MHC Recognition Research Articles

Liver NK cells expressing TRAIL are toxic against self hepatocytes in mice.

Although it is known that activation of natural killer (NK) cells causes liver injury, the mechanisms underlying NK cell-induced killing of self-hepatocytes are not clear. We demonstrated that liver NK cells have cytotoxicity against normal syngeneic hepatocytes in mice. Polyinosinic-polycytidylic acid (poly I:C) treatment enhanced hepatocyte toxicity of liver NK cells but not that of spleen NK cells. Unlike NK cells in other tissues, approximately 30%-40% of liver NK cells constitutively express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). An in vitro NK cell cytotoxic assay revealed that hepatocyte toxicity of liver NK cells from both naïve and poly I:C-treated mice was inhibited partially by an anti-TRAIL monoclonal antibody (mAb) alone and completely by the combination with anti-Fas ligand (FasL) mAb and a perforin inhibitor, concanamycin A, indicating contribution of TRAIL to NK cell-mediated hepatocyte toxicity. The majority of TRAIL(+) NK cells lacked expression of Ly-49 inhibitory receptors recognizing self-major histocompatibility complex class I, indicating a propensity to targeting self-hepatocytes. Poly I:C treatment significantly upregulated the expression of Ly-49 receptors on TRAIL(-) NK cells. This might be a compensatory mechanism to protect self-class I-expressing cells from activated NK cell-mediated killing. However, such compensatory alteration was not seen at all in the TRAIL(+) NK cell fraction. Thus, liver TRAIL(+) NK cells have less capacity for self-recognition, and this might be involved in NK cell-dependent self-hepatocyte toxicity. In conclusion, our findings are consistent with a model in which TRAIL-expressing NK cells play a critical role in self-hepatocyte killing through poor recognition of MHC.

MHC Recognition by Hapten-Specific HLA-A2-Restricted CD8+ CTL

T cell recognition by peptide-specific alphabeta TCRs involves not only recognition of the peptide, but also recognition of multiple molecular features on the surface of the MHC molecule to which the peptide has been bound. We have previously shown that TCRs that are specific for five different peptides presented by HLA-A2 recognize similar molecular features on the surface of the alpha1 and alpha2 helices of the HLA-A2 molecule. We next asked whether these same molecular features of the HLA-A2 molecule would be recognized by hapten-specific HLA-A2-restricted TCRs, given that hapten-specific T cells frequently show reduced MHC dependence/restriction. The results show that a panel of CD8+ CTL that are specific for the hapten DNP bound to two different peptides presented by HLA-A2 do the following: 1) show stringent MHC restriction, and 2) are largely affected by the same mutations on the HLA-A2 molecule that affected recognition by peptide-specific CTL. A small subset of this panel of CD8+ CTL can recognize a mutant HLA-A2 molecule in the absence of hapten. These data suggest that TCR recognition of a divergent repertoire of ligands presented by HLA-A2 is largely dependent upon common structural elements in the central portion of the peptide-binding site.

Allogeneic and syngeneic class I MHC complexes drive the association of CD8 and TCR on 2C T cells

In most cases, cytotoxic T cell activation is dependent on the interaction of the T cell receptor (TCR) and CD8 with MHC class I molecules. In the CD8 + T cell system based on the mouse cytotoxic T cell clone 2C, recognition of the allogeneic MHC L d exhibits a less significant role for CD8 than recognition of the syngeneic MHC K b. Here, we examined whether this difference is related to the relative abilities of the two pepMHC complexes to drive the association of CD8 and TCR on the T cell surface. We show that both the syngeneic and allogeneic pepMHC induced association of CD8 and TCR, as revealed by fluorescence resonance energy transfer (FRET). Thus, the orientation of the syngeneic and allogeneic ligands when bound to the same TCR both allow CD8 to be recruited to the TCR complex. The conserved diagonal orientation of TCRs on different pepMHC ligands may facilitate such associations. The FRET results are consistent with the known binding properties and the CD8 involvement of the two different TCR:pepMHC interactions.

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Structure of the Epstein-Barr Virus gp42 Protein Bound to the MHC Class II Receptor HLA-DR1

Epstein-Barr virus (EBV) causes infectious mononucleosis, establishes long-term latent infections, and is associated with a variety of human tumors. The EBV gp42 glycoprotein binds MHC class II molecules, playing a critical role in infection of B lymphocytes. EBV gp42 belongs to the C-type lectin superfamily, with homology to NK receptors of the immune system. We report the crystal structure of gp42 bound to the human MHC class II molecule HLA-DR1. The gp42 binds HLA-DR1 using a surface site that is distinct from the canonical lectin and NK receptor ligand binding sites. At the canonical ligand binding site, gp42 forms a large hydrophobic groove, which could interact with other ligands necessary for EBV entry, providing a mechanism for coupling MHC recognition and membrane fusion.

Flexibility in MHC and TCR recognition: degenerate specificity at the T cell level in the recognition of promiscuous Th epitopes exhibiting no primary sequence homology.

Recognition of peptide Ags by T cells through the TCR can be highly specific. In this report we show the degeneracy of Ag recognition at both MHC and TCR levels. We present evidence that unrelated promiscuous Th cell epitopes from various protein sources exhibit sufficient structural homology, despite minimal structural identity, to elicit cross-reactive proliferative responses at the bulk T cell level. This epitopic mimicry was also observed when peptide (CS.T3(378-395) and TT(830-844))-specific CD4+ T cell lines and T cell hybridoma clones were used in proliferation and Ag presentation assays. A scrambled CS.T3(378-395) peptide did not show any proliferation, indicating that the specificity of the cross-reactive responses may be linked with the primary structure of the peptides. Blocking of CS.T3(378-395)-specific CD4+ T cell proliferation by anti-MHC class II mAb showed that recognition of promiscuous T cell epitopes is largely in association with MHC class II molecules. These findings suggest that promiscuous Th epitopes may be useful in designing peptide-based vaccine constructs. At the same time these results show that at the T cell level there may be a great deal of immunological cross-reactivity between heterologous pathogens, and because of this the host's response to a pathogen may be modified by its previous experience with other unrelated pathogens.

MHC recognition in thymic development: distinct, parallel pathways for survival and lineage commitment.

The molecular events triggered by MHC recognition and how they lead to the emergence of mature CD4 and CD8 lineage thymocytes are not yet understood. To address these questions, we have examined what signals are necessary to drive the development of CD8 lineage thymocytes in TCRalpha(-) mice in which TCR/MHC engagement cannot occur. We find that the combination of constitutive Notch activity and constitutive Bcl-2 expression are necessary and sufficient to allow the appearance of mature CD8 lineage thymocytes in TCRalpha(-) mice. In addition, Notch activity alone in TCRalpha(-) mice can induce the up-regulation of HES1, suggesting that thymocytes are competent to respond to Notch signaling in the absence of MHC recognition. These data indicate that survival and lineage commitment represent distinct, parallel pathways that occur as a consequence of MHC recognition, both of which are necessary for the development of mature CD8 lineage T cells.

Positive impact of inhibitory Ly49 receptor-MHC class I interaction on NK cell development.

NK cells can kill MHC-different or MHC-deficient but not syngeneic MHC-expressing target cells. This MHC class I-specific tolerance is acquired during NK cell development. MHC recognition by murine NK cells largely depends on clonally distributed Ly49 family receptors, which inhibit NK cell function upon ligand engagement. We investigated whether these receptors play a role for the development of NK cells and provide evidence that the expression of a Ly49 receptor transgene on developing NK cells endowed these cells with a significant developmental advantage over NK cells lacking such a receptor, but only if the relevant MHC ligand was present in the environment. The data suggest that the transgenic Ly49 receptor accelerates and/or rescues the development of NK cells which would otherwise fail to acquire sufficient numbers of self-MHC-specific receptors. Interestingly, the positive effect on NK cell development is most prominent when the MHC ligand is simultaneously present on both hemopoietic and nonhemopoietic cells. These findings correlate with functional data showing that MHC class I ligand on all cells is required to generate functionally mature NK cells capable of reacting to cells lacking the respective MHC ligand. We conclude that the engagement of inhibitory MHC receptors during NK cell development provides signals that are important for further NK cell differentiation and/or maturation.

Molecular basis for recognition of an arthritic peptide and a foreign epitope on distinct MHC molecules by a single TCR.

KRN TCR transgenic T cells recognize two self-MHC molecules: a foreign peptide, bovine RNase 42-56, on I-Ak and an autoantigen, glucose-6-phosphate isomerase 282-294, on I-Ag7. Because the latter recognition event initiates a disease closely resembling human rheumatoid arthritis, we investigated the structural basis of this pathogenic TCR's dual specificity. While peptide recognition is altered to a minor degree between the MHC molecules, we show that the receptor's cross-reactivity critically depends upon a TCR contact residue completely conserved in the foreign and self peptides. Further, the altered recognition of peptide derives from discrete differences on the MHC recognition surfaces and not the disparate binding grooves. This work provides a detailed structural comparison of an autoreactive TCR's interactions with naturally occurring peptides on distinct MHC molecules. The capacity to interact with multiple self-MHCs in this manner increases the number of potentially pathogenic self-interactions available to a T cell.

Differential recognition of MHC class I molecules of xeno-/allo-endothelial cells by human NK cells

Using human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial cells (PAEC) as target cells, human peripheral blood NK cells (PBNK) and NK92 cells as effector cells, the differential cytotoxicities of NK cells to allo- and xeno-endothelial cells were studied. The influence of MHC class I molecules on the cytotoxicity of human NK cells was assayed using acid treatment, and blockades of MHC class I antigens, CD94 and KIR (NKB1). The results indicated that the killing of PAEC by the two kinds of NK cells is higher than that of HUVEC. After acid treatment, the cytotoxicity of the two kinds of NK cells to PAEC and HUVEC is significantly enhanced, but the magnitude of the enhancement is different. The enhancement of NK killing to acid treated HUVEC is much greater than that to PAEC. Blockade of CD94 mAb did not alter the NK cytotoxicity, while blockade of NKB1 mAb enhanced the cytotoxicity of PBNK to HUVEC and PAEC by 95% and 29% respectively. The results above suggested that the differential recognition of MHC I molecules of xeno-endothelial cells by human NK cells could be the major reason for higher NK cytotoxicity to PAEC. KIR might be the primary molecule that transduced inhibitory signals when endothelial cells were injured by NK cells.

The Role of CD8α′ in the CD4 Versus CD8 Lineage Choice

AbstractDuring thymic development the recognition of MHC proteins by developing thymocytes influences their lineage commitment, such that recognition of class I MHC leads to CD8 T cell development, whereas recognition of class II MHC leads to CD4 T cell development. The coreceptors CD8 and CD4 may contribute to these different outcomes through interactions with class I and class II MHC, respectively, and through interactions with the tyrosine kinase p56lck (Lck) via their cytoplasmic domains. In this paper we provide evidence that an alternatively spliced form of CD8 that cannot interact with Lck (CD8α′) can influence the CD4 vs CD8 lineage decision. Constitutive expression of a CD8 minigene transgene that encodes both CD8α and CD8α′ restores CD8 T cell development in CD8α mutant mice, but fails to permit the development of mismatched CD4 T cells bearing class I-specific TCRs. These results indicate that CD8α′ favors the development of CD8-lineage T cells, perhaps by reducing Lck activity upon class I MHC recognition in the thymus.

The Mode of Ligand Recognition by Two Peptide:MHC Class I-Specific Monoclonal Antibodies

The Ig superfamily members TCR and B cell receptor (BCR) share high structural and amino acid homology, yet interact with Ags in a distinct manner. The overall shape of the TCR ligand is rather constant, with the variation coming from the MHC polymorphism and the peptide heterogeneity. Consequently, the TCR alpha- and beta-chains form a relatively flat ligand-binding site that interacts with the peptide:MHC (pep:MHC) ligand in a fixed diagonal orientation relative to the MHC alpha-helices, with the alpha- and beta-chains of the TCR contacting the N and C termini of the pep:MHC complex, respectively. By contrast, the shape of BCR ligands varies dramatically, and the BCR exhibits much greater variability of the Ag-binding site. The mAbs 25-D1.16 (D1) and 22-C5.9 (C5), specific for the OVA-8:H-2Kb complex, allowed us to directly compare how TCR and BCR approach the same ligand. To that effect, we mapped D1 and C5 footprints over the OVA-8:H-2Kb complex. Using peptide variants and mutant MHC molecules, we show that the D1 and C5 contacts with the OVA-8:Kb complex C terminus overlap with the TCR beta-chain footprint, but that this footprint also extends to the regions of the molecule not contacted by the TCR. These studies suggest that D1 and C5 exhibit a hybrid mode of pep:MHC recognition, in part similar to that of the TCR beta-chain and in part similar to the conventional anti-MHC Ab.

Allele-specific and peptide-dependent recognition of swine leukocyte antigen class I by human cytotoxic T-cell clones.

The T-cell mediated immune responses play a major role in xenograft rejection. However, the mechanisms behind human T-cell recognition of porcine xenoantigens remain to be elucidated. Human CD8+ T-cell lines were generated against porcine aortic endothelial cells (PAECs) from y/y and z/z haplotypes of Yucatan inbred swine. T-cell clones were obtained by limiting dilution. The human T-cell receptor (TCR)-swine leukocyte antigen (SLA) class I interaction was characterized. The human CD8+ T-cell mediated direct recognition of PAECs was SLA haplotype-specific. The haplotype specificity was restricted by the SLA class I allelic polymorphism. To characterize the role of SLA-bound peptides in the human TCR-SLA class I interaction, we stripped peptides from SLA molecules by a brief acid treatment. Using z/z-specific CD8+ T cells as effectors, we demonstrated that the acid-treatment, which stripped SLA molecules of bound peptides, decreased the lysis of PAECs by 72%. Addition of peptides eluted from affinity purified z/z SLA class I molecules, but not from the irrelevant y/y SLA class I, restored the lysis of acid-treated z/z PAECs. In addition, the lysis of a human HLA class I negative cell line, 721.221, transfected with a relevant SLA class I allele derived from the z/z haplotype, was significantly increased with the addition of relevant z/z peptides. These experiments indicated that both SLA class I and bound peptides were required for recognition by human CD8+ T cells. Cloning studies identified two groups of xenoreactive T-cell clones. Group I clones recognized distinct porcine peptides in the context of SLA class I molecules, whereas group II clones recognized human endogenous cross-reactive peptides presented by SLA class I. Our results demonstrated that, despite the differences in MHC molecules between species, human T-cell recognition of porcine MHC is similar to direct allo-recognition, that is, human TCR recognizes xenogeneic SLA-peptide complexes.

Regulation of T cell fate by Notch.

The transmembrane receptor Notch participates in diverse cell fate decisions throughout embryonic development. Notch receptors and their ligands are expressed in the mammalian thymus, raising the possibility that Notch could regulate T cell fate decisions. Expression of a constitutively activated form of Notch in developing thymocytes causes thymocytes normally destined for the CD4 lineage to adopt the CD8 lineage instead. This suggests that Notch activity normally acts to direct CD4+CD8+ precursors to the CD8 lineage. The choice between CD4 and CD8 T cell fates is also controlled by MHC recognition during positive selection, implying that recognition of class I or II MHC might regulate Notch signaling. Possible models for the regulation of Notch by MHC recognition during CD4 versus CD8 lineage determination are discussed.

Fine Specificity and MHC Restriction of Trinitrophenyl- Specific CTL

In this study, the fine specificity and MHC restriction of a CTL response specific to the trinitrophenyl (TNP) hapten was analyzed. Based on the structure of peptide/Kb complexes and ternary TCR/Ag/MHC complexes, four TNP peptides, two octamers, and two nonamers were chosen for eliciting anti-TNP CTL responses. Hapten was conjugated at position 4 in the octamers and at position 5 in the nonamers, positions which should allow engagement of the hapten by TCRs. Potent CTL activity for each of the TNP peptides was obtained that was highly hapten-specific; however, there were considerable differences in the extent of cross-reactivity with other TNP peptides, with the octamers generating more cross-reactive CTL than the nonamers. MHC restriction analysis suggested that anti-hapten responses were less dependent on MHC recognition than anti-peptide responses. This was evidenced by the relative ease of detecting cross-reactivity to haptenated peptides presented by allo-MHC and by the relative insensitivity of anti-hapten vs anti-peptide CTL to mutations in the Kb molecule at potential TCR interaction sites. One potential explanation for this insensitivity to MHC mutation was the finding that the anti-hapten response appeared to be of higher avidity, since a > 100-fold difference in the amount of Ag required to sensitize target cells was found between these two types of Ags.

Indirect T Cell Allorecognition and Alloantibody-Mediated Rejection of MHC Class I-Disparate Heart Grafts

Recent studies in the rat have identified a role for T cell-dependent alloantibody in rejection of MHC class I-disparate allografts. RT1Aa-disparate PVG.R8 heart grafts are rejected acutely in naive, and hyperacutely in sensitized, PVG.RTIu recipients by CD4 T cell-dependent alloantibody. Here, we examined the T cell Ag recognition pathways responsible and show that direct injection into skeletal muscle of plasmid DNA, encoding a water-soluble form of the RT1Aa MHC class I heavy chain (pcmu-tAa), stimulates IgG2b cytotoxic alloantibody and markedly accelerates rejection of PVG.R8 heart grafts (median survival time 2 days). pcmu-tAa injection did not induce CTL to Aa, arguing against direct allorecognition of soluble Aa. Treatment with mAbs confirmed that the alloimmune response to pcmu-tAa injection depended on CD4, not CD8, T cells. Priming T cells for indirect allorecognition by injection of 15-mer peptides spanning the alpha1 and alpha2 domains of Aa failed to stimulate anti-Aa Ab but caused an accelerated Ab response to a PVG.R8 heart and a modest acceleration in graft rejection (median survival time 4 days). These results suggest that both soluble MHC class I and allopeptides prime CD4 T cells by the indirect pathway, but that soluble class I is a more effective immunogen for humoral alloimmunity because its tertiary protein structure provides B cell epitopes. We propose that priming humoral alloimmunity, like CTL priming, requires recognition of intact MHC on donor cells, but essential T cell help can be provided by CD4 T cells recognizing allogeneic class I exclusively by the indirect pathway.

DIRECT RECOGNITION OF MINIATURES SWINE ENDOTHELIAL CELLS BY HUMAN CD8 T CELLS INVOLVES BOTH XENOGENEIC SLA CLASS I MOLECULE AND ASSOCIATED PEPTIDES

78 The theoretical basis of xenorecognition is poorly defined. It is not known how the disparity in MHC molecules between species such as human and porcine affects the TCR MHC interaction. Human anti-miniature swine xeno responses is of particular interest because of the potential clinical use of miniature swine as an organ donor in human transplantation. We generated human anti-miniature swine cytolytic T cell line which showed swine haplotype restricted lysis of porcine endothelial cells (PAEC) from z/z haplotype. In this communication we demonstrate that; 1) human CD8 T cells directly recognize and differentiate swine leukocyte antigens (SLA) expressed on porcine endothelial cells (PAEC), 2) human CD8 is directly involved in the interaction with SLA class I molecules, and 3) xenogeneic peptides associated with the SLA molecules are also required for this interaction. To demonstrate to role of xenogeneic peptides in this xenorecognition, first we stripped peptides from SLA molecules by brief citric acid (pH 3.0) treatment. Untreated z/z PAEC was lysed 25% at E:T 8:1; z/z PAEC stripped of peptide was lysed 11%. Peptides eluted from affinity purified z/z SLA class I molecules but not from the irrelevant y/y SLA class I molecules, restored the lysis of the target cells. Second, peptides eluted from z/z SLA class I molecule but not from the irrelevant y/y SLA class I molecule induced the lysis of human class I null 721.221 and 721.220 cell lines transfected with a SLA class I heavy chain from the z/z haplotype. The above experiments indicates that both the xenopeptides and SLA class I alleles confer specificity for the human CD8 mediated lysis of porcine cells. T cell clones derived from the T cell line also showed similar results. In conclusion, our results demonstrated that despite the difference in MHC molecules between species, recognition of porcine MHC by human T cells is similar to direct allo-recognition, i.e. human TCR recognizes xenogeneic MHC-peptide complex.

DIRECT RECOGNITION OF MINIATURE SWINE ENDOTHELIAL CELLS BY HUMAN CD8 T CELLS INVOLVES BOTH XENOGENEIC SLA CLASS I MOLECULE AND ASSOCIATED PEPTIDES.

78 The theoretical basis of xenorecognition is poorly defined. It is not known how the disparity in MHC molecules between species such as human and porcine affects the TCR MHC interaction. Human anti-miniature swine xeno responses is of particular interest because of the potential clinical use of miniature swine as an organ donor in human transplantation. We generated human anti-miniature swine cytolytic T cell line which showed swine haplotype restricted lysis of porcine endothelial cells (PAEC) from z/z haplotype. In this communication we demonstrate that; 1) human CD8 T cells directly recognize and differentiate swine leukocyte antigens (SLA) expressed on porcine endothelial cells (PAEC), 2) human CD8 is directly involved in the interaction with SLA class I molecules, and 3) xenogeneic peptides associated with the SLA molecules are also required for this interaction. To demonstrate to role of xenogeneic peptides in this xenorecognition, first we stripped peptides from SLA molecules by brief citric acid (pH 3.0) treatment. Untreated z/z PAEC was lysed 25% at E:T 8:1; z/z PAEC stripped of peptide was lysed 11%. Peptides eluted from affinity purified z/z SLA class I molecules but not from the irrelevant y/y SLA class I molecules, restored the lysis of the target cells. Second, peptides eluted from z/z SLA class I molecule but not from the irrelevant y/y SLA class I molecule induced the lysis of human class I null 721.221 and 721.220 cell lines transfected with a SLA class I heavy chain from the z/z haplotype. The above experiments indicates that both the xenopeptides and SLA class I alleles confer specificity for the human CD8 mediated lysis of porcine cells. T cell clones derived from the T cell line also showed similar results. In conclusion, our results demonstrated that despite the difference in MHC molecules between species, recognition of porcine MHC by human T cells is similar to direct allo-recognition, i.e. human TCR recognizes xenogeneic MHC-peptide complex.

Effect of Physical Stress on Immune Function in Cell Lines: B7-2 Molecules Contribute to Augmented Antigen Presentation Induced by Heat Shock

T cell activation occurs following the recognition of MHC II–antigen complexes on antigen-presenting cells (APCs) and in conjunction with binding of costimulatory moleclues such as B7. The heat sho...

CYTOKINE MODULATION OF TH1/TH2 PHENOTYPE DIFFERENTIATION IN DIRECTLY ALLORESPONSIVE CD4+ HUMAN T CELLS1

Organ graft rejection is caused by the recognition of allogeneic MHC molecules by recipient T cells by two different pathways. The indirect pathway of alloreactivity requires the presentation of MHC peptides from the graft by autologous APC, as with conventional antigen. The direct pathway, on the other hand, requires the recognition of foreign MHC on foreign cells. The regulatory mechanisms for this component of alloreactivity have not been extensively studied. We show here that the T cell response activated by alloantigens in the direct pathway is similarly constrained and modulated by cytokines, as has been shown for classic antigen presentation. Thus, the inclusion of IL-2 or TGF-beta in MLC performed with purified responder T cells resulted in outgrowth of cells secreting IL-2 and IFN-gamma, whereas addition of IL-4, IL-10, or anti-TGF-beta encouraged outgrowth of cells secreting IL-4 and IL-10. T cells alloactivated via the direct pathway and then cloned in IL-2 alone secreted IL-4 and IL-10 as well as IFN-gamma and IL-2 (Th0 phenotype). Established clones remained susceptible to cytokine modulation, such that IL-4 and IL-10 decreased their secretion of IL-2 and IFN-gamma, whereas TGF-beta suppressed IL-4 and IL-10 secretion. The first alterations of Th0 toward Th1 or Th2 phenotypes could already be observed after only a very brief exposure to cytokines of 48 hr, followed by extended culture with IL-2 alone. These results confirm that human T cells with Th1 and Th2 phenotypes, recognizing alloantigen via the direct pathway, derive from the same IL-2-secreting precursor and can be manipulated by cytokines in an analogous fashion to conventional antigen-reactive cells. These findings may have implications for manipulating the direct pathway of alloantigen recognition in human organ transplantation.