MHC Ia Research Articles

MHC Ib-restricted T Cells Generate the Dominant Memory T Cell Response in Early Stages of Mycobacterium tuberculosisInfection

Abstract Unconventional T cells, which recognize monomorphic MHC Ib molecules, such as Qa1, Qa2, M3, CD1, and MR1 have been implicated in the primary response to Mycobacterium tuberculosis(Mtb) infection. However, whether these T cells play a role and which MHC Ib molecules participate in the memory response against Mtbinfection remains unknown. To determine the optimal immunization strategy, we vaccinated mice that lack MHC Ia (K b−/−D b−/−) with an attenuated MtbH37Rv strain via intravenous (IV) or subcutaneous (SC) injection. We found both routes of immunization induced a comparable MHC Ib-restricted CD8 +T cell response after virulent MtbH37Rv re-challenge. Since bacteria were cleared through the SC but not the IV route, we selected SC as our primary route of immunization. MHC Ib-restricted CD8 +T cells in vaccinated mice produced multiple proinflammatory cytokines, expanded faster after Mtbchallenge than those in unvaccinated mice, and recognized several Mtbprotein antigens. Importantly, the memory response of MHC Ib-restricted CD8 +T cells was more dominant than that of MHC Ia-restricted CD8 +T cells at early stages of infection. We also observed an increased percentage and number of Qa1, Qa2 and M3-restricted CD8 +T cells in vaccinated mice in the early stages of infection, suggesting that these MHC Ib molecules can elicit MHC Ib-restricted memory CD8 +T cell response. While CD1d-restricted NKT cells did not differ significantly between vaccinated and non-vaccinated mice, the activation kinetics of MR1-restricted T cells was accelerated in vaccinated mice. Overall, we show that MHC Ib-restricted T cells memory response can play an important role at the early stages of Mtbinfection thus should be included in subunit vaccine formulation. Supported by grants from NIH (R01AI141083)

Immune surveillance of immune surveillance

The peptide repertoire presented by classical MHC Ia molecules is critical for immune surveillance of intracellular pathogens and cancer. The presentation of this normal peptide repertoire is regulated by ERAAP, the ER aminopeptidase associated with antigen processing. Changes in ERAAP function cause profound changes in the presented peptides that can potentially cause autoimmunity or alter the efficacy of immunotherapy of cancer. Thus, an unusual subset of CD8+ T cells monitors normal ERAAP function. Cells with compromised ERAAP activity present the QFL ligand that contains a highly conserved FL9 peptide presented by Qa-1b, a non-classical MHC Ib molecule expressed. The QFL-specific CD8+ T cells bear highly conserved αβ TCRs and have distinct anatomical locations as well as functions. Together these features implicate QFL-specific cells in carrying out immune surveillance of the MHC Ia immune surveillance pathway.

Immune responses to Vibrio vulnificus formalin-killed vaccine and ghost vaccine in Scophthalmus maximus.

In this research, Vibrio vulnificus formalin-killed (FKCs) vaccine and ghost (VVGs) vaccine were successfully developed, and shown to prevent vibriosis of Scophthalmus maximus resulting from V. vulnificus. The antibody titre of FKCs and VVGs vaccine was 1: 28 and 1: 211 . The RPS of FKCs and VVGs vaccine was 60% and 80%. In order to improve the understanding of vaccine protection mechanism, transcriptome data was used to analyse the immune response of S. maximus infected with V. vulnificus after vaccination with FKCs and VVGs vaccine. In the SmCon and SmIV groups, a series of innate immune-related genes were upregulated (such as, TLR5, Tp12, AP-1 and IL-1β) or downregulated (such as, CASP6 and CASP8), which suggested that the immune protection mechanism induced by inactivated vaccine was similar to that of autoimmune response. In the SmIV and SmGho group, a number of innate and adaptive immune-related genes (such as, STAT1, IFN-γ and MHC Ia) were activated, in which the expression of these genes was higher in SmGho, and VVGs vaccine induced stronger innate and acquired immune responses. In conclusion, the results lay a foundation for further study on the molecular mechanisms of immune protection induced by VVGs vaccine and FKCs vaccine.

Dimorphism of HLA-E and its Disease Association.

HLA-E is a nonclassical member of the major histocompatibility complex class I gene locus. HLA-E protein shares a high level of homology with MHC Ia classical proteins: it has similar tertiary structure, associates with β2-microglobulin, and is able to present peptides to cytotoxic lymphocytes. The main function of HLA-E under normal conditions is to present peptides derived from the leader sequences of classical HLA class I proteins, thus serving for monitoring of expression of these molecules performed by cytotoxic lymphocytes. However, opposite to multiallelic classical MHC I genes, HLA-E in fact has only two alleles—HLA-E*01:01 and HLA-E*01:03—which differ by one nonsynonymous amino acid substitution at position 107, resulting in an arginine in HLA-E*01:01 (HLA-ER) and glycine in HLA-E*01:03 (HLA-EG). In contrast to HLA-ER, HLA-EG has higher affinity to peptide, higher surface expression, and higher thermal stability of the corresponding protein, and it is more ancient than HLA-ER, though both alleles are presented in human populations in nearly equal frequencies. In the current review, we aimed to uncover the reason of the expansion of the younger allele, HLA-ER, by analysis of associations of both HLA-E alleles with a number of diseases, including viral and bacterial infections, cancer, and autoimmune disorders.

Protective Immunity Induced by DNA Vaccination against Ranavirus Infection in Chinese Giant Salamander Andrias davidianus.

Andrias davidianus ranavirus (ADRV) is an emerging viral pathogen that causes severe systemic hemorrhagic disease in Chinese giant salamanders. There is an urgent need for developing an effective vaccine against this fatal disease. In this study, DNA vaccines containing the ADRV 2L gene (pcDNA-2L) and the 58L gene (pcDNA-58L) were respectively constructed, and their immune protective effects were evaluated in Chinese giant salamanders. In vitro and in vivo expression of the vaccine plasmids were confirmed in transfected cells and muscle tissues of vaccinated Chinese giant salamanders by using immunoblot analysis or RT-PCR. Following ADRV challenge, the Chinese giant salamanders vaccinated with pcDNA-2L showed a relative percent survival (RPS) of 66.7%, which was significant higher than that in Chinese giant salamanders immunized with pcDNA-58L (RPS of 3.3%). Moreover, the specific antibody against ADRV was detected in Chinese giant salamanders vaccinated with pcDNA-2L at 14 and 21 days post-vaccination by indirect enzyme-linked immunosorbent assay (ELISA). Transcriptional analysis revealed that the expression levels of immune-related genes including type I interferon (IFN), myxovirus resistance (Mx), major histocompatibility complex class IA (MHC IA), and immunoglobulin M (IgM) were strongly up-regulated after vaccination with pcDNA-2L. Furthermore, vaccination with pcDNA-2L significantly suppressed the virus replication, which was seen by a low viral load in the spleen of Chinese giant salamander survivals after ADRV challenge. These results indicated that pcDNA-2L could induce a significant innate immune response and an adaptive immune response involving both humoral and cell-mediated immunity that conferred effective protection against ADRV infection, and might be a potential vaccine candidate for controlling ADRV disease in Chinese giant salamanders.

Nonclassical MHC Ib-restricted CD8+ T Cells Recognize Mycobacterium tuberculosis-Derived Protein Antigens and Contribute to Protection Against Infection.

MHC Ib-restricted CD8+ T cells have been implicated in host defense against Mycobacterium tuberculosis (Mtb) infection. However, the relative contribution of various MHC Ib-restricted T cell populations to anti-mycobacterial immunity remains elusive. In this study, we used mice that lack MHC Ia (Kb-/-Db-/-), MHC Ia/H2-M3 (Kb-/-Db-/-M3-/-), or β2m (β2m-/-) to study the role of M3-restricted and other MHC Ib-restricted T cells in immunity against Mtb. Unlike their dominant role in Listeria infection, we found that M3-restricted CD8+ T cells only represented a small proportion of the CD8+ T cells responding to Mtb infection. Non-M3, MHC Ib-restricted CD8+ T cells expanded preferentially in the lungs of Mtb-infected Kb-/-Db-/-M3-/- mice, exhibited polyfunctional capacities and conferred protection against Mtb. These MHC Ib-restricted CD8+ T cells recognized several Mtb-derived protein antigens at a higher frequency than MHC Ia-restricted CD8+ T cells. The presentation of Mtb antigens to MHC Ib-restricted CD8+ T cells was mostly β2m-dependent but TAP-independent. Interestingly, a large proportion of Mtb-specific MHC Ib-restricted CD8+ T cells in Kb-/-Db-/-M3-/- mice were Qa-2-restricted while no considerable numbers of MR1 or CD1-restricted Mtb-specific CD8+ T cells were detected. Our findings indicate that nonclassical CD8+ T cells other than the known M3, CD1, and MR1-restricted CD8+ T cells contribute to host immune responses against Mtb infection. Targeting these MHC Ib-restricted CD8+ T cells would facilitate the design of better Mtb vaccines with broader coverage across MHC haplotypes due to the limited polymorphism of MHC class Ib molecules.

Nonclassical MHC Ib-restricted CD8+ T cells recognize Mycobacterium tuberculosis-derived protein antigens and contribute to protection against infection (MPF6P.744)

Abstract MHC Ib-restricted CD8+ T cells have been implicated to contribute to host defense during Mycobacteria tuberculosis (Mtb) infection, but the relative contribution of various MHC Ib-restricted responses has yet to be defined. In this study, we used mice that lack MHC Ia (Kb-/-Db-/-, DKO), MHC Ia/M3 (Kb-/-Db-/-M3-/-, TKO), or β2m (β2m-/-) to study the role of M3-restricted and other MHC Ib-restricted T cells in immunity against Mtb infection. Unlike their role in Listeria infection, we found that M3-restricted CD8+ T cells only represent a small proportion of the CD8+ T cells responding to Mtb infection. This finding highlights the differential role of various MHC Ib-restricted responses in immunity against distinct microbial pathogens. Non-M3, MHC Ib-restricted CD8+ T cells expanded preferentially in the lungs of TKO mice and expressed higher level of KLRG1 compared to MHC Ia-restricted CD8+ T cells. These CD8+ T cells produced IFNγ, TNFα and IL-2 upon stimulation with Mtb-derived protein antigens in a β2m-dependent but TAP-independent manner. TKO mice had similar bacterial loads in tissues as DKO and wildtype mice, but lower bacterial loads than β2m-/- mice. Our findings indicate non-M3, MHC Ib-restricted CD8+ T cells can specifically recognize Mtb protein antigens and contribute to host immune response to Mtb. Targeting these MHC Ib-restricted CD8+ T cells would facilitate the design of better vaccines against Mtb, due to the limited polymorphism of MHC class Ib molecules.

Extensive diversification of MHC in Chinese giant salamanders Andrias davidianus (Anda-MHC) reveals novel splice variants

A series of MHC alleles (including 26 class IA, 27 class IIA, and 17 class IIB) were identified from Chinese giant salamander Andrias davidianus (Anda-MHC). These genes are similar to classical MHC molecules in terms of characteristic domains, functional residues, deduced tertiary structures and genetic diversity. The majority of variation between alleles is found in the putative peptide-binding region (PBR), which is driven by positive Darwinian selection. The coexistence of two isoforms in MHC IA, IIA, and IIB alleles are shown: one full-length transcript and one novel splice variant. Despite lake of the external domains, these variants exhibit similar subcellular localization with the full-length transcripts. Moreover, the expression of MHC isoforms are up-regulated upon in vivo and in vitro stimulation with Andrias davidianus ranavirus (ADRV), suggesting their potential roles in the immune response. The results provide insights into understanding MHC variation and function in this ancient and endangered urodele amphibian.

Murine MHC class Ib gene T11: a functional paralog of T23/Qa-1? (106.42)

Abstract MHC class Ia molecules, H2-K/D/L in mouse, select CD8 T cells during development and present peptides to appropriate CD8 T cells in immune responses. Mouse MHC class Ib H2-Q, T, M regions contain more than 20 protein-encoding genes, but only few have been studied. Qa-1, encoded by H2-T23 gene, is one of the best-studied MHC class Ib. Qa-1 presents peptide generated from MHC Ia to NKG2A/CD94 receptors and provides inhibitory signal to NK cells. Here, we report the study of another MHC class Ib gene, H2-T11. T11 is 95% identical in DNA level and 87% identical in protein level to T23. T11 mRNA was detected in spleen, thymus and uterus in B6 mice, and its expression pattern is more limited than that of T23. Recombinant T11 was refolded with β2m, with or without the exogenous Qdm peptides. The refolded T11 had a typical secondary structure of MHC class I proteins, shown by circular dichroism analysis. The refolded T11 bound Qdm in vitro and could be recognized by a Qa-1 specific mAb. T11 was ectopically expressed on the surface of several cell lines, including a Tap deficient one. The T11 expressing cells could not present insulin to Qa-1 restricted, insulin-specific T cell hybridoma, in contrast to T23 expressing cells. Our results indicated that despite a high similarity to T23, T11 might have different function from T23/Qa-1. We are in the process of eluting naturally bound peptides from T11 expressing cells, and using T11/Qdm tetramers to look for its potential ligand(s).

Differential ability of H-2 class Ia genes to effectively direct anti viral immunity (105.53)

Abstract MHC class Ia molecules are regulators of antiviral immunity and are thought to function similarly in the presentation of self and antigenic peptides to T cells. However, differences in natural polymorphisms found among class Ia loci in humans and mice indicate these genes may not function equivalently. We used transgenic mice to evaluate the equivalence of MHC Ia gene function in directing cellular immunity against the picornavirus, Theiler’s encephalomyelitis virus, and found that effective antiviral immunity was promoted by H-2D transgenes but not by H-2K in this system. Because transgenes evaluated in our study encoded the peptide binding domain of the Db antigen presenting molecule expressed in the context of the Db or Kb genetic framework, our findings indicate that structural features distinguishing these genes mapping outside of these coding sequences determine ability to direct effective viral immunity. Irrespective of which transgene was expressed, the mice developed robust anti viral T cell responses against the immunodominant TMEV epitope and the T cells were recruited to infected tissue. However, only when the Db antigen binding domain was expressed in the context of the H-2D genetic framework was virus cleared. Our findings suggest that differences in tissue specific class I gene expression at the site of infection is a key factor in this differential function of class Ia genes. Such difference could underlie natural variations in class Ia polymorphism.

Isoforms of the nonclassical class I MHC antigen H2-Q5 are enriched in brain and encode Qdm peptide

Although the human nonclassical class Ib major histocompatibility complex (Mhc) locus, HLA-G, is known to act as an immune suppressor in immune-privileged sites, little is currently known regarding participation of the rodent class Ib Mhc in similar pathways. Here, we investigated the expression properties of the mouse nonclassical Mhc H2-Q5(k) gene, previously detected in tumors and tissues associated with pregnancy. We find that H2-Q5(k) is alternatively spliced into multiple novel isoforms in a wide panel of C3H tissues. Unlike other known class I MHC, it is most highly transcribed in the brain, where the classical class Ia Mhc products are scarce. The truncated isoforms are selectively enriched in sites of immune privilege and are translated into cell surface proteins in neural crest-derived transfected cells. Furthermore, we present data supporting a model whereby Q5(k) isoforms serve an immune-protective role by donating their Qdm leader peptide to Qa-1, in a pathway homologous to the HLA-G leader fragment binding HLA-E and inhibiting CD94/NKG2A-positive cytotoxic cells. In addition, we report a previously unknown homolog of H2-Q5(k) in the C57BL/6 mouse, which encodes Qdm, but is transcribed solely into noncanonical isoforms. Collectively, these studies demonstrate that H2-Q5(k), and its homologous class I-like H2(b) gene may play tissue-specific roles in regulating immune surveillance.

The role of MHC Ib in selecting functional CD8 T cell during acute LCMV infection (36.38)

Abstract Conventional MHC Ia & II molecules (H-2K/D/L & I-A/E in mouse) select α/β CD8 & CD4 T cell development, respectively. Although unconventional MHC class Ib, mainly encoded in H-2 Q, T & M regions in mouse, contains more than 20 MHC Ib genes, their functions remain largely unknown. We developed a mouse model, in which 3 genes, Kb, Db & CIITA were deleted, thus called triple knock-out (TKO) mouse. In TKO, CD8 T cells are selected only by MHC Ib. Previously we have shown that α/β CD8 T cells develop well in TKO. Functionally, we observe that TKO mice mount a protective immune response against in vivo acute infections of LCMV. The protection is CD8 T cell mediated & β2m dependent. Rapid production of IFNγ and Granzyme B is detected in expended CD8 cells. Intriguingly, TKO mice do not appear to mount a memory response to a second infection. We report that splenocytes, from in vivo LCMV primed TKO, can be restimulated in vitro, by LCMV infected peritoneal macrophages or bone marrow derived dendritic cells, to produce IFNγ, but little IL-2. This MHC Ib-dependent response appears to be partially mediated by Qa-2. Therefore we have established an in vitro system to analyze MHC Ib-restricted T cell responses to LCMV & we are attempting to map the MHC Ib restriction element(s) and target epitopes. Collectively, our results suggest that MHC Ib can select functional CD8 T cells & these T cells participate in, at least under some circumstances, protective immune responses to pathogens.

Protracted Contraction and Memory T Cell Differentiation in Mice Deficient in TNF and Perforin (87.6)

Abstract Following infection, pathogen-specific T cells undergo an expansion in numbers followed by contraction to relatively stable memory numbers. Work from others suggests that CD8 T cell responses to infection are altered in mice lacking TNF receptors or perforin. To understand how TNF regulates T cell responses we infected perforin/TNF-double deficient (DKO) mice, perforin-deficient (PKO) mice, and TNF-deficient (TKO) mice with Listeria monocytogenes (LM) and analyzed the MHC Ia-, MHC Ib-, and MHC II-restricted T cell responses. DKO mice exhibited a delay in contraction of MHC Ia- and MHC II-restricted T cells compared to WT mice. All three T cell responses exhibited delayed acquisition of CD62L, a central memory marker, in both DKO and TKO mice. DKO mice have a prolonged infection of LM, however, this did not account for the delay in contraction of the T cell populations, which still occurred in mice treated with antibiotics to eliminate the prolonged infection. These data suggest that TNF and perforin regulate antigen-specific T cell homeostasis, while TNF regulates the phenotype of antigen-specific T cells. Ongoing studies are addressing the mechanisms underlying delayed contraction and whether additional memory characteristics are altered by TNF deficiency.

Colocalization of Glial Fibrillary Acidic Protein, Metallothionein, and MHC II in Human, Rat, NOD/SCID, and Nude Mouse Skin Keratinocytes and Fibroblasts

The expression of glial fibrillary acidic protein (GFAP) by perivascular cells of many mammalian organs suggests an as yet unknown function of this intermediate filament protein in the maintenance of homeostasis and vascular permeability at the blood-tissue interface. Although a similar situation may exist at the air-tissue interface, the cellular distribution of GFAP in skin tissue has never been demonstrated. To approach this issue, we have employed immunofluorescence and Western blotting techniques to detect GFAP in skin sections of young and adult humans, normal rodents, and two types of mutant mice, as well as in rat lung sections, and in cultured human keratinocytes and fibroblasts. Colocalization with antigens known to be associated with GFAP in other tissues was also tested. Epidermal and hair follicle keratinocytes and dermal fibroblasts showed distinct staining for GFAP as well as colocalization with alpha-actin, metallothionein, and antigens of the class-II major histocompatibility complex (MHC II). GFAP was also identified in rat alveolar fibroblasts which, in common with keratinocytes, form part of the air-tissue interface. GFAP was upregulated together with MHC II in nude mice but was barely detectable in the skin of non-obese diabetic severe combined immunodeficiency mice, suggesting a possible involvement in antigen-presenting functions. The intriguing distribution of a common set of antigens both in certain cells of the integumentary system and at the blood-tissue interfaces of internal organs suggests the involvement of these proteins in universal mechanisms controlling tissue homeostasis and protection.

Recognition of H-2Kb by Ly49Q suggests a role for class Ia MHC regulation of plasmacytoid dendritic cell function

Ly49Q is a member of the polymorphic Ly49 family of NK cell receptors that displays both a high degree of conservation and a unique expression pattern restricted to myeloid lineage cells, including plasmacytoid dendritic cells (pDC). The function and ligand specificity of Ly49Q are unknown. Here, we use reporter cell analysis to demonstrate that a high-affinity ligand for Ly49Q is present on H-2b, but not H-2d, H-2k, H-2q, or H-2a-derived tumor cells and normal cells ex vivo. The ligand is peptide-dependent and MHC Ia-like, as revealed by its functional absence on cells deficient in TAP-1, β2m, or H-2KbDb expression. Furthermore, Ly49Q is specific for H-2Kb, as the receptor binds peptide-loaded H-2Kb but not H-2Db complexes, and Ly49Q recognition can be blocked using anti-Kb but not anti-Db mAb. Greater soluble H-2Kb binding to ligand-deficient pDC also suggests cis interactions of Ly49Q and H-2Kb. These results demonstrate that Ly49Q efficiently binds H-2Kb ligand, and suggest that pDC function, like that of NK cells, is regulated by classical MHC Ia molecules. MHC recognition capability by pDC has important implications for the role of this cell type during innate immune responses.

Can Fibroblasts Be Reprogrammed into Macrophages?.

Previous work showed that B cell precursors can be reprogrammed into functional macrophages by the enforced expression of the bZip transcription factor C/EBPalpha. The efficient activation of myelomonocytic genes, such as Mac-1, required the co-operation with endogenous PU.1 (Xie et al. 2004), reflecting the fact that many myelomonocytic genes are regulated by a combination of the two transcription factors. We therefore asked: Is C/EBPa and PU.1 sufficient to convert non-hematopoietic cells into macrophages? To test this, NIH-3T3 cells were co-infected with PU.1-GFP and C/EBPa-hCD4 retrovirusesor control vectors encoding the indicators GFP and hCD4 only. Uninfected cells in the retrovirus treated cultures served as additional controls. Our results showed that ~25% of the PU.1 only infected cells express Mac-1 and that this percentage could be increased ~3 fold by co-expression with C/EBPa. In addition, most cells also expressed CD45 and some expressed F4/80 antigen. The PU.1 infected and the double infected cells, but not the C/EBPa only infected cells, also expressed a number of other myelomonocytic genes as detected by RT-PCR. These included CSF-1R (M-CSFR), GM-CSF Ralpha, Lysozyme, CD32, PYK2 as well as endogenous PU.1. The PU.1 induced reprogramming of fibroblasts required the DNA binding and transcription activation domains, but not the PEST domain of the transcription factor. To test whether the reprogrammed cells have functional macrophage properties, we generated two stable cell lines co-expressing C/EBPa and PU.1 delta PEST (wild type PU.1 is toxic in long-term cultures). These cells were morphologically altered, ingested carboxylated particles, and expressed functional Fc-gamma receptors but were unable to phagocytize antibody coated red blood cells. Remarkably, the two cells lines acquired CSF-1 dependence for growth. In accordance with this finding they exhibited a 10–15 fold reduction of CSF-1 production compared to NIH3T3 cells. The response observed was not restricted to fibroblast cell lines since both embryonic and adult fibroblasts could also be partially reprogrammed by co-infection with PU.1 and C/EBPa in that they expressed Mac-1, CD45, F4/80 and IA MHC antigens. In conclusion, enforced expression of PU.1 and C/EBPa converts fibroblasts into macrophage like cells, indicating that the combination of these two transcription factors is sufficient to regulate the majority of genes that define the myelomonocytic phenotype.

Polymorphism, natural selection, and structural modeling of class Ia MHC in the African clawed frog (Xenopus laevis)

In the African clawed frog (Xenopus laevis), two deeply divergent allelic lineages of multiple genes of the class I MHC region have been discovered. For the MHC class I UAA locus, functional differences and the molecular basis for lineages maintenance are unknown. Alleles of linked class I region genes also exhibit strong disequilibrium with specific MHC alleles, but the underlying cause is not clear. We use MHC class Ia sequence data to estimate substitution rates and investigate structural differences between allelic lineages from protein models. Results indicate the operation of natural selection, and differences in the steric properties in the F pocket of the peptide-binding region among lineages. Variability in this pocket likely enables allelic lineages to bind very different sets of peptides and to interact differently with MHC chaperones in the endoplasmic reticulum. These results constitute evidence of the molecular evolutionary basis for 1) the maintenance of allelic lineages, 2) functional differences among lineages, and 3) strong linkage disequilibrium of allelic variants of class I region genes in X. laevis.

Mechanisms of immune privilege in the eye and hair follicle.

It has been recognized for over a century that the eye is endowed with remarkable properties that permit the long-term survival of foreign tumor and tissue grafts that are normally rejected at extraocular sites. This ocular immune privilege was originally attributed to a putative sequestration of antigens in the eye as a result of the conspicuous absence of intraocular lymphatic drainage channels. In the last 30 years, a sizeable body of information indicates that ocular immune privilege is a product of multiple anatomical, physiological, and immunoregulatory processes. Ocular tissues and fluids express a wide variety of anti-inflammatory and immunosuppressive molecules, including CD95L (FasL), transforming growth factor-beta, macrophage migration inhibitory factor, alpha-melanocyte-stimulating hormone, calcitonin gene-related peptide, somatostatin, and complement regulatory proteins. Moreover, antigens entering the anterior chamber of the eye evoke a unique form of immune deviation that culminates in the antigen-specific suppression of TH1 immune responses. Finally, the intraocular milieu contains both cell membrane and soluble factors that inhibit both the adaptive and innate immune systems. The hair follicle is also recognized for its immune privilege. Like the anterior chamber of the eye, it produces anti-inflammatory and immunosuppressive cytokines, such as transforming growth factor-beta and adrenocorticotrophic hormone. The cells of the hair follicle display limited expression of class Ia MHC molecules and, like cells that line the anterior chamber of the eye, are protected against CD8+ cytotoxic T lymphocyte attack. Gaining a better understanding of the immune privilege of the hair follicle may provide insights into the regulation and pathogenesis of immune-mediated diseases of the skin.

Peptide binding characteristics of the non-classical class Ib MHC molecule HLA-E assessed by a recombinant random peptide approach

BackgroundIncreasing evidence suggests that the effect of HLA-E on Natural Killer (NK) cell activity can be affected by the nature of the peptides bound to this non-classical, MHC class Ib molecule. However, its reduced cell surface expression, and until recently, the lack of specific monoclonal antibodies hinder studying the peptide-binding specificity HLA-E.ResultsAn in vitro refolding system was used to assess binding of recombinant HLA-E to either specific peptides or a nonamer random peptide library. Peptides eluted from HLA-E molecules refolded around the nonamer library were then used to determine a binding motif for HLA-E. Hydrophobic and non-charged amino acids were found to predominate along the peptide motif, with a leucine anchor at P9, but surprisingly there was no methionine preference at P2, as suggested by previous studies.ConclusionsCompared to the results obtained with rat classical class Ia MHC molecules, RT1-A1c and RT1-Au, HLA-E appears to refold around a random peptide library to reduced but detectable levels, suggesting that this molecule's specificity is tight but probably not as exquisite as has been previously suggested. This, and a previous report that it can associate with synthetic peptides carrying a viral sequence, suggests that HLA-E, similar to its mouse counterpart (Qa-1b), could possibly bind peptides different from MHC class I leader peptides and present them to T lymphocytes.

Expression of a nonclassical MHC class Ib molecule in the eye.

MHC class Ia molecules are absent, or expressed at low levels, on cells lining the anterior chamber of the eye, an immune-privileged site. Although the scarcity of class Ia MHC antigens may protect cells from T cell-mediated tissue injury, it may also render them vulnerable to natural killer cell-mediated cytolysis. There is growing evidence that MHC class Ib molecules share similar functions to class Ia. In this study, we examine the expression and distribution of Qa-2, one of the best-characterized murine MHC class Ib molecules in the eye. The transcription of Qa-2 mRNA in whole eye and eye-derived cells was analyzed by sensitive and specific RNase protection and reverse transcription-polymerase chain reaction assays. Immunohistochemistry, flow cytometry, and ELISA were used to determine whether Qa-2 was expressed as cell surface proteins. Expression levels of Qa-2 were monitored in resting cells and cells stimulated with interferon-gamma. Expression of membrane-bound and soluble Qa-2 isoforms was detected in various tissues of the eye, including cell subsets lining the anterior chamber. Immunohistological staining revealed Qa-2 expression on corneal epithelium as well as endothelium, iris ciliary bodies, and retina. These observations were confirmed by analysis of cultured, eye-derived cells. Qa-2 expression was inducible by interferon-gamma. Qa-2 was not detected in lens cells. The results demonstrate that membrane-bound and soluble MHC class Ib molecules are expressed by eye cells. Expression of Qa-2 in the corneal endothelium and other substructures lining the anterior chamber suggests that this class Ib protein may contribute to the immune-privileged status of the anterior chamber.