Cytotoxic T lymphocyte response to minor H-42a alloantigen in H-42b mice: clonal inactivation of the precursor cytotoxic T lymphocytes by veto-like spleen cells that express the H-42a antigen

When (B10.BR X CWB)F1 (BWF1; H-2k/b) mice carrying the H-42b allele at the minor H-42 locus were injected with H-42a C3H.SW (CSW; H-2b) or C3H (H-2k) spleen cells (SC), self-H-2Kb restricted anti-H-42a pCTL in the BWF1 recipients were primed and differentiated to anti-H-42a CTL after in vitro stimulation with (B10.BR X CSW)F1 (BSF1; H-2k/b, H-42b/a) SC. In contrast, anti-H-42a pCTL in H-42b mice were inactivated by injection with H-42-congenic H-42a SC, and stable anti-H-42a CTL tolerance was induced. Preference of H-2Kb restriction of anti-H-42a CTL was strict, and self-H-2Kb-restricted anti-H-42a CTL did not lyse target cells carrying H-42a antigen in the context of H-2Kbm1. Involvement of suppressor cells in the anti-H-42a CTL tolerance was ruled out by the present cell transfer study and the previous cell-mixing in vitro study. Notably, treatment with anti-Thy-1.2 antibody (Ab) plus complement (C) wiped out the ability of CSW SC in the priming of anti-H-42a pCTL of BWF1 mice but left that of C3H SC unaffected, and injection of the anti-Thy-1.2 Ab plus C-treated CSW SC induced anti-H-42a CTL tolerance in the BWF1 recipients. Furthermore, H-42a/b, I-Ab/bm12 [CSW X B6.CH-2bm12 (bm12)]F1 SC could not prime anti-H-42a pCTL in H-42b, I-Ab (CWB X B6)F1 recipients, whereas H-42a/b, I-Ab (CSW X B6)F1 SC primed anti-H-42a pCTL in H-42b, I-Ab/bm12 (CWB X bm12)F1 recipients. The unresponsiveness of anti-H-42a pCTL in H-42b mice to H-42-congenic H-42a SC was sometimes corrected by immunization of H-42b female mice with H-42-congenic H-42a male SC. Taking all of the results together, we propose the following. Unresponsiveness of anti-H-42a pCTL in H-42b mice to H-42-congenic H-42a SC is caused by "veto cells" contained in the antigenic H-42a SC. Anti-H-42a pCTL in the H-42b recipients directly interacting with H-42-congenic H-42a SC, which bear H-42a antigen and H-2Kb restriction element, are inactivated or vetoed.(ABSTRACT TRUNCATED AT 400 WORDS)     

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)     

Modulation of F1 cytotoxic potentials by GvHR. Host- and donor-derived cytotoxic lymphocytes arise in the unirradiated F1 host spleens under the condition of GvHR-associated immunosuppression

As an approach to dissect complex cellular events that lead to GvHR-associated immune disorders, we followed cytotoxic activities, including NK cytotoxicity, in the spleens of unirradiated F1 hosts undergoing GvHR induced by parental spleen cells. Spleen cells of (B10 X DBA/2)F1 or (B10 X AKR/J)F1 hosts undergoing GvHR induced by parental B10 spleen cells displayed a prompt and marked increase in NK cell activity within 36 hr, and the heightened activity lasted until day 8. The activity then declined abruptly and disappeared on day 12 of GvHR. Inversely, donor B10-derived CTL specifically directed to the opposite parental alloantigens of the F1 hosts emerged in these F1 host spleens on day 8, and the CTL activity reached a peak on day 12 when the host NK cell activity disappeared. During the period that the donor-derived anti-host CTL were present, these F1 host spleen cells lost not only NK cell activity but also the ability to mount in vitro CTL responses. In contrast, the respective F1 strain mice undergoing GvHR induced by the parental DBA/2 or AKR/J spleen cells showed only transient but marked increases in NK cell activity during the initial 36 hr, and then the activity decreased gradually to return to the normal level on day 10. In such GvHR F1 host spleens, donor-derived CTL could never be detected, and the spleen cells showed normal in vitro CTL responsiveness during the entire observation period of 16 days. These results are discussed from the viewpoint of genetically defined cellular events that lead to the GvHR-associated immune disorders.     

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.     

Modulation of F1 cytotoxic potentials by GvHR: role and mode of action of non-MHC genes that determine the hybrid resistance to GvHR-associated suppression of F1 cytotoxic potential

The resistance of unirradiated F1 mice against graft-vs-host reaction (GvHR) induced by lymphocytes from certain parental strains is apparently a violation of the basic law in classical transplantation immunity. To explore genetic mechanisms of this peculiar phenomenon, GvHR-associated immunosuppression was examined on various kinds of F1 mice undergoing GvHR induced by parental lymphocytes. In F1 mice raised by crossing DBA/2 mice with various H-2-congeneic B10-series strains, parental lymphocytes having non-H-2 genetic background of DBA (DBA/2 and DBA/1) invariably could not induce GvHR-associated immunosuppression, irrespective of the H-2 haplotype incompatibility involved, whereas lymphocytes of the partner parental strain induced the immunosuppression. The number of the relevant loci in the DBA non-H-2 was assessed to be three recessive loci by examination of the capability to induce the GvHR-associated immunosuppression on lymphocytes from individual (B 10.D2 X DBA/2)F1 X DBA/2 backcross mice. On the other hand, in F1 mice raised by crossing C3H/He or AKR/J mice with various H-2-congeneic B10-series strains, parental lymphocytes of H-2k haplotype, irrespective of their non-H-2 haplotype, invariably could not induce the GvHR-associated immunosuppression. Furthermore, it was revealed that non-H-2 genes of parental C3H or AKR incorporated in the F1 mice determine the resistance of the F1 mice against the H-2k-induced GvHR. The results of examination of the resistance on individual (B10 X [B10.BR X C3H/He]F1) and (B10 X [B10.BR X AKR/J]F1) mice suggested that three non-H-2 loci of C3H/He or two non-2 loci of AKR/J incorporated in F1 hybrids could determine the resistance of the respective F1 mice.     

H-40, an antigen controlled by an Igh-linked gene and recognized by cytotoxic T lymphocytes. II. Recognition of H-40 as a tumor antigen in leukemic animals

C.B-20 (Ighb) but not (C.B-20 X BALB/c)F1 mice reject BCL1, a sIg+ tumor that spontaneously arose in an Igh congenic BALB/c (Igha) mouse. C.B-20 immune T cells from mice immunized with either BCL1 or BALB/c splenocytes adoptively transfer tumor protection to sublethally irradiated C.B-20 but not BALB/c or (BALB/c X C.B-20)F1 mice. These data suggest that BALB/c and BCL1 share an antigen, which if present in the host prevents the immune cells from eradicating the tumor. The antigen is controlled by H-40, a gene that maps to the C end of the Igh complex, telomeric to Tsu and in the region of Pre-1. The ability of H-40 to act as a tumor antigen for other BALB/c tumors inoculated into C.B-20 hosts was investigated. H-40 did not elicit rejection of P1798 (T lymphoma), Meth A (fibrosarcoma), or MOPC-315 (alpha, lambda myeloma) tumor cells. C.B-20 mice that previously rejected BCL1, however, showed partial resistance to a low challenge dose of the MOPC-104E (mu, lambda myeloma) tumor. These data suggest that H-40 has a differential degree of expression on BALB/c tumor cells. The ability of the adoptively transferred cells to confer protection against BCL1 is abrogated by pretreatment of the cells with anti-Lyt-1 or anti-Lyt-2 antibodies. However, an admixture of anti-Lyt-1- and anti-Lyt-2-treated cells provided protection. These data, together with the results detected by cytotoxic T lymphocyte (CTL) activity in vitro, indicate that H-40 can serve as a target antigen for tumor rejection by CTL in allogeneic hosts. The implications of the results for allogeneic bone marrow transplantation into leukemic individuals who benefit from a graft vs leukemia effect are discussed.     

Abrogation of hybrid resistance to bone marrow engraftment by graft-vs-host-induced immune deficiency

Lethally irradiated F1 mice, heterozygous at the hematopoietic histocompatibility locus Hh-1, which is linked with H-2Db, reject bone marrow grafts from H-2b parents. This hybrid resistance (HR) is reduced by prior injection of H-2b parental spleen cells. Because injection of parental spleen cells produces a profound suppression of F1 immune functions, we investigated whether parental-induced abrogation of HR was due to graft-vs-host-induced immune deficiency (GVHID). HR was assessed by quantifying engraftment of H-2b bone marrow in F1 mice with the use of splenic [125I]IUdR uptake; GVHID, by the ability of F1 spleen cells to generate cytotoxic T lymphocytes (CTL) in vitro. We observed a correlation in the time course and spleen cell dose dependence between loss of HR and GVHID. Both GVHID and loss of HR were dependent on injection of parental T cells; nude or T-depleted spleen cells were ineffective. The injection of B10 recombinant congenic spleens into (B10 X B10.A)F1 mice, before grafting with B10 marrow, demonstrated that only those disparities in major histocompatibility antigens that generated GVH would result in loss of HR. Thus, spleens from (B10 X B10.A(2R]F1 mice (Class I disparity only) did not induce GVHID or affect HR, whereas (B10 X B10.A(5R))F1 spleens (Class I and II disparity) abrogated CTL generation and HR completely. GVHID produced by a class II only disparity, as in (B10 X B10.A(5R))F1 spleens injected into (B6bm12 X B10.A(5R))F1 mice, was also sufficient to markedly reduce HR to B10 bone marrow. This evidence that GVHID can modulate hematopoietic graft rejection may be relevant to the mechanisms of natural resistance to marrow grafts in man.     

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.     

Idiotype-specific T lymphocytes. I. Regulation of antibody production by idiotype-specific H-2-restricted T lymphocytes

Cytotoxic T lymphocytes (CTL) specific for MOPC-104E myeloma cells of BALB/c origin could be induced in BALB/c, (BALB/c X BALB.B)F1, and (BALB/c X BALB.K)F1 mice. (BALB/c X BALB.B)F1 CTL activity specific for MOPC-104E was effectively inhibited by anti-H-2d but not by anti-H-2b alloantiserum. However, the activity was hardly blocked by specific anti-idiotypic antibodies to MOPC-104E. For further analysis of the recognition of idiotype on target cells by CTL, the effect of those lymphocytes on anti-dextran B1355S antibody-producing B lymphocytes, which have a cross-reactive idiotype to MOPC-104E, was investigated. Lymphocytes from the CTL population did inhibit antibody production by dextran-immune spleen cells, but those from the CTL population specific for irrelevant myeloma cells (MOPC-167) did not. The (BALB/c X BALB.K)F1 CTL population suppressed the antibody production of BALB/c but not of BALB.K. This indicates that F1 cells can preferentially see H-2 antigens of immunizing myeloma cells on target B lymphocytes. The inhibition of antibody production was antigen specific and was only restricted to the PFC that were inhibitable by anti-idiotypic antibodies. The surface phenotypes of the cells that inhibited the antibody production were Thy-1+, Lyt-1-, Lyt-2+, and I-J-. These results strongly suggest that CTL specific for MOPC-104E recognize self H-2 antigens simultaneously with idiotypic determinants on B lymphocytes. Possible immunoregulatory roles of idiotype-specific CTL on antibody production systems are also suggested.     

Analysis of Ir gene control of cytotoxic response to hapten-modified self: helper T cells specific for a sulfhydryl hapten can substitute for an anti-TNP-H-2b self helper cell defect

Helper T cells specific for N-iodoacetyl-N'-(5-sulfonic 1-naphthyl) ethylene diamine (I-AED) were generated in (C56BL/6 X C3H/He)F1 mice by immunization with I-AED-modified syngeneic cells (AED-self). The requirements for activation of hapten-induced helper cells were investigated. The results demonstrated that activation of AED and trinitrophenyl- (TNP) helper cells was strictly hapten specific. In addition, F1 AEd-helpers could be activated efficiently by either I-AED-modified H-2b or H-2k self components to enhance the anti-AED self-CTL responses. This contrasts with the previous findings demonstrating the failure of TNP-H-2b self to activate F1 TNP-helper cells. After AED-helpers were activated, they were capable of augmenting sensitization of cytotoxic T cells (CTL) against TNP-self. These results indicate that although the activation of hapten-reactive helper cells is antigen (hapten)-specific, the subsequent helper activity, as determined by augmentation of CTL responses against another hapten, is antigen nonspecific. Since helper function was antigen nonspecific, F1 AED-helper cells activated by AED-H-2b or AED-H-2k self were tested for their ability to augment the F1 and anti-TNP-H-2b CTL response. The results indicate that the Ir gene defect in the ability of F1 spleen cells to respond to TNP-H-2b self could not be corrected by these helper cells. These results are discussed in the light of Ir gene controlled differences in the activation of AED and TNP-helper cells and possible models for augmenting CTL responses against various antigens in strains that generate marginal helper activity to TNP-self.     

Augmentation of syngeneic tumor-specific immunity by semiallogeneic cell hybrids

Hybrid cell lines were established from fusions between lipopolysaccharide- (LPS) stimulated C57BL/6J spleen cells and MPC-11 tumor cells (45.6TG1.7, abbreviated M45), and were tested for their ability to immunize semiallogeneic mice against a parental tumor challenge. These hybrids were tumorigenic in syngeneic (BALB/c X C57BL/6J) F1 (CB6F1) mice but did not grow in semiallogeneic (BALB/c X A/J) F1 (CAF1) mice. All hybrids express both parental major histocompatibility antigens (H-2b and H-2d) as detected by indirect immunofluorescence and by their ability to function as either stimulators or targets for allogeneic cytotoxic lymphocytes (CTL). M45 tumor-associated antigens (TAA) were expressed on the hybrid surface as shown by their ability to act as either stimulators or targets for syngeneic CTL specific for M45 TAA. Immunization of semiallogeneic CAF1 mice with the hybrids i.p. followed by a challenge with M45 tumor cells resulted in extended survival when compared to untreated mice or animals immunized i.p. with M45 tumor cells. This immunity was specific and was not due to an allogeneic effect; immunization with an unrelated H-2bd tumor, 70Z/3, or H-2bd B6D2F1 spleen cells or with semiallogeneic spleen cells plus M45 did not protect mice from M45 challenge. Interestingly, prophylactic priming with semiallogeneic hybrid tumor cells or parental myeloma cells led to M45-specific CTL and "help" for an in vitro CTL response; however, the degree of CTL priming by hybrid tumors was not augmented when compared to the level of CTL achieved with parental tumor alone. Hence, stimulation of CTL activity per se by hybrid tumor cells cannot explain the protective effect of hybrid tumor immunization. These studies nevertheless confirm that semiallogeneic hybrids, which we show express TAA and alloantigens, can be used to immunize mice against a lethal syngeneic myeloma tumor challenge.     

In vivo priming of mouse CTL precursors directed to product of a newly defined minor H-42 locus is under a novel control of class II MHC gene

In a previous study, we discovered a new mouse minor histocompatibility antigen encoded by a locus at 8.5 cM apart from the H-2 complex, and we have since named the locus H-42. One allele of H-42, which is named H-42a, had been elucidated, but the other alleles, which we tentatively named H-42b, have not been elucidated. In the present study, we explored MHC control on the anti-H-42a cytotoxic T lymphocyte (CTL) responsiveness in H-42b mice. In vivo immunization (i.v. injection) of H-42b mice with 5 to 30 X 10(6) spleen cells (SC) bearing allogeneic H-42a antigen but carrying H-2 complex (mouse MHC) matched with the H-42b mice failed to prime anti-H-42a CTL but induced stable and specific anti-H-42a CTL unresponsiveness, i.e., tolerance, in the H-42b recipient mice. In contrast, H-2 heterozygous H-42b F1 mice injected with SC bearing H-42a alloantigen on either of the parental H-2 haplotypes were effectively primed to generate anti-H-42a CTL. Exploration of the region or subregion in the H-2 complex of H-42a donor SC that should be compatible with H-42b recipient mice for the induction of their anti-H-42a CTL tolerance demonstrated that the compatibility at I region, most probably I-A subregion, but not at K, S, or D region, determined the induction of the tolerance. MHC class II compatible H-42a skin graft (SG) to H-42b mice, however, consistently primed the anti-H-42a CTL in the H-42b recipients. These results were discussed in several aspects, including uniqueness of MHC class II control on the CTL response to minor H-42a antigen, possibility of inactivation of responding anti-H-42a precursor CTL or helper T cells in H-42b mice by encountering the veto cells present in MHC class II-matched H-42a SC population, and significance of the present observations as a mechanism of CTL tolerance to self-components.     

Autoimmune syndrome after induction of neonatal tolerance to alloantigens: effects of in vivo treatment with anti-T cell subset monoclonal antibodies

BALB/c (H-2d) mice rendered tolerant to h-2b alloantigens by neonatal injection of semiallogeneic (C57BL/6 X BALB/c)F1 spleen cells develop autoimmune features due to an abnormal activation of persisting F1 donor B cells. The role of T cells in this autoimmune syndrome was studied by in vivo treatment of tolerant mice with anti-L3T4(GK-1.5) or anti-Ly-2 (H-35-17.2) monoclonal antibodies. The treatment of tolerant mice from day 2 to day 21 of life with anti-L3T4 MAb completely prevented the occurrence of circulating immune complexes of anti-ssDNA anti-Sm and anti-hapten (FITC) IgG antibodies as well as the glomerular deposition of Ig that were usually seen in untreated tolerant mice. This effect persisted for at least 6 wk after stopping this treatment. When the injections of anti-L3T4 MAb were delayed until day 15 of life, a very significant decrease of the autoimmune manifestations was still observed. Treatment of tolerant mice with anti-Ly-2 MAb during the same period had no effects on the autoimmune disease as compared with untreated tolerant mice. No effects on the maintenance of tolerance vs H-2b alloantigens were observed after treatment with anti-L3T4 MAb, as followed by the decrease of CTL and CTL-p alloreactivity and by the persistence of F1 donor B cells, indicated by the presence of Ig bearing the Ighb donor allotype. These results suggest the existence of interactions between L3T4+ T cells and persisting autoreactive B cells from F1 donor origin in the development of the autoimmune syndrome after neonatal induction of transplantation tolerance.     

Cytotoxic T lymphocyte response to minor H-43a alloantigen in H-43b mice. Privileged H-2Kb restriction to the response is not due to immunodominance or epistatic effect but due to Ir gene function of H-2Kb itself

Previous study demonstrated that anti-H-43a cytotoxic T lymphocyte (CTL) response of H-43b CWB (H-2b) stain carrying non-major histocompatability complex (MHC) genes of C3H and F1 strains raised by crossing CWB with various H-43b strains was restricted exclusively by self H-2Kb (Kb). In the present study, newly produced C3W strain (H-2k, H-43b), which is H-43-congenic to C3H/HeN (H-2k, H-43a), was used as H-43b mice, and possibility of immunodominance of Kb was examined. No anti-H-43a CTL response could be induced in C3W strain and F1 strains raised by crossing C3W with other H-43b strains not carrying Kb. Thus, the possibility of immunodominance of Kb over the other MHC class I alleles could not be supported. We also examined possibility of epistatic effect of I region genes and non-MHC genes on the Kb restriction. (C3W x C57BL/6)F1(I-Ak/b) and (C3W x B6.CH-2bm12)F1(I-Ak/bm12)mice showed equally anti-H-43a CTL response restricted exclusively by self Kb, and (C3W x B10.MBR)F1(Ik/k) mice also showed anti-H-43a CTL response restricted solely by self Kb. Cold target competition experiments demonstrated that H-43b C57BL/10 or A.BY mice, which do not have non-MHC genes of C3H mounted anti-H-43a CTL response restricted solely by self Kb. Thus, no relation of I region genes or non-MHC genes to the Kb restriction was shown. All the results indicate that H-43b mouse strains, including F1, can not achieve anti-H-43a CTL response unless they carry Kb allele. Notably, (C3W x C57BL/6)F1 mice mounted self Kb-restricted anti-H-43a CTL response, whereas (C3W x B6.CH-2bm1)F1 mice carrying mutated Kb could not mount anti-H-43a CTL response at all. These findings indicate strongly that Kb itself is classical Ir gene of anti-H-43a CTL response and directs self Kb restriction of the response.     

Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus. I. Generation and recognition of virus strains and H-2b mutants

Cytotoxic T lymphocytes (CTL) were induced in C57BL/6 and (C57BL/6 X DBA/2)F1 mice after immunization with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV-Arm) and were cloned by limiting dilution in vitro. The cytotoxic activity of these clones was LCMV specific and H-2 restricted. All clones induced in C57BL/6 (H-2b) mice with LCMV-Arm lysed target cells infected with each of five distinct strains of LCMV (Arm, Traub , WE, Pasteur, and UBC ), suggesting recognition of common regions of viral proteins in association with H-2b molecules. In contrast, one clone obtained from (B6 X D2)F1 mice and restricted to the H-2d haplotype only lysed cells infected with one of three strains of virus (Arm, Traub , WE) but not two others (Pasteur, UBC ), suggesting recognition of variable regions of viral proteins in the context of H-2d molecules. To assess the fine specificity for H-2 molecules, we tested H-2Kb-restricted CTL clones for their ability to kill LCMV-infected target cells bearing mutations in their H-2Kb, and we tested clones presumed to be restricted to the H-2Db region for their ability to all LCMV targets cells bearing a mutation in the H-2Db region. Several different patterns of killing of the mutant targets were observed, indicating that a number of different epitopes on the H-2b molecules were used as restricting determinants for LCMV antigen recognition by CTL. Thus, cross-reactive viral determinants were recognized in the context of several different restricting determinants. Mutations in the N or C1 domains of the H-2 molecule affected recognition by a single LCMV specific CTL clone. One implication of this result is that CTL recognize a conformational determinant on the H-2 molecule formed by the association of virus antigen(s) with H-2. An alternate explanation is that one site on the H-2 molecule is involved in the interaction of viral antigens with H-2, whereas another may serve as a binding site for the CTL receptor.     

Cell surface expression of cytotoxic T lymphocyte-defined, AKR/Gross leukemia virus-associated tumor antigens by normal AKR.H-2b splenic B cells

We previously described a system in which H-2Kb-restricted C57BL/6 (B6) cytotoxic T lymphocytes (CTL) could be raised that were specific for tumors, such as the thymic lymphoma AKR.H-2b SL1, that were induced by endogenous AKR/Gross murine leukemia virus and that expressed the Gross cell surface antigen. In this study, certain normal lymphoid cells from AKR.H-2b mice were also found to express target antigens defined by such anti-AKR/Gross virus CTL. AKR.H-2b spleen, but surprisingly not thymus, cells stimulated the production of anti-AKR/Gross virus CTL when employed at either the in vivo priming phase or the in vitro restimulation phase of anti-viral CTL induction. This selective stimulation by spleen vs thymus cells was not dependent on the age of the mice over the range (3 to 28 wk) tested. Both AKR.H-2b spleen and thymus cells, however, were able to stimulate the generation of H-2-restricted B6 anti-AKR minor histocompatibility (H) antigen-specific CTL. Thus, AKR.H-2b spleen cells appeared to display the same sets (minor H and virus-associated) of cell surface antigens recognized by CTL as the AKR.H-2b SL1 tumor, whereas AKR.H-2b thymocytes were selectively missing the virus-associated target antigens, a situation analogous to that of cl. 18-5, a variant subclone of AKR.H-2b SL1 insusceptible to anti-AKR/Gross virus CTL. Like AKR.H-2b thymocytes, neither AKR spleen cells or thymocytes nor B6.GIX + thymocytes were able to stimulate the generation of anti-AKR/Gross virus CTL from primed B6 responder cell populations. In contrast, both T cell-enriched and B cell-enriched preparations derived from AKR.H-2b spleen cells were able to stimulate at the in vitro phase of induction, although B cell-enriched preparations were considerably more efficient. The discordant results obtained with AKR.H-2b spleen cells vs thymocytes were confirmed and extended in experiments in which these cells were employed as target cells to directly assess the cell surface expression of virus-associated, CTL-defined antigens. Thus, AKR.H-2b spleen cells, but not thymocytes, were recognized by anti-AKR/Gross virus CTL when fresh normal cells were tested as unlabeled competitive inhibitors, or when mitogen blasts were tested as labeled targets. Fresh or lipopolysaccharide-stimulated B cell-enriched spleen cells were as efficiently recognized as unseparated spleen cell preparations. Unexpectedly, fresh or Lens culinaris hemagglutinin-stimulated T cell-enriched spleen cell preparations, although susceptible to anti-minor H CTL, were almost as poor as targets for anti-viral CTL as were thymocytes. Together, these results demonstrate the H-2-restricted expression of CTL-defined, endogenous, AKR/Gross virus-associated target antigens by normal AKR.H-2b splenic B cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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

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

Genetic control of the induction of cytolytic T lymphocyte responses to AKR/Gross viral leukemias. II. Negative control by the Fv-1 locus in AKR mice of responder H-2b haplotype

To assess whether the presence of a responder H-2b haplotype would be sufficient to allow mice of nonresponder "high leukemic" phenotype to generate syngeneic anti-AKR/Gross virus cytolytic T lymphocytes (CTL), the AKR.H-2b strain was examined. Although capable of mounting vigorous apparent anti-minor histocompatibility-specific CTL responses, AKR.H-2b mice failed to produce anti-viral CTL after a variety of stimulation protocols. In contrast, the "doubly congenic" AKR.H-2b:Fv-1b strain was able to respond with substantial levels of H-2-restricted anti-AKR/Gross virus CTL activity. These results indicated that Fv-1n alleles could exert negative epistatic control over responder H-2b-encoded gene(s). Because the B6.Fv-1n congenic was also able to generate anti-viral CTL indistinguishable from the prototype B6 strain, however, it was apparent that other genes of AKR background were required for the Fv-1n-mediated inhibition in AKR.H-2b mice. The mechanism by which Fv-1 intereacted with other genes to override positive H-2b control appeared to be related to the expression of the CTL-defined, virus-associated antigens by normal AKR.H-2b cells. Thus, AKR.H-2b spleen cells but not thymus cells were able to stimulate the production of B6 anti-AKR/Gross virus CTL and were recognized as target cells by such anti-viral CTL. In contrast, both spleen cells and thymocytes from AKR.H-2b:Fv-1b mice were negative when tested as stimulator or target cells in these assays. In addition, AKR.H-2b but not AKR.H-2b:Fv-1b spleen cells were shown to display serologically defined gp70 determinants and the Gross cell surface antigen. Taking these data together, it appeared that the inhibition of anti-viral CTL responsiveness might be due to tolerance induced by the cell surface expression of virus-associated antigens by normal AKR.H-2b cells. Widespread display of viral antigens, in turn, may have been due to the permissive effects of Fv-1n on the spread of the early arising N-ecotropic, endogenous AKR leukemia virus controlled by other background genes. In this context, the implications of the multi-gene control of anti-AKR/Gross virus CTL production are discussed with respect to the induction of spontaneous leukemia in the high incidence AKR strain.     

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.