The CD8+ T cell repertoire in beta 2-microglobulin-deficient mice is biased towards reactivity against self-major histocompatibility class I.

Beta 2-Microglobulin-deficient (beta 2m -/-) mice are reported to lack cell surface expression of major histocompatibility complex (MHC) class I molecules, CD8+ T cells, and the ability to mount MHC class I-specific T cell responses. We have observed that beta 2m -/- mice possess CD8+ T cells that can be induced to perform strong allospecific cytotoxic responses against nonself-MHC class I by in vivo priming. We report that these beta 2m -/- cytotoxic T lymphocyte (CTL) differ from those induced in beta 2m-positive littermates in that they cross-react and kill cells expressing self-MHC class I at normal ligand density with beta 2m. beta 2m -/- CTL could even be induced in primary mixed lymphocyte culture by self-MHC class I expressing stimulator cells, whereas allogeneic stimulator cells failed to elicit a response under similar conditions. Cells with a reduced cell surface MHC class I expression were less sensitive, while syngeneic beta 2m -/- cells were resistant to the beta 2m -/- CTL. This antiself-MHC reactivity could not be induced when beta 2m -/- T cells matured in an environment with normal MHC class I expression in bone marrow chimeric mice. Antiself-MHC reactivity was also observed against human peptide loading-deficient cells expressing the appropriate murine class I molecules, suggesting that affinity to self-MHC class I may occur irrespective of peptide content. The results fit with a model where positive and negative selection of CD8+ T cells in beta 2m -/- mice is mediated by low levels of MHC class I free heavy chains. In this model, low ligand density on selecting cells leads to positive selection of rare T cells that bind to low levels of MHC class I free heavy chains, resulting in a very small peripheral CD8+ compartment. Due to low density of the selecting ligand, negative selection does not remove T cells recognizing beta 2m-positive cells expressing self-MHC class I at normal ligand density, which generates a T cell repertoire that would be autoreactive in a beta 2m-positive littermate. The first "MHC deficient" animals thus paradoxically provide a tool for direct demonstration and analysis of self MHC bias in the T cell repertoire.

Alteration of the natural killer repertoire in H-2 transgenic mice: specificity of rapid lymphoma cell clearance determined by the H-2 phenotype of the target.

The mechanism behind natural tumor resistance conveyed by a H-2Dd transgene to C57Bl/6 (B6) mice was investigated. Transgenic D8 mice were more efficient than control mice in natural killer (NK) cell mediated rapid elimination of intravenously inoculated radiolabeled lymphoma cells of B6 origin, such as RBL-5. There was no difference between D8 and B6 mice when elimination of YAC-1 targets was monitored. The effect of the transgene on the NK repertoire was related to the H-2 phenotype of the target: the differential elimination of RBL-5 lymphoma cells in D8 and B6 mice was not seen when a H-2 deficient variant of this line was used (efficiently eliminated in both genotypes), nor was it seen with a H-2Dd transfectant (surviving in both genotypes). The data show that a MHC class I transgene can directly control natural killing in vivo by altering the repertoire rather than the general levels of NK activity. Since the NK mediated elimination seen after introduction of a novel gene in the host was neutralized by introducing the same gene (H-2Dd), but not an unrelated class I gene (H-2Dp), in the tumor, the data support the concept of NK surveillance against missing self. This combined transgenic/transfectant system may serve as a tool for a molecular dissection of the interactions between NK cells and their targets in vivo.

Recruitment and activation of natural killer (NK) cells in vivo determined by the target cell phenotype. An adaptive component of NK cell-mediated responses.

Natural killer (NK) cells can spontaneously lyse certain virally infected and transformed cells. However, early in immune responses NK cells are further activated and recruited to tissue sites where they perform effector functions. This process is dependent on cytokines, but it is unclear if it is regulated by NK cell recognition of susceptible target cells. We show here that infiltration of activated NK cells into the peritoneal cavity in response to tumor cells is controlled by the tumor major histocompatibility complex (MHC) class I phenotype. Tumor cells lacking appropriate MHC class I expression induced NK cell infiltration, cytotoxic activation, and induction of transcription of interferon gamma in NK cells. The induction of these responses was inhibited by restoration of tumor cell MHC class I expression. The NK cells responding to MHC class I-deficient tumor cells were approximately 10 times as active as endogenous NK cells on a per cell basis. Although these effector cells showed a typical NK specificity in that they preferentially killed MHC class I-deficient cells, this specificity was even more distinct during induction of the intraperitoneal response. Observations are discussed in relation to a possible adaptive component of the NK response, i.e., recruitment/activation in response to challenges that only NK cells are able to neutralize.

A pathogenic role for myelin-specific CD8(+) T cells in a model for multiple sclerosis.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) characterized by plaques of infiltrating CD4(+) and CD8(+) T cells. Studies of MS and experimental autoimmune encephalomyelitis (EAE), an animal model of MS, focus on the contribution of CD4(+) myelin-specific T cells. The role of CD8(+) myelin-specific T cells in mediating EAE or MS has not been described previously. Here, we demonstrate that myelin-specific CD8(+) T cells induce severe CNS autoimmunity in mice. The pathology and clinical symptoms in CD8(+) T cell-mediated CNS autoimmunity demonstrate similarities to MS not seen in myelin-specific CD4(+) T cell-mediated EAE. These data suggest that myelin-specific CD8(+) T cells could function as effector cells in the pathogenesis of MS.

Natural resistance against lymphoma grafts conveyed by H-2Dd transgene to C57BL mice.

The H-2Dd transgenic strain D8 on C57BL background was more resistant to subcutaneous challenge of RBL-5 lymphoma cells than B6 controls. The direct role of the H-2Dd antigen was investigated by the use of (D8 x B6)F1 crosses and (D8 B6) x B6 backcrosses. The latter showed cosegregation with regard to Dd antigen expression and lymphoma resistance, both of which were inherited in a pattern consistent with control by a single dominant gene. The rejection potential in (D8 x B6)F1 mice appeared as strong as that seen in crosses between B6 and MHC congenic mice (on B10 background) carrying H-2Dd. The lymphoma resistance could be abrogated by treatment with anti-asialo GM1 antiserum or anti-NK 1.1 mAb, indicating a role for NK cells.

Prevention of allogeneic bone marrow graft rejection by H-2 transgene in donor mice.

Rejection of bone marrow grafts in irradiated mice is mediated by natural killer (NK) cells and is controlled by genes linked to the major histocompatibility complex (MHC). It has, however, not been possible to identify the genes or their products. An MHC class I (Dd) transgene introduced in C57BL donors prevented the rejection of their bone marrow by NK cells in irradiated allogeneic and F1 hybrid mice expressing the Dd gene. Conversely, H-2Dd transgenic C57BL recipients acquired the ability to reject bone marrow from C57BL donors but not from H-2Dd transgenic C57BL donors. These results provide formal evidence that NK cells are part of a system capable of rejecting cells because they lack normal genes of the host type, in contrast to T cells, which recognize cells that contain abnormal or novel sequences of non-host type.

Effects of dimethyl sulfoxide treatment on H-2 expression and susceptibility to NK- or cytotoxic T-lymphocyte-mediated lysis of the YAC-1 lymphoma and its beta 2-microglobulin-deficient variant.

The effects of dimethyl sulfoxide (DMSO) on H-2 expression and susceptibility to NK- and cytotoxic T-lymphocyte (CTL)-mediated lysis in the murine T-cell lymphoma YAC-1 and its beta-2-microglobulin (beta 2m)-deficient variant were studied. Fluorescence-activated cell sorter analysis revealed induction of H-2Kk and beta 2m 3 days after culture of YAC-1 with DMSO, whereas optimal H-2Dd induction required more than 1 week. H-2Kk and H-2Dd induction by DMSO was equal to pretreatment of YAC-1 cells with 50-100 and 10-20 U/ml interferon (IFN)-gamma, respectively, but the T-cell differentiation antigens Lyt-1, Lyt-2, Thy-1, and L3T4 remained unaffected. DMSO protected YAC-1 cells from NK lysis as efficiently as 10-20 U IFN/ml, whereas susceptibility to anti-H-2a-, H-2Kk-, and H-2Dd-specific CTLs was augmented as in IFN-treated YAC-1 cells. In contrast, the beta 2m-deficient variant, which remained H-2 negative at the cell surface after DMSO treatment, also remained NK sensitive. Thus DMSO can induce H-2 expression and alter the sensitivity of murine lymphoma cells to different effector cells.

Dendritic cells lose ability to present protein antigen after stimulating antigen-specific T cell responses, despite upregulation of MHC class II expression.

Immature dendritic cells (DC) take up, process and present protein antigens; mature DC are specialized for stimulating primary T cell responses with increased expression of MHC class II and co-stimulatory molecules, but are incapable of processing and presenting soluble protein. The current study examined whether maturation of DC is triggered by T cell recognition of antigens presented by immature DC. Human DC derived from CD34+ progenitor cells by culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-6 (IL-6) in serum-free medium could prime naive CD4+ T cells to keyhole limpet hemocyanin (KLH) and ovalbumin (OVA). The cultured DC retained the ability to prime T cells to native protein for at least 15 days. To test for changes in DC function after participation in an immune response, DC were co-cultured with either allogeneic or autologous CD4+ T cells. DC co-cultured with autologous T cells retained the ability to prime T cells to intact protein antigens. By contrast, DC which had previously stimulated an allogeneic T cell response lost ability to prime T cells to soluble proteins. However, such <> induced a MLR and stimulated peptide-specific primary CD4+ T cell responses. This indicated that <> did not die or lose the ability to prime, but lost the ability to process and present subsequent antigens. Following participation in T cell activation, DC increased surface expression of MHC class II, co-stimulatory molecules CD40 and B7.2, and the intercellular adhesion molecule-1 (ICAM-1). In addition, our data suggest that interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) are involved in this T cell-mediated DC maturation.

Antigen presentation and the association of class-I molecules.

We have identified two mutant cell lines which are not able to present epitopes of influenza virus synthesized in the cytoplasm but can present the same epitope when exposed to it as a peptide in the extracellular medium. The cell lines also have a defect in class-I assembly, with reduced expression of assembled alpha chain: beta 2M heterodimers at their cell surface. This led to the suggestion that the two traits were the result of the same mutation and that stable assembly of class-I molecules is dependent on peptide binding. Consistent with this idea was the finding that exposure to specific peptides in the extracellular fluid promotes stable association of class-I heavy chains with beta 2M and restores expression of class-I at the cell surface. We have gone on to show that stable assembly of class-I molecules can be supported in detergent extracts of the mutant cells when specific peptides are added. Peptides stabilized a conformational change in the class-I heavy chain and association with beta 2M by binding to the complexes. This effect is apparent at peptide concentrations around 100-fold lower than required in "peptide feeding" experiments with whole cells. We have also demonstrated that the conformational change induced in heavy chain is influenced by the concentration of beta 2M, and consequently have been able to demonstrate the formation of empty class-I molecules.

Cutting edge: myelin basic protein-specific cytotoxic T cell tolerance is maintained in vivo by a single dominant epitope in H-2k mice.

Multiple sclerosis (MS) is believed to be an autoimmune disease mediated by T cells specific for CNS Ags. MS lesions contain both CD4+ and CD8+ T lymphocytes. The contribution of CD4+ T cells to CNS autoimmune disease has been extensively studied in an animal model of MS, experimental autoimmune encephalomyelitis. However, little is known about the role of autoreactive CD8+ cytotoxic T cells in MS or experimental autoimmune encephalomyelitis. We demonstrate here that myelin basic protein (MBP) is processed in vivo by the MHC class I pathway leading to a MBP79-87/Kk complex. The recognition of this complex by MBP-specific cytotoxic T cells leads to a high degree of tolerance in vivo. This study is the first to show that the pool of self-reactive lymphocytes specific for MBP contain MHC class I-restricted T cells whose response is regulated in vivo by the induction of tolerance.

The RMA-S lymphoma mutant; consequences of a peptide loading defect on immunological recognition and graft rejection.

Experiments with mutant cell lines have underscored the role for peptide in maintenance of the structure of major histocompatibility complex (MHC) class-I molecules. The class-I molecules act as receptors for antigenic and "self" peptides derived from degraded intracellular synthesized proteins. The class-I/peptide trimeric complex is transported to the cell surface where it is scrutinized by cytotoxic T lymphocytes (CTL). The murine RMA-S mutant cell lines have a defect in class-I assembly and express markedly reduced levels of class-I molecules at the cell surface. The mutation is consistent with a defect in peptide transport from the cytosol to the place of assembly with class-I molecules. Addition of synthetic peptides to RMA-S cells and RMA-S cell lysates stimulates assembly of the class-I molecules and indicates that peptide plays a crucial role in attaining the class-I structure. Recent findings have demonstrated that class-I heavy clains (HCs) and beta 2 microglobulin (beta 2m) can assemble in absence of synthetic peptides, forming presumably "empty" (non-peptide-containing) class-I dimeric complexes, in RMA-S cells cultured at reduced temperature. The few class-I molecules present on RMA-S cultured at physiological temperature share the phenotype of "empty" class-I molecules induced at reduced temperature. This finding has allowed the re-interpretation of earlier studies and opened new ways to analyze the interaction between MHC-class-I molecules and different effector cells such as allo-specific CTLs, class-I-restricted CTLs and natural killer (NK) cells. In addition, recent data also suggest that RMA-S represents an attractive model for examining direct class-I-peptide interactions on intact cells or in cell lysates.

Afferent and efferent cellular interactions in natural resistance directed against MHC class I deficient tumor grafts.

H-2-deficient variants, selected from the murine lymphoma RBL-5, were recently shown to be less tumorigenic in syngeneic mice compared with the corresponding H-2-positive wild-type cell line. The present study focused on the detailed cellular interactions leading to the specific rejection of H-2-deficient cells in vivo. In vivo experiments with mixed tumor inocula, measuring either tumor outgrowth or rapid elimination, showed that the presence of H-2-positive lymphoma cells did not suppress the rejection of H-2-deficient cells. Conversely, H-2-deficient cells did not activate a rejection of H-2-positive cells. H-2-deficient cells were selectively eliminated even when they were present in a 10-fold excess compared with the H-2-positive lymphoma cells in the same inoculum. In vitro, H-2-deficient cells were more sensitive to killing by spleen cells from mice treated with interferon or natural killer-inducing agents. The presence of cold H-2-deficient cells did not activate the killing of H-2-positive cells, and H-2-positive cells did not inhibit killing of H-2-deficient cells. Such mixing experiments revealed a more efficient cold target inhibition by the H-2-deficient cells, although no difference was seen between these and the H-2-positive wild-type cells in target binding assays. When Thy-1.2+, nylon wool adherent or phagocytic populations were removed from normal spleen effectors, the difference in spontaneous cell-mediated cytotoxicity between the H-2-deficient and the H-2-positive cells persisted. This result was also obtained when spleen cell effectors from nude mice or asialo GM1+ effectors, positively enriched by fluorescence-activated cell sorter, were used. The results show that the selectivity in the rejection of H-2-deficient cells was not determined in the afferent arm alone. The present data fit with a previously proposed model where natural killer cells would bind equally well to both major histocompatibility complex class I negative and class I positive target cells, but only the latter would be able to present a postbinding inhibitory signal allowing them to escape killing which results in outgrowth in vivo and poor cold target competition in vitro.

Effect of IFN-gamma treatment and in vivo passage of murine tumor cell lines on their sensitivity to lymphokine-activated killer (LAK) cell lysis in vitro; association with H-2 expression on the target cells.

Interferon-gamma (IFN-gamma) treatment or in vivo passage of the murine YAC-1 lymphoma resulted in reduced sensitivity to in vitro lysis by syngeneic murine spleen cells cultured in rIL-2 (LAK-cells). IFN-gamma treatment also rendered the murine B16 melanoma less sensitive to lysis by syngeneic LAK cells, whereas in vivo passage did not alter LAK sensitivity. The reduction in sensitivity to lysis correlated with enhanced expression of cell surface H-2 on the target cells. The possible role of H-2 was studied with a beta 2-microglobulin-deficient, and thus H-2-deficient, variant of the YAC-1 lymphoma. This variant line remained H-2 negative even after IFN-gamma treatment or in vivo passage, and was highly sensitive to LAK-cell-mediated lysis, even after IFN-gamma treatment or in vivo passage. The present results are discussed in relation to IFN-gamma and in vivo induced modulation of MHC class-1 molecules on target cells and the possible consequences for interaction with activated as well as "natural" effector cells.

A mutant cell in which association of class I heavy and light chains is induced by viral peptides.

Association of the Db heavy chain with beta 2-microglobulin and expression of Db and Kb at the surface of the cell are induced by specific peptides in the mutant RMA-S. Association of antigenic peptides with the binding site of class I molecules may be required for correct folding of the heavy chain, association with beta 2-microglobulin, and transport of the antigen-MHC complex to the cell surface.

Allorecognition by NK cells: nonself or no self?

The issue of antigen recognition by NK cells is complex, fascinating and, as yet, unresolved. This article reviews recent research on the repertoire of human NK cell clones for the recognition of different allogeneic cells, and summarizes the studies, most of which have been performed in mice, that implicate the MHC in NK cell recognition. It goes on to provide a common conceptual framework within which these different systems may be understood.

Association of class I major histocompatibility heavy and light chains induced by viral peptides.

We describe a cell in which association of a major histocompatibility complex class I heavy chain with beta 2-microglobulin is induced by a peptide derived from influenza nucleoprotein. Association of antigenic peptides with the binding site of class I molecules may be required for correct folding of the heavy chain, association with beta 2-microglobulin and transport of the antigen-MHC complex to the cell surface.

Expression of a tolerizing tumor antigen in peripheral tissue does not preclude recovery of high-affinity CD8+ T cells or CTL immunotherapy of tumors expressing the antigen.

Transgenic (TG) mice were generated selectively expressing the gag protein of Friend murine leukemia virus (FMuLV) in the liver. FMuLV(gag) is also expressed by the FBL leukemia, and is the immunodominant tumor Ag of the CD8(+) T cell response in C57BL/6 mice. gag-TG mice expressing FMuLV(gag) in the liver were tolerant to the protein and failed to generate a CTL response to either FBL or FMuLV(gag). This tolerance reflected anergy rather than deletion, as CTL responsiveness could be recovered after four cycles of in vitro stimulation. Adoptively transferred gag-specific T cells were not anergized in gag-TG recipients, as revealed by antitumor activity in vivo. Also, such T cells did not induce detectable autoimmune injury in gag-TG liver cells. These results suggest that the requirements for a tissue Ag to provide a tolerizing stimulus are distinct from those for being the target of a T cell-mediated autoimmune response and that the requirements for induction and maintenance of peripheral tolerance are distinct for naive and primed T cells. That anergic T cells reactive with tumor-associated Ags can be recovered by repetitive in vitro stimulation and can mediate tumor therapy suggests strategies that use such Ags to generate CTL for adoptive immunotherapy should be further developed.

Enhanced H-2 expression and T-cell-dependent rejection after intracerebral transplantation of the murine lymphoma YAC-1.

The relationship between MHC class I (H-2) expression and tumorigenicity was investigated after intracerebral inoculation of the murine lymphoma YAC-1 and its H-2 negative variant, A.H-2-. YAC-1 was less tumorigenic than A.H-2- in normal as well as NK-depleted syngeneic A/Sn mice. However, in T-cell-depleted syngeneic mice YAC-1 was as tumorigenic as A.H-2-. Following intracerebral growth, the H-2 expression of YAC-1 was markedly enhanced in a similar fashion as after intraperitoneal passage. The A.H-2- variant remained H-2 negative after intracranial passage. The H-2 negative variant cells were not rejected from the brain even when intermixed with wild-type YAC-1 cells prior to intracerebral inoculation, excluding an "innocent bystander" effect. In vitro, the intracerebrally passaged YAC-1 line showed enhanced sensitivity to lysis by H-2 Kk Dd (H-2a) specific CTLs but decreased sensitivity to NK cells. The A.H-2- line was unchanged. Our data suggest that the lack of H-2 molecules may facilitate the growth of antigenic tumor cells in the brain due to escape from T-cell-mediated immunosurveillance. Our data also suggest, in line with other recent findings, that intracerebrally growing tumor cells are sheltered from NK cell-mediated rejection.

Inhibition of natural killer cell-mediated bone marrow graft rejection by allogeneic major histocompatibility complex class I, but not class II molecules.

The role of major histocompatibility complex (MHC) class I and class II molecules in natural killer (NK) cell-mediated rejection of allogeneic, semisyngeneic and MHC-matched bone marrow grafts was investigated. The use of beta 2-microglobulin (beta 2m) -/- and beta 2m +/- mice as bone marrow donors to MHC-mismatched recipients allowed an analysis of whether the presence of semi-syngeneic and allogeneic MHC class I gene products would be triggering, protective or neutral, in relation to NK cell-mediated rejection. Loss of beta 2m did not allow H-2b bone marrow cells to escape from NK cell-mediated rejection in allogeneic (BALB/c) or semi-allogeneic (H-2Dd transgenic C57BL/6) mice. On the contrary, it led to stronger rejection, as reflected by the inability of a larger bone marrow cell inoculum to overcome rejection by the H-2-mismatched recipients. In H-2-matched recipients, loss of beta 2m in the graft led to a switch from engraftment to rejection. At the recipient level, loss of beta 2m led to loss of the capability to reject H-2-matched beta 2m-deficient as well as allogeneic grafts. When MHC class II-deficient mice were used as donors, the response was the same as that against donors of normal MHC phenotype: allogeneic and semi-syngeneic grafts were rejected by NK cells, while syngeneic grafts were accepted. These data suggest a model in which allogeneic class I molecules on the target cell offer partial protection, while certain syngeneic class I molecules give full protection from NK cell-mediated rejection of bone marrow cells. There was no evidence for a role of MHC class II molecules in this system.

Recognition of beta 2-microglobulin-negative (beta 2m-) T-cell blasts by natural killer cells from normal but not from beta 2m- mice: nonresponsiveness controlled by beta 2m- bone marrow in chimeric mice.

The role of major histocompatibility complex (MHC) class I expression in control of the sensitivity of normal cells to natural killer (NK) cells was studied by the use of mutant mice made deficient for expression of beta 2-microglobulin (beta 2m) through homologous recombination in embryonal stem cells. T-cell blasts from beta 2m-deficient (beta 2m -/-) mice were killed by NK cells from normal mice in vitro, while beta 2m +/- blasts were resistant. The beta 2m defect also affected the NK effector cell repertoire: NK cells from beta 2m -/- mice failed to kill beta 2m -/- blasts, while they retained the ability to kill the prototype NK cell target lymphoma YAC-1, although at reduced levels. The inability to recognize beta 2m -/- blasts could be transferred with beta 2m -/- bone marrow to irradiated beta 2m-expressing mice. In contrast, the development of CD8+ T cells (deficient in beta 2m -/- mice) was restored in such chimera. These results indicate that loss of MHC class I/beta 2m expression is sufficient to render normal cells sensitive to NK cells, and that the same defect in the hemopoietic system of a mouse renders its NK cells tolerant to beta 2m-deficient but otherwise normal cells. In the beta 2m -/- mice, NK cells may be selected or educated by other bone marrow cells to tolerate the MHC class I deficiency. Alternatively, the specificity may be controlled directly by the class I molecules on the NK cells themselves.