Non-mutational neoantigens in disease.

The ability of mammals to mount adaptive immune responses culminating with the establishment of immunological memory is predicated on the ability of the mature T cell repertoire to recognize antigenic peptides presented by syngeneic MHC class I and II molecules. Although it is widely believed that mature T cells are highly skewed towards the recognition of antigenic peptides originating from genetically diverse (for example, foreign or mutated) protein-coding regions, preclinical and clinical data rather demonstrate that novel antigenic determinants efficiently recognized by mature T cells can emerge from a variety of non-mutational mechanisms. In this Review, we describe various mechanisms that underlie the formation of bona fide non-mutational neoantigens, such as epitope mimicry, upregulation of cryptic epitopes, usage of non-canonical initiation codons, alternative RNA splicing, and defective ribosomal RNA processing, as well as both enzymatic and non-enzymatic post-translational protein modifications. Moreover, we discuss the implications of the immune recognition of non-mutational neoantigens for human disease.

Mechanisms of Allosteric Inhibition of Insulin-Regulated Aminopeptidase.

Inhibition of Insulin-Regulated Aminopeptidase is being actively explored for the treatment of several human diseases and several classes of inhibitors have been developed although no clinical applications have been reported yet. Here, we combine enzymological analysis with x-ray crystallography to investigate the mechanism employed by two of the most studied inhibitors of IRAP, an aryl sulfonamide and a 2-amino-4H-benzopyran named HFI-419. Although both compounds have been hypothesized to target the enzyme's active site by competitive mechanisms, we discovered that they instead target previously unidentified proximal allosteric sites and utilize non-competitive inhibition mechanisms. X-ray crystallographic analysis demonstrated that the aryl sulfonamide stabilizes the closed, more active, conformation of the enzyme whereas HFI-419 locks the enzyme in a semi-open, and likely less active, conformation. HFI-419 potency is substrate-dependent and fails to effectively block the degradation of the physiological substrate cyclic peptide oxytocin. Our findings demonstrate alternative mechanisms for inhibiting IRAP through allosteric sites and conformational restricting and suggest that the pharmacology of HFI-419 may be more complicated than initially considered. Such conformation-specific interactions between IRAP and small molecules can be exploited for the design of more effective second-generation allosteric inhibitors.

CD4 Effector TCR Avidity for Peptide on APC Determines the Level of Memory Generated.

Initial TCR affinity for peptide Ag is known to impact the generation of memory; however, its contributions later, when effectors must again recognize Ag at 5-8 d postinfection to become memory, is unclear. We examined whether the effector TCR affinity for peptide at this "effector checkpoint" dictates the extent of memory and degree of protection against rechallenge. We made an influenza A virus nucleoprotein (NP)-specific TCR transgenic mouse strain, FluNP, and generated NP-peptide variants that are presented by MHC class II to bind to the FluNP TCR over a broad range of avidity. To evaluate the impact of avidity in vivo, we primed naive donor FluNP in influenza A virus-infected host mice, purified donor effectors at the checkpoint, and cotransferred them with the range of peptides pulsed on activated APCs into second uninfected hosts. Higher-avidity peptides yielded higher numbers of FluNP memory cells in spleen and most dramatically in lung and draining lymph nodes and induced better protection against lethal influenza infection. Avidity determined memory cell number, not cytokine profile, and already impacted donor cell number within several days of transfer. We previously found that autocrine IL-2 production at the checkpoint prevents default effector apoptosis and supports memory formation. Here, we find that peptide avidity determines the level of IL-2 produced by these effectors and that IL-2Rα expression by the APCs enhances memory formation, suggesting that transpresentation of IL-2 by APCs further amplifies IL-2 availability. Secondary memory generation was also avidity dependent. We propose that this regulatory pathway selects CD4 effectors of highest affinity to progress to memory.

PDIA3 epitope-driven immune autoreactivity contributes to hepatic damage in type 2 diabetes.

A diet rich in saturated fat and carbohydrates causes low-grade chronic inflammation in several organs, including the liver, ultimately driving nonalcoholic steatohepatitis. In this setting, environment-driven lipotoxicity and glucotoxicity induce liver damage, which promotes dendritic cell activation and generates a major histocompatibility complex class II (MHC-II) immunopeptidome enriched with peptides derived from proteins involved in cellular metabolism, oxidative phosphorylation, and the stress responses. Here, we demonstrated that lipotoxicity and glucotoxicity, as driven by a high-fat and high-fructose (HFHF) diet, promoted MHC-II presentation of nested T and B cell epitopes from protein disulfide isomerase family A member 3 (PDIA3), which is involved in immunogenic cell death. Increased MHC-II presentation of PDIA3 peptides was associated with antigen-specific proliferation of hepatic CD4+ immune infiltrates and isotype switch of anti-PDIA3 antibodies from IgM to IgG3, indicative of cellular and humoral PDIA3 autoreactivity. Passive transfer of PDIA3-specific T cells or PDIA3-specific antibodies also exacerbated hepatocyte death, as determined by increased hepatic transaminases detected in the sera of mice subjected to an HFHF but not control diet. Increased humoral responses to PDIA3 were also observed in patients with chronic inflammatory liver conditions, including autoimmune hepatitis, primary biliary cholangitis, and type 2 diabetes. Together, our data indicated that metabolic insults caused by an HFHF diet elicited liver damage and promoted pathogenic immune autoreactivity driven by T and B cell PDIA3 epitopes.

Phenylsulfamoyl Benzoic Acid Inhibitor of ERAP2 with a Novel Mode of Inhibition.

ERAP1 and ERAP2 are endoplasmic reticulum zinc-binding aminopeptidases that play crucial roles in processing peptides for loading onto class I major histocompatibility complex proteins. These enzymes are therapeutic targets in cancer and autoimmune disorders. The discovery of inhibitors specific to ERAP1 or ERAP2 has been challenging due to the similarity in their active site residues and domain architectures. Here, we identify 4-methoxy-3-{[2-piperidin-1-yl-4-(trifluoromethyl) phenyl] sulfamoyl} benzoic acid (compound 61) as a novel inhibitor of ERAP2 and determine the crystal structure of ERAP2 bound to compound 61. Compound 61 binds near the catalytic center of ERAP2, at a distinct site from previously known peptidomimetic inhibitors, and inhibits by an uncompetitive mechanism. Surprisingly, for ERAP1, compound 61 was found to activate model substrate hydrolysis, similarly to the previously characterized 5-trifluoromethyl regioisomer of compound 61, known as compound 3. We characterized the specificity determinants of ERAP1 and ERAP2 that control the binding of compounds 3 and 61. At the active site of ERAP1, Lys380 in the S1' pocket is a key determinant for the binding of both compounds 3 and 61. At the allosteric site, ERAP1 binds either compound, leading to the activation of model substrate hydrolysis. Although ERAP2 substrate hydrolysis is not activated by either compound, the mutation of His904 to alanine reveals a cryptic allosteric site that allows for the activation by compound 3. Thus, we have identified selectivity determinants in the active and allosteric sites of ERAP2 that govern the binding of two similar compounds, which potentially could be exploited to develop more potent and specific inhibitors.

SepA Enhances Shigella Invasion of Epithelial Cells by Degrading Alpha-1 Antitrypsin and Producing a Neutrophil Chemoattractant.

Shigella spp. are highly adapted pathogens that cause bacillary dysentery in human and nonhuman primates. An unusual feature of Shigella pathogenesis is that this organism invades the colonic epithelia from the basolateral pole. Therefore, it has evolved the ability to disrupt the intestinal epithelial barrier to reach the basolateral surface. We have shown previously that the secreted serine protease A (SepA), which belongs to the family of serine protease autotransporters of Enterobacteriaceae, is responsible for the initial destabilization of the intestinal epithelial barrier that facilitates Shigella invasion. However, the mechanisms used by SepA to regulate this process remain unknown. To investigate the protein targets cleaved by SepA in the intestinal epithelium, we incubated a sample of homogenized human colon with purified SepA or with a catalytically inactive mutant of this protease. We discovered that SepA targets an array of 18 different proteins, including alpha-1 antitrypsin (AAT), a major circulating serine proteinase inhibitor in humans. In contrast to other serine proteases, SepA cleaved AAT without forming an inhibiting complex, which resulted in the generation of a neutrophil chemoattractant. We demonstrated that the products of the AAT-SepA reaction induce a mild but significant increase in neutrophil transepithelial migration in vitro. Moreover, the presence of AAT during Shigella infection stimulated neutrophil migration and dramatically enhanced the number of bacteria invading the intestinal epithelium in a SepA-dependent manner. We conclude that by cleaving AAT, SepA releases a chemoattractant that promotes neutrophil migration, which in turn disrupts the intestinal epithelial barrier to enable Shigella invasion. IMPORTANCEShigella is the second leading cause of diarrheal death globally. In this study, we identified the host protein targets of SepA, Shigella's major protein secreted in culture. We demonstrated that by cleaving AAT, a serine protease inhibitor important to protect surrounding tissue at inflammatory sites, SepA releases a neutrophil chemoattractant that enhances Shigella invasion. Moreover, SepA degraded AAT without becoming inhibited by the cleaved product, and SepA catalytic activity was enhanced at higher concentrations of AAT. Activation of SepA by an excess of AAT may be physiologically relevant at the early stages of Shigella infection, when the amount of synthesized SepA is very low compared to the concentration of AAT in the intestinal lumen. This observation may also help to explain the adeptness of Shigella infectivity at low dose, despite the requirement of reaching the basolateral side to invade and colonize the colonic epithelium.

Conformational dynamics linked to domain closure and substrate binding explain the ERAP1 allosteric regulation mechanism.

The endoplasmic-reticulum aminopeptidase ERAP1 processes antigenic peptides for loading on MHC-I proteins and recognition by CD8 T cells as they survey the body for infection and malignancy. Crystal structures have revealed ERAP1 in either open or closed conformations, but whether these occur in solution and are involved in catalysis is not clear. Here, we assess ERAP1 conformational states in solution in the presence of substrates, allosteric activators, and inhibitors by small-angle X-ray scattering. We also characterize changes in protein conformation by X-ray crystallography, and we localize alternate C-terminal binding sites by chemical crosslinking. Structural and enzymatic data suggest that the structural reconfigurations of ERAP1 active site are physically linked to domain closure and are promoted by binding of long peptide substrates. These results clarify steps required for ERAP1 catalysis, demonstrate the importance of conformational dynamics within the catalytic cycle, and provide a mechanism for the observed allosteric regulation and Lys/Arg528 polymorphism disease association.

A protocol for qualitative and quantitative measurement of endosomal processing using hot spot analysis.

A detailed quantification of antigen processing by endosomal compartments provides important information on the pattern of protein fragmentation. Here, we describe a protocol that combines gradient purified endosomes, incubated with antigens, followed by hot spot analysis of MS/MS-sequenced peptides. The analysis identifies differences in endosomal antigen processing by dendritic cells under diverse experimental conditions. For complete details on the use and execution of this protocol, please refer to Clement et al. (2021).

Distinguishing Signal From Noise in Immunopeptidome Studies of Limiting-Abundance Biological Samples: Peptides Presented by I-Ab in C57BL/6 Mouse Thymus.

Antigen presentation by MHC-II proteins in the thymus is central to selection of CD4 T cells, but analysis of the full repertoire of presented peptides responsible for positive and negative selection is complicated by the low abundance of antigen presenting cells. A key challenge in analysis of limiting abundance immunopeptidomes by mass spectrometry is distinguishing true MHC-binding peptides from co-eluting non-specifically bound peptides present in the mixture eluted from immunoaffinity-purified MHC molecules. Herein we tested several approaches to minimize the impact of non-specific background peptides, including analyzing eluates from isotype-control antibody-conjugated beads, considering only peptides present in nested sets, and using predicted binding motif analysis to identify core epitopes. We evaluated these methods using well-understood human cell line samples, and then applied them to analysis of the I-Ab presented immunopeptidome of the thymus of C57BL/6 mice, comparing this to the more easily characterized splenic B cell and dendritic cell populations. We identified a total of 3473 unique peptides eluted from the various tissues, using a data dependent acquisition strategy with a false-discovery rate of <1%. The immunopeptidomes presented in thymus as compared to splenic B cells and DCs identified shared and tissue-specific epitopes. A broader length distribution was observed for peptides presented in the thymus as compared to splenic B cells or DCs. Detailed analysis of 61 differentially presented peptides indicated a wider distribution of I-Ab binding affinities in thymus as compared to splenic B cells. These results suggest different constraints on antigen processing and presentation pathways in central versus peripheral tissues.

Pleiotropic consequences of metabolic stress for the major histocompatibility complex class II molecule antigen processing and presentation machinery.

Hyperglycemia and hyperlipidemia are often observed in individuals with type II diabetes (T2D) and related mouse models. One dysmetabolic biochemical consequence is the non-enzymatic reaction between sugars, lipids, and proteins, favoring protein glycation, glycoxidation, and lipoxidation. Here, we identified oxidative alterations in key components of the major histocompatibility complex (MHC) class II molecule antigen processing and presentation machinery in vivo under conditions of hyperglycemia-induced metabolic stress. These modifications were linked to epitope-specific changes in endosomal processing efficiency, MHC class II-peptide binding, and DM editing activity. Moreover, we observed some quantitative and qualitative changes in the MHC class II immunopeptidome of Ob/Ob mice on a high-fat diet compared with controls, including changes in the presentation of an apolipoprotein B100 peptide associated previously with T2D and metabolic syndrome-related clinical complications. These findings highlight a link between glycation reactions and altered MHC class II antigen presentation that may contribute to T2D complications.

CDR3α drives selection of the immunodominant Epstein Barr virus (EBV) BRLF1-specific CD8 T cell receptor repertoire in primary infection.

The T cell receptor (TCR) repertoire is an essential component of the CD8 T-cell immune response. Here, we seek to investigate factors that drive selection of TCR repertoires specific to the HLA-A2-restricted immunodominant epitope BRLF1109-117 (YVLDHLIVV) over the course of primary Epstein Barr virus (EBV) infection. Using single-cell paired TCRαß sequencing of tetramer sorted CD8 T cells ex vivo, we show at the clonal level that recognition of the HLA-A2-restricted BRLF1 (YVL-BR, BRLF-1109) epitope is mainly driven by the TCRα chain. For the first time, we identify a CDR3α (complementarity determining region 3 α) motif, KDTDKL, resulting from an obligate AV8.1-AJ34 pairing that was shared by all four individuals studied. This observation coupled with the fact that this public AV8.1-KDTDKL-AJ34 TCR pairs with multiple different TCRß chains within the same donor (median 4; range: 1-9), suggests that there are some unique structural features of the interaction between the YVL-BR/MHC and the AV8.1-KDTDKL-AJ34 TCR that leads to this high level of selection. Newly developed TCR motif algorithms identified a lysine at position 1 of the CDR3α motif that is highly conserved and likely important for antigen recognition. Crystal structure analysis of the YVL-BR/HLA-A2 complex revealed that the MHC-bound peptide bulges at position 4, exposing a negatively charged aspartic acid that may interact with the positively charged lysine of CDR3α. TCR cloning and site-directed mutagenesis of the CDR3α lysine ablated YVL-BR-tetramer staining and substantially reduced CD69 upregulation on TCR mutant-transduced cells following antigen-specific stimulation. Reduced activation of T cells expressing this CDR3 motif was also observed following exposure to mutated (D4A) peptide. In summary, we show that a highly public TCR repertoire to an immunodominant epitope of a common human virus is almost completely selected on the basis of CDR3α and provide a likely structural basis for the selection. These studies emphasize the importance of examining TCRα, as well as TCRß, in understanding the CD8 T cell receptor repertoire.

Inactivating mutations and X-ray crystal structure of the tumor suppressor OPCML reveal cancer-associated functions.

OPCML, a tumor suppressor gene, is frequently silenced epigenetically in ovarian and other cancers. Here we report, by analysis of databases of tumor sequences, the observation of OPCML somatic missense mutations from various tumor types and the impact of these mutations on OPCML function, by solving the X-ray crystal structure of this glycoprotein to 2.65 Å resolution. OPCML consists of an extended arrangement of three immunoglobulin-like domains and homodimerizes via a network of contacts between membrane-distal domains. We report the generation of a panel of OPCML variants with representative clinical mutations and demonstrate clear phenotypic effects in vitro and in vivo including changes to anchorage-independent growth, interaction with activated cognate receptor tyrosine kinases, cellular migration, invasion in vitro and tumor growth in vivo. Our results suggest that clinically occurring somatic missense mutations in OPCML have the potential to contribute to tumorigenesis in a variety of cancers.

Recognition of Listeria Infection by Germline Elements of the Vγ1.1 Vδ6.3 TCR.

γδNKT cells are an abundant γδT cell population with restricted Vγ1.1 Vδ6.3 gene usage and phenotypic and functional similarity to conventional αß-invariant NKT cells. The γδNKT population responds to Listeria infections, but specific ligands are not known. In this work, we studied the CDR3 requirements of the γδNKT TCR, Vγ1.1Vδ6.3 for recognizing naive macrophages, and macrophages infected with Listeria We expressed four different variants of the Vγ1.1Vδ6.3 TCR in TCR-deficient hybridomas, one with germline-encoded sequences and three with nongermline-encoded sequences. All of the hybridomas were activated when cultured in the presence of macrophages, and the activation was increased when the macrophages were infected with Listeria This indicates that these TCRs can recognize a self-ligand present in macrophages and suggests that the ligand is modified or upregulated when the cells are infected with Listeria One of the three nongermline-encoded Vγ1.1 variants induced a lower activation level compared with the other variants tested in this study, suggesting that recognition of the Listeria-induced ligand involves the CDR3γ region of the TCR.

MHC-I peptide binding activity assessed by exchange after cleavage of peptide covalently linked to ß2-microglobulin.

A common approach to measuring binding constants involves combining receptor and ligand and measuring the distribution of bound and free states after equilibration. For class I major histocompatibility (MHC-I) proteins, which bind short peptides for presentation to T cells, this approach is precluded by instability of peptide-free protein. Here we develop a method wherein a weakly-binding peptide covalently attached to the N-terminus of the MHC-I ß2m subunit is released from the peptide binding site after proteolytic cleavage of the linker. The resultant protein is able to bind added peptide. A direct binding assay and method for estimation of peptide binding constant (Kd) are described, in which fluorescence polarization is used to follow peptide binding. A competition binding assay and method for estimation of inhibitor binding constant (Ki) using the same principle also are also described. The method uses a cubic equation to relate observed binding to probe concentration, probe Kd, inhibitor concentration, and inhibitor Ki under general reaction conditions without assumptions relating to relative binding affinities or concentrations. We also delineate advantages of this approach compared to the Cheng-Prusoff and Munson-Rodbard approaches for estimation of Ki using competition binding data.

Editing the immunopeptidome of melanoma cells using a potent inhibitor of endoplasmic reticulum aminopeptidase 1 (ERAP1).

The efficacy of cancer immunotherapy, including treatment with immune-checkpoint inhibitors, often is limited by ineffective presentation of antigenic peptides that elicit T-cell-mediated anti-tumor cytotoxic responses. Manipulation of antigen presentation pathways is an emerging approach for enhancing the immunogenicity of tumors in immunotherapy settings. ER aminopeptidase 1 (ERAP1) is an intracellular enzyme that trims peptides as part of the system that generates peptides for binding to MHC class I molecules (MHC-I). We hypothesized that pharmacological inhibition of ERAP1 in cells could regulate the cellular immunopeptidome. To test this hypothesis, we treated A375 melanoma cells with a recently developed potent ERAP1 inhibitor and analyzed the presented MHC-I peptide repertoire by isolating MHC-I, eluting bound peptides, and identifying them using capillary chromatography and tandem mass spectrometry (LC-MS/MS). Although the inhibitor did not reduce cell-surface MHC-I expression, it induced qualitative and quantitative changes in the presented peptidomes. Specifically, inhibitor treatment altered presentation of about half of the total 3204 identified peptides, including about one third of the peptides predicted to bind tightly to MHC-I. Inhibitor treatment altered the length distribution of eluted peptides without change in the basic binding motifs. Surprisingly, inhibitor treatment enhanced the average predicted MHC-I binding affinity, by reducing presentation of sub-optimal long peptides and increasing presentation of many high-affinity 9-12mers, suggesting that baseline ERAP1 activity in this cell line is destructive for many potential epitopes. Our results suggest that chemical inhibition of ERAP1 may be a viable approach for manipulating the immunopeptidome of cancer.

Naturally processed HLA-DR3-restricted HHV-6B peptides are recognized broadly with polyfunctional and cytotoxic CD4 T-cell responses.

Human herpes virus 6B (HHV-6B) is a widespread virus that infects most people early in infancy and establishes a chronic life-long infection with periodic reactivation. CD4 T cells have been implicated in control of HHV-6B, but antigenic targets and functional characteristics of the CD4 T-cell response are poorly understood. We identified 25 naturally processed MHC-II peptides, derived from six different HHV-6B proteins, and showed that they were recognized by CD4 T-cell responses in HLA-matched donors. The peptides were identified by mass spectrometry after elution from HLA-DR molecules isolated from HHV-6B-infected T cells. The peptides showed strong binding to matched HLA alleles and elicited recall T-cell responses in vitro. T-cell lines expanded in vitro were used for functional characterization of the response. Responding cells were mainly CD3+ CD4+ , produced IFN-γ, TNF-α, and low levels of IL-2, alone or in combination, highlighting the presence of polyfunctional T cells in the overall response. Many of the responding cells mobilized CD107a, stored granzyme B, and mediated specific killing of peptide-pulsed target cells. These results highlight a potential role for polyfunctional cytotoxic CD4 T cells in the long-term control of HHV-6B infection.

Molecular pathways for antigenic peptide generation by ER aminopeptidase 1.

Endoplasmic Reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that can generate or destroy potential peptide ligands for MHC class I molecules. ERAP1 activity influences the cell-surface immunopeptidome and epitope immunodominance patterns but in complex and poorly understood manners. Two main distinct pathways have been proposed to account for ERAP1's effects on the nature and quantity of MHCI-bound peptides: i) ERAP1 trims peptides in solution, generating the correct length for binding to MHCI or overtrimming peptides so that they are too short to bind, and ii) ERAP1 trims peptides while they are partially bound onto MHCI in manner that leaves the peptide amino terminus accessible. For both pathways, once an appropriate length peptide is generated it could bind conventionally to MHCI, competing with further trimming by ERAP1. The two pathways, although not necessarily mutually exclusive, provide distinct vantage points for understanding of the rules behind the generation of the immunopeptidome. Resolution of the mechanistic details of ERAP1-mediated antigenic peptide generation can have important consequences for pharmacological efforts to regulate the immunopeptidome for therapeutic applications, and for understanding association of ERAP1 alleles with susceptibility to autoimmune disease and cancer. We review current evidence in support of these two pathways and discuss their relative importance and potential complementarity.

HLA-DO Modulates the Diversity of the MHC-II Self-peptidome.

Presentation of antigenic peptides on MHC-II molecules is essential for tolerance to self and for initiation of immune responses against foreign antigens. DO (HLA-DO in humans, H2-O in mice) is a nonclassical MHC-II protein that has been implicated in control of autoimmunity and regulation of neutralizing antibody responses to viruses. These effects likely are related to a role of DO in selecting MHC-II epitopes, but previous studies examining the effect of DO on presentation of selected CD4 T cell epitopes have been contradictory. To understand how DO modulates MHC-II antigen presentation, we characterized the full spectrum of peptides presented by MHC-II molecules expressed by DO-sufficient and DO-deficient antigen-presenting cells in vivo and in vitro using quantitative mass spectrometry approaches. We found that DO controlled the diversity of the presented peptide repertoire, with a subset of peptides presented only when DO was expressed. Antigen-presenting cells express another nonclassical MHC-II protein, DM, which acts as a peptide editor by preferentially catalyzing the exchange of less stable MHC-II peptide complexes, and which is inhibited when bound to DO. Peptides presented uniquely in the presence of DO were sensitive to DM-mediated exchange, suggesting that decreased DM editing was responsible for the increased diversity. DO-deficient mice mounted CD4 T cell responses against wild-type antigen-presenting cells, but not vice versa, indicating that DO-dependent alterations in the MHC-II peptidome could be recognized by circulating T cells. These data suggest that cell-specific and regulated expression of HLA-DO serves to fine-tune MHC-II peptidomes, in order to enhance self-tolerance to a wide spectrum of epitopes while allowing focused presentation of immunodominant epitopes during an immune response.

The N-terminal region of photocleavable peptides that bind HLA-DR1 determines the kinetics of fragment release.

Major Histocompatibility Complex class II (MHC-II) molecules bind peptides and present them to receptors on CD4+ T cells as part of the immune system's surveillance of pathogens and malignancy. In the absence of peptide, MHC-II equilibrates between peptide-receptive and peptide-averse conformations. The conversion between these forms has been postulated to be important in regulating cellular antigen presentation but has been difficult to study. In order to generate the MHC-II molecule HLA-DR1 in the peptide-receptive form, we designed and tested a series of photocleavable peptides that included the UV-sensitive 3-amino-3-(2-nitrophenyl)-propionate amino acid analog. They were intended to bind tightly to the HLA-DR1 MHC molecule, but to generate low-affinity fragments after UV exposure that would be released to yield HLA-DR1 in the peptide-receptive conformation. We were able to identify photocleavable peptides that bound tightly to HLA-DR1 and generated the peptide-receptive conformation after UV exposure. However, slow release of photocleaved peptide fragments from the binding site limited the rate of binding of an incoming labeled peptide and complicated kinetic measurements of the individual steps of the overall peptide binding reaction. Modification of the N-terminal region of the photocleavable peptide to reduce MHC-II pocket or H-bonding interactions allowed for generation of the peptide receptive form immediately after UV exposure with peptide fragments neither retained within the site nor interfering with binding of an incoming peptide. However this was achieved only at the expense of a substantial reduction in overall peptide binding affinity, and these peptides had such weak interaction with HLA-DR1 that they were easily exchanged by incoming peptide without UV exposure. These results show that photocleavable peptides can be used to generate peptide-receptive HLA-DR1 and to facilitate peptide exchange in generation of specific peptide-MHC-II complexes, but that usage of these peptides for kinetic studies can be constrained by slow fragment release.