Search

Type 1 diabetes (T1D) is an autoimmune disease characterized by T cell-mediated destruction of insulin-producing pancreatic beta cells. In both humans and the non-obese diabetic (NOD) mouse model of T1D, class II MHC alleles are the primary determinant of disease susceptibility. However, class I MHC genes also influence risk. These findings are consistent with the requirement for both CD4(+) and CD8(+) T cells in the pathogenesis of T1D. Although a large body of work has permitted the identification of multiple mechanisms to explain the diabetes-protective effect of particular class II MHC alleles, studies examining the protective influence of class I alleles are lacking. Here, we explored this question by performing biochemical and structural analyses of the murine class I MHC molecule H-2K(wm7), which exerts a diabetes-protective effect in NOD mice. We have found that H-2K(wm7) molecules are predominantly occupied by the single self-peptide VNDIFERI, derived from the ubiquitous protein histone H2B. This unexpected finding suggests that the inability of H-2K(wm7) to support T1D development could be due, at least in part, to the failure of peptides from critical beta-cell antigens to adequately compete for binding and be presented to T cells. Predominant presentation of a single peptide would also be expected to influence T-cell selection, potentially leading to a reduced ability to select a diabetogenic CD8(+) T-cell repertoire. The report that one of the predominant peptides bound by T1D-protective HLA-A*31 is histone derived suggests the potential translation of our findings to human diabetes-protective class I MHC molecules.

Regulation of dendritic cell (DC) function is critical for maintaining self-tolerance and preventing autoimmunity. The dendritic cell-specific transmembrane protein (DC-STAMP) plays a key role in cell-cell fusion of osteoclasts and foreign body giant cells, but though originally identified in DCs, its specific roles there remain undefined. Here, we report that aged DC-STAMP-deficient mice display several systemic autoimmune symptoms such as spontaneous lymphoproliferation, splenomegaly associated with infiltration of T cells in several organs and increased serum anti-double-stranded DNA antibody production. Although a lack of DC-STAMP did not inhibit DC differentiation or proliferation, antigen presentation activity of DC-STAMP-deficient DCs was significantly up-regulated in both class I and II pathways through increased phagocytotic activity compared with wild-type DCs, an activity likely leading to autoimmunity. Our results indicate that DC-STAMP is required for proper regulation of DC activity and maintenance of immune self-tolerance.

In the course of viral infection, the immune system exploits only a fraction of the available CTL repertoire and focuses on a few of a myriad of potentially antigenic peptides. This phenomenon, known as immunodominance, depends on a number of factors, including antigen processing and transport, MHC binding, competition for antigen-presenting cells, availability of the CD8 T cell repertoire and other mechanisms that function largely by restricting the immune response. Here we elucidate a novel mechanism that increases the immunodominance of the epitope rather by enhancing the immune response. Using a peptide-specific MHC-restricted mAb and functional assays of CTL activation, we show that T cells with high avidity for the immunodominant, H-2D(d) restricted, P18-I10 epitope expand rapidly following immunization, and this expansion in turn determines the level of the P18-I10 epitope immunodominance. This proliferation has little dependence on the number of MHC-peptide complexes. Since most self-reactive T cells of high avidity are depleted in the thymus, the selection of immunodominant epitopes based on the expansion of high-avidity T cells in the periphery reduces the potential for autoimmunity.

NK cells monitor expression of MHC class I by inhibitory receptors and preferentially kill cells that lose or down-regulate MHC class I expression. One possible mechanism by which tumor cells evade NK cell killing is continued expression of appropriate MHC class I ligands to engage inhibitory receptors on NK cells. We show here that small-mol.-wt blockers against the mouse inhibitory NK cell receptor Ly49A enhance NK cell killing of such tumor cells. We identified Ly49A-binding peptides by selecting phages with the capacity to bind recombinant Ly49A expressed in Escherichia coli from a phage display random peptide library. The Ly49A-binding peptides could also bind Ly49A expressed on mammalian cells. Importantly, the Ly49A-binding peptides blocked Ly49A recognition of its MHC class I ligands H-2Dd and H-2Dk. Moreover, blockade of Ly49A by the peptides enhanced cytotoxicity of Ly49A+ NK cells towards H-2Dd-expressing tumor cells. These results clearly indicate effectiveness of small-mol.-wt blockers of inhibitory NK cell receptors in enhancing NK cell-mediated killing of tumor cells that are otherwise resistant because of MHC class I expression.

The mouse inhibitory NK cell receptor Ly49A recognizes the mouse MHC class I molecule H-2D(k). The present study focuses on the species specificity of beta(2)-microglobulin (beta(2)m), an invariant component of MHC class I, in the interaction between Ly49A and H-2D(k). Transfection of the beta(2)m-defective mouse cell line R1E/TL8x.1 with human (h) beta(2)m induced cell-surface expression of H-2D(k), but failed to protect the cells from killing by Ly49A(+) NK cells. In contrast, the cells transfected with mouse (m) beta(2)m were protected from killing by Ly49A(+) NK cells. These data indicate that Ly49A distinguishes mbeta(2)m from hbeta(2)m when it recognizes the H-2D(k) complexes. To identify the species-specific determinant of beta(2)m required for Ly49A recognition of H-2D(k), we prepared a panel of mbeta(2)m mutants and tested the H-2D(k) that included each of the beta(2)m mutants for its capacity to engage Ly49A on NK cells. Ly49A failed to functionally recognize the H-2D(k) that included the mbeta(2)m with K3R and Q29G mutations. Moreover, Ly49A was able to recognize the H-2D(k) that included the hbeta(2)m with R3K and G29Q mutations. These data indicate that Lys3 and Gln29 consist of the central part of the species-specific determinant of beta(2)m required for Ly49A recognition of H-2D(k). The two residues are conserved in the mouse and the rat, in which NK cells use Ly49 family molecules as the receptors specific for MHC class I. These results suggest functional importance of beta(2)m in NK cell recognition of target cells.

Class I histocompatibility molecules, consisting of a heavy chain, beta2-microglobulin and peptide, are assembled in the endoplasmic reticulum (ER) with the assistance of several molecular chaperones and accessory proteins. Peptide binding occurs when assembling class I molecules associate with a loading complex consisting of the transporter associated with antigen processing (TAP) peptide transporter, tapasin, ERp57 and calreticulin (CRT)/calnexin. To assess the physical organization of this complex, we generated a series of mutants in the murine H-2Dd heavy chain and assessed their association with components of the complex. Seven mutations, clustered between amino acids 122 and 136 in the heavy chain alpha2 domain plus one mutation at position 222 in the alpha3 domain, resulted in loss of interaction with tapasin. Association with TAP was always lost simultaneously, supporting the view that tapasin acts as an obligatory bridge between class I molecules and TAP. Compared with previous studies on the HLA-A2 molecule, some differences in points of tapasin interaction were observed. Failure of the H-2Dd mutants to bind tapasin resulted in low cell-surface expression and altered intracellular transport. Most mutants retained a substantial degree of peptide loading, consistent with the view that although tapasin may promote peptide binding to class I, it is not required. A surprising observation was that all mutants lacking tapasin interaction retained normal association with CRT. This contrasts with previous observations on other class I molecules and, combined with differences in tapasin interaction, suggests that the organization of the ER peptide-loading complex can vary depending on the specific class I molecule examined.

The molecular event governing the assembly of the MHC class I heavy chain-beta(2)-microglobulin-peptide complex is still not fully understood. In order to characterize the transport properties of MHC class I molecules, several truncated H-2K(b) genes were constructed and expressed in COS7 cells. Surprisingly, the expressed soluble molecule containing connecting peptide (CP) segment (sK(b)(CP)) did not secrete as efficiently as the one without CP (sK(b)(CYT)). When the sK(b)(CP) gene was transfected into a calnexin-deficient cell line CEM.NK(R), the amount of soluble K(b) molecules in the supernatant was comparable with sK(b)(CYT)-transfected CEM.NK(R). To further demonstrate the different transport of sK(b)(CP) and sK(b)(CYT) within living cells, we attached green fluorescent protein (GFP) to the C-termini of both molecules and, as a comparison, to the full-length transmembrane counterpart (mK(b)-GFP). While the mK(b)-GFP-transfected cells showed the green fluorescence in the reticular network and the nuclear envelope, sK(b)(CP)-GFP showed obviously lump fluorescence of high intensity within cells. However, the distribution of sK(b)(CYT)-GFP was fairly uniform. Furthermore, GFP-tagged molecules allow us to analyze their interaction with other proteins in a direct, simple and quantitative method, designated immunofluorescence precipitation. The results showed that 60% of sK(b)(CP)-GFP molecules were associated with calnexin, while <10% with tapasin. Taken together with the results from sK(b)(CYT)-GFP and mK(b)-GFP, it is reasonable to deduce that the CP segment is involved in the association of class I molecules with calnexin and the transmembrane region might play a dynamic role in the dissociation from calnexin. The suggested kinetic association of class I molecules with calnexin is likely to contribute to the different maturation rate between several class I alleles.

The transcription of the MHC class I genes is regulated by interaction of cis-elements, located in the 5' genomic flanking regions, with sequence-specific trans-factors. We have identified a cis-regulatory element, 5'-TGACGCG-3', of the H-2D(d) gene. This cyclic adenosine-3',5'-monophosphate regulatory element (CRE)-like sequence, named H-2 binding factor 1 (H-2 BF1) binding motif, is highly conserved among species. In addition, we found that homo- and heterodimers of activation transcription factor 1 (ATF-1) and CRE binding protein (CREB) associate with the H-2 BF1 binding motif and activate transcription of the H-2D(d) gene. Here we demonstrate that a homologue of ATF-1, originally isolated and designated ATF-1DN, acts as a dominant repressor, blocking the ability of wild-type ATF-1 and CREB to bind to the H-2 BF1 probe in electrophoretic mobility shift assays (EMSA). We have utilized this molecule to analyze the participation of the H-2 BF1 complexes, consisting of the H-2 BF1 binding motif and ATF-1/CREB trans-factors, in the physiological regulation of MHC class I expression in tissue culture cells. A human epidermoid carcinoma cell line, A431, was transfected with ATF-1DN and clones expressing the gene transcripts were selected. When analyzed in the EMSA, nuclear proteins prepared from these clones exhibited a decreased shift of the H-2 BF1 probe corresponding to the levels of the ATF-1DN gene expression. Additionally, MHC class I expression of cells with reduced H-2 BF1 activity was significantly higher than in control cells lacking ATF-1DN. These findings indicate that in these carcinoma cells, the H-2 BF1 complexes negatively regulate the constitutive expression of MHC class I.

Ly49 receptors are inhibitory receptors expressed on subsets of both NK cells and NK1.1(+) T cells. The function of these receptors on NK cells is believed to be important in maintaining self-tolerance, yet their role on T cells is unclear. In this report we investigated how an Ly49A transgene alters T and NK cell development in an in vivo environment, where a ligand for Ly49A is expressed. Ly49A transgenic mice that co-expressed an MHC ligand for Ly49A, H-2D(d), developed a severe inflammatory disorder that resulted in death within the first weeks of age. T cells expressing forbidden TCR V(beta) chains were found both in the thymus and periphery of transgenic mice, while non-transgenic littermates had successfully deleted these T cell subsets. These data indicate that the expression of Ly49A on T cells could alter T cell selection and allow survival of potentially self-reactive T cells.

The two-signal model states that activation of naive T cells requires a signal 1 stimulus through the TCR and a co-stimulatory signal 2. By contrast, signal 1 alone is sufficient for pre-activated T cells. Recently, however, it has been shown that under certain conditions T cells can bypass the requirement for co-stimulation. For example, CD28-deficient mice, when immunized with lymphocytic choriomeningitis virus, mount a vigorous cytotoxic T lymphocyte response and clear the virus. As a continuous effort to unravel the mechanisms of T cell activation, we previously reported activation of hybridoma T cells by recombinant single-chain MHC molecules in the absence of antigen-presenting cells. In such reconstitution experiments, since the signals delivered to the T cells are well controlled, the contribution of any known or unknown signals can be ruled out. In the present study, we analyzed the requirements for activation of naive T cells by using splenocytes from TCR transgenic mice as a source of responding cells. We observed that naive CD8+ T cells are fully activated by signal 1 alone, but that co-stimulation lowers their activation threshold. Previously activated T cells are fully responsive, even when the first stimulation was performed in the absence of co-stimulation. They display a low activation threshold and are insensitive to co-stimulation. The physiological relevance of this finding and its consequences for immunotherapy as well as for our understanding of self-tolerance are discussed.

Tumor cells can have different morphologic or metabolic phenotypes and display genetic instability. Thus they could also vary in their ability to present epitopes to the immune system. We have analyzed the presentation of H-2 Kb- and Db-restricted cytotoxic T lymphocyte (CTL) epitopes of a tumor-associated antigen by three cell lines derived from hepatocarcinomas developed in vivo by mice transgenic for SV40 T targeted to the liver. SV40 T is the obvious tumor-specific antigen and epitopes derived from this antigen were therefore studied. The study included four already known epitopes that can be presented by SV40-transformed kidney cells and two new CTL epitopes that were identified in the present work. CTL lines specific for each epitope were obtained from C57BL/6 mice and were used to map the presentation of SV40 T peptides by the hepatocarcinoma cells. These tumor cells were derived from the same tissue, induced by the same agent and all naturally presented peptide p232-240 from p53. Despite these common features, they all had different patterns of spontaneous presentation of SV40 T CTL epitopes. The mechanisms underlying this disparity are discussed, together with the possible consequences for establishing immunotherapeutic strategies.

The immunodominant ovalbumin257-264 (OVA-8, SIINFEKL) and herpes simplex virus gB496-503 (HSV-8, SSIEFARL) peptides share 50% amino acid identity (residues P1, P3, P5 and P8) and bind with comparable efficacy to the murine MHC-encoded class I molecule H-2Kb. However, these two peptides bind differently to H-2Kbm8, a natural H-2Kb variant with a substitution in four amino acids on the floor of the peptide-binding site; HSV-8 binds with high and OVA-8 with a relatively low efficacy. To investigate which of the non-homologous peptide residues were responsible for this differential binding, we used substituted peptide variants and the class I thermodynamic stabilization assay. Variation at the solvent-exposed peptide residues P6 and P7 did not appreciably influence binding. By contrast, variation at the buried P2 and, surprisingly, at the solvent-exposed P4 residue was found to be important. Transplantation of the HSV-8 P2 or P4 residues onto the OVA-8 backbone created variant peptides O2S (P2I-->S) and O4E (P4N-->E) that bound considerably better to H-2Kbm8 than OVA-8. Furthermore, the double-substituted peptide, O2S4E, bound even better, revealing a cooperative effect of the two residues. The reciprocally substituted peptides H2I and H4N, generated by grafting the OVA-8 P2 and P4 residues onto the HSV-8 backbone respectively, bound to H-2Kbm8 slightly worse than HSV-8 but the double-substituted peptide H2I4N bound as poorly as OVA-8. Effects exerted by the P4 residue, which is solvent accessible and therefore available for the TCR contact, demonstrated that exposed peptide residues can, in certain situations, influence not only the TCR contact but also MHC-peptide binding.

Motifs of peptides naturally associated with H-2Ek and Ed molecules were determined by (I) pool sequencing of natural ligand mixtures and (II) sequencing of individual natural ligands followed by their alignment to the basic motif suggested by pool sequencing. The data reveal nine amino acid motifs with interaction sites at relative positions P1, P4, P6 and P9, with specificities that are identical at some but different at other anchor positions between Ed and Ek motifs, illustrating the different requirements for peptides to be presented by these two MHC molecules. The anchors with the most restricted specificity are P1 and P9. P1 is aliphatic for Ek and predominantly aromatic for Ed. P9 is positively charged for both molecules. P4 and P6 show a totally different amino acid preference between Ek and Ed ligand motifs. An alignment of Ed and Ek protein sequences to the recently reported HLA-DR1 pocket residues is in agreement with observed anchor residues in Ek and Ed motifs, thus confirming the predicted similarity of mouse class II E molecules with human DR molecules. Furthermore, this alignment was extended to the putative pockets of class II Eb and Ek molecules, and allowed, together with sequence information of previously identified natural ligands of Eb and Ec molecules, a prediction of their respective motifs. The information obtained by this study should be useful to identify putative class II E epitopes in proteins and to design peptides for blocking class II E molecules.

HLA-DRA transgenic (tg) mice on H-2d background were constructed to study assembly, expression and function of DR alpha: E beta class II heterodimers when an alternate E alpha chain is available. Cytofluorimetric analysis and immunoprecipitation studies demonstrate that the majority (90%) of E beta d molecules on class II-positive splenocytes from DRA-tg mice are associated with DR alpha rather than E alpha chains. To characterize the functional role of the interspecies as compared with the wild-type I-E molecules, MHC restriction and T cell epitope immunodominance of synthetic peptides spanning the entire sequence of 65 kDa heat shock protein (hsp) from Mycobacterium tuberculosis were determined in hsp-primed DRA-tg and DBA/2 mice. A similar pattern of responsiveness was observed in both strains, but hsp epitopes recalled a higher response in DRA-tg as compared with DBA/2 mice. A panel of T cell hybridomas specific for two hsp peptides or a hen egg white lysozyme peptide presented by both DR alpha: E beta d and E alpha d: E beta d was studied in detail. Surprisingly, DR alpha: E beta d dimers present these peptides more efficiently than E alpha d: E beta d, even when the TCR was selected in mice expressing only E alpha d: E beta d molecules. The higher efficiency of antigen presentation by DR alpha: E beta d dimers does not appear to depend on increased binding affinity for peptides, as demonstrated by competition for antigen presentation, nor on increased efficiency in the interaction with CD4 molecules. Rather, the higher efficiency of antigen presentation could be explained by a more effective ligand-TCR interaction. This is consistent with molecular modeling based on the class II structure, indicating that 16 out of 17 substitutions between the first domain of E alpha d and DR alpha chains ile outside the peptide binding groove and are potentially available for interaction with the TCR.

Single alanine substitutions were introduced into the CDR1 region of the beta chain of a Kd-restricted TCR. Mutants and wild-type TCR were attached to the zeta chain of the CD3 complex and expressed at the surface of a rat basophil cell line. Transfectants were tested for the binding of purified soluble Kd-peptide complexes. With this experimental system, accessory molecules are unlikely to play a major role and the contribution of each residue to the interaction can be addressed. Results show that all positions in the CDR1 region are involved in the binding to the Kd-peptide complex but at varying degrees. These effects are discussed in relation to a molecular model of the TCR. Comparison of these results with previous data obtained in a T cell hybridoma system suggests the existence of a threshold in the TCR affinity necessary for mature T cell activation.

A panel of 15 single alanine substitutions on the floor of the peptide binding groove of the murine class I histocompatibility molecule H-2Kd has been analyzed. All but two mutant molecules were expressed on the cell surface, and were tested for peptide binding and presentation to specific cytotoxic T lymphocytes. Eleven out of 13 mutant molecules appeared to be functionally altered. Five of the substituted residues were involved in the presentation of all peptides tested. Three participated in the presentation of certain peptides but not others. Three other residues participated in epitope formation through indirect interactions. Only two mutations had no detectable effect.

The extent of peripheral clonal deletion of the T cell antigen receptor (TCR) from a CD8-dependent cytotoxic T lymphocyte of H-2k origin with alloreactivity for H-2Kb was measured in a TCR transgenic (Tg) model by use of a clonotype-specific mAb (anti-Ti mAb). Deletion varied depending on whether the TCR Tg was expressed in mice heterozygous (H-2k x b) or homozygous (H-2b x b) for the H-2Kb antigen. CD8 surface staining and functional analyses of peripheral T cells stimulated with polyclonal activators in bulk culture or by limiting dilution confirmed that in the H-2b x b mice few Ti+ cells were generated as measured by anti-Ti mAb-dependent target cell killing; while such cells could readily be detected in the H-2k x b mice. The latter maintained non-responsiveness to H-2Kb, as measured by cytolysis of H-2Kb-expressing tumor target cells, due to their down-regulation of surface CD8 expression. The question of whether the difference between H-2b x b and H-2k x b mice was due to the influence of positive selection imposed by the k haplotype in the H-2k x b hybrid, or to the lower antigen density in heterozygous than homozygous mice was addressed by analysis of H-2b x d Tg mice, H-2d being a non-selecting haplotype. Results obtained were similar for H-2b x d and H-2k x b Tg mice, suggesting that the density of the H-2Kb antigen may be one of the parameters controlling the extent of clonal deletion in the thymus.

The RMA-S mutant T cell line is defective in H-2b restricted antigen presentation and has markedly reduced surface expression of Kb and Db. We examined RMA-S for the expression of the medial class I histocompatibility antigens Qa1b and Mta. While RMA-S targets varied in their susceptibility to lysis by cytotoxic T lymphocytes (CTL) specific for Qa1b, Mta levels were detectable but consistently low compared to the parent RMA cell line. Addition of synthetic ND1 alpha 1-26 or ND1 alpha 1-17 peptides that mimic MTF alpha (the ligand of Mta) increased killing of RMA-S by anti-Mta alpha CTL to levels comparable to or better than RMA, with 300 nM peptide being fully effective. None of the MTF peptides increased the killing of RMA-S by anti-H-2b or anti-Qa1b CTL, even at the highest (1 microM) peptide concentrations. RMA-S cells treated with 100 microM of either the ND1 alpha 4-26 or ND1 alpha 1-26 peptides showed a small increase in the fluorescent staining for beta 2-microglobulin but not for H-2Kb or H-2Db. These results show that Mta and Qa1b, although affected, are not obliterated by the defect in RMA-S cells; that the association of MTF peptides with HMT is exclusive; and that MTF enters the endoplasmic reticulum in the same fashion as other endogenous peptides.