Characterization of effector lymphocytes associated with immunity to murine sarcoma virus (MSV) induced tumors. I. Physical properties of cytolytic T lymphocytes generated in vitro and of their immediate progenitors.

Cytolytic T lymphocytes (CTL) were generated in secondary mixed leukocyte-tumor cell cultures (MLTC) with syngeneic RB1-5 tumor cells as stimulating cells and with responding spleen cells from regressor mice that had rejected a murine sarcoma virus (MSV)-induced tumor. CTL precursor cells were found to be exclusively of thymic origin and non-T cells were apparently not required for CTL generation. When the size variations of CTL from syngeneic MLTC were analyzed by velocity sedimentation it appeared that a transition from small precursor cells to larger effector cells occurred during the first 5 days in culture; this change in cell size was then followed by a shift toward small-sized cells. Furthermore, the CTL generated in syngeneic MLTC in the MSV tumor immune system were compared with those CTL obtained in allogeneic mixed leukocyte cultures (MLC) and were shown to exhibit fundamental similarities.     

Inhibition of cytolytic T lymphocyte maturation with ornithine, arginine, and putrescine

L-Ornithine was shown to inhibit the development of cytolytic T lymphocytes (CTL) in mixed lymphocyte cultures (MLC). Lymphokines were unable to reverse the suppressive effect, and cytotoxic activity was not revealed by coupling ornithine-inhibited MLC cells to target cells with phytohemagglutinin (PHA). If addition of ornithine to MLC were delayed, sensitivity of CTL to inhibition was reduced after 24 hr and lost by 48 hr. Suppression of CTL development was not due to a toxic effect. MLC washed free of ornithine after 3 days produced detectable cytolytic activity within 24 hr of secondary culture, and to the same degree as the uninhibited MLC control within 48 hr. Cytotoxic cells generated in secondary cultures were Lyt-2+, did not kill the natural killer-sensitive YAC-1 cell line, and were shown to be antigen-specific by virtue of the findings that cytolysis and cold target inhibition were observed only with cells carrying the original, inducing H-2 haplotype. Cytolysis of target cells by normal CTL effector cells was not inhibited by L-ornithine. MLC depleted of accessory cells so that CTL activation was dependent upon addition of lymphokines remained susceptible to inhibition by ornithine. Our findings indicate that in the ornithine-inhibited MLC, CTL precursors undergo clonal expansion, but their maturation is arrested at a precytolytic stage. L-Arginine and putrescine also suppressed generation of CTL in primary MLC, and cells recovered from arginine- and putrescine-inhibited MLC developed control levels of CTL within 48 hr of secondary culture. Inhibition by putrescine was observed in tissue culture medium supplemented with human serum but not with fetal calf serum, presumably due to the presence of diamine oxidase activity in fetal calf serum. Similar to ornithine, the suppressive effects of arginine and putrescine on T lymphocytes were apparently selective for CTL because they did not inhibit mitogen activation with concanavalin A or the production of interleukin 2 and interleukin 3. These findings are consistent with a hypothesis that the inhibitory effects of ornithine, arginine, and putrescine are mediated by polyamines, and exerted on the differentiative stage of CTL development.     

Interleukin-7 mediates the generation and expansion of murine allosensitized and antitumor CTL.

Interleukin-7 (IL-7) is a 25-kDa cytokine that was initially described as a pre-B cell growth factor and more recently has been shown to cause T cell proliferation. We have investigated the in vitro effects of IL-7 on mature T cells to include the generation and further expansion of allospecific and antitumor CTL. B6 anti-DBA allospecific CTL were generated in the presence of IL-7, IL-2, the combination IL-7 plus IL-2, or no cytokine. IL-7 alone or when combined with IL-2 enhanced the generation of allospecific CTL. To evaluate the proliferative effects of IL-7, 4-day B6 anti-DBA cultures were cultured in IL-7, IL-2, or no cytokine. Cell proliferation and duration of growth of cells cultured in IL-7 were significantly greater than cells cultured in IL-2 or in the absence of cytokine. Allospecific cytolytic activity was maintained during proliferation in IL-7 to a maximum of 60 days. In contrast with the ability of IL-2 to generate LAK cells, murine splenocytes cultured at varying doses of IL-7 (1 to 10,000 ng/ml), resulted in minimal LAK cell activity. The effect of IL-7 in the generation of CTL with antitumor activity was also studied. Seven days after footpad injection of MCA 203 or 205 sarcoma, draining lymph nodes (DLN) were harvested and restimulated in vitro with MCA 203 or 205, respectively, and maintained in culture with either IL-7, IL-2, the combination of IL-7 plus IL-2, or no cytokine. After 10 days in culture, cells generated in IL-7 or IL-2 exhibited similar cytotoxicity against the syngeneic autologous MCA tumor. IL-7 generated cells, however, showed specificity when tested by 51Cr release which was not seen with IL-2-generated cells. Cells generated in IL-7 plus IL-2 were more cytolytic than cells cultured with either cytokine alone. To further define the mechanism of action of IL-7 in antitumor CTL cultures, a monoclonal antibody, S4B6.1, capable of blocking murine-specific IL-2 was employed. The partial inhibition by this mAb of the generation of antitumor CTL demonstrated that IL-7 acted, in part, by an IL-2-dependent mechanism. Finally, IL-7 cultures restimulated at Day 11 with autologous MCA 203 showed greater proliferation than IL-2 cultures and remained lytic at Day 21 of culture.(ABSTRACT TRUNCATED AT 400 WORDS)     

Generation of cytotoxic T lymphocytes in vitro. VI. Effect of cell density on response in mixed leukocyte cultures.

Reexposure of day 14 murine mixed leukocyte culture (MLC) populations to the original irradiated allogeneic stimulating spleen cells has previously been found to result in the ratpid generation of cytolytic T lymphocytes (CTL) associated with a net increase in cultured cell number. Under the experimental conditions used, day 5 MLC cells appeared unable to respond to the allogeneic stimulus. In order to characterize further the development of the potential for anamnestic reactivity during the course of MLC, C57BL/6 spleen cells were incubated with irradiated (1000 rads) DBA/2 spleen cells (primary MLC) for up to 3 weeks. At various time intervals after the onset of the primary MLC, the surviving cells were collected and reexposed, at varying cell concentrations, to irradiated DBA/2 spleen cells (secondary MLC). At daily intervals thereafter, CTL activity was assessed using a quantitative 51Cr-release assay system. A paradoxic effect of responding cell concentration on generation of CTL activity was observed; relatively greater increase in CTL activity was observed as the concentration of responding cells was decreased over a 100-fold range. This effect was more pronounced with responding cells reexposed to antigen after primary MLC for 20 days, but was observed even with normal cells. The apparent unresponsiveness of day 5 MLC cells to alloantigen restimulation could be overcome by simple dilution of responding cells. Cytotoxic activity at the time of restimulation with antigen seems to be a major factor determining the magnitude of the secondary response. Since intact cells bearing alloantigens are required for the generation of CTL in MLC, residual cytotoxic cells reduce the effective antigenic stimulus by destroying stimulating cells. This effect of concentration of responding cells on generation of CTL in MLC complicates interpretation of experiments investigating the role of "inhibitor" and "helper" cell in cell-mediated immune responses occurring in vitro. Under optimal conditions, the highest CTL activity and the largest increase in total cell number was observed 4 days after restimulation of day 10 MLC cells. On a per cell basis, the lytic activity was up to 4 times greater than that observed at the peak of a primary response, and the number of viable cells recovered was nearly 20 times higher than that at the onset. Such secondary MLC are thus a convenient source of lymphoid cells selected primarily on the basis of proliferation induced by alloantigens.     

Immunoregulation by mouse T-cell clones. I. Suppression and amplification of cytotoxic responses by cloned H-Y-specific cytolytic T lymphocytes

H-Y-specific and H-2Db-restricted, Lyt-1-2+ T-cell clones ( CTLL ) with graded specific cytotoxic activities on male C57BL/6 (B6) target cells ( 1E3 , ; 2C5 , ++; 2A5 , +, 3E6 , +/-) were tested for their capacity to inhibit the generation of H-Y-specific cytotoxic T lymphocytes (CTL) in vitro. Addition of irradiated lymphocytes of CTLL 1E3 and CTLL 3E6 but not those of CTLL 2A5 or CTLL 2C5 abolished the generation of CTL from in vivo primed H-Y-specific precursor cells (CTLP) when added to fresh mixed-lymphocyte cultures (MLC). Exogenous sources of T-cell growth factors (TCGF) did not overcome suppression. Rather the presence of TCGF resulted in a further enhancement of suppressive activities in CTLL 1E3 and 3E6 and the induction of similar activities in cells from CTLL 2A5 and 2C5 , which by themselves were not inhibitory. Moreover when added to similar MLC on Day 1 instead of Day 0, only irradiated cells of CTLL 3E6 but not those of the other three CTLL were suppressive. Induction of suppressive activities in H-Y-specific CTLL was independent of the appropriate male stimulator cells since it was also observed in MLC induced by irrelevant antigens (H-2, trinitrophenol). Furthermore at low cell numbers, irradiated lymphocytes from any of the CTLL consistently enhanced CTL activities generated from H-Y-specific CTLP. This augmenting activity, which was not TCGF, could be transferred by soluble mediators present in antigen-sensitized CTLL cultures. Thus, these data indicate (i) that cytotoxic effector cells can function as suppressor cells in the generation of CTL, (ii) that the cytotoxic activity of cloned CTL does not correlate with their capacity to suppress CTL responses, (iii) that the inhibition of CTL responses by CTLL is not due to simple consumption of T-cell growth factors produced in MLC, and (iv) that different CTL clones may interfere with the generation of CTL at different stages of their maturation. Moreover, the experiments suggest an antigen-independent enhancement of suppression by the interaction of CTL with lymphokines. Together with the augmenting activity evoked by cloned CTL the data provide strong evidence for the expression of multiple immunological functions by one particular subset of T cells and suggest that cytotoxic effector cells can differentially regulate the maturation and/or clonal expression of their precursor cells.     

Linear density gradient separation of human lymphocyte subsets. II. Characterization of cells responding in secondary MLC and CML.

Lymphocytes were separated on linear density gradients (LDG) after they had been sensitized in vitro against allogeneic cells and had reverted to small cells. Cells from individual density fractions were restimulated with autologous, specific, or third-party cells and assayed 48 hr later for their response in secondary mixed leukocyte culture (MLC) and cell-mediated lympholysis (CML). Memory cells capable of responding in secondary MLC were broadly distributed and found in both heavy and light fractions. The various density classes of memory cells differed with respect to the degree of their specificity for the restimulating cells. In secondary MLC the greatest specificity for the originally sensitizing cells and the least cross-reactivity for third-party cells were primarily features of light- and medium-density cells. Memory killer cells for CML were fairly homogeneously grouped. Following restimulation, killers were enriched in light to medium fractions also, as was previously seen at the peak of the response on Day 6.     

Effect of synthetic beta-carotene on the formation of cytolytic T-lymphocytes

The effect of intraperitoneal injection of beta-carotene in different doses on the formation of cytolytic T lymphocytes (CTL) in a one-way mixed lymphocyte culture (MLC) of allogeneic mice was studied. The maximal cytotoxic activity of lymphocytes was attained in the MLC with splenocytes of mice which received 10 mg/kg beta-carotene 6 days before experimentation. The correlation was studied between the beta-carotene ability to stimulate CTL formation and antineoplastic activity. It was discovered that injection of beta-carotene in doses and times provoking maximal CTL induction had no effect on the animals' lifespan and the size of transplanted sarcoma 180.     

Immunologically specific cytolytic activity induced in long-term mixed leukocyte culture cells by concanavalin A.

Stimulation of cells from long-term primary MLC with Con A resulted in the generation of CTL activity comparable in magnitude to that induced by reexposure of the cells to the original stimulating cellular antigen. CTL generated by stimulation of long-term MLC cells with ConA had lytic activity specific for the original stimulating alloantigen used in primary MLC. The pattern of stimulation of long-term MLC cells with Con A differed from that of restimulation with alloantigen in that there was no detectable CTL activity the first 24 hr after Con A stimulation and the peak lytic activity occurred later. Unlike restimulation with alloantigen early lytic activity after Con A stimulation was dependent on DNA synthesis. PHA also proved to be an effective agent for stimulating cytolytic activity in long-term MLC cells. The response to PHA was comparable in magnitude to that generated by Con A. Stimulation of long-term MLC cells with T cell mitogens gave decreased cell recoveries relative to restimulation with alloantigen, however, the lytic activity per cells recovered was generally greater in the mitogen-stimulated cultures.     

Inhibition of T cell-mediated cytolysis by 2-deoxy-D-glucose (2-DG): differential effect of 2-DG on effector cells isolated early or late after alloantigenic stimulation in vitro.

The effect of the hexose analogue 2-deoxy-D-glucose (2-DG) on the functional activity of various populations of cytolytic T lymphocytes (CTL) has been compared. Under aerobic conditions, CTL harvested at the peak of the response (day 4) in primary or secondary mixed leukocyte cultures (MLC) were much more readily inhibited by 2-DG that CTL obtained from MLC at later times (day 11 to 18) or from the peritoneal cavity of alloimmune mice. Quantitatively, 0.4 mM 2-DG was sufficpient to inhibit cytolysis by 50% in day 4 CTL populatons, whereas 25 mM had little or no effect on day 11 to 18 CTL. Evidence was obtained that inhibition of cytolysis by 2-DG under these conditions was accompanied by a parallel inhibition of effector:target cell binding. In contradistinction to these findings, the cytolytic activity of both day 4 and day 11 MLC cells was readily inhibited by 2-DG under conditions where cell respiration was blocked by sodium azide. Furthermore, uptake of radiolabeled 2-DG was observed under aerobic conditions in both day 4 and day 11 MLC cells. These results strongly suggest that inhibition of cytolysis by 2-DG under aerobic conditions is mediated via a direct effect on CTL which is independent of the consequences of energy depletion. An indirect method by which CTL may be inhibited by 2-DG is suggested.     

Dissociation of differentiation and proliferation in the primary induction of cytolytic T lymphocytes by alloantigens

The requirement for DNA synthesis in the induction of cytolytic T lymphocytes (CTL) by alloantigens has been investigated. C57BL/6 splenic T cells purified by passage on nylon wool columns were stimulated in vitro in mixed leukocyte culture (MLC) and assayed for cytotoxicity against 51Cr-labeled target cells. With this system, CTL activity was detectable after 24 hr of MLC and reached high levels after 48 hr. Addition of cytosine arabinoside (ARA-C) or hydroxyurea to such cultures at concentrations that were sufficient to inhibit DNA synthesis by greater than 98% did not reduce CTL activity measured after 24 hr; however, the increase in activity that occurred between 24 and 48 hr in control cultures was strongly reduced (or abolished) by these drugs. Velocity sedimentation analysis of MLC cells activated for 48 hr in the presence of ARA-C further revealed that CTL precursor lymphocytes had enlarged into medium- to large-sized CTL under these conditions. These studies provide direct evidence that the primary induction of CTL by alloantigens can be dissociated into a differentiation step, which occurs within 24 hr in the absence of DNA synthesis and is accompanied by blast transformation, and a subsequent proliferation.     

Generation of cytolytic T lymphocytes in vitro. VIII. failure of anti-RS antisera to inhibit the generation of cytolytic T lymphocytes or cytolytic T lymphocyte activity.

Antibody reactive with "recognition structures" (RS) of mouse lymphoid cells for alloantigens (anti-RS) was prepared by immunization of F1 hybrid mice with parentalstrain lymphoid cells or with antibody produced in one parental strain against alloantigens of the other parental strain. Such antisera prevented generation of the "product of antigenic recognition" (PAR) that is produced within a few hours in cultures prepared with a mixture of lymphoid cells from genetically disparate mice. However, treatment of responding lymphoid cells with anti-RS sera and complement did not inhibit generation of cytolytic T lymphocytes (CTL) in mixed lymphocyte cultures (MLC). Treatment of cells obtained from MLC with anti-RS sera and complement failed to inhibit cytolytic activity of such cells for specific alloantigens.     

Regulatory mechanisms in cytotoxic T lymphocyte development. II. Dissociation of in vitro cytotoxic T-lymphocyte and suppressor T-cell activities with L-ornithine

L-ornithine was found to differentially affect the induction of allospecific cytotoxic T lymphocytes (CTL) and suppressor T cells (Ts). At a concentration of 10 mM, ornithine inhibited the development of CTL in a mixed-leukocyte culture (MLC). This same population of cells suppressed the generation of CTL when irradiated and cocultured with fresh syngeneic lymphocytes and alloantigen. Suppression was mediated by Lyt-1-2+ cells and was antigen specific. Suppression was abrogated when IL-2 (10 U/ml) was added to the cocultures, but could not be reversed by increasing the antigen dose. Ornithine was not toxic to CTL precursors but rather arrested their development. Cells from MLC plus ornithine developed CTL activity within 2 days of transfer to secondary cultures in the absence of ornithine. Development of CTL effector cells (CTLe) was augmented by but did not require exogenous IL-2. Generation of CTLe from the MLC plus ornithine population was radiation sensitive and could be inhibited by reexposure to ornithine, even in the presence of IL-2. Thus, Lyt-1-2+ T cells allostimulated in vitro in MLC plus ornithine and lacking CTL activity convey radiation-resistant, antigen-specific suppression.     

Three different signals are required for the induction of cytolytic T lymphocytes from resting precursors

The requirement for the signals in induction of cytolytic T lymphocytes (CTL) has been investigated. C57BL/6 X CBA/T6 F1 spleen cells stimulated with the lectin leukoagglutinin (L-A) failed to show CTL activity in a PHA-facilitated assay, although L-A-activated splenic T cells were able to respond to T cell growth factor (TCGF). Concanavalin A (Con A) on the other hand was able to induce cytolytic activity from CTL-P, as well as to render splenic T cells responsive to TCGF. Furthermore, L-A-activated splenic T cells could generate cytolytic activity upon subsequent culture in secondary mixed leukocyte culture supernatant (2 degrees MLC SN). In contrast, EL-4-derived SN (EL-4 SN) was unable to induce cytolytic activity from L-A-activated spleen cells. In addition, proliferation of L-A-activated spleen cells cultured in EL-4 SN was similar to those cultured in 2 degrees MLC SN. Nonactivated spleen cells were totally unresponsive to both SN in proliferation and generation of CTL. Analysis of T cell clones for the production of a factor necessary for induction of cytolytic activity revealed that both cytolytic and noncytolytic T cell clones were able to produce a factor(s) for the generation of cytolytic activity from L-A-activated T cells. On the other hand, SN from certain antigen-stimulated T cell clones produced factors capable of inducing cytocytic activity by L-A-activated T cells only in the presence of EL-4 SN. Neither EL-4 SN nor cloned T cell SN alone had such a capacity. The nature of the necessary lymphokines in the SN from the clone cells or from the EL-4 is unknown. In the case of the EL-4 SN, it is not known whether the presence of TCGF plays a role or whether that role is perhaps more differentiative than proliferative. This study provides evidence that the induction of CTL from CTL-P can be dissociated into activation, which is required to render T cells responsive to second signals, and differentiation, which is mediated by two different factors.     

Modulation of in vitro immune responses by monoclonal antibody to T200 antigen

The effects of monoclonal antibody to the T200 antigen on murine mixed-lymphocyte cultures (MLC) and on the generation of alloreactive cytotoxic T lymphocytes (CTL) are investigated. Addition of monoclonal anti-T200 without complement to MLC results in a late suppression of the proliferative response preceded in some cases by an early enhancement. These modulations require the presence of allogeneic stimulator cells; no effects are seen when antibody is added to responders alone. A similar effect is seen on the generation of CTL. Compared to controls without antibody, cultures carried out in the presence of anti-T200 show reduced levels of cytotoxicity measured against allogeneic targets by Day 5. The kinetics of the suppressive effects differ from those seen with anti-Lyt-2, and no suppressive effects are seen with monoclonal antibodies to other cell surface molecules.     

Human T cell subpopulations defined by a monoclonal antibody. II. Evidence for cell cooperation in the response to alloantigens and generation of cytolytic cells

It has been shown previously that the 5/9 monoclonal antibody defines a small T cell subpopulation in human peripheral blood that includes all the cells responsible for proliferation to tetanus toxoid and to alloantigens as well as the helper cells for B cell differentiation. In the present study, human peripheral blood T cells were fractionated according to their reactivity with the 5/9 monoclonal antibody and stimulated in mixed lymphocyte culture (MLC). In spite of a strong proliferative response in MLC, 5/9+ cells generated no cytolytic activity against PHA-activated lymphocytes bearing the stimulating alloantigens (CTL activity) or against the K562 human cell line (NK activity). The precursors of these cytolytic effector cells were present in the 5/9- fraction. However, 5/9+ cells or soluble factors derived from 5/9+ cells were needed to induce 5/9- cells to respond in MLC and develop cytolytic activity. Both 5/9+ and 5/9- cell populations, upon MLC stimulation, were able to lyse L1210 mouse lymphoma cells in the presence of specific antibodies (ADCC).     

Generation of cytotoxic T lymphocytes in vitro. VII. Suppressive effect of irradiated MLC cells on CTL response.

Irradiated cells obtained from MLC at the peak of the CTL response caused profound suppression of generation of CTL when added in small numbers at the initiation of primary MLC prepared with normal spleen cells. The inhibitory activity of the MLC cells was not affected by irradiation (1000 rads) but was abolished by treatment with anti-theta serum and complement. The suppression was immunologically specific. The response of A (H-2a) spleen cells toward C3H (H-2k) alloantigens was suppressed by irradiated MLC cells obtained from MLC prepared with A spleen cells and irradiated C3H-stimulating cells, whereas the response of A spleen cells toward DBA/2 (H-2d) alloantigens was affected relatively little. However, if irradiated C3H X DBA/2 F1 hybrid spleen cells were used to stimulate A spleen cells in MLC, addition of irradiated MLC cells having cytotoxic activity toward C3H antigens abolished the response to both C3H and DBA/2 antigens. The response to DBA/2 antigens was much less affected when a mixture of irradiated C3H and DBA/2 spleen cells was used as stimulating cells. Thus, the presence of MLC cells having cytotoxic activity toward one alloantigen abolished the response to another non-cross reacting antigen only when both antigens were present on the same F1 hybrid-stimulating cells. This suppression of generation of CTL by irradiated MLC cells apparently involves inactivation of alloantigen-bearing stimulating cells as a result of residual cytotoxic activity of the irradiated MLC cells. This mechanism may be active during the decline in CTL activity noted in the normal immune response in vivo and in vitro.     

Inhibition of murine suppressor cell function by progesterone

The effects of progesterone on murine suppressor cell function generated in allogeneic MLCs were investigated. BALB/c splenic lymphocytes stimulated in vitro with C3H/He cells significantly suppressed the proliferative response of BALB/c lymphocytes in a secondary MLC. This suppression was highly specific for the sensitizing alloantigens since the suppressor cells had no effect on the proliferative response of BALB/c lymphocytes to third-party alloantigens. In addition, BALB/c lymphocytes stimulated with syngeneic cells were observed to nonspecifically suppress the MLC response to a lesser extent. One to 10 micrograms/ml progesterone added at initiation to suppressor cell generating cultures diminished the ability of both alloantigen specific and nonspecific suppressor cell populations to suppress the proliferative response of homologous lymphocytes to alloantigens. Experiments with pyrilamine, an antihistamine, which blocks cytotoxic T lymphocyte (CTL) generation, suggests that progesterone has a direct inhibitory effect on suppressor cell function independent of its ability to block CTL induction. The effects of progesterone on suppressor cells were not due to shifts in peak response time in MLC or induction of radiosensitive cells in progesterone-treated cultures. Estradiol at doses between 5 and 10 micrograms/ml, and cortisol at dose of 1 microgram/ml, also significantly inhibited suppressor cell function. These results suggest that the steroid hormone milieu within the placenta may effect the activity of allogeneic or nonspecific suppressor cell activity.     

Alloantigen-induced activation of rat splenocytes is regulated by the oxidative metabolism of L-arginine

Activated macrophages have been demonstrated to metabolize the amino acid L-arginine by the oxidative pathway to produce nitric oxide, citrulline, and NO2-/NO3-. Nitric oxide has been shown to be cytostatic for tumor targets and to inhibit the mitochondrial respiration and other functions of the macrophages that produce it. Addition of NG monomethyl-L-arginine (NMA), a competitive inhibitor of oxidative L-arginine metabolism, to rat splenocyte (SPL) MLC results in allospecific lymphocyte proliferation and CTL induction. In the absence of NMA, neither proliferation nor CTL induction is observed. Citrulline and NO2-/NO3- levels in the supernatants of rat SPL MLC are decreased in the presence of NMA compared with cultures without NMA. NMA also augments the proliferation and CTL induction in mouse SPL MLC. Detectable levels of cytokines able to induce T cell proliferation were present in supernatants of rat SPL MLC without NMA on days 1 to 5 of culture. Supernatants of cultures with NMA contained detectable levels of cytokines on days 1 to 3 and undetectable levels by days 4 and 5 of culture, concomitant with the observed lymphocyte proliferation and presumed depletion of cytokines. Thus, inhibition of rat SPL proliferation to alloantigen seems not to be caused by the lack of production of cytokines able to induce T cell proliferation. The inhibition of proliferation and CTL induction in rat SPL cultures may be caused by a direct effect of the cytostatic products of oxidative L-arginine metabolism on lymphocyte proliferation, or by an indirect deleterious effect on the mitochondrial respiration and viability of macrophages that oxidatively metabolize L-arginine. Alternatively, diversion of L-arginine to the oxidative pathway may affect production of polyamines that are necessary for cell growth and proliferation.     

Cellular interactions in the cytotoxic T lymphocyte response to herpes simplex virus antigens: differential antigen activation requirements for the helper T lymphocyte and cytotoxic T lymphocyte precursors

The role and induction requirements of helper T lymphocyte responses to herpes simplex virus type 1 (HSV-1) was examined. Splenocytes from mice that had been primed in vivo with infectious HSV-1 can be restimulated in vitro with live or partially UV-inactivated HSV-1 to generate high levels of herpes virus-specific cytotoxic T lymphocyte (CTL) activity. By comparison, naive splenocytes or splenocytes taken from mice primed with heat-inactivated HSV-1 failed to generate CTL after in vitro viral stimulation. In addition, infectious HSV-primed splenocytes can be rendered unresponsive to secondary in vitro restimulation by pretreatment with anti-Lyt-1 antiserum plus complement. Spleen cells were taken from mice that had been primed and restimulated in vivo with infectious HSV-1. Two days after the second priming, splenocytes were prepared and irradiated. These cells were capable of assisting in the generation of CTL to varying degrees in all of the above unresponsive populations of cells. The irradiated cells did not produce detectable levels of CTL activity when cultured alone with antigen. Also, if the irradiated splenocytes were treated with anti-Lyt-1 plus complement before their addition to cultures, all restorative activity was ablated. In contrast, irradiated splenocytes from mice that had been primed and restimulated in vivo with either heat-inactivated or UV-inactivated HSV-1 were unable to provide help to naive or helper-depleted cultures. The failure to supply helper activity appears not to involve the preferential activation of suppressor cells, as evidenced by cell mixing experiments and the addition of concentrated, antigen-stimulated spleen cell supernatant fluids to secondary anti-HSV-1 splenocyte cultures. Proliferative assays using interleukin 2- (IL 2) dependent cell lines as a measure of relative helper activity indicated that the inactivated forms of HSV-1 were incapable of effectively enlisting helper activity. These experiments therefore suggest that the observed failure of heat-inactivated or UV-inactivated HSV-1 preparations to induce anti-HSV CTL responses reflects the inability of the HSV-1-specific subset of helper T lymphocytes to recognize these forms of the antigen.     

Ecto-ATPase activity in cytolytic T-lymphocytes. Protection from the cytolytic effects of extracellular ATP

Addition of ATP or ATP analog to the incubation media is shown to result in cell death in experiments with different cultured cell lines as evidenced by the results of several independent assays, both in the absence or presence of extracellular Ca2+. Cytolytic T-lymphocyte (CTL) clone itself was not only resistant to cytolytic effects of ATP, but was able to "rescue" antigen-nonbearing 51Cr-labeled cells from lytic effects of extracellular ATP (but not from lytic effects of adenosine 5'-thiotriphosphate) when present during assay. To test whether the resistance of CTL to ATP is due to a high activity of ecto-ATPase, four independent assays of ATPase activity were utilized to demonstrate the presence and relatively high activity of the ecto-ATPase(s) on CTL surface. Studies of substrate specificity of CTL ecto-ATPase suggest that there is more than one nucleoside 5'-triphosphatase on the surface of CTL. The enzyme(s) activity is Ca2+- and Mg2+-dependent and in this respect is similar to recently described hepatic cells ecto-ATPase. We tested effects of known ATP-binding site-specific reagents fluorescein 5'-isothiocyanate (FITC) and 5'-fluorosulfonylbenzoyladenosine (FSBA) to find covalent modification procedures to be used in studies of functional role of ecto-ATPase. FSBA, but not FITC, inhibits lymphocyte ecto-ATPase but addition of ATP together with FSBA protects ecto-ATPase activity. Inactivation of CTL ecto-ATPase by pretreatment with FSBA makes CTL susceptible to lytic effects of extracellular ATP, as was hypothesized for the functional role of this enzyme in CTL.