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Within each individual, the adaptive immune system generates a repertoire of cells expressing receptors capable of recognizing diverse potential pathogens. The theoretical diversity of the T-cell receptor (TCR) repertoire exceeds the actual size of the T-cell population in an individual by several orders of magnitude - making the observation of identical TCRs in different individuals extremely improbable if all receptors were equally likely. Despite this disparity between the theoretical and the realized diversity of the repertoire, these 'public' receptor sequences have been identified in autoimmune, cancer and pathogen interaction contexts. Biased generation processes explain the presence of public TCRs in the naive repertoire, but do not adequately explain the different abundances of these public TCRs. We investigate and characterize the distribution of genomic TCR-β sequences of naive CD8+ T cells from three genetically identical mice, comparing non-productive (non-functional sequences) and productive sequences. We find public TCR-β sequences at higher abundances compared with unshared sequences in the productive, but not in the non-productive, repertoire. We show that neutral processes such as recombination biases, codon degeneracy and generation probability do not fully account for these differences, and conclude that thymic or peripheral selection plays an important role in increasing the abundances of public TCR-β sequences., (© 2021 John Wiley & Sons Ltd.)

T cells must display diversity regarding both the cell state and T-cell receptor (TCR) repertoire to provide effective immunity against pathogens; however, the generation and evolution of cellular T-cell heterogeneity in the adaptive immune system remains unclear. In the present study, a combination of multiplex PCR and immune repertoire sequencing (IR-seq) was used for a standardized analysis of the TCR β-chain repertoire of CD4 + naive, CD4 + memory, CD8 + naive and CD8 + memory T cells. We showed that the T-cell subsets could be distinguished from each another with regard to the TCR β-chain (TCR-β) diversity, CDR3 length distribution and TRBV usage, which could be observed both in the preselection and in the post-selection repertoire. Moreover, the Dβ-Jβ and Vβ-Dβ combination patterns at the initial recombination step, template-independent insertion of nucleotides and inter-subset overlap were consistent between the pre- and post-selection repertoires, with a remarkably positive correlation. Taken together, these results support differentiation of the CD4 + and CD8 + T-cell subsets prior to thymic selection, and these differences survived both positive and negative selection. In conclusion, these findings provide deeper insight into the generation and evolution of TCR repertoire generation., (© 2020 John Wiley & Sons Ltd.)

EBV is one of the most common viruses found in humans and is prototypic of a persistent viral infection characterized by periods of latency. Across many HLA class I molecules, the latent-specific CD8 + T cell response is focused on epitopes derived from the EBNA-3 protein family. In the case of HLA-B*07:02 restriction, a highly frequent class I allele, the T cell response is dominated by an epitope spanning residues 379-387 of EBNA-3 (RPPIFIRRL [EBV RPP ]). However, little is known about either the TCR repertoire specific for this epitope or the molecular basis for this observed immunodominance. The EBV RPP CD8 + T cell response was common among both EBV-seropositive HLA-B*07:02 + healthy and immunocompromised individuals. Similar TCRs were identified in EBV RPP -specific CD8 + T cell repertoires across multiple HLA-B7 + individuals, indicating a shared Ag-driven bias in TCR usage. In particular, TRBV4-1 and TRAV38 usage was observed in five out of six individuals studied. In this study, we report the crystal structure of a TRBV4-1 + TCR-HLA-B*07:02/EBV RPP complex, which provides a molecular basis for the observed TRBV4-1 bias. These findings enhance our understanding of the CD8 + T cell response toward a common EBV determinant in HLA-B*07:02 + individuals., (Copyright © 2020 by The American Association of Immunologists, Inc.)

In multiple tumor types, prediction of response to immune therapies relates to the presence, distribution and activation state of tumor infiltrating lymphocytes (TILs). Although such therapies are, to date, unsuccessful in gliomas, little is known on the immune contexture of TILs in these tumors. We assessed whether low and high-grade glioma (LGG and HGG, grade II and IV respectively) differ with respect to number, location and tumor reactivity of TILs; as well as expression of molecules involved in the trafficking and activation of T cells. Intra-tumoral CD8 T cells were quantified by flow cytometry (LGG: n = 12; HGG: n = 8) and immunofluorescence (LGG: n = 28; HGG: n = 28). Neoantigen load and expression of Cancer Germline Antigens (CGAs) were assessed using whole exome sequencing and RNA-seq. TIL-derived DNA was sequenced and the variable domain of the TCRβ chain was classified according to IMGT nomenclature. QPCR was used to determine expression of T cell-related genes. CD8 T cell numbers were significantly lower in LGG and, in contrast to HGG, mainly remained in close vicinity to blood vessels. This was accompanied by lower expression of chemo-attractants CXCL9, CXCL10 and adhesion molecule ICAM1. We did not observe a difference in the number of expressed neoantigens or CGAs, nor in diversity of TCR-Vβ gene usage. In summary, LGG have lower numbers of intra-tumoral CD8 T cells compared to HGG, potentially linked to decreased T cell trafficking. We have found no evidence for distinct tumor reactivity of T cells in either tumor type. The near absence of TILs in LGG suggest that, at present, checkpoint inhibitors are unlikely to have clinical efficacy in this tumor type.

Allergic contact dermatitis caused by contact sensitizers is a T-cell-mediated inflammatory skin disease. The most prevalent contact allergens is nickel. Whereas, memory T cells from nickel-allergic patients are well-characterized, little is known concerning nickel-specific naïve T-cell repertoire. The purpose of this study was to identify and quantify naïve CD4+ and CD8+ T cells recognizing nickel in the general population. Using a T-cell priming in vitro assay based on autologous co-cultures between naïve T cells and dendritic cells loaded with nickel, we were able to detect a naïve CD4+ and CD8+ T-cell repertoire for nickel in 10/11 and 7/8 of the tested donors. We calculated a mean frequency of 0.49 nickel-specific naïve CD4+ T cells and 0.37 nickel-specific naïve CD8+ T cells per million of circulating naïve T cells. The activation of these specific T cells requires MHC molecules and alongside IFN-γ production, some nickel-specific T-cells were able to produce granzyme-B. Interestingly, nickel-specific naïve CD4+ and CD8+ T cells showed a low rate of cross-reactivity with cobalt, another metallic hapten, frequently mixed with nickel in many alloys. Moreover, naïve CD4+ T cells showed a polyclonal TCRβ composition and the presence of highly expanded clones with an enrichment and/or preferentially expansion of some TRBV genes that was donor and T-cell specific. Our results contribute to a better understanding of the mechanism of immunization to nickel and propose the T-cell priming assay as a useful tool to identify antigen-specific naïve T cells.

Systemic lupus is characterized by the expansion of a self-reactive repertoire of B cells and CD4 cells that together promote IgG Ab production against common nuclear Ags. Although several studies have suggested roles for CD8 + T cells in lupus, the full contribution of these lymphocytes to disease remains undefined. In particular, few studies have examined TCR clonotypes of the CD8 pool in lupus. We previously described activated but nonpathogenic CD8 + T cells in a mouse model of systemic autoimmune disease triggered by increased copy number of the tlr7 gene (TLR7tg mice), in which some of these T cells accumulate in the brain. In this article, we report, through the analysis of TCRβ sequences, that CD8 cells from TLR7tg animals are strongly selected for a small number of clones, some of them reaching 30% of the repertoire, compared with less than 0.4% for the top clone in any wild type mice. High frequency clones are variable in sequence among individual TLR7tg mice and are distinct from top clones in the control animals, whereas CDR3 sequences of spleen and brain-resident T cells from the same TLR7tg animals have perfect concordance. These results suggest that top CD8 clones are selected in stochastic fashion in each animal but limit further diversification, and that brain-infiltrating CD8 cells in TLR7tg mice are not selected by a common tissue Ag. This kind of extreme clonal dominance and narrowing of the CD8 + repertoire might impair anti-viral responses and should be considered as an additional detrimental feature of chronic autoimmune disease.

With age, the immune system becomes less effective, causing increased susceptibility to infection. Chronic CMV infection further impairs immune function and is associated with increased mortality in the elderly. CMV exposure elicits massive CD8 + T cell clonal expansions and diminishes the cytotoxic T cell response to subsequent infections, leading to the hypothesis that to maintain homeostasis, T cell clones are expelled from the repertoire, reducing T cell repertoire diversity and diminishing the ability to combat new infections. However, in humans, the impact of CMV infection on the structure and diversity of the underlying T cell repertoire remains uncharacterized. Using TCR β-chain immunosequencing, we observed that the proportion of the peripheral blood T cell repertoire composed of the most numerous 0.1% of clones is larger in the CMV seropositive and gradually increases with age. We found that the T cell repertoire in the elderly grows to accommodate CMV-driven clonal expansions while preserving its underlying diversity and clonal structure. Our observations suggest that the maintenance of large CMV-reactive T cell clones throughout life does not compromise the underlying repertoire. Alternatively, we propose that the diminished immunity in elderly individuals with CMV is due to alterations in cellular function rather than a reduction in CD8 + T cell repertoire diversity., (Copyright © 2019 The Authors.)

Superantigens (SAgs) produced by Staphylococcus aureus at high concentrations induce proliferation of T cells bearing specific TCR Vβ sequences and massive cytokinemia that cause toxic shock syndrome. However, the biological relevance of SAgs produced at very low concentrations during asymptomatic colonization or chronic infections is not understood. In this study, we demonstrate that suboptimal stimulation of human PBMCs with a low concentration (1 ng/ml) of staphylococcal enterotoxin C1, at which half-maximal T cell proliferation was observed, induced CD8 + CD25 + T cells expressing markers related to regulatory T cells (Tregs), such as IFN-γ, IL-10, TGF-β, FOXP3, CD28, CTLA4, TNFR2, CD45RO, and HLA-DR. Importantly, these CD8 + CD25 + T cells suppressed responder cell proliferation mediated in contact-dependent and soluble factor-dependent manners, involving galectin-1 and granzymes, respectively. In contrast, optimal stimulation of human PBMCs with a high concentration (1 μg/ml) of staphylococcal enterotoxin C1, at which maximal T cell proliferation was observed, also induced similar expression of markers related to Tregs, including FOXP3 in CD8 + CD25 + cells, but these T cells were not functionally immunosuppressive. We further demonstrated that SAg-induced TCR Vβ-restricted and MHC class II-restricted expansion of immunosuppressive CD8 + CD25 + T cells is independent of CD4 + T cells. Our results suggest that the concentration of SAg strongly affects the functional characteristics of activated T cells, and low concentrations of SAg produced during asymptomatic colonization or chronic S. aureus infection induce immunosuppressive CD8 + Tregs, potentially promoting colonization, propagation, and invasion of S. aureus in the host., (Copyright © 2018 by The American Association of Immunologists, Inc.)

The concept of immunological surveillance implies that immunogenic variants of tumor cells arising in the organism can be recognized by the immune system. Tumor progression is provided by somatic evolution of tumor cells under the pressure of the immune system. The loss of MHC Class I molecules on the surface of tumor cells is one of the most known outcomes of immune selection. This study developed a model of immune selection based on the immune response of TCR 1d1 single β-chain transgenic B10.D2(R101) (K(d)I(d)D(b)) mice to allogeneic EL4 (H-2(b)) thymoma cells. In wild-type B10.D2(R101) mice, immunization with EL4 cells induced a vigorous CTL response targeted to the H-2K(b) molecule and results in full rejection of the tumor cells. In contrast, transgenic mice developed a compromised proliferative response in mixed-lymphocyte response assays and were unable to reject transplanted allogeneic EL4 cells. During the immune response to EL4 cells, CD8(+) T-lymphocytes with endogenous β-chains accumulated predominantly in the spleen of transgenic mice and only a small part of the T-lymphocytes expressing transgenic β-chains became CD8(+)CD44(+)CD62L(-) effectors. Then, instead of a full elimination of tumor cells as in wild-type mice, a reproducible prolonged equilibrium phase and subsequent escape was observed in transgenic mice that resulted in death of 90% of the mice in 40-60 days after grafting. Prolonged exposure of tumor cells to the pressure of the immune system in transgenic mice in vivo resulted in a stable loss of H-2K(b) molecules on the EL4 cell surface. Genetic manipulation of the T-lymphocyte repertoire was sufficient to reproduce the classic pattern of interactions between tumor cells and the immune system, usually observed in reliable syngeneic models of anti-tumor immunity. This newly-developed model could be used in further studies of immunoregulatory circuits common for transplantational and anti-tumor immune responses.

We report a case of T cell prolymphocytic leukemia (T-PLL) involving blast transformation. At the initial diagnosis, most peripheral blood cells demonstrated proliferation of indolent T cell small cell variants, i.e., small to medium prolymphocytes with inconspicuous nucleoli and a normal karyotype. These cells were positive for surface CD4, CD5, and CD7, and cytoplasmic CD3, but negative for surface CD3 and CD8 and cytoplasmic terminal deoxynucleotidyl transferase (TdT). The T cell receptor (TCR) Cβ1 gene was rearranged in the cells. Large prolymphocytes with prominent nucleoli, irregular nuclei, and cytoplasmic vacuoles that exhibited chromosome 8 trisomy were observed about 1.5 years later. The CD4+CD8- single positive effector memory T cells transformed into surface CD4+CD8+ double positive precursor T cells. The clonal TCR gene rearrangement patterns of these cells were identical throughout the clinical course, suggesting clonal blast transformation. The CD4+CD8+ cells demonstrated increased chromosome 8 trisomy combined with complex chromosome abnormalities with t(14;14)(q11.2;q32) containing a 14q32 chromosome after transformation. T cell leukemia 1a (TCL1a) (14q32.1) may be implicated in this case. The TCL1a oncoprotein is expressed in approximately 70% of T-PLL cases. The disease gradually developed resistance to chemotherapy, and the patient died of the disease. It is known that indolent T-PLL can become aggressive. Therefore, similar transformations may occur in other aggressive T-PLL cases, particularly those involving trisomy 8 and TCL1a.

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 role of adaptive immunity in the development or progression of chronic obstructive pulmonary disease (COPD) remains undefined. Recently, the presence of autoantibodies and autoreactive T cells has been demonstrated in COPD patients. In addition, oligoclonal expansions of lung T cells have been observed in COPD patients, but the overlapping incidence of infections, tumors, and cigarette smoke exposure obscures the antigenic stimulus. We analyzed the TCR Vbeta repertoire of CD4 and CD8 T cells purified from the lungs and spleens of mice chronically exposed to cigarette smoke. In a mouse model of COPD, we demonstrate that chronic cigarette smoke exposure causes oligoclonal expansions of T cells isolated from the lungs, but not spleens. TCR Vbeta repertoire analyses revealed oligoclonal expansions predominantly occurred in lung CD8 T cells, with preferential usage of Vbeta7, Vbeta9, Vbeta13, and Vbeta14. Using nucleotide sequence analysis based on Jbeta analyses, we demonstrate selection of CDR3 amino acid motifs, which strongly suggests Ag-driven oligoclonal T cell expansion. Analysis of the lung TCR Vbeta repertoire of mice with cigarette smoke-induced emphysema, which had undergone smoking cessation for 6 mo, revealed that oligoclonal expansions persisted. This study formally demonstrates that chronic cigarette smoke exposure, alone, causes a persistent adaptive T cell immune response. These findings have important implications for therapeutic approaches in the treatment of COPD, and provide insight into potential mechanisms involved in disease pathogenesis.

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.

T cells that survive thymic selection express a diverse array of unique heterodimeric alphabeta TCRs that mediate peptide-MHC Ag recognition. The proportion of the total T cell repertoire that expresses a particular Vbeta protein may be determined by a variety of factors: 1) germline preference for use of particular Vbeta genes; 2) allelic effects on the expression of different Vbeta genes; and 3) HLA effects on the expression of different Vbeta genes (acting via thymic selection and/or peripheral mechanisms). In this study, we show that Vbeta usage by human CD4(+) and CD8(+) T cells in neonatal and adult donors is highly correlated between unrelated individuals, suggesting that a large proportion of the observed pattern of Vbeta expression is determined by factors intrinsic to the TCR-beta locus. The presence of identical TCR alleles (within an individual) leads to a significantly better correlation between CD4(+) and CD8(+) T cells with respect to Vbeta expression; these effects are, however, relatively minor. The sharing of HLA alleles between individuals also leads to an increased correlation between their Vbeta expression patterns, although this did not reach statistical significance. We therefore conclude that the correlation in Vbeta expression patterns between CD4(+) and CD8(+) T cells can be explained predominantly by germline TCR-beta locus factors and not TCR-beta allelic or HLA effects.

Background & Aims: Biliary atresia is an inflammatory, fibrosclerosing neonatal cholangiopathy, characterized by a periductal infiltrate composed of CD4(+) and CD8(+) T cells. The pathogenesis of this disease has been proposed to involve a virus-induced, subsequent autoreactive T cell-mediated bile duct injury. Antigen-specific T-cell immunity involves clonal expansion of T cells expressing similar T-cell receptor (TCR) variable regions of the beta-chain (Vbeta). We hypothesized that the T cells in biliary atresia tissue expressed related TCRs, suggesting that the expansion was in direct response to antigenic stimulation., Methods: The TCR Vbeta repertoire of T cells from the liver, extrahepatic bile duct remnants, and peripheral blood of biliary atresia and other cholestatic disease controls were characterized by fluorescent-activated cell sorter analysis, and TCR junctional region nucleotide sequencing was performed on expanded TCR Vbeta regions to confirm oligoclonality., Results: FACS analysis revealed Vbeta subset expansions of CD4(+) and CD8(+) T cells from the liver or bile duct remnant in all patients with biliary atresia and only 1 control. The CD4(+) TCR expansions were limited to Vbeta3, -5, -9, and -12 T-cell subsets and the CD8(+) TCR Vbeta expansions were predominantly Vbeta20. Each Vbeta subset expansion was composed of oligoclonal populations of T cells., Conclusions: Biliary atresia is associated with oligoclonal expansions of CD4(+) and CD8(+) T cells within liver and extrahepatic bile duct remnant tissues, indicating the presence of activated T cells reacting to specific antigenic stimulation. Future studies entail identifying the specific antigen(s) responsible for T-cell activation and bile duct injury.

Dominant epitope-specific CD8(+) T-lymphocyte responses play a central role in controlling viral spread. We explored the basis for the development of this focused immune response in simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys through the use of two dominant (p11C and p199RY) and two subdominant (p68A and p56A) epitopes. Using real-time PCR to quantitate T-cell receptor (TCR) variable region beta (Vbeta) family usage, we show that CD8(+) T-lymphocyte populations specific for dominant epitopes are characterized by a diverse Vbeta repertoire, whereas those specific for subdominant epitopes employ a dramatically more focused Vbeta repertoire. We also demonstrate that dominant epitope-specific CD8(+) T lymphocytes employ TCRs with multiple CDR3 lengths, whereas subdominant epitope-specific cells employ TCRs with a more restricted CDR3 length. Thus, the relative dominance of an epitope-specific CD8(+) T-lymphocyte response reflects the clonal diversity of that response. These findings suggest that the limited clonal repertoire of subdominant epitope-specific CD8(+) T-lymphocyte populations may limit the ability of these epitope-specific T-lymphocyte populations to expand and therefore limit the ability of these cell populations to contribute to the control of viral replication.

Functional T cells are critical to host defense against infection. It has been reported that functional T cells as determined by their cytokine production represent antigen-specific T cells in infectious disease models. In this study, we enumerated Histoplasma-specific interferon gamma-producing cells in bulk splenocyte culture and showed that infection with Histoplasma capsulatum, an intracellular pathogen of the macrophage, activated both CD4 and CD8 T cells. The magnitude of CD8 T cell response was lower than CD4 T cell, but the expansion and contraction of both cell types followed the same kinetics. Over 90% of interferon gamma-producing CD4 T cells and >85% of CD8 T cells expressed CD44(hi) phenotype. The strong correlation between interferon gamma production and CD44(hi) expression was observed not only at the peak of response but also throughout the course of infection. Moreover, a broad spectrum of Vbeta populations responded to systemic as well as pulmonary infections, suggesting no obvious T cell receptor bias in primary immune response to histoplasmosis. While each Vbeta population contributed to interferon gamma production, several specific Vbeta populations made up higher percentages of interferon gamma-producing cells. Our study laid the groundwork for further investigations in immune response to histoplasmosis.

Graft-versus-host disease (GVHD) can be induced in lethally irradiated mice after allogeneic bone marrow transplantation between major histocompatibility complex-matched strains expressing multiple minor histocompatibility antigen differences. In the B6 --> BALB.B irradiation model, both CD4(+) and CD8(+) donor T cells have the capacity to mediate lethal GVHD. Previously, CDR3-size spectratyping was used to analyze these T-cell responses at a single early time point (day 5) after transplantation and revealed clonal or oligoclonal expansions of the V beta 2, 4, and 6 to 14 families for the CD4(+) response and of the V beta 4, 6, 8 to 11, and 14 families for the B6 CD8(+) response. Appropriate positive selection of these T-cell receptor V beta-skewed CD4(+) and CD8(+) T-cell subsets and their subsequent transfer into lethally irradiated BALB.B recipients resulted in fatal GVHD induction. In contrast, BALB.B mice transplanted with nonskewed V beta CD4(+) T cells survived, with minimal symptoms of GVHD. This study was undertaken to investigate the evolution of the donor/antihost minor histocompatibility antigen T-cell repertoire responses throughout the course of GVHD development. The results indicated that a number of V beta families were consistently involved throughout the course of GVHD, whereas some V beta families exhibited skewed expansions only in either the early or late stages of disease. In addition, sequence analysis of relevant representative skewed CDR3 bands from the CD4(+) V beta 11(+) and the CD8(+) V beta 14(+) families, both of which exhibited strong consistent responses, demonstrated increased use of the J beta 2.5 and J beta 2.4 segments, respectively, thus identifying the T-cell receptor specificities involved.

Owing to ordered, stage-specific T-cell receptor (TCR) gene rearrangements and cell division during T-cell development, small cohorts of "half-sibling" T cells sharing an ancestral TCR VDJbeta rearrangement but expressing different TCR alpha-locus rearrangements may be selected into the mature T-cell repertoire. We wondered whether different alphabetaTCRs expressed by T cells from the same ancestral VDJbeta cohort might be capable of recognizing the same foreign peptide-major histocompatibility complex complex (pMHC). By a combined flow cytometric and single-cell polymerase chain reaction (PCR) approach to analyze TCRs selected by the previously defined foreign antigen, pCW3170-179/H-2Kd, we were able to identify cohorts of half-sibling antigen-specific CD8 T cells after their expansion in immunized mice. We amplified residual DJbeta rearrangements as clonal markers to confirm that the shared VDJbeta sequences represent ancestral rearrangements rather than identical but independent ones. An intriguing explanation of our findings would be that only a very limited repertoire of TCR alpha-chains is selected to pair with a given TCR beta-chain during T-cell development.

The T cell receptor (TCR) repertoires of 24 human immunodeficiency virus (HIV) type 1-infected children were determined by flow cytometry in combination with sequencing of the highly variable TCR complementarity-determining region 3, permitting a quantitative and qualitative assessment of TCR repertoire. Expanded subsets of CD8(+) cells expressing a particular TCR beta-chain variable region were more commonly identified in HIV-1-infected children than in healthy control subjects (75% vs. 13.5%; P<.0001). Older age and lower percentage of CD4(+) cells were correlated with expansions. Oligoclonal populations occupied 71%-95% of each expanded subset, and predominant clones had high absolute counts. There was evidence of functional differentiation to CD28(-) effector cytotoxic T lymphocytes, and cells bearing identical TCRs were identified in both CD28(+) and CD28(-) cell populations. HIV-1 specificity was observed for expanded clones. Children with expansions were not more likely to have increased numbers of CD8(+) T cells, a finding consistent with the possibility that the CD8(+) TCR repertoire has limited diversity.

The authors describe two pediatric cases of large granular lymphocytosis presenting early in the second decade of life with neutropenia and sepsis. They are among the youngest described in the literature. This report focuses on the advantages of detailed immunophenotypic and molecular analysis and highlights some of the controversies and uncertainties in the management of these patients, particularly the choice of immunosuppressive therapy. Immunosuppressive therapy in the two children described in this report resulted in improvement of neutropenia and clinical status, but this was not accompanied by the disappearance of the clonal population.

A minor component (about 25%) of lymphocytes in Hodgkin's disease (HD) are CD8(+) T cells. It is unclear whether the presence of these cells reflects an antitumor cytotoxic response. The goal of the present study was to investigate clonal composition and the T cell receptor (TCR) beta repertoire of the CD8(+) T cell population in HD. Single CD8(+) cells were micromanipulated from frozen tissue sections of lymph nodes affected by primary HD and subjected to single target amplification of TCRbeta gene rearrangements. Sequence analysis of the V region genes revealed the presence of expanded CD8(+) T cell clones in all three cases analyzed. Most of these clonal expansions accounted for less than 10% of the CD8(+) T cell population. In one case, 30% of the CD8(+) T cells belonged to one or two clones. Comparison of V region sequences, however, did not provide evidence that the micromanipulated CD8(+) cells were sampled from a population that was selected for particular antigen specificities. No obvious biases in TCR Vbeta and Jbeta gene segment usage or CDR3 length distribution were found. Similarities of CDR3 amino acid sequences as found in selected CDR3 structures were rare. These results suggest that, like CD4(+) T cells, CD8(+) T cells may also be recruited into the tumor tissue in an antigen-nonspecific manner.

Using 50 samples of umbilical cord blood lymphocytes from Japanese donors, we analysed two human T-cell receptor beta variable (TCRBV) genes, BV6S4 and BV6S5, for their polymorphism, usage frequencies and CD4/CD8 skewness. They showed contrasting CD4/CD8 skewness, BV6S4 to CD8+ T cells and BV6S5 to CD4+ T cells. Genotyping of the BV6S4 alleles (A1, A2 and A3) revealed two of the six possible BV6S4 genotypes, A1/A2 and A2/A2. Among the two BV6S4 genotypes, no significant difference was detected in usage frequency or CD4/CD8 skewness. On the other hand, genotyping of the BV6S5 alleles (A1 and A2) revealed all three possible BV6S5 genotypes, A1/A1, A1/A2 and A2/A2, and the gene usage frequency was high, in the order A1/A1 > A1/A2 > A2/A2. These results indicate that the amino acid substitutions in BV6S5 (S36R and G70E) are in some way associated with the expression level of this gene. In the analysis of CD4/CD8 skewness, the three BV6S5 genotypes had similar skewness, indicating that A1 alleles are expressed more frequently than A2 alleles in both CD4+ and CD8+ T-cell populations. Although BV6S5 exhibits marked skewness to CD4+ T cells, our results indicate that the higher expression of A1 alleles is not associated with the increased ratio of CD4+ T cells.

While numerous studies have analyzed the shaping of T cell repertoires by self or foreign peptides, little is known on the influence of commensal self peptides derived from the intestinal flora (IF). Here, we have analyzed naive and immune repertoires in mice devoid of IF [germ-free (GF) mice]. First, by means of an extensive CDR3beta sequencing strategy, we show that the naive peripheral CD8 T cell repertoire does not exhibit a major imprint of IF antigens. Second, using MHC-peptide tetramers, CDR3beta length distribution analyses and TCR sequencing, we show that cytotoxic T lymphocyte (CTL) responses specific for two distinct epitopes are quasi-identical in normal and GF mice. Our findings indicate that, in general, peptides derived from the intestinal microflora have little if any influence on CTL responses in the mouse.

Background: Because of several studies, idiopathic nephrotic syndrome (INS) of childhood is suspected to have an immunologic pathogenesis with T cells playing a major role. To investigate this hypothesis further, we studied the diversity of the CDR3 region of the T-cell receptor (TCR) beta-chain from peripheral T cells isolated from patients with INS., Methods: The study was performed over a three-year period to obtain longitudinal data on the repertoire of peripheral T cells. mRNA from peripheral mononuclear cells (PBMCs) of seven INS patients and two healthy controls (NHD) was prepared and analyzed for CDR3 length polymorphism of TCR beta-chain by spectratyping., Results: All INS patients presented individually skewed spectratype histograms in at least one Vbeta-family. Patients suffering from a frequent relapsing course of INS or a focal global sclerosis showed some alterations to persist in all samples isolated in the observation period (up to 3 years). In addition, sequence analyses of the beta-chain of the TCR CDR3 region confirmed clonal expansion of peripheral T cells in those patients who had displayed spectratype alterations., Conclusions: The data give strong evidence for an direct involvement of CD8+ T cells in the complicated course of INS.

The introduction of cloned T cell receptor (TCR) genes into bone marrow cells could provide a way to increase the frequency of tumor- or pathogen-specific cytotoxic T lymphocyte (CTL) precursors. We demonstrate here the ability of a retroviral vector to direct expression of a Valpha15/Vbeta13 MHC class I-restricted TCR in lethally irradiated mice reconstituted with transduced bone marrow cells. We have detected retroviral-mediated TCR expression by flow cytometry 6-19 weeks after transplantation in C57L (Vbeta13(-/-)) and Rag1(-/-) bone marrow-reconstituted mice, and in C57BL/6 hosts reconstituted with transduced C57BL/6-Rag1(-/-) bone marrow. Southern analysis confirmed the presence of integrated provirus and revealed that the frequency of transduction is greater than the frequency of cell surface TCR expression. Although TCR expression on Vbeta13+ transduced cells is lower than endogenous TCR levels, it is largely confined to CD4+CD8+ (thymus) and CD8+ (thymus and spleen) T cells. In Rag1(-/-) mice, which display a developmental arrest of thymocytes at the immature CD4-CD8- stage, retrovirus-mediated TCR expression selectively rescues CD4+CD8+ and CD8+ populations. These results indicate that the ectopically expressed TCR is functional during T cell development. Furthermore, we have observed Vbeta13+ TCR expression by up to 13% of peripheral CD8+ T cells in C57L and C57BL/6 hosts. This represents a substantial increase relative to total Vbeta13 frequency in normal C57BL/6 mice (3-5%), and an even greater increase over the estimated frequency of CTL precursors of a defined specificity (10(-5)-10(-4)). Our findings indicate that TCR gene transfer can be used to develop new approaches to immunotherapy, and provide the basis for further studies examining the contribution of retrovirus-mediated TCR expression to an antigen-specific CTL response.

The T cell receptor (TCR) variable (V) gene repertoire was analyzed in patients with monoclonal gammopathy of undetermined significance (MGUS) (n = 17), multiple myeloma (MM) stage I (n = 16), MM stages II/III (n = 31) and age-matched controls (n = 27) by immunofluorescence and flow cytometry using a panel of mouse monoclonal antibodies (mAb) (n = 10) against TCR V alpha and V beta gene products. T cell expansion was defined as a value > or = thrice the normal median value for each respective TCR V mAb. Fifty-three percent of all patients displayed CD8+ expansion(s) as compared to 30% of age-matched controls (p < 0.001). Within the CD4 subset, 18% of the patients displayed T cell expansion(s) in comparison to 11% of the controls (not significant). Interestingly, the CD8+ expansion(s) were more frequently noted in patients with a low tumor burden (MGUS/MMI) (73%) as compared to those with advanced disease (MM II/III) (32% and control donors (30%) (p < 0.01). Likewise, multiple CD8+ expansions (two or more) were more common in MGUS/MM I patients than in MM II/III and controls (p < 0.01). The T cell expansions were stable over time in patients with a stable disease. A high degree of clonality of the expansions was detected by TCR CDR3 fragment length analysis, determination of J beta gene usage and nucleotide sequencing. The frequent finding of oligoclonal CD8+ T cell expansions in patients with a low tumor mass, but not in patients with advanced disease justifies further work in order to identify the relevance of expanded CD8+ T cells. In one patient with T cell reactivity against the autologous myeloma idiotype, two expansions within the CD8 population (V beta 3 and V beta 5.2 respectively) displayed no reactivity against the idiotype. Instead, idiotype recognition was confined to a CD8 non-expanded V beta 22+ T cell population, with a highly restricted TCR usage (CDR3 fragment length analysis).

Inoculation with replicating virus leads to an increase in T cell numbers within lymph nodes that drain the site of infection. This increase has been associated with a nonspecific proliferation of bystander cells, with only a minority thought to be directed to the infectious agent. Such an assumption is largely based on precursor cytotoxic T lymphocyte (CTL) estimations using limiting dilution analysis. Recently, studies using more advanced molecular approaches have suggested that such functionally derived precursor frequencies considerably underestimate the proportion of T cells specific for the antigen under investigation. We have defined T cell receptor sequences characteristic of CTL populations directed to a dominant determinant of the herpes simplex virus (HSV) glycoprotein B (gB). In this investigation, we used this receptor signature as a probe to directly monitor changes occurring within lymph nodes draining the sites of active infection with HSV. We found that although lymph node CD8+ T cell numbers increase as a consequence of HSV infection, the majority of these cells are small resting cells that are not enriched for gB-specific receptors. In contrast, a significant proportion of activated T cells are highly enriched for CTL bearing gB-specific receptors. Our results are therefore consistent with a nonspecific migration of CTL precursors into the lymph nodes draining the site of infection, followed by the activation and proliferation of the antigen-specific subset that normally makes up a small proportion of the naive T cell repertoire.