Lipid‐mediated presentation of MHC class II molecules guides thymocytes to the CD4 lineage

Previous studies on the MHC class‐specific differentiation of CD4⁺CD8⁺ thymocytes into CD4⁺ and CD8⁺ T cells have focused on the role of coreceptor molecules. However, CD4 and CD8 T cells develop according to their MHC class specificities even in these mice lacking coreceptors. This study investigated the possibility that lineage is determined not only by coreceptors, but is also guided by the way how MHC molecules are presented. MHC class II molecules possess a highly conserved Cys in their transmembrane domain, which is palmitoylated and thereby associates with lipid rafts, whereas neither palmitoylation nor raft association was observed with MHC class I molecules. The generation of CD4 T cells was impaired and that of CD8 T cells was augmented when the rafts on the thymic epithelial cells were disrupted. This was due to the conversion of MHC class II‐specific thymocytes from the CD4 lineage to CD8. The ability of I‐A^d molecule to associate with rafts was lost when its transmembrane Cys was replaced. The development of DO11.10 thymocytes recognizing this mutant I‐A^dm was converted from CD4 to CD8. These results suggest that the CD4 lineage commitment is directed by the raft‐associated presentation of MHC class II molecules.     

Mapping and restriction of a dominant viral CD4+ T cell core epitope by both MHC class I and MHC class II

Virus-specific CD4(+) T cells contribute to effective virus control through a multiplicity of mechanisms including direct effector functions as well as "help" for B cell and CD8(+) T cell responses. Here, we have used the lymphocytic choriomeningitis virus (LCMV) system to assess the minimal constraints of a dominant antiviral CD4(+) T cell response. We report that the core epitope derived from the LCMV glycoprotein (GP) is 11 amino acids in length and provides optimal recognition by epitope-specific CD4(+) T cells. Surprisingly, this epitope is also recognized by LCMV-specific CD8(+) T cells and thus constitutes a unique viral determinant with dual MHC class I- and II-restriction.     

rGM—CSF对U937细胞系膜MHC分子及CD86分子表达的作用

本实验研究了人重组ＧＭ－ＣＳＦ对Ｕ９３７人单核细胞样细胞系ＨＬＡ和ＣＤ８６分子表达的调节作用，将Ｕ９３７细胞在ｒＧＭ－ＣＳＦ１０μｇ／Ｌ培养１ｄ，ＨＬＡ－ＤＲ分子的表达率为８３％，对照组为９１％，培养ｄ表达率为５１％，对照组为７８％，培养５ｄ，表达率为５３％，对照组为８１％，Ｕ９３７细胞在ｒＧＭ－ＣＳＦ１０μｇ／Ｌ浓度下培养１ｄ，ＣＤ８６分子表达率为２％，对照组为１．６％，培养３ｄ，表达率为３．９     

The Qa-1b molecule binds to a large subpopulation of murine NK cells

Recent studies on human NK cells have demonstrated that the NK cell CD94/NKG2 receptors bind to the nonclassical MHC class I molecule HLA-E. A functional CD94/NKG2 complex has not yet been identified in rodents, but cDNA encoding rat and mouse CD94 and NKG2 have recently been cloned, suggesting that CD94/NKG2 receptors may exist in species other than man. The mouse nonclassical MHC class I molecule Qa-1 shares several features with HLA-E. This suggests that Qa-1 may be similarly recognized by murine NK cells. To study the ability of Qa-1 to bind to murine NK cells, we have produced a soluble tetrameric form of Qa-1^b . In the present study, we demonstrate that Qa-1^b tetramers distinctly bind to a large subset of fresh or IL-2-activated NK1.1⁺ /CD3⁻ splenocytes independently of the expression of Ly49 inhibitory receptors. Binding occurs whether NK cells have evolved in an MHC class I-expressing or in an MHC class I-deficient environment. Our data suggest the existence of a Qa-1-recognizing structure on a large subpopulation of murine NK cells that may be similar to the human CD94/NKG2 heterodimeric complex.     

Conformational changes in MHC class I molecules

In this article we review the role of MHC conformation, including peptide-induced MHC conformation, in forming antibody (Ab), T-cell receptor (TCR), and natural killer (NK) cell receptor epitopes. Abs recognize conformational major histocompatibility (MHC) epitopes that often are influenced by the identity of MHC-bound peptide. Diverse TCRs recognize a common docking site on peptide/MHC complexes and directly contact peptide. Human NK cell inhibitory receptors (KIR) appear to recognize limited regions of the HLA α₁ helix. DX9+ KIR specifically focus on HLA-B residues 82 and 83. However, NK cells recognize much broader regions of HLA class I molecules and are sensitive to bound peptides. Thus, several classes of lymphocyte receptors are peptidespecific. Peptide specificity could be the result of direct contact with the receptor, or to conformational shifts in MHC residues that interact with both receptor and bound peptide.     

Mushroom plant workers experience a shift towards a T helper type 2 dominant state: contribution of innate immunity to spore antigen

Contemporary mushroom factories are places where there is a substantial risk of the occurrence of respiratory allergy. The aims of this investigation were to estimate its causative agents and to evaluate the contribution of innate immune response in mushroom workers who cultivate Hypsizigus marmoreus (Bunashimeji). Cross-sectional and follow-up studies were performed in the factory. We investigated CD1b, CD3, CD4, CD8, CD14, CD45RO, CD62L and CD161 expression in peripheral blood mononuclear cells (PBMC) by flow cytometry, and serum levels of interleukin (IL-2), IL-4, granulocyte-macrophage colony stimulating factor (GM-CSF), IL-13 and interferon (IFN)-gamma by enzyme-linked immunosorbent assay (ELISA). Co-culture experiments of PBMC with spore extracts were also performed. Percentages of CD1b+ monocytes, natural killer (NK), NK T and CD4+ T cells were increased in the workers compared with controls. Increases in Th2 type cells, Th2/Th1 ratio and serum IL-13 and decreased IFN-gamma were detected, indicating a Th2-biased status of the workers. The follow-up study showed that monocytes and NK cells increased soon after employment while CD4+ T, Th2 and NK T cells increased gradually as employment time lengthened. Serum precipitating antibody to the mushroom antigen could be detected at a later stage. Co-cultivation of PBMC with the spore extracts induced much higher CD1b expression, and suppressed secretion of Th1 cytokine in culture supernatants. These results indicate that the mushroom antigen contains highly immunogenic substances which stimulate PBMC into a Th2-biased in vivo status, and innate immune cells might also play a critical role in developing respiratory allergy in mushroom workers.     

Rescue of cytotoxic function in the CD8alpha knockout mouse by removal of MHC class II

CD8 plays an important role in the activity of cytolytic T cells (CTL). However, whether or not CD8 is required for the development of CTL has not been clearly determined. Cytotoxic activity in the CD8alpha knockout mouse is difficult to induce, and has only been demonstrated against allogenic MHC targets. The lack of cytotoxicity may result from impaired lineage commitment of CTL in the absence of CD8, or diminished competitiveness during selection against (unimpaired) development of CD4(+) T cells on MHC class II (MHC II). To differentiate between these possibilities, we have generated a double-knockout mouse (MHC II(-/-)CD8alpha(-/-)). In MHC II(-/-)CD8alpha(-/-) mice, developing MHC class I (MHC I)-reactive thymocytes cannot rely upon CD8 for selection, but they also cannot be overwhelmed by efficient selection of MHC II-reactive thymocytes. In this mouse, a large, heterogeneous population of peripheral coreceptor double-negative (DN) and CD4(+) T cells develops. Peripheral DN T cells are fully functional CTL. They display cytolytic activity against allogeneic MHC, and against syngeneic MHC following lymphocytic choriomeningitis virus (LCMV) infection. Cells from LCMV-infected mice bind more MHC I tetramer at lower concentrations than their wild-type CTL counterparts. These results demonstrate unequivocally that CD8 is not required for commitment of thymocytes to the CTL lineage.     

The role of tetraspanin CD63 in antigen presentation via MHC class II

Interactions between MHC class II (MHC II)-positive APCs and CD4(+) T cells are central to adaptive immune responses. Using an Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell line (LCL) as MHC II-positive APCs and CD4(+) T-cell clones specific for two endogenously expressed EBV antigens, we found that shRNA knockdown of the tetraspanin protein CD63 in LCL cells consistently led to increased CD4(+) T-cell recognition. This effect was not due to enhanced antigen processing nor to changes in MHC II expression since CD63 knockdown did not influence the amount or dimerization of MHC II in LCL cells. We therefore investigated the possible involvement of exosomes, small MHC II- and tetraspanin-abundant vesicles which are secreted by LCL cells and which we found could themselves activate the CD4(+) T-cell clones in an MHC II-dependent manner. While equal loadings of exosomes purified from the control and CD63(low) LCLs stimulated T cells to a comparable degree, we found that exosome production significantly increased following CD63-knockdown, suggesting that this may underlie the greater T-cell stimulatory capacity of the CD63(low) LCLs. Taken together, our data reveal a new insight into the mechanisms by which tetraspanins are involved in the regulation of MHC II-dependent T-cell stimulation.     

The ILT2(LIR1) and CD94/NKG2A NK cell receptors respectively recognize HLA-G1 and HLA-E molecules co-expressed on target cells

Previous studies on NK recognition of HLA-G1 employed as targets 721.221 transfectants (.221-G1) that unknowingly co-expressed the HLA-E molecule, subsequently found to be a major ligand for the CD94/NKG2 receptors. In the present study we re-evaluated the relative role played by CD94/NKG2 and ILT2(LIR1) molecules in recognition of HLA-G1 by NK clones. We employed as targets .221-G1 cells and a surface HLA-E-negative transfectant, .221-G1(E_neg), generated by site-directed mutagenesis of the HLA-G1 leader sequence. The antagonistic effects of receptor- (i.e. CD94/NKG2A, ILT2) and ligand-specific mAb (i.e. HLA-G, HLA-E) were assessed. In addition, binding of an ILT2-Ig fusion protein to the .221-AEH, expressing only HLA-E, and the .221-G1(E_neg) transfectants was analyzed. Our data demonstrate that NK recognition of cells expressing HLA-G1 involves at least two non-overlapping receptor-ligand systems: the CD94/NKG2 interaction with HLA-E, and the engagement of the ILT2(LIR1) receptor by HLA-G1 molecules.     

Application of nine-color flow cytometry for detailed studies of the phenotypic complexity and functional heterogeneity of human lymphocyte subsets

Innate and adaptive cellular immunity is initiated, directed and regulated by a vast array of cell surface receptors. Attempts to harness the cellular immune system in translational settings such as immunotherapy and vaccine development require tools to accurately describe and isolate lymphocytes with specific characteristics. One such tool, flow cytometry, is undergoing a revolution in instrumentation and reagents, providing opportunities for high resolution phenotypic and functional analysis of lymphocytes. Here, we demonstrate how nine-color flow cytometry can be adapted, optimized and applied to investigate the phenotypic complexity and functional heterogeneity of human lymphocyte subsets. We provide examples of studies of adaptive T cell responses against viruses, as well as the assessment of CD1d-restricted NKT cells and NK cells. We discuss the importance of this technology for detailed investigations of lymphocyte subsets in studies of infectious diseases and cancer.     

Improved methods for predicting peptide binding affinity to MHC class II molecules

Major histocompatibility complex class II (MHC‐II) molecules are expressed on the surface of professional antigen‐presenting cells where they display peptides to T helper cells, which orchestrate the onset and outcome of many host immune responses. Understanding which peptides will be presented by the MHC‐II molecule is therefore important for understanding the activation of T helper cells and can be used to identify T‐cell epitopes. We here present updated versions of two MHC–II–peptide binding affinity prediction methods, NetMHCII and NetMHCIIpan. These were constructed using an extended data set of quantitative MHC–peptide binding affinity data obtained from the Immune Epitope Database covering HLA‐DR, HLA‐DQ, HLA‐DP and H‐2 mouse molecules. We show that training with this extended data set improved the performance for peptide binding predictions for both methods. Both methods are publicly available at www.cbs.dtu.dk/services/NetMHCII-2.3 and www.cbs.dtu.dk/services/NetMHCIIpan-3.2 .     

NKG2D performs two functions in invariant NKT cells: direct TCR-independent activation of NK-like cytolysis and co-stimulation of activation by CD1d

Invariant NKT cells are important in the activation and regulation of immune responses. They can also function as CD1d-restricted killer cells. However, the role of activating innate NK-cell receptors expressed on NKT cells in triggering cytolytic function is poorly characterized. Here, we initially confirmed that the cellular stress-ligand receptor NKG2D is expressed on CD4- NKT cells, whereas most CD4+ NKT cells lack this receptor. Interestingly, NKG2D+ NKT cells frequently expressed perforin, and both NKG2D and perforin localized at the site of contact with NKG2D ligand-expressing target cells. CD4- NKT cells degranulated in response to NKG2D engagement in a redirected activation assay independent of stimulation via their invariant TCR. NKT cells killed P815 cells coated with anti-NKG2D mAb and CD1d-negative K562 tumor target cells in an NKG2D-dependent manner. Furthermore, NKG2D engagement co-stimulated TCR-mediated NKT-cell activation in response to endogenous CD1d-presented ligands or suboptimal levels of anti-CD3 triggering. These data indicate that the CD4- subset of human NKT cells can mediate direct lysis of target cells via NKG2D engagement independent of CD1d, and that NKG2D also functions as a co-stimulatory receptor in these cells. NKG2D thus plays both a direct and a co-stimulatory role in the activation of NKT cells.     

Enhanced immunogenicity of CTL antigens through mutation of the CD8 binding MHC class I invariant region

CD8(+) cytotoxic T lymphocytes (CTL) are key determinants of immunity to intracellular pathogens and neoplastic cells. Recognition of specific antigens in the form of peptide-MHC class I complexes (pMHCI) presented on the target cell surface is mediated by T cell receptor (TCR) engagement. The CD8 coreceptor binds to invariant domains of pMHCI and facilitates antigen recognition. Here, we investigate the biological effects of a Q115E substitution in the alpha2 domain of human leukocyte antigen (HLA)-A*0201 that enhances CD8 binding by approximately 50% without altering TCR/pMHCI interactions. Soluble and cell surface-expressed forms of Q115E HLA-A*0201 exhibit enhanced recognition by CTL without loss of specificity. These CD8-enhanced antigens induce greater CD3 zeta chain phosphorylation in cognate CTL leading to substantial increases in cytokine production, proliferation and priming of naive T cells. This effect provides a fundamental new mechanism with which to enhance cellular immunity to specific T cell antigens.     

MHC class I immune evasion in MCMV infection

Murine cytomegalovirus (MCMV) is a well-studied model of natural β-herpesvirus infection. However, many questions remain regarding its control by and evasion of the immune response it generates. CD8 and CD4 T cells have both unique and redundant roles in control of the virus that differ based on the immunocompetence of the infected mice. MCMV encodes major histocompatibility complex (MHC) class I immune evasion genes that can have an impact in vitro, but their role in infection of immunocompetent mice has been difficult to identify. This review addresses the evidence for their in vivo function and suggests why they may be evolutionarily conserved.     

MHC structure and autoimmune T cell repertoire development

Recent work has continued to clarify the relationship between MHC structure and thymic selection that leads to peripheral T cell repertoire development in the pathogenesis of autoimmune diseases. Particular attention has been focused on the nonobese diabetic model of autoimmune diabetes, in which a unique MHC class II molecule (I-Ag7) plays a central role. In the past year, reports on the biochemistry of I-Ag7-combined with analysis of the role of I-Ag7 in T cell repertoire selection--support a model of defective thymic selection as the basis of the association between particular MHC molecules and autoimmune diseases. Analogous work has been done on the structure of the human MHC disease-susceptible and -resistant alleles, DQA1*0301 DQB1*0302 and DQA1*0102 DQB1*0602, and their effect on autoimmune repertoire selection. Comparison of these results (in naturally occurring, spontaneous autoimmune human and murine diabetes), with results in a variety of transgenic and knockout models, has produced an integrated view of how avidity considerations in repertoire selection in the thymus could affect predisposition towards autoimmunity.     

T cell differentiation and generation of the antigen-specific T cell repertoire in man: observations in MHC class II deficiency

The circulating T cell pool of an MHC class II-deficient patient was shown to lack the MHC class II-specific T cell functions. This was demonstrated by the absence of MHC class Il-specific alloreactive T cells and a substantially decreased number of circulating CD4⁺ lymphocytes. The patient's T cells did respond to an allostimulus, although the restriction pattern of this reaction remains speculative. The function and distribution of peripheral T cell subsets from the patient resemble findings in MHC class II-deficient mice, which also lack interaction of T cell precursors with MHC class Il-bearing accessory cells during thymic differentiation. Our data support the concept that Tcell differentiation in humans is similar, and that the human MHC-restricted Tcell repertoire depends on prior interaction of T cell precursors with self MHC.     

Impaired expression of MHC class II molecules in response to interferon-gamma (IFN-γ) on human thymoma neoplastic epithelial cells

A human thymoma is a neoplasm derived from the thymic epithelial cell, and is well known for its association with autoimmune diseases, especially myasthenia gravis. The neoplastic epithelial cells of thymoma clearly retain thymic epithelial functions, but the development of T cells in thymoma is somewhat impaired. In this study, we quantified by flow cytometry the in vitro expression of MHC molecules on neoplastic epithelial cells precultured with IFN-γ. While MHC class I expression was comparable with that on normal thymic epithelial cells, the level of MHC class II molecules on neoplastic epithelial cells was lower than in controls, and also varied greatly from case to case. Additionally, there was a significant positive correlation between the expression level of MHC class II and the proportion of mature CD3⁺ cells in the CD4⁺CD8^? subset. Thus, accumulation of CD3^?CD4⁺CD8^? cells in thymoma may result from impaired expression of the MHC class II molecules, suggesting that the function of the neoplastic epithelial cells might determine the maturation and the positively selected repertoire of T cells in thymomas.     

PA28 and the proteasome immunosubunits play a central and independent role in the production of MHC class I-binding peptides in vivo

Proteasomes play a fundamental role in the processing of intracellular antigens into peptides that bind to MHC class I molecules for the presentation of CD8(+) T cells. Three IFN-γ-inducible catalytic proteasome (immuno)subunits as well as the IFN-γ-inducible proteasome activator PA28 dramatically accelerate the generation of a subset of MHC class I-presented antigenic peptides. To determine whether these IFN-γ-inducible proteasome components play a compounded role in antigen processing, we generated mice lacking both PA28 and immunosubunits β5i/LMP7 and β2i/MECL-1. Analyses of MHC class I cell-surface levels ex vivo demonstrated that PA28 deficiency reduced the production of MHC class I-binding peptides both in cells with and without immunosubunits, in the latter cells further decreasing an already diminished production of MHC ligands in the absence of immunoproteasomes. In contrast, the immunosubunits but not PA28 appeared to be of critical importance for the induction of CD8(+) T-cell responses to multiple dominant Influenza and Listeria-derived epitopes. Taken together, our data demonstrate that PA28 and the proteasome immunosubunits use fundamentally different mechanisms to enhance the supply of MHC class I-binding peptides; however, only the immunosubunit-imposed effects on proteolytic epitope processing appear to have substantial influence on the specificity of pathogen-specific CD8(+) T-cell responses.