA syndrome, with continuous pro-inflammatory cytokine activation and a reduced anti-inflammatory response.
Abstract: The immunological environment of leukemic blasts in the bone marrow might play a decisive role in determining an individual's risk for relapse. In order to identify potential predictors of relapse and to elucidate the mechanisms of immune control of leukemic blasts we examined the expression of cytokines, costimulatory molecules and members of the TNF family in leukemic marrow samples in a prospective study. Samples from 49 consecutive pediatric patients with B cell precursor acute lymphocytic leukemia (BCP ALL) were analyzed by semiquantitative RT-PCR. We identified interleukin (IL)-10 expression as a significant adverse prognostic indicator in childhood BCP-ALL. The event free survival (EFS) of patients expressing IL-10 mRNA in high quantity was significantly lower compared with patients expressing low IL-10 mRNA. Taqman RT-PCR of sorted cell populations showed that IL-10 mRNA was synthetized almost exclusively by NK or T cells. In addition, we found an increased expression of IL-1, IL-4, CD86 and VEGF mRNA in patients with late relapses. Possibly, ALL cells mediate a Th2 shift through increased expression of CD86 and thereby influence the individual relapse risk. These findings emphasize the role of the immune system for the outcome of childhood ALL.
Abstract: Adoptive transfer of polyclonal CD4(+)CD25(+) regulatory T cells (Treg) can tolerize transplantation alloresponses. Treg are activated via their specific TCR, but the antigen specificity of wild-type Treg remains elusive, and therefore controlling potency and duration of Treg activity in the transplantation setting is still not feasible. In this study, we used murine graft-versus-host disease (GVHD) as a model system to show that antigen-specific Treg suppress the response of T effector cells to alloantigens in vitro and prevent GVHD in vivo. The suppressive potential of antigen-specific Treg was much greater than that of polyclonal Treg. To acquire large numbers of antigen-specific Treg, we transduced CD4(+)CD25(-) cells with foxp3, and found that these foxp3-induced Treg suppress alloresponses in vitro and prevent GVHD in vivo as effectively as naturally derived CD4(+)CD25(+) Treg. Furthermore, we used an antigen-specific CD4 Th1 clone as a source of foxp3-induced Treg after transduction with foxp3, and found those Treg to effectively prevent GVHD in an antigen-dependent manner. The findings of this study provide a basis for the concept that the onset and potency of the suppression by Treg can be regulated, and suggest a novel approach to enhance the feasibility and effectiveness of inducing tolerance by Treg as an adoptive immunotherapy in transplantation.
Abstract: Successful hematopoietic cell transplantation (HCT) from an allogeneic donor ideally should produce tolerance to recipient alloantigens while preserving anti-infectious and antitumor immunity. Rapamycin together with costimulation blockade can induce tolerance in organ allograft models by inhibiting G(1) --> S-phase progression and promoting T-cell apoptosis. In contrast to blocking costimulation through CD28, administration of agonistic CD28-specific antibody 37.51 partially prevents lethal graft-versus-host disease (GVHD) by selective depletion of alloreactive T cells in mice. We hypothesized that combining rapamycin with agonistic CD28 treatment would improve GVHD control by tolerizing a small subset of alloreactive T cells that might escape effects of the CD28-specific antibody. A short course of rapamycin plus agonistic CD28 treatment showed synergism at suboptimal doses, was highly effective in preventing lethal GVHD, and was superior to rapamycin plus CD28 blockade in a major histocompatibility complex class I- and II-mismatched HCT model. The combination treatment reduced the number of proliferating, alloreactive cells in the recipient, promoted donor B- and T-cell reconstitution, and reduced inflammatory cytokine levels. Administration of rapamycin plus agonistic CD28 antibodies offers a promising new therapeutic approach to facilitate tolerance after HCT.
Abstract: Intracellular detection of cytokines via fluorescent antibody staining and flow cytometry has quickly become a standard method in experimental immunology. However, in pediatrics most studies have been hampered by the exclusion of healthy control individuals or have been skewed by neglecting to observe age-dependent differences in cytokine production. We therefore intended to establish normal values for different age groups and to describe the age-dependent development of cytokine profiles. Whole blood from 46 healthy children and 33 adults was analyzed by flow cytometry after stimulation with PMA, ionomycin and Mmonensin, and staining with anti-cytokine and surface antibodies. In the pediatric population, we found a significant positive correlation between age and intracellular cytokine levels of IFN-gamma, IL-2, IL-4 and TNF-alpha in CD4+ cells, as well as for IFN-gamma and TNF-alpha in CD8+ cells. In adulthood, no such striking trend could be detected, but significant correlation was found for IL-10 in CD4+ cells and IFN-gamma in CD8+ cells as well as for TNF-alpha in both cell subgroups. We present here the first systematic analysis of intracellular cytokine production in normal, healthy children between the ages of 0 to 18 years compared to results in adults. These data may provide a reference basis for the study of cytokine secretion patterns, and they also demonstrate a significant maturation of the T-cell cytokine production capacity from birth to adulthood.
Abstract: Hyper-IgE syndrome is a rare primary immunodeficiency of unknown etiology characterized by recurrent infections of the skin and respiratory system, chronic eczema, elevated total serum IgE, and a variety of associated skeletal symptoms. Recent reports about susceptibility to pyogenic bacterial infections and high IgE levels in patients and animals with defects in toll-like receptor (TLR) signaling pathways prompted us to search for TLR signaling defects as an underlying cause of hyper-IgE syndrome. Blood samples from six patients with hyper-IgE syndrome were analyzed for serum cytokine levels, intracellular cytokine production in T cells after stimulation with PMA/ionomycin, and cytokine production from peripheral blood mononuclear cells stimulated by TLR ligands and bacterial products including LPS (TLR4), peptidoglycan (TLR2), PolyIC (TLR3), R848 (TLR7/8), CpG-A, and CpG-B (TLR9), zymosan and heat killed Listeria monocytogenes. All results were compared to data from healthy controls. A reduction in IFN-gamma, IL-2, and TNF-alpha producing T cells after PMA stimulation suggested a reduced inflammatory T cell response in patients with hyper-IgE syndrome. Increased serum levels of IL-5 indicated a concomitant Th2 shift. However, normal production of cytokines (TNF-alpha, IL-6, IL-10, IFN-alpha, IP-10) and upregulation of CD86 on B cells and monocytes after TLR stimulation made a defect in TLR signaling pathways highly unlikely. In summary, our data confirmed an imbalance in T cell responses of patients with hyper-IgE syndrome as previously described but showed no indication for an underlying defect in toll-like receptor signaling.
Abstract: Administration of an agonistic anti-CD28 mAb paradoxically inhibits donor T cell expansion and prevents graft-versus-host disease (GVHD) in mice. Here we examined the mechanism of anti-CD28-mediated immunosuppression and found that anti-CD28 mAb activated, rather than blocked, CD28-mediated signaling in vivo. Anti-CD28 treatment prevented GVHD by selectively depleting alloantigen-activated donor T cells through apoptosis but spared the T cells that did not recognize recipient alloantigens. Overexpression of Bcl-x(L) did not protect T cells from depletion and did not affect GVHD prevention after anti-CD28 treatment. Depletion of activated T cells mediated through CD28 did not depend on the expression of death receptors Fas and TNF receptors type I and II, but both the depletion of activated T cells and the suppressive effect of anti-CD28 mAb on GVHD lethality required donor-derived IFN-gamma production. This study demonstrates that agonistic Ab's specific for the CD28 costimulatory molecule may be used as novel therapeutic agents to abrogate pathogenic T cell responses by selective depletion of activated T cells.
Abstract: Alpha-mannosidosis (alpha-mannosidosis) is a lysosomal storage disease characterized by accumulation of oligosaccharides in various tissues leading to symptoms such as coarse facial features, dysostosis multiplex, hearing disabilities, mental developmental delay and skeletal involvement (dysostosis multiplex). Without treatment, the severe infantile onset form of this autosomal recessive disease leads to progressive neurodegeneration and sometimes to early death. Stem cell transplantation has been shown to be an effective treatment. In the five patients published so far, correction of skeletal abnormalities and improvement of neuropsychological capabilities have been observed. We report the first patient who received a T-cell-depleted peripheral blood stem cell transplantation (PBSCT) for alpha-mannosidosis. The diagnosis of alpha-mannosidosis was made at the age of 14 months. At the age of 24 months, he underwent PBSCT with T-cell depletion by CD34-positive selection from his HLA phenotypically identical mother. Conditioning was carried out with busulfan (20 mg/kg), cyclophosphamide (200 mg/kg), OKT3 and methylprednisolone. The patient is alive and well 27 months after PBSCT and has made significant developmental progress. The pattern of urinary oligosaccharides has returned to almost normal. CD34-positive-selected PBSCT is a feasible option to reduce risk for GVHD for these patients.
Abstract: The Yersinia outer surface protein invasin binds to beta1 integrins on target cells and has been shown to trigger phagocytic uptake by macrophages. Here, we investigated the role of the actin regulator Wiskott-Aldrich syndrome protein (WASp), its effector the Arp2/3 complex and the Rho-GTPases CDC42Hs, Rac and Rho in invasin/beta1 integrin-triggered phagocytosis. During uptake of invasin-coated latex beads, the alpha5beta1 integrin, WASp and the Arp2/3 complex were recruited to the developing actin-rich phagocytic cups in primary human macrophages. Blockage of beta1 integrins by specific antibodies, inhibition of Arp2/3 function by microinjection of inhibitors or the use of WASp knockout macrophages inhibited phagocytic cup formation and uptake. Furthermore, microinjection of the dominant negative GTPase mutants N17CDC42Hs, N17Rac or the Rho-specific inhibitor C3-transferase into macrophages greatly attenuated invasin-induced formation of cups. These data suggest that during invasin-triggered phagocytosis beta1 integrins activate actin polymerization via CDC42Hs, its effector WASp and the Arp2/3 complex. The contribution of Rac and Rho to phagocytic cup formation also suggests a complex interplay between different Rho GTPases during phagocytosis of pathogens.
