Coordinated V gamma and V delta gene segment rearrangements in human T cell receptor gamma/delta+ lymphocytes

Monoclonal antibodies (mAb) were used to characterize a panel (n = 46) of T cell receptor (TcR) gamma/delta+ T cell clones. Three of these antibodies have been described to react with specific variable region-encoded protein products and can therefore be used to detect functional gene rearrangements. The majority of peripheral blood-derived clones (43 out of 45) expressed the epitopes recognized by mAb BB3, encoded by the V delta 2 gene segment and mAb Ti gamma A, encoded by the V gamma 9 gene segment. These clones lacked the antigenic determinant recognized by mAb delta-TCS-1, encoded by the V delta 1 gene segment. The other two peripheral blood-derived clones and an ascites-derived clone were Ti gamma A-, BB3- and delta-TCS-1+. Biochemical analysis revealed that all Ti gamma A+, BB3+ T cell clones expressed the disulfide-linked form of the receptor. The two peripheral blood-derived delta-TCS-1+ T cell clones expressed the nondisulfide-linked form whereas the ascites-derived delta-TCS-1+ clone, AK119 expressed the disulfide-linked form of the TcR gamma/delta heterodimer. This indicates that V delta 1-encoded delta chains can be associated either with a C gamma 1- or a C gamma 2-encoded gamma chain. The preferential use of certain V gamma and V delta gene segments suggests the existence of a limited combinatorial diversity in TcR gamma/delta heterodimers, i.e. Ti gamma A+ (V gamma 9), BB3+ (V delta 2) and delta-TCS-1- disulfide-linked heterodimers and Ti gamma A-, BB3- and delta-TCS-1+ (V delta 1) disulfide- or non disulfide-linked forms.     

Subpopulations of human peripheral T gamma delta lymphocytes

In the search for the genes encoding the alpha and beta chains of the T-cell receptor, Tonegawa et al. discovered a third class of rearranging T-cell specific genes. This finding led them to postulate the existence of additional forms of T-cell receptors. In this article, Frédéric Tribel and Thierry Hercend briefly discuss recent results, which may help in the delineation of human peripheral blood gamma delta+ subsets based on the molecular structure of this receptor.     

Cloned CD3+ TcR alpha/beta- Ti gamma A- peripheral blood lymphocytes compared to the Ti gamma A+ counterparts: structural differences of the gamma/delta receptor and functional heterogeneity

We have assessed the organization of T cell gamma rearranging genes (TRG) in circulating TcR gamma/delta+ lymphocytes which do not express V gamma 9-encoded Ti gamma A+ gamma chain. Following purification of the minor TcR gamma/delta+ Ti gamma A- fraction, cloned cell lines were developed from peripheral blood of 5 individuals. Out of the 26 clones studied, only 3 TcR gamma/delta+ Ti gamma A- cells were found to express a disulfide-linked C1-encoded gamma chain. The remaining 23 Ti gamma A- clones with a C2-encoded nondisulfide-linked receptor were found to display rearrangements of various V genes to J2 segments on both chromosomes; there was no predominance of a unique rearrangement even though the TRG-V3 and -V4 genes belonging to subgroup I were frequently employed. Together, these findings further strengthen the hypothesis that lymphocytes with a C gamma 1 encoded chain are produced earlier in T cell ontogeny than the C gamma 2 counterparts. The "non-major histocompatibility complex (MHC) requiring" (i.e., "natural killer-like") cytotoxicity mediated by many TcR gamma/delta+ Ti gamma A- cells appeared to be very low as compared to that of Ti gamma A+ clones. Yet, treatment by the OKT3 monoclonal antibody revealed a strong lytic potential in the Ti gamma A- lymphocytes with little, if any, natural killer-like activity. Thus, with respect to the latter function, a substantial heterogeneity is found in cells expressing distinct gamma chains. In an attempt to characterize undefined specificities of Ti gamma A- lymphocytes, they were screened against a panel of Epstein-Barr virus-transformed B cell lines homozygous for HLA-DR1 to DR10 determinants; one of the clones was found to recognize DR7. In light of reports from other groups describing class I-related specificities, it is apparent that TcR gamma/delta+ lymphocytes are able, like the TcR alpha/beta+, to recognize and kill target cells through either an MHC-dependent (with involvement of either class I or class II gene products) or a non-MHC-requiring pathway.     

A major fraction of human intraepithelial lymphocytes simultaneously expresses the gamma/delta T cell receptor, the CD8 accessory molecule and preferentially uses the V delta 1 gene segment

The frequency of T cell receptor (TcR) type and the variable gene segment expression in human intraepithelial lymphocytes (IEL) from the large intestinal mucosa were studied by flow cytometry and immunohistochemistry, and compared to those in peripheral blood lymphocytes (PBL) - or lamina propria lymphocytes (LPL). Employing anti-gamma/delta TcR and anti-alpha/beta TcR monoclonal antibodies (mAb), flow cytometric analysis revealed that a large fraction of IEL (37%) are gamma/delta T cells, whereas within LPL and PBL gamma/delta T cells comprise a minor population (4.6% and 3.8% respectively). At these sites the number of gamma/delta T cells labeled with anti-CD8 mAb were 58.3% (IEL), 43.3% (LPL) and 24.4% (PBL). In situ staining of serial sections of large intestine confirmed these results. Hence, these data suggest a selective accumulation of CD8+ gamma/delta T cells in the human epithelium of the large intestine. Furthermore, analysis of gamma/delta TcR bearing IEL+ disclosed a marked preponderance of cells using the V delta 1 gene segment, whereas gamma/delta TcR+ PBL preferentially express V delta 2. Strikingly, the majority of these V delta 1+ IEL bear the CD8 molecule on their surface. These results are taken as evidence for a selective localization of V delta 1+ CD8+ gamma/delta T cells in the epithelium of the large intestine.     

Restricted diversity of V gamma 9-JP rearrangements in unstimulated human gamma/delta T lymphocytes.

The diversity of human peripheral blood gamma/delta T cells is known to be limited by the preferential use of V genes coding for V gamma 9 (usually linked to JP) and V delta 2. We show that the diversity of these cells is further limited at the junctional region. First, an identical rearrangement is found in 10%-30% of all gamma/delta T cells which contain V gamma 9-JP rearrangements. Second, the vast majority of V gamma 9-JP rearrangements which are different from this predominant sequence have, nevertheless, the same length or code for variable regions whose length differs by only one amino acid (+/- 1). Overall, 30%-50% of V gamma 9-JP rearrangements have a junctional region which encodes for a peptide with the amino acid sequence E VX EL, in which EV is predominantly, but not exclusively, encoded by the germ-line V gamma 9 sequence and EL is encoded by JP. The X amino acid is variable, but a glutamine is over-represented. The diversity of the V gamma 9-JP repertoire is fairly constant in different individuals and at different ages, including before, during and after the post-natal expansion of peripheral blood gamma/delta T cells.     

T-cell receptor V delta gene usage by tumour reactive gamma delta T lymphocytes infiltrating human lung cancer.

In seven human adenocarcinomas and a non-neoplastic granulomatous disease of the lung, gamma delta+ infiltrating lymphocytes (TIL) could be isolated and selectively expanded in vitro upon culture in interleukin-2 (IL-2), without any additional stimuli, indicating a prior activation in vivo. In most cases gamma delta TIL were predominantly V delta 1+, despite a normal V delta 2:V delta 1 ratio in paired peripheral blood lymphocytes, suggesting a possible expansion of this subset in response to localized antigens/superantigens. Moreover, in five patients it was possible to identify a V delta 1- V delta 2- TIL population which by polymerase chain reaction (PCR) analysis was shown to be heterogeneous as V delta gene usage, inclusive of V delta 3,4,5,6,7 and 8. Of note, these V delta regions have not been found in peripheral blood so far. Finally, in all cases, gamma delta TIL displayed killing activity of the autologous tumour, which appeared to be more restricted in the case of V delta 1+ cells. Altogether, these findings suggest a preferential expansion, at the tumour site, of V delta 1+ cells and of cells expressing V delta genes other than V delta 2.     

Cross-talk between V beta 8+ and gamma delta+ T lymphocytes in contact sensitivity.

We have previously reported that T lymphocytes proliferating in vitro to the hapten trinitrochlorobenzene (TNCB) exhibit a very restricted V beta gene usage and response to TNCB is limited to T-cell receptors (TCR) composed of V beta 8.2 in combination with V alpha 3.2, V alpha 8 and V alpha 10. This paper investigates the role played by T lymphocytes expressing the V beta 8.2 gene segment in the contact sensitivity (CS) reaction to TNCB in the intact mouse and in its passive transfer into naive recipient mice. Mice injected with monoclonal antibodies to V beta 8 are unable to develop CS upon immunization with TNCB and 4-day TNCB-immune lymph node cells from mice that had been depleted in vivo or in vitro of V beta 8+ T lymphocytes fail to transfer CS. However, when separated V beta 8+ and V beta 8- cells were used for passive transfer, it was found that V beta 8+ T lymphocytes failed to transfer CS when given alone to recipient mice and a V beta 8- population was absolutely required. Further analysis revealed that within the V beta 8- population, T lymphocytes expressing the gamma delta TCR were fundamental to allow transfer of the CS reaction. These gamma delta cells were found to be antigen non-specific, genetically unrestricted and to rearrange the V gamma 3 gene segment. This indicates that transfer of the CS reaction requires cross-talk between V beta 8+ and gamma delta+ T lymphocytes, thus confirming our previous results obtained using TNCB-specific T-cell lines. Time-course experiments showed that V beta 8+ lymphocytes taken 4-24 days after immunization with TNCB were able to proliferate and produce interleukin-2 (IL-2) in response to the specific antigen in vitro. Similar time-course experiments were then undertaken using the passive transfer of the CS reaction system. The results obtained confirm that TNCB-specific V beta 8+ T lymphocytes are present in the lymph nodes of immunized mice from day 4 to day 24, and reveal that gamma delta+ T lymphocytes are active for a very short period of time, i.e. days 4 and 5 after immunization. In fact, TNCB-specific V beta 8+ cells are able to transfer CS when taken 4-24 days after immunization, providing the accompanying V beta 8- or gamma delta+ T lymphocyte are obtained 4 days after immunization. In contrast, injection of V beta 8+ T lymphocytes together with V beta 8- or gamma delta+ T lymphocytes that had been taken 2 or 6 days after immunization, failed to transfer significant CS into recipient mice. Taken together, our results confirm that cross-talk between V beta 8+ and gamma delta+ T lymphocytes is necessary for full development of the CS reaction and may explain why the CS reaction in the intact mouse lasts up to 21 days after immunization while the ability of immune lymph node cells to transfer CS is limited to days 4 and 5 after immunization.     

Selective lysis of the autologous tumor by delta TCS1+ gamma/delta+ tumor-infiltrating lymphocytes from human lung carcinomas.

Two distinct non-overlapping populations of TcR1+ (gamma/delta) T cells have been described: the first, bearing the disulfide-linked gamma/delta heterodimer, is predominant in the peripheral blood; the second, expressing the non-disulfide-linked form of TcR1, is mostly confined to epithelial tissues (lung, gut, skin). TcR1+ lymphocytes may be cytotoxic and could be involved in anti-tumor immunity, especially against tumors at epithelial sites. Freshly derived tumor-infiltrating lymphocytes (TIL) obtained from two patients with lung cancer were enriched in CD3+WT31- cells. The percentage of this subset substantially increased upon culture in the presence of interleukin 2. These cells were TcR1+ as demonstrated by immunofluorescence and immunoprecipitation. In one case only 40% of this population reacted with delta TCS1 mAb, that recognizes the non-disulfide-linked form of TcR1, and co-expressed the CD8 antigen. Cultured TcR1+ TIL were able to kill fresh autologous tumor cells, K-562 and, to a lesser extent, some natural killer-resistant cell lines and allogeneic lung tumor cells in 4-h 51Cr-release cytotoxicity assays. The fractionated delta TCS1+ TIL lysed only autologous tumor cells and K-562, whereas the lytic activity against all the other targets was confined to the delta TCS1- subset. Moreover, the autotumor cytotoxicity was inhibited by anti-HLA class I but not by anti-CD1c or anti-LFA-1 mAb, suggesting that killing of the autologous tumor cells and non-major histocompatibility complex-restricted cytotoxicity are mediated by different mechanisms.     

Subset-specific, uniform activation among V gamma 6/V delta 1+ gamma delta T cells elicited by inflammation

The V gamma 6/V delta 1(+) cells, the second murine gamma delta T cell subset to arise in the thymus, express a nearly invariant T cell receptor (TCR), colonize select tissues, and expand preferentially in other tissues during inflammation. These cells are thought to help in regulating the inflammatory response. Until now, V gamma 6/V delta 1(+) cells have only been detectable indirectly, by expression of V gamma 6-encoding mRNA. Here, we report that 17D1, a monoclonal antibody, which detects the related epidermis-associated V gamma 5/V delta 1(+) TCR, will also bind the V gamma 6/V delta 1(+) cells if their TCR is first complexed to an anti-C delta antibody. Features of this special condition for recognition suggest the possibility that an alternate structure exists for the V gamma 6/V delta 1 TCR, which is stabilized upon binding to the anti-C delta antibody. Using the 17D1 antibody as means to track this gamma delta T cell subset by flow cytometry, we discovered that the response of V gamma 6/V delta 1(+) cells during inflammation often far exceeds that of other subsets and that the responding V gamma 6/V delta 1(+) cells display a strikingly uniform activation/memory phenotype compared with other gamma delta T cell subsets.     

Ti gamma A + delta TCS1+ T cells in human thymus. Analysis of the T cell receptor.

TcR1+ T cells in peripheral blood have been shown to express in an exclusive fashion either the Ti gamma A or the delta TCS1 epitope. Here, we characterize a subset of TcR1+ T cells in fresh thymus co-expressing the Ti gamma A and delta TCS1 epitopes. TcR1dim and TcR1bright clones can be distinguished. Biochemical and molecular studies on both types of clones generated from these thymocytes reveal a unique T cell receptor structure characterized by the use of a V gamma 9/C gamma 2-encoded 55-kDa gamma chain nondisulfide linked to a V delta 1-encoded delta chain.     

Identification of a molecule uniquely expressed on a gamma/delta TCR+ subset within bovine intestinal intraepithelial lymphocytes.

An antigen has been identified, recognized by a novel monoclonal antibody CC45, which is expressed by a subpopulation of bovine gamma/delta T-cell receptor-positive (gamma/delta TCR+) T cells restricted in their distribution to the intestinal epithelium. This subset of intestinal intraepithelial lymphocytes (iIEL) which represented 8-29% of gamma/delta TCR+ T cells in the gut epithelium expressed CD45, CD3 and L-selectin; most of these cells were CD2- and CD8-. Electron microscopic studies of CC45+ cells revealed that they were large mononuclear leucocytes containing numerous mitochondria and smooth vesicles; a proportion of these contained membrane-bound dense granules. Immunoprecipitation of 125I-labelled iIEL analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing and non-reducing conditions revealed polypeptides of 60,000 and 200,000 molecular weights, respectively indicating that the antigen, which appears distinct from molecules described in other species, is expressed on the cell surface as a complex.     

V gamma 9V delta 2 T cells in human legionellosis

In humans, expansion of circulating Vgamma9Vdelta2 T cells seems to be a pathophysiological denominator shared by protozoan and intracellular bacterial diseases. The assumption was tested here on legionellosis, a condition conforming to the category but not yet described with respect to gammadelta T cells. Levels of Vgamma9Vdelta2 T cells in peripheral blood were measured at various intervals in 14 subjects undergoing a Pontiac fever-like disease, shown by serological investigation to be caused by Legionella micdadei. In samples obtained 4 to 6 days after the onset of the disease, the mean percentage (+/- the standard deviation) of Vgamma9Vdelta2+ T cells among CD3+ cells was 1.0% +/- 0.5%, compared to 5.0% +/- 3.9% in healthy control subjects (P < 0.001). Thereafter, a pronounced increase occurred and at 2 to 7 weeks after onset, mean peak levels were as high as approximately equal to 15%. During the next 6 months, values slowly declined, although without reaching the normal range. Percentages of gammadelta+ T cells expressing tumor necrosis factor alpha or gamma interferon in response to phorbol myristate acetate were assayed in vitro. At 14 to 16 days after the onset of disease, the expression of both cytokines was increased (P < 0.01), whereas at 5 to 7 weeks, the expression of tumor necrosis factor alpha was decreased (P < 0.05), possibly reflecting modulation of an inflammatory response. In conclusion, Pontiac fever was found to be associated with a pronounced and long-lasting expansion of Vgamma9Vdelta2 T cells, implying that the subset may also be pathophysiologically important in a mild and transient form of intracellular bacterial diseases. Surprisingly, the expansion was preceded by a depletion of circulatory Vgamma9Vdelta2 T cells. Possibly, Vgamma9Vdelta2 T cells are initially recruited to a site of infection before they expand in response to antigen and occur in high numbers in blood.     

Opportunist mycobacteria express ligands that stimulate production of human V gamma 9V delta 2 T lymphocytes.

Human gamma delta T cells are known to respond at high frequencies to pathogenic mycobacteria. Here we show that opportunistic strains of mycobacteria share with pathogenic mycobacteria the ability to trigger at high frequencies human V gamma 9V delta 2 T-cell-receptor-positive T lymphocytes. Stimulating ligands were present in part in a low-molecular-weight fraction of lysates from opportunistic mycobacteria, as has been found for pathogenic strains. These results support the view that postnatal exposure to ever-present opportunistic mycobacteria may be a driving force for the numerical expansion of the V gamma 9V delta 2 T-cell subset in adolescence.     

Selective death of T cell receptor gamma/delta+ intraepithelial lymphocytes by apoptosis

In mice, the majority of T cells expressing the gamma/delta T cell receptor (TcR) are found at mucosal surfaces, especially the intestinal epithelium. Here we show that in vitro, the majority of TcR gamma/delta+ intraepithelial lymphocytes, but not TcR alpha/beta+ intraepithelial lymphocytes, undergo rapid and selective programmed cell death by apoptosis.     

Induction of human TCR gamma delta + and TCR gamma delta-CD2+CD3- double negative lymphocytes by bacterial stimulation

When human blood mononuclear cells (MNC) were incubated with heat-killed bacteria, proliferation of MNC was observed 5 days after stimulation, showing a peak on day 7. Interestingly, the bioassay of the culture supernatant and Northern blot analysis of mRNA demonstrated that no IL-2 production was associated with these proliferative responses. The induced lymphoblasts consisted predominantly of TCR gamma delta + (22.4 +/- 9.3%) and TCR gamma delta-CD2+CD3-(33.2 +/- 11.8%) double negative lymphocytes (n = 10), which were initially minor populations (less than 10%) in freshly isolated MNC. The prominent induction of TCR gamma delta + cells was confirmed by Northern blot analysis. TCR gamma delta + cells induced by bacterial stimulation seemed to generate from lymphocytes lacking the apparent expression of gamma delta TCR. The inducing capability for double negative cells is present in a large number of species of bacteria, especially Gram-positive bacteria. Gel filtration analysis of ultrasonicated filtrates of Staphylococcus aureus and Streptococcus pyogenes revealed that a substance with an Mr of 25-26 kd could be substituted for whole bacterial particles in the cell proliferative responses. In contrast to the purified protein derivative (PPD)-induced response, the response described here was inducible in the cord blood of neonates who had not yet been exposed to the corresponding bacterial infection. The physicochemical properties of the sonicated filtrates were different from those of PPD. These results suggested that the present phenomenon may be nonspecific, polyclonal (or oligoclonal) activation of TCR gamma delta + and TCR gamma delta -CD2+CD3- cells by bacterial stimulation.     

Non-random migration of CD4+, CD8+ and gamma delta+T19+ lymphocytes through peripheral lymph nodes.

The experiments described in this paper have examined the migration of three fluorochrome-labelled T-lymphocyte subsets (CD4+, CD8+ and gamma delta+T19+) on a single passage from blood to lymph, through prescapular lymph nodes. Lymphocytes obtained from prescapular efferent lymph were labelled in vitro with fluorochrome and returned to the blood of the same animal. Over the next 2 days, lymph was continuously monitored and the cells in all collections, including the one used for intravenous infusion, were phenotyped and analysed by flow cytometry. Significant differences in the subset ratios between the infused, starting population and the recirculated population indicated that CD4+ and gamma delta+T19+ lymphocytes are extracted by a resting lymph node at the same rate and that both are extracted at a faster rate than CD8+ lymphocytes. The results presented here also suggest that a unique subset of gamma delta+T19+ lymphocytes may be present in blood that does not recirculate through peripheral lymph nodes.     

Plasmodium falciparum merozoites primarily stimulate the V gamma 9 subset of human gamma/delta T cells.

Peripheral blood-derived T cells from six unprimed caucasian donors were tested for the in vitro reactivity to Plasmodium falciparum merozoites (PFM). Without exception vigorous proliferative responses were observed within the donors tested. The frequency of PFM-reactive T cells ranged from 1/150-1/300. Phenotypic analysis of peripheral blood lymphocytes cultured in the presence of PFM revealed the preferential outgrowth of gamma/delta T cells, which represented within 7 days about 70% of the reactive T cell blasts. All reactive gamma/delta T cell blasts displayed the V gamma 9+ TcR phenotype. We conclude that human gamma/delta T cells respond vigorously to PFM, and that this property is confined to V gamma 9+ T cell subset.