Clonal dominance and selection for similar complementarity determining region 3 motifs among T lymphocytes responding to the HLA-DR3-associated Mycobacterium leprae heat shock protein 65-kd peptide 3-13.

In order to establish whether specific MHC class II-peptide complexes are capable of selecting TCR V regions, we investigated in detail the TCR beta chain used in the recognition of HLA-DR3 restricted hsp65 peptide 3-13 in a tuberculoid leprosy patient. Using RT-PCR, a clear dominance of the TCRBV5 gene family was observed in a hsp65 peptide 3-13-specific T-cell line; however, not in fresh, unstimulated PBMCs, PHA-stimulated PBMCs, or a T-cell line specific for tetanus toxoid. DNA sequence analysis of the TCR V regions, comprising TCRBV5 genes, derived from the hsp65 peptide 3-13-specific T-cell line revealed the exclusive usage of the TCRBV55S1 gene segment and a predominance of one V-D-J gene rearrangement, which is indicative of clonal expansion of these T lymphocytes. Additional highly similar V-D-J gene rearrangements were detected at a low level in this hsp65 peptide 3-13 specific T-cell line. These conserved junctional regions (CDR3 regions) could not be detected within the TCRBV5 gene family of fresh PBMCs, PHA-stimulated PBMCs, hsp65, and tetanus-toxoid-specific T-cell lines from this patient. The observations in this tuberculoid leprosy patient reveal that an HLA class-II-restricted T-cell response results in selection of TCRBV regions which are highly similar in amino acid composition to the CDR3 region within the expanding TCRBV regions.

T-cell receptor V-gene usage in synovial fluid lymphocytes of patients with chronic arthritis.

In this study we analyzed the usage frequencies of the TCR V-gene segments by alpha beta+ T cells present in synovial fluid of 17 patients with chronic arthritis, including rheumatoid arthritis. The results of this study, obtained from semiquantitative PCR analyses, showed that in all patients most of the TCR V alpha- and V beta-gene segments could be detected both in fresh PBMCs and in fresh SFMCs. The relative frequencies of use of these V-region genes were variable between the different patients. Although there was some skewing of increased usage frequencies of particular TCR V alpha and V beta genes among SFMC-derived TCRs when compared with PBMCs, we could not correlate such increased TCR V-gene usage with the inflammation in the joints as a disease-specific marker.

Differential usage of T cell receptor V gene segments in CD4+ and CD8+ subsets of T lymphocytes in monozygotic twins.

The TCR confers immunity by the specific recognition of foreign Ag peptides in the context of self-MHC molecules. The mechanisms controlling TCR selection and repertoire generation are not clearly understood and seem to occur in an apparently random, (self) Ag-driven manner. To address the question to what extent the TCR repertoire is randomly shaped or genetically predetermined, we have analyzed the alpha beta TCR repertoire of the CD4+ and CD8+ subsets of peripheral blood lymphocyte cultures of monozygotic twins by using the polymerase chain reaction technique with TCR V region gene family-specific oligonucleotide primers. Our studies demonstrate that there is high concordance in the overall patterns of V gene usage within a pair of twins, particularly in V beta usage (mean V beta CD4+ R2 = 0.869 and CD8+ R2 = 0.833) and to a lesser extent V alpha usage (mean V alpha CD4+ R2 = 0.621 and CD8+ R2 = 0.627); whereas the patterns between unrelated individuals show more variability. This study has also demonstrated that the V alpha and V beta genes are not randomly used within the CD4+ and CD8+ subsets. We observed significant preferential skewing of several V alpha or V beta gene families to either the CD4+ or CD8+ subset in the majority of individuals analyzed (p-value range = 0.0476 to < 0.001). In particular, V alpha 11, 17, 22, and V beta 3, 9, 12, 18 were skewed to the CD4+ subset; whereas V alpha 2, 6, 12, 15, 20 and V beta 7, 14, 17 were skewed to the CD8+ subset. Furthermore, a number of the V genes showed patterns of skewing consistent only within a pair of twins. In three pairs of twins, V beta 2 was skewed to the CD4+ subset, whereas the fourth pair used almost equal frequencies of V beta 2 in both subsets. This observation was made for the V beta 2, 4, 5, 6, 8, 19 and V alpha 7, 16, 18, 21 families. Finally, the ratio of the relative V gene usage frequency that could be observed within an individual was conserved within the sets of twins; for instance, the relative amount of V beta 2 to that of V beta 3 was higher in both individuals of one set of twins, whereas it was lower in all of the other three sets. Together these observations suggest that the predominant influence shaping the TCR repertoire is genetically predetermined, of which, HLA-predicted selection mechanisms exerted during thymic maturation might be contributing factors.

Limited restriction in the TCR-alpha beta V region usage of antigen-specific clones. Recognition of myelin basic protein (amino acids 84-102) and Mycobacterium bovis 65-kDa heat shock protein (amino acids 3-13) by T cell clones established from peripheral blood mononuclear cells of monozygotic twins and HLA-identical individuals.

We have analyzed the TCR-alpha beta repertoire specific for a given peptide/MHC complex by using pairs of HLA-identical individuals ranging from monozygotic twins to unrelated individuals to examine the contribution of genetic background and HLA expression in shaping the potential response to a single antigenic epitope. This panel has been previously defined, demonstrating that the concordance of the peripheral TCR-alpha beta repertoires directly correlates to the level of relation and HLA identity. We have analyzed peptide-specific T cell clones derived from T cell lines from these individuals specific for MHC class II-restricted peptides: Mycobacterium bovis 65-kDa heat shock protein (65-kDa hsp) amino acids (aa) 3-13 (DR3-restricted), and myelin basic protein aa 84-102 (DR2-restricted). DNA sequence analysis was used to determine the composition of the TCR-alpha beta V regions. Although the overall TCR-alpha beta repertoires between individuals were diverse, an intra-individual limited restriction was evident. There was also a limited conservation in the response to the different peptides: high frequencies of V beta 2, 4, 7, 19, V alpha 21, and J alpha 17 responded to the MBP aa84-102, whereas these V/J regions were limited or absent in the 65-kDa hsp aa3-13 repertoire. Similarly, V beta 5.1 and J alpha 9 were increased in the 65-kDa hsp aa3-13 repertoire. Within the CDR3s, motifs could be identified that were similar between twins. Furthermore, one of these motifs resembled CDR3s previously found in corresponding animal models. Similarities could also be seen in the CDR3s of T cell clones sharing V gene usage and peptide specificity. Thus, the in vitro response to antigenic peptides seems to be quite heterogeneous overall and individual specific.

Skewing of TCR V genes to CD4+ and CD8+ subsets of T lymphocytes is not determined by amino acid composition of CDR3.

TCR V genes show differing expression patterns, termed skewing, in CD4+ and CD8+ subsets of T lymphocytes. To determine which elements of the TCR V regions contribute to these observed TCR V gene skewing patterns, we have performed an in-depth analysis, taking advantage of RT-PCR and DNA sequencing, which was focused on the multi-member TCRBV6 gene family. These studies allowed us to evaluate the contributions of the various elements, that constitute the TCR beta chain variable region, to the observed TCR V gene skewing patterns. The results of these analyses revealed that within the TCRBV6 family individual members exhibited differing skewing patterns, i.e. TCRB6S7 was significantly skewed towards the CD4+ T cell subset, whereas TCRBV6S5 was significantly skewed towards the CD8+ subset. Scrutiny of the usage of TCRBV6 family members in combination with TCRBJ gene usage and amino acid composition of CDR3 did not reveal obvious structural characteristics which would explain the differing skewing patterns between TCRBV6S7 and TCRBV6S5. Further examination of these TCR V regions showed that the CDR1 and 2 regions within these TCRBV elements were composed of different amino acids. These observations suggests that these components contribute to the observed TCR V gene skewing patterns.

Dominant rearrangements among human tumor-infiltrating lymphocytes. Analysis of T-cells derived from 32 patients with melanoma, lung, and renal cell carcinoma.

Dominant rearrangements of T-cell receptor (TCR) beta-chain genes are reported among tumor-infiltrating lymphocytes (TIL). After interleukin-2 expansion of TIL from renal and lung carcinoma and melanoma biopsy tissues, rearrangements of TCR beta-chain genes were analyzed by Southern blotting. Nongermline restriction fragments, indicating dominant rearrangements, were detected among the TIL from all 6 patients with renal cell carcinoma, 17 of 20 patients with melanoma, and 3 of 6 patients with lung tumors. The restriction-fragment sizes of these dominant rearrangements were heterogeneous among the various patients. Rearrangements into C beta 1 were more common than C beta 2 rearrangements. Phenotypic analyses indicated that dominant rearrangements occurred in both CD4 and CD8 predominant TIL populations. The TIL populations that were extracted were expanded to derive large cell numbers suitable for in vivo transfer in an interleukin-2 and TIL immunotherapy program. The data indicated that the cells delivered to these patients usually were characterized by dominant populations of T-cells with selective TCR gene rearrangements. The significance of selective TCR use requires evaluation of the function and specificity of the TIL comprising these dominant populations both in their native in vivo setting and in the context of therapeutic transfer.

Evidence for selective in vivo expansion of synovial tissue-infiltrating CD4+ CD45RO+ T lymphocytes on the basis of CDR3 diversity.

In this study we have analyzed the TCR V alpha and V beta regions at the DNA level in the CD4+CD45RO+ memory T cell population of synovial tissue infiltrating T lymphocytes of three rheumatoid arthritis (RA) patients and one patient with chronic arthritis. Cell lines of CD4+CD45RO+, CD4+CD45RO-, CD8+CD45RO+ and CD8+CD45RO- T lymphocyte populations were generated following FACS cell sorting of freshly isolated synovial tissue mononuclear cell infiltrates (STMC) and of freshly isolated peripheral blood mononuclear cells (PBMC) of these patients. The phenotypic and molecular analyses have revealed the following. (i) The TCR repertoires of tissue infiltrating T lymphocytes in the various subsets were extensive on the basis of TCR V gene family usage. (ii) Furthermore, each patient displayed individual specific TCR V gene expression patterns in the various STMC and PBMC derived T cell subsets. However, the majority of these arthritis patients manifested increased expression of multiple TCR V gene families in the synovial tissue derived CD4+CD45RO+ T cell population when compared with the peripheral blood derived CD4+CD45RO+ subset. Of these gene families, we found enhanced expression of the TCR V alpha 7 and V beta 11 gene segments in synovial tissue to be shared by all four patients analyzed. (iii) Nucleotide sequence analysis of the CDR3 regions of a number of TCR V regions in the CD4+CD45RO+ T cell subsets has revealed that the CDR3 regions comprised within synovial tissue derived TCR V regions differed from those found in peripheral blood derived TCR V regions. These differences in CDR3 diversity might be the consequence of a specific interaction with particular MHC-peptide complexes expressed at the site of inflammation. (iv) The CDR3 region analysis also showed individual specific amino acid motifs within the N-D-N regions of all analyzed TCR V beta genes derived from PBMC as well as STMC.

Conserved TcR beta chain usage for a single MHC class II-restricted heat shock protein peptide.

Previously we have shown that the T-cell response against the HLA-DR3 (17)-restricted heat shock protein (Mr 65 000)-derived peptide amino acids (aa) 3-13 (hsp65 aa 3-13) is recognized by the exclusive usage of the TRBV5 gene as well as a conserved CDR3 region in a tuberculoid leprosy patient. In the present study we analyzed the TcR of T-cell clones specific for hsp65 aa 3-13 derived from three healthy individuals with a response level similar to that of the leprosy patient. We show that unlike the tuberculoid leprosy patient, healthy high responders have a diverse T-cell response to hsp65 aa 3-13. However, a striking observation was made: even though high responders have a diverse specific TcR repertoire, TRBV5-expressing clones from two healthy individuals could be isolated that were nearly identical to a dominant clone in the tuberculoid leprosy patient. In conclusion, the data show that restriction of TcR specific for an antigen correlates with the presence of that antigen in disease. However, the preferred TcR can also be detected in healthy high responders. A natural infection in vivo, as with the tuberculoid leprosy patient, may be responsible for the observed trimming and preferential outgrowth of a certain TcR.

Molecular analysis of the T-cell receptor beta-chain repertoire in early rheumatoid arthritis: heterogeneous TCRBV gene usage with shared amino acid profiles in CDR3 regions of T lymphocytes in multiple synovial tissue needle biopsies from the same joint.

To gain insight into the nature of the immune response with respect to accumulation and composition of the T-cell receptor (TCR) repertoire in synovial tissue in rheumatoid arthritis (RA), we have determined the nucleotide sequence of TCRBV regions transcribed by T lymphocytes derived from synovial tissue. Synovial tissue was obtained by needle biopsies from three different sites of the same joint in two early RA patients. We found that the TCRBV region repertoire among synovial tissue-infiltrating mononuclear cells was heterogeneous when the different biopsies taken from each patient were compared. However, DNA sequence analysis of TCRBV rearrangements of synovial T lymphocytes showed conserved amino acid usage profiles in the CDR3 domains of different TCRBV regions, which exhibited an individual specific character. These CDR3 motifs were not present in paired samples of peripheral blood. The existence of homologous CDR3 amino acid profiles within the TCRBV regions derived from synovial tissue is indicative of an antigen-driven immune response.

Intrauterine transfusions affect fetal T-cell immunity.

Intrauterine transfusion (IUT) therapy is the treatment of choice in severe hemolytic disease of the fetus. This treatment automatically implies the introduction of alloantigens in the fetal circulation, which might potentially influence the unprimed fetal immune system. The present study provides evidence that the fetal immune system is indeed prone to modulations of the T-cell receptor BV (TCRBV) repertoire as a result of IUT treatment. Most notably, IUT therapy affects the composition of the CD4+ repertoire, whereas this effect may be obscured in the CD8+ subset. The CD8+ subset was found to be influenced by alterations of the TCRBV repertoire both in IUT patients and controls, suggesting that modulations in this subset could be the result of developmental influences. A more detailed analysis on the composition of the individual TCRBV families was performed by evaluating the distribution of the complementarity determining region 3 (CDR3) size lengths of [32P]-radiolabeled TCRBV transcripts. Using this technique, referred to as spectratyping, only marginal changes were observed in the CD4+ and CD8+ subset during the course of treatment and gestational development of both IUT-treated patients and controls. Therefore, the alterations in the overall TCRBV repertoire were of a quantitative rather than a qualitative nature. To evaluate whether the observed alterations in TCRBV usage-frequencies were a reflection of an allo-reactive response, a primed lymphocyte test (PLT) was performed in 3 IUT-treated patients. We observed that IUT, performed as early as 23 weeks of gestation, may induce the establishment of memory T cells against the IUT donor. However, there was no association between the observed changes in TCRBV repertoire and the magnitude of the secondary allo-reactive response.

Affinity, specificity and T-cell-receptor diversity of melanoma-specific CTL generated in vitro against a single tyrosinase epitope.

MHC-class-I-restricted cytotoxic T lymphocytes (CTL) specific for tumor-associated antigens expressed by malignant cells are important components of the immune response against cancer. Recently, tumor-specific CTL could be generated in vitro, with responding lymphocytes from the blood of healthy blood donors. In the present study, we confirm that peptide-specific stimulation in vitro can induce high-affinity CTL capable of recognizing tumor cells expressing the appropriate tumor antigen. These tyrosinase-specific CTL display a restricted usage of TCRAV and TCRBV gene segments but of diverse CDR3 regions, resulting in a distinct fine-specificity for each CTL clone. This suggests that, similar to in vivo priming, peptide-pulsed APC are capable of stimulating a T-cell response in vitro expressing a limited TCR repertoire against autologous tumors. The generated CTL can recognize their target structure with high affinity, and this correlates in part with tumor-cell lysis. This methodology may be used to treat melanoma patients with infusion of ex vivo-induced and -expanded CTL.