Multigene DNA priming-boosting vaccines protect macaques from acute CD4+-T-cell depletion after simian-human immunodeficiency virus SHIV89.6P mucosal challenge.

We evaluated four priming-boosting vaccine regimens for the highly pathogenic simian human immunodeficiency virus SHIV89.6P in Macaca nemestrina. Each regimen included gene gun delivery of a DNA vaccine expressing all SHIV89.6 genes plus Env gp160 of SHIV89.6P. Additional components were two recombinant vaccinia viruses, expressing SHIV89.6 Gag-Pol or Env gp160, and inactivated SHIV89.6 virus. We compared (i) DNA priming/DNA boosting, (ii) DNA priming/inactivated virus boosting, (iii) DNA priming/vaccinia virus boosting, and (iv) vaccinia virus priming/DNA boosting versus sham vaccines in groups of 6 macaques. Prechallenge antibody responses to Env and Gag were strongest in the groups that received vaccinia virus priming or boosting. Cellular immunity to SHIV89.6 peptides was measured by enzyme-linked immunospot assay; strong responses to Gag and Env were found in 9 of 12 vaccinia virus vaccinees and 1 of 6 DNA-primed/inactivated-virus-boosted animals. Vaccinated macaques were challenged intrarectally with 50 50% animal infectious doses of SHIV89.6P 3 weeks after the last immunization. All animals became infected. Five of six DNA-vaccinated and 5 of 6 DNA-primed/particle-boosted animals, as well as all 6 controls, experienced severe CD4(+)-T-cell loss in the first 3 weeks after infection. In contrast, DNA priming/vaccinia virus boosting and vaccinia virus priming/DNA boosting vaccines both protected animals from disease: 11 of 12 macaques had no loss of CD4(+) T cells or moderate declines. Virus loads in plasma at the set point were significantly lower in vaccinia virus-primed/DNA-boosted animals versus controls (P = 0.03). We conclude that multigene vaccines delivered by a combination of vaccinia virus and gene gun-delivered DNA were effective against SHIV89.6P viral challenge in M. nemestrina.

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.

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.

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.

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.

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.

Host MHC class I gene control of NK-cell specificity in the mouse.

The missing self model predicts that NK cells adapt somatically to the type as well as levels of MHC class I products expressed by their host. Transgenic and gene knock-out mice have provided conclusive evidence that MHC class I genes control specificity and tolerance of NK cells. The article describes this control and discusses the possible mechanisms behind it, starting from a genetic model to study how natural resistance to tumors is influenced by MHC class I expression in the host as well as in the target cells. Data on host gene regulation of NK-cell functional specificity as well as Ly49 receptor expression are reviewed, leading up to the central question: how does the system develop and maintain "useful" NK cells, while avoiding "harmful" and "useless" ones? The available data can be fitted within each of two mutually none-exclusive models: cellular adaptation and clonal selection. Recent studies supporting cellular adaptation bring the focus on different possibilities within this general mechanism, such as anergy, receptor calibration and, most importantly, whether the specificity of each NK cell is permanently fixed or subject to continuous regulation.

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.

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.

Major histocompatibility complex class I-specific and -restricted killing of beta 2-microglobulin-deficient cells by CD8+ cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTLs) recognize major histocompatibility complex (MHC) class I molecules, normally composed of a heavy chain, a beta 2-microglobulin (beta 2m), and peptide antigens. beta 2m is considered essential for the assembly and intracellular transport of MHC class I molecules as well as their peptide presentation to CTLs. Contrary to this dogma, we now report the generation of allospecific and restricted CD8+ and TCR alpha beta+ CTLs (where TCR is T-cell receptor) capable of killing beta 2m-deficient cells. Such CTLs were obtained by priming mice with live allogeneic beta 2m- spleen cells or mutant lymphoma cells producing MHC class I protein but no detectable beta 2m. Although both beta 2m- and beta 2m-expressing lymphoma cells were rejected in allogeneic mice, only the former were efficient inducers of CTLs recognizing beta 2m- cells. These CTLs were MHC class I (H-2Kb or Db)-specific and CD8-dependent and did not require serum as a source of external beta 2m in the culture. They could be induced across major and minor histocompatibility barriers. The H-2-restricted CTLs generated in the latter case failed to kill the antigen-processing-deficient target RMA-S cells. The results show that MHC class I heavy chains in beta 2m- cells can be transported to the cell surface and act as antigens or antigen-presenting molecules to allospecific and MHC-restricted CTLs.

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.

Different types of allospecific CTL clones identified by their ability to recognize peptide loading-defective target cells.

Allospecific immune responses against the MHC of another individual are remarkably strong, due t a high number of responding T cell clones. Although it has been demonstrated that some allospecific cytotoxic T lymphocytes (CTL) recognize peptides presented by allogeneic MHC class I molecules, it has remained unclear whether MHC molecules can be recognized directly. We used the H-2b-derived murine lymphoma mutant RMA-S, which has a defect affecting peptide loading of class I molecules, to test whether recognition by allospecific CTL always requires the presence of peptides. Three types of anti-H-2Kb CTL clones can be distinguished by their ability to lyse RMA-S target cells. Type A CTL clones efficiently lyse these target cells, the lysis by type B CTL clones is inefficient, and type C clones fail to lyse RMA-S. Up-regulation of the levels of H-2Kb density improved lysis by type B clones, but did not lead to lysis by type C clones. Some type A and B CTL clones apparently can recognize class I molecules devoid of peptides, while others are likely to recognize peptides which are not affected by the presentation defect of RMA-S. We suggest that type C clones are specific for peptides which are not presented by the mutant cells. The results show that the majority of alloreactive CTL recognize peptide/MHC complexes, while some CTL behave as if they can recognize class I molecules in the absence of MHC-bound peptides.

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.

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.

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.

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.

Empty MHC class I molecules come out in the cold.

Major histocompatibility complex (MHC) class I molecules present antigen by transporting peptides from intracellularly degraded proteins to the cell surface for scrutiny by cytotoxic T cells. Recent work suggests that peptide binding may be required for efficient assembly and intracellular transport of MHC class I molecules, but it is not clear whether class I molecules can ever assemble in the absence of peptide. We report here that culture of the murine lymphoma mutant cell line RMA-S at reduced temperature (19-33 degrees C) promotes assembly, and results in a high level of cell surface expression of H-2/beta 2-microglobulin complexes that do not present endogenous antigens, and are labile at 37 degrees C. They can be stabilized at 37 degrees C by exposure to specific peptides known to interact with H-2Kb or Db. Our findings suggest that, in the absence of peptides, class I molecules can assemble but are unstable at body temperature. The induction of such molecules at reduced temperature opens new ways to analyse the nature of MHC class I peptide interactions at the cell surface.

Restoration of H-2b expression and processing of endogenous antigens in the MHC class I pathway by fusion of a lymphoma mutant to L cells of the H-2k haplotype.

The RMA-S lymphoma mutant cannot process and present antigens to H-2-restricted cytotoxic T lymphocytes. It synthesizes major histocompatibility complex class I heavy (H-2KbDb) and light beta 2-microglobulin (beta 2mb) chains of normal size and charge, but only a fraction of these assemble and reach the cell surface. As a first step investigating the genetic defect of this line, we have fused it to a L cell fibroblast line (H-2KkDk/beta 2ma). The fusion restored H-2Kb, Db and beta 2mb expression as well as the ability to process and present internally derived (minor histocompatibility and influenza virus nucleoprotein) antigens in RMA-S. This shows that the mutation(s) responsible for the phenotype of RMA-S is (are) not located within the MHC class I heavy and light chain genes. Other cellular factors, derived from the L cell fusion partner, can control antigen processing and transport of MHC class I molecules. These findings are discussed in relation to the observation that assembly and transport of MHC class I molecules can be induced in the mutant by H-2b-restricted peptides. The recessive nature of the defect and its independence of MHC class I genes in the mutant has important implications for future transfection studies, of this and similar mutants, aiming at establishing cells containing non-assembled MHC class I molecules of different alleles and identifying the gene(s) controlling processing of endogenous antigens.