Induction of TCR Vbeta-specific CD8+ CTLs by TCR Vbeta-derived peptides bound to HLA-E

Previous studies have identified murine and human regulatory CD8+ T cells specific for TCR-Vbeta families expressed on autologous activated CD4+ T cells. In the mouse, these regulatory CD8+ T cells were shown to be restricted by the MHC class Ib molecule, Qa-1. In the present study, we asked whether HLA-E, the human functional equivalent of Qa-1, binds Vbeta peptides and whether the HLA-E/Vbeta-peptide complex induces and restricts human CD8+ CTLs. We first created stable HLA-E gene transfectants of the C1R cell line (C1R-E). Two putative HLA-E binding nonapeptides identified in human TCR Vbeta1 and Vbeta2 chains (SLELGDSAL and LLLGPGSGL, respectively) were shown to bind to HLA-E. CD8+ T cells could be primed in vitro by C1R-E cells loaded with the Vbeta1 (C1R-E/V1) or Vbeta2 (C1R-E/V2) peptide to preferentially kill C1R-E cells loaded with the respective inducing Vbeta peptide, compared with targets loaded with the other peptides. Priming CD8+ T cells with untreated C1R-E cells did not induce Vbeta-specific CTLs. Of perhaps more physiological relevance was the finding that the CD8+ CTLs primed by C1R-E/V1 also preferentially killed activated autologous TCR Vbeta1+. Similar results were observed in reciprocal experiments using C1R-E/V2 for priming. Furthermore, anti-CD8 and anti-MHC class I mAbs inhibited this Vbeta-specific killing of C1R-E and CD4+ T cell targets. Taken together, the data provide evidence that certain TCR-Vbeta peptides can be presented by HLA-E to further induce Vbeta-specific CD8+ CTLs.     

Preferential usage of TCR-Vbeta17 by peripheral and cutaneous T cells in nickel-induced contact dermatitis.

Nickel (Ni) is one of the most common contact sensitizers in man, and Ni-induced contact dermatitis is considered as a model of hapten-induced delayed type hypersensitivity. Previous studies indicated that Ni-reactive T cells derived from Ni-allergic individuals preferentially express distinct TCR-Vbeta chains. However, data on the TCR-Vbeta repertoire of Ni-responsive T cells are not consistent. Therefore, the aim of this study was to identify the TCR-Vbeta receptors of Ni-responsive peripheral and cutaneous T cells in a cohort of 17 donors with Ni-induced contact dermatitis in comparison with those of 6 healthy controls. Peripheral NiSO(4)-responsive T lymphocytes showed a significant overexpression of TCR-Vbeta17 and the frequency of TCR-Vbeta17(+) T cells correlated significantly with the in vitro reactivity of PBMC to NiSO(4). In addition, the cutaneous infiltrate of Ni-induced patch test reactions consisted primarily of Vbeta17(+) T cells. The majority of patch test-derived NiSO(4)-responsive T cells of three allergic donors were TCR-Vbeta17(+), whereas patch test-derived NiSO(4) unresponsive T cells of four additional donors did not express TCR-Vbeta17. Skin-derived Ni-responsive T cell lines from three donors uniformly secreted the Th2 cytokine, IL-5, but no IFN-gamma or IL-10. These in vitro and in vivo findings strongly suggest that T cells with a restricted TCR-Vbeta repertoire, i.e., Vbeta17, predominate in NiSO(4)-induced contact dermatitis and may be crucial in the effector phase of Ni hypersensitivity.     

Analysis of T-cell receptor Vbeta gene from infiltrating T cells in insulitis and myocarditis in encephalomyocarditis virus-infected BALB/C mice

Encephalomyocarditis (EMC) virus induces insulin-dependent diabetes and myocarditis in several strains of mice. The T-cell receptor (TCR) Vbeta genes of infiltrating T cells in the pancreas and myocardium of BALB/C mice infected with EMC virus D-variant (EMC-D virus) were analyzed. Using a nested two-step polymerase chain reaction (PCR), TCR Vbeta cDNAs were cloned and sequenced. Two and four kinds of TCR Vbeta clones were obtained from T cells infiltrating into the pancreas and myocardium of BALB/C mice infected with EMC-D virus, respectively. The infiltrating lymphocytes in the diabetic mice expressed Vbeta 8.1, 8.2, and 8.3 genes predominantly. Previously, the use of Vbeta 8.2 has been reported in autoimmune diseases such as murine experimental allergic encephalomyelitis (EAE) and non-obese diabetic (NOD) mouse. This study suggests that mice infected with EMC virus are a useful animal model for autoimmune diseases such as insulin-dependent diabetes.     

Active specific immunotherapy with hapten-modified autologous melanoma cell vaccine

 We have developed a novel approach to cancer immunotherapy – an autologous whole-cell vaccine modified with the hapten dinitrophenyl (DNP). This approach elicits significant inflammatory responses in metastatic sites and some objective tumor responses. Post-surgical adjuvant immunotherapy with DNP-modified melanoma vaccine in a setting of micrometastatic disease produces significant survival prolongation in stage III melanoma patients. Histologically, the inflammatory responses of the tumor consist of infiltration by lymphocytes, the majority of which are CD8⁺, HLA-DR⁺ T cells. T cells from these lesions tend to have mRNA for interferon γ. T cell receptor analysis suggests that the tumor-infiltrating T cells are clonally expanded. DNP-modified vaccine also induces T cells in the peripheral blood, which respond to DNP-modified autologous cells in a hapten-specific, MHC-restricted manner. Moreover, a T cell line generated from these lymphocytes responded to only a single HPLC fraction of MHC-associated, DNP-modified tumor peptides. Since inflammatory responses in metastases were not consistently associated with dramatic tumor regression, we considered the possibility of immunosuppression at the tumor site. We found that mRNA for the anti-inflammatory cytokine, interleukin-10 (IL-10) is expressed in most metastatic melanoma tissues and subsequently demonstrated that IL-10 protein is produced by melanoma cells. Thus the efficacy of DNP vaccine could be further enhanced by inhibition of IL-10 production or binding. Finally, we expect these results obtained with melanoma to be applicable to other human cancers. Received: 6 August 1996 / Accepted: 20 September 1996     

Successful TCR-based immunotherapy for autoimmune myocarditis with DNA vaccines after rapid identification of pathogenic TCR

The identification of TCRs of autoimmune disease-inducing T cells within a short period of time is a key factor for designing TCR-based immunotherapy during the course of the disease. In this study, we show that experimental autoimmune carditis-associated TCRs, Vbeta8.2 and Vbeta10, were determined by complementarity-determining region 3 (CDR3)-spectratyping analysis and subsequent sequencing of the CDR3 region of spectratype-derived TCR clones. Immunotherapy targeting both Vbeta8.2 and Vbeta10 TCRs using mAbs and DNA vaccines significantly reduced the histological severity of experimental autoimmune carditis and completely suppressed the inflammation in some animals. Since depletion or suppression of one of two types of effector cells does not improve the severity of the disease significantly, combined TCR-based immunotherapy should be considered as a primary therapy for T cell-mediated autoimmune diseases. TCR-based immunotherapy after rapid identification of autoimmune disease-associated TCRs by CDR3 spectratyping can be applicable, not only to animal, but also to human autoimmune diseases whose pathomechanism is poorly understood.     

Self-reactive T cell receptor-reactive CD8+ T cells inhibit T cell lymphoma growth in vivo

Syngenic C57BL/6 mice (H-2(b)) vaccinated with mitomycin C-treated L12R4 T lymphoma cells develop protective immunity toward the MHC class II-negative tumor cells. In the present study, we characterize the nature, mode of function, and specificity of the effector cells in this immunity. These cells are TCR-specific CD8(+) T lymphocytes with effector function in vitro as well as in vivo upon transfer to naive mice. They produce high levels of IFN-gamma and TNF-alpha, but little or no IL-4. By means of TCRbeta-negative variant L12R4 cells, P3.3, and TCR-Vbeta2 cDNA-transfected and TCR-Vbeta2-expressing P3.3 lymphoma cells, we found that a significant part of the effector T cells are specific for the Vbeta12 region. The growth inhibition of L12R4 cells in vitro was inhibited by anti-H-2, anti-K(b), and anti-D(b) mAb. Furthermore, vaccination with Vbeta12 peptide p67-78, which binds to both K(b) and D(b) MHC class I molecules, induces partial protection against L12R4 T lymphoma cells. Thus, self-reactive TCR-Vbeta-specific, K(b)-, or D(b)-restricted CD8(+) T cells mediate inhibition of T cell lymphoma growth in vitro and in vivo.     

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.     

Frequent contribution of T cell clonotypes with public TCR features to the chronic response against a dominant EBV-derived epitope: application to direct detection of their molecular imprint on the human peripheral T cell repertoire

In an attempt to provide a global picture of the TCR repertoire diversity of a chronic T cell response against a common Ag, we performed an extensive TCR analysis of cells reactive against a dominant HLA-A2-restricted EBV epitope (hereafter referred to as GLC/A2), obtained after sorting PBL or synovial fluid lymphocytes from EBV-seropositive individuals using MHC/peptide multimers. Although TCR beta-chain diversity of GLC/A2+ T cells was extensive and varied greatly from one donor to another, we identified in most cell lines several recurrent Vbeta subsets (Vbeta2, Vbeta4, and Vbeta16 positive) with highly conserved TCRbeta complementarity-determining region 3 (CDR3) length and junctional motifs, which represented from 11 to 98% (mean, 50%) of GLC/A2-reactive cells. While TCR beta-chains expressed by these subsets showed limited CDR1, CDR2, and CDR3 homology among themselves, their TCR alpha-chains comprised the same TCRAV region, thus suggesting hierarchical contribution of TCR alpha-chain vs TCR beta-chain CDR to recognition of this particular MHC/peptide complex. The common occurrence of T cell clonotypes with public TCR features within GLC/A2-specific T cells allowed their direct detection within unsorted PBL using ad hoc clonotypic primers. These results, which suggest an unexpectedly high contribution of public clonotypes to the TCR repertoire against a dominant epitope, have several implications for the follow-up and modulation of T cell-mediated immunity.     

Persistence of dominant T cell clones in accepted solid organ transplants

Donor/recipient MHC class II matching is beneficial to the survival of allogeneic kidneys in humans and swine. In the latter, tolerance to class I-disparate grafts can be induced by a short course of immunosuppression, a peripheral mechanism that implicates regulatory T cells. Absence of treatment will lead to prompt rejection. Rejected grafts are infiltrated by dominant alloaggressive T cells, whereas there is still speculation on the specificity and function of T cells invading accepted tissues. To characterize the TCR repertoire of graft-infiltrating T cells (GITC) in accepted kidneys, we have used the RT-PCR-based spectratyping technique to assess the length polymorphism of the porcine TCRbeta chain complementary-determining region 3 (CDR3). Results show that T cells infiltrating accepted kidneys (n = 5) express a restricted polymorphism of the CDR3 length, whereas PBL from the same animal have the polymorphic distribution of CDR3 lengths found in naive animals; that the skewed Vbeta repertoire in accepted grafts involved distinct Vbeta subfamilies in otherwise MHC-identical recipient animals; that GITC clonal dominance is not caused by immunosuppression because a second kidney, accepted without drug treatment, exhibits the same TCR Vbeta CDR3 profiles than those detected in the first graft; and that intragraft clonal dominance intensifies with time, indicating progressive preeminence of nonaggressive GITC clones. Collectively, these data represent the first example, in a preclinical model, of the emergence of nonaggressive intragraft clones, which may be involved in the induction/maintenance of local tolerance to allogeneic tissues.     

The protective immune response to heat shock protein 60 of Histoplasma capsulatum is mediated by a subset of V beta 8.1/8.2+ T cells

Immunization with recombinant heat shock protein 60 (rHsp60) from Histoplasma capsulatum or a region of the protein designated fragment 3 (F3) confers protection from a subsequent challenge in mice. To determine the T cell repertoire involved in the response to Hsp60, T cell clones from C57BL/6 mice immunized with rHsp60 were generated and examined for Vbeta usage by flow cytometry and RT-PCR. Vbeta8.1/8.2(+) T cells were preferentially expanded; other clones bore Vbeta4, -6, or -11. When Vbeta8.1/8.2(+) cells were depleted in mice, Vbeta4(+) T cell clones were almost exclusively isolated. Measurement of cytokine production demonstrated that nine of 16 Vbeta8.1/8.2(+) clones were Th1, while only three of 13 non-Vbeta8.1/8.2(+) clones were Th1. In mice immunized with rHsp60, depletion of Vbeta8.1/8.2(+), but not Vbeta6(+) plus Vbeta7(+), T cells completely abolished the protective efficacy of Hsp60 to lethal and sublethal challenges. Examination of the TCR revealed that a subset of Vbeta8.1/2(+) clones that produced IFN-gamma and were reactive to F3 shared a common CDR3 sequence, DGGQG. Transfer of these T cell clones into TCR alpha/beta(-/-) or IFN-gamma(-/-) mice significantly improved survival, while transfer of other Vbeta8.1/8.2(+) clones that were F3 reactive but were Th2 or clones that were not reactive to F3 but were Th1 did not confer protection. These data indicate that a distinct subset of Vbeta8.1/8.2(+) T cells is crucial for the generation of a protective response to rHsp60.     

Effect of the dose and composition of an autologous hapten-modified melanoma vaccine on the development of delayed-type hypersensitivity responses

We have reported that treatment of melanoma patients with a vaccine consisting of autologous tumor cells modified with the hapten, dinitrophenyl (DNP) and preceded by low-dose cyclophosphamide induces delayed-type hypersensitivity (DTH) to autologous, unmodified tumor cells and that this response is a significant predictor of survival. We analyzed the vaccines prepared for 284 patients who were treated following resection of regional or distant metastases to find out whether the dose and composition determined the immunological response. A positive DTH response (> or =5 mm induration) to unmodified autologous tumor cells was induced in 57% of the patients (median: 5 mm; range: 0-22 mm). Regression analysis showed no significant association between the magnitude of DTH and the number of live (trypan blue exclusion) melanoma cells per dose over a dosage range of 0.5-25.0 x 10(6). Surprisingly, there was a small but significant positive relationship between the mean number of dead cells in the vaccines of a given patient and that patient's maximum DTH to unmodified melanoma cells. Only 37% of patients whose vaccines contained >50% live cells developed DTH, as compared with 69% and 65% of patients whose vaccines contained 26% to 50% or < or =25% live cells, respectively. Thus, it appears that dead tumor cells contribute to the immunogenicity of the DNP vaccine, but other factors such as the administration schedule may be more important determinants of immunological and clinical outcome.     

Reactivity and regulatory properties of human anti-idiotypic antibodies induced by T cell vaccination

Immunization with irradiated autoreactive T cells (T cell vaccination) induces anti-idiotypic T cell responses that preferentially recognize complementarity-determining region 3 sequences, contributing to clonal depletion of autoreactive T cells. However, it remains unknown whether T cell vaccination elicits anti-idiotypic humoral responses and whether the anti-idiotypic Abs play a similar role in the regulatory mechanism induced by T cell vaccination. In this study we examined the occurrence, the reactivity pattern, and the regulatory role of anti-idiotypic Abs elicited by T cell vaccination in patients with multiple sclerosis. We demonstrated for the first time that B cells producing anti-idiotypic Abs could be isolated from vaccinated patients. These EBV-transformed B cell lines were selected for specific reactivity to a 20-mer TCR peptide incorporating a common complementarity-determining region 3 sequence of the immunizing T cell clones. The resulting anti-idiotypic Abs were found to react with the original immunizing T cell clones and exhibit an inhibitory effect on their proliferation. The findings suggest that anti-idiotypic Ab responses can be induced by T cell vaccination in humans and that their regulatory properties are likely to contribute to the suppression of myelin basic protein-reactive T cells in vaccinated patients. The study has important implications in our understanding of the regulatory role of the anti-idiotypic humoral responses induced by T cell vaccination.     

V beta 6+ T cells are obligatory for vaccine-induced immunity to Histoplasma capsulatum

We examined TCR usage to a protective fragment of heat shock protein 60 from the fungus, Histoplasma capsulatum. Nearly 90% of T cell clones from C57BL/6 mice vaccinated with this protein were Vbeta6+; the remainder were Vbeta14+. Amino acid motifs of the CDR3 region from Vbeta6+ cells were predominantly IxGGG, IGG, or SxxGG, whereas it was uniformly SFSGG for Vbeta14+ clones. Short term T cell lines from Vbeta6+-depleted mice failed to recognize Ag, and no T cell clones could be generated. To determine whether Vbeta6+ cells were functionally important, we eliminated them during vaccination. Depletion of Vbeta6+ cells abrogated protection in vivo and upon adoptive transfer of cells into TCR alphabeta(-/-) mice. Transfer of a Vbeta6+, but not a Vbeta14+, clone into TCR alphabeta(-/-) mice prolonged survival. Cytokine generation by Ag-stimulated splenocytes from immunized mice depleted of Vbeta6+ cells was similar to that of controls. The efficacy of the Vbeta6+ clone was associated with elevated production of IFN-gamma, TNF-alpha, and GM-CSF compared with that of the Vbeta14+ clone. More Vbeta6+ cells were present in lungs and spleens of TCR alphabeta(-/-) on day 3 postinfection compared with Vbeta14+ cells. Thus, a single Vbeta family was essential for vaccine-induced immunity. Moreover, the mechanism by which Vbeta6+ contributed to protective immunity differed between unfractionated splenocytes and T cell clones.     

Influence of the route of infection on development of T-cell receptor beta-chain repertoires of reovirus-specific cytotoxic T lymphocytes

It is well established that the route of infection affects the nature of the adaptive immune response. However, little is known about the effects of the route of exposure on development of cytotoxic T-lymphocyte (CTL) responses. Alternative antigen-presenting cell populations, tissue-restricted expression of class I major histocompatibility complex-encoded molecules, and unique T-cell receptor (TCR)-bearing cells in mucosal tissues could influence the selection and expansion of responder T cells. This study addresses the question of whether the route of virus infection affects the selection and expansion of subpopulations of virus-specific CTLs. Mice were infected orally or in the hind footpads with reovirus, and the repertoires of TCR beta-chains expressed on virus-specific CD8(+) T cells in Peyer's patches or lymph nodes and spleens were examined. CD8(+) cells expressing the variable gene segment of the TCR beta-chain 6 (Vbeta6) expanded in the spleens of mice infected by either route and in CTL lines established from the spleens and draining lymphoid tissues. Adoptively transferred Vbeta6(+) CD8(+) T cells from orally or parenterally infected donors expanded in reovirus-infected severe combined immunodeficient recipient mice and mediated cytotoxicity ex vivo. Furthermore, recovered Vbeta6(+) cells were enriched for clones utilizing uniform complementarity-determining region 3 (CDR3) lengths. However, sequencing of CDR3beta regions from Vbeta6(+) CD8(+) cells indicated that Jbeta gene segment usage is significantly more restricted in CTLs from orally infected mice, suggesting that the route of infection affects selection and/or subsequent expansion of virus-specific CTLs.     

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.     

Role of the complementarity-determining region 3 (CDR3) of the TCR-beta chains associated with the V alpha 14 semi-invariant TCR alpha-chain in the selection of CD4+ NK T Cells

The NK1.1(+)TCRalphabeta(int) CD4(+), or double negative T cells (NK T cells) consist of a mixture of CD1d-restricted and CD1d-unrestricted cells. The relationships between CD4(+)NK1.1(+) T cells and conventional T cells are not understood. To compare their respective TCR repertoires, NK1.1(+)TCRalphabeta(int), CD4(+) T cells have been sorted out of the thymus, liver, spleen, and bone marrow of C57BL/6 mice. Molecular analysis showed that thymus and liver used predominantly the Valpha14-Jalpha281 and Vbeta 2, 7, and 8 segments. These cells are CD1d restricted and obey the original definition of NK T cells. The complementarity-determining region 3 (CDR3) sequences of the TCR Vbeta8.2-Jbeta2.5 chain of liver and thymus CD4(+) NK T cells were determined and compared with those of the same rearrangements of conventional CD4(+) T cells. No amino acid sequence or usage characteristic of NK T cells could be evidenced: the Vbeta8.2-Jbeta2.5 diversity regions being primarily the same in NK T and in T cells. No clonal expansion of the beta-chains was observed in thymus and liver CD1d-restricted CD4(+)NK T cells, suggesting the absence of acute or chronic Ag-driven stimulation. Molecular analysis of the TCR used by Valpha14-Jalpha281 transgenic mice on a Calpha(-/-) background showed that the alpha-chain can associate with beta-chains using any Vbeta segment, except in NK T cells in which it paired predominately with Vbeta 2, 7, and 8(+) beta-chains. The structure of the TCR of NK T cells thus reflects the affinity for the CD1d molecule rather than a structural constraint leading to the association of the invariant alpha-chain with a distinctive subset of Vbeta segment.