Molecular mimicry mediated by MHC class Ib molecules after infection with gram-negative pathogens.

The development of many autoimmune diseases has been etiologically linked to exposure to infectious agents. For example, a subset of patients with a history of Salmonella infection develop reactive arthritis. The persistence of bacterial antigen in arthritic tissue and the isolation of Salmonella or Yersinia reactive CD8+ T cells from the joints of patients with reactive arthritis support the etiological link between Gram-negative bacterial infection and autoimmune disease. Models proposed to account for the link between infection and autoimmunity include inflammation-induced presentation of cryptic self-epitopes, antigen persistence and molecular mimicry. Several studies support molecular mimicry as a mechanism for the involvement of class II epitopes in infectious disease-induced self-reactivity. Here, we have identified an immunodominant epitope derived from the S. typhimurium GroEL molecule. This epitope is presented by the mouse H2-T23-encoded class Ib molecule Qa-1 and was recognized by CD8+ cytotoxic T lymphocytes induced after natural infection. S. typhimurium-stimulated cytotoxic T lymphocytes recognizing the GroEL epitope cross-reacted with a peptide derived from mouse heat shock protein 60 and recognized stressed macrophages. Our results indicate involvement of MHC class Ib molecules in infection-induced autoimmune recognition and indicate a mechanism for the etiological link between Gram-negative bacterial infection and autoimmunity.

Qa-2, H-2K, and H-2D alloantigens evolved from a common ancestral gene.

The Tla region located on the murine 17th chromosome controls several serologically defined cell surface antigens. These antigens, referred to as Qa-1-5 and TL, are expressed on a variety of hematopoeitic cell populations. In the present studies we have immunoprecipitated isotopically labeled Qa-2 and H-2 molecules from mitogen-stimulated B6 spleen cells. Sequential immunoprecipitation experiments have shown that the determinants recognized by alpha Qa-2, alpha H-2Kb, and alpha H-2Db alloantisera reside on separate molecular species. Comparative mapping of the arginine-labeled tryptic peptides from Qa-2, H-2Kb, and H-2Db molecules indicate that Qa-2 is structurally distinct but that there is considerable structural homology; 21-43% of the Qa-2 peptides co-chromatograph with peptides derived from H-2Db and H-2Kb, respectively. Similar levels of homology are observed when Qa-2 is compared with H-2Kk or H-2Dd. The results show that the Qa-2 alloantigen is encoded by a locus separate from the loci encoding H-2K or H-2D alloantigens, but that the Qa-2, H-2K, and H-2D alloantigens are sufficiently related at the primary structural level to indicate that they evolved from a common primordial gene.

Transporters associated with antigen processing (TAP)-independent presentation of soluble insulin to alpha/beta T cells by the class Ib gene product, Qa-1(b).

T cell hybridomas isolated from nonresponder H-2(b) mice immunized with pork insulin were stimulated by insulin in the presence of major histocompatibility complex (MHC)-unmatched antigen presenting cells. The restriction element used by these CD4(-) T cells was mapped to an oligomorphic MHC class Ib protein encoded in the T region and identified as Qa-1(b) using transfectants. The antigenic determinant was localized to the insulin B chain, and experiments with truncated peptides suggested that it is unexpectedly long, comprising most or all of the 30 amino acid B chain. The antigen processing pathway used to present insulin to the Qa-1(b)- restricted T cells does not require transporters associated with antigen processing (TAP), and it is inhibited by chloroquine. A wide variety of cell lines from different tissues efficiently present soluble insulin to Qa-1(b)-restricted T cells, and insulin presentation is not enhanced by phagocytic stimuli. Our results demonstrate that Qa-1(b) can function to present exogenous protein to T cells in a manner similar to MHC class II molecules. Therefore, this class Ib protein may have access to a novel antigen processing pathway that is not available to class Ia molecules.

Physiological regulation of the immunological synapse by agrin.

T cell activation is dependent on both a primary signal delivered through the T cell receptor and a secondary costimulatory signal mediated by coreceptors. Although controversial, costimulation is thought to act through the specific redistribution and clustering of membrane and intracellular kinase-rich lipid raft microdomains at the contact site between T cells and antigen-presenting cells. This site has been termed the immunological synapse. Endogenous mediators of raft clustering in lymphocytes have not been identified, although they are essential for T cell activation. We now demonstrate that agrin, an aggregating protein crucial for formation of the neuromuscular junction, is also expressed in lymphocytes and is important in reorganization of membrane lipid microdomains and setting the threshold for T cell signaling. Our data show that agrin induces the aggregation of signaling proteins and the creation of signaling domains in both immune and nervous systems through a common lipid raft pathway.

Lymphocyte apoptosis: mediation by increased type 3 inositol 1,4,5-trisphosphate receptor.

B and T lymphocytes undergoing apoptosis in response to anti-immunoglobulin M antibodies and dexamethasone, respectively, were found to have increased amounts of messenger RNA for the inositol 1,4,5-trisphosphate receptor (IP3R) and increased amounts of IP3R protein. Immunohistochemical analysis revealed that the augmented receptor population was localized to the plasma membrane. Type 3 IP3R (IP3R3) was selectively increased during apoptosis, with no enhancement of type 1 IP3R (IP3R1). Expression of IP3R3 antisense constructs in S49 T cells blocked dexamethasone-induced apoptosis, whereas IP3R3 sense, IP3R1 sense, or IP3R1 antisense control constructs did not block cell death. Thus, the increases in IP3R3 may be causally related to apoptosis.

High-fat diet modulates non-CD1d-restricted natural killer T cells and regulatory T cells in mouse colon and exacerbates experimental colitis.

Environmental factors such as diet are known to play important roles in inflammatory bowel disease (IBD). Epidemiological studies have indicated that a high-fat diet is a risk factor for IBD. In addition, the balance between effector T cells (T(eff)) and regulatory T cells (T(reg)) contributes to the pathogenesis of mucosal inflammation. The aim of this study was to understand the mechanisms by which a high-fat diet can regulate susceptibility to intestinal inflammation. Wild-type C57BL/6 mice were fed either a commercial high-fat diet or a normal diet, then exposed to dextran sulphate sodium (DSS) to induce colonic inflammation. Intraepithelial lymphocytes (IEL) were isolated from the colon, and their phenotype and cytokine profile were analysed by flow cytometry. Mice receiving the high-fat diet were more susceptible to DSS-induced colitis. They had higher numbers of non-CD1d-restricted natural killer (NK) T cells in the colonic IEL, when compared to mice fed a normal diet. These cells expressed tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma, which are up-regulated by high-fat diets. Mice fed the high-fat diet also had decreased levels of colonic T(reg). Depletion of colonic NK T cells or adoptive transfer of T(reg) reduced the DSS colitis in these mice, and reduced the colonic expression of TNF-alpha and IFN-gamma. We conclude that a high-fat diet can increase non-CD1d-restricted NK T cells and decrease T(reg) in the colonic IEL population. This altered colonic IEL population leads to increased susceptibility to DSS-induced colitis. This effect may help to explain how environmental factors can increase the susceptibility to IBD.

Recognition of tumor cells by the innate immune system.

There has been a rapid increase in our understanding of the cellular components of the innate immune system, the receptors used to distinguish changes in homeostasis, and how these components integrate into an anti-tumor effector response. Recently, significant progress has been made in the identification of ligands for receptors that activate NK cells, and the results have implications for the recognition of tumor cells.

Tumor defense by murine cytotoxic T cells specific for peptide bound to nonclassical MHC class I.

Cytotoxic T Cells (CTLs) can exhibit considerable antitumor activity. Thus far, the characterized tumor peptide antigens recognized by CTLs are all presented by classical MHC class Ia molecules [human lymphocyte antigen A (HLA-A), HLA-B, and HLA-C in humans and H-2K, H-2D, and H-2L in mice]. Here we show that CTLs recognized peptides presented by nonclassical MHC class Ib molecule Qa-1b expressed by tumor cells. These CTLs conferred in vivo protection by delaying the growth of Qa-1b-expressing B78H1 melanoma cells pulsed with Qa-1b-binding peptides Cw4L or B35L and injected s.c. in C57BL/6 mice. A hierarchy of the peptides was found with regard to their ability to trigger CTLs; Cw4L stimulated a strong CTL response. The closely related and cross-reactive peptide B35L induced a weaker CTL response but was still efficient in sensitizing the target cells. Finally, Qa-1b-expressing melanoma cells without exogenous peptides were not immunogenic but possibly expressed endogenous cross-reactive antigenic peptides. The data are compatible with earlier findings that CTL activation requires relatively strong peptide antigens, whereas subsequent effector functions are also mediated by weak peptide analogues. In conclusion, CTLs mediated tumor immunity through the recognition of peptides presented by nonclassical MHC class Ib molecules. The identification of similar CTLs in humans may facilitate the vaccination of cancer patients because MHC class Ib/peptide complexes are much less polymorphic than MHC class Ia/peptide complexes.

Biosynthesis of glycophospholipid bound and secreted murine class I Qa-2 polypeptides.

Murine T cells synthesize and express a cell-surface glycophospholipid anchored 40 kDa and a secreted water-soluble 39 kDa Qa-2 polypeptide. We have examined the biosynthetic pathways which lead to the production of the membrane-bound and water-soluble isoforms of the Qa-2 molecule. Using the detergent TX-114, both detergent (membrane)-bound and soluble Qa-2 polypeptides can be identified in cell lysates and can be distinguished by charge and molecular weight. Two membrane-bound forms, a 40-kDa Endo H resistant cell-surface form and a 38 kDa-Endo H sensitive form can be identified, both of which can be biosynthetically labeled with 3H-ethanolamine and can be converted to water soluble forms by digestion with a phosphatidylinositol specific phospholipase C. In addition, several water soluble polypeptides at 39, 37, 35 kDa, and a minor species at 33 kDa were identified, none of which radiolabel with 3H-ethanolamine. While the 39-kDa polypeptide was Endo H resistant, the other isoforms were sensitive to Endo H digestion. Pulse chase experiments and molecular weights of the deglycosylated core polypeptides suggest a precursor to product relationship between the intracellular water-soluble species and the mature 39-kDa secreted Qa-2 molecule. This relationship is supported by the observation that murine L cells transfected with the Qa-2 encoding class I gene Q7 fail to express membrane-bound Qa-2 molecules yet synthesize both intracellular water-soluble and secreted Qa-2 molecules. These findings argue for a pathway in which secreted soluble Qa-2 molecules are derived from intracellular precursors.

Physical and molecular genetic analysis of Qa-2 antigen expression: multiple factors controlling cell surface levels.

We have examined the surface expression of Qa-2 on lymphocyte subpopulations and on splenocytes from inbred mouse strains by a radioactive binding assay using purified anti-Qa-2 antibodies and antibody fragments (Fab). Quantitative measurements by Scatchard analysis revealed that spleen cells from Qa-2high mice express (4-5) x 10(4) Qa-2 molecules/cell, whereas T lymphocytes have as high as (7-8) x 10(4) molecules/cell. In addition, it was determined that B lymphocytes express (5-6) x 10(3) molecules on their cell surface. The Qa-2 levels on anti-CD3-activated T cells is 1.0 x 10(5) molecules/cell. Previous experiments have shown that the quantity of Qa-2 varies in a strain-specific fashion and may be classified into three groups: Qa-2high, Qa-2medium and Qa-2low. Our results indicated that Qa-2high strains express (4-5) x 10(4) Qa-2 molecules/cell, Qa-2medium strains (B6-K2, B10.A, A/J, BALB/c and DBA/2) express (1-1.7) x 10(4) molecules/cell, and Qa-2low strains (SWR/J and DBA/1) express no more than 6 x 10(3) molecules/cell. Detection of Q7 or Q9 mRNA by the polymerase chain reaction revealed that Qa-2high strains express two functional Qa-2 enconding genes, Q7 and Q9, whereas Qa-2medium and Qa-2low strains express either Q7 or Q9. These results strongly suggest that Qa-2 gene dosage contributes in part to the variation of Qa-2 levels on the cell surface.

Molecular weight diversity among murine class I antigens: both the mature cell surface forms and the unglycosylated polypeptides vary significantly in molecular weight.

The molecular weights of the fully glycosylated cell surface form and the unglycosylated polypeptide of five murine class I antigens (H-2Kb, Db, TL, Qa-1.1, and Qa-2) were compared by SDS-PAGE. Significant molecular weight diversity was observed for both forms among these molecules. The size of the fully glycosylated forms ranged from approximately 52,000 daltons (H-2Db) to 41,000 daltons (Qa-2), whereas the unglycosylated polypeptides ranged from 43,000 daltons (H-2Kb and TL) to 33,000 daltons (Qa-2). The magnitude of the size variation observed in the unglycosylated polypeptides implies that there are differences in the gene organization, RNA processing or post-translational modifications of various class I glycoproteins.

The involvement of class Ib molecules in the host response to infection with Salmonella and its relevance to autoimmunity.

Class I molecules with limited polymorphism have been implicated in the host response to infectious agents. Following infection with Salmonella typhimurium, mice develop a CD8+ CTL response that specifically recognizes bacteria infected cells. An immunodominant component of the CTL response recognizes a peptide epitope derived from the Salmonella GroEL molecule that is presented by the non-polymorphic MHC class Ib molecule Qa-1. T cells recognizing the bacterial peptide also cross-recognize a homologous peptide from the mammalian hsp60 molecule. Since Qa-1 has a functional equivalent in humans, this observation may be relevant not only to the host response involved in clearing infection but also in understanding the link between infection with Gram-negative pathogens and autoimmune disease.

Alloreactive cytotoxic T cells recognize minor transplantation antigens presented by major histocompatibility complex class Ib molecules.

BACKGROUND: Cytotoxic T lymphocytes (CTLs) contribute to the rejection of transplanted tissues through two pathways: first, by direct recognition of foreign graft major histocompatibility complex (MHC) class I molecules; and second, by recognition of foreign graft-derived peptides presented by classical MHC class Ia molecules that are matched between graft and donor. However, a number of observations suggest that additional categories of CTL recognition patterns may exist, but they remain to be defined molecularly. METHODS: Previous studies showed that the murine nonclassical MHC molecule H2 M3 may be involved in allorecognition. We investigated whether other members of nonclassical MHC class Ib, namely Qa1 and Qa2, may be recognized. Alloreactive CTLs were generated from mice mismatched for non-MHC and/or MHC genetic backgrounds and tested using various target cells, including cells transfected with Qa1 or Qa2. Furthermore, candidate peptides were synthesized and used to generate CTLs specific for peptide presented by Qa1 or Qa2. RESULTS: The experiments demonstrate that allogeneic and xenogeneic peptides were recognized by CTLs when presented on shared nonclassical MHC class Ib Qa1 and Qa2 molecules. CONCLUSIONS: The results confirm that MHC class Ib molecules present peptides to CTLs. This potentially important alloreactivity pathway may be functional between most individuals because sharing of MHC class Ib alleles is frequent.

Fetal ascitic fluid: a new source of lymphocytes for rapid chromosomal analysis.

BACKGROUND: The cells found in ascites can be processed like amniotic fluid for fetal karyotyping. We have characterized these cells and used them for a rapid cytogenetic result. CASES: Three patients presented with massive fetal ascites detected by sonography. Samples of ascitic fluid were obtained at fetal paracentesis. Cells from the fluid were cultured using standard methods for fetal blood, and were compared with fetal blood lymphocytes and amniocytes. The length of time in culture, chromosome morphology, and mitotic index of ascitic fluid cells were equivalent to those of fetal blood. In the third case, we performed immunophenotyping on the ascitic fluid cells. CONCLUSION: Ascitic fluid contains lymphocytes that permit rapid chromosomal analysis within 96 hours.

Association of a nonsynonymous single-nucleotide polymorphism of matrix metalloproteinase 9 with giant cell arteritis.

OBJECTIVE: Giant cell arteritis (GCA) is the most common type of primary vasculitis. Matrix metalloproteinase 9 (MMP-9) is present in arterial lesions of GCA and may be involved in its pathogenesis. We investigated whether certain genotypes of 4 single-nucleotide polymorphisms (SNPs) of MMP-9 are overrepresented in patients with histologically confirmed GCA. METHODS: Four SNPs of MMP-9, rs3918242 in the promoter region and 3 nonsynonymous coding SNPs (rs3918252, rs17576, and rs2250889) were genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis in 58 white patients for whom there was a clinical suspicion of GCA. Thirty of these patients had histologically confirmed GCA (group 1), and 28 patients had negative results of a temporal artery biopsy for GCA (group 2). Estimates of the genotype distributions of each of these SNPs in a white population were determined using publicly available genotype data for a panel of 23 individuals (group 3). RESULTS: Although 1 SNP was monomorphic in all 3 groups, we observed statistically significant differences in the genotype distributions for rs2250889 between group 1 and group 2 (P = 0.005) and between group 1 and group 3 (P = 0.009), but not between groups 2 and 3 (P = 0.965). CONCLUSION: These data derived from a sample of patients with GCA suggest that the G allele of MMP-9 polymorphism rs2250889 is overrepresented in patients with histologically confirmed GCA. Clearly, larger sample sizes will be necessary to confirm this suggestive association and better characterize a possible linkage disequilibrium structure among polymorphisms.

Heat shock proteins can regulate expression of the Tla region-encoded class Ib molecule Qa-1.

T cells recognize foreign antigens in association with the highly polymorphic class I and class II molecules encoded in the major histocompatibility complex (MHC). In addition to these highly polymorphic molecules, the murine MHC also encodes, in the Qa/Tla region, several less polymorphic structures referred to as class I-like or class Ib molecules. Although no specific function has been assigned to these molecules, their overall structural similarities to the classical class I molecules and their association with beta 2-microglobulin suggest a role in antigen recognition. Recent data have suggested that the class Ib molecule Qa-1 may be involved in antigen presentation to T cells expressing gamma delta receptors. In addition, several reports have demonstrated that gamma delta T cells can respond to mycobacterial heat shock proteins. We report that transfection of a mouse fibroblast line with gene T23b leads to the surface expression of a molecule that is structurally identical to lymphocyte Qa-1b. In the transfected cells the predominant Qa-1 species was present in an immature intracellular form. The expression of mature cell surface Qa-1 was dramatically and selectively increased following heat shock. Furthermore, the addition of a tryptic digest of Mycobacterium bovis 65-kDa heat shock protein stabilized the surface expression of Qa-1b. These observations suggest that the Qa-1 molecule may be involved in the presentation of heat shock protein-derived peptides to the immune system.