Cloned cytolytic T cells can suppress primary cytotoxic responses directed against them

In vivo and in vitro, murine peripheral T cells can suppress or "veto" the activation of cytotoxic T lymphocytes directed against antigens presented by those T cells. This suppression is antigen-specific and H-2-restricted. The recognition event initiating this suppression appears to be unidirectional; precursors of cytotoxic T lymphocytes recognize the antigen-bearing veto cell and are thereby inactivated--the veto cell need not recognize the CTL precursor. We show here that 3/3 cytolytic T cell clones can exert veto activity in vitro on normal spleen cells which do not bear antigens the T cell clones can recognize. This suppression results in greatly diminished cytotoxic activity generated during a primary 5-day mixed lymphocyte culture against antigens which the veto cell expresses, but not against third-party antigens present in the same culture. In this same system, a noncytolytic T cell clone will not serve as a source of veto cells. Secondary cytotoxic responses are relatively resistant to the veto cell activity of cloned cytolytic T cells. The cloned veto cells do not suppress the generation of cytotoxic activity directed against antigens they recognize (and presumably carry over via antigen-specific receptors). Cold target competition during the cytotoxic assay has been eliminated as a possible mechanism for T cell clone-induced suppression, and suppression cannot be reversed by the addition to the mixed lymphocyte cultures of supernatants from concanavalin A-activated spleen cells. It is suggested that this mechanism of inactivating primary cytotoxic T lymphocyte responses could play an important role in the maintenance of self-tolerance and in the induction and maintenance of tolerance to allografts.     

Cyclosporine A and peripheral tolerance. Inhibition of veto cell-mediated clonal deletion of postthymic precursor cytotoxic T lymphocytes.

Veto cell-mediated suppression of CTL responses has been proposed as one mechanism by which self tolerance is maintained in mature T cell populations. We have reported that murine bone marrow cells cultured in the presence of high-dose IL-2 (activated bone marrow cells) mediate strong veto suppressor function in vitro and in vivo, and that such veto activity is effected through clonal deletion of cytotoxic T cell precursors. In our studies, we have determined that bone marrow cell populations from athymic NCr-nu mice (H-2d) mediate strong veto cell activity without exposure to exogenous IL-2 in vitro. To examine mechanisms by which these naturally occurring veto cell populations in BM suppress precursor CTL (pCTL) responses, we used as a responding cell population in MLC, spleen cells of transgenic mice expressing at high frequency TCR specific for H-2 Ld encoded Ag with stimulation by H-2d-expressing cells in culture. Flow cytometric analysis was performed by staining the responding MLC cell population with the mAb 1B2 specific for the transgene-encoded TCR and determined changes of 1B2+ T cells. Such experiments demonstrated that the anti-H-2d cytotoxic response by these cell populations was specifically suppressed by NCr-nu (H-2d) bone marrow, and that 1B2+ pCTL were in fact specifically deleted from the responding cell population by incubation with such naturally occurring veto cell populations expressing the appropriate target Ag. In addition, to further understand the interactions of pCTL and veto cells and possible contributions by the latter to peripheral tolerance, we evaluated the effect of cyclosporine A (CsA) on veto cell-mediated suppression of pCTL of the transgenic mice. CsA inhibited veto cell-mediated suppression of cytotoxic T cell responses, and this inhibition correlated with a lack of clonal deletion of pCTL by veto cells in the presence of CsA. Furthermore, CsA exerted its effect through pCTL and not through veto cells, indicating that pCTL may play an active role in their own deletion by veto cells.     

Suppressor T cells, distinct from "veto cells," are induced by alloantigen priming and mediate transferable suppression of cytotoxic T lymphocyte responses in vivo

Primary and secondary cytotoxic T lymphocyte responses to minor alloantigens can be suppressed by priming host mice with a high dose (10(8) cells) of alloantigenic donor spleen cells (SC). Such suppression is antigen specific and transferable into secondary hosts with T cells. One interpretation of this is that antigen-specific host suppressor T cells (Ts) are activated. Alternatively, donor Lyt-2+ T cells, introduced in the priming inoculum, may inactivate host CTL precursors (CTLp) that recognize the priming (donor) alloantigens. Donor cells that act in this way are termed veto T cells. The experiments described here exclude veto T cell participation in transferable alloantigen-specific suppression, and demonstrate the operation of an alloantigen-specific host-derived T suppressor (Ts) cell. The origin of the Ts has been studied directly by using Thy-1-disparate BALB/c mice. The cell responsible for the transfer of suppression of a secondary CTL response to B10 minors was of the host Thy-1 allotype, and so originated in the host spleen and was not introduced in the priming inoculum. Secondly, antigen-specific Ts generated in CBA female mice against B10 minors could act on CTL responses to an unequivocally non-cross-reactive-third party antigen (H-Y), provided the two antigens were expressed on the same cell membrane. Such third-party suppression is incompatible with the operation of veto T cells. Depletion of Thy-1.2+ or Lyt-2+ cells from the suppression-inducing donor SC inoculum did not abrogate suppression induction in BALB/c mice; instead, suppression was enhanced. The demonstration of veto cell activity in similarly primed mice by other groups of investigators indicates that both types of suppression may operate. However, our results show that only antigen-specific Ts can mediate the transferable suppression of CTL responses to alloantigens.     

Veto cell H-2 antigens: veto cell activity is restricted by determinants encoded by K, D, and I MHC regions

Responder cells from primary syngeneic and allogeneic one-way mixed-lymphocyte cultures (MLC) specifically inhibit the development of cytotoxic T lymphocytes (CTL) directed against the major histocompatibility complex (MHC) antigens of the MLC responder cells. This special kind of suppressor activity is known as veto suppression. Ia+ cells with veto activity obtained from H-2 recombinant mouse strains were shown to downregulate alloantigen (class II)-specific helper activity for class I-specific CTL development in a primary MLC provided that the veto cells expressed the same I-E alpha subregion as the MLC stimulator cells. The veto-induced suppression of allo-help was prevented by the addition of supernatant from concanavalin A-stimulated spleen cells (Con A-SN) and was inhibited considerably by very high amounts of recombinant interleukin-2 (IL-2). In the presence of Con A-SN, CTL precursors recognizing either the K end or the D end of the veto cell MHC were found to be inactivated. Thus, our results indicate that MLC responder cells include active veto cells expressing Ia region-encoded restriction elements for allospecific T helper cells, as well as K- or D-encoded restriction elements for allospecific T cytotoxic cells.     

Reversal of phorbol ester-mediated reduction of cloned T lymphocyte cytolysis by interleukin 2

During previous studies on the regulation of cloned T lymphocyte function, we observed that murine cytotoxic T lymphocyte (CTL) clones progressively lose the ability to lyse appropriate target cells during prolonged (24 to 48 hr) incubation with the tumor promoter phorbol myristic acetate (PMA). We further observed that the cytolytic function of PMA-treated CTL clones can be restored by incubation with secondary MLC supernatant (2 degrees MLC SN), a potent source of cytokines. We now report our observations on the nature of the cytokine(s) responsible for recovery of CTL activity. Like 2 degrees MLC SN, the lectin-induced SN from a cloned helper T cell and the lectin-induced SN from a T cell hybridoma can restore cytolytic activity to cloned CTL treated with PMA. In contrast, supernatants from L929 cells, WEHI-3 cells, and P388D1 cells fail to restore cytolytic activity to similarly treated cloned CTL. These data suggest that IL 2 and/or gamma-IFN, but not CSF-1, CSF-GM, IL 3, or IL 1, can influence expression of cytolysis by cloned CTL. Furthermore, highly purified IL 2 can restore cytolytic activity, even when cytosine arabinoside is present to inhibit clonal expansion. Our studies indicate that cytolysis is a reversible function of cloned CTL, and that cytolysis may not necessarily represent an end-stage feature of CTL maturation. Our studies further show that IL 2 is both necessary and sufficient for resumption of cytolytic function by "deactivated" CTL. As such, these observations suggest that IL 2 can regulate not only T cell proliferation but also the expression of cytolysis by some cytolytic T cell populations.     

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.     

The role of Ca2+ in activation of mature cytotoxic T lymphocytes for lysis

We carried out a detailed analysis of the requirement for Ca2+ in the lysis of target cells by cloned cytotoxic T lymphocytes (CTL). In direct, antigen-specific lysis we always observed an influx of Ca2+ into the CTL concomitant with target cell binding. However, we never observed an increase in CTL Ca2+ content during lectin-mediated lysis, or nonspecific lysis by phorbol myristate acetate-induced CTL. We found that in all three types of lysis (direct, lectin-mediated lysis, C or phorbol myristate acetate-induced) the requirement for Ca2+ in lysis was dictated by the target cell used; the same CTL can kill one target cell in the absence of detectable Ca2+, and absolutely require Ca2+ for the lysis of another target cell. Target cell killing, when it occurred in the absence of Ca2+, was accompanied by microtubule organizing center reorientation in the CTL, showing that this function is not uniformly Ca2+ dependent. These results provide further evidence that Ca2+ is not always required for activation of the lytic pathway in CTL, although Ca2+ may be absolutely required for other CTL functions such as interleukin production or expression of the interleukin 2 receptor.     

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.     

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

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

The mechanism of cell-mediated cytotoxicity. II. The apparent biochemical requirements for cytolysis are influenced by the source and frequency of murine cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTL) were generated in allogeneic mixed-lymphocyte cultures (MLC) with the responder cells from immunized mice (secondary (II°) MLC.) These CTL could be maintained as proliferating cells for 2 to 4 weeks by culturing in a medium with T-cell growth factor (TCGF) activity and were 10 to 30 times as active as effectors obtained directly from immunized mice or those generated in primary (I°) MLC. Cytolysis by CTL from II° MLC was refractory to inhibition by azide, cyanide, or cycloheximide in contrast to previous reports that these agents were inhibitory. However, cytolysis by CTL from II° MLC was sensitive to azide when sufficient normal spleen cells were added to reduce the apparent CTL frequency to approximate that of the less active populations. Cycloheximide (Cx) partially inhibited cytolysis by CTL from immune spleen cells as shown earlier by others. However, CTL from II° MLC remained refractory to Cx even when diluted with normal spleen cells. These data show that CTL populations from various sources have both qualitative and quantitative differences that may alter their apparent biochemical requirements for CML. These considerations must be taken into account when attempting to define the minimal requirements for CTL-mediated killing.     

Regulation of primary cytotoxic T lymphocyte responses generated during mixed leukocyte culture with H-2d identical Qa-1-disparate cells

Cytotoxic lymphocyte (CTL) responses are not usually generated during primary mixed leukocyte culture (MLC) with H-2 identical cells. Thus NZB mice are unusual in that their spleen cells do mount CTL responses during primary MLC with H-2d identical stimulator cells; the predominant target antigen for these NZB responses is Qa-1b. Considering the numerous immunoregulatory defects in NZB mice, we postulated that these NZB anti-Qa-1 primary CTL responses were due to an abnormality in T suppressor cell activity. Cellular interactions capable of suppressing NZB anti-Qa-1 primary CTL responses were investigated by using one-way and two-way MLC with spleen cells from NZB mice and other H-2d strains. Although H-2d identical one-way MLC with the use of NZB responders resulted in substantial CTL responses, only minimal CTL responses were detected from two-way MLC with the use of NZB spleen cells plus nonirradiated spleen cells from other H-2d mice. Thus the presence of non-NZB spleen cells in the two-way H-2d identical MLC prevented the generation of NZB CTL. Noncytotoxic mechanisms were implicated in the suppression of the NZB CTL responses during two-way MLC, because only minimal CTL activity was generated when NZB spleen cells were cultured with semiallogeneic, H-2d identical (e.g., NZB X BALB) F1 spleen cells. The observed suppression could be abrogated with as little as 100 rad gamma-irradiation to the non-NZB spleen cells. The phenotype of these highly radiosensitive spleen cells was Thy-1+, Lyt-1+, Lyt-2-, L3T4+. The functional presence of these cells in the spleens of semiallogeneic, H-2d identical F1 mice indicated that their deficiency in NZB mice was a recessive trait. These data suggest that NZB mice lack an L3T4+ cell present in the spleens of normal mice that is capable of suppressing primary anti-Qa-1 CTL responses. This model system should facilitate additional investigations of the cellular interactions and immunoregulatory mechanisms responsible for controlling primary CTL responses against non-H-2K/D class I alloantigens. The model may also provide insight into the immunoregulatory defects of autoimmune NZB mice.     

Veto activity of activated bone marrow does not require perforin and Fas ligand

Veto cells suppress generation of CD8(+) T cell immune responses in an antigen-specific manner, with specificity dictated by antigens on the veto cell surface. Activated bone marrow (ABM) veto cells belong to the NK cell type lineage and veto by clonally deleting antigen-specific precursor cytotoxic T cell lymphocyte (CTL). In vitro cytotoxicity of ABM depends largely on the perforin/granzyme and Fas/Fas ligand pathways. Utilizing perforin-deficient and functional Fas ligand-deficient gld mice as a source of ABM and functional Fas-deficient lpr mice as a source of precursor CTL, we demonstrate in this study that ABM cells utilize a perforin- and Fas-independent pathway to veto allogeneic cell-mediated cytotoxic responses. We also show that ABM cells mediate perforin- and Fas-independent veto activity even in an 8-h clonal deletion assay. We conclude that ABM veto activity does not require the two primary pathways of cell-mediated death.     

Resistance of primary CD8+ cytotoxic T lymphocytes to lysis by cytotoxic granules from cloned T cell lines

Recent evidence has shown that cloned, murine CTL cell lines are resistant to the cytotoxic components of the toxic granules they release upon specific interaction with their target cells. Inasmuch as the resistance might be due to selection in culture over many months by repeated exposure to these cytolytic components (which are released repeatedly as a result of the cultured CTL being periodically stimulated by target cells), we asked whether primary CTL are also resistant. The primary CTL were elicited in vivo by i.p. injection of allogeneic tumor cells or in vitro by 5- to 6-day MLC or by 48-h exposure to the lectin Con A. The responding cells were separated into purified CD8+ (i.e., CD4-, CD8+) and purified CD4+ (i.e., CD4+, CD8-) T cell populations that were analyzed for cytolytic activity and for resistance to lysis by toxic secretory granules derived from cloned CTL cell lines. The CD8+ T cells were highly cytolytic and relatively resistant; they retained their cytolytic activity and were lysed to a minimal extent (0 to 10%) by quantities of isolated granules that lysed 80 to 90% of the P815 tumor cell line (tested as a representative standard cell line). The CD4+ T cells, in contrast, had only minimal cytolytic activity and were far more susceptible to granule-mediated lysis. Although the resistance of primary CD8+ T cells is impressive, it is not as pronounced as the resistance of the cloned CTL cell lines, indicating that during long-term culture there is some selection for increased resistance to granule-mediated lysis. In contrast to T cells (especially CD8+ T cells), Ia+ macrophages, isolated from primary immune peritoneal exudates, were highly susceptible to granule-mediated lysis.     

Two roles for CD4 cells in the control of the generation of cytotoxic T lymphocytes.

The generation of CTL against Qa-1 Ag in C57BL/6 (B6) (Qa-1b) and B6.Tlaa (Qa-1a) congenic strains requires in vivo priming with the Qa-1 alloantigen together with a helper Ag, such as H-Y. The primed precursors obtained from these female mice generate Qa-1-specific CTL activity upon culture in vitro. Although the presence of the H-Y helper Ag is not required for the in vitro sensitization, no response occurs in the absence of CD4 cells. Addition of unprimed B6.Tlaa CD4 cells from Qa-1 incompatible radiation bone marrow chimeras (B6.Tlaa----B6), that are presumably tolerant to Qa-1b, provide helper activity for Qa-1b-specific CTL. This indicates that although CD4 cells are obligatory for the Qa-1 response, they need not be specific for alloantigens on the APC to generate helper activity in in vitro cultures. Addition of unirradiated B6 CD8-depleted spleen cells to CD4-depleted B6.Tlaa anti-B6 cultures in the presence of either B6.Tlaa CD4 cells or rIL-2 prevents the generation of Qa-1 specific CTL. This inhibition is not due to an anti-idiotypic Ts cell since B6.Tlaa----B6 chimeric cells do not suppress an anti-Qa-1b response. Rather, this finding is consistent with that of a veto cell mechanism. To determine whether CD4 cells themselves exhibit veto activity, highly purified CD4 populations were tested for their ability to inhibit the generation of Qa-1-specific CTL. CD4 cells precultured for 2 to 3 days with Con A and rIL-2 specifically inhibit CTL activity whereas resting cells do not, similar to that noted for CD8 veto cells. The relative efficiency of activated CD4 cells is greater than that of resting NK cells but is less than that of activated CD8 or NK cells. Thus, CD4 cells not only provide helper activity for CTL precursors, but also act as veto cells to prevent the generation of CTL activity.     

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.     

A novel cytotoxic T-cell clone that exhibits differential modes of recognition in the proliferative and cytolytic phase

A T-cell clone (1G8-H7) cytotoxic to P815Y mastocytoma (H-2d) has been established from spleen cells of a C3H/He mouse (H-2k) primed with P815Y cells by means of in vitro stimulation with irradiated C3H.H-2o(H-2KdDk) spleen cells. The clone 1G8-H7 was an interleukin 2 (IL-2)-dependent and H-2Kd antigen-dependent CTL clone and it killed P815Y cells but not Concanavalin A-induced spleen blast cells bearing H-2Kd antigen. The involvement of H-2Kd antigen in the cytolytic recognition mechanism was shown by the inhibition of lysis by anti-H-2Kd monoclonal antibody and also by the cold inhibition experiment that employed H-2Kd-bearing spleen cells. Comparison of cytotoxic activities between 1G8-H7 and Kd-specific CTL clones showed that the killing of P815Y cells by clone 1G8-H7 was not explained by the susceptibility to cell-mediated cytolysis of P815Y cells. These results suggest that H-2Kd antigen on the stimulating cell is sufficient to deliver a proliferation signal in the proliferative phase of this clone, but in the cytolytic phase an additional interaction with surface structure on the target cell other than that with H-2Kd antigen is required for the induction of cytolysis. Possible elucidations for the differential modes of recognition are discussed.     

Cell-mediated inhibition of proliferation and activation of alloreactive cytotoxic lymphocytes: maintenance of response potential of precursors and dissociation between proliferation and effector function of activated cytotoxic lymphocytes

Adherent layers of macrophages (M phi-c) generated in vitro from splenic precursors inhibit lymphoproliferative responses to mitogen and to alloantigen without inhibiting the production of interleukin-2 (IL-2). Analysis of spleen cells stimulated for 48 hr in the presence of M phi-c indicated that both blastogenesis (increased cell mass) and expression of IL-2 receptors (7D4 determinants) were reduced. Analysis of BrdU incorporation (frequency of S-phase cells) and total cellular DNA revealed that the M phi-c inhibited the progression from G1 to S phase of cell cycle. The M phi-c not only inhibited the proliferative response to alloantigen but also prevented the generation of alloreactive cytotoxic T cells. The M phi-c were shown not to inhibit CTL responses by eliminating the stimulators or by inactivating precursors or inducing suppressors. The M phi-c were affecting the induction of CTL activity since the M phi-c did not affect the expression of cytolytic activity by activated CTL. The M phi-c did inhibit the proliferation of the activated CTL, suggesting that although cytolytic activity can be expressed in G1 phase of cell cycle, the activation of cytolytic activity in CTL-P may require a G1 to S phase transition. The cells recovered from 5-day MLC incubated in the presence of M phi-c were fully capable of generating a subsequent CTL response. This is in contrast to cells recovered from unstimulated cultures (no M phi-c) which have lost the ability to generate CTL responses. The M phi-c therefore prevent the generation of CTL responses in a totally reversible fashion, so as to allow activation and proliferation of CTL-P which have been removed from the influence of the M phi-c. These observations are discussed in the context of the currently hypothesized role of tissue macrophages in microenvironmental regulation.     

Requirement for three distinct lymphokines for the induction of cytotoxic T lymphocytes from thymocytes

The induction of cytotoxic T lymphocytes (CTL) from CTL precursors requires a combination of antigen and lymphokine signals. To investigate lymphokine requirements for CTL generation, we used an assay in which helper T cell and accessory cell-depleted spleen cells or whole thymocytes were cultured with lectin (Con A) and lymphokines. This culture was followed by assessment of lectin-dependent cytolysis. High concentrations of recombinant interleukin 2 (R-IL 2) (100 U/ml) alone were not sufficient for lectin-mediated CTL induction from thymocytes, whereas 20 to 100 U/ml of R-IL 2 alone could induce a significant lectin-mediated CTL response from accessory cell-depleted spleen cells. Using thymocytes as responders, we found purified or recombinant interferon-gamma (IFN-gamma) did not cause cytolytic activity either in the absence of or in the presence of R-IL 2. However, supernatant from Con A-stimulated rat spleen cells (rat Con A SN) in combination with R-IL 2 could induce cytolytic activity, suggesting that several factors are required for CTL induction. Con A SN was fractionated by gel filtration and the fractions were tested for ability to induce CTL. In the presence of a low level of R-IL 2 (5 U/ml), fractions with a Mr of approximately 31,000 could induce CTL, and this activity was referred to as CTL differentiation factor (CDF). The peak fractions containing CDF activity did not have detectable IL 1, IL 2, IFN-gamma, or CSF activity. However, by add-back experiments and the use of blocking antibodies, a monoclonal antibody against the IL 2 receptor or antibodies against murine IFN-gamma, we demonstrated that CTL induction from mature thymocytes (L3T4-, Lyt-2+) requires CDF activity in addition to IL 2 and IFN-gamma.     

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.     

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.