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HLA-A*02:01-negative tissues as determined by HLA-typing do not stain with BB7.2 or RL21A.

Supplementary Figure Legends for S1-S3.

HLA-DQ donor-specific antibodies (DSA) are the most prevalent type of DSA after renal transplantation and have been associated with eplet mismatches between donor and recipient HLA. Eplets are theoretically defined configurations of surface exposed amino acids on HLA molecules that require verification to confirm that they can be recognized by alloantibodies and are therefore clinically relevant. In this study, we isolated HLA-DQ specific memory B cells from immunized individuals by using biotinylated HLA-DQ monomers to generate 15 recombinant human HLA-DQ specific monoclonal antibodies (mAb) with six distinct specificities. Single antigen bead reactivity patterns were analyzed with HLA-EMMA to identify amino acids that were uniquely shared by the reactive HLA alleles to define functional epitopes which were mapped to known eplets. The HLA-DQB1*03:01-specific mAb LB_DQB0301_A and the HLA-DQB1*03-specific mAb LB_DQB0303_C supported the antibody-verification of eplets 45EV and 55PP respectively, while mAbs LB_DQB0402_A and LB_DQB0602_B verified eplet 55R on HLA-DQB1*04/05/06. For three mAbs, multiple uniquely shared amino acid configurations were identified, warranting further studies to define the inducing functional epitope and corresponding eplet. Our unique set of HLA-DQ specific mAbs will be further expanded and will facilitate the in-depth analysis of HLA-DQ epitopes, which is relevant for further studies of HLA-DQ alloantibody pathogenicity in transplantation.

Neoantigens are novel peptide sequences resulting from somatic mutations in tumors that upon loading onto major histocompatibility complex (MHC) molecules allow recognition by T cells. Accurate neoantigen identification is thus critical for designing cancer vaccines and predicting response to immunotherapies. Neoantigen identification and prioritization relies on correctly inferring whether the presenting peptide sequence can successfully induce an immune response. As the majority of somatic mutations are SNVs, changes between wildtype and mutant peptide are subtle and require cautious interpretation. An important, yet underappreciated, variable in neoantigen-prediction pipelines is the mutation position within the peptide relative to its anchor positions for the patient’s specific HLA alleles. While a subset of peptide positions are presented to the T-cell receptor for recognition, others are responsible for anchoring to the MHC, making these positional considerations critical for predicting T-cell responses. However, a systematic method for determining anchor locations for the wide range of HLA alleles present in the population and application of these to evaluate MT/WT peptide pairs arising in tumors has not been reported. As a result, many neoantigen studies have either failed to adequately consider this crucial factor or have used conventional assumptions to guide their neoantigen identification process. Here, we provide a computational workflow for predicting anchor locations for a wide range of HLA alleles, using a reference dataset generated from clinical and The Cancer Genome Atlas (TCGA) patient samples. We calculated high probability anchor positions for different peptide lengths for over 300 common HLA alleles. Analysis of these results showed clusters of different anchor trends among the HLA alleles analyzed. A subset of these HLA anchor results were orthogonally validated using protein crystallography structures. Analysis of 923 tumor samples showed that 7-41% of neoantigen candidates were potentially misclassified in the neoantigen selection process and can be rescued using allele-specific knowledge of anchor positions. These anchor predictions are currently undergoing experimental validation using both peptide-MHC stability assays as well as fluorescence-based competition binding assays. By incorporating our anchor prediction results into neoantigen prediction pipelines, such as pVACtools, we hope to formalize and streamline the identification process for relevant clinical studies. Citation Format: Huiming Xia, Joshua McMichael, Michelle Becker-Hapak, Onyinyechi C. Onyeador, Rico Buchli, Ethan McClain, Patrick Pence, Suangson Supabphol, Megan M. Richters, Anamika Basu, Cody A. Ramirez, Cristina Puig-Saus, Kelsy C. Cotto, Jasreet Hundal, Susanna Kiwala, S. Peter Goedegebuure, Tanner M. Johanns, Gavin P. Dunn, Antoni Ribas, Christopher A. Miller, William Gillanders, Todd A. Fehniger, Obi L. Griffith, Malachi Griffith. Computational prediction of MHC anchor locations guide neoantigen prediction and prioritization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5639.

Neoantigens are novel peptide sequences resulting from sources such as somatic mutations in tumors. Upon loading onto major histocompatibility complex (MHC) molecules, they can trigger recognition by T cells. Accurate neoantigen identification is thus critical for both designing cancer vaccines and predicting response to immunotherapies. Neoantigen identification and prioritization relies on correctly predicting whether the presenting peptide sequence can successfully induce an immune response. As the majority of somatic mutations are single nucleotide variants, changes between wildtype and mutated peptides are typically subtle and require cautious interpretation. A potentially underappreciated variable in neoantigen-prediction pipelines is the mutation position within the peptide relative to its anchor positions for the patient’s specific MHC molecules. While a subset of peptide positions are presented to the T-cell receptor for recognition, others are responsible for anchoring to the MHC, making these positional considerations critical for predicting T-cell responses. We computationally predicted high probability anchor positions for different peptide lengths for 328 common HLA alleles and identified unique anchoring patterns among them. Analysis of 923 tumor samples shows that 6-38% of neoantigen candidates are potentially misclassified and can be rescued using allelespecific knowledge of anchor positions. A subset of anchor results were orthogonally validated using protein crystallography structures. Representative anchor trends were experimentally validated using peptide-MHC stability assays and competition binding assays. By incorporating our anchor prediction results into neoantigen prediction pipelines, we hope to formalize, streamline and improve the identification process for relevant clinical studies.One Sentence SummaryNeoantigen prediction accuracy is significantly influenced by the mutation position within the neoantigen and its relative position to the patient’s allele-specific MHC anchor locations.

HLA specific antibodies vary in their pathogenicity and this is likely to be the net effect of constant chain usage, quantity, specificity, and affinity. Here we have measured the affinity of human monoclonal antibodies for a range of HLA proteins. Purified antibodies and ligands allowed dynamic interactions to be measured directly by surface plasmon resonance. Physiochemical differences between pairs of ligands were quantified using electrostatic mismatch and hydrophobic mismatch scores. \ud \ud All antibodies were characterized by fast on-rates and slow off rates but with a wide range of association rates (kon, 3.63-24.25 × 10(5) per mol per second) and dissociation rates (koff, 0.99-10.93 × 10(-3) per second). Dissociation constants (KD) ranged from 5.9 × 10(-10) M to 3.0 × 10(-8) M. SN320G6 has approximately a twenty-fold greater affinity for HLA A2 compared with SN607D8, but has a similar affinity for HLA-A2 and B57. In contrast, SN607D8 has greater than a twofold greater affinity for HLA-A2 compared with A68. Similarly, WK1D12 has about a threefold greater affinity for HLA-B27 compared with B7. The higher affinity interactions correlate with the specificity of stimulating antigen. This is the first study to directly measure the binding kinetics and affinity constants for human alloantibodies against HLA.

Cytotoxic T lymphocytes (CTL) limit influenza virus replication and prevent morbidity and mortality upon recognition of HLA class I presented epitopes on the surface of virus infected cells, yet the number and origin of the viral epitopes that decorate the infected cell are unknown. To understand the presentation of influenza virus ligands by human MHC class I molecules, HLA-B*0702-presented viral peptides were directly identified following influenza infection. After transfection with soluble class I molecules, peptide ligands unique to infected cells were eluted from isolated MHC molecules and identified by comparative mass spectrometry (MS). Then CTL were gathered following infection with influenza and viral peptides were tested for immune recognition. We found that the class I molecule B*0702 presents 3–6 viral ligands following infection with different strains of influenza. Peptide ligands derived from the internal viral nucleoprotein (NP418–426and NP473–481) and from the internal viral polymerase subunit PB1 (PB1329–337) were presented by B*0702 following infection with each of 3 different influenza strains; ligands NP418–426, NP473–481, and PB1329–337derived from internal viral proteins were consistently revealed by class I HLA. In contrast, ligands derived from hemagglutinin (HA) and matrix protein (M1) were presented intermittently on a strain-by-strain basis. When tested for immune recognition, HLA-B*0702 transgenic mice responded to NP418–426and PB1329–337consistently and NP473–481intermittently while ligands from HA and M1 were not recognized. These data demonstrate an emerging pattern whereby class I HLA reveal a handful of internal viral ligands and whereby CTL recognize consistently presented influenza ligands.

Dendritic cells (DCs) are highly specialized antigen-presenting cells of the immune system that are efficient at presenting peptide-antigen for the activation of T cells and are often the cell type of choice for vaccine targeting by virtue of high expression levels of MHC and costimulatory molecules. Since the level of peptide-MHC complex significantly influences stimulation of T cells, a proof-of-concept potency assay was developed to directly examine the presentation and density of MHC class I peptides derived from the processing of a model tumor antigen, (hCGbeta), on the surface of DCs. In this study we first generated antibodies (TCR mimics or TCRm) to two peptide-HLA-A*0201 epitopes derived from hCGbeta designated as TMT (40-48) and GVL (47-55). Characterization of each TCRm by ELISA and flow cytometric analysis, demonstrated specific binding to soluble recombinant HLA-A2 protein and HLA-A2.1+ T2 cells loaded with relevant peptide. TCRm reactive against the TMT and GVL epitopes blocked granzyme-B production by peptide-specific cytotoxic T lymphocytes (CTL) lines further supporting their recognition specificity. For the assessment of antigen presentation function, human immature monocyte-derived DCs (iDCs) were treated with the mannose receptor targeting vaccine, B11-hCGbeta and matured with Poly I:C. The TMT and GVL epitope reactive CTL lines responded to vaccine-treated but not vehicle-treated mature DCs (mDCs) with TMT and GVL TCRm specifically blocking IFN-gamma production. The TCRm were then used to directly confirm specific peptide-MHC complexes on mDCs. TCRm staining of vaccine-treated mDCs showed detection of the TMT and GVL peptide-HLA-A2 complexes. These findings demonstrate that TCRms may be important tools for determining the potency of DC-based vaccines.

T cells recognize cancer cells via HLA/peptide complexes, and when disease overtakes these immune mechanisms, immunotherapy can exogenously target these same HLA/peptide surface markers. We previously identified an HLA-A2–presented peptide derived from macrophage migration inhibitory factor (MIF) and generated antibody RL21A against this HLA-A2/MIF complex. The objective of the current study was to assess the potential for targeting the HLA-A2/MIF complex in ovarian cancer. First, MIF peptide FLSELTQQL was eluted from the HLA-A2 of the human cancerous ovarian cell lines SKOV3, A2780, OV90, and FHIOSE118hi and detected by mass spectrometry. By flow cytometry, RL21A was shown to specifically stain these four cell lines in the context of HLA-A2. Next, partially matched HLA-A*02:01+ ovarian cancer (n = 27) and normal fallopian tube (n = 24) tissues were stained with RL21A by immunohistochemistry to assess differential HLA-A2/MIF complex expression. Ovarian tumor tissues revealed significantly increased RL21A staining compared with normal fallopian tube epithelium (P < 0.0001), with minimal staining of normal stroma and blood vessels (P < 0.0001 and P < 0.001 compared with tumor cells) suggesting a therapeutic window. We then demonstrated the anticancer activity of toxin-bound RL21A via the dose-dependent killing of ovarian cancer cells. In summary, MIF-derived peptide FLSELTQQL is HLA-A2–presented and recognized by RL21A on ovarian cancer cell lines and patient tumor tissues, and targeting of this HLA-A2/MIF complex with toxin-bound RL21A can induce ovarian cancer cell death. These results suggest that the HLA-A2/MIF complex should be further explored as a cell-surface target for ovarian cancer immunotherapy. Mol Cancer Ther; 15(2); 313–22. ©2015 AACR.

For the quantification of HLA-specific memory B cells from peripheral blood of sensitized individuals, a limited number of methods are available. However, none of these are capable of detecting memory B cells directed at HLA class II molecules. Since the majority of antibodies that occur after transplantation appear to be specific for HLA class II, our aim was to develop an assay to detect and quantify HLA class II-specific memory B cells from peripheral blood. By using biotinylated soluble HLA class II molecules as detection agent, we were able to develop an HLA class II-specific memory B cell ELISPOT assay. The assay was validated using B cell-derived hybridomas that produce human monoclonal antibodies directed at specific HLA class II molecules. In pregnancy-immunized females, we found memory B cell frequencies ranging from 25 to 756 spots per 10(6) B cells specific for the immunizing paternal HLA class II molecules, whereas in non-immunized males no significant spot formation was detected. Here, we present a novel ELISPOT assay for quantifying HLA class II-specific memory B cells from peripheral blood. This technique provides a unique tool for monitoring the HLA class II-specific memory B cell pool in sensitized transplant recipients.

There is currently a significant interest in the identification and validation of HLA-restricted CTL epitopes, which are thought to have important implications for the development of preventive and/or therapeutic applications in bacterial or viral infections, autoimmune diseases, and cancer. To better facilitate epitope discovery and validation, we present a cell- and radioisotope-free HLA-A⁎0201 assay system which relies upon fluorescence polarization. The assay has the advantage of allowing real-time measurements in solution without separation steps. In this report, we directed our efforts towards enhancing the sensitivity and reproducibility of the assay by conducting an in-depth analysis of parameters critical for standardization. Initial experiments demonstrated that the attachment of a fluorescence moiety at positions 5 and 8 for 9-mers and positions 5 and 6 for 10-mers, respectively, does not interfere with ligand binding to soluble HLA-A⁎0201. In addition, it was found that their binding to HLA-A⁎0201 was very effective showing high affinity binding with Kd's between 10.7 to 21.8 nM and binding capacities of up to 37%. In order to deliver maximized responses, factors such as the regulation of thermal HLA activation parameters to initiate peptide exchange as well as the specific adjustment of assay components were identified. Overall, the results obtained clearly demonstrate high accuracy, sensitivity and reproducibility of the FP-based assay approach. With the need for both increased throughput and miniaturized volumes, this fully homogenous, fluorescent-type binding assay is expected to be useful for routine analysis of peptide binding to MHC class I as well as class II molecules.

HIV-specific CTL play a central role in immune control of HIV. The basis for understanding the success or failure of this immune response requires identification of the specific epitopes targeted by CTL. However, in populations most severely affected by the global epidemic, this fundamental knowledge is hindered by the lack of characterization of many of the HLA class I alleles highly prevalent in such populations. Overall, the peptide-binding motif has been determined for a small minority (9%) of HLA class I alleles, with a strong bias toward those alleles prevalent in Caucasoid populations. These studies therefore set out to define, in a South African Zulu/Xhosa population at the epicenter of the epidemic, the epitopes presented by alleles highly prevalent, but for which the peptide-binding motif had not been characterized. Using a method of motif inference, epitopes presented by four such alleles prevalent in the Zulu/Xhosa population of Durban, South Africa, namely, B*3910, B*4201, B*8101, and Cw*1801, are described. Importantly, this approach may additionally facilitate optimization of epitopes in certain instances where conflicting reports in the literature exist regarding the peptide-binding motif, such as for HLA-A*2902, also highly prevalent in southern African populations. These data indicate that the previously anomalous position of HLA-A*2902 among HLA-A alleles, outside any recognized HLA-A supertype, is artifactual, and the true position of the A*2902 motif overlaps those of the A1 and A24 supertypes.

Various approaches are currently proposed to successfully develop therapies for the prevention and treatment of infectious diseases and cancer. One of the most promising approaches is the development of vaccines that elicit cytotoxic T lymphocyte (CTL) responses. Consequently, identification and exact definition of molecular parameters involved in peptide-MHC class-I interactions of putative CTL epitopes are of prime importance for the development of immunomodulating compounds. To better facilitate epitope discovery, we developed and validated a novel state-of-the-art biochemical HLA-A0201 assay, which is comprised of technologically advanced cutting edge reagents. The technique is based on competition and uses a FITC-labeled reference peptide and highly purified soluble HLA-A0201 molecules to quantitatively measure the binding capacity of nonlabeled peptide candidates. Detection by fluorescence polarization allows real-time measurement of binding ratios without separation steps. During standardization, the problem of assay parameter variation is discussed, showing the dramatic influence of HLA and reference peptide concentrations as well as the choice of the reference peptide itself on IC(50) determinations. For validation, a panel of 15 well-defined HLA-A0201 ligands from various sources covering a broad range of binding affinities was tested. Binding data were used to compare against pre-existing quantitative assay systems. The results obtained demonstrated significant correlation among assay procedures, suggesting that the application of fluorescence polarization in combination with recombinant sHLA molecules is highly advantageous for the accurate assessment of peptide binding. Furthermore, the assay also features high-throughput screening capacity, providing uniquely efficient means of identifying and evaluating immune target molecules.

Sperm protein 17 (Sp17) is a highly antigenic, testes-specific protein whose known function is to bind sperm to the zona pellucida. However, the Sp17 gene has been recently detected in normal non-testes tissues and malignant neoplasias. As the role of Sp17 in non-testes tissues is unknown, the characterization of the Sp17 gene in highly proliferating tissues may provide further insight into the regulation and alternative function of Sp17. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify the Sp17-1 transcript in multiple normal human tissues and cancer cell lines. Similarly, the Sp17-2 gene was examined by PCR. In addition, Northern and Western blot analyses were used to detect Sp17 mRNA and protein expression. The Sp17-1a and Sp17-1b transcripts were amplified from cancer cell lines. Similarly, an Sp17-2 transcript was also detected in cancer cell lines. Furthermore, Northern blot analysis revealed Sp17 mRNA expression in all cancer cell lines examined. However, Sp17 protein expression was not detected. The differential detection of the Sp17 transcripts in cancer cell lines as compared to normal non-testes tissues, suggests a potential pathogenic role for Sp17 in diseased cells. Moreover, the Sp17-2 transcript may be a marker for highly proliferating cells. Collectively, these data implicate Sp17 as a cancer testis antigen.

Sp17 was initially thought to be a sperm specific protein involved in the interaction of the spermatozoon with the oocyte's surrounding extracellular glycoprotein matrix. Recent reports, however, indicate that Sp17 expression is neither testis-specific nor is it exclusively used for binding to the zona pellucida of the oocyte. In this study, we provide comprehensive characterization of the genomic structure of Sp17. We identified an intron-containing gene (Sp17-1) containing five exonic and four intronic sequences. Analysis of Sp17 transcripts using rapid amplification of DNA complementary to RNA (cDNA) ends (RACE) and polymerase chain reaction (PCR) techniques showed the presence of alternative polyadenylation resulting in the production of varying lengths of mRNAs as well as the usage of different transcriptional start sites. Moreover, an earlier description of the human Sp17 mRNA describing a splice variant could not be confirmed. Comparison to mouse Sp17 gene organization demonstrated a high degree of conservation, suggesting selective evolutionary pressure for this protein to retain a conserved gene architecture. Additionally, we identified a second gene (Sp17-2), whose most striking characteristic was the complete absence of introns. This Sp17-2 gene has likely arisen by reverse transcription (RT) of a spliced Sp17-1 mRNA with subsequent integration into the human genome. Its open reading frame (ORF) is interrupted by stop codons, giving rise to a pseudogene. Furthermore, Southern blot analysis of human genomic DNA indicated the possibility of additional Sp17 species within the human genome.

Aim The primary focus of HLA antibody testing for transplant patients is to assess a given patient’s potential risk for graft loss by determining the immunological status before and after transplantation. Since the physiological responses of the adaptive immune system against an HLA target is highly variable and antibody composition is distinct to each individual, accurate determination of specificities, titer and strengths of antibodies is of uttermost importance. Although MFI values provide a pragmatic approach in ranking responses, the method makes no attempt to explain titer and affinity influences in evaluating a response. To overcome such limitations, our goal was to demonstrate that titration analysis will allow better interpretation of individual antibody-HLA target interactions which will result in a better indicative risk assessment than MFI values. Methods By introduction of recombinant technology, soluble HLA ABC locus alleles were produced by a mammalian expression system and utilized to create a unique 120 single antigen assay. Utilizing this novel assay, a dose-response model was evaluated by performing multiple serological titration experiments probing the interaction of single HLA antigens with HLA antibodies. Results Results showed that the assay follows the principles of the dose-response concept not only demonstrating the binding of HLA antibodies as effectors to specific HLA targets with sigmoidal functions but also delivering potency values of their interaction. Potency analysis is a clinically relevant evaluation approach to determine titer in combination with strength of HLA antibodies independent of antibody efficacy levels. Reducing dose-response data to relative half maximum effect values showed the advantages of a fine-tuned data output by decreasing titration complexity, allowing semi-quantitative analysis and becoming independent of MFI values. Conclusions We believe that dose-response information will greatly support the identification of factors that can contribute to the individual characterization of sera antibodies and offer the potential of direct physical meaning that will resonate with clinical outcomes. This technique will likely lead to a better assessment of HLA titers and generate new insights for pre- and post-transplant monitoring.

Aim The identification of prognostic parameters for the clinical course of disease states is of considerable scientific and therapeutic relevance. Serum levels of soluble HLA (sHLA) class I molecules have been advocated at various occasions possessing such predictive potential. However, lack of quantitation of sHLA levels, unavailability of appropriate assay standards and small population sizes investigated have dominantly contributed to the inability to establish the physiological importance of the presence of sHLA in body fluids and to make accurate disease associations. In this work, we have redesigned the assay approach for sHLA class I detection and introduced optimized assay conditions including a sHLA standard of highest purity and structural integrity. Methods Applying a standardized detection methodology, serum samples were screened from one hundred normal volunteer blood donors with known HLA-phenotypes. In addition, plasmapheresis samples from various patients were utilized for large-scale sHLA purification using a W6/32-affinity column. Eluted probes were further analyzed applying multiple assay strategies. Results Our findings showed an average serum level of sHLA class I in normal individuals of 494 ng/ml [190–1402 ng/ml]. No significant differences in sHLA serum levels were observed between different blood types or between male and female donors. Closer investigation of the sHLA content of several plasmapheresis samples indicated that more than 75% of sHLA is complexed with other proteins and not available in free form. HLA specificity testing using mAb matched the determined allotype of the patients confirming the self-origin of the purified sHLA. Conclusions The design and development of assay methods is crucial to meet analytical performance requirements for intact sHLA quantitation. Extraction of sHLA from human blood is highly feasible and offers a valid approach for the elucidation of the relevance of sHLA-mediated immunoregulation. Analysis of HLA-complexed antibody species or just the simple presence of elevated sHLA amounts can provide a source of information to establish a foundation for unique diagnostic approaches to gain insights into immunopathological processes for transplantation and for diseases such as cancer, autoimmunity, allergy, and viral diseases.

The present study was designed to identify and characterize muscarinic acetylcholine receptors in normal human melanocytes. We used subtype-specific oligonucleotide primers to localize the five genetically defined mAChR mRNAs (m1 through m5) by reverse transcription-polymerase chain reaction. These experiments showed that all five mAChR subtype mRNAs are expressed in melanocytes. The PCR products were verified by restriction analysis and Southern blotting. Receptors were visualized in cultures of normal human melanocytes and specimens of normal human skin by subtype-specific rabbit anti-receptor polyclonal antibodies. Radioligand binding assays with the lipophilic drug [3H]quinuclidinyl benzilate demonstrated approximately 9000 high affinity binding sites/cell. Micromolar concentrations of muscarine or carbachol transiently increased intracellular Ca2+, which could be attenuated by atropine, demonstrating coupling of the receptors to mobilization of intracellular free Ca2+. Lower concentrations of muscarine induced spontaneous repetitive spike-like increases of intracellular Ca2+ which is characteristic for the activation of muscarinic receptors. These results indicate that normal human skin melanocytes express the m1, m2, m3, m4, and m5 subtypes of classic muscarinic acetylcholine receptors on their cell membrane and that these receptors regulate the concentration of intracellular free Ca2+, which may play an important physiologic role in melanocyte behavior and skin pigmentation.

Previous studies have demonstrated that muscarinic acetylcholine receptors (mAChRs) are expressed by human skin fibroblasts (HSF). We have identified the molecular subtypes of these receptors by reverse transcription-polymerase chain reaction (RT-PCR), using m1-m5 subtype-specific primers. These experiments showed that only mRNAs for m2, m4, and m5 mAChR subtypes are present in HSF. The RT-PCR products were characterized by restriction analysis and Southern blotting. Northern blot analysis showed the presence of m2 and m4 mAChR RNA. Rabbit antibodies were raised using a synthetic peptide as immunogen corresponding to the C-terminus of the m2 protein and were used to visualize fibroblast mAChRs. Cell membranes of HSF in cell culture and specimens of normal human skin had a unique staining pattern specific for anti-m2 antibody, as well as for antibodies against m4 and m5. In Western blots of fibroblast proteins, the antibodies visualized the m2 receptor at 65 kDa, m4 at 70 kDa, and m5 at 95 kDa. The function of fibroblast mAChRs was examined by measuring muscarinic effects on intracellular free Ca2+ concentration ([Ca2+]i). Muscarine increased transiently [Ca2+]i in cultured HSF. This effect could be abolished by the muscarinic antagonist atropine. Thus, the results of this study showed that HSF express m2, m4, and m5 mAChR subtypes, and that fibroblast mAChRs are coupled to the regulation of [Ca2+]i. J. Cell. Biochem. 74:264–277, 1999. © 1999 Wiley-Liss, Inc.

Acetylcholine mediates cell-to-cell communications in the skin. Human epidermal keratinocytes respond to acetylcholine via two classes of cell-surface receptors, the nicotinic and the muscarinic cholinergic receptors. High affinity muscarinic acetylcholine receptors (mAChR) have been found on keratinocyte cell surfaces at high density. These receptors mediate effects of muscarinic drugs on keratinocyte viability, proliferation, adhesion, lateral migration, and differentiation. In this study, we investigated the molecular structure of keratinocyte mAChR and their location in human epidermis. Polymerase chain reaction amplification of cDNA sequences uniquely present within the third cytoplasmic loop of each subtype demonstrated the expression of the m1, m3, m4, and m5 mAChR subtypes. To visualize these mAChR, we raised rabbit anti-sera to synthetic peptide analogs of the carboxyl terminal regions of each subtype. The antibodies selectively bound to keratinocyte mAChR subtypes in immunoblotting membranes and epidermis, both of which could be abolished by preincubating the anti-serum with the peptide used for immunization. The immunofluorescent staining patterns produced by each antibody in the epidermis suggested that the profile of keratinocyte mAChR changes during epidermal turnover. The semiquantitative analysis of fluorescence revealed that basal cells predominantly expressed m3, prickle cells had equally high levels of m4 and m5, and granular cells mostly possessed m1. Thus, the results of this study demonstrate for the first time the presence of m1, m3, m4, and m5 mAChR in epidermal keratinocytes. Because keratinocytes express a unique combination of mAChR subtypes at each stage of their development in the epidermis, each receptor may regulate a specific cell function. Hence, a single cytotransmitter, acetylcholine, and muscarinic drugs may exert different biologic effects on keratinocytes at different stages of their maturation.

The U21 gene product from human herpesvirus 7 binds to and redirects class I major histocompatibility complex (MHC) molecules to a lysosomal compartment. The molecular mechanism by which U21 reroutes class I MHC molecules to lysosomes is not known. Here, we have reconstituted the interaction between purified soluble U21 and class I MHC molecules, suggesting that U21 does not require additional cellular proteins to interact with class I MHC molecules. Our results demonstrate that U21, itself predicted to contain an MHC class I-like protein fold, interacts tightly with class I MHC molecules as a tetramer, in a 4:2 stoichiometry. These observations have helped to elucidate a refined model describing the mechanism by which U21 escorts class I MHC molecules to the lysosomal compartment. IMPORTANCE In this report, we show that the human herpesvirus 7 (HHV-7) immunoevasin U21, itself a class I MHC-like protein, binds with high affinity to class I MHC molecules as a tetramer and escorts them to lysosomes, where they are degraded. While many class I MHC-like molecules have been described in detail, this unusual viral class I-like protein functions as a tetramer, associating with class I MHC molecules in a 4:2 ratio, illuminating a functional significance of homooligomerization of a class I MHC-like protein.

Identification of CD8 + cytotoxic T lymphocyte (CTL) epitopes has traditionally relied upon testing of overlapping peptide libraries for their reactivity with T cells in vitro . Here, we pursued deep ligand sequencing (DLS) as an alternative method of directly identifying those ligands that are epitopes presented to CTLs by the class I human leukocyte antigens (HLA) of infected cells. Soluble class I HLA-A*11:01 (sHLA) was gathered from HIV-1 NL4-3-infected human CD4 + SUP-T1 cells. HLA-A*11:01 harvested from infected cells was immunoaffinity purified and acid boiled to release heavy and light chains from peptide ligands that were then recovered by size-exclusion filtration. The ligands were first fractionated by high-pH high-pressure liquid chromatography and then subjected to separation by nano-liquid chromatography (nano-LC)–mass spectrometry (MS) at low pH. Approximately 10 million ions were selected for sequencing by tandem mass spectrometry (MS/MS). HLA-A*11:01 ligand sequences were determined with PEAKS software and confirmed by comparison to spectra generated from synthetic peptides. DLS identified 42 viral ligands presented by HLA-A*11:01, and 37 of these were previously undetected. These data demonstrate that (i) HIV-1 Gag and Nef are extensively sampled, (ii) ligand length variants are prevalent, particularly within Gag and Nef hot spots where ligand sequences overlap, (iii) noncanonical ligands are T cell reactive, and (iv) HIV-1 ligands are derived from de novo synthesis rather than endocytic sampling. Next-generation immunotherapies must factor these nascent HIV-1 ligand length variants and the finding that CTL-reactive epitopes may be absent during infection of CD4 + T cells into strategies designed to enhance T cell immunity. IMPORTANCE HIV-1 epitopes catalogued by the Los Alamos National Laboratory (LANL) have yielded limited success in vaccine trials. Because the HLA of infected cells have not previously been assessed for HIV-1 ligands, the objective here was to directly characterize the viral ligands that mark infected cells. Recovery of HLA-presented peptides from HIV-1-infected CD4 + T cells and interrogation of the peptide cargo by mass spectrometric DLS show that typical and atypical viral ligands are efficiently presented by HLA and targeted by human CTLs. Nef and Gag ligands dominate the infected cell's antigenic profile, largely due to extensive ligand sampling from select hot spots within these viral proteins. Also, HIV-1 ligands are often longer than expected, and these length variants are quite antigenic. These findings emphasize that an HLA-based view of HIV-1 ligand presentation to CTLs provides previously unrealized information that may enhance the development of immune therapies and vaccines.

Immunizing events including pregnancy, transfusions, and transplantation promote strong alloantibody responses to HLA. Such alloantibodies to HLA preclude organ transplantation, foster hyperacute rejection, and contribute to chronic transplant failure. Diagnostic antibody-screening assays detect alloreactive antibodies, yet key attributes including antibody concentration and isotype remain largely unexplored. The goal here was to provide a detailed profile of allogeneic antibodies to class II HLA. Methodologically, alloantibodies were purified from sensitized patient sera using an HLA-DR11 immunoaffinity column and subsequently categorized. Antibodies to DR11 were found to fix complement, exist at a median serum concentration of 2.3 μg/mL, consist of all isotypes, and isotypes IgG2, IgM, and IgE were elevated. Because multimeric isotypes can confound diagnostic determinations of antibody concentration, IgM and IgA isotypes were removed and DR11-IgG tested alone. Despite removal of multimeric isotypes, patient-to-patient antibody concentrations did not correlate with MFI values. In conclusion, allogeneic antibody responses to DR11 are comprised of all antibody isotypes at differing proportions, these combined isotypes fix complement at nominal serum concentrations, and enhancements other than the removal of IgM and IgA multimeric isotypes may be required if MFI is to be used as a means of determining anti-HLA serum antibody concentrations in diagnostic clinical assays.

The recent West Nile virus (WNV) outbreak in the United States underscores the importance of understanding human immune responses to this pathogen. Via the presentation of viral peptide ligands at the cell surface, class I HLA mediate the T cell recognition and killing of WNV infected cells. At this time, there are two key unknowns in regards to understanding protective T cell immunity: 1) the number of viral ligands presented by the HLA of infected cells, and 2) the distribution of T cell responses to these available HLA/viral complexes. Here, comparative mass spectroscopy was applied to determine the number of WNV peptides presented by the HLA-A*11:01 of infected cells after which T cell responses to these HLA/WNV complexes were assessed. Six viral peptides derived from capsid, NS3, NS4b, and NS5 were presented. When T cells from infected individuals were tested for reactivity to these six viral ligands, polyfunctional T cells were focused on the GTL9 WNV capsid peptide, ligands from NS3, NS4b, and NS5 were less immunogenic, and two ligands were largely inert, demonstrating that class I HLA reduce the WNV polyprotein to a handful of immune targets and that polyfunctional T cells recognize infections by zeroing in on particular HLA/WNV epitopes. Such dominant HLA/peptide epitopes are poised to drive the development of WNV vaccines that elicit protective T cells as well as providing key antigens for immunoassays that establish correlates of viral immunity.

Several aminotransferases with kynurenine aminotransferase (KAT) activity are able to convert L-kynurenine into kynurenic acid, a putative endogenous modulator of glutamatergic neurotransmission. In the rat, one of the described KAT isoforms has been found to correspond to glutamine transaminase K. In addition, rat kidney alpha-aminoadipate aminotransferase (AadAT) also shows KAT activity. In this report, we describe the isolation of a cDNA clone encoding the soluble form of this aminotransferase isoenzyme from rat (KAT/AadAT). Degenerate oligonucleotides were designed from the amino acid sequences of rat kidney KAT/AadAT tryptic peptides for use as primers for reverse transcription-polymerase chain reaction of rat kidney RNA. The resulting polymerase chain reaction fragment was used to screen a rat kidney cDNA library and to isolate a cDNA clone encoding KAT/AadAT. Analysis of the combined DNA sequences indicated the presence of a single 1275-base pair open reading frame coding for a soluble protein of 425 amino acid residues. KAT/AadAT appears to be structurally homologous to aspartate aminotransferase in the pyridoxal 5'-phosphate binding domain. RNA blot analysis of rat tissues, including brain, revealed a single species of KAT/AadAT mRNA of approximately 2.1 kilobases. HEK-293 cells transfected with the KAT/AadAT cDNA exhibited both KAT and AadAT activities with enzymatic properties similar to those reported for the rat native protein.

Aim Dendritic Cells (DC) mediate anti-tumor immunity, and DC-based immunotherapies represent a promising way to fight cancer. However, questions about the optimal manufacturing process and delivery strategies still remain. So far, DC-based immunotherapies have only been tested for maturational state and expression of select CD markers. However, these characteristics do not establish successful peptide loading or actual presentation of peptides by HLA. To date, class I HLA presentation of peptides on loaded DC has only been confirmed using indirect immune monitoring techniques such as ELISPOT and tetramer staining. Here, we present the first quantitative assay designed for ICT-107, a DC immunotherapy being tested in glioblastoma, that directly assesses peptide presentation on pulsed DC. This assay may prove valuable for monitoring quality and potency of DC-based immunotherapies via a reliable, reproducible and straightforward method. Methods We generated a T cell receptor mimic monoclonal antibody (TCRm), RL13A, specific for HLA-A∗01:01 in complex with MAGE-1 peptide (EADPTGHSY; “EAD9”). Next, mature DC from three different HLA-A1+ patients were pulsed with 20 μg/ml of EAD9 peptide. Loaded DC were then stained with PE-labeled RL13A. QuantiBRITE PE-Beads were used to establish a standard curve so that the number of HLA-A1/EAD9 complexes could subsequently be determined. Results Flow cytometric staining of peptide-pulsed DC with RL13A showed a clear shift in MFI compared to patient-matched, unpulsed DC. Using the QuantiBRITE PE-bead standard curve, the number of HLA-A1/EAD9 on the surface of ICT-107 was determined. All 3 lots of ICT-107 presented a comparable number of HLA-A1/EAD complexes (1200–2500 copies) on the surface. Conclusions The data from ICT-107 provide proof of concept that an HLA-specific, quantitative assay can be used to directly confirm presentation of the peptides on DC-based immunotherapies. TCRm staining enables detection as well as quantification of HLA/peptide complexes presented by peptide-pulsed DC, and the assay can now be used to characterize the DC immunotherapy, ICT-107. Future applications for this assay include both the optimization of the manufacturing process and serving as a release assay for HLA-A1+ DC-based therapeutics containing the MAGE-1 EAD9 peptide. R. Buchli: Employee; Company/Organization; Pure MHC LLC. W.H. Hildebrand: Scientific/Medical Advisor; Company/Organization; Pure MHC LLC.

Aim Mapping anti-HLA antibody epitopes has commonly been done by comparing antibody reactivity patterns to a panel of naturally occurring HLA allelic variants such as the solid phase HLA antibody screening panels. Information including the donor’s immunizing alleles and the recipient-derived HLA further helps to refine HLA antibody epitopes. While these methods are inexpensive and widely available, they represent an indirect means of mapping those amino acids on HLA that are critical to antibody recognition and binding. Here we test a complementary direct approach to measure the ‘foot print’ of an antibody at it interacts with HLA. A recently developed method called protein painting was used to map the interaction of mouse monoclonal antibody BB7.2 with HLA-A*02:01. Methods Briefly, purified soluble HLA-A*02:01 was incubated with and without BB7.2. Protein mixtures were incubated with the trypsin inhibitor Remazol Brilliant Blue (RBB), trypsin digested, and tryptic peptide fragments of HLA-A*02:01 were analyzed using LCMS. In this manner BBB protects solvent accessible Arg and Lys residues from trypsin digestion. When HLA-A*02:01 is first incubated with BB7.2, Arg and Lys residues covered by the antibody are not painted by BBB and therefore they remain trypsin susceptible. Results A mass spectrometric analysis of the resulting trypic peptides revealed that BB7.2 protects R44, R97, R108, R111, R131, and K144 from painting. These residues are primarily located on lateral edge of the alpha-2 alpha helix as shown on the three dimensional structure of HLA-A*02:01. In a solid phase assay, BB7.2 reacts exclusively with A2 and A28 allomorphs, data that identifies the well defined 144TKH epitope. The protein painting data shown here are consistent with the 144TKH epitope as K144 was protected from RBB binding by BB7.2. Conclusion We demonstrate a new method for direct mapping of anti-HLA antibody epitopes that is complementary to solid phase assays. Furthermore, protein painting reveals not only the critical residues for antibody binding, but the surrounding amino acids covered by the antibody. R. Buchli : Employee; Company/Organization; Pure Protein LLC. W. Hildebrand: Scientific/Medical Advisor; Company/Organization; Pure Protein LLC.

Background: Abacavir drug hypersensitivity in HIV-treated patients is associated with HLA-B� 57:01 expression. To understand the immunochemistry of abacavir drug reactions, we investigated the effects of abacavir on 1) HLA-B� 57:01 epitope-binding in vitro and 2) the quality and quantity of self-peptides presented by HLA-B� 57:01 from abacavir-treated cells. Design and methods: An HLA-B� 57:01 specific epitope binding assay was developed to test for effects of abacavir, didanosine or flucloxacillin on self-peptide binding. To examine whether abacavir alters the peptide repertoire in HLA-B� 57:01, a B-cell line secreting soluble HLA (sHLA) was cultured in the presence or absence of abacavir, peptides were eluted from purified HLA, and the peptide epitopes comparatively mapped by mass spectroscopy to identify drug-unique peptides. Results: Abacavir, but not didansosine or flucloxacillin, enhanced binding of the FITC labeled self-peptide LF9 to HLA-B� 57:01 in a dose dependent manner. Endogenous peptides isolated from abacavir-treated HLA-B� 57:01 B-cells showed amino acid sequence differences compared with peptides from untreated cells. Novel druginduced (DI)-peptides lacked typical carboxyl(C)-terminal amino-acids characteristic of the HLA-B� 57:01 peptide motif and instead contained predominantly Isoleucine or Leucine residues. DI-peptides bind to soluble HLA-B� 57:01 with high affinity that was not altered by abacavir addition. Conclusion: Our results support a model of drug-induced autoimmunity in which abacaviraltersthequantity andquality of self-peptideloadinginto HLA-B� 57:01.Druginduced loading of novel self-peptides into HLA, possibly by abacavir either altering the binding cleft or modifying the peptide loading complex, generates an array of neo

Elevated levels of human leukocyte antigen (HLA) proteins have been reported in several pathologic conditions that are associated with increased concentrations of white blood cells (e.g., infection, inflammation, and lymphoproliferative disorders). The mechanisms by which HLA proteins are solubilized from cell membranes are insufficiently understood. We hypothesized that HLA proteins may be cleaved from cell membranes by insufficiently inhibited leukocytic elastase, as expected in alpha-1 antitrypsin deficiency (A1ATD), resulting in elevated plasma levels of soluble HLA (sHLA) proteins. Using an enzyme-linked immunosorbent assay, we measured sHLA II levels in the peripheral blood of patients with A1ATD with or without co-existing chronic obstructive pulmonary disease (COPD), with COPD only, and in a control group. Mean (±SD) sHLA II plasma levels were 110 ± 200 pg/mL in patients with A1ATD and COPD (Group 1), 10 ± 30 pg/mL in patients with COPD without A1ATD (Group 2), 70 ± 90 pg/mL in patients with A1ATD without COPD (Group 3), and 10 ± 30 pg/mL in healthy donors (Group 4). Soluble HLA II plasma levels were significantly higher in Group 1 (P = .001) and Group 3 (P = .002) versus Group 4. Our preliminary results suggest that leukocytic elastase and probably other proteinases solubilize HLA proteins from cell membranes. This mechanism would operate in inflammation with elevated leukocytic elastase levels but more so with inflammation and A1ATD, where elastase would be insufficiently inhibited. If this mechanism is verified, plasma sHLA levels could potentially be used to measure cell damage due to proteinases and, therefore, for monitoring the therapeutic efficacy of alpha-1 antitrypsin (A1AT) augmentation therapy.

Aim The measurement of rejection-causing HLA Abs in serological samples of HLA sensitized individuals has clinical relevance in the evaluation of pre- and post-transplant patients. However, due to the increasing demand for more accurate and meaningful data, the determination of MFI values alone is not sufficient anymore to evaluate a patient’s risk potential. In this study, we aimed to demonstrate the enormous complexity of Ab patterns in serological specimen. Methods To visualize variations and different levels of HLA Ab patterns, several investigative tools were applied including HLA Ab-detection assays, neutralization experiments, sera titration analysis and comparative information from human monoclonal Ab profiles derived from single B cells. Results In tier 1, neutralization experiments clearly demonstrated that original response patterns could be deconvoluted into individual sub-patterns and single alleles identified as primary cause of an immunizing event. Depending on exposure, one or several antigens were generally involved. Tier 2 patterns were originated from a single epitope harbored by the immunizing antigen of origin that naturally cross-reacts with related epitope-sharing antigens. Currently, over 100 human monoclonal Ab patterns were collected defining such epitopes and being used to further resolve Tier 1 patterns by a pattern recognition algorithm. Tier 3 involves either individual epitopes shared among related antigens or Abs that recognize a specific HLA/Ab interaction but not necessarily at the same affinity. Tier 4 includes diverse epitope patterns that appear using different isotype-recognizing detection systems. Tier 5 refers to experiments conducted under a time interval, visualizing the appearance/disappearance of different Ab species in time. Conclusion We believe that the knowledge gained about the apparent complexity of specific immune patterns will help us to reach the next chapter of patient care and contribute to a better risk management in Ab-related graft rejection.

Aim Some earlier studies indicated that monoclonal Abs can be neutralized and inhibition measured with a bioassay design structured to reduce 95–100% of the maximal signal at 100 μ g/ml sHLA if operated in the mid linear range of the response signal. The aim of this investigation was to determine whether the theoretical concepts describing the basic nature of neutralization of HLA antibodies could be applied to more complex serological specimen resulting in clinically useful information. Methods Sera specimens were tested by individually adding different combinations of sHLA molecules as neutralizing agents prior to response measurement. After subtraction of the resulting inhibitory profile from the non-treated reference profile, a neutralization profile was generated, visualizing Ab responses directly related to the applied neutralizer allele. Results As shown, sHLA-A ∗ 02:01 was able to 100% neutralize A2-related responses, leaving other HLA/antibody interactions untouched. In this example, when also subtracting B ∗ 08:01 and B ∗ 49:01, a near complete inhibition of the reference profile was achieved. Minor assay-to-assay background fluctuations of 10% can generally be observed. Significantly, HLA antigens considered directly responsible for the immunizing event generally showed a complete inhibition of the reference profile, whereas partial inhibitions were seen as indicator of HLA antibody species present with different antigen/epitope correlation. Conclusion Creating a neutralizer profile subtracting the inhibitory profile from the reference profile is most helpful for better analytical assessment of signal patterns, delivering a positive view of the blocking event, ultimately allowing better interpretation of antibody populations. As demonstrated, complex reactivity patterns could be reduced to more simplifying outputs. We believe that reductive profiling techniques will greatly assist in obtaining more accurate and meaningful data in the assessment of risk for immunologic rejection.

To escape immune recognition, viruses acquire amino acid substitutions in class I human leukocyte antigen (HLA)-presented cytotoxic T-lymphocyte (CTL) epitopes. Such viral escape mutations may (i) prevent peptide processing, (ii) diminish class I HLA binding, or (iii) alter T-cell recognition. Because residues 418 to 426 of the hypervariable influenza A virus nucleoprotein (NP418-426) epitope are consistently bound by class I HLA and presented to CTL, we assessed the impact that intraepitope sequence variability has upon T-cell recognition. CTL elicited by intranasal influenza virus infection were tested for their cross-recognition of 20 natural NP418-426epitope variants. Six of the variant epitopes, of both H1N1 and H3N2 origin, were cross-recognized by CTL while the remaining NP418-426epitope variants escaped targeting. A pattern emerged whereby variability at position 5 (P5) within the epitope reduced T-cell recognition, changes at P4 or P6 enabled CTL escape, and a mutation at P8 enhanced T-cell recognition. These data demonstrate that substitutions at P4 and/or P6 facilitate influenza virus escape from T-cell recognition and provide a model for the number, nature, and location of viral mutations that influence T-cell cross-recognition.

Cellular immune mechanisms detect and destroy cancerous and infected cells via the human leukocyte antigen (HLA) class I molecules that present peptides of intracellular origin on the surface of all nucleated cells. The identification of novel, tumor-specific epitopes is a critical step in the development of immunotherapeutics for breast cancer. To directly identify peptide epitopes unique to cancerous cells, secreted human class I HLA molecules (sHLA) were constructed by deletion of the transmembrane and cytoplasmic domain of HLA A*0201. The resulting sHLA-A*0201 was transferred and expressed in breast cancer cell lines MCF-7, MDA-MB-231, and BT-20 as well as in the immortal, nontumorigenic cell line MCF10A. Stable transfectants were seeded into bioreactors for production of > 25 mg of sHLA-A*0201. Peptides eluted from affinity purified sHLA were analyzed by mass spectroscopy. Comparative analysis of HLA-A*0201 peptides revealed 5 previously uncharacterized epitopes uniquely presented on breast cancer cells. These peptides were derived from intracellular proteins with either well-defined or putative roles in breast cancer development and progression: Cyclin Dependent Kinase 2 (Cdk2), Ornithine Decarboxylase (ODC1), Kinetochore Associated 2 (KNTC2 or HEC1), Macrophage Migration Inhibitory Factor (MIF), and Exosome Component 6 (EXOSC6). Cellular recognition of the MIF, KNTC2, EXOSC6, and Cdk2 peptides by circulating CD8+ cells was demonstrated by tetramer staining and IFN-gamma ELISPOT. The identification and characterization of peptides unique to the class I of breast cancer cells provide putative targets for the development of immune diagnostic tools and therapeutics.

Cytotoxic T lymphocytes (CTL) play an important role in the control and elimination of infection by West Nile virus (WNV), yet the class I human leukocyte antigen (HLA)-presented peptide epitopes that enable CTL recognition of WNV-infected cells remain uncharacterized. The goals of this work were first to discover the peptide epitopes that distinguish the class I HLA of WNV-infected cells and then to test the T cell reactivity of newly discovered WNV epitopes. To discover WNV-immune epitopes, class I HLA was harvested from WNV (NY99 strain)-infected and uninfected HeLa cells. Then peptide epitopes were eluted from affinity-purified HLA, and peptide epitopes from infected and uninfected cells were comparatively mapped by mass spectroscopy. Six virus-derived peptides from five different viral proteins (E, NS2b, NS3, NS4b, and NS5) were discovered as unique to HLA-A*0201 of infected cells, demonstrating that the peptides sampled by class I HLA are distributed widely throughout the WNV proteome. When tested with CTL from infected individuals, one dominant WNV target was apparent, two epitopes were subdominant, and three demonstrated little CTL reactivity. Finally, a sequence comparison of these epitopes with the hundreds of viral isolates shows that HLA-A*0201 presents epitopes derived from conserved regions of the virus. Detection and recovery from WNV infection are therefore functions of the ability of class I HLA molecules to reveal conserved WNV epitopes to an intact cellular immune system that subsequently recognizes infected cells.

Major histocompatibility complex (MHC) class I molecules bind and display peptide antigens on the cell surface. CD8(+) T lymphocytes recognize peptides in association with class I proteins to initiate a cytotoxic immune response. To understand the specificity of such immune responses and to facilitate the development of therapies for disease, it is important to identify MHC-presented peptides. In this study, platelets, easily obtainable and often associated with immune-mediated disease, were selected to identify MHC class I-associated peptides. MHC-associated peptides presented on platelets of normal individuals and individuals with idiopathic thrombocytopenic purpura (ITP) were characterized. ITP is characterized by the premature immune destruction of platelets. It is associated with the production of antiplatelet autoantibodies, most often targeting platelet membrane GPIIb/IIIa or GPIb/IX. In addition to characterizing five fully and several partially sequenced peptides from platelets, the peptide GPRGA(L/I)S(L/I)(L/I) was identified from four of the five ITP patients. The anchor motif of this peptide correlates with the presence of the HLA-B7 allele. A BLAST search identified this peptide as GPIb (4-12). In conclusion, platelets from normal and ITP individuals can present peptides from general cellular proteins and platelet specific proteins, such as GPIb, to the immune system via MHC class I.

Dendritic cells (DCs) loaded with viral peptides are a potential form of immunotherapy of human immunodeficiency virus type 1 (HIV-1) infection. We show that DCs derived from blood monocytes of subjects with chronic HIV-1 infection on combination antiretroviral drug therapy have increases in expression of HLA, T-cell coreceptor, and T-cell activation molecules in response to the DC maturation factor CD40L comparable to those from uninfected persons. Mature DCs (mDCs) loaded with HLA A*0201-restricted viral peptides of the optimal length (9-mer) were more efficient at activating antiviral CD8+T cells than were immature DCs or peptide alone. Optimal presentation of these exogenous peptides required uptake and vesicular trafficking and was comparable in DCs derived from HIV-1-infected and uninfected persons. Furthermore, DCs from HIV-1-infected and uninfected persons had similar capacities to process viral peptides with C-terminal and N-terminal extensions through their proteasomal and cytosolic pathways, respectively. We conclude that DCs derived from HIV-1-infected persons have similar abilities to process exogenous peptides for presentation to CD8+T cells as those from uninfected persons. This conclusion supports the use of DCs loaded with synthetic peptides in immunotherapy of HIV-1 infection.

Measuring the interaction of class I human leukocyte antigens (HLA) and their peptide epitopes acts as a guide for the development of vaccines, diagnostics, and immune-based therapies. Here, we report the development of a sensitive biochemical assay that relies upon fluorescence polarization to indicate peptide interactions with recombinant soluble HLA proteins. It is a cell- and radioisotope-free assay that has the advantage of allowing the direct, real-time measurement of the ratio between free and bound peptide ligand in solution without separation steps. Peptide/HLA assay parameters were established using several HLA A*0201-specific fluorescein isothiocyanate-labeled peptides. Optimal loading of synthetic peptides into fully assembled soluble HLA-A*0201 complexes was enabled by thermal destabilization at 53 degrees C for 15 min, demonstrating that efficient peptide exchange does not require the removal of endogenous peptides from the reaction environment. An optimal ratio of three beta-2 microglobulin molecules per single HLA heavy chain was determined to maximize peptide binding. Kinetic binding studies indicate that soluble HLA-A*0201/peptide interactions are characterized by a range of moderate k(on) values (1 x 10(4) to 8.7 x 10(4) M(-1) s(-1)) and slow k(off) values (1.9 x 10(-4) to 4.3 x 10(-4) s(-1)), consistent with parameters for native HLA molecules. Testing of the A*0201-specific peptides with 48 additional class I molecules demonstrates that the unique peptide binding behavior of individual HLA molecules is maintained in the assay. This assay therefore represents a versatile tool for characterizing the binding of peptide epitopes during the development of class I HLA-based vaccines and immune therapies.

MHC class I molecules present host- and pathogen-derived peptides for immune surveillance. Much attention is given to the search for viral and tumor nonself peptide epitopes, yet the question remains, “What is self?” Analyses of Edman motifs and of small sets of individual peptides suggest that the class I self repertoire consists of thousands of different peptides. However, there exists no systematic characterization of this self-peptide backdrop, causing the definition of class I-presented self to remain largely hypothetical. To better understand the breadth and nature of self proteins sampled by class I HLA, we sequenced >200 endogenously loaded HLA-B*1801 peptides from a human B cell line. Peptide-source proteins, ranging from actin-related protein 6 to zinc finger protein 147, possessed an assortment of biological and molecular functions. Major categories included binding proteins, catalytic proteins, and proteins involved in cell metabolism, growth, and maintenance. Genetically, peptides encoded by all chromosomes were presented. Statistical comparison of proteins presented by class I vs the human proteome provides empiric evidence that the range of proteins sampled by class I is relatively unbiased, with the exception of RNA-binding proteins that are over-represented in the class I peptide repertoire. These data show that, in this cell line, class I-presented self peptides represent a comprehensive and balanced summary of the proteomic content of the cell. Importantly, virus- and tumor-induced changes in virtually any cellular compartment or to any chromosome can be expected to be presented by class I molecules for immune recognition.

Class I MHC molecules bind intracellular peptides for presentation to cytotoxic T lymphocytes. Identification of peptides presented by class I molecules during infection is therefore a priority for detecting and targeting intracellular pathogens. To understand which host-encoded peptides distinguish HIV-infected cells, we have developed a mass spectrometric approach to characterize HLA-B*0702 peptides unique to or up-regulated on infected T cells. In this study, we identify 15 host proteins that are differentially presented on infected human T cells. Peptides with increased expression on HIV-infected cells were derived from multiple categories of cellular proteins including RNA binding proteins and cell cycle regulatory proteins. Therefore, comprehensive analysis of the B*0702 peptide repertoire demonstrates that marked differences in host protein presentation occur after HIV infection.

Aim Successful pairing of a transplant recipient with a donor organ depends on accurate prediction of risk factors for immunologic rejection. As such, there is much interest in accurate clinical measurement of HLA Ab specificities in recipient sera. However, it has become apparent that current methodologies are still surrounded by shortcomings. Here we offer a new solution that is based on recent advancements made in the application of microarray technology in clinical diagnostics, to increase the precision and sensitivity of HLA Ab serology. The aim of this study was to demonstrate the coupling of recombinant soluble HLA proteins to a novel microarray technology as a new, simplified, inexpensive and accurate approach to transplant serology. Methods In order to validate and optimize sHLA microarray performance, multiple 12 × 12 microarrays were fabricated by microfluidic spotting of highly purified soluble HLA class I and II molecules under a range of conditions chosen to optimize protein stabilization on the microarray surface. Of particular interest were the evaluation of maximal signal and background intensities, signal uniformity between different HLA alleles and the structural integrity of the HLA proteins subsequent to microfluidic attachment to the microarray surface. Results HLA protein printed in this array system yielded >65,000 MFI with essentially no background signal. Signal uniformity among alleles tested was consistently below a 10% spread. As assessed by structure-specific antibodies HC-10 and W6/32, it was determined that the native dimer configuration predominated subsequent to microarray printing. Accuracy and reproducibility required for a low cost HLA antibody-screening array was validated with over 200 samples, resulting in a high degree of concordance with known monoclonal Ab patterns and established clinical reference sera. Conclusion We have successfully demonstrated that the combination of microarray and soluble HLA technology has resulted in a simple, accurate, and low cost method for high-performance multiplexed HLA Ab detection, which with additional development in progress will be ready to accommodate very high resolution HLA Ab screening, at time of transplantation and as a routine follow-up test to monitor the earliest stages of graft rejection.

Aim Rhesus macaques are a commonly used primate model for the study of viral infection. The class I MHC of these animals has been well studied at the genetic level. However functional ligandome characteristics such as peptide binding motif and length distribution have been elucidated in only a handful of macaque MHC (Mamu). Historically, Mamu ligandome characteristics have mimicked HLA-B despite the MAMU-A designation. Here we selected a high-frequency, uncharacterized, allele Mamu-A1∗004:01 to define the ligandome characteristics of this allele for comparison to HLA class I. Method Soluble MAMU-A1∗004:01 and HLA-B57:01 constructs were created and transfected these into the class I deficient cell line 721.221. sMHC harvested from transfected cells was affinity purified and ligand pools were isolated with an acid boil followed by 3 kDa cut-off ultrafiltration. Ligand pools were separated with two-dimentional LCMS. Data was acquired with a triple TOF mass spectrometer in information-dependant acquisition mode. Peptide sequences from the resulting MS2 fragment spectra were identified using the PEAKS and Mascot algorithms at a False Discovery Rate (FDR) of 1%. Results In this study, we identified 1447 ligands for Mamu-A1∗004. The peptide binding motif for MAMU-A1∗004 shows the P2 anchor as S, T, A and a PΩ anchor of W or F. This motif is strikingly similar to the HLA-B∗57:01 peptide binding motif. Indeed a comparison of the Mamu-A1∗004 and HLA-B∗57:01 eluted ligands from the same cell line shows that 32% of the Mamu-A1∗004 ligands are shared/identical with HLA-B∗57:01 ligands. Further, we established a Mamu class I peptide binding assay and demonstrated that known HLA-B57 HIV ligands bind to MAMU-A1∗004 with the same affinities. Although these allomorphs are similar in their motif, the length preferences are different: Compared to HLA-B57, Mamu-A1∗004 prefers nonamers and shows less tolerance for long (>13 amino acids) ligands. Conclusion Here we found that a common Rhesus macaque allele Mamu-A1∗004 is functionally homologous to HLA-B∗57:01. In humans, HLA-B∗57:01 is considered to be an important protective allele in HIV infections. These data enable the study of an HLA-B∗57:01-like molecule in non-human primate models of SIV infection. C. McMurtrey: Consultant; Company/Organization; Pure Protein LLC. R. Buchli: Employee; Company/Organization; Pure Protein LLC. W. Hildebrand: Scientific/Medical Advisor; Company/Organization; Pure Protein LLC.