Mouse Hepa-1 tumor is rejected by H-2Db-restricted CTL despite decreased MHC class I antigen expression

It has recently been hypothesized that tumor cells with reduced levels of MHC class I antigens are more susceptible to NK-mediated lysis and are rejected by NK cells, whereas tumor cells with normal levels of class I are rejected by tumor-specific CTL. We have tested this hypothesis using a mouse hepatoma system. The Hepa-1 tumor is a spontaneous H-2Kb loss variant that arose from the BW7756 tumor, when BW7756 was adapted to growth in culture. Our studies have shown that despite the loss of H-2Kb antigen, Hepa-1 is not more susceptible to NK lysis than its H-2Kb-transfected variants. These studies also suggested that NK cells were not responsible for rejection of the Hepa-1 tumor. The Hepa-1 tumor, therefore, appears to contradict the hypothesized linkage of MHC levels and NK susceptibility. Because NK cells are not involved in immunity to this tumor, we have sought to identify the effector cell responsible for Hepa-1 rejection. Cytotoxic T lymphocyte assays demonstrate that in vitro, Hepa-1 cells are lysed by Hepa-1-specific H-2Db-restricted CD4-CD8+ T lymphocytes. Footpad assays demonstrate that in vivo, Hepa-1 rejection requires CD4+CD8- and CD4-CD8+ Hepa-1-primed splenocytes. These results indicate that immunity to Hepa-1 is T cell mediated. Hepa-1 is therefore an example of an unusual tumor in that down-regulation of MHC class I antigen expression is associated with increased CTL susceptibility.     

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.     

Cellular stimuli for rejection of sarcoma I tumor allografts by BALB/c recipient mice

We have evaluated the role of passenger leukocytes in Sarcoma I (SaI) tumor allograft rejection by BALB/c recipient mice. SaI tumor cells grown in tissue culture expressed low levels of class I MHC antigens as determined by flow cytometry. Ascites-derived tumor cells contained additional cell populations of A/J host origin which expressed high levels of class I and class II alloantigens. Tissue culture-derived SaI grafts grew progressively or were rejected in delayed fashion by some BALB/c hosts. In contrast, ascites-derived tumor allografts were rejected rapidly by 100% of recipient mice. Passenger cells appear to be responsible for these striking differences in immunogenicity because the addition of allogeneic A/J splenocytes to the tissue culture form of SaI caused rapid rejection. When tissue culture- and ascites-derived tumors simultaneously were grafted on opposite shoulders of recipient mice, both tumors were rejected. These data indicate that passenger cells provide the primary stimulus for SaI tumor allograft rejection by BALB/c mice. The mechanism of SaI enhancement by anti-Ia antibodies may be analogous to the prolonged survival of endocrine grafts after depletion of Ia+ passenger leukocytes.     

H-2Kb antigen expression has no effect on natural killer susceptibility and tumorigenicity of a murine hepatoma

Recent reports suggested a correlation between decreased expression of tumor cell MHC class I Ag and increased susceptibility to NK cells. These studies led to the hypothesis that tumor cells displaying reduced levels of MHC class I Ag have reduced tumorigenicity in vivo because they are eliminated from the host by endogenous NK cells. The present studies use the murine hepatoma BW7756 and a spontaneous H-2Kb loss variant, Hepa-1, to test this hypothesis. The parental BW7756 tumor is highly malignant in syngeneic C57L/J hosts while Hepa-1 cells do not give rise to tumors, suggesting that the loss of H-2Kb Ag expression correlates with decreased tumorigenicity and NK susceptibility. Hepa-1 cells were therefore transfected with an H-2Kb gene to generate H-2Kb Ag expressing clones. The resulting clones were tested for tumorigenicity. Syngeneic or NK-deficient C57BL/6-beige/beige mice challenged with Hepa-1 or the H-2Kb transfectants rejected the cells, suggesting that reexpression of H-2Kb Ag does not restore tumorigenicity and that NK cells are not involved in Hepa-1 rejection. In vitro H-2Kb Ag-negative and -positive Hepa-1 cells are equally susceptible to tilorone-boosted NK cells, indicating that MHC class I Ag expression also does not affect in vitro NK susceptibility. Tumor challenged athymic nude and sublethally irradiated syngeneic mice develop tumors demonstrating that T cells are probably responsible for rejection of the Hepa-1 tumor, and that H-2Kb Ag expression has no effect on rejection. Inasmuch as the expression of H-2Kb Ag on Hepa-1 cells does not effect tumorigenicity or in vitro NK susceptibility, the previously reported association between reduced MHC class I Ag levels and increased NK susceptibility is not universally applicable.     

Differential susceptibility of cells expressing allogeneic MHC or viral antigen to killing by antigen-specific CTL

CD8(+) cytotoxic T lymphocytes (CTLs) generated by immunization with allogeneic cells or viral infection are able to lyse allogeneic or virally infected in vitro cells (e.g., lymphoma and mastocytoma). In contrast, it is reported that CD8(+) T cells are not essential for allograft rejection (e.g., heart and skin), and that clearance of influenza or the Sendai virus from virus-infected respiratory epithelium is normal or only slightly delayed after a primary viral challenge of CD8-knockout mice. To address this controversy, we generated H-2(d)-specific CD8(+) CTLs by a mixed lymphocyte culture and examined the susceptibility of a panel of H-2(d) cells to CTL lysis. KLN205 squamous cell carcinoma, Meth A fibrosarcoma, and BALB/c skin components were found to be resistant to CTL-mediated lysis. This resistance did not appear to be related to a reduced expression of MHC class I molecules, and all these cells could block the recognition of H-2(d) targets by CTLs in cold target inhibition assays. We extended our observation by persistently infecting the same panel of cell lines with defective-interfering Sendai virus particles. The Meth A and KLN205 lines infected with a variant Sendai virus were resistant to lysis by Sendai virus-specific CTLs. The Sendai virus-infected Meth A and KLN205 lines were able to block the lysis of Sendai virus-infected targets by CTLs in cold target inhibition assays. Taken together, these results suggest that not all in vivo tissues may be sensitive to CTL lysis.     

Introduction of soluble protein into the class I pathway of antigen processing and presentation

In order to investigate how peptides associate with class I major histocompatibility complex (MHC) glycoproteins intracellularly, we generated cytotoxic T lymphocytes (CTL) specific for a readily available soluble protein in association with class I. C57BL/6 (H-2b) mice immunized against a syngeneic tumor cell transfected with chicken ovalbumin (OVA) cDNA gave rise to H-2Kb-restricted CTL specific for the OVA258-276 peptide. This synthetic peptide and CNBr fragments of OVA (242-285 and 242-273) were able to target H-2b cells for lysis by the CTL in a 3 hr assay. Cells incubated with native OVA for up to 24 hr did not become sensitized for recognition and lysis. However, when OVA was introduced directly into the cytoplasm of cells by the osmotic lysis of pinosomes, the Kb restricted determinant formed readily.     

Serpin-6 expression protects embryonic stem cells from lysis by antigen-specific CTL

The immune response to embryonic stem (ES) cells is still poorly understood. In this study, we addressed the adaptive cellular immune response to undifferentiated and differentiated ES cells infected with lymphocytic choriomeningitis virus (LCMV), a vertically transmitted pathogen in mice and humans. In contrast to the prevailing view, we found that undifferentiated and differentiated murine ES cells express MHC class I molecules, although at low levels. When cocultured with LCMV-infected ES cells, syngeneic but not allogeneic LCMV-specific CTL secrete IFN-gamma. Strikingly, LCMV-specific CTL do not efficiently kill LCMV-infected ES cells. ES cells showed high-level expression of the serine protease inhibitor 6, an endogenous inhibitor of the CTL-derived cytotoxic effector molecule granzyme B. Down-regulation of serpin-6 by RNA interference sensitized ES cells for CTL-induced cell death. The results of this study suggest that LCMV-infected murine ES cells present viral Ags and are recognized by LCMV-specific CTL in a MHC class I-restricted manner, yet resist CTL-mediated lysis through high-level expression of serine protease inhibitor 6.     

Resistance to H-2-restricted but not to allo-H2-specific graft and cytotoxic T lymphocyte responses in lymphoma mutant

The lymphoma mutant RMA-S escaped graft rejection after transplantation over a minor histocompatibility barrier, whereas it was rejected in H-2 allogeneic mice. The parental control line was rejected in both situations. The mutant, which had been selected against MHC class I molecules retained 5 to 10% of the wild-type H-2Db, Kb, and beta 2-microglobulin expression on the cell surface. It remained sensitive to allo-H-2b CTL in vitro, but was completely resistant to minor histocompatibility antigen-specific, H-2b-restricted CTL. It was equally resistant to other H-2b-restricted responses against internally derived Ag, such as tumor-specific CTL or a CTL clone specific for the influenza virus nucleoprotein. The results indicate a target cell defect that selectively abolishes the sensitivity to H-2-restricted CTL directed against internally processed Ag. This appears sufficient to shift the transplantation response over a minor histocompatibility Ag barrier from rejection to acceptance. There are two possible explanations for the results: 1) a block in the MHC class I-directed pathway for internal Ag processing, and 2) subthreshold H-2/Ag ligand density in relation to triggering requirements of restricted CTL. Regardless of the type of defect, the results demonstrate a difference between allo-H-2-specific and H-2-restricted CTL recognition at the level of the target cell.     

Cell-mediated lympholysis responses against autologous cells modified with haptenic sulfhydryl reagents. IV. Self-determinants recognized by wild-type anti-H-2Kb and H-2Db-restricted cytotoxic T cells specific for sulfhydryl and amino-reactive haptens are absent in certain H-2 mutant strains

Virus-specific H-2-restricted cytotoxic T cells (CTL) have been found to discriminate between wild-type and mutant class I molecules. The only results reported concerning a hapten-self model, however, indicate that TNP-specific CTL do not discriminate between wild-type and mutant self determinants (7). In the present study, hapten-specific CTL generated against N-iodoacetyl-N'-(5-sulfonic-1-naphthyl) ethylene diamine-modified syngeneic cells (AED-self) were used to determine whether a hapten that is known to react with different cell surface sites than TNP can induce CTL that distinguish mutant H-2K and D molecules from those of wild type. The findings of this study indicate that H-2Kb-AED-self cytotoxic effector cells can discriminate between self-determinants of H-2Kb wild-type and the H-2bm1 and H-2bm11 mutants, but not between wild-type and the H-2bm6 and H-2bm9 mutants. H-2Db-AED-self effector cells were also found to discriminate between self-determinants of H-2Db wild-type and the H-2bm13 and H-2bm14 mutants. Furthermore, cold target competition experiments indicated that the bm1 and bm11 Kb products also lack some determinants recognized by anti-wild-type Kb TNP-specific CTL. These findings provide the first demonstration that hapten-self-specific effectors can detect alterations in H-2 mutant class I molecules. The results in the present report also support the hypothesis that haptens do not have to derivatize H-2 molecules in order to form antigens recognized by H-2-restricted CTL. These findings are discussed with respect to the involvement of self-determinants on MHC and non-MHC cell surface molecules.     

Retrovirus-induced changes in major histocompatibility complex antigen expression influence susceptibility to lysis by cytotoxic T lymphocytes

Retrovirus infection of murine fibroblasts was found to alter the expression of major histocompatibility complex (MHC) antigens. Fibroblasts infected with Moloney murine leukemia virus (M-MuLV) exhibited up to a 10-fold increase in cell surface expression of all three class I MHC antigens. Increases in MHC expression resulted in the increased susceptibility of M-MuLV-infected cells to lysis by allospecific cytotoxic T lymphocytes (CTL). M-MuLV appears to exert its effect at the genomic level, because mRNA specific for class I antigens, as well as beta 2-microglobulin, show a fourfold increase. Fibroblasts infected with the Moloney sarcoma virus (MSV):M-MuLV complex show no increase in MHC antigen expression or class I mRNA synthesis, suggesting that co-infection with MSV inhibits M-MuLV enhancement of MHC gene expression. Quantitative differences in class I antigen expression on virus-infected cells were also found to influence the susceptibility of infected cells to lysis by H-2-restricted, virus-specific CTL. Differential lysis of infected cells expressing varied levels of class I antigens by M-MuLV-specific bulk CTL populations and CTL clones suggests that individual clones may have different quantitative requirements for class I antigen expression. The MSV inhibition of MHC expression could be reversed by interferon-gamma. Treatment of MSV:M-MuLV-infected fibroblasts with interferon-gamma increased their susceptibility to lysis by both allogeneic and syngeneic CTL. The data suggest that interferon-gamma may function in the host's immune response to viral infections by enhancing MHC antigen expression, thereby increasing the susceptibility of virus-infected cells to lysis by H-2-restricted, virus-specific CTL.     

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.     

The alpha-3 domain of class I MHC proteins influences cytotoxic T lymphocyte recognition of antigenic determinants located within the alpha-1 and alpha-2 domains

An interspecies class I MHC molecule, Kb1+2/A2 (in which the alpha-1 and alpha-2 domains of the H-2Kb molecule have been linked to the alpha-3, transmembrane and intracytoplasmic domains of the HLA-A2 molecule) has been expressed on both human and mouse target cells by gene transfer. Maintenance of serologic determinants has been demonstrated. However, decreased lysis by allospecific CTL populations of cell lines that expressed a hybrid interspecies class I molecule, Kb1+2/A2, as compared with lines that expressed the native Ag, H-2Kb, has been described. An analysis with a limited panel of H-2Kb allospecific clones demonstrated that not all H-2Kb-specific CTL can lyse cells that express Kb1+2/A2 Ag. This suggested that the reduction of lysis by CTL populations was due to the loss of specific alloreactive clones in the population. Each clone used in this study was then defined as having high or low affinity characteristics. No correlation between the affinity of the CTL and the ability to recognize the interspecies hybrid molecule could be shown. Rather, these data suggest that antigenic determinants that are located within the polymorphic domains, alpha-1 and alpha-2, may be conformationally influenced by the alpha-3 domain.     

Target cell lysis by cytotoxic T lymphocytes redirected by antibody-coated polystyrene beads

Cytotoxic T lymphocytes were found to mediate rapid lysis of target cells not normally recognized in the presence of small polystyrene beads coated with a combination of anti-T3 and antitarget cell antibodies. Lysis was not seen with beads bearing one of these antibodies alone, nor with a mixture of two types of beads each coated with a single antibody. The effector cells mediating this lysis include long term allospecific human CTL, and both human and mouse CTL clones recognizing mouse class I MHC Kb Ag. TNP-modified mouse tumor cells, a human lymphoblastoid line, and human red cells were found to be good targets for this cytotoxicity. Polystyrene beads with diameters of 3 to 15 mu caused target lysis, with a dose-response curve which typically went through a maximum and declined at high bead numbers. Maximal bead-redirected lysis by CTL was less efficient than that mediated by soluble antibody heteroconjugates of the same two antibodies. Bead-redirected target lysis was calcium dependent. These results are interpreted as a form of bystander lysis induced by the beads, since the target cell membrane is not directly crosslinked to the region of CTL activation. These observations thus favor a mechanism of lysis involving the polarized secretion of a locally acting lytic agent by CTL.     

Failure or success in the restoration of virus-specific cytotoxic T lymphocyte response defects by dendritic cells

C57BL/6 (B6, H-2b) mice are CTL responders to both Sendai virus and Moloney leukemia virus. In the former response the H-2Kb class I MHC molecule is used as CTL restriction element, in the latter response the H-2Db molecule. B6 dendritic cells (DC) are superior in the presentation of Sendai virus Ag to CTL in comparison with B6 normal spleen cells. Con A blasts have even less capacity to present viral Ag than NSC, and LPS blasts show an intermediate capacity to present viral Ag. H-2Kb mutant bm1 mice do not generate a CTL response to Sendai virus, but respond to Moloney leukemia virus, as demonstrated by undetectable CTL precursors to Sendai virus and a normal CTL precursor frequency to Moloney virus. Compared to B6 mice, other H-2Kb mutant mice show decreased Sendai virus-specific CTL precursor frequencies in a hierarchy reflecting the response in bulk culture. The Sendai virus-specific CTL response defect of bm1 mice was not restored by highly potent Sendai virus-infected DC as APC for in vivo priming and/or in vitro restimulation. In mirror image to H-2Kb mutant bm1 mice, H-2Db mutant bm14 mice do not generate a CTL response to Moloney virus, but respond normally to Sendai virus. This specific CTL response defect was restored by syngeneic Moloney virus-infected DC for in vitro restimulation. This response was Kb restricted indicating that the Dbm14 molecule remained largely defective and that a dormant Kb repertoire was aroused after optimal Ag presentation by DC. In conclusion, DC very effectively present viral Ag to CTL. However, their capacity to restore MHC class I determined specific CTL response defects probably requires at least some ability of a particular MHC class I/virus combination to associate and thus form an immunogenic complex.     

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.     

Tumor allograft rejection is mainly mediated by CD8+ cytotoxic T lymphocytes stimulated with class I alloantigens in cooperation with CD4+ helper T cells recognizing class II alloantigens

Presence of the three major pathways (self-Ia restricted, allo-K/D restricted, and allo-Ia restricted pathways) in generating class I-restricted CTL has been reported. The present study was conducted in order to clarify which of the three is the main pathway in mediating tumor allograft rejection. One million EL-4 tumor cells derived from C57BL/6 (B6;H-2b) were inoculated into the various strains of mice that were genetically different from B6. Class I (K/D) Ag-disparate but IA Ag-matched B6.C-H-2bm1 (bm1;Kbm1, IAb, IE-, Db) mice or B10.A (5R) (5R; b, b, k, d) mice could not reject 1 x 10(6) EL-4 tumor cells in spite of the strong generation of CTL against the B6 Ag, suggesting the inability of the self-Ia restricted pathway and the allo-K/D restricted pathway in rejecting tumor allografts. The strains of mice being capable of rejecting EL-4 tumor were disparate from B6 mice in both class I and class II (IA) Ag, suggesting the importance of the allo-Ia restricted pathway in rejecting tumor allografts. To generate CTL against Kb Ag via the allo-Ia restricted pathway in the bm1 mice, 2 x 10(7) B6.H-2bm12 (bm12; b, bm12, -, b) spleen cells were injected into the bm1 mice as a supplementary source of allogeneic APC that possibly raise CTL through CD4+ Th cells of bm1 origin. These bm1 mice became capable of rejecting 1 x 10(6) EL-4 tumor cells. The same was observed in the combination of bm12----B10.A (5R) (b, b, k, d) mice. To further elucidate the role of the class II restricted CD4+ Th cells, anti-CD4 antibody was repeatedly i.v. administered into the C3H/He (C3H; H-2k) or the DBA/2 (DBA; H-2d) mice on days 0, 1, and 4. Injection of anti-CD4 antibody led 1 x 10(6) EL-4 tumor cells to grow and kill the C3H and DBA mice. These results suggest that the main effector CTL pathway involved in tumor allograft rejection is allo-Ia restricted pathway where CD8+ precursor CTL were stimulated by the class II-restricted CD4+ Th cells.     

Antigen presentation of a modified tumor-derived peptide by tumor infiltrating lymphocytes

CD8+ T-lymphocytes recognize peptides in the context of major histocompatibility complex (MHC) class I antigens. Upon activation, these cells differentiate into effector cytotoxic T lymphocytes (CTL) and no longer require formal antigen presentation by professional antigen presenting cells (APC). Subsequently, any cell expressing MHC class I/cognate peptide can stimulate CTL. Using TIL specific for a melanoma antigen-derived peptide, IMDQVPFSV (g209 2M), we sought to determine whether these CTL could present peptide to each other. Our findings demonstrate that peptide presentation of the g209 2M peptide epitope by TIL is comparable to conventional methods of using T2 cells as APC. We report here that CTL are capable of self-presentation of antigenic peptide to neighboring CTL resulting in IFN-gamma secretion, proliferation, and lysis of peptide-loaded CTL. These results demonstrate that human TIL possess both APC functions as well as cytotoxic functions and that this phenomenon could influence CTL activity elicited by immunotherapy.     

Inhibition of an allospecific T cell hybridoma by soluble class I proteins and peptides: estimation of the affinity of a T cell receptor for MHC

To investigate the molecular basis of the interaction between the T cell receptor and the MHC class I antigen in an allogeneic response, a soluble counterpart of the murine class I molecule, H-2Kb, was genetically engineered. Cells secreting this soluble molecule, H-2Kb/Q10b, inhibited stimulation of an H-2Kb-reactive T cell hybridoma by cells transfected with H-2Kbm10, a weak stimulus, but not by H-2Kb- or H-2Kbm6-transfected cells. Soluble purified H-2Kb/Q10b protein also blocked T cell stimulation. In addition, a peptide from the wild-type H-2Kb molecule spanning the region of the bm10 mutation specifically inhibited activation of the T cell hybridoma by H-2Kbm10 cells, thus suggesting that amino acid residues 163-174 of H-2Kb define a region important for T cell receptor binding. An estimate for the Kd of the T cell receptor for soluble H-2Kb/Q10b was 10(-7) M, while the Kd for soluble peptide 163-174 was 10(-4) M.     

Increased sensitivity to MHC-nonrestricted lysis of BL6 melanoma cells by transfection with class I H-2Kb gene

An H-2Kb- negative clone of BL6 melanoma (BL6-8) was transfected with neor, H-2Kb, or H-2IAk genes. In an 18-h cytotoxicity assay clones with high levels of H-2Kb Ag expression were found more sensitive to lysis by spleen cells of syngenic and allogeneic mice than H-2Kb low clones. NK cells were involved in the lysis of H-2Kb+ BL6 melanoma clones, with spleen cell cytotoxicity of mice increased after poly I:C stimulation or decreased after pretreatment with anti-asialo GM1 serum or NK1.1 mAb. Anti-TNF Ab were also able to reduce the cytotoxicity of normal spleen cells and completely abolished the cytotoxicity of the NK-depleted spleen cells suggesting involvement of NC cells in lysis of H-2Kb+ BL6 melanoma clones. Increase in sensitivity of H-2Kb+ BL6 cells to natural cell-mediated cytotoxicity was associated with the appearance of NK recognizable determinants as assessed by the cold target inhibition assay. All BL6 clones, irrespective of sensitivity to natural cell-mediated cytotoxicity, showed high sensitivity to lysis by LGL-derived granules. In contrast, all H-2Kb low BL6 clones were resistant and all H-2Kb highly positive clones were sensitive to lysis by TNF-alpha. When an H-2Kb highly positive clone was selected in vitro for resistance to TNF, it concomitantly showed increased resistance to cytotoxicity by spleen cells, confirming the importance of TNF in spleen cell cytotoxicity against H-2Kb+ melanoma cells. Taken together, the data indicate that class I H-2Kb but not class II H-2IAk gene product could increase the sensitivity of BL6 cells to lysis by NK and natural cytotoxic cells as well as TNF. We hypothesize that these effects could be due to pleiotropic effects of H-2Kb gene products on various biologic properties of BL6 melanoma cells some of which may be more directly involved in regulation of tumor cell sensitivity to lysis by NK and/or natural cytotoxic cells.