In vivo reexpression of H-2 antigens in B16 melanoma cells

We have previously shown that B16-A (H-2b) murine melanoma cells, when cultured in vitro for more than ten passages, have an undetectable or reduced expression of H-2Kb and Db antigens, respectively. We have now studied the possibility to restore H-2 expression (measured by quantitative antisera absorption) in B16-A cells either by a limited (30 days) period of in vivo growth or by treatment with immune interferon. In vivo transplants in allogeneic H-2k or H-2d mice and in H-2-compatible but Mls and multiple non-H-2 loci incompatible mice restored the normal expression of Kb and Db antigens. Cells obtained from tumors grown in syngeneic or in minor histocompatibility antigens-allogeneic mice showed only a weak increase in Db antigens. Such induction of H-2 expression appears to be mediated by the host's immune system, since (1) cells obtained from tumors grown in allogeneic BALB/c nude mice expressed much lower levels of H-2 antigens than those from tumors grown in normal BALB/c mice, and (2) it was possible to induce H-2 expression by growing B16 cells in syngeneic C57Bl/6 mice previously allostimulated with unrelated BALB/c tumor. In vitro treatment with immune interferon restored the expression of both Kb and Db antigens. We hypothesize that H-2 reexpression on B16 cells grown in allogeneic hosts could take place via the nonspecific components of the immune response, such as immune interferon.     

Interferon-mediated enhancement of metastasis. Are MHC antigens involved?

The relationship between major histocompatibility complex (MHC) antigens and metastasis was investigated on B16 melanoma variants. B16 cell lines express low amounts of murine MHC (H-2) antigens. A high expression can be induced in line B16-A by in vitro treatment with immune interferon (IFN-gamma) or by in vivo transplant in allogeneic mice. The increase of H-2 antigens correlated with an enhancement of lung colonization in young syngeneic mice. The higher metastatic capacity of B16-A cells with induced high levels of H-2 antigens was observed also in adult mice and in young mice pretreated with cyclophosphamide. These results were confirmed investigating the behaviour of a mutant B16 clone (B78H1) which was selectively resistant to the H-2-inducing action of IFN-gamma: lung colonization ability was not increased by IFN pretreatment. The study of variants derived from individual B16-A lung colonies revealed a wide range of H-2 levels. Variants with a low expression had a low colonization ability; one out of two variants with a high H-2 expression also was poorly colonizing. IFN-gamma-mediated H-2 expression appeared to act as an enhancer, rather than a determinant of B16 metastatic capacity.     

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.     

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.     

Growth of transplantable melanoma and leukaemia and prevention of virus-induced leukaemia in long lived radiation chimeras constructed with unmanipulated bone marrow.

Haemopoietic radiation chimeras across the H-2 barrier (BALB/c----C57Bl/6; H-2d----H-2b chimeras and vice versa) have been studied for their capacity to suppress the growth, or to reject, transplantable B16 melanotic melanoma and radiation leukaemia virus-induced, transplantable leukaemia. Also, radiation leukaemia virus (RadLV) obtained from the thymus of leukaemic C57Bl/6 mice was injected i.p. into established chimeras (H-2d----H-2b). As expected, long lived, graft versus host disease free allogeneic chimeras constructed with intact bone marrow were unable to reject the tumours both when recipients were BALB/c----C57Bl/6 or C57Bl/6----BALB/c chimeras. However, also inoculation of a large number of immunocompetent cells from normal BALB/c mice into BALB/c----C57Bl/6 chimeras, failed to promote a rejection of the tumours. On the contrary, the same amount of syngeneic (BALB/c) immunocompetent cells prevented growth of melanoma when transferred into athymic nude BALB/c mice, while the tumour grew unimpaired in untreated athymic nude BALB/c mice. The same type of H-2d----H-2b chimeras displayed complete resistance to inculation of leukaemogenic H-2b restricted RadLV while all H-2b----H-2b, syngeneically reconstituted mice developed disseminated leukaemia. These findings demonstrate that: (a) a powerful suppressive principle operates in the chimeras which does not allow effector function and anti-tumour activity of passively transferred normal, mature T cells from resistant BALB/c mice. Thus, no H-2 restriction of donor T cells can be advocated for suppression of anti-tumour effector functions in the chimeras. (b) New donor (BALB/c, H-2d) marrow character in the H-2d----H-2b chimeras prevents expression of the H-2b restricted viral activity and leukaemogenic transformation and/or proliferation.     

Enhancement of sheep erythrocyte (SRBC)-induced pleurisy in non-sensitized mice by cyclophosphamide: demonstration of natural cell-mediated immune reactivity to SRBC.

Intrapleural injection of 2 x 10(8) sheep erythrocytes (SRBC) into normal C57BL/6N (B6) mice produced a low but significant exudate leucocyte reaction, which was delayed in onset and mononuclear cell dominant. However, administration of 20-200 mg/kg cyclophosphamide (CY) in mice induced a dose-dependent enhancement of this reaction. In contrast, erythrocytes of syngeneic and allogeneic mice, rats, guinea-pigs rabbits and humans showed no or slight enhancement in CY-treated B6 mice. Maximal enhancement was observed on day 6 after CY treatment, but the reactions fell on day 7 and gradually decreased thereafter. B6 mice from specific pathogenfree colonies also showed strong reactions on day 6 after CY treatment. B6 mice demonstrated the highest reactions and C3H/He (C3H) mice were intermediate, while BALB/c mice showed very low responses. F1 hybrid (BALB/c x B6) mice showed an intermediate response as compared with the parent strains. The enhanced reactions reached their peak at 24 hr after SRBC injection and mainly consisted of macrophages and polymorphs. The enhancement could be successfully transferred to naive syngeneic mice by viable spleen cells from CY-treated mice but not by sera. The mediator cells could not bind to plastic petri dishes or a nylon-wool column, and were sensitive to anti-Thy-1.2 + C treatment . These results thus indicated that the enhancement was induced by the cell-mediated immune (CMI) mechanisms. The appearance of natural CMI reactivity to SRBC, and its role in relation to immunological reactions is discussed.     

Antigenic specificity of the cytolytic T lymphocyte (CTL) response to murine sarcoma virus-induced tumors. I. Preferential reactivity of in vitro generated secondary CTL with syngeneic tumor cells.

Incubation of spleen cells from mice having rejected a Moloney sarcoma virus (MSV)-induced tumor with syngeneic irradiated lymphoma or sarcoma cells bearing MSV-associated antigens in secondary mixed leukocyte-tumor cell cultures (MLTC) resulted in the generation of highly active cytolytic T lymphocytes (CTL) specifically directed against syngeneic target cells bearing MSV-associated antigens. When MSV-immune spleen cells from C57BL/6 (H-2b) and BALB/c(H-2d) mice were compared with respect to their ability to generate CTL in syngeneic secondary MLTC, it was found that both lymphoid cell populations were equally able to mount an anamnestic CTL response to MSV-associated antigens as assessed by a short-term 21Cr release assay. However, quantitative analysis of the activity of both CTL populations on either H-2b or H-2d tumor cells indicated that target cells sharing the same major histocompatibility complex (MHC) as the effector cells were lysed 10- to 100-fold more efficiently than allogeneic target cells. As suggested by the results of inhibition experiments using mixtures of 51Cr-labeled and unlabeled target cells, preferential lysis of syngeneic versus allogeneic tumor cells might be related to the establishment of effective adhesions between the former and CTL. Direct evidence for the role of MHC in determining the antigenic specificity of CTL directed against MSV-associated antigens was provided by results obtained using MSV-immune spleen cells from congenic resistant mice. Furthermore, studies of the response of F1 (H-2b/d) hybrid mice showed that stimulation of immune spleen cells with tumor cells from one parental strain or the other in secondary MLTC resulted in the generation of CTL capable of lysing tumor target cells of the same perental strain as the stimulating cells, but not of the other. The results thus suggested the presence of two sets of CTL precursor cells in F1 MSV-immune spleens, each set responding exclusively to tumor antigens associated with only one of the two parental phenotypes.     

Absence of major histocompatibility complex class I on neural stem cells does not permit natural killer cell killing and prevents recognition by alloreactive cytotoxic T lymphocytes in vitro

Potential applications of neural stem cells (NSCs) for transplantation requires understanding myosin heavy chain (MHC) expression and the ability of T cells and natural killer (NK) cells to recognize this progenitor population. Cells from the cortices of day-13 embryonic (E13) B6 (H-2(b)) mice were explanted and cultured to expand NSCs. Analysis of P2-P17-cultured cells using anti-MHC class I/II monoclonal antibodies (mAbs) showed marginal expression of both products. Although recombinant murine interferon-gamma (rmIFN gamma) exposure did not alter the multipotential capacity of these stem cells, titration of mrIFN gamma NSC cultures demonstrated that MHC molecules could be strongly upregulated after addition of 3 ng/ml rmIFN gamma for 60 hours. To assess the susceptibility of NSCs with low or absent versus high levels of MHC expression to lysis by cytotoxic T lymphocyte (CTL) and NK populations, untreated and rmIFN gamma-treated NSC target cells were examined. Untreated NSCs were not recognized by BALB/c (H-2(d)) allospecific anti-H-2(b) CTL, consistent with the mAb findings; however, upregulation of MHC products on both early and later passaged NSCs resulted in their efficient lysis by CTL. NK cells were prepared from syngeneic B6 or allogeneic BALB/c mice. Although NK cells effectively killed control YAC-1 target cells, these effectors did not kill MHC-deficient (or expressing) NSC targets. Thus, similar to hematopoietic, embryonic, and mesenchymal stem cell populations, unmanipulated NSCs are not readily killed by T and NK cells. These findings suggest that following transplant into syngeneic or allogeneic recipients, NSCs may exhibit diminished susceptibility to clearance by host T- and NK-cell populations.     

Semi-allogeneic vaccines and tumor-induced immune tolerance

Experimental results from studies with inbred mice and their syngeneic tumors indicated that the inoculation of semi-allogeneic cell hybrids (derived from the fusion between syngeneic tumor cells and an allogeneic cell line) protects the animal host from a subsequent lethal challenge with unmodified syngeneic tumor cells. Semi-allogeneic somatic cell hybrids were generated by the fusion of EL-4 T lymphoma cells (H-2^b) and BALB/c-derived renal adenocarcinoma RAG cells (H-2^d). Cell hybrids were injected intra-peritoneally (i.p.) in C57BL/6 mice (H-2^b) before challenging the mice with a tumorigenic dose of EL-4 cells. Semi-allogeneic tumor cell hybrids could not form a tumor in the animal host because they expressed allogeneic determinants (H-2^d) and were rejected as a transplant. However, they conferred protection against a tumorigenic challenge of EL-4 cells compared to control mice that were mock-vaccinated with i.p.-injected phosphate-buffered saline (PBS) and in which EL-4 lymphomas grew rapidly to a large size in the peritoneal cavity. Screening of spleen-derived RNA by means of focused microarray technology showed up-regulation of genes involved in the Th-1-type immune response and in the activation of dendritic antigen-presenting cells (APC). The results of our studies confirm the role of APC in mediating the immune protection induced by semi-allogeneic vaccines by activating a Th-1 response; these studies also reveal that semi-allogeneic vaccines are able to interfere with or even block the tumor-mediated induction of immune tolerance, a key mechanism underlying the suppression of anti-tumor immunity in the immune competent host.     

Influence of class I H-2 gene expression on local tumor growth. Description of a model obtained from clones derived from a solid BALB/c tumor

A BALB/c methylcholanthrene-induced tumor was studied for class I and II H-2 expression. GR9 was adapted to tissue culture without any passage in vivo to avoid immunoselection mechanisms. Forty-three clones were obtained and typed for H-2 antigens. A variety of techniques were used for typing, for example, 51Cr release assay, radiobinding assay; absorption with anti H-2 monoclonal antibodies or H-2 allo-antisera and alloreacting cytotoxic T lymphocytes. A heterogeneity of H-2 expression was detected in these clones with clones H-2 K+/D+, to clones H-2K-D-. This heterogeneity was also evident when several GR9 clones were tested for in vivo growth in syngeneic BALB/c mice. Two clones, A7 and B9, were selected for further studies and injected into syngeneic animals to measure local tumor growth. A7 (K+/D+) was rather immunogenic while B9 (K-/D-) was not. These differences were probably due to an immunoresponse, since both clones grow similarly in irradiated syngeneic BALB/c mice. Immunoprecipitations of H-2 antigens and SDS-PAGE analysis confirmed the results obtained in serology. Three isoenzymes were also determined in GR9 clones and their electrophoretic mobility was always identical to that obtained in normal BALB/c tissues. These results suggest that K and D molecules are important structures for an immunoresponse against tumor-associated antigens.     

Rat thymic epithelium positively selects mouse T cells with specificity for rat MHC class II antigens but fails to induce detectable tolerance in the mouse T cells to the rat MHC antigens.

BALB/c (H-2d) nude mice were grafted with allogeneic AKR/J (H-2k) or xenogeneic (ACI-N rat, RT1av1) fetal thymuses which were depleted of hemopoietic cells by incubating with 2'-deoxyguanosine (2'dGuo) in vitro prior to grafting. The nylon-wool-passed LN T cells from nude mice grafted with 2'dGuo-treated AKR/J thymus showed a poor proliferative response to B10BR (H-2k) stimulator cells, confirming that mouse thymic epithelium has the capacity to induce tolerance against the mouse MHC antigens on the thymic epithelium. On the other hand, the nylon-wool-passed LN T cells from nude mice grafted with untreated or 2'dGuo-treated ACI/N rat thymus showed significant proliferative responses to ACI/N, which can be blocked by anti-rat MHC class II mAb, whereas the nylon-wool-passed LN T cells from nude mice grafted with syngeneic thymus hardly responded to the xenogeneic stimulator cells. These results suggest that rat thymic stromal cells including thymic epithelium can not induce detectable tolerance in mouse T cells to rat MHC antigens; but rat thymic epithelium may positively select mouse T cells with specificity for rat MHC class II antigens, resulting in a mouse T cell repertoire with strong xeno-reactivity.     

Serological analysis of H-2 antigens expression on the cell surface of selected mouse tumors

Studies on histocompatibility antigens expression on different selected mouse tumor cells revealed very variable pattern. No qualitative alterations in H-2 antigens expression on the cells of the following tumors: RL male 1 and MP26a of BALB/c (H-2d) mice, ASL-1 and RADA-1 of A (H-2a) mice, EL-4 and E male G2 of C57B1/6 (H-2b) mice, K36 and AKSL-4 of AKR (H-2k) mice could be detected. However, the quantitative differences in H-2 antigens expression on some of these tumors were observed.     

Host-Reactive Cytotoxic T Lymphocyte Precursors in Long-lived Fully Allogeneic Mouse Bone Marrow Chimeras

Fully H-2-incompatible chimeric mice were constructed by grafting lethally (950 rad) irradiated germ-free (GF) CBA (H2k) mice with anti-Thy 1 antibody plus complement-treated allogeneic C57Bl/6 (B6) (H2b) bone marrow cells. These chimeric mice were kept for more than 11 months, either under GF conditions or under barrier-sustained specific-pathogen-free (SPF) conditions. Controls included nonirradiated, nontransplanted, sex- and age-matched CBA and B6 mice raised under SPF conditions, and syngeneic chimeric mice of the CBA----CBA type kept under GF and SPF conditions. All chimeric mice were completely repopulated with donor-type lymphoid cells and showed no clinical or histological evidence of graft-versus-host disease. From the fully allogeneic chimeric mice, we enumerated the numbers of splenic cytotoxic T lymphocyte precursors (CTL-p) that could be clonally expanded under limiting dilution conditions in response to third-party alloantigens, or nonmodified and trinitrophenyl (TNP)-modified stimulator cells bearing host or donor H-2 antigens. The existence of high numbers of alloreactive and host- or donor-type H-2-restricted TNP-specific CTL-p in the spleens of fully allogeneic chimeras indicated almost normal immunocompetence. The surprising finding, however, was that large numbers of host (CBA)-reactive splenic CTL-p were inducible under limiting dilution conditions in healthy long-lived allogeneic chimeras, although these chimeric mice were devoid of any histological or clinical signs of graft-versus-host disease.     

Definition of alien H-2 determinants on a Friend leukaemia by analysis of alloreactivity of CTL from primary MLC

A Friend virus-induced tumour of BALB/c (H-2d) origin, HFL/d, was examined for the expression of alien H-2 antigens. The alloantigens on HFL/d were typed by generating CTL in primary MLC with HFL/d as stimulator and measuring reactivity to targets with known H-2 antigens, and confirmed by assessing recognition of HFL/d targets by CTL generated in primary MLC with stimulators expressing known H-2 antigens. Potential cross-reactivities between alloantigens were analysed by cold-target inhibition experiments. BALB/c cells stimulated with HFL/d lysed H-2b targets, and BALB/c anti-H-2b CTL lysed HFL/d; analysis with recombinant haplotypes demonstrated both H-2Kb and H-2Db alien antigens antigens on HFL/d. C57BL/6 (H-2b) cells stimulated with HFL/D recognized H-2Kd, H-2Dd, and an additional determinant unique to HFL/d. (BALB/c x B6)F1 cells also recognised a unique HFL/d determinant not of H-2b or H-2d origin. These unique determinants, which induced a strong cytotoxic response in primary MLC, were not shared by BALB/c or B6 tumours induced by cross-reactive FMR viruses. Thus, HFL/d expressed the K and D antigens of its strain of origin, two typed alien H-2 antigens, and at lest one other untyped antigen which may represent an additional H-2 determinant. These studies further demonstrate the utility of examining the reactivity of CTL generated in primary MLC to probe for the presence of alien H-2 antigens.     

供体脾细胞输注诱导小鼠移植耐受及其机理的研究

目的以持续供体脾细胞输注的方法建立异基因嵌合体动物模型,并探讨移植耐受形成的机制。方法BALB/c（H-2     

Vaccination with allogeneic dendritic cells fused to carcinoma cells induces antitumor immunity in MUC1 transgenic mice.

Fusions of autologous tumor cells with allogeneic dendritic cells (DC) represent an approach for the induction of antitumor immunity. In the present studies, we investigated the antitumor effects of vaccinating MUC1-transgenic (MUC1.Tg) mice with MC38/MUC1 carcinoma cells fused to allogeneic DC from BALB/c mice (allo-DC, H-2(d)) or syngeneic DC from C57BL/6 mice (syn-DC, H-2(b)). Both allo and syn fusion cells (FC/MUC1) expressed MHC class II, costimulatory molecules, and the MUC1 antigen. Allo-FC/MUC1 exhibited dual expression of MHC class I haplotypes (H-2(d)/H-2(b))and MUC1 antigen. By contrast, only H-2(b) and MUC1 antigen were expressed by syn-FC/MUC1. CTLs from MUC1.Tg mice immunized with allo- or syn-FC/MUC1 fusion cells lysed MC38/MUC1 targets. Moreover, immunization with allo- or syn-FC/MUC1 was effective in eliminating established MUC1-positive pulmonary metastases in MUC1.Tg mice. These results indicate that immunization of MUC1.Tg mice with syn- or allo-FC/MUC1 is effective in reversing immunologic unresponsiveness to MUC1 antigen and inducing immunity against MUC1-positive tumors. The findings in the present study have broader clinical implications for fusion cell vaccines.     

H-2 antigen-specific cytotoxic T cells induced by concanavalin A: estimation of their relative frequency.

Specific and nonspecific lysis of DBA/2 (H-2d) mastocytoma cells, P815, by concanavalin A (Con A)-induced cytotoxic T cells was studied. In the assay for nonspecific lysis, phytohemagglutinin (PHA) was present to glue the target and killer cells together. We have presented evidence previously to show that PHA reveals only, and all, cytotoxic T cells. In the assay for specific lysis the only glue present was specific receptors on a fraction of the killer cells and surface antigens of P815. We show that when PHA was present, Con A-induced cells which were syngeneic, semi-syngeneic, or allogeneic, lysed P815 very efficiently in a 4-h 51Cr release assay. However, only Con A-induced T cells which were allogeneic and did not carry H-2d lysed P815 when the assay was carried out in the absence of PHA. In an experiment with two target cells, Con A-induced B10 (H-2b) T cells lysed B10.D2 (H-2d) targets specifically but did not lyse B10 targets, while Con A-induced B10.D2 T cells lysed B10 targets specifically but not B10.D2 targets. Furthermore, Con A-induced B6 (H-2b) T cells from normal mice lysed P815 specifically but Con A-induced B6 T cells from irradiated F1 (B6 x BALB/c) (H-2b/d) mice reconstituted with B6 bone marrow did not lyse P815 specifically. A fraction of Con A-induced T cells therefore appear to bear specific surface receptors for nonself H-2 coded structures. We describe conditions of assay and a new method of plotting the results such that nonspecific (PHA-revealed) and specific (PHA-independent) cytotoxicity can be quantitatively compared. We conclude that 1-4% of the total Con A-induced cytotoxic effector T cells are directed against any particular foreign H-2 haplotype. This is the first estimate of the relative frequency of antigen-reactive cytotoxic T cells.     

A syngeneic anti tumor serum recognizing a complex H-2 alloantigen

A methylcholanthrene induced tumor of BALBc (H-2d) origin had a high rate of spontaneous regression when transplanted into syngeneic animals. The tumor induced in BALB/c mice iso-antibodies with high anti-tumor cytotoxic activity. A specificity analysis of such BALB/c anti MCG4 sera revealed that the antibodies were directed against a tumor antigen which is very similar to H-2- alloantigens (e.g. H-2.5) expressed on normal cells of certain foreign mouse strains. The sera also reacted with nine out of ten B10.W congenic strains bearing H-2wi haplotypes derived from wild mice. Whether the tumor antigen is identical with foreign H-2 antigens or only cross-reactive cannot be decided at present.     

Modification of the anti-tumour immune response by suppressor gene products of lymphoid cells.

Immunization of mice with BALB/c spleen cells leads to the production of effector lymphocytes which are cytostatic in in vitro assays to tumours of the same haplotype or carrying cross-reacting antigens. Immunization with B10.D2, a strain H-2 identical with BALB/c, does not generate cytostatic effector cells, nor does immunization with the F1 hybrids between B10.D2 and BALB/c. Analysis of the progeny of backcrosses of the F1 hybrids to BALB/c gave evidence that the suppressive effect of B10.D2 immunization is controlled by a single gene. Spleen cells from mice immunized with BALB/c or B10.D2 cultured in vitro with the corresponding stimulator cells yielded soluble factors in the supernatants that were respectively capable of amplifying or suppressing the in vitro cytostatic effect. Such experiments revealed that the inhibition of cytostasis caused by immunization with B10.D2 is not at the sensitization but at the effector phase of the assay. Possible mechanisms of action of this suppressor gene are discussed.     

COINCIDENT EFFECT IN THE GRAFT-VERSUS-HOST REACTION OF BALB/c LYMPHOID CELLS DERIVED FROM MICE IMMUNE EITHER TO ALLOGENEIC NORMAL TISSUE OR TO SYNGENEIC CHEMICALLYINDUCED FIBROSARCOMATA

By using the graft-versus-host reaction (GVHR) based on the spleen enlargement test of Simonsen (1962), lymphocytes from BALB/c mice immune to syngeneic ST2 or ST5 sarcomata were shown to behave like BALB/c anti-DBA/2 lymphocytes in specifically increasing the GVHR in (BALB/c × DBA/2)F₁ newborns as compared with non-immune BALB/c cells. No such effect was evident when BALB/c anti-ST2 or anti-ST5 lymphoid cells were given to (BALB/c × C3Hf)F₁ and to (BALB/c × AKR)F₁ mice; anti-ST5 lymphocytes were also ineffective into (BALB/c × C57BL/6)F₁ newborns in which anti-ST2 lymphocytes were able to increase the GVHR over the background induced by non-immune BALB/c cells. A third immunogenic BALB/c fibrosarcoma C-1 was able to activate syngeneic lymphocytes which gave an augmented GVHR in (BALB/c × C3Hf)F₁ and (BALB/c × AKR)F₁ but not in the other hybrids. The efficacy of BALB/c anti-ST2 or -ST5 lymphocytes in reproducing a BALB/c anti-DBA/2 like anamnestic response in (BALB/c × DBA/2)F₁ mice support previous findings indicating that non-H-2 alien histocompatibility antigens of the DBA/2 strain, are expressed on these tumour cells. The GVHR induced by lymphoid cells immune to C-1 tumour are also in keeping with serological and transplantation studies (reported elsewhere) indicating the expression of foreign H-2^k antigens on this sarcoma.