Cell Receptor Beta Chain Gene Research Articles

Longitudinal Profiling of the T Cell Compartment in Patients with Chronic Lymphocytic Leukemia Treated with Venetoclax

Venetoclax (ven), an inhibitor of the BCL2 anti-apoptotic protein, has been proven remarkably effective in the treatment of patients with chronic lymphocytic leukemia (CLL), where the malignant B cells typically overexpress BCL2. BCL2 is also expressed by various bystander cells in the CLL microenvironment, including T cells. Hence, investigating the effects of ven treatment on T cells is highly relevant, especially considering that distinct T cell subpopulations exhibit different sensitivity to anti-apoptotic signaling blockade. Herein we interrogated the molecular and phenotypic profiles of the T cell compartment in 1 treatment-naïve and 15 relapsed/refractory CLL patients who received ven either as monotherapy (n=12) or in combination with an anti-CD20 antibody (obinutuzumab, n=1; rituximab, n=3). We studied peripheral blood samples collected prior to ven initiation and after 5 months of treatment, from which we isolated T cell subpopulations (CD3+ T cells n=8; CD4+ and CD8+ T cells, n=8). Longitudinal analysis of pre/post-treatment samples by flow cytometry (n=7) revealed a significant (p=0.01) decrease in the numbers of central memory (CD45RO+/CCR7+) CD4+ (21.8% vs 8.8%) and CD8+ cells (21.4% vs 9.7%) under ven; in contrast, a significant (p=0.01) increase of CD8+ effector memory cells (CD45RO+/CCR7-) was observed (27.8% vs 32.9%). Profiling of the T cell receptor beta (TRB) chain gene repertoire overtime (n=15) was performed by next generation sequencing (NGS). Clonotypes (i.e. TRBV-TRBD-TRBJ gene rearrangements with unique pairs of TRBV genes and identical CDR3 amino acid sequences) were calculated and clonality was estimated as the average cumulative frequency of the 10 most frequent clonotypes per sample (ACF-10). A significantly (p=0.01) more diverse repertoire was identified in CD4+ vs CD8+ cells at both pre- and post-treatment timepoints (average numbers of clonotypes pre-/post-treatment: CD4+ cells, 16,908/13,768; CD8+ cells, 4,437/3,633). Expanded clonotypes corresponding to oligoclonal expansions were documented in all examined subpopulations at both timepoints. However, significantly (p=0.01) more pronounced skewing was observed in CD8+ vs CD4+ cells (pre-/post-treatment ACF-10 values: 64.4%/70.6% vs 31.4%/42.2%, respectively). Repertoire comparisons overtime highlighted a fraction of T clonotypes that were retained after treatment in all examined subpopulations. Of note, repertoire conservation was significantly (p=0.01) greater in CD8+ vs CD4+ T cells [retained pre-treatment clonotypes: 7,102/31,063 (28%) vs 11,525/118,359 (12%), respectively]. Transcriptome profiling of CD4+ and CD8+ cells pre-/post-treatment was performed using the Oncomine Immune Response Research Assay (395 genes) in 5 cases attaining a complete response after treatment. While the transcriptomes of CD4+ cells remained essentially unaltered, significant differences were identified in CD8+ cells, where 19 genes were found to be downregulated (adj.p<0.05, fold change>2). These genes are implicated in functions such as immune response (e.g. TLR7, TLR9, CD40), regulation of immune system (e.g. HLA genes, CIITA, TNFAIP8) and lymphocyte activation pathways (e.g. ICOSLG, CEACAM1, CXCR5), with at least some of these having immunosuppressive functions (e.g. BTLA and CTLA4 encoding inhibitory receptors associated with T cell exhaustion). In conclusion, ven treatment led to increased numbers of CD8+ effector subpopulations along with prominent clonal expansions and transcriptional rewiring that could conceivably contribute to clinical response. Downregulation of immune-related genes implicated in core regulatory/activation pathways supports immune recovery by ven, offering a rationale for future combination strategies aiming to increase the depth of clinical response.

T Cell Epitope Prediction within the Clonotypic Immunoglobulin Heavy and Light Chains in Patients with Multiple Myeloma

Despite remarkable therapeutic advances in recent years, multiple myeloma (MM) remains incurable, hence representing an unmet clinical need. Therapeutic approaches based on induced T cell anti-tumor immunity towards cancer eradication show promising results in many hematologic malignancies, including multiple myeloma (MM), highlighting the need for comprehensive understanding of the implicated mechanisms. Similar to other mature B cell malignancies, immunoglobulin (IG) gene rearrangements in MM lead to the expression of unique, novel peptide sequences that can be used as neoantigens, conceivably representing a potential attractive target for cancer-specific immune responses. Here we sought to explore this possibility by performing ad hoc prediction of putative T-cell class I/II neoepitopes contained within the MM clonotypic IG gene rearrangements. To that end, we investigated 16 newly diagnosed patients with MM from whom we isolated the peripheral blood and/or bone marrow mononuclear cell (PBMC/BMMC) fraction as well as CD138+ myeloma cells, CD4+ and CD8+ T cells. Clonotypic IG heavy and light chain gene rearrangements were RT-PCR amplified on RNA extracted from the PBMC/BMMC fraction using subgroup-specific leader primers for the IGHV/IGK/LV gene and universal primers annealing to the constant domain (IGHG, IGHM, IGKC, IGLC), in order to produce the full-length V-(D)-J gene rearrangement sequence, plus the start of the constant domain. The corresponding sequences were determined by bidirectional Sanger sequencing. Moreover, T cell receptor beta (TRB) chain gene rearrangements were RT-PCR amplified on RNA extracted from CD4+ and CD8+ T cells and then subjected to paired-end next generation sequencing (NGS). The deduced amino acid sequences of the IG heavy and light chain variable/constant domains were subsequently parsed in peptides and subjected to bioinformatics analysis for the identification of putative T-cell class I/II neoepitopes using the NetMHCpan and NetMHIICpan softwares. The rank score was calculated, considering the 4-digit HLA-A, -B, -C and DRB1 typing for each individual patient. High- and medium-binding peptides (rank score <2%) were selected. Prediction of the binding specificity of T Cell Receptors (TR) to MHC-peptide complexes (pMHCs) was performed by the ERGO-II tool. The rank score was calculated, considering the peptides resulted by NetMHCpan/ NetMHIICpan, the HLA, and the clonotypes for each patient (AUC>0.8). Exact matches to germline and/or proteome databases were excluded. Overall, 757,263 TRΒ distinct clonotypes were assessed (median= 18,244 /sample). All patients displayed oligoclonal T cell expansions in both CD4+ and CD8+ T cells, albeit these were significantly (p<0.001) more pronounced in the latter. Both T cell subpopulations displayed skewed TRBV and TRBJ gene repertoires (7 TRBV genes and 3 TRBJ genes accounting for about 40% and 59% of the repertoire, respectively). Overall, 1,219 predicted neoepitopes were identified. All patients had predicted CD4+ and CD8 + T-cell epitopes within the MM clonotypic IG, either in the heavy chain (16/16 pts, n=459 epitopes) or the light chain (16/16 pts, n=760 epitopes). There was no statistically significant difference in the rank score of peptides involving the complementarity determining region 3 (CDR3) vs. all other IG regions. Interestingly, 998/1,219 (82%) peptides with strong binding affinity resulted from IG gene sequence motifs outside the CDR3. Most of these peptides (853/1,219, 75%) resulted from somatic hypermutations (SHM) across the IG variable domain. Overall, 51,574 clonotypes of CD4+ T cells and 35,751 clonotypes of CD8+ T cells were in silico predicted by ERGO to have strong binding affinity. Relevant to mention, 528 clonotypes with high binding score were found to be shared between CD4+ T cells of all patients and 209 clonotypes between CD8+ T cells of all patients and, thus, were deemed as public. In conclusion, in silico prediction identified a significant number of putative T-cell class I/II neoepitopes contained within the clonotypic IG of MM patients. Many of the identified peptides derive from SHM, implying that the SHM which shapes the MM BcR IG repertoire may produce immunogenic CD4+/CD8+ T cell epitopes. Their actual immunogenicity has to be tested in ex vivo studies, currently underway by our group.

Immunoglobulin and T Cell Receptor β Chain Gene Rearrangement Analysis of Ocular Adnexal Lymphoid Neoplasms: Clinical and Biologic Implications

We investigated the organization of the immunoglobulin heavy chain (IgH) and the T cell receptor beta chain (T beta) gene loci in 20 ocular adnexal and four extraocular lymphoid neoplasms obtained from 18 patients presenting with an ocular adnexal lymphoid neoplasm. Fifteen ocular adnexal and four extraocular lymphoid neoplasms occurring in 13 patients were classified by morphological examination and immunophenotypic analysis as monoclonal B cell lymphomas. Each one of these 19 lymphoid neoplasms exhibited clonal IgH gene rearrangements upon hybridization of EcoRI- or HindIII-digested DNA to a heavy-chain joining region (JH)-specific DNA probe. The bilateral ocular adnexal monoclonal B cell neoplasms occurring simultaneously in two individuals exhibited identical clonal IgH gene rearrangements, which indicated their derivation from an identical B cell clone. The ocular adnexal and the extraocular monoclonal B cell neoplasms occurring in two of three patients also exhibited identical clonal IgH gene rearrangements, which suggested that they too were derived from an identical B cell clone. Five ocular adnexal lymphoid neoplasms were classified by morphological examination and immunophenotypic analysis as benign, polyclonal pseudolymphomas. Three of these five ocular adnexal lymphoid neoplasms exhibited clonal IgH gene rearrangements, which suggested the presence of monoclonal B cell populations that escaped detection by morphological and immunophenotypic examination. None of the 24 pathological samples exhibited clonal T beta gene rearrangements upon hybridization of EcoRI- or BamHI-digested DNA to a T beta gene DNA probe. The results of these studies demonstrate the value of Southern blot hybridization analysis for clonal IgH and T beta gene rearrangements in the diagnosis, classification, and investigation of extranodal lymphoid neoplasms originating and/or presenting in the ocular adnexa.

Cytotoxic peripheral T cell lymphoma arising in a patient with nodular lymphocyte predominant Hodgkin lymphoma: a case report

In this paper, we describe a case of nodular lymphocyte predominant Hodgkin lymphoma with the subsequent development of a peripheral T cell lymphoma. This case is unusual in that the sheets of atypical and small to intermediate-sized T cells in the diffuse component were CD8 positive and expressed cytotoxic proteins. The diagnosis of peripheral T cell lymphoma was supported by the demonstration of a clonal T cell receptor beta chain gene rearrangement by Southern blot analysis. Peripheral T cell lymphoma with a cytotoxic phenotype is a rare entity with an aggressive clinical behavior. As such, this report emphasizes the need to consider a diagnosis of coexisting peripheral T cell lymphoma in cases of nodular lymphocyte predominant Hodgkin lymphoma with atypical features, such as few or poorly defined B cell macronodules and diffuse T cell areas. The examination of both T cell receptor gamma and beta chain gene rearrangements should be performed to confirm such cases.

Detection of Putative T cell Clones Using T cell Receptor β Chain Gene Clonality Assay in Korean Patients with Aplastic Anemia

We analyzed T cell receptor beta chain (TCRB) gene to investigate the presence of putative T cell clones and its clinicopathologic implications in Korean patients with aplastic anemia (AA). Twenty-nine bone marrow specimens were collected from 20 AA patients, 19 specimens from initial diagnosis and 10 from follow-up. T cell clonality assay was performed using IdentiClone TCRB Gene Clonality Assay kit (InVivoScirbe Technology, USA) and automatic genetic analyzer. Patients' clinical information and laboratory parameters were also analyzed. Five patients had definitive underlying factors related with aplastic anemia, such as hepatitis B virus (4 cases) and benzene exposure (1 case). Putative T cell clones were detected in bone marrow specimens of 11 (58%) out of 19 patients at diagnosis. The location of putative T cell clones of TCRB gene (diversity region, Dbeta; joining region, Jbeta; variable region, Vbeta) was distributed in Dbeta2+Jbeta2 (6 cases), Dbeta1+Jbeta1 (3 cases), Vbeta+Jbeta1 (2 cases), and Dbeta1+Jbeta2 (2 cases). Interestingly, among seven patients who underwent stem cell transplantation, five patients with no T cell clones detected at diagnosis developed new T cell clones during the follow-up. Putative pathogenetic T cell clones were detected in most of AA patients in the current study. T cell clonality assay would be useful for investigating the pathophysiology of acquired AA.

Maintenance and characterization of an Epstein Barr virus-infected CD56-negative T cell lymphoma.

T cell lymphoma carrying Epstein Barr virus (EBV+TL) is very rare among Western countries while it is much more common among Japanese. Here we report an EBV+TL which has been maintained for years by the use of mice with severe combined immune deficiency (SCID) mice. Lymphoma was obtained from a 55‐year‐old male suffering from oculomotor nerve palsy and lymphadenopathy. A small piece of biopsied tumor was transplanted into SCID mice and the lymphoma has been maintained for over 3 years with passages every 2–3 weeks. The maintained lymphoma, termed as TMS24, and the original lymphoma cells showed identical phenotype and genotype, including diffuse medium‐sized cell morphology lacking granules, suppressor/cytotoxic immunophenotype and identical T cell receptor β‐chain gene rearrangement mode. Further, both were shown to carry an identical EBV clone in terms of the number of terminal repeats and the latency II‐type restricted gene expression profile. Cytogenetically, TMS24 retained two characteristic chromosomal translocations of t(l;18)(q32;q21) and t(6;12)(p21;q24). Since only one cell line with such characters has been reported previously, TMS24 should be useful for detailed analysis of EBV+TL.

Linkage analysis in multiple sclerosis of chromosomal regions syntenic to experimental autoimmune disease loci

Multiple sclerosis is a demyelinating disorder of the central nervous system with a putative autoimmune aetiology in which several genes are thought to be involved. Four published genomic screens have confirmed that a gene influencing MS resides within or close to the HLA class II region in 6p21. Still, this locus is likely to confer only a part of the genetic susceptibility in MS. Further, all four studies identified a number of other regions with possible linkage. We have investigated eight chromosomal intervals syntenic to loci of importance for experimental autoimmune model diseases in the rat in 74 Swedish MS families. Possible linkage (a non-parametric linkage NPL score of 1.16 by GENEHUNTER computer package) was observed with markers in 12p13.3, a region syntenic to the rat Oia2 locus which is importance for oil induced arthritis (OIA). Four markers in the T cell receptor beta chain gene region in 7q35 showed possible linkage (highest NPL score of 1.16). This locus is syntenic to the rat Cia3 locus (collagen induced arthritis). These two loci at least partially overlap with chromosomal regions showing indicative evidence for linkage in the previous MS genomic screens. Indeed, both Oia2 and Cia3 were recently found to be linked also with experimental autoimmune encephalomyelitis, a commonly used model for MS. Markers in 2p12, 3p25, 10q11.23, 17q21-25, 19q13.1, and 22q12-13 failed to provide evidence for linkage. We conclude that evidence is amounting that 12p13-12 and 7q34-36 may harbour genes with an importance for MS. The synteny with experimental loci may eventually facilitate their identification.

Structure and diversity of the TCR alpha-chain in a teleost fish.

T cell receptor beta-chain genes are well characterized in representatives of most vertebrate phyla, from sharks to mammals, but the molecular structure of complete TCR alpha-chains has not yet been established in cold-blooded vertebrates. We used a PCR approach to isolate cDNAs encoding putative teleost fish (Oncorhynchus mykiss, rainbow trout) TCR alpha-chains. Eight V alpha segments were identified, belonging to six different families, and the best amino acid sequence identity scores for these trout V alpha were all provided by mammalian V alpha or V delta sequences. Twenty-four (60.1 %) of the 39 analyzed V alpha segments belong to the V alpha 2 family, which has limited homology with mammalian V alpha/delta sequences and with the human V pre-B sequence. A total of 32 different J alpha segments were identified from 40 J alpha regions sequenced, suggesting that a large repertoire of J alpha segments is a characteristic of most vertebrates. The structural properties of the TCR alpha-chain complementarity-determining region 3 loop are well conserved between trout and mammals, suggesting that this region has been under continuous selective pressure in jawed vertebrate evolution. The trout C alpha segment has conserved N-terminal and transmembrane domains, but the C alpha intercysteine distance contains only 40 residues, significantly smaller as compared with mammals (49-56 residues). The conserved features of teleost fish TCR beta- and alpha-chains with their mammalian equivalents suggest that TCR-alpha beta receptors were still present in the common Devonian ancestors of modern teleost fish and mammals, about 450 million years ago.

Time course analysis of α+β+ T cell clones during normal pregnancy

During normal pregnancy, the fetus continues to mature inside the uterus without rejection. Inherited paternal antigens could be targeted by the maternal immune system. These reactions are believed to play a role in a number of habitual abortions. However, the precise maternal mechanisms preventing fetal tissue rejection are not well understood. Maternal T cells should recognize fetal antigens, so it is conceivable that antigen-specific T cell response to fetal antigens would occur by proliferation and accumulation of certain T cell clones in the pregnant mother. To elucidate the maternal immune response to the fetus we investigated the clonality of expanded T cells in peripheral blood lymphocytes in ten normal pregnant women. We employed reverse transcriptase-polymerase chain reaction for T cell receptor beta chain gene and subsequently analyzed the PCR product by single-strand conformation polymorphism analysis. A large number of distinctly expanded T cell clones were detected during pregnancy. These accumulations were observed as early as the ninth to tenth week post-conception and reached a maximum during the second trimester, suggesting the existence of dynamic antigen-specific T cell responses in the pregnant mother. However, after the 30th week of gestation, nearly all expanded T cell clones disappeared before parturition and the degree of clonality reached almost normal levels. Our results clearly indicate the existence of dynamic maternal T cell responses during pregnancy.

Evidence for a streptococcal superantigen-driven process in acute guttate psoriasis.

Recent studies have suggested that T cells play a critical role in the pathogenesis of psoriasis. Guttate psoriasis is a well-defined form of psoriasis frequently associated with streptococcal throat infection. This study tested the hypothesis that T cells in acute guttate psoriasis skin lesions may be activated by streptococcal superantigens. Peripheral blood as well as lesional and perilesional skin biopsies were analyzed for T cell receptor V beta repertoire using monoclonal antibodies against 10 different V beta families. Skin biopsies from all patients with acute guttate psoriasis, but not skin biopsies from patients with acute atopic dermatitis or inflammatory skin lesions induced in normal subjects with sodium lauryl sulfate, demonstrated selective accumulation of V beta 2+ T cells (P < 0.05). The expansion of V beta 2+ T cells occurred in both the CD4+ and the CD8+ T cell subsets. Sequence analysis of T cell receptor beta chain genes of V beta 2-expressing T cells from skin biopsies of patients with guttate psoriasis showed extensive junctional region diversity that is more compatible with a superantigen rather than a conventional (nominal) antigen-driven T cell response. All streptococcal isolates from patients with guttate psoriasis secreted streptococcal pyrogenic exotoxin C, a superantigen known to stimulate marked V beta 2+ T cell expansion. These data support the concept that acute guttate psoriasis is associated with superantigenic stimulation of T cells triggered by streptococcal superantigen(s).

The polymerase chain reaction in the demonstration of monoclonality in T cell lymphomas.

To evaluate polymerase chain reaction (PCR) amplification of T cell receptor (TCR) beta and gamma chain genes as a means of demonstrating monoclonality in T cell lymphomas using histological samples; to compare the performance of PCR with Southern blot analysis. TCR-beta, TCR-gamma and immunoglobulin heavy chain (IGH) genes were analysed using PCR in 55 cases of T cell lymphoma (28 frozen tissue and 27 paraffin wax embedded samples), diagnosed using morphological and immunohistochemical criteria. The 28 frozen samples were subjected to Southern blot analysis using TCR-beta, TCR-gamma and IGH gene probes. Twenty five B cell lymphomas and 21 non-neoplastic lymphoid tissue samples were used as controls. Using TCR-beta PCR, monoclonality was detected in 24 (44%) of 55 T cell lymphomas compared with 43 (78%) of 55 using TCR-gamma PCR and in 82% with both techniques. Five (9%) of 55 T cell lymphomas were IGH PCR positive. None of the non-neoplastic lymphoid control samples were PCR positive. All B cell lymphomas showed a polyclonal pattern with TCR-beta PCR while a single B cell lymphoma was positive using TCR-gamma primers. With TCR-beta PCR, a monoclonal result was seen in 12 (43%) of 28 frozen samples of T cell lymphoma, compared with 23 (82%) of 28 using Southern blot analysis. With TCR-gamma PCR, 19 (68%) of 28 frozen tissue samples were positive, compared with 26 (93%) of 28 using Southern blot analysis. A single case showed IGH rearrangement by Southern blot analysis. TCR-gamma PCR should be the method of choice for analysis of clonality in paraffin wax embedded sections of lymphoproliferative lesions, as TCR-beta PCR has a high false negative rate. Southern blot analysis remains the most successful technique when sufficient fresh tissue samples and resources are available.

T cell receptor gene recombination patterns and mechanisms: cell death, rescue, and T cell production.

The antigen-specific receptors of T and B lymphocytes are generated by somatic recombination between noncontiguous gene segments encoding the variable portions of these molecules. The semirandom nature of this process, while desirable for the generation of diversity, has been thought to exact a high price in terms of sterile (out-of-frame) products. Historically, the majority of T lymphocytes generated in mammals were thought to be useless, either because they generated such sterile rearrangements or because the receptors generated did not appropriately recognize self-molecules (i.e., positive and negative selection). In the studies described here, we characterize the onset of T cell receptor (TCR) alpha and beta chain gene rearrangements and quantitate their progression throughout T cell development. The results show that T cell production efficiency is enhanced through (a) rearrangement of TCR-beta chain genes early during T cell development, with selective expansion of those cells possessing in-frame rearrangements; (b) deletion of sterile rearrangements at the TCR-alpha chain locus through ordered (proximal to distal) sequential recombination; and (c) modification of nonselectable alpha/beta heterodimer specificities through generation and expression of new TCR-alpha chains. In addition, we demonstrate strict correlations between successful TCR-beta gene rearrangement, the onset of TCR-alpha gene rearrangement, rapid cell division, and programmed cell death, which together serve to maintain cell turnover and homeostasis during T cell development.

Dual T cell receptor beta chain expression on human T lymphocytes.

Allelic exclusion of lymphocyte antigen receptor chains has been hypothesized as a mechanism developed by the immune system to ensure efficient lymphocyte repertoire selection and tight control of lymphocyte specificity. It was effectively shown to be operative for both the immunoglobulin (Ig) and the T cell receptor (TCR) beta chain genes. Our present observations suggest that close to 1% of human T lymphocytes escape this allelic control, and express two surface TCR beta chains with distinct superantigenic reactivities. Since this high frequency of dual beta chain expressors did not result in any dramatic immune dysregulations, these results question the need for a mechanism ensuring clonal monospecificity through allelic exclusion.

Structure and specificity of T cell receptors expressed by potentially pathogenic anti-DNA autoantibody-inducing T cells in human lupus.

The production of potentially pathogenic anti-DNA autoantibodies in SLE is driven by special, autoimmune T helper (Th) cells. Herein, we sequenced the T cell receptor (TCR) alpha and beta chain genes expressed by 42 autoimmune Th lines from lupus patients that were mostly CD4+ and represented the strongest inducers of such autoantibodies. These autoimmune TCRs displayed a recurrent motif of highly charged residues in their CDR3 loops that were contributed by N-nucleotide additions and also positioned there by the recombination process. Furthermore, Th lines from four of the five patients showed a marked increase in the usage of the V alpha 8 gene family. Several independent Th lines expressed identical TCR alpha and/or beta chain sequences indicating again antigenic selection. 10 of these Th lines could be tested further for antigenic specificity. 4 of the 10 pathogenic anti-DNA autoantibody-inducing Th lines responded to the non-histone chromosomal protein HMG and two responded to nucleosomal histone proteins; all presented by HLA-DR molecules. Another Th line responded to purified DNA more than nucleosomes. Thus, these autoimmune Th cells of lupus patients respond to charged epitopes in various DNA-binding nucleoproteins that are probably processed and presented by the anti-DNA B cells they selectively help.

A genetically determined insertion/deletion related polymorphism in human T cell receptor beta chain (TCRB) includes functional variable gene segments.

Polymorphism in the human T cell receptor beta chain (TCRB) gene complex includes haplotypes with different numbers of TCRBV genes. An insertion/deletion related polymorphism (IDRP) in the human TCRBV region was found to involve TCRBV gene segments. Inserted TCRB haplotypes contain an additional 21.5 kb in which three TCRBV genes are encoded, members of the TCRBV7, TCRBV9, and TCRBV13 families. Two TCRBV gene segments were present only in inserted haplotypes; one of these, TCRBV7S3, is a functional gene and the other, TCRBV9S2(P), is a pseudogene because of an inframe termination colon. In addition, inserted haplotypes contain two identical copies of the TCRBV13S2 gene, whereas deleted haplotypes have only one copy. Deleted haplotypes could be subdivided into two types, deleted*1 and deleted*2, on the basis of sequence variations in TCRBV6S7 and TCRBV13S2 genes. Both deleted*1 and deleted*2 haplotypes contained the same number of TCRBV genes; both contain 60 genes of which 50 are functional, whereas, inserted haplotypes contained 63 genes of which 52 are functional. Comparisons of inserted region sequences with the homologous region in a deleted haplotype, and with sequences surrounding related TCRBV genes, revealed patterns of similarity that suggest insertion as well as deletion events have occurred in the evolution of the TCRBV gene complex. These data indicate that the genomic TCR repertoire is expanded in individuals who have inserted TCRBV haplotypes. The presence of additional TCRBV genes or, alternatively, the absence of certain TCRBV genes may have an impact upon immune responses and susceptibility to autoimmune diseases.

T cell receptor beta chain gene polymorphisms in Graves' disease.

Graves' disease, or subgroups of such patients with thyroglobulin antibodies or HLA-DR3, have been associated with a restriction fragment length polymorphism of the T cell receptor beta chain constant region gene but not all studies are in agreement. We have examined 58 patients with Graves' disease and found no abnormal distribution of this polymorphism compared to 33 controls. There was also no significant association with the presence of thyroglobulin antibodies or HLA-DR3, even when the results were pooled with previously collected data (N = 90 patients with TG antibody results and N = 128 patients with HLA-DR types). We also found no abnormal distribution of three polymorphisms of the T cell receptor beta chain variable region genes, using probes which span over 600 kb of this complex. These results argue against an association of these allelic variations of the T cell receptor beta chain with Graves' disease.

Characterization of T cell repertoire changes in acute Kawasaki disease.

Kawasaki disease (KD) is an acute multisystem vasculitis of unknown etiology that is associated with marked activation of T cells and monocyte/macrophages. Using a quantitative polymerase chain reaction (PCR) technique, we recently found that the acute phase of KD is associated with the expansion of T cells expressing the V beta 2 and V beta 8.1 gene segments. In the present work, we used a newly developed anti-V beta 2 monoclonal antibody (mAb) and studied a new group of KD patients to extend our previous PCR results. Immunofluorescence analysis confirmed that V beta 2-bearing T cells are selectively increased in patients with acute KD. The increase occurred primarily in the CD4 T cell subset. The percentages of V beta 2+ T cells as determined by mAb reactivity and flow cytometry correlated linearly with V beta expression as quantitated by PCR. However, T cells from acute KD patients appeared to express proportionately higher levels of V beta 2 transcripts per cell as compared with healthy controls or convalescent KD patients. Sequence analysis of T cell receptor beta chain genes of V beta 2 and V beta 8.1 expressing T cells from acute KD patients showed extensive junctional region diversity. These data showing polyclonal expansion of V beta 2+ and V beta 8+ T cells in acute KD provide additional insight into the immunopathogenesis of this disease.

T cell receptor (TCR) beta chain homodimers on the surface of immature but not mature alpha, gamma, delta chain deficient T cell lines.

Transfected T cell receptor (TCR) beta chain genes are expressed as homodimers on the surface of immature (Sci/ET27F) but not on mature (58 alpha-beta-) T cell lines which lack TCR alpha, gamma and delta chains. The homodimer on Sci/ET27F cells is tightly bound to CD3 delta and CD3 epsilon while the association with CD3 gamma and CD3 zeta proteins is rather weak. Crosslinking of the TCR beta homodimers resulted in a strong and rapid calcium flux. In 58 alpha-beta- T cells the beta TCR chain could be easily visualized intracellularly but was not transported to the cell surface. The Scid cell lines considerably facilitate the molecular analysis of early differentiation events in the thymus which are likely to be regulated by the beta TCR homodimer.

Lymphocyte subsets associated with T cell receptor beta-chain gene rearrangement in patients with rheumatoid arthritis and neutropenia.

To determine the incidence of a clonal lymphoid disease in patients with chronic rheumatoid arthritis (RA) and neutropenia. Lymphocytes from 23 RA patients with either current neutropenia or a history of this complication were studied. Eight patients had a clonal rearrangement of the T cell receptor beta-chain gene. Phenotypically, they showed a distinctive pattern characterized by an inverted CD4+:CD8+ cell ratio and an increased number and percentage of CD57+/CD8+ and CD3+/DR+ lymphocytes. None had evidence of a lymphoid malignancy. Among RA patients with neutropenia, there is a subset who have a subclinical disease resembling T gamma lymphoproliferative disease.

Differential lysis of tumor target cells displayed by lymphokine activated killer (LAK) cell clones.

Lymphokine-activated killer (LAK) cells exhibit major histocompatibility complex (MHC) unrestricted cytolysis against a wide variety of fresh and cultured tumor cells. Because previous work from our laboratory suggested that trypsin treatment of unseparated populations of LAK cells had a differential effect on lysis of different tumors, in this report we analyzed the lytic specificity of LAK cell clones against a panel of three different targets: MCA, B16 and YAC-1. We found that 21 out of the 24 analyzed murine spleen and bone marrow clones killed a combination of two, but not all three, of these tumor cells. Determinations of the phenotype of 10 LAK cell clones showed six with rearrangements for the T cell receptor (TCR) beta chain gene, suggesting a T cell origin, and four with germ line configurations for the TCR beta and delta chain genes, a result consistent with a non-T cell lineage. This cloning procedure provided an experimental tool to develop new procedures of adaptive immunotherapy.