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.

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.

CD4+ TCRalphabeta T cells are critically involved in the control of experimental murine cysticercosis in C57BL/6J mice.

Taenia crassiceps cysticerci develop in the peritoneal cavity of BALB/cAnN mice and, to a lesser extent, in C57BL/6J mice. The mechanisms involved in the immunity to this murine cysticercosis seem to be mainly mediated by T cells. To gain further insight into the mechanisms of cysticercal immunity, the susceptibility of mice deficient in different immunologically relevant genes was compared with that of the respective wild type. Mice were classified according to the parasite load and survival after infection: highly susceptible (HS), with an increased parasite load and mortality rate (CD4-/-, TCRalpha-/-, TCRbeta-/-, RAG1-/-), susceptible, with only increased parasite load (TCRdelta-/-, BALB/cAnN), and relatively resistant, with a lower number of parasites (CD8-/-, WT). Neither specific proliferative response nor Th2 cytokine or antibody responses were observed in HS mice. These data strongly suggest that CD4+TCRalphabeta+ T cells have a critical role in the control of T. crassiceps murine cysticercosis.

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.

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.

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.

Host immune response to intracellular bacteria: A role for MHC-linked class-Ib antigen-presenting molecules.

MHC-linked class-Ib molecules are a subfamily of class-I molecules that display limited genetic polymorphism. At one time these molecules were considered to have an enigmatic function. However, recent studies have shown that MHC-linked class-Ib molecules can function as antigen presentation structures that bind bacteria-derived epitopes for recognition by CD8+ effector T cells. This role for class-Ib molecules has been demonstrated across broad classes of intracellular bacteria including Listeria moncytogenes, Salmonella typhimurium, and Mycobacterium tuberculosis. Additionally, evidence is emerging that MHC-linked class-Ib molecules also serve an integral role as recognition elements for NK cells as well as several TCR alpha/beta and TCR gamma/delta T-cell subsets. Thus, MHC-linked class-Ib molecules contribute to the host immune response by serving as antigen presentation molecules and recognition ligands in both the innate and adaptive immune response to infection. In this review, we will attempt to summarize the work that supports a role for MHC-linked class-Ib molecules in the host response to infection with intracellular bacteria.

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.

Single H2Kb, H2Db and double H2KbDb knockout mice: peripheral CD8+ T cell repertoire and anti-lymphocytic choriomeningitis virus cytolytic responses.

Single H2Kb, H2Db and double H2KbDb homozygous knockout (KO) mice were generated and their peripheral CD8+ T cell repertoires compared to that of C57BL/6 (B6) mice. Limited (10-20%, H2Db), substantial (30-50%, H2Kb) and profound (90%, H2KbDb) reduction of peripheral CD8+ T cells was observed in KO mice, without Vbeta diversity alteration. Classical class Ia molecules therefore ensure most but not all of the peripheral CD8+ T cell repertoire education. As expected, H2Kb but also H2Db KO mice developed choriomeningitis following intracranial infection by lymphocytic choriomeningitis virus with the same kinetics, lethality and CD8+ cell implication as wild-type B6 mice. By contrast, H2KbDb (class Ia-Ib+) KO mice survived. Choriomeningitis of H2Db KO mice was linked to the development of a subdominant (in normal B6 mice) H2Kb-restricted cytotoxic T lymphocyte response. Mice expressing a restricted set of histocompatibility class I molecules should represent useful tools to evaluate the immunological potentials of individual MHC class I molecules.

T cell responses to Gram-negative intracellular bacterial pathogens: a role for CD8+ T cells in immunity to Salmonella infection and the involvement of MHC class Ib molecules.

Despite being a major group of intracellular pathogens, the role of class I-restricted T cells in the clearance of Gram-negative bacteria is not resolved. Using a murine typhoid model, a role for class I-restricted T cells in the immune response to the Gram-negative pathogen Salmonella typhimurium is revealed. Class I-deficient beta2-microglobulin-/- mice show increased susceptibility to infection with S. typhimurium. Following infection, CD8+ CTLs specific for Salmonella-infected targets can be readily detected. The Salmonella-specific CTLs recognize infected H-2-mismatched targets, suggesting the involvement of shared class Ib molecules. Studies using transfectants expressing defined class Ia and class Ib molecules indicate the involvement of the class Ib molecule, Qa-1. Ab-blocking studies and the measurement of bacteria-specific CTL frequencies identified Qa-1 as a dominant restricting element. The Qa-1-restricted CTL recognition depends on TAP and proteasome functions. Surprisingly, Qa-1-restricted CTLs recognized cells infected with other closely related Gram-negative bacteria. Taken together, these observations indicate that Salmonella-specific CTLs recognize a cross-reactive epitope presented by Qa-1 molecules and, as such, may be novel targets for vaccine development.

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.

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.

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.

Constitutive and regulated expression of the class IB molecule Qa-1 in pancreatic beta cells.

Enhanced major histocompatibility complex (MHC) class I expression is a prominent early feature of pancreatic beta-cell pathology in autoimmune diabetes. The number and nature of class I MHC loci expressed by beta cells are generally undefined and potentially critical to the onset and progression of insulitis. Mounting evidence indicates that the non-classical MHC class IB molecule Qa-1, encoded by H2-T23, is capable of presenting antigens to alpha beta and gamma delta T cells and that lymphocytes restricted to Qa-1 may contribute immunoregulatory functions. We compared the expression of Qa-1 and MHC class IA in a beta-cell line (beta TC6-F7) before and after treatment with the insulitic cytokine interferon-gamma (IFN-gamma). Similar to MHC class IA, Qa-1 was expressed constitutively at a low level in beta TC6-F7 cells, with both T23b mRNA and cell surface Qa-1b being up-regulated following 24-hr treatment with mouse IFN-gamma. Based on binding characteristics established for the predominant Qa-1-binding peptide, Qa-1 determinant modifier (Qdm), we also examined the possibility that Qa-1 binding peptides may be encoded in the preproinsulin leader sequence. One nonarmeric peptide (Ins II: ALWMRFLPL) derived from the preproinsulin II leader sequence was recognized by a Qa-1b-specific cytotoxic T-lymphocyte (CTL) clone. Specific binding of Ins II to Qa-1b was confirmed by a CTL peptide-blocking assay. Demonstration of IFN-gamma-regulated Qa-1 expression in beta cells and identification of a Qa-1-binding peptide in the preproinsulin leader sequence invoke further consideration of possible roles of Qa-1 in the progression of islet inflammation.

Flow cytometric analysis of major histocompatibility complex (MHC) class II antigen expression on brain cells from Borna disease virus-infected rats without an intervening in vitro culture step.

Borna disease virus (BDV) infects astrocytes in the Lewis rat brain. BDV-infected astrocytes have been shown to express MHC class II in vitro but not in vivo. Using a sensitive fluorescence-activated cytometric technique, we now report the detection of MHC class II on freshly harvested S100-positive cells from BDV-infected rat brain, without an intervening in vitro culture step. These data support the hypothesis that astrocytes from BDV-infected rats express MHC class II on their surface and, thus, are potential participants in the encephalitic response to BDV infection.

Dominance of a single peptide bound to the class I(B) molecule, Qa-1b.

One of the hallmarks of class I(A) molecules is their ability to bind and present a wide array of peptides to CD8 T cells. This diversity is consistent with their ability to restrict a variety of pathogenic peptide epitopes as well as elicit strong transplantation responses. In contrast, class I(B) molecules appear to be involved in presentation of pathogenic epitopes to a relatively lesser extent as well as play a minor role in transplantation responses. Here we have examined the peptides bound and presented by the class I(B) molecule Qa-1b in order to determine if their diversity was similar to that reported for class I(A) Ags. First, we show that bulk-cultured anti-Qa-1b CTL predominantly recognize a single peptide (Qdm) derived from the leader segment of class I(A) alloantigens. These CTL are peptide specific and reflect the activity of previously described CTL clones. Second, we find approximately 4.6 x 10(4) copies of the Qdm peptide/cell. Most of the peptide is Qa-1b associated since the recovery of this peptide from anti-Qa-1b immunoprecipitates is approximately 75% of that seen in whole cell extracts and no detectable activity is observed in Kb or Db extracts from H-2b lymphoblasts. Third, the expression of Qa-1b on lymphoblasts is approximately 1 to 1.25 x 10(4) molecules/cell indicating that the Qdm peptide must be derived from both cell membrane and intracellular compartments. Finally, examination of the diversity of peptides associated with Qa-1b as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry indicates few detectable peptide species associated with this molecule. Taken together, Qa-1b appears to predominantly bind a single peptide species that is recognized by alloreactive CD8 T cells. This feature may account, in part, for the class I(B) properties of this molecule.

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.