Effect of in vivo activation of natural killer (NK) cells by a tilorone analogue on the survival of mice injected intravenously with different experimental murine tumours

We studied the effect of a tilorone analogue (RMI 10,874DA) and anti-asialo GM₁ serum on the survival of BALB/c and C57B1/6 mice after i.v. injections of different syngeneic murine tumour cells. Tumour lines used were different clones from chemically (GR9 wild type, GR9.B9, B7.1.B4, B7.1.B5, B7.2.38), and ultraviolet light (GRUV3)-induced sarcomas; B16 melanoma and LSTRA and YC8 lymphomas. Pretreatment of mice with tilorone inhibited metastatic colonization and increased survival significantly in all cases. In some tumour systems, the effect was attenuated when high numbers of cells were injected. Abrogation of NK cells with anti-asialo GM₁ serum significantly decreased (in all tumours and at different cell doses) survival in comparison with untreated mice injected with tumours, regardless of cell dose used. These results clearly suggest that NK cell activation in vivo by the tilorone analogue we tested prolongs survival and inhibits metastasis formation in mice, even when pretreatment consists of a single dose of the analogue.     

Influence of H-2K transfection on susceptibility of fibrosarcoma tumor cells to natural killer (NK) cells

In this study, we have used the T-10 fibrosarcoma tumor cells to further analyze the relationship between metastatic competence, expression of H-2K antigens and susceptibility to lysis by virus augmented NK cells in vitro. Our results show an inverse correlation between metastatic properties of the original T-10 clones, IC9 and IE7, and susceptibility in vitro to lysis by virus-augmented NK cells. Restoration by transfection of expression of H-2K genes (H-2K^b or H-2K^k) led to the alteration of the metastatic phenotype of the tumors cells, yet had minor influence on the putative susceptibility of these clones to NK. These observations suggest that expression of MHC gene products, while affecting metastases, does not exclusively determine sensitivity to NK cells.     

Anti-Thy-1 Response of H-2f/H-2f Heterozygotes: An Unusual Case of Genetic Control

Anti-Thy-1 responsiveness of H-2 homozygous and H-2/H-2^f heterozygous mice was studied Good responsiveness appeared to be independent of H-2 phenotype of responder but was influenced by the phenotype of the donor. These results were incompatible with the concept of Ir-Thy-1 genes controlling the response to cell-free Thy-1 in these mice In contrast, the results were indicative of the response to the cell-bound form of the Thy-1 antigen. It is proposed that good anti-Thy-1 response may reflect the presence of clones capable of recognizing the Thy-1 antigen in the context of or in association with incompatible class I H-2 molecules     

NK sensitivity, H-2 expression and metastatic potential: analysis of H-2Dk gene transfected fibrosarcoma cells

We have used the 3-Methylcholanthrene induced T-10 fibrosarcoma tumour cell system (H-2b xH-2k)F1 to elucidate the possible correlation between metastatic potential, expression of individual H-2 antigens and susceptibility to NK cells. Transfection of the non-metastatic and NK sensitive IC9 cells (Db+, Dk-, Kb-, Kk-) with the H-2Dk gene, altered the metastatic phenotype of the parental cells, yet had no effect on the susceptibility of these tumour cells to lysis by NK and did not elicit a specific CTL response in syngeneic hosts. Variants of the metastatic and NK resistant IE7 clone (Db+, Dk+, Kb-, Kk-), lacking H-2Dk, were selected by treatment with monoclonal anti H-2Dk antibodies and complement. These variants were sensitive to NK and poorly or non metastatic. Retransfection of 'Dk' 'loss' variants with the H-2Dk gene, resulted in the isolation of several clones expressing a wide range of metastatic phenotypes but maintained sensitivity to NK. These results indicate that the H-2D region of the MHC and or closely linked genes may be involved in the complex interrelationship between target susceptibility to NK and metastasis.     

Original H-2d and alien H-2k-like antigens of a BALB/c fibrosarcoma as defined by in vitro and in vivo studies of cell-mediated immunity

The present study examines the immunosensitivity and the immunogenicity of both original H-2^d and alien H-2^k-like antigens of the BALB/c (H-2^d) fibrosarcoma C-1 as detected by in vitro and in vivo cell-mediated cytotoxicity (CMC) assays. It was found that ⁵¹Cr-labeled C-1 cells were lysed in vitro by C 57 BL/6 anti-H-2^d lymphocytes. The specificity of this reaction was shown by cold inhibition experiments in which the anti-H-2^d cytotoxic activity on YC8 (H-2^d) targets was inhibited by unlabeled YC8 or C-1 but not by C3UR11 (H-2^k) tumor cells. Both D^d- and K^d-encoded antigens were recognized by appropriate cytotoxic effectors. The immunogenicity of H-2^d antigens of C-1 was revealed by the ability of C 57 BL/6 anti-C-1 lymphocytes to lyse YC8 targets. The expression of H-2^k-like alien alloantigens on C-1 was indicated by the finding that anti-H-2^k cytotoxic T lymphocytes (CTL), generated by culturing BALB/c spleen cells immune to BALB.K (H-2^k), C3Hf (H-2^k) or A (H-2^a = H-2^k/d) tissues with the cells of the same strain used for immunization, lysed C-1 targets. The cytotoxicity of these anti-H-2^k CTL against C 3 UR11 (H-2^k) targets could be specifically inhibited by cold C 3 UR 11 or C-1 cells but not by two other BALB/c tumors. Using recombinant H-2-congenic mice, it was shown that both D^k and K^k antigens were recognized by CTL on C-1 cells. The immunogenicity of the H-2^k-like antigens, however, could not be detected in vitro. In fact, effector spleen cells from BALB/c mice immune to C-1 did not develop any detectable cytotoxicity against C 3 UR 11 targets as assayed either by a direct in vitro test or after in vitro restimulation with C-1 sarcoma cells. A similar experimental design was adopted in Winn assays carried out by mixing spleen cells of BALB/c immune mice with either C-1 or C3 UR 11 targets and injecting the mixtures in BALB/c or hybrid recipients. These in vivo tests revealed the presence of both H-2^d (K^d and D^d) and H-2^k-like (K^k and D^k) antigens on C-1. At variance with the in vitro CMC assays, however, the Winn assay also detected the immunogenicity of the H-2^k alien antigens, since BALB/c anti-C-1 spleen cells were able to significantly reduce the growth of C 3 UR 11 lymphoma cells in (BALB/c × C 3 Hf)F₁ hosts.     

Influence of glycosylphosphatidylinositol-linked H-2Dd molecules on target cell protection and natural killer cell specificity in transgenic mice

The expression of certain major histocompatibility complex (MHC) class I ligands on target cells is one important determinate of their susceptibility to lysis by natural killer (NK) cells. NK cells express receptor molecules that bind to MHC class I. Upon binding to their MHC class I ligand, the NK cell is presumed to receive a signal through its receptor that inhibits lysis. It is unclear what role the MHC class I molecules of the effector and target cells play in signaling to the NK cell. We have investigated the role of the cytoplasmic and transmembrane domains of MHC class I molecules by producing a glycosylphosphatidylinositol (GPI)-linked H-2D^d molecule. The GPI-linked H-2D^d molecule is recognized by H-2D^d-specific antibodies and cytotoxic T lymphocytes. Expression of the GPI-linked H-2D^d molecule on H-2^b tumor cells resulted in protection of the tumor cells after transplantation into D8 mice (H-2^b, H-2D^d) from rejection by NK cells. In addition, NK cells from mice expressing the GPI-linked H-2D^d molecule as a transgene were able to kill nontransgenic H-2^b lymphoblast target cells. The GPI-linked MHC class I molecule was able to alter NK cell specificity at the target and effector cell levels. Thus, the expression of the cytoplasmic and transmembrane domains of MHC class I molecules are not necessary for protection and alteration of NK cell specificity.     

IMPAIRED H-2 EXPRESSION IN B 16 MELANOMA VARIANTS

We studied the expression of H-2^b alloantigens in three different B16 melanoma lines cultured 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-2^b immune lymphocytes and absorption of anti-H-2^b antisera activity. The B16-F1 line was able to efficiently kill allogeneic hosts, could not be lysed by anti-H-2^b cytotoxic effectors and did not express any serologically detectable amount of H-2^b 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-2^b cytotoxic effectors and the H-2K^b antigens became undetectable. The expression of H-2D^b was reduced, although at a lower degree. Similar data were obtained with B 16-B cells, which after 10 in vitro passages grew and killed allogeneic hosts, showed a decreased sensitivity to cytotoxic anti-H-2^b effectors and a very low expression of the K region antigens. The results indicate that H-2 expression is altered in B 16 melanoma lines and this may influence the different metastatic capacity of such cells.     

Expression of ganglioside GM3 and H-2 antigens in clones with different metastatic and growth potentials isolated from Lewis lung carcinoma (3LL) cell line

In view of the evidence that cell expression of gangliosides in several tumors is positively involved in the metastatic phenotype, Lewis lung carcinoma (3LL) cell line, expressing GM3 as the major ganglioside, was analysed for the cell surface expression of GM3. An indirect immunofluorescence assay, using a M2590 monoclonal antibody recognizing GM3, was used for this purpose. Since the parental 3LL cells consist of heterogenous subpopulations differing in the degrees of GM3 expression, we have developed clones of this cell line with different degrees of metastatic potentials by using anin vitro non-selective procedure in order to investigate whether the expression of GM3 is associated with metastatic potential. The degree of cell surface expression of GM3 among the clones correlated well with their total cellular content of this ganglioside. However, we were unable to confirm the report of increased level of GM3 in high metastatic 3LL clones, nor did a decreased level correlate with weak metastatic ability. In our recent work, an inhibitor of glucosylceramide synthase,d-threo-l-phenyl-2-decanoylamino-3-morpholino-l-propanol (DPDMP), was found to decrease the levels of all cellular glucosphingolipids and cause the accumulation of the precursors of glucosylceramide. The present study does not, however, rule out the possible involvement of this lipid family in metastatic dissemination, since treatment of 3LL cells with D-PDMP resulted in significant inhibition of their experimental metastatic potential. Clones expressing very low GM3 grew slowly in culture dishes, suggesting that GM3 may have a regulatory role in cell proliferation. The low metastatic clones expressed high levels of H-2K^b antigen, while the expression of the same antigen on the high metastatic clones was relatively low, confirming the previous observation of this tumor system. Moreover, a clone showing the lowest tumorigenic potency revealed both a high cell surface expression of H-2K^b and a high H-2K^b/H-2D^b ratio.     

H-2K RESTRICTION OF THE T CELL-MEDIATED LYSIS OF A CHEMICALLY-INDUCED BALB/c FIBROSARCOMA

Previous work has shown that a cytotoxic T lymphocyte (CTL) immune response of syngeneic mice immunized with a chemically-induced BALB/c (H-2^d) fibrosarcoma was directed against an individual tumour-associated antigen. To see whether this reaction was restricted by products of the major histocompatibility complex (MHC), anti-H-2 alloantisera to K or D antigens were used to interfere with the CTL-mediated immune response. Antisera to K^d but not to D^d antigens inhibited the lytic activity of CTL against fibrosarcoma cells. In addition, the study of the CTL response in F₁→ P antitumour immunized chimeric mice showed that antitumour cytotoxicity developed only when F₁ and parental host shared the K^d region. Both experiments strongly indicate that recognition of the individual tumour-associated antigen of the BALB/c fibrosarcoma is restricted by the products of H-2K^d genes.     

Control of metastatic properties of BL6 melanoma cells by H-2Kb gene: immunological and nonimmunological mechanisms

The effect of class I H-2 antigen expression on the metastatic properties of BL6 melanoma cells was investigated. The BL6-8 clone isolated from the highly metastatic BL6 melanoma did not express H-2K b gene. Following transfection with the H-2K^b gene, BL6-8 cells displayed a low metastatic potential in the immunocompetent as well as immunosuppressed (X-irradiated) or triple-immunodeficient mice with impaired T, B and natural killer (NK) cells function. The expression of H-2K^b gene and the low metastatic ability of transfected BL6 melanoma cells were associated with appearance of cell membrane soybean agglutinin (SBA) and Griffonia simplicifolia 1B₄ (GS1B₄) lectin-binding carbohydrataes. These alterations in cell surface carbohydrates were found to be a result of reduction in sialylation of SBA binding sites and upregulation of the 1.3 galactosyltransferase (1.3GT) gene. To assess the importance of H-2K^b-induced alterations in cell surface carbohydrates for metastasis formation, BL6-8 melanoma cells were transfected with H-2K^b gene without neo^r gene cotransfection and selected for adherence to SBA-lectin-conjugated agarose beads. The transfected clones that expressed SBA and GS1B₄ lectin-binding carbohydrates were low metastatic. Further analysis of these clones showed that presence of SBA and GS1B₄ lectin-binding carbohydrates rather than expression of H-2K^b molecules per se might be responsible for low metastatic potentials of H-2K^b-transfected cells in the immunocompromized mice. Studies of the possible mechanisms responsible for low metastatic ability of H-2K^b-transfected melanoma cells revealed that these cells displayed a reduced ability to adhere to murine pulmonary endothelial cells as well as to laminin and collagen IV. We hypothesized that the observed nonimmunological effects of H-2K^b gene in BL6 melanoma cells is a result of an interaction between the H-2K^b gene and B16 melanoma-specific ecotropic retrovirus. It results in inhibition of this retrovirus production with consecutive alteration in the expression of cellular genes controlling cell surface glycosylation and adhesion properties essential for the metastatic phenotype of BL6 melanoma.     

EVIDENCE OF ONLY ONE H-2D/L - RELATED GLYCOPROTEIN IN H-2dm1 MUTANT CELLS

Analyses of the H-2D/L-related glycoproteins from dm1 mutant cell extracts by sequential immunoprecipitation, by SDS gel electrophoresis and by tryptic peptide mapping indicate that dm1 cells express only a single glycoprotein with H-2D/L-related determinants. In contrast to the four H-2D/L-related antigens identified for the parental d haplotype viz. H-2D^d, H-2M^d, H-2L^d and H-2R^d, separate and distinguishable “H-2D^dm1”, “H-2M^dm1”, “H-2L^dm1” and “H-2R^dm1” glycoprotein counterparts are apparently lacking in the dm1 mutant haplotype. Only a single H-2D/L-related glycoprotein is identified in dm1 extracts by standard serological methods and this glycoprotein is designated H-2D/L^dm1 because of its H-2D^d/H-2L^d hybrid characteristics, as recently shown by Burnside and colleagues (1984). Thus, the seemingly complex phenotype of the dm1 mutant appears to originate primarily from one molecule having properties of two (or more) molecules of the parental haplotype.     

The ability of H-2Dd molecule to affect natural resistance to hemopoietic allografts is an intrinsic property shared by Ddm1 but not Ld

F₁ hybrid resistance (HR) to parental bone marrow grafts is mediated by natural killer (NK) cells, and thought to be controlled by the non-class I hemopoietic histocompatibility (Hh) genes linked to the major histocompatibility complex (MHC). However, as in the in vitro NK cytotoxicity against hemopoietic targets, expression of certain class I MHC molecules does affect HR, although mechanisms underlying such an effect are not understood. In this study, we examine the relevance of the “self/non-self” property of class I molecules and the molecular domains responsible for this function. H-2^b/Hh-1^b lymphoma cells were transfected with class 1 H-2D^d or L^d gene, and its effect on the Hh-1 phenotype was examined by testing the transfectant's ability to competitively inhibit the in vivo rejection of parental H-2^b/Hh-1^b bone marrow grafts by irradiated F₁ hybrid hosts. Multiple independent clones of transfectants show that the genomic or cDNA of the D^d gene, but not of L^d, renders the Hh-1^b-positive cells incapable of inhibiting HR in F₁ mice, although both genes belong to the same region of the same haplotype. The same effect could be observed not only in H-2^b/d F₁ mice for which D^d and L^d are self, but also in H-2^b/k F₁ mice for which both D^d and L^d are non-self. Thus, this function of the D^d molecule is an intrinsic property, not necessarily related to its self/non-self characteristic relative to the effector cells. Furthermore, given the nature of the assay used in this study, the results favor a “target interference” model as the underlying mechanism of the D^d effect. To locate the relevant domain(s) of the D^d molecule, mutant D^dm1 gene was tested and found to have the same effect as the non-mutant D^d. D^dm1 is a hybrid molecule between D^d and L^d, sharing with D^d only the α1 domain and a portion of the α2 domain. The two N-terminal domains of D^dm1 differ from those of D^d by three amino acid substitutions, two of which affect the molecules' peptide-binding properties.     

Dominant allele-specific regulation of expression of H-2Kk gene products revealed by somatic cell hybridization

A study has been made of the H-2 profiles of the AKR thymoma K36 and two series of somatic cell hybrids derived from it. Despite expressing good amounts of the H-2D ^k product, the H-2K^k antigen was barely detectable in this tumor, approximately only 1% of that expressed by a comparable AKR lymphoma, 339. The isoelectric focusing profile of the H-2K^k product immunoprecipitated from K36 was found to be identical to that from normal AKR lymphocytes. The phenotype of K36 therefore results from regulatory constraints. Fusion ofK36 with normal K-2^k lymphocytes resulted in hybrids expressing the H-2D^k product well but having a drastically reduced expression of the H-2K^k product. Fusion of K36 with normal H-2^b lymphocytes resulted in good expression of the H-2D^k and H-2D^b alleles in addition to good expression of the H-2K^b products. The expression of the H-2K^k antigen remained marginal. The data suggest a suppressive mechanism which is trans-acting and dominant and specific for the H-2K^k allele.     

Analysis of the Developmental Potential of Conditionally Immortal Marrow-Derived Mesenchymal Progenitor Cells Isolated from the H-2Kb-tsA58 Transgenic Mouse

All nucleated cells from the adult transgenic mouse H-2K^b-tsA58 harbor the temperature-sensitive mutant gene for SV40 large T-antigen. Bone marrow cells from this transgenic mouse were isolated, expanded and cloned in vitro under conditions permissive to the expression of stable T-antigen. Clones of marrow-derived mesenchymal progenitor cells were tested in vitro and in vivo for their capacity to differentiate into mature mesenchymal phenotypes of bone, cartilage, muscle, adipose, and hematopoietic support cells (termed “stromacytes”). Mono-, bi-, and tri-potential clones were identified that were able to differentiate into bone, adipose and stromacyte phenotypes. A mixed population of cells showed some chondrocytic potential in vivo, however, no evidence of cartilage matrix production was detected in vitro for any of the immortomouse clones. These results support the hypothesis that marrow contains multiple progenitor cells that are part of a mesenchymal lineage.     

α1 / α2 domains of H-2Dd,but not H-2Ld,induce “missing self” reactivity in vivo – No effect of H-2Ld on protection against NK cells expressing the inhibitory receptor Ly49G2

Introduction of the MHC class I transgene H-2D^d on C57BL / 6 (B6) background conveys NK cell-mediated “missing self” reactivity against transgene-negative cells, and down-regulates expression of the inhibitory receptors Ly49A and Ly49G2 in NK cells. We here present an analysis of transgenic mice expressing chimeric H-2D^d / L^d MHC class I transgenes, and show that the α1 / α2 domains of H-2D^d were necessary and sufficient to induce “missing self” recognition and to down-modulate Ly49A and Ly49G2 receptors. In contrast, transgenes containing the α1 / α2 domains of H-2L^d induced none of these changes, suggesting that not all MHC class I alleles in a host necessarily take part in NK cell education. The lack of effect of the α1 / α2 domains of H-2L^d on NK cell specificity was surprising, considering that both H-2L^d and H-2D^d have been reported to interact with Ly49G2. Therefore, the role of H-2L^d for protection against NK cells expressing Ly49G2 was re-investigated in a transfection system. In contradiction to earlier reports, we show that H-2D^d, but not H-2L^d, abolished killing by sorted Ly49G2⁺ NK cells, indicating that H-2L^d does not inhibit NK cells via the Ly49G2 receptor.     

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.     

Lack of F1 anti-parental resistance in H-2b/d F1 hybrids devoid of β2-microglobulin

F₁ hybrid mice often reject parental hematopoietic grafts, a phenomenon known as hybrid resistance. Hybrid resistance is mediated by natural killer (NK) cells and although the molecular interactions responsible for this phenomenon are largely unknown, one hypothesis suggests that parental cells are rejected because they fail to express a complete set of host major histocompatibility complex (MHC) class I molecules. Inherent in this theory is that NK cells in the F₁ hybrid are instructed by self MHC class I molecules to form an NK cell repertoire capable of reacting against cells lacking these self MHC class I molecules. Here, we show that C57BL/6 x DBA/2 mice (H-2^b/d) devoid of β₂-microglobulin (β₂m) are incapable of rejecting β₂m?/? parental C57BL/6 cells (H-2^b) both in vivo and in vitro. From this, we conclude that the development of an NK cell repertoire, at least in F₁ mice of the H-2^b/d haplotype, requires expression of MHC class I molecules complexed with β₂m.     

Vβ11+ T-lymphocyte expansion by toxic shock syndrome toxin-1 differs in mice bearing H-2q versus H-2b haplotypes

We have recently demonstrated that toxic shock syndrome toxin-1 (TSST-1) expanded Vβ11⁺ T lymphocytes contribute to Staphylococcus aureus arthritis and sepsis-induced mortality. Interestingly, Vβ11⁺ T-cell mediated joint pathology varies in different mouse strains. In this study, we characterized the in vitro pattern of Vβ11⁺ T-cell expansion by TSST-1 in mice with various genetic backgrounds. Mice expressing major histocompatibility complex (MHC) class II I-E molecules did not expand Vβ11⁺ T cells upon stimulation with TSST-1. Using B10 congeneic I-E negative mouse strains, we found that the TSST-1-expanded Vβ11⁺ T cells in B10Q (H-2^q) and B10M (H-2^f) mice but not in B10B (H-2^b) mice. Antigen-presenting cells (APC) from B10Q mice, L cells and lymphoma cell line transfected with a q gene did not restore the deficient Vβ11⁺ T-cell expansion by TSST-1 in purified T cells from B10B mice. In contrast, I-A^b APC were able to stimulate Vβ11⁺ T cells from H-2^q mice. Furthermore, Vβ11⁺ T cells in H-2^b mice did expand when exposed to staphylococcal enterotoxin A (SEA). These findings suggest that the T-cell repertoire, skewed by clonal deletion and inactivation of self-reactive T cells, accounts for the different magnitude of Vβ11⁺ T-cell expansion among the different mouse strains.     

H-2 genetic control of the response of T lymphocytes to insulins. Priming of nonresponder mice by forbidden variants of specific antigenic determinants

H-2 gene control of the response of T lymphocytes to antigenic determinants of ungulate insulins has been studied. We injected H-2-congenic mice with different insulins emulsified in complete Freund's adjuvant and measured the proliferative response in vitro of the draining lymph node cells to various related insulins. Two groups of determinants were antigenic: the A chain loop determinant present on bovine and sheep insulins, but absent on pig insulin, and a pig-associated determinant(s) also shared by bovine and sheep insulins. The responses to these two sets of determinants were controlled by H-2 genes as follows: (a) The pig-associated determinant(s) was antigenic for H-2^d but not for H-2′ or H-2^k mice. (b) The A chain loop determinant was immunodominant over the pig-associated determinant(s) for H-2^dmice. In the presence of the A chain loop determinant, H-2^d mice did not respond to the pig-associated determinant(s) on the same molecule. (c) H-2^d mice did not distinguish between the bovine and sheep variants of the A chain loop that differed by substitution of one amino acid. (d) In contrast, H-2^k mice responded only to the sheep variant, while H-2^b mice responded primarily to the bovine variant of the A chain loop determinant. (e) However, injection of H-2^b mice with “forbidden” sheep insulin primed them for an in vitro response to the “permitted” bovine variant of the A chain loop determinant. Similarly, some H-2^k mice injected with forbidden bovine insulin responded in vitro to the permitted sheep variant of the A chain loop determinant. Thus, H-2 gene products decided whether the immune response would express an affinity for the specific immunizing antigen itself and/or for a closely related variant.     

ROUS SARCOMA VIRUS-INDUCED TUMOURS IN MICE: II. CONTRIBUTION OF H-2 AND NON-H-2 ALLOANTIGEN BARRIERS TO TUMOUR IMMUNOGENICITY IN VIVO

The features of the immune recognition of a murine fibrosarcoma induced by Rous sarcoma virus were tested in histocompatible and histoincompatible mice. No evidence of a genetic regulation of spontaneous reactivity to tumour-associated antigens was found in various histocompatible F₁ hybrids. Incompatibility in multiple minor histocompatibility antigens triggers a host reaction incapable of causing tumour rejection in some cases. The growth rate of incipient tumours is unaffected, whereas that of already visible tumoour masses is significantly delayed. Admixture to the challenge of inactivated leukocytes bearing the same minor histocompatibility antigens as the tumour triggers a significantly stronger reaction. The reaction of hosts incompatible in the H-2K or H-2DL regions is quite efficient. However, the intensity of the immune reaction to H-2DL^s antigens displayed by tumour cells is markedly dependent on the alleles of genes located in the central regions of the H-2 complex.