Selection of T lymphocytes bearing limited T-cell receptor beta chains in the response to a human pathogen.

Delayed-type hypersensitivity (DTH) is a classic measure of T-cell responsiveness to foreign antigen. To estimate the extent of the T-cell repertoire in the DTH response to a human pathogen, we measured T-cell receptor (TCR) beta-chain variable-region (V beta) gene usage in reversal reactions in leprosy. Reversal reactions represent naturally occurring DTH responses in leprosy, in which augmentation of T-cell responses to Mycobacterium leprae is concomitant with clearance of bacilli from lesions. T cells using the V beta 6-, V beta 12-, V beta 14-, and V beta 19-encoded TCRs were strikingly overrepresented in the lesions of patients as compared to blood and pre-DTH lesions from the same individuals. Furthermore, these data indicate a possible association between the predominant expression of a V beta gene segment in lesions and the major histocompatibility complex class II haplotype of the individual. V beta 6 was prominent in the lesions of four patients who were DR15, a marker of resistance in leprosy infection. Sequence analysis of V beta 6 TCRs showed frequent use of V beta 6.1 and J beta 2.7 gene segments and a conserved amino acid motif in the V-J junction in a reversal-reaction lesion, but not in blood from the same patient. The limited TCR repertoire expressed by the infiltrating T cells suggests that a limited set of antigens is recognized in the DTH response to a human pathogen. We suggest that the mechanism by which major histocompatibility complex haplotype influences DTH in this disease involves the presentation of specific peptides, with subsequent selection of specific TCRs followed by local oligoclonal expansion.     

Oligoclonal expansion of CD8+ CD57+ T cells with restricted T-cell receptor beta chain variability after bone marrow transplantation

A major expansion of CD8+57+ lymphocytes expressing an alpha-beta T- cell receptor (TCR) is frequent after bone marrow transplantation (BMT). We examined the clonality of the TCR beta gene repertoire in these expanded CD8+57+ cells after allogeneic or autologous BMT. We performed a polymerase chain reaction (PCR) analysis of the V beta chain usage in CD8+57+ cells purified from nine BMT recipients with a series of oligonucleotides specific for 20 V beta gene families. PCR products from selected TCR beta gene rearrangements were sequenced. The CD8+57+ cells from eight of nine patients used a restricted set of V beta families, with a marked predominance of two to three V beta gene families per patient, whereas the control autologous CD57- subset expressed the whole 20 V beta families. A direct sequencing analysis confirmed the V beta 16 and V beta 17 clonality in six patients, showing a striking homology in the CDR3 sequences of the V beta 16 products. The CD8+57+ cells, but not the CD57- cells, displayed an oligoclonal pattern of TCR rearrangements as shown by PCR analysis of TCR gamma gene rearrangements. Such an oligoclonal expansion of CD8+57+ cells, using a restricted set of the V beta gene families, may result from a specific TCR stimulation of a limited number of T-cell clones in BMT recipients.     

Interindividual conservation of T-cell receptor beta chain variable regions by minor histocompatibility antigen-specific HLA-A*0201- restricted cytotoxic T-cell clones

Minor histocompatibility antigens (mHags) are involved in the induction of graft-versus-host disease (GVHD) after HLA-identical bone marrow transplantation. Previously, we isolated a series of HLA-A*0201- restricted cytotoxic T-cell (CTL) clones specific for the same mHag HA- 1 from peripheral blood of three unrelated patients who were suffering from GVHD. We have now analyzed the composition of the T-cell receptor (TCR) V regions of 12 of these mHag HA-1-specific HLA-A*0201-restricted CTL clones by DNA sequencing of the alpha and beta chains. Of these 12 clones, derived from three unrelated individuals, five independent TCR alpha V- and beta V-region sequences were established. The TCR alpha chains were composed of varying TCR alpha V and TCR alpha J genes with no obvious similarities in structure in the N regions. However, the TCR beta chains all used the TCR beta V6S9 gene segment, and showed remarkable similarities within the N-D-N regions; ie, three independent beta-chain sequences (originating from donors Ha and Gy) shared a leucine/valine amino acid pair, whereas the other two (originating from donors Ha and Wi) shared a serine/threonine pair, all at positions 99 and 100 of the TCR beta V region. In conclusion, the TCR analysis of HA- 1 mHag-specific CTL clones has shown that the HA-1 mHag/HLA-A*0201 complex selects for highly similar TCR beta V regions.     

Changes in the pattern of TCR V beta repertoire expression after bone marrow transplant is linked to the HLA haplotype in humans

Neutrophil, monocyte, natural killer- and B-cell number and function are rapidly restored after bone marrow transplant (BMT), whereas T-cell reconstitution is often quite delayed. Our hypothesis was that V beta T-cell receptor (TCR) repertoire diversity among recipients of allogeneic BMT is influenced by the expression of major and minor HLA antigens in the host. The study population comprised unmanipulated and CD34(+)-selected allogeneic bone marrow grafts, autologous peripheral blood stem cell transplants and recipients of volunteer unrelated donor (VUD) bone marrow transplants. Using flow cytometry, the relative frequencies of 18 V beta TCR families were determined and ranked for each time point studied. Comparisons and correlations were made between paired blood samples obtained within a single patient over time, and between donors and their recipients. The pattern of the V beta TCR repertoire from allogeneic recipients and their HLA-matched donors was very similar, with a correlation coefficient (CC) of 0.59. This similarity was not as marked in VUD pairs (CC = 0.32). By 3 months after transplant, the pattern of the V beta TCR repertoire in recipients of HLA-matched sibling transplants was more similar to the pattern seen in pretransplant recipients than to the donor pattern (CC = 0.40 vs. 0.31). Our data suggest that both major and minor HLA antigens influence V beta TCR repertoire diversity and reconstitution after BMT.     

Analysis of T-cell receptor variability in transplanted patients with acute graft-versus-host disease.

T lymphocytes play a pivotal role in graft-versus-host disease (GVHD) and largely contribute to the graft-versus-leukemia (GVL) effect. Most mature T lymphocytes specifically recognize antigens through the alpha/beta T-cell receptor (TCR). Each alpha/beta TCR chain includes a constant region and a variable region, the latter being encoded by V-J alpha or V-D-J beta rearranged gene segments. To better characterize T cells involved in GVHD, V alpha and V beta gene segment usage was analyzed, after cDNA amplification, in peripheral blood mononuclear cells (PBMC) and skin samples from three patients with grade II cutaneous GVHD. At time of GVHD diagnosis (days 11, 22, and 25), when first signs of engraftment were detectable, virtually all V alpha and V beta subfamilies were represented in PBMC RNAs of the three recipients. These results suggest that diversified TCR gene segment expression is observed early after allogenic bone marrow transplantation (alloBMT). Lymphocytes infiltrating GVHD skin also expressed a large series of V alpha and V beta subfamily specificities. However, analysis of the V alpha and V beta amplified products showed substantial differences between PBMC and the skin lymphocyte RNAs. These observations indicate that a large variety of T lymphocytes are present at the disease site, while some of them may be specifically amplified or decreased in response to minor histocompatibility antigens (miHA). Further characterization of the latter T-cell subpopulations should lead to a better understanding of human in vivo responses directed at miHA.     

Role of CXCR3-induced donor T-cell migration in acute GVHD

OBJECTIVE: The chemokine receptor CXCR3 has an important role in the migration of effector T cells. To investigate the role of CXCR3 on donor cells in acute graft vs host disease (GVHD) we used a well-defined experimental bone marrow transplantation (BMT) model where acute GVHD is mediated by donor CD8(+) T cells against minor histocompatibility antigens. METHODS; Lethally irradiated C3H.SW recipients were transplanted from either wild-type B6 or CXCR3(-/-) B6 donors. Donor T-cell expansion was analyzed in the spleen and small intestine of recipients by FACS. Donor T-cell function was analyzed by cytokine secretion. The severity of acute GVHD was assessed by histopathological analysis of intestine and liver, GVHD clinical scores, and survival after BMT. RESULTS: Significantly higher numbers of donor CD8(+) CXCR3(-/-) T cells were found in the spleen on days +7 and +14 compared to donor wild-type T cells. By contrast, the number of CD8(+) T cells in the small bowel of BMT recipients from CXCR3(-/-) donors was sevenfold lower than from wild-type donors. Systemic concentrations of INF-gamma and TNF-alpha were equivalent between groups. Animals that received CXCR3(-/-) donor T cells demonstrated diminished GI tract and liver damage and showed improved survival after BMT compared to recipients of wild-type donor cells (43% vs 0%, p<0.001). CONCLUSION: The migration of donor CD8(+) T cells to GVHD target organs such as the intestine depends on the expression of CXCR3 and contributes significantly to GVHD damage and overall mortality.     

Structure of human T-cell receptors specific for an immunodominant myelin basic protein peptide: positioning of T-cell receptors on HLA-DR2/peptide complexes.

T-cell receptors (TCRs) recognize peptide bound within the relatively conserved structural framework of major histocompatibility complex (MHC) class I or class II molecules but can discriminate between closely related MHC molecules. The structural basis for the specificity of ternary complex formation by the TCR and MHC/peptide complexes was examined for myelin basic protein (MBP)-specific T-cell clones restricted by different DR2 subtypes. Conserved features of this system allowed a model for positioning of the TCR on DR2/peptide complexes to be developed: (i) The DR2 subtypes that presented the immunodominant MBP peptide differed only at a few polymorphic positions of the DR beta chain. (ii) TCR recognition of a polymorphic residue on the helical portion of the DR beta chain (position DR beta 67) was important in determining the MHC restriction. (iii) The TCR variable region (V) alpha 3.1 gene segment was used by all of the T-cell clones. TCR V beta usage was more diverse but correlated with the MHC restriction--i.e., with the polymorphic DR beta chains. (iv) Two clones with conserved TCR alpha chains but different TCR beta chains had a different MHC restriction but a similar peptide specificity. The difference in MHC restriction between these T-cell clones appeared due to recognition of a cluster of polymorphic DR beta-chain residues (DR beta 67-71). MBP-(85-99)-specific TCRs therefore appeared to be positioned on the DR2/peptide complex such that the TCR beta chain contacted the polymorphic DR beta-chain helix while the conserved TCR alpha chain contacted the nonpolymorphic DR alpha chain.     

Efficient identification of T-cell clones associated with graft-versus-host disease in target tissue allows for subsequent detection in peripheral blood

Graft-versus-host disease (GVHD) causes severe morbidity and mortality in allogeneic haematopoietic stem cell transplantation (HSCT) because of destruction of recipient tissues by donor alloreactive T cells. We hypothesized that GVHD-specific T-cell clones are expanded within affected tissue of HSCT patients and can also be detected in blood at the time of active disease. A multiplex polymerase chain reaction (PCR) was used to amplify T-cell receptor (TCR) variable beta (VB) chain rearrangements in skin biopsies from eight allogeneic HSCT patients. Molecular analysis of the complementarity-determining region 3 (CDR3) of amplified products defined expanded, potentially disease-associated 'clonotypes' and enabled the design of clonotype-specific PCR assays. We detected immunodominant clones in seven of eight GVHD-positive skin biopsies. In serial skin biopsies from the same patient, the identical clone was found in each biopsy. In a patient who underwent two successive HSCTs from different donors, distinct clones were identified for each engraftment. Using clonotypic PCR assays, individual tissue-derived clones could be identified in peripheral blood samples obtained during active GVHD. We hypothesize that clonotypic sequences derived from target tissue can serve as markers for GVHD and may have utility in diagnosis and monitoring response to therapy, as well as enable future therapies targeted against pathogenic clones.     

Long-term expression of a T-cell receptor beta-chain gene in mice reconstituted with retrovirus-infected hematopoietic stem cells.

To determine the feasibility of retrovirus-mediated gene transfer into stem cells for studying T-cell development, we constructed a high-titer retrovirus vector containing the neomycin phosphotransferase (neo) gene and a murine T-cell receptor (TCR) beta-chain gene with the V beta 6 variable segment. The TCR gene was placed under the control of the human beta-actin promoter and enhancer. Bone marrow cells pretreated with 5-fluorouracil were infected by coculturing with psi-2 virus-producing cells in the presence of recombinant interleukins 1, 2, 4, and 6 as well as interleukin 3 from WEHI-3 conditioned medium. The infected cells were transplanted into irradiated mice, and expression of the exogenous V beta 6 gene was examined with a V beta 6-specific monoclonal antibody, RNase protection, and polymerase chain reaction amplification. Three of seven mice expressed the retroviral TCR gene on the surface of a significant proportion of mature T cells 5-6 months after transplantation. In mice analyzed less than 1 month after transplantation, up to 30% of mature T cells expressed V beta 6 TCRs, an increase of at least 20% above the level of endogenous V beta 6 expression. DNA analysis revealed that pluripotent hematopoietic stem cells were infected by the retroviral vector in a long-term reconstituted mouse that showed increased V beta 6 expression.     

Molecular analysis of T-cell receptor V beta chains of human T-cell chronic lymphocytic leukemia does not show intraclonal variability: implications for immunotherapy

Human T-cell chronic lymphocytic leukemia (T-cell CLL) is a heterogeneous disease characterized by a monoclonal malignant proliferation of T cells in which the T-cell receptors (TCRs) can be, when expressed, considered to be membrane tumor-specific antigens. Owing to the increasing number of available monoclonal antihuman TCR reagents, it could be of interest to evaluate the feasibility of anti- TCR treatment during T-cell CLL. To test the therapeutic potentiality of anti-TCR monoclonal antibodies, we first analyzed the intraclonal variability in two terminally ill patients suffering from TCR alpha beta-positive cell CLL bearing different immunophenotypes. The cDNA corresponding to the variable regions of the TCR beta chains originating from the malignant T cells were amplified, cloned into M13 phages, and sequenced. The sequence analysis of multiple independent clones showed no intraclonal variability, with no evidence for ongoing hypermutation in the V beta region genes. The relevance of these findings with regard to an anti-V beta therapy and the comparison with similar analysis during B-cell monoclonal lymphoproliferations are discussed.     

Redirection of antileukemic reactivity of peripheral T lymphocytes using gene transfer of minor histocompatibility antigen HA-2-specific T-cell receptor complexes expressing a conserved alpha joining region

Donor-derived T lymphocytes directed against minor histocompatibility antigens (mHags) exclusively expressed on cells of the hematopoietic lineages can eliminate hematologic malignancies. Transfer of T-cell receptors (TCRs) directed against these mHags into T lymphocytes may provide a strategy to generate antileukemic T cells. To investigate the feasibility of this strategy the TCR usage of mHag HA-2-specific T-cell clones was characterized. Thirteen different types of HA-2-specific T-cell clones were detected, expressing TCRs with diversity in TCR alpha- and beta-chain usage, however, containing in the TCR alpha chain a single conserved gene segment J alpha 42, indicating that J alpha 42 is involved in HA-2-specific recognition. We transferred various HA-2 TCRs into T lymphocytes from HLA-A2-positive HA-2-negative individuals resulting in T cells with redirected cytolytic activity against HA-2-expressing target cells. Transfer of chimeric TCRs demonstrated that the HA-2 specificity is not only determined by the J alpha 42 region but also by the N-region of the alpha chain and the CDR3 region of the beta chain. Finally, when HA-2 TCRs were transferred into T cells from HLA-A2-negative donors, the HA-2 TCR-modified T cells exerted potent antileukemic reactivity without signs of anti-HLA-A2 alloreactivity. These results indicate that HA-2 TCR transfer may be used as an alternative strategy to generate HA-2-specific T cells to treat hematologic malignancies of HLA-A2-positive, HA-2-expressing patients that received transplants from HLA-A2-matched or -mismatched donors.     

T-cell regeneration after bone marrow transplantation: differential CD45 isoform expression on thymic-derived versus thymic-independent progeny

To study the source of regenerated T cells after bone marrow transplantation (BMT), lethally irradiated thymectomized and thymus- bearing C57BL/6 (Thy 1.2+) mice were injected with syngeneic T-cell depleted bone marrow (TCD BM) cells and graded numbers of congenic B6/Thy 1.1+ lymph node (LN) cells. LN cell expansion was the predominant source for T-cell regeneration in thymectomized hosts but was minimal in thymus-bearing hosts. Analysis of T-cell receptor (TCR) expression on LN progeny showed a diverse V beta repertoire. Therefore, peripheral T-cell progenitors exist within V beta families, but expansion of these progenitors after BMT is downregulated in the presence of a functional thymus. CD4+ cells derived from BM versus LN in thymus-bearing hosts displayed differential CD44 and CD45 isoform expression. BM-derived cells were primarily CD45RB+CD44lo and LN derived cells were nearly exclusively CD45RB- CD44hi. In thymectomized hosts, BM, host, and LN CD4+ progeny were CD45RB- CD44hi. We conclude that T-cell regeneration via peripheral T-cell progenitors predominates in hosts lacking thymic function and gives rise to T cells that display a "memory" phenotype. In contrast, the ability to generate sizable populations of "naive" type T cells after BMT appears limited to the prethymic progenitor pool and could serve as a marker for thymic regenerative capacity.     

T-cell receptor usage by melanoma-specific clonal and highly oligoclonal tumor-infiltrating lymphocyte lines.

Tumor-infiltrating lymphocytes (TIL) obtained from human melanomas can specifically lyse autologous tumor in vitro and mediate tumor regression in vivo. To develop more effective therapeutic reagents and to further understand the T-cell response to tumors, the diversity of T-cell receptors (TCRs) involved in melanoma antigen recognition has been studied. The TCR variable (V) genes, joining (J) segments, and N diversity regions used by five clonal lines and one highly oligoclonal, melanoma-specific, CD8+ TIL line were examined utilizing PCR amplification with V gene subfamily-specific primers and anchor PCR. The TIL lysed multiple allogeneic melanomas expressing matched surface major histocompatibility complex class I molecules. TCR analysis confirmed the clonal nature of the TIL lines; however, the TCR repertoire was diverse. Even among the three HLA-A2 restricted TIL (TIL 1200, TIL F2-2, and TIL-5), no common V gene usage was found. Comparison of the third complementarity-determining regions of the TCRs from the HLA-A2 restricted TIL revealed no homology. Results presented here identify T-cell clonotypes that recognize epitopes on highly prevalent, shared melanoma tumor-associated antigens presented in the context of HLA-B55, HLA-A1, and HLA-A2. These T cells and the antigens they recognize represent important components for the design of new immunotherapies for patients with advanced melanoma.     

Engineered secreted T-cell receptor alpha beta heterodimers.

We have produced a soluble form of a mouse alpha beta T-cell antigen receptor (TCR) by shuffling its variable (V) and constant (C) domains to the C region of an immunoglobulin kappa light chain. These chimeric molecules composed of V alpha C alpha C kappa and V beta C beta C kappa chains were efficiently secreted (up to 1 micrograms/ml) by transfected myeloma cells as noncovalent heterodimers of about 95-kDa molecular mass. In the absence of direct binding measurement, we have refined the epitopic analysis of the soluble V alpha C alpha C kappa-V beta C beta C kappa dimers and shown that they react with an anti-clonotypic antibody and two antibodies directed to the C domain of the TCR alpha and beta chains. Conversely, we have raised three distinct monoclonal antibodies against the soluble TCR heterodimers and shown that they recognize surface-expressed TCRs. Two of these antibodies were found to react specifically with the products of the V alpha 2 (V delta 8) and V beta 2 gene segments, respectively. When considered together, these data suggest that these soluble TCR molecules are folded in a conformation indistinguishable from that which they assume at the cell surface.     

Selective outgrowth of CD45RO+ V gamma 9+/V delta 2+ T-cell receptor gamma/delta T cells early after bone marrow transplantation.

Before and after bone marrow transplantation (BMT) for hematologic malignancies, peripheral blood mononuclear cells from 10 patients were obtained. The relative and absolute numbers of CD3+ T-cell receptor gamma delta+ (TCR gamma delta+) cells, as defined by the reaction of monoclonal antibodies (MoAbs) directed against CD3 and the TCR gamma delta (anti-TCR gamma delta-1), were determined. Before transplantation, eight of nine patients tested had less than 10% CD3+TCR gamma delta+ cells. Consistent increased numbers of gamma delta cells up to eightfold the pretransplant level can be seen in four of nine patients tested within the first 4 months after BMT. The large majority of early posttransplant gamma delta and alpha beta T cells express the CD45RO antigen, which is usually expressed on "memory" cells only. The V-region usage of the TCR gamma delta+ T cells was analyzed using fresh mononuclear cells and MoAbs against known V gamma and V delta regions. For more detailed analysis, CD3+TCR gamma delta+ cells were sorted and cultured in bulk and cloned. Using fresh cells and bulk cultures, mainly V gamma 9+V delta 1-V delta 2+ cells were found during engraftment. Only after 6 weeks post-BMT, V gamma 9-V delta 1+V delta 2- cells appear. Analysis of the V gamma and V delta usage at the clonal level confirmed the observation that early after BMT only V gamma 9+V delta 2+ cells are present, whereas gamma delta T-cell clones expressing other gamma delta TCR phenotypes can only be detected 4 to 6 weeks post-BMT. The predominance of V gamma 9+ cells during early engraftment could be explained by several mechanisms: (A) sequential rearrangements during T-cell development, leading to an early wave of V gamma 9+ cells, or (B) selective outgrowth of preexisting V gamma 9+V delta 2+CD45RO+ TCR gamma delta cells in the bone marrow graft, possibly as a result of antigen driven expansion due to exposure to environmental antigens.