Listeria monocytogenes-infected hepatocytes are targets of major histocompatibility complex class Ib-restricted antilisterial cytotoxic T lymphocytes

Subclinical infection of BALB/c mice with the intracellular bacterial pathogen Listeria monocytogenes results in the development of protective antilisterial immunity. L. monocytogenes can infect hepatocytes, and antilisterial cytotoxic T lymphocytes (CTL) lyse Listeria-infected hepatocytes in a major histocompatibility complex (MHC) class Ia-restricted manner. It remained to be determined whether L. monocytogenes-infected hepatocytes are susceptible to MHC class Ib-restricted cytolysis. In this study, we showed that hepatocytes express MHC class Ib molecule Qa-1(b) mRNA and protein. We further showed that Listeria-infected hepatocytes are susceptible to MHC class Ib-restricted cytolysis, since C57BL/6-derived Listeria-infected hepatocytes were lysed by BALB/c-derived antilisterial CTL. These results establish that Listeria-infected hepatocytes are susceptible to cytolysis by MHC class Ib restricted Listeria-specific CTL.     

Peptide-specific cytotoxic T lymphocytes restricted by nonself major histocompatibility complex class I molecules: reagents for tumor immunotherapy

Studies in melanoma patients have revealed that self proteins can function as targets for tumor-reactive cytotoxic T lymphocytes (CTL). One group of self proteins MAGE, BAGE, and GAGE are normally only expressed in testis and placenta, whilst another group of CTL recognized proteins are melanocyte-specific differentiation antigens. In this study we have investigated whether CTL can be raised against a ubiquitously expressed self protein, mdm-2, which is frequently overexpressed in tumors. The observation that T-cell tolerance is self major histocompatibility complex-restricted was exploited to generate CTL specific for an mdm-2 derived peptide presented by nonself major histocompatibility complex class I molecules. Thus, the allo-restricted T-cell repertoire of H-2d mice was used to isolate CTL specific for the mdm100 peptide presented by allogeneic H-2Kb class I molecules. In vitro, these CTL discriminated between transformed and normal cells, killing specifically Kb-positive melanoma and lymphoma tumors but not Kb-expressing dendritic cells. In vivo, the CTL showed antitumor activity and delayed the growth of melanoma as well as lymphoma tumors in H-2b recipient mice. These experiments show that it is possible to circumvent T-cell tolerance to ubiquitously expressed self antigens, and to target CTL responses against tumors expressing elevated levels of structurally unaltered proteins.     

Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatibility complex class I-peptide complexes

This study describes the construction of soluble major histocompatibility complexes consisting of the mouse class I molecule, H-2Db, chemically biotinylated beta2 microglobulin and a peptide epitope derived from the glycoprotein (GP; amino acids 33-41) of lymphocytic choriomeningitis virus (LCMV). Tetrameric class I complexes, which were produced by mixing the class I complexes with phycoerythrin-labeled neutravidin, permitted direct analysis of virus-specific cytotoxic T lymphocytes (CTLs) by flow cytometry. This technique was validated by (a) staining CD8+ cells in the spleens of transgenic mice that express a T cell receptor (TCR) specific for H-2Db in association with peptide GP33-41, and (b) by staining virus-specific CTLs in the cerebrospinal fluid of C57BL/6 (B6) mice that had been infected intracranially with LCMV-DOCILE. Staining of spleen cells isolated from B6 mice revealed that up to 40% of CD8(+) T cells were GP33 tetramer+ during the initial phase of LCMV infection. In contrast, GP33 tetramers did not stain CD8+ T cells isolated from the spleens of B6 mice that had been infected 2 mo previously with LCMV above the background levels found in naive mice. The fate of virus-specific CTLs was analyzed during the acute phase of infection in mice challenged both intracranially and intravenously with a high or low dose of LCMV-DOCILE. The results of the study show that the outcome of infection by LCMV is determined by antigen load alone. Furthermore, the data indicate that deletion of virus-specific CTLs in the presence of excessive antigen is preceded by TCR downregulation and is dependent upon perforin.     

In vivo administration of major histocompatibility complex class I-specific peptides from influenza virus induces specific cytotoxic T cell hyporesponsiveness

We have been investigating the immunogenicity of two class I major histocompatibility complex-specific peptides with a sequence derived from influenza virus nucleoprotein specific for Kd and one for Db. Peptide-modified splenocytes are unable to immunize for a primary cytotoxic T (Tc) cell response in vivo, or secondary response in vitro. Peptide-modified stimulator cells can boost virus-primed splenocytes for a strong secondary response in vitro. Animals primed with syngeneic peptide-modified splenocytes upon challenge with virus in vivo do not generate strong secondary Tc cell responses on day 3 after challenge in contrast to virus primed animals. Day 6 responses of virus-challenged, peptide-primed animals are reduced as compared to unprimed mice. This hyporesponsiveness is independent of CD8+ T cells in the priming population and can be elicited with tumor cell lines. The data are discussed in the framework of the two-signal model of immune induction.     

Human cytomegalovirus-infected cells have unstable assembly of major histocompatibility complex class I complexes and are resistant to lysis by cytotoxic T lymphocytes

Viruses which cause persistence in the naturally infected host are predicted to have evolved immune evasion mechanisms. Human cytomegalovirus (HCMV) causes significant morbidity and mortality in immunocompromised patients yet persists without clinical manifestations in seropositive individuals who have normal immune function. We report that HCMV infection in vitro impairs major histocompatibility complex class I (MHC-I) assembly accompanied by resistance to killing by cytotoxic CD8+ T lymphocytes. Pulse-chase metabolic labelling experiments show that MHC-I complexes continue to be assembled by both uninfected and HCMV-infected cells. However, MHC-I molecules are unstable in HCMV-infected cells and are rapidly broken down. Endoglycosidase H treatment of immunoprecipitates indicates that the breakdown of MHC-I complexes in HCMV-infected cells occurs primarily in a pre-Golgi compartment. Interference with normal MHC-I assembly and expression, if relevant in vivo, may have implications for the restriction of the diversity of the CD8+ cytotoxic T lymphocyte repertoire directed against HCMV antigens and may be an important mechanism of viral persistence.     

Assembly of an abundant endogenous major histocompatibility complex class II/peptide complex in class II compartments

To identify the intracellular site(s) of formation of an endogenous class II/peptide complex in a human B cell line, we employed kinetic pulse-chase labeling experiments followed by subcellular fractionation by Percoll density gradient centrifugation and immunogold labeling on ultrathin cryosections. For direct demonstration of assembly of such complexes, we used the monoclonal antibody YAe, which detects an endogenous complex of the mouse class II molecule I-Ab with a 17-amino acid peptide derived from the alpha chain of HLA-DR (DR alpha52-68). We show that in human B lymphocytes, these class II/peptide complexes assemble and transiently accumulate in major histocompatibility complex class II-enriched compartments before reaching the cell surface.     

Molecular docking of superantigens with class II major histocompatibility complex proteins

The molecular recognition of two superantigens with class II major histocompatibility complex molecules was simulated by using protein-protein docking. Superantigens studied were staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin-1 (TSST-1) in their crystallographic assemblies with HLA-DR1. Rigid-body docking was performed sampling configurational space of the interfacial surfaces by employing a strategy of partitioning the contact regions on HLA-DR1 into separate molecular recognition units. Scoring of docked conformations was based on an electrostatic continuum model evaluated with the finite-difference Poisson-Boltzmann method. Estimates of nonpolar contributions were derived from the buried molecular surface areas. We found for both superantigens that docking the HLA-DR1 surface complementary with the SEB and TSST-1 contact regions containing a homologous hydrophobic surface loop provided sufficient recognition for the reconstitution of native-like conformers exhibiting the highest-scoring free energies. For the SEB complex, the calculations were successful in reproducing the total association free energy. A comparison of the free-energy determinants of the conserved hydrophobic contact residue indicates functional similarity between the two proteins for this interface. Though both superantigens share a common global association mode, differences in binding topology distinguish the conformational specificities underlying recognition.     

Minimum structural requirements for peptide presentation by major histocompatibility complex class II molecules: implications in induction of autoimmunity

The precise mechanisms of failure of immunological tolerance to self proteins are not known. Major histocompatibility complex (MHC) susceptibility alleles, the target peptides, and T cells with anti-self reactivity must be present to cause autoimmune diseases. Experimental autoimmune encephalomyelitis (EAE) is a murine model of a human autoimmune disease, multiple sclerosis. In EAE, residues 1-11 of myelin basic protein (MBP) are the dominant disease-inducing determinants in PL/J and (PL/J x SJL/J)F1 mice. Here we report that a six-residue peptide (five of them native) of MBP can induce EAE. Using peptide analogues of the MBP-(1-11) peptide, we demonstrate that only four native MBP residues are required to stimulate MBP-specific T cells. Therefore, this study demonstrates lower minimum structural requirements for effective antigen presentation by MHC class II molecules. Many viral and bacterial proteins share short runs of amino acid similarity with host self proteins, a phenomenon known as molecular mimicry. Since a six-residue peptide can sensitize MBP-specific T cells to cause EAE, these results define a minimum sequence identity for molecular mimicry in autoimmunity.     

The T/B cell interaction involved in induction of the mouse IgG2ab suppression is restricted by major histocompatibility complex class I, but not class II molecules

To determine the major histocompatibility complex (MHC) restriction of the T/ B cell interaction involved in a negative regulation of Ig production, we used mouse model of T cell-induced IgG2ab suppression in vivo. Normal or specifically triggered T splenocytes from mice of the Igha haplotype, when neonatally transferred into histocompatible Igha/b heterozygotes, are able to induce a specific and total suppression of the IgG2ab allotype. Nevertheless, only transfer of IgG2ab-primed Igha T splenocytes induces this suppression in Ighb/b homozygous congenic mice in which the whole IgG2a isotype production is inhibited. This suppression is chronically maintained by CD8+ T cells, but can be experimentally reversed. We have established that the suppression induction required a CD4+CD8+ T cell cooperation and operated via the recognition by the involved TCR of C gamma 2ab-derived peptides presented by the target B cells in an MHC haplotype-restricted manner. Here, by using Ighb mice genetically deficient for MHC class I (beta 2-microglobulin%, or beta 2m%) or class II (I-A beta%) molecules, we demonstrate functionally that the suppression induction implicates an MHC class I-, but not class II-restricted interaction. Indeed, the anti-IgG2ab T cells transferred into Ighb H-2b I-A beta% mice carry out the suppression process normally, while in Ighb H-2b beta 2m% recipients, their suppression induction capacity is significantly inhibited. Moreover, the C gamma 2ab 103-118 peptide, identified as the sole C gamma 2ab-derived peptide able to amplify the anti-IgG2ab T cell reactivity in Igha H-2b mice, is also able to stabilize the H-2Db, but not the H-2Kb class I molecules at the surface of RMA-S (TAP2-, H-2b) cells. These results indicate that, despite the CD4+/CD8+ T cell cooperation during the induction phase of suppression only MHC class I molecule expression is required at the surface of IgG2ab+ B cells for suppression establishment.     

Expression of major histocompatibility complex class II antigens in the canine intestine

Immunohistochemical techniques were used to assess major histocompatibility complex (MHC) class II expression by enterocytes and lamina propria cells in the canine intestinal tract. Duodenal enterocyte class II expression was faint and limited to the lower crypt region whereas jejunal and ileal enterocyte expression was stronger, being present in both crypt and villus areas. Enterocyte staining was of greatest intensity in crypts adjacent to Peyer's patches and intense membrane staining of most Peyer's patch lymphocytes was also seen. Enterocyte MHC class II expression in the colon was largely limited to the lower crypt region. Within the lamina propria, of all intestinal sites examined, a heterogeneous population of cells were MHC class II positive and these had morphological features of macrophages and dendritic cells. Lymphocytes, plasma cells, fibroblasts and vascular endothelium were not stained. Definition of constitutive expression of MHC class II within the canine intestine may be important in identifying upregulation of this molecule in inflammatory bowel diseases.     

Specific effects of estrogen on growth factor and major histocompatibility complex class II antigen expression in rat aortic allograft

OBJECTIVE: Transplant arteriosclerosis is the major determinant for long-term survival of cardiac transplants. Estradiol treatment inhibits transplant arteriosclerosis. The objective of this study is to determine, in the absence of immunosuppression, the temporal effect of estradiol treatment on the expression of insulin-like growth factor, platelet-derived growth factor, basic fibroblast growth factor, and major histocompatibility complex class II antigen in rat aortic allografts. METHODS: Orthotopic abdominal aortic allograft transplantation was performed in male rats with Brown-Norway rats used as donors and Lewis rats as recipients. The recipients (n = 50) were treated with estradiol 20 micrograms/kg per day or placebo by osmotic minipump for 2 days before the operation and until they were put to death on postoperative days 1, 3, 7, 14, or 21. The allografts were harvested and insulin-like growth factor, platelet-derived growth factor, basic fibroblast growth factor, and major histocompatibility complex class II antigen expression were determined by immunohistochemical staining. Myointimal thickening was measured by morphometric analysis. RESULTS: In the placebo-treated group, insulin-like growth factor protein progressively increased in all three layers of the allograft, whereas platelet-derived growth factor protein peaked at day 3 and basic fibroblast growth factor protein increased only moderately. Estradiol treatment inhibited the continuous increase in insulin-like growth factor expression, the peak in platelet-derived growth factor expression at day 3, the moderate-basic fibroblast growth factor increase at day 21, and major histocompatibility complex class II antigen expression in all three layers of the allograft at day 21. Intimal thickening of allografts from estradiol-treated recipients was twofold to threefold less than that of the placebo-treated recipients at day 21. CONCLUSION: The development of transplant arteriosclerosis is associated with an early alloimmune response involving sustained increase in insulin-like growth factor expression. Estradiol treatment of the recipient inhibits transplant arteriosclerosis and suppresses insulin-like growth factor and major histocompatibility complex class II antigen expression but not platelet-derived growth factor or basic fibroblast growth factor in all three layers of the allograft during the early posttransplantation alloimmune rejection phase.     

Structural requirements for major histocompatibility complex class II invariant chain endocytosis and lysosomal targeting

The invariant chain (Ii) targets newly synthesized major histocompatibility complex class II complexes to a lysosome-like compartment. Previously, we demonstrated that both the cytoplasmic tail (CT) and transmembrane (TM) domains of Ii were sufficient for this targeting and that the CT contains two di-leucine signals, 3DQRDLI8 and 12EQLPML17 (Odorizzi, C. G., Trowbridge, I. S., Xue, L., Hopkins, C. R., Davis, C. D., and Collawn, J. F. (1994) J. Cell Biol. 126, 317-330). In the present study, we examined the relationship between signals required for endocytosis and those required for lysosomal targeting by analyzing Ii-transferrin receptor chimeras in quantitative transport assays. Analysis of the Ii CT signals indicates that although 3DQRDLI8 is necessary and sufficient for endocytosis, either di-leucine signal is sufficient for lysosomal targeting. Deletions between the two signals reduced endocytosis without affecting lysosomal targeting. Transplantation of the DQRDLI sequence in place of the EQLPML signal produced a chimera that trafficked normally, suggesting that this di-leucine sequence coded for an independent structural motif. Structure-function analysis of the Ii TM region showed that when Ii TM residues 11-19 and 20-29 were individually substituted for the corresponding regions in the wild-type transferrin receptor, lysosomal targeting was dramatically enhanced, whereas endocytosis remained unchanged. Our results therefore demonstrate that the structural requirements for Ii endocytosis and lysosomal targeting are different.     

Gamma delta TCR-bearing intraepithelial lymphocytes regulate class II major histocompatibility complex molecule expression on the mouse small intestinal epithelium

OBJECTIVE: To examine the hypothesis that addition of therapeutic plasma exchange (TPEX) to an immunosuppressive drug regimen increases that regimen's efficacy to halt the progression of chronic progressive multiple sclerosis (CPMS). METHODS: The literature was searched for prospective controlled clinical trials evaluating the efficacy of TPEX in CPMS. Six studies were eligible for meta-analysis. Their results were combined, using Cochran's and Peto's methods. Three outcome measures were studied: 1) the change in Kurtzke's disability status scale (DSS) scores, 2) the relative odds of neurologic decline by 1 or more DSS grades, and 3) the relative odds of neurologic improvement by 1 or more DSS grades, in the treatment versus the comparison group of patients. Reported results of neurologic evaluations at 6, 12, 24, and 36 months of follow-up were analyzed separately. RESULTS: TPEX significantly (P < .05) reduced the proportion of patients who experienced neurologic decline (by 1 or more DSS grades) at 12 months of follow-up (relative odds of decline = 0.441, 95% confidence interval = 0.210-0.929). CONCLUSIONS: There is a need for further clinical research into the possibility of a beneficial effect of TPEX in patients with CPMS likely to experience neurologic decline over the ensuring 12 months. Targeting treatment to a particular subgroup of CPMS patients may be necessary for TPEX to prove effective.     

Correlation between lymphocyte-induced donor-specific tolerance and donor cell recirculation. Role of class I and class II major histocompatibility complex

Intravenous infusion of mice with viable allogeneic lymphocytes can produce donor-specific enhancement of skin graft survival, but only if the injected lymphocytes can persist in the host's recirculating lymphocyte pool for at least 3 days. We have investigated the relative roles of class I and class II MHC for C57BL/6 mice infused with lymphoid cells from co-isogenic strains mutated at class I MHC (bm1) or class II MHC (bm12), and for A.TH lymphoid cells infused into C3H (class I different, class II identical) or A.TH (class II different, class I identical). Injected cells differing from the host at class I MHC, but not at class II MHC, can be rapidly removed by host natural immune mechanisms (probably NK cells). Persistence is favored if the injected cells also carry host class I MHC, i.e., tolerance is more readily induced by injecting F1 (A x B) into A rather than B into A, consistent with the "missing self" hypothesis of NK recognition, with class I MHC being the relevant self-marker. Injected cells differing from the host at class II MHC but not at class I MHC always persist for at least 3 days, even when class I-different cells are being actively removed.     

Recombinant modified vaccinia virus Ankara-simian immunodeficiency virus gag pol elicits cytotoxic T lymphocytes in rhesus monkeys detected by a major histocompatibility complex class I/peptide tetramer

The utility of modified vaccinia virus Ankara (MVA) as a vector for eliciting AIDS virus-specific cytotoxic T lymphocytes (CTL) was explored in the simian immunodeficiency virus (SIV)/rhesus monkey model. After two intramuscular immunizations with recombinant MVA-SIVSM gag pol, the monkeys developed a Gag epitope-specific CTL response readily detected in peripheral blood lymphocytes by using a functional killing assay. Moreover, those immunizations also elicited a population of CD8+ T lymphocytes in the peripheral blood that bound a specific major histocompatibility complex class I/peptide tetramer. These Gag epitope-specific CD8+ T lymphocytes also were demonstrated by using both functional and tetramer-binding assays in lymph nodes of the immunized monkeys. These observations suggest that MVA may prove a useful vector for an HIV-1 vaccine. They also suggest that tetramer staining may be a useful technology for monitoring CTL generation in vaccine trials in nonhuman primates and in humans.     

Analysis of the role of variation of major histocompatibility complex class II expression on nonobese diabetic (NOD) peripheral T cell response

The current paradigm of major histocompatibility complex (MHC) and disease association suggests that efficient binding of autoantigens by disease-associated MHC molecules leads to a T cell-mediated immune response and resultant autoimmune sequelae. The data presented below offer a different model for this association of MHC with autoimmune diabetes. We used several mouse lines expressing different levels of I-Ag7 and I-Ak on the nonobese diabetic (NOD) background to evaluate the role of MHC class II in the previously described NOD T cell autoproliferation. The ratio of I-Ag7 to I-Ak expression correlated with the peripheral T cell autoproliferative phenotype in the mice studied. T cells from the NOD, [NOD x NOD. I-Anull]F1, and NOD I-Ak transgenic mice demonstrated autoproliferative responses (after priming with self-peptides), whereas the NOD.H2(h4) (containing I-Ak) congenic and [NOD x NOD. H2(h4) congenic]F1 mice did not. Analysis of CD4(+) NOD I-Ak transgenic primed lymph node cells showed that autoreactive CD4(+) T cells in the NOD I-Ak transgenic mice were restricted exclusively by I-Ag7. Considered in the context of the avidity theory of T cell activation and selection, the reported poor peptide binding capacity of NOD I-Ag7 suggested a new hypothesis to explain the effects of MHC class II expression on the peripheral autoimmune repertoire in NOD mice. This new explanation suggests that the association of MHC with diabetes results from "altered" thymic selection in which high affinity self-reactive (potentially autoreactive) T cells escape negative selection. This model offers an explanation for the requirement of homozygous MHC class II expression in NOD mice (and in humans) in susceptibility to insulin-dependent diabetes mellitus.     

Fc gamma receptor II dependency of enhanced presentation of major histocompatibility complex class II peptides by a B cell lymphoma

Here we show that the B cell lymphoma A20.292 is capable of enhanced antigen presentation to CD4+ T cells in the presence of specific antibodies. This enhancement was inhibited by anti-Fc gamma receptor (R) antibodies, suggesting that it might be due to preferential uptake of the antigen/antibody complex through the Fc gamma RII receptor. However, immunoprecipitation studies revealed that the FcR of A20.292 cells was of the B cell type, Fc gamma RIIb1, which is not thought to be able to internalize antigen/antibody complexes via clathrin-coated pits. It was considered unlikely that A20.292 had an altered form of the B cell Fc gamma R (RIIb1) receptor that enabled internalization, since similar enhancing effects were also observed using an Fc gamma RII cell line that had been transfected with Fc gamma RIIb1. To reconcile these findings with the expression of Fc gamma RIIb1, it is postulated that immune complexes are concentrated on the cell surface by the Fc gamma RIIb1 and are thus available for preferential uptake by random fluid-phase endocytosis. This results in more efficient generation of the epitopes recognized by these T cell hybridomas.     

Invariant chain cleavage and peptide loading in major histocompatibility complex class II vesicles

B lymphocytes contain a novel population of endocytic vesicles involved in the transport of newly synthesized major histocompatibility complex (MHC) class II alpha beta chains and alpha beta peptide complexes to the cell surface. We now present evidence that these class II-enriched vesicles (CIIV) are also likely to be a site for the loading of immunogenic peptides onto MHC molecules. We used the serine protease inhibitor leupeptin to accumulate naturally occurring intermediates in the degradation of alpha beta-invariant chain complexes and to slow the intracellular transport of class II molecules. As expected, leupeptin caused an accumulation of Ii chain and class II molecules (I-A(d)) in endosomes and lysosomes. More importantly, however, it enhanced the selective accumulation of a 10-kD invariant chain fragment associated with sodium dodecyl sulfate (SDS)-labile (empty) alpha beta dimers in CIIV. This was followed by the dissociation of the 10-kD fragment, formation of SDS-stable (peptide-loaded) alpha beta dimers, and their subsequent appearance at the cell surface. Thus, CIIV are likely to serve as a specialized site, distinct from endosomes and lysosomes, that hosts the final steps in the dissociation of invariant chain from class II molecules and the loading of antigen-derived peptides onto newly synthesized alpha beta dimers.