Phenotypic and functional analysis of gamma delta T cell receptor-positive murine dendritic epidermal clones

Thy-1+ dendritic cells isolated from the epidermis of normal mice (dEC)3 bear the gamma delta TCR associated with the CD3 complex. We have analyzed the effects of antibodies directed against the TCR complex, Ly-6C, and Thy-1, as well as pharmacologic agents which have been shown to activate T cells without engagement of the TCR complex, on levels of intracellular free calcium, activation of protein kinase C, cytolysis, IL-2R expression, and secretion of lymphokines by dEC clones. We have found that the dEC cells express a fully functional TCR complex which can function to transmit signals upon perturbation leading to an increase in IL-2R expression, release of lymphokines, and cytolytic activity. These results indicate that the gamma delta TCR+ dEC are capable of responding to activation signals in the same manner as mature alpha beta TCR+ cells and suggests that they may play a functional role in the skin.     

Functional and phenotypic differences between CD4+ and CD4- T cell receptor-gamma delta clones from peripheral blood.

CD4+ TCR-gamma delta+ T cells comprise a very small subset of TCR-gamma delta+ T cells. CD4+ TCR gamma delta+ T cell clones were established to study the phenotypical and functional characteristics of these cells. Thirty-four CD4+ TCR-gamma delta+ T cell clones were established after sorting CD4+ T cells from a pre-expanded TCR-gamma delta+ T cell population. These clones as well as the CD4- TCR-gamma delta+ T cells from the same donor used V gamma 2 and V delta 2. In a second cloning experiment CD4+ TCR-gamma delta+ T cells were cloned directly from freshly isolated TCR-gamma delta+ T cells using a cloning device coupled to a FACS sorter. Forty-three clones were obtained, which all expressed CD4 and TCR-gamma delta. Eleven of these clones used V delta 1 and three of them coexpressed V gamma 2. The other CD4+ TCR-gamma delta+ T cell clones used both V delta 2 and V gamma 2. CD4+ TCR-gamma delta+ T cell clones expressed CD28 irrespective of the V gamma or V delta usage, and were CD11b negative. Three CD4-CD8+ TCR-gamma delta+ clones expressed CD8 alpha but not CD8 beta and were CD11b positive. CD28 expression among CD4-CD8+ and CD4-CD8- was variable but lower than on CD4+ T cell clones. CD4- TCR-gamma delta+ T cell clones using V gamma 2 and V delta 2 specifically lyse the Burkitt lymphoma cell line Daudi and secrete low levels of IFN-gamma and granulocyte-macrophage-CSF upon stimulation with Daudi. In contrast, most CD4+ T cell clones that use V gamma 2 and V delta 2 had a very low lytic activity against Daudi cells and secrete high levels of IFN-gamma and granulocyte-macrophage-CSF after stimulation with Daudi cells. The NK-sensitive cell line K562 was killed efficiently by the CD4- TCR-gamma delta+ T cell clones, but not by CD4+ TCR-gamma delta+ T cell clones, and could not induce cytokine secretion in CD4+ or CD4- T cell clones. CD4+ TCR-gamma delta+ T cell clones, but not the CD4- clones, could provide bystander cognate T cell help for production of IgG, IgM, and IgA in the presence of IL-2 and IgE in the presence of IL-4. Thus, CD4+ TCR-gamma delta+ T cells are similar to CD4+ TCR-alpha beta+ T cells in their abilities to secrete high levels of cytokines and to provide T cell help in antibody production.(ABSTRACT TRUNCATED AT 400 WORDS)     

MHC-unrestricted cytotoxic and proliferative responses of two distinct human gamma/delta T cell subsets to Daudi cells.

Human V gamma 9/V delta 2 T cells, the major subset of gamma/delta T cells in peripheral blood of adults, mediate proliferative and cytotoxic responses to Daudi Burkitt's lymphoma cells without previous in vitro exposure to Daudi. Our experiments show that some gamma/delta T cells coexpressing V gamma 9 and V delta 1 genes also react to Daudi cells in cytotoxic and proliferative assays. Expression of V gamma 9 is not sufficient for the recognition of Daudi cells because most gamma/delta T cells expressing V delta 1 paired with V gamma 9 or other V gamma genes neither kill Daudi cells nor proliferate to Daudi. V gamma 9/V delta 2 T cells do not proliferate to other cell lines such as K562 or Molt4 that are sensitive to MHC-unrestricted cytolysis by NK cells and by most IL-2-activated gamma/delta T cell clones. Cold target inhibition assays demonstrate that Daudi cells are stronger inhibitors than K562 and Molt4 of MHC-unrestricted lysis by V gamma 9/V delta 2 clones. However, cold Daudi cells are relatively weaker inhibitors of MHC-unrestricted lysis by NK cell clones, most gamma/delta T cell clones expressing V delta 1 and alpha/beta T cell clones. Thus, recognition by V gamma 9/V delta 2 T cells and certain V gamma 9/V delta 1 T cells of Daudi appears to involve a specific triggering pathway that is distinct from recognition by these gamma/delta T cells of Molt4, K562, and other target cells. NK cell clones and most other gamma/delta and alpha/beta T cell clones derived from the same normal volunteer blood donors do not show this specific interaction with Daudi cells. These data show that distinct subsets of human gamma/delta T cells recognize Daudi cells and support the idea that the gamma/delta TCR may be directly involved.     

Systematic development of distinct T cell receptor-gamma delta T cell subsets during fetal ontogeny

To elucidate the developmental pattern and diversity of murine cluster of differentiation (CD)3-associated TCR-gamma delta heterodimers, adult and fetal thymocytes were examined for cell-surface expression of various gamma- and delta-encoded TCR. Biochemical analysis, using antisera specific for distinct C gamma gene products, revealed the presence of T cells expressing C gamma 1 and/or C gamma 4 heterodimers in adult and fetal CD4- CD8- thymocyte populations. Although CD4-CD8- thymocyte populations express both C gamma 1 and C gamma 4 TCR-gamma delta heterodimers early in fetal thymus development, the relative level of C gamma 4-expressing T cells was significantly lower than previously observed in peripheral lymphoid organs. In addition, biochemical studies revealed the presence of TCR-gamma delta heterodimer(s) expressed during fetal ontogeny which were not detected in adult thymocyte or peripheral lymphoid populations. Studies of N-glycosylation patterns of one of these heterodimers suggested that it contained a rearranged V gamma 3/C gamma 1 gene product. To examine in detail individual TCR-gamma delta heterodimers, a panel of TCR-gamma delta expressing hybridomas was prepared. Biochemical analysis at the clonal level revealed that indeed three distinct TCR-gamma delta heterodimers were present at day 16 of fetal thymus development, with TCR-gamma-chains most likely encoded by V gamma 2/C gamma 1, V gamma 3/C gamma 1, and V gamma/C gamma 4. Together these findings suggest an ordered development of TCR-gamma delta T cells in the thymus and selective expression of distinct TCR-gamma delta subsets in peripheral lymphoid organs such as spleen and lymph nodes.     

Functional gamma delta T-lymphocyte defect associated with human immunodeficiency virus infections.

BACKGROUND: Antiviral cellular immune responses may influence immunological homeostasis in HIV-infected persons. Recent data indicate that V gamma 9/V delta 2 T lymphocytes display potent cytotoxic activities against human cells infected with certain viruses including HIV. Understanding the role of gamma delta T cells in the course of HIV infection may be helpful for designing novel treatment strategies for HIV-associated disorders. MATERIALS AND METHODS: The constitutive recognition of Daudi cells and monoethyl pyrophosphate (Etpp) by peripheral blood V gamma 9/V delta 2 T cells was assessed using a proliferation assay. The cytotoxicity of Daudi-stimulated lymphocyte populations was measured by chromium release assays. The HIV infectivity for gamma delta T cell clones was determined by measuring the levels of HIV p24 in cell supernatants. The effect of in vitro HIV-infection on cytokine mRNA production by gamma delta T cell clones was assessed by PCR. RESULTS: The constitutive proliferative responses of peripheral blood V gamma 9/V delta 2 T cells and the lytic functions of Daudi-expanded lymphoid cells from HIV+ persons were substantially diminished in comparison with those of HIV-seronegative persons. These alterations were present in asymptomatic HIV+ persons prior to substantial alpha beta CD4+ T cell loss. Productive HIV infection of gamma delta T cells in vitro had no measurable effect either on their proliferative response to Daudi stimuli or on the expression of cytokine mRNAs for IFN-gamma, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-13. CONCLUSIONS: The constitutive responsiveness of V gamma 9/V delta 2 T lymphocytes to Daudi and Etpp is severely altered in HIV+ persons. HIV infection of gamma delta T cells in vitro does not substantially change their cytokine expression or antigenic response.     

Human V gamma 9-V delta 2 T cell receptor-gamma delta lymphocytes show specificity to Daudi Burkitt's lymphoma cells

Peripheral blood TCR-gamma delta cells with different functional V gamma or V delta gene rearrangements represent two nonoverlapping subsets. The major subset uses the V gamma 9 and the V delta 2 gene segments and the minor subset the V delta 1 gene segments in its functional TCR rearrangement. Upon in vitro activation, these TCR-gamma delta lymphocytes display MHC-unrestricted lytic activity, against a wide variety of tumor cells of distinct histologic origin. Here we show that fresh TCR-gamma delta lymphocytes that express a V gamma 9-V delta 2 encoded TCR display a specific proliferative response to Daudi, Burkitt's lymphoma cells. Moreover, cloned V gamma 9-V delta 2 lymphocytes show the capacity to lyse Daudi cells, whereas none of the cloned V gamma 1 TCR-gamma delta lymphocytes shows such specificity. Nucleotide diversity at the V-D-J junction of the TCR-V delta 2 gene did not contribute to this Daudi cell specificity. Comparison of the MHC-unrestricted cytolytic capacities of the V gamma 9-V delta 2 and the V delta 1 clones using a panel of distinct types of tumor target cells showed that on average, the level of MHC unrestricted lysis of V gamma 9-V delta 2 clones against these tumor cells exceeded that of V delta 1 clones. However, in contrast to all these tumor cell lines, only the Daudi cells showed such an absolute distinction in susceptibility to lysis by V gamma 9-V delta 2 and V delta 1 clones. V gamma 9-V delta 2 clones that were generated with a stimulator cell other than Daudi did not lyse their stimulator cells but nevertheless showed specific cytolysis of Daudi cells. The specific proliferation to and cytolysis of Daudi cells of the entire V gamma 9-V delta 2 subpopulation of TCR-gamma delta lymphocytes is reminiscent of a superantigen response.     

Different roles of the CD2 and LFA-1 T-cell co-receptors for regulating cytotoxic, proliferative, and cytokine responses of human V gamma 9/V delta 2 T cells

BACKGROUND: Human V gamma 9/V delta 2 T lymphocytes recognize nonpeptidic antigens in a manner distinct from the classical antigen recognition by alpha beta T cells. The apparent lack of major histocompatibility (MHC) restriction and antigen processing allows very fast responses against pathogenic insults. To address the potential functional requirement for accessory molecules, we investigated the roles of the CD2 and lymphocyte function-associated antigen (LFA)-1 T-cell co-receptors in antigen-induced activities of human V gamma 9/V delta 2 T-cell clones. MATERIALS AND METHODS: Human peripheral blood V gamma 9/V delta 2 T lymphocytes were cloned and their cytotoxicity against Daudi lymphoma was measured by a standard 51Cr-release assay. The responses of V gamma 9/V delta 2 T lymphocytes to nonpeptidic antigens were assessed using DNA synthesis and cytokine ELISA assays. Monoclonal antibodies specific for various molecules with potential T-cell accessory functions were utilized in blocking assays. RESULTS: All of our V gamma 9/V delta 2 T-cell clones displayed the Th1 phenotype. The anti-LFA-1 antibody strongly inhibited the cytotoxicity of V gamma 9/V delta 2 T cells against Daudi B-cell lymphoma; whereas, it had no influence on the antigen-induced cytokine release or proliferation. In contrast, antibodies against CD2 and LFA-3 had no effect on the lytic activity of V gamma 9/V delta 2 T cells, but strongly inhibited the cytokine release and proliferation. However, the CD2-LFA-3 interaction was not an absolute requirement for the cytokine release and the DNA synthetic activity of antigen-stimulated V gamma 9/V delta 2 T cells, since the inhibitory effect could be reversed by addition of exogenous interleukin 2 (IL-2). CONCLUSIONS: These novel observations indicate that the signals generated by different accessory molecules and IL-2 can contribute in an integrated fashion to the regulation of V gamma 9/V delta 2 T cells. These interactions may be important for the effectiveness of V gamma 9/V delta 2 T-cell responses.     

A developmental switch in thymic lymphocyte maturation potential occurs at the level of hematopoietic stem cells

Hematopoietic stem cells (HSCs) isolated from mouse fetal liver, like adult HSCs, are Thy-1lo Lin- Sca-1+. Donor-derived V gamma 3+ T cells were detected in fetal thymic lobes repopulated in vitro with fetal liver HSCs, but not in those with adult bone marrow HSCs. Single clonogenic fetal HSCs gave rise to thymic progeny that include V gamma 3+, other gamma delta+, and alpha beta+ T cells. No V gamma 3+ T cells were detected in adult thymus injected intrathymically with either fetal or adult HSCs. These results support the hypothesis that only fetal HSCs have the capacity to differentiate into V gamma 3+ T cells in the fetal thymic microenvironment and that the developmental potential of HSCs may change during ontogeny.     

T cell antigen receptor engagement and specificity in the recognition of stress-inducible MHC class I-related chains by human epithelial gamma delta T cells

Human gamma delta T cells with the TCR variable region V(delta)1 occur mainly in epithelia and respond to stress-induced expression of the MHC class I-related chains A and B, which have no function in Ag presentation. MIC function as ligands for NKG2D-DAP10, an activating receptor complex that triggers NK cells, costimulates CD8 alpha beta and V(gamma)9V(delta)2 gamma delta T cells, and is required for stimulation of V(delta)1 gamma delta T cells. It is unresolved, however, whether triggering of V(delta)1 gamma delta TCRs is also mediated by MIC or by unidentified cell surface components. Soluble MICA tetramers were used as a binding reagent to demonstrate specific interactions with various V(delta)1 gamma delta TCRs expressed on transfectants of a T cell line selected for lack of NKG2D. Tetramer binding was restricted to TCRs derived from responder T cell clones classified as reactive against a broad range of MIC-expressing target cells and was abrogated when TCRs were composed of mismatched gamma- and delta-chains. These results and the inability of V(delta)1 gamma delta T cells to respond to target cells expressing the ULBP/N2DL ligands of NKG2D, which are highly divergent from MIC, indicate that MIC delivers both the TCR-dependent signal 1 and the NKG2D-dependent costimulatory signal 2. This dual function may serve to prevent erroneous gamma delta T cell activation by cross-reactive cell surface determinants.     

Allelic exclusion at the TCR delta locus and commitment to gamma delta lineage: different modalities apply to distinct human gamma delta subsets

Expression of a beta-chain, as a pre-TCR, in T cell precursors prevents further rearrangements on the alternate beta allele through a strict allelic exclusion process and enables precursors to undergo differentiation. However, whether allelic exclusion applies to the TCR delta locus is unknown and the role of the gamma delta TCR in gamma delta lineage commitment is still unclear. Through the analysis of the rearrangement status of the TCR gamma, delta, and beta loci in human gamma delta T cell clones, expressing either the TCR V delta 1 or V delta 2 variable regions, we show that the rate of partial rearrangements at the delta locus is consistent with an allelic exclusion process. The overrepresentation of clones with two functional TCR gamma chains indicates that a gamma delta TCR selection process is required for the commitment of T cell precursors to the gamma delta lineage. Finally, while complete TCR beta rearrangements were observed in several V delta 2 T cell clones, these were seldom found in V delta 1 cells. This suggests a competitive alpha beta/gamma delta lineage commitment in the former subset and a precommitment to the gamma delta lineage in the latter. We propose that these distinct behaviors are related to the developmental stage at which rearrangements occur, as suggested by the patterns of accessibility to recombination sites that characterize the V delta 1 and V delta 2 subsets.     

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.     

IL-4-producing gamma delta T cells that express a very restricted TCR repertoire are preferentially localized in liver and spleen.

IL-4-producing gamma delta thymocytes in normal mice belong to a distinct subset of gamma delta T cells characterized by low expression of Thy-1. This gamma delta thymocyte subset shares a number of phenotypic and functional properties with the NK T cell population. Thy-1dull gamma delta thymocytes in DBA/2 mice express a restricted repertoire of TCRs that are composed of the V gamma 1 gene product mainly associated with the V delta 6.4 chain and exhibit limited junctional sequence diversity. Using mice transgenic for a rearranged V gamma 1J gamma 4C gamma 4 chain and a novel mAb (9D3) specific for the V delta 6.3 and V delta 6.4 murine TCR delta chains, we have analyzed the peripheral localization and functional properties of gamma delta T cells displaying a similarly restricted TCR repertoire. In transgenic mice, IL-4 production by peripheral gamma delta T cells was confined to the gamma delta+9D3+ subset, which contains cells with a TCR repertoire similar to that found in Thy-1dull gamma delta thymocytes. In normal DBA/2 mice such cells represent close to half of the gamma delta T cells present in the liver and around 20% of the splenic gamma delta T cells.     

Activation requirements of newborn thymic gamma delta T cells.

We have analyzed the requirements for the induction of proliferative responses by thymic CD4-CD8- gamma delta T cells. Enriched populations of CD4-CD8- thymocytes from newborn mice, purified by negative selection with anti-CD4, anti-CD8, and anti-TCR alpha beta mAbs were found to contain approximately 20% gamma delta T cells that were p55IL-2R-. When these cells were cultured with a panel of lymphokines (IL-1, -2, -4, and -7), a small response was observed to some of the cytokines tested individually; however, combinations of certain lymphokines (IL-1 + 2, IL-1 + 7, and IL-2 + 7) were found to induce significant proliferation and the selective outgrowth (75-90%) of gamma delta T cells. These cells were IL-2R+, remained CD4-, yet expressed variable levels of CD8. A limited analysis with specific anti-V gamma and V delta mAb suggested that there had not been a selective expansion of preexisting V gamma 2, V gamma 3, or V delta 4 populations in response to the stimulatory lymphokine combinations. Thymic CD4-CD8- gamma delta T cells were unresponsive to stimulation with immobilized anti-pan gamma delta mAb alone. However, in the presence of immobilized anti-pan gamma delta mAb and IL-1, IL-2, or IL-7, but not IL-4, a vigorous proliferative response was observed. Phenotypic analysis showed that 80 to 95% of the proliferating cells were polyclonally expanded gamma delta T cells, expressed the p55IL-2R, and the majority remained CD4-CD8-. Blocking studies with anti-IL-2R mAb showed that stimulation with anti-pan gamma delta + IL-1, but not anti-pan gamma delta + IL-7 was dependent on endogenously produced IL-2. Collectively, these studies suggest that the activation requirements of newborn thymic gamma delta T cells differ markedly from alpha beta T cells in that gamma delta T cells 1) respond to combinations of cytokines in the absence of TCR cross-linking, 2) can respond to TCR cross-linking in the presence of exogenous cytokines, 3) but are unable to activate endogenous cytokine production solely in the presence of TCR cross-linking.     

Human T cell clones with gamma/delta and alpha/beta receptors are differently stimulated by monoclonal antibodies to CD2

Requirements for stimulating autocrine proliferation of human T cell clones expressing either alpha/beta or gamma/delta antigen receptors via the "alternative" CD2 pathway have been examined using a large set of monoclonal antibodies (mAb). In the presence of autologous accessory cells (AC, B-lymphoblastoid cell lines) 2 of 13 single CD2 mAb (CLB-T11.1/1 and 6F10.3) stimulated proliferation of gamma/delta but not alpha/beta cells. Interleukin (IL) 1 or IL 6 did not substitute for AC in stimulating gamma/delta clones. Addition of CD28 mAb YTH 913.12 with the CD2 mAb did not result in stimulation of any alpha/beta clones. In the absence of AC, none of the CD2 mAb singly could stimulate any T cell clones, but pairs of mAb directed to different epitopes of CD2 (CLB-T11.1/1 + CLB- T11.2/1 or 6F10.3 + 39C1.5) stimulated both alpha/beta and gamma/delta clones. In both cases, stimulation was reduced by the presence of CD3 mAb. These results confirm that the established AC-independent alternative pathway of T cell activation, which requires binding of two separate epitopes of CD2, operates in both gamma/delta and alpha/beta T cells, and further suggest that an additional pathway initiated by binding of a single CD2 epitope in the presence of AC is exclusively operational in gamma/delta cells.     

CD16 on human gamma delta T lymphocytes: expression, function, and specificity for mouse IgG isotypes.

We examined the expression, the signal transduction capacity and mouse IgG-isotype specificity of CD16 on human gamma delta T cells. CD16 is expressed by the majority of gamma delta T cells in peripheral blood and by part of the gamma delta T cell clones. The amount of CD16 expressed on gamma delta T cell clones varied considerably with passaging of the cells, but was always significantly less than on freshly isolated gamma delta T cells. Like CD16 on CD3- CD16+ natural killer (NK) cells, CD16 on gamma delta T cells can act as an activation site triggering cytotoxic activity. CD16+ gamma delta T cell clones exerted antibody-dependent cellular cytotoxicity (ADCC) which could be blocked by anti-CD16 mAb. ADCC activity of gamma delta T cell clones was also inhibited by anti-CD3 mAb, suggesting a functional linkage between the CD16 and CD3 activation pathways. MAb directed against CD16 induced lysis of Fc gamma R+ target cells by CD16+ gamma delta T cell clones. The mouse IgG-isotype specificity of CD16 on gamma delta T cells was analyzed using isotype switch variants of a murine anti-glycophorin A mAb in EA rosette assays, and was found to be identical to that of CD16 on CD3- CD16+ NK cells, i.e., highest affinity for mIgG2a, intermediate affinity for mIgG2b, and undetectable binding of mIgG1-sensitized erythrocytes. CD16 was partly modulated from the cell surface of both gamma delta T cells and NK cells after rosette formation with mIgG2a-sensitized erythrocytes, indicating that the rosette formation was indeed mediated via the CD16 molecule.     

Patterns of T cell receptor gamma gene rearrangements in human CD3+ clones derived from WT31- or Leu7+ cells in relation to non-MHC-restricted cytotoxic activity

Clones were obtained from human peripheral blood WT31-, WT31-CD4-8-, CD4-8- or Leu 7+ cells in the presence of interleukin 2 and phytohaemagglutinin. Almost all clones were CD3+, about 50% were CD4-8- and all clones tested derived from WT31- remained WT31-, indicating that they were expressing a gamma/delta heterodimer in association with CD3. Some clones derived from CD4-8- cells expressing CD3 were WT31- and some were WT31+. All CD3+ clones had T cell receptor (TCR) gamma gene rearrangements; most also had their TCR beta genes rearranged, including all clones derived from Leu 7+ cells. TCR gamma gene rearrangements were noted involving all five known J segments. There was a tendency for V gene segments from the VII and VIII subgroups to be rearranged to J gamma 2 less often than those from the more 5' VI subgroup. Two clones definitely had one rearrangement to C gamma 1 and one to C gamma 2. When clones derived from WT31- cells were considered, the only obvious relationship which emerged was that all clones with both chromosomes rearranged to C gamma 2 had low or negligible cytotoxic activity against natural killer (NK)-sensitive and NK-resistant targets. Several of these clones were expressing CD8 on about 30% of cells. Most clones with rearrangements involving only C gamma 1 had high non-MHC-restricted cytotoxicity while those with at least one C gamma 1 rearrangement had either high or low activity. The only exceptions noted were a clone with a single V9JP rearrangement and a clone with a V9JP and a VI/IIIJP1 rearrangement, which both had low activity. A similar pattern was also found with most clones derived from Leu 7+ cells. The data are consistent with the participation of most types of disulphide-linked (C gamma 1) gamma/delta heterodimers in non-MHC-restricted cytotoxic activity mediated by CD3+ gamma/delta + T cell clones.