Shaping the T-cell repertoire in the periphery

Selection of T cells does not end with events in the thymus, but continues in extrathymic tissues and for the life of the organism. In this review, we examine how self-reactive T cells are rendered harmless and the processes that select for T cells that are most efficient at combating pathogens. The implications of peripheral T-cell selection for the immune response as animals age are discussed as is the critical role of dendritic cells in directing T-cell differentiation.     

CD4 promotes breadth in the TCR repertoire

A diverse population of MHC class II-restricted CD4 lineage T cells develops in mice that lack expression of the CD4 molecule. In this study, we show that the TCR repertoire selected in the absence of CD4 is distinct, but still overlapping in its properties with that selected in the presence of CD4. Immunization of mice lacking CD4 caused the clonal expansion of T cells that showed less breadth in the range of Ag-binding properties exhibited by their TCRs. Specifically, the CD4-deficient Ag-specific TCR repertoire was depleted of TCRs that demonstrated low-affinity binding to their ligands. The data thus suggest a key role for CD4 in broadening the TCR repertoire by potentiating productive TCR signaling and clonal expansion in response to the engagement of low-affinity antigenic ligands.     

Evolution of the antigen-specific CD8+ TCR repertoire across the life span: evidence for clonal homogenization of the old TCR repertoire

Defects in T cell responses against pathogens and reduced diversity of TCRs have been described at both extremes of the life span. Yet, we still lack information on how Ag-specific T cell populations are maintained and/or altered from birth to old age. In this study, for the first time to our knowledge, we provide insight into Ag-specific TCR repertoire changes over the life span at the single-cell level. We have examined the TCR diversity of the primary CD8(+) T cell response to the immunodominant HSV-1 epitope HSV glycoprotein B 495-502 (HSV gB(498-505); SSIEFARL) (gB-8p) in neonatal, adult, and old C57BL/6 mice. The global distinctive features of the gB-8p-specific TCR repertoire were preserved in mice of different ages. However, both old and especially neonatal mice exhibited significant decreases in TCR diversity compared with that of adult mice. Still, although the neonatal Ag-specific repertoire comprised expectedly shorter germline-biased CDR3β lengths, the repertoire was surprisingly complex, and only a minority of responding cells lacked random nucleotide additions. Changes with aging included increased use of the already dominant TCRVβ10 family, a trend for lower content of the TCR containing the germline WG motif in the CDR3, and a remarkable sharing of one dominant clonotype between individual old mice, implying operation of selective mechanisms. Implications for the rational design of vaccines for neonates and the elderly are discussed.     

The proteoglycan repertoire of lymphoid cells

Proteoglycans have been studied to a limited extent in lymphoid cells. In this study we have investigated the expression of proteoglycans in B-cells, CD4+ T-cells, CD8+ T-cells, natural killer cells, as well as in nine different cell lines established from patients with lymphoid malignancies. Serglycin was the major proteoglycan expressed at mRNA level by the primary lymphocytes. None of the syndecans or glycpicans was detected at mRNA level in the primary lymphocytes, except for syndecan-4 in CD4+ T-cells and CD8+ T-cells. All lymphoid cell lines expressed serglycin mRNA, as well as one or several members of the syndecan and glypican families. Further, increased synthesis of proteoglycans was found in the cell lines compared to the primary lymphocytes, as well as the presence of heparan sulfate on the cell surface of five of the cells lines. Western blot analysis showed a close correlation between serglycin mRNA level and expression of serglycin core protein. Our results show that serglycin is a major proteoglycan in all the normal lymphoid cells and that these cells carry little, or none, proteoglycans on the cell surface. Serglycin was also a major proteoglycan in the malignant lymphoid cells, but these also expressed one or more types of cell surface proteoglycans. Thus, malignant transformation of lymphoid cells may be followed by increased synthesis of proteoglycans and expression of cell surface proteoglycans.     

CDR3 spectratyping analysis of the TCR repertoire in myasthenia gravis

Because myasthenia gravis (MG) is an autoimmune disease mediated by Abs specific for the acetylcholine receptor, helper T cells play a role in Ab production. In this study, we have performed large-scale cross-sectional and longitudinal TCR studies by CDR3 spectratyping using PBL and thymus tissues from MG patients. We found that there was no preferential usage of any particular TCR beta-chains that was identical among MG patients. However, the longitudinal study clearly demonstrated that one or more TCR Vbeta expansions persisted frequently in MG patients. Importantly, persistent TCR expansions correlated with clinical severity and high anti-acetylcholine receptor Ab titer. Finally, examinations of T cells expressing CXCR5, i.e., follicular B-helper T cells, revealed that spectratype expansions in MG patients were detected mainly in the CD4+ CXCR5+ T cell populations, whereas CD8+ T cells were the major source of clonal expansion in healthy subjects. These findings suggest that persistent clonal expansions of T cells in MG patients are associated with the development and maintenance of MG. Close examination of pathogenic T cells in MG provides useful information to elucidate the pathogenesis and to estimate the disease status.     

RNA in the periphery of rapidly proliferating mouse lymphoid cells

RNA in the peripheries of various populations of lymph node cells (LNC) has been evaluated by measuring the electrophoretic mobilities of cells, before and after treatment with active or inactivated ribonucleases. Three different populations of LNC were studied: (1) “resting” normal age control LNC; (2) “syngeneic” LNC from irradiated (C3H × C57BL)F₁ or C3H mice four to six days following transplantation of syngeneic spleen cells; such cells were progeny of lymphopoietic progenitor cells of the spleen; and (3) “allogeneic” LNC from irradiated (C3H × C57BL)F₁ mice four to six days after grafting C3H (parental) spleen cells; such cells were progeny of lymphopoietic progenitor cells, but also alloantigen-sensitive cells of the spleen which proliferate in response to the host's alloantigens (a “graft-versus-host” immunological reaction). Whereas the normal LNC had no detectable peripheral RNA, the allogeneic and syngeneic LNC did, i.e., ribonuclease reduced their mean electrophoretic mobilities by 13.6 and 9.2 per cent, respectively. Since both allogeneic and syngeneic LNC had peripheral RNA, no specific correlation could be made with immunological activity. ³H-uridine and ¹⁴C-thymidine incorporation into lymph nodes was greatest in allogeneic, intermediate in syngeneic and least in age control lymph nodes, indicating a “population shift” in the spleen cell chimeras toward relatively immature, rapidly proliferating cells, which had a relatively high rate of RNA synthesis. Thus, rapidly proliferating lymphoid cells do have RNA in their peripheries, but its relation to specific immunological function has yet to be ascertained.     

Use of the allogeneic TCR repertoire to enhance anti-tumor immunity

It is well established that antigen-specific T lymphocytes can inhibit tumor growth in humans and in mice, leading to complete tumor elimination in some cases. However, in many cases T cell immunity is unable to successfully control tumor progression. Since tumors are derived from normal tissues, most antigens are shared with normal tissues, although expression levels are usually elevated in malignant cells. Nevertheless, low-level expression in normal cells can be sufficient to render autologous T cells tolerant and thus unable to mount effective immune responses against tumors. Here, we review how allogeneic T cells can be used to isolate T cells that effectively recognise and kill tumor cells, but not normal cells with low level of antigen expression. The TCR of allogeneic T cells can be introduced into patient T cells to equip them with anti-tumor specificity that may not be present in the autologous T cell repertoire.     

Comprehensive analysis of the functional TCR repertoire at the single-cell level

A Vβ TCR repertoire is analyzed for understanding the T-cell population in the immune response. However, the TCR repertoire of the Vα-Vβ pair is difficult to analyze because no suitable analytical method is available. Here, we have applied the single-cell 5′-RACE method for amplifying TCR cDNAs from single T-cells and analyzed the repertoire of Vα-Vβ pairs in human T-cells that responded to a superantigen, SEB. We found that the TCR Vβ profile of the SEB-stimulated CD4⁺ T-cells was in accordance with the previous reports, that the TCR Vα profile also exhibited a prominent difference, and that the TCR Vα-Vβ pairs of the SEB-responding T-cells were promiscuous. We have also found a significant dual TCRα expression in single T-cells. This is the first report of a comprehensive analysis of the functional repertoire of Vα-Vβ pairs at the single T-cell level. This novel method may contribute to TCR-based immunotherapeutics.     

Complementarity-determining region 3 spectratyping analysis of the TCR repertoire in multiple sclerosis

Multiple sclerosis (MS) is considered to be an autoimmune disease mediated by T cells reactive with Ags in the CNS. Therefore, it has been postulated that neuroantigen-reactive T cells bearing particular types of TCRs are expanded clonally during the course of the disease. However, there is a controversy with regard to the TCR usage by T cells associated with the development of MS. By the use of complementarity-determining region 3 spectratyping analysis that is shown to be a useful tool for identification of pathogenic TCR in autoimmune disease models, we tried to demonstrate that spectratype was T cells bearing particular types of TCR are activated in MS patients. Consequently, it was found that Vbeta5.2 were often oligoclonally expanded in peripheral blood of MS patients, but not of healthy subjects. Sequence analysis of the complementarity-determining region 3 region of spectratype-derived TCR clones revealed that the predominant TCR clone was different from patient to patient, but that similar results were obtained in a patient examined at different time points. More importantly, examination of cerebrospinal fluid T cells and longitudinal studies of PBLs from selected patients revealed that Vbeta5.2 expansion was detectable in the majority of patients examined. These findings suggest that Vbeta5.2 spectratype expansion is associated with the development of MS and that TCR-based immunotherapy can be applicable to MS patients if the TCR activation pattern of each patient is determined at different stages of the disease.     

CD4+ TCR repertoire heterogeneity in Schistosoma mansoni-induced granulomas

The hallmark of Schistosoma mansoni infection is the formation of liver granulomas around deposited ova. The initiation of granuloma formation is T cell-dependent since granulomas are not formed in their absence. We investigated whether a few T cells arrive to initiate the inflammatory lesion and subsequently expand locally, or whether a large repertoire of systemically activated T cells home to the delayed type hypersensitivity reaction induced by the ova. The TCR repertoire of single granulomas from the same liver were analyzed by PCR using Vbeta-specific primers and CDR3 analysis. Each granuloma has a very diverse TCR repertoire indicating that most of the T cells recruited to these lesions are activated systemically. At the same time, sequence analysis of individually sized CDR3 products from single granuloma indicate that a fraction of T cells expand locally at the lesion site. Using TCR transgenic mice containing a pigeon cytochrome c-specific T cell population or lymphocytic choriomeningitis virus infection tracked with lymphocytic choriomeningitis virus-specific tetramers, we demonstrated that nonspecific T cells home to the granuloma if they are activated. However, recombinase-activating gene 2(-/-) pigeon cytochrome c-specific TCR transgenic mice fail to form granulomas in response to S. mansoni ova even after T cell activation, suggesting a requirement for egg-specific T cells in the initiation of these inflammatory lesions. Understanding the mechanism of T cell recruitment into granulomas has important implications for the rational design of immunotherapies for granulomatous diseases.     

Size estimate of the alpha beta TCR repertoire of naive mouse splenocytes

The diversity of the T cell repertoire of mature T splenocytes is generated, in the thymus, by pairing of alpha and beta variable domains of the alpha beta TCR and by the rearrangements of various gene segments encoding these domains. In the periphery, it results from competition between various T cell subpopulations including recent thymic migrants and long-lived T cells. Quantitative data on the actual size of the T cell repertoire are lacking. Using PCR methods and extensive sequencing, we have measured for the first time the size of the TCR-alpha beta repertoire of naive mouse T splenocytes. There are 5-8 x 105 different nucleotide sequences of BV chains in the whole spleen of young adult mice. We have also determined the size of the BV repertoire in a subpopulation of AV2+ T splenocytes, which allows us to provide a minimum estimate of the alpha beta repertoire. We find that the mouse spleen harbors about 2 x 106 clones of about 10 cells each. This figure, although orders of magnitude smaller than the maximum theoretical diversity (estimated up to 1015), is still large enough to maintain a high functional diversity.     

Comprehensive analysis of TCR repertoire in COVID-19 using single cell sequencing

T-cell receptor (TCR) is crucial in T cell-mediated virus clearance. To date, TCR bias has been observed in various diseases. However, studies on the TCR repertoire of COVID-19 patients are lacking. Here, we used single-cell V(D)J sequencing to conduct comparative analyses of TCR repertoire between 12 COVID-19 patients and 6 healthy controls, as well as other virus-infected samples. We observed distinct T cell clonal expansion in COVID-19. Further analysis of VJ gene combination revealed 6 VJ pairs significantly increased, while 139 pairs significantly decreased in COVID-19 patients. When considering the VJ combination of α and β chains at the same time, the combination with the highest frequency on COVID-19 was TRAV12-2-J27-TRBV7-9-J2-3. Besides, preferential usage of V and J gene segments was also observed in samples infected by different viruses. Our study provides novel insights on TCR in COVID-19, which contribute to our understanding of the immune response induced by SARS-CoV-2.     

Immunodominance in the TCR repertoire of a [corrected] TCR peptide-specific CD4+ Treg population that controls experimental autoimmune encephalomyelitis

Immunodominance in self-Ag-reactive pathogenic CD4(+) T cells has been well established in several experimental models. Although it is clear that regulatory lymphocytes (Treg) play a crucial role in the control of autoreactive cells, it is still not clear whether immunodominant CD4(+) Treg clones are also involved in control of autoreactivity. We have shown that TCR-peptide-reactive CD4(+) and CD8(+) Treg play an important role in the spontaneous recovery and resistance from reinduction of experimental autoimmune encephalomyelitis in B10.PL mice. We report, by sequencing of the TCR alpha- and beta-chain associated with CD4(+) Treg, that the TCR repertoire is limited and the majority of CD4(+) Treg use the TCR Vbeta14 and Valpha4 gene segments. Interestingly, sequencing and spectratyping data of cloned and polyclonal Treg populations revealed that a dominant public CD4(+) Treg clonotype expressing Vbeta14-Jbeta1.2 with a CDR3 length of 7 aa exists in the naive peripheral repertoire and is expanded during the course of recovery from experimental autoimmune encephalomyelitis. Furthermore, a higher frequency of CD4(+) Treg clones in the naive repertoire correlates with less severity and more rapid spontaneous recovery from disease in parental B10.PL or PL/J and (B10.PL x PL/J)F(1) mice. These findings suggest that unlike the Ag-nonspecific, diverse TCR repertoire among the CD25(+)CD4(+) Treg population, TCR-peptide-reactive CD4(+) Treg involved in negative feedback regulation of autoimmunity use a highly limited TCR V-gene repertoire. Thus, a selective set of immunodominant Treg as well as pathogenic T cell clones can be targeted for potential intervention in autoimmune disease conditions.     

The TCR repertoire of an immunodominant CD8+ T lymphocyte population

The TCR repertoire of an epitope-specific CD8(+) T cell population remains poorly characterized. To determine the breadth of the TCR repertoire of a CD8(+) T cell population that recognizes a dominant epitope of the AIDS virus, the CD8(+) T cells recognizing the tetrameric Mamu-A*01/p11C(,CM) complex were isolated from simian immunodeficiency virus (SIV)-infected Mamu-A*01(+) rhesus monkeys. This CD8(+) T cell population exhibited selected usage of TCR V beta families and complementarity-determining region 3 (CDR3) segments. Although the epitope-specific CD8(+) T cell response was clearly polyclonal, a dominance of selected V beta(+) cell subpopulations and clones was seen in the TCR repertoire. Interestingly, some of the selected V beta(+) cell subpopulations and clones maintained their dominance in the TCR repertoire over time after infection with SIV of macaques. Other V beta(+) cell subpopulations declined over time in their relative representation and were replaced by newly evolving clones that became dominant. The present study provides molecular evidence indicating that the TCR repertoire shaped by a single viral epitope is dominated at any point in time by selected V beta(+) cell subpopulations and clones and suggests that dominant V beta(+) cell subpopulations and clones can either be stable or evolve during a chronic infection.     

Prevalent role of TCR alpha-chain in the selection of the preimmune repertoire specific for a human tumor-associated self-antigen

The specificity of recognition of pMHC complexes by T lymphocytes is determined by the V regions of the TCR alpha- and beta-chains. Recent experimental evidence has suggested that Ag-specific TCR repertoires may exhibit a more V alpha- than V beta-restricted usage. Whether V alpha usage is narrowed during immune responses to Ag or if, on the contrary, restricted V alpha usage is already defined at the early stages of TCR repertoire selection, however, has remained unexplored. Here, we analyzed V and CDR3 TCR regions of single circulating naive T cells specifically detected ex vivo and isolated with HLA-A2/melan-A peptide multimers. Similarly to what was previously observed for melan-A-specific Ag-experienced T cells, we found a relatively wide V beta usage, but a preferential V alpha 2.1 usage. Restricted V alpha 2.1 usage was also found among single CD8(+) A2/melan-A multimer(+) thymocytes, indicating that V alpha-restricted selection takes place in the thymus. V alpha 2.1 usage, however, was independent from functional avidity of Ag recognition. Thus, interaction of the pMHC complex with selected V alpha-chains contributes to set the broad Ag specificity, as underlined by preferential binding of A2/melan-A multimers to V alpha 2.1-bearing TCRs, whereas functional outcomes result from the sum of these with other interactions between pMHC complex and TCR.     

Quantification of repertoire diversity of influenza-specific epitopes with predominant public or private TCR usage

The H-2Db-restricted CD8 T cell immune response to influenza A is directed at two well-described epitopes, nucleoprotein 366 (NP366) and acid polymerase 224 (PA224). The responses to the two epitopes are very different. The epitope NP366-specific response is dominated by TCR clonotypes that are public (shared by most mice), whereas the epitope PA224-specific response is private (unique within each infected animal). In addition to being public, the NP366-specific response is dominated by a few clonotypes, when T cell clonotypes expressing the Vbeta8.3 element are analyzed. Herein, we show that this response is similarly public when the NP366+Vbeta4+ CD8 T cell response is analyzed. Furthermore, to determine whether these features resulted in differences in total TCR diversity in the NP366+ and PA224+ responses, we quantified the number of different CD8 T clonotypes responding to each epitope. We calculated that 50-550 clonotypes recognized each epitope in individual mice. Thus, although the character of the response to the two epitopes appeared to be different (private and diverse vs public and dominated by a few clonotypes), similar numbers of precursor cells responded to both epitopes and this number was of similar magnitude to that previously reported for other viral CD8 T cell epitopes. Therefore, even in CD8 T cell responses that appear to be oligoclonotypic, the total response is highly diverse.     

Analysis of the TCR beta variable gene repertoire in chimpanzees: identification of functional homologs to human pseudogenes

Chimpanzees are used for a variety of disease models such as hepatitis C virus (HCV) infection, where Ag-specific T cells are thought to be critical for resolution of infection. The variable segments of the TCR alphabeta genes are polymorphic and contain putative binding sites for MHC class I and II molecules. In this study, we performed a comprehensive analysis of genes that comprise the TCR beta variable gene (TCRBV) repertoire of the common chimpanzee Pan troglodytes. We identified 42 P. troglodytes TCRBV sequences representative of 25 known human TCRBV families. BV5, BV6, and BV7 are multigene TCRBV families in humans and homologs of most family members were found in the chimpanzee TCRBV repertoire. Some of the chimpanzee TCRBV sequences were identical with their human counterparts at the amino acid level. Notably four successfully rearranged TCRBV sequences in the chimpanzees corresponded to human pseudogenes. One of these TCR sequences was used by a cell line directed against a viral CTL epitope in an HCV-infected animal indicating the functionality of this V region in the context of immune defense against pathogens. These data indicate that some TCRBV genes maintained in the chimpanzee have been lost in humans within a brief evolutionary time frame despite remarkable conservation of the chimpanzee and human TCRBV repertoires. Our results predict that the diversity of TCR clonotypes responding to pathogens like HCV will be very similar in both species and will facilitate a molecular dissection of the immune response in chimpanzee models of human diseases.     

Role of commensal bacteria in development of gut-associated lymphoid tissues and preimmune antibody repertoire

Intestinal bacteria are required for development of gut-associated lymphoid tissues (GALT), which mediate a variety of host immune functions, such as mucosal immunity and oral tolerance. In rabbits, the intestinal microflora are also required for developing the preimmune Ab repertoire by promoting somatic diversification of Ig genes in B cells that have migrated to GALT. We studied the mechanism of bacteria-induced GALT development. Bacteria were introduced into rabbits in which the appendix had been rendered germfree by microsurgery (we refer to these rabbits as germfree-appendix rabbits). We then identified specific members of the intestinal flora that promote GALT development. The combination of Bacteroides fragilis and Bacillus subtilis consistently promoted GALT development and led to development of the preimmune Ab repertoire, as shown by an increase in somatic diversification of VDJ-C micro genes in appendix B cells. Neither species alone consistently induced GALT development, nor did Clostridium subterminale, Escherichia coli, or Staphylococcus epidermidis. B. fragilis, which by itself is immunogenic, did not promote GALT development; hence, GALT development in rabbits does not appear to be the result of an Ag-specific immune response. To identify bacterial pathways required for GALT development, we introduced B. fragilis along with stress-response mutants of B. subtilis into germfree-appendix rabbits. We identified two Spo0A-controlled stress responses, sporulation and secretion of the protein YqxM, which are required for GALT development. We conclude that specific members of the commensal, intestinal flora drive GALT development through a specific subset of stress responses.