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.     

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.     

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.     

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.     

Chronic modulation of the TCR repertoire in the lymphoid periphery

Using TCR V beta 5 transgenic mice as a model system, we demonstrate that the induction of peripheral tolerance can mold the TCR repertoire throughout adult life. In these mice, three distinct populations of peripheral T cells are affected by chronic selective events in the lymphoid periphery. First, CD4+V beta 5+ T cells are deleted in the lymphoid periphery by superantigens encoded by mouse mammary tumor viruses-8 and -9 in an MHC class II-dependent manner. Second, mature CD8+V beta 5+ T cells transit through a CD8lowV beta 5low deletional intermediate during tolerance induction by a process that depends upon neither mouse mammary tumor virus-encoded superantigens nor MHC class II expression. Third, a population of CD4-CD8-V beta 5+ T cells arises in the lymphoid periphery in an age-dependent manner. We analyzed the TCR V alpha repertoire of each of these cellular compartments in both V beta 5 transgenic and nontransgenic C57BL/6 mice as a function of age. This analysis revealed age-related changes in the expression of V alpha families among different cellular compartments, highlighting the dynamic state of the peripheral immune repertoire. Our work indicates that the chronic processes maintaining peripheral T cell tolerance can dramatically shape the available TCR repertoire.     

T cell receptor delta repertoire in inflamed and noninflamed colon of patients with IBD analyzed by CDR3 spectratyping

Gamma/delta T cells might play an important role in autoimmune conditions like inflammatory bowel disease (IBD). In the present study, we characterized the T cell receptor (TCR)-delta repertoire by complementarity determining region 3 (CDR3) spectratyping in the inflamed and noninflamed mucosa and in the peripheral blood of subjects with Crohn's disease and ulcerative colitis. In contrast to previously published data about alpha/beta T cells, we rarely found oligoclonal expansions of gamma/delta T cells specific only for the inflamed mucosa. The same dominant gamma/delta T cell expansions were also present in the noninflamed colon. Furthermore, the peripheral gamma/delta TCR repertoire was oligoclonal but clearly distinct from that in the inflamed intestine. Thus our results do not support a role for antigen-specific gamma/delta T cells in IBD, and dominant gamma/delta T cells of the peripheral blood are not likely to be derived from the inflamed gut. However, in several patients, the TCR-delta-repertoire was highly diversified, whereas in others we observed a loss of dominant gamma/delta T cell clones when inflamed and noninflamed mucosa were compared. In conclusion, those changes indicate that gamma/delta T cells might play an important role in a subset of patients with IBD.     

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.     

Limited pattern of TCR delta chain gene rearrangement on the RNA level in multiple sclerosis

Susceptibility to multiple sclerosis (MS) is most likely affected by a number of genes, including HLA and T-cell receptor (TCR) genes. T cells expressing gamma/delta receptors seem to contribute to autoagression in MS, as evidenced by their localization in the MS plaques in the brain. The aim of this study was to analyse the TCRdelta chain gene rearrangement at the RNA (cDNA) level and compare to the DNA pattern rearrangement. TCRdelta gene rearrangement was analysed in MS patients and healthy individuals with the use of primers specific for Vdelta1-6 and Jdelta1 genes (at the DNA level) and specific for Vdelta1-6 and Cdelta1 genes (at the cDNA level). The size of PCR products was analysed on agarose gel and by ALF-Express (Pharmacia). Additionally, the lymphocyte surface immunophenotype was studied with specific monoclonal antibodies. At the DNA level a restricted pattern of Vdelta3-Jdelta1 and Vdelta5-Jdelta1 was found only in MS patients. Contrary to DNA, mono-, oligoclonal RNA (cDNA) rearrangements were limited to Vdelta1-Cdelta1, Vdelta2-Cdelta1 and Vdelta3-Cdelta1 only in MS patients as well. Surface immunophenotype analysis revealed in MS a much higher frequency of activated gamma/delta T lymphocytes, i.e. expressing HLA-DR and CD25. An elevated level of CD56 positive cells in MS was recorded. Mono-oligoclonal pattern of TCRdelta gene rearrangement at the RNA level, along with increase in activated gamma/delta T cells, strongly argue for a significant role of gamma/delta T lymphocytes in the pathogenesis of MS.     

Distortion of the self-reactive IgG antibody repertoire in multiple sclerosis as a new diagnostic tool

To date, none of the myelin-associated Ag targets definitively discriminates between the immune response observed in multiple sclerosis (MS) patients and healthy subjects. However, it has been shown recently that analysis of global immune Ab profiles such as natural autoantibody reactivities can help to distinguish between normal individuals and patients suffering from various immune diseases. The aim of our study was to compare the global IgG immune response against brain self-Ags in sera from 82 MS patients and 27 healthy subjects. The analysis of the immune profiles was performed by Western blotting, and data were subjected to linear discriminant analysis. Particular patterns of IgG reactivity were found in healthy subjects, Sj?gren patients, and MS patients. Moreover, this approach separated the three clinical forms of MS with a high concordance rate with the clinical data (kappa value, 77.8%). Our study suggests, for the first time, that serum IgG Ab repertoires are able to distinguish MS patients. In addition, our data suggest that patterns of IgG reactivity could model the pathological processes underlying the various forms of MS. Further characterization of such discriminant Ags could provide useful information regarding their potent role in pathogenesis or regulatory processes in MS.     

Single-cell repertoire analysis demonstrates that clonal expansion is a prominent feature of the B cell response in multiple sclerosis cerebrospinal fluid

Single-cell RT-PCR was used to sample CD19(+) B cell repertoires in cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) or viral meningitis. Analysis of amplified Ab H and L chain products served to identify the rearranged germline segment and J segment, and to determine the degree of homology for the H and L chain sequence of individual B cells. The B cell repertoire of viral meningitis CSF was predominantly polyclonal, whereas B cell clonal expansion was a prominent feature of the IgG repertoire in three of four MS patients. Two dominant clonal populations in one MS CSF accounted for approximately 70% of the IgG H chain V regions sequenced, while the corresponding IgM repertoires were more heterogeneous. One clonal B cell population revealed multiple L chain rearrangements, raising the possibility of a role for receptor editing in shaping the B cell response in some MS patients. The most immediate implications of identifying rearranged Ig sequences in MS B cells is the potential to accurately recreate recombinant Abs from these overrepresented H and L chains that can be used to discover the relevant Ag(s) in MS.     

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.     

High incidence of spontaneous disease in an HLA-DR15 and TCR transgenic multiple sclerosis model

Multiple sclerosis (MS) is thought to involve CD4 T cell recognition of self myelin, many studies focusing on a pathogenic role for anti-myelin, HLA-DR15-restricted T cells. In experimental allergic encephalomyelitis, it is known which epitopes trigger disease and that disease is associated with determinant spread of T cell reactivity. Characterization of these events in human MS is critical for the development of peptide immunotherapies, but it has been difficult to define the role of determinant spread or define which epitopes might be involved. In this study, we report humanized transgenic mice, strongly expressing HLA-DR15 with an MS-derived TCR; even on a RAG-2 wild-type background, mice spontaneously develop paralysis. Disease, involving demyelination and axonal degeneration, correlates with inter- and intramolecular spread of the T cell response to HLA-DR15-restricted epitopes of myelin basic protein, myelin oligodendrocyte glycoprotein, and alphaB-crystallin. Spread is reproducible and progressive, with two of the epitopes commonly described in responses of HLA-DR15 patients. The fact that this pattern is reiterated as a consequence of CNS tissue damage in mice demonstrates the value of the transgenic model in supplying an in vivo disease context for the human responses. This model, encompassing pathologically relevant, spontaneous disease with the presentation of myelin epitopes in the context of HLA-DR15, should offer new insights and predictions about T cell responses during MS as well as a more stringent test bed for immunotherapies.     

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.     

TCR zeta mRNA with an alternatively spliced 3'-untranslated region detected in systemic lupus erythematosus patients leads to the down-regulation of TCR zeta and TCR/CD3 complex

The reduction or absence of TCR zeta-chain (zeta) expression in systemic lupus erythematosus (SLE) patients is thought to be related to the pathogenesis of SLE. Recently, we reported the predominant expression of zeta mRNA containing an alternatively spliced 3'-untranslated region (3'UTR; zetamRNA/as-3'UTR) and a reduction in the expression of zeta mRNA containing the wild-type 3'UTR (zetamRNA/w-3'UTR) in T cells from SLE patients. Here we show that AS3'UTR mutants (MA5.8 cells deficient in zeta protein that have been transfected with zetamRNA/as-3'UTR) exhibit a reduction in the expression of TCR/CD3 complex and zeta protein on their cell surface as well as a reduction in the production of IL-2 after stimulation with anti-CD3 Ab compared with that in wild-type 3'UTR mutants (MA5.8 cells transfected with zetamRNA/w-3'UTR). Furthermore, the real-time PCR analyses demonstrated that the half-life of zetamRNA/as-3'UTR in AS3'UTR mutants (3 h) was much shorter than that of zetamRNA/w-3'UTR in wild-type 3'UTR mutants (15 h). Thus, the lower stability of zetamRNA/as-3'UTR, which is predominant in SLE T cells, may be responsible for the reduced expression of the TCR/CD3 complex, including zeta protein, in SLE T cells.     

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.     

Lack of TIM-3 immunoregulation in multiple sclerosis

Multiple sclerosis (MS) is an inflammatory disease of the CNS white matter associated with T cell infiltrates and alterations of immune functions that can be measured in the peripheral immune system. TIM-3 has been identified as a central regulator of IFN-gamma-secreting type 1 Th (Th1) cells and immune tolerance. In this study, using a newly generated mAb against human TIM-3, we examined TIM-3 function on ex vivo CD4(+) T cells isolated from the circulation of healthy subjects and patients with MS. Blocking TIM-3 during T cell stimulation significantly enhanced IFN-gamma secretion in control subjects but had no effect in untreated patients with MS, demonstrating a defect in TIM-3 immunoregulation. Treatment with glatiramer acetate or IFN-beta reversed this functional defect. Reduced levels and altered kinetics of T cell TIM-3 expression, which was restored in treated patients, is one mechanism that can explain the loss of TIM-3 regulation of T cell function in untreated patients with MS. These data provide functional, mechanistic data for dysregulated TIM-3 immunoregulation in a human autoimmune disease and suggest that approved therapies for the treatment of MS may function in part by restoring TIM-3 immunoregulation of T cell function.     

Gene microarray analysis of multiple sclerosis lesions

Multiple sclerosis (MS) is an autoimmune disease affecting central nervous system white matter. The cause is unknown. It is thought that environmental factors trigger an immune response against myelin antigens in a genetically susceptible individual. The characteristic lesion of MS seen in the brain is a plaque, an area of inflammation, demyelination and glial reaction or ‘sclerosis’. Several recent studies have examined gene expression in MS plaques on a large scale using microarray technology. The involvement of immune-related genes has been confirmed, and many new genes not previously associated with MS lesions have been identified. Microarray studies are significant in identifying potential new targets for therapy.