Peripheral Repertoire Research Articles

Mechanisms of plasticity in the peripheral T cell receptor repertoire (115.15)

Abstract We recently reported a method for amplifying paired T cell receptor (TCR) αβ CDR3 regions from single cells in mice and have developed a similar protocol for human cells. Using this technique, we have discovered a surprising plasticity in peripheral TCR repertoire, particularly under conditions of inflammation. This plasticity includes two major mechanisms: TCR modulation and TCR revision. Modulation consists of the up or down regulation of secondary allele expression, almost exclusively from the TCRα locus. Revision results in changes to the amino acid sequence of the TCR by de novo rearrangement. Here we describe the upregulation of both processes in CD8+ T cells derived from influenza-infected lungs when compared to circulating naïve T cells. In particular, instances of modulation and dual chain expression are elevated over the course of a primary influenza T cell response, whereas in recall responses memory cells express only a single allele of each TCR chain. In repeated infections, revised TCRs are observed by using out of frame TCR sequences as an internal, lineage specific, barcode. The relative contribution of each mechanism to the activation of autoreactive T cells is assessed in genetic models.

Regenerative capacity of adult cortical thymic epithelial cells

Involution of the thymus is accompanied by a decline in the number of thymic epithelial cells (TECs) and a severely restricted peripheral repertoire of T-cell specificities. TECs are essential for T-cell differentiation; they originate from a bipotent progenitor that gives rise to cells of cortical (cTEC) and medullary (mTEC) phenotypes, via compartment-specific progenitors. Upon acute selective near-total ablation during embryogenesis, regeneration of TECs fails, suggesting that losses from the pool of TEC progenitors are not compensated. However, it is unclear whether this is also true for the compartment-specific progenitors. The decline of cTECs is a prominent feature of thymic involution. Because cTECs support early stages of T-cell development and hence determine the overall lymphopoietic capacity of the thymus, it is possible that the lack of sustained regenerative capacity of cTEC progenitor cells underlies the process of thymic involution. Here, we examine this hypothesis by cell-type-specific conditional ablation of cTECs. Expression of the human diphtheria toxin receptor (hDTR) gene under the regulatory influence of the chemokine receptor Ccx-ckr1 gene renders cTECs sensitive to the cytotoxic effects of diphtheria toxin (DT). As expected, DT treatment of preadolescent and adult mice led to a dramatic loss of cTECs, accompanied by a rapid demise of immature thymocytes. Unexpectedly, however, the cTEC compartment regenerated after cessation of treatment, accompanied by the restoration of T-cell development. These findings provide the basis for the development of targeted interventions unlocking the latent regenerative potential of cTECs to counter thymic involution.

Inhibitory Spleen-Derived Anti-ADAMTS13 Antibodies Are Characterized by a Limited Number of Variable Heavy Chain CDR3 Signatures in Patients with Relapsing Acquired Thrombotic Thromobocytopenic Purpura

Abstract 194 Background.Acquired Thrombotic thromobcytopenic purpura (TTP) is the result of autoantibodies neutralizing and-or accelerating clearance of ADAMTS13. We have previously isolated mononuclear cells of two spleens donated to our laboratory by patients (A and B) splenectomized for frequently relapsing TTP to generate human monoclonal anti-ADAMTS13 antibodies. Preliminary characterization of the patient’s entire IgG4 repertoire using phage display technology and Epstein Barr virus (EBV) transformation of switched memory B cells (CD19+, CD27+, IgG+) showed that the memory repertoire of Rituximab-resistant (B) and non-resistant (A) anti-ADAMTS13 IgG used the same restricted VH germline genes, namely VH1-3 (55%), VH1-69 (17%), VH3-30 (7%) and 4–28 (21%). In contrast, peripheral blood-derived IgG1 anti-ADAMTS13 antibody repertoire from 2 acquired TTP patients, inhibiting ADAMTS13 activity by maximal 15–40% used VH1-69 germline gene segment only (Luken et al, JTH 2006 and Pos et al, JTH 2009). To evaluate if the generated spleen-derived monoclonal anti-ADAMTS13 antibodies are of pathological relevance we started with their functional characterization. Methods.Both patients (A and B) were suffering from acute TTP with high titers of anti-ADAMTS13-Ab and therefore needed to be splenectomized because of frequent relapses, which occurred even after several courses of Rituximab (B). Both patients have remained in remission since splenectomy. Previous screening of the IgGk-IgGλ IgG4-Fab phage-display libraries and EBV-transformed clones on recombinant ADAMTS13 by ELISA revealed high ADAMTS13 specificity for 16/34 and 13/234 antibodies, respectively. Functional characterization of the phage-display clones included (a) determination of their inhbitiory capacity by functional FRETS inhibitor assay, (b) assessment of their affinity performed by either fluid-phase competition ELISA and/or plasma surface resonance (Biacore), and (c) epitope mapping by dot blot analysis testing their recognition of recombinant full-length wildtype ADAMTS13 and a truncated ADAMTS13 variant. Results.Detailed sequence analysis of all human monoclonal anti-ADAMTS13 analyzed showed seven unique CDR3 signatures in the variable heavy region of all clones generated using both techniques (phage display and EBV-transformation), three of which were seen in both patients. These CDR3 signatures are different from signatures found in peripheral blood-derived antibodies (Luken et al, JTH 2006 and Pos et al, Blood 2010). Strikingly, the anti-ADAMTS13 antibodies of EBV-transformed memory B cells used λ light chain exclusively, whereas the phage-display-derived clones showed usage of either κ- (A: 6/11 and B: 3/5) or λ-light chain (A: 5/11 and B: 2/5). Anti-ADAMTS13 IgG in supernatants of EBV-immortalized switched memory B-cell clones were mainly of subclass IgG4 (A: 60%; B: 25%) and IgG1 (A: 40%; B: 75%) thus IgG1 being the prevailing subclass in patient B, who relapsed despite several courses of Rituximab. Functional characterization of the anti-ADAMTS13 IgG4 antibodies revealed that 15/16 phage display clones are strongly inhibitory, showing 0–100% (A) and 60–100% (B) inhibition at 200 nM and even 0–92% (A) and 0–16% (B) inhibition at 20 nM. This represents a 5–6 times increase of inhibitory capacity when compared to previouse findingy by others, in the range of 2×10−7-7×10−10 M (A) and 1×10−9-6×10−10M (B) as measured by Biacore or/and competition ELISA. Epitope mapping revealed that 3/16 clones recognized a novel epitope at the C-terminus of ADAMTS13, in contrast to the previously reported N-terminally located spacer domain. Conclusions.The identification of several spleen-derived anti-ADAMTS13 clones displaying highly similar CDR3 variable heavy chains, in 2 unrelated acquired TTP patients is striking and novel. Our data points at the discovery of unique CDR3 signatures that characterize the pathological relevant, inhibitory antibodies paving thus the road to define their triggering ADAMTS13 epitope(s), while providing insight into the mode of success of splenectomy in relapsing TTP patients. Further functional characterization of the anti-ADAMTS13 EBV-derived clones and confirmation of our data by analysis of the immunological memory from two additional spleens are underway. Disclosures:No relevant conflicts of interest to declare.

Chronic B Cell Deficiency from Birth Prevents Age-Related Alterations in the B Lineage

Aging is accompanied by a decline in B lymphopoiesis in the bone marrow and accumulation of long-lived B cells in the periphery. The mechanisms underlying these changes are unclear. To explore whether aging in the B lineage is subjected to homeostatic regulation, we used mutant mice bearing chronic B cell deficiency from birth. We show that chronic B cell deficiency from birth, resulting from impaired maturation (CD19(-/-) and CD74(-/-)) or reduced survival (baff-r(-/-)), prevents age-related changes in the B lineage. Thus, frequencies of early and late hematopoietic stem cells, B lymphopoiesis, and the rate of B cell production do not substantially change with age in these mice, as opposed to wild-type mice where kinetic experiments indicate that the output from the bone marrow is impaired. Further, we found that long-lived B cells did not accumulate and peripheral repertoire was not altered with age in these mice. Collectively, our results suggest that aging in the B lineage is not autonomously progressing but subjected to homeostatic regulation.

Anti-idiotypic Fab Fragments Image a Conserved N-terminal Epitope Patch of Grass Pollen Allergen Phl p 1

BACKGROUND AND AIMS: Naturally occurring anti-idiotypic antibodies structurally mimic the original antibody epitope. Anti-idiotypes, therefore, are interesting tools for the portrayal of conformational B-cell epitopes of allergens. In this study we used this strategy particularly for major timothy grass pollen (Phleum pratense) allergen Phl p 1. METHODS AND RESULTS: We used a combinatorial phage display library constructed from the peripheral IgG repertoire of a grass pollen allergic patient which was supposed to contain anti-idiotypic Fab specificities. Using purified anti-Phl p 1 IgG for biopanning, several Fab displaying phage clones could be isolated. 100 amplified colonies were screened for their binding capacity to anti-Phl p 1-specific antibodies, finally resulting in four distinct Fab clones according to sequence analysis. Interestingly, heavy chains of all clones derived from the same germ line sequence and showed high homology in their CDRs. Projecting their sequence information on the surface of the natural allergen Phl p 1 (PDB ID: 1N10) indicated matches on the N-terminal domain of the homo-dimeric allergen, including the bridging region between the two monomers. The resulting epitope patches were formed by spatially distant sections of the primary allergen sequence. CONCLUSION: In this study we report that anti-idiotypic specificities towards anti-Phl p 1 IgG, selected from a Fab library of a grass pollen allergic patient, mimic a conformational epitope patch being distinct from a previously reported IgE epitope area.

Mechanisms of tolerance

Mechanisms of tolerance

Immune tolerance in multiple sclerosis

Multiple sclerosis is believed to be mediated by T cells specific for myelin antigens that circulate harmlessly in the periphery of healthy individuals until they are erroneously activated by an environmental stimulus. Upon activation, the T cells enter the central nervous system and orchestrate an immune response against myelin. To understand the initial steps in the pathogenesis of multiple sclerosis, it is important to identify the mechanisms that maintain T-cell tolerance to myelin antigens and to understand how some myelin-specific T cells escape tolerance and what conditions lead to their activation. Central tolerance strongly shapes the peripheral repertoire of myelin-specific T cells, as most myelin-specific T cells are eliminated by clonal deletion in the thymus. Self-reactive T cells that escape central tolerance are generally capable only of low-avidity interactions with antigen-presenting cells. Despite the low avidity of these interactions, peripheral tolerance mechanisms are required to prevent spontaneous autoimmunity. Multiple peripheral tolerance mechanisms for myelin-specific T cells have been identified, the most important of which appears to be regulatory T cells. While most studies have focused on CD4(+) myelin-specific T cells, interesting differences in tolerance mechanisms and the conditions that abrogate these mechanisms have recently been described for CD8(+) myelin-specific T cells.

Autoimmunity-associated LYP regulates T cell receptor signaling and thymus development (148.21)

Abstract A single nucleotide polymorphism (SNP) in the PTPN22 gene, which encodes the lymphoid tyrosine phophatase LYP, is associated with increased risk of Rheumatoid Arthritis and other autoimmune diseases. LYP inhibits T cell receptor (TCR) signaling by dephosphorylation of Lck and Zap70. The disease associated LYP R620W (LypW) protein caused by the missense C1858T SNP in PTPN22 appears to exert “gain-of-function” effects upon TCR activation. Here we show that LypW negatively regulates TCR-induced intracellular Ca2+ flux, tyrosine phosphorylation, MAPK activation, and NFAT response element transactivation in Jurkat cells. The effects of LYP on TCR signaling are dependent on its enzymatic function. Studies from ptpn22−/− mice shows that PTPN22 regulates thymocyte positive selection. To evaluate effects of LypW expression on lymphocyte selection and function, we generated BAC transgenic mice bearing either the major human PTPN22 allele (LypR) or LypW. In contrast to intact positive selection in LypW H-Y TCR transgenic female mice, we observed impaired negative selection in LypW, but not LypR, H-Y male mice. These findings suggest that susceptibility to autoimmune diseases in LypW-carrying people may result from enhanced presence of autoreactive T cell in the peripheral repertoire, due to impaired thymocyte deletion. Together, our data suggest a thymic developmental mechanism whereby a “gain of function” Lyp variant can predispose to autoimmune disease.

Fas (CD95/APO-1) limits the expansion of T lymphocytes in an environment of limited T-cell antigen receptor/MHC contacts

Fas-deficient mice (Fas(lpr/lpr)) and humans have profoundly dysregulated T lymphocyte homeostasis, which manifests as an accumulation of CD4(+) and CD8(+) T cells as well as an unusual population of CD4(-)CD8(-)TCRαβ(+) T cells. To date, no unifying model has explained both the increased T-cell numbers and the origin of the CD4(-)CD8(-)TCRαβ(+) T cells. As Fas(lpr/lpr) mice raised in a germ-free environment still manifest lymphadenopathy, we considered that this process is primarily driven by recurrent low-avidity TCR signaling in response to self-peptide/MHC as occurs during homeostatic proliferation. In these studies, we developed two independent systems to decrease the number of self-peptide/MHC contacts. First, expression of MHC class I was reduced in OT-I TCR transgenic mice. Although OT-I Fas(lpr/lpr) mice did not develop lymphadenopathy characteristic of Fas(lpr/lpr) mice, in the absence of MHC class I, OT-I Fas(lpr/lpr) T cells accumulated as both CD8(+) and CD4(-)CD8(-) T cells. In the second system, re-expression of β(2)m limited to thymic cortical epithelial cells of Fas(lpr/lpr) β(2)m-deficient mice yielded a model in which polyclonal CD8(+) thymocytes entered a peripheral environment devoid of MHC class I. These mice accumulated significantly greater numbers of CD4(-)CD8(-)TCRαβ(+) T cells than conventional Fas(lpr/lpr) mice. Thus, Fas shapes the peripheral T-cell repertoire by regulating the survival of a subset of T cells proliferating in response to limited self-peptide/MHC contacts.

Thymic Self-Antigen Expression for the Design of a Negative/Tolerogenic Self-Vaccine against Type 1 Diabetes

Before being able to react against infectious non-self-antigens, the immune system has to be educated in the recognition and tolerance of neuroendocrine proteins, and this critical process essentially takes place in the thymus. The development of the autoimmune diabetogenic response results from a thymus dysfunction in programming central self-tolerance to pancreatic insulin-secreting islet β cells, leading to the breakdown of immune homeostasis with an enrichment of islet β cell reactive effector T cells and a deficiency of β cell-specific natural regulatory T cells (nTreg) in the peripheral T-lymphocyte repertoire. Insulin-like growth factor 2 (IGF-2) is the dominant member of the insulin family expressed during fetal life by the thymic epithelium under the control of the autoimmune regulator (AIRE) gene/protein. Based on the close homology and cross-tolerance between insulin, the primary T1D autoantigen, and IGF-2, the dominant self-antigen of the insulin family, a novel type of vaccination, so-called “negative/tolerogenic self-vaccination”, is currently developed for prevention and cure of T1D. If this approach were found to be effective for reprogramming immunological tolerance in T1D, it could pave the way for the design of negative self-vaccines against autoimmune endocrine diseases, as well as other organ-specific autoimmune diseases.

Transcription Factor Network Regulating CD+CD8+ Thymocyte Survival

More than 80% of thymocytes are CD4(+)CD8(+) double positive (DP) cells subject to positive/ negative selection. The lifespan of DP thymocytes is critical in shaping the peripheral T-cell repertoire essential for mounting immune responses against foreign, but not self, antigens. During T-cell maturation, if the first round of T-cell receptor (TCR) α chain rearrangement fails to generate a productive T-cell receptor, DP cells start another round of α chain rearrangement until positive selection or cell death intervenes. Thus, the lifespan of DP cells determines how many rounds of α chain rearrangement can be carried out, and influences the likelihood of completing positive selection. The antiapoptotic protein Bcl-x(L) is the ultimate effector regulating DP cell survival, and several transcription factors critical for T-cell development, such as TCF-1, E proteins, c-Myb, and RORγt, regulate DP survival via a Bcl-x(L)-dependent pathway. However, the relationship between these transcription factors in this process is largely unclear. Recent results are revealing an interactive network among these critical factors during regulation of DP thymocyte survival. This review will discuss how these transcription factors potentially work together to control DP thymocyte survival that is critical for successful completion of T-cell development.

HLA-Dependent Immune Selection Pressure on the Idiotype Is Predominantly Driven by Somatic Hypermutation In Malignant Lymphoma but Also Modulates Vh Usage In Normal B Cells

AbstractAbstract 3132The idiotype (Id) of malignant lymphomas is a tumor-specific antigen and has been evaluated for active immunotherapy predominantly in follicular lymphoma (FL). We have previously described “reverse immunology” evidence that HLA class I-dependent and hence presumably T-cell mediated selection pressure restricts the growth of malignant FL clones with comparatively highly immunogenic Ids (Strothmeyer et al., Blood, epub 2010). This effect was mostly attributable to the Id CDRs. We therefore hypothesized that somatic hypermutation, a constitutively active process in FL, is the mechanism that creates immunogenic Id epitopes and enables T-cell mediated immunosurveillance. To test this hypothesis, we analyzed Id of FL that utilized the same Vh gene but arose in individuals with different HLA types (IGHV3-23*01, n=9 patients: IGHV3-7*01, n=3). For every represented HLA type, an “immunogenicity landscape” mapping was performed by plotting the ten most immunogenic peptides of the germ-line Vh sequence in comparison to FL Id peptides in the order of their HLA binding strength as predicted by the BIMAS algorithm for the respective patient. Neither changes in the order of peptides nor major differences in their binding scores were observed. Parallel plotting of Vh-related Ids from different HLA types revealed novel peptides with higher binding scores within and in addition to the ten highest ranking epitopes (Figures). Comparisons of these peptides with the germ-line and the index patient's sequence directly demonstrated that somatic hypermutation creates peptides that would have strongly bound to HLA type of the index patient but were immunologically neutral in the patient of origin. Occurrence of such highly immunogenic peptides by somatic hypermutation would hence not be tolerated by an FL patient's immune system. To explore whether this mechanism also operates in other lymphoma types, we analyzed predicted HLA-binding Id peptides from mantle cell (n=20) and marginal zone (n=12) lymphomas. Like in FL, the median sum of the 20 peptides with the highest BIMAS scores were to various degrees less immunogenic on the autologous HLA type than on allogeneic HLA types (ratio of autologous to allogeneic BIMAS sum scores: FL: 0.63; MCL: 0.78; MZL: 0.87). In subanalyses of the CDR, these differences were observed only in FL and MCL but not in MZL (CDR1: FL 0.40, MCL 0.55. MZL 1.39; CDR2: FL 0.25, MCL 0.36; MZL 1.54: CDR3: FL 0.43, MCL 0.53, MZL 1.28).To investigate whether HLA-dependent immunosurveillance acts on normal B-cell receptors, we compared BIMAS sum scores of 96 non-clonal Ids present in diagnostic biopsies of 29 FL and MCL patients. These Ids represent bona fide contaminating, healthy B cells. No significant differential immunogenicity existed for the entire Vh sequences or individual CDR regions, regardless of their mutational status. We therefore randomly sequenced B-cell receptors from two mouse strains with an identical B6 genetic background but a congenic H-2 complex (C57BL/6ByJ: H-2b; 422 sequences; B6-H2d/bByJ: H-2d; 412 sequences). In these SPF-bred mice, 61% and 71% of sequences, respectively, were unmutated. Significantly higher immunogenicity was observed in the FR1-3 regions of C57BL/6ByJ compared to B6-H2d/bByJ Ids on H-2kd (p=0.04). Also, several Vh genes were significantly (Fisher's exact test) less frequent in mice expressing the MHC type with stronger binding to Id-derived peptides of the corresponding germ-line Vh sequence. Overall, the ratio of predicted MHC binding strength of the 20 highest scoring peptides from 59 represented germ-line Vh genes correlated to the relative frequency of representation of these genes in the peripheral B-cell repertoire (p=0.016, Pearson correlation). Collectively, these results indicate that somatic hypermutation is the predominant mechanism that enables T-cell mediated control of malignant B-cell clones in FL but possibly also MCL on the basis of the HLA binding of Id-derived peptides. In addition, HLA binding strength of germ-line-encoded Vh peptides appears to exert a subtle modulation of the primary B cell repertoire by MHC class I-restricted T cells. These findings strengthen the theoretical basis of active Id-directed immunotherapy of B-cell lymphomas with mutated antigen receptors. In addition, they may also lead to a novel explanation for the association of HLA type with aberrant functions of the nonmalignant immune system. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Analysis of the peripheral T‐cell repertoire in kidney transplant patients

The long-term stability of renal grafts depends on the absence of chronic rejection. As T cells play a key role in rejection processes, analyzing the T-cell repertoire may be useful for understanding graft function outcomes. We have therefore investigated the power of a new statistical tool, used to analyze the peripheral blood TCR repertoire, for determining immunological differences in a group of 229 stable renal transplant patients undergoing immunosuppression. Despite selecting the patients according to stringent criteria, the patients displayed heterogeneous T-cell repertoire usage, ranging from unbiased to highly selected TCR repertoires; a skewed TCR repertoire correlating with an increase in the CD8(+) /CD4(+) T-cell ratio. T-cell repertoire patterns were compared in patients with clinically opposing outcomes i.e. stable drug-free operationally tolerant recipients and patients with the "suspicious" form of humoral chronic rejection and were found significantly different, from polyclonal to highly selected TCR repertoires, respectively. Moreover, a selected TCR repertoire was found to positively correlate with the Banff score grade. Collectively, these data suggest that TCR repertoire categorization might be included in the calculation of a composite score for the follow-up of patients after kidney transplantation.

Natural killer T cells constitutively expressing the interleukin‐2 receptor α chain early in life are primed to respond to lower antigenic stimulation

Invariant natural killer T (iNKT) cells are known to constitutively express the high affinity interleukin-2 receptor α chain (CD25) in neonates, but the functional consequence of this phenotype is unknown. Here, we show that high numbers of CD25-expressing iNKT cells are present early in gestation and represent a significant proportion of the developing immune system. Despite their activated phenotype, neonatal iNKT cells express high levels of the Krüppel-like factor-2, a transcription factor associated with quiescent T cells, and require de novo T-cell receptor and CD28 co-stimulation to proliferate. In contrast to bona fide CD4/CD25-expressing regulatory T cells, neonatal iNKT cells do not suppress T-cell responses, indicating that they do not represent an immunosuppressive cell subset. Evidence that neonatal iNKT cells respond to dramatically reduced amounts of CD1d-restricted antigen compared with adult iNKT cells or T cells, and that their proliferation can be induced in the absence of early interleukin-2 suggest that constitutive expression of CD25 'primes' neonatal iNKT cells to respond rapidly to low amounts of antigen. This unique phenotype, which is distinct from adult iNKT cells, as well as other CD25-expressing activated T or regulatory T cells, may be important to ensure stability of a structurally limited peripheral iNKT-cell repertoire early in life.

Immune Reconstitution Following Rabbit Antithymocyte Globulin

Depletional induction therapies are routinely used to prevent acute rejection and improve transplant outcome. The effects of depleting agents on T-cell subsets and subsequent T-cell reconstitution are incompletely defined. We used flow cytometry to examine the effects of rabbit antithymocyte globulin (rATG) on the peripheral T-cell repertoire of pediatric and adult renal transplant recipients. We found that while rATG effectively depleted CD45RA+CD27+ naïve and CD45RO+CD27+ central memory CD4+ T cells, it had little effect on CD45RO+CD27- CD4+ effector memory or CD45RA+CD31-, CD45RO+CD27+ and CD45RO+CD27- CD8+ T cell subsets. When we performed a kinetic analysis of CD31+ recent thymic emigrants and CD45RA+/RO+ T cells, we found evidence for both thymopoiesis and homeostatic proliferation contributing to immune reconstitution. We additionally examined the impact of rATG on peripheral CD4+Foxp3+ T cells. We found that in adults, administration of rATG-induced peripheral expansion and new thymic emigration of T cells with a Treg phenotype, while CD4+Foxp3+ T cells of thymic origin predominated in children, providing the first evidence that rATG induces Treg in vivo. Collectively our data indicate that rATG alters the balance of regulatory to memory effector T cells posttransplant, providing an explanation for how it positively impacts transplant outcome.

B-lymphocyte homeostasis and BLyS-directed immunotherapy in transplantation

Current strategies for immunotherapy after transplantation are primarily T-lymphocyte directed and effectively abrogate acute rejection. However, the reality of chronic allograft rejection attests to the fact that transplantation tolerance remains an elusive goal. Donor-specific antibodies are considered the primary cause of chronic rejection. When naive, alloreactive B-cells encounter alloantigen and are activated, a resilient "sensitized" state, characterized by the presence of high-affinity antibody, is established. Here, we will delineate findings that support transient B-lymphocyte depletion therapy at the time of transplantation to preempt sensitization by eliminating alloreactive specificities from the recipient B-cell pool (ie, "repertoire remodeling"). Recent advances in our understanding of B-lymphocyte homeostasis provide novel targets for immunomodulation in transplantation. Specifically, the tumor necrosis factor-related cytokine BLyS is the dominant survival factor for "tolerance-susceptible" transitional and "preimmune" mature follicular B-cells. The transitional phenotype is the intermediate through which all newly formed B-cells pass before maturing into the follicular subset, which is responsible for mounting an alloantigen-specific antibody response. Systemic BLyS levels dictate the stringency of negative selection during peripheral B-cell repertoire development. Thus, targeting BLyS will likely provide an opportunity for repertoire-directed therapy to eliminate alloreactive B-cell specificities in transplant recipients, a requirement for the achievement of humoral tolerance and prevention of chronic rejection. In this review, the fundamentals of preimmune B-cell selection, homeostasis, and activation will be described. Furthermore, new and current B-lymphocyte-directed therapy for antibody-mediated rejection and the highly sensitized state will be discussed. Overall, our objective is to propose a rational approach for induction of humoral transplantation tolerance by remodeling the primary B-cell repertoire of the allograft recipient.

Thymic self-antigens for the design of a negative/tolerogenic self-vaccination against type 1 diabetes

Before being able to react against infectious nonself-antigens, the immune system has to be educated in the recognition and tolerance of neuroendocrine proteins and this critical process takes place only in the thymus. The development of the autoimmune diabetogenic response results from a thymus dysfunction in programing central self-tolerance to pancreatic insulin-secreting islet beta cells, leading to the breakdown of immune homeostasis with an enrichment of islet beta-cell reactive effector T cells and a deficiency of beta-cell specific natural regulatory T cells (nTregs) in the peripheral T-lymphocyte repertoire. Insulin-like growth factor 2 (IGF-2) is the dominant member of the insulin family expressed during fetal life by the thymic epithelium under the control of the autoimmune regulator (AIRE) gene/protein. The very low degree of insulin gene transcription in normal murine and human thymus explains why the insulin protein is poorly tolerogenic as demonstrated in many studies, including the failure of all clinical trials that have attempted immune tolerance to islet beta cells via various methods of insulin administration. On the basis of the close homology and crosstolerance between insulin, the primary T1D autoantigen, and IGF-2, the dominant self-antigen of the insulin family, a novel type of vaccination, so-called 'negative/tolerogenic self-vaccination', is currently being developed for the prevention and cure of T1D. If this approach were found to be effective for reprograming immunological tolerance in T1D, it could pave the way for the design of other self-vaccines against autoimmune endocrine diseases, as well as other organ-specific autoimmune diseases.

The feature of clonal expansion of TCR Vβ repertoire, thymic recent output function and TCRζ chain expression in patients with immune thrombocytopenic purpura

In order to identify the feature of T cells immune status in idiopathic/immune thrombocytopenic purpura (ITP) patients, TCR Vbeta repertoire, T-cell receptor (TCR) excision DNA circles (TRECs) and TCRzeta chain gene expression were examined. Reverse-transcriptase polymerase chain reaction, genescan and real-time PCR techniques were used to analyze. DNA and cDNA from peripheral blood mononuclear cells (PBMCs) of 18 ITP patients were investigated and 25 normal individuals served as control. The results showed that only 4-11 Vbeta subfamilies could be detected in each ITP case. The most frequently expressed Vbeta subfamilies were Vbeta2 and Vbeta3, while 11 Vbeta subfamilies were absent. Clonal expansion of Vbeta T cells were found in all patients. The most frequent clonal expansion T cell was Vbeta21. The number of TRECs in ITP patients (2.60 +/- 2.99 copies/1000 PBMCs) was not significant different, compared with control (3.76 +/- 3.42 copies/1000 PBMCs). The expression level of TCRzeta gene was significantly lower in ITP samples than those in control (P = 0.017). In summary, T cells in ITP patients were characterized by restricted expression of TCR Vbeta subfamilies and clonal expansion predominant in Vbeta21. The alteration of peripheral TCR Vbeta repertoire pattern may not relate to the thymic, recent output function in ITP patients. Our report is the first description of decreased TCRzeta mRNA level in the majority of ITP patients.

Narrowing of T‐cell receptor beta variable repertoire during symptomatic herpesvirus infection in transplant patients

Primary infection or recrudescence of latent virus infection in transplant recipients can be manifested either as asymptomatic or symptomatic disease. Here we show that symptomatic human cytomegalovirus (HCMV) or Epstein-Barr virus (EBV) infection or recrudescence following solid organ transplantation (SOT) was coincident with a dramatic skewing of T-cell receptor beta variable (TRBV) repertoire, with expansions of monoclonal/oligoclonal clonotypes. As the clinical symptoms resolved, the peripheral blood repertoire reverted to a more diverse distribution. In contrast, SOT recipients with asymptomatic or no viral infection or recrudescence showed minimal or no skewing of the T-cell receptor repertoire to maintain peripheral blood repertoire diversity. More importantly, we show that large monoclonal/oligoclonal repertoire expansions are associated with the loss of HCMV-specific T-cell function observed in SOT patients undergoing symptomatic viral infection or recrudescence, whereas SOT recipients who maintain peripheral blood TRBV repertoire diversity and functional antigen-specific T-cell responses can resist clinical symptomatic disease in spite of high levels of viral load.

The feature of TRGV and TRDV repertoire distribution and clonality in patients with immune thrombocytopenic purpura

Chronic idiopathic (immune) thrombocytopenic purpura (ITP) is an autoimmune disorder in which anti-platelet antibodies induce platelet destruction due to an imbalanced immune response. Recently, data indicated the γδ+T cells may play an important role in autoimmune disease. Our previous study has shown the restricted expression of TRBV subfamilies and the alteration of peripheral TRBV repertoire pattern in the majority of ITP patients. In the present study, we further analyze the feature of TRGV and TRDV repertoire distribution and clonality in patients with ITP. The CDR3 size of three TRGV and eight TRDV subfamily genes were analyzed in peripheral blood mononuclear cells (PBMCs) from 11 cases with ITP, using RT-PCR and GeneScan techniques. To determine the expression level of TRGV subfamily genes, quantitative analysis of TRGV I–III subfamilies was performed by real-time PCR. TRGV I–III subfamilies could be detected in the most samples from ITP as well as in healthy controls. However, clonal expansion of TRGV was identified in five cases with ITP, which displayed polyclonality in all of samples from healthy controls. The expression level of all TRGV I–III subfamilies in ITP was significantly lower than that from healthy controls (p=0·048, 0·001, 0·035, respectively). The expression pattern of TRGV I–III repertoire in ITP was TRGV I>TRGV III>TRGV II, in contrast, TRGV II>TRGV I>TRGV II was found in healthy controls. TRDV 1 and TRDV 2 could be detected in most samples from ITP as well as in healthy controls, whereas TRDV 3 could be detected in only two out of 11 cases with ITP, which could be found in 90% of healthy controls (p=0·02). Oligoclonally expanded TRDV 1 and TRDV 2 T cells could be identified in the half of the ITP samples, with similar results in healthy control. In conclusion, the alteration of peripheral TRGV and TRDV repertoire pattern might play a role in the pathogenesis of immune-mediated platelet destruction in some cases with ITP.