The immunodominant major histocompatibility complex class I-restricted antigen of a murine colon tumor derives from an endogenous retroviral gene product.

Tumors express peptide antigens capable of being recognized by tumor-specific cytotoxic T lymphocytes (CTL). Immunization of mice with a carcinogen-induced colorectal tumor, CT26, engineered to secrete granulocyte/macrophage colony-stimulating factor, routinely generated both short-term and long-term CTL lines that not only lysed the parental tumor in vitro, but also cured mice of established tumor following adoptive transfer in vivo. When either short-term or long-term CTL lines were used to screen peptides isolated from CT26, one reverse-phase high performance liquid chromatography peptide fraction consistently sensitized a surrogate target for specific lysis. The bioactivity remained localized within one fraction following multiple purification procedures, indicating that virtually all of the CT26-specific CTL recognized a single peptide. This result contrasts with other tumor systems, where multiple bioactive peptide fractions have been detected. The bioactive peptide was identified as a nonmutated nonamer derived from the envelope protein (gp70) of an endogenous ecotropic murine leukemia provirus. Adoptive transfer with CTL lines specific for this antigen demonstrated that this epitope represents a potent tumor rejection antigen. The selective expression of this antigen in multiple non-viral-induced tumors provides evidence for a unique class of shared immunodominant tumor associated antigens as targets for antitumor immunity.

An increased risk of Crohn's disease in individuals who inherit the HLA class II DRB3*0301 allele.

The role of inflammatory T cells in Crohn's disease suggests that inherited variations in major histocompatibility complex (MHC) class II genes may be of pathogenetic importance in inflammatory bowel disease. The absence of consistent and strong associations with MHC class II genes in Caucasian patients with inflammatory bowel disease probably reflects the use of less precise typing approaches and the failure to type certain loci by any means. A PCR-sequence-specific oligonucleotide-based approach was used to type individual alleles of the HLA class II DRB1, DRB3, DRB4, and DRB5 loci in 40 patients with ulcerative colitis, 42 Crohn's disease patients, and 93 ethnically matched healthy controls. Detailed molecular typing of the above alleles has previously not been reported in patients with inflammatory bowel disease. A highly significant positive association with the HLA-DRB3*0301 allele was observed in patients with Crohn's disease (P = 0.0004) but not in patients with ulcerative colitis. The relative risk for this association was 7.04. Other less significant HLA class II associations were also noted in patients with Crohn's disease. One of these associations involved the HLA-DRB1*1302 allele, which is known to be in linkage disequilibrium with HLA-DRB3*0301. These data suggest that a single allele of an infrequently typed HLA class II locus is strongly associated with Crohn's disease and that MHC class II molecules may be important in its pathogenesis.

Major histocompatibility complex class I molecule serves as a ligand for presentation of the superantigen staphylococcal enterotoxin B to T cells.

Superantigens, such as staphylococcal enterotoxin B (SEB), elicit a strong proliferative response in T cells when presented in the context of major histocompatibility complex (MHC) class II molecules. We observed a similar T-cell response, when MHC class II-negative epidermal cell lines were employed as antigen-presenting cells. Immunoprecipitation studies indicated that the ligand to which SEB bound had a molecular mass of 46 kDa. Radiolabeled SEB could be immunoprecipitated from isolated membrane proteins on the SCC13 epidermal cell line with a monoclonal antibody directed against the MHC class I molecule, and transfection of the K-562 cell line with MHC class I molecules showed a 75% increased SEB-binding capacity compared with the nontransfected MHC class I- and class II-negative counterpart. In functional studies, antibodies to the MHC class I molecule inhibited T-cell proliferation by at least 50%. From these studies, we conclude that MHC class I molecules on malignant squamous cell carcinomas serve as ligands for SEB, which, given the appropriate costimulatory signals, is sufficient to allow for superantigen-induced T-cell proliferation.

Contrasting histories of avian and mammalian Mhc genes revealed by class II B sequences from songbirds.

To explore the evolutionary dynamics of genes in the major histocompatibility complex (Mhc) in nonmammalian vertebrates, we have amplified complete sequences of the polymorphic second (beta1) and third (beta2) exons of class II beta chain genes of songbirds. The pattern of nucleotide substitution in the antigen-binding site of sequences cloned from three behaviorally and phylogenetically divergent songbirds [scrub jays Aphelocoma coerulescens), red-winged blackbirds (Agelaius phoeniceus), and house finches (Carpodacus mexicanus) reveals that class II B genes of songbirds are subject to the same types of diversifying forces as those observed at mammalian class II loci. By contrast, the tree of avian class II B genes reveals that orthologous relationships have not been retained as in placental mammals and that, unlike class II genes in mammals, genes in songbirds and chickens have had very recent common ancestors within their respective groups. Thus, whereas the selective forces diversifying class II B genes of birds are likely similar to those in mammals, their long-term evolutionary dynamics appear to be characterized by much higher rates of concerted evolution.

Two distinct proteolytic processes in the generation of a major histocompatibility complex class I-presented peptide

Although cellular proteins degraded by proteasomes are the source of most antigenic peptides presented on major histocompatibility complex class I molecules, it is unknown whether the eight- to nine-residue peptides that fit in the binding groove of class I molecules are directly produced by proteasomes alone in vivo. If the eight-residue peptide SIINFEKL from chicken ovalbumin is extended by one or several residues at its C terminus and microinjected into cells or expressed from a minigene, it is processed and presented on major histocompatibility complex class I. However, processing and presentation are inhibited by proteasome inhibitors, such as lactacystin. In contrast, when SIINFEKL is extended by 2 to 25 residues at its N terminus, its presentation is not blocked by proteasome inhibitors. N-terminal processing also can occur when the extended peptide is cotranslationally inserted into the endoplasmic reticulum. Thus, two different proteolytic steps in the generation of an chicken ovalbumin-presented peptide can be distinguished. Cleavage by the proteasome defines the proper C terminus, whereas distinct peptidase(s) in the cytosol or endoplasmic reticulum may generate the appropriate N terminus from extended peptides.

Heat shock fusion proteins as vehicles for antigen delivery into the major histocompatibility complex class I presentation pathway

Mice immunized with heat shock proteins (hsps) isolated from mouse tumor cells (donor cells) produce CD8 cytotoxic T lymphocytes (CTL) that recognize donor cell peptides in association with the major histocompatibility complex (MHC) class I proteins of the responding mouse. The CTL are induced apparently because peptides noncovalently associated with the isolated hsp molecules can enter the MHC class I antigen processing pathway of professional antigen-presenting cells. Using a recombinant heat shock fusion protein with a large fragment of ovalbumin covalently linked to mycobacterial hsp70, we show here that when the soluble fusion protein was injected without adjuvant into H-2b mice, CTL were produced that recognized an ovalbumin-derived peptide, SIINFEKL, in association with Kb. The peptide is known to arise from natural processing of ovalbumin in H-2b mouse cells, and CTL from the ovalbumin-hsp70-immunized mice and a highly effective CTL clone (4G3) raised against ovalbumin-expressing EL4 tumor cells (EG7-OVA) were equally effective in terms of the concentration of SIINFEKL required for half-maximal lysis in a CTL assay. The mice were also protected against lethal challenge with ovalbumin-expressing melanoma tumor cells. Because large protein fragments or whole proteins serving as fusion partners can be cleaved into short peptides in the MHC class I processing pathway, hsp fusion proteins of the type described here are promising candidates for vaccines aimed at eliciting CD8 CTL in populations of MHC-disparate individuals.

Identification and characterization of a retinoid-induced class II tumor suppressor/growth regulatory gene

Retinoids, synthetic and natural analogs of retinoic acid, exhibit potent growth inhibitory and cell differentiation activities that account for their beneficial effects in treating hyperproliferative diseases such as psoriasis, actinic keratosis, and certain neoplasias. Tazarotene is a synthetic retinoid that is used in the clinic for the treatment of psoriasis. To better understand the mechanism of retinoid action in the treatment of hyperproliferative diseases, we used a long-range differential display–PCR to isolate retinoid-responsive genes from primary human keratinocytes. We have identified a cDNA, tazarotene-induced gene 3 (TIG3; Retinoic Acid Receptor Responder 3) showing significant homology to the class II tumor suppressor gene, H-rev 107. Tazarotene treatment increases TIG3 expression in primary human keratinocytes and in vivo in psoriatic lesions. Increased TIG3 expression is correlated with decreased proliferation. TIG3 is expressed in a number of tissues, and expression is reduced in cancer cell lines and some primary tumors. In breast cancer cell lines, retinoid-dependent TIG3 induction is observed in lines that are growth suppressed by retinoids but not in nonresponsive lines. Transient over-expression of TIG3 in T47D or Chinese hamster ovary cells inhibits colony expansion. Finally, studies in 293 cells expressing TIG3 linked to an inducible promoter demonstrated decreased proliferation with increased TIG3 levels. These studies suggest that TIG3 may be a growth regulator that mediates some of the growth suppressive effects of retinoids.

Evolutionary instability of the major histocompatibility complex class I loci in New World primates

Homologues of the human major histocompatibility complex (MHC) HLA-A, -B, -E, -F, and -G loci are present in all the Catarrhini (Old World primates, apes, and humans), and some of their allelic lineages have survived several speciation events. Analysis of 26 MHC class I cDNAs from seven different genera of New World primates revealed that the Callitrichinae (tamarins and marmosets) are an exception to these rules of MHC stability. In gene trees of primate MHC class I genes, sequences from the Callitrichinae cluster in a genus-specific fashion, whereas in the other genera of New World primates, as in the Catarrhini, they cluster in a transgeneric way. The genus-specific clustering of the Callitrichinae cDNAs indicates that there is no orthology between MHC class I loci in genera of this phyletic group. Additionally, the Callitrichinae genera exhibit limited variability of their MHC class I genes, in contrast to the high variability displayed by all other primates. Each Callitrichinae genus, therefore, expresses its own set of MHC class I genes, suggesting that an unusually high rate of turnover of loci occurs in this subfamily. The limited variability of MHC class I genes in the Callitrichinae is likely the result of the recent origin of these loci.

Gene Conversion of Major Histocompatibility Complex Genes in the Mouse Spermatogenesis is a Premeiotic Event

The molecular genetic mechanism of gene conversion in higher eukaryotes remains unknown. We find it of considerable interest to determine when during spermatogenesis gene conversion occurs. We have therefore purified pachytene spermatocytes and haploid spermatocytes from adult mice and analyzed these fractions for the presence of gene conversion products resulting from the transfer between the major histocompatibility complex class II genes Ebd and Abk in a polymerase chain reaction assay. We have further isolated spermatogenic cells from prepubescent mice and analyzed them for the presence of the same gene conversion products. We can detect gene conversion products in testis cells as early as in 8-d-old mice where the only existing spermatogenic cells are spermatogonia. The frequency of gene conversion products remains the same as the cells reach meiosis in 18-d-old mice, and is unchanged after meiosis is completed in haploid spermatocytes. Gene conversion of this specific fragment therefore appears to be a premeiotic event and, consequently, relies on genetic mechanisms other than normal meiotic recombination.

Identification of HLA class II-restricted determinants of Mycobacterium tuberculosis-derived proteins by using HLA-transgenic, class II-deficient mice

T helper 1 cells play a major role in protective immunity against mycobacterial pathogens. Since the antigen (Ag) specificity of CD4+ human T cells is strongly controlled by HLA class II polymorphism, the immunogenic potential of candidate Ags needs to be defined in the context of HLA polymorphism. We have taken advantage of class II-deficient (Ab0) mice, transgenic for either HLA-DRA/B1*0301 (DR3) or HLA-DQB1*0302/DQA*0301 (DQ8) alleles. In these animals, all CD4+ T cells are restricted by the HLA molecule. We reported previously that human DR3-restricted T cells frequently recognize heat shock protein (hsp)65 of Mycobacterium tuberculosis, and only a single hsp65 epitope, p1–20. DR3.Ab0 mice, immunized with bacillus Calmette–Guérin or hsp65, developed T cell responses to M. tuberculosis, and recognized the same hsp65 epitope, p1–20. Hsp65-immunized DQ8.Ab0 mice mounted a strong response to bacillus Calmette–Guérin but not to p1–20. Instead, we identified three new DQ8-restricted T cell epitopes in the regions 171–200, 311–340, and 411–440. DR3.Ab0 mice immunized with a second major M. tuberculosis protein, Ag85 (composed of 85A, 85B, and 85C), also developed T cell responses against only one determinant, 85B p51–70, that was identified in this study. Importantly, subsequent analysis of human T cell responses revealed that HLA-DR3+, Ag85-reactive individuals recognize exactly the same peptide epitope as DR3.Ab0 mice. Strikingly, both DR3-restricted T cell epitopes represent the best DR3-binding sequences in hsp65 and 85B, revealing a strong association between peptide-immunodominance and HLA binding affinity. Immunization of DR3.Ab0 with the immunodominant peptides p1–20 and p51–70 induced T cell reactivity to M. tuberculosis. Thus, for two different Ags, T cells from DR3.Ab0 mice and HLA-DR3+ humans recognize the same immunodominant determinants. Our data support the use of HLA-transgenic mice in identifying human T cell determinants for the design of new vaccines.

An in vitro study of the dynamic features of the major histocompatibility complex class I complex relevant to its role as a versatile peptide-receptive molecule

The major histocompatibility complex class I complex consists of a heavy chain and a light chain (β2-microglobulin, β2m), which assemble with a short endogenously derived peptide in the endoplasmic reticulum. The class I peptide can be directly exchanged, either at the cell surface or, as recently described, in vesicles of the endocytic compartments, thus allowing exogenous peptides to enter the class I presentation pathway. To probe the interactions between the components of the class I molecule, we analyzed the exchange of peptide and β2m by using purified, recombinant H2-Kb/peptide complexes in a cell-free in vitro system. The exchange of competitor peptide was primarily dependent on the off-rate of the original peptide in the class I binding groove. Peptide exchange was not enhanced by the presence of exogenous β2m, as exchange occurred to the same extent in its absence. Thus, the exchange of peptide and β2m are independent events. The exchange rate of β2m also was not affected by the dissociation rates of the original peptides. Furthermore, peptides could substantially exchange into class I molecules over a pH range of 5.5 to 7.5, conditions prevalent in certain endocytic compartments. We conclude that the dynamic properties of the components of class I molecules explain its function as a highly peptide-receptive molecule. The major histocompatibility complex class I can readily receive peptides independent of the presence of exogenous β2m, even at a low pH. Such properties are relevant to class I peptide acquisition, which can occur at the cell surface, as well as in specialized endosomes.

The Export of Major Histocompatibility Complex Class I Molecules from the Endoplasmic Reticulum of Rat Brown Adipose Cells Is Acutely Stimulated by Insulin

Major histocompatibility complex class I (MHC-I) molecules have been implicated in several nonimmunological functions including the regulation and intracellular trafficking of the insulin-responsive glucose transporter GLUT4. We have used confocal microscopy to compare the effects of insulin on the intracellular trafficking of MHC-I and GLUT4 in freshly isolated rat brown adipose cells. We also used a recombinant vaccinia virus (rVV) to express influenza virus hemagglutinin (HA) as a generic integral membrane glycoprotein to distinguish global versus specific enhancement of protein export from the endoplasmic reticulum (ER) in response to insulin. In the absence of insulin, MHC-I molecules largely colocalize with the ER-resident protein calnexin and remain distinct from intracellular pools of GLUT4. Surprisingly, insulin induces the rapid export of MHC-I molecules from the ER with a concomitant approximately three-fold increase in their level on the cell surface. This ER export is blocked by brefeldin A and wortmannin but is unaffected by cytochalasin D, indicating that insulin stimulates the rapid transport of MHC-I molecules from the ER to the plasma membrane via the Golgi complex in a phosphatidyl-inositol 3-kinase–dependent and actin-independent manner. We further show that the effect of insulin on MHC-I molecules is selective, because insulin does not affect the intracellular distribution or cell-surface localization of rVV-expressed HA. These results demonstrate that in rat brown adipose cells MHC-I molecule export from the ER is stimulated by insulin and provide the first evidence that the trafficking of MHC-I molecules is acutely regulated by a hormone.

CD4+ T cell recognition of MHC class II-restricted epitopes from NY-ESO-1 presented by a prevalent HLA DP4 allele: Association with NY-ESO-1 antibody production

NY-ESO-1 is a tumor-specific shared antigen with distinctive immunogenicity. Both CD8+ T cells and class-switched Ab responses have been detected from patients with cancer. In this study, a CD4+ T cell line was generated from peripheral blood mononuclear cells of a melanoma patient and was shown to recognize NY-ESO-1 peptides presented by HLA-DP4, a dominant MHC class II allele expressed in 43–70% of Caucasians. The ESO p157–170 peptide containing the core region of DP4-restricted T cell epitope was present in a number of tumor cell lines tested and found to be recognized by both CD4+ T cells as well as HLA-A2-restricted CD8+ T cells. Thus, the ESO p157–170 epitope represents a potential candidate for cancer vaccines aimed at generating both CD4+ and CD8+ T cell responses. More importantly, 16 of 17 melanoma patients who developed Ab against NY-ESO-1 were found to be HLA-DP4-positive. CD4+ T cells specific for the NY-ESO-1 epitopes were generated from 5 of 6 melanoma patients with NY-ESO-1 Ab. In contrast, no specific DP4-restricted T cells were generated from two patients without detectable NY-ESO-1 Ab. These results suggested that NY-ESO-1-specific DP4-restricted CD4+ T cells were closely associated with NY-ESO-1 Ab observed in melanoma patients and might play an important role in providing help for activating B cells for NY-ESO-1-specific Ab production.

Dual HLA class I and class II restricted recognition of alloreactive T lymphocytes mediated by a single T cell receptor complex

The alloreactive human T cell clone MBM15 was found to exhibit dual specificity recognizing both an antigen in the context of the HLA class I A2 molecule and an antigen in the context of the HLA class II DR1. We demonstrated that the dual reactivity that was mediated via a single clonal T cell population depended on specific peptide binding. For complete recognition of the HLA-A2-restricted specificity the interaction of CD8 with HLA class I is essential. Interestingly, interaction of the CD8 molecule with HLA class I contributed to the HLA-DR1-restricted specificity. T cell clone MBM15 expressed two in-frame T cell receptor (TCR) Vα transcripts (Vα1 and Vα2) and one TCR Vβ transcript (Vβ13). To elucidate whether two TCR complexes were responsible for the dual recognition or one complex, cytotoxic T cells were transduced with retroviral vectors encoding the different TCR chains. Only T cells transduced with the TCR Vα1Vβ13 combination specifically recognized both the HLA-A2+ and HLA-DR1+ target cells, whereas the Vα2Vβ13 combination did not result in a TCR on the cell surface. Thus a single TCRαβ complex can have dual specificity, recognizing both a peptide in the context of HLA class I as well as a peptide in the context of HLA class II. Transactivation of T cells by an unrelated antigen in the context of HLA class II may evoke an HLA class I-specific T cell response. We propose that this finding may have major implications for immunotherapeutic interventions and insight into the development of autoimmune diseases.

Human cytomegalovirus US3 impairs transport and maturation of major histocompatibility complex class I heavy chains.

The human cytomegalovirus (HCMV) early glycoprotein products of the US11 and US2 open reading frames cause increased turnover of major histocompatibility complex (MHC) class I heavy chains. Since US2 is homologous to another HCMV gene (US3), we hypothesized that the US3 gene product also may affect MHC class I expression. In cells constitutively expressing the HCMV US3 gene, MHC class I heavy chains formed a stable complex with beta 2-microglobulin. However, maturation of the N-linked glycan of MHC class I heavy chains was impaired in US3+ cells. The glycoprotein product of US3 (gpUS3) occurs mostly in a high-mannose form and coimmunoprecipitates with beta 2-microglobulin associated class I heavy chains. Mature class I molecules were detected at steady state on the surface of US3+ cells, as in control cells. Substantial perinuclear accumulation of heavy chains was observed in US3+ cells. The data suggest that gpUS3 impairs egress of MHC class I heavy chains from the endoplasmic reticulum.