Immature and advanced patterns of T cell receptor gene rearrangement among lymphocytes in splenic culture

Bulk populations and 39 hybridomas from splenic Con A cultures were analyzed for rearrangements among TCR genes: alpha, beta, gamma, and delta. Patterns were categorized to reveal general rules governing gene rearrangement within the activated adult peripheral population. Many patterns of gene rearrangement were consistent with previous studies of T cell lines. Additional points of interest were the following: 1) A large proportion of Con A-stimulated splenic cells bore no TCR gene rearrangements. 2) One splenic hybridoma exhibited an unusual gene pattern, with rearrangements, at alpha and beta, but not J gamma 1 or J gamma 2 loci. 3) Multiple gamma rearrangements were noted other than V1.2-J2 and V2-J1. 4) One hybridoma exhibited TCR gene rearrangements typical of day 14 to 15 fetal thymocytes, as well as rearrangements at immunoglobulin gene loci. 5) Among hybridomas with J alpha rearrangements, homologous chromosomes exhibited rearrangements at similar positions along the J alpha locus.     

Gamma delta T cell receptor analysis supports a role for HSP 70 selection of lymphocytes in multiple sclerosis lesions.

BACKGROUND: Interactions between gamma delta T cells and heat shock proteins (HSP) have been proposed as contributing factors in a number of diseases of possible autoimmune etiology but definitive evidence to support this hypothesis has been lacking. In multiple sclerosis (MS), a chronic inflammatory neurologic disease, HSP and gamma delta T cells are known to colocalize in brain lesions. Analysis of T cell receptor (TCR) gene usage in these lesions has detected evidence of clonality within both the V delta 2-J delta 1 and V delta 2-J delta 3 populations of gamma delta T cells. In our own studies, using direct sequence analysis, a dominant V delta 2-J delta 3 TCR sequence was found in 9 MS brain samples, suggesting a response to a common antigen. In this report, we have examined gamma delta T cell receptor gene usage in MS peripheral blood T cell lines selected for reactivity to HSP 70. MATERIALS AND METHODS: TCR rearrangement patterns for V delta 2-J delta 1 and V delta 2-J delta 3 were studied using the polymerase chain reaction (PCR) and a direct sequencing technique in populations of peripheral blood mononuclear cells (PBMC) cultured with Mycobacterium tuberculosis (M. tuberculosis) purified protein derivative (PPD) and then selected for reactivity to a 70-kD heat shock protein (HSP70). Cells were obtained from health donors, patients with MS, and patients with tuberculosis (TB). PCR products were subjected to direct sequence analysis to look for evidence for clonality within these T cell lines and to define the sequence of the V-D-J (CDR3) region of the TCR. RESULTS: In freshly isolated PBMC, both V delta 2-J delta 1 and V delta 2-J delta 3 gene rearrangement patterns were detected, whereas in HSP70+ T cell lines the predominant delta chain rearrangement pattern was V delta 2-J delta 3. Direct sequence analyses indicated that in cells reactive with HSP70 the V delta 2-J delta 3 sequences were usually oligoclonal and used D delta 3 exclusively. In four of four MS and two of three TB patients, the oligoclonal sequences in the HSP70+ T cell lines were identical to one another and to a dominant sequence previously detected in MS brain lesions. In two of three HSP70+ T cell lines from healthy controls, the oligoclonal sequences differed from those found in both groups of patients but were identical to one another except for a small region of heterogeneity in the second N region. In contrast, in freshly isolated PBMC or in PPD+HSP70- T cell lines, the V delta 2-J delta 3 gene rearrangement patterns were usually polyclonal and dominant sequences were rarely identified. CONCLUSIONS: These results support the conclusion that a subpopulation of gamma delta T cells in MS lesions are responding to HSP 70 and that non-CNS-specific antigens contribute to the pathogenesis of MS.     

Rearranged beta T cell receptor genes in a helper T cell clone specific for lysozyme: no correlation between V beta and MHC restriction

The helper T cell clone 3H.25 is specific for hen egg white lysozyme and the class II MHC molecule I-Ab. This TH cell has three rearrangements in the beta-chain gene family-a V beta-D beta-J beta 1 and a D beta 2-J beta 2 rearrangement on one homolog and a D beta 1-J beta 2 rearrangement on the other. These observations demonstrate that this functional T lymphocyte expresses only a single V beta gene segment and, accordingly, exhibits allelic exclusion of beta-chain gene expression. The rearranged 3H.25 V beta gene segment is the same as that expressed in a T helper cell specific for cytochrome c and an I-Ek MHC molecule. Thus, there is no simple correlation between the V beta gene segment and antigen specificity or MHC restriction.     

T cell receptor gamma gene status of human alpha/beta+ and gamma/delta+ T cell clones: absence of V9JP rearrangements in alpha/beta+ clones is not a result of a lack of rearrangements involving more 5' J gamma segments

T cell receptor (TCR) gamma gene rearrangements were examined in panels of human T cell clones expressing TCR alpha/beta or gamma/delta heterodimers. Over half of the alpha/beta+ clones had both chromosomes rearranged to C gamma 2 but this was the case for only 20% of the gamma/delta+ clones. While more than half of the gamma/delta+ clones showed a V9JP rearrangement, this configuration was absent from all 49 alpha/beta+ clones analysed. However, this was not a result of all rearrangements being to the more 3' J gamma genes as 11 alpha/beta+ clones had rearrangement(s) to JP1, the most 5' J gamma gene segment. Both alpha/beta+ and gamma/delta+ clones showed a similar pattern of V gamma gene usage in rearrangements to J gamma 1 or J gamma 2 with a lower proportion of the more 3' genes being rearranged to J gamma 2 than for the more 5' genes. Several alpha/beta+ and several gamma/delta+ clones had noncoordinate patterns of rearrangement involving both C gamma 1 and C gamma 2. Eleven out of fourteen CD8+ clones tested had both chromosomes rearranged to C gamma 2 whereas all clones derived from CD4-8- cells and having unconventional phenotypes (CD4-8- or CD4+8+) had at least one C gamma 1 rearrangement. Twelve out of twenty-seven CD4+ clones also had this pattern, suggesting that CD4-8+ clones had a tendency to utilize more 3' J gamma gene segments than CD4+ clones. There was some evidence for interdonor variation in the proportions of TCR gamma rearrangements to C gamma 1 or C gamma 2 in alpha/beta+ clones as well as gamma/delta+ clones. The results illustrate the unique nature of the V9JP rearrangement in gamma/delta+ clones and the possible use of a sequential mechanism of TCR gamma gene rearrangements during T cell differentiation is discussed.     

Identification of distinct T cell receptor (TCR)-gamma delta heterodimers using an anti-TCR-gamma variable region serum

T cell hybridomas were generated from CD3+, CD4-, CD8- splenocytes and fetal thymocytes. V gamma 1-expressing proteins present on these murine TCR-gamma delta hybridomas were identified by using an anti-TCR V gamma 1 peptide serum. This antiserum specifically immunoprecipitated 41-kDa TCR V gamma-C gamma 4 chains and 31-kDa TCR V gamma-C gamma 1/2 chains from distinct heterodimers expressed on the TCR-gamma delta T cell hybridomas. The RNA from a hybridoma with a 31-kDa TCR-gamma chain hybridized with a V gamma 1 probe but failed to hybridize with a V gamma 2 probe. In contrast, the RNA from a hybridoma with a 32-kDa TCR-gamma chain hybridized with a V gamma 2 probe. This 32-kDa TCR-gamma chain was not immunoprecipitated by the anti-V gamma 1 serum. These data were consistent with the conclusion that the 31-kDa protein was the product of a V gamma 1 to C gamma 2 rearrangement, whereas the 32-kDa protein was the product of a V gamma 2 to C gamma 1 rearrangement. Furthermore, Southern analyses confirmed that the 32-kDa protein was the product of a V gamma 1.2-J gamma 2 rearrangement, and all three of the 41-kDa TCR-gamma chains were the results of V gamma 1.1-J gamma 4 rearrangements. This was the first demonstration at the clonal level of TCR-gamma proteins which use members of the V gamma 1 gene family, as well as the C gamma 2 constant region. Additional biochemical analyses of the TCR-gamma and -delta proteins from three independently derived C gamma 4-bearing T cell hybridomas suggested that most of the molecular mass diversity observed in the bulk subpopulation of peripheral C gamma 4-containing heterodimers may be contributed by the TCR-delta chains.     

V gamma 3 T cell receptor rearrangement and expression in the adult thymus.

Rearrangement and expression of the V gamma 3-J gamma 1 TCR has been found in murine dendritic epidermal cells (DEC) and fetal thymus. By using the polymerase chain reaction technique, V gamma 3-J gamma 1 rearrangements and RNA expression were detected in the murine adult thymus. Individual genomic and cDNA junctions were cloned and sequenced. In genomic DNA, 55% (16/29) of V gamma 3-J gamma 1 junctional sequences had N regions ranging in length from 1 to 12 nucleotides resulting in considerable junctional diversity. Only 5% (2/42) of cDNA sequences had N regions. The canonical DEC sequence represented 36% (15/42) of the cDNA sequences. Thus, fetal-type V gamma 3-J gamma 1 rearrangements lacking N regions were preferentially expressed in adult thymocytes, some of which may be DEC precursors. The developmental stages in which V gamma 3-J gamma 1 rearrangements are generated were studied by using polymerase chain reaction to detect circular rearrangement products. Active V gamma 3-J gamma 1 rearrangement was detected in thymuses from fetal, newborn, and 2-wk-old mice but not in 5-wk or 8-wk-old (adult) mice. V gamma 2, one of the most common V gamma rearrangements in the adult, was found to be actively rearranging to J gamma 1 in the adult thymus. However, V gamma 2-V gamma 3 replacement rearrangement was not found. These results support the hypotheses that adult thymocytes with rearranged V gamma 3-J gamma 1 are persistent from earlier developmental stages and represent a separate lineage from those with V gamma 2-J gamma 1 rearrangements.     

Human T cell gamma-chain gene rearrangements in acute lymphoid and nonlymphoid leukemia: comparison with the T cell receptor beta-chain gene

Rearrangement of germ-line genes coding for T and B cell antigen receptor molecules is an early event in lymphoid development which eventually leads to the generation of clonal diversity in receptor-positive lymphocytes. Three T cell-associated rearranging genes have been described. Two, T alpha and T beta, code for the two polypeptide chains that form the T cell receptor heterodimer. The function of the third gene, the gamma-gene (T gamma), is not known. To learn more about the behavior of T gamma during lymphoid ontogeny, we compared rearrangement of T gamma and T beta genes in leukemic cells arrested at varied stages of lymphoid and myeloid development. We analyzed 38 fresh cell lines and 15 established cell lines from a total of 53 leukemic patients. Cells were immunophenotyped with a panel of monoclonal antibodies recognizing T-, B-, or myeloid-associated surface markers. Sixteen T-lineage cases were studied; 15 displayed both T beta and T gamma rearrangements. The exception (germ-line for T beta and T gamma) was an immature CD2(T11)+, CD3(T3)-, CD7(3A1)+, CD1(T6)+, CD5(T101)+ phenotype. Fourteen non-T non-B leukemias were analyzed; eight were germ-line for both T beta and T gamma, four had rearrangements involving both T beta and T gamma, and two were germ-line for T beta and rearranged to T gamma. Four cases with acute biphenotypic leukemia were studied; two had rearrangements of T beta and T gamma, and two were germ-line for both genes. Cells from nonlymphocytic leukemias were studied in 19 cases. All were found to be germ-line for both T beta and T gamma. Fifty-one of 53 genomic DNA samples were concordant for T gamma and T beta rearrangement. These results indicate that rearrangement of T gamma can occur in leukemic cells of B cell as well as T cell precursor origin, as has been reported previously for T beta.     

Preferential use of a T cell receptor V beta gene by acetylcholine receptor reactive T cells from myasthenia gravis-susceptible mice.

Experimental autoimmune myasthenia gravis (EAMG) is an important model for testing current concepts in autoimmunity and novel immunotherapies for autoimmune diseases. The EAMG autoantigen, acethylcholine receptor (AChR), is structurally and immunologically complex, a potential obstacle to the application of therapeutic strategies aimed at oligoclonal T cell populations. Inasmuch as we had previously shown that the clonal heterogeneity of T cell epitope recognition in EAMG was unexpectedly limited, we examined TCR V beta expression. AChR primed lymph node T cells and established AChR reactive T cell clones from EAMG-susceptible C57BL/6 (B6; H-2b, Mls-1b) mice showed preferential utilization of the TCR V beta 6 segment of the TCR. After in vivo priming and in vitro restimulation for 7 days with AChR or a synthetic peptide bearing an immunodominant epitope, V beta 6 expressing lymph node cells (LNC) were expanded several-fold, accounting for up to 75% of recovered viable CD4+ cells. The LNC of B6.C-H-2bm12 (bm12; H-2bm12, Mls-1b) mice, which proliferated in response to AChR but not to the B6 immunodominant peptide, failed to expand V beta 6+ cells. Inasmuch as nonimmune bm12 and B6 animals had similar numbers of V beta 6+ LNC (4-5%), this suggested that structural requirements for TCR recognition of Ag/MHC complexes dictated V beta usage. Results concerning peptide reactivity and V beta 6 expression among T cells from (B6 x bm12)F1 animals also suggested that structure-function relationships, rather than negative selection or tolerance, accounted for the strain differences between B6 and bm12. To examine the potential effects of thymic negative selection of V beta 6+ cells on the T cell response to AChR, CB6F1 (H-2bxd, Mls-1b; V beta 6-expressing) and B6D2F1 (H-2bxd, Mls-1axb; V beta 6-deleting) strains were analyzed for AChR and peptide reactivity and V beta 6 expression. Both F1 strains responded well to AChR but the response of B6D2F1 mice to peptide was significantly reduced compared to CB6F1. Short and long term cultures of peptide-reactive B6D2F1 LNC showed no expansion of residual V beta 6+ cells, although similar cultures of CB6F1 LNC were composed of more than 60% V beta 6+ cells. The results from the F1 strains further indicated that the T cell repertoire for peptide was highly constrained and that non-V beta 6 expressing cells could only partially overcome Mls-mediated negative selection of V beta 6+ TCR capable of recognizing peptide.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence that the intervening sequence was excised as a linear molecule during D beta-J beta rearrangement in T-cell receptor beta chain gene loci

In a previous paper (Proc. Natl. Acad. Sci. USA 84: 4264, 1987) we reported an unusual DNA rearrangement in T-cell receptor beta chain gene loci in cells from a patient with human T-cell leukemia. A D beta 1-J beta 2.3 junction was found on one chromosome, while the other chromosome kept the germline configuration. Although the DNA fragment located between the D beta 1 and J beta 2.3 loci should have disappeared from the cells, it was found on chromosome 6 as an inserted segment. We have now determined the nucleotide sequences bordering both sides of the inserted segment. The signal sequence for D beta-J beta rearrangement at the 5' side of J beta 1.2 gene seems to have been used for the insertion. The 3' end of the inserted segment corresponded to the edge of the signal heptamer at the 5' side of J beta 2.3 which was used for the initial D beta 1-J beta 2.3 joining. This indicates that, during D beta-J beta rearrangement, the intervening sequence was excised as a linear molecule.     

Monte Carlo simulations of voltage-driven translocation of a signal sequence

CD1d-deficient (CD1d-/-) mouse lymphocytes were analyzed to classify the natural killer T (NKT) cells without reactivity to CD1d. The cells bearing a V(alpha)19.1-J(alpha)26 (AV19-AJ33) invariant TCR alpha chain, originally found in the peripheral blood lymphocytes, were demonstrated to be abundant in the NK1.1+ but not NK1.1- T cell population isolated from CD1d-/- mice. Moreover, more than half (11/21) of the hybrid cell lines established from CD1d-/- NKT cells expressed the V(alpha)19.1-J(alpha)26 invariant TCR alpha chain. The expression of the invariant V(alpha)19.1-J(alpha)26 mRNA was absent in beta2-microglobulin-deficient mice. Collectively, the present findings suggest the presence of a second NKT cell repertoire characterized by an invariant TCR alpha chain (V(alpha)19.1-J(alpha)26) that is selected by an MHC class I-like molecule other than CD1d.     

Cytokine network regulating terminal maturation of human bone marrow B cells capable of spontaneous and high rate Ig secretion in vitro.

Human bone marrow (BM) B cells capable of spontaneous and high rate Ig secretion for 14 days in vitro have been described previously. We have shown recently that Ig secretion by these BM cells depends on stromal adherent BM cell-derived factors identified as IL-6 and fibronectin. Our report shows that the endogenous generation of IL-1 beta and TNF-alpha in serum-containing cultures of BM mononuclear cells (BMMC) is also involved in the control of Ig-secreting cells, because their blockade with specific antibodies markedly reduced Ig production. Further experiments revealed that IL-1 beta and TNF-alpha acted by regulating IL-6 production, as can be deduced from the following findings: 1) the inhibition of Ig secretion caused by either anti-IL-1 beta or anti-TNF-alpha antibodies could be reversed by exogenous IL-6; 2) the addition of either of these antibodies inhibited endogenous IL-6 production in BMMC cultures; 3) IL-1 beta plus TNF-alpha, but neither one alone, restored complete IL-6 and Ig production by BMMC in serum-free cultures. Moreover, adherent, but not nonadherent, BM cells were responsible for endogenous IL-1 beta and TNF-alpha secretion. Finally, IL-1 beta plus TNF-alpha induced the production of IL-6, but not of Ig, by adherent BM cells. Neither IL-6 nor Ig production was induced by adding this cytokine combination to nonadherent BM cell cultures, despite the fact that this fraction contained all the Ig-secreting cells. However, the addition of IL-6 restored Ig secretion in this cell fraction. These results suggest that IL-1 beta and TNF-alpha produced by adherent BM cells synergistically induce early IL-6 generation, which, in turn, drives BM B cell producers into the high rate Ig-secreting state.     

CD3+4-8- alpha beta T cell population with biased T cell receptor V gene usage. Presence in bone marrow and possible involvement of IL-3 for their extrathymic development.

Analysis of TCR of a series of CD4-8- (double negative; DN) alpha beta T cell lines induced with IL-3 revealed that their V gene usage was biased for V alpha 4 and V beta 2. This has been confirmed in the primary short-term cultures. Thus, IL-3 induced the generation of DN alpha beta T cells with predominant V beta 2 gene expression from the CD4+/CD8+ T cell-depleted spleen or bone marrow (BM) cells of both normal and nude BALB/c mice within 10 days. It was further indicated that the V beta 2+ beta-chain genes contained few junctional N regions in both IL-3-induced primary DN alpha beta T cells and continuous lines. Search for the in vivo counterpart of in vitro IL-3-induced DN alpha beta T cells revealed that BM, but not spleens, of normal BALB/c and B6 mice did contain a significant proportion of DN alpha beta T cells, and that the majority of them expressed V beta 2+ beta-chain genes with few junctional N regions. The presence of V beta 2+ DN alpha beta T cells was similarly observed in the BM of BALB/c nude mice, but their proportion varied markedly among various strains of mice, which was not linked to H-2 haplotypes. The results indicated that V beta 2+ DN alpha beta T cells in the BM represented one of the thymus-independent T cell populations, whose development was under the major histocompatibility Ag complex-unlinked genetic control. TCR of these T cells were shown to be functional as judged by the proliferative response to anti-V beta 2 antibody. Taken together, present results suggested that IL-3 could induce differentiation and/or proliferation of DN alpha beta T cells with uniquely limited repertoire, which existed preferentially in BM in vivo, and implied the possible involvement of extrathymic endogenous ligands as a positive selection force.     

Expansion of large granular lymphocytes in IL-2-driven 14-day-old fetal thymocytes in organ culture

These experiments were designed to evaluate the role of cytokines in early T cell development within the thymus. By using a thymic organ culture model, we have studied the influence of high dose of IL-2 (10 to 1000 IU/ml) on the cell populations that are generated during 12 days starting from a thymic rudiment of 14-day-old mouse embryo. The IL-2 treatment resulted in the expansion of Thy-1+/-, CD4-, CD8-, CD3-, Fc gamma RII+, CD5 (Lyt-1)-, HSA-, Pgp- 1+, Mel-14- population. These cells had the morphology of large granular lymphocytes and displayed broad cytotoxic activity. In addition, IL-2-treated organ cultures had a dramatic decrease in CD4+CD8+ thymocytes, a marked reduction in TCR-alpha beta+ thymocytes--even more pronounced in the TCR-V beta 6+ and TCR-V beta 8+ thymocytes--and no significant changes in the number of TCR-gamma delta+ as compared to control organ cultures.     

The repertoire of killer cell Ig-like receptor and CD94:NKG2A receptors in T cells: clones sharing identical alpha beta TCR rearrangement express highly diverse killer cell Ig-like receptor patterns

Killer cell Ig-like receptor (KIR) and CD94:NKG2A molecules were first defined as human NK cell receptors (NKR), but now are known to be expressed and to function on subpopulations of T cells. Here the repertoires of KIR and CD94:NKG2A expression by T cells from two donors were examined and compared with their previously defined NK cell repertoires. T cell clones generated from peripheral blood of both donors expressed multiple NKR in different combinations and used the range of receptors expressed by NK cells. In both donors alpha beta T cells less frequently expressed the inhibitory receptors CD94:NKG2A and KIR2DL1 than either gamma delta T cells or NK cells. In contrast to NK cells, not all NKR(+) T cells expressed an inhibitory receptor for autologous HLA class I. This lack of specific inhibitory NKR was especially apparent on alpha beta T cells of one donor. Overall, alpha beta T cells exhibited a distinct pattern of NKR expression different from that of gamma delta T and NK cells, which expressed highly similar NKR repertoires. In one donor, analysis of TCR rearrangement revealed a dominant subset of NKR(+) T cells sharing identical TCR alpha- and beta-chains. Remarkably, among 55 T cell clones sharing the same TCR alpha beta rearrangement 18 different KIR phenotypes were seen, suggesting that KIR expression was initiated subsequently to TCR rearrangement.     

Development of T cells in vitro from precursors in mouse bone marrow

Bone marrow cells from 6- to 8-week-old athymic nude mice were depleted of nylon-wool adherent cells and cultured in vitro at low cell numbers (300 cells/well) in medium supplemented with a supernatant from a thymoma cell line. About 1% of cultured cells grew. Pooled cultures contained cells expressing CD3 (52%), CD4 (37%), CD8 (11%), Thy 1.2 (72%), MAC-1 (43%) and J11d (86%) but no cells expressing sIg. They also contained cells expressing mRNA for the alpha, beta, gamma, and delta chains of the T cell receptor as assessed with C region probes using a sensitive dot blot assay. These cells appear to develop from progenitors which are CD3-. When pooled Day 10 cultures were depleted of nylon-wool adherent cells, the remaining cells were nearly all J11d+, Thy 1.2+, MAC-1-, CD3+, and either CD4+CD8+; CD4+CD8-; CD4-CD8+, or CD4-CD8-; i.e., their surface marker patterns were reminiscent of those of thymocytes. We conclude that our culture system is enabling bone marrow precursors to commence differentiation down the T cell lineage in the absence of a thymic environment.