Delta Repertoire Research Articles

3D structures inferred from cDNA clones identify the CD1D-Restricted γδ T cell receptor in dromedaries.

The Camelidae species occupy an important immunological niche within the humoral as well as cell mediated immune response. Although recent studies have highlighted that the somatic hypermutation (SHM) shapes the T cell receptor gamma (TRG) and delta (TRD) repertoire in Camelus dromedarius, it is still unclear how γδ T cells use the TRG/TRD receptors and their respective variable V-GAMMA and V-DELTA domains to recognize antigen in an antibody-like fashion. Here we report about 3D structural analyses of the human and dromedary γδ T cell receptor. First, we have estimated the interaction energies at the interface within the human crystallized paired TRG/TRD chains and quantified interaction energies within the same human TRG/TRD chains in complex with the CD1D, an RPI-MH1-LIKE antigen presenting glycoprotein. Then, we used the human TRG/TRD-CD1D complex as template for the 3D structure of the dromedary TRG/TRD-CD1D complex and for guiding the 3D human/dromedary comparative analysis. The choice of mutated TRG alternatively combined with mutated TRD cDNA clones originating from the spleen of one single dromedary was crucial to quantify the strength of the interactions at the protein-protein interface between the paired C. dromedarius TRG and TRD V-domains and between the C. dromedarius TRG/TRD V-domains and CD1D G-domains. Interacting amino acids located in the V-domain Complementarity Determining Regions (CDR) and Framework Regions (FR) according to the IMGT unique numbering for V-domains were identified. The resulting 3D dromedary TRG V-GAMMA combined with TRD V-DELTA protein complexes allowed to deduce the most stable gamma/delta chains pairings and to propose a candidate CD1D-restricted γδ T cell receptor complex.

Prenatal development of the porcine TCR delta repertoire: dominant expression of an invariant T cell receptor Vdelta3-Jdelta3 chain.

The prenatal development of the porcine gamma/delta TCR repertoire was studied by complementarity-determining region 3 (CDR3) spectratyping and sequencing of TRDV1-DV5 transcripts. Specimens from the small and large intestine, spleen, thymus, liver, bone marrow and PBMC from fetal piglets between 38 and 114 days of gestation (DG) were examined. The TCR delta repertoire was highly restricted early in gestation (DG38-DG57) and an invariant TRDV3 transcript, lacking the N/D region, was found in different fetuses throughout gestation and dominated the TRDV3 repertoires of all organs at mid gestation ( approximately DG55). Near the end of gestation, this invariant TRDV3 transcript was absent from the thymus but was still present, in a less dominant manner, in the intestine and spleen. The average CDR3 length of all Vdelta subgroups increased with ontogeny, suggesting an increase in activity of TdT. Thus, the persistence of fetal gamma/delta T cells expressing an invariant TRDV3 chain throughout development is especially surprising since TdT is active early in gestation in swine. We speculate that these gamma/delta T cells might have been selectively expanded by (self)-ligands and may have an important function throughout fetal development.

Compartmentalization g/d T Cells and Their Putative Role in Mucosal Immunity

gamma/delta T cells are an enigmatic group of cells and their functions still remain unknown. The epithelial-associated gamma/delta T cells, which are abundant at mucosal surfaces, are ideally situated to contribute to the initial stages of the immune response. Recent evidence suggests that they recognize stress-induced self-antigens which would enable a homogeneous population of gamma/delta T cells to monitor multiple insults to the epithelium. This could explain the observed oligoclonality and homogeneous distribution of cells carrying identical TCR within mucosal surfaces. However, the analysis of the TCR delta repertoire from different mucosal surfaces indicated that gamma/delta T cells are highly compartmentalized. Thus, gamma/delta T cells are not one homogeneous group of cells which recognize the same (stress-induced) self-antigens, but consist of different subsets that are likely to have distinct functions. It is possible that gamma/delta T cells interact with antigens that are specific for that organ or recognize foreign antigens which are limited to that site. In addition it was shown that gamma/delta T cells can have opposite functions and be proinflammatory or promote epithelial healing. This review focuses on the distribution and repertoire of mucosal gamma/delta T cells and discusses what is currently known about the functions of these cells. Furthermore, their potential role in inflammatory bowel disease is examined.

Distinct TCR delta repertoires are present in the cutaneous lesions and inflamed duodenum of patients with dermatitis herpetiformis.

Intraepithelial gammadelta T cells are increased in the inflamed small bowel and are also found in increased numbers in cutaneous lesions from patients with dermatitis herpetiformis (DH). Thus, these cells might play an important role in the pathogenesis of the disease. We investigated the T-cell receptor (TCR) delta repertoire in involved and non-involved skin and compared it with the TCR delta repertoire of the inflamed duodenum and peripheral blood of the same patients. An identical TCR delta repertoire in the small bowel and in the cutaneous lesions would suggest a migration of antigen-specific gammadelta T cells from the intestine to the skin which cross-react with cutaneous antigens. T-cell receptor DV1-DV3 transcripts were amplified by reverse transcriptase (RT)-PCR and analyzed by complementarity determining region 3 (CDR3) size spectratyping and nucleotide sequencing. Our results indicate that the cutaneous TCR delta repertoires were oligoclonal and identical dominant gammadelta T-cell clones were present in the involved and non-involved skin. Furthermore, the TCR delta repertoire of the skin was distinct from that in the small bowel. The peripheral blood exhibited a restricted TCR delta repertoire, which differed from that in the intestine and skin. Thus, cutaneous gammadelta T cells are not specifically expanded within the involved skin and are unlikely to be derived from the inflamed duodenum.

V delta repertoire during thymic ontogeny suggests three novel waves of gamma delta TCR expression.

Taking advantage of a PCR technique that allows amplification of all variable region genes with equal efficiency, we defined three novel waves of TCR delta-chain transcription during thymic ontogeny. The canonical DV101-D2-J2 rearrangement was confined to a narrow window from days 14 to 18 of gestation, indicating that the postulated two consecutive gamma delta precursor waves bearing this canonical DV101 rearrangement will coincide on day 16. Neonatal delta-chain transcripts used a second wave of diverse V alpha gene segments that are exclusively located in the delta locus-proximal gene cluster of intermingled single members of different V alpha subfamilies. In the adult, only expression of a clan of three homologous subfamilies, ADV7, DV104, and ADV17, persists. The members of the ADV7 subfamily are also scattered across the alpha locus, but their usage does not show the position-dependent bias of the other V alpha-to-delta rearrangements.

Expression of the mucosal γδ T cell receptor V region repertoire in patients with IgA nephropathy

IgA nephropathy (IgAN) is characterized by the deposition of IgA in the glomerular mesangium and often leads to progressive renal dysfunction and kidney failure. We have previously shown that the mesangial IgA is likely to derive from the bone marrow plasma cells, and suggested that a primary abnormality within the mucosal immune system may underly the pathogenesis of IgAN. This study has analyzed the T cell receptor (TCR) variable (V) region expression by gamma delta T cells in the intestinal mucosa of patients with IgAN. The V gamma and V delta usage of TCR transcripts was determined using a semiquantitative reverse transcriptase-PCR protocol. Primers specific for the four human V gamma and six V delta subfamilies were used each with a constant (C) gamma or C delta specific primer, and the PCR-amplified TCR transcripts were detected by Southern blotting and oligonucleotide hybridization. gamma delta TCR V region expression was determined in gut biopsies and peripheral blood of 11 patients with IgAN, and the TCR V gamma and V delta repertoires were compared to those in gut and peripheral blood of 11 control individuals. gamma delta T cells in normal blood predominantly expressed V gamma 2 (V gamma 9 gene) and V delta 2 gene segments whereas those in normal gut mainly expressed V gamma 3 and V delta 3. In IgAN patients, V delta 2 was also the predominant V delta gene utilized by peripheral blood gamma delta T cells, however, we observed a predominance of V gamma 3 and reduced V gamma 2 usage by these cells. gamma delta T cells in the gut of IgAN patients mainly used V gamma 3 and V delta 1. While the gamma and delta TCR V region repertoires did not differ significantly between the peripheral blood of patients and controls, there were significant differences in V gamma and V delta repertoire expression between IgAN and control gut biopsies. V gamma 3 gene expression was significantly decreased in IgAN gut compared to control gut (P = 0.023). In addition, there was a significant decrease in V delta 3 gene expression in IgAN gut compared to control gut (P = 0.043). These findings indicate that a subpopulation of gamma delta T cells, which represent the majority of gamma delta T cells in normal gut mucosa, are significantly diminished in the gut of patients with IgAN. This suggests that a "hole" in the mucosal gamma delta T cell repertoire may play a fundamental role in contributing to the pathogenesis of IgAN.

Characterisation of gamma delta T cells in renal cell carcinoma patients by polymerase chain reaction analysis of T cell receptor transcripts.

Renal cell carcinomas (RCC) contain tumour infiltrating lymphocytes (TIL) but these are essentially immunosuppressed in that they do not generate effective antitumour immune responses in vivo. These TIL comprise predominantly alpha beta T cells, although gamma delta T cells are also present. The repertoire of gamma delta T cells in RCC, however, has not been fully investigated. To identify the gamma delta T cell populations infiltrating RCC, this study has characterised the gamma delta T cell receptor (TCR) repertoire expression in these tumours and compared this to autologous normal kidney and autologous peripheral blood. A semi-quantitative reverse transcriptase/polymerase chain reaction technique was used for amplification of rearranged TCR V-C mRNA transcripts. Primers specific for the four human TCR V gamma and six V delta subfamilies were used, each in conjunction with a primer specific for either the C gamma or C delta region. The specificity of the PCR products was confirmed by Southern blotting and hybridisation with an internal C region probe. A densitometry score was assigned to each DNA band and the level of V gene expression was determined as ratio of C delta gene expression. The gamma delta TCR expression in each sample was determined as a ratio of C delta: glyceraldehyde phosphate dehydrogenase densitometry score. This demonstrated that TCR C delta gene expression was significantly higher in RCC compared to normal kidney (P < 0.019), suggesting a selective infiltration of gamma delta T cells into the tumour. Furthermore, we observed differences in the TCR V gamma and V delta repertoires between RCC and peripheral blood. V gamma l expression was significantly decreased (P < 0.043) whereas there was an over-representation of V gamma 4 transcripts (P < 0.028) in RCC compared to blood. A significant reduction in expression of both V delta 1 (P < 0.028) and V delta 3 (P < 0.051) was also observed within kidney tumour compared to peripheral blood. These findings show that expression of the gamma delta TCR repertoire in RCC differs from that in peripheral blood and normal kidney.

Restricted T cell receptor delta chain genes repertoire in peripheral blood of multiple sclerosis patients

Multiple sclerosis (MS) is a chronic inflammatory disease characterized by focal demyelination of central nervous system (CNS). Susceptibility to MS is thought to be affected by multiple genes including HLA and T cell receptor (TCR) genes. In view of the recent evidence, that in addition to α/β T lymphocytes also γ/δ T cells may have autoreactive potential, TCR delta repertoire in peripheral blood of MS patients has been studied. TCR delta repertoire, as assessed by Vδ‐Jδ rearrangements, has been analysed in 13 MS cases and in 30 healthy individuals by seminested PCR technique. Oligonucleotide primers specific for six Vδ regions and for Jδ1 gene were used for amplification of Vδ‐Jδ junctional region responsible for the diversity of γ/δ TCR. In the majority of MS patients PAGE analysis of Vδ1‐Jδ1, Vδ3‐Jδ1 and Vδ5‐Jδ1 rearrangements showed single‐band or two‐band pattern. The most striking result has been observed in Vδ5‐Jδ1 rearrangement, where in nine cases studied single band and in four patients two bands have been found. In all but one MS cases multi‐band pattern of Vδ2‐Jδ1 rearrangement was obtained. None of the 13 MS patients showed single‐band rearrangement pattern of Vδ4‐Jδ1 and Vδ6‐Jδl. Contrary to the MS group almost all healthy individuals produced smear‐like or multi‐band pattern of Vδ1‐Vδ5 to Jδ1 rearrangements. On the basis of the banding pattern produced by Vδ‐Jδ rearrangement in MS, it can be suggested that T lymphocytes had undergone clonal expansion in vivo, most likely due to stimulation by antigen related to CNS. In particular a very consistent single‐band pattern of Vδ5‐Jδ1 rearrangement observed in almost all MS patients studied, argues very strongly for a significant role of γ/δ T cells with Vδ5 rearrangement in the pathogenesis of MS. However, it cannot be excluded that the observed patterns of TCR δ gene rearrangement in MS patients may represent secondary changes to CNS damage.

T cell receptor γδ repertoire is skewed in cerebrospinal fluid of multiple sclerosis patients: molecular and functional analyses of antigen‐reactive γδ clones

To study the relevance of gamma delta T cells in multiple sclerosis (MS) we analyzed the T cell receptor (TCR) gamma delta repertoire and the antigen reactivity of gamma delta clones isolated from cerebrospinal fluid (CSF). In T cell cultures derived from CSF we found an increased percentage of V delta 1+ cells as compared to peripheral blood of the same donors. Phenotypic analysis of cells from MS CSF with V gamma- and V delta-specific monoclonal antibodies (mAb) showed that the V delta 1 chain is most frequently associated with gamma chains belonging to the V gamma 1 family. Sequence analysis of TCR genes revealed heterogeneity of junctional regions in both delta and gamma genes indicating polyclonal expansion. gamma delta clones were established and some recognized glioblastoma, astrocytoma or monocytic cell lines. Stimulation with these targets induced serine esterase release and lymphokine expression characteristic of the TH0-like phenotype. Remarkably, these tumor-reactive gamma delta cells were not detected in the peripheral blood using PCR oligotyping, but were found in other CSF lines independently established from the same MS patient. Altogether, these results demonstrate that in the CSF there is a skewed TCR gamma delta repertoire and suggest that gamma delta cells reacting against brain-derived antigens might have been locally expanded.

T cell receptor gamma delta repertoire in HIV-1-infected individuals.

While V gamma 9/V delta 2 cells dominate among peripheral blood gamma delta T cells in healthy adults, the majority of gamma delta T cells in most HIV-1-infected individuals express V delta 1. We asked whether these elevated levels of V delta 1 T cells were due to clonal expansion. Three-color flow cytometry with monoclonal antibodies against V gamma 2/V gamma 3/V gamma 4, V gamma 4 and V gamma 9 was used to investigate V gamma usage in 27 patients with elevated numbers of V delta 1 T cells. While the relative proportion of V gamma 9 cells among gamma delta T cells was significantly reduced in HIV-1+ individuals (10 +/- 11% vs. 80 +/- 17%, p < 0.001), the fraction of gamma delta T cells using V gamma 5 or V gamma 8 was significantly increased (54 +/- 15% vs. 7 +/- 11%, p < 0.001). In 1 patient, 76% of the V delta 1 cells expressed V gamma 2 or V gamma 3, suggesting clonality of the V delta 1 population. In line with this assumption, analysis of the V delta 1-J delta junctional regions by reverse transcription-polymerase chain reaction (RT-PCR) resulted in products of only one junctional length, as demonstrated by electrophoresis on denaturing gels, and 12 out of 16 (75%) in-frame junctional sequences were identical in this patient. In other HIV-1+ patients, RT-PCR resulted in products of several distinct sizes, also indicating a highly restricted repertoire. After sequencing the V delta 1-J delta junctional regions of 3 additional patients, we found repeated but patient-specific in-frame junctions accounting for 10-30% of the sequenced clones. However, limited V delta 1-J delta junctional diversity was also seen in healthy donors. RT-PCR products from 10 healthy individuals resulted in distinct bands on denaturing gels. In 1 of them exhibiting a single prominent band, 10 out of 17 (58%) sequenced junctions were identical. Two other healthy donors displayed 2/14 and 5/18 identical junctional sequences, respectively. Taken together, our results reveal significant alterations of V gamma usage in HIV-1+ patients, while the V delta 1 junctional repertoire is similarly restricted in HIV-1+ and HIV-1- individuals. Therefore, these data argue against an obligatory clonal expansion of V delta 1-expressing cells during HIV-1 infection.

Peripheral T cell receptor gamma delta variable gene repertoire maps to the T cell receptor loci and is influenced by positive selection.

Although the mechanisms that determine TCR-alpha beta V gene repertoire are well studied, the genetic influences involved in TCR-gamma delta repertoire development are unclear. Unlike the TCR-gamma delta populations that localize in epithelial tissues, the circulating peripheral TCR-gamma delta V region repertoire is quite diverse. Previous studies have shown that three TCR-gamma chains and at least six TCR-V delta genes are expressed by splenic TCR-gamma delta cells. However, the relative frequency of individual gamma delta subsets among genetically diverse mice has not been determined. Therefore, the repertoire of TCR-gamma delta cells was examined using anti-TCR V region specific mAb against V gamma 2 and V delta 4 on TCR-gamma delta + cells from total splenocytes. We found that there was a strain-specific variation in TCR-gamma delta usage. The frequency of V gamma 2 expression in different strains varied from 54 to 12%, and the frequency of V delta 4 expression in different strains varied from 38 to 10%. However, the level of V delta 4 and V gamma 2 expression for an individual strain was highly consistent from experiment to experiment. F1 analysis between parental strains that differed in relative frequency of either V gamma 2+ or V delta 4+ cells revealed that high expression was genetically dominant, suggesting that positive selection events play a major role in the peripheral gamma delta repertoire. Variations in the levels of V gamma 2+ cells and V delta 4+ cells was not associated with Mls or MHC haplotype. Analysis of recombinant inbred strains revealed that high V delta 4 expression mapped to the TCR-gamma locus, while high V gamma 2 expression was influenced by the TCR-delta locus. Back-cross analysis confirmed that the TCR loci dominantly influenced the level of V delta 4+ cells and V gamma 2+ cells; however, there was clear evidence that multiple genes affect the TCR-gamma delta repertoire.

Characterization of gamma delta T lymphocytes at the maternal-fetal interface.

A specific subset of gamma delta T lymphocytes bearing a V gamma 6V delta 1-encoded TCR is known to populate the normal nonpregnant murine uterine and vaginal epithelium. However, gamma delta T lymphocytes residing at the maternal-fetal interface during pregnancy have not yet been investigated. Using mAb and cytofluorographic analysis, we analyzed gamma delta TCR-bearing lymphocytes obtained from placental/decidual tissues of allogeneic (C57B1/10 X BALB/c and BALB/c X C57B1/10) pregnancies. Gestations were analyzed at several time points during the second half of pregnancy, with lymphocytes from both maternal spleen and nonpregnant C57B1/10 uteri analyzed as controls. We found that all gamma delta T lymphocytes at the maternal-fetal interface are maternally derived. Relative to the total T lymphocyte population, the percentage of gamma delta TCR-bearing T lymphocytes at the maternal-fetal interface is enriched three- to four-fold compared with maternal spleen, and twofold compared with nonpregnant uteri. Although one-third of gamma delta T lymphocytes from pregnant animals express a cell-surface marker associated with activation (IL-2R), gamma delta cells from uteri of nonpregnant mice fail to express IL-2R. In terms of absolute numbers, we estimate that reproductive-tract gamma delta T cells are increased nearly 100-fold in pregnant animals compared with nonpregnant animals. To characterize the TCR-gamma delta repertoire in the placenta/decidua, we generated 21 TCR-gamma delta-bearing hybridomas from lymphocytes in this tissue. Analysis of these hybridomas revealed at least six distinct gamma delta receptor types expressed at the maternal-fetal interface, with V gamma 6V delta 1 encoded TCR representing the predominant population. As specific resident constituents of the reproductive tract, gamma delta T lymphocytes may be involved in regulating a variety of physiologic and pathophysiologic events in reproductive biology.

Developmental regulation of D beta reading frame and junctional diversity in T cell receptor-beta transcripts from human thymus.

During fetal life, both Ig and TCR-gamma delta repertoires are enriched for a specific set of Ag receptors with limited diversity. In order to test the hypothesis that diversification of the human TCR-beta repertoire also follows a developmental program, we examined TCR-beta DJ and VDJ transcripts from fetal and adult thymi. A consistent bias for D beta 1.1 and J beta 1.1 was present in DJ transcripts amplified from 8-wk gestation thymi. Although it is possible to splice and translate D beta 1.1 in all three reading frames, 8-wk gestation fetal DJ transcripts were enriched for D beta 1.1 spliced to J beta 1.1 in reading frame one. Preference for D beta 1.1 reading frame one and for use of J beta 1.1 dwindled with increasing gestational age. Reading frame bias was not affected by the insertion of non-germ-line-encoded nucleotides (N regions) or by the site of gene splicing. In contrast, choice of reading frame and use of J beta 1.1 appeared random in VDJ transcripts from the same 8-wk gestation and adult thymic samples. Among the occasional VDJ transcripts that contained D beta 1.1-J beta 1.1 rearrangements, use of D beta reading frame one was rare. Up to 75% of DJ and VDJ transcripts at 8-wk gestation lacked N regions. Both the percentage of DJ and VDJ transcripts with N regions and the average number of inserted nucleotides per transcript increased with gestational age. Primarily as a result of enhanced N region addition, both the length and sequence diversity of the VDJ junctions increased markedly from 8-wk gestation to adult life with a concomitant increase in the potential size of the TCR-alpha beta repertoire. Terminal deoxynucleotidyl-transferase (TdT), the enzyme associated with N region addition, has not been detected in thymus before 19 wk of gestation. However, we were able to amplify TdT mRNA from all fetal thymi examined. This suggests that TdT may be expressed as early as 8 wk gestation, although at low levels. The early fetal thymus appears enriched for a population of thymocytes that produce minimal quantities of TdT, express a special set of TCR-beta DJ transcripts, and generate a TCR-beta repertoire with limited diversity. These findings support the hypothesis that limitations in the diversity of the TCR repertoire imposed at the time of gene rearrangement contribute to the immaturity of the fetal immune response.

Use of variable human V delta genes to create functional T cell receptor alpha chain transcripts.

Previous studies of the human T cell receptor delta genes identified five commonly used V delta segments distinct from any of the known V alpha genes. To define better the relationship between the T cell receptor delta and alpha repertoires we amplified cDNA obtained by polyclonally activated lymphocytes with a common 3' antisense C alpha-specific primer and with five different 5' sense V delta family-specific primers. Amplified products were detected in staphylococcal enterotoxin C2, staphylococcal enterotoxin E, phytohemagglutinin, concanavalin A, anti-CD3 and anti-V beta 8-activated cells, although each cell population expressed a selective pattern of V delta genes. Sequence analysis revealed that each of the known V delta genes can productively rearrange to J alpha segments to produce functional V delta-J alpha-C alpha transcripts. These results argue strongly against the notion that the human V delta and V alpha repertoires are distinct. They further suggest that the restricted delta repertoire observed in many gamma/delta clones results from selection rather than from controlled rearrangements at the T cell receptor alpha/delta locus.

T cell receptor delta-gene expression and diversity in the mouse spleen.

Thirteen T cell hybridomas and two cell lines expressing gamma delta TCR were generated from B10 and B6 CD3+CD4-CD8- splenic T cells. At least three different types of gamma-chains (V gamma 2-C gamma 1, C gamma 2, and C gamma 4) were shown to be expressed in these cells. Analysis of V delta gene segment usage in these hybridomas was performed by Northern blot hybridizations using probes specific for the V delta 1 through V delta 6 gene families and electrophoretic analysis of polymerase chain reaction amplified DNA obtained using V region-specific oligonucleotide primers for the V delta gene families as well as for V alpha 10, V alpha 4, and V alpha 11. These analyses revealed that V delta 5 gene segments are used by a high percentage of splenic gamma delta CD4-CD8- T cells (53% of the cells in our panel). Other V delta segments expressed by our panel of cells were V delta 2, V delta 4, V delta 6, and V alpha 10, indicating that the V delta repertoire expressed in the spleen is similar, but possibly not identical to the adult thymus repertoire. Sequence analysis of the V-D-J joinings of the delta-chain messages revealed substantial diversity, indicating that the delta-chain repertoire expressed in peripheral lymphoid organs uses a significant portion of the potential diversity predicted for these chains. These results demonstrate that, in spite of the low numbers of gamma delta T cells in peripheral lymphoid organs, the diversity of their TCR is extensive enough to play an important role in the immune response.

Non‐random expression of T cell receptor γ and δ variable gene segments in functional T lymphocyte clones from human peripheral blood

Human T cell receptor (TcR) gamma delta displays a variety of protein forms. Disulfide-linked (type 1) or non disulfide-linked (type 2) receptors occur, with gamma chains encoded by the C gamma 1 or the C gamma 2 gene segment, respectively. Exon 2 of C gamma 2 may either be duplicated or triplicated (type 2a or 2b receptors). TcR gamma chains differ in molecular mass and charge between type 1 and type 2 receptors. The delta chains as well as the gamma chains have different structural properties between receptor types. This cannot be due to the use of different C delta gene segments, since the genome encodes only one. To understand the genetic basis of this dichotomy in gamma/delta combinations, rearrangement and expression of V gamma, J gamma, C gamma and V delta gene segments were determined in TcR gamma/delta+ clones derived randomly from peripheral blood of normal donors. Most clones used C gamma 1, a minority C gamma 2. The different protein properties of receptor types could be explained by the non-random expression of V gamma (J gamma) and V delta gene segments. Type 1 receptors preferentially used gamma chains encoded by the V gamma 9 and J gamma 1.2 gene segments together with delta chains encoded by V delta 2. In type 2a receptors, V gamma 9 was not predominant; often other V gamma gene segments were employed, but then in high frequency in coordination with V delta 1. Reactivity of the clones with monoclonal antibodies anti-Ti gamma A, BB3 and delta-TCS-1 correlated with the expression of the V gamma 9, V delta 2 and V delta 1 gene segments, respectively. Therefore, V gamma and V delta use in TcR gamma/delta+ cells from peripheral blood of eight healthy individuals, including the two donors of the clones, could be determined tentatively by double immunofluorescence. Indeed, the V gamma 9-V delta 2 combination was predominant, while the V gamma 9-V delta 1 and particularly the V gamma 9-"V delta other" combination was rare. These data indicate that the TcR gamma delta repertoire in peripheral blood of normal individuals is largely dependent on junctional diversity and suggest that selection of receptors occurs.

T-cell receptor delta-chain diversity in peripheral lymphocytes.

A small percentage (approximately 5%) of the cells in the adult thymus expresses a heterodimeric receptor, gamma delta, that exhibits extensive clonal diversity. The specificity and function of these cells are unclear. Furthermore, it is not known if their role in the immune system is primarily one that operates within the thymus during the selection of the T-cell repertoire or if they function primarily in an antigen-recognition capacity in the peripheral lymphoid system. To examine if gamma delta+ T cells in the periphery are as diverse as those in the thymus, we used the polymerase chain reaction to amplify delta-chain transcripts from polyclonal populations of thymic and splenic lymphocytes (the latter were derived from allogeneic mixed lymphocyte cultures). The nucleotide sequences of delta chains from the spleen, like those from the thymus, were all different. Most of the diversity was present in the region between the variable (V) and joining (J) gene segments and was generated through the use of the two known diversity (D) elements, D delta 1 and D delta 2, and by the addition or deletion of bases at the V delta D delta 1, D delta 1D delta 2, and D delta 2J delta junctions. The extensive gamma delta repertoire among peripheral cells suggests that they have the potential to recognize an array of ligands that could be as diverse as those recognized by alpha beta+ cells. The amplification strategy described here can be used to analyze rapidly the diversity exhibited by any of the members of the immunoglobulin-like gene families that undergo rearrangement.

A novel gamma delta T cell receptor for antigen adds limited diversity to the gamma delta repertoire in adult thymus.

The surface expression of CD3-associated TCR chains on hybridoma cell lines derived from adult gamma delta thymocytes was analyzed. These cell lines were unusual, in that a) they expressed a surface heterodimer consisting of a 40- and a 42-kDa chain, i.e., comprised of chains different from any previously reported gamma delta-TCR all of which express C gamma 1- or C gamma 2-encoded gamma-chains; b) their CD3-associated TCR could not be categorized as alpha beta-TCR dimers, despite the similarities in m.w. of the TCR chains, because full size 1.3-kb beta-chain mRNA capable of encoding a functional beta-chain could not be detected in these cells; c) neither of the receptor chains could be precipitated with anti-C gamma 1C gamma 2-peptide antisera. Biochemical analysis demonstrated that the 42-kDa delta-chain is a novel chain, which differs from any reported delta-chains in size, charge and number of glycosylation sites. Collectively, the data on analysis of the 40-kDa chain strongly suggest that it represents a gamma-chain encoded for by the C gamma 4 locus, protein products of which have not yet been reported in the thymus. This gamma-chain was also unique, in that its isoelectric point was much lower than that of other gamma-chains. The gamma- and delta-chains on these C gamma 4-expressing hybridomas were indistinguishable from one another in size and charge (as determined by nonequivalent pH gradient electrophoresis/SDS-PAGE analysis and analysis after endoglycosidase treatment). Because the cell lines were randomly chosen from large panels of hybridomas, these results may well imply strikingly nonrandom pairing of thymocyte-derived C gamma 4 chains and the delta-chains reported here. Thus, only limited additional gamma delta repertoire diversity may be generated by availability of this gamma delta-TCR in the thymus.