Four sulfhydryl-modifying compounds cause different structural damage but similar functional damage in murine lymphocytes

Four thiol-modifying compounds were used to inhibit murine lymphocyte mitogenesis. The compounds were a copper sulfate/O-phenanthroline complex (CuP) to oxidize surface thiols, N-ethyl maleimide (NEM) to alkylate surface and intracellular thiols, D,L-buthionine-S,R-sulfoximine (BSO) to prevent synthesis of glutathione, and hydrogen peroxide, which reacts with various cellular constituents, including sulfhydryls. Splenic lymphocytes were incubated with one of the four compounds, washed, and then stimulated with the B cell mitogen, LPS, or the T cell mitogen, Con A. In spite of their differing chemical reactivities and differing effects on cell viability, lipids, and total, protein, and non-protein thiols, the four sulfhydryl-modifying compounds had very similar effects on the kinetics and inhibition of lymphocyte growth. All compounds had complex effects on mitogenesis, causing enhanced, delayed, or inhibited tritiated thymidine incorporation. Although the total thiol contents of untreated T cells and B cells were found to be equivalent, the LPS response consistently was inhibited by lower concentrations than the Con A response, suggesting that B cells were more sensitive than T cells to thiol modification. To compare compounds the efficiency of inhibition was determined by functionally relating reductions in mitogenesis with reductions in thiol content of the cells. The compounds differed in inhibitory efficiency; thus, damage to some thiols must be more important than damage to others. CuP ablated mitogenesis with the least change in thiol content. Therefore, surface sulfhydryls appear critical in lymphocyte mitogenesis. With all compounds inhibition of mitogenesis occurred over a very narrow range of thiol content, suggesting that the thiols important in inhibition were few in number relative to the total thiol content of the cell.     

The role of low molecular weight thiols in T lymphocyte proliferation and IL-2 secretion

Glutathione (GSH) is an abundant intracellular tripeptide that has been implicated as an important regulator of T cell proliferation. The effect of pharmacological regulators of GSH and other thiols on murine T cell signaling, proliferation, and intracellular thiol levels was examined. l-Buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH synthesis, markedly reduced GSH levels and blocked T cell proliferation without significant effect on cell viability. N-acetylcysteine markedly enhanced T cell proliferation without affecting GSH levels. Cotreatment of T cells with N-acetylcysteine and BSO failed to restore GSH levels, but completely restored the proliferative response. Both 2-ME and l-cysteine also reversed the BSO inhibition of T cell proliferation. Intracellular l-cysteine levels were reduced with BSO treatment and restored with cotreatment with NAC or l-cysteine. However, 2-ME completely reversed the BSO inhibition of proliferation without increasing intracellular cysteine levels. Therefore, neither GSH nor cysteine is singularly critical in limiting T cell proliferation. Reducing equivalents from free thiols were required because oxidation of the thiol moiety completely abolished the effect. Furthermore, BSO did not change the expression of surface activation markers, but effectively blocked IL-2 and IL-6 secretion. Importantly, exogenous IL-2 completely overcame BSO-induced block of T cell proliferation. These results demonstrate that T cell proliferation is regulated by thiol-sensitive pathway involving IL-2.     

Modulation of T-cell functions: I. Effect of 2-mercaptoethanol and macrophages on T-cell proliferation

The in vitro effects of 2-mercaptoethanol (2-ME), macrophages (MØ), and concanavalin A (Con A) on the proliferation of normal spleen cells (NSC), MØ-depleted spleen cells (DSC), T cells, T-cell subpopulations, and B cells were assessed by [³H]thymidine incorporation. 2-ME alone was consistently shown not to be mitogenic for purified T cells; however, 2-ME enhanced the early (Days 1 and 2) Con A (2 μg/ml)-induced response of NSC, DSC, and T-cell preparations, but depressed the late response (Days 4 and 5). 2-ME alone was mitogenic for purified B-cells, as reported previously; and the 2-ME-induced B-cell response was inhibited by Con A. Preincubation of T cells with 2-ME was sufficient for enhanced Con A responsiveness; however, if 2-ME was added 24 hr after the initiation of culture, no alteration of the Con A-induced response was observed. Ly-2,3⁺ T cells were unresponsive to Con A (0.3–20 μg/ml), but the addition of 2-ME or peritoneal cells enhanced the Con A responsiveness of Ly-2,3⁺ T cells over 200-fold. Ly-1⁺ T cells responded with a similar doseresponse and kinetic profile as unselected T cells. Although Ly-1⁺ T cells responded to Con A, unlike Ly-2, 3⁺ T cells, extensive removal of MØ significantly reduced the Con A-induced responsiveness of the Ly-1⁺ T cells. The reactivities of Ly-1⁺ and Ly-2,3⁺ DSC could be reconstituted by the addition of MØ or 2-ME; however, the kinetic response of Ly-1⁺ T cells peaked on Day 2–3, and Ly-2,3⁺ T cells had a delayed response which peaked on Day 4–5. The results indicated that (i) 2-ME and/or MØ accelerate the response kinetics of T-cells to Con A; (ii) T-cell subpopulations have differential requirements for MØ and/or 2-ME in the response to Con A; (iii) T-cell subpopulations exhibit differential dose responsiveness to Con A; and (iiii) 2-ME alters Con A responsiveness by a direct effect on T cells.     

The proliferative response of rat T cells to calcium ionophores increases with age

Splenocytes and T cells from both old and young rats proliferate to A23187 and ionomycin, and this response increases 3- to 10-fold in aged animals. Augmented responsiveness to ionomycin occurs in the absence of any defect in Con A-induced proliferation of T lymphocytes of aged rats and is dependent upon the addition of thiol compounds to the tissue culture medium. Augmented proliferative responses to ionomycin precede the significant but much smaller decline (30 to 40%) in Con A-induced proliferative responsiveness of splenocytes, which is evident only when rats reach 24 months of age. Heightened proliferation to calcium ionophores is not caused by a greater ability of T lymphocytes from aged rats to increase [Ca2+]i in response to ionomycin. The increased proliferative response of lymphocytes from aged rats to ionomycin occurs in the absence of detectable amounts of secreted IL 2 or IL 4. The ionophore response is a much more sensitive biomarker of age than the decline in Con A-induced proliferative responses of lymphocytes and identifies an activity of T lymphocytes that increases rather than decreases during the aging process.     

Differential inhibition of human T-lymphocyte activation by maleimide probes

Cellular thiols are known to be involved in lymphocyte activation, differentiation, and growth. In theory, alkylation of selective cellular thiols could be used to regulate specific processes in the activation sequence by inactivating particular enzymes or structural proteins, although to date specific alkylating probes have not been reported. N-Ethylmaleimide (NEM) is a lipophilic sulfhydryl-alkylating agent that is known to block the in vitro proliferative response of T lymphocytes. NEM (10 microM) was found to be fully inhibitory in PHA, Con A, and MLC assays only when added prior to or simultaneously with the mitogens or allogeneic cells; the addition of NEM only 15 sec after stimulating the cells with PHA resulted in a loss of greater than 50% of the inhibitory activity. The addition of 50 microM 2-ME 10 min after treating the cells with NEM failed to block the inhibitory effect. NEM (10-20 microM) had no adverse effect on lymphocyte viability, but completely blocked lymphocyte agglutination in response to mitogens or allogeneic cells. The lymphocytes overcame the inhibitory effects of NEM after 48 hr in both the PHA and MLC experiments. Resumption of the proliferative response was associated with the onset of agglutination in the PHA assay. In experiments using various analogs of NEM, we noted that the presence of a nonpolar N-linked side group was necessary for inhibitory activity. Pretreatment of PBMC with NEM decreased the total cellular thiols by 50% and blocked proliferation by 99%, whereas N-hydroxymaleimide decreased the total cellular thiols by 38% but had no effect on the proliferative response. The additional 12% of the cellular thiols that react with NEM, but not NHM, account for the inhibitory effect of NEM on lymphocyte proliferation. These findings suggest that selective cellular thiols are critical for T-cell activation.     

Effects of concanavalin A-induced cells on the proliferative response of T cells. Concanavalin A-induced suppressor and amplifier cells to the proliferative response of human T cells to trinitrophenyl-modified autologous lymphocytes.

Effects of Con A-induced human mononuclear cells on the proliferative response of peripheral T cells were examined by using TNP-modified autologous lymphocytes as stimulator cells. Cells induced by incubation with Con A contained both suppressor cells and amplifier cells. The former were induced from nylon wool-nonadherent T cells and these precursor cells were sensitive to mitomycin treatment. On the other hand, amplifier precursor cells were nylon wool-nonadherent T cells and were resistant to mitomycin treatment. Cell proliferation was required for the induction of suppressor cells but not for the induction of amplifier cells. Con A-induced suppressor effector cells were both nylon wool-adherent and nonadherent cells, on the contrary, Con A-induced amplifier effector cells were nonadherent cells. A small number of macrophages enhanced the suppressive activity of nonadherent T cells when added at the induction phase of suppressor T cells.     

Suppression of leukocyte inhibitory factor (LIF) production and [3H]thymidine incorporation by concanavalin A-activated mononuclear cells.

The capacity of human mononuclear (MN) cells pretreated with concanavalin A (Con A) to suppress the activity of fresh phytohemagglutinin (PHA)-pulsed mononuclear cells was assessed. Con A-pretreated MN cells suppressed leukocyte inhibitory factor (LIF) activity in supernatants of PHA-pulsed cell cultures and [³H]thymidine incorporation by these cells. Suppression was obtained in both allogeneic and autologous systems with mitomycin-treated, irradiated, or untreated Con A-induced cells. Lymphocytes from two patients that, following treatment with Con A, did not suppress mitogen-induced proliferative response of normal cells also did not suppress LIF production.     

Splenic and inguinal lymph node T cells of aged mice respond differently to polyclonal and antigen-specific stimuli.

Numerous changes have been reported to occur in T cell responsiveness of mice with increasing age. However, most of these studies have examined polyclonal stimulation of spleen cells from a limited number of mouse strains. This study investigated the influence of genetic background, source of lymphocytes, and type of stimulus on age-associated changes in T cells response. Con A-induced proliferation and IL-2 and IFN-gamma production by splenic lymphocytes (SL) was significantly greater in CBA/Ca mice compared to C57BL/6 mice, regardless of age. SL of both strains exhibited the predicted age-dependent decline in proliferative response and an increase in IFN-gamma production in response to Con A. In contrast, however, only SL from C57BL/6 mice demonstrated the predicted age-dependent decline in Con A-induced IL-2 production; Con A-induced SL of young and aged CBA/Ca mice produced comparable amounts of IL-2. Differences in age-associated responses to Con A were also observed between SL and inguinal lymph node (ILN) cells of CBA/Ca mice. In contrast to SL, ILN cells demonstrated an increased proliferative response to Con A. However, lymphokine production by Con A-stimulated ILN cells from aged CBA/Ca mice was similar to that of Con A-stimulated SL from aged CBA/Ca mice. To determine if aged ILN T cells respond similarly to polyclonal and antigen-specific stimuli, keyhole limpet hemocyanin (KLH) responses of T cells isolated from ILN of aged and young CBA/Ca mice were examined. KLH-specific T cells from aged mice cultured with KLH-pulsed macrophages (M phi) from aged mice were significantly reduced in their ability to proliferate compared to KLH-specific T cells of young mice cultured with young KLH-pulsed M phi. In contrast to the expected results, the defect was not at the level of the T cells; proliferation of young T cells cultured with aged KLH-pulsed M phi was equivalent to the proliferation of aged T cells cultured with aged M phi. These results suggest that aging has differential effects on polyclonal and antigen-specific T cell proliferation and on polyclonal stimulation of T cells isolated from different lymphoid organs and from different strains of mice.     

Con A-inducible suppression of MLC: evidence for mediation by the TH2 + T cell subset in man.

Human peripheral lymphoid cells pretreated with Concanavalin A for 48 hr can markedly suppress the proliferative response of untreated autologous lymphoid cells in MLC. Isolation studies with Sephadex G-200 anti-F(ab')2 affinity chromatography, nylon adherence, and E rosetting indicate that the Con A-induced suppressor cell is a T cell. Further fractionation into TH2+ and TH2- cell subsets with an equine-anti TH2 serum show that both subsets can be activated by Con A to an equivalent degree. After activation only the TH2+ subset can suppress autologous responder cells in MLC. The TH2- subset, which comprises 80% of peripheral human T cells, although induced by Con A to proliferate, cannot itself suppress the MLC response. Nevertheless, the TH2- subset can be shown to modulate the generation of suppressor TH2+ cells at 24 hr but not at 48 hr. These studies support the notion that the Con A-induced suppressor cell is confined to a distinct T cell subset in man and that T-T interactions are important in the overall expression of the immune response.     

Immune oxidative injury induced in mice exposed to normobaric O2: effects of thiol compounds on the splenic cell sulfhydryl content and Con A proliferative response

In vivo exposure of mice to normobaric O2 depresses the cellular immune response by a mechanism that remains unknown. In vitro oxidative injury leads to decreased sulfhydryl groups (SH) in lymphocytes. To determine whether in vivo exposure to O2 would have similar effects, we measured the SH content in spleen cells both from mice that had been exposed to normobaric O2 (O2 SC) and from controls exposed to ambient air (Air SC). The SH content of the fresh O2 SC was slightly decreased, whereas after 48 hr of culture, the SH content and the proliferative response of these cells were found to vary with the type and concentration of thiol or disulfide compounds added to the culture medium. Under standard culture conditions, i.e., RPMI 1640 medium containing 0.41 mM half-cystine, the SH content in O2 SC decreased sharply to about 10 and 20% that of Air SC in the absence or presence of Con A (2 micrograms/ml), respectively. Under these culture conditions, the proliferative response of O2 SC was 20.5% +/- 3.2 of Air SC. In cystine-free RPMI 1640 medium supplemented with various concentrations of L-cystine, L-cystine and 2-mercaptoethanol (2-ME), L-cysteine, or reduced glutathione (GSH), the proliferative response to Con A and the SH content of the O2 SC varied in parallel and were correlated (p less than 0.01). Half-cystine (0.41 mM) plus 2-ME (5 X 10(-5) M) or L-cysteine alone (4 mM) completely protected the SH content of O2 SC and induced a proliferative response 82% +/- 6 that of the controls. In cystine-free RPMI 1640 medium supplemented with GSH (4 mM), the SH content and proliferative response of O2 SC were 79 and 67.5% of Air SC, respectively. Other concentrations of these compounds were less effective. Oxygen scavengers such as SOD, catalase, mannitol, and vitamin E did not protect against the decrease of the O2 SC. The induced oxidative cellular damage might be related in part to a membrane lipid peroxidative process. These data show that in vivo exposure of mice to normobaric O2 induced lesions in splenic cells manifested under standard culture conditions by a decrease in both SH content and Con A proliferative response. The extent of these alterations could be modulated by variations of the thiol environment. Protection of the SH content correlated with protection of the proliferative response of the O2 SC.     

Swainsonine, an inhibitor of mannosidase II during glycoprotein processing, enhances concanavalin A-induced T cell proliferation and interleukin 2 receptor expression exclusively via the T cell receptor complex.

The T cell receptor (TCR) is a disulfide-linked heterodimer consisting of both complex and high-mannose types of N-linked oligosaccharides. The objective of the present investigation was to examine the effect of altered oligosaccharide structure on the expression and function of the TCR. Human mononuclear lymphocytes (MNL) were treated with castanospermine (CAST) or swainsonine (SW), inhibitors of glucosidase I or mannosidase II, respectively. Treatment with these inhibitors does not prevent glycosylation, but results in synthesis of glycoproteins with high-mannose or hybrid types of oligosaccharides. Treatment of MNL with CAST (1000-10 microM) or SW (100-1 microM) for up to 72 hr had no effect on cell surface expression of of the TCR. SW potentiated Con A-induced T cell proliferation without effecting anti-CD3 (OKT3) or alloantigen-induced proliferation. CAST had no effect on Con A, anti-CD3, or alloantigen-induced T cell proliferation. The T cell proliferative response to Con A in the presence of SW was completely eliminated in the presence of monoclonal anti-TCR antibodies. Monoclonal anti-CD2, -CD3, -CD4, -CD8, or isotypic control monoclonal antibodies had no effect on SW enhancement of T cell proliferation. SW treatment potentiated Con A-induced MNL expression of both the alpha and beta subunits of the IL 2R. This effect was also specifically blocked by anti-TCR monoclonal antibodies. These results demonstrate that selective changes in the glycosylation state of the TCR complex can alter mitogen recognition and subsequent cellular activation.     

Selective effects of interferon on distinct sites of the T lymphocyte triggering process

Lectin- and antigen-induced proliferation of murine T cells consists of two major events, namely, a rapid induction of susceptibility to growth factors and a later-occurring, accessory cell-dependent production of T cell growth factors (TCGF). The mechanism by which interferon (IFN) inhibits T cell responses was studied accordingly. A decrease of Con A-induced proliferation was observed in the presence of increasing amounts of IFN. The reduced proliferative response in such cultures was found to be due to an accumulation of cells in the G0/G1 phase of the cell cycle. Furthermore, the results show that IFN did not inhibit the early events in T cell triggering, because the acquisition of responsiveness of resting T cells to TCGF was unaltered in the presence of IFN, nor did it interfere with production of TCGF. In contrast, IFN was found to interfere with the TCGF-dependent T cell blast growth. Cytofluorometric analysis of the proliferative phase revealed that IFN exerts its effect on T cells, which have entered the proliferative cycle, by a postmitotic accumulation in G0/G1, thus reducing the proliferating population. The results demonstrate that IFN primarily affects the later phase of proliferative activity after T cell triggering, leaving the helper cell functions untouched.     

Trypanosoma cruzi: maintenance of parasite-specific T cell responses in lymph nodes during the acute phase of the infection

Mice infected with 5 x 10(3) forms of Trypanosoma cruzi showed a transient, but severe impairment of in vitro spleen cell responses to parasite antigens and to Concanavalin A (Con A). In contrast, inguinal and periaortic lymph node (LN) cells displayed high parasite-specific proliferative responses and only a partial reduction of the Con A-induced proliferation during the acute and chronic phases of infection. Lymphocytes that underwent blastic transformation in T. cruzi-stimulated cell cultures were of the L3T4+ phenotype. Suppression of spleen cell responses occurred in the acute phase whether mice were infected with high (3 x 10(5] or low (5 x 10(3] doses of T. cruzi by intraperitoneal or subcutaneous route. Suppression of the T. cruzi-specific proliferative response of LN cells was only observed in mice infected with high subcutaneous inocula. This suppression, however, was restricted to the LNs draining the site of inoculation without affecting distant LNs. Supernatants from parasite-stimulated proliferating LN cells displayed low or undetectable T cell growth factor (TCGF) activity, in contrast with the high TCGF levels found in supernatants of the same cells stimulated with Con A. Low levels of TCGF were also detected in cultures of LN cells from mice immunized with T. cruzi extracts. Neither the T. cruzi antigen used for in vitro stimulation nor the LN cell supernatants from infected mice inhibited TCGF activity. These findings indicate that (1) parasite-specific responses are present in the LN compartment throughout the acute phase of T. cruzi infection in mice and (2) the proliferative response of L3T4+ LN cells from infected mice to T. cruzi antigens is not associated with a high TCGF secretory response.     

Regulation of T cell proliferation by IL-7

The regulation of murine T cell proliferation by IL-7 was investigated. Highly purified resting splenic T cells were induced to proliferate in a short term assay by IL-7 in the presence of the comitogen, Con A. The proliferation of these resting T cells showed both IL-2-dependent and -independent components as determined by the susceptibility of the response to the blocking effects of anti-IL-2 mAb. Furthermore, IL-7 was found to augment the Con A-induced production of IL-2 and expression of IL-2R by resting splenic T cells. In contrast, Con A blasts and long term, Ag-dependent cloned T cells proliferated in response to IL-7 independently of any involvement of IL-2. Finally, differences were observed between IL-7 and IL-6 with regard to the regulation of T cell growth and activation. As with IL-7, IL-6 stimulated resting splenic T cells to proliferate in the presence of comitogen. However, in contrast to IL-7, IL-6 failed to stimulate the proliferation of Con A blasts or T cell clones and did not augment the Con A-induced expression of IL-2R on resting T cells.     

Intermediate Mr cytosolic components potentiate hepatic 5''-deiodinase activation by thiols.

The role in the activation of microsomal 5'-deiodinase (5'-DI) of rat hepatic cytosolic components of Mr approx. 13,000 (Fraction B) was studied in the presence of various concentrations of thiol compounds such as dithiothreitol (DTT), dihydrolipoamide (DHLA), GSH, and 2-mercaptoethanol (2-ME). Although Fraction B (which was prepared by gel filtration to exclude GSH and GSSG) had no intrinsic 5'-DI activity, could not stimulate microsomal 5'-DI activity in the absence of added thiol and did not contain GSH as a mixed disulphide, it could produce a 3-fold increase in the maximal deiodinase activity achievable with DTT as well as other thiols, with the order being the same as the activation potency of these thiols in the absence of Fraction B (i.e. DHLA greater than DTT greater than 2-ME greater than GSH). These observations suggest that: a component of cytosolic Fraction B, designated 'deiodination factor B' (DFB), operates as an efficient intermediary to enhance activation of microsomal 5'-DI by thiols through a mechanism independent of GSH; thiols may participate in a non-specific thiol-disulphide exchange with inactive (oxidized) DFB to convert it into an active form that contains one or more thiol groups and is more effective than GSH or other thiols in facilitating the re-activation of inactive (oxidized) microsomal 5'-DI thiol (ESI) to its active state (ESH).     

Increased cytolytic T lymphocyte activity induced by 2-mercaptoethanol in mixed leukocyte cultures: kinetics and possible mechanisms of action.

The 20- to 50-fold increase in cytolytic T lymphocyte (CTL) activity caused by the addition of 50 muM 2-mercaptoethanol (2-ME) at the onset of a one-way murine mixed leukocyte culture (MLC) between C57BL/6 and DBA/2 splenic lymphocytes appears to be unrelated to early events in the culture: if 2-ME was present for the first 24 hr of culture only, there was no increase on day 4, but if addition of 2-ME was delayed until the last 24 hr of culture, the CTL activity was almost as high as that of cultures that were exposed to 2-ME for the entire 4-day culture period. The increase of CTL activity caused by delayed addition of 2-ME ("2-ME rescue") was used to investigate the mechanism by which the thiol induces differentiation of CTL from precursor cells. 2-ME rescue was mimicked by two other thiols, dithiothreitol and cysteamine phosphate, but at higher concentrations. Because the latter compound has no free sulhydryl group until it diffuses into cells and is enzymatically dephosphorylated, we conclude that thiols may increase the differentiation of CTL from precursor cells by an intracellular process involving free sulphydryl groups rather than by interaction with membrane sulfhydryls or destruction of inhibitor cells or their products. Cell separation experiments indicated that 2-ME rescue was independent of the presence of B lymphocytes and of adherent cells (macrophages) and was restricted to a subpopulation of T lymphocytes that developed into large lymphoid precursor cells during the first 3 days in culture even without 2-ME. The development of this subpopulation required DNA synthesis between 24 nad 72 hr after the onset of MLC. When 2-ME was added to day-3 MLC, CTL activity increased slightly as early as 4 hr later, but the major increase occurred during the second half of the 24 hr "rescue"period. Because this increase was inhibited by cytosine arabinoside (ARA-C), it seems likely that DNA synthesis is associated with and may be required for the differentiation of large precursor lymphoid cells into CTL after the addition of 2-ME.     

Expression of increased immunogenicity by thiol-releasing tumor variants.

Even moderate variations of the extracellular cysteine concentration were previously shown to affect T cell functions in vitro despite high concentrations of cystine. We therefore analyzed the membrane transport activities of T cells for cysteine and cystine, and the role of low molecular weight thiol in T cell-mediated host responses against a T cell tumor in vivo. A series of T cell clones and tumors including the highly malignant lymphoma L5178Y ESb and its strongly immunogenic variant ESb-D was found to express extremely weak transport activity for cystine but strong transport activity for cysteine. However, not all cells showed the expected requirement for cysteine (or 2-mercaptoethanol (2-ME)) in the culture medium. One group of clones and tumors including the malignant ESb-lymphoma did not respond to changes of extracellular cystine concentrations and was strongly thiol dependent. This group released only little acid soluble thiol (cysteine) if grown in cystine-containing cultures. The other T cell lines, in contrast, were able to maintain high intracellular GSH levels and DNA synthesis activity in cystine-containing culture medium without cystein or 2-ME and released substantial amounts of thiol. This group included the immunogenic ESb-D line. Additional thiol-releasing ESb variants were obtained by culturing large numbers of L5178Y ESb tumor cells in cultures without cysteine or 2-ME. All of these ESb variants showed a significantly decreased tumorigenicity and some of them induced cytotoxic and protective host responses even against the malignant ESb parent tumor. Taken together, our experiments suggest that the host response against a tumor may be limited in certain cases by the failure of the stimulator (i.e., the tumor) cell to deliver sufficient amounts of cysteine to the responding T cells.     

Simultaneous suppression of allogeneic cytolytic activity and stimulation of lectin-dependent cytolytic activity by con A.

It is well known that when mouse lymphocytes are cultured with irradiated allogeneic stimulator lymphocytes, T cells capable of mediating specific cytolysis are generated. We noted that when the stimulator cells were pretreated with concanavalin A (Con A), the generation of T cell-mediated specific lysis (TSL) is largely abolished, but large amounts of T cell-mediated lectin-dependent lytic (LDL) activity are nevertheless produced. Data are presented indicating that generation of TSL is inhibited by suppressor cells which are generated by the Con A in the culture.One possible source of the LDL would be a specific clonal response to Con A-altered H-2 molecules. Evidence concerning this was equivocal since the amount of lectin required tor expression of the LDL is suffcient to cause non-specific T cell-mediated lysis of any target cell type, making inoperative the conventional tests for H-2 restricted recognition.Use of dead cell fragments for stimulation in MLC has previously been reported to produce LDL without TSL, but Con A-induced premature death of the stimulator cells was ruled out as a source of the LDL in our cultures.Another possible source of LDL would be suppressor-induced blockade of killer cell differentiation at a hypothetical stage expressing LDL but not TSL. However, delayed addition of Con A induced suppressor cells to M LC's never allowed generation of LDL when TSL was suppressed. Therefore, such a blocked differentiation mechanism did not contribute significantly to the LDL produced.The LDL activity results largely from Con A-induced polyclonal activation of cytolytic progenitor cells. It is shown that generation of LDL by Con A is able to be suppressed by Con A-induced suppressor cells added at the initiation of culture. However, in cultures in which Con A is simultaneously generating LDL and suppressor activities, the LDL is generated rapidly (in 2 days) and apparently passes the suppressable stage before the suppressor cells become active.     

Virus-induced interferon alpha/beta (IFN-alpha/beta) production by T cells and by Th1 and Th2 helper T cell clones: a study of the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta on functions of different T cell populations

Spleen cells, resting T cells, activated T cells, and T cell clones characterized as type 1 (Th1) and type 2 (Th2) were investigated for their ability to produce interferon (IFN) following in vitro culture with Newcastle disease virus (NDV). All of the above cell populations, including both Th1 and Th2 T cell clones, produced high levels of IFN following in vitro culture with NDV. This IFN was characterized as a mixture of IFN-alpha and IFN-beta with IFN-alpha being the predominate species of IFN contained in the mixture. IL-2 greatly enhanced the production of IFN-alpha/beta by all cell populations in response to NDV. These different T cell populations responded very differently to the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta. IFN-alpha/beta was shown to be a potent inhibitor of Con A or IL-2-induced proliferation of different T cell populations. This inhibition was not associated with a reduction in lymphokine production since spleen cells or Th1 T cell clones cultured with Con A and IFN-alpha/beta had no decrease in IL-2 or IFN-gamma production when compared to Con A-stimulated control cultures. IFN-gamma had little to no inhibitory activity on Con A-induced proliferation of spleen cells. In fact, Con A-induced proliferation was usually enhanced by IFN-gamma when nylon wool-enriched T cells were assessed. Different results were observed when IFN-gamma and IFN-alpha/beta were investigated for their ability to inhibit IL-2-induced proliferation of different T helper cell clones. IFN-gamma and IFN-alpha/beta were both capable of inhibiting IL-2-induced proliferation of T cell clones characterized as type 2 (Th2). In contrast, IFN-gamma had no effect on IL-2-induced proliferation of Th1 clones. IFN-alpha/beta, however, inhibited IL-2-induced proliferative responses of both Th1 and Th2 T cell clones. These results document the facts that (1) IFN-gamma and IFN-alpha/beta differ in their immunoregulatory actions, (2) different T cell subpopulations vary in their susceptibility to IFN-gamma regulation, and (3) virus induction of IFN-alpha/beta appears to be a ubiquitous function associated with different T cell populations.     

The effect of changes in thiol subcompartments on T-cell colony formation and cell cycle progression: relevance to AIDS.

Recently, it has been shown that intra- and extracellular thiol levels are significantly lower than normal even in the relatively early stages of human immunodeficiency virus (HIV) infection. It is plausible that this deficiency could contribute both to the loss of T-cell function and the ability to replenish T cells associated with HIV infection. We had previously reported that the T-cell colony-forming cell (T-CFC) is impaired in HIV infection and that it can be enhanced with the thiol compounds 2-mercaptoethanol (2-ME) and N-acetylcysteine (NAC). In this study, the effect of the thiol-depleting reagents buthionine sulfoximine, cyclohexene-1-one, and copper phenanthroline on T-CFC formation and cell cycle progression was determined in HIV+ subject and/or controls. All three reagents inhibited T-CFC formation and cell cycle progression with a suggestion that colony formation by cells from HIV+ subjects was more sensitive to the effects of thiol depletion. 2-ME and NAC enhanced effect of NAC did not appear to involve increased protein kinase C translocation. Our results suggest that oxidation of membrane thiols, as well as depletion of intracellular glutathione, inhibits T-CFC formation as well as cell cycle progression for mitogen-stimulated cells in bulk culture.