Antigen-presenting T cells. II. Clonal responses of alloreactive and virus-specific self-restricted human cytotoxic T cell responses stimulated by T lymphoblasts

The capacity of various stimulator cell types to present alloantigens or viral antigens to resting human CD8+ cytotoxic lymphocyte precursors (CLP) was analyzed in a limiting dilution culture system. Cell sorter-separated T lymphoblasts of both CD4+ and CD8+ phenotypes but not resting T cells were found to efficiently stimulate the clonal development of allogeneic CD8+ CLP. Thus, 5000 CD4+ T lymphoblasts activated as many (one out of 200 to one out of 300) allogeneic CLP as 50,000 peripheral blood mononuclear stimulator cells. This potent stimulator activity was found in CD4+ and CD8+ T lymphoblasts activated by mitogen, anti-T3 monoclonal antibody, or mixed leukocyte reactions. Cytotoxic T cells generated in this system were highly specific for HLA class I antigens. Furthermore, T lymphoblasts infected with mumps virus efficiently induced development of autologous CLP into CTL clones that were virus specific and self-HLA restricted, as shown by split-well analysis. The possible in vivo significance of antigen-presenting T lymphoblasts is discussed.     

Human cytotoxic lymphocytes. IV. Frequency and clonal specificity of CD8+CD16-(Leu2+Leu11-) and CD16+CD3-(Leu11+Leu4-) cytotoxic lymphocyte precursors activated by alloantigen or K562 stimulator cells

Cell sorter-purified CD8+CD16- (Leu2+Leu11-) cytotoxic T cell precursors and CD16+CD3-(Leu11+Leu4-) natural killer (NK) cells were cultured under limiting dilution (LD) conditions with allogeneic stimulator cells or with K562 tumor cells in the presence of exogenous interleukin 2. One out of 100-200 alloantigen-stimulated Leu2+ T cells clonally developed into an alloantigen-specific cytotoxic T cell, but only 1 out of 500-3400 of these cells lysed NK-susceptible K562 target cells. In contrast, 1 out of 2-35 alloantigen-stimulated Leu11+ precursor cells developed into an effector cell that lysed K562, but less than 1 out of 500 of these cells lysed allogeneic Con A blast targets. However, clonal activation of Leu11+ precursor cells under LD conditions did not require alloantigenic stimulator cells. Comparable high frequencies (f = 1/3 to 1/28) of anti-K562 cytotoxic lymphocyte precursors were thus measured when Leu11+ precursor cells were cultured on autologous or K562 feeder cells. As shown by a split culture approach, the vast majority of alloantigen-activated Leu2+ effector cells were highly specific for the stimulating alloantigen (i.e., they did not lyse K562), while the majority of Leu11+ microcultures lysed K562 tumor cells but neither autologous nor allogeneic Con A blast targets. On a quantitative basis, these data show that CD8+CD16- T cells and CD16+CD3-NK cells are two mutually exclusive lymphocyte populations which clonally develop into cytotoxic effector cells specific for alloantigen or K562 target cells, respectively.     

Activation of human T lymphocytes by 12-O-tetradecanoylphorbol-13-acetate: role of accessory cells and interaction with lectins and allogeneic cells

Stimulatory effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on human T lymphocytes have been investigated. TPA was found to stimulate highly purified T cells (obtained by a three-step isolation procedure involving plastic adherence, nylon wool passage and Ig-anti-Ig column passage) in the absence of accessory cells (stimulation index of 5 to 10), whereas phytohemaglutinin (PHA) and concanavalin A (Con A) did not. This response was, however, increased by the addition of autologous adherent cells. Addition of TPA, but not adherent cells, induced T-cell proliferation in response to the nonmitogenic lectin, wheat germ agglutinin (WGA), while both adherent cells and TPA restored T-cell proliferation to mitogenic lectins such as PHA and Con A. Furthermore, TPA greatly increased the mixed-lymphocyte response of purified T cells to otherwise nonstimulating allogeneic cells such as T lymphocytes or tumor cells from some patients with chronic lymphocytic leukemia. These results suggest that TPA can directly act on human T cells to render them reactive to a variety of stimuli.     

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.     

The WD repeat protein FAN regulates lysosome size independent from abnormal downregulation/membrane recruitment of protein kinase C

FAN (factor associated with neutral sphingomyelinase [N-SMase] activation) exhibits striking structural homologies to Lyst (lysosomal trafficking regulator), a BEACH protein whose inactivation causes formation of giant lysosomes/Chediak-Higashi syndrome. Here, we show that cells lacking FAN show a statistically significant increase in lysosome size (although less pronounced as Lyst), pointing to previously unrecognized functions of FAN in regulation of the lysosomal compartment. Since FAN regulates activation of N-SMase in complex with receptor for activated C-kinase (RACK)1, a scaffolding protein that recruits and stabilizes activated protein kinase C (PKC) isotypes at cellular membranes, and since an abnormal (calpain-mediated) downregulation/membrane recruitment of PKC has been linked to the defects observed in Lyst-deficient cells, we assessed whether PKC is also of relevance in FAN signaling. Our results demonstrate that activation of PKC is not required for regulation of N-SMase by FAN/RACK1. Conversely, activation of PKC and recruitment/stabilization by RACK1 occurs uniformly in the presence or absence of FAN (and equally, Lyst). Furthermore, regulation of lysosome size by FAN is not coupled to an abnormal downregulation/membrane recruitment of PKC by calpain. Identical results were obtained for Lyst, questioning the previously reported relevance of PKC for formation of giant lysosomes and in Chediak-Higashi syndrome. In summary, FAN mediates activation of N-SMase as well as regulation of lysosome size by signaling pathways that operate independent from activation/membrane recruitment of PKC.     

Regulation of T-cell death-associated gene 51 (TDAG51) expression in human T-cells

T-cell death-associated gene 51 (TDAG51) has been described to regulate T-cell receptor/CD3-dependent induction of CD95/Fas and subsequent activation-induced cell death (AICD) in a murine T-cell hybridoma. Using well-defined pharmacological inhibitors, we investigated the regulation of TDAG51 expression in human T-cells and the correlation with cell death. TDAG51 was induced in resting T-cells, lymphoid cell lines and AICD-susceptible as well as AICD-resistant T-cell clones, and induction was inhibited by MAP-kinase inhibitors and PKC inhibitor G?6983. No correlation between the effects of inhibitors on TDAG51 expression and cell death was observed. The constitutive TDAG51 expression in five pancreatic carcinoma cell lines was reduced by MAP-kinase inhibitors but not by G?6983. Furthermore, the inducible overexpression of TDAG51 in TetOn Jurkat cells did not modulate cellular proliferation, phorbolester/ionomycin-induced growth arrest, or the expression of various cell surface molecules. Our results indicate that the expression of TDAG51 in human T-cells does not correlate with AICD.     

The adapter protein Nck: Role of individual SH3 and SH2 binding modules for protein interactions in T lymphocytes

Nck is a ubiquitously expressed, primarily cytosolic adapter protein consisting of one SH2 domain and three SH3 domains. It links receptor and nonreceptor tyrosine kinases to actin cytoskeleton reorganizing proteins. In T lymphocytes, Nck is a crucial component of signaling pathways for T cell activation and effector function. It recruits actin remodeling proteins to T cell receptor (TCR)‐associated activation clusters and thereby initiates changes in cell polarity and morphology. Moreover, Nck is crucial for the TCR‐induced mobilization of secretory vesicles to the cytotoxic immunological synapse. To identify the interactome of Nck in human T cells, we performed a systematic screen for interaction partners in untreated or pervanadate‐treated cells. We used GST fusion proteins containing full length Nck, the combined SH3 domains or the individual SH3 and SH2 domains to precipitate putative Nck interactors from cellular lysates. Protein bands were excised from gels, processed by tryptic in‐gel digestion and analyzed by mass spectrometry. Using this approach, we confirmed previously established interactions (e.g., with Slp76, CD3ε, WASP, and WIPF1) and identified several novel putative Nck‐binding proteins. We subsequently verified the SH2 domain binding to the actin‐binding protein HIP55 and to FYB/ADAP, and the SH3‐mediated binding to the nuclear proteins SFPQ/NONO. Using laser scanning microscopy, we provide new evidence for a nuclear localization of Nck in human T cells. Our data highlight the fundamental role of Nck in the TCR‐to‐cytoskeleton crosstalk and point to yet unknown nuclear functions of Nck also in T lymphocytes.     

Characterization of tumor reactivity of human V gamma 9V delta 2 gamma delta T cells in vitro and in SCID mice in vivo

Human Vgamma9Vdelta2 gammadelta T cells are selectively activated by bacterial phosphoantigens and aminobisphosphonates and exert potent cytotoxicity toward various tumor cells. In this study we have characterized the cytotoxic reactivity of gammadelta T cell lines established from healthy donors by stimulation with aminobisphosphonate alendronate toward melanoma MeWo and pancreatic adenocarcinomas Colo357 and PancTu1 lines in vitro and in vivo upon adoptive transfer into SCID mice. Lysis of all tumor cells was enhanced when gammadelta effector cells were preactivated with phosphoantigens. Recognition of MeWo was TCR dependent, as shown by anti-TCR Ab blockade, whereas only the phosphoantigen-mediated increased, but not the basal, lysis of Colo357 and PancTu1 was inhibited by anti-TCR Ab. Furthermore, lysis of Colo357, but not that of MeWo or PancTu1, was completely inhibited by the pan-caspase inhibitor zVAD, indicating different recognition and effector mechanisms involved in the gammadelta T cell/tumor cell interactions. Upon transfer into SCID mice, alendronate-activated gammadelta T cells given together with IL-2 and alendronate significantly prolonged the survival of SCID mice inoculated with human tumor cells. The best results were thus obtained when gammadelta T cells were repetitively given five times over a period of 30 days. With this protocol, human gammadelta T cells prolonged the mean survival of mice inoculated with MeWo melanoma from 28.5 to 87.3 days (p < 0.0001) and in the case of PancTu1 adenocarcinoma from 23.0 to 48.4 days (p < 0.0001). We conclude that an effective gammadelta T cell-based immunotherapy might require activation of endogenous gammadelta T cells with aminobisphosphonate (or phosphoantigen) and IL-2, followed by adoptive transfer of in vitro expanded gammadelta T cells.