Identification of a novel UTY-encoded minor histocompatibility antigen.

Minor histocompatibility antigens (mHags) encoded by the Y-chromosome (H-Y-mHags) are known to play a pivotal role in allogeneic haematopoietic cell transplantation (HCT) involving female donors and male recipients. We present a new H-Y-mHag, YYNAFHWAI (UTY(139-147)), encoded by the UTY gene and presented by HLA-A*24:02. Briefly, short peptide stretches encompassing multiple putative H-Y-mHags were designed using a bioinformatics predictor of peptide-HLA binding, NetMHCpan. These peptides were used to screen for peptide-specific HLA-restricted T cell responses in peripheral blood mononuclear cells obtained post-HCT from male recipients of female donor grafts. In one of these recipients, a CD8+ T cell response was observed against a peptide stretch encoded by the UTY gene. Another bioinformatics tool, HLArestrictor, was used to identify the optimal peptide and HLA-restriction element. Using peptide/HLA tetramers, the specificity of the CD8+ T cell response was successfully validated as being HLA-A*24:02-restricted and directed against the male UTY(139-147) peptide. Functional analysis of these T cells demonstrated male UTY(139-147) peptide-specific cytokine secretion (IFNγ, TNFα and MIP-1ß) and cytotoxic degranulation (CD107a). In contrast, no responses were seen when the T cells were stimulated with patient tumour cells alone. CD8+ T cells specific for this new H-Y-mHag were found in three of five HLA-A*24:02-positive male recipients of female donor HCT grafts available for this study.

T cell responses affected by aminopeptidase N (CD13)-mediated trimming of major histocompatibility complex class II-bound peptides.

Endocytosed protein antigens are believed to be fragmented in what appears to be a balance between proteolysis and MHC-mediated epitope protection, and the resulting peptide-MHC complexes are transported to the surface of the antigen-presenting cells (APC) and presented to T cells. The events that lead to antigenic peptide generation and the compartments where antigen processing takes place remains somewhat enigmatic. The importance of intracellular antigen processing has been well established; however, it is unclear whether additional processing occurs at the APC surface. To follow antigen processing, we have identified a pair of T cell hybridomas that recognize a long vs. a short version of the same epitope. We have used prefixed APC and various protease inhibitors to demonstrate that the APC surface has a considerable potential for antigen processing. Specific antibodies further identified the exopeptidase Aminopeptidase N (APN, CD13) as one of the enzymes involved in the observed cell-surface antigen processing. The NH2-terminal end of the longer peptide could, even while bound to major histocompatibility complex (MHC) class II molecules, be digested by APN with dramatic consequences for T cell antigen recognition. This could be demonstrated both in cell-free systems using purified reagents and in cellular systems. Thus, MHC class II and APN may act in concert to generate the final T cell epitopes.

Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis.

Phage that display a surface peptide with the NGR sequence motif home selectively to tumor vasculature in vivo. A drug coupled to an NGR peptide has more potent antitumor effects than the free drug [W. Arap et al., Science (Washington DC), 279: 377-380, 1998]. We show here that the receptor for the NGR peptides in tumor vasculature is aminopeptidase N (APN; also called CD13). NGR phage specifically bound to immunocaptured APN and to cells engineered to express APN on their surface. Antibodies against APN inhibited in vivo tumor homing by the NGR phage. Immunohistochemical staining showed that APN expression is up-regulated in endothelial cells within mouse and human tumors. In another tissue that undergoes angiogenesis, corpus luteum, blood vessels also expressed APN, but APN was not detected in blood vessels of various other normal tissues stained under the same conditions. APN antagonists specifically inhibited angiogenesis in chorioallantoic membranes and in the retina and suppressed tumor growth. Thus, APN is involved in angiogenesis and can serve as a target for delivering drugs into tumors and for inhibiting angiogenesis.

Receptor-ligand interactions measured by an improved spun column chromatography technique. A high efficiency and high throughput size separation method.

Size exclusion chromatography may under the right circumstances be an easy and powerful way to measure in solution the interaction between a receptor an dits ligand. Spun column chromatography is a fast size exclusion technique of increasing popularity, however, little information exists on the method development essential to obtain efficient separation in particular when used for analytical purposes. In this paper we describe a systematic approach to select the optimal parameters for spun column separation including a simple modification of the technique whereby the spun columns are eluted by high-speed gradient centrifugation. This modification is easy to implement and it considerably improves spun column performance. We hypothesize that the high-speed centrifugation step leads to the release of additional buffer which assists in the complete elution of excluded molecules while the gradient centrifugation helps to achieve equilibrium across the gel matrix during the elution. The new method has been used successfully for several different receptor-ligand interactions, and this paper describes a general approach on how to develop new applications of the technique.

Longer peptide can be accommodated in the MHC class I binding site by a protrusion mechanism.

According to current consensus, CD8(+) T cell responses are focused upon short peptide sequences (8-11 amino acids) presented by MHC class I molecules. This size restriction is thought to operate mostly at the level of peptide-MHC class I interaction. Crystal structures have shown that the free N and C termini of a bound peptide interact through hydrogen bonding networks to conserved residues at either end of the class I binding site. Accordingly, it is thought that the termini are fixed and that only minor variations in peptide size are possible through a central bulging mechanism. We find that this consensus view is not always correct as some peptide-MHC class I interaction will accept significant extensions. Furthermore, our results indicate that in some cases protrusion, rather than bulging, may be the mechanism of extension. Depending upon the particular peptide-MHC combination in question, such extensions can occur at either the N or C terminus (but never both at the same time). Finally, we show that MHC and T cell in some cases can detect the identity of the extension, i.e. that extensions may be part of the specificity of the T cell immune response. We suggest that such extensions may play a physiological role.

Peptide binding specificity of major histocompatibility complex class I resolved into an array of apparently independent subspecificities: quantitation by peptide libraries and improved prediction of binding.

Considerable interest has focused on understanding how major histocompatibility complex (MHC) specificity is generated and characterizing the specificity of MHC molecules with the ultimate goal being to predict peptide binding. We have used a strategy where all possible peptides of a particular size are distributed into positional scanning combinatorial peptide libraries (PSCPL) to develop a highly efficient, universal and unbiased approach to address MHC specificity. The PSCPL approach appeared qualitatively and quantitatively superior to other currently used strategies. The average effect of any amino acid in each position was quantitated, allowing a detailed description of extended peptide binding motifs including primary and secondary anchor residues. It also identified disfavored residues which were found to be surprisingly important in shaping MHC class I specificity. Assuming that MHC class I specificity is the result of largely independently acting subsites, the binding of unknown peptides could be predicted. Conversely, this argues that MHC class I specificities consist of an array of subspecificities acting in a combinatorial mode.

A recombinant antibody with the antigen-specific, major histocompatibility complex-restricted specificity of T cells.

Specific recognition of peptide/major histocompatibility complex (MHC) molecule complexes by the T-cell receptor is a key reaction in the specific immune response. Antibodies against peptide/MHC complexes would therefore be valuable tools in studying MHC function and T-cell recognition and might lead to novel approaches in immunotherapy. However, it has proven difficult to generate antibodies with the specificity of T cells by conventional hybridoma techniques. Here we report that the phage display technology is a feasible alternative to generate antibodies recognizing specific, predetermined peptide/MHC complexes.

Modeling the interactions of a peptide-major histocompatibility class I ligand with its receptors. II. Cross-reaction between a monoclonal antibody and two alpha beta T cell receptors.

The recombinant antibody, pSAN13.4.1, has a unique T cell like specificity; it binds an Influenza Hemagglutinin octapeptide (Ha255-262) in an MHC (H-2Kk)-restricted manner, and a detailed comparison of the fine specificity of pSAN13.4.1 with the fine specificity of two Ha255-262-specific, H-2Kk-restricted T cell hybridomas has supported this contention. A three-dimensional model of pSAN13.4.1 has been derived by homology modeling techniques. Subsequently, the structure of the pSAN13.4.1 antibody in complex with the antigenic Ha-Kk ligand was derived after a flexible and automated docking of the MHC-peptide pair into the Fab combining site. Interestingly, the most energetically favored binding mode shows numerous analogies to the recently determined recognition of class I MHC-peptide complexes by alpha beta T cell receptors (TCRs). The pSAN13.4.1 also binds diagonally across the MHC binding groove but is more deeply anchored to the peptide-MHC (pep/MHC) ligand than TCRs, notably through numerous interactions of its heavy chain. The present model accounts well for the experimentally determined binding affinity of a set of 144 single amino acid substituted Ha analogues and the observed shared specificity between the pSAN antibody and two different T cell receptors for the Ha-Kk antigenic ligand. Analogies and differences between Fab and TCR recognition are explained by dissecting the binding role of each chain of the immune receptors as well as the contribution of all peptide amino acids.

Phage display of peptide / major histocompatibility class I complexes.

Major histocompatibility complex class I (MHC-I) molecules sample peptides from the intracellular environment and present them to cytotoxic T cells (CTL). To establish a selection system, and, thereby, enable a library approach to identify the specificities involved (that of the MHC-I for peptides and subsequently that ot the T cell receptor for peptide-MHC-I complex), we have fused a single chain peptide-MHC-I complex to the phage minor coat protein, gpIII, and displayed it on filamentous phage. Expression of peptide-MHC-I complexes was shown with relevant conformation-specific monoclonal antibodies and, more importantly, with a unique "T cell receptor-like" (i. e. peptide-specific, MHC-I-restricted) antibody. Thus, properly assembled and folded peptide-MHC-I complexes can be displayed on filamentous phage. Despite the successful display, interaction with T cells could not be demonstrated.

Measurement of peptide-MHC interactions in solution using the spin column filtration assay.

This unit describes how peptide-MHC complexes can be generated in vitro using affinity-purified MHC and synthetic peptide. The unit first describes how the interaction between peptide and MHC interaction can be measured in an accurate, quantitative biochemical assay. This procedure has been optimized for efficient separation of free peptide and MHC-bound peptide through a novel principle, termed "gradient centrifugation." The first two support protocols describe how to set up a biochemical fluid-phase binding reaction between peptide and MHC class I and class II, respectively. Also, an alternative procedure for setting up a biochemical fluid phase binding reaction between beta(2)m and MHC class I is included. Finally a more versatile inhibition assay is described. The assay is simple and robust, and has several advantages compared to the classical gel-filtration assay, including increased sensitivity and throughput. It also demands fewer resources both in terms of unique reagents and labor, and it generates less hazardous waste. Thus, the spin column gel-filtration assay is ideal for routine work.

Preformed purified peptide/major histocompatibility class I complexes are potent stimulators of class I-restricted T cell hybridomas.

A panel of antigen-specific, major histocompatibility complex class I-restricted T cell hybridomas has been generated to examine the capacity of peptide/class I complexes to stimulate T cells at the molecular level. Peptide/class I complexes were generated in detergent solution, purified and quantitated. Latex particles were subsequently coated with known amounts of preformed complexes and used to stimulate the T cell hybridomas. Stimulation was specific, i.e. only the appropriate peptide/class I combination were stimulatory, and quite sensitive, i.e. as little as 300 complexes per bead could be detected by the T cells. Preformed complexes were about 500,000 times more potent than free peptide in terms of T cell stimulation, demonstrating the physiological relevancy of the biochemically generated complexes. Surprisingly, the majority (including the most sensitive of the hybridomas) had lost CD8 expression, suggesting that antigen-specific stimulation of class I-restricted T cell hybridomas, as assessed by IL-2 release, does not depend on CD8.

Modeling the interactions of a peptide-major histocompatibility class I ligand with its receptors. I. Recognition by two alpha beta T cell receptors.

A three-dimensional model of the complex between an Influenza Hemagglutinin peptide, Ha255-262, and its restricting element, the mouse major histocompatibility complex (MHC) class I molecule, Kk, was built by homology modeling and subsequently refined by simulated annealing and restrained molecular dynamics. Next, three-dimensional models of two different T cell receptors (TCRs) both specific for the Ha255-262/Kk complex were generated based on previously published TCR X-ray structures. Finally, guided by the recently published X-ray structures of ternary TCR/peptide/MHC-I complexes, the TCR models were successfully docked into the Ha255-262/Kk model. We have previously used a systematic and exhaustive panel of 144 single amino acid substituted analogs to analyze both MHC binding and T cell recognition of the parental viral peptide. This large body of experimental data was used to evaluate the models. They were found to account well for the experimentally obtained data, lending considerable support to the proposed models and suggesting a universal docking mode for alpha beta TCRs to MHC-peptide complexes. Such models may also be useful in guiding future rational experimentation.

pH dependence of MHC class I-restricted peptide presentation.

The function of MHC class I molecules is to bind and present antigenic peptides to cytotoxic T cells. Here, we report that class I-restricted peptide presentation is strongly pH dependent. The presentation of some peptides was enhanced at acidic pH, whereas the presentation of others was inhibited. Biochemical peptide-MHC class I binding assays demonstrated that peptide-MHC class I complexes are more stable at neutral pH than at acidic pH. We suggest that acid-dependent peptide dissociation can generate empty class I molecules and that the resulting binding potential can be exploited by a subset of peptide-MHC class I combinations, in some cases leading to considerable peptide exchange. We further speculate that the relative instability of peptide-class I complexes under acidic conditions may affect the outcome of class I-restricted Ag presentation, as less stably associated peptides may dissociate from class I during passage of the acidic trans-Golgi network, and therefore may not be presented. Finally, our results may in part explain how endocytosed proteins can be presented by MHC class I molecules to cytotoxic T cells.

The interaction of beta 2-microglobulin (beta 2m) with mouse class I major histocompatibility antigens and its ability to support peptide binding. A comparison of human and mouse beta 2m.

The function of major histocompatibility complex (MHC) class I molecules is to sample peptides derived from intracellular proteins and to present these peptides to CD8+ cytotoxic T lymphocytes. In this paper, biochemical assays addressing MHC class I binding of both peptide and beta 2-microglobulin (beta 2m) have been used to examine the assembly of the trimolecular MHC class I/beta 2m/peptide complex. Recombinant human beta 2m and mouse beta 2ma have been generated to compare the binding of the two beta 2m to mouse class I. It is frequently assumed that human beta 2m binds to mouse class I heavy chain with a much higher affinity than mouse beta 2m itself. We find that human beta 2m only binds to mouse class I heavy chain with slightly (about 3-fold) higher affinity than mouse beta 2m. In addition, we compared the effect of the two beta 2m upon peptide binding to mouse class I. The ability of human beta 2m to support peptide binding correlated well with its ability to saturate mouse class I heavy chains. Surprisingly, mouse beta 2m only facilitated peptide binding when mouse beta 2m was used in excess (about 20-fold) of what was needed to saturate the class I heavy chains. The inefficiency of mouse beta 2m to support peptide binding could not be attributed to a reduced affinity of mouse beta 2m/MHC class I complexes for peptides or to a reduction in the fraction of mouse beta 2m/MHC class I molecules participating in peptide binding. We have previously shown that only a minor fraction of class I molecules are involved in peptide binding, whereas most of class I molecules are involved in beta 2m binding. We propose that mouse beta 2m interacts with the minor peptide binding (i.e. the "empty") fraction with a lower affinity than human beta 2m does, whereas mouse and human beta 2m interact with the major peptide-occupied fraction with almost similar affinities. This would explain why mouse beta 2m is less efficient than human beta 2m in generating the peptide binding moiety, and identifies the empty MHC class I heavy chain as the molecule that binds human beta 2m preferentially.

Shared fine specificity between T-cell receptors and an antibody recognizing a peptide/major histocompatibility class I complex.

Cytotoxic T cells recognize mosaic structures consisting of target peptides embedded within self-major histocompatibility complex (MHC) class I molecules. This structure has been described in great detail for several peptide-MHC complexes. In contrast, how T-cell receptors recognize peptide-MHC complexes have been less well characterized. We have used a complete set of singly substituted analogs of a mouse MHC class I, Kk-restricted peptide, influenza hemagglutinin (Ha)255-262, to address the binding specificity of this MHC molecule. Using the same peptide-MHC complexes we determined the fine specificity of two Ha255-262-specific, Kk-restricted T cells, and of a unique antibody, pSAN, specific for the same peptide-MHC complex. Independently, a model of the Ha255-262-Kk complex was generated through homology modeling and molecular mechanics refinement. The functional data and the model corroborated each other showing that peptide residues 1, 3, 4, 6, and 7 were exposed on the MHC surface and recognized by the T cells. Thus, the majority, and perhaps all, of the side chains of the non-primary anchor residues may be available for T-cell recognition, and contribute to the stringent specificity of T cells. A striking similarity between the specificity of the T cells and that of the pSAN antibody was found and most of the peptide residues, which could be recognized by the T cells, could also be recognized by the antibody.

Quantitative predictions of peptide binding to MHC class I molecules using specificity matrices and anchor-stratified calibrations.

Peptides are key immune targets. They are generated by fragmentation of antigenic proteins, selected by major histocompatibility complex (MHC) molecules and subsequently presented to T cells. One of the most selective requirements is that of peptide binding to MHC. Accurate descriptions and predictions of peptide-MHC interactions are therefore important. Quantitative matrices representing MHC class I specificity can be used to search any query protein for the presence of MHC binding peptides. Assuming that each peptide residue contributes to binding in an additive and sequence independent manner, such "crude" matrix-driven predictions can be expressed as a quantitative estimates of binding strength. Crude matrix-driven predictions are reasonably uniform (i.e. precise), however, there is a general tendency towards overestimating binding (i.e. being inaccurate). To evaluate and possibly improve predictions, we have measured the MHC class I binding of a large number of peptides. In an attempt to further improve predictions and to include sequence dependency, we subdivided the panel of peptides according to whether the peptides had zero, one or two primary anchor residues. This allowed us to define unique anchor-stratified calibrations, which led to predictions of improved precision and accuracy.