Immunity to melanoma in mice immunized with transfected allogeneic mouse fibroblasts expressing melanoma-associated antigens

Summary Transfection of genomic DNA from B16 mouse melanoma into LM(TK^–) fibroblasts led to the generation of several clones of transfected cells that strongly expressed B 16 melanoma-associated antigens (MAA). The transfected cells retained their H-2^k markers and served as allogeneic cells with expressive MAA in C57BL/6 mice, syngeneic with the melanoma. The cells were capable of eliciting primary anti-B16 immune responses in vitro in spleen cells from C57BL/6 mice. Immunization of C57BL/6 mice with the transfected cells led to the generation of anti-B16 cytotoxic activity in spleen cells, and C57BL/6 mice immunized with the MAA-positive transfected cells were partially resistant to a lethal challenge with B16 melanoma cells. Under similar conditions, B16 cells were nonimmunogenic. Therefore, transfected allogeneic LM(TK^–) fibroblast cells expressing MAA served as more potent anti-melanoma immunogens than the parental B16 tumor cells themselves.     

MHC antigen expression by melanomas recovered from mice treated with allogeneic mouse fibroblasts genetically modified for interleukin-2 secretion and the expression of melanoma-associated antigens

Recent approaches toward the immunotherapy of neoplastic disease involve the introduction of expression-competent genes for interleukin-2 (IL-2) into autologous malignant cells. Treatment of tumor-bearing experimental animals with the IL-2-secreting cells successfully induces partial and at times complete remissions. In most instances, however, although delayed, progressive tumor growth continues. Here, certain of the characteristic of B16 melanomas (H-2^b) persisting in C57BL/6 mice (H-2^b) treated with an IL-2-secreting, melanoma-antigen-positive cellular immunogen (RLBA-IL-2 cells) are described. Unlike the melanoma cells first injected, B16 cells recovered from mice treated with RLBA-IL-2 cells were deficient in the experssion of MHC class I, but not class II determinants. Deficient MHC class I expression correlated with the cells' resistance to cytotoxic T lymphocytes (CTL) from the spleens of mice immunized with RLBA-IL-2 cells. Melanomas persisting in mice treated with non-IL-2-secreting, melanoma-antigen-positive cell constructs (RLBA-ZipNeo cells) were also deficient in the expression of MHC class I determinants, and the melanoma cells were resistant to CTL from mice immunized with RLBA-ZipNeo cells. Thus, the expression of melanoma-associated antigens rather than IL-2-secretion correlated with deficient MHC class I expression by the persistent melanomas. This point was substantiated by the expression of MHC class I antigens by melanomas persisting in mice treated with IL-2-secreting, melanoma-antigen-negative LM cells (LM-IL-2); it was equivalent to that of melanomas in untreated mice. The involvement of MHC class I antigens in the immune resistance of persistent melanoma cells from mice treated with the melanoma-autigen-positive immunogens was indicated by the effect of interferon (IFN) orN-methyl-N-nitro-N-nitrosoguanidine (MNNG) on the susceptibility of the cells to anti-melanoma CTL. Treatment of the resistant melanomas with IFN or MNNG stimulated MHC class I antigen expression and restored the cells' sensitivity to CTL from mice immunized with IL-2-secreting or nonsecreting, melanoma-antigen-positive cellular immunogens. Prior treatment of the treated cells with antibodies to MHC class I determinants inhibited the cells' susceptibility to CTL from mice immunized with RLBA-IL-2 cells.     

Cytotoxic activity toward mouse melanoma following immunization of mice with transfected cells expressing a human melanoma-associated antigen

Summary B78H1 is a mouse melanoma cell line that is weakly antigenic in syngeneic mice. In an attempt to augment their immunogenicity, B78H1 cells were transfected with genomic DNA from a line of human melanoma cells expressing a 96-kDa melanoma-associated antigen (ICAM-1). A selective co-amplification procedure was employed that generated a population of transfected cells (Ui11) that expressed fivefold higher quantities of the melanoma-associated antigen than the cells from which the DNA was obtained. To test the transfected cells' relative capacity to generate a cellular immune response against B78H1 cells, Ui11 cells and B78H1 cells were administered (in parallel) to syngeneic C57BL/6 mice, susceptible to the growth of the melanoma. Each cell line (lethally iradiated beforehand) was injected intraperitoneally at weekly intervals into the mice. After two or three injections, a standard chromium-release assay was employed to detect the presence of cellular immunity toward B78H1 cells. The population of spleen cells from mice immunized with the transfected melanoma cells exhibited higher levels of cytotoxicity toward B78H1 cells than spleen cells from mice immunized with equivalent numbers of nontransfected cells. This observation is consistent with the notion that the transfected human melanoma-associated antigen acted as a second antigen capable of potentiating cellular immune responses against the weakly immunogenic determinants of the mouse melanoma cells. The introduction of genes for foreign antigens into weakly antigenic tumor cells may generate immunogens that can lead to augmented anti-tumor cellular immune responses.     

Rejection of B16 melanoma induced by expression of a transfected major histocompatibility complex class I gene.

Transfection of a functional major histocompatibility complex class I gene into certain tumor cells, induced by oncogenic viruses or chemical carcinogens, can effectively abrogate their tumorigenic activity. Since experimentally induced tumors possess strong tumor-specific transplantation antigens, expression of cell surface class I antigens may present the tumor cells to appropriate immune effector cells. Most spontaneously arising tumors do not possess tumor-specific transplantation antigens, and their tumorigenicity may not be affected by the expression of a transfected class I gene. We demonstrate that the poorly immunogenic B16-BL6 melanoma can be rendered nontumorigenic in syngeneic mice by the expression of the class I H-2K antigen but not the class II I-A antigen. Furthermore, the poorly tumorigenic, class I-expressing B16-BL6-transfected cells can effectively immunize syngeneic C57BL/6 mice against the highly tumorigenic, class I-deficient B16-BL6 parental cells. Our success in experimentally manipulating the tumorigenicity of a spontaneously derived neoplasm offers hope for a potential modality for the effective treatment of human cancer.     

Resistance to 402AX teratocarcinoma involves immunity to minor histocompatibility antigens

The 402AX teratocarcinoma is a 12/J-derived mouse major histocompatibility complex (MHC) antigen negative tumor that is induced to express H-2^b class I antigens during rejection. Resistance to 402AX by MHC allogeneic and syngeneic mice is immunologically mediated and involves the recognition of tumor-associated antigens (TAA) in the context of induced MHC class I antigens. The current studies were undertaken to define the 402AX TAAs. Reconstitution of irradiated susceptible hosts (129/J) with 402AX-primed resistant spleen cells (C57BL/6) results in acute graft-versus-host disease, suggesting that tumor-primed C57BL/6 splenocytes are reactive to tumor genotype (129/J) minor histocompatibility (Hm) antigens. C57BL/6 anti-129/J effector cells, although not directly cytotoxic for 402AX cells, are specifically cold target inhibited by 402AX cells. Genetically susceptible hosts (C3H.SW) immunized to 129/J Hm antigens by skin grafting become resistant to an i.p. challenge of 402AX cells. These results suggest that 129/J Hm antigens may be the TAAs recognized during genetically controlled rejection of the 402AX teratocarcinoma.     

The biological effects of syngeneic and allogeneic cytokine-expressing prophylactic whole cell vaccines and the influence of irradiation in a murine melanoma model

Allogeneic whole tumour cell vaccines are inherently practical compared with autologous vaccines. Cell lines are derived from allogeneic tumour, grown in bulk and then administered as a vaccine to the patient, following irradiation, which not only prevents any replication but also enhances antigen presentation. Protection is believed to occur through the presentation of antigens shared between the syngeneic and allogeneic tumours. Although cytokine-transfected tumour whole cell vaccines have been used clinically, little data is available comparing the effects of immunomodulatory cytokine-transfection directly on the same cells when used as both an allogeneic and autologous vaccine. To address this, weakly immunogenic B16-F10 (H-2^b) murine melanoma was transfected to secrete either GM-CSF, IL-4 or IL-7. Prophylactic vaccination of both syngeneic C57/BL6 (H-2^b) (B6) and allogeneic C3H/Hej (H-2^k) (C3H) mice showed the effects of transfected cytokine varied between models. Both GM-CSF and IL-7 significantly (P<0.05) increased the levels of protection within syngeneic B6 mice, but had a diminished effect (P>0.05) within C3H allogeneic mice. Allogeneic B16-F10 cells and syngeneic K1735 cells generated CTL against K1735 suggesting cross-reactive immunity. Using cells labeled with fluorescent dye we demonstrate that irradiated vaccines, of either syngeneic or allogeneic origin, appear to generate potent immune responses and fragments of either vaccine remain at the injection site for up to 9 days. This study shows that protection can be enhanced in vivo by using transfected cytokine, but suggests that irradiated whole cell vaccines, of either tissue-type, are rapidly processed. This leads to the conclusion that the cytokine effects are transient and thus transfection with cytokine may be of limited long-term use in situ.     

Augmentation of syngeneic tumor-specific immunity by semiallogeneic cell hybrids

Hybrid cell lines were established from fusions between lipopolysaccharide- (LPS) stimulated C57BL/6J spleen cells and MPC-11 tumor cells (45.6TG1.7, abbreviated M45), and were tested for their ability to immunize semiallogeneic mice against a parental tumor challenge. These hybrids were tumorigenic in syngeneic (BALB/c X C57BL/6J) F1 (CB6F1) mice but did not grow in semiallogeneic (BALB/c X A/J) F1 (CAF1) mice. All hybrids express both parental major histocompatibility antigens (H-2b and H-2d) as detected by indirect immunofluorescence and by their ability to function as either stimulators or targets for allogeneic cytotoxic lymphocytes (CTL). M45 tumor-associated antigens (TAA) were expressed on the hybrid surface as shown by their ability to act as either stimulators or targets for syngeneic CTL specific for M45 TAA. Immunization of semiallogeneic CAF1 mice with the hybrids i.p. followed by a challenge with M45 tumor cells resulted in extended survival when compared to untreated mice or animals immunized i.p. with M45 tumor cells. This immunity was specific and was not due to an allogeneic effect; immunization with an unrelated H-2bd tumor, 70Z/3, or H-2bd B6D2F1 spleen cells or with semiallogeneic spleen cells plus M45 did not protect mice from M45 challenge. Interestingly, prophylactic priming with semiallogeneic hybrid tumor cells or parental myeloma cells led to M45-specific CTL and "help" for an in vitro CTL response; however, the degree of CTL priming by hybrid tumors was not augmented when compared to the level of CTL achieved with parental tumor alone. Hence, stimulation of CTL activity per se by hybrid tumor cells cannot explain the protective effect of hybrid tumor immunization. These studies nevertheless confirm that semiallogeneic hybrids, which we show express TAA and alloantigens, can be used to immunize mice against a lethal syngeneic myeloma tumor challenge.     

Tumor rejection mediated by transfection with allogeneic class I histocompatibility gene

Non-self class I histocompatibility Ag can act as strong alloantigens and be recognized as distinct targets by CTL. To study the possibility of using allograft rejection to generate tumor-specific immunity, we have introduced an allogeneic class I histocompatibility gene, the H-2Kb gene, into a k haplotype tumor, K36.16, by DNA-mediated gene transfer. The K36.16 tumor grows readily and does not confer protective immunity in AKR mice. A total of 37 H-2Kb-transfected K36.16 clones (Kb/K36.16) was isolated and studied individually. The Kb/K36.16 clones were found to differ significantly in the amount of the exogenous H-2Kb antigens expressed on their cell surface. Moreover, as a result of the transfection, the level of expression of the endogenous H-2Dk Ag was also altered when compared to that of the parental K36.16 tumor cells. All the Kb/K36.16 clones that were positive for the H-2Kb Ag were rejected by the semisyngeneic AKR mice. Moreover, some of these Kb/K36.16 clones were also rejected by syngeneic (AKR x C57BL/10)F1 mice. In consequence of immunization with the Kb/K36.16 clones, the AKR and F1 mice were able to survive a subsequent challenge of the wild-type, unmodified, parental K36.16 tumor cells. More importantly, some of these Kb/K36.16 clones demonstrated an active and specific immunotherapeutic effect, and they were able to eradicate the growth of the parental K36.16 tumor cells in AKR mice. This observation therefore reinforces the feasibility of using DNA-mediated gene transfer as a molecular approach to abrogate tumor growth.     

Transfection and expression of syngeneic H-2 genes does not reduce malignancy of H-2 negative teratocarcinoma cells in the autologous host

Rejection of the MHC class I negative 402AX teratocarcinoma is accompanied by induction of tumor cell-encoded H-2K and H-2D antigens by the genetically resistant host. To determine whether MHC antigen expression is required for 402AX rejection, we have prepared H-2Db-transfected 402AX cells (402AX/Db). Transfectants express high levels of H-2Db, most of which is not associated with beta 2-microglobulin. MHC syngeneic and allogeneic mice susceptible to 402AX are resistant to 402AX/Db, suggesting that MHC class I antigen expression is required for tumor rejection. Autologous 129 hosts, however, are susceptible to 402AX/Db. 402AX cells transfected with the H-2Kb gene (402AX/Kb) are also lethal in the autologous 129/J host, but rejected by MHC syngeneic and allogeneic mice. Non-129 strain 402AX-susceptible mice pre-immunized with 402AX/Db or simultaneously challenged with 402AX/Db plus 402AX are immune to 402AX. Mice immunized with 402AX/Db produce MHC class I induction factor. 402AX/Db and 402AX cells are lysed equally by natural killer cells, indicating that in 402AX cells the expression of class I antigens is unrelated to NK susceptibility. These studies confirm the requirement for class I expression in 402AX immunity, but demonstrate that in the autologous host immunity requires additional factors beyond class I antigen expression.     

Characterization of newly isolated monoclonal antibodies against MHC of a Japanese wild mouse

We have already developed nine B10.MOL congenic strains carrying H-2 haplotypes derived from Japanese wild mice, Mus musculus molossinus, with the C57BL/10 genetic background. To obtain monoclonal antibodies against the H-2 antigen of the Japanese wild mouse, we carried out cell fusion using spleen cells from the animal immunized with one of the B10.MOL strains, B10.MOL-SGR (H-2 ^wm7). As a result, 19 hybridomas producing monoclonal antibodies were produced. Analysis with the intro-H-2 recombinants derived from B10.MOL-SGR indicated that 8 of them reacted with the class I and II with the class II molecule. The class I antibodies were tested for their cross -reactivities on wild mice and on the panels of standard inbred and B10.MOL strains. Most of the antibodies reacted with both the Japanese wild mice and the other subspecies, including standard inbred, while two antibodies highly specific for the donor H-2K region reacted with only three wild-derived mice, two M. m. molossinus from Anj o and Shizuoka, Japan, and one M. m. domesticus from Pigeon, Canada. In addition, all of the other four antibodies reactive with the K antigen of B10.MOL-SGR also reacted with the same three wild mice. The wild mice belonging to different subspecies might share very similar H-2K antigenic determinants in spite of their genetic and geographical remoteness.     

Genetic control of murine T cell proliferative responses to Mycobacterium leprae. V. Evidence for cross-reactivity between host antigens and Mycobacterium leprae

T cell proliferative responses to Mycobacterium leprae were measured by immunization of mice at the base of the tail with Ag and challenging lymphocytes from draining lymph nodes in culture with M. leprae. C57BL/10J and B10.BR mice were identified as low responder mice and the congenic strains B10.M, B10.Q, and B10.AKM as high responders whereas F1 (high x low) hybrid mice were found to be low responders. The cellular basis of low responsiveness did not appear to result from a defect in Ag-presenting cells or the activation of suppressor T cells by M. leprae. The influence of the environment in which T cells developed on responsiveness to M. leprae was analyzed in chimeric mice prepared by irradiating F1(C57BL/10J x B10.M) mice and reconstituting with bone marrow from C57BL/10J, B10.M, or F1 donors. Six weeks later, chimeric mice were immunized with M. leprae, lymph node cells were subsequently prepared, and H-2 phenotyped and challenged in culture with M. leprae Ag. T cell proliferative responses were found to be low in all cases, similar to those observed using lymph node cells from F1 hybrid mice. These results suggested that high responder B10.M lymphocytes developing in the irradiated F1 mice became tolerized to antigenic determinants found on M. leprae. This implied cross-reactive epitopes existed between some mouse strains and M. leprae. Low responsiveness to M. leprae in low responder and F1 hybrid mice may result from tolerance to H-2-encoded Ag that show cross-reactivity with M. leprae.     

Treatment of breast cancer with fibroblasts transfected with DNA from breast cancer cells.

This investigation was based on the hypothesis that weakly immunogenic, breast cancer-associated Ags, the products of mutant or dysregulated genes in the malignant cells, will be expressed in a highly immunogenic form by semiallogeneic IL-2-secreting fibroblasts transfected with DNA from breast cancer cells. (Classic studies indicate that transfection of genomic DNA can stably alter both the genotype and the phenotype of the cells that take up the exogenous DNA.) To investigate this question, we transfected LM mouse fibroblasts (H-2k) modified to secrete IL-2 with genomic DNA from a breast adenocarcinoma that arose spontaneously in a C3H/He mouse (H-2k). To increase their nonspecific immunogenic properties, the fibroblasts were also modified before transfection to express allogeneic MHC determinants (H-2Kb). Afterward, the IL-2-secreting semiallogeneic cells were cotransfected with DNA from the spontaneous breast neoplasm, along with a plasmid (pHyg) conferring resistance to hygromycin. Pooled colonies of hygromycin-resistant cells were then tested in C3H/He mice for their immunotherapeutic properties against the growth of the breast neoplasm. The results indicated that tumor-bearing mice immunized with the transfected cells survived significantly longer than mice in various control groups. Similar beneficial effects were seen in C57BL/6 mice injected with a syngeneic breast carcinoma cell line (EO771) and semiallogeneic, IL-2-secreting fibroblasts transfected with DNA from EO771 cells. The immunity was mediated by CD8+ T cells since immunized mice depleted of CD8+ cells failed to resist tumor growth.     

Genetic control of murine immune responses to larval Dirofilaria immitis

Previous studies have demonstrated that BALB/c mice, immunized against infection with Dirofilaria immitis, were capable of killing a significant percentage of challenge larvae found within diffusion chambers. The percentage of larvae killed by immunized mice was, however, less than in immunized dogs and unlike immunized dogs, mice were unable to retard the development of the surviving larvae. The objective of the present study was to test 3 inbred strains of mice to determine whether a higher level of protective immunity would develop in these hosts and if larval growth retardation would occur. DBA/2J and C57BL/6J mice and their F1 hybrids B6D2F1/J were used in these studies; it was determined that there were differences in susceptibility among the 3 strains but no difference in ability to eliminate larvae from challenge infections. Growth retardation was seen in larvae recovered from immunized DBA/2J and C57BL/6J mice but not in B6D2F1/J. No difference was noted between immune and control mice in the cell types found in the diffusion chambers. The predominant cell types seen were mononuclear macrophages, multinucleate syncytial cells, and neutrophils. Antibody responses to soluble third- and fourth-stage larval antigens and larval excretory/secretory antigens were measured. Although antibodies to all 3 antigen groups were found in higher concentrations in immunized mice than in their respective controls, only antibody responses to soluble L-3 antigens provided a clear correlation with protective immunity.     

Demonstration and isolation of murine melanoma-associated antigenic surface proteins

Melanomas are antigenic and induce the formation of antibodies in both syngeneic and xenogeneic species. The nature of melanoma-associated antigens remains problematic, however. We found that xenogeneic (goat) antiserum to the mouse (C57BL/6) B16 melanoma, following appropriate absorptions with nonmelanoma cells, showed specificity for melanoma-associated surface antigens of B16 and one other murine melanoma. The antibody to B16 did not react with histocompatibility antigens, mouse-specific xenoantigens, viral antigens or melanin. The IgG fraction of the goat antibody was cross-linked covalently to protein A-Sepharose using dimethylsuberimidate. This immunoadsorbent was used to isolate shed antigens from cultures in which B16 cells had been grown and from detergent extracts of biosynthetically labeled (³H-leucine) B16 cells. The immune-affinity purified antigen preparation contained two major components of apparent molecular weight 60,000 and 50,000 daltons as assessed by SDS-polyacrylamide gel electrophoresis. Immunization of rabbits with immune-affinity purified B16 antigens induced antibodies which bound specifically to B16 cells.     

Ubiquitin-fusion degradation pathway: a new strategy for inducing CD8 cells specific for mycobacterial HSP65

The ubiquitin-proteasome system (UPS) plays an indispensable role in inducing MHC class I-restricted CD8⁺ T cells. In this study, we exploited UPS to induce CD8⁺ T cells specific for mycobacterial HSP65 (mHSP65), one of the leading vaccine candidates against infection with Mycobacterium tuberculosis. A chimeric DNA termed pU-HSP65 encoding a fusion protein between murine ubiquitin and mHSP65 was constructed, and C57BL/6 (B6) mice were immunized with the DNA using gene gun bombardment. Mice immunized with the chimeric DNA acquired potent resistance against challenge with the syngeneic B16F1 melanoma cells transfected with the mHSP65 gene (HSP65/B16F1), compared with those immunized with DNA encoding only mHSP65. Splenocytes from the former group of mice showed a higher grade of cytotoxic activity against HSP65/B16F1 cells and contained a larger number of granzyme B- or IFN-γ-producing CD8⁺ T cells compared with those from the latter group of mice.     

Beneficial therapeutic effects with different particulate structures of murine polyomavirus VP1-coat protein carrying self or non-self CD8 T cell epitopes against murine melanoma

Polyomavirus-like-particles (PLPs) are empty, non-replicative, non-infectious particles that represent a potent antigen-delivery system against malignant disease. Protective anti-tumour immunity can be induced under therapy conditions by subcutaneous (s.c.) treatment with particulate antigenic structures like chimerical polyomavirus-pentamers (PPs). These PPs displaying an immunodominant H-2K^b-restricted ovalbumin (OVA)_257-264 epitope evoked nearly complete tumour remission in MO5 (B16-OVA) melanoma-bearing C57BL/6 mice by two s.c. applications in a weekly interval. The immunotherapeutic intervention started at day 4 after melanoma implant. Furthermore, 40% of melanoma-bearing mice vaccinated with heterologous PPs carrying a H-2K^b-restricted cytotoxic T lymphocyte (CTL) epitope derived from of tyrosinase-related protein 2 (TRP2) survived similar treatment conditions. However, a late immunotherapeutic onset at day 10 post melanoma inoculation revealed no significant differences between the therapeutic values (40–60% survival) of VP1-OVA_252-270 and VP1-TRP2_180-192 PPs, respectively. These experiments underlined the capacity of PPs to break T cell tolerance against a differentially expressed self-antigen. As a correlate for preventive and therapeutic immunity against MO5 melanoma the number of OVA_257-264- or TRP2_180-188-specific CD8 T cells were significantly increased within the splenocyte population of treated mice as measured by H-2K^b-OVA_257-264-PE tetramer staining or appropriate ELISPOT assays, respectively. These results reveal that heterologous PLPs and even chimerical PPs represent highly efficient antigen carriers for inducing CTL responses underlining their potential as immunotherapeutics against cancer.This article is a symposium paper from the second international conference Strategies for Immune Therapy, 29 February--3 March 2004, Würzburg, Germany, summarized by G. Pawelec and C. Gouttefangeas.     

Induction of antitumor immunity with dendritic cells transduced with adenovirus vector-encoding endogenous tumor-associated antigens.

Dendritic cells (DCs) are professional Ag-presenting cells that are being considered as potential immunotherapeutic agents to promote host immune responses against tumor Ags. In this study, recombinant adenovirus (Ad) vectors encoding melanoma-associated Ags were used to transduce murine DCs, which were then tested for their ability to activate CTL and induce protective immunity against B16 melanoma tumor cells. Immunization of C57BL/6 mice with DCs transduced with Ad vector encoding the hugp100 melanoma Ag (Ad2/hugp100) elicited the development of gp100-specific CTLs capable of lysing syngeneic fibroblasts transduced with Ad2/hugp100, as well as B16 cells expressing endogenous murine gp100. The induction of gp100-specific CTLs was associated with long term protection against lethal s.c. challenge with B16 cells. It was also possible to induce effective immunity against a murine melanoma self Ag, tyrosinase-related protein-2, using DCs transduced with Ad vector encoding the Ag. The level of antitumor protection achieved was dependent on the dose of DCs and required CD4+ T cell activity. Importantly, immunization with Ad vector-transduced DCs was not impaired in mice that had been preimmunized against Ad to mimic the immune status of the general human population. Finally, DC-based immunization also afforded partial protection against established B16 tumor cells, and the inhibition of tumor growth was improved by simultaneous immunization against two melanoma-associated Ags as opposed to either one alone. Taken together, these results support the concept of cancer immunotherapy using DCs transduced with Ad vectors encoding tumor-associated Ags.     

Genetic control of the target structures recognized in hybrid resistance

Hybrid resistance of lethally irradiated (C57BL/6 × DBA/2)F₁ and (C57BL/10 × C3H)F₁ hybrid mice to the engraftment of parental C57BL/6 or C57BL/10 bone marrow cells is controlled by the H-2-linked Hh-1 locus. This resistance can be specifically blocked or inhibited by the injection of irradiated spleen cells from lethally irradiated, marrow reconstituted donor mice of certain strains. By testing the ability of regenerating spleen cells from various donor strains to block the resistance, we studied the genetic requirements for the expression of putative cell-surface structures recognized in hybrid resistance to H-2^b marrow cells. Strains of mice bearing informative intra-H-2 or H-2/ Qa-Tla recombinant haplotypes provided evidence that the Hh-1 locus is located telomeric to the H-2S region complement loci and centromeric to the H-2D region class I locus in the H-2 ^b chromosome. Two mutations that affect the class I H-2D ^b gene have no effect on Hh-1 ^b gene expression. The H-2D region of the H-2 ^S haplotype contains an allele of the Hh-1 locus indistinguishable from that of the H-2D ^b region, as judged by the phenotypes of relevant strains and F₁ hybrids. Collectively these data indicate that the Hh-1 locus is distinct from the class I H-2D (L) locus in the H-2 ^b or H-2 ^s genome, and favor the view that the expression or recognition of the relevant determinants is not associated with class I gene products.Abbreviations used in this paper BM(C) bone marrow (cells) - CML cell-mediated lympholysis - CTL cytotoxic T lymphocytes - FBS fetal bovine serum - HBSS Hanks' balanced salt solution - SC spleen cells from irradiated, bone marrow-reconstituted mice Address correspondence to: Dr. I. Najamura, Department of Pathology, School of Medicine, State University of New York at Buffalo, Buffalo, NY 14214, USA     

Characterization of B16 melanoma-specific cytotoxic T lymphocytes

A B16 melanoma-specific CD8⁺ T cell line (AB1) was established from the spleen cells of C57BL/6 mice cured of B16 melanoma with interleukin (IL)-12 treatment. The AB1 line exclusively used T cell receptor V_β11. The AB1 cells exhibited a cytolytic activity against both syngeneic B16 melanoma and allogeneic P815 mastocytoma, whereas a cold inhibition assay revealed specificity of the AB1 cells against B16 melanoma. Their lostability to kill a class I loss variant of B16 melanoma was restored by the transfection of H-2K^b gene. In addition, their interferon (IFN)-γ production was significantly suppressed by the addition of anti-H-2K^b monoclonal antibody, and RT-PCR analysis showed that the AB1 line expressed the mRNA encoding IFN-γ, but not IL-4 or IL-10. The experiment using synthetic peptides of tyrosinase-related protein-2 (TRP-2) revealed that the AB1 cells could recognize TRP-2_181–188 peptide. Moreover, the AB1 cells showed an in vivo antitumor effect against established pulmonary metastases of B16 melanoma. Overall, these results indicate that the Tc1-type V_β11 ⁺ AB1 cells exert an antitumor activity against syngeneic B16 melanoma through recognition of TRP-2_181–188 peptide in an H-2K^b-restricted manner. Received: 4 June 1998 / Accepted: 21 July 1998     

Anti-bacterial immunity to Listeria monocytogenes in allogeneic bone marrow chimera in mice

Protection and delayed-type hypersensitivity (DTH) to the facultative intracellular bacterium Listeria monocytogenes (L.m.) were studied in allogeneic and syngeneic bone marrow chimeras. Lethally irradiated AKR (H-2k) mice were successfully reconstituted with marrow cells from C57BL/10 (B10) (H-2b), B10 H-2-recombinant strains or syngeneic mice. Irradiated AKR mice reconstituted with marrow cells from H-2-compatible B10.BR mice, [BR----AKR], as well as syngeneic marrow cells, [AKR----AKR], showed a normal level of responsiveness to the challenge stimulation with the listeria antigens when DTH was evaluated by footpad reactions. These mice also showed vigorous activities in acquired resistance to the L.m. By contrast, chimeric mice that had total or partial histoincompatibility at the H-2 determinants between donor and recipient, [B10----AKR], [B10.AQR----AKR], [B10.A(4R)----AKR], or [B10.A(5R)----AKR], were almost completely unresponsive in DTH and antibacterial immunity. However, when [B10----AKR] H-2-incompatible chimeras had been immunized with killed L.m. before challenge with live L.m., these mice manifested considerable DTH and resistance to L.m. These observations suggest that compatibility at the entire MHC between donor and recipient is required for bone marrow chimeras to be able to manifest DTH and protection against L.m. after a short-term immunization schedule. However, this requirement is overcome by a preceding or more prolonged period of immunization with L.m. antigens. These antigens, together with marrow-derived antigen-presenting cells, can then stimulate and expand cell populations that are restricted to the MHC (H-2) products of the donor type.