Quantitative analysis of the immune response to mouse non-MHC transplantation antigens in vivo: the H60 histocompatibility antigen dominates over all others

Minor histocompatibility Ags (minor H Ags) are substantial impediments to MHC-matched solid tissue and bone marrow transplantation. From an antigenic standpoint, transplantation between MHC-matched individuals has the potential to be remarkably complex. To determine the extent to which the immune response is simplified by the phenomenon of immunodominance, we used peptide/MHC tetramers based on recently discovered minor H Ags (H60, H13, and HY) and monitored in vivo CD8 T cell responses of female C57BL/6 mice primed with MHC-matched, but background-disparate, male BALB.B cells. CD8 T cells against H60 overwhelmed responses to the H13 and HY throughout primary and secondary challenge. H60 immunodominance was an inherent quality, overcoming a lower memory precursor frequency compared with that of H13 and evoking a T cell response with diverse TCRV beta usage. IFN-gamma staining examining congenically defined minor H Ags extended H60 dominance over additional minor H Ags, H28, H4, and H7. These four minor H Ags accounted for up to 85% of the CD8 T cell response, but H60 stood out as the major contributor. These findings show that immunodominance applies to antigenically complex transplantation settings in vivo and that the responses to the H60 minor H Ag dominates in this model. We suggest that immunodominant minor H Ags are those that result from the absence of a self analog.     

Duration of alloantigen presentation and avidity of T cell antigen recognition correlate with immunodominance of CTL response to minor histocompatibility antigens

CD8 T lymphocytes (CTL) responsive to immunodominant minor histocompatibility (minor H) Ags are thought to play a disproportionate role in allograft rejection in MHC-identical solid and bone marrow transplant settings. Although many studies have addressed the mechanisms underlying immunodominance in models of infectious diseases, cancer immunotherapy, and allograft immunity, key issues regarding the molecular basis of immunodominance remain poorly understood. In this study, we exploit the minor H Ag system to understand the relationship of the various biochemical parameters of Ag presentation and recognition to immunodominance. We show that the duration of individual minor H Ag presentation and the avidity of T cell Ag recognition influence the magnitude and, hence, the immunodominance of the CTL response to minor H Ags. These properties of CTL Ag presentation and recognition that contribute to immunodominance have implications not only for tissue transplantation, but also for autoimmunity and tumor vaccine design.     

Minor antigen h60-mediated aplastic anemia is ameliorated by immunosuppression and the infusion of regulatory T cells

Human bone marrow (BM) failure mediated by the immune system can be modeled in mice. In the present study, infusion of lymph node (LN) cells from C57BL/6 mice into C.B10-H2(b)/LilMcd (C.B10) recipients that are mismatched at multiple minor histocompatibility Ags, including the immunodominant Ag H60, produced fatal aplastic anemia. Declining blood counts correlated with marked expansion and activation of CD8 T cells specific for the immunodominant minor histocompatibility Ag H60. Infusion of LN cells from H60-matched donors did not produce BM failure in C.B10 mice, whereas isolated H60-specific CTL were cytotoxic for normal C.B10 BM cells in vitro. Treatment with the immunosuppressive drug cyclosporine abolished H60-specific T cell expansion and rescued animals from fatal pancytopenia. The development of BM failure was associated with a significant increase in activated CD4+CD25+ T cells that did not express intracellular FoxP3, whereas inclusion of normal CD4+CD25+ regulatory T cells in combination with C57BL/6 LN cells aborted H60-specific T cell expansion and prevented BM destruction. Thus, a single minor histocompatibility Ag H60 mismatch can trigger an immune response leading to massive BM destruction. Immunosuppressive drug treatment or enhancement of regulatory T cell function abrogated this pathophysiology and protected animals from the development of BM failure.     

Primary vascularization of the graft determines the immunodominance of murine minor H antigens during organ transplantation

Grafts can be rejected even when matched for MHC because of differences in the minor histocompatibility Ags (mH-Ags). H4- and H60-derived epitopes are known as immunodominant mH-Ags in H2(b)-compatible BALB.B to C57BL/6 transplantation settings. Although multiple explanations have been provided to explain immunodominance of Ags, the role of vascularization of the graft is yet to be determined. In this study, we used heart (vascularized) and skin (nonvascularized) transplantations to determine the role of primary vascularization of the graft. A higher IFN-γ response toward H60 peptide occurs in heart recipients. In contrast, a higher IFN-γ response was generated against H4 peptide in skin transplant recipients. Peptide-loaded tetramer staining revealed a distinct antigenic hierarchy between heart and skin transplantation: H60-specific CD8(+) T cells were the most abundant after heart transplantation, whereas H4-specific CD8(+) T cells were more abundant after skin graft. Neither the tissue-specific distribution of mH-Ags nor the draining lymph node-derived dendritic cells correlated with the observed immunodominance. Interestingly, non-primarily vascularized cardiac allografts mimicked skin grafts in the observed immunodominance, and H60 immunodominance was observed in primarily vascularized skin grafts. However, T cell depletion from the BALB.B donor prior to cardiac allograft induces H4 immunodominance in vascularized cardiac allograft. Collectively, our data suggest that immediate transmigration of donor T cells via primary vascularization is responsible for the immunodominance of H60 mH-Ag in organ and tissue transplantation.     

Adoptive transfer of minor histocompatibility antigen-specific T lymphocytes eradicates leukemia cells without causing graft-versus-host disease.

Adoptive transfer of T cells reactive to minor histocompatibility antigens has the unmatched ability to eradicate malignant hematopoietic cells. Unfortunately, its use is hampered by the associated graft-versus-host disease. The critical issue of a possible dissociation of the antileukemic effect and graft-versus-host disease by targeting specific minor histocompatibility antigens remains unresolved because of the unknown nature and number of minor histocompatibility antigens necessary or sufficient to elicit anti-leukemic activity and graft-versus-host disease. We found that injection of T lymphocytes primed against a single major histocompatibility complex class I-restricted immunodominant minor histocompatibility antigen (B6dom1) caused no graft-versus-host disease but produced a curative anti-leukemic response. Avoidance of graft-versus-host disease required that no other host-reactive T cells be co-injected with T cells primed with B6dom1. Here we show that effective and non-toxic immunotherapy of hematologic malignancies can be achieved by targeting a single immunodominant minor histocompatibility antigen.     

Increased positive selection of B1 cells and reduced B cell tolerance to intracellular antigens in c1q-deficient mice

Inherited deficiency of early components of the classical complement pathway is strongly associated with the targeting of intracellular self Ags in systemic lupus erythematosus, but the reasons for this association are debated. In this study, we show that C1q deficiency increases the positive selection of B1b B cells and IgM autoantibodies by an intracellular self Ag, which is exposed on dying cells, and decreases the negative selection of autoreactive conventional B cells by the same Ag. These effects are specific to intracellular Ag because C1q deficiency does not affect negative selection by extracellular self Ag or increase the positive selection of naive B cells. The B1-derived IgM autoantibody binds to the intracellular Ag when it is expressed on dying cells, leading to fixation of C1q and clearance of cells by phagocytosis. These findings suggest that the positive selection of autoreactive B1 cells by self Ags may contribute to the IgM and C1q-dependent clearance of dying cells in a feedback loop that limits exposure of conventional B cells to immunogenic self Ags. We show that exposure of intracellular Ag leads to the activation of conventional B cells, when there is a source of T cell help in vivo.     

The model B6(dom1) minor histocompatibility antigen is encoded by a mouse homolog of the yeast STT3 gene

The B6(dom1) minor histocompatibility antigen (MiHA) is a model antigen, since it is both the epitome of an immunodominant epitope and an ideal target for adoptive cancer immunotherapy. Based on DNA sequencing and MS/MS analyses, we report that B6(dom1) corresponds to amino acids 770-778 (KAPDNRETL) of a protein we propose to call SIMP (source of immunodominant MHC-associated peptides) that is encoded by a mouse homolog of the yeast STT3gene. STT3, a member of the oligosaccharyltransferase complex, is essential for cell proliferation. Phenotypic and genotypic analyses among eight strains of mice revealed a precise correlation between susceptibility or resistance to B6(dom1)-specific cytotoxic T lymphocytes (CTLs) and the presence of a Glu vs Asp amino acid at position 776 of the SIMP protein, respectively. Strikingly, while the difference in the amino acid sequence 770-778 encoded by the two SIMP alleles represents a very conservative substitution, these allelic peptides were not crossreactive at the CTL level, and both peptides were immunodominant when presented to mice homozygous for the opposite allele. In addition, we have cloned a human ortholog of SIMP whose predicted protein shares 97% amino acid identity with mouse SIMP. These results strengthen the concept that MHC class-I-associated MiHAs originate as a consequence of rare polymorphisms among highly conserved genes. Furthermore, the notion that a peptide differing from a self analog by a single methylene group can be immunodominant has implications regarding our understanding of the mechanisms of immunodominance.     

From affinity selection to kinetic selection in Germinal Centre modelling

Affinity maturation is an evolutionary process by which the affinity of antibodies (Abs) against specific antigens (Ags) increases through rounds of B-cell proliferation, somatic hypermutation, and positive selection in germinal centres (GC). The positive selection of B cells depends on affinity, but the underlying mechanisms of affinity discrimination and affinity-based selection are not well understood. It has been suggested that selection in GC depends on both rapid binding of B-cell receptors (BcRs) to Ags which is kinetically favourable and tight binding of BcRs to Ags, which is thermodynamically favourable; however, it has not been shown whether a selection bias for kinetic properties is present in the GC. To investigate the GC selection bias towards rapid and tight binding, we developed an agent-based model of GC and compared the evolution of founder B cells with initially identical low affinities but with different association/dissociation rates for Ag presented by follicular dendritic cells in three Ag collection mechanisms. We compared an Ag collection mechanism based on association/dissociation rates of B-cell interaction with presented Ag, which includes a probabilistic rupture of bonds between the B-cell and Ag (Scenario-1) with a reference scenario based on an affinity-based Ag collection mechanism (Scenario-0). Simulations showed that the mechanism of Ag collection affects the GC dynamics and the GC outputs concerning fast/slow (un)binding of B cells to FDC-presented Ags. In particular, clones with lower dissociation rates outcompete clones with higher association rates in Scenario-1, while remaining B cells from clones with higher association rates reach higher affinities. Accordingly, plasma cell and memory B cell populations were biased towards B-cell clones with lower dissociation rates. Without such probabilistic ruptures during the Ag extraction process (Scenario-2), the selective advantage for clones with very low dissociation rates diminished, and the affinity maturation level of all clones decreased to the reference level.     

Cutting edge: the minor histocompatibility antigen H60 peptide interacts with both H-2Kb and NKG2D

Minor histocompatibility Ags elicit cell-mediated immune responses and graft rejection in individuals receiving MHC-matched tissues. H60 represents a dominant Ag that elicits a strong CTL response in C57BL/6 mice immunized against BALB.B. An 8-aa peptide in the H60 protein is presented by H-2K(b) and this is recognized by the TCR as an alloantigen. The intact H60 glycoprotein is a ligand for the costimulatory NKG2D receptor that is expressed by activated CD8(+) T cells. Thus, H60 may provide both an allogeneic peptide and its own costimulation. We show that mutation of an H-2K(b)-binding anchor residue in the H60 peptide completely abrogates binding of H60 glycoprotein to NKG2D and a synthetic H60 peptide partially blocks the binding of NKG2D to its ligand. Ligands of the human NKG2D receptor are remarkably polymorphic, suggesting that these may also serve as minor histocompatibility Ags.     

The in vivo fate of APCs displaying minor H antigen and/or MHC differences is regulated by CTLs specific for immunodominant class I-associated epitopes

The goal of this work was to evaluate the fate of APCs following interactions with T cells in unprimed mice with a normal T cell repertoire. We elaborated a model in which male adherent peritoneal mononuclear cells were injected into the foreleg footpads of naive female recipients mismatched for either minor or major histocompatibility Ags. At various times after injection, APC numbers in the draining (axillary and brachial) lymph nodes were assessed using a Ube1y gene-specific PCR assay. Our experimental model was designed so that the number of APCs expressing the priming epitope was similar to what is observed under real life conditions. Thus, early after injection, the frequency of afferent lymph-derived APCs expressing the priming epitope was in the range of 101-102/106 lymph node cells. We found that APCs presenting some, but not all, nonself epitopes were killed rapidly after entrance into the lymph nodes. Rapid elimination of APCs occurred following interactions with MHC class I-restricted, but not class II-restricted, T cells and was observed when APCs presented an immunodominant (B6dom1/H7a), but not a nondominant (HY), epitope. Killing of APCs was mediated partly, but not exclusively, by perforin-dependent process. We propose that killing of APCs by CTLs specific for immunodominant MHC class I-restricted epitopes may be instrumental in regulating the intensity, duration, and diversity of T cell responses.     

The role of the CD19/CD21 complex in B cell processing and presentation of complement-tagged antigens.

The CD19/CD21 complex is an essential B cell coreceptor that functions synergistically to enhance signaling through the B cell Ag receptor in response to T cell-dependent, complement-tagged Ags. In this study, we use a recombinant protein containing three tandemly arranged copies of C3d and the Ag hen egg lysozyme, shown to be a highly effective immunogen in vivo, to evaluate the role of the CD19/CD21 complex in Ag processing in B cells. Evidence is provided that coengagement of the CD19/CD21 complex results in more rapid and efficient production of antigenic peptide/class II complexes as compared with B cell Ag receptor-mediated processing alone. The CD19/CD21 complex does not itself target complement-tagged Ags for processing, but rather appears to influence B cell Ag processing through its signaling function. The ability of the CD19/CD21 complex to augment processing may be an important element of the mechanism by which the CD19/CD21 complex functions to promote B cell responses to T cell-dependent complement-tagged Ags in vivo.     

Identification of BING-4 cancer antigen translated from an alternative open reading frame of a gene in the extended MHC class II region using lymphocytes from a patient with a durable complete regression following immunotherapy

Multiple human cancer Ags have been identified, although little is known concerning which would be most effectively used in cancer immunotherapy. To gain insight into the selection of appropriate Ags, the immunologic reactivity of a patient who had a durable complete regression of melanoma metastases was measured. PBMCs were directly cloned using the monoclonal anti-CD3 Ab OKT3 and IL-2 without any bias introduced by previous culture. A lymphocyte clone recognized a previously unknown shared melanoma Ag that was identified as the BING-4 protein encoded in a gene-rich region of the extended class II MHC. The HLA-A2-restricted BING-4 immunodominant peptide was translated from a 10-aa-long alternative open reading frame. In vitro sensitization against this peptide generated lymphocytes reactive against HLA-A2(+) melanomas. Real-time semiquantitative RT-PCR analysis revealed that 8 of 15 melanoma cell lines overexpressed BING-4, and this correlated with recognition by lymphocytes. Overexpression was not found in normal tissues or other tumor types. Thus, BING-4 represents another candidate Ag for possible use in the immunotherapy of patients with melanoma.     

Vaccination with a Melan-A peptide selects an oligoclonal T cell population with increased functional avidity and tumor reactivity

Both the underlying molecular mechanisms and the kinetics of TCR repertoire selection following vaccination against tumor Ags in humans have remained largely unexplored. To gain insight into these questions, we performed a functional and structural longitudinal analysis of the TCR of circulating CD8(+) T cells specific for the HLA-A2-restricted immunodominant epitope from the melanocyte differentiation Ag Melan-A in a melanoma patient who developed a vigorous and sustained Ag-specific T cell response following vaccination with the corresponding synthetic peptide. We observed an increase in functional avidity of Ag recognition and in tumor reactivity in the postimmune Melan-A-specific populations as compared with the preimmune blood sample. Improved Ag recognition correlated with an increase in the t(1/2) of peptide/MHC interaction with the TCR as assessed by kinetic analysis of A2/Melan-A peptide multimer staining decay. Ex vivo analysis of the clonal composition of Melan-A-specific CD8(+) T cells at different time points during vaccination revealed that the response was the result of asynchronous expansion of several distinct T cell clones. Some of these T cell clones were also identified at a metastatic tumor site. Collectively, these data show that tumor peptide-driven immune stimulation leads to the selection of high-avidity T cell clones of increased tumor reactivity that independently evolve within oligoclonal populations.     

Elucidation and role of critical residues of immunodominant peptide associated with T cell-mediated parasitic disease.

Granulomatous inflammation in schistosomiasis is strictly dependent on CD4+ Th lymphocytes sensitized to egg Ags, but its intensity is genetically regulated. C3H and CBA (H-2k) are strains of mice that develop large granulomas; they also strongly respond to the major egg Ag Sm-p40. We now show that the immunodominant epitope recognized by CD4+ Th cells from infected H-2k mice is confined to 13-mer peptide 234-246 (PKSDNQIKAVPAS), which elicits an I-Ak-restricted Th1-type response. Using a panel of alanine-monosubstituted peptides, we identified Asp237 as the main contact residue with I-Ak. On the other hand, three TCR contact residues were essential to stimulate epitope-specific T cell hybridomas: for two hybridomas these were Asn238, Gln239, and Lys241; and for one, Asn238, Lys241, and Pro244. In one instance, alanine substitution for Gln239 generated an antagonist that blocked subsequent stimulation with wild-type peptide. Most importantly, replacement of Asn238, Gln239, or Lys241 caused a profound loss of polyclonal CD4+ T cell reactivity from schistosome-infected mice. This study identifies the critical residues of immunodominant peptide 234-246 involved in the T cell response against the Sm-p40 egg Ag and suggests that suitable altered peptides may be capable of precipitating its down-regulation.     

MHC class II antigen processing in B cells: accelerated intracellular targeting of antigens.

Processing and presentation by Ag-specific B cells is initiated by Ag binding to the B cell Ag receptor (BCR). Cross-linking of the BCR by Ag results in a rapid targeting of the BCR and bound Ag to the MHC class II peptide loading compartment (IIPLC). This accelerated delivery of Ag may be essential in vivo during periods of rapid Ag-driven B cell expansion and T cell-dependent selection. Here, we use both immunoelectron microscopy and a nondisruptive protein chemical polymerization method to define the intracellular pathway of the targeting of Ags by the BCR. We show that following cross-linking, the BCR is rapidly transported through transferrin receptor-containing early endosomes to a LAMP-1+, beta-hexosaminadase+, multivesicular compartment that is an active site of peptide-class II complex assembly, containing both class II-invariant chain complexes in the process of invariant chain proteolytic removal as well as mature peptide-class II complexes. The BCR enters the class II-containing compartment as an intact mIg/Igalpha/Igbeta complex bound to Ag. The pathway by which the BCR targets Ag to the IIPLC appears not to be identical to that by which Ags taken up by fluid phase pinocytosis traffick, suggesting that the accelerated BCR pathway may be specialized and potentially independently regulated.     

Immunodominance in the graft-vs-host disease T cell response to minor histocompatibility antigens

Immunodominance controls the generation of CTL in the C57BL/6By (B6) anti-BALB.B H-2b-matched strain combination. Despite the potential of responding to numerous individual minor histocompatibility (H) Ag on BALB.B APC, the focus of the CTL response is largely specific for only a limited number of target Ag. These minor H Ag could be distinguished by their differential expression on a panel of target cells from the CXB recombinant inbred strains, the E, G, I, J, and K (all H-2b), which express different composites of the original BALB minor H Ag. A hierarchy was observed in which first-order immunodominant Ag were present on both CXBK and CXBG cells, whereas second-order dominant Ag were found on CXBE, CXBJ, and CXBI cells. To test whether immunodominance also plays a role in the development of lethal graft-vs-host disease (GVHD) directed to multiple minor H Ag, B6 T cells were transplanted along with T cell depleted bone marrow, to irradiated (825 rad) recipients of either the BALB.B or CXB recombinant inbred strains. The results indicate that a hierarchy of immunodominance does exist in GVHD, but it differs from that predicted from the in vitro CTL studies. GVHD was observed in BALB.B, CXBE, CXBI, and CXBJ recipients, but not in CXBG and CXBK recipients. Presensitization of B6 donor mice to CXBG or CXBK splenocytes 3 wk before transplant did not significantly increase the overall GVHD potential in the corresponding CXBG or CXBK recipients. Evidence for second-order immunodominance was provided by the transfer of CXBE T cells and ATBM to irradiated CXBG and BALB.B recipients with resultant, potent GVHD.     

Processing and presentation of a mycobacterial antigen 85B epitope by murine macrophages is dependent on the phagosomal acquisition of vacuolar proton ATPase and in situ activation of cathepsin D

Mycobacterium tuberculosis (strain H37Rv) and bacillus Calmette-Guérin (BCG) vaccine inhibit phagosome maturation in macrophages and their effect on processing, and presentation of a secreted Ag85 complex B protein, Ag85B, by mouse macrophages was analyzed. Macrophages were infected with GFP-expressing mycobacterial strains and analyzed for in situ localization of vacuolar proton ATPase (v-ATPase) and cathepsin D (Cat D) using Western blot analysis and immunofluorescence. H37Rv and BCG phagosomes excluded the v-ATPase and maintained neutral pH while the attenuated H37Ra strain acquired v-ATPase and acidified. Mycobacterial phagosomes acquired Cat D, although strains BCG and H37Rv phagosomes contained the inactive 46-kDa form, whereas H37Ra phagosomes had the active 30-kDa form. Infected macrophages were overlaid with a T cell hybridoma specific for an Ag85B epitope complexed with MHC class II. Coincident with active Cat D, H37Ra-infected macrophages presented the epitope to T cells inducing IL-2, whereas H37Rv- and BCG-infected macrophages were less efficient in IL-2 induction. Bafilomycin inhibited the induction of macrophage-induced IL-2 from T cells indicating that v-ATPase was essential for macrophage processing of Ag85B. Furthermore, the small interfering RNA interference of Cat D synthesis resulted in a marked decrease in the levels of macrophage-induced IL-2. Thus, a v-ATPase-dependent phagosomal activation of Cat D was required for the generation of an Ag85B epitope by macrophages. Reduced processing of Ag85B by H37Rv- and BCG-infected macrophages suggests that phagosome maturation arrest interferes with the efficient processing of Ags in macrophages. Because Ag85B is immunodominant, this state may lead to a decreased ability of the wild-type as well as the BCG vaccine to induce protective immunity.     

Striking immunodominance hierarchy of naturally occurring CD8+ and CD4+ T cell responses to tumor antigen NY-ESO-1

Immunodominance has been well-demonstrated in many antiviral and antibacterial systems, but much less so in the setting of immune responses against cancer. Tumor Ag-specific CD8+ T cells keep cancer cells in check via immunosurveillance and shape tumor development through immunoediting. Because most tumor Ags are self Ags, the breadth and depth of antitumor immune responses have not been well-appreciated. To design and develop antitumor vaccines, it is important to understand the immunodominance hierarchy and its underlying mechanisms, and to identify the most immunodominant tumor Ag-specific T cells. We have comprehensively analyzed spontaneous cellular immune responses of one individual and show that multiple tumor Ags are targeted by the patient's immune system, especially the "cancer-testis" tumor Ag NY-ESO-1. The pattern of anti-NY-ESO-1 T cell responses in this patient closely resembles the classical broad yet hierarchical antiviral immunity and was confirmed in a second subject.     

How H13 histocompatibility peptides differing by a single methyl group and lacking conventional MHC binding anchor motifs determine self-nonself discrimination.

The mouse H13 minor histocompatibility (H) Ag, originally detected as a barrier to allograft transplants, is remarkable in that rejection is a consequence of an extremely subtle interchange, P4(Val/Ile), in a nonamer H2-D(b)-bound peptide. Moreover, H13 peptides lack the canonical P5(Asn) central anchor residue normally considered important for forming a peptide/MHC complex. To understand how these noncanonical peptide pMHC complexes form physiologically active TCR ligands, crystal structures of allelic H13 pD(b) complexes and a P5(Asn) anchored pD(b) analog were solved to high resolution. The structures show that the basis of TCRs to distinguish self from nonself H13 peptides is their ability to distinguish a single solvent-exposed methyl group. In addition, the structures demonstrate that there is no need for H13 peptides to derive any stabilization from interactions within the central C pocket to generate fully functional pMHC complexes. These results provide a structural explanation for a classical non-MHC-encoded H Ag, and they call into question the requirement for contact between anchor residues and the major MHC binding pockets in vaccine design.     

The H4b minor histocompatibility antigen is caused by a combination of genetically determined and posttranslational modifications

Minor histocompatibility (H) Ag disparities result in graft-vs-host disease and chronic solid allograft rejection in MHC-identical donor-recipient combinations. Minor H Ags are self protein-derived peptides presented by MHC class I molecules. Most arise as a consequence of allelic variation in the bound peptide (p) that results in TCR recognizing the p/MHC as foreign. We used a combinational peptide screening approach to identify the immune dominant H2K(b)-restricted epitope defining the mouse H4(b) minor H Ag. H4(b) is a consequence of a P3 threonine to isoleucine change in the MHC-bound peptide derived from epithelial membrane protein-3. This allelic variation also leads to phosphorylation of the H4(b) but not the H4(a) epitope. Further, ex vivo CD8(+) T lymphocytes bind phosphorylated Ag tetramers with high efficiency. Although we document the above process in the minor H Ag system, posttranslational modifications made possible by subtle amino acid changes could also contribute to immunogenicity and immune dominance in tumor immunotherapeutic settings.