Mouse Hepa-1 tumor is rejected by H-2Db-restricted CTL despite decreased MHC class I antigen expression

It has recently been hypothesized that tumor cells with reduced levels of MHC class I antigens are more susceptible to NK-mediated lysis and are rejected by NK cells, whereas tumor cells with normal levels of class I are rejected by tumor-specific CTL. We have tested this hypothesis using a mouse hepatoma system. The Hepa-1 tumor is a spontaneous H-2Kb loss variant that arose from the BW7756 tumor, when BW7756 was adapted to growth in culture. Our studies have shown that despite the loss of H-2Kb antigen, Hepa-1 is not more susceptible to NK lysis than its H-2Kb-transfected variants. These studies also suggested that NK cells were not responsible for rejection of the Hepa-1 tumor. The Hepa-1 tumor, therefore, appears to contradict the hypothesized linkage of MHC levels and NK susceptibility. Because NK cells are not involved in immunity to this tumor, we have sought to identify the effector cell responsible for Hepa-1 rejection. Cytotoxic T lymphocyte assays demonstrate that in vitro, Hepa-1 cells are lysed by Hepa-1-specific H-2Db-restricted CD4-CD8+ T lymphocytes. Footpad assays demonstrate that in vivo, Hepa-1 rejection requires CD4+CD8- and CD4-CD8+ Hepa-1-primed splenocytes. These results indicate that immunity to Hepa-1 is T cell mediated. Hepa-1 is therefore an example of an unusual tumor in that down-regulation of MHC class I antigen expression is associated with increased CTL susceptibility.     

MHC class I endosomal and lysosomal trafficking coincides with exogenous antigen loading in dendritic cells

Background

Cross-presentation by dendritic cells (DCs) is a crucial prerequisite for effective priming of cytotoxic T-cell responses against bacterial, viral and tumor antigens; however, this antigen presentation pathway remains poorly defined.

Methodology/Principal Findings

In order to develop a comprehensive understanding of this process, we tested the hypothesis that the internalization of MHC class I molecules (MHC-I) from the cell surface is directly involved in cross-presentation pathway and the loading of antigenic peptides. Here we provide the first examination of the internalization of MHC-I in DCs and we demonstrate that the cytoplasmic domain of MHC-I appears to act as an addressin domain to route MHC-I to both endosomal and lysosomal compartments of DCs, where it is demonstrated that loading of peptides derived from exogenously-derived proteins occurs. Furthermore, by chasing MHC-I from the cell surface of normal and transgenic DCs expressing mutant forms of MHC-I, we observe that a tyrosine-based endocytic trafficking motif is required for the constitutive internalization of MHC-I molecules from the cell surface into early endosomes and subsequently deep into lysosomal peptide-loading compartments. Finally, our data support the concept that multiple pathways of peptide loading of cross-presented antigens may exist depending on the chemical nature and size of the antigen requiring processing.

Conclusions/Significance

We conclude that DCs have ‘hijacked’ and adapted a common vacuolar/endocytic intracellular trafficking pathway to facilitate MHC I access to the endosomal and lysosomal compartments where antigen processing and loading and antigen cross-presentation takes place.     

Human neonatal dendritic cells are competent in MHC class I antigen processing and presentation

Neonates are clearly more susceptible to severe disease following infection with a variety of pathogens than are adults. However, the causes for this are unclear and are often attributed to immunological immaturity. While several aspects of immunity differ between adults and neonates, the capacity of dendritic cells in neonates to process and present antigen to CD8+ T cells remains to be addressed. We used human CD8+ T cell clones to compare the ability of neonatal and adult monocyte-derived dendritic cells to present or process and present antigen using the MHC class I pathway. Specifically, we assessed the ability of dendritic cells to present antigenic peptide, present an HLA-E-restricted antigen, process and present an MHC class I-restricted antigen through the classical MHC class I pathway, and cross present cell-associated antigen via MHC class I. We found no defect in neonatal dendritic cells to perform any of these processing and presentation functions and conclude that the MHC class I antigen processing and presentation pathway is functional in neonatal dendritic cells and hence may not account for the diminished control of pathogens.     

Endosomal sorting of MHC class II determines antigen presentation by dendritic cells

Dendritic cells (DCs) initiate primary immune responses by presenting pathogen-derived antigens in association with major histocompatibility Class II molecules (MHC II) to T cells. In DCs, MHC II is constitutively synthesized and loaded at endosomes with peptides from hydrolyzed endogenous proteins or exogenously acquired antigens. Whether peptide loaded MHC II (MHC II-p) is subsequently recruited to and stably expressed at the plasma membrane or degraded in lysosomes is determined by the status of the DC. In immature DCs, MHC II-p is ubiquitinated after peptide loading, driving its sorting to the luminal vesicles of multivesicular bodies. These luminal vesicles, and the MHC II-p they carry, are delivered to lysosomes for degradation. MHC II-p is inefficiently ubiquitinated in DCs that are activated by pathogens or inflammatory stimuli, thus allowing its transfer to and stable expression at the plasma membrane.     

Induction of impaired antitumor immunity by fusion of MHC class II-deficient dendritic cells with tumor cells

To dissect the role of Ag presentation through MHC class I and/or II pathways by dendritic cell (DC)-tumor fusion cells, we have created various types of DC-tumor fusion cells by alternating fusion cell partners. Fusions of MC38/MUC1 carcinoma cells with DC from wild-type (WT-DC), MHC class I knockout (IKO-DC), class II knockout (IIKO-DC), or class I and II knockout (I/IIKO-DC) mice created WTDC-fusion cells (FC), IKO-FC, IIKO-FC, and I/IIKO-FC, respectively. MHC class II- and MUC1-positive fusion cells were constructed by fusion of B16/MUC1 melanoma cells with IKO-DC (IKO/B16-FC). Immunization of MUC1 transgenic mice with 5 x 10(5) WTDC-FC, IKO-FC, IIKO-FC, or I/IIKO-FC provided 100, 91.7, 61.5, and 15.4% protection, respectively, against tumor challenge with MC38/MUC1 cells. In contrast, all mice immunized with irradiated MC38/MUC1 tumor cells or WT-DC developed tumors. One group of mice was immunized with 5 x 10(5) IKO/B16-FC and then challenged with B16/Ia(+)/MUC1 on one flank and MC38/MUC1 on the other flank. Immunization of these mice with IKO/B16-FC resulted in 100 and 78.6% protection against B16/Ia(+)/MUC1 and MC38/MUC1 tumor challenge, respectively. The antitumor immunity induced by immunization with IKO/B16-FC was able to inhibit the growth of MHC class II-negative tumor. In addition, in vivo results correlated with the induction of Ag-specific CTL. Collectively, the data indicate that MHC class II Ag presentation targeting activation of CD4 T cells is indispensable for antitumor immunity.     

Reorganization of multivesicular bodies regulates MHC class II antigen presentation by dendritic cells

Immature dendritic cells (DCs) sample their environment for antigens and after stimulation present peptide associated with major histocompatibility complex class II (MHC II) to naive T cells. We have studied the intracellular trafficking of MHC II in cultured DCs. In immature cells, the majority of MHC II was stored intracellularly at the internal vesicles of multivesicular bodies (MVBs). In contrast, DM, an accessory molecule required for peptide loading, was located predominantly at the limiting membrane of MVBs. After stimulation, the internal vesicles carrying MHC II were transferred to the limiting membrane of the MVB, bringing MHC II and DM to the same membrane domain. Concomitantly, the MVBs transformed into long tubular organelles that extended into the periphery of the cells. Vesicles that were formed at the tips of these tubules nonselectively incorporated MHC II and DM and presumably mediated transport to the plasma membrane. We propose that in maturing DCs, the reorganization of MVBs is fundamental for the timing of MHC II antigen loading and transport to the plasma membrane.     

Presentation and binding affinity of equine infectious anemia virus CTL envelope and matrix protein epitopes by an expressed equine classical MHC class I molecule

Control of a naturally occurring lentivirus, equine infectious anemia virus (EIAV), occurs in most infected horses and involves MHC class I-restricted, virus-specific CTL. Two minimal 12-aa epitopes, Env-RW12 and Gag-GW12, were evaluated for presentation by target cells from horses with an equine lymphocyte Ag-A1 (ELA-A1) haplotype. Fifteen of 15 presented Env-RW12 to CTL, whereas 11 of 15 presented Gag-GW12. To determine whether these epitopes were presented by different molecules, MHC class I genes were identified in cDNA clones from Arabian horse A2152, which presented both epitopes. This horse was selected because it is heterozygous for the SCID trait and is used to breed heterozygous females. Offspring with SCID are used as recipients for CTL adoptive transfer, and normal offspring are used for CTL induction. Four classical and three putative nonclassical full-length MHC class I genes were found. Human 721.221 cells transduced with retroviral vectors expressing each gene had equine MHC class I on their surface. Following peptide pulsing, only cells expressing classical MHC class I molecule 7-6 presented Env-RW12 and Gag-GW12 to CTL. Unlabeled peptide inhibition of (125)I-labeled Env-RW12 binding to 7-6-transduced cells demonstrated that Env-RW12 affinity was 15-fold higher than Gag-GW12 affinity. Inhibition with truncated Env-RW12 demonstrated that amino acid positions 1 and 12 were necessary for binding, and single substitutions identified positions 2 and 3 as possible primary anchor residues. Since MHC class I 7-6 presented both epitopes, outbred horses with this allele can be immunized with these epitopes to optimize CTL responses and evaluate their effectiveness against lentiviral challenge.     

MHC class I antigen processing of an adenovirus CTL epitope is linked to the levels of immunoproteasomes in infected cells

Proteasomes are the major source for the generation of peptides bound by MHC class I molecules. To study the functional relevance of the IFN-gamma-inducible proteasome subunits low molecular mass protein 2 (LMP2), LMP7, and mouse embryonal cell (MEC) ligand 1 in Ag processing and concomitantly that of immunoproteasomes, we established the tetracycline-regulated mouse cell line MEC217, allowing the titrable formation of immunoproteasomes. Infection of MEC217 cells with Adenovirus type 5 (Ad5) and analysis of Ag presentation with Ad5-specific CTL showed that cells containing immunoproteasomes processed the viral early 1B protein (E1B)-derived epitope E1B192-200 with increased efficiency, thus allowing a faster detection of viral entry in induced cells. Importantly, optimal CTL activation was already achieved at submaximal immunosubunit expression. In contrast, digestion of E1B-polypeptide with purified proteasomes in vitro yielded E1B192-200 at quantities that were proportional to the relative contents of immunosubunits. Our data provide evidence that the IFN-gamma-inducible proteasome subunits, when present at relatively low levels as at initial stages of infection, already increase the efficiency of antigenic peptide generation and thereby enhance MHC class I Ag processing in infected cells.     

Induction of MHC class I presentation of exogenous antigen by dendritic cells is controlled by CD4+ T cells engaging class II molecules in cholesterol-rich domains.

We investigated interactions between CD4+ T cells and dendritic cells (DC) necessary for presentation of exogenous Ag by DC to CD8+ T cells. CD4+ T cells responding to their cognate Ag presented by MHC class II molecules of DC were necessary for induction of CD8+ T cell responses to MHC class I-associated Ag, but their ability to do so depended on the manner in which class II-peptide complexes were formed. DC derived from short-term mouse bone marrow culture efficiently took up Ag encapsulated in IgG FcR-targeted liposomes and stimulated CD4+ T cell responses to Ag-derived peptides associated with class II molecules. This CD4+ T cell-DC interaction resulted in expression by the DC of complexes of class I molecules and peptides from the Ag delivered in liposomes and permitted expression of the activation marker CD69 and cytotoxic responses by naive CD8+ T cells. However, while free peptides in solution loaded onto DC class II molecules could stimulate IL-2 production by CD4+ T cells as efficiently as peptides derived from endocytosed Ag, they could not stimulate induction of cytotoxic responses by CD8+ T cells to Ag delivered in liposomes into the same DC. Signals requiring class II molecules loaded with endocytosed Ag, but not free peptide, were inhibited by methyl-beta-cyclodextrin, which depletes cell membrane cholesterol. CD4+ T cell signals thus require class II molecules in cholesterol-rich domains of DC for induction of CD8+ T cell responses to exogenous Ag by inducing DC to process this Ag for class I presentation.     

Presentation of the self antigen myelin basic protein by dendritic cells leads to experimental autoimmune encephalomyelitis.

Bone marrow (BM)-derived dendritic cells (DC) are potent stimulators of naive CD4+ T cell activation. Because DC are efficient at Ag processing and could potentially present self Ags, we investigated the role of DC in the presentation of an encephalitogenic peptide from myelin basic protein (Ac1-11) in the induction of experimental autoimmune encephalomyelitis (EAE). To determine if DC could prime for EAE, we transferred DC pulsed with Ac1-11 or with medium alone into irradiated mice in combination with CD4+ T cells isolated from a mouse transgenic for a TCR specific for Ac1-11 + I-Au. Mice transferred with Ac1-11-pulsed DC developed EAE 7-10 days later, whereas mice receiving medium-pulsed DC did not. By day 15, all mice given peptide-loaded DC had signs of tail and hind limb paralysis, and by day 20 infiltration of Ac1-11-specific CD4+ T cells was detected in the brain parenchyma. We also demonstrated interactions between Ac1-11-pulsed DC and Ac1-11-specific T cells in the lymph nodes 24 h following adoptive transfer of both cell populations. These data show that DC can efficiently present the self Ag myelin basic protein Ac1-11 to Ag-specific T cells in the periphery of mice to induce EAE.     

Regulation of MHC class I transport in human dendritic cells and the dendritic-like cell line KG-1

Dendritic cells (DCs) progress through distinct maturational phases; immature DCs capture Ag while mature DCs are optimized for Ag presentation. Proper control of immunity requires regulated compartmentalization of MHC class II molecules. We report that DCs also regulate MHC class I trafficking throughout maturation. Although mature human DCs express high levels of surface MHC class I, immature DCs exhibit lower surface levels while retaining MHC class I-peptide complexes in the Golgi. A cell line, KG-1, behaves similarly. We confirm the similarity of KG-1 to DCs by demonstrating its capacity to present exogenous Ags in an MHC class I-restricted fashion to CD8(+) T cell hybridomas, a phenomenon called cross-presentation. Biochemical characterization of MHC class I trafficking throughout maturation showed that, in early KG-1 dendritic-like cells, surface arrival of MHC class I-peptide complexes is delayed by their retention in the Golgi. In mature dendritic-like cells, these complexes relocate to the surface and their stability increases, concomitant with up-regulation of costimulatory molecules. Maturation induces qualitative changes in the MHC class I-associated peptide repertoire demonstrated by increased thermostability. The differential processing of MHC class I throughout maturation may prevent premature immune activation while promoting T cell responses in lymph nodes to Ags acquired at sites of inflammation.     

Retrovirally transduced human dendritic cells can generate T cells recognizing multiple MHC class I and class II epitopes from the melanoma antigen glycoprotein 100

Involvement of tumor-Ag specific CD4(+) and CD8(+) T cells could be critical in the generation of an effective immunotherapy for cancer. In an attempt to optimize the T cell response against defined tumor Ags, we previously developed a method allowing transgene expression in human dendritic cells (DCs) using retroviral vectors. One advantage of using gene-modified DCs is the potential ability to generate CD8(+) T cells against multiple class I-restricted epitopes within the Ag, thereby eliciting a broad antitumor immune response. To test this, we generated tumor-reactive CD8(+) T cells with DCs transduced with the melanoma Ag gp100, for which a number of HLA-A2-restricted epitopes have been described. Using gp100-transduced DCs, we were indeed able to raise T cells recognizing three distinct HLA-A2 epitopes within the Ag, gp100(154-162), gp100(209-217), and gp100(280-288). We next tested the ability of transduced DCs to raise class II-restricted CD4(+) T cells. Interestingly, stimulation with gp100-transduced DCs resulted in the generation of CD4(+) T cells specific for a novel HLA-DRbeta1*0701-restricted epitope of gp100. The minimal determinant of this epitope was defined as gp100(174-190) (TGRAMLGTHTMEVTVYH). These observations suggest that retrovirally transduced DCs have the capacity to present multiple MHC class I- and class II-restricted peptides derived from a tumor Ag, thereby eliciting a robust immune response against that Ag.     

Characterization of the signaling function of MHC class II molecules during antigen presentation by B cells.

In addition to their role as peptide binding proteins, MHC class II proteins can also function as signal transducing molecules. Recent work using B cells expressing genetically engineered truncated MHC class II molecules has suggested that signaling through the cytoplasmic domains of these proteins plays an important role in the generation of signals required for the activation of some T cell hybrids. Treatment of truncated Ia-expressing B cells with cAMP-elevating agents corrects the deficiency in Ag presentation by these cells. We report that the MHC class II-mediated signal appears to act by a mechanism that increases the efficiency of Ag presentation by B cells thereby lowering the amount of specific Ag required for T cell activation. We further show that the induction of the cAMP-induced signal in B cells is inhibited by cycloheximide and cytochalasin A, implicating protein synthesis as well as cytoskeletal rearrangements in Ag presentation to accessory signal- dependent hybrids. In contrast, these agents do not block Ag presentation to a T cell hybrid previously shown not to require the cAMP-induced signal for activation. The signal-dependent T hybrid is additionally dependent on LFA-1-ICAM-1 interaction for activation, whereas the signal-independent hybrid is not. These observations suggest the existence of two types of T cell hybrid with respect to their requirements for activation: those that require only the recognition of MHC class II-peptide complexes without accessory signals, as shown by their ability to respond to purified Ia on planar membranes, and those that, in addition to recognition of MHC II/Ag, require LFA-1-ICAM-1 interaction and the delivery of additional signal(s) induced in the B cell via signal transduction through MHC class II molecules.     

Augmentation of MHC class I antigen presentation via heat shock protein expression by hyperthermia

Heat shock proteins are recognized as significant participants in immune reactions. In this study, we have demonstrated that the cell surface presentation of MHC class I antigen was increased in tandem with increased heat shock protein 70 (HSP70) expression and the immunogenicity of rat T-9 glioma cells was enhanced by hyperthermia. T-9 cells showed growth inhibition for 24 h after the heat treatment at 43 degrees C for 1 h in vitro, but then resumed a normal growth rate. HSP70 expression reached a maximum at 24 h after heating. Flow cytometric analysis revealed a significant increase in MHC class I antigen on the surface of the heated cells. The augmentation of MHC class I surface expression started 24 h after heating and reached a maximum 48 h after heating. The expression of other immunologic mediators, such as intracellular adhesion molecule-1 (ICAM-1) and MHC class II antigens, did not increase. In an in vivo experiment using immunocompetent syngeneic rats (F344), growth of the heated T-9 cells, with augmentation of MHC class I antigen surface expression, was significantly inhibited, while the cells grew progressively in nude rats (F344/N Jcl-rnu). Furthermore, compared with lymphocytes from non-immunized (PBS only injection) rats or rats injected with non-heated T-9 cells, the splenic lymphocytes of the rats in which the heated T-9 cells were injected displayed specific cytotoxicity against T-9 cells. These results suggest that HSP70 is an important modulator of tumor cell immunogenicity, and that hyperthermic treatment of tumor cells can induce the host antitumor immunity via the expression of HSP70. These results may benefit further efforts on developing novel cancer immunotherapies based on hyperthermia.     

CD1 molecules efficiently present antigen in immature dendritic cells and traffic independently of MHC class II during dendritic cell maturation

Upon exposure to Ag and inflammatory stimuli, dendritic cells (DCs) undergo a series of dynamic cellular events, referred to as DC maturation, that involve facilitated peptide Ag loading onto MHC class II molecules and their subsequent transport to the cell surface. Besides MHC molecules, human DCs prominently express molecules of the CD1 family (CD1a, -b, -c, and -d) and mediate CD1-dependent presentation of lipid and glycolipid Ags to T cells, but the impact of DC maturation upon CD1 trafficking and Ag presentation is unknown. Using monocyte-derived immature DCs and those stimulated with TNF-alpha for maturation, we observed that none of the CD1 isoforms underwent changes in intracellular trafficking that mimicked MHC class II molecules during DC maturation. In contrast to the striking increase in surface expression of MHC class II on mature DCs, the surface expression of CD1 molecules was either increased only slightly (for CD1b and CD1c) or decreased (for CD1a). In addition, unlike MHC class II, DC maturation-associated transport from lysosomes to the plasma membrane was not readily detected for CD1b despite the fact that both molecules were prominently expressed in the same MIIC lysosomal compartments before maturation. Consistent with this, DCs efficiently presented CD1b-restricted lipid Ags to specific T cells similarly in immature and mature DCs. Thus, DC maturation-independent pathways for lipid Ag presentation by CD1 may play a crucial role in host defense, even before DCs are able to induce maximum activation of peptide Ag-specific T cells.     

Recombinant tumor-associated MUC1 glycoprotein impairs the differentiation and function of dendritic cells

Tumors exploit several strategies to evade immune recognition, including the production of a large number of immunosuppressive factors, which leads to reduced numbers and impaired functions of dendritic cells (DCs) in the vicinity of tumors. We have investigated whether a mucin released by tumor cells could be involved in causing these immunomodulating effects on DCs. We used a recombinant purified form of the MUC1 glycoprotein, an epithelial associated mucin that is overexpressed, aberrantly glycosylated, and shed during cancer transformation. The O-glycosylation profile of the recombinant MUC1 glycoprotein (ST-MUC1) resembled that expressed by epithelial tumors in vivo, consisting of large numbers of sialylated core 1 (sialyl-T, ST) oligosaccharides. When cultured in the presence of ST-MUC1, human monocyte-derived DCs displayed a modified phenotype with decreased expression of costimulatory molecules (CD86, CD40), Ag-presenting molecules (DR and CD1d), and differentiation markers (CD83). In contrast, markers associated with an immature phenotype, CD1a and CD206 (mannose receptor), were increased. This effect was already evident at day 4 of DC culture and was dose dependent. The modified phenotype of DCs corresponded to an altered balance in IL-12/IL-10 cytokine production, with DC expressing an IL-10(high)IL-12(low) phenotype after exposure to ST-MUC1. These DCs were defective in their ability to induce immune responses in both allogeneic and autologous settings, as detected in proliferation and ELISPOT assays. The altered DC differentiation and Ag presentation function induced by the soluble sialylated tumor-associated mucin may represent a mechanism by which epithelial tumors can escape immunosurveillance.     

The mechanism of unresponsiveness to circulating tumor antigen MUC1 is a block in intracellular sorting and processing by dendritic cells

Immunity to tumor Ags in patients is typically weak and not therapeutic. We have identified a new mechanism by which potentially immunogenic glycoprotein tumor Ags, such as MUC1, fail to stimulate strong immune responses. MUC1 is a heavily glycosylated membrane protein that is also present in soluble form in sera and ascites of cancer patients. We show that this soluble protein is readily taken up by dendritic cells (DC), but is not transported to late endosomes or MHC class II compartments for processing and binding to class II MHC. MUC1 uptake is mediated by the mannose receptor, and the protein is then retained long term in early endosomes without degradation. Long-term retention of MUC1 does not interfere with the ability of DC to process and present other Ags. We also demonstrate inhibited processing of another important glycoprotein tumor Ag, HER-2/neu. This may, therefore, be a frequent obstacle to presentation of tumor Ags and an important consideration in the design of cancer vaccines. It should be possible to overcome this obstacle by providing DC with a form of tumor Ag that can be better processed. For MUC1 we show that a 140-aa-long synthetic peptide is very efficiently processed by DC.     

NMR study on the interaction between MHC class I protein and its antigen peptide

A major histcompatibility complex (MHC) class I protein H-2K(b) was expressed in a large scale as a fusion protein with thioredoxin and hexahistidine at the N-terminus to analyze the interaction with the antigen peptide SIYRYYGL. NMR spectra of the peptide in the mixture solution with the protein showed very broad signals, indicating the obviously clear existence of the dynamic interaction between the class I protein and the antigen peptide. The interaction of the protein and peptide was discussed as well as the surrounding atmosphere of the peptide in the complex.     

Alloreactive and syngeneic CTL are comparably dependent on interaction with MHC class I alpha-helical residues.

The molecular basis for the difference in the strength of T cell responses to self vs alloantigens is unknown, but may reflect how T cells are selected in the thymus. Because T cells with a high affinity for foreign as opposed to self MHC molecules are able to mature, it has been proposed that alloreactive T cells may be more strongly dependent upon interaction with MHC residues than are self-restricted T cells. This study was undertaken to rigorously address this hypothesis. Whereas other studies have compared self vs alloantigen recognition of different MHC alleles by a single T cell clone, we have compared self vs alloantigen recognition of a single MHC allele, H-2Ld, by a large panel of self-restricted and alloreactive T cell clones. Target cells expressing Ld molecules mutated at several different potential TCR contact residues were analyzed to determine which residues are important for recognition by self-restricted vs alloreactive T cells. We unequivocally demonstrate that self-restricted and alloreactive T cells do not differ, but rather are comparably dependent on interaction with MHC residues. Importantly, both self-restricted and alloreactive T cells are dependent upon the same MHC residues as primary contacts and, in addition, share a common recognition pattern of Ld. Furthermore, our analysis enables us to provide a model for allotype-specific T cell recognition of Ld vs Kb class I molecules.