Immunosuppressive activity of murine newborn spleen cells. I. Selective inhibition of in vitro lymphocyte activation.

Cell-mediated immune responses to newborn lymphocyte alloantigens were investiated using mitogen activation, mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML). Spleen cells from 1- to 5-day-old (C57BL/6 × Balb/c) F₁ mice co-cultured with maternal strain (BALB/c) splenocytes did not affect DNA synthesis of maternal strain cells in the presence of concanavalin A or phytohemagglutinin. Newborn cells did inhibit the lipopolysaccharide response of maternal strain lymphocytes and these cells also depressed DNA synthesis when added to MLR cultures of BALB/c and C57BL/6 spleen cells. Newborn cells expressed poor stimulatory capacity in semiallogeneic MLR and also caused marked inhibition of DNA synthesis when added to semiallogeneic MLR containing BALB/c (responder) and CB6F₁ adult splenocytes (stimulator). The suppression of MLR by neonatal cells persisted for the first 2 weeks of life and was associated with a soluble factor released during culture. The suppressive activity was almost completely abrogated after depleting the T-cells from newborn splenocytes. However, these same cells did not interfere with the in vitro generation of cytotoxic lymphocytes in the CML assay. The selective immunosuppressive properties of newborn spleen cells may be important during pregancy by protecting the immunologically alien fetus from rejection by the mother.     

H-2 homology requirements for secondary cell-mediated lympholysis and miced lymphocyte reactions to TNP-modified syngeneic lymphocytes.

Secondary cell-mediated lympholysis (CML) and mixed lymphocyte reactions (MLR) were generated in a tissue culture system against trinitrophenyl (TNP)-modified murine syngeneic spleen cells. H-2 homology between primary and secondary TNP-modified stimulating cells was required in order to restimulate in the secondary CML. Strong proliferative responses (MLR) were detected only in the secondary cultures, for which H-2 homology was also required between TNP-modified primary and secondary immunogens. Intra-H-2 mapping for the secondary MLR indicated that the relevant regions of homology were I, D, and K and/or I-A. Homology throughout the entire major histocompatibility complex or at K plus I-A gave stronger MLR than did cultures in which there was homology between the primary and secondary phases at I or D only.     

Effect of cyclosporin-A, antimalarial drug, on lymphocyte proliferation of Balb/c mice in vitro

Cyclosporin-A(CsA) caused inhibition of lymphocyte proliferation at higher doses (5 and 10 micrograms/ml) compared to controls. When spleen cells were preincubated with high doses of CsA and washed, the normal lymphocyte response to stimulation with mitogen (concanavalin-A) and lymphokine (interleukin-2) was not affected. The results indicate that CsA's suppressive effect at higher doses, was a temporary one and potential use of CsA to control parasitic infections should be examined.     

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.     

Certain characteristics of human lymphocyte migration inhibitory factors (LyMIF) and the effect of ampicillin on their appearance in unstimulated and PHA-stimulated cultures

Supernatants of human blood mononuclear cells stimulated with PHA, contained factors inhibitory for in vitro migration of human lymphocytes and granulocytes. After ultrafiltration of supernatants through Amicon PM-10 some stimulatory activity appeared in the bottom fraction. Sephadex G-100 gel filtration chromatography of supernatants showed three zones of lymphocyte migration inhibitory activity: In the range of molecules of m.w. 35 000-90 000, heat-stable; Factors of m.w. from several to from ten to twenty thousand daltons, heat unstable; Low molecular weight substances, resistant to heat. The possible relationship of these factors to lymphotoxins, soluble lymphocyte T receptors for SRBC, lymphocyte chemotactic factor and prostaglandins is discussed. Ampicillin in doses of 10, 20 and 50 micrograms/ml potentiated both the development of lymphocyte migration inhibitory factors and the production of factors with an opposing effect (stimulating lymphocyte migration).     

Effect of ribonucleases on cell-mediated lympholysis reaction and on GM-CFC colonies in bone marrow culture

Natural dimer of bovine seminal ribonuclease (AS RNase) suppressed markedly DNA synthesis in allogeneic mixed lymphocyte culture (MLC) of normal human lymphocytes and simultaneously inhibited induction of cytotoxic effector cells within the sensitization phase of indirect cell-mediated lympholysis (CML) reaction. The last purification step of the AS RNase isolation procedure did not increase the suppressive activity of AS RNase compared to a less purified preparation (ZS RNase), thus, the later preparation was mostly used. ZS RNase (10 micrograms/ml) caused 50% inhibition of MLC reaction whereas pancreatic ribonuclease (A RNase) was 10 times less effective. The suppressive effect of RNases added in the beginning of the sensitization phase of the CML reaction correlated with that observed in the MLC reaction. The concentrations of ZS RNase (10 micrograms/ml), A RNase (100 micrograms/ml), and additionally tested cyclosporin A (0.5 microgram/ml) resulted in nearly total abrogation of cytolysis in CML. ZS RNase added after the sensitization of effector cells did not influence their cytolytic action on target cells within the destruction phase of CML. Natural killer and killer cell activities in normal peripheral lymphocytes were not inhibited by ZS RNase at the concentration of 330 micrograms/ml. ZS RNase (20 micrograms/ml), cocultivated 1 h with normal human bone marrow cells and then washed off, enhanced formation of GM-CFC colonies in semisolid agar culture up to 200%. Simultaneously tested antilymphocyte globulin increased the number of GM-CFC colonies at the average of 128%. This stimulating effect on colony formation appeared also in bone marrow culture of patients suffering with various hematological disorders. The possibility of utilizing the preparations gained from seminal plasma in clinical bone marrow transplantation is discussed.     

Differential effect of cyclosporin A on the expression of T and B lymphocyte activation antigens

We have used a monoclonal antibody (Mab) to the interleukin 2 (IL 2) receptor as well as a Mab to the transferrin receptor to analyze the effects of cyclosporin A (CsA) on the induction and expression of these activation antigens on mitogen-stimulated murine T and B lymphocytes. The same concentration (0.25 microgram/ml) of CsA that produced optimal inhibition of the T cell proliferative response to concanavalin A (Con A) was also very effective at inhibiting IL 2 production and the induction of IL 2 responsiveness, as well as the expression of the IL 2 and transferrin receptors when measured 72 hr after mitogen activation. Surprisingly, CsA only minimally inhibited expression of these receptors 24 hr after the addition of mitogen; however, T cell blasts recovered from these cultures failed to respond to IL 2 even though IL 2 receptor expression was only modestly decreased. These results suggest that inhibition of the maturation of receptor expression is secondary to an early effect of CsA in blocking the induction of IL 2 responsiveness or to an arrest in the sequence of events required for maturation of T cells that bear high densities of these receptors. In contrast to the results observed with T lymphocytes, CsA had no effect on the B cell proliferative response to lipopolysaccharide (LPS) or on the induction of the IL 2 and transferrin receptors on activated B lymphocytes.     

Inhibition of normal lymphocyte responses by cell membranes

Cell membranes bearing the appropriate antigen are known to stimulate a variety of cell-mediated immune responses. This report confirms that tumor cell membranes at doses of 2-5 micrograms protein/ml will stimulate in vitro generation of allogeneic cytotoxic T lymphocytes (CTL). However, higher doses (50-100 micrograms protein/ml) of the same membranes completely abrogate the generation of lytic activity. Responding lymphocytes are inhibited by membranes from either syngeneic or allogeneic cells. The inhibition appears to act at a proliferative or differentiation step in the generation of the CTL response, since membranes are known to have little direct effect on the lytic phase of CTL activity. Similar doses of membranes also inhibit LPS-induced B-cell proliferation. B-Cell proliferation is inhibited equally well by allogeneic and syngeneic membranes, and membranes from normal spleen cells are as inhibitory as tumor cell membranes. The inhibitory activity copurifies with the plasma membrane. The results raise important considerations regarding the use of subcellular forms of antigen in studies of lymphocyte recognition. In addition, these data suggest that cell-cell contacts might provide signals regulating the proliferation of lymphocytes.     

Immunosuppressive properties of murine trophoblast

The modification of the immunological response by murine trophoblast cells of different sources was investigated using the mixed lymphocyte reaction (MLR) and the cell mediated lympholysis (CML) test. MLR between C57BL (H-2b) stimulator splenocytes (mitomycin C treated in the unidirectional MLR) and BALB/c (H-2d) responder lymph node cells were markedly suppressed by trophoblast of ectoplacental cone (EPC) and placental origin. The same in vitro effect was observed with supernatants (SN) of trophoblast cells and with supernatants of blastocysts. Addition of anti-progesterone serum (APS), anti-testosterone serum (RAT), and anti-immunoglobulin serum (RAHIg) in serial dilutions to the trophoblast-MLR system revealed that the immunosuppressive effect of trophoblast giant cells and trophoblast giant cell culture supernatants can be abolished with APS. Identical results were obtained with APS added to immunosuppressive doses of progesterone. CML between C57BL responder lymph node cells and mitomycin C-treated BALB/c stimulator spleen cells was also markedly suppressed when trophoblast of EPC origin was added. A similar suppression of cytotoxic T-cell induction was seen when progesterone was added to the system. The immunosuppressive action of trophoblast as detected in vitro is likely to play an important role in the maintainance of pregnancy by protecting the semiallogeneic conceptus against immune aggression by the maternal immune system.     

Lysozyme-induced inhibition of the lymphocyte response to mitogenic lectins

Both human lysozyme (HL) and hen egg white lysozyme (HEWL) inhibited the proliferative response of peripheral blood lymphocytes to T cell mitogens such as the lectins phytohemagglutinin and concanavalin A. This inhibition was observed both when HL or HEWL was added to the lymphocyte cultures in combination with phytohemagglutinin or concanavalin A and when lymphocytes were pretreated with either lysozyme and extensively washed prior to culture with mitogens. Under both conditions, the effects were strictly dose dependent; the lysozyme concentrations yielding maximal inhibitory effect were 5 micrograms/ml for HL and 1 microgram/ml for HEWL, while both lower and higher concentrations were less effective. Specific antilysozyme rabbit sera completely prevented the inhibitory effects of both HL and HEWL on the proliferative response of lymphocytes to phytohemagglutin or concanavalin A. Chitotriose (a lysozyme inhibitor) caused a strong reduction in the inhibitory effects of the two lysozymes on the lymphocyte response to either lectin. HL and HEWL also were found to markedly inhibit the polyclonal B cell proliferation and differentiation induced by pokeweed mitogen and T cells. A less marked inhibition was also obtained when T cells, but not B cells, were pretreated with HL or HEWL. Again, as in the experiments with T cell mitogens, the effects were dose dependent and 5 micrograms/ml HL and 1 microgram/ml HEWL proved to be the most effective concentrations. The possible mechanisms by which lysozyme inhibits the lymphocyte response to mitogenic lectins are considered and discussed. The enzymatic activity seemed to perform an essential function, as shown by the loss of effect when the heat- or trypsin-inactivated lysozymes were used and by the fact that only the enzymatically active compound, among certain semisynthetic derivatives of HEWL, inhibited the lymphocyte response to the mitogens. However, the cationic properties of the lysozyme molecule appeared to be essential too, since enzymes with a similar specificity of action showed effects similar to those observed with HL or HEWL only when they carried a strong positive charge. It is suggested that lysozyme, which is naturally secreted by monocytes and macrophages, might interact with lymphocyte surface receptor sites and participate in the complex mononuclear phagocyte-lymphocyte interactions and in the modulation of lymphocyte activation.     

A phorbol ester (TPA) can replace macrophages in human lymphocyte cultures stimulated with a mitogen but not with an antigen

A culture system was developed in which human peripheral blood mononuclear cells (PBM) depleted of macrophages did not proliferate in response to the lectin mitogen PHA or to the soluble antigen of tetanus toxoid. These cells were able to respond to both mitogen and antigen if purified autologous macrophages were added back to the culture. The response to PHA was partially restored by supplementing the cultures with supernatants from LPS-stimulated macrophages or with partially purified human interleukin 1 (IL 1). The response to tetanus was not restored by reconstitution with these materials. The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), has been shown to have IL 1-like effects in other species and is a polyclonal activator of human T and B lymphocytes. In this study, we tested the ability of TPA to replace macrophages in human lymphocyte cultures stimulated with mitogen or with antigen. Small doses of TPA (50 ng/ml) completely replaced macrophages in the PHA-stimulated cultures; however, in doses of up to 400 ng/ml, TPA was not able to replace macrophages in cultures stimulated with tetanus. Thus, TPA appears to mimic the macrophage-replacing ability of soluble factors (IL 1, macrophage supernatants) in the triggering of human lymphocytes.     

Alloantigen-specific suppressor T cells are not inhibited by cyclosporin A, but do require IL 2 for activation

Alloantigen-specific suppressor T cells are activated from normal murine spleen cells in mixed lymphocyte reactions (MLR). These T cells are radioresistant and suppress the activation of cytotoxic T lymphocytes (CTL) in second primary MLR cultures. This report demonstrates that cyclosporin A (CsA) blocks the activation of these suppressor cells at a dose of 1 microgram/ml. However, reconstitution of CsA blocked cultures with IL 2 restores the activation of the suppressor T cells, but fails to significantly restore the activation of CTL in these same cultures. This differential activation requirement was used to establish T cell lines that demonstrate enriched suppressor cell activity but depletion of CTL activity. These findings are discussed in terms of the mechanism of action of CsA in these distinct T cell subsets and the relevance to models of allograft unresponsiveness.     

Induction by an immunogenic immunomodulating agent of nonspecific T cell suppression of lymphocyte responsiveness in MLR but not of antibody production

Summary Spleen cells derived from BALB/c mice that had been repeatedly immunized with the methanol extraction residue (MER) fraction of tubercle bacilli exhibited a depressed capacity to act as responder cells in allogeneic and syngeneic mixed lymphocyte reactions (MLR). Previously reported studies revealed that such spleen cells are also defective in the in vitro generation of antibodies. In order to determine the nature of the cells responsible for the depressed MLR reactivity, purified populations of splenic macrophages, B lymphocytes, T lymphocytes originating from normal and from MER-immunized mice, and cell culture supernatants were added to MLR mixtures consisting of normal mouse splenocytes. Macrophages originating from MER-immunized mice and their culture supernatants exerted a significantly higher suppressive effect on MLR than that of corresponding preparation from normal mice. Splenic T cells originating from MER-immunized mice and their supernatants also significantly suppressed the MLR response. However, the same T cell populations that were inhibitory in MLR failed to suppress the in vitro generation of antibodies against sheep red blood cells in the presence of either MER or 2-mercaptoethanol. These and previously reported findings indicate that a nonspecific immunomodulating agent, MER, can, under certain conditions of treatment, elicit the induction of nonspecific suppressor T cells for MLR but not for antibody production, and, accordingly, can inhibit cellular and humoral immunological responsiveness by different mechanisms.     

Helper factor production in murine secondary syngeneic mixed leukocyte reactions

Culture supernatants of murine thymocytes or spleen cells responding in a secondary syngeneic mixed leukocyte reaction (SMLR) were studied for their biologic effects on cell-mediated immune responses in vitro. Such supernatants contained helper factor(s) that facilitated the development of alloantigen-specific cytotoxic T lymphocyte (CTL) responses from thymocyte precursors. Thymocytes, but not spleen cells, required activation by allogeneic effect factor (AEF) in primary culture in order to proliferate and produce biologically active mediator(s) during a secondary SMLR. The same culture supernatants possessed, in some instances, weak T cell growth factor (TCGF; IL 2) activity. However, TCGF activity could be dissociated from helper factor(s) active in the CTL induction assay because some culture supernatants that had potent helper activity were devoid of TCGF activity. This lack of TCGF activity was not due to a lower degree of sensitivity of the TCGF assay or to the presence of a selective TCGF inhibitor in the SMLR-derived supernatants, indicating that the helper factor(s) studied is distinct from TCGF. Production of immunoregulatory lymphokines during the SMLR may serve as a physiologically relevant model for studying the role of T cell-derived lymphokines in immunoregulation.     

Suppression of phytohemagglutinin-stimulated lymphocyte blastogenesis by ovine uterine milk protein

Two basic glycoproteins (UTM-P) with molecular weights of 57,000 and 59,000 were purified from ovine uterine milk collected on Days 125 and 130 of pregnancy. The UTM-P were evaluated for immunosuppressive activity in phytohemagglutinin (PHA)-treated, mixed lymphocyte (MLC) and resting lymphocyte (RLC) cultures. For PHA and RLC cultures, UTM-P (2.5 to 800 micrograms UTM-P/ml) were added to 1 X 10(6) lymphocytes and 0.8 micrograms of PHA (for PHA cultures only), while for the MLC, UTM-P (50 to 1600 micrograms UTM-P/ml) were added to 5 X 10(5) lymphocytes combined from each of two ewes. Following [3H] thymidine addition, cells were later harvested for determination of thymidine incorporation. Lymphocyte blastogenesis was suppressed by UTM-P in PHA (R2 = 0.32 to 0.92, P less than 0.01 to 0.001), MLC (R2 = 0.8, P less than 0.001) and RLC (R2 = 0.65, P les than 0.01) experiments. To determine reversibility, PHA-treated lymphocytes were incubated with UTM-P for 6, 12 or 24 h, then washed to remove surface UTM-P. Incubation was continued in the presence of PHA as with other experiments. Exposure of lymphocytes to UTM-P for 6 or 12 h did not result in suppression of blastogenesis, whereas exposure for 24 h was sufficient for suppression (P less than 0.01). In an additional experiment, UTM-P were added to PHA-treated cultures at 0, 6, 12 or 24 h. Suppression (P less than 0.01) of blastogenesis was observed for each time period. Immunosuppressive activity was not mediated by overall cytotoxicity and was not affected by routine handling and storage of UTM-P. Data from these experiments provide one explanation for tolerance of the conceptus allograft during defined stages of ovine pregnancy.     

Lymphotoxin production by human lymphocytes. I. Study of the supernatants of phytohemagglutinin stimulated human lymphocyte cultures]

Lymphotoxin was found to be present in supernatants from 22 human lymphocytes cultures stimulated with phytohemagglutinin in a dose of 5 and 10 microgram/ml. The lymphocytes were obtained from the peripheral blood of 6 apparently healthy persons. Lymphotoxin activity was determined by simple and objective method, i.e. by staining the target cells (mouse L-cells) monolayer with crystal violet, with the following determination of optic densities of the L-cells lysates at 570 nm in the spectrophotometer. As revealed, 1 : 5 dilutions of the supernatants from the lymphocyte cultures incubated for 48 hours inhibited the L-cells growth by from 40 to 60%. With further incubation of the cultures (up to 72 and 96 hours) the cytotoxicity of their supernatants for the target cells showed no increase, whereas the blasttransformation index reaches the maximal value by 72nd incubation hour. Supernatants from unstimulated lymphocyte cultures failed to produce any cytotoxic effect on L-cells.     

Functional analysis of cloned macrophage hybridomas. IV. Induction and inhibition of mixed lymphocyte responses

A series of macrophage (M phi) hybridomas were generated by fusion of drug-marked P388D1 (H-2d) tumor cells with CKB (H-2k) splenic adherent cells. The ability of this panel of cloned M phi hybridomas expressing various levels of surface Ia antigens to induce allogeneic mixed lymphocytes responses (MLR) was examined. All MLR stimulatory M phi hybridomas expressed surface Ia antigens. However, some Ia+ and all Ia- M phi hybridomas were unable to induce vigorous MLR responses. Furthermore, even after induction of surface Ia antigen expression with Con A supernatants (Con A Sn) or purified interferon-gamma, the nonstimulatory M phi hybridomas remained ineffective at inducing strong MLR proliferative responses. Furthermore, addition of the latter M phi hybridoma clones (both with and without Con A Sn treatment) to conventional MLR cultures resulted in inhibition of MLR responses. The series of inhibitory M phi hybridomas secreted normal levels of IL 1 upon stimulation with lipopolysaccharide. After surface Ia induction with Con A Sn, the inhibitory M phi hybridomas could stimulate secretion of IL 2 and expression of IL 2 receptors. Moreover, although they inhibited conventional MLR responses, IL 2 production and IL 2 receptor expression were not significantly inhibited. Addition of these M phi hybridomas 24 to 48 hr after initiation of MLR response also inhibited MLR proliferation. The results indicated that the group of inhibitory M phi hybridomas can inhibit MLR responses after IL 2 secretion and acquisition of IL 2 receptors. Finally, this inhibitory activity has been maintained during 1 yr of continuous in vitro culture, and the hybridomas represent a stable "homogeneous" subpopulation of inhibitory macrophages. Thus, the inhibitory phenotype appears to reflect arrest at a distinct differentiation stage.     

Suppression of cell-mediated immune responses after total lymphoid irradiation (TLI). I. Characterization of suppressor cells of the mixed lymphocyte reaction

Total lymphoid irradiation (TLI) was administered to (BALB/c X C57BL/6)F1 mice in eight daily doses of 200 rad (total 1600 rad). Spleen cells isolated from mice after treatment with TLI do not respond to alloantigens in vitro in a one-way mixed lymphocyte reaction (MLR), but normal reactivity recovers after approximately 2 mo. Radioresistant, antigen-nonspecific suppressor cells are documented in the spleens of TLI-treated mice immediately after radiotherapy, but suppressive capacity gradually disappears within 30 days. After TLI, the spleen is repopulated with large cells, the proportion of which is greatest at a time when theta-bearing cells are still depleted. Radioresistant suppression is mediated predominantly by the large cell subset and is thymus independent. Suppressor function can be abolished by lethal physicochemical procedures including formaldehyde fixation, multiple freeze-thawing, and heating to 56 degrees C, and it cannot be conferred by supernatants of TLI-suppressed MLR suspensions. Suppression cannot be overcome by adding various cell factors including T cell growth factor (TCGF) and lymphocyte-activating factor (LAF), nor is it affected by a prostaglandin inhibitor. Equally potent radioresistant suppressive activity is documented by co-culturing cells derived from other sources enriched in large, immature hematopoietic cells, including fetal liver cells and bone marrow cells obtained from normal and congenitally athymic mice. The presence of a large cell population and MLR suppressor function is also documented in the spleens of mice treated with single dose or fractionated doses of lethal whole body irradiation, followed by reconstitution with bone marrow cells obtained from normal mice. The data suggest that MLR suppressor cells, which are large, immature and predominantly radioresistant, can be induced after a short and well-tolerated TLI regimen.     

Development of precytotoxic T cells in cyclosporine-suppressed mixed lymphocyte reactions

Cyclosporine (CsA) blocked the generation of cytolytic activity in a primary MLR of mouse spleen cells. As expected from the known mechanism of action of this drug, it also blocked the accumulation of IL-2 during the MLR. Addition of human rIL-2 did not overcome the inhibition of CTL generation, even when it was added daily to keep its level similar to that produced in a normal MLR. Daily addition was necessary, because the CsA-inhibited MLR consumed IL-2, either by utilization or degradation. The outcome of a 5-day MLR in the presence of CsA (CsA-MLR) depended on whether or not IL-2 was continuously present. In the presence of IL-2, there was no generation of CTL activity, probably because such cultures contained IL-2-dependent suppressive elements described previously. However, when day 5 CsA-MLR cells generated in the absence of IL-2 were washed and recultured with human rIL-2, there was a burst of CTL activity, with a more than 50-fold increase in alloantigen-specific cytotoxicity within 24 to 48 h. This increase is not explainable simply by the proliferation of existing effector CTL. The noncytotoxic cells produced in an MLR in the presence of CsA, and which can be rapidly activated to cytotoxic effector cells by IL-2, are termed "precursor-effector CTL" (peCTL). They could be detected by day 3 of a primary CsA-MLR culture. Their conversion to effector CTL by IL-2 was not inhibited by CsA. Exposure of peCTL to IL-4 also generated CTL activity, to a somewhat lesser degree than IL-2, but the IL-4-induced activation was inhibited by CsA, suggesting that it depended on the induction of another CsA-sensitive lymphokine. The intracellular levels of mRNA encoding the CTL-specific serine esterases CCP1 and CCP2 (granzymes B and C, respectively) increased rapidly during the IL-2-driven conversion of peCTL to effector CTL. This study demonstrates that in the presence of CsA precursors for CTL can accumulate, and that these can be rapidly converted to cytotoxic effector cells by IL-2.     

Viral inhibition of lymphocyte mitogenesis: interference with the synthesis of functionally active T cell growth factor (TCGF) activity and reversal of inhibition by the addition of same

We have investigated the mechanisms whereby co-incubation of several types of virus particles with human lymphoid cells in the presence of T cell lectins leads to inhibition of the proliferative response that otherwise ensues. The data indicate that, in the absence of infection, such inhibition can be reversed by the addition to cultures of relatively high concentrations of fluids rich in T cell growth factor (TCGF) activity. The ability of these fluids to achieve such reversal of inhibition is both concentration- and time-dependent. Addition of the factor to virus co-incubated cells more than 26 hr after culture initiation does not restore responsiveness. We have also shown that virus co-incubated cultures are deficient with respect to their ability to synthesize detectable levels of TCGF activity in the presence of phytohemagglutinin. In contrast, the use of relatively dilute virus preparations (less than 10 particles per cell) permits partial responsiveness to lectin as well as the synthesis of moderate levels of TCGF. These finding suggest that viral inhibition of lymphocyte mitogenesis is mediated directly or indirectly by interference with the synthesis of functionally active TCGF activity.