Diversification, expression, and gamma delta T cell recognition of evolutionarily distant members of the MIC family of major histocompatibility complex class I-related molecules

Distant relatives of major histocompatibility complex (MHC) class I molecules, human MICA and MICB, function as stress-induced antigens that are broadly recognized by intestinal epithelial gamma delta T cells. They may thus play a central role in the immune surveillance of damaged, infected, or otherwise stressed intestinal epithelial cells. However, the generality of this system in evolution and the mode of recognition of MICA and MICB are undefined. Analysis of cDNA sequences from various primate species defined translation products that are homologous to MICA and MICB. All of the MIC polypeptides have common characteristics, although they are extraordinarily diverse. The most notable alterations are several deletions and frequent amino acid substitutions in the putative alpha-helical regions of the alpha1 alpha2 domains. However, the primate MIC molecules were expressed on the surfaces of normal and transfected cells. Moreover, despite their sharing of relatively few identical amino acids in potentially accessible regions of their alpha1 alpha2 domains, they were recognized by diverse human intestinal epithelial gamma delta T cells that are restricted by MICA and MICB. Thus, MIC molecules represent a family of MHC proteins that are structurally diverse yet appear to be functionally conserved. The promiscuous mode of gamma delta T cell recognition of these antigens may be explained by their sharing of a single conserved interaction site.     

HIV-1 Nef protein protects infected primary cells against killing by cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTLs) lyse virally infected cells that display viral peptide epitopes in association with major histocompatibility complex (MHC) class I molecules on the cell surface. However, despite a strong CTL response directed against viral epitopes, untreated people infected with the human immunodeficiency virus (HIV-1) develop AIDS. To resolve this enigma, we have examined the ability of CTLs to recognize and kill infected primary T lymphocytes. We found that CTLs inefficiently lysed primary cells infected with HIV-1 if the viral nef gene product was expressed. Resistance of infected cells to CTL killing correlated with nef-mediated downregulation of MHC class I and could be overcome by adding an excess of the relevant HIV-1 epitope as soluble peptide. Thus, Nef protected infected cells by reducing the epitope density on their surface. This effect of nef may allow evasion of CTL lysis by HIV-1-infected cells.     

Isolation and characterization of murine clonogenic osteoclast progenitors by cell surface phenotype analysis

MHC class I molecules bind short peptides for presentation to CD8+ T cells. The determination of the three-dimensional structure of various MHC class I complexes has revealed that both ends of the peptide binding site are composed of polar residues conserved among all human and murine MHC class I sequences, which act to lock the ends of the peptide into the groove. In the rat, however, differences in these important residues occur, suggesting the possibility that certain rat MHC class I molecules may be able to bind and present longer peptides. Here we have studied the peptide length preferences of two rat MHC class Ia molecules expressed in the TAP2-deficient mouse cell line RMA-S: RT1-A1c, which carries unusual key residues at both ends of the groove, and RT1.Aa which carries the canonical residues. Temperature-dependent peptide stabilization assays were performed using synthetic random peptide libraries of different lengths (7-15 amino acids) and successful stabilization was determined by FACS analysis. Results for two naturally expressed mouse MHC class I molecules revealed different length preferences (H2-Kb, 8-13-mer and H2-Db, 9-15-mer peptides). The rat MHC class Ia molecule, RT1-Aa, revealed a preference for 9-15-mer peptides, whereas RT1-A1c showed a more stringent preference for 9-12-mer peptides, thereby ruling out the hypothesis that unusual residues in rat MHC molecules allow binding of longer peptides.     

Immunodominance of a single CTL epitope in a primate species with limited MHC class I polymorphism

MHC class I molecules play a crucial role in immunity to viral infections by presenting viral peptides to cytotoxic T lymphocytes. One of the hallmarks of MHC class I genes in outbred populations is their extraordinary polymorphism, yet the significance of this diversity is poorly understood. Certain species with reduced MHC class I diversity, such as the cotton-top tamarin (Saguinus oedipus), are more susceptible to fatal viral infections. To explore the relationship between this primate's limited MHC class I diversity and its susceptibility to viruses, we infected five cotton-top tamarins with influenza virus. Every tamarin recognized the same immunodominant CTL epitope of the influenza nucleoprotein. Surprisingly, this nucleoprotein peptide was bound by Saoe-G*08, an MHC class I molecule expressed by every cotton-top tamarin. Two tamarins also made a subdominant response to an epitope of the matrix (M1) protein. This peptide appeared to be bound by another common MHC class I molecule. With the exception of an additional subdominant response to the polymerase (PB2) protein in one individual, no other influenza-specific CTL responses were detected. In populations or species with limited MHC class I polymorphism like the cotton-top tamarin, a dependence on shared MHC class I molecules may enhance susceptibility to viral infection, since viruses that evade MHC class I-restricted recognition in one individual will likely evade recognition in the majority of individuals.     

Population pharmacokinetics of vancomycin in Japanese adult patients

MICA is a new polymorphic gene in the HLA region expressed in epithelial cell lines and gastrointestinal epithelium. Little is yet known about the MICA protein, and the pattern of its expression by freshly isolated cells has not been established. In the present experiments, we used antibodies raised in rabbits against alpha1 and alpha2 domain-peptides to study the expression of MICA. By western blot and immunoprecipitation, we detected a band of 62 000 Mr in various cell lines (THP-1, U937, HeLa, A431, Raji, MOLT-4, and HUV-EC-C) and in freshly isolated keratinocytes, endothelial cells, and monocytes but not in CD4+ and CD8+ T cells, and CD19+ cells (B lymphocytes). It was not possible to up-regulate the expression of MICA in different cells by stimulation with gamma-interferon, but the expression of MICA was induced in phytohemagglutinin-stimulated T cells. We confirmed that MICA is expressed at the cell surface by flow cytometry. Results of immunoprecipitation studies of beta2-microglobulin (beta2m)- or MICA-depleted, metabolically labeled HeLa cells indicated that MICA was not associated with beta2m. Although the function of MICA is still unknown, its restricted pattern of tissue expression, the fact that it is expressed on the cell surface, and its polymorphic nature suggest that this new molecule, encoded close to HLA class I, may play a role in the interaction between epithelial cells and cells of the immune system.     

Peptide-induced conformational changes in class I molecules. Direct detection by flow cytometry

Activation of a T cell in response to peptide bound to class I MHC occurs by the sum of interactions across the area of contact between the TCR, the peptide, and class I MHC. It has been observed recently that substitution of the peptide residue at a position that is not accessible from the exterior of the class I molecule modulates T cell responses, raising the possibility that there may be indirect structural effects in the peptide-class I complex as a consequence of peptide binding. This report describes the use of mAbs to probe the conformation of the alpha 1 and alpha 2 domains of the mouse class I molecule Kb when bound to ovalbumin peptide and a panel of 19 peptide analogues that differ at position 2 (P2). By crystallographic data, side chains of this position are buried in the Ag binding cleft and have no direct access to the TCR. Substitution of position 2 results in a measurable change in conformation of the class I molecule, a change that correlates with the ability to stimulate T cells. This leads to a model that T cell activation by the peptide-class I complex may occur in three ways: 1) direct interaction of the TCR with the class I heavy chain, 2) direct interaction of the TCR with solvent-accessible peptide side chains, and 3) indirect interaction of peptide with TCR mediated via conformational perturbations in the class I complex.     

Histophysiological observations on the external auditory meatus, middle, and inner ear of the Weddell seal (Leptonychotes weddelli)

The assembly assay for peptide binding to class I major histocompatibility complex (MHC) is based on the ability to stabilise MHC class I molecules from mutant cell lines by the addition of suitable peptides. Such cell lines lack a functional transporter associated with antigen presentation (TAP) and as a result accumulate empty, unstable class I molecules in the ER. These dissociate rapidly in cell lysates unless they are stabilised by the addition of an appropriate binding peptide during lysis. The extent of stabilisation of class I molecules is directly related to the binding affinity of the added peptide. However, some MHC class I molecules, including HLA-B * 2705 and H-2Kk are unusually stable in their peptide-receptive state making them inappropriate for analysis using this assay or assays which depend on the ability of peptides to stabilise MHC class I molecules at the cell surface. Here we present an improved method that permits reliable measurements of peptide binding to such class I MHC molecules that are unusually stable in the absence of peptide. Cells are lysed in the presence of peptide and incubated at 4 degrees C. After 2 h, during which peptide binding to empty MHC molecules occurs, the lysate is heated to a temperature which preferentially destabilises those MHC molecules that remain empty. We have used this technique to assay peptide binding to HLA-B * 2705, as well as to the murine allele H-2Kk which also displays a stable phenotype when transfected into TAP-deficient T2 cells and show that this method represents a marked improvement over previous methods in terms of lower background signal and higher recovery of peptide bound molecules.     

Regulation of MHC class I and II gene transcription: differences and similarities

Major histocompatibility complex (MHC) molecules serve as peptide receptors. These peptides are derived from processed cellular or extra-cellular antigens. The MHC gene complex encodes two major classes of molecules, MHC class I and class II, whose function is to present peptides to CD8+ (cytotoxic) and CD4+ (helper) T cells, respectively. The genes encoding both classes of MHC molecules seem to originate from a common ancestral gene. One of the hallmarks of the MHC is its extensive polymorphism which displays locus and allele-specific characteristics among the various MHC class I and class II genes. Because of its central role in immunosurveillance and in various disease states, the MHC is one of the best studied genetic systems. This review addresses several aspects of MHC class I and class II gene regulation in human and in particular, the contribution to the constitutive and cytokine-induced expression of MHC class I and II genes of MHC class-specific regulatory elements and regulatory elements which apparently are shared by the promoters of MHC class I and class II genes.     

Tumour cell vaccines that secrete interleukin-2 (IL-2) and interferon gamma (IFN gamma) are recognised by T cells while resisting destruction by natural killer (NK) cells

The inoculation into mice of genetically engineered tumour cells that secrete IL-2 or IFN gamma results in rejection, while unmodified parental tumour cells grow progressively. In vivo studies demonstrated synergy between IL-2 and IFN gamma leading to the rejection of the transduced tumour cells. IL-2 is required for T cell proliferation and differentiation. IFN gamma induced the upregulation of MHC class I molecules that present peptides to CD8+ T cells. Furthermore, IFN gamma can correct defects in antigen processing. Thus, for T cells, IL-2/IFN gamma-secreting double cytokine tumour cell vaccines might serve as class I+ peptide/antigen presenting depots for developing effector cells. In contrast to T cells, NK cells exert spontaneous killing and kill class I+ targets less well than those that are class I-. For this reason, they may actually have a detrimental effect by destroying a class I+ tumour cell vaccine before adequate T cell stimulation occurs. Based upon this rationale, we tested the hypothesis that an unrecognised benefit of increased class I expression by tumour cells in response to IFN gamma secretion would be to enable cytokine-secreting vaccine cells to resist destruction by NK cells. Our results demonstrated that T cells recognised tumour cells secreting IFN gamma better than those secreting IL-2. NK cells, in contrast, were inhibited by tumour cells that secreted IFN gamma, but not by those that secreted IL-2. The findings suggest that, in addition to upregulating adhesion molecules, MHC molecules, and correcting defects in antigen presentation pathways, IFN gamma secretion may protect tumour cell vaccines from early NK-mediated destruction, keeping them available for T cell priming.     

Comparison of the roles of CD8 alpha alpha and CD8 alpha beta in interaction with MHC class I

The CD8 molecule is expressed either as an alpha/alpha homodimer or an alpha/beta heterodimer on thymocytes and cytotoxic T cells, and functions as a coreceptor in concert with TCR for binding the MHC class I/peptide complex. Although CD8alpha/beta heterodimers have been shown to be more effective coreceptors, the precise role of the beta-chain in TCR-mediated thymic maturation and T cell activation is not understood. To understand the role of CD8beta in mediating CD8/MHC class I interaction, we examined whether cell surface CD8alpha/beta heterodimer promotes better cell-cell adhesion with MHC class I than the CD8alpha/alpha homodimer. The abilities of different forms of CD8 to adhere to MHC class I were measured with a cell-cell binding assay. Using a wild-type CD8beta and -alpha, we found that CD8alphabeta heterodimers did not mediate greater cell-cell adhesion than CD8alphaalpha homodimers. Furthermore, we found that chimeric CD8beta-alpha homodimers afforded no detectable binding. These results do not support the idea that CD8alphabeta binding to MHC class I is greater than that of CD8alphaalpha. Rather, they point to an alternative explanation in which CD8beta may play an role in promoting CD8/TCR interaction and/or in signaling/regulatory pathways.     

Cutting edge: MHC class I triggering by a novel cell surface ligand costimulates proliferation of activated human T cells

BY55 is a human cell surface molecule whose expression is restricted to NK cells, a subset of circulating CD8+ T lymphocytes, and all intestinal intraepithelial T lymphocytes. Here, we report that BY55 is a novel NK receptor showing broad specificity for both classical and nonclassical MHC class I molecules, and that optimal binding requires a prior aggregation of MHC class I complexes. Using BY55 transfectants, we have identified functional consequences of MHC class I/ligand interactions for the class I-bearing cell. The triggering of MHC class I molecules on human T cell clones by BY55 delivered a potent proliferative signal in the presence of soluble CD3 mAb. The costimulatory signal provided by MHC class I ligation was only seen in activated, and not resting, peripheral blood T cells. This observation represents an additional and/or alternative pathway to CD28 costimulation and may be of particular relevance in memory T cells lacking CD28, such as intestinal intraepithelial T lymphocytes, which are CD28- but BY55+.     

Heteroclitic immunization induces tumor immunity

In tumor transplantation models in mice, cytotoxic T lymphocytes (CTLs) are typically the primary effector cells. CTLs recognize major histocompatibility complex (MHC) class I-associated peptides expressed by tumors, leading to tumor rejection. Peptides presented by cancer cells can originate from viral proteins, normal self-proteins regulated during differentiation, or altered proteins derived from genetic alterations. However, many tumor peptides recognized by CTLs are poor immunogens, unable to induce activation and differentiation of effector CTLs. We used MHC binding motifs and the knowledge of class I:peptide:TCR structure to design heteroclitic CTL vaccines that exploit the expression of poorly immunogenic tumor peptides. The in vivo potency of this approach was demonstrated using viral and self-(differentiation) antigens as models. First, a synthetic variant of a viral antigen was expressed as a tumor antigen, and heteroclitic immunization with peptides and DNA was used to protect against tumor challenge and elicit regression of 3-d tumors. Second, a peptide from a relevant self-antigen of the tyrosinase family expressed by melanoma cells was used to design a heteroclitic peptide vaccine that successfully induced tumor protection. These results establish the in vivo applicability of heteroclitic immunization against tumors, including immunity to poorly immunogenic self-proteins.     

The cytoplasmic and the transmembrane domains are not sufficient for class I MHC signal transduction

Class I MHC molecules deliver activation signals to T cells. To analyze the role of the cytoplasmic and the transmembrane (TM) domains of class I MHC molecules in T cell activation, Jurkat cells were transfected with genes for truncated class I MHC molecules which had only four intracytoplasmic amino acids and no potential phosphorylation sites or native molecules or both. Cross-linking either the native or the truncated molecules induced IL-2 production even under limiting stimulation conditions of low engagement of the stimulating mAb. Moreover, direct comparison of transfected truncated and native class I MHC molecules expressed on the same cell revealed significant stimulation induced by cross-linking the truncated molecules, despite low expression. In addition, truncated class I MHC molecules were as able to synergize with CD3, CD2, or CD28 initiated IL-2 production as native molecules. In further experiments, hybrid constructs made of the extracellular portion of the murine CD8 alpha chain and of the TM and the intracytoplasmic domains of H-2Kk class I MHC molecule were transfected into Jurkat T cells. The expression of the transfected hybrid molecules was comparable to that of the native HLA-B7 molecules. Cross-linking the intact monomorphic HLA-A,B,C epitope or the polymorphic HLA-B7 epitope induced IL-2 production upon costimulation with PMA. In contrast, cross-linking the hybrid molecules generated neither an increase in intracellular calcium concentration ([Ca2+]i) nor stimulated IL-2 production. By contrast, cross-linking intact murine class I MHC molecules induced [Ca2+]i, signal and IL-2 production in transfected Jurkat cells. The data therefore indicate that unlike many other signaling molecules, signaling via class I MHC molecules does not involve the cytoplasmic and the TM portions of the molecule, but rather class I MHC signal transduction is likely to be mediated by the extracellular domain of the molecule.     

MHC class I genes in a New World primate, the cotton-top tamarin (Saguinus oedipus), have evolved by an active process of loci turnover

Lymphocytes of a New World primate, the cotton-top tamarin (Saguinus oedipus), express classical G-related major histocompatibility complex (MHC) class I molecules with unusually limited polymorphism and variability. Three G-related loci, an F locus, an E locus, and two pseudogenes (So-N1 and So-N3) have been identified by cDNA library screening and extensive PCR analysis of both cDNA and genomic DNA from the cotton-top tamarin. Furthermore, each genus of the subfamily Callitrichinae (tamarins and marmosets) appears to express its own unique set of MHC class I genes, likely due to a rapid turnover of loci. The rapid emergence of unique MHC class I genes in the Callitrichinae genera, resulting from an active process of duplication and inactivation of loci, may account for the limited diversity of the MHC class I genes in the cotton-top tamarin. To determine the nature of the entire complement of MHC class I genes in the cotton-top tamarin, we synthesized a genomic DNA library and screened it with MHC class I-specific probes. We isolated nine new MHC class I pseudogenes from this library. These newly isolated tamarin G-related MHC class I pseudogenes are not closely related to any of their functional counterparts in the tamarin, suggesting that they do not share a recent common ancestral gene with the tamarin's currently expressed MHC class I loci. In addition, these tamarin sequences display a high rate of nonsynonymous substitutions in their putative peptide binding region. This indicates that the genes from which they have derived were likely subject to positive selection and, therefore, were once functional. Our data support the notion that an extremely high rate of loci turnover is largely responsible for the limited diversity of the MHC class I genes in the cotton-top tamarin.     

RT1-U: identification of a novel, active, class Ib alloantigen of the rat MHC

In common with other mammalian species, the laboratory rat (Rattus norvegicus) expresses MHC class I molecules that have been categorized as either classical (class Ia) or nonclassical (class Ib). This distinction separates the class Ia molecules that play a conventional role in peptide Ag presentation to CD8 T cells from the others, whose function is unconventional or undefined. The class Ia molecules are encoded by the RT1-A region of the rat MHC, while the RT1-C/E/M region encodes up to 60 other class I genes or gene fragments, a number of which are known to be expressed (or to be expressible). Here we report upon novel MHC class Ib genes of the rat that we have expression cloned using new monoclonal alloantibodies and which we term RT1-U. The products detected by these Abs were readily identifiable by two-dimensional analysis of immunoprecipitates and were shown to be distinct from the class Ia products. Cellular studies of these molecules indicate that they function efficiently as targets for cytotoxic killing by appropriately raised polyclonal alloreactive CTL populations. The sequences of these class Ib genes group together in phylogenetic analysis, suggesting a unique locus or family. The combined serological, CTL, and sequence data all indicate that these products are genetically polymorphic.     

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.     

Metabolism of the insulin secretagogue methyl succinate by pancreatic islets

Presentation of antigenic peptides by major histocompatibility complex (MHC) class I molecules requires MHC-encoded molecules of the adenosine triphosphate binding cassette (ABC) family. Defects in these proteins represent a potential risk, since they are essential links in the machinery of T cell-mediated surveillance which continuously scrutinizes peptide samples of cellular proteins. Nevertheless, transfection of the mouse lymphoma mutant RMA-S with the rat ABC gene mtp2a (homologue to mouse HAM2 and human RING11), commonly termed TAP-2 genes, led to a marked increase in tumor outgrowth potential in vivo. This occurred despite restored antigen presentation and sensitivity to cytotoxic T lymphocytes, and was found to be due to escape from natural killer (NK) cell-mediated rejection. It has previously been proposed that adequate expression of self-MHC class I is one important mechanism to avoid elimination by NK cells. Our data argue that a defect in the machinery responsible for processing and loading of peptides into MHC class I molecules is sufficient to render cells sensitive to elimination by NK cells. The latter thus appear to function as a surveillance of the peptide surveillance machinery.     

NK cells can recognize different forms of class I MHC

NK recognition and lysis of targets are mediated by activation receptor(s) whose effects may be over-ridden by inhibitory receptors recognizing class I MHC on the target. Incubation of normal lymphoblasts with a peptide that can bind to their class I MHC renders them sensitive to lysis by syngeneic NK cells. By binding to class I MHC, the peptide alters or masks the target structure recognized by an inhibitory NK receptor(s). This target structure is most likely an "empty" dimer of class I heavy chain and beta2m as opposed to a "full" class I trimer formed by binding of specific peptide that is recognized by CTL.