Induction and characterization of minor histocompatibility antigens. Specific primary cytotoxic T lymphocyte responses in vitro

A definite cytotoxic activity was developed in a BALB/c (H-2d) anti-DBA/2 primary mixed leukocyte culture (MLC), which received interleukin 2 (IL-2) on day 3 of culture. This cytotoxic activity was minor histocompatibility antigens (MIHA)-specific at the stimulator level, and was not developed in a syngeneic (BALB/c anti-BALB/c) MLC. The addition of IL-2 on day 3 of culture was crucial; no or very weak cytotoxic activity was developed in MLC receiving IL-2 on day 0 or on both day 0 and day 3. Only appropriate MIHA-allogeneic tumor cells were lysed as the target of the cytotoxic activity. The cytotoxic activity seemed MIHA-specific also at the target level; it lysed tumor cells of DBA/2 mouse origin but not those of BALB/c (syngeneic) origin. Phenotypes of the cytotoxic effector cell were Thy-1+ Lyt-2+. We concluded from these results that MIHA-specific cytotoxic T lymphocytes (CTL) were generated in the MIHA-allogeneic primary MLC. In this newly developed system, we studied genetic and antigenic requirements for primary anti-MIHA CTL responses in vitro. We demonstrated; among spleen cells (SC) of seven B10 H-2-congenic strains only SC of B10.D2 strain whose major histocompatibility complex (MHC) (H-2d) was compatible with the responder MHC effectively stimulated responder BALB/c (H-2d) SC for an anti-MIHA (DBA-C57BL-common) CTL response. Similarly, only SC of two out of seven C x B recombinant inbred strains (C x B.H and C x B.D), which were compatible at the MHC with responder SC, activated responder BALB/c SC for the response. The possibility that cells responding to H-2 alloantigens suppressed the anti-MIHA response was ruled out. Additional experiments showed that compatibility at the H-2K-end or the H-2D-end of the MHC was sufficient for a definite anti-MIHA response. These provided formal evidence that primary anti-MIHA CTL responses in vitro were MHC-restricted at the stimulator level. We then showed that sonication-disrupted SC or Sephadex G-10 column-passed nonadherent SC failed to stimulate responder SC for a primary anti-MIHA CTL response, whereas G-10-passed nonadherent SC responded well to adherent stimulator cells. Further study demonstrated that Ia+ adherent cells were the most active cell type as stimulator. Finally, we confirmed that the primary anti-MIHA CTL responses to adherent stimulator cells was MHC-restricted.     

Suppression of antibody responses in allogeneic mice by products of lymphoid tissue. II. Lack of antigenic specificity and immunogenetic requirements of allogeneic suppressive factor (ASF).

Mice were irradiated, infused with thymocytes and immunized with a variety of antigens, i.e., sheep or horse red blood cells (SRBC or HRBC), diphtheria toxoid (DT) or bovine gamma-globulin (BGG). The spleen cells (T.Spleen cells) were harvested 5 days later and cellfree extracts were prepared. The extracts contained an allogeneic suppressive factor (ASF) that was capable of inhibiting IgM antibody responses of allogeneic or semi-allogeneic unirradiated mice. ASF had to be injected within 24 hr of immunization to be effective and a single injection delayed, rather than abolished, the antibody response at the cellular level. However, daily injections of ASF resulted in persistent suppression of antibody response. ASF activity was antigen nonspecific, i.e., the antigen used to stimulate ASF production did not have to be the same as the antigen used to test for ASF activity. C3H T.Spleen extracts were even immunosuppressive when prepared by exposure to C3BF1 alloantigens only; such extracts suppressed antibody responses of C3BF1 and DBA/2 mice. C3H ASF was removed from extracts after incubation with C3BF1 spleen cells but not after incubation with C3H spleen cells. C3BF1 spleen cells which had been preincubated with C3H ASF were unable to generate antibody-forming cells upon transfer to irradiated C3BF1 host mice. This suggests that the ASF molecule may be or include receptors for alloantigens. The immunogenetic requirements for ASF activity were evaluated by injecting extracts from C3H, C57BL, C3BF and BALB/c T.Spleen cells into C3H, CBA, C57BL, BALB/c, DBA/2, A or C3H.A recipient mice. All extracts tested had ASF activity. However, all allogeneic recipients were not suppressed by the extract material. The suppressive activity of ASF seemed to require two (or more) antigenic differences between donors and recipients of extract material, an H-2K or I antigen difference and a second antigen difference, possibility Ig-1. In the limited numbers of strain combinations tested, T.Spleen extracts suppressed IgM antibody response only if exposed to H-2 and Ig-1 antigens, e.g., BALB/c (H-2d, Ig-1a) ASF suppressed A (H-2a, Ig-1e) but not C3H.A (H-2a, Ig-1a) or DBA/2 (H-2d, Ig-1c). Separate ASF molecules may react with separate antigens on the cell surface, i.e., with H-2 and gammaG2a. Alternatively, one ASF molecule may react with two structurally associated antigens. If the latter is correct, it is conceivable that the beta2-microglobulin which is non-covalently linked to the major component of H-2 molecules expresses allotypic antigens coded for by Ig-1 and beta2-microglobulin is one of the antigens recognized by ASF.     

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.     

Cell-mediated immune responses in vitro. II. Simultaneous generation of cytotoxic lymphocyte responses to two sets of alloantigens of limited cross-reactivity.

The conditions for generation of simultaneous and independent cytotoxic lymphocyte (CL) responses to each of two sets of alloantigens of limited cross-reactivity by mouse spleen cells in vitro have been investigated. Responder spleen cells were incubated with mitomycin C-treated C57BL/6 (H-2b) or DBA/2 (H-2d) stimulator spleen cells and day 5 CL responses were assayed with 51Cr-labeled EL-4 leukemia (H-2b) and P815 mastocytoma (H-2d) as target cells. Spleen cells from mice of the various H-2 haplotypes tested differed greatly in their ability to develop specific CL responses against alloantigens on the stimulator spleen cells and in the degree of cross-reactive cytotoxic activity against target cells bearing alloantigens not present on the stimulator spleen cells. In contrast to the other strains examined, DBA/1 (H-2q) spleen cells developed specific CL responses to either H-2b or H-2d alloantigens without exhibiting significant cross-reactive activity on the inappropriate target cell. The CL responses to H-2b and H-2d alloantigens by DBA/1 spleen cells were comparable in magnitude and had similar stimulator cell-dose requirements. Further, DBA/1 spleen cells developed CL responses of normal magnitude simultaneously against both target cells when incubated with both mitomycin C-treated C57BL/6 and DBA/2 stimulator cells.     

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.     

Mechanism of mastocytoma-mediated suppression of lymphocyte reactivity.

Previously we have shown that mitomycin-treated P-815 (H-2d) cells (P-815m) inhibit the in vitro response of C57BL/6 spleen cells to mitogens and DBA/2 alloantigens. The present data indicate that P-815m cells also inhibit the response of C57BL/6 spleen cells to AKR (H-2k)-stimulating cells and that the inhibition does not appear to be the result of crowding. Subpopulations of spleen cells obtained by density gradient centrifucation or after renoval of glass-adherent cells are all sensitive to the inhibitory effects of P-815m. We also show that P-815m cells appears to operate both in vitro and in vivo. The experiments characterize the suppression of MLC reactivity by cell-free preparations of sonicated P-815 mastocytoma cells. The results suggest that the inhibition is not removed by ultracentrifugation, ultra-violet irradiation, or dialysis of the sonicate. P-815 sonicates subjected to chloroform extraction, heating at 56 degrees C, 0.1 mu filtration, or treatment with anit-minute virus of mice serum are still inhibitory. This inhibition is resistant to RNAase treatment but destroyed by pronase. P-815 sonicate suppresses the response of C57BL/6 spleen cells to both DBA/2 and AKR-stimulating spleen. Inhibition of MLC reactivity is only seen if P-815 sonicate is added within the first 48 hr after the initiation of culture. These results indicate that the inhibition of MLC by sonicates of P-815 cells is due to a nondialyzable protein whose effects are not H-2 restricted and are acting on the early phase of sensitization.     

Frequency and cross-reactivity od cytolytic T lymphocyte precursors reacting against male alloantigens

It is well established that cytotoxic T lymphocytes (CTL) specific for the male minor histocompatibility antigen (H-Y) are generated by restimulation in vitro of in vivo primed spleen cells from C57BL/6 (H-2b) female mice with syngeneic male spleen cells. When tested on target cells from H-2 different strains, the male-specific C57BL/6 CTL populations exhibited significant lysis of DBA/2 (H-2d), A (H-2a), but not C3H (H-2k), male and female target cells. In an attempt to document this cross-reactivity further at the clonal level, a sensitive technique of limiting dilution analysis was used to determine the specificity of C57BL/6 individual CTL precursors (CTL-P) reactive against the male antigen. The mean frequency of anti-H-Y CTL-P in spleens of primed female mice was about 1/3500. Between one-third to one-tenth of these CTL-P produced a progeny that cross-reacted with H-2d (allogeneic) female target cells. These findings were confirmed by the analysis of the reactivity pattern exhibited by male-specific CTL clones derived by limiting dilution. Of 99 clones tested, 13 were found to cross-react with female DBA/2 target cells. These results thus indicate that a relatively large proportion (greater than 10%) of H-2b CTL-P directed against the H-Y antigen cross-react with target cells expressing H-2d alloantigens in the absence of H-Y antigen.     

Limiting dilution analysis of alloantigen-reactive T lymphocytes. III. Effect of priming on precursor frequencies.

The effect of specific priming with alloantigens on the frequency of cytolytic T lymphocyte precursors (CTL-P) has been investigated. Alloimmune lymphoid cells were obtained from the spleen of C57BL/6 (H-2b) mice primed with DBA/2 (H-2d) tumor cells or from 14-day unidirectional mixed leukocyte cultures (C57BL/6 anti-DBA/2). CTL-P frequencies directed against H-2d alloantigens were estimated by limiting dilution analysis in a sensitive micro MLC system. Under these conditions, an apparent increase of 3 to 4-fold in CTL-P frequency was observed in alloimmune (as compared with normal) C57BL/6 spleen cells. Evidence was obtained suggesting that this increase was specific for the priming alloantigens. A much greater increase in CTL-P frequency (25 to 100-fold) was observed after alloimmunization of C57BL/6 spleen cells in unidirectional MLC. Under the latter conditions, 5 to 20% of the surviving splenic MLC cells could be identified operationally as CTL-P. A similar enrichment in CTL-P frequency was obtained when lymph node, peripheral blood, or thymus cells were cultured for 14 days in MLC. These studies provide direct evidence that the pool of specific CTL-P can be expanded after alloimmunization. Furthermore, the very high frequencies observed after in vitro priming indicate that this system should be particularly useful for future studies of the progeny of individual CTL-P.     

Pretreatment with minor histocompatibility antigens prevents the development of functional CTL helper cell activity

We have been studying the regulation of allogeneic cytotoxic cells (CTL) in vivo. CBA/J (H-2k, mls d) responder mice are unable to develop CTL after an allogeneic footpad immunization if they are pretreated i.p. with spleen cells from either C3H/HeN (H-2k, mls c) or B10.BR (H-2k, mls b) mice. These mouse strain combinations are H-2 compatible but differ at the Mls and other minor histocompatibility loci. We reported that this state of CTL unresponsiveness is specific and that the allogeneic cells used for footpad immunization and the pretreatment strain must share both minor antigens and part of the MHC. In this paper, we describe some of the characteristic features of this CTL unresponsiveness. The CBA host plays an active role and appears to down-regulate its subsequent response against minor antigens after the initial pretreatment. This statement is based on the following: 1) The inhibition of in vivo CTL generation can be achieved by injecting F1 or irradiated C3H cells, i.e., under conditions where GVHD was not a factor; and 2) the state of unresponsiveness is abolished by host treatment with cyclophosphamide. In addition, we demonstrate that the lack of CTL development in pretreated responder animals is the result of impaired helper cell activity. Draining LNC from unresponsive mice can become functionally cytolytic if cultured in a Con A-activated spleen cell supernatant. However, normal CTL responses were not restored after adult thymectomy or splenectomy. Thus, the state of CTL inhibition that is induced by the minor antigen pretreatment is the result of a host-mediated regulatory circuit.     

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.     

Limiting dilution analysis of alloantigen-reactive T lymphocytes. IV. High frequency of cytolytic T lymphocyte precursor cells in MLC blasts separated by velocity sedimentation

A sensitive limiting dilution microculture system was used to obtain minimal estimates of the frequency of cytolytic T lymphocyte precursor cells (CTL-P) directed against DBA/2 alloantigens, after priming of spleen cells in unidirectional mixed leukocyte cultures (MLC, C57BL/6 anti-DBA/2). The mean CTL-P frequency in day 4 to 5 MLC populations was found to be approximately 50- to 100-fold greater than the frequency in normal spleen, and up to 25% of the cells present in such MLC could be identified operationally as CTL-P. Even higher frequencies (up to 50%) of CTL-P were obtained in a population of large-sized cells separated from day 4 MLC by velocity sedimentation. Furthermore, since a strikingly quantitative correlation was observed between CTL activity and CTL-P frequency in such separated MLC populations, it is likely that mature CTL in MLC are not end cells, but can further proliferate and thus behave operationally as CTL-P.     

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.     

Differential expression of the repertoire in in vitro and in vivo responses to minor alloantigens

The response to multiple minor alloantigens in the secondary mixed lymphocyte culture (MLC) between mouse strains that are identical at the major histocompatibility complex (MHC) generally yields effector cytotoxic T lymphocytes (CTL) which show cross-reactive killing of most or all third-party mouse strains which also share MHC haplotypes. We have investigated the clonal diversity of CTL responses in vivo versus in vitro by examination of such cross-reactions, using CTL effector cells derived from a primary response, an in vivo secondary response, and an in vitro secondary MLC. CTL from these three responses were assayed on a panel of H-2' targets. Restimulation of antigen-primed spleen cells in vitro yielded CTL which were strongly cross-reactive on all targets, whereas the in vivo responses were much less so. We conclude that the set of clones which become cytotoxic effectors in vivo is much less diverse than the set which is primed on a first encounter with antigen and that powerful constraints must therefore operate on the specificity of in vivo responses to non-MHC antigens.     

Transplantation antigen expression on murine trophoblast detection by induction of specific alloimmunity

Cell-mediated immune responses to murine embryonic trophoblast cells were investigated using lymphocyte trophoblast cultures (LTC) and cell-mediated lympholysis (CML). Spleen cells from CBA (H-2^k) or C57BL/6 (H-2^b) mice hyperimmunized with 3.5-day-old Balb/c (H-2^d) blastocysts did not undergo DNA synthesis after in vitro exposure to Balb/c blastocyst outgrowths nor were cytotoxic lymphocytes (CTL) generated against H-2^d alloantigens. Splenocytes from Balb/c mice presensitized with semiallogeneic (Balb/c female × C57BL/6 male) trophoblast cells derived from 17- to 20-day placental tissue expressed a weak proliferative response in the presence of semiallogeneic placental trophoblast and produced a moderate number of CTL against H-2^b (paternal strain) alloantigens when compared to mixed lymphocyte cultures (MLC) between Balb/c responder and semiallogeneic (stimulator) spleen cells. CTL were also generated in vitro after splenocytes from Balb/c mice hyperimmunized with semiallogeneic spleen cells were restimulated in vitro with placental trophoblast cells. These studies showing that early-stage trophoblast cells fail to evoke transplantation immunity and placental trophoblast is capable of generating alloimmunity only after combined in vivo hyperimmunization with in vitro restimulation suggest that these trophoblast cells are poorly immunogenic due in part to the relatively weak functional expression of major transplantation antigens.     

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.     

Cellular requirements for the generation of primary cell-mediated lympholysis responses to Qa-1 antigens

Mixed leukocyte cultures (MLC) between NZB responder spleen cells and Qa-1-disparate stimulator spleen cells were employed to determine the cellular requirements for the generation of primary anti-Qa-1 cell-mediated lympholysis (CML) responses. Although primary anti-Qa-1 cytotoxic lymphocytes (CTL) were generated during H-2-homologous stimulation, anti-Qa-1 CTL were not detectable from MLC in which the stimulators were H-2 allogeneic. Anti-Qa-1 CTL also were not generated from MLC in which the stimulators were semiallogeneic. Thus, H-2 identity between responder and stimulator cells was not sufficient to permit the generation of primary anti-Qa-1 CTL when H-2 disparity was also present. The capacity for H-2 disparity to prevent anti-Qa-1 CML responses was further demonstrated in MLC containing both H-2-allogeneic and H-2-homologous stimulator cells. Therefore, in subsequent studies we employed NZB responders and H-2-homologous, Qa-1-disparate stimulators. When various subpopulations of stimulator cells were studied for their ability to induce anti-Qa-1 CTL, nylon wool-adherent cells were found to be potent stimulators, but nylon wool-nonadherent cells were not. Furthermore, depletion of macrophages from the stimulator population abrogated the generation of anti-Qa-1 CML responses, despite the presence of responder macrophages in the culture. In contrast, all fractionated subpopulations stimulated anti-H-2 CML responses. When macrophage-enriched cells were used as stimulators, anti-Qa-1 CTL could be generated with approximately 80-fold fewer stimulator cells than when unfractionated splenocytes were used as stimulators. These findings indicated that stimulator macrophages were essential for the generation of primary anti-Qa-1 CTL. Direct evidence for macrophage expression of Qa-1-antigens was obtained by using a Qa-1b-specific CTL clone. These studies provide i) the first evidence for Qa-1 expression on macrophages, ii) a basis for comparison of the cellular interactions necessary to generate CTL against H-2K/D-encoded vs Qa-1-encoded class 1 antigens, and iii) a model for investigating the mechanisms responsible for the immunodominance of H-2K/D alloantigens.     

The effects of cyclophosphamide on in vitro cytotoxic responses to a syngeneic tumour

Summary We have studied the effects of treating DBA/2 mice with high doses of cyclophosphamide upon their subsequent ability to generate cytotoxic cells in vitro against syngeneic tumour antigens or alloantigens. High doses of cyclophosphamide (100–200 mg/kg body weight) eliminated the response to both antigens. The addition of normal DBA/2 thymocytes into these cultures restored the response to allogeneic cells but not to tumour cells. The anti-tumour response could be restored by the addition of interleukin 2 to the cultures. Treatment with high doses of cyclophosphamide decreased the number of anti-tumour cytotoxic cell precursors in the spleen, but did not affect the capacity of bulk cultures of spleen cells to produce interleukin 2 when stimulated with the mitogen concanavalin A.Abbreviations CY Cyclophosphamide - CTL cytotoxic T cells - CTLp precursor cytotoxic T cells - IL2 interleukin 2 - Con A concanavalin A - FCS fetal calf serum     

Comparison of immune responsiveness in mice after single or multiple donor-specific transfusions

Immune responsiveness was compared in B6AF1 mice after one, two, three, or four donor-specific DBA/2 blood transfusions (DST). Ten days after the last transfusion, the spleen cells of transfused mice were assayed for direct lymphocyte-mediated cytotoxicity, for the ability to respond in mixed lymphocyte culture (MLC) and cell-mediated lymphocytotoxic (CML) assays to DBA/2 and C3H/He antigens, and for the ability to inhibit the MLC and CML response of normal B6AF1 to DBA/2 and C3H/He antigens. Immune responsiveness was also tested in B6AF1 2 to 80 days after a single DBA/2 DST. The MLC response of transfused mice was specifically suppressed to the blood donor after both single and multiple transfusions. The CML response to DBA/2 was suppressed after a single DST, but returned to normal after multiple transfusions. Spleen cells from transfused mice did not inhibit the MLC response of normal B6AF1 mice to DBA/2 or C3H/He antigens after one or two transfusions regardless of time tested, but were able to inhibit the response to both stimulators after three or more transfusions. The MLC response remained specifically suppressed to the blood donor for as long as 80 days after a single DST, while the CML response was suppressed up to 50 days after transfusion, but had returned to normal by 80 days.     

Differential responsiveness to MIs locus antigens in graft-versus-host and host-versus-graft reactions

After (semi)allogeneic transplantation of lymphoid cells into lethally irradiated mice, the development of anti-host directed T effector cells can be demonstrated by means of a simple delayed-type hypersensitivity (DTH) assay. Using this assay we have shown that in H-2 compatible combinations Mls locus antigens can induce the generation of such T effector cells during a graft-versus-host (GvH) reaction. Other non-H-2 alloantigens are probably of minor importance. The capacity of Mls locus antigens to induce distinct anti-host DTH reactivity correlated with the capacity to induce a one-way mixed lymphocyte culture (MLC) response. Mls^a and Mls^c locus antigens initiated a positive MLC response as well as distinct GvH-related DTH reactivity. On the other hand, in the combination DBA/2 versus (BALB/c × DBA/2) F₁, the Mls^b locus antigen was not able to initiate in vitro proliferation, a lack of response which coincided with a marginal and short-lasting GvH-related DTH reactivity. In contrast, the host-versus-graft (HvG) DTH reaction of BALB/c and DBA/2 mice to subcutaneously injected (BALB/c × DBA/2) F₁ spleen cells was equally strong. Here antigens other than those coded for by the Mls locus were mainly responsible for the antigraft DTH response. These results suggest that T effector cells generated in GvH and HvG reactions are specific for largely different sets of minor histocompatibility antigens, with a selective stimulation by Mls locus antigens under GvH conditions.