Membranes of activated CD4+ T cells expressing T cell receptor (TcR) alpha beta or TcR gamma delta induce IgE synthesis by human B cells in the presence of interleukin-4.

In the present study it is demonstrated that human B cells can be induced to switch to IgE production following a contact-mediated signal provided by activated T cell receptor (TcR) gamma delta+, CD4+ and TcR alpha beta+, CD4+ T cell clones and interleukin (IL)-4. The signal provided by these T cell clones was antigen nonspecific, indicating that the TcR alpha beta/CD3 or TcR gamma delta/CD3 complexes were not involved in these T-B cell interactions. Activated TcR alpha beta+, CD8+, and TcR gamma delta+, CD4-CD8-, or resting CD4+ T cell clones were ineffective. Intact TcR alpha beta+ or TcR gamma delta+, CD4+ T cell clones could be replaced by plasma membrane-enriched fractions isolated from these activated CD4+ T cell clones. In contrast, membranes isolated from resting TcR alpha beta+, CD4+, TcR gamma delta+, CD4+ T cell clones or an Epstein-Barr virus (EBV)-transformed B cell line (EBV-LCL) failed to provide the costimulatory signal that, in addition to IL-4, is required for induction of IgE synthesis. As described for intact CD4+ T cells, CD4+ T cell membranes induced purified surface IgM+ B cells to switch to IgG4- and IgE- but not to IgA-producing cells, excluding the possibility of a preferential outgrowth of IgG4- and IgE-committed B cells. The membrane activity was inhibited by protease or heat treatment. Induction of IgE synthesis by B cells co-cultured with both TcR alpha beta+, CD4+ and TcR gamma delta+, CD4+ T cell clones and membrane preparations of these cells was blocked by anti-class II major histocompatibility complex (MHC) monoclonal antibodies (mAb), whereas various anti-CD4 mAb had differential blocking effects. Murine L cells, or EBV-LCL transfected with CD4 could not replace CD4+ T cell clones. These results indicate that, although CD4 and class II MHC antigens are required for productive CD4+ T cell clone-B cell interactions, an additional signal, provided by a membrane associated (glyco)protein that is induced by activation of both TcR alpha beta and TcR gamma delta, CD4+ T cells, is needed for induction of IgE production in the presence of IL-4.     

Functional expression of CD28 on T cell antigen receptor gamma/delta-bearing T lymphocytes

T lymphocytes express a T cell antigen receptor (TcR) complex composed of either an TcR alpha/beta or TcR gamma/delta heterodimer in noncovalent association with the CD3 glycoproteins. CD28, a 44-kDa disulfide-linked homodimer, is present on the surface of the majority of TcR alpha/beta-bearing T lymphocytes. Monoclonal antibodies (mAb) directed against CD28 potentiate activation signals delivered through the CD3/TcR alpha/beta complex. Herein, we demonstrate that CD28 is expressed on approximately 40%-60% of TcR gamma/delta-bearing T lymphocytes in most donors. Anti-CD28 mAb substantially augmented proliferative signals delivered through the TcR gamma/delta, demonstrating the presence of functional CD28 molecules on TcR gamma/delta-bearing T lymphocytes. The majority of TcR gamma/delta+ thymocytes also expressed CD28.     

Phenotype, activation and lymphokine secretion by gamma/delta T lymphocytes from schistosomiasis and carcinoma of the urinary bladder.

In humans the majority of the CD3+ T cells usually express an alpha/beta T cell receptor (TcR) and a minority express a gamma/delta TcR. The CD3+ TcR alpha/beta and CD3+ TcR gamma/delta cells from blood of the patients with schistosomiasis with carcinoma of the urinary bladder (SCB) were analyzed for phenotype, activation, secretion of interleukin 2 (IL 2). B cell growth factor (BCGF) and B cell differentiation factor (BCDF), as well as for autologous (AMLR) and allogeneic (MLR) mixed lymphocyte reaction. Patients with SCB had a highly increased percentage of CD3+ TcR gamma/delta and a decreased percentage of CD3+ TcR alpha/beta T cells in their circulation. These CD3+ TcR gamma/delta T cells expressed the CD25 (IL 2 receptor), CD38, CD71 (transferrin receptor) and HLA-DR activation antigens at a higher intensity after in vitro stimulation with recombinant IL 2, phytohemagglutinin and soluble egg antigen (from Schistosoma haematobium). The SCB patients' CD3+ TcR gamma/delta T cells were highly deficient in secretion of IL 2 but produced highly elevated levels of BCGF and BCDF. On the contrary, both BCGF and BCDF activities of the CD3+ TcR alpha/beta T cells were decreased. Moreover, CD3+ TcR gamma/delta T cells demonstrated highly deficient AMLR and MLR activity. These observations suggest a possible role of CD3+ TcR gamma/delta T cells in the immune response and the disease pathogenesis in human schistosomiasis infections.     

Selective expression of CD8 alpha (Ly-2) subunit on activated thymic gamma/delta cells

Although most cells of the T cell receptor (TcR) gamma/delta lineage are CD4-CD8-, some gamma/delta cells express CD8. We show here that mouse thymic gamma/delta cells express CD8 following in vitro activation by concanavalin A. Staining with monoclonal and polyclonal antibodies to the CD8 alpha (Ly-2) and CD8 beta (Ly-3) subunits indicates that only CD8 alpha is expressed by these activated TcR gamma/delta cells. Furthermore, immunoprecipitation and reduced/nonreduced gel analysis reveals that thymic gamma/delta cells express CD8 alpha as a homodimer. In contrast, similarly activated TcR alpha/beta cells express a conventional CD8 alpha/CD8 beta heterodimer.     

Existence of mature human CD4+ T cells with genuine class I restriction.

A human T cell receptor (TcR) alpha/beta CD4+CD8-T cell clone (R416) is reactive with the minor histocompatibility antigen H-Y in the context of major histocompatibility complex (MHC) class I and not class II molecules. Therewith clone R416 violates the so-called specificity association of mature TcR alpha/beta+ T cells. R416 displays H-Y-specific, HLA-A2-restricted proliferation as well as cytotoxicity in vitro. Its fine specificity is identical to that of a classical H-Y-reactive CD4-CD8+ MHC class I-restricted CTL clone, showing that CTL expressing either CD4 or CD8 can display identical antigenic specificities. Exploiting the MHC class I restriction of this CD4+ T cells clone, it was found that interaction of CD4 with non-TcR-bound MHC class II molecules does not contribute to antigen specific activation of these CD4+ T cells. This coreceptor-mismatched T cell clone was not generated in vitro but obtained by expansion of CD8-depleted peripheral blood mononuclear cells of a female who had been immunized against H-Y. The existence of such MHC class I-restricted mature TcR alpha/beta+ T cells expressing CD4 and not CD8 is relevant because it indicates that the generally accepted model for thymic selection, in which the TcR specificity alone determines CD4/CD8 expression of mature thymocytes, may not be absolute.     

Phenotypical and Functional Characterization of Small Intestinal TcRγδ+ T Cells in Coeliac Disease

Increased numbers of TcR gamma delta + T cells are present in the small intestinal epithelium of patients with coeliac disease (CoD). Their function, however, is unknown. In order to facilitate detailed functional studies, intestinal gamma delta T cells have been isolated from small intestinal biopsies of patients with CoD (n = 18) and controls (n = 14). As expected, increased numbers of V delta 1+ TcR gamma delta + T cells were detected in freshly isolated intraepithelial cell suspensions (IEL) from CoD patients. Also, in the in vitro expanded IEL T-cell populations from CoD patients the numbers of V delta 1+ TcR gamma delta + T cells were increased compared with similar cell cultures from control patients. From IEL cultures derived from six CoD patients, 107 T-cell clones were generated by limiting dilution and analysed. Sixty of these clones were either CD4 or CD8 positive TcR alpha beta + clones. The remaining 47 clones expressed the TcR gamma delta. Further phenotypical analysis of the gamma delta T-cell clones indicated that the TcR gamma delta + T-cell population in the small intestinal epithelium of CoD patients is heterogeneous: four TcR gamma delta phenotypes could be detected and, although the majority of the TcR gamma delta + T cells were CD4 CD8, gamma delta T-cell clones expressing either a CD8 alpha alpha homodimer, a CD8 alpha beta heterodimer or CD4 were also identified. In contrast to the TCR alpha beta + IEL, most TcR gamma delta + IEL were CD5 negative. Furthermore, biochemical analysis indicated that the increase in V delta 1+ gamma delta T cells in the small intestinal epithelium of CoD patients was not the result of a monoclonal expansion. The small intestinal epithelium-derived gamma delta T-cell clones were functional in vitro since the majority of these clones were able to lyse target cell lines such as K562. Molt4 and Daudi. These novel findings therefore indicate that the gamma delta T cells in the small intestine of CoD patients represent a heterogeneous population and that such cells are functional in vitro. The isolation and the in vitro propagation and cloning of these cells may open new avenues for the study of the putative immune mechanisms leading to coeliac disease.     

Monoclonal antibodies which react with the T cell receptor gamma/delta recognize different subsets of CD3+WT31- T lymphocytes

A polyclonal CD3+4-8-WT31- cell line (termed SFG) was utilized for mice immunization in order to produce monoclonal antibodies (mAb) specific for the T cell receptor (TcR) gamma/delta. Hybrid supernatants were screened for their ability to induce SFG cells (but not conventional TcR alpha/beta + CTL lines) to kill the murine Fc receptor-positive P815 target cell line. Three hybrids, termed G1, A13 and F11, were isolated according to this screening. By indirect immunofluorescence G1 mAb reacted with 65% of SFG cells, while A13 stained 26% and F11 75% of cells. Double-fluorescence analysis revealed that G1 and A13 mAb identify two distinct, non-overlapping subsets of cells present in the SFG cell line. The reactivity of the mAb was also analyzed on a panel of representative TcR gamma/delta clones. G1 mAb reacted with 5 clones, that were also stained by the previously described BB3 mAb (recognizing the disulfide-linked form of TcR gamma/delta). These clones failed to react with A13 and delta-TCS-1 mAb (the latter of which is known to react with a non-disulfide-linked form of TcR gamma/delta). Out of six clones that reacted with A13 mAb, four were also delta-TCS-1+, whereas two were delta-TCS-1- and none of them reacted with G1, (or BB3) mAb. In contrast to the mAb above, F11 brightly stained the G1+A13- clones and more weakly the G1-A13+ clones. Moreover, F11 efficiently triggered both types of clones to kill the P815 target cells while G1 and A13 were able to trigger only G1+ or A13+ clones, respectively. None of the mAb above reacted with a large number of CD3+WT31+ clones. Antibody-induced surface antigen modulation experiments indicated that molecules recognized by G1, A13 and F11 were physically associated on cell surface with CD3 determinants. In addition, immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (performed on 125I-surface-labeled TcR gamma/delta+ clones) revealed that molecules recognized by G1, A13 and F11 displayed an apparent mol. wt. corresponding to that of CD3-associated TcR molecules, immunoprecipitated by anti-CD3 mAb from the same clones.     

Isolation and characterization of human peripheral blood CD4+ T cell clones expressing gamma delta T cell receptors.

Rare T cell clones bearing both CD4 and gamma delta T cell receptors (TcR gamma delta) were obtained from human peripheral blood by cell sorting using anti-CD4 and anti-TCR delta 1 antibodies. All the clones established were reactive with anti-TcR gamma delta 1 antibody, whereas only about 20% of the clones showed reactivity with anti-delta TCS1 antibody. Unlike most CD4+ T cells bearing TcR alpha beta, all the clones tested showed lectin-dependent and anti-CD3 antibody-redirected cytolytic activity. About 60% of the clones exhibited natural killer cell-like activity. Immunoprecipitation analysis of TcR gamma delta showed that each clone expressed either a disulfide-linked or non-disulfide-linked heterodimer consisting of 37-44-kDa TcR gamma and TcR delta chains.     

Identification and characterization of rat T cell subpopulations expressing T cell receptors alpha/beta and gamma/delta

Peripheral lymphoid organs of the rat were investigated for the presence of lymphocytes that expressed the pan-T cell markers CD5 and OX-52 but not the T cell receptor (TcR) alpha/beta. Two such populations were identified: 2% to 4% of lymphocytes in adult spleen, lymph nodes and peripheral blood are CD5+ TcR alpha/beta- and express the OX-52 antigen at the same density as TcR alpha/beta+ T-cells. About 90% of these cells are CD8+. A second population is CD5-, CD8+ and OX-52low. Radioimmunoprecipitation from digitonin lysates of surface-labeled cells with an anti-CD3 antiserum showed that the CD5+, but not the CD5- population of TcR alpha/beta- cells expresses a CD3-associated disulfide-linked cell surface molecule of about 100 kDa apparent mol. mass. Upon reduction, one major band, migrating with 48 kDa was observed. A band of the same size was obtained with an anti-human delta chain peptide antiserum, indicating that the CD3-associated non-TcR alpha/beta molecule is the rat TcR gamma/delta. Functional assays showed that most, if not all natural killer (NK) cell activity is present in the CD5(-)-OX-52low population. Reactivity to foreign major histocompatibility complex (MHC) antigens in mixed lymphocyte reaction was exclusively found in TcR alpha/beta+ splenic T cells. It is concluded that rat gamma/delta T cells in the spleen do not contain a high frequency of cells with specificity for foreign MHC antigens. The seeding of the periphery with alpha/beta and the presumptive gamma/delta T cells was followed from birth. Most prominently in the spleen, alpha/beta T cells reached adult levels much later than gamma/delta T cells. Taken together, these findings demonstrate the expression of the TcR gamma/delta on a minor population of peripheral rat T cells with the predominant phenotype CD4-CD8+ that has no NK cell activity when freshly isolated and does not contain a high frequency of alloreactive cells.     

Morphological features of cloned lymphocytes expressing gamma/delta T cell receptors

We have analyzed the morphological characteristics of human T lymphocytes bearing CD3-associated T cell receptor (TcR) gamma and delta chains. BB3 and delta-TCS1 monoclonal antibodies (mAb) were used to identify two distinct, nonoverlapping populations of TcR gamma/delta + cells which express the products of V delta 2 and V delta 1 gene segments, respectively. In the peripheral blood, most V delta 1+ (delta TCS-1+) lymphocytes express the non-disulfide-linked form of receptor whereas V delta 2+ (BB3+) cells express the disulfide-linked form. The majority of cloned TcR gamma/delta + cells exhibit a growth pattern different from that of conventional TcR alpha/beta + cells as they adhere promptly to surfaces and undergo morphological changes which can be summarized as follows: cells spread on the surface, form a distinct uropod and, in the final phase of adherence, emit long filopodia ending with adhesion plaques. Immunofluorescence studies of TcR gamma/delta + clones demonstrated the presence of submembraneous actin microfilaments and actin-binding protein confirming that these cells are capable of active motility which is related to the propensity of TcR gamma/delta + cells to home to epithelia. Scanning electron microscope analyses of effector/target cell conjugates showed that in TcR gamma/delta + cells the region of the uropodia next to the cell body is responsible for the binding to tumor target cells. Interestingly, immunofluorescence analyses revealed that LFA-1 molecules are predominantly distributed in the uropodium whereas they are virtually absent in the cell bodies. These morphological characteristics of TcR gamma/delta + cells may pertain to defensive mechanisms the mucosal level.     

Expression of functional CD8alpha Beta heterodimer on rat gamma delta T cells does not correlate with the CDR3 length of the TCR delta chain predicted for MHC class I-restricted antigen recognition

In accordance with their lack of MHC restriction, most mouse and human gamma delta T cells express neither the CD4 nor CD8(alpha beta) coreceptor. In striking contrast, up to 80% of splenic rat gamma delta T cells express the CD8alpha beta isoform of CD8, which for the alpha beta T cell subset serves as a marker for MHC class I-restricted cells. We compared CD8 on alpha beta and gamma delta T cells with regard to co-stimulatory function and correlation of CD8 expression with TCRDV usage and CDR3delta length. In both subsets, CD8 acted as a co-stimulatory molecule in vitro and was found to bind the kinase lck efficiently. No differences between the CDR3delta length spectra of CD8+ and CD8- gamma delta T cells or between unselected thymic and peripheral gamma delta T cells were found. As seen in man and mice, CDR3delta were rather long, a structural feature which can be expected to interfere with an alpha beta TCR-like mode of MHC class I binding. In summary, CD8 expressed by rat gamma delta T cells is a molecule with the potential to act as a coreceptor, but its expression gives no indication for antigen recognition analogous to that of MHC class I-restricted alpha beta T cells.     

Phenotypical heterogeneity among human T cell receptor gamma/delta-expressing clones derived from peripheral blood

Human T cell clones expressing the T cell receptor (TcR) gamma/delta were isolated from peripheral blood lymphocytes of two unrelated donors. The TcR gamma/delta+ clones derived from one of these donors were all of the Ti gamma A+, delta-TCS1-, BB3+ phenotype indicating the exclusive use of the V gamma 9 and V delta 3 gene segments. In contrast, the T cell clones derived from the second donor were either Ti gamma A+, delta-TCS1-, BB3+:Ti gamma A-, delta-TCS1+, BB3- or Ti gamma A-, delta-TCS1-, BB3-. The delta-TCS1 determinant was expressed on both nondisulfide- and disulfide-linked TcR gamma/delta. Northern blot and DNA sequence analysis indicated that the Ti gamma A-, delta-TCS1-, BB3- clones do use the V delta 1 gene segment demonstrating that the delta-TCS1 monoclonal antibody does not react with all TcR gamma/delta using this particular gene segment. In contrast to the delta-TCS1+ T cell clones, the V delta 1+ delta-TCS1- T cell clones were found to express V delta 1 in conjunction with the J delta 3 gene segment suggesting that this particular V delta 1-J delta 3 combination is not recognized by the delta-TCS1 monoclonal antibody. In T cell clones derived from one individual the V delta 1 gene segment was found to be expressed with either J delta 1, J delta 2 or J delta 3. Heterogeneity among the 18 clones was detected with respect to the expression of the CD4, CD5 and CD8 antigens: one clone was CD4+, nine clones were CD5+ and two clones were CD8+. Thus, in this panel of clones, heterogeneity exists both with regard to CD antigen expression and the TcR gamma/delta phenotype. Also, our results indicate that the delta-TCS1 monoclonal antibody does not react with all TcR gamma/delta using the V delta 1 gene segment.     

Growth-dependent variation of major histocompatibility complex (MHC) restriction and expression of Ly-2 and CD3/alpha/beta T cell receptor in cloned cytotoxic T cells

The specificity of major histocompatibility complex (MHC)-restricted antigen recognition by cytotoxic T cells (CTL) has been clearly correlated to the alpha/beta T cell receptor (TcR) complex on the T cell surface. Occasional changes in the specificity of in vitro cultivated CTL clones, therefore, have been suspected to result from alterations of the genes coding for the TcR alpha and/or beta chain. Here we demonstrate that pronounced variations in the stringency of MHC restriction, previously reported to occur during long-term culture of 2,4,6-trinitrophenyl (TNP)-specific CTL clones, may occur rapidly in a growth-dependent, reversible manner, i.e. without structural TcR variation. Several H-2b TNP-specific CTL clones were shown to possess strong cross-reactivity for H-2k TNP target cells when seeded at low cell numbers, but exhibit reduced or undetectable cross-reaction to H-2k TNP in high-density cultures. Another clone revealed "heteroclitic" properties with significantly stronger cytotoxic activity towards allogeneic (H-2k) than syngeneic (H-2b) TNP-modified target cells. In this case dilute cultures appeared as exclusively allo-MHC restricted, whereas dense cultures were allo/self cross-restricted. In all instances these phenomena were accompanied by cell density-dependent quantitative changes in the expression of Ly-2 and T cell antigen receptor. CTL from dilute cultures had at least 2-fold higher surface concentrations of Ly-2 and CD3 antigens than cells from dense cultures while other surface markers such as Thy-1 or LFA-1 were completely identical. No such effects were observed for CTL clones exhibiting cell density-independent specificity patterns. We conclude from these findings that (a) the stringency of MHC restriction specificity may be significantly affected by the amount of expressed TcR and/or Ly-2 molecules, (b) CTL possess mechanisms to regulate Ly-2 and TcR expression and, hence, their MHC-restricted antigen recognition, and (c) the ability to regulate Ly-2 and TcR expression may be altered during prolonged culture of a CTL clone.     

Autoreactive T cells in normal mice: unrestricted recognition of self peptides on dendritic cell I-A molecules by CD4-CD8- T cell receptor alpha/beta+ T cell clones expressing V beta 8.1 gene segments

CD4-CD8- double-negative (DN) and CD4+CD8- T cell clones were derived from splenic precursors resistant to killing by anti-Thy-1, -CD5, -CD4 and -CD8 monoclonal antibodies and complement. Both DN and CD4+ clones express functional T cell receptor (TcR) alpha/beta and exhibit strong autoreactivity in vitro. DN cells can be induced to proliferate by dendritic cells (DC) of all haplotypes tested, although this activation is inhibited by antibodies specific for I-A determinants expressed on the stimulatory DC. In contrast, CD4+ clones only respond to syngeneic or I-Ad-compatible DC. Both DN and CD4+ autoreactive clones do not proliferate when cultured with class II+ H-2d normal or tumor macrophages and B cell lines or with class II-transfected L cells, suggesting that these cells recognize self peptides only present on the surface of DC. Despite their phenotype resembling that of immature thymocytes and their inability to interact directly with B lymphocytes, DN cloned T cells, like CD4+ T cells, exhibit nonspecific helper functions and can induce polyclonal B cell proliferation and differentiation. DN TcR alpha/beta+ peripheral T cells represent, like TcR gamma/delta+ lymphocytes, a new T cell subset physiological role whose remains to be defined.     

Recognition of a particular HLA-DQ heterodimer by a human gamma/delta T cell clone

Peripheral blood mononuclear cells from a single donor were depleted of T cell receptor (TcR) alpha/beta T cells, stimulated with allogeneic cells, and gamma/delta T lymphocyte clones (TLC) were isolated by limiting dilution. Five TLC were cytotoxic against B-lymphoblastoid cell lines from the stimulating cell donor, and demonstrated a restricted allospecificity in panel cell studies. One of these, gamma/delta TLC RNG-135, was studied in more detail. Its phenotype was CD3+ TcR alpha/beta - TcR gamma/delta + BB3- delta TCS1+ CD4- CD8-. Inhibition experiments using monoclonal antibodies indicated that the cytotoxicity of TLC RNG-135 was mediated through the TcR gamma/delta, and directed against an HLA-DQ molecule. In extended panel cell experiments, this gamma/delta TLC only lysed cells carrying the DQA1*0501 and DQB1*0301 genes, either in cis position (on the DR5, DQw7 haplotype), or in trans position (the donor of the stimulating cells, DR3,4; DQw2, w7). Thus, it appears that gamma/delta T cells may recognize a particular HLA-DQ alpha/beta heterodimer, which may be encoded by DQA1 and B1 genes both in cis and trans position.     

Human TcR gamma delta+ lymphocyte response on primary exposure to Plasmodium falciparum.

In 29 patients experiencing their first P. falciparum malarial attack, blood levels of TcR gamma delta+ lymphocytes were studied from the onset of infection to up to 6-9 months later. Blood TcR gamma delta+ lymphocytes, revealed using the TcR delta 1 monoclonal antibody (MoAb), were increased both in absolute and relative numbers. Alterations lasted for up to 3-4 months following the attack. A Ti gamma A/BB3 reactive V gamma 9 subset was preferentially amplified. In vitro, TcR gamma delta+ lymphocytes from both malaria-sensitized and unprimed donors responded to P. falciparum schizont extract (PFSE). PFSE-stimulated polyclonal T cell lines consisted principally in TcR gamma delta+ cells with a Ti gamma A+/BB3+ phenotype. Several TcR gamma delta+ T cell clones obtained from patients recovering from acute malarial attack were maintained in the presence of PFSE and autologous irradiated PBL. They belong to the V gamma 9 subset. In long-term cultures, TcR gamma delta+ clones progressively lost their capacity to react to PFSE antigen while they were able to proliferate and to exert cytotoxic activity in response to autologous TcR alpha beta+, PFSE-specific T lymphocyte clones. This suggests that regulatory interactions occur between activated TcR gamma delta+ and TcR alpha beta+ cells generated by P. falciparum. Sequential variations in blood TcR gamma delta+ and TcR alpha beta+ lymphocyte levels after primary exposure to P. falciparum suggest that such regulatory interactions may occur in vivo.     

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.