
==== Front
Mol Cell Proteomics
Mol Cell Proteomics
Molecular & Cellular Proteomics : MCP
1535-9476
1535-9484
American Society for Biochemistry and Molecular Biology

S1535-9476(24)00113-0
10.1016/j.mcpro.2024.100823
100823
Perspective
Special Issue: Celebrating the Career of Donald F. Hunt: Perspective
Innovations Toward Immunopeptidomics
Abelin Jennifer G. jabelin@broadinstitute.org
jenn.abelin@gmail.com
1∗
Cox Andrea L. 23
1 Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA
2 Johns Hopkins Bloomberg School of Public Health, W. Harry Feinstone Department of Molecular Microbiology and Immunology, Baltimore, Maryland, USA
3 Division of Infectious Diseases, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
∗ For correspondence: Jennifer G. Abelin jabelin@broadinstitute.orgjenn.abelin@gmail.com
31 7 2024
9 2024
31 7 2024
23 9 1008236 5 2024
24 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Over the past 30 years, immunopeptidomics has grown alongside improvements in mass spectrometry technology, genomics, transcriptomics, T cell receptor sequencing, and immunological assays to identify and characterize the targets of activated T cells. Together, multiple research groups with expertise in immunology, biochemistry, chemistry, and peptide mass spectrometry have come together to enable the isolation and sequence identification of endogenous major histocompatibility complex (MHC)-bound peptides. The idea to apply highly sensitive mass spectrometry techniques to study the landscape of peptide antigens presented by cell surface MHCs was innovative and continues to be successfully used and improved upon to deepen our understanding of how peptide antigens are processed and presented to T cells. Multiple research groups were involved in this bringing immunopeptidomics to the forefront of translational research, and we will highlight the contributions of one of the earliest developers, Professor Donald F. Hunt, and his research group at the University of Virginia. The Hunt laboratory applied cutting edge mass spectroscopy–based immunopeptidomics to study cancer, autoimmunity, transplant rejection, and infectious diseases. Across these diverse research areas, the Hunt laboratory and collaborators would characterize previously unknown MHC peptide–binding motifs and identify immunologically active antigens using ultra sensitive mass spectrometry techniques. Amazingly, many of the MHC-bound peptide antigens discovered in collaborations with the Hunt laboratory were sequenced by mass spectrometry before the completion of the human genome using manual de novo sequencing. In this perspective article, we will chronicle the work of the Hunt laboratory and their many collaborators that would be a major part of the foundation for mass spectrometry–based immunopeptidomics and its application to immunology research.

Graphical Abstract

Highlights

• Donald F. Hunt pioneered the use of mass spectrometry to sequence MHC-bound peptides.

• The Hunt lab and collaborators applied immunopeptidomics to study diverse diseases.

• Immunopeptidomics is used to study cancer, autoimmunity, and infectious diseases.

• We thank Donald F. Hunt for his contributions to the field of immunopeptidomics.

In Brief

In celebration of the career of Donald F. Hunt, we chronicle the contributions of Don, the Hunt laboratory and their collaborators to the field of mass spectrometry–based immunopeptidomics in this perspective article.

Keywords

immunopeptidomics
HLA
MHC
antigen
peptidomics
Abbreviations

ETD electron-transfer dissociation

FETD front-end ETD

HLA human leukocyte antigen

MART-1 melanoma-associated antigen recognized by T cell

MHC major histocompatibility complex

MS mass spectrometry

pMHC MHC-bound peptide

PTM posttranslational modifications

TAA tumor-associated antigen

TAP transporter associated with antigen processing
==== Body
pmcThe discovery that peptide antigens are presented to T cells by cell surface proteins expressed from the major histocompatibility complex (MHC), or human leukocyte antigen (HLA) in humans (1, 2, 3, 4, 5, 6), has spurred efforts to characterize the populations of MHC-bound peptides (pMHCs) unique to diverse cell types and disease states. Although the term immunopeptidome (7) was first applied years after the earliest efforts to sequence pMHC, we will use it to describe the application of mass spectrometry (MS) to identify the sequences of pMHC. The field of immunopeptidomics is at the forefront of discovery again, in part due to revitalized interest in T cell targeting immunotherapies (8) and in rationalized vaccine design that was sparked by the global coronavirus disease 2019 pandemic (9). These events have been accompanied by exponential improvement in MS technology, which has enabled hundreds of immunopeptidomics studies focused on improving our understanding the rules of peptide antigen processing and presentation and the direct identification of disease specific pMHCs for the development of immunotherapeutics.

While many research groups have contributed to our overall understanding of the immunopeptidome (10, 11, 12), this perspective article will highlight the contributions of Professor Donald F. Hunt (Don), members of his laboratory, and his collaborators, especially his longtime collaborators Craig Slingluff and Victor Engelhard at the University of Virginia. Although we have not named all of the collaborators in the research described for the sake of being concise, the research was in many cases initiated by collaborators and in every case depended on the expertise and hard work of many individual contributors. Prior to the seminal 1992 Science articles that describe the sequencing of peptides bound to HLA-A∗02 (13, 14) and the murine MHC-II molecule I-Ad (15), the Hunt laboratory was developing and applying at the time the most sensitive (femtomolar detection) MS techniques to sequence peptides and proteins. Their expertise and the foresight to pair microcapillary liquid chromatography–electrospray ionization with tandem MS led to the advance of sequencing femtomole amounts of peptides isolated from endogenously processed and presented pMHC. This was a spectacular achievement, and one that would enable many MS-based immunopeptidomics methods in use today.

Don's vision to juxtapose next generation proteomic technology development in combination with highly collaborative science to address important biological questions facilitated rapid advancements in immunopeptidomics. This vision continues to serve a model for how MS-based research can play a major role in translational medicine. It is an honor to write this perspective, and we are humbled that many of the immunopeptidomics research areas initially investigated by the Hunt laboratory and colleagues remain active today. Most importantly, we want to thank Don for his contributions to the field of immunopeptidomics and for his unwavering commitment to the training and support for the future generation of mass spectrometrists.

1990 to 2000

In 1986, the Hunt laboratory published their influential peptide sequencing paper (16) leveraging tandem MS on a triple-quadrupole mass spectrometer to sequence peptides from apolipoprotein B digested with cyanogen bromide and trypsin. A few years later, John B. Fenn reported on electrospray ionization (17), which was a key breakthrough that revolutionized MS and was awarded the 2002 Nobel Prize in Chemistry. In 1991, Rammensee and colleagues (18) reported the sequence motifs of naturally processed peptides from immunopurified pMHC from three murine MHC and from human HLA-A∗02 using HPLC fractionation and Edman degradation. This importantly revealed distinct allele-specific peptide motifs, but did not provide individual peptide sequences. Building upon these innovations in both MS technology and our understanding that peptides that bind to MHC molecules require specific amino acid motifs, the Hunt Laboratory and colleagues demonstrated in 1992 that endogenously processed pMHC could be directly sequenced at the individual peptide level using online microcapillary liquid chromatography paired with electrospray tandem MS (13, 15). These rapid proteomic developments led to the characterization of novel allele-specific and minor histocompatibility antigens and a de novo–sequenced neoantigen, all of which remain active areas of research in the field of immunopeptidomics today.

Learning the Rules of MHC Peptide Processing and Presentation

The application of the MS techniques pioneered in the Hunt Lab to directly sequence peptides eluted from HLA-A∗01:01 molecules created the tools to define other MHC peptide–binding motifs (13, 19, 20). This MS-based approach was also used to sequence peptides bound to class II MHCs in collaboration with Alessandro Sette and colleagues, who generated in vitro MHC-binding data using synthetic peptides derived from nested sets. Their data suggested that the peptide binding groove on class II molecules was open at both ends, as was subsequently confirmed via an orthogonal X-ray crystallography approach (15, 21). Additionally, a nonclassical MHC molecule targeted by gamma delta T cells, T10b, was found by Kaliyaperumal et al. not to associate with polymorphic peptides typical of classical MHC class I molecules and was expressed in a functionally active form in the absence of peptides on the cell surface, highlighting that not all T cell responses are directed at pMHC (22). Immunopeptidomics was also used to advance our understanding of peptide processing and presentation pathways (23, 24, 25, 26, 27, 28).

As immunopeptidomics was in its earliest days, discoveries in the field of immunology continued to provide opportunities for this technology to be utilized. For example, through mutation of peptides at anchor residues, the amino acids that interact with the MHC molecule and assessing binding of peptides of varying lengths, Chen et al. established that MHC molecules accommodate the same peptide in different conformations, increasing the chances for recognition of a given peptide by a randomly generated T cell receptor (26). With this new understanding of MHC peptide presentation, the Hunt lab was involved with multiple collaborations aimed at characterizing MHC-presented peptides from cells with mutations in pathways potentially implicated in antigen presentation. A series of immunopeptidomics studies of naturally processed peptides bound by class II MHC molecules by normal and mutant antigen-presenting cells helped define the role of invariant chain in the assembly, intracellular transport, and function of MHC class II molecules (23, 24, 29). Similarly, immunopeptidomics applied by Henderson et al. to cells lacking transporter associated with antigen processing (TAP) demonstrated that TAP is essential for normal, broad MHC peptide presentation on the cell surface due to its role in peptide delivery from the cytosol into the lumen of the endoplasmic reticulum, where peptides are loaded on class I MHC molecules (23, 24, 29). These influential works helped define basic rules for peptide presentation to T cells.

Immunopeptidomics studies that characterized normal cells in part sparked the quest to identify specific peptide targets of CD8+ T cells, particularly those present on malignant cells (30, 31, 32, 33, 34, 35). While immunotherapy is a central and essential component of effective cancer therapy today, the field was still emerging in the 1990s. There was evidence of spontaneous immune control of melanoma and Slingluff et al. demonstrated that pMHC on human melanoma cell lines were recognized by CD8+ T cells isolated from multiple patients with melanoma (36). The hope was that the identification of the peptides targeted by T cells might advance understanding of the immune response to tumors and identify a way to enhance specific targeting of melanoma. The subsequent search to find among the tens of thousands of pMHC on the cell surface, the single shared epitope targeted by T cells from melanoma patients was the proverbial needle in the haystack. HPLC was applied, but the similar size, shape, and hydrophobicity of the peptides made it impossible to separate the peptides into fractions containing few enough peptides to sequence. As he often did through decades of partnership with Don, Dr Jeffery Shabanowitz (Jeff) applied his technical expertise to solve the problem by creating an online microcapillary HPLC column effluent splitter that deposited peptide fractions through a capillary representing one-sixth of the column effluent into micro wells containing culture media to assess for T cell recognition, while the remaining material passed through a second capillary into the electrospray ionization source and mass spectra on each of the peptide fractions deposited into the wells were obtained (Fig. 1) (30). Through this novel technique, Cox et al. sequenced the recognized peptide, found to be derived from the melanocyte protein premelanosome protein, also known as glycoprotein 100 (gp100) (30). Present in normal melanocytes, this peptide is a tumor-associated antigen (TAA), which are derived from overexpressed proteins which may also be present in normal cells at low levels. The identification of the gp100 TAA was consistent with prior observations that T cells specific for melanoma could in some cases kills normal melanocytes and that spontaneous remission of melanoma was in some cases accompanied by vitiligo, skin depigmentation caused by T cell destruction of normal melanocytes (30). Additional protein premelanosome protein and melanoma-associated antigen recognized by T cells (MART-1) were identified as TAA targets of melanoma as specific T cells (32, 34, 37). Skipper et al. further demonstrated that a posttranslationally modified tyrosinase peptide, an aspartic acid (D) was found instead of the expected asparagine (N), was targeted by melanoma-specific T cells, establishing that TAAs can also arise from posttranslational modifications (PTMs) of normal host proteins (32).Fig. 1 Overview of the splitter workflow that enabled simultaneous immunopeptidomics and immunogenicity testing of HLA peptide fractions. A, microcapillary LC-MS/MS system with splitter to direct the HLA peptide effluent simultaneously to the mass spectrometer and to wells of a microtiter plate for downstream T cell reactivity assays. B, to create a zero dead volume splitter, a PRP-1 (Hamilton) microcapillary HPLC column (100 um by 22 cm) was connected to two small capillaries of different lengths and interior diameters (25 um and 40 um ID, Polymicro Technologies). The HPLC column was eluted into the union and 1/6 of the material was deposited into 50 uL of culture media in microtiter plate wells. The larger of the two capillaries directed the remaining 5/6 of the material was used for LC-MS/MS analysis on a Finnigan-MAT triple quadrupole mass spectrometer. C, example MS1 spectra of a peptide fraction shown in (D) that activated patient T cells. HLA, HLA, human leukocyte antigen; MS, mass spectrometry.

The splitter approach was then successfully applied to identification of additional tumor antigens across multiple collaborations. For example, Slingluff et al. isolated pMHC and confirmed the existence of a CD8+ T cell response against autologous human squamous cell cancer of the lung in 1994 (38). Two years later, Huang et al. determined that the immunodominant MHC class I–restricted antigen of a murine colon tumor derives from an endogenous retroviral gene product, another class of tumor antigens (33). In the year that followed, Dubey et al. confirmed that neoantigen recognition can result in tumor control with the identification of a single amino acid substitution in the DEAD box helicase p68 responsible for T cell–mediated tumor rejection in a mouse model (39). Neoantigens are the result of DNA mutations that produce amino acid changes unique to the tumor that can serve as the target of tumor-specific T cell responses. In 1998, subsequent identification of peptide epitopes for lung cancer–specific CD8+ T cells by Hogan et al. serendipitously revealed single amino acid mutations in the host proteins such as elongation factor 2, demonstrating the existence of neoantigens recognized by T cells that lyse lung cancer cells (35). Amazingly, this neoantigen was identified prior to the completion of the human genome and confirmed using targeted PCR and DNA sequencing of the elongation factor 2 gene (39). The sum of this work led to better understanding of different classes of T cell targets on tumors, and today neoantigen vaccines and other forms of T cell targeting immunotherapy are currently being pursued (40, 41).

During the 1990 s, the Hunt laboratory also significantly advanced our understanding of tolerance, autoreactivity, xenoreactivity, and alloreactivity (42, 43, 44, 45, 46, 47). Central to current understanding of how thymic selection gives rise to self tolerant T cells capable of MHC-restricted antigen recognition, Hu et al. directly examined how peptide/MHC ligands expressed on thymic epithelial cells trigger positive selection of CD8+ T cells (46). This provided an explanation for how the specific recognition of a limited repertoire of thymic self-peptides selects a diverse repertoire of T cells. Crotzer et al. demonstrated that epitope abundance did not determine immunodominance of T cell responses (48). Den Haan and colleagues identified the peptide and MHC that defined the minor histocompatibility antigen HA-2, isolated from a patient with severe graft versus host disease after HLA-identical bone marrow transplantation. Wang and colleagues identified the target of an alloreactive T cell clone, demonstrating that peptide was present on the surface at the density of several other epitopes presented by HLA-A∗02:01 to self MHC-restricted T cells. In addition, the alloreactive T cell had an affinity similar to that of many self MHC-restricted T cells. This showed that alloreactive responses can be directed against antigenic determinants of low abundance and are not limited by a lower affinity of T cells for nonself MHC molecules. Wang and colleagues also showed that alloreactivity was not just driven by recognition of foreign MHC but required peptides bound to the foreign MHC (47). These findings collectively advanced our understanding of normal T cell development, autoimmunity, graft versus host disease, and other T cell–mediated disease in organ and bone marrow transplantation.

2000 to 2010

The 2000 s was a decade of astonishing scientific and technological achievements. The human genome was reported in 2003, which enabled more accurate peptide mapping using both manual de novo sequencing and database searching algorithms. MS technology for peptide sequencing was also moving from low resolution mass measurements to high resolution with the development of the Thermo LTQ-FT and the Orbitrap, both of which entered the Hunt laboratory between 2002 and 2006. The combination of high resolution accurate mass measurements and the genomic sequences of protein coding genes accelerated immunopeptidomics research through the use of tools, such as MASCOT (49, 50) to support peptide sequencing. Additionally, the Hunt laboratory’s invention of electron-transfer dissociation (ETD) in 2004 (51), a method of fragmenting multiply charged peptide ions that maintains labile PTMs, would be applied to characterize phosphorylated pMHC (52). Overall, this decade encompassed immunopeptidomics advances in our understanding of antigen presentation in the context of autoimmunity, cancer, viral, and bacterial infections by the Hunt laboratory and colleagues.

Understanding Antigen Presentation and TAAs

Investigating the processing pathway that governs pMHC presentation is critical for deepening our understanding of pMHC presentation to T cells. The Hunt laboratory was able to leverage MS-based immunopeptidomics to help address knowledge gaps in the field of pMHC antigen presentation. During the early 2000s, the assembly, transport, and peptide binding to MHC-II molecules was relatively well characterized, however the proteolytic processing steps involved in MHC-II presentation were not well understood. Therefore, the Hunt laboratory and collaborators began efforts to sequence the MHC-II immunopeptidome from a cell line that was homozygous for HLA-DR∗04:01, which enabled confident assignments of peptides to HLA heterodimers (53). These data suggested that most MHC-II–binding peptides begin as longer sequences that are proteolytically cleaved at both the amino and C-terminal ends after the initial binding. The Hunt laboratory also explored aspects of MHC-I presentation, including collaborating on a study demonstration that tapasin helps to stabilize the peptide-free MHC-I molecules and can increase the diversity of the immunopeptidome (54). Beyond understanding the mechanisms behind antigen presentation, the Hunt laboratory also utilized immunopeptidomics to better directly characterize TAAs presented in the context of melanoma (55, 56, 57), PTMs on pMHC (58), and minor histocompatibility antigens involved in transplant rejection (59). These early studies established that immunopeptidomics can be utilized to learn the rules of MHC-I and MHC-II peptide presentation and identify antigens that can be used as immunotherapeutic targets.

Phosphorylated HLA Peptides as Tumor Antigens

In 1998, Hogan et al. identified the first example of a naturally processed and presented phosphorylated pMHC (35). This was a serendipitous finding, as this peptide was discovered while using the splitter approach to identify antigens that activate T cells in lung cancer when it was not yet broadly accepted that phosphorylated peptides would survive the MHC class I processing and presentation pathway. Shortly after, Andersen and colleagues reported that synthetic phosphorylated MHC-I peptides could be transported by TAP into the endoplasmic reticulum, where they were able to bind to MHC molecules and be recognized by cytotoxic T cells (60). To directly explore the landscape of naturally presented phosphorylated pMHC, the Hunt laboratory and their collaborators in the Engelhard laboratory would go on to develop and apply immobilized metal affinity chromatography enrichment to enrich and characterize phosphorylated pMHC in the immunopeptidome (61). Over the next decade, they would continue this collaboration to directly identify phosphorylated pMHC and provide support that these posttranslationally modified peptides, which could be presented both by MHC-I and MHC-II (52), were putative targets for future immunotherapies (62, 63).

Beyond Cancer: Immunopeptidomics of Autoimmune and Infectious Diseases

A majority of the immunopeptidomics studies at this time were cancer focused, however the Hunt laboratory demonstrated that this MS-based technology could also be used to improve our understanding of the antigens involved in autoimmune diseases. This included studies of multiple sclerosis (64) and diabetes (65, 66). Many autoimmune diseases display immune responses that are organ-specific, and identifying the pMHC that elicit these self-directed T cell responses can facilitate the development of future immunotherapies. Similarly, immunopeptidomics can also be leveraged to define and better understand the antigens involved in infectious disease, leading to better vaccine and immune targeting therapies. The Hunt laboratory also used immunopeptidomics to investigate how pMHC abundance impacts the immunodominance of Epstein–Barr virus transformed cells (48), as well as identifying class I pMHC derived from Mycobacterium tuberculosis (67). These early applications of immunopeptidomics to autoimmune and infectious disease established the value of this technology for improving our understanding of disease biology.

2010 to 2020

Advances in MS instrumentation continued throughout the 2010s alongside a reinvigorated era for immunotherapy with the FDA approval of checkpoint inhibitors (8). In the Hunt laboratory, the development ETD continued with the implementation of front-end ETD (FETD) (68) in 2008 with in-house modifications to the Thermo Orbitrap Classic and LTQ-FT instruments and able to be applied to immunopeptidomics (52, 69) because of its increased robustness. In 2011, a Thermo prototype FETD was installed on the Velos Pro and the commercially available FETD Thermo Fusion Tribrid arrived in 2014. All of these instrument platforms improved pMHC identification rates due to the ability to collect high-resolution MS1 and MS2 data and increases in acquisition speed and overall sensitivity. In addition, the Hunt laboratory leveraged sequential collision-induced dissociation and ETD spectra of each peptide precursor for de novo sequencing, as the complementary information in each increased sequence coverage. This is especially true for class II pMHC that are longer and have higher charge states, making them well suited for ETD fragmentation (51, 70).

During this time, the observation that higher mutation burden tumors had stronger responses to checkpoint blockade was made (71, 72), suggesting that neoantigens, pMHC-containing somatic mutations, were likely playing a role. Thus, many academic and industry groups became interested in using MS-based immunopeptidomics to assist in the identification of cancer-specific pMHC targets, especially neoantigens. This led to numerous partnerships between academic and industry groups, including the Hunt laboratory and a biotechnology company Agenus in 2015 that was interested in cancer-associated PTM pMHC that could be leveraged as immunotherapeutic targets. During the 2010 s, the Hunt laboratory identified novel PTM pMHC, helped to elucidate a mechanism for pMHC mediated drug hypersensitivity, and applied immunopeptidomics to primary human tumor samples.

Continuing to Learn Rules of Antigen Processing and Presentation

The Hunt laboratory furthered the use of MS-based immunopeptidomics to learn the peptide binding rules of MHCs that had not been profiled previously. This included human MHC alleles, like understudied MHC-C alleles (73), and multiple studies of primate pMHC, as these animals are valuable models of human disease because they exhibit similar symptoms of disease progression (74, 75). The study of nonhuman MHC molecules and their immunopeptidomes remains an important area of research, as animal models such as primates may be required for preclinical studies that are needed to show safety of some immunotherapies (76). We anticipate that as reagents evolve, immunopeptidomics will be applied to reveal the peptide binding rules of MHC molecules of other species to improve our understanding of disease biology and enable the development of new immunotherapeutics.

MHC Peptide Mediated Drug Hypersensitivity

Understanding the mechanisms behind drug-induced immune adverse events, which can include inflammation, pulmonary symptoms, and gastrointestinal, endocrine, and dermatologic toxicities, is crucial for being able to predict which patients may experience adverse events and improve the safety profiles of drugs and immunotherapies. Interestingly, clinical data has suggested some immune adverse responses are linked to specific HLA alleles. Because of this, exploring the immunopeptidomes of drug-treated and control samples may reveal which antigens are involved in these adverse events. One such example is the the antiviral drug abacavir and its association with adverse reactions in patients expressing HLA-B∗57:01 (77, 78). The Hunt laboratory applied immunopeptidomics to abacavir-treated samples and determined that the pMHC repertoire was significantly changed upon drug treatment (79). These data helped to demonstrate that abacavir can bind within the peptide-being groove of HLA-B∗57:01, altering the self immunopeptidome, which results in an adverse immune response similar to alloreactive T cell responses. Additionally, the group helped to demonstrate a similar effect on the immunopeptidome of acyclovir-treated cells expressing HLA-B∗57:01 (80) and nevirapine-treated cells expressing a group of HLA-C alleles sharing a common binding groove F pocket with HLA-C∗04:01 and class II alleles which share the HLA-DR∗B1-P4 pocket (81). Although nevirapine significantly altered the immunopeptidome, leading to adverse immune responses, acyclovir minimally altered without adverse effects. Therefore, these two extremes can be used to help establish thresholds to drug induced changes of the immunopeptidome that can be used to develop future assays, which are likely to include immunopeptidomics, to predict adverse immune events mediated by changes in MHC peptide presentation.

Posttranslationally Modified MHC Peptides Presented on Primary Tumor Samples

Although the Hunt laboratory had identified PTM pMHC on numerous cancer cell lines by this time, they applied their MHC phosphopeptide sequencing technology to primary tumor samples for the first time in 2013 (69, 82, 83). This coincided with an increased rate of detection from 10s to 100s of PTM peptides per sample. They also continued to work with their collaborators to combine structural studies to understand the antigenicity of tumor associated phosphorylated pMHC identified using immunopeptidomics (84), providing additional evidence that dysregulated signaling in cancer cells leads to tumor-associated phosphorylated pMHC presentation, and these tumor-associated peptides represent putative immunotherapeutic targets. The immunopeptidomics research article in this special issue by Mahoney et al. describes the most recent advances of the Hunt laboratory in the isolation and identification of posttranslationally modified pMHC.

Most immunopeptidome studies, including the cancer-associated phosphorylated pMHC investigations, were focused on MHC-I, yet CD4+ T cells and their MHC-II targets also play a role in antitumor responses. In 2009, it was first demonstrated that CD4+ T cells can specifically respond to tumor-specific phosphorylated pMHC, including a phosphorylated MART-1 presented by HLA-DRB1∗01:0152. These findings led to a study that elucidated the structural basis for the presentation of phosphorylated MHC-II peptides by solving the structure of a phosphorylated MART-1 peptides bound to HLA-DR1 in combination with measurements of peptide-MHC affinity and T cell recognition of truncated and substituted pMART-1 peptides (85). Novel PTMs presented by MHC-II were also discovered by the Hunt Laboratory including the characterization of glycosylated and disulfide-bonded peptides presented on the surface of cancer cells (86). The landscape of MHC-II immunopeptidome that contains PTMs, and how these PTMs change during disease progression and treatment remains an understudied area of immunopeptidomics research, likely due to the low level of many PTMs and the lack of robust enrichment techniques with low sample input requirements. We anticipate that as MS instrumentation becomes more sensitive and biochemical approaches are developed to improve the identification of PTMs from samples with minimal peptide input, we will continue to learn how PTMs on pMHC impacts T cell recognition in different diseases.

Paving the Road for Immunopeptidomics

In January 2023, Don retired with Emeritus from his role as a mentor and University Professor of Chemistry and Pathology at the University of Virginia after over 50 years of teaching. His retirement exemplified his personality, quiet yet profound. We can confidently say that our time in the Hunt laboratory was wonderful. We were always supported, encouraged, and provided with the ultimate academic freedom to pursue our research passions.

Impact on the Authors

A. L. C.: Under the phenomenal support, guidance, and creativity of Don and Jeff, I found my passion for discovery spanning basic to translational research and continue an academic research career focused on defining determinants of human immune responses with the goal of changing patient care. The rigor of the training and the science performed in the Hunt lab cannot be underestimated. Don once told me, “Every scientist discovers something important in their career. Some even realize it.” Don’s extraordinary approach to doing science facilitated careful assessment of all data obtained with an open mind and embraced the concept that the experiment that does not come out the way you expected may be the most informative of all. I arrived in the lab at the nascency of what would become a revolution in immunology driven by the Hunt lab and collaborators and remain immensely grateful to have been trained by Don, a truly visionary giant.

J. G. A.: I found a lifelong passion in immunopeptidomics and its application to translational research with the support of Don and Jeff. Don would stop by my desk regularly with his vintage avocado green coffee cup and sit on a wooden lab stool to ask me questions about whatever project was ongoing. Sometimes he would give me unmarked tubes from his pockets and tell me that I needed to confirm the peptide sequences of synthetic peptides that were planned to be used for an immunology experiment by a collaborator, and not to worry, I could de novo sequence what was in these tubes without any prior knowledge. I was never sure if this was a test or if he forgot which peptide was in which tube after he picked them up. What I am sure of is that Don, his longtime colleague Jeff, and the members of the Hunt laboratory were exactly what I needed at the time. They provided me with the highest level of MS training and gave me the freedom and resources to apply these skills to immunopeptidomics. I am both lucky and proud to be a trainee of the Hunt laboratory and thrilled that I can continue to do research in the area of immunopeptidomics, and I never miss an opportunity to tell others that this is the case.

As I look back at the multiple decades of immunopeptidomics research in which the Hunt laboratory was involved, I realize that many ongoing immunopeptidomics efforts have a connection to their early studies. Along these lines, a colleague once said that the Hunt laboratory paved the road that I now treat like a super highway. This statement always stuck with me for multiple reasons. It is clearly true, but also conveys a shift away from smaller scale immunopeptidome studies (<10 samples per study) that leveraged manual de novo sequencing to high-throughput immunopeptidomics (>100 samples per study) that rely on personalized protein database searches. This is something that I maintain torn about, as Don once told me that he would never do high-throughput immunopeptidomics, although he was supportive of those doing these types of studies. Don understood the benefit of high-throughput immunopeptidomics, but always put a higher value on the precision of manually confirming all the pMHC identified to be sure the research question was being confidently addressed. I believe that Don’s vision for the future of immunopeptidomics is that disease associated PTM peptides, specifically phosphopeptides, can be utilized either as biomarkers or in the development of immunotherapies (87), and that the MS technology will continue to play an essential role is defining HLA presented therapeutic targets.

Summary

The field of immunopeptidomics has benefited greatly from the research of Don Hunt’s laboratory, and it should, in return, be mindful of the lessons that can be learned and the discoveries that can be made from small cohort immunopeptidomics studies that leverage manual spectra interpretation aimed at deepening our understanding of MHC peptide processing and presentation. We anticipate that the area of immunopeptidomics will continue to grow over the next decades and are immensely grateful to Don, his laboratory, and collaborators who helped to pave that way for the future of MS-based immunopeptidomics research.

Conflict of interest

J. G. A. is a paid consultant of Enara Bio. The other author declares no competing interests.

Acknowledgments

We thank Jeffrey Shabanowitz for the editorial guidance and the opportunity to contribute to this special issue. We acknowledge that the graphical abstract was made using BioRender.

Author contributions

J. G. A. and A. L. C. writing–review and editing; J. G. A. and A. L. C. writing–original draft; J. G. A. and A. L. C. conceptualization.
==== Refs
References

1 Bjorkman P.J. Saper M.A. Samraoui B. Bennett W.S. Strominger J.L. Wiley D.C. The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens Nature 329 1987 512 518 2443855
2 Townsend A.R. Rothbard J. Gotch F.M. Bahadur G. Wraith D. McMichael A.J. The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides Cell 44 1986 959 968 2420472
3 Wabuke-Bunoti M.A. Fan D.P. Isolation and characterization of a CNBr cleavage peptide of influenza viral hemagglutinin stimulatory for mouse cytolytic T lymphocytes J. Immunol. 130 1983 2386 2391 6601150
4 Wabuke-Bunoti M.A. Taku A. Garman R. Fan D.P. Stimulation of anti-influenza cytolytic T lymphocytes by a synthetic peptide of the influenza hemagglutinin can be modulated by at least three independent helper factors J. Immunol. 133 1984 2186 2193 6206155
5 Guertin D.P. Fan D.P. Stimulation of cytolytic T cells by isolated viral peptides and HN protein coupled to agarose beads Nature 283 1980 308 311 6153234
6 Wabuke-Bunoti M.A. Taku A. Fan D.P. Kent S. Webster R.G. Cytolytic T lymphocyte and antibody responses to synthetic peptides of influenza virus hemagglutinin J. Immunol. 133 1984 2194 2201 6206156
7 Istrail S. Florea L. Halldórsson B.V. Kohlbacher O. Schwartz R.S. Yap V.B. Comparative immunopeptidomics of humans and their pathogens Proc. Natl. Acad. Sci. U. S. A. 101 2004 13268 13272 15326311
8 Couzin-Frankel J. Breakthrough of the year 2013. Cancer immunotherapy Science 342 2013 1432 1433 24357284
9 Barouch D.H. Covid-19 vaccines - immunity, variants, boosters N. Engl. J. Med. 387 2022 1011 1020 36044620
10 Yewdell J.W. MHC class I immunopeptidome: past, present, and future Mol. Cell Proteomics 21 2022 100230
11 Thibault P. Perreault C. Immunopeptidomics: Reading the immune signal that defines self from nonself Mol. Cell Proteomics 21 2022 100234
12 Zhang B. Bassani-Sternberg M. Current perspectives on mass spectrometry-based immunopeptidomics: the computational angle to tumor antigen discovery J. Immunother. Cancer 11 2023 e007073
13 Hunt D.F. Henderson R.A. Shabanowitz J. Sakaguchi K. Michel H. Sevilir N. Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry Science 255 1992 1261 1263 1546328
14 Stern L.J. Characterizing MHC-associated peptides by mass spectrometry J. Immunol. 179 2007 2667 2668 17709475
15 Hunt D.F. Michel H. Dickinson T.A. Shabanowitz J. Cox A.L. Sakaguchi K. Peptides presented to the immune system by the murine class II major histocompatibility complex molecule I-Ad Science 256 1992 1817 1820 1319610
16 Hunt D.F. Yates J.R. 3rd Shabanowitz J. Winston S. Hauer C.R. Protein sequencing by tandem mass spectrometry Proc. Natl. Acad. Sci. U. S. A. 83 1986 6233 6237 3462691
17 Fenn J.B. Mann M. Meng C.K. Wong S.F. Whitehouse C.M. Electrospray ionization for mass spectrometry of large biomolecules Science 246 1989 64 71 2675315
18 Falk K. Rötzschke O. Stevanović S. Jung G. Rammensee H.G. Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules Nature 351 1991 290 296 1709722
19 Huczko E.L. Bodnar W.M. Benjamin D. Sakaguchi K. Zhu N.Z. Shabanowitz J. Characteristics of endogenous peptides eluted from the class I MHC molecule HLA-B7 determined by mass spectrometry and computer modeling J. Immunol. 151 1993 2572 2587 8360479
20 Kubo R.T. Sette A. Grey H.M. Appella E. Sakaguchi K. Zhu N.Z. Definition of specific peptide motifs for four major HLA-A alleles J. Immunol. 152 1994 3913 3924 8144960
21 Stern L.J. Brown J.H. Jardetzky T.S. Gorga J.C. Urban R.G. Strominger J.L. Wiley D.C. Crystal structure of the human class II MHC protein HLA-DR1 complexed with an influenza virus peptide Nature 368 1994 215 221 8145819
22 Kaliyaperumal A. Falchetto R. Cox A. Dick R. Shabanowitz J. Chien Y.H. Functional expression and recognition of nonclassical MHC class I T10b is not peptide-dependent J. Immunol. 155 1995 2379 2386 7650372
23 Henderson R.A. Michel H. Sakaguchi K. Shabanowitz J. Appella E. Hunt D.F. Engelhard V.H. HLA-A2.1-associated peptides from a mutant cell line: a second pathway of antigen presentation Science 255 1992 1264 1266 1546329
24 Sette A. Ceman S. Kubo R.T. Sakaguchi K. Appella E. Hunt D.F. Invariant chain peptides in most HLA-DR molecules of an antigen-processing mutant Science 258 1992 1801 1804 1465617
25 Engelhard V.H. Appella E. Benjamin D.C. Bodnar W.M. Cox A.L. Chen Y. Mass spectrometric analysis of peptides associated with the human class I MHC molecules HLA-A2.1 and HLA-B7 and identification of structural features that determine binding Chem. Immunol. 57 1993 39 62 8260089
26 Chen Y. Sidney J. Southwood S. Cox A.L. Sakaguchi K. Henderson R.A. Naturally processed peptides longer than nine amino acid residues bind to the class I MHC molecule HLA-A2.1 with high affinity and in different conformations J. Immunol. 152 1994 2874 2881 8144888
27 Davenport M.P. Smith K.J. Barouch D. Reid S.W. Bodnar W.M. Willis A.C. HLA class I binding motifs derived from random peptide libraries differ at the COOH terminus from those of eluted peptides J. Exp. Med. 185 1997 367 371 9016886
28 Meadows L. Wang W. den Haan J.M. Blokland E. Reinhardus C. Drijfhout J.W. The HLA-A∗0201-restricted H-Y antigen contains a posttranslationally modified cysteine that significantly affects T cell recognition Immunity 6 1997 273 281 9075928
29 Sette A. DeMars R. Grey H.M. Oseroff C. Southwood S. Appella E. Isolation and characterization of naturally processed peptides bound by class II molecules and peptides presented by normal and mutant antigen-presenting cells Chem. Immunol. 57 1993 152 165 8260087
30 Cox A.L. Skipper J. Chen Y. Henderson R.A. Darrow T.L. Shabanowitz J. Identification of a peptide recognized by five melanoma-specific human cytotoxic T cell lines Science 264 1994 716 719 7513441
31 Slingluff C.L. Jr. Hunt D.F. Engelhard V.H. Direct analysis of tumor-associated peptide antigens Curr. Opin. Immunol. 6 1994 733 740 7530012
32 Skipper J.C. Hendrickson R.C. Gulden P.H. Brichard V. Van Pel A. Chen Y. An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins J. Exp. Med. 183 1996 527 534 8627164
33 Huang A.Y. Gulden P.H. Woods A.S. Thomas M.C. Tong C.D. Wang W. The immunodominant major histocompatibility complex class I-restricted antigen of a murine colon tumor derives from an endogenous retroviral gene product Proc. Natl. Acad. Sci. U. S. A. 93 1996 9730 9735 8790399
34 Skipper J.C. Kittlesen D.J. Hendrickson R.C. Deacon D.D. Harthun N.L. Wagner S.N. Shared epitopes for HLA-A3-restricted melanoma-reactive human CTL include a naturally processed epitope from Pmel-17/gp100 J. Immunol. 157 1996 5027 5033 8943411
35 Hogan K.T. Eisinger D.P. Cupp S.B. Lekstrom K.J. Deacon D.D. Shabanowitz J. The peptide recognized by HLA-A68.2-restricted, squamous cell carcinoma of the lung-specific cytotoxic T lymphocytes is derived from a mutated elongation factor 2 gene Cancer Res. 58 1998 5144 5150 9823325
36 Slingluff C.L. Jr. Cox A.L. Henderson R.A. Hunt D.F. Engelhard V.H. Recognition of human melanoma cells by HLA-A2.1-restricted cytotoxic T lymphocytes is mediated by at least six shared peptide epitopes J. Immunol. 150 1993 2955 2963 7681084
37 Skipper J.C. Gulden P.H. Hendrickson R.C. Harthun N. Caldwell J.A. Shabanowitz J. Mass-spectrometric evaluation of HLA-A∗0201-associated peptides identifies dominant naturally processed forms of CTL epitopes from MART-1 and gp100 Int. J. Cancer 82 1999 669 677 10417764
38 Slingluff C.L. Cox A.L. Stover J.M. Moore M.M. Hunt D.F. Engelhard V.H. Cytotoxic T-lymphocyte response to autologous human squamous cell cancer of the lung: epitope reconstitution with peptides extracted from HLA-Aw68 Cancer Res. 54 1994 2731 2737 7513255
39 Dubey P. Hendrickson R.C. Meredith S.C. Siegel C.T. Shabanowitz J. Skipper J.C. The immunodominant antigen of an ultraviolet-induced regressor tumor is generated by a somatic point mutation in the DEAD box helicase p68 J. Exp. Med. 185 1997 695 705 9034148
40 Blass E. Ott P.A. Advances in the development of personalized neoantigen-based therapeutic cancer vaccines Nat. Rev. Clin. Oncol. 18 2021 215 229 33473220
41 Fritsch E.F. Burkhardt U.E. Hacohen N. Wu C.J. Personal neoantigen cancer vaccines: a road not fully paved Cancer Immunol. Res. 8 2020 1465 1469 33262163
42 Henderson R.A. Cox A.L. Sakaguchi K. Appella E. Shabanowitz J. Hunt D.F. Engelhard V.H. Direct identification of an endogenous peptide recognized by multiple HLA-A2.1-specific cytotoxic T cells Proc. Natl. Acad. Sci. U. S. A. 90 1993 10275 10279 7694286
43 den Haan J.M. Sherman N.E. Blokland E. Huczko E. Koning F. Drijfhout J.W. Identification of a graft versus host disease-associated human minor histocompatibility antigen Science 268 1995 1476 1480 7539551
44 den Haan J.M. Meadows L.M. Wang W. Pool J. Blokland E. Bishop T.L. The minor histocompatibility antigen HA-1: a diallelic gene with a single amino acid polymorphism Science 279 1998 1054 1057 9461441
45 Wang W. Gulden P.H. Pierce R.A. Shabanowitz J. Man S.T. Hunt D.F. A naturally processed peptide presented by HLA-A∗0201 is expressed at low abundance and recognized by an alloreactive CD8+ cytotoxic T cell with apparent high affinity J. Immunol. 158 1997 5797 5804 9190931
46 Hu Q. Bazemore Walker C.R. Girao C. Opferman J.T. Sun J. Shabanowitz J. Specific recognition of thymic self-peptides induces the positive selection of cytotoxic T lymphocytes Immunity 7 1997 221 231 9285407
47 Wang W. Man S. Gulden P.H. Hunt D.F. Engelhard V.H. Class I-restricted alloreactive cytotoxic T lymphocytes recognize a complex array of specific MHC-associated peptides J. Immunol. 160 1998 1091 1097 9570521
48 Crotzer V.L. Christian R.E. Brooks J.M. Shabanowitz J. Settlage R.E. Marto J.A. Immunodominance among EBV-derived epitopes restricted by HLA-B27 does not correlate with epitope abundance in EBV-transformed B-lymphoblastoid cell lines J. Immunol. 164 2000 6120 6129 10843661
49 Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Probability-based protein identification by searching sequence databases using mass spectrometry data Electrophoresis 20 1999 3551 3567 10612281
50 Koenig T. Menze B.H. Kirchner M. Monigatti F. Parker K.C. Patterson T. Robust prediction of the MASCOT score for an improved quality assessment in mass spectrometric proteomics J. Proteome Res. 7 2008 3708 3717 18707158
51 Syka J.E.P. Coon J.J. Schroeder M.J. Shabanowitz J. Hunt D.F. Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry Proc. Natl. Acad. Sci. U. S. A. 101 2004 9528 9533 15210983
52 Depontieu F.R. Qian J. Zarling A.L. McMiller T.L. Salay T.M. Norris A. Identification of tumor-associated, MHC class II-restricted phosphopeptides as targets for immunotherapy Proc. Natl. Acad. Sci. U. S. A. 106 2009 12073 12078 19581576
53 Lippolis J.D. White F.M. Marto J.A. Luckey C.J. Bullock T.N.J. Shabanowitz J. Analysis of MHC class II antigen processing by quantitation of peptides that constitute nested sets J. Immunol. 169 2002 5089 5097 12391225
54 Zarling A.L. Luckey C.J. Marto J.A. White F.M. Brame C.J. Evans A.M. Tapasin is a facilitator, not an editor, of class I MHC peptide binding J. Immunol. 171 2003 5287 5295 14607930
55 Slingluff C.L. Colella T.A. Thompson L. Graham D.D. Skipper J.C. Caldwell J. Melanomas with concordant loss of multiple melanocytic differentiation proteins: immune escape that may be overcome by targeting unique or undefined antigens Cancer Immunol. Immunother. 48 2000 661 672 10752474
56 Hogan K.T. Sutton J.N. Chu K.U. Busby J.A.C. Shabanowitz J. Hunt D.F. Slingluff C.L. Use of selected reaction monitoring mass spectrometry for the detection of specific MHC class I peptide antigens on A3 supertype family members Cancer Immunol. Immunother. 54 2005 359 371 15378283
57 Altrich-VanLith M.L. Ostankovitch M. Polefrone J.M. Mosse C.A. Shabanowitz J. Hunt D.F. Engelhard V.H. Processing of a class I-restricted epitope from tyrosinase requires peptide N-glycanase and the cooperative action of endoplasmic reticulum aminopeptidase 1 and cytosolic proteases J. Immunol. 177 2006 5440 5450 17015730
58 Thompson L.W. Hogan K.T. Caldwell J.A. Pierce R.A. Hendrickson R.C. Deacon D.H. Preventing the spontaneous modification of an HLA-A2-restricted peptide at an N-terminal glutamine or an internal cysteine residue enhances peptide antigenicity J. Immunother. 27 2004 177 183 15076134
59 Brickner A.G. Evans A.M. Mito J.K. Xuereb S.M. Feng X. Nishida T. The PANE1 gene encodes a novel human minor histocompatibility antigen that is selectively expressed in B-lymphoid cells and B-CLL Blood 107 2006 3779 3786 16391015
60 Andersen M.H. Bonfill J.E. Neisig A. Arsequell G. Sondergaard I. Valencia G. Phosphorylated peptides can be transported by TAP molecules, presented by class I MHC molecules, and recognized by phosphopeptide-specific CTL J. Immunol. 163 1999 3812 3818 10490979
61 Zarling A.L. Ficarro S.B. White F.M. Shabanowitz J. Hunt D.F. Engelhard V.H. Phosphorylated peptides are naturally processed and presented by major histocompatibility complex class I molecules in vivo J. Exp. Med. 192 2000 1755 1762 11120772
62 Zarling A.L. Polefrone J.M. Evans A.M. Mikesh L.M. Shabanowitz J. Lewis S.T. Identification of class I MHC-associated phosphopeptides as targets for cancer immunotherapy Proc. Natl. Acad. Sci. U. S. A. 103 2006 14889 14894 17001009
63 Mohammed F. Cobbold M. Zarling A.L. Salim M. Barrett-Wilt G.A. Shabanowitz J. Phosphorylation-dependent interaction between antigenic peptides and MHC class I: a molecular basis for the presentation of transformed self Nat. Immunol. 9 2008 1236 1243 18836451
64 Seamons A. Sutton J. Bai D. Baird E. Bonn N. Kafsack B.F.C. Competition between two MHC binding registers in a single peptide processed from myelin basic protein influences tolerance and susceptibility to autoimmunity J. Exp. Med. 197 2003 1391 1397 12756272
65 Nepom G.T. Lippolis J.D. White F.M. Masewicz S. Marto J.A. Herman A. Identification and modulation of a naturally processed T cell epitope from the diabetes-associated autoantigen human glutamic acid decarboxylase 65 (hGAD65) Proc. Natl. Acad. Sci. U. S. A. 98 2001 1763 1768 11172025
66 Lieberman S.M. Evans A.M. Han B. Takaki T. Vinnitskaya Y. Caldwell J.A. Identification of the beta cell antigen targeted by a prevalent population of pathogenic CD8+ T cells in autoimmune diabetes Proc. Natl. Acad. Sci. U. S. A. 100 2003 8384 8388 12815107
67 Flyer D.C. Ramakrishna V. Miller C. Myers H. McDaniel M. Root K. Identification by mass spectrometry of CD8(+)-T-cell Mycobacterium tuberculosis epitopes within the Rv0341 gene product Infect. Immun. 70 2002 2926 2932 12010981
68 Earley L. Anderson L.C. Bai D.L. Mullen C. Syka J.E.P. English A.M. Front-end electron transfer dissociation: a new ionization source Anal. Chem. 85 2013 8385 8390 23909443
69 Cobbold M. De La Peña H. Norris A. Polefrone J.M. Qian J. English A.M. MHC class I-associated phosphopeptides are the targets of memory-like immunity in leukemia Sci. Transl. Med. 5 2013 203ra125
70 Hunt D.F. Shabanowitz J. Bai D.L. Peptide sequence analysis by electron transfer dissociation mass spectrometry: a web-based tutorial J. Am. Soc. Mass Spectrom. 26 2015 1256 1258 25821049
71 Yarchoan M. Hopkins A. Jaffee E.M. Tumor mutational burden and response rate to PD-1 inhibition N. Engl. J. Med. 377 2017 2500 2501 29262275
72 Lawrence M.S. Stojanov P. Polak P. Kryukov G.V. Cibulskis K. Sivachenko A. Mutational heterogeneity in cancer and the search for new cancer-associated genes Nature 499 2013 214 218 23770567
73 Sim M.J.W. Malaker S.A. Khan A. Stowell J.M. Shabanowitz J. Peterson M.E. Canonical and cross-reactive binding of NK cell inhibitory receptors to HLA-C allotypes is dictated by peptides bound to HLA-C Front. Immunol. 8 2017 193 28352266
74 Moore C. Sidney J. English A.M. Wriston A. Hunt D.F. Shabanowitz J. Identification of the peptide-binding motif recognized by the pigtail macaque class I MHC molecule Mane-A1∗082:01 (Mane A∗0301) Immunogenetics 64 2012 461 468 22278177
75 Mothé B.R. Southwood S. Sidney J. English A.M. Wriston A. Hoof I. Peptide-binding motifs associated with MHC molecules common in Chinese rhesus macaques are analogous to those of human HLA supertypes and include HLA-B27-like alleles Immunogenetics 65 2013 371 386 23417323
76 Deycmar S. Gomes B. Charo J. Ceppi M. Cline J.M. Spontaneous, naturally occurring cancers in non-human primates as a translational model for cancer immunotherapy J. Immunother. Cancer 11 2023 e005514 36593067
77 Rauch A. Nolan D. Martin A. McKinnon E. Almeida C. Mallal S. Prospective genetic screening decreases the incidence of abacavir hypersensitivity reactions in the Western Australian HIV cohort study Clin. Infect. Dis. 43 2006 99 102 16758424
78 Chessman D. Kostenko L. Lethborg T. Purcell A.W. Williamson N.A. Chen Z. Human leukocyte antigen class I-restricted activation of CD8+ T cells provides the immunogenetic basis of a systemic drug hypersensitivity Immunity 28 2008 822 832 18549801
79 Ostrov D.A. Grant B.J. Pompeu Y.A. Sidney J. Harndahl M. Southwood S. Drug hypersensitivity caused by alteration of the MHC-presented self-peptide repertoire Proc. Natl. Acad. Sci. U. S. A. 109 2012 9959 9964 22645359
80 Metushi I.G. Wriston A. Banerjee P. Gohlke B.O. English A.M. Lucas A. Acyclovir has low but detectable influence on HLA-B∗57:01 specificity without inducing hypersensitivity PLoS One 10 2015 e0124878
81 Pavlos R. McKinnon E.J. Ostrov D.A. Peters B. Buus S. Koelle D. Shared peptide binding of HLA Class I and II alleles associate with cutaneous nevirapine hypersensitivity and identify novel risk alleles Sci. Rep. 7 2017 8653 28819312
82 Abelin J.G. Trantham P.D. Penny S.A. Patterson A.M. Ward S.T. Hildebrand W.H. Complementary IMAC enrichment methods for HLA-associated phosphopeptide identification by mass spectrometry Nat. Protoc. 10 2015 1308 1318 26247297
83 Penny S.A. Abelin J.G. Malaker S.A. Myers P.T. Saeed A.Z. Steadman L.G. Tumor infiltrating lymphocytes target HLA-I phosphopeptides derived from cancer signaling in colorectal cancer Front. Immunol. 12 2021 723566
84 Mohammed F. Stones D.H. Zarling A.L. Willcox C.R. Shabanowitz J. Cummings K.L. The antigenic identity of human class I MHC phosphopeptides is critically dependent upon phosphorylation status Oncotarget 8 2017 54160 54172 28903331
85 Li Y. Depontieu F.R. Sidney J. Salay T.M. Engelhard V.H. Hunt D.F. Structural basis for the presentation of tumor-associated MHC class II-restricted phosphopeptides to CD4+ T cells J. Mol. Biol. 399 2010 596 603 20417641
86 Malaker S.A. Ferracane M.J. Depontieu F.R. Zarling A.L. Shabanowitz J. Bai D.L. Identification and characterization of complex glycosylated peptides presented by the MHC class II processing pathway in melanoma J. Proteome Res. 16 2017 228 237 27550523
87 Mahoney K.E. Shabanowitz J. Hunt D.F. MHC phosphopeptides: Promising targets for immunotherapy of cancer and other chronic diseases Mol. Cell Proteomics 20 2021 100112
