CXCR4 acts as a costimulator during thymic beta-selection.

Passage through the beta-selection developmental checkpoint requires productive rearrangement of segments of the T cell antigen receptor-beta gene (Tcrb) and formation of a pre-TCR on the surface of CD4(-)CD8(-) thymocytes. How other receptors influence betabeta-selection is less well understood. Here we define a new role for the chemokine receptor CXCR4 during T cell development. CXCR4 functionally associated with the pre-TCR and influenced beta-selection by regulating the steady-state localization of immature thymocytes in thymic subregions, by facilitating optimal pre-TCR-induced survival signals, and by promoting thymocyte proliferation. We also characterize functionally relevant signaling molecules downstream of CXCR4 and the pre-TCR in thymocytes. Our data designate CXCR4 as a costimulator of the pre-TCR during beta-selection.

Two Successive Inversional Vß Rearrangements on a Single Tcrb Allele Can Contribute to the TCRß Repertoire.

Mammalian TCRß loci contain 30 Vß gene segments upstream and in the same transcriptional orientation as two DJCß clusters, and a downstream Vß (TRBV31) in the opposite orientation. The textbook view is upstream Vßs rearrange only by deletion and TRBV31 rearranges only by inversion to create VßDJCß genes. In this study, we show in mice that upstream Vßs recombine through inversion to the DJCß2 cluster on alleles carrying a preassembled Trbv31-DJCß1 gene. When this gene is in-frame, Trbv5 evades TCRß-signaled feedback inhibition and recombines by inversion to the DJCß2 cluster, creating αß T cells that express assembled Trbv5-DJCß2 genes. On alleles with an out-of-frame Trbv31-DJCß1 gene, most upstream Vßs recombine at low levels and promote αß T cell development, albeit with preferential expansion of Trbv1-DJß2 rearrangements. Finally, we show wild-type Tcrb alleles produce mature αß T cells that express upstream Vß peptides in surface TCRs and carry Trbv31-DJß2 rearrangements. Our study indicates two successive inversional Vß-to-DJß rearrangements on the same allele can contribute to the TCRß repertoire.

T cell receptor sequencing of early-stage breast cancer tumors identifies altered clonal structure of the T cell repertoire.

Tumor-infiltrating T cells play an important role in many cancers, and can improve prognosis and yield therapeutic targets. We characterized T cells infiltrating both breast cancer tumors and the surrounding normal breast tissue to identify T cells specific to each, as well as their abundance in peripheral blood. Using immune profiling of the T cell beta-chain repertoire in 16 patients with early-stage breast cancer, we show that the clonal structure of the tumor is significantly different from adjacent breast tissue, with the tumor containing ∼2.5-fold greater density of T cells and higher clonality compared with normal breast. The clonal structure of T cells in blood and normal breast is more similar than between blood and tumor, and could be used to distinguish tumor from normal breast tissue in 14 of 16 patients. Many T cell sequences overlap between tissue and blood from the same patient, including ∼50% of T cells between tumor and normal breast. Both tumor and normal breast contain high-abundance "enriched" sequences that are absent or of low abundance in the other tissue. Many of these T cells are either not detected or detected with very low frequency in the blood, suggesting the existence of separate compartments of T cells in both tumor and normal breast. Enriched T cell sequences are typically unique to each patient, but a subset is shared between many different patients. We show that many of these are commonly generated sequences, and thus unlikely to play an important role in the tumor microenvironment.

T-cell repertoires in refractory coeliac disease.

OBJECTIVE: Refractory coeliac disease (RCD) is a potentially hazardous complication of coeliac disease (CD). In contrast to RCD type I, RCD type II is a precursor entity of enteropathy-associated T-cell lymphoma (EATL), which is associated with clonally expanding T-cells that are also found in the sequentially developing EATL. Using high-throughput sequencing (HTS), we aimed to establish the small-intestinal T-cell repertoire (TCR) in CD and RCD to unravel the role of distinct T-cell clonotypes in RCD pathogenesis. DESIGN: DNA extracted from duodenal mucosa specimens of controls (n=9), active coeliacs (n=10), coeliacs on a gluten-free diet (n=9), RCD type I (n=8), RCD type II (n=8) and unclassified Marsh I cases (n=3) collected from 2002 to 2013 was examined by TCRß-complementarity-determining regions 3 (CDR3) multiplex PCR followed by HTS of the amplicons. RESULTS: On average, 106 sequence reads per sample were generated consisting of up to 900 individual TCRß rearrangements. In RCD type II, the most frequent clonotypes (ie, sequence reads with identical CDR3) represent in average 42.6% of all TCRß rearrangements, which was significantly higher than in controls (6.8%; p<0.01) or RCD type I (6.7%; p<0.01). Repeat endoscopies in individual patients revealed stability of clonotypes for up to several years without clinical symptoms of EATL. Dominant clonotypes identified in individual patients with RCD type II were unique and not related between patients. CD-associated, gliadin-dependent CDR3 motifs were only detectable at low frequencies. CONCLUSIONS: TCRß-HTS analysis unravels the TCR in CD and allows detailed analysis of individual TCRß rearrangements. Dominant TCRß sequences identified in patients with RCD type II are unique and not homologous to known gliadin-specific TCR sequences, supporting the assumption that these clonal T-cells expand independent of gluten stimulation.

Comprehensive assessment of T-cell repertoire following autologous hematopoietic stem cell transplantation for treatment of type 1 diabetes using high-throughput sequencing.

OBJECTIVE: We aimed to investigate T-cell receptor (TCR) repertoires in type 1 diabetes (T1D) patients receiving autologous hematopoietic stem cell transplantation (AHSCT) treatment. METHODS: High-throughput deep TCR beta (TCRB) chain sequencing was performed to assess millions of individual TCRs in five T1D patients receiving AHSCT treatment and another five patients receiving insulin treatment during 12 months of follow-up. RESULTS: No significant changes in TCRB sequence reads, complementarity-determining region 3 (CDR3) sequences, or the usage of TCRB VJ gene-segments were observed at 12 months after AHSCT. Compared with the baseline, the usage of TCRB VJ gene-segments at 12 months decreased in the insulin treatment group (1836.4 ± 437.7 vs 2763.6 ± 390.6, P = 0.015), and the change rates were larger than those undergoing AHSCT (-0.62 ± 0.16 vs 0.06 ± 0.45, P = 0.002). Changes in the TCR repertoire were smaller after AHSCT than those with insulin treatment (P = 2.2*10-32 ). TCRBV 7-7/TCRBJ 2-5 was depleted after AHSCT while expanded with insulin treatment. TCRBV 12-4, TCRBV 10-3, TCRBV 12-3/TCRBJ 1-2 were expanded after AHSCT while ablated with insulin treatment. CONCLUSIONS: We found that AHSCT is safe without reduction in the diversity of TCR repertoires and TCR repertoires tend to be more stable after AHSCT. Furthermore, these four candidate TCRBV/TCRBJ gene usages on CDR3 regions may act as therapeutic targets and biomarkers.

A circulating reservoir of pathogenic-like CD4+ T cells shares a genetic and phenotypic signature with the inflamed synovial micro-environment.

OBJECTIVES: Systemic immunological processes are profoundly shaped by the micro-environments where antigen recognition occurs. Identifying molecular signatures distinctive of such processes is pivotal to understand pathogenic immune responses and manipulate them for therapeutic purposes. Unfortunately, direct investigation of peripheral tissues, enriched in pathogenic T cells, is often impossible or imposingly invasive in humans. Conversely, blood is easily accessible, but pathogenic signatures are diluted systemically as a result of the strict compartmentalisation of immune responses. In this work, we aimed at defining immune mediators shared between the bloodstream and the synovial micro-environment, and relevant for disease activity in autoimmune arthritis. METHODS: CD4(+) T cells from blood and synovium of patients with juvenile idiopathic arthritis (JIA) were immunophenotyped by flow cytometry. The TCR repertoire of a circulating subset showing similarity with the synovium was analysed through next-generation sequencing of TCR ß-chain CDR3 to confirm enrichment in synovial clonotypes. Finally, clinical relevance was established by monitoring the size of this subset in the blood of patients with JIA and rheumatoid arthritis (RA). RESULTS: We identified a small subset of circulating CD4(+) T cells replicating the phenotypical signature of lymphocytes infiltrating the inflamed synovium. These circulating pathogenic-like lymphocytes (CPLs) were enriched in synovial clonotypes and they exhibited strong production of pro-inflammatory cytokines. Importantly, CPLs were expanded in patients with JIA, who did not respond to therapy, and also correlated with disease activity in patients with RA. CONCLUSIONS: CPLs provide an accessible reservoir of pathogenic cells recirculating into the bloodstream and correlating with disease activity, to be exploited for diagnostic and research purposes.

Unifying model for molecular determinants of the preselection Vß repertoire.

The primary antigen receptor repertoire is sculpted by the process of V(D)J recombination, which must strike a balance between diversification and favoring gene segments with specialized functions. The precise determinants of how often gene segments are chosen to complete variable region coding exons remain elusive. We quantified Vß use in the preselection Tcrb repertoire and report relative contributions of 13 distinct features that may shape their recombination efficiencies, including transcription, chromatin environment, spatial proximity to their DßJß targets, and predicted quality of recombination signal sequences (RSSs). We show that, in contrast to functional Vß gene segments, all pseudo-Vß segments are sequestered in transcriptionally silent chromatin, which effectively suppresses wasteful recombination. Importantly, computational analyses provide a unifying model, revealing a minimum set of five parameters that are predictive of Vß use, dominated by chromatin modifications associated with transcription, but largely independent of precise spatial proximity to DßJß clusters. This learned model-building strategy may be useful in predicting the relative contributions of epigenetic, spatial, and RSS features in shaping preselection V repertoires at other antigen receptor loci. Ultimately, such models may also predict how designed or naturally occurring alterations of these loci perturb the preselection use of variable gene segments.

Transcriptional immune response of cage-cultured Pacific bluefin tuna during infection by two Cardicola blood fluke species.

Infections by two blood fluke species, Cardicola orientalis and Cardicola opisthorchis, currently present the greatest disease concern for the sea-cage culture of Pacific bluefin tuna (PBT) - a species of high global economic importance and ecological concern. In this study, we aimed to rapidly, quantitatively, and differentially identify infections by these two parasite species in cultured PBT as well as identify potential host immune responses. Using real-time qPCR, we were successful in quantitatively detecting parasite-specific DNA from within host blood, gill, and heart tissues; positively identifying parasitic infections 44 days earlier than microscopy methods previously employed. Both gill and heart became heavily infected by both parasite species in PBT within two months of sea-cage culture, which was only mitigated by the administration of anthelmintic praziquantel. Nevertheless, fish were observed to mount an organ specific transcriptive immune response during infection that mirrored the relative quantity of pathogenic load. In heart, significant (3-6 fold) increases in IgM, MHC2, TCRß, and IL-8 transcription was observed in infected fish relative to uninfected controls; whereas in the gills only IgM transcription was observed to be induced (11 fold) by infection. Interestingly, the relative quantity of IgM transcription was highly correlated to the relative abundance of C. orientalis but not C. opisthorchis DNA in the gill samples, even though this organ showed high prevalence of DNA from both parasite species. Taken together, these findings indicate that although ineffective at combating infection during primary exposure, a cellular immune response is mounted in PBT as a potential rejoinder to future Cardicola exposure, particularly against C. orientalis. Although future investigation into antibody effectiveness will be needed, this work provides valuable preliminary insight into host responsiveness to Cardicola infection as well as additional support for the need of anthelmintic treatment following primary parasite exposure during PBT culture.

Differential regulation of proximal and distal Vbeta segments upstream of a functional VDJbeta1 rearrangement upon beta-selection.

Developmental stage-specific regulation of transcriptional accessibility helps control V(D)J recombination. Vß segments on unrearranged TCRß alleles are accessible in CD4(-)/CD8(-) (double-negative [DN]) thymocytes, when they recombine, and inaccessible in CD4(+)/CD8(+) (double-positive [DP]) thymocytes, when they do not rearrange. Downregulation of Vß accessibility on unrearranged alleles is linked with Lat-dependent ß-selection signals that inhibit Vß rearrangement, stimulate Ccnd3-driven proliferation, and promote DN-to-DP differentiation. Transcription and recombination of Vßs on VDJß-rearranged alleles in DN cells has not been studied; Vßs upstream of functional VDJß rearrangements have been found to remain accessible, yet not recombine, in DP cells. To elucidate contributions of ß-selection signals in regulating Vß transcription and recombination on VDJß-rearranged alleles, we analyzed wild-type, Ccnd3(-/-), and Lat(-/-) mice containing a preassembled functional Vß1DJCß1 (Vß1(NT)) gene. Vß10 segments located just upstream of this VDJCß1 gene were the predominant germline Vßs that rearranged in Vß1(NT/NT) and Vß1(NT/NT)Ccnd3(-/-) thymocytes, whereas Vß4 and Vß16 segments located further upstream rearranged at similar levels as Vß10 in Vß1(NT/NT)Lat(-/-) DN cells. We previously showed that Vß4 and Vß16, but not Vß10, are transcribed on Vß1(NT) alleles in DP thymocytes; we now demonstrate that Vß4, Vß16, and Vß10 are transcribed at similar levels in Vß1(NT/NT)Lat(-/-) DN cells. These observations indicate that suppression of Vß rearrangements is not dependent on Ccnd3-driven proliferation, and DN residence can influence the repertoire of Vßs that recombine on alleles containing an assembled VDJCß1 gene. Our findings also reveal that ß-selection can differentially silence rearrangement of germline Vß segments located proximal and distal to functional VDJß genes.

A barrier-type insulator forms a boundary between active and inactive chromatin at the murine TCRß locus.

In CD4(-)CD8(-) double-negative thymocytes, the murine Tcrb locus is composed of alternating blocks of active and inactive chromatin containing Tcrb gene segments and trypsinogen genes, respectively. Although chromatin structure is appreciated to be critical for regulated recombination and expression of Tcrb gene segments, the molecular mechanisms that maintain the integrity of these differentially regulated Tcrb locus chromatin domains are not understood. We localized a boundary between active and inactive chromatin by mapping chromatin modifications across the interval extending from Prss2 (the most 3' trypsinogen gene) to D(ß)1. This boundary, located 6 kb upstream of D(ß)1, is characterized by a transition from repressive (histone H3 lysine 9 dimethylation [H3K9me2]) to active (histone H3 acetylation [H3ac]) chromatin and is marked by a peak of histone H3 lysine 4 dimethylation (H3K4me2) that colocalizes with a retroviral long terminal repeat (LTR). Histone H3 lysine 4 dimethylation is retained and histone H3 lysine 9 dimethylation fails to spread past the LTR even on alleles lacking the Tcrb enhancer (E(ß)) suggesting that these features may be determined by the local DNA sequence. Notably, we found that LTR-containing DNA functions as a barrier-type insulator that can protect a transgene from negative chromosomal position effects. We propose that, in vivo, the LTR blocks the spread of heterochromatin, and thereby helps to maintain the integrity of the E(ß)-regulated chromatin domain. We also identified low-abundance, E(ß)-dependent transcripts that initiate at the border of the LTR and an adjacent long interspersed element. We speculate that this transcription, which extends across D(ß), J(ß) and C(ß) gene segments, may play an additional role promoting initial opening of the E(ß)-regulated chromatin domain.

Circulating activated and effector memory T cells are associated with calcification and clonal expansions in bicuspid and tricuspid valves of calcific aortic stenosis.

We sought to delineate further the immunological significance of T lymphocytes infiltrating the valve leaflets in calcific aortic stenosis (CAS) and determine whether there were associated alterations in circulating T cells. Using clonotypic TCR ß-chain length and sequence analysis we confirmed that the repertoire of tricuspid CAS valves contains numerous expanded T cell clones with varying degrees of additional polyclonality, which was greatest in cases with severe calcification. We now report a similar proportion of clonal expansions in the much younger bicuspid valve CAS cases. Peripheral blood flow cytometry revealed elevations in HLA-DR(+) activated CD8 cells and in the CD8(+)CD28(null)CD57(+) memory-effector subset that were significantly greater in both bicuspid and tricuspid CAS cases with more severe valve calcification. Lesser increases of CD4(+)CD28(null) T cells were identified, principally in cases with concurrent atherosclerotic disease. Upon immunostaining the CD8 T cells in all valves were mainly CD28(null), and CD8 T cell percentages were greatest in valves with oligoclonal repertoires. T cell clones identified by their clonotypic sequence as expanded in the valve were also found expanded in the circulating blood CD28(null)CD8(+) T cells and to a lesser degree in the CD8(+)CD28(+) subset, directly supporting the relationship between immunologic events in the blood and the valve. The results suggest that an ongoing systemic adaptive immune response is occurring in cases with bicuspid and tricuspid CAS, involving circulating CD8 T cell activation, clonal expansion, and differentiation to a memory-effector phenotype, with trafficking of T cells in expanded clones between blood and the valve.

A structural basis for selection and cross-species reactivity of the semi-invariant NKT cell receptor in CD1d/glycolipid recognition.

Little is known regarding the basis for selection of the semi-invariant alphabeta T cell receptor (TCR) expressed by natural killer T (NKT) cells or how this mediates recognition of CD1d-glycolipid complexes. We have determined the structures of two human NKT TCRs that differ in their CDR3beta composition and length. Both TCRs contain a conserved, positively charged pocket at the ligand interface that is lined by residues from the invariant TCR alpha- and semi-invariant beta-chains. The cavity is centrally located and ideally suited to interact with the exposed glycosyl head group of glycolipid antigens. Sequences common to mouse and human invariant NKT TCRs reveal a contiguous conserved "hot spot" that provides a basis for the reactivity of NKT cells across species. Structural and functional data suggest that the CDR3beta loop provides a plasticity mechanism that accommodates recognition of a variety of glycolipid antigens presented by CD1d. We propose a model of NKT TCR-CD1d-glycolipid interaction in which the invariant CDR3alpha loop is predicted to play a major role in determining the inherent bias toward CD1d. The findings define a structural basis for the selection of the semi-invariant alphabeta TCR and the unique antigen specificity of NKT cells.

Mechanism of T cell tolerance induced by myeloid-derived suppressor cells.

Ag-specific T cell tolerance plays a critical role in tumor escape. Recent studies implicated myeloid-derived suppressor cells (MDSCs) in the induction of CD8(+) T cell tolerance in tumor-bearing hosts. However, the mechanism of this phenomenon remained unclear. We have found that incubation of Ag-specific CD8(+) T cells, with peptide-loaded MDSCs, did not induce signaling downstream of TCR. However, it prevented subsequent signaling from peptide-loaded dendritic cells. Using double TCR transgenic CD8(+) T cells, we have demonstrated that MDSC induced tolerance to only the peptide, which was presented by MDSCs. T cell response to the peptide specific to the other TCR was not affected. Incubation of MDSCs with Ag-specific CD8(+) T cells caused nitration of the molecules on the surface of CD8(+) T cells, localized to the site of physical interaction between MDSC and T cells, which involves preferentially only TCR specific for the peptide presented by MDSCs. Postincubation with MDSCs, only nitrotyrosine-positive CD8(+) T cells demonstrated profound nonresponsiveness to the specific peptide, whereas nitrotyrosine-negative CD8(+) T cells responded normally to that stimulation. MDSCs caused dissociation between TCR and CD3zeta molecules, disrupting TCR complexes on T cells. Thus, these data describe a novel mechanism of Ag-specific CD8(+) T cell tolerance in cancer.

Public TCR use by herpes simplex virus-2-specific human CD8 CTLs.

Recombination of germline TCR alpha and beta genes generates polypeptide receptors for MHC peptide. Ag exposure during long-term herpes simplex infections may shape the T cell repertoire over time. We investigated the CD8 T cell response to HSV-2 in chronically infected individuals by sequencing the hypervariable regions encoding TCR alpha and beta polypeptides from T cell clones recognizing virion protein 22 aa 49-57, an immunodominant epitope. The most commonly detected TCRBV gene segment, found in four of five subjects and in 12 of 50 independently derived T cell clones, was TCRBV12-4. Nineteen to seventy-two percent of tetramer-binding cells in PBMCs were stained ex vivo with a TCRBV12 mAb. Three alpha-chain and three beta-chain public TCR sequences were shared between individuals. Public heterodimers were also detected. Promiscuous pairing of a specific TCRVA1-1 sequence with several different TCRB polypeptides was observed, implying a dominant structural role for the TCRA chain for these clonotypes. Functional avidity for cytotoxicity and IFN-gamma release was relatively invariant, except for one subject with both high avidity and unique TCR sequences and lower HSV-2 shedding. These data indicate that the CD8 response to a dominant alpha-herpesvirus epitope converges on preferred TCR sequences with relatively constant functional avidity.

Evaluation of T-Cell receptor diversity in pediatric patients with minimal change nephrotic syndrome.

AIMS: To further elucidate the clinical relevance of T-cell abnormalities in minimal change nephrotic syndrome (MCNS), and to predict the consequences of MCNS, we studied T-cell receptor (TCR) diversity by analyzing CDR3 size distribution and the frequency of Vß repertoire usage. METHODS: Participants comprised 36 pediatric patients with MCNS. 18 were frequent relapsers (FRs) and/or steroid-dependent (SD) and 18 were non-frequent relapsers (NFRs). Serial changes in TCR Vß repertoires were analyzed for these two groups of patients. Frequencies of Vß repertoire usage were determined by flow cytometry, and TCR CDR3 length distribution was analyzed by GeneScan. RESULTS: In NFRs, abnormalities in the distribution of Vß repertoires were few in both CD4+ and CD8+ T cells. In FRs/ SD patients, patterns were normal in CD4+ T cells, while selected Vß repertoires were significantly increased in CD8+ T cells in some patients. Furthermore, TCR diversity was significantly reduced in CD8+ T cells in FRs/SD patients, as shown by marked skewing of CDR3 size distributions. Of note was the finding that some FRs/SD patients showed improvements in the initially abnormal TCR diversity with improvement in clinical symptoms, eventually becoming NFRs. CONCLUSION: Analysis of TCR diversity may delineate the subgroup of FRs/SD patients and provide a rationale for early intervention with immunosuppressive therapy for these patients.

New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling.

To gain more insight into initiation and regulation of T cell receptor (TCR) gene rearrangement during human T cell development, we analyzed TCR gene rearrangements by quantitative PCR analysis in nine consecutive T cell developmental stages, including CD34+ lin- cord blood cells as a reference. The same stages were used for gene expression profiling using DNA microarrays. We show that TCR loci rearrange in a highly ordered way (TCRD-TCRG-TCRB-TCRA) and that the initiating Ddelta2-Ddelta3 rearrangement occurs at the most immature CD34+CD38-CD1a- stage. TCRB rearrangement starts at the CD34+CD38+CD1a- stage and complete in-frame TCRB rearrangements were first detected in the immature single positive stage. TCRB rearrangement data together with the PTCRA (pTalpha) expression pattern show that human TCRbeta-selection occurs at the CD34+CD38+CD1a+ stage. By combining the TCR rearrangement data with gene expression data, we identified candidate factors for the initiation/regulation of TCR recombination. Our data demonstrate that a number of key events occur earlier than assumed previously; therefore, human T cell development is much more similar to murine T cell development than reported before.

Fixing an irrelevant TCR alpha chain reveals the importance of TCR beta diversity for optimal TCR alpha beta pairing and function of virus-specific CD8+ T cells.

TCR repertoire diversity can influence the efficacy of CD8(+) T-cell populations, with greater breadth eliciting better protection. We analyzed TCR beta diversity and functional capacity for influenza-specific CD8(+) T cells expressing a single TCR alpha chain. Mice (A7) transgenic for the H2K(b)OVA(257-264)-specific V alpha 2.7 TCR were challenged with influenza to determine how fixing this "irrelevant" TCR alpha affects the "public" and restricted D(b)NP(366) (+)CD8(+) versus the "private" and diverse D(b)PA(224) (+)CD8(+) responses. Though both D(b)NP(366) (+)CD8(+) and D(b)PA(224) (+)CD8(+) sets are generated in virus-primed A7 mice, the constrained D(b)NP(366) (+)CD8(+) population lacked the characteristic, public TCRV beta 8.3, and consequently was reduced in magnitude and pMHC-I avidity. For the more diverse D(b)PA(224) (+)CD8(+) T cells, this particular forcing led to a narrowing and higher TCR beta conservation of the dominant V beta 7, though the responses were of comparable magnitude to C57BL/6J controls. Interestingly, although both the TCR beta diversity and the cytokine profiles were reduced for the D(b)NP(366) (+)CD8(+) and D(b)PA(224) (+)CD8(+) sets in spleen, the latter measure of polyfunctionality was comparable for T cells recovered from the infected lungs of A7 and control mice. Even "sub-optimal" TCR alpha beta pairs can operate effectively when exposed in a milieu of high virus load. Thus, TCR beta diversity is important for optimal TCR alpha beta pairing and function when TCR alpha is limiting.

The composition of a primary T cell response is largely determined by the timing of recruitment of individual T cell clones.

Primary T cell responses rely on the recruitment and proliferation of antigen-specific T cell precursors. The extent of expansion of each individual T cell clone may depend on (a) its frequency before immunization, (b) its proliferative capacity, and (c) the time at which it first encounters its cognate antigen. In this report, we have analyzed the relative contribution of each of these parameters to the shaping of immune repertoires in the T cell response specific for the epitope 170-179 derived from HLA-Cw3 and presented by Kd. By means of hemisplenectomy, we compared immune and naive repertoires in the same animal and found that the frequency of all expanded T cell clones was extremely low before immunization. In particular, the most expanded clones did not derive from high-frequency precursors. In addition, recruited T cells were found to proliferate at the same rate, irrespective of their T cell antigen receptor sequence. Finally, we showed that only T cells that encounter the antigen at early time points account for a significant part of the specific response. Therefore, the contribution of a T cell clone to the immune response is mostly determined by the time of its entry into the immune repertoire, i.e., the time of first cell division after antigen encounter.

T cell-independent spontaneous loss of tolerance by anti-double-stranded DNA B cells in C57BL/6 mice.

Systemic lupus erythematosus is characterized by loss of tolerance to DNA and other nuclear Ags. To understand the role of T cells in the breaking of tolerance, an anti-DNA site-specific transgenic model of spontaneous lupus, B6x56R, was studied. T cells were eliminated by crossing B6x56R with CD4(-/)(-) or TCRbeta(-/-)delta(-/-) mice, and the effects on anti-dsDNA serum levels, numbers of anti-dsDNA Ab-secreting cells, and isotypes of anti-dsDNA were analyzed. In addition, the development and activation of B cells in these mice were examined. Surprisingly, the presence of T cells made little difference in the development and character of the serum anti-dsDNA Ab in B6x56R mice. At 1 mo of age, anti-dsDNA Abs were somewhat lower in mice deficient in alphabeta and gammadelta T cells. Levels of Abs later were not affected by T cells, nor was autoantibody class switching. B cell activation was somewhat diminished in T cell-deficient mice. Thus, in the B6 background, the presence of an anti-dsDNA transgene led the production of autoantibodies with a specificity and isotype characteristic of murine systemic lupus erythematosus with little influence from T cells. TLR9 also did not appear to play a role. Although we do not yet understand the mechanism of this failure of immunoregulation, these results suggest that similar processes may influence autoimmunity associated with clinical disease.

TCR beta-chain sharing in human CD8+ T cell responses to cytomegalovirus and EBV.

The CD8(+) TCR repertoires specific for many immunogenic epitopes of CMV and EBV are dominated by a few TCR clonotypes and involve public TCRs that are shared between many MHC-matched individuals. In previous studies, we demonstrated that the observed sharing of epitope-specific TCRbeta chains between individuals is strongly associated with TCRbeta production frequency, and that a process of convergent recombination facilitates the more efficient production of some TCRbeta sequences. In this study, we analyzed a total of 2836 TCRbeta sequences from 23 CMV-infected and 10 EBV-infected individuals to investigate the factors that influence the sharing of TCRbeta sequences in the CD8(+) T cell responses to two immunodominant HLA-A*0201-restricted epitopes from these viruses. The most shared TCRbeta amino acid sequences were found to have two features that indicate efficient TCRbeta production, as follows: 1) they required fewer nucleotide additions, and 2) they were encoded by a greater variety of nucleotide sequences. We used simulations of random V(D)J recombination to demonstrate that the in silico TCRbeta production frequency was predictive of the extent to which both TCRbeta nucleotide and amino acid sequences were shared in vivo. These results suggest that TCRbeta production frequency plays an important role in the interindividual sharing of TCRbeta sequences within CD8(+) T cell responses specific for CMV and EBV.