Expression and function of asialo GM1 in alloreactive cytotoxic T lymphocytes

In the present study we examined asialo GM1 (AsGM1) expression and its function in alloreactive cytotoxic T lymphocytes (CTL). We consistently found that the cytotoxic activity of bulk culture-derived allo-CTL was susceptible to the treatment of anti-AsGM1 (alpha AsGM1) plus complement. To further determine whether the expression of AsGM1 was maintained in CTL, we examined cloned T cells. The expression of AsGM1 in the T cell clones was assessed by their susceptibility to lysis by alpha AsGM1 plus complement and the reduction or abrogation of their cytotoxic activity by this treatment. It was found that, with one exception, all Lyt-2+, Thy-1+ CTL clones were AsGM1+ (seven out of eight), independent of their class specificity (class I or class II). In contrast, all Thy-1+, L3T4+ CTL (2) or helper T cell (4) clones AsGM1-. These findings suggested that there was a close association between the expression of AsGM1 and the expression of Lyt-2. The cytotoxic reaction of the anti-class I MHC CTL clones that expressed AsGM1 was blocked by alpha AsGM1 or alpha Lyt-2 antibody. The Lyt-2+, AsGM1+ anti-class II MHC CTL clone-mediated lysis was inhibited by alpha AsGM1. Addition of AsGM1 in micelle form (AsGM1-M) alone also blocked the cytotoxic reactions. Addition of other structurally similar but antigenically different glycolipids or other non-AsGM1-containing liposome preparations did not affect CTL-mediated cytotoxicity. Furthermore, adding both alpha AsGM1 and AsGM1-M together at proper doses inhibited the blocking effect (deblocking) of either alone, and other structurally similar glycolipids did not inhibit the blocking. The deblocking was specific, since AsGM1-M did not affect the blocking by alpha Lyt-2. These findings indicate that not only is AsGM1 expressed in a majority of Lyt-2+ CTL clones, but it may also be involved in the CTL- target interaction to mediate lytic reaction.     

Differential expression of asialo GM1 on alloreactive cytotoxic T lymphocytes and lymphokine-activated killer cells

The present study was undertaken to examine the differential expression of asialo GM1 (AsGM1) on the responding cells and effectors of alloreactive cytotoxic T lymphocytes (CTL) and lymphokine-induced activated killers (LAK). It was found that AsGM1 was expressed on the 3-day-cultured LAK effectors. Its expression gradually disappeared to the extent that AsGM1 became undetectable after 5 to 6 days of culturing. In contrast, AsGM1 was detected on 3-day CTL generated in mixed-lymphocyte cultures (bulk cultures); however, the levels of AsGM1 expression remained the same for at least 7 days. When examining the expression of AsGM1 on the responding cells, the reciprocal results were obtained. AsGM1 was expressed the LAK responders, but we were unable to demonstrate AsGM1 on CTL responders. Depletion of AsGM1+ cells from the responding population reduced subsequent CTL responses; however, CTL responses could be restored by adding conditioned media containing both interleukin 2 (IL-2) and other helper-T-cell factors and could not be restored by purified IL-2 alone adding at comparable doses. Reconstituting the AsGM1-depleted responders with Lyt-2-depleted splenocytes also restored the CTL response. Furthermore, depletion of AsGM1 cells from the responding population did not reduce the precursor frequency of allo-CTL, whereas the precursor frequency of LAK cells was reduced 42-fold. These findings show that the reduction of CTL responses after depletion of AsGM1+ cells was not due to the removal of precursors; instead, the defect appeared to be in the helper population. We further found that the helper defect was not due to impaired IL-2 production, because the endogenous production of IL-2 AsGM1-depleted responders was not reduced. Therefore, AsGM1+ cells may play a role in the helper pathway other than IL-2 production.     

Resistance of lymphokine-activated T lymphocytes to cell-mediated cytotoxicity

We observed that lymphokine-activated T lymphocytes, obtained in short- and long-term cultures following stimulation with recombinant interleukin-2 (rIL-2), are resistant to cell-mediated cytotoxicity. In particular, lymphokine-activated killer (LAK) cells do not undergo self-lysis or lysis by alloreactive cytotoxic T lymphocytes (CTL), in line with recent reports concerning CTL clones. Similar findings were further confirmed in a lectin-dependent cell cytotoxicity assay. LAK cell lysis resistance was not due to an inability to recognize itself, since inactivated LAK cells used as cold competitors inhibited tumor cell lysis in a dose-dependent manner. In contrast, the addition on Day 0 of irradiated LAK cells or alloreactive CTL, as well as a CTL clone having LAK-like activity to rIL-2-stimulated cultures abrogated or strongly reduced LAK cell generation. Therefore, LAK cell precursors were most likely susceptible to the lytic activity of differentiated cytotoxic cells, as no inhibition was detected when cell to cell contact was prevented by using a diffusible chamber culture system. These findings, on the whole, suggest that the emergence of the lysis-resistant phenotype is most likely the result of a selective process occurring in vitro that leads to the elimination of lysis-susceptible lymphocytes present in culture.     

Heterogeneity of long-term cultured activated killer cells induced by anti-T3 antibody

The present study has characterized the short term and long term cultured murine-activated killer (AK) cells that are induced by antibody directed against the epsilon-chain of T3 complex. The conventional lymphokine AK (LAK) cells were generated by culturing normal B6 spleen cells with purified human rIL-2. The alpha T3-induced AK cells (T3AK) were induced by culturing normal B6 spleen cells with alpha T3 and were then maintained in culture medium supplemented with human rIL-2 and/or alpha T3. After initial activation with alpha T3, lymphocyte proliferation and generation of cytotoxic effectors (T3AK) were noted, and these events were related to the endogenous production of IL-2 and IL-4. Addition of alpha IL-2 and/or alpha IL-4 suppressed both the proliferative response and the cytotoxic response induced by alpha T3. In comparing the T3AK cells with the conventional LAK cells, there were many similarities as well as some distinct differences. Both cells displayed a similar cytotoxic spectrum against a variety of tumor targets. The T3AK cells usually gave much higher levels of cytotoxic activity against susceptible targets. However, the susceptibility of different tumor targets to conventional LAK cells and T3AK cells varied. The time course for the generation of lytic activity also differed between the conventional LAK and T3AK cells. One distinct difference was their ability to survive in vitro. The conventional LAK cells survived in culture for only 1 wk. The T3AK cells could survive for at least 4 to 5 wk with active growth. The serologic phenotype of the LAK precursors was asialo GM1 (AsGM1+) cells, but the T3AK precursors could be either AsGM1+ or AsGM1-, depending on the target cell. The LAK effectors were both Lyt-2+ and Lyt-2-, but the short-term T3AK effectors were exclusively Lyt-2+. The long term T3AK cells (cultured for more than 2 wk) were found to consist of Lyt-2+ and Lyt-2- cells, and these subsets of T3AK cells showed different target specificities. These findings demonstrate the heterogeneity of LAK and T3AK cells, and this heterogeneous property may contribute to their diversity in specificity against different tumor targets and thus may affect their effectiveness in the immunotherapy of cancer.     

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit T cell-mediated cytotoxicity by inhibiting Fas ligand expression

We reported recently that the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) protect CD4+ T cells against Ag-induced apoptosis by down-regulating the expression of Fas ligand (FasL). Because the cytotoxic activity of CD8+ CTLs is mediated through two mechanisms, which involve the perforin/granzyme and the FasL/Fas pathways, in this study we investigated the effects of VIP/PACAP on the generation and activity of allogeneic CTLs, of CD8+ T1 and T2 effector cells and of alloreactive peritoneal exudate cytotoxic T cells (PEL) generated in vivo. VIP/PACAP did not affect perforin/granzyme-mediated cytotoxicity, perforin gene expression, or granzyme B enzymatic activity, but drastically inhibited FasL/Fas-mediated cytotoxicity against allogeneic or syngeneic Fas-bearing targets. VIP/PACAP inhibit CTL generation, but not the activity of competent CTLs. The inhibition is associated with a profound down-regulation of FasL expression, and these effects are mediated through both VPAC1 and VPAC2 receptors. VIP/PACAP inhibit the FasL/Fas-mediated cytotoxicity of T1 effectors and do not affect T2 cytotoxicity, which is entirely perforin/granzyme mediated. Similar effects were observed in vivo. Both the FasL/Fas-mediated cytotoxicity and FasL expression of cytotoxic allogeneic PELs generated in vivo in the presence of VIP or PACAP were significantly reduced. We conclude that, similar to their effect on CD4+ T cells, the two structurally related neuropeptides inhibit FasL expression in CD8+ cytotoxic T cells and the subsequent lysis of Fas-bearing target cells.     

Augmentation by anti-T3 antibody of the lymphokine-activated killer cell-mediated cytotoxicity

This study showed that a mAb (145-2C11) against the T3 epsilon-chain of the TCR complex augmented the cytotoxic activity of the lymphokine-activated killer (LAK) effectors. The LAK cells were induced by culturing normal spleen cells with purified human rIL-2. Adding alpha T3 at the effector phase of the cytotoxic reactions augmented the LAK-mediated cytotoxicity. The alpha T3-augmented LAK killing was seen only with tumor targets, and there was no increase of killing against Con A-induced lymphoblasts. The augmentation effect was dose dependent on both the amounts of alpha T3 and the number of LAK cells added. A very low concentration of alpha T3 (1/10,000 dilution of culture supernatants) was sufficient to induce alpha T3-augmented LAK-mediated cytotoxicity. Human rIL-2 at 10 to 30 U/ml was sufficient to generate LAK cells for maximal alpha T3 augmentation, whereas 300 to 1000 U/ml of IL-2 were needed to generate maximal LAK activity when tested in the absence of alpha T3. LAK cells generated for longer periods of time showed a progressive increase of alpha T3-augmented cytotoxicity. For some targets, the alpha T3-augmented LAK killing was FcR dependent as evidenced by the ability of alpha FcR mAb 2.4G2 to inhibit, and for others it was not inhibited. The alpha T3-augmented killing did not correlate with the FcR expression on target cells as defined by 2.4G2. The LAK cells were both Lyt-2+ and Lyt-2-, but the LAK cells involved in alpha T3-augmented killing were exclusively Lyt-2+. Preincubation of LAK cells with alpha T3, but not preincubation of targets with alpha T3, resulted in augmented killing suggesting that the alpha T3 effect was unrelated to an antibody-dependent cell-mediated cytotoxicity. Our findings indicate that alpha T3 is a potent reagent to augment the cytotoxic reaction of LAK cells. These results suggested that a relationship might exist between the T3 complex and the cytotoxic activity of a subpopulation of Lyt-2+ LAK cells.     

Lymphokine-activated cell-associated antigen involved in broad-reactive killer cell-mediated cytotoxicity

Spleen cells from rats which had been hyperimmunized with mouse lymphokine-activated killer (LAK) cells, were fused with the mouse myeloma cell line, P3 X 63 Ag8.653. Antibodies secreted by 1500 cultures were selected by their blocking effect on LAK cell-mediated cytotoxicity in the absence of complement. Two monoclonal antibodies (KBA4 and KBA6) greatly inhibited the cytotoxic activity of LAK cells, which were induced from mouse spleen cells by culture with recombinant human interleukin 2 (r-IL-2). These antibodies also blocked the cytotoxic activity of natural killer (NK) cells, but activated macrophages (A-M phi) were only slightly sensitive to them. However, no effect of the antibodies on the cytotoxic activity of cytotoxic T lymphocytes (CTL) was detected. These data suggest that the specific antigen, lymphokine-activated cell-associated (LAA) antigen, defined by these monoclonal antibodies may be associated with the recognition mechanisms of broad-reactive killer (BRK) cell-mediated cytotoxicity. The observation that low levels of LAA antigen are distributed in all lymphoid cells and that it was significantly enhanced by treatment of the cells with r-IL-2 suggests that the antigen may be involved in lymphocyte-activation mechanisms. We also found that the LAA antigen consists of two distinct polypeptides with Mr of 180,000 and 95,000 Da, which are similar to that of LFA 1 antigen. However, the biological characteristics of LAA antigen did not coincide with those of LFA 1. Therefore, KBA MAb may recognize a carbohydrate epitope distinct from that of LFA 1.     

Disparate primary and secondary allospecific CD8+ T cell cytolytic effector function in the presence or absence of host CD4+ T cells

The role of CD4+ T cells in promoting CD8+ T cell effector activity in response to transplant Ags in vivo has not been reported. We used a hepatocellular allograft model known to initiate both CD4-dependent and CD4-independent rejection responses to investigate the contribution of CD4+ T cells to the development, function, and persistence of allospecific CD8+ T cell effectors in vivo. Complete MHC-mismatched hepatocellular allografts were transplanted into C57BL/6 (CD4-sufficient) or CD4 knockout (CD4-deficient) hosts. The development of in vivo allospecific cytotoxicity was determined by clearance of CFSE-labeled target cells. CD8+ T cell cytotoxic effector activity was enhanced in response to allogeneic hepatocellular grafts with a greater magnitude of allocytotoxicity and a prolonged persistence of CTL effector activity in CD4-sufficient hosts compared with CD4-deficient hosts. Cytolytic activity was mediated by CD8+ T cells in both recipient groups. In response to a second hepatocyte transplant, rejection kinetics were enhanced in both CD4-sufficient and CD4-deficient hepatocyte recipients. However, only CD4-sufficient hosts developed recall CTL responses with an augmented magnitude and persistence of allocytotoxicity in comparison with primary CTL responses. These studies show important functional differences between alloreactive CD8+ T cell cytolytic effectors that mature in vivo in the presence or absence of CD4+ T cells.     

Lectin-dependent and anti-CD3 induced cytotoxicity are preferentially mediated by peripheral blood cytotoxic T lymphocytes expressing Leu-7 antigen

Monoclonal antibodies against the CD3 antigen and certain lectins can induce interleukin 2 dependent antigen-specific T cell clones to mediate non-antigen specific cytotoxicity. On the basis of this observation, we predicted that it may be possible to identify cytotoxic T lymphocytes (CTL) in peripheral blood without knowing the antigen specificity of these in vivo primed CTL. By using this strategy, peripheral blood lymphocytes were separated into low and high-density fractions on Percoll gradients and were tested for cytotoxic activity in the presence or absence of concanavalin A (Con A) or anti-Leu-4 antibody. Lectin-dependent cellular cytotoxicity (LDCC) and anti-CD3 induced cytotoxicity against both natural killer (NK)-insensitive and NK-sensitive targets were exclusively mediated by low-density CD3+ T lymphocytes. Additional studies indicated that low-density CD3+ T lymphocytes co-expressing Leu-7 antigen preferentially mediated this activity, although in some individuals, significant activity was also observed in the low-density T cells lacking Leu-7. In contrast, high-density CD3+ T lymphocytes and CD16+ (Leu-11+) NK cells (both Leu-7 and Leu-7+) did not mediate nonantigen-specific cytotoxicity under these conditions. The finding that NK cell-mediated cytotoxicity was unaffected by these lectins refutes the hypothesis that lectin-dependent cellular cytotoxicity is simply a result of effector and target agglutination. T cell-mediated cytotoxicity was both lectin and antibody specific. Phytohemagglutinin, Con A, and pokeweed mitogen induced cytolytic activity in the Leu-7+ T cells, whereas wheat germ agglutinin did not. Of the antibodies against T cell-associated differentiation antigens (anti-Leu-2,3,4, and 5), only anti-Leu-4 induced cytotoxicity. This anti-CD3-induced cytotoxicity was essentially completely inhibited by the presence of anti-LFA-1 or anti-CD2 monoclonal antibodies, implicating these molecules in the triggering process. A proportion of the CD3+, Leu-7+ CTL expressed HLA-DR antigens, indicating possible in vivo activation. Because previous clinical studies have indicated that lymphocytes with this phenotype may be elevated in clinical situations associated with immunosuppression and chronic viral infection, this unique subset of CD3+ T lymphocytes may represent a population of in vivo primed CTL possibly against viral antigens.     

Phenotypic and functional heterogeneity of thymic and splenic lymphokine activated killer cells relative to ornithine sensitivity

We have previously reported the selective inhibition of cytotoxic T lymphocytes (CTL) by 10 mM ornithine (ORN) relative to natural killer (NK) cell-derived lymphokine activated killer cells (LAK). To determine if this were due to differences in the progenitor cells or the type of stimulus, we used cortisone-resistant thymocytes (CRT) as a source of mature T cells for induction of LAK and CTL, and compared the results with spleen. Thymic and splenic CTL precursors (CTLp) from C57B1/6 (B6) mice were CD8+, ASGM1-, ORN sensitive. Splenic LAK precursors (LAKp) were CD8-, ASGM1+, ORN resistant when assayed against both YAC-1 and P815 tumor targets. In contrast, CRT-derived LAKp were CD8-, ASGM1+, ORN resistant against YAC-1, whereas LAKp against P815 were CD8+, ASGM1+, ORN sensitive. ORN sensitivity was also observed among CTL and LAK in DBA/2 mice and was associated with CD8+ phenotype. Therefore, our initial observation of differential ORN sensitivity in CTL vs LAK was a function of the progenitor cells; furthermore, CD8+ cytolytic cells are ORN sensitive whether activated by antigen (CTL) or IL-2 (T-LAK).     

Effects of beta-2 microglobulin anti-sense oligonucleotides on sensitivity of HER2/neu oncogene-expressing and nonexpressing target cells to lymphocyte-mediated lysis.

The mechanism by which HER2/neu overexpressing tumor cells resist NK, LAK, and LDCC cytotoxic lymphocytes was investigated. Resistance was not explained by a delay in kinetics of lysis, concurrent resistance to TNF, or a diminished expression of the transferrin receptor. HLA-class I expression, however, was markedly elevated compared to HER2 nonexpressing targets suggesting a reason for resistance. To test the role of class I, we selectively decreased expression by incubation of targets with beta-2 microglobulin anti-sense oligonucleotides. Anti-sense-treated HER2+ targets, displaying levels of class I comparable to HER2- targets, were still markedly resistant to cytotoxic effectors. Down-regulation of class I expression in HER2- carcinoma cells also had no effect on sensitivity to cytotoxicity by anti-sense treatment of Raji and U937 targets resulted in enhanced sensitivity to NK and LAK effectors but not to T cells mediating LDCC. These data indicate resistance to cytotoxicity in HER2-expressing targets cannot be solely explained by heightened expression of class I. The data also support the concept that class I expression regulates sensitivity to NK and LAK cells (but not LDCC effectors) in selected targets.     

Regulation of the cytotoxic activity of alloreactive cytotoxic T lymphocytes by helper cells and lymphokines

The cytotoxic activity of alloreactive cytotoxic T lymphocytes (CTL) was maintained and augmented by transferring cells from a 5-day mixed lymphocyte culture MLC into a host culture (HC) containing indomethacin, freshly explanted normal spleen cells, and peritoneal cells which were syngeneic to the MLC cells. The MLC cells used in the transfer experiments were generated by culturing untreated H-2b splenic responders with irradiated H-2d stimulators, or were generated by culturing Lyt-2-depleted H-2b splenic responders with irradiated H-2d stimulators. The allo-CTL were found to be derived from the donor MLC (first culture) when unfractionated MLC cells were transferred into a host (second) culture and incubated for 5 days. In contrast, the allo-CTL were derived from host culture cells when Lyt-2-depleted MLC cells were transferred and the combined cultures incubated for 5 days. In the former case, the augmentation of MLC-derived cytotoxicity did not result from nonspecific expansion of all donor T cells; instead it was mediated by lymphokine(s), distinct from IL-2, produced by helper T cells generated in host culture, which appeared to selectively expand the antigen-specific CTL or to increase the cytotoxic activity of these CTL. The helper T cells were Thy-1+, L3T4+, and Lyt-2-. These findings indicate that antigen-nonspecific help was provided by helper cells or helper factors (lymphokines) generated in the host culture, which maintained and augmented the cytotoxic activity of the fully generated allo-CTL. This helper effect was also seen in the induction of primary allo-CTL responses which could be generated with fewer stimulating cells and with a stronger cytotoxic response at different R/S ratios tested. The generation of allo-CTL in second culture following transfer of Lyt-2-depleted MLC cells to host cultures appears to have involved antigen carryover from the MLC; however, antigen carryover alone was not sufficient. It appears that in the absence of Lyt-2+ suppressor T cells, antigen-specific help might be generated in donor cultures (Lyt-2-depleted MLC) which promoted or recruited the generation of antigen-specific CTL in host culture.     

Induction of LAK-like cells in the peritoneal cavity of mice by inactivated Candida albicans

We have investigated the effect of multiple administrations of inactivated Candida albicans (CA) cells on induction of non-MHC-restricted antitumor cytotoxic responses both in normal and congenitally athymic (nude) mice. Intraperitoneal inoculation of CD2F1 mice with five doses of 2 x 10(7) CA cells over a 2-week interval was associated with the induction of peritoneal exudate cells (PEC) that mediated natural killer cell activity. These cells, in contrast to those elicited by a single dose of CA, killed both NK-sensitive and NK-resistant tumor target cells in vitro. This broad-spectrum, antitumor cytotoxicity peaked 1 day after the last injection of CA, and decreased to control values within 6 (NK-resistant) or 14 (NK-sensitive target cells) days. Cytotoxicity could be recalled to a high level by a boosting injection of CA or a major mannoprotein-soluble antigen (MP) from the Candida cell wall, given 30 days after multiple CA treatment. Upon a 24-hr in vitro incubation, CA-induced peritoneal immunoeffectors lost their killing activity unless human recombinant interleukin-2 (rIL-2) was added to cultures. The non-MHC-restricted cytotoxic PEC activity induced by CA was mainly associated with nonadherent, nonphagocytic large granular lymphocytes (LGL) which exhibited the following phenotypes: (i) asialo GM1+, Lyt 2.2-, and partially Thy 1.2+ (effectors active against NK-sensitive targets) and (ii) asialo GM1+, Lyt 2.2-, and Thy 1.2+ (effectors active against NK-resistant targets). Nude mice also responded to multiple CA inoculations by displaying high cytotoxic activity against NK-sensitive targets and significant cytotoxicity against NK-resistant targets. This cytotoxicity could be recalled on Day +30, and the cytotoxic effectors involved were highly sensitive to anti-asialo GM1 plus complement treatment. Overall, the results add further experimental evidence to the wide range of immunomodulatory properties possessed by C. albicans, and demonstrate that the majority of antitumor cytotoxic activity induced by fungal cells was due to lymphokine-activated killer (LAK)-like effectors.     

Interleukin 2 induces human acute lymphocytic leukemia cells to manifest lymphokine-activated-killer (LAK) cytotoxicity

Lymphokine-activated killer cells (LAK) were originally distinguished from natural killers (NK) and cytotoxic T lymphocytes. Recently, however, IL 2-activated NK cells were suggested as the major source of LAK reactivity in human peripheral blood (PBL). Because certain T cell acute lymphoblastic leukemia (T-ALL) cells are phenotypically similar to LAK precursors, we have asked whether these leukemic cells can be induced toward LAK-cytotoxicity and express NK reactivity before stimulation. Five out of seven T-ALL preparations were induced by IL 2 to kill target cells. The cytotoxicity of the leukemic-LAK cells resembled that of normal LAK effectors as they lysed efficiently the NK-resistant target Daudi, as well as fresh human sarcoma, carcinoma, and renal cancer cells but not normal PBL. The ALL-LAK precursors phenotype was T3-, T4-, T8-, and T11+, similar to most normal LAK precursors. In contrast to normal PBL that generated LAK effectors when their proliferation was inhibited, the irradiated, nonproliferating T-ALL leukemic cells did not respond to IL 2. Therefore, the T-ALL LAK cytotoxicity was attributed to the leukemic cells rather than to residual normal lymphocytes. The IL 2-responding T-ALL cells did not express autonomous NK type cytotoxicity, suggesting that they reflect LAK precursors of non-NK origin. The homogeneous leukemic preparations with inducible LAK cytotoxicity described herein provide a model system for studying normal LAK cells.     

Regulation of lymphokine-activated killer activity and pore-forming protein gene expression in human peripheral blood CD8+ T lymphocytes. Inhibition by transforming growth factor-beta.

The effect of transforming growth factor-beta 1 (TGF-beta) on activation-induced CD8+ T cell cytotoxicity and gene expression was investigated. TGF-beta was demonstrated to inhibit pore-forming protein (PFP) mRNA expression and total benzoyloxycarbonyl-L-lysine thiobenzyl ester esterase activity in CD8+ T cells cultured with IL-2 and OKT3 mAb for 6 to 18 days. Consistently, in the absence or presence of TGF-beta, the PFP mRNA expression and lymphokine-activated killer (LAK) activity of CD8+ T cells were closely correlated. The inhibitory effects of TGF-beta on both CD8+ T cell PFP mRNA expression and LAK activity were reversible by removal of TGF-beta from the culture. Expression of lymphokines, adhesion/recognition molecules, and activated p55 IL-2R, previously implicated in the lytic mechanism of cytotoxic lymphocytes, either was not detectable or did not correlate with TGF-beta inhibition of LAK activity. In addition, independently of effector/target cell binding, the lectin- or heteroconjugated antibody-dependent cellular cytotoxicity of IL-2/OKT3 mAb-activated CD8+ T cells was inhibited by preculture with TGF-beta. TGF-beta also inhibited the rapid activation-induced expression of PFP mRNA and cytotoxic potential in resting T cells, thereby indicating that the effect of TGF-beta was independent of T cell proliferation. TGF-beta inhibition of CD8+ T cell PFP mRNA expression and cytotoxic potential was TGF-beta dose dependent; however, a variety of activation stimuli (including IL-2, IL-6, and OKT3 mAb) were all similarly inhibited by TGF-beta. Therefore, TGF-beta may be an important general regulator of CD8+ T cell cytotoxic function, in particular by suppressing expression of PFP, a major cytolytic protein implicated in the lytic function of cytotoxic lymphocytes.     

A homogeneous population of lymphokine-activated killer (LAK) cells is incapable of killing virus-, bacteria-, or parasite-infected macrophages

Previous reports have suggested a role for natural killer (NK) cells in directly lysing host cells infected with bacteria and other intracellular microorganisms. Here, we determined the inability of a highly homogeneous population of lymphokine activated killer (LAK) cells to kill macrophages infected with the following intracellular parasites: Mycobacterium avium, Listeria monocytogenes, Legionella pneumophila, Toxoplasma gondii, and Trypanosoma cruzi. In parallel cytotoxicity assays, LAK cells lysed the tumor targets YAC-1 and P815 effectively. Furthermore, we were able to demonstrate that influenza-specific cytotoxic T lymphocytes (CTL), but not LAK cells, were efficient killers of influenza virus-infected macrophages.     

Studies on cytotoxicity generated in human mixed lymphocyte culture

Summary High levels of cytotoxic activity against the natural killer (NK) cell-sensitive target K562 and the NK-resistant target UCLA-SO-M14 (M14) can be generated in vitro either by mixed lymphocyte culture (MLC) or by culture of lymphocytes in interleukin 2 (IL2) (lymphokine activated killer (LAK) cells). The purpose of this study was to identify similarities and differences between MLC-LAK and IL2-LAK cells and allospecific cytotoxic T cells. Induction of cytotoxicity against K562 and M14 in both culture systems was inhibited by antibodies specific either for IL2 or the Tac IL2 receptor. Like NK effector cells, the precursors for the MLC-LAK cells were low density large lymphocytes. However these precursors differed from the large granular lymphocytes that mediated NK cytolysis in sensitivity to the toxic lysosomotropic agent L-leucine methyl ester (LME). The resistance of the MLC-LAK precursors to LME indicated that the precursors included large agranular lymphocytes. Although interferon-gamma (IFN-gamma) is produced in MLC and in IL2 containing cultures, it is not required for induction of either type of cytotoxic activity. Neutralization of IFN-gamma in MLC-and IL2-containing cultures with specific antibodies had no effect on the induction of cytotoxic activities. Both allospecific cytotoxic T lymphocyte (CTL) and LAK activities were enhanced by IL2 and IFN-gamma at the effector cell stage. However, the mechanism of cytolysis was different in the two systems. NK- and MLC-induced LAK activities were independent of CD3-T cell receptor complex while CTL activity was blocked by monoclonal antibodies specific for the CD3 antigen. These results suggest that NK and the in vitro induced LAK cytotoxicities are a family of related functions that differ from CTL. Furthermore, MLC-induced and IL2-induced cytotoxicities against K562 and M14 appear to be identical.This work was supported by NIH grant CA34442     

Lymphokine-activated killer cells are generated before classical cytotoxic T lymphocytes after bone marrow transplantation in mice

Lymphokine-activated killer (LAK) cells are demonstrable within 2 wk after syngeneic or allogeneic (H-2-compatible) bone marrow transplantation in mice. Classical cytotoxic T lymphocytes (CTL) are not active until at least 4 wk after transplant. Both LAK cells and CTL bear the Thy-1 marker and do not possess the murine natural killer cell marker asialo GM.     

T helper cells in cytotoxic T lymphocyte development: role of L3T4(+)-dependent and -independent T helper cell pathways in virus-specific and alloreactive cytotoxic T lymphocyte responses

I have compared the requirements for T helper (Th) cell function during the generation of virus-specific and alloreactive cytotoxic thymus (T)-derived lymphocyte (CTL) responses. Restimulation of vesicular stomatitis virus (VSV)-immune T cells (VSV memory CTLs) with VSV-infected stimulators resulted in the generation of class I-restricted, VSV-specific CTLs. Progression of VSV memory CTLs (Lyt-1-2+) into VSV-specific CTLs required inductive signals derived from VSV-induced, Lyt-1+2- Th cells because: (i) cultures depleted by negative selection of Lyt-1+ T cells failed to generate CTLs; (ii) titration of VSV memory CTLs into a limiting dilution (LD) microculture system depleted of Th cells generated curves which were not consistent with a single limiting cell type; (iii) LD analysis of VSV memory CTLs did produce single-hit curves in the presence of Lyt-1+2- T cells sensitized against VSV; and (iv) monoclonal anti-L3T4 antibody completely abrogated CTL generation against VSV. Similar results were also obtained with Sendai virus (SV), a member of the paramyxovirus family. The notion that a class II-restricted, L3T4+ Th cell plays an obligatory role in the generation of CTLs against these viruses is also supported by the observation that purified T cell lymphoblasts (class II antigen negative) failed to function as antigen-presenting cells for CTL responses against VSV and SV. T cell lymphoblasts were efficiently lysed by class I-restricted, anti-VSV and -SV CTLs, indicating that activated T cells expressed the appropriate viral peptides for CTL recognition. Furthermore, heterogeneity in the VSV-induced Th cell population was detected by LD analysis, suggesting that at least two types of Th cells were required for the generation of an anti-VSV CTL response. VSV-induced Th cell function could not simply be replaced by exogenous IL-2 because this lymphokine induced cytotoxic cells that had the characteristics of lymphokine-activated killer (LAK) cells and not anti-viral CTLs. In contrast, CTL responses against allogeneic determinants could not be completely blocked with antibodies against L3T4 and depletion of L3T4+ cells did not prevent the generation of alloreactive CTLs in cultures stimulated with allogeneic spleen cells or activated T cell lymphoblasts. Thus, these studies demonstrate an obligatory requirement for an L3T4-dependent Th cell pathway for CTL responses against viruses such as VSV and SV; whereas, CTL responses against allogeneic determinants can utilize an L3T4-independent pathway.     

Human vascular endothelial cells stimulate memory but not naive CD8+ T cells to differentiate into CTL retaining an early activation phenotype

Endothelial cell (EC)-selective alloreactive CTL may mediate alloimmune vascular injury. In the present study, EC-selective CTL were generated in cocultures of purified human CD8+ T cells with allogeneic EC and were compared with conventional CTL against corresponding B lymphoblastoid cells (BLC). EC caused activation and expansion of memory but not naive CD8+ T cells, which differentiated into EC-selective CTL that retained high surface expression of CD69, CD25, and CD62L and displayed low intracellular perforin content. In contrast, BLC-stimulated CTL could be generated from naive or memory CD8+ T cells and showed a more mature phenotype (low CD69, CD25, and CD62L with higher levels of perforin). The expansion of alloreactive T cells by EC stimulation was 5- to 20-fold less effective than in corresponding BLC-stimulated cultures, accounting for a reduction in the assayable cytotoxicity of individual microcultures. In these IL-2-supplemented cocultures, no effect on CTL generation or phenotype was observed by mAb blocking of costimulation provided by LFA-3, ICAM-1, or CD40, by addition of comitogenic anti-CD28 mAb, or by preactivation of EC with CD40 ligand. Cyclosporine inhibited CTL expansion and cytotoxicity similarly in both EC- and BLC-stimulated cultures but did not affect the phenotype of those CTL that did emerge. This study extends the characterization of endothelium as an immunoregulatory cell type distinct from conventional APC and may explain why graft rejection within the arterial intima, an anatomic compartment in which EC may be the primary type of APC, is separable from rejection in the graft parenchyma.