Induction of lysis by T cell receptor gamma delta+/CD3+ T lymphocytes via CD2 requires triggering via the T11.1 epitope only

The requirements for activation of the lytic machinery through CD2 of TCR gamma delta+/CD3+ cells were examined, by utilizing bispecific heteroconjugates containing anti-CD2 mAb cross-linked to anti-DNP. Contrary to the CD2 activation requirements in TCR alpha beta+/CD3+ cells, cytotoxic activity in TCR gamma delta+/CD3+ clones and TCR-/CD3- NK cell clones can be induced by heteroconjugates containing a single anti-CD2 (OKT11.1) mAb. Activation of TCR gamma delta+/CD3+ cells via CD2 is independent of heteroconjugates binding to CD16 (Fc gamma RIII), because heteroconjugates prepared from Fab fragments induced equal levels of lysis. Moreover, anti-CD16 mAb did not inhibit triggering via CD2 in TCR gamma delta+/CD3+ cells. In TCR-/CD3- NK cells, however, induction of cytotoxicity via CD2 is co-dependent on interplay with CD16. Anti-CD3 mAb blocked the anti-CD2 x anti-DNP heteroconjugate-induced cytotoxicity of TCR gamma delta+/CD3+ cells, indicating a functional linkage between CD2 and CD3 on these cells. We conclude that induction of lysis via CD2 shows qualitatively different activation requirements in TCR gamma delta+/CD3+, TCR alpha beta+/CD3+ CTL and TCR-/CD3- NK cells.     

Expression of murine IL-2 receptor beta-chain on thymic and splenic lymphocyte subpopulations as revealed by the IL-2-induced proliferative response in human IL-2 receptor alpha-chain transgenic mice

Lymphocytes from the human (h) IL-2R alpha chain transgenic mice (TGM) constitutively express high affinity binding sites for hIL-2, consisting of transgenic h-IL-2R alpha and endogenous murine IL-2R beta, and therefore easily proliferate in vitro in response to hIL-2. Our study was undertaken to clarify the hIL-2-responsive lymphocyte subsets in the TGM, which should most likely reflect the normal distribution of m IL-2R beta expression. In both thymus and spleen, the majority of expanded cells by hIL-2 was CD3+CD4-CD8+ TCR alpha beta+ cells. The proliferation of CD4+ cells was not observed at all from either organ despite the expression of transgenic hIL-2R alpha. Potent cellular proliferation was also observed from the thymocytes that had been depleted of CD8+ cells, the expanded cells consisting of CD3- (15-40%) and CD3+ populations (60-85%). Among CD3+ cells, approximately the half portion expressed TCR alpha beta, whereas the other half was suggested to express TCR gamma delta. A variable portion (5-20%) of the CD3+ cells expressed CD8 (Lyt-2) in the absence of Lyt-3, and the CD3+CD8+ cells were confined preferentially to the TCR alpha beta- (TCR gamma delta+) population. In the culture of splenocytes depleted of CD8+ cells, however, the proliferated cells were mostly CD3-CD4-CD8-TCR-Mac1-, whereas a minor portion (10-30%) was CD3+CD4-CD8-TCR alpha beta- (TCR gamma delta+. Analysis of TCR genes at both DNA and mRNA levels confirmed the phenotypical observations. These results strongly suggested that IL-2R beta was constitutively and selectively expressed on the primary murine thymocytes and splenic T and NK cells, except for CD4+ cells in both organs.     

Cytokine production by mature and immature CD4-CD8- T cells. Alpha beta-T cell receptor+ CD4-CD8- T cells produce IL-4.

This study follows our previous investigation describing the production of four cytokines (IL-2, IL-4, IFN-gamma, and TNF-alpha) by subsets of thymocytes defined by the expression of CD3, 4, 8, and 25. Here we investigate in greater detail subpopulations of CD4-CD8- double negative (DN) thymocytes. First we divided immature CD25-CD4-CD8-CD3- (CD25- triple negative) (TN) thymocytes into CD44+ and CD44- subsets. The CD44+ population includes very immature precursor T cells and produced high titers of IL-2, TNF-alpha, and IFN-gamma upon activation with calcium ionophore and phorbol ester. In contrast, the CD44- subset of CD25- TN thymocytes did not produce any of the cytokines studied under similar activation conditions. This observation indicates that the latter subset, which differentiates spontaneously in vitro into CD4+CD8+, already resembles CD4+CD8+ thymocytes (which do not produce any of the tested cytokines). We also subdivided the more mature CD3+ DN thymocytes into TCR-alpha beta- and TCR-gamma delta-bearing subsets. These cells produced cytokines upon activation with solid phase anti-CD3 mAb. gamma delta TCR+ DN thymocytes produced IL-2, IFN-gamma and TNF-alpha, whereas alpha beta TCR+ DN thymocytes produced IL-4, IFN-gamma, and TNF-alpha but not IL-2. We then studied alpha beta TCR+ DN T cells isolated from the spleen and found a similar cytokine production profile. Furthermore, splenic alpha beta TCR+ DN cells showed a TCR V beta gene expression profile reminiscent of alpha beta TCR+ DN thymocytes (predominant use of V beta 8.2). These observations suggest that at least some alpha beta TCR+ DN splenocytes are derived from alpha beta TCR+ DN thymocytes and also raises the possibility that these cells may play a role in the development of Th2 responses through their production of IL-4.     

Signal transduction of gamma/delta T cell antigen receptor with a novel mitogenic anti-delta antibody

Human T lymphocytes express either alpha/beta- or gamma/delta-TCR in association with the CD3 complex. We have isolated a mAb, delta TCS1, that immunoprecipitated the gamma/delta-TCR heterodimer from cell lysates of Peer and Molt-13 leukemia cell lines. After dissociation of the gamma- and delta-chains of TCR by treatment with SDS, delta TCS1 specifically immunoprecipitated the delta-chain. This antibody bound to the surface of other gamma/delta-positive T cell lines and clones and was able to stimulate the proliferation of a minor cell population (0.9 to 4.0%) of resting human PBL. Upon binding to gamma/delta-TCR-bearing Molt-13 cells and PBL, delta TCS1 elicited a fura-2 Caa+ signal indicating that the gamma/delta-receptor is functionally similar to the alpha/beta-heterodimer. These data indicate that the delta TCS1 antibody recognizes an epitope on TCR delta-chain and its mitogenic activity should be useful in characterizing the functional properties of human gamma/delta-positive T lymphocytes.     

Regulatory role of peritoneal NK1.1+ alpha beta T cells in IL-12 production during Salmonella infection.

NK1.1+ alpha beta T cells emerge in the peritoneal cavity after an i.p. infection with Salmonella choleraesuis in mice. To elucidate the role of the NK1.1+ alpha beta T cells during murine salmonellosis, mice lacking NK1.1+ alpha beta T cells by disruption of TCR beta (TCR beta-/-), beta 2m (beta 2m-/-), or J alpha 281 (J alpha 281-/-) gene were i.p. inoculated with S. choleraesuis. The peritoneal exudate T cells in wild type (wt) mice on day 3 after infection produced IL-4 upon TCR alpha beta stimulation, whereas those in TCR beta-/-, beta 2m-/-, or J alpha 281-/- mice showed no IL-4 production upon the stimulation, indicating that NK1.1+ alpha beta T cells are the main source of IL-4 production at the early phase of Salmonella infection. Neutralization of endogenous IL-4 by administration of anti-IL-4 mAb to wt mice reduced the number of Salmonella accompanied by increased IL-12 production by macrophages after Salmonella infection. The IL-12 production by the peritoneal macrophages was significantly augmented in mice lacking NK1.1+ alpha beta T cells after Salmonella infection accompanied by increased serum IFN-gamma level. The aberrantly increased IL-12 production in infected TCR beta-/- or J alpha 281-/- mice was suppressed by adoptive transfer of T cells containing NK1.1+ alpha beta T cells but not by the transfer of T cells depleted of NK1.1+ alpha beta T cells or T cells from J alpha 281-/- mice. Taken together, it is suggested that NK1. 1+ alpha beta T cells eliciting IL-4 have a regulatory function in the IL-12 production by macrophages at the early phase of Salmonella infection.     

Novel function for intestinal intraepithelial lymphocytes. Murine CD3+, gamma/delta TCR+ T cells produce IFN-gamma and IL-5.

Intestinal intraepithelial lymphocytes (IEL) from mice are greater than 80% CD3+ T cells and could be separated into four subsets according to expression of CD4 and CD8. In our studies designed to assess the functions of IEL, namely, cytokine production, it was important to initially characterize the various subsets of T cells that reside in IEL. The major subset was CD4-, CD8+ (75% of CD3+ T cells), which contained approximately 45 to 65% gamma/delta TCR+ and 35 to 45% alpha/beta TCR+ T cells. Approximately 7.5% of IEL T cells were CD4-, CD8- (double negative) and gamma/delta+ population. On the other hand, CD4+, CD8+ (double positive) and CD4+, CD8- fractions represented 10% and 7.5% of CD3+ T cells, respectively, which were all alpha/beta TCR+. Inasmuch as CD3+, CD4-, CD8+ T cells are a major subset of IEL which contain both gamma/delta TCR or alpha/beta TCR-bearing cells, the present study was focused on the capability of this subset of IEL T cells to produce the cytokines IFN-gamma and IL-5. Both gamma/delta TCR+ and alpha/beta TCR+ IEL spontaneously produced IFN-gamma and IL-5, although higher frequencies of cytokine spot-forming cells were associated with the alpha/beta TCR+ subset. Approximately 30% of CD8+, gamma/delta TCR+ cells produced both cytokines, whereas approximately 90% of alpha/beta TCR+ T cells produced either IFN-gamma or IL-5. Both gamma/delta TCR+ and alpha/beta TCR+ IEL possessed large quantities of cytokine-specific mRNA, clearly showing that these IEL were programmed for cytokine production. When IEL were activated with anti-gamma/delta or anti-CD8 antibodies, higher numbers of IFN-gamma and IL-5 spot-forming cells were noted. The present study has provided direct evidence that a major function of IEL involves cytokine production, and this is the first evidence that gamma/delta TCR+ cells in IEL possess the capability of producing both IL-5 and IFN-gamma.     

Differential expression of CD8 alpha and CD8 beta associated with MHC-restricted and non-MHC-restricted cytolytic effector cells

The differential expression of the alpha and beta chains of the CD8 glycoprotein was examined in three functionally distinct cytolytic effector cell populations: (i) T cells (CD3+ CD56-), (ii) NK cells (CD56+ CD3-), and (iii) non-MHC-restricted T cells (CD56+ CD3+). Twenty-four percent of T cells were CD8+, and they consistently coexpressed both CD8 alpha and CD8 beta. Moreover, CD8+ T cells uniformly expressed high-density CD8 alpha. Forty percent of NK cells were CD8+ but the vast majority (approximately 75%) expressed only CD8 alpha without CD8 beta. In addition, CD8+ NK cells uniformly expressed low-density CD8 alpha. In comparison, 75% of non-MHC-restricted T lymphocytes were CD8+ but they displayed an intermediate phenotype: 60% coexpressed CD8 alpha and CD8 beta while 40% expressed only CD8 alpha. Within this population, CD8 alpha was expressed at high density, similar to that of T cells. Following IL-2 activation, enhancement of non-MHC-restricted cytotoxicity was not associated with any changes in either the quantitative or qualitative pattern of expression of CD8 alpha or CD8 beta by these cells. Addition of either anti-CD8 alpha or anti-CD8 beta mAb did not alter non-MHC-restricted cytotoxicity of either CD56+ CD3- or CD56+ CD3+ effector cells. However, within the CD56+ cell population, non-MHC-restricted cytotoxicity was almost entirely found within the CD8- and CD8 alpha + beta- populations, and both subsets displayed a similar level of killing. In contrast, CD8 alpha+ beta+ cells exhibited very little non-MHC-restricted cytotoxicity. Thus, the coexpression of CD8 alpha and CD8 beta in conjunction with the TCR/CD3 complex appears to characterize MHC restricted cells while the expression of CD8 alpha alone is associated with non-MHC-restricted cytotoxicity. Taken together, these findings suggest that neither CD8 alpha nor CD8 beta is involved in the initial phases of target cell binding or recognition during NK cell-mediated lysis. However, the selective expression of CD8 alpha by a large fraction of non-MHC-restricted effector cells suggests that this antigen may play a different functional role in this unique subset of cytolytic lymphocytes.     

T cell receptor/CD3-signaling induces death by apoptosis in human T cell receptor gamma delta + T cells.

mAb directed against the TCR/CD3 complex activate resting T cells. However, TCR/CD3 signaling induces death by apoptosis in immature (CD4+CD8+) murine thymocytes and certain transformed leukemic T cell lines. Here we show that anti-TCR and anti-CD3 mAb induce growth arrest of cloned TCR-gamma delta + T cells in the presence of IL-2. In the absence of exogenous IL-2, however, the very same anti-TCR/CD3 mAb stimulated gamma delta (+)-clones to proliferation and IL-2 production. In the presence of exogenous IL-2, anti-TCR/CD3 mAb induced the degradation of DNA into oligosomal bands of approximately 200 bp length in cloned gamma delta + T cells. This pattern of DNA fragmentation is characteristic for the programmed cell death termed apoptosis. These results demonstrate that TCR/CD3 signaling can induce cell death in cloned gamma delta + T cells. In addition, this report is the first to show that apoptosis triggered by TCR/CD3 signaling is not restricted to CD4+CD8+ immature thymocytes and transformed leukemic T cell lines but can be also observed with IL-2-dependent normal (i.e., TCR-gamma delta +) T cells.     

Distribution of T cells bearing different forms of the T cell receptor gamma/delta in normal and pathological human tissues

Frozen sections from normal and pathologic human tissues were immunostained by the APAAP technique with three mAb directed against different epitopes of the TCR gamma delta; TCR delta 1 which binds to all cells bearing the TCR gamma delta; BB3 and delta TCS1 which, by immunoprecipitation studies, appear to react respectively with the disulfide-linked and nondisulfide-linked form of the TCR gamma delta. In normal thymus, TCR delta 1+ cells accounted for approximately 2% of the CD3+ thymocytes and were about three times more numerous in the medulla than in the cortex. TCR delta 1+ cells were mostly constituted by the delta TCS1 reactive subset (average ratio delta TCS1/BB3: 3.7). In the tonsil, the TCR delta 1+ cells (about 3% of CD3+ elements) were mainly located in the interfollicular area, where they frequently tended to arrange around high endothelium venules. In most samples, TCR delta 1+ cells were distributed beneath to the tonsil epithelium. Unlike thymus, the majority of TCR delta 1+ cells were usually constituted by the BB3-reactive subset (average BB3/delta TCS1 ratio: 2.0). A similar predominance of BB3+ over delta TCS1+ cells was also observed in normal peripheral blood. The spleen was the organ with the highest concentration of TCR delta 1+ cells that, like in the thymus, were mostly represented by delta TCS1+ elements. Noteworthy, the TCR delta 1+ cells were preferentially located in the splenic sinusoids while TCR alpha beta-bearing lymphocytes mostly occupied the periarteriolar sheaths of penicilliary arteries. The majority of neoplastic T cell proliferations studied lacked to express the TCR gamma delta. Two cases of beta F1-(TCR alpha beta-) T lymphoblastic lymphoma, however, were TCR gamma delta+ (delta TCS1+/BB3-). Both of them showed a stage II cortical phenotype, e.g., CD1+/CD3+/CD4+/CD8+/TCR delta 1+. Among inflammatory conditions, an increase of BB3+ cells was observed in close association with necrotic areas in cases of Kikuchi's and tuberculous lymphadenitis. The significance of this finding is under study.     

IL-2 and a contact-mediated signal provided by TCR alpha beta + or TCR gamma delta + CD4+ T cells induce polyclonal Ig production by committed human B cells. Enhancement by IL-5, specific inhibition of IgA synthesis by IL-4.

To study the role of T cells in T-B cell interactions resulting in isotype production, autologous purified human splenic B and T cells were cocultured in the presence of IL-2 and Con A. Under these conditions high amounts of IgM, IgG, and IgA were secreted. B cell help was provided by autologous CD4+ T cells whereas autologous CD8+ T cells were ineffective. Moreover, CD8+ T cells suppressed Ig production when added to B cells cocultured with CD4+ T cells. Autologous CD4+ T cells could be replaced by allogeneic activated TCR gamma delta,CD4+ or TCR alpha beta,CD4+ T cell clones with nonrelevant specificities, indicating that the TCR is not involved in these T-B cell interactions. In contrast, resting CD4+ T cell clones, activated CD8+, or TCR gamma delta,CD4-,CD8- T cell clones failed to induce IL-2-dependent Ig synthesis. CD4+ T-B cell interaction required cell-cell contact. Separation of the CD4+ T and B cells by semiporous membranes or replacement of the CD4+ T cells by their culture supernatants did not result in Ig synthesis. However, intact activated TCR alpha beta or TCR gamma delta,CD4+ T cell clones could be replaced by plasma membrane preparations of these cells. Ig synthesis was blocked by mAb against class II MHC and CD4. These data indicate that in addition to CD4 and class II MHC Ag a membrane-associated determinant expressed on both TCR alpha beta or TCR gamma delta,CD4+ T cells after activation is required for productive T-B cell interactions resulting in Ig synthesis. Ig production was also blocked by mAb against IL-2 and the IL-2R molecules Tac and p75 but not by anti-IL-4 or anti-IL-5 mAb. The CD4+ T cell clones and IL-2 stimulated surface IgM-IgG+ and IgM-IgA+, but not IgM+IgG- or IgM+IgA- B cells to secrete IgG and IgA, respectively, indicating that they induced a selective expansion of IgG- and IgA-committed B cells rather than isotype switching in Ig noncommitted B cells. Induction of Ig production by CD4+ T cell clones and IL-2 was modulated by other cytokines. IL-5 and transforming growth factor-beta enhanced, or blocked, respectively, the production of all isotypes in a dose-dependent fashion. Interestingly, IL-4 specifically blocked IgA production in this culture system, indicating that IL-4 inhibits only antibody production by IgA-committed B cells.     

Identification of a CD2- CD3+ T cell receptor-gamma+ peripheral blood lymphocyte subpopulation

We have identified, in a healthy individual, a sub-population of human peripheral lymphocytes which surface express a CD3-TCR-gamma complex recognized by anti-Ti gamma A mAb, while being unreactive with a phycoerythrin-conjugated anti-CD2 antibody with T11/1 specificity. Further immunofluorescence analyses performed on uncultured cells indicated that such a putative CD2-CD3+ phenotype was restricted to a fraction of those T lymphocytes which carry a surface receptor of the "second family" (gamma/delta). The actual lack of CD2 expression was confirmed by a subsequent series of cloning experiments which showed that none of the three well characterized CD2 epitopic clusters, namely T11/1, T11/2, and T11/3, were detectable on the surface of the relevant cells. The cultured CD2-, CD3+/TCR gamma + lymphocytes were found to display, as well as their CD2+ counterparts, both non-MHC-restricted cytotoxic function and proliferative responses induced via the gamma receptor complex. In contrast, the proliferative capacity of the CD2-, CD3+/TCR-gamma + cells observed in a culture system designed for in vitro expansion of lymphocytes with undefined specificity was extremely limited. This may relate to an impaired interaction of the CD2- cloned lymphocytes with lymphocyte function-associated (LFA)3+ irradiated cells present in the feeder layer. Further characterization of such minor CD2- T lymphocytes subsets may help to better understand the biologic relevance of the CD2/LFA3 pathway of cell-cell interaction.     

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.     

Long-term culture growth of CD4-CD8- lymphocytes exhibiting elevated non-MHC-restricted cytotoxic activity.

We have developed a culture system for "long-term" growth of human lymphokine-activated killer (LAK) cells exhibiting an elevated, wide-spectrum anti-tumor cytotoxicity. The system allows the exponential growth of monocyte- and B-lymphocyte-depleted CD4-CD8- lymphocytes in the presence of human AB serum and recombinant human interleukin-2 (IL-2) (2 x 10(2) U/ml) combined with interleukin (IL-1) beta (50 ng/ml). After 21 days in culture, these cells undergo massive amplification (i.e., the cell yield rises up to 30-120 times the starting values), and exhibit a marked anti-tumor cytotoxic activity against a panel of natural killer (NK)-resistant tumor cell lines. Interestingly, this activity correlates with the high level of perforin RNA. The membrane phenotypes of the final cell population, assessed by a panel of monoclonal antibodies (MoAbs) indicate a mixed population comprising two cell types in variable proportions (i) NKH-1+, T cell receptor (TCR) alpha/beta-, TCR gamma/delta-, CD3-, Leu 23+; (ii) NKH-(+), TCR alpha/beta-, TCR gamma/delta+, CD3+, Leu 23+. This culture system may provide a tool for cellular and molecular studies on the mechanisms of anti-tumor cytotoxicity, as well as the basis for new adoptive immunotherapy protocols in advanced cancers.     

IL-7 up-regulates IL-4 production by splenic NK1.1+ and NK1.1- MHC class I-like/CD1-dependent CD4+ T cells.

NK T cells are an unusual subset of T lymphocytes. They express NK1. 1 Ag, are CD1 restricted, and highly skewed toward Vbeta8 for their TCR usage. They express the unique potential to produce large amounts of IL-4 and IFN-gamma immediately upon TCR cross-linking. We previously showed in the thymus that the NK T subset requires IL-7 for its functional maturation. In this study, we analyzed whether IL-7 was capable of regulating the production of IL-4 and IFN-gamma by the discrete NK T subset of CD4+ cells in the periphery. Two hours after injection of IL-7 into mice, or after a 4-h exposure to IL-7 in vitro, IL-4 production by CD4+ cells in response to anti-TCR-alphabeta is markedly increased. In contrast, IFN-gamma production remains essentially unchanged. In beta2-microglobulin- and CD1-deficient mice, which lack NK T cells, IL-7 treatment does not reestablish normal levels of IL-4 by CD4+ T cells. Moreover, we observe that in wild-type mice, the memory phenotype (CD62L-CD44+) CD4+ T cells responsible for IL-4 production are not only NK1.1+ cells, but also NK1.1- cells. This NK1.1-IL-4-producing subset shares three important characteristics with NK T cells: 1) Vbeta8 skewing; 2) CD1 restriction as demonstrated by their absence in CD1-deficient mice and relative overexpression in MHC II null mice; 3) sensitivity to IL-7 in terms of IL-4 production. In conclusion, the present study provides evidence that CD4+MHC class I-like-dependent T cell populations include not only NK1.1+ cells, but also NK1.1- cells, and that these two subsets are biased toward IL-4 production by IL-7.     

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.     

CD4-CD8- human T cells: phenotypic heterogeneity and activation requirements of freshly isolated "double-negative" T cells

In the present study we have analyzed the in vitro activation requirements of freshly isolated CD4-CD8- "double-negative" (DN) human peripheral blood T cells. DN cells were isolated from E+ cells by removal of CD4+, CD8+, and CD16+ cells through consecutive steps of C'-mediated lysis and panning. While the majority (79.0 +/- 12.0%) of DN cells were TCR gamma delta+ as shown by staining with mAb TCR delta-1, a minor fraction (6.7 +/- 4.7%) expressed TCR alpha beta as revealed by staining with mAb BMA031. Within the gamma delta+ DN fraction, most cells reacted with mAb Ti gamma A which delineates a V gamma 9JPC gamma 1 epitope, whereas a minor fraction stained with mAb delta TCS-1 which identifies a V delta 1J delta 1 epitope. Functional studies performed at low cell number (1000) per microculture indicated that DN cells can be activated by anti-CD3 mAb, PHA and allogeneic stimulator cells, provided that exogenous growth factors are supplied. Both rIl-2 and rIl-4 acted as efficient growth factors for DN cells, and a synergistic stimulatory effect of rIl-2 and rIl-4 was observed when DN cells were cocultured with allogeneic LCL stimulator cells. As compared to unseparated E+ cells, isolated DN responder cells had a reduced capacity to secrete Il-2 upon PHA stimulation in the presence of LCL feeder cells. The majority of DN cells maintained their CD3+ CD4-CD8- phenotype upon coculture with allogeneic LCL stimulator cells. These data demonstrate that CD3+ DN cells in human peripheral blood are heterogeneous with respect to TCR expression. In addition, they show that freshly isolated DN cells are deficient in Il-2 production but may be normally stimulated by anti-CD3, PHA, or alloantigen if exogenous growth factors (rIL-2 and/or rIl-4) are provided.     

In vitro and in vivo activation of murine gamma/delta T cells induces the expression of IgA, IgM, and IgG Fc receptors.

The present studies examined resting and activated murine gamma/delta T lymphocytes, in vitro and in vivo, for surface expression of FcR. Polyclonal gamma/delta TCR+ lymphocytes selectively grown from the spleen and intestine of normal mice did not express FcR when the cells were in a resting state, but when cells were activated with anti-CD3 antibody virtually all of the splenic gamma/delta lymphocytes and a large subpopulation of the intestinal gamma/delta lymphocytes expressed IgA and IgM FcR. This was confirmed by using transgenic mice. Resting gamma/delta TCR+ lymphocytes from the spleen, thymus, lymph node, and blood of gamma/delta TCR transgenic mice did not express FcR for any of the five major classes of Ig H chains. Activation of the gamma/delta TCR+ cells via the CD3/TCR complex induced high levels of IgM and IgA FcR and low levels of IgG FcR. Finally, in hepatic granulomas of schistosome-infected mice, activated gamma/delta TCR+ cells are present and express high levels of IgA and IgM FcR and low levels of IgG FcR. These investigations establish that transition of gamma/delta TCR+ lymphocytes from a resting to an activated state (triggered via the T3Ti TCR complex) is accompanied by the induction of surface membrane receptors specific for Ig H chain isotypes. The activation-linked expression of FcR on gamma/delta TCR+ lymphocytes provides potential mechanisms for coupling the functional activities of gamma/delta T lymphocytes with immune mechanisms that involve Ig molecules, such as antibody-dependent cellular cytotoxicity.     

A third sublineage of avian T cells can be identified with a T cell receptor-3-specific antibody

Avian homologues of mammalian gamma delta and alpha beta TCR, termed TCR1 and TCR2, have been identified in the chicken with specific mAb. A third TCR, dubbed TCR3, has been identified on a subpopulation of T cells that lack the TCR1 or TCR2 epitopes. We have now produced a mAb that identifies this TCR3 molecule. The anti-TCR3 antibody immunoprecipitates a CD3-associated heterodimer with a relative Mr of 88,000, composed of 48,000 and 40,000 disulfide-linked chains. The Mr 40,000 chains of TCR3 and TCR2 exhibited the same isoelectric points of 5.6 to 6.5 and had core proteins of 34,000. Although the Mr 48,000 chain of TCR3 and the Mr 50,000 chain of TCR2 had the same basic isoelectric point of 6.2 to 7.6, their core proteins were different in size, 31,000 vs 29,000. Immunofluorescence analysis reveals that the TCR3 was present on all of the CD3+ T cells not identified by antibodies specific for TCR1 or TCR2. Thymocytes that expressed the surface CD3/TCR3 complex at relatively low levels were predominantly CD4+ and CD8+, whereas those with higher levels of surface CD3/TCR3 were predominantly CD4+ and CD8+ singles. Mature TCR3+ cells in the periphery were also either CD4+ (80%) or CD8+ (20%). The TCR1+, TCR2+, and TCR3+ subsets of T cells were generated sequentially in the thymus and seeded to the periphery in the same order. Intrathymic development of the TCR3+ cells was selectively inhibited by embryonic treatment with the anti-TCR3 mAb. The pattern of histologic localization of TCR3+ cells in the periphery was similar to the TCR2 subset of cells except that the TCR3+ cells were rarely seen in the intestine. Cross-reactivity patterns of the anti-chicken TCR antibodies suggested that other gallinaceous species share the three types of TCR. We conclude that TCR2 and TCR3 in gallinaceous birds may represent alpha beta subfamilies of TCR that are sequentially expressed on developmentally discrete sublines of T cells.     

gamma delta T cells in the human intestine express surface markers of activation and are preferentially located in the epithelium

We have investigated the expression of the alpha beta and the gamma delta T cell receptor (TCR) in the human intestine. By immunohistology we found that 39% of CD3+ intraepithelial lymphocytes (IEL) expressed the gamma delta TCR compared to 3% of CD3+ lamina propria lymphocytes (LPL). Cytofluorometric analysis of isolated cells revealed a significantly higher proportion of gamma delta T cells among CD3+ IEL compared to LPL and peripheral blood lymphocytes. This was due to an increase in both CD8+ (low density) and CD4-CD8- gamma delta T cells in IEL. Most alpha beta IEL expressed high-density CD8. About 30% of both IEL and LPL expressed CD25 (alpha-chain of the IL-2 receptor). HML-1 expression was detected on nearly all IEL and on 27% of LPL. CD25 and HML-1 were preferentially expressed on intestinal alpha beta and gamma delta T cells, respectively. Thus, human gamma delta T cells are located preferentially in the gut epithelium and are phenotypically different from alpha beta T cells, which constitute the majority of both LPL and IEL.