Large-scale purification of murine I-Ak and I-Ek antigens and characterization of the purified proteins

Detailed analysis of the role of la antigens in T-cell activation and of the structural characteristics of these molecules will require isolation of relatively large amounts of these antigens in serologically active form. We have purified murine Ia antigens on a large scale by affinity chromatography using monoclonal antibodies coupled to Sepharose 4B. Both I-A^k and I-E^k were isolated by sequential passage of cell lysate over columns prepared using specific monoclonal antibodies. Elution of the bound antigens required high pH (11–12) but, nonetheless, the purified material was 50–75% serologically active. Using LPS-stimulated spleen cells or B-lymphocyte tumor cells as starting material, 0.5 mg of each antigen can be readily purified. Based on antigen yields, it can be estimated that normal B-cells have about the same surface density of Class I and Class II MHC antigens. LPS blasts, in contrast, have normal levels of Class I antigen but 3–5 times higher levels of Class II antigens. We have now purified I-A1' and I-E1' from a number of different cell sources and have noted differences in both the mol. wts of the α- and β-chains and in their apparent associations with cytoskeletal components. Proteins having the same apparent mol. wts as actin and myosin co-purify with both I-A^k and I-E^k antigens from various sources. These proteins do not co-purify with H-2K and D molecules obtained by similar methods, suggesting that Ia antigens may specifically interact with cytoskeletal elements.     

Flow cytometric analysis of specific binding of soluble Ia by I-region restricted alloactivated T cells

An antigen binding assay has been developed for quantitation by flow cytometry of vesicular and soluble Ia binding by alloactivated T cells. Binding of stimulator membrane vesicles was detected by anti-Ly-6.2 or anti-Ia monoclonal antibodies coupled to fluorescent latex beads. Vesicle binding by an I-A^k specific A.TH anti-A.TL T cell line occurred via I-A^k molecules, in that (a) vesicles expressing I-A^k molecules bound much more effectively than vesicles of H-2^b,q strains, and (b) inhibition of H-2^k vesicle binding occurred with anti-I-A^k, but not anti-K^k, anti-E^k, or anti-D^k antibodies. T cell receptor/Ia interactions were directly studied by inhibition of H-2^k vesicle binding by T cells with partially purified Ia glycoproteins. Inhibition of binding occurred via Ia molecules since (a) affinity column partially purified allogeneic I-A^k molecules inhibited binding much more effectively than syngeneic I-A^s molecules and (b) depletion of I-A^k but not E^k molecules in Ia^k containing glycoprotein fractions abrogated the inhibitory effect. The ability of this method to detect specific binding of soluble Ia with antigen activated T cells makes it a useful tool for studying interaction of membrane free major histocompatibility complex (MHC) products with native T cell receptor.     

Cross-reactions of Ia antigens between mouse and man

Murine alloantisera with specificity for the I-region of the MHC were found to cross-react with human B lymphocytes. By using antisera selected for subregion specificity and including monoclonal antibodies, antisera directed to I-E/C^k products were shown to have the greatest cross-reactivity with human cells. In the reciprocal direction, human anti-DRw alloantisera were also found to contain antibodies that reacted with murine B lymphocytes and by using congenic strains this reaction was shown to be MHC restricted and was of the “Ia type.” By studying appropriate intra-H-2 recombinant strains, the reactivity could be mapped to the I-E subregion (especially to I-E^k). In addition, antibodies to the murine I-E^k subregion could specifically block the reaction of the two DRw antisera examined; whereas, anti-I-A^k antisera were without effect. These studies further highlight the unique cross-reactions between two highly polymorphic gene products in mouse and man, and demonstrate that the I-E     

Monoclonal antibodies to hemagglutinin and to H-2 inhibit the cross-reactive cytotoxic T cell populations induced by influenza

The target antigens recognized by the cross-reactive population of cytotoxic T cells on A/USSR/90/77 (H 1N 1) influenza virus-infected cells was studied with monoclonal antibodies to the H-2^k antigen and viral hemagglutinin. The cytotoxic killing of virus- infected cells was differentially inhibited by three monoclonal antibodies to H-2^k. Synergistic inhibition of cytotoxic activity was obtained with monoclonal antibodies to H-2 and to some antigenic determinants on the hemagglutinin melecule of A/USSR but not with others. Since target cells infected with other subtypes of influenza A viruses were not inhibited by any of the monoclonal antibodies to AIUSSR hemagglutinin, it is suggested that the sites recognized by the cross-reactive cytotoxic T cells were satirically inhibited by the anti-A/USSR monoclonal antibodies. The results suggest that at least one of the target antigens recognized by the cross-reactive population of cytotoxic T cells is located on the hemagglutinin molecule. The present observations are in agreement with cytotoxic T cell recognition of closely situated viral and H- 2 determinants.     

Monoclonal antibody detection of carbohydrate-defined and protein-defined H-2Kk antigens

Three different monoclonal anti-H-2K^k antibodies, 27R9, 30R3 and 11-4 were examined for the biochemical nature of the antigenic determinants they recognize. When these were compared on the basis of their sensitivity to pronase and various glycosidases, 27R9 was shown to bind to protein-defined H-2K^k antigens, while 30R3 and 11-4 bound to H-2 antigens defined by carbohydrate. From sugar inhibition studies, and treatments with specific glycosidases, d-mannose appears to be the immunodominant sugar involved in the antigenic site recognized by 30R3, while several sugars, namely sialic acid, d-mannose and α-and β-linked d-galactose would appear to be components of the antigenic site bound by 11-4. The carbohydrate determinants appear to be present on glycolipid molecules, since both the 30R3 and 11-4 antibodies could be inhibited by glycolipid extracts from spleen cells of the appropriate H-2 haplotype, as well as from several other strains of mice previously shown to be cross-reactive targets for these antibodies. This finding is supported by evidence that the molecule carrying the protein-defined antigen is distinct from that carrying the carbohydrate-defined antigens. The results are discussed in the light of current information on the nature of glycolipid Ia antigens, as well as the role of H-2 antigens in T-cell interactions.     

Gene complementation in the T lymphocyte proliferative response to poly(Glu56Lys35Phe9)nFunctional evidence for a restriction element coded for by both the I-A and I-E subregions

The T lymphocyte proliferative response to poly(Glu⁵⁶Lys³⁵Phe⁹)_n (GLΦ) is under the control of two immune response genes, Ir-GLΦ-β and Ir-GLΦ-α, mapping in the I-A and I-E/C subregions of the major histocompatibility complex, respectively. Previous studies have demonstrated that in order to generate a response to GLΦ, both gene products must be expressed in the antigen-presenting cell (APC) but that neither responder allele has to be present in the responding T lymphocyte, provided that the T cell has matured in a responder environment. These results suggested that both gene products function as restricting elements in GLΦ presentation by APC. In this report, we provide further evidence for this model from experiments designed to test histocompatibility restrictions in antigen presentation at the I-E/C subregion. Genetic identity at the I-A subregion between T cells and APC was required for GLΦ presentation. To assess the requirements at I-E/C, B10. A(5R) T cells (I-A^b, I-E^k) primed to GLΦ were stimulated in vitro with GLΦ-pulsed spleen cells from F₁ hybrids between C57BL/10 (B10: I-A^b, I-E^b) which made the cells compatible at I-A, and a variety of B10 congenics bearing other H-2 haplotypes. Although none of the parental spleen cells could present GLΦ to B10.A(5R) T cells, spleen cells from F₁ hybrids between B10 and strains possessing H-2I of k, d, p and r presented GLΦ, whereas hybrids with strains possessing H-2I of f, q and s failed to present. This pattern of complementation for GLΦ presentation could not be explained on the basis of the responder status of the I-E/C donating parental haplotypes nor by invoking inhibitory stimuli from mixed lymphocyte reactions induced by the F_l APC. Rather, the pattern correlated with the presence of the serologic marker Ia.7 coded for by the I-E subregion of the complementing parental haplotype and the possession of an I-E-encoded a chain which has been shown by peptide mapping to be very similar in strains bearing the k, d, p and r haplotypes. These results suggest that the restriction element involved in the presentation of GLΦ to B10.A(5R) T cells is composed of a β chain encoded in I-A^b and an a chain encoded in I-E for which the allelic products of the k, d, p and r haplotypes are functionally equivalent. This correlation between structure and function represents the strongest evidence so far that Ia antigen-bearing molecules are the Ir gene products.     

Lympho-stromal interactions in the thymus: medullary thymocytes react with I-A determinants on autochthonous thymic stimulator cells

Primary (1^o) and secondary (2^o) syngeneic mixed lymphocyte culture (SMLC) reactions in autochthonous murine thymus cell cultures (peanut agglutinin-unreactive) or syngeneic mixed thymus-spleen cell cultures were studied. The 1^o thymic SMLC responder cells were Thy-1⁺, Lyt-1⁺, Lyt-2^?. The 1^o thymic syngeneic mixed lymphocyte reaction (SMLR) could be inhibited with monoclonal antibodies directed against determinants Ia.m1 and Ia.m6, in the absence of complement. Positive selection of thymic SMLC cells from 1^o cultures was achieved by enriching reactive lymphoblasts on discontinuous Percoll density gradients. The 2^o memory populations were obtained by allowing the selected lymphoblasts to revert back to small resting 2^o cells in the absence of SMLC stimulation. The thymic 2^o SMLC populations were highly enriched in their activity and were almost exclusively specific for self stimulator cells. The 2^o thymic SMLC cells were found to be Thy-1⁺, membrane immunoglobulin-negative, H-2K/D⁺, I-A^?, I-E^? and I-J^?. They were hardly lysed by anti-Lyt-1 antibodies and not at all lysed by monoclonal antibodies detecting Qat-4 and 5, and Lyt-2 antigens, in the presence of complement. Stimulator cells were found in fast-sedimenting or low-density fractions of thymus. They expressed H-2K/D, I-A-, and I-E antigens but lacked membrane immunoglobulin and Thy-1. Not all I region determinants seemed to be of equal importance in the 2^o thymic SMLC: I-A subregion compatibility between responding and stimulating cells was a minimal requirement for the induction of 2^o thymic SMLC responses. Although several relevant anti I-region antibodies reacted with the stimulator cells in the presence of C, these and other anti-Ia antibodies varied considerably in their capacity to block 1^o and 2^o autochthonous thymic SMLR in the absence of complement. Our finding that the thymus contains the responder as well as the stimulator cells capable of interacting in an autochthonous proliferative reaction will be discussed with regard to its implications for the intrathymic differentiation of T lymphocytes.     

Radioautographic Analysis of Surface Markers on Decidual Cells Shared by Cells of the Lymphomyeloid Tissues

ABSTRACT: Stromal type decidual cells recovered from the murine decidua by a mild collagenase dispersion procedure contain immunoregulatory cells whose ultimate precursors may originate from the bone marrow. To explore the familial relationship of these cells with other cells of the immune system, a battery of cell surface markers recognized on lymphomyeloid cells were examined and quantitated at the morphological level on typical stromal type decidual cells of the dispersed CBA mouse decidua at 8–14 days of syngeneic pregnancy, using a sensitive radioautographic technique. Cells were either labeled directly by exposure to ¹²⁵I-labeled monoclonal antibodies against Thy-1, Mac-1, or Lyt antigens or indirectly by a sequential exposure to monoclonal anti-I-A^k (Ia. 17) or monospecific anti-I-J^k antibodies and ¹²⁵I-labeled Protein A. Decidual cells were found to be Thy-1± (13–73% positive, the incidence rising with advancing gestation in the decidua basalis), Mac-1 ± (present on 6–11% on day 8 and 17–32% on day 12), I-A^?, 1-J^?, and Lyt^?. Macrophages within the decidua were Thy-1^?, Lyt^?, Mac-1 ⁺, and I-A± (present on 5–61% of cells, the incidence rising with advancing gestation). These surface properties of decidual cells along with the presence of FcR, an absence of C₃R, and the presence of a unique marker Dec-1 reported by us earlier, distinguish them from most other stromal type immunoregulatory cells such as Langerhans cells (I-A⁺, FcR⁺, C₃R⁺, Mac-1^?, Thy-1^?), follicular dendritic cells (I-A⁺, FcR⁺, Thy-1^?, Mac-1^?, Dec-1^?), and dendritic reticular cells (I-A⁺, FcR^?, C₃R^?, Thy-1^?, Lyt^?, Mac-1). However, a substantial subpopulation of decidual cells share a few properties (1-A^?,Thy-1⁺) with another marrow derived stromal type cell in the epidermis termed as epidermal dendritic cells that remain to be investigated for other surface markers such as Mac-1, Dec-1, and FcR.     

Inhibition of killer-target cell interaction by monoclonal anti-H-2 antibodies.

The sites on target cells with which cytotoxic T lymphocytes interact were characterized using three different monoclonal BALB/c anti-CBA antibodies derived from plasma cell hybrids. The antibodies all reacted with H-2K^k and appeared to recognize public specificities H-2.5,11 and 25, respectively. Allogeneic killing directed at products of H-2K^k was inhibited by all three antibodies, irrespective of the H-2 haplotype of the responder; cytotoxicity directed at products of another allele, H-2K^d, or of the H-2 D region on the same target cell was not affected. The antibodies did not inhibit killer cells carrying H-2K^k. Cytotoxic reactions against minor histocompatibility antigens, including the male-specific antigen H-Y, were also blocked by all three monoclonal antibodies when restricted by H-2K^k, but not when restricted by H-2D on the same target cell. Cytotoxic T lymphocytes thus appear to interact with their target via the same, serologically defined H-2K^k molecule which carries public specificities, whether they recognize it as an alloantigen or as self. This argues against the existence of separate H-2 K-encoded molecules recognized by killer cells only and against H-2 specific modifications of minor histocompatibility antigens as the basis of H-2 restriction. One of the antibodies, 27 R9, which reacted with H-2K^k and H-2′ and was thought to recognize specificity H-2.25, showed a weak cytotoxic reaction but bound with a high titer to H-2 D^k, a reaction that has not previously been described. This antibody selectively and with a very high titer inhibited male-specific cytotoxicity restricted by H-2D^k, but did not significantly interfere with allogeneic killing against products of the H-2 D^k region nor apparently with H-2D^k- restricted cytotoxicity specific for other minor antigens. The results suggest the existence of at least two different restriction elements controlled by the H-2D^k region.     

Immune recognition of insulin by H-2b mice: the mutation in the I-A gene of the B6.C-H-2bm12 mouse alters the self-I-A-restricted T cell repertoire

The response to heterologous insulin in H-2^b mice is restricted to the A chain loop determinant(s) of beef insulin. The recognition of this specificity requires the expression of the immune response (Ir) gene epitope Ia. W39 which is absent from the I-A^b mutant B6.C-H-2^bm12 (bm12) mice. This restriction could reflect the inability of H-2^b antigen-presenting cells (APC) to present other insulin determinants or may reflect “self-major histocompatibility complex”-dependent influences on the generation of the T cell repertoire. To assess these possibilities we analyzed the genetic control and fine specificity of the insulin-specific T cell repertoire of H-2^b mice by fusing the AKR thymoma BW5147 with T cells of C57BL/6 mice which had been immunized in vivo and challenged in vitro with beef insulin. The cloned hybridomas that we have produced respond to APC either alone or in conjunction with insulin by the production of interleukin 2. The insulin-specific hybridomas vary in their fine specificity such that some clones recognize a determinant(s) shared by beef, sheep and pork insulin and the isolated B chain, while other clones recognize a determinant(s) shared by beef and sheep insulin only, likely to involve amino acids 8 and/or 10 of the A chain loop. The presentation of insulin to these hybridomas is restricted by I-A^b, but not by Ia.W39. This analysis revealed that the insulin-specific immune potential in H-2^b mice is of greater scope than previously defined and led us to consider, whether insulin nonresponder bm12 mice also possess a latent insulin-specific immune potential. Our study of the insulin-specific immune recognition by bm12 mice shows that these nonresponders do possess insulin-specific T cell clones. Despite the fact that the I-A^b and I-A^bm12 gene products differ only by 3 amino acids, insulin-specific C57BL/6 and bm12 hybridomas are restricted to recognize exogenous antigen only in the context of C57BL/6 and bm12 APC, respectively. Furthermore, upon direct analysis of autoreactive subclones, a similar although not complete, restriction was observed. The implications of these findings for understanding the mechanism of Ir gene control are discussed.     

A promiscuous T cell hybridoma restricted to various I-A molecules

In a previous study, we identified T cell receptor and major histocompatibility complex (MHC) contact sites on the pigeon cytochrome c p43-58 peptide. Positions 46 and 54 of p43-58 were shown to be the MHC-binding sites. Specific amino acids were identified on the MHC-binding sites which bound to the relevant I-A molecule. In the present study, using NOD (I-A^g7) mice, we established a T cell hybridoma, NOE33-1-2, specific for a p43-58 analog 46R50E54A with arginine (R) and alanine (A) at positions 46 and 54, respectively. Interestingly, NOE 33-1-2 recognized 46R50E54A in the presence of not only I-A^g7, but also I-A^{d, s, u and v}. In contrast to previous reports that promiscuous T cells were able to recognize peptide antigens with various HLA-DR or I-E molecules consist of monomorphic α and polymorphic β chains, the promiscuous T cell clone NOE33-1-2 recognized peptides with various I-A molecules lacking the monomorphic chain.     

Some fixation reagents reduce or abolish the detectability of Ia-antigen and HLA-DR on cells

The effect of paraformaldehyde (PF), glutaraldehyde (GT), methanol (ME), ethanol (ET) and acetone (AC) fixation on the detectability of Ia antigens on murine and rat peritoneal exudate (PE) and resident peritoneal (RP) macrophages (MØ), and on detectability of HLA-DR antigens on human blood leukocytes (HBL) and human splenic MØ (HSMØ) was examined. Ia-antigen on MØ from H-2^k mice was detected by a rosetting assay using erythrocytes (E) to which a monoclonal antibody (MoAb) reactive to Ia.2 (E anti-Ia.2) had been coupled, and by the direct binding of ¹²⁵I-labeled anti-Ia.2. The antigen was detected on Wistar/Furth (W/Fu) rat RPMØ and splenocytes (SC) by rosetting with E coupled with a MoAb to the murine determinate Ia.17, which cross-reacts with an Ia-like molecule on cells from the W/Fu strain. HLA-DR framework determinants were detected on HBL and HSMØ by the binding of ¹²⁵I-labeled MoAb and by an avidin-biotinylated peroxidase procedure. Exposure of murine PEMØ or RPMØ to 1% PF or 0.5% GT for 15 min at room temperature reduced ¹²⁵I-anti-Ia.2 binding and E anti-Ia.2 rosetting by at least 60%; the radioimmunoassay was more affected by the fixatives than was the rosetting assay. Further, PEMØ were more sensitive to the effect of PF fixation than were RPMØ. Treatment of freshly isolated RPMØ with 1% PF reduced the proportion of Ia-bearing cells detected by the rosetting assay by > 50%. Culturing alone did not affect the detectability of Ia on RPMØ as assessed by the rosetting test, but cultured RPMØ were more sensitive to the effects of PF fixation than fresh cells except when lymphokine from Con A-stimulated murine SC was included in the culture medium. Similar losses of HLA-DR were recorded when HBL and HSMØ were exposed to PF, GT, ME or ET, but brief (< 20 s) treatment with cold AC did not appreciably reduce antigen detectability. Procedures in which fixation takes place after the primary antibody binding step did not result in an appreciable loss of detectable Ia. Thus, commonly used fixatives affect the detectability of Ia and Ia-like antigens on a variety of cells. Results obtained from assays on cells treated prior to the primary antibody binding step, therefore, must be interpreted with caution.     

Structure of Ia antigens from the rat. Mouse alloantisera demonstrate at least two distinct molecular species

Ia antigens isolated from spleen cells of rats and mice are composed of two polypeptide chains, designated α and β. Mouse alloantisera specific for the I-A^k and I-E^k subregions react with two distinct groups of rat Ia antigens, designated A-like and E- like, respectively. Two-dimensional gel electrophoresis and peptide map analysis demonstrate that the A-like antigens of rat are distinct from the E-like antigens. Both rat Ia antigens react with alloantiserum produced in rats congenic for the major histocompatibility complex (MHC). These results demonstrate for the first time that two distinct Ia antigens are present in the rat. Accordingly, the rat, like the mouse, may have Ia antigens encoded by at least two sub regions of the rat MHC. The existence of multiple Ia gene products in rats is revealed by chemical techniques even in the absence of formal genetic evidence of more than one I subregion in the rat.     

Monoclonal mouse anti-I-Ak and anti-I-Ek antibodies cross-reacting with HLA-DR supertypic and subtypic determinants rather than classical DR allelic specificities

Thirty monoclonal alloantibodies (mAB) against mouse Iak antigens have been derived by fusion of mouse myeloma and spleen cells from A.TH (Ks Is Dd) mice immune to A.TL (Ks Ik Dd) lymphoid cells. Analysis of: (i) their reactivity (using 125I labelled protein A cell binding or cytotoxicity assays) on lymphoid cells from selected mouse strains with recombinant H-2 haplotypes; and (ii) the spatial arrangement of the specificities detected on the Iak molecules (studied by means of competitive inhibition of binding of radio-labelled monoclonal antibodies), permitted the identification of various epitopes present either on the I-Ak molecules (some of which were apparently identical to the conventional Ia.2, Ia.1 and Ia.19 specificities), or the I-Ek molecule (some being apparently analogous to the Ia.7 specificity) or on both I-Ak and I-Ek products. These mAB were tested in two different panels of human T and B lymphocytes. Panel (a) consisted of 28 Caucasian unrelated individuals, highly selected with regard to HLA-DR specificities, while panel (b) concerned 53 random HLA-A, B, C, DR typed individuals. The standard complement dependent lymphocytotoxicity microtechnique of histocompatibility workshop VIII was used throughout. All mAB were negative on resting T cells. Testing on B cells produced three patterns: 1) ten mAB did not react with any B cell tested; 2) four mAb reacted with all the panel cells; 3) sixteen mAb reacted with different sets of the panel indicating identification of polymorphic determinants. However, the strength of positivity obtained with a majority of single mAb varied considerably in the panel, suggesting identification of cross-reactive determinants. This necessitated the use of individual assignment criteria for each mAb. Following this procedure, 8 mAb were ascertained as reacting with HLA-DR supertypic determinants, 6 with associations to MT1, MT2, or both. Eight mAb reacted with HLA-DR subtypic determinants (more restricted than a classical DR allele). No mAb were ascertained, reacting exquisitely with acknowledged HLA-DR allelic specificities.     

Major histocompatibility complex regulation of T helper functions mapped to a peptide C terminus that controls ligand density

The functional status (Th1- versus Th2-like) of CD4 T cells primed against human collagen type IV (hCol IV) or a single 30mer peptide from the α2 chain of this molecule is predicted by the major histocompatibility complex (MHC) class II (I-A) genotype of the responding mice. H-2^s mice elicit Th1-like cell-mediated responses to these antigens, whereas Th2-like humoral responses are primed in H-2^b,d,k mice. We now report that the ability of MHC to dictate T helper function in this system depends upon a single amino acid of the minimal α2(IV) peptide. The C terminus of this minimal (12mer) peptide is -G-G-P-K, which is predicted to form a β-turn. The present data demonstrate that the terminal lysine (K) stabilizes the immunogens full biological effects necessary for exclusive cellmediated responses in H-2^s mice. The lysine-truncated (11mer) peptide with otherwise identical sequence effectively primes T helper function in both H-2^b and H-2^s genotypes. Most importantly, our direct analysis of these peptides' presentation by live antigen-presenting cells (APC) reveals that the 12mer is bound at a log higher density on H-2^s APC than on H-2^b APC, and that the 11mer is presented at an equally low relative density on APC from both genotypes. In vitro analyses of 12mer/11mer cross-reactive Th clones demonstrate that I-A^s restricted clones require about 1–2 log lower doses of 12mer peptide than 11mer peptide to stimulate equivalent thymidine incorporation and cytokine release. By contrast, I-A^b-restricted (12mer/11mer cross-reactive) Th clones show no preference for the 12mer and require relatively high peptide doses similar to those required to stimulate the I-A^s clones with the 11mer peptide. Thus, the peptide dose requirements of Th clones reflect the high density of presentation associated with the 12mer: I-A^s ligand. Taken together, the results directly support the role of ligand density as an important control point in the functional decision of CD4 T cells.     

Tumorigenicity of mouse T lymphoma cells is controlled by the level of major histocompatibility complex class I H-2Kk antigens

We have previously found that an increased tumorigenicity and spontaneous metastatic potential of BW5147-derived T lymphoma cells was associated with a decrease in major histocompatibility complex (MHC) class I H-2K^k antigen expression. This suggested that H-2K^k antigens may control the tumorigenic potential of BW T lymphoma cells. Our current experiments aimed to prove this association by specifically altering H-2K^k expression by gene transfection. Transfected cells expressing a high level of H-2K^k antigens were significantly less tumorigenic and metastatic after subcutaneous inoculation. However, there was selectionin vivo for cells expressing a reduced level of H-2K^k antigens, which concomitantly led to an increased tumorigenicity. These data further confirmed the strong association between H-2K^k expression and tumorigenicity. We subsequently tested whether the immune system is implicated in this phenomenon by inoculating the H-2K^k transfectants into irradiated, immunocompromised recipients. Our results indicate that the reduced tumorigenicity of the BW H-2K^k transfectants is due to an immune rejection mechanism, mediated by CD8⁺ immune effector cells, as revealed byin vivo depletion experiments with anti-CD8 antibodies. Hence, we hereby demonstrated that H-2K^k antigens increased the immunogenicity of BW cells, via a CD8-dependent mechanism, which consequently reduced their tumorigenicity.     

INACTIVATION OF SPECIFIC ANTI-H-2 SUPPRESSOR T CELLS BY ANTISERA TO I-J AND I-C SUBREGION PRODUCTS OF THE H-2 COMPLEX

Two antisera to Ia antigens, products of the H-2 complex I-C^d and I-J^kE^k subregions, respectively, have been obtained by immunization of the F₁ hybrids of recombinant strains of mice. These antisera are shown to display a 50% cytotoxic effect in vitro, in the presence of complement, upon lymphocyte populations immune to the H-2-complex antigens and enriched for specific suppressor T cells (SSTC) by fractionation on a monolayer of target cells. The specificity of anti-Ia cytotoxins is shown by cross-antibody absorption with T and B cells of mice originating from the recombinant H-2 haplotypes and bearing either particular I-C^d, I-J^k and I-K^k antigens, or their combinations. Anti-I-C^d cytotoxins were found to react with both B and T cells, but at a different rate, and the anti-I-J^kE^k serum contains two antibody types directed to I-E^k and I-J^k products, respectively, the latter being able to react preferently with T cells. Although both antisera do inactivate the in vitro SSTC function in the presence of complement to a similar degree, the inactivating action of the anti-I-C^d serum, but not that of the anti-I-J^kE^k serum, occurs without complement. SSTC are shown to bear both Ia-antigens, I-J and I-C, as shown by both inactivation of the anti-suppressor effect of the antisera absorbed with spleen cells of different H-2 origin, and variation of the H-2 origin of SSTC pretreated with the intact antisera. It is suggested that these two markers, located on the same SSTC, function differently in SSTC immune to the H-2 antigens, and I-C antigen expression on the SSTC surface is presumed to be required for their interaction with the inhibited responder T cells proliferating in MLC.     

ORIGINAL H-2d AND FOREIGN H - 2k - LIKE ANTIGENS ARE INDEPENDENT ENTITIES ON A CHEMICALLY INDUCED SARCOMA

We have previously shown that a methylcholanthrene-induced sarcoma of BALB/c strain (C-1) expressed, in addition to its original H-2^d antigens, foreign H-2^k-like determinants. In the present study the relationship between H-2^d and H-2^k-like antigens was examined by in vitro and in vivo assays. Cultured tumour cells were exposed in the cold to either a monospecific (D-23, D-25, D-1) or polyspecific (BALB/c anti-C3Hf) alloantiserum directed to H-2^k specificities and then used to absorb the cytotoxic activity of either monospecific (D-31) or a polyspecific (C3Hf anti-BALB/c) alloantisera to H-2^d antigens (blocking test). The opposite was also done, i.e. tumour cells were coated with anti-H-2^d sera and used to absorb the cytotoxicity of anti-H-2^k sera. No reciprocal interference was found between the two H-2-different antigens in the absorption of related alloantisera. Suspensions of irradiated C-1 tumour cells were exposed in vitro to either anti-H-2^d or anti-H-2^k antisera and then used to immunize either syngeneic BALB/c or allogeneic C3Hf mice. The coating of immunizing neoplastic cells with BALB/c anti-C3Hf serum prevented anti-C3Hf (anti-H-2^k) antibody production in BALB/c mice without affecting anti-BALB/c (anti-H-2^d) antibody development in C3Hf animals; coating of H-2^d antigens on tumour cells strongly reduced anti-BALB/c but not anti-C3Hf antibody production. Both in vitro and in vivo experiments indicated that foreign H-2^k-like determinants are physically separated from the wild H-2^d antigens on the C-1 sarcoma cells.     

The presence of a common idiotype in anti-H-2 immune sera as detected by anti-idiotype to a monoclonal anti-H-2 antibody

To study the idiotypes (Id) of the humoral response to H-2L^d antigens, xenogeneic antisera were produced to three independent monoclonal antibodies, recognizing L^d determinants H-2.64 or 65. The reactivity of each of these anti-Id was specific for the immunizing antibody, no cross-reactions among the three anti-L hybridomas being detectable. Antisera produced in BALB/c H-2^dm2 mice hyperimmunized with BALB/c tissue were examined for the presence of cross-reactive Id (Id^CR). When the ability of these anti-Id to inhibit the binding of alloantibodies to L^d splenic antigens was tested, a broadly shared Id was detected by one anti-Id (anti-23-10-1S). The shared Id represented between one-fourth and one-half of the total anti-L^d humoral response and was found in every BALB/c H-2^dm2 anti-BALB/c serum tested. The Id^CR was limited to those alloantibodies reacting with the determinant H-2.65 which corresponds with the serologic reactivity of monoclonal antibody 23-10-1S. The other two anti-Id did not detect components of the anti-L^d humoral response, even though one of these was made against monoclonal antibody 30-5-7S which also detects serologic specificity H-2.65. The detection of a Id^CR in anti-L^d responses stands in contrast to our previous failure to detect Id^CR in anti-H-2K^k responses. Among the possible reasons for this contrast are (a) the fact that 23-10-1S is an IgM antibody, whereas the anti-H-2K^k antibodies studies were IgG; (b) the chance occurrence of anti-H-2 hybridomas with dominant Id and (c) the possibility that the anti-L^d antibody repertoire may be less heterogeneous than that directed to K^k antigens.