Specificity of anti-H-2 class I antibodies induced by syngeneic immunization with Sendai virus-treated cells is regulated by the mouse MHC and viral antigens. No evidence for MHC-restricted virus-specific antibodies

In a previous study, we searched for Sendai virus (SV)-specific antibodies that were restricted in their binding by self-major histocompatability complex (MHC) antigens. In C57BL/6 (B6; H-2b) mice, most of the sera obtained after i.p. injections with syngeneic SV-coated (SV+) spleen cells contained auto- and alloreactive lymphocytotoxic antibodies directed against H-2 class I molecules, but no viral-specific, MHC-restricted antibodies. Here we report that syngeneic immunization with SV+ cells regularly induced H-2-specific antibodies in various mouse strains. From a total of 12 strains tested, only the B10.S (H-2s) strain appeared to be a low responder. The immune responses are of two types: (i) mice of some strains produce autoreactive antibodies and a broad variety of alloreactive antibodies; and (ii) mice of some strains produce only narrow or widely alloreactive antibodies. Because most of the strains differ only in the H-2 region, the patterns observed are regulated by the MHC. To locate the genes involved in the induction of H-2-specific antibodies more precisely, two B6 mutant strains, bm1 (Kb mutant) and bm13 (Db mutant), were immunized with syngeneic SV+ cells. The results suggest that the H-2Db region plays an important role in the induction and specificity of the lymphocytotoxic H-2 class I-specific antibodies present in sera of H-2b mice after syngeneic immunization with SV+ cells. The role of SV in the induction of H-2-specific antibodies was studied in B6 mice after injections of syngeneic cells coated with liposomes bearing the F and HN proteins of SV. The results suggest that SV surface glycoproteins as well as internal proteins are directly involved in regulating the specificity of anti-H-2 antibodies present in sera after syngeneic immunization with SV+ cells. This study does not support the concept that antigen-specific, MHC-restricted antibodies are a part of the B-cell repertoire.     

SPECIFICITY OF ANTI-H-2 CLASS I ANTIBODIES INDUCED BY SYNGENEIC IMMUNIZATION WITH SENDAI VIRUS-TREATED CELLS IS REGULATED BY THE MOUSE MHC AND VIRAL ANTIGENS. NO EVIDENCE FOR MHC-RESTRICTED VIRUS- SPECIFIC ANTIBODIES

In a previous study, we searched for Sendai virus (SV)-specific antibodies that were restricted in their binding by self-major histocompatability complex (MHC) antigens. In C57BL/6 (B6; H-2^b) mice, most of the sera obtained after i.p. injections with syngeneic SV-coated (SV⁺) spleen cells contained auto- and alloreactive lymphocytotoxic antibodies directed against H-2 class I molecules, but no viral-specific, MHC-restricted antibodies. Here we report that syngeneic immunization with SV⁺ cells regularly induced H-2-specific antibodies in various mouse strains. From a total of 12 strains tested, only the B10.S (H-2^s) strain appeared to be a low responder. The immune responses are of two types: (i) mice of some strains produce autoreactive antibodies and a broad variety of alloreactive antibodies; and (ii) mice of some strains produce only narrow or widely alloreactive antibodies. Because most of the strains differ only in the H-2 region, the patterns observed are regulated by the MHC. To locate the genes involved in the induction of H-2-specific antibodies more precisely, two B6 mutant strains, bml (K^b mutant) and bm13 (D^b mutant), were immunized with syngeneic SV⁺ cells. The results suggest that the H-2D^b region plays an important role in the induction and specificity of the lymphocytotoxic H-2 class I-specific antibodies present in sera of H-2^b mice after syngeneic immunization with SV⁺ cells.     

Generation of virus specific cytotoxic T cells in vitro. III. Spleen cells stimulated by viral antigens generate alloreactive cytotoxic T cells.

Cytotoxic T lymphocytes (CTL) from DBA/2 strain mice primed with Sendai virus (SV) in vivo were activated by secondary stimulation of spleen cells with viral antigens in vitro and analyzed for their target antigen specificity. These effector cells lysed syngeneic Sendai virus infected target cells, marginally a variety of non-infected targets and had a strong cytotoxic effect on H-2b targets. Studies on the antigenic requirements revealed that all SV preparations which generated specific CTL also induced the alloreactive populations. Similar results were found in the response to Newcastle disease virus (NDV) and some influenza A viruses; all these viruses were mitogenic for lymphocytes. Experiments on the cellular requirements indicated that virus specific and alloreactive cells can be separated by their requirements for help and for restimulation. By competition experiments both activities could be attributed to clearly separable T cell subpopulations. The induction mechanism of alloreactive T cells by viral antigens is discussed.     

H-2 influence on the production of real but not of apparent H-2-specific antibodies induced by the association of syngeneic class I heavy chains with bovine beta 2-microglobulin.

Immunogenic properties of class I molecules resulting from the association of mouse class I heavy chains with a xenogeneic beta 2-microglobulin (beta 2-m) were investigated by studying the antibody response of mice of injections to syngeneic Con A lymphoblasts, induced in conditions allowing the replacement of endogenous beta 2-m by exogenously added bovine beta 2-m provided by fetal calf serum (FCS-Con A blasts). Lymphocytotoxic antibodies were regularly produced and according to their specificities they could be divided into two types: antibodies whose reactivity was (1) dependent on and (2) independent of the presence of bovine beta 2-m on target cells. Although both types displayed an H-2 dependent polymorphic reaction pattern, only antibodies recognizing class I molecules without bovine beta 2-m can be considered as real H-2-specific antibodies. The others are only apparent H-2-specific antibodies: their polymorphic reaction pattern is dependent both on the presence of bovine beta 2-m on the surface of target cells and on their H-2 haplotype. A comparison of the antibody response of mice with various H-2 haplotypes to injections of syngeneic FCS-Con A blasts showed no significant difference in the induction of bovine beta 2-m-dependent antibodies (apparent H-2-specific) among the mice from all strains tested (H-2b, H-2p, H-2q, and H-2s). Unexpectedly, for most strains more than 60% of the immunized mice produced also beta 2-m-independent antibodies (real H-2-specific), with the exception of H-2q mice, in which only 30% of sera were positive. The real H-2-specific antibody response is of two types: some mice (H-2p and H-2s) produced antibodies only reactive with allogeneic target cells whereas others (H-2b and H-2q) produced in addition antibodies that were reactive with syngeneic cells. Thus H-2 appears to play an important role in the induction and specificity of the lymphocytotoxic H-2-specific antibodies induced upon immunization with cells expressing syngeneic class I heavy chains associated with bovine beta 2-m.     

Association between H-2 antigens and thyroglobulin influences the immunogenicity of thyroglobulin in mice

To ascertain the role of H-2 in the immunogenicity of thyroglobulin, congenic strains of mice, B10.BR (H-2k, high-responder) and B10.D2 (H-2d, low-responder) were immunized with purified thyroglobulin (from B10.BR and B10.D2) which was or was not passed through affinity columns of Sepharose coupled to anti-H-2k or anti-H-2d sera. Thyroglobulin absorbed with the same anti-H-2 as its source did not induce thyroglobulin antibodies or thyroid infiltrates in mice of the same strain and induced thyroid lesions but no antibodies in mice of the opposite strain. These experiments suggest that thyroglobulin, an autoantigen, is associated with syngeneic histocompatibility antigens in vivo and this association is important for the (auto) antigenicity of thyroglobulin.     

Association Between H-2 Antigens and Thyroglobulin Influences the Immunogenicity of Thyroglobulin in Mice

To ascertain the role of H-2 in the immunogenicity of thyroglobulin, congenic strains of mice, B10.BR (H-2^khigh-responder) and B10.02 (H-2^d, low-responder) were immunized with purified thyroglobulin (from B10.BR and B10.D2) which was or was not passed through affinity columns of Sepharose coupled to anti-H-2^k or anti-H-2^d sera. Thyroglobulin absorbed with the same anti-H-2 as its source did not induce thyroglobulin antibodies or thyroid infiltrates in mice of the same strain and induced thyroid lesions but no antibodies in mice of the opposite strain. These experiments suggest that thyroglobulin, an autoantigen, is associated with syngeneic histocompatibility antigens in vivo and this association is important for the (auto) antigenicity of thyroglobulin.     

Cell-mediated cytotoxicity against Sendai-virus-infected cells.

After injection of Sendai virus, a parainfluenza virus type I, mice generate cytotoxic lymphocytes which lyse specifically Sendai-virus-infected target cells in vitro. Their action is not inhibited by specific antibody in vitro. Killer cell activity appears 4 days after infection, reaches a maximum on the 7th day and disappears on the 14th to 16th day. Decrease of cytotoxic cell activity is correlated with an increase of haemagglutinating antibodies. The cytotoxic effector cell could be characterized as a thymus-derived cell, there is no specific activity in antibody-dependent cell-mediated cytolysis (ADCC). The degree of cytotoxic effector cell activity is only slightly influenced by the dose of injected infective virus. Using different syngeneic Sendai-virus-infected cells as targets for cell-mediated cytotoxicity, a tumor line was not lysed by cytotoxic lymphocytes in spite of viral surface antigens. Preliminary experiments were performed to demonstrate the H-2 gene restriction of the cytotoxic interaction. Using macrophages and tumor cells as targets only syngeneic infected target cells were lysed.     

Association Between H-2 Antigens and Thyroglobulin Influences the Immunogenicity of Thyroglobulin in Mice

To ascertain the role of H-2 in the immunogenicity of thyroglobulin, congenic strains of mice, B10.BR (H-2^khigh-responder) and B10.02 (H-2^d, low-responder) were immunized with purified thyroglobulin (from B10.BR and B10.D2) which was or was not passed through affinity columns of Sepharose coupled to anti-H-2^k or anti-H-2^d sera. Thyroglobulin absorbed with the same anti-H-2 as its source did not induce thyroglobulin antibodies or thyroid infiltrates in mice of the same strain and induced thyroid lesions but no antibodies in mice of the opposite strain. These experiments suggest that thyroglobulin, an autoantigen, is associated with syngeneic histocompatibility antigens in vivo and this association is important for the (auto) antigenicity of thyroglobulin.     

Shared determinants between virus-infected and trinitrophenyl-conjugated H-2-identical target cells detected in cell-mediated lympholysis.

Infection of H-2-identical mice with either lymphocytic choriomeningitis (LCM) virus, vaccinia virus, or paramyxo (Sendai) virus resulted in the generation of specifically sensitized cytotoxic T lymphocytes (CTL). CTL generated in vitro against 2,4,6-trinitrophenyl (TNP)-conjugated syngeneic stimulator cells were specifically cytotoxic for TNP-conjugated H-2K (D) region identical targets. Both LCM and vaccinia-induced CTL, however, were found to be strongly cytotoxic towards TNP-conjugated, H-2K(D) region-identical target cells. In contrast, Sendai virus-induced CTL did not lyse TNP-conjugated, syngeneic target cells. Inhibition experiments using cold targets suggested that shared antigenic determinants can be detected on either LCM virus-infected and TNP-conjugated targets, which are not present on the cell surface of normal target cells.     

Impaired H-2 expression in B16 melanoma variants

We studied the expression of H-2b alloantigens in three different B16 melanoma lines cultures in vitro. Cell lines were B16-F1 and two cell cultures (named B16-A and B16-B) newly derived from two different in vivo sublines of B16 melanoma. The assays used were in vivo tumour growth in allogeneic (BALB/c and B10.BR) as compared to syngeneic mice, in vitro cell-mediated cytotoxicity by anti-H-2b immune lymphocytes and absorption of anti-H-2b antisera activity. The B16-F1 line was able to efficiently kill allogeneic hosts, could not be lysed by anti-H-2b cytotoxic effectors and did not express any serologically detectable amount of H-2b alloantigens. The B16-A line was H-2 positive during the early in vitro passages, then, at the 8th-10th passages, it acquired the capacity to kill allogeneic hosts, lost the sensitivity to anti-H-2b cytotoxic effectors and the H-2Kb antigens became undetectable The expression of H-2Db was reduced, although at a lower degree. Similar data were obtained with B16-B cells, which after 10 in vitro passages grew and killed allogeneic hosts, showed a decreased sensitivity to cytotoxic anti-H-2b effectors and a very low expression of the K region antigens. The results indicate that H-2 expression is altered in B16 melanoma lines and this may influence the different metastatic capacity of such cells.     

Alloantigen-specific regulation of cytotoxic T cell responses is mediated through the induction of clonal anergy of CD8+ T cells.

Priming mice with an alloantigen before immunization with this same alloantigen presented in association with a second one on an F1 stimulator cell inhibits the induction of cytotoxic response directed against the second alloantigen. This inhibition is associated with the induction of a strong cytotoxic T lymphocyte (CTL) response against the first priming alloantigen. For example, a specific suppression of anti-H-2b CTL responses could be induced in C3H/He mice (H-2k) by priming them with H-2d spleen cells before immunization with F1 (H-2dxb) spleen cells. In the present study, we have analyzed the mechanisms underlying this specific suppression of CTL responses. We have demonstrated that the reduction of H-2b-specific CTL responses is reflected by a decrease in the frequency of effector cells specific for H-2b antigen. However, there was no difference in the frequencies of precursor CTL in control and suppressed mice excluding clonal deletion as the mechanism maintaining low responsiveness. Co-culture experiments have shown that the suppression of anti-H-2b CTL responses was not due to suppressor cells but to the failure of CD8+ T cells of suppressed mice to collaborate with normal helper CD4+ T cells. The suppression was therefore ascribed to a functional impairment (clonal anergy) of the CD8+ T cell subset.     

Restricted blocking of cytotoxic T-cell function by anti-H-2K/D antibodies

Antibodies specific for H-2K and H-2D, the murine major histocompatibility complex (MHC)-encoded class I antigens, can block cytotoxic T (Tc)-cell function. Antibodies specific for the Tc cell and not the target cell have been used to map inhibition to the effector cell, suggesting a role for class I antigens in Tc-cell function. These antibody effects have been demonstrated for both alloreactive and MHC-restricted Tc cells, but inhibition has only been revealed by measuring cytotoxicity in a short-term assay. Using the neutral red assay for cytotoxicity, blocking effects evident after a 1.5-hr assay were lost by 2.5 hr. For some Tc-cell responses, only anti-H-2K antibodies have been found to be inhibitory, despite evidence of the expression of both H-2K and H-2D molecules on these cells. Some Tc-cell populations can be blocked by antibodies specific for both the H-2K and H-2D molecules. B10.A(4R) anti-Sendai Tc cells can be inhibited by anti-H-2Kk antibodies, but five different anti-H-2Db antibodies have been ineffective inhibitors. In contrast, B10.A(4R) anti-ectromelia Tc cells can be inhibited very effectively by each of these anti-H-2Db antibodies, as well as by anti-H-2Kk antibodies. Anti-H-2 antibodies also inhibit the function of cloned alloreactive Tc-cell lines such that the inhibitory capacity of antibodies specific for K versus D determinants appears to be consistent and specific for each Tc-cell line. A long-term Tc-cell clone, AR1, has been inhibited specifically by anti-H-2Kb and not anti-H-2Db antibodies, suggesting a clonally 'restricted' phenomenon.     

Damage in B2m genes and DNA methylation of H-2 genes are involved in loss of expression of class I MHC products on the membrane of LR.4, a cell line derivative of the T-cell lymphoma L5178Y.

We have isolated an H-2 deficient cell line (LR.4) from the T-cell lymphoma L5178Y which grew without restrictions in the peritoneal cavity of different inbred strains of mice. The use of polyclonal anti-H-2 antiserum and complement indicated that LR.4 cells did not express class I determinants on the cell membrane. Southern blots of genomic DNA of LR.4 cells showed that B2m genes were severely damaged and that class I H-2 genes were extensively methylated. Consequently, LR.4 cells failed to transcribe mRNAs for both B2m and class I H-2 genes. On the other hand, specific immunity to LR.4 was demonstrated in C57BL/6J mice since, in subsequent challenges with either LR.4 or EL4.4, LR.4 did not grow, whereas EL4.4 grew and killed the mice. In C57BL/6J mice, rejection of LR.4 was accompanied by the production of cytotoxic antibodies. The immune response induced in C57BL/6J mice was determined by non-H-2 antigenic determinants in LR.4 cells.     

Thymic immune response gene function in radiation chimeras reconstituted with purified hemopoietic stem cells

Thymectomized (C57BL/6[B6] X bm1)F1 mice and thymectomized (B6 X bm12)F1 mice were engrafted with neonatal parental thymus of either B6 type [H-2b mouse, Sendai virus cytotoxic T cell (Tc) responder] or bm1 type (H-2Kb mutant, Sendai virus Tc nonresponder) and B6 type (H-Y Tc responder) or bm12 type (H-2 I-Ab mutant, H-Y Tc nonresponder), respectively. All mice were irradiated and reconstituted with highly purified syngeneic pluripotent hemopoietic stem cells. All types of thymus engraftment resulted in a restored T cell immunocompetence. The Tc reaction to Sendai virus in (B6 X bm1)F1 mice engrafted with both responder type B6 and nonresponder, type bm1 neonatal thymus allowed maturation of Sendai-specific, H-2Kb-restricted Tc. For the Tc reaction to H-Y, only responder type B6 thymus restored the Tc response, whereas this was not achieved with nonresponder type bm12 thymuses. We conclude from this study that in this radiation stem cell chimera system the radioresistant component of the thymus dictates major histocompatibility complex (MHC) specificity and immune response phenotype of T cells restricted to class II MHC molecules but not of T cells restricted to class I MHC molecules.     

Immunogenicity of H-2 positive and H-2 negative clones of a mouse tumour, GR9

The GR9 tumour was induced with methylcholanthrene in a BALB/c mouse, adapted to tissue culture and cloned without any passage in vivo GR9 clones were typed for H-2 with three monoclonal antibodies that define H-2Kd + Dd, Kd and Dd antigens. A great heterogeneity of H-2d expression was found from clones which were Kd and Dd positive to clones Kd and Dd negative. These results were confirmed for A7 and B9 clones using immunoprecipitation with anti-H-2D.4 and anti-H-2K.31 alloantisera and SDS-PAGE analysis. In addition, the number of chromosomes per cell was heterogeneous amongst the clones, ranging from 38 +/- 2 to pseudotetraploid clones which have 75 +/- 2 chromosomes. GR9 clones were injected into syngeneic BALB/c mice to measure local tumour growth. We found that the growth correlated with the amount of H-2 antigen expressed, i.e. clones with low H-2d expression were highly malignant while clones with normal H-2d expression were highly immunogenic. Finally we found that BALB/c mice immunized against A7 (Kd, Dd-positive) protected against A7, as expected, but surprisingly also against B9 (Kd, Dd-negative).     

DAMAGE IN B2m GENES AND DNA METHYLATION OF H-2 GENES ARE INVOLVED IN LOSS OF EXPRESSION OF CLASS I MHC PRODUCTS ON THE MEMBRANE OF LR.4, A CELL LINE DERIVATIVE OF THE T-CELL LYMPHOMA L5178Y

We have isolated an H-2 deficient cell line (LR.4) from the T-cell lymphoma L5178Y which grew without restrictions in the peritoneal cavity of different inbred strains of mice. The use of polyclonal anti-H-2 antiserum and complement indicated that LR.4 cells did not express class I determinants on the cell membrane. Southern blots of genomic DNA of LR.4 cells showed that B2m genes were severely damaged and that class I H-2 genes were extensively methylated. Consequently, LR.4 cells failed to transcribe mRNAs for both B2m and class I H-2 genes. On the other hand, specific immunity to LR.4 was demonstrated in C57BL/6J mice since, in subsequent challenges with either LR.4 or EL4.4, LR.4 did not grow, whereas EL4.4 grew and killed the mice. In C57BL/6J mice, rejection of LR.4 was accompanied by the production of cytotoxic antibodies. The immune response induced in C57BL/6J mice was determined by non-H-2 antigenic determinants in LR.4 cells.     

UNEXPECTED LYMPHO-CYTOTOXIC REACTIONS OF ANTI-H-2 SERA ON NORMAL LYMPH-NODE CELLS: ARE THEY DUE TO ALTERED H-2 STRUCTURES OR ANTI-VIRAL ANTIBODIES?

When testing the serum of an individual anti-H-2 immunized mouse (B10 x A.SW)F₁ anti-B10.M by the routine micro-lymphocytotoxicity test on lymph-node cells, unexpected antibodies were found. The most striking finding was that after absorption of anti-H-2.8 antibodies with B10.A(2R) (K^k) cells, antibodies remained which reacted with AKR, B10.AKM and B10.A V + mice while B10.A V-, B10.BR and C3H mice were negative. While all these strains share the K^k allele, only the positively reacting strains express high titres of infectious RNA turnover viruses. Unexpected reactions were observed also with H-2^d, H-2^j and H-2^r cells and absorption experiments indicated two or three antibody populations. These reactions could be interpreted by two different possibilities: (1) anti-H-2 antibodies react with virus-altered H-2 structures; and (2) antiviral antibodies react with H-2 structures complexed with viruses. These possibilities should be taken into account when H-2 sera are tested on tumour or virus-infected cells.     

Ontogeny of the erythrocyte-dependent IgM antibody responses to cell membrane antigens

Our previous experiments characterized the T-cell independent type 2 B-cell responses to cell membrane antigens that are controlled by two donor cell types with different antigen-presenting(AP) activities. We here report about the ontogeny of this novel type of responses with special reference to the mutual relation of the development among two AP activities and their acceptor functions. The responses of mice to H-2^d antigens on allogeneic cells and hapten (fluorescein isothiocyanate) antigens on syngeneic cells were examined in parallel. The positive AP activity displayed by red blood cells(RBC) for induction of antihapten responses was fully developed in the fetus, although H-2^d antigens on the RBC for induction of anti-H-2^d responses was immature in mice under 7 days old. In contrast, the negative AP activity displayed by spleen cells(B cells) for inhibition of the RBC-dependent anti-hapten and anti-H-2^d responses was first developed in mice about 3 weeks old. The B cell functions accepting the positive and negative AP activities were also matured by that time. The possible significance of these findings in the physiology and pathology of the unique responses was discussed.     

Peptide loading of empty major histocompatibility complex molecules on RMA-S cells allows the induction of primary cytotoxic T lymphocyte responses.

The antigen processing-defective mutant cell line RMA-S expresses at the cell surface major histocompatibility complex (MHC) class I molecules devoid of peptide that can be efficiently loaded with exogenous immunogenic peptides. We now report that viral peptide-loaded RMA-S cells, unlike parental RMA cells, can induce primary cytotoxic T lymphocyte (CTL) responses in vitro, in a T helper cell-independent fashion. This was shown for an H-2Kb-binding peptide of Sendai virus nucleoprotein and an H-2Db-binding peptide of adenovirus type 5 E1A protein with responding spleen cells of C57BL/6 mice, the strain of origin of RMA and RMA-S cells. Primary Sendai peptide-induced CTL lyse both peptide-loaded and virus-infected cells. Pre-culture of RMA-S cells at low temperature (22 degrees - 26 degrees C), which increases the amount of empty MHC class I molecules at the cell surface, decreases the peptide concentrations required for the induction of primary CTL responses. Primary peptide-specific CTL responses induced by peptide-loaded RMA-S cells are CD4+ cell- and MHC class II+ cell-independent. CTL response induction is blocked by the presence of anti-CD8 monoclonal antibody during culture. Direct peptide binding studies confirm the efficient loading of empty MHC molecules on RMA-S cells with peptide and show 2.5-fold more peptide bound per RMA-S cell compared to RMA cells. An additional factor explaining the difference in primary response induction between RMA and RMA-S cells is related to the CD8 dependence of these responses. MHC class I molecules occupied with irrelevant peptides (a majority present on RMA, largely absent on RMA-S) may interfere in the interaction of the CD8 molecule with relevant MHC/peptide complexes. The results delineate a novel strategy of peptide based in vitro immunization to elicit CD8+ cytotoxic T cell responses.