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.     

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.     

Teratocarcinoma cell MHC antigen expression is regulated in vitro by a soluble noninterferon factor

The 402AX murine teratocarcinoma is a spontaneous testicular tumor of 129 (H-2b) origin which does not express MHC encoded antigens. Rejection of this tumor is immunologically mediated and the tumor cells are induced in vivo to synthesize H-2b antigens when passaged in genetically resistant host mice. The present studies demonstrate that serum from tumor primed genetically resistant host mice can induce tumor cell MHC antigen expression in vitro as measured by indirect immunofluorescence using monoclonal antibodies. The inducing factor is specific for 402AX tumor cells and is not interferon as shown by the lack of response of the 402AX tumor to gamma interferon, and the absence of significant interferon activity in inducer serum. These studies demonstrate another factor independent of interferon that can induce MHC class I antigen expression on tumor cells.     

L3T4+ T-cell-independent reactivity of Lyt2+ T cells in vivo

The aim of this study was to analyze in vivo the L3T4+ T-cell-subset-independent reactivity of Lyt2+ T cells toward transplantation alloantigens. To this end, we depleted normal mice of L3T4+ T cells by injection of monoclonal antibodies to the L3T4 antigen. This procedure not only led phenotypically to a disappearance of L3T4+ T cells, but also effectively abolished reactivity toward class II MHC antigens in vitro and in vivo. However, L3T4+ T-cell-depleted mice still reacted to class I MHC alloantigens in vivo: after immunization with class I MHC alloantigens Il-2 receptor-bearing T cells appeared in the draining lymph nodes, and developed antigen-specific cytolytic activity. Moreover, upon in vivo priming the frequencies of class I MHC-specific precursors of Il-2-producing and cytolytic Lyt2+ T lymphocytes increased up to 20-fold. L3T4+ T-cell-depleted mice rejected class I MHC-bearing skin grafts promptly. We conclude that not only in vitro but also in vivo Lyt2+ T cells remain reactive toward class I MHC antigens in the absence of L3T4+ T helper cells.     

Multiple splenic lymphoid cell subpopulations regulate H-2 antigen expression on teratocarcinoma cells in vivo

Undifferentiated murine 402AX teratocarcinoma cells do not express MHC antigens when passaged in vitro or in vivo in genetically susceptible host mice. When passaged in vivo in genetically resistant mice, however, the tumor cells become H-2b antigen positive regardless of the H-2 haplotype of the resistant host mouse. The present studies use monoclonal anti-H-2b antibodies to corroborate these earlier findings, which were performed with conventional antisera. Previous studies have established that host bone marrow plus lymphoid cells from resistant primed donors regulate tumor cell H-2b antigen expression. Using bone marrow and mature lymphoid cell reconstitution techniques, the present studies indicate that splenic Ig- cells from genetically resistant host mice are the most efficient lymphoid cell subpopulation in tumor cell H-2b antigen induction. Ig+ spleen cells also reconstitute the capacity to induce teratocarcinoma cell H-2 antigens but are less effective than Ig- spleen cells. Tumor cell H-2 antigen induction in C57BL/6 beige mice is impaired compared to C57BL/6 hosts, which suggests that host NK cells may also be involved in tumor cell H-2 antigen induction. Reconstitution of lethally irradiated resistant hosts for teratocarcinoma cell H-2 antigen expression requires bone marrow plus resistant primed lymphoid cell subpopulations; bone marrow alone is insufficient. These results indicate that multiple splenic lymphoid cell subpopulations requiring a radiosensitive host environment and/or factor for differentiation regulate teratocarcinoma 402AX H-2b antigen expression in vivo in genetically resistant mice.     

Immune recognition by cytotoxic T lymphocytes of minor histocompatibility antigens expressed on a murine colon carcinoma line

We have characterized in vivo and in vitro responses of mice to the BALB/c-derived carcinoma, C26. BALB/c mice were highly susceptible, in a dose-dependent fashion, to local tumor development following subcutaneous injection of C26. Other strains of mice, including allogeneic strains and major histocompatibility complex compatible strains of different minor histocompatibility (H) backgrounds, were resistant to C26-induced tumors. The basis for resistance of mice to C26 was studied using an in vitro-derived C26 line as target cells in microcytotoxicity assays, and as a source of antigen for in vivo priming. An H-2d-specific alloreactive cytotoxic T lymphocyte (CTL) line was isolated from C57BL/6 mice primed with C26, demonstrating the expression, and immune recognition, of MHC class I antigens on C26. C26 also expressed minor H antigens of BALB background as demonstrated by the ability of CTL specific for BALB minor H antigens to selectively lyse C26. Conversely, minor H antigens on C26 were immunogenic across a minor H barrier as demonstrated by the ability to raise anti-minor H CTL to C26 from minor H disparate strains. Collectively, those experiments indicate that C26 may be useful for immunologic and biochemical studies of murine minor H antigens, and for in vivo and in vitro studies of local immunity.     

The unique killing of embryo-derived teratocarcinoma cells by nonactivated murine macrophages is not due to a lack of H-2 antigen expression.

It is well documented that activated macrophages, but not nonactivated ones, kill tumor cells in vitro without damaging normal cells. We, however, have previously shown that embryo-derived teratocarcinoma cells (F9, P19, PCC4) are efficiently killed by nonactivated macrophages as well as by activated ones. Whereas other tumor cells are killed extracellularly by macrophages, we found that F9 teratocarcinoma cells are phagocytosed alive by macrophages and subsequently killed intracellularly by a process dependent on intact lysosomal function. Neither the H-2 antigens nor the mRNAs for the alpha-chain and beta 2-microglobulin are detectable in embryo-derived teratocarcinoma cells. An obvious explanation for this unique killing is that the nonactivated macrophages recognize and kill these cells due to their lack of class I MHC antigen expression, assuming that class I MHC gene products on the target cells switch off the cytolytic machinery of nonactivated macrophages. Our present findings demonstrate that there is no correlation between H-2 antigen expression on tumor cells and their susceptibility to killing by macrophages. Retinoic acid-differentiated F9 cells and P19 cells expressing H-2 antigen after exposure to MAF (IFN-gamma) were sensitive to the killing by nonactivated macrophages. Hybrids that arose from fusion of P19 teratocarcinoma cells with embryonal normal fibroblasts (C57BL/6), which displayed the morphology of embryonal carcinoma stem cells and expressed H-2 antigens, were also sensitive to the killing by nonactivated macrophages. On the other hand, the H-2-negative testicular 402AX teratocarcinoma cells and K1735P melanoma cells were both resistant to the killing by nonactivated macrophages. We concluded that the unique killing of embryo-derived teratocarcinoma cells by nonactivated murine macrophages is not related to a lack of H-2 antigen expression.     

Target cell heterogeneity in murine leukemia virus infection. III. Identification of susceptible Lyt 1+ and resistant Lyt 2+ T-cell subsets following in vitro infection with Friend murine leukemia virus

The susceptibility of splenic T-cell subpopulations to productive infection with Friend murine leukemia virus was determined after in vitro infection and stimulation with Con A. Con A enhanced the number of productively infected cells in unseparated spleen cells as well as in T-cell-enriched spleen cell fractions. Splenic T cells were fractionated into Lyt 1+ and Lyt 2+ subpopulations using both positive and negative selection techniques; susceptible splenic T cells were recovered in the Lyt 1+ fraction and specific cytotoxic treatment with anti-Lyt 1 antibody and complement reduced the number of infectious center-producing cells by greater than 87%. In marked contrast, Lyt 2+ splenic T cells were resistant to productive infection by Friend murine leukemia virus in vitro.     

Characterization of an endogenous Lyt2+ T-suppressor-cell population regulating autoreactive T cells in vitro and in vivo

Autoreactive T cells have been defined by their capacity to respond to self-Ia antigens expressed on non-T cells. Several recent studies have suggested that these cells may play important immunoregulatory functions. However, it is not clear what regulates the responsiveness of autoreactive T cells and why such cells are not demonstrably stimulated in vivo, where they are in the constant presence of self-Ia antigens. In the present study we examined the role of T suppressor (Ts) cells in regulating autoreactive T cells. We observed that enhanced autoreactivity occurred in vitro when Lyt2+ T cells were depleted from the responding and/or stimulating spleen cells in a syngeneic mixed-lymphocyte reaction. Similarly, addition of irradiated Lyt2+ T cells but not L3T4+ T cells inhibited the response of L3T4+ T cells to self-Ia antigens. The activity of the suppressor cells was specific to the autoreactive T cells since antigen-specific and alloreactive T-cell proliferation were not inhibited. Furthermore, depletion of Lyt2+ T cells by in vivo treatment of mice with anti-Lyt2 monoclonal antibodies caused enhanced endogenous proliferation of lymph node and splenic T cells and increased the T-cell response to self-Ia antigens in vitro. These studies, therefore, suggest that T-cell tolerance to self-Ia antigens in vivo may be maintained by naturally occurring Lyt2+ Ts. Mice having enhanced autoreactivity may provide a useful tool to address the role of autoreactive T cells in the immune response to foreign antigens and in the pathogenesis of autoimmune diseases.     

Relationship among function, phenotype, and specificity in primary allospecific T cell populations: identification of phenotypically identical but functionally distinct primary T cell subsets that differ in their recognition of MHC class I and class II allodeterminants

The goal of the present study was to evaluate the relationship among function, Lyt phenotype, and MHC recognition specificity in primary allospecific T cell populations. By using Lyt-2+ and L3T4+ T cells obtained from the same responder populations, we assessed the ability of T cells of each phenotype to generate cytotoxic effector cells (CTL) and IL 2-secreting helper T cells in response to either class I or class II MHC allodeterminants. It was found that a discordance between Lyt phenotype and MHC recognition specificity does exist in primary allospecific T cells, but only in one T cell subpopulation with limited functional potential: namely, Lyt-2+ T cells with cytotoxic, but not helper, function that recognize class II MHC alloantigens. Target cell lysis by these Lyt-2+ class II-allospecific CTL was inhibited by anti-Ia monoclonal antibodies (mAb), but not anti-Lyt-2 mAb, indicating that they recognized class II MHC determinants as their "restriction" specificity and not as their "nominal" specificity even though they were Lyt-2+. A second allospecific T cell subset with limited functional potential was also identified but whose Lyt phenotype and MHC restriction specificity were not discordant: namely, an L3T4+ T cell subset with helper, but not cytotoxic, function specific for class I MHC allodeterminants presented in the context of self-Ia. Thus, the present study demonstrates that primary allospecific T cell populations contain phenotypically identical subpopulations of helper and effector cells that express fundamentally different MHC recognition specificities. Because the recognition specificities expressed by mature T cells reflect the selection pressures they encountered during their differentiation into functional competence, these findings suggest that functionally distinct but phenotypically identical T cell subsets may be selected independently of one another during ontogeny. Thus, the existence of Lyt-2+ CTL specific for class II allodeterminants can be explained by the hypothesis that the association of Lyt phenotype with MHC recognition specificity results from the process of thymic selection that these Lyt-2+ effector cells avoid.     

T cells targeted against a single minor histocompatibility antigen can cure solid tumors

T cells responsive to minor histocompatibility (H) antigens are extremely effective in curing leukemia but it remains unknown whether they can eradicate solid tumors. We report that injection of CD8(+) T cells primed against the immunodominant H7(a) minor H antigen can cure established melanomas in mice. Tumor rejection was initiated by preferential extravasation at the tumor site of interferon (IFN)-gamma-producing H7(a)-specific T cells. Intratumoral release of IFN-gamma had two crucial effects: inhibition of tumor angiogenesis and upregulation of major histocompatibility complex (MHC) class I expression on tumor cells. Despite ubiquitous expression of H7(a), dissemination of a few H7(a)-specific T cells in extralymphoid organs caused neither graft-versus-host disease (GVHD) nor vitiligo because host nonhematopoietic cells were protected by their low expression of MHC class I. Our preclinical model yields unique insights into how minor H antigen-based immunotherapy could be used to treat human solid tumors.     

Distinct effects of interferon-gamma and MHC class I surface antigen levels on resistance of the K562 tumor cell line to natural killer-mediated lysis

Various investigators have examined the relationship between tumor cell susceptibility to natural killer (NK) cell lysis and the expression of HLA class I antigens on the tumor cell. There is controversy as to whether or not an inverse relationship exists, and if so, the basis of the relationship between these two phenomena remains undefined. To address these questions, the genomic clones for two HLA antigens were transfected into the erythroleukemia cell line K562, a cell line that is used as the standard to assess human NK and major histocompatibility complex (MHC) nonrestricted cytolysis. Susceptibility to NK lysis was not affected by the de novo expression of HLA antigens on the K562 after DNA mediated gene transfer. Interferon-gamma (IFN-gamma) treatment of K562 induced levels of MHC class I antigen surface expression comparable to those found on the transfected cells; however, the IFN-gamma-treated cells were resistant to NK lysis. When very high levels of surface HLA antigens were induced on the transfectants, a potential effect of class I MHC expression on K562 lysis could be discerned that was distinct from the resistance to NK lysis induced by IFN-gamma-treatment.     

Increase in the ability of human cancer cells to induce cytotoxic T lymphocytes by ultraviolet irradiation

The roles of ultraviolet-B (UV) radiation in the immunogenicity of human cancer cells have not been fully studied. We have investigated the effects of UV radiation on metastatic melanoma and renal cell carcinoma cells with regard to MHC antigen expression and the ability to induce cytotoxic T lymphocyte (CTL) activity in peripheral blood mononuclear cells (PBMC) or tumor-infiltrating lymphocytes (TIL) against untreated autologous tumor cells. UV radiation respectively decreased or increased MHC class I expression of freshly isolated tumor cells or cultured tumor cells, and also decreased MHC class I expression of starved cultured tumor cells. It increased the ability of both freshly isolated and cultured tumor cells to induce CTL activity from PBMC against untreated autologous tumor cells. UV-irradiated subclones that were more susceptible to CTL lysis were more potent for CTL induction from TIL than either an untreated parental clone or a UV-irradiated subclone that was resistant to CTL lysis. In summary, UV radiation increased the ability of tumor cells to induce CTL activity without a corresponding effect on MHC antigen expression.This work was supported in part by a grant CA47891 from the National Cancer Institute, USA, a grant-in-aid of the comprehensive 10-years strategy for cancer control from ministry of a Health and Welfare, Japan, and the Ishibashi Research Fund, Japan     

Stimulation of splenic T cells on syngeneic monolayers of epidermal basal cells (EBC) in mice. Regulation by Lyt 1+ and Lyt 2+ cell subsets

Syngeneic proliferative response of splenic T cells against monolayers of epidermal basal cells (EBC) was obtained with C57BL/6 and DBA/2 mice. Optimal response, as assessed by [³H]thymidine uptake, occurred on the 6th day of coculture. The level of [^3H]thymidine uptake by unseparated spleen cells was lower than by fractionated T cells from C57BL/6 mice, and null for DBA/2 mice. It was not significantly different when lymphocytes were cocultured with syngeneic or allogeneic epidermal cells. Ia antigens did not appear to be involved in the syngeneic response, since it was not prevented by pretreating stimulator monolayers with monoclonal anti-Ia^k antibody or by adding this antibody directly to the cultures. When the proliferative responses of separated Lyt 1⁺ and Lyt 2⁺ cell subsets were compared, the prominent role of Lyt 1⁺ cells was demonstrated. Enhancement of the T-cell reactivity by eliminating Lyt 2⁺ cells and suppression of the response of a constant number of Lyt 1⁺ cells by adding Lyt 2⁺ cells suggested that Lyt 2⁺ cells could suppress and modulate the Lyt 1⁺ cell proliferation.     

MHC class I antigens,immune surveillance,and tumor immune escape

Oncogenic transformation in human and experimental animals is not necessarily followed by the appearance of a tumor mass. The immune system of the host can recognize tumor antigens by the presentation of small antigenic peptides to the receptor of cytotoxic T-lymphocytes (CTLs) and reject the nascent tumor. However, cancer cells can sometimes escape these specific T-cell immune responses in the course of somatic (genetic and phenotypic) clonal evolution. Among the tumor immune escape mechanisms described to date, the alterations in the expression of major histocompatibility complex (MHC) molecules play a crucial step in tumor development due to the role of MHC antigens in antigen presentation to T-lymphocytes and the regulation of natural killer cell (NK) cell function. In this work, we have (1) updated information on the mechanisms that allow CTLs to recognize tumor antigens after antigen processing by transformed cells, (2) described the altered MHC class I phenotypes that are commonly found in human tumors, (3) summarized the molecular mechanisms responsible for MHC class I alteration in human tumors, (4) provided evidence that these altered human leukocyte antigens (HLA) class I phenotypes are detectable as result of a T-cell immunoselection of HLA class I-deficient variants by an immunecompetent host, and (5) presented data indicating the MHC class I phenotype and the immunogenicity of experimental metastatic tumors change drastically when tumors develop in immunodeficient mice.     

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.     

Rejection of allogeneic tumor is not determined by host responses to MHC class I molecules and is mediated by CD4-CD8+ T lymphocytes that are not lytic for the tumor

In previous studies, the murine SaI (A/J derived, KkDd) sarcoma was transfected with the allogeneic MHC class I H-2Kb gene, and expressed high levels of H-2Kb antigen. Contrary to expectations, the tumor cells expressing the alloantigen (SKB3.1M tumor cells) were not rejected by autologous A/J mice. Because these results contradict the laws of transplantation immunology, the present studies were undertaken to examine the immunogenicity of SKB3.1M and SaI cells in allogeneic hosts. Similar to SKB3.1M, SaI cells are lethal in some allogeneic strains, despite tumor-host MHC class I incompatibilities. Tumor challenges of SKB3.1M and SaI cells, however induce MHC class I-specific antibodies and CTL in both tumor-resistant and -susceptible hosts. Although the tumors induce specific CTL, tumor cells are not lysed in vitro by these CTL, suggesting that the tumor cells are resistant to CTL-mediated lysis. Since growth of these tumors does not follow the classical rules of allograft transplantation, and because the tumor is not susceptible to CTL-mediated lysis, we have used Winn assays to identify the effector lymphocyte(s) responsible for SaI rejection. Depletion studies demonstrate that the effector cell is a CD4-CD8+ T lymphocyte. Collectively these studies suggest that the host's response to MHC class I alloantigens of SKB3.1M and SaI cells does not determine tumor rejection, and that effector cells other than classically defined CTL, but with the CD4-CD8+ phenotype, can mediate tumor-specific immunity.     

Diabetes resistance/susceptibility in T cells of nonobese diabetic mice conferred by MHC and MHC-linked genes

Polymorphism of MHC and MHC-linked genes is tightly associated with susceptibility to type 1 diabetes (T1D) in human and animal models. Despite the extensive studies, however, the role of MHC and MHC-linked genes expressed by T cells on T1D susceptibility remains unclear. Because T cells develop from TCR(-) thymic precursor (pre-T) cells that undergo MHC restriction mediated by thymic stroma cells, we reconstituted the T cell compartment of NOD.scid-RIP-B7.1 mice using pre-T cells isolated from NOD, NOR, AKR, and C57BL/6 (B6) mice. T1D developed rapidly in the mice reconstituted with pre-T cells derived from NOD or NOR donors. In contrast, most of the NOD.scid-RIP-B7.1 mice reconstituted with pre-T cells from AKR or B6 donors were free of T1D. Further analysis revealed that genes within MHC locus of AKR or B6 origin reduced incidence of T1D in the reconstituted NOD.scid-RIP-B7.1 mice. The expression of MHC class I genes of k, but not b haplotype, in T cells conferred T1D resistance. Replacement of an interval near the distal end of the D region in T cells of B6 origin with an identical allele of 129.S6 origin resulted in T1D development in the reconstituted mice. These results provide evidence that the expression of MHC class I and MHC-linked genes in T cells of NOD mice indeed contributes to T1D susceptibility, while expression of specific resistance alleles of MHC or MHC-linked genes in T cells alone would effectively reduce or even prevent T1D.