Tumor cells engineered to express major histocompatibility complex class II molecules induce T helper cell-dependent responses that protect mice from normally lethal doses of unmodified tumor cells.

Tumor cells typically fail to stimulate protective immune response in the autochthonous host. This does not appear to be the result of either inadequate antigenicity or failure to express a normal complement of major histocompatibility complex (MHC) class 1 molecules. To investigate if tumor cells fail to stimulate protective immunity because they fail to activate adequate numbers of T helper cells, we transfected murine fibrosarcoma and melanoma cells with genes encoding syngeneic and allogeneic MHC class II molecules. Fibrosarcoma cells expressing either type of MHC class II molecules failed to induce tumors in syngeneic mice and stimulated T helper cell-dependent antitumor immune responses that protected mice from subsequent challenge with untransfected tumor cells. The antitumor response involved both CD4+ and CD8+ T cells, and appeared to be dependent on at least low levels of innate tumor cell immunogenicity.

Fibrosarcoma cells expressing allogeneic MHC Class II antigens induce protective antitumor immunity.

The initiation of effective immune responses usually requires presentation of Ags by MHC class I and class II molecules. Although most tumors express MHC class I molecules, MHC class II molecule expression is generally limited to specialized APCs. One reason spontaneous tumors may fail to elicit effective immune responses is that tumor Ags are inefficiently presented by APCs, and adequate T cell-mediated help is not generated. To test this hypothesis, we investigated the possibility of enhancing Th cell stimulation by inducing expression of MHC class II molecules on tumor cells. We transfected a murine fibrosarcoma, Sa1N, with the genes encoding allogeneic (I-Ad) or syngeneic (I-Ak) MHC class II molecules. We then compared the tumorigenic and immunogeneic potential of these transfectants to parental Sa1N tumor cells. Subcutaneous injection of allogeneic or syngeneic transfectants resulted in dramatically fewer tumors than injection of unmodified fibrosarcoma cells, and mice inoculated with MHC class II gene-transfected cells were resistant to subsequent challenge with parental Sa1N cells. Rejection of allogeneic MHC class II Ag+ tumor cells could be mediated by either CD4+ or CD8+ T cells, whereas rejection of secondary challenge with wild-type Sa1N tumor cells required both T cell subsets. These results demonstrate that allogeneic, as well as syngeneic, MHC class II Ag+ tumor cells can stimulate protective antitumor immunity.

Protective antitumor immunity induced by immunization with MHC class II gene-transfected tumor cells is unrelated to MHC class II expression.

A/JCr mice reject Sa1N fibrosarcoma cells genetically engineered to express major histocompatibility complex (MHC) class II molecules and are highly resistant to subsequent challenge with unmodified Sa1N cells. In this report we examine the mechanism by which this protective antitumor immunity is induced. We found that MHC class II antigen-positive tumor cells were no more effective than irradiated, MHC class II antigen-negative cells at inducing secondary protective immunity. Additionally, therapeutic immunization with MHC class II antigen-positive tumor cells had no effect on the growth of admixed Sa1N cells or preexisting Sa1N tumors. Based on these observations, we conclude that the MHC class II antigen-induced immune response is not related to Sa1N-specific antitumor immunity.

Flow cytometric analysis of transport activity in lymphocytes electroporated with a fluorescent organic anion dye.

Organic anion transport in polarized epithelia and macrophages has previously been studied by monitoring the efflux of fluorescent organic anion dyes from cells. We adapted this strategy to the study organic anion transport in lymphocytes. Cloned lymphoma cells and normal and activated human T cells were loaded with a membrane-impermeant, organic anion dye (Lucifer Yellow) by electroporation. Dye efflux in lymphocytes was rapid, energy-dependent, and inhibitable by organic anion transporter inhibitors. Dye efflux could not be attributed to the effects of electroporation. In addition, electroporated, dye-loaded T helper cells retained the ability to properly respond to specific antigen. Thus, dye loss occurred in viable, functionally competent cells. These experiments demonstrate that electroporation is an effective means of loading cells with Lucifer Yellow, and that lymphocytes possess organic anion transporters that are functionally similar to those previously described for secretory epithelia and macrophages.

Selective impairment of humoral immunity in feline leukemia virus-induced immunodeficiency.

We used a panel of in vitro assays to investigate the nature of immune dysfunction in cats infected with FeLV-FAIDS, a naturally occurring, molecularly cloned feline leukemia virus (FeLV) isolate which induces a fatal immunodeficiency syndrome in infected cats. During the asymptomatic period preceding immunodeficiency disease, we were unable to detect any deficits in concanavalin A-induced blastogenesis, xenogeneic mixed-lymphocyte reaction assays, stimulation of lymphocytes by soluble protein antigen, and cytotoxic T lymphocyte assays. However, during this period humoral immune responses in the FeLV-FAIDS-infected cats were dramatically impaired. As early as 9 weeks after virus inoculation, the ability to mount either an IgM or IgG response to soluble protein antigens was lost. Neither B cell function, as assessed by lipopolysaccharide-induced blastogenesis or circulating B cell numbers, as assessed by immunofluorescence, differed between infected and control cats. These results suggest that FeLV-FAIDS infection may impair a subpopulation of T helper cells, that provides help for the production of antibody. Consistent with earlier observations of cats naturally infected with FeLV, our results indicate that early impairment of humoral immunity is an important component of the immunodeficiency syndrome induced by FeLV in cats.

Cells of chemically induced tumors differentially express major histocompatibility complex class I antigens.

Several recent studies have indicated that alterations in expression of major histocompatibility complex (MHC) antigens by tumor cells affects the ability of the host to mount an effective antitumor immune response. To investigate whether newly induced tumors frequently exhibit altered MHC antigen expression, we used methylcholanthrene to induce a series of tumors and elevated MHC antigen expression by these cells. The tumors exhibited a variety of MHC phenotypes in vitro. The nature of their phenotypes suggested that these cells were, in fact, capable of independent and abnormal regulation of MHC class 1 genes. However, when maintained in vivo, these same tumor cells expressed measurable levels of all of the appropriate MHC class I antigens. Thus, newly induced tumor cells are capable of abnormal MHC class I antigen expression. However, there was no obvious correlation between the phenotypes exhibited by these tumor cells in vitro and either their phenotype or their tumorigenic potential in vivo.

Development of an enzyme-linked immunosorbent assay to detect IgG, IgM, and complement (C3) on canine erythrocytes.

An ELISA was used to detect IgG, IgM, and complement (C3) on the surface of canine erythrocytes. Erythrocytes were placed in wells of a microtitration plate and incubated with affinity purified, alkaline phosphatase-conjugated anti-canine IgG, IgM, or C3. Results of the ELISA were compared with the direct antiglobulin test (DAT) by preparing standard reference curves from canine blood type A erythrocytes that had been incubated with serial dilutions (1:2 to 1:8, 192) of canine anti-A serum. The ELISA detected increased erythrocyte-bound immunoglobulin and complement at two- to fourfold dilutions greater than those required for positive results with the DAT. The ELISA required small sample and reagent volumes and detected lower concentrations of immune components than did the DAT.

Spontaneous interferon production by pulmonary leukocytes is associated with lentivirus-induced lymphoid interstitial pneumonia.

Ovine lentiviruses share genome sequence, structural features, and replicative mechanisms with HIV, the etiologic agent of AIDS. A lamb model of lentivirus-induced lymphoid interstitial pneumonia, comparable to lymphoid interstitial pneumonia associated with pediatric AIDS, was used to investigate production of leukocyte-soluble mediators. Lentivirus-infected lambs and adult sheep with severe lymphoid interstitial pneumonia had significantly elevated levels of spontaneous interferon (IFN) production from pulmonary leukocytes compared with ovine lentiviruses-infected animals with mild or no lesions of lymphoid interstitial pneumonia or non-infected controls. However, peripheral blood mononuclear cells from lentivirus-infected lambs did not spontaneously release significant amounts of IFN. IFN production by pulmonary lymph node lymphocytes was enhanced in the presence of lentivirus-infected alveolar macrophages. Animals with lentivirus-induced disease and spontaneous IFN production had enhanced virus replication within tissues. The ovine lentiviruses-induced IFN had a m.w. of between 25,000 and 35,000 and was resistant to freeze/thawing procedures. The IFN activity was sensitive to trypsin and stable to low pH and heat. IFN with similar physical and biochemical properties was produced when ovine lentiviruses was added to control leukocyte cultures. IL-2 and PGE2 production and responses to mitogen by pulmonary lymph node lymphocytes of lentivirus-diseased lambs were not statistically different from control animals. Increased local production of IFN in lentivirus-infected host tissues may serve to accelerate the entry of leukocytes into virus-induced lesions promoting cell-mediated tissue damage and also provide increased numbers of cells for virus replication.

Soluble factors produced during an immune response regulate Ia antigen expression by murine adenocarcinoma and fibrosarcoma cells.

LT-85 is an alveologenic adenocarcinoma of C3Hf/HeN mice. Comparisons of the in vitro and in vivo surface properties of these cells revealed that under normal conditions, they expressed I-A and I-E antigens iv vivo only. By using clonally derived cells, it was established that this phenomenon was not due to the selection of an Ia antigen-positive tumor cell subpopulation, but resulted from phenotypic conversion of Ia antigen-negative tumor cells. These tumor cells and 1053 cells (a fibrosarcoma of C3H/HeN MTV- mice) could, however, be induced to express I-A, I-E, and much higher levels of H-2 antigens in vitro by co-culturing them with spleen cells from LT-85 tumor-bearing C3H/HeN MTV- mice. In vitro induction of Ia and H-2 antigens did not result from contaminating splenocytes or from antigen transfer, because splenocytes from BALB/c (H-2d) mice immunized with A/J (H-2k/d) cells were able to induce the expression of Iak antigens by both tumor cell lines. It was found that this phenomenon was neither H-2-restricted nor antigen-specific. The results clearly indicated, however, that an immune response was required to generate phenotypic conversion of the tumor cells, both in vivo and in vitro. It was further found that soluble, rather than cellular, factors produced during an immune response induced the expression of Ia antigens by LT-85 and 1053 tumor cells. In contrast to what has been reported about the induction of Ia antigens on macrophages and normal epithelial and endothelial cells, the induction of Ia antigens on LT-85 and 1053 cells did not appear to require T cells, and did not involve gamma-interferon. These findings demonstrate that some tumor cells are capable of altering their MHC antigen phenotype in response to factors produced during an immune response in vivo or in vitro. Because of the involvement of Ia antigens in several aspects of immune phenomena, the ability of tumor cells to differentially express Ia antigens in response to environmental factors may have profound effects on host-tumor interactions. Furthermore, the differences seen in the phenotypes of tumor cells grown in vitro and in vivo suggest that in vitro methodologies of tumor cell characterization may not present a complete picture of the natural state of the tumor cell surface.

Identification of Ia antigens on the murine adenocarcinoma LT-85.

Antigens associated with the H-2Kk and I-Ak regions of the major histocompatibility complex have been identified with monoclonal antibodies on an in vivo grown murine alveologenic adenocarcinoma, LT-85. Immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated differences in the molecular structure of I-Ak region-coded antigens between LT-85 and host C3H/HeN mammary tumor virus-negative (MTV-) or autologous C3HfB/HeN splenocytes. Ia antigens derived from LT-85 tumor cells exhibited both an increased heterogeneity in the alpha-chain and a lower apparent molecular weight in the beta-chain. Tryptic peptide mapping of the I-Ak antigen alpha- and beta-chains derived from C3H/HeN MTV- mice and LT-85 tumor cells revealed a single peptide difference in the beta-chains of these antigens. Results obtained with neuraminidase-treated I-Ak beta-chains indicated that this difference was not due to sialic and content. Maintenance of LT-85 in vitro, even for short periods, resulted in the loss of these I-Ak antigens. However, this loss of I-Ak antigen expression was fully reversible with in vivo growth.

Biochemical evidence for expression of a semi-allogeneic, H-2 antigen by a murine adenocarcinoma.

LT-85 is an alveolegenic adenocarcinoma induced in mutant C3HfB/HeN (C3Hf) mice. This tumor, however, grows preferentially in allogeneic, wild-type C3H/HeN (C3H) mice. The tumor-associated transplantation antigen has been mapped to the K end of the major histocompatibility complex. H-2K antigens were isolated from detergent extracts of LT-85 cells by immunoprecipitation with monoclonal antibody. The tryptic peptides of these antigens were compared, by using high-pressure liquid chromatography, with the tryptic peptides of H-2K antigens isolated from syngeneic mutant C3Hf and ancestral wild-type C3H spleen cells. We found that the H-2K antigens of the LT-85 tumor cells were very similar to, but distinct from, those present on syngeneic C3Hf lymphoid cells. We also found, however, that the H-2K antigens of LT-85 tumor cells were clearly different from the H-2K antigens of allogeneic C3H spleen cells. The H-2K antigens of LT-85 cells are therefore foreign to syngeneic C3Hf cells, but do not represent expression by the tumor cells of the allogeneic H-2K antigens expressed by normal C3H cells. Furthermore, the nature of the differences observed between the H-2K antigens of LT-85 cells and C3Hf and C3H spleen cells strongly suggests that the structure of the H-2K molecule of LT-85 cells is identical in some regions to the H-2K molecule of C3Hf cells, and in other regions to the H-2K molecule of C3H cells.

Biochemical comparison of MHC antigens isolated from mutant C3HfB/HeN and parent C3H/HeN mice.

H-2K antigen heavy chains were isolated from C3HfB/HeN and C3H/HeN spleen cells by immunoprecipitation and high pressure liquid chromatography and were compared by tryptic peptide mapping. Glycopeptides obtained from 3H-glucosamine-labeled H-2Kk (C3H) and H-2Kkv1 (C3Hf) heavy chains were found to be identical, indicating that previously observed differences between the two molecules are due to alterations in amino acid sequence. I-A subregion antigen alpha- and beta-chains were also isolated from C3H and C3Hf spleen cells by immunoprecipitation and SDS-polyacrylamide gel electrophoresis, and were compared by tryptic peptide mapping analyses. Material isolated from 3H-glucosamine-labeled cells revealed that I-A alpha- and beta-chains were glycosylated, and antigens from C3H and C3Hf mice contained identical glycopeptides. Similar analyses of I-A antigen alpha- and beta-chains isolated from C3H and C3Hf spleen cells labeled with 3H-arginine or 3H-lysine revealed no differences in the amino acid sequences of the I-A antigens of these two strains. These findings indicate that all of the unique immunologic properties of mutant C3Hf cells can be attributed to modifications of the amino acid sequence of the MHC K locus gene product.

Low responsiveness to Dk or Db plus vaccinia virus or to Kk plus lymphocytic choriomeningitis virus assessed by availability of D or K products.

The possibility was examined that K or D regulated responsiveness of virus specific cytotoxic T cells was due to the virus-specific and differential effects on expression and/or accessibility of H-2K or D products on virus-infected target cells. Cells from established lines of fibroblasts kept in tissue culture, uninfected, infected with either vaccinia virus, with lymphocytic choriomeningitis virus (LCMV) or with both LCMV plus vaccinia virus, were compared with respect to expression of Kk, Dk and Db. H-2K or D expression was assessed by: 1) absorption of defined anti-K or anti-D antisera: 2) susceptibility to alloreactive cytotoxic T cells; and 3) susceptibility to K or D restricted virus-specific cytotoxic T cells. In all 3 tests, no virus-specific effect on Kk, Dk or Db expression on all target cells (uninfected, LCMV, or vaccinia virus infected, or doubly infected) was noticed. Most importantly, H-2k target cells infected with both LCMV plus vaccinia virus, that were not lysed by low-responder Dk-restricted vaccinia specific T cells were lysed by high-responder Dk restricted LCMV-immune T cells; in contrast, these targets were lysed by Kk-restricted vaccinia specific T cells but only poorly by Kk-restricted LCMV-immune T cells. Thus, expression or accessibility of Dk could not have limited responsiveness to vaccinia virus on this target cell since Dk was accessible to Dk-restricted LCMV-specific T cells; a similar argument can be made for the accessibility of Kk. These experiments suggest that expression and/or accessibility of Kk, Dk or Db on target cells does not explain the virus- and K or D dependent responsiveness differences of virus-specific effector T cells.

Biochemical comparison of H-2K antigen isolated from C3HfB/HeN and C3H/HeN mice.

The H-2K glycoproteins were isolated from spleen cells of C3H/HeN and C3HfB/Hen mice and compared by tryptic peptide mapping techniques. The two antigens were found to be very similar in that more than 90 percent of detectable peptides appeared identical. However, two lysine-containing peptide present in tryptic digests of H-2K antigens isolated from C3H mice were absent from tryptic digests of H-2K antigens isolated from C3Hf mice. This was probably not the result of altered glucosylation since neuramindase digestion demonstrated that the disparate peptides were not glycopeptides but most probably resulted from substitution of one or two amino acids in the H-2K molecule of C3HfB/HeN mice. These differences were small but significant and demonstrated that H-2Kk (C3H) and H-2Kkv1 (C3Hf) antigens are structurally distinct. This is compatible with the observed reciprocal skin-graft rejection, MLR, and generation of cytotoxic T lymphocytes between the two strains. The significance of this finding in conjunction with what is known about properties of 1-ethyl-1-nitrosourea-induced tumors of C3Hf mice is discussed.

Cross-reactivity between human and murine lymphocyte antigens. IV. Reactivity of H-2 alloantisera with HLA-A, B antigens.

The anti-H-2 alloantiserum D-32 [(B10.A(2R) x C3H.SW) anti-C3H] is cytolytic to human lymphocytes. Fab2 blocking assays, indirect immunoprecipitation and sequential immunoprecipitation experiments showed that the anti-H-2 alloantiserum D-32 recognizes antigenic determinants which are expressed on the heavy chain of subpopulations of HLA-A, B antigens. These determinants are different from those defining the serological polymorphism of the HLA-A, B, C system, are the same as or spatially close to those recognized by the anti-HLA-A, B monoclonal antibody Q6/64 and are expressed on rabbit, rat or guinea pig lymphocytes.

Human and murine tumours: changes in cell surface structures coded by the major histocompatibility complex region.

Serological and structural changes of surface markers involved in immune reactions may occur in human and murine tumour systems. Thus nine out of twenty-one human tumour cell lines and SV40-transformed fibroblasts differed from autologous lymphoblastoid cells or fibroblasts in their reactivity with HLA alloantisera. H-2 antigens isolated from the murine tumour cells 6C3HED and TP1422, undergo structural changes. An alien HLA-B7 was detected in sera from two melanoma patients. The serologic activity of H-2 antigens was significantly increased in the serum and ascites fluid of tumour bearing mice. Additionally, human SV40-transformed fibroblasts acquire receptors for monkey red blood cells and the murine lymphosarcoma cells 6C3HED express receptors for sheep red blood cells.

Cross-reacting xenoantibodies to the heavy chain of H-2 and HLA-A,B antigens: serologic and immunochemical characterization.

H-2 cross-reactive antibodies present in HLA xenoantisera were purified by absorption on and elution from murine cells. Antibodies isolated from one of these antisera, 78-E48, were found to mediate complement-dependent lysis of both human and murine lymphoid cells but not of Daudi cells or of human lymphoid cells coated with Fab2 fragments from a cow anti-human beta 2-microglobulin (beta 2 m) antiserum. In indirect immunoprecipitation analyses 78-E48 reacted only with proteins of approximately 45,000 and 12,000 MW present in NP-40 extracts of both human and murine lymphoid cells. Sequential precipitation experiments with rabbit anti-human beta 2 m and allo-anti-H-2Kk, Iak sera established that these proteins were in fact H-2 and HLA-A,B antigens. It was also found that 78-E48 reacted only with the heavy chain of HLA-A,b and H-2 antigens, since this eluate was unreactive with beta 2 m in a radioimmunoassay, and its capacity to immunoprecipitate the 45,000 and 12,000 MW proteins from human cell extracts was unaffected by prior reaction with purified human beta 2 m. These data show for the first time that H-2 and HLA-A,B antigens share properties that probably depend upon their teritary structure.