IMMUNOGENETIC AND IMMUNOCHEMICAL STUDIES OF H-2 ANTIGENS OF FOREIGN HAPLOTYPES ON TUMOUR CELLS

The polymorphism of histocompatibility antigens is usually explained by classical Mendelian laws, which allow the inheritance of one allele per locus. Thus each individual would express two antigens for each locus of the pair of relevant homologous chromosomes, one from each parent. Another explanation is that the structural genes of all the different haplotypes are all in tandem. Regulatory genes would then determine the polymorphism by mimicking Mendelian inheritance–allowing only the relevant gene products for a particular haplotype to be expressed. If correct, one might expect the mechanism to fail sometimes, and silent genes of foreign haplotypes to be derepressed, allowing H-2 antigens of the wrong haplotype to become expressed. In the light of this hypothesis (Martin, 1975; Amos, 1971; Bodmer, 1975; Festenstein, 1978; Garrido et al., 1976a,b), the original findings in our laboratory of extra foreign ‘H-2-like’ determinants on tumour cells passaged in vitro and in vivo with and without virus are considered rather important (Festenstein, 1978; Garrido et al., 1976a,b); not only because they could lead to a better understanding of the genetic basis of MHS polymorphism, but also because of its implication for the surveillance of tumours (Garrido et al., 1976b; Gomard et al., 1977; Blank et al., 1976; Parmiani & Invernizzi, 1975) and microbially damaged cells (Doherty & Zinkernagel, 1975). These extra determinants were tested by various serological techniques and assays of cell mediated immunity.     

MULTIPLE FOREIGN NON-H-2 DETERMINANTS ON THE SURFACE OF A CHEMICALLY-INDUCED MURINE SARCOMA*

By immunizing BALB/c (H-2^d mice against normal tissues from C57BL/6J (H-2^b), C3Hf (H-2^k) and DBA/2 (H-2^d), but not from AKR (H-2^k) strains, resistance was induced to the subsequent challenge of the ‘syngeneic’ methyl-cholanthrene-induced BALB/c sarcoma ST5; lymph node cells from allo-immune BALB/c mice were also able to exert a parallel cytotoxic effect against in vitro cultured ST5 cells. The involvement of foreign H-2 specificities in the observed cross-reactions was ruled out by absorptions of H-2 monospecific sera and by the interallelic combinations used, thus suggesting that non-H-2 histocompatibility antigens were responsible for the above findings. By using the indirect isotopic antiglobulin assay, BALB/c anti-C57BL/6J and anti-C3Hf polyspecific sera were found to bind specifically to cultured ST5 cells. C57BL/6J and C3Hf, but not DBA/2, lymph node cells were able to absorb the anti-ST5 activity of the anti-C57BL/6J serum. These results indicated that ST5 cells expressed on their surface at least two different sets of foreign non-H-2 antigens: one shared by C57BL/6J and C3Hf tissues, and detected by both cell-mediated and serological techniques; the other one belonging to DBA/2 tissues, and revealed mainly at the cell-mediated level.     

RELATIONSHIPS BETWEEN H-2 AND VIRAL ANTIGENS IN MURINE ONCORNAVIRUS-INDUCED TUMOURS*

Cytolytic T lymphocytes (CTL) from murine sarcoma virus (MSV) or Friend leukaemia virus (FLV) inoculated mice lyse syngeneic much more efficiently than allogeneic FMRGi+ lymphoma cells. By comparing the cytolysis of various H-2 different ⁵¹Cr lymphomas by CTL from several inbred and congenic lines differing at H-2, and by competition experiments using unlabelled cells, one can demonstrate that this phenomenon is due to an H-2 barrier. H-2^b/H-2^d hybrid-anti-MSV-CTL immunized by H-2^b, H-2^d or H-2^b/H-2^d tumours lyse only FMRGi+ lymphomas of the same H-2, and their activity for a given target is inhibited only by H-2-identical competitive cells. H-2 antigens are therefore directly involved in the interaction between tumour cells and immune CTL which probably react with an 'H-2 modified' antigen of the tumour cells surface. The use of CTL from intra-H-2 recombinant lines shows that H-2D and probably H-2K molecules are involved, but vary according to the tumour cells. A possible role of the I region is discussed as well as the implications of these results in immunosurveillance against viral neoplasia.     

VARIABLE EXPRESSION AND IMMUNOGENICITY OF AN H-2K-CODED ALLOANTIGEN ON MURINE TUMOURS

The K region of the H-2 major histocompatibility complex (MHC) of mice of H-2K^k haplotype codes for two distinct alloantigens. One of these alloantigens, designated k-common, is expressed by C3HfB/HeN mice (C3Hf). The other alloantigen, designated k-unique, is not expressed by C3Hf mice. The H-2 haplotype of C3Hf mice has been classified as kv1 and the variant antigen distinguishing this strain from mice of H-2K^k haplotype has been designated kv1-unique. Several transplacentally-induced lung tumours of C3Hf mice express the k-unique, rather than the expected kv1-unique, antigen. The immunogenicity of the k-unique antigen on C3Hf-derived lung tumours varies with different tumours. In particular, the capacity of the k-unique antigen to induce radioresistant immunity in C3Hf mice appears to be lost on long term cultured tumour cells even though the tumour remains susceptible to in vivo immune responses directed against the k-unique antigen. Alterations in expression and in immunogenicity of unique H-2-coded antigens may dictate the nature and efficacy of immune surveillance of autochthanous tumours.     

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.     

H-2D ANTIGENS RELEASED BY THYMOCYTES AND CELL ADHESION

The identity and complete purification of mouse Thymocyte Interaction Modulation Factor (T IMF) is described. Use of silver-stained PAGE methods shows that previous methods of purification yield preparations containing two protein or glycoprotein bands. T IMF activity from H-2^k mice can be bound to 15.5.5 monoclonal antibody columns (anti H-2 D^k) but not to 11.4.1 columns (anti H-2 K^k). The activity can be recovered from 15.5.5 columns and runs on PAGE as a single band at approximately 34,000 Daltons. This evidence together with previous evidence relating the activity to H-2 D locus argues that T IMF is a soluble H-2 D antigen fragment equivalent to a papainized H-2 fragment. Additional evidence is presented on an improved method of assay of T IMF activity, on its inactivation by enzymes and serine-esterase inhibitors and of its effect on syngeneic leucocytes and macrophages. It is also shown that T IMF is not appreciably toxic for cells.     

H-2d-LIKE SPECIFICITIES ON GARDNER (H-2k) TUMOUR DETECTED IN A RESTRICTED ANTI-TUMOUR REACTION

Cytostatis of the H-2^d tumour LSTRA by H-2-restricted effector lymphocytes was inhibited by antisera against H-2.4 and H-2.31 but not by antisera against public specificities or non-H-2 antigens. The unexpected reaction of the same effector cells against Gardner tumour (H-2^k) was also shown to be inhibited by a combination of antisera against H-2.4 and H-2.31 but not by each antiserum used separately. The inhibitory capacity of these antisera was removed by absorption with B10.D2 but not with B10 lymphocytes. This indicated the presence of H-2^d-like specificities on gardner tumour which could function as self-recognition structures in an H-2K^d and H-2D^d restricted system.     

THE ROLE OF H-2 AND H-2-LIKE DETERMINANTS IN SYNGENEIC AND ALLOGENEIC CYTOSTASIS

Specific sensitization against H-2 determinants was effected by immunizing allogeneic mice with spleen and lymph node cells in H-2 congenic combinations. Lymph nodes from the sensitized and non-sensitized mice were respectively cultured together with H-2 syngeneic tumour cell lines. The growth and viability of the tumour cells was subsequently measured by the amount of radiothymidine incorporation. If the tumour cells incorporated less isotope when cultured with the immune cells than with the normal cells this was termed ‘cytostasis’. To identify the H-2 genes controlling the sensitization phase in the cytostasis assay, we studied the effect on different transplantable tumour target cells of lymphoid cells from mice sensitized against different congenic spleen cells. The results suggest that the cytostasis assay can measure an in vitro specific response to H-2-incompatible sensitizing antigens, and that I-B incompatibility, together with K and/or D, is essential to produce effectors. Furthermore, H-2 allogeneic sensitization could induce cytostasis even against tumour cells syngeneic for the H-2 halotype of the responder strain. The implications of these findings are discussed.     

STUDIES ON H-2 SPECIFICITIES ON MOUSE TUMOUR CELLS BY A NEW MICRORADIOASSAY*

Seven mouse tumour cell lines of different aetiology and origin were tested for the expression of surface alloantigens using twenty-four well defined H-2 allo-antisera and anti-Thy 1.2. We used a new radioassay that involves antibody-complement treatment of the tumour target cells followed by postlabelling the surviving tumour cells with ¹⁴C-thymidine. With a relatively high frequency the anti-H-2 sera were reacting differently with the tumour cells than with respective tive syngeneic lymphoid cells. Thirty-six anomalous reactions out of 129 investigated were detected. Absorbtion experiments performed with H-2 antigen positive or negative lymphoid cells revealed a striking similarity between these extra-specificities and H-2 specificities of foreign haplotypes. The results are discussed in relation to the biological importance of the extra-specificities, both with regard to origin (derepression) and function (transplantation antigen properties).     

RHODIOLA SACHALINESIS INDUCES THE EXPRESSION OF INDUCIBLE NITRIC OXIDE SYNTHASE GENE BY MURINE FETAL HEPATOCYTES (BNL CL.2)

We have examined the effect of the aqueous extract of Rhodiola sachalinensis root (RSE), a traditional herbal medicine, on nitric oxide (NO) synthesis in murine fetal hepatocytes (BNL CL.2) by measuring the stable end-product nitrite and the mRNA of inducible NO synthase (iNOS). Interferon-gamma (IFN-γ) by itself failed to induce NO synthesis in BNL CL.2 cells. RSE also did not elicit NO synthesis at concentrations up to 1000 μg/ml, but dose- and time-dependently induced NO synthesis in the presence of IFN-γ in BNL CL.2 cells. Whereas RSE or IFN-γ failed to induce detectable levels of iNOS mRNA, a combination of RSE and IFN-γ markedly induced iNOS mRNA in BNL CL.2 cells. Thus, we found that RSE triggered IFN-γ-primed BNL CL.2 cells to synthesize NO by inducing iNOS gene expression. The capability of RSE to induce NO synthesis might be related to the therapeutic efficacy of RSE on the liver diseases.     

CELLULAR AND MOLECULAR HETEROGENEITY OF H-2Kk ANTIGENS

Up to 70% of mouse spleen cells expressing H-2K^k alloantigens reacted with the monoclonal anti-H-2K^k antibody 11-4.1 in an indirect rosetting assay. When the cells that rosetted with the monoclonal antibody 11-4.1 were removed by density centrifugation, the residual unreactive cells reacted with the polyclonal anti-H-2K^k alloantiserum D23 but not with the monoclonal antibody 11-4.1 indicating the existence of two cell populations. In sequential immunoprecipitation experimens, the glycoprotein fraction of NP40 extracts from H-2K^k cells, after removal of antigens reacting with the monoclonal antibody 11-4.1 still contained structures reactive with the alloantiserum. Therefore, there are at least two antigenically distinct H-2K^k molecules which are differentially expressed on two subpopulations of spleen cells.     

CD4单克隆抗体在体内诱导小鼠胸腺细胞凋亡

为研究抗ＣＤ４ ＭｃＡｂ引起胸腺细胞减少的机理，本课题探讨了ＣＤ４ ＭｃＡｂ诱导胸腺细胞发生凋亡的可能性。小鼠注射抗ＣＤ４ ＭｃＡｂ后，皮质胸腺细胞体积缩小，染色质凝聚；ＰＩ染色后经流式细胞计测定，显示大量的亚二倍体细胞（凋亡细胞，Ａｐｏｐｔｏｔｉｃ ｃｅｌｌｓ），与正常小鼠相比Ｐ〈０．００１；片断化ＤＮＡ的百分比也明显高于正常小鼠，Ｐ〈０．０１。片断化ＤＮＡ在凝胶电泳上呈现典型的阶梯现象。细胞内     

GENETIC REGULATION OF THE EXPRESSION OF H-2K.5 ANTIGEN ON ERYTHROID CELLS BY H-2K END

Difference in the amounts of H-2K.5 antigens present on erythrocytes was observed, using quantitative absorption method, among several B10 congenic strains. However, no such variation was seen on the lymphocytes and lung cells. The variability in the amount of this antigen on the erythrocyte surface was primarily dependent on the haplotype of the H-2K end in the B10 recombinant strains examined. This suggests that the regulator of H-2 expression on erythrocytes is in this region. Further genetic analysis confirmed that this regulation functions in the cis-position. Finally, the H-2K.5 antigenic activity was expressed on the reticulocytes of all strains tested, but appeared lower in the type 2 group indicating that the regulation begins early in erythropoietic differentiation and eventually results in a complete loss of detectable activity.     

不同毒力株弓形虫速殖子排泄分泌抗原（ESA）对小鼠自然杀伤细胞（NK）作用的比较

为观察不同毒力株弓形虫排泄分泌抗原（Excretory—Secretory antigen,ESA）对小鼠NK细胞的作用,将18只雌性C57BL／6J小鼠随机分为3组,各组每鼠分别腹腔注射刚地弓形虫RH株ESA200μL（含ESA0．002mg）,PRU株ESA200μL（含ESA0．002mg）和PBS200μL。于注射后6天取脾,流式细胞仪检测脾脏NK细胞的比例,并用乳酸脱氢酶（Lactate dehydrogenase,LDH）法检测各组NK细胞的杀伤活性,ELISA检测血清IFN-γ水平。结果显示,RH株ESA处理组NK细胞比例（5．974-0．26）％,PRU株ESA处理组NK细胞比例（3．32±0．29）％,两组均明显高于PBS对照组（3．084-0．39）％（P〈0．001）,其中RH株ESA处理组明显高于PRU株ESA处理组,差异有统计学意义（P〈0．001）;不同毒力株ESA处理的小鼠NK细胞杀伤能力较对照组有明显的升高,3组间差异有统计学意义（P〈0．01）;不同毒力株ESA处理后小鼠血清的IFN．1水平升高,与PBS组相比差异有统计学意义（P〈0．01）,其中RH组小鼠血清中IFN-1浓度较PRU组升高更为明显,差异存在统计学意义（P〈0．05）。结果表明,弓形虫ESA有活化小鼠NK细胞的作用,且RH株ESA的作用强于PRU株ESA。     

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.     

用抗生物素蛋白—生物素酶标染色法探讨两种胚胎抗原对结肠癌的诊断价值

本文用新近发展起来的抗生物素蛋白-生物素酶标染色法(ABC 法)探讨癌胚抗原(CEA)与特定时期胚胎抗原(SSEA-1)在结肠癌中的分布及其对结肠癌的诊断价值。结果显示CEA 存在于几乎所有结肠癌病例(29/30),但也可在约40%左右的正常结肠粘膜表面上皮或腺上皮细胞膜上出现。SSEA-1存在于约84%的结肠癌病例,但仅微量存在于约20%正常腺隐窝基底部的上皮细胞膜。两者在结肠癌细胞及正常结肠上皮细胞膜上的分布情况也不同。认为用 ABC 法检测这两种抗原,特别是 SSEA-1在结肠癌中的呈现情况可能有助于临床诊断。本文并对 ABC 法略加讨论。     

GENE EXPRESSION OF A REGION OF CHROMOSOME 17 DURING MURINE SPERMATOGENESIS

The presence of H-2-linked gene products on spermatozoa and their time of appearance during spermatogenesis was determined. Thymus leukaemia antigen specificities 1, 2 and 3 could not be detected on spermatozoa by absorption of the antisera. Immunofluorescent studies with anti-Sip sera did not reveal any specific reactivity with target spermatozoa. In contrast, H-2D antigens were present on somatic as well as germ line components in testes so the time of their first appearance during spermatogenesis could not be precisely specified. Cell separation experiments indicate that H-2D antigens are present on pachytene spermatocytes and increased in quantity on spermatids. The sperm-specific isoenzyme of phosphoglycerate kinase, Pgk-2, appears at a later stage of spermatogenesis than do the H-2 region antigens.     

ABSORPTION ANALYSIS OF H-2D AND K ANTIGENS ON SPERMATOZOA

The presence of H-2D and K antigens on mouse spermatozoa has been investigated by absorption followed by testing on the proper target lymphocytes. It is concluded that, in addition to Ia antigens, H-2D and K antigens are indeed expressed on mouse sperm cells.     

SEROLOGICAL AND IMMUNOCHEMICAL STUDIES OF H-2 ALLOSPECIFICITIES ON k36, A SYNGENEIC TUMOUR OF AKR

Expression of H-2 antigenic specificities on K36, a spontaneous leukaemia originating from AKR (H-2^k) mice, was studied by serology and immunochemistry. Two ascites lines of the tumour, as well as a tissue culture adjusted and cloned tumour line, were used in these studies with similar results being obtained. K36 expresses on its cell surface D-region encoded H-2K antigens but does not express K-region encoded H-2K alloantigens. It also expresses on its cell membrane, H-2 specificities of foreign haplotypes not present on normal AKR lymphoid cells. The molecular basis of the H-2D^d specificity on K36 (H-2^k was analysed by immunoprecipitation and polyacrylamide gel electrophoresis. The specificity was shown to be present on a glycoprotein of apparent molecular weight 45,000. However, antisera against the H-2D^d private specificity (H-2.4) precipitate additional glycoprotein of 45,000D and also 70,000D. In tryptic peptide maps of the isolated 45,000D fraction precipitated by anti-H-2.4 serum from radiolabelled K36 glycoprotein, all H-2D^d specific peptides were present in the same quantitative ratio. This is consistent with the structural identity of the foreign H-2D^d from the K36 tumour with normal H-2D^d and supports the hypothesis of a regulator system controlling the H-2 allelism. Under certain circumstances such a system could cause suppression of one and derepression of the other H-2 gene products.