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.     

CD3- bone marrow cells augment the generation of cytotoxic T lymphocytes showing a preference for the X-chromosome linked gene product of stimulator cells

Responder cells, composed of both a limited number of nylon wool-passed lymph node (NW-LN) cells and an excess number of CD3+ cell-depleted bone marrow (CD3- BM) cells from the same strain of mice, were stimulated with allogeneic spleen cells in vitro. The CD3- BM cells augmented the generation of allogeneic major histocompatibility complex (MHC) class I specific cytotoxic T lymphocytes (CTL) from NW-LN cells. C3H/He (H-2k, C3H background) responder cells were stimulated with either B10.D2 (H-2d, B10 background) or BALB/c (H-2d, BALB background) spleen cells. In the former stimulation, the CTL induced lysed B10.D2 target cells more efficiently than the BALB/c cells. Furthermore, these CTL lysed more (B10.D2 x BALB/c) F1 male target cells than (BALB/c x B10.D2) F1 male. In the latter stimulation, the CTL lysed more BALB/c than B10.D2 cells, and more (BALB/c) x B10.D2) F1 male than (B10.D2 x BALB/c) F1 male. The reciprocal mixed lymphocyte cultures (MLC) were carried out, in which BALB/c responder cells were stimulated with either C3H/He or B10.BR (H-2k, B10 background) spleen cells. In the former stimulation, the CTL induced lysed more C3H/He or (C3H/He x B10.BR) F1 male target cells than B10.BR or (B10.BR x C3H/He) F1 male, and in the latter, the reciprocal results were obtained. These results suggested that the CTL induced had a preference for the X-chromosome linked gene products (Xlgp), besides the specificity for the allogeneic MHC class I, of the mice used as stimulator.     

Immune dysfunction associated with graft-vs-host reaction in mice transplanted across minor histocompatibility barriers. II. Reversible defect in T-dependent antibody responses

B cell and Th cell functions were assessed in mice undergoing a graft-vs-host reaction (GvHR) in response to minor histocompatibility Ag by using the plaque-forming cell (PFC) response to the T-independent Ag TNP-Brucella abortus and the T-dependent Ag TNP-SRBC. Bone marrow plus spleen cells from B10.D2 mice were transplanted into lethally irradiated B10.D2 (syngeneic recipient) or H-2d-compatible BALB/c (allogeneic recipient) to produce a chronic form of GvHR. BALB/c recipients of an allogeneic transplant demonstrated a marked and proportional lymphoid depletion of the spleen with normal percentages of B cells, T cells, and CD4+ and CD8+ T cell subsets. Mice with GvHR made normal numbers of PFC/10(5) spleen cells in response to the T-independent Ag, but a significantly depressed number of PFC/10(5) spleen cells to the T-dependent Ag compared with normal B10.D2 mice and with irradiated B10.D2 recipients of syngeneic B10.D2 marrow plus spleen cells. Mice undergoing the minor Ag GvHR made significantly larger numbers of PFC/10(5) spleen cells after secondary immunization with TNP-SRBC compared with controls. In vitro assays demonstrated that B cells from mice with GvHR responded to T help from normal B10.D2 mice and that T cells from mice with GvHR provided help to normal B cells after in vivo immunization. These data demonstrate that radiation chimeras with GvHR in response to minor histocompatibility Ag have relatively normal B cell function and an apparent defect in T helper cell function that is reversible by immunization with appropriate Ag.     

Graft-vs-host reactions (GVHR) across minor murine histocompatibility barriers. II. Development of natural suppressor cell activity

We explored the immunoincompetence of mice undergoing a chronic graft-vs-host reaction (GVHR) across minor histocompatibility barriers. BALB/c and B10.D2 mice are H-2d and mls b, and differ only with regard to minor histocompatibility antigens (MiHA). A large number of BALB/c mice were unirradiated or were irradiated with 300, 600, or 900 R. They then were injected with 5 X 10(7) spleen cells from either allogeneic B10.D2 or syngeneic BALB/c mice. The spleen cells from these recipient mice were assayed at various times post-irradiation/injection for their proliferative response to Con A and LPS, their ability to suppress the mitogen responses of normal spleen cells, and for the genetic specificity of this suppression. Spleen cells from BALB/c mice that had received 600 or 900 R (but not 0 or 300 R), and allogeneic B10.D2 lymphocytes, became very hyporesponsive to mitogens and became suppressive in vitro by days 7 to 10 post-irradiation/injection. These phenomena persisted for the entire 49 days of the experiment. After an initial period of splenomegaly, the spleens of these mice gradually became depleted of viable lymphocytes. Initial characterization of suppressor cells found in the spleens of GVH mice showed that they were not removed by treatment with anti-Thy-1.2 plus complement. GVH suppressors also were not adherent to plates coated with antiserum directed towards murine Ig. In addition, these cells did not adhere to plastic plates. Thus, we believe that the suppressor cells found in mice undergoing GVHD across MiHA are not mature T cells, B cells, or macrophages, but belong to a class of suppressor cells termed natural suppressor (NS). Genetic analysis of NS cell activity showed that as early as 10 days post-irradiation/injection, NS cells inhibited mitogen responses of all mouse strains tested, the exception being the relative difficulty in suppressing the LPS response of B10.D2 (syngeneic with donor cells). By day 42, this had developed into an almost complete inability to suppress a B10.D2 LPS response, although at this time NS cells were still capable of inhibiting all the other mitogen responses of all strains tested, including the Con A response of B10.D2 spleen cells. Moderate amounts of mitogen unresponsiveness and suppressor activity were seen in the syngeneic groups (BALB/c----BALB/c) but only if recipients received 600 or 900 R. This was a transient phenomenon that was maximal at day 14, and which we believe to be a similar but less severe degree of immunoincompetence when compared with that seen with allogeneic stimulation in the B10.D2----BALB/c GVH model.(ABSTRACT TRUNCATED AT 400 WORDS)     

Antigen-dependent proliferation of cloned continuous lines of H-2-restricted influenza virus-specific cytotoxic T lymphocytes

The antigenic requirements for in vitro proliferation of several cloned continuous lines of H-2-restricted influenza virus-specific cytotoxic T lymphocytes (CTL) has been examined. The cloned CTL lines were established from individual splenic CTL precursors obtained from A/JAPAN/305/57 (H2N2)-immune F1 (C57BL/6 X BALB/c) donors. The lines were isolated (by limiting dilution in liquid culture) and expanded in the presence of A/JAPAN/305/57-infected irradiated syngeneic (F1) spleen cells and T cell growth factor (TCGF) of rat spleen origin. Optimal proliferation (and long-term in vitro cultivation) of these H-2-restricted CTL lines required both specific antigenic stimulation in the form of virus-infected syngeneic spleen cells and an exogenous source of TCGF. In addition, the antigenic requirements for proliferation of these lines directly reflected the pattern of H-2-restricted influenza virus-specific recognition at the level of target cell recognition and lysis.     

Requirement of I-E molecule for thymocyte apoptosis induced by staphylococcal enterotoxin B in vivo

In vivo administration of bacterial superantigen staphylococcal enterotoxin B (SEB) to BALB/c mice led to thymus atrophy resulting from thymocyte apoptosis. In this study, we demonstrated that SEB induced a substantial reduction in thymocyte numbers in BALB/c, B10. D2 (H-2(d) haplotype), B10.BR, C3H/HeJ, C3H/HeN (H-2(k)), and (BALB/c x B6)F1 (H-2(dxb)), but caused little or no effect in I-E- strains such as B6, B10, A.BY (H-2(b)), and A.SW (H-2(s)) mice. Elimination of CD4(+)CD8(+) cells predominantly accounted for the thymocyte loss, although the numbers of other subpopulations may also be reduced. Thymocyte apoptosis was shown by an increase in the level of DNA fragmentation in BALB/c but not in B6 mice after SEB administration. Treatment with anti-I-Ed monoclonal antibody to BALB/c mice blocked SEB-induced thymocyte apoptosis when anti-I-Ad exerted less effect. In contrast to SEB, staphylococcal enterotoxin A led to comparable levels of thymus atrophy in BALB/c and B6 mice. Studies on the surface marker expression indicated that CD25 expression was upregulated on BALB/c mouse thymocytes but with only a moderate increase in B6 mice. The CD4(+)CD8(+) cells were the major (>90%) population that expressed elevated levels of CD25 in BALB/c mice. An increase in the expression of TCRalphabeta, CD3, and CD69 surface markers was also observed on thymocytes from BALB/c mice, but not from I-E- strains. The differential response of I-E+ and I-E- mice to SEB may be exploited as a model for the study of apoptosis in the thymus.     

Effect of graft-versus-host disease on anti-tumor immunity

BCL1, a spontaneous B cell leukemia of BALB/c origin, is rejected by C.B-20 (Ighb, H-40b) but not BALB/c (Igha, H-40a) mice. Adoptive transfer of C.B-20 anti-BCL1 effector cells specific for the minor histocompatibility Ag H-40a protects irradiated C.B-20 but not BALB/c recipients. Because C.B-20 donor cells could potentially generate graft-vs-host disease (GVHD) in BALB/c recipients, we investigated the possibility that GVHD prevents the anti-tumor effect. GVHD was induced in (C.B-20 X B10.D2)F1 [H-2d, H-40b X H-2d,H-40b] recipients after injection of B10.D2-primed C.B-20 donor cells. GVHD was indicated by the histologic appearance of tissue sections from C.B-20----F1 livers, target organs of GVHD, which showed a marked mononuclear cell infiltrate around the portal tracts and central veins. In addition, splenic lymphocytes from these mice had altered CD4/CD8 ratios and were unable to respond to the polyclonal activators Con A and LPS. The mitogen unresponsiveness was at least partially due to the presence of a suppressor cell, because proliferation of normal spleen cells to Con A and LPS was suppressed upon addition of C.B-20----F1 spleen cells. Further immune dysfunction was evident by the inability of T cells from mice with GVHD to generate a CTL response to H-2 alloantigens. Addition of C.B-20----F1 spleen cells to F1 responder cells at the induction of culture did not prevent generation of CTL, indicating that a suppressor cell was not responsible for the lack of CTL activity. In this setting of GVHD, F1 recipients were able to reject BCL1 upon adoptive transfer of C.B-20 anti-BCL1 effector cells. These data indicate that GVHD-induced immune dysfunction does not inhibit the activity of antileukemia T cells.     

Investigations into the nature of Igh-V region-restricted T cell interactions by using antibodies to antigens on methylcholanthrene-induced sarcomas. I. Analysis of an Igh-V-restricted suppressor-inducer factor

We have previously demonstrated the relationship between antigens on BALB/c methylcholanthrene (MC)-induced fibrosarcomas and T cell regulatory molecules by using a variety of antisera raised to these sarcomas in BALB/c and BALB/c X C57BL/6 (CB6F1) mice. One such pool of antiserum, a CB6F1 anti-CMS 4 (Pool XIV) serum, was used to investigate the nature of the T cell regulatory structures recognized by these antibodies. Pool XIV antiserum was capable of blocking the induction of feedback suppression by Ly-1 TsiF, an SRBC-specific suppressor T cell factor secreted by Ly-1+, 2- I-J+ T cells. Ly-1 TsiF induces suppression by interacting with an Ly-1+,2+ I-J+ T cell target. Successful interaction of Ly-1 TsiF with its target cell requires genetic homology between inducer and target cells at the variable region of the immunoglobulin heavy chain gene complex (Igh-V). The addition of Pool XIV antiserum to primary in vitro anti-SRBC cultures resulted in blocking the ability of Ly-1 TsiF from Igha (BALB/c) and Ighj (CBA/J) mice to induce suppression on syngeneic cells, whereas suppression induced by Ly-1 TsiF in Ighb (B6), Ighc (DBA/2), Ighd (A/J), and Ighe (AKR) mice are unaffected by addition of the Pool XIV antiserum. The ability of Pool XIV antiserum to block Ly-1 TsiF activity is linked to the Igh region, because Pool XIV antiserum can block Ly-1 TsiF from BALB/c (H-2d, Igha) and the Igh congenic B.C9 (H-2b, Igha) while not affecting Ly-1 TsiF activity on B6 (H-2b, Ighb) or its Igh congenic C.B20 (H-2d, Ighb). In CB6F1 animals, Pool XIV antiserum could block the ability of CB6F1 Ly-1 TsiF to suppress BALB/c spleen cells but not B6 spleen cells. Conversely, Pool XIV antiserum could block the ability of BALB/c Ly-1 TsiF to suppress CB6F1 spleen cells, whereas B6 Ly-1 TsiF showed normal suppressive activity in the presence of Pool XIV antiserum. In contrast, Pool XIV was capable of blocking the ability of Ly-1 TsiF from BALB/c into CB6F1 bone marrow chimeras (BMC) to suppress both BALB/c and B6 mice, whereas the activity of Ly-1 TsiF from B6 into CB6F1 BMC on BALB/c or B6 spleen cells was unaffected by the addition of Pool XIV antiserum. We then investigated the molecular nature of the molecule recognized by Pool XIV antiserum on the Ly-1 TsiF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alloantigen-induced cytotoxicity against syngeneic tumor cells: analysis at the clonal level

It has been shown that peritoneal exudate cells (PEC) from BALB/c mice immunized with minor histocompatibility antigens presented by DBA/2 or B10.D2 spleen cells are capable of lysing syngeneic YC8 tumor cells in a 4-hr 51Cr-release assay. In this study, we employed limiting dilution analysis to determine the frequency of CTL precursors (CTL-P) reactive against both the specific DBA/2 (or P815) target and the syngeneic tumor YC8. The mean frequency of anti-DBA/2 CTL-P in PEC from BALB/c mice immunized with DBA/2 was 1/302. Between one-third and one-fifth of limiting dilution microcultures that exhibited lytic activity against DBA/2 lymphoblasts (or P815) were also able to lyse YC8. No lysis of YC8 was observed in the absence of a parallel lysis on DBA/2 lymphoblasts or P815 target cells. T cell clones, derived by micromanipulation from microcultures selected for cytotoxic activity against YC8 and/or P815, maintained either the specific anti-allogeneic or the doubly reactive ( antiallogeneic plus anti-syngeneic tumor) phenotype. Fourteen clones (six specific and eight doubly reactive) were tested for cytotoxic activity on a panel of target cells with different haplotypes. All showed H-2-restricted specificity for minor histocompatibility antigens shared by DBA/2 and B10.D2. The restriction element for some of the clones mapped in the K region of the H-2 complex, whereas for other clones the restriction element mapped in the D region; both K- and D-restricted clones were able to lyse YC8. When the clones that exhibited lysis on YC8 were tested on two other BALB/c tumor targets, LSTRA, a Moloney virus induced lymphoma, and RL male-1, a radiation induced lymphoma, two of seven were found to lyse all three syngeneic tumor targets equally well, but not syngeneic BALB/c blasts. These clones were functionally categorized as conventional CTL because they were unable to proliferate when cultured with antigen in the absence of exogenous lymphokines, and were unable to produce lymphokine with IL 2 activity when stimulated by the appropriate splenocytes. When tested in vivo in a Winn assay, a strong anti-tumor activity against YC8 was exerted by the anti-DBA/2 clones DY4 -3 and DY16 -3. These clones lysed both YC8 and the immunizing target cells in vitro. No in vivo effect in neutralizing YC8 tumor growth was observed with clone D2-1, a clone that lysed DBA/2 targets but not YC8 in vitro.     

The generation of cytolytic activity in mixed leukocyte cultures: cortisone-resistant thymocytes are deficient in helper T lymphocytes

Cortisone-resistant (CR) thymocytes did not generate cytolytic activity toward H-2 K or D alloantigen unless they were also stimulated by H-2 I or non-H-2 alloantigens, even though spleen cells generated brisk cytolytic activity toward H-2 K or D alone. Backstimulation by stimulating strain T lymphocytes accounted for neither the response of spleen cells toward H-2 K or D alloantigen nor the response of CR thymocytes to a full set of alloantigens. In addition, lack of non-T accessory cells did not account for the CR thymocyte pattern of reactivity. Rather, CR thymocytes appeared to be relatively deficient in helper T lymphocytes (HTL). CR thymocytes generated specific cytolytic activity toward H-2 D alloantigen when T cell growth factors (TCGF) or cloned alloreactive helper T lymphocytes were added to culture. CR thymocytes contained fewer HTL precursors detected at limit dilution than spleen cells did. Thus spleen cells generated cytolytic activity toward class I alloantigens alone, but under the same culture conditions CR thymocytes had to be stimulated by both class I and class II alloantigens. Class II alloantigens may be required to stimulate cytolytic activity only under culture conditions in which class I-reactive HTL are not sufficient to provide a minimal threshold of help.     

Augmentation of killer activity of murine spleen cells against allogeneic and syngeneic tumor cells by inoculation of alloantigen in vivo

After C57BL/6 (B6) mice were inoculated with BALB/c spleen cells via tail vein, kinetics of cytotoxic activities in the B6 mice against sensitizing alloantigens (H-2d) and against syngeneic antigens were investigated using, as target cells, P815 mastocytoma cells (H-2d) and B16 melanoma cells (H-2b). Cytotoxic activity against P815 in the B6 spleen cells reached a peak 3 days after alloantigen inoculation, decreased drastically on day 5 and rose again thereafter. The profile of anti-B16 cytotoxic activity was similar to that of anti-P815 activity. The cytotoxic activity against P815 was inhibited partially by cold B16, but that against B16 was not inhibited by cold P815. Surface phenotype of cytotoxic cells against P815 was Lyt2+, Thy1+, Asialo GM1+ and that of cytotoxic cells against B16 was Lyt2-, Thy1+/-, and Asialo GM1+. The results indicate that inoculation of B6 mice with allogeneic BALB/c spleen cells induce two types of cytotoxic cells; one is similar to lymphokine-activated killer (LAK) cells and the other is activated natural killer cells.     

Cellular and antigenic requirements for production of mixed leukocyte reaction suppressor factor

When murine spleen cells, alloantigen-sensitized previously in vivo, are incubated with spleen cells bearing the sensitizing alloantigens, a supernatant factor is produced that inhibits 3H-thymidine incorporation by responding lymphocytes in the mixed leukocyte reaction. This study evaluates the cellular and antigenic requirements during restimulation for elaboration of this suppressor factor, MLR-TsF. BALB/c spleen cells, sensitized to C57BL/6 (B6) alloantigens in vivo, produced MLR-TsF when cultured with B6 spleen cells in vitro, despite depletion of Sephadex G-10-adherent cells from factor-producing cells, stimulator cells, or from both populations. T cells were not required within the stimulating population, but a requirement for viable stimulator cells was demonstrated when heat-killed or glutaraldehyde-fixed stimulator cells failed to induce MLR-TsF production. The alloantigenic requirements for MLR-TsF production were addressed by 2 approaches. Treatment of stimulator cells with appropriate anti-I region antisera and complement did not affect MLR-TsF production, demonstrating that an absolute requirement for cells expressing I region determinants did not exist. However, spleen cells primed against entire H-2 haplotype differences produced significant quantities of MLR-TsF when they were restimulated with spleen cells homologous to the priming cells in only the I region, in the K and D regions, or in the D region alone. The additive nature of subregion-specific restimulation suggests that distinct subpopulations of K, I, and D region-specific MLR-Ts comprise the MLR-Ts population primed to entire H-2 haplotype differences.     

Thymocyte and macrophage interactions: separation of murine thymocyte subsets and enrichment of syngeneic cell-responding thymocytes by adsorption to macrophage monolayers

The spontaneous binding of murine thymocytes to macrophage monolayers was employed to separate thymocytes into macrophage-unbound and -bound subsets, and the functional reactivities of these two subpopulations were examined. Macrophage-unbound thymocytes were found to be enriched in functional subsets reactive to semi-allogeneic and allogeneic stimulating spleen cells by proliferation in mixed leukocyte culture (MLC). Furthermore, macrophage-unbound thymocytes were frequently found to respond to syngeneic spleen cells. This syngeneic proliferative response showed both memory and specificity upon subsequent restimulation and thus seems to represent a syngeneic mixed leukocyte reaction (SMLR). Syngeneic responding thymocytes were also found to produce interleukin 2 when cultured with syngeneic but not allogeneic stimulator cells. In contrast, macrophage-bound thymocytes showed greatly reduced proliferative responses to allogeneic stimulators and no responses to syngeneic stimulators. The macrophage-bound thymocyte subset was not enriched in detectable suppressive activity; proliferative responses of macrophage-unbound thymocytes to either allogeneic or syngeneic cells were neither suppressed nor enhanced when macrophage-unbound thymocytes were added to the cultures. Thus, the macrophage-unbound subset seems to be enriched in functionally mature thymocytes and the macrophage-bound subset appears to be enriched in functionally immature thymocytes. This functional separation of thymocytes by macrophage adherence also correlated well with thymocyte subpopulations separated by bovine serum albumin density gradients; the low density mature thymocytes showed enhanced responses to both allogeneic and syngeneic stimulators, whereas the high density immature cells were unresponsive. This correlation was supported further by binding studies in which T cell tumor lines derived from C57BL/6 mice were used. ERLD tumor cells, which are similar to cortical immature thymocytes in both enzymatic and surface antigenic markers, were found to bind readily to macrophages, whereas both EL-4 and E male G2 tumor cells, with characteristics of mature thymocytes, bound to macrophages poorly. The binding of thymocytes and ERLD tumor cells to macrophages was not genetically restricted. We speculate that thymocyte binding to macrophages may play a critical role during the functional maturation of thymocytes.     

Roles of I-E molecule and CD28 costimulation in induction of suppression by staphylococcal enterotoxin B in vivo.

Exposure to bacterial superantigens leads to the induction of a subsequent state of immune hyporesponsiveness. Using a transwell coculture system, a previous report demonstrated that splenocytes from staphylococcal enterotoxin B (SEB)-injected BALB/c mice secreted soluble mediators to suppress the proliferative response of naive syngeneic splenocytes to SEB stimulation. We show in the present study that, in contrast to the suppressive effect induced by SEB in BALB/c (H-2(d) haplotype), MRL(+/+), and MRL-lpr/lpr (H-2(k)) mice, SEB-primed splenocytes from I-E(-) strains such as B6, B10, A.BY (H-2(b)), and A.SW (H-2(s)) mice failed to inhibit the CD25 expression and the proliferative activity of their syngeneic naive responder splenocytes. Further results revealed that the SEB-primed cells from BALB/c, but not B6, mice inhibited the CD25 expression and proliferation of naive responder cells from either BALB/c or B6 mice, indicating the critical regulatory role of the effector cells. Unlike SEB, staphylococcal enterotoxin A induced profound suppression in both BALB/c and B6 mice. Moreover, the suppressive competence of SEB-primed splenocytes was diminished in CD28-deficient BALB/c mice. Taken together, our results indicate that when SEB is used as a stimulator in vivo, both the I-E molecule and CD28 costimulation are required for the induction of regulatory cells bearing suppressive activity.     

Recognition of Rous sarcoma virus-induced tumor antigens by cytotoxic T lymphocytes (CTL): studies on specificity of killing by CTL employing H-2 congenic and recombinant mouse tumor cells

The specificities of cytotoxic T lymphocytes (CTL) were studied for the analysis of CTL against tumor-specific cell surface antigen(s) (TSSA) of non-virus-producing tumor cells induced by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) in B10 congenic and recombinant mice. Eight CTL clones were established from immune spleen cells of B10.A(5R) mice. These clones demonstrated six patterns of cytotoxic reactivity in vitro: Two clones showed H-2 restriction in tumor cell lysis. Two other clones had the capacity to lyse syngeneic, H-2K-compatible B10 and H-2-incompatible B10.A(4R) tumor cells, but not YAC-1 cells. One clone had cytotoxic activity against syngeneic, H-2D-compatible B10.D2 tumor cells and YAC-1 cells, but not against H-2-incompatible tumor cells. One clone had cytotoxic activity against syngeneic and YAC-1 tumor cells, but not against either H-2-compatible or H-2-incompatible tumor cells. One clone had lytic activity to syngeneic, H-2-compatible, H-2-incompatible, and YAC-1 tumor cells. Another clone killed H-2-incompatible B10.A(4R) tumor and YAC-1 cells, but not syngeneic or H-2-compatible tumor cells. All these clones strongly expressed surface Thy-1.2 antigens, whereas the expression of Lyt-1.2 and Lyt-2.2 antigens was different from clone to clone. These results demonstrate heterogeneity of both lytic specificity and phenotype of CTL against RSV-induced mouse tumor cells, suggesting the existence of multiple antigenic sites on the RSV TSSA recognized by CTL populations.     

Immunoregulatory pathways in adult responder mice. II. Regulation of delayed-type hypersensitivity responses by GAT-specific suppressor factors present in GAT-tolerant adult responder mice

We studied the effects of T cell extracts from adult responder BALB/c mice tolerized with poly(Glu60Ala30Tyr10) (GAT)-coupled syngeneic spleen cells (GAT-SP) on delayed-type hypersensitivity (DTH), T cell-proliferative (Tprlf), and plaque-forming cell (PFC) responses. Adult responder mice injected i.v. with GAT-SP develop Lyt-1-2+ suppressor T cells (Ts), which suppress the induction of GAT-specific DTH and PFC, but not Tprlf responses. Sonicates from these Ts contain an afferent-acting, soluble factor(s) (GAT-TsFdh) that specifically suppresses the same responses as the intact Ts (i.e., DTH and PFC, but not Tprlf). Immunosorbent chromatography studies were employed to determine the molecular nature of the suppressive material active on both cellular and humoral responses. In both assay systems, GAT-TsFdh was found to bear determinants encoded by the I subregion of the H-2 complex and a receptor(s) for GAT. BALB/c-derived GAT-TsFdh suppressed the induction of GAT DTH in syngeneic BALB/c and H-2-compatible B10.D2, but not in allogeneic C57BL/6 or CBA/Cum, suggesting a possible H-2 restriction in the suppression. It was also shown that one target of functional regulation by GAT-TsFdh is the T helper cell for DTH responses (DTH-Th). The results suggest that similar Ts and TsF regulate humoral and cell-mediated responses, perhaps by affecting a target common to both pathways (e.g., the T helper cell). The resistance of Tprlf responses to suppression by GAT-TsFdh indicates that the effector DTH-Th target is not a major component of the proliferative response. These data are discussed with respect to GAT-specific TsF-regulating PFC responses, which have been identified in nonresponders and in responders tolerized as neonates with GAT.     

Defective T helper activity in the spleen of BALB/c mice immune to a syngeneic fibrosarcoma

Summary BALB/c mice were immunized with the syngeneic 3-methylcholanthrene-induced fibrosarcoma CA-2 by the growth and excision method. When lymphoid cells from different organs of these tumor-free mice were tested in a direct ⁵¹Cr-release assay, peritoneal exudate cells but not spleen cells displayed specific cytotoxicity against the syngeneic tumor target. A cytotoxic response could be obtained by tumor-immune spleen cells when cultured in a mixed lymphocyte tumor cell culture (MLTC) at high but not low density although at the same effector/stimulator ratio. Lack of cytotoxic activity in low density MLTC was not due to an impairment of cytotoxic precursors since cytotoxicity was rescued by adding exogenous interleukin-2 in experimental conditions in which no lymphokine-activated killer cells could develop relevant anti-CA-2 lysis. When low density MLTC were supplemented with either 800 R-irradiated cells or nonirradiated, negatively selected Lyt 1⁺ cells from the same immune mice, induction of a cytotoxic response against CA-2 occurred and interleukin-2 production became detectable. Additional studies indicated that spleen cells of CA-2-immune mice were also impaired in their ability to provide help to syngeneic thymocytes for the generation of cytotoxic T lymphocytes against C57BL/6J alloantigens. Dilution effect of helper cells due to immunization procedures was excluded since spleen cells of mice immunized against another BALB/c tumor, the YC8 lymphoma, or against DBA/2 minor histocompatibility antigens provided good help to thymocytes against the same alloantigens. These results indicate that tumor-immune animals may also have selective T helper defects in an important lymphoid organ like spleen.     

Effects of costimulator on immune responses in vitro.

We recently described a factor, costimulator, that is required for the concanavalin A-induced proliferation of CBA mouse thymocytes in vitro (see Reference 1). Using the costimulator dependence of mouse thymocytes as an assay, we have now determined that spleen cells from congenitally athymic (nude) BALB/c mice do not produce costimulator in response to Con A, and spleen cells depleted of Thy 1-positive cells do not respond to it in the presence of Con A. Thus, costimulator both requires thymus-derived (Thy 1+ lymphocytes for its production and has an effect on this type of cell. (However, the costimulator-producing and responsive cells may be different.) Purified costimulator preparations are a source of the required second component for the stimulation of adult, CBA/J thymic lymphocytes by PHA, normally a poor mitogen for these cells. They also enhance the level of DNA synthesis in a mixed leukocyte reaction, and the specific generation of cytotoxic lymphocytes to allogeneic tumor cells in vitro. Costimulator is not H-2 restricted in its effects, and it is produced in mixed leukocyte reactions. Finally, it has been possible to grow normal, primary thymic lymphocytes in culture for about 20 days by adding partially purified costimulator to the cultures.     

"Physiological interaction" does not explain the H-2 requirement for recognition of virus-infected cells by cytotoxic T cells.

B10.A (H-2Kk, H-2Dd) ectromelia-immune T cells from secondary responses in vitro were protent killers of both infected L929 (H-2Kk H-2Dk) and infected P-815 (H-2Kd, H-2Db) target cells. Specific competition with unlabelled targets showed that two separate T cell subsets were responsible for lysis of infected L929 and infected P-815 cells. One hypothesis to account for this (a form of "physiological interaction") is that T cells which kill one target e.g. infected L929) display only one out of two possible self-complementary recognition structures, in this example the H-2Kk alloantigen, not H-2Dd, whereas T cells that lyse infected P-815 targets display only H-2Dd, not H-2Kk. This hypothesis was tested and seems untenable because of the following results: A.TH (H-2Ks, H-2Dd) ectromelia-immune, secondary cytotoxic T cells which killed infected SJL/J (H-2Ks, H-2Ds) targets were themselves inactivated by pre-incubation with SJL/J cytotoxic T cells generated in one-way mixed lymphocyte reaction (MLR) against BALB/c (H-2Kd, H-2Dd). A.TL (H-2Ks, H-2Dd) ectromelia-immune secondary cytotoxic T cells which killed infected BALB/c targets were themselves inactivated by BALB/c cytotoxic T cells generated in MLR against SJL/J. Thus, virus-immune T cells which lyse infected targets by virtue of shared H-2K are also displaying H-2D alloantigen, and vice versa.     

Dissociation of H-2 recognition by antibody and cytotoxic T cells of a cloned murine leukemia cell line

The differential expression of H-2 specificities recognized by antibody and by cytotoxic T lymphocytes (CTL) has been studied using a clone (FY7) of the C57BL/6 leukemia cell line FBL-3 (H-2b/H-2b). Unlike C57BL/10 spleen cells, EL-4 lymphoma cells and Y57-2C leukemia cells (all H-2b/H-2b), FY7 failed to induce the primary in vitro generation of anti-H-2b CTL by (B10.A x A)F1 (H-2a/H-2a) or B10.D2 x BALB/c)F1 (H-2d/H-2d) responder spleen cells. In addition, FY7 was not lysed by, and did not competitively inhibit anti-H-2b CTL. Quantitative absorption tests with H-2Kb and H-2Db antisera revealed that FY7 expressed these antigens in quantitatively similar amounts to EL-4. The H-2Kb product of FY7 appeared to be identical with that of C57BL/10 spleen cells both in apparent molecular weight and isoelectric point. Yet FY7 failed to inhibit anti-H-2Kb CTL competitively in a cold target inhibition assay. Possible mechanisms are discussed for the lack of T-lymphocyte recognition of the H-2Kb-gene product expressed by FY7.