Method for increasing the peptide repertoire presented by MHC proteins on a mammalian cell

Abstract

Provided herein is a method for increasing the peptide repertoire presented by MHC proteins on a mammalian cell. In some embodiments, the method may comprise modifying the cell to express a modified ERAP2 protein, e.g., an ERAP2 protein that lacks an aminopeptidase activity. Nucleic acids encoding the modified ERAP2 protein and various methods of treatment are provided.

Description

[0001]SF2024-175-2 METHOD FORINCREASING THEPEPTIDEREPERTOIREPRESENTED BYMHC PROTEINS ON AMAMMALIANCELLSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under Grant No. R01 AI136972 awarded by the National Institutes of Health. The government has certain rights in the invention. INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS ASEQUENCELISTINGXML FILEA Sequence Listing is provided herewith as a Sequence Listing XML, “UCSF- 795WO_SEQLIST.xml” created on March 10, 2025 and having a size of 4,170 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety. BACKGROUND Major histocompatibility complex (MHC) class I and class II proteins play a pivotal role in the adaptive branch of the immune system. Both classes of proteins share the task of presenting peptides on the cell surface for recognition by T cells. Immunogenic peptide–MHC class I (pMHCI) complexes are presented on nucleated cells and are recognized by cytotoxic CD8+ T cells. The presentation of pMHCII by antigen-presenting cells (e.g., dendritic cells (DCs), macrophages, or B cells), on the other hand, can activate CD4+ T cells, leading to the coordination and regulation of effector cells. These interactions are mediated by the T cell receptor. New strategies for altering the repertoire of peptides that are presented by MHC proteins are therefore of great clinical value. SUMMARY Provided herein is a method for increasing the peptide repertoire presented by MHC proteins on a mammalian cell. In some embodiments, the method may comprise modifying the cell to express a modified ERAP2 protein, e.g., an ERAP2 protein that lacks aminopeptidase activity, such as an ERAP2 protein in which the aminopeptidase activity has been inactivated by one or more amino acid substitutions, insertions or deletions, or an ERAP2 protein that lacks an SF2024-175-2 aminopeptidase domain. Nucleic acids encoding the modified ERAP2 protein, a vaccine and various methods of treatment are also provided. Without wishing to be bound to any particular theory, it is thought that the modified ERAP2 protein may interfere with the processing of peptides prior to loading on the MHC (via dominant negative interactions or by sequestering substrate peptides from the wild type ERAP1 and / or ERAP2 proteins, which are catalytically active, for example), which results in an increased peptide repertoire in the MHC proteins. Additional embodiments and other features, advantages and variations may become apparent in view of the description that follows below. BRIEF DESCRIPTION OF THE FIGURES The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way. FIGS. 1A-1E: Evolution, geographic distribution, and disease association of ERAP2 haplotypes. (FIG. 1A) Linkage disequilibrium (upper) and tube (lower) plots demonstrating strong linkage disequilibrium (LD) of polymorphisms at the ERAP2 locus with segregation into haplotypes A, B1, and B2 in the pooled data from 1000 Genomes, Human Genome Diversity Project, and Simons Genome Diversity Project. Colored exons are predicted to be translated. (FIG. 1B) Intron-exon diagrams of gene products produced by ERAP2 haplotypes in unstimulated cells. Inset highlights the splice-site variant rs2248374 and intronic variant rs2548535 that separate ERAP2-A and ERAP2-B1 / B2, and a tandem repeat (rs1581849348) that distinguishes ERAP2-B1 and ERAP2-B2. (FIG. 1C) Geographic distribution of ERAP2 haplotypes in databases from FIG. 1A. (FIG. 1D) Inferred ancestral recombination graph of ERAP2 haplotypes, calculated using tree sequence format of Kelleher et al. for 3601 modern human genomes, 4 Neanderthal genomes (3 high coverage) and 1 high coverage Denisovan genome, with key branch points highlighted. (FIG. 1E) Risk for specific disease phenotypes for ERAP2-A and ERAP2-B1 in African Americans and Europeans. FIGS. 2A-2E: Induction of ERAP2Iso3 by nucleic acid sensing. (FIG. 2A) Intron-exon diagrams of full length (ERAP2Iso1) and truncated (ERAP2Iso3 / 4) isoforms and location of primers SF2024-175-2 used for quantifying expression of each isoform, total gene expression, or allele specific expression. Shaded exons are predicted to be translated. (FIG. 2B) Estimated expression of ERAP2Iso1 and ERAP2Iso3 / 4 from bulk RNA-seq in monocyte-derived dendritic cells left unstimulated or stimulated with IAV for 10hrs in individuals of African ancestry separated by genotype. (FIG. 2C) Boxplots showing ERAP2Iso3 / 4 expression measured by qPCR in monocytes, MDMs, and MoDCs left unstimulated or stimulated with IAV for 10hrs for individuals who are homozygous for A or B2. (FIG. 2D) Boxplots showing ERAP2Iso1(top) and ERAP2Iso3 (bottom) expression in monocyte-derived macrophages from individuals with the indicated ERAP2 genotypes and stimulated with PAMP agonists. (FIG. 2E) Ratio of ERAP2Iso3 to ERAP2Iso1expression determined by allele-specific expression in monocyte-derived macrophages from AB2heterozygous individuals stimulated with the indicated PAMP agonists. FIGS. 3A-3E: Identification of causal allele controlling ERAP2Iso3 / 4 expression. (FIG. 3A) Chromatin accessibility profiles by bulk ATAC-seq at the ERAP2 locus in monocyte- derived macrophages infected with IAV. Inset shows region surrounding candidate cis- regulatory element 1 (CRE1) containing the tandem repeat and linked SNPs. (FIG. 3B) Constructs overlapping each peak for haplotypes A, B1, or B2 from FIG. 3A. (FIG. 3C) Pseudobulk chromatin accessibility of a region capturing the TR and rs2548535 estimated from single-cell ATAC-seq of monocytes as a function of ERAP2 genotype across 350 healthy donors of four different genetic ancestries. (FIG. 3D) Luciferase reporter gene expression in U937 cells transfected with constructs from FIG. 3B unstimulated or infected with IAV for 18hrs. (FIG. 3E) Luciferase reporter gene expression in IAV-infected U937 cells transfected with all possible combinations of the tandem repeat, rs2548535, and rs2161657. FIGS. 4A-4D: ERAP2Iso3 elicits differentiated antigen processing in vitro. (FIG. 4A) Activity of human ERAP1, ERAP2Iso1, and ERAP2Iso3against known preferred residue for each wildtype enzyme. (FIG. 4B) Relative cleavage of X-pNA (X=indicated amino acid) substrate by full-length aminopeptidases human ERAP1 (hERAP1), ERAP2Iso1, and murine ERAP1 (mERAP1) with or without ERAP2Iso3. (FIG. 4C) Dose dependent alteration of hERAP1, ERAP2Iso1, and mERAP1 enzymatic activity against leucine or arginine residues by ERAP2Iso3. (FIG. 4D) Processing of ER-targeted peptide substrates by full-length ERAP2Iso1 with or without ERAP2Iso3coexpression in mERAP1 / TAP deficient fibroblasts. SF2024-175-2 FIGS. 5A-5H: ERAP2Iso3 alters antigen peptide repertoire in vivo. (FIG. 5A) Generation of transgenic ERAP2Iso1 [Tg(ERAP2Iso1)] and ERAP2Iso3 [Tg(ERAP2Iso3)] mice using CRISPR / Cas9 genome editing followed by homology directed repair (HDR) of C57 / Bl6 at the Rosa26 locus. (FIG. 5B) Ubiquitous expression of ERAP2Iso1and ERAP2Iso3across various tissues in wildtype (B6) and transgenics. (FIG. 5C) Frequency of B and T cells amongst CD45+splenocytes between wildtype (B6) and transgenic animals. (FIG. 5D) Expression of MHC-I and MHC-II molecules in B cells and dendritic cells (DCs) in wildtype (B6) and transgenic animals. (FIG. 5E) Representative antigen specific CD8 T cell responses generated by immunizing wildtype mice with APCs from Tg(ERAP2Iso3) (top) or immunizing Tg(ERAP2Iso3) mice with APCs from wildtype (bottom). (FIG. 5F) Summary of antigen specific CD8 T cell responses across multiple animals between wildtype and ERAP2Iso3(left) or between wildtype and ERAP1 knockout (ERAP1KO) (right). (FIG. 5G) Representative antigen specific CD8 T cell responses generated by immunizing wildtype mice with APCs from Tg(ERAP2Iso1). (FIG. 5H) Summary of antigen specific CD8 T cell responses across multiple animals between wildtype and ERAP2Iso1. FIG. 6 is a series of graphs showing that ERAP2Iso3 can alter the antigen repertoire recognized by CD4 T cells. DEFINITIONS As used herein, the terms "treatment," "treating," “treat” and the like, refer to obtaining a desired pharmacologic and / or physiologic effect and / or a response related to the treatment. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which can be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. A “therapeutically effective amount” or “efficacious amount” refers to the amount of an agent (including biologic agents, such as cells), or combined amounts of two agents, that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such SF2024-175-2 treatment for the disease. The “therapeutically effective amount” will vary depending on the agent(s), the disease and its severity and the age, weight, etc., of the subject to be treated. The terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein, refer to a mammal, including, but not limited to, murines (e.g., rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), lagomorphs, etc. In some cases, the individual is a human. In some cases, the individual is a non- human primate. In some cases, the individual is a rodent, e.g., a rat or a mouse. In some cases, the individual is a lagomorph, e.g., a rabbit. The term “operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. Operably linked nucleic acid sequences may but need not necessarily be adjacent. For example, in some instances a coding sequence operably linked to a promoter may be adjacent to the promoter. In some instances, a coding sequence operably linked to a promoter may be separated by one or more intervening sequences, including coding and non-coding sequences. Also, in some instances, more than two sequences may be operably linked including but not limited to e.g., where two or more coding sequences are operably linked to a single promoter. DETAILEDDESCRIPTIONBefore the methods and compositions of the present disclosure are described in greater detail, it is to be understood that the methods and compositions are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the methods and compositions will be limited only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the methods and compositions. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed SF2024-175-2 within the methods and compositions, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods and compositions. Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions belong. Although any methods and compositions similar or equivalent to those described herein can also be used in the practice or testing of the methods and compositions, representative illustrative methods and compositions are now described. All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and / or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present methods and compositions are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed. It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. It is appreciated that certain features of the methods and compositions, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods and compositions, which SF2024-175-2 are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present methods and compositions and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. As noted above, provided herein in a method for increasing the peptide repertoire presented by MHC proteins on a mammalian cell. In some embodiments, the method may comprise modifying the cell to express a modified ERAP2 (endoplasmic reticulum aminopeptidase 2) protein. Modified ERAP2 proteins ERAP2 (which may be alternatively referred to as LRAP or L-RAP in other publications) is a zinc metalloaminopeptidase of the M1 protease family that resides in the endoplasmic reticulum and functions in N-terminal trimming antigenic epitopes for presentation by major histocompatibility complex (MHC) class I molecules. This protein is known to hydrolyze N- terminal amino acids of proteins or peptide substrates so the MHC class I can present them to CD8+ T cells. The human ERAP2 protein is encoded by Genbank Gene ID 64167. There are several isoforms of ERAP2, include: isoform 1 (which is 960 amino acids in length and defined by Genbank accession nos. NP_071745.1 and GID11641261), isoform 2 (which is 915 amino acids in length and defined by Genbank accession nos. NP_001316158.1 and GID1042779813) and isoform 3 (which is 350 amino acids in length and defined by Genbank accession nos. NP_001316162.1 and GID1042779815). See, e.g., Saulle et al Cells. 20209: 1951. Isoform 1 of ERAP2 has been crystallized (see, e.g., Papakyriakou et al, Front Immunol. 202213: 863529 and SF2024-175-2 Birtley et al Biochemistry 201251: 286-95). There are four domains in ERAP2 Isoform 1: domains I, II, III, and IV. Isoform 1 of ERAP2 dimerizes with ERAP1, which interaction is believed to be mediated through the N terminal domain. The following amino acid sequence defines ERAP2 Isoform 1. In this sequence, the N terminal domain (I) is shown in bold, the aminopeptidase domain (i.e., the catalytic domain) (II) is in italics, the hinge domain (III) is shown in bold italics and the C terminal domain (IV) is underlined. The underlined Y of the aminopeptidase domain is the active site of the enzyme. ERAP2Iso1 (SEQ ID NO: 1: MFHSSAMVNSHRKPMFNIHRGFYCLTAILPQICICSQFSVPSSYHFTEDPGAFPV ATNGERFPWQELRLPSVVIPLHYDLFVHPNLTSLDFVASEKIEVLVSNATQFIIL HSKDLEITNATLQSEEDSRYMKPGKELKVLSYPAHEQIALLVPEKLTPHLKYYVA MDFQAKLGDGFEGFYKSTYRTLGGETRILAVTDFEPTQARMAFPCFDEPLFKANF SIKIRRESRHIALSNMPKVKTIELEGGLLEDHFETTVKMSTYLVAYIVCDFHSLS GFTSSGVKVSIYASPDKRNQTHYALQASLKLLDFYEKYFDIYYPLSKLDLIAIPD FAPGAMENWGLITYRETSLLFDPKTSSASDKLWVTRVIAHELAHQWFGNLVTMEW WNDIWLKEGFAKYMELIAVNATYPELQFDDYFLNVCFEVITKDSLNSSRPISKPA ETPTQIQEMFDEVSYNKGACILNMLKDFLGEEKFQKGIIQYLKKFSYRNAKNDDL WSSLSNSCLESDFTSGGVCHSDPKMTSNMLAFLGENAEVKEMMTTWTLQKGIPLL VVKQDGCSLRLQQERFLQGVFQEDPEWRALQERYLWHIPLTYSTSSSNVIHRHIL KSKTDTLDLPEKTSWVKFNVDSNGYYIVHYEGHGWDQLITQLNQNHTLLRPKDRV GLIHDVFQLVGAGRLTLDKALDMTYYLQHETSSPALLEGLSYLESFYHMMDRRNI SDISENLKRYLLQYFKPVIDRQSWSDKGSVWDRMLRSALLKLACDLNHAPCIQKA AELFSQWMESSGKLNIPTDVLKIVYSVGAQTTAGWNYLLEQYELSMSSAEQNKIL YALSTSKHQEKLLKLIELGMEGKVIKTQNLAALLHAIARRPKGQQLAWDFVRENW THLLKKFDLGSYDIRMIISGTTAHFSSKDKLQEVKLFFESLEAQGSHLDIFQTVL ETITKNIKWLEKNLPTLRTWLMVNT* The following amino acid sequence defines ERAP2 Isoform 3. Relative to ERAP2 Isoform 1, ERAP2 Isoform 3 has a truncated N-terrminus (i.e., it lacks the N-terminal 545 amino acids of ERAP2 Isoform 1). Relative to ERAP2 Isoform 1, ERAP2 Isoform 3 lacks the N- terminal domain (i) and the aminopeptidase domain (i.e., the catalytic domain) (II). The following amino acid sequence defines ERAP2 Isoform 3. In this sequence, the hinge domain (III) is shown in bold italics and the C terminal domain (IV) is underlined. ERAP2Iso3 (SEQ ID NO: 2): MMTTWTLQKGIPLLVVKQDGCSLRLQQERFLQGVFQEDPEWRALQERYLWHIPLT YSTSSSNVIHRHILKSKTDTLDLPEKTSWVKFNVDSNGYYIVHYEGHGWDQLITQ LNQNHTLLRPKDRVGLIHDVFQLVGAGRLTLDKALDMTYYLQHETSSPALLEGLS YLESFYHMMDRRNISDISENLKRYLLQYFKPVIDRQSWSDKGSVWDRMLRSALLK LACDLNHAPCIQKAAELFSQWMESSGKLNIPTDVLKIVYSVGAQTTAGWNYLLEQ SF2024-175-2 YELSMSSAEQNKILYALSTSKHQEKLLKLIELGMEGKVIKTQNLAALLHAIARRP KGQQLAWDFVRENWTHLLKKFDLGSYDIRMIISGTTAHFSSKDKLQEVKLFFESL EAQGSHLDIFQTVLETITKNIKWLEKNLPTLRTWLMVNT* The results described in the experimental section of this disclosure were obtained using ERAP2 isoform 3 and, as such, in some embodiments, the modified ERAP2 protein may be aminopeptidase-deficient, e.g., may be a modified ERAP2 protein comprising an aminopeptidase domain that has been inactivated by one or more amino acid substitutions, insertions or deletions (which may include one or more an amino acid substitutions at the active site) or a modified ERAP2 protein that lacks an aminopeptidase domain (i.e., an ERAP2 protein in which the aminopeptidase domain has been removed by a truncation, deletion or insertion, e.g., a deletion of at least 10, at least 50, at least 100, at least 200, at least 300, at least 400 or at least 500 amino acids which, in some embodiments, may be at the N-terminal end. In some embodiments, the modified ERAP2 may be capable of dimerizing with ERAP1. In other embodiments, the modified ERAP2 may be incapable of dimerizing with ERAP1. In any embodiment, a modified ERAP2 protein may contain a sequence of at least 100 amino acids, at least 200 amino acids, at least 300 amino acids, at least 400 amino acids or 424 amino acids that is at least 90%, at least 95%, or at least 98% identical to the amino acid sequence of the sequence of ERAP2 isoform 3, as set forth above. Cells In some embodiments, the modified mammalian cell may be a cancer cell. In other embodiments, the cell is a non-cancer cell, e.g., an antigen presenting cell such as a dendritic cell. Almost all cell types can present antigens in some way. Dedicated or “professional” antigen-presenting cells include macrophages, B cells and dendritic cells, which present foreign antigens to helper T cells, while virus-infected cells (or cancer cells) can present antigens originating inside the cell to cytotoxic T cells. In some embodiments and depending on the type of cell, the MHC proteins that present the increased peptide repertoire are MHC Class I proteins. In other embodiments, the MHC proteins that present the increased peptide repertoire are MHC Class II proteins. There are two types of antigen-presenting cell: professional and non-professional. Professional antigen-presenting cells express MHC class II molecules along with co-stimulatory molecules and pattern recognition receptors. Non-professional antigen-presenting cells express MHC class I molecules. SF2024-175-2 T cells must be activated before they can expand and perform their function. T cell activation is achieved by interacting with professional antigen-presenting cells, which present an antigen recognized by a T cell receptor. The antigen-presenting cells most commonly involved in T cells activation are dendritic cells. T cells cannot recognize (and therefore cannot respond to) soluble antigens; they can only recognize and respond to antigen that has been processed and presented by cells in using MHC proteins. Helper T cells recognize exogenous antigen presented on MHC class II whereas cytotoxic T cells can recognize endogenous antigen presented on MHC class I. Most cells in the body can present antigen to CD8+ cytotoxic T cells via MHC class I. Professional antigen-presenting cells can stimulate CD4+ helper T cells as well as cytotoxic T cells. Professional antigen-presenting cells “specialize” in presenting antigens to T cells. These cells are very efficient at internalizing antigens (e.g., by phagocytosis (for macrophages) or receptor-mediated endocytosis (B cells)), processing the antigen into fragments and then displaying those peptides (bound to a class II MHC) on their membrane. The T cell recognizes and interacts with the antigen-class II MHC complex on the membrane of the antigen-presenting cell. An additional co-stimulatory signal is then produced by the antigen-presenting cell, leading to activation of the T cell. The expression of co-stimulatory molecules and MHC class II are defining features of professional antigen-presenting cells. All professional antigen-presenting cells also express MHC class I molecules as well. Professional antigen-presenting cells include dendritic cells, macrophages and B cells. Dendritic cells are necessary for activation of naive T cells. Dendritic cells present antigen to both helper and cytotoxic T cells. They can also perform cross-presentation, a process by which they present exogenous antigen on MHC class I molecules to cytotoxic T cells. Cross- presentation allows for the activation of these T cells. Dendritic cells also play a role in peripheral tolerance, which contributes to prevention of auto-immune disease. Before they encounter a foreign antigen, dendritic cells express very low levels of MHC class II and co-stimulatory molecules on their cell surface. Immature dendritic cells are ineffective at presenting antigen to T helper cells. After a dendritic cell’s receptors recognize a pathogen-associated molecular pattern, antigen is phagocytosed and the dendritic cell becomes activated, upregulating the expression of MHC class II molecules. At this point, the cell also upregulates several co-stimulatory molecules required for T cell activation, including CD40 and SF2024-175-2 B7. The latter can interact with CD28 on the surface of a CD4+ T cell. At this point, the dendritic cell becomes a fully mature professional APC and it moves from the tissue to lymph nodes, where it encounters and activates T cells. Macrophages, on the other hand, can be stimulated by T cell secretion of interferon. After this activation, macrophages express MHC class II and co-stimulatory molecules, including the B7 complex and can present phagocytosed peptide fragments to helper T cells. Activation of these cells can assist pathogen-infected macrophages in clearing the infection. These cells circulate in the blood and enter affected sites and differentiate from monocytes to macrophages. Macrophage surrounds the site of infection or tissue damage and internalize peptides by phagocytosis. B cells internalize antigen that binds to their B cell receptor and present the antigen to helper T cells. Unlike T cells, B cells recognize soluble antigen (via the B cell receptor). The cells can process the antigen and present peptides using MHC class II molecules. When a T helper cell with a T cell receptor specific for that peptide binds to the B cell, the B cell marker CD40 binds to CD40L on the T cell surface. When activated by a T cell, a B cell can undergo antibody isotype switching, affinity maturation, as well as formation of memory cells. Non-professional antigen presenting cells include all nucleated cell types in the body. These cells make use of an MHC class I molecule that is coupled to beta-2 microglobulin to display endogenous peptides on the cell membrane. These peptides originate within the cell itself, in contrast to the exogenous antigen displayed by professional APCs using MHC class II molecules. Cytotoxic T cells are able to interact with endogenous antigen presented using an MHC class I molecule. Non-professional APCs do not typically express MHC class II molecules. However, antigen presentation to CD4+ cells via MHC class II is not restricted to the classically professional APCs. Other leukocytes, including granulocytes such as mast cells and neutrophils, can be induced to do so, as can endothelial and epithelial cells under certain circumstances. Methods of modifying cells The cells may be modified to express a modified ERAP2 protein by any suitable method. For example, the modifying may be done by modifying the cell to express a nucleic acid encoding the modified ERAP2 protein in the cell, e.g., by introducing an RNA or DNA encoding the modified ERAP2 protein in the cell. In these embodiments, the RNA or DNA is in a plasmid or viral vector. SF2024-175-2 In these embodiments, the transgene may introduced to the cell via a plasmid vector, retroviral replicating vector (RRV), a minicircle, a retroviral vector, retroviral replicating vector, adeno-associated virus (AAV), adenoviral or lentiviral vector, or encapsulated RNA, for example. In some embodiments, the modified ERAP2 may be encoded by a retroviral replicating vector (RRV). Retroviral replicating vectors are non-lytic replication-competent gamma retroviral vectors and are sometimes referred to as a replication-competent retrovirus (RCR). Such vectors have been reviewed in a variety of publications, including Logg et al (Methods Mol. Biol. 2004246:499-525), Kubo et al (Cancer Gene Therapy 201926: 128–135), Logg et al (Methods Enzymol. 2012507: 199–228), Lewis et al (J Virol. 199468:510-6) and Chen et al (Int. J. Mol. Sci. 202021: 1433). These vectors have a stringent requirement for cell division and their ability to integrate stably into the genome of cancer cells, without immediate cytolysis, contributes to long-lasting therapeutic efficacy. Thus, retroviral replicating vectors selectively infect and replicate in the tumor environment. They spread through the tumor when infected tumor cells produce infectious virus that buds off from the infected cell and spreads to neighboring replicating tumor cells. In any embodiment, the retroviral replicating vector may be a murine leukemia virus (MLV) retroviral viral vector. In other embodiments, the cell may be modified by directly introducing the aminopeptidase-deficient ERAP2 protein into the cell, e.g., using any of the methods described in Lee et al (Theranostics 20199: 3280–3292). In other embodiments, the cell may be modified by modifying the genome of the cell using a tailored endonucleases such as but not limited to a meganuclease, a TAL effector nuclease, or a CRISPR / Cas9-based system including but not limited to base or prime editors. For example, the ERAP2 gene could be modified so that it encodes the modified ERAP2 protein. Alternative, a coding sequence for the modified ERAP2 protein could be introduced elsewhere in the genome. The cell may modified in vitro (e.g., using a cultured mammalian cell), ex vivo (on a cell that has been removed from a mammal, e.g., a primary cell or a progenitor thereof), or in vivo (where the cell is inside a mammal). The latter embodiments may be implemented using an AAV, VLP or LNP delivery system. Hamilton et al (Nat. Biotechnol. 2024 doi: 10.1038 / s41587- 023-02085-z (epub ahead of print)) and Banskota et al (Cell 2022185: 250–265) describe how SF2024-175-2 CRISPR / Cas9-based therapeutics can be delivered to cells in vivo. Nucleic acids A recombinant nucleic acid comprising a coding sequence for the modified ERAP2 protein. In some embodiments, the nucleic acid may be a plasmid or viral vector, as described above, and, in some cases, the coding sequence is operably linked to an inducible, tissue-specific or constitutive promoter, particularly a promoter that is active in target cells, where the promoter is not the ERAP2 promoter. Vaccines Cellular vaccines may produced by obtaining autologous professional APCs (e.g., dendritic cells or monocytes) from a subject via apheresis. In these embodiments, the monocytes may have to be first differentiated into dendritic cells. Subsequently, the APCs (typically dendritic cells) are matured and loaded with tumor antigen (particularly from the subject, or recombinant antigens if the subject’s tumor is associated with a particular mutation). Finally, the APCs (again, typically dendritic cells) are administrated to the subject. In these embodiments, natural circulating dendritic cells or monocytes may be isolated from autologous peripheral blood mononuclear cells obtained by apheresis. In case of monocytes, ex vivo differentiation into dendritic cells is required. Both natural circulating dendritic cells and monocyte-derived dendritic cells are matured as this is important for effective T-cell activation. Maturation is associated with functional and morphological changes in dendritic cells. Following maturation, dendritic cells show enhanced expression of major histocompatibility complexes I and II, co- stimulatory molecules and increased capability of cytokine production. During the process of vaccine manufacturing, dendritic cells are loaded with relevant tumor antigen(s) to induce a tumor-specific immune response in the patient. As with the other steps in the process of manufacturing DC, several methods to load DC with antigen exist (see, e.g., Sabado et al Cell Res. 201727:74–95; Ott et al Nature 2017547: 217–21). After quality control, the vaccine may be administered to the patient. See, e.g., Fu et al (Vaccines (Basel). 20208: 706), Palucka (Immunity. 201339: 38–48) and Calmeiro et al (J. Immunother. Cancer. 20197:238) among many others. A similar approach could be used for autoimmune disease. As such, in some embodiments, the method may comprise contacting the APCs with antigens from normal cells, and then administering the APCs to a subject. SF2024-175-2 In these embodiments, the APCs (particularly dendritic cells) will have been modified to express a modified ERAP2 protein, thereby stimulating a wider range of immune responses in the subject. In vivo and ex vivo methods Also provided is a method for increasing the peptide repertoire presented by MHC proteins in a subject. This method may comprise modifying a cell in the subject to express the modified ERAP2. This may be done using any of the methods described above, e.g., by administering a nucleic acid encoding the modified ERAP2 (e.g., an encapsulated RNA, a viral vector or a plasmid vector) to the subject. In these embodiments, nucleic acid may be administered locally to the site at which the peptide repertoire is going to be increased, e.g., into a tumor, e.g., by direct injection into the tumor. In other embodiments, the administering may be systemic, e.g., intravenous. Also provided is a method of increasing the peptide repertoire presented by MHC proteins in cells ex vivo. This method may comprise removing cells from the subject and modifying the cells to express a modified ERAP2 protein, as described herein. In some embodiments, the method may involve re-introducing the cells back into the subject which, in some embodiments, may additionally comprise exposing the cells to other cells before re- introducing them back into the subject. In any of these embodiments, the cells may antigen- presenting cells (APCs) (e.g., dendritic cells) or a progenitor thereof (e.g., a hematopoietic stem cell (HSCs), which is multipotent precursor that have self-renewal capacity and the ability to regenerate all of the different cell types that comprise the blood-forming system. In any of these embodiments, the subject may have cancer, an autoimmune disease, an infection, or any other disease or condition in which increasing the peptide repertoire presented by MHC proteins would be beneficial. In cancer, an increased peptide repertoire presented by MHC proteins would provide more opportunity for cytotoxic T cells to recognize cancer cells and become activated. In autoimmune disease, an increased peptide repertoire presented by MHC proteins would provide more opportunity for T helper cells to recognize cells as being ‘self” and dampen the immune system. In these embodiments, the cells may be a treatment for cancer. In these embodiments, the subject may have a solid tumor, e.g., a carcinoma or a sarcoma. If the solid tumor is a carcinoma, in certain embodiments, the carcinoma is a basal cell carcinoma, squamous cell carcinoma, renal SF2024-175-2 cell carcinoma, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, or adenocarcinoma. Carcinomas that can treated by a method disclosed herein include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma, including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma. Sarcomas that can be treated by a method disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas. Other solid tumors that can be treated by a method disclosed herein include, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma, including primary tumors and, in certain cases, metastases. For example, the solid tumor may be colon cancer, bladder cancer, prostate cancer, breast cancer, lung cancer, or mesothelioma. In other embodiments, In these embodiments, the cells may be a treatment for an autoimmune disease and, as such, the subject may have a disease or condition that has local inflammation as part of its sequela, e.g., cardiovascular disease (which results in athleroscerotic plaque formation) and irritable bowel disease (IBD), although many others are known. In some embodiments, the subject may have an autoimmune disease. In these embodiments, the BTTS may have an extracellular binding domain that binds to cells in the inflamed tissue, e.g., to a disease-specific antigen or to an organ / tissue specific antigen. Autoimmune diseases include, but are not limited to, achalasia, Addison’s disease, adult still's disease, agammaglobulinemia, SF2024-175-2 alopecia areata, amyloidosis, ankylosing spondylitis, anti-gbm / anti-tbm nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal & neuronal neuropathy, Baló disease, Behcet’s disease, benign mucosal pemphigoid, bullous pemphigoid, Castleman disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy, chronic recurrent multifocal osteomyelitis, Churg-Strauss syndrome or eosinophilic granulomatosis, cicatricial pemphigoid, Cogan’s syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, Crest syndrome, Crohn’s disease, dermatitis herpetiformis, dermatomyositis, Devic’s disease (neuromyelitis optica), discoid lupus, Dressler’s syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, goodpasture’s syndrome, granulomatosis with polyangiitis, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis or pemphigoid gestationis, hidradenitis suppurativa (acne inversa), hypogammalglobulinemia, iga nephropathy, IGg4-related sclerosing disease, immune thrombocytopenic purpura (itp), inclusion body myositis (ibm), interstitial cystitis (ic), juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis (jm), kawasaki disease, lambert-eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear iga disease (lad), lupus, lyme disease chronic, meniere’s disease, microscopic polyangiitis (mpa), mixed connective tissue disease (mctd), Mooren’s ulcer, Mucha-Habermann disease, multifocal motor neuropathy, multiple sclerosis, myasthenia gravis, myelin oligodendrocyte glycoprotein antibody disorder, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism, pandas, paraneoplastic cerebellar degeneration (pcd), paroxysmal nocturnal hemoglobinuria, Parry Romberg syndrome, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, Poems syndrome, polyarteritis nodosa, polyglandular syndromes type i, ii, iii, polymyalgia rheumatica, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, primary biliary cholangitis, primary SF2024-175-2 sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia, pyoderma gangrenosum, Raynaud’s phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, schmidt syndrome, scleritis, scleroderma, sjögren’s syndrome, sperm & testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis, Susac’s syndrome, sympathetic ophthalmia, Takayasu’s arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura, thyroid eye disease, Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, ulcerative colitis, undifferentiated connective tissue disease, uveitis, vasculitis, vitiligo, and Vogt-Koyanagi-Harada disease. Combination therapies The present method of treatment may comprise co-administration of at least one additional therapeutic agent, e.g., an anti-inflammatory treatment, an anti-cancer treatment, or an anti-infection treatment such as an antibiotic or anti-viral. By “co-administration” it is meant that both the transgene and at least one additional therapeutic agent are administered to an individual, although not necessarily at the same time, in order to achieve a therapeutic effect that is the result of having administered both the nucleic acid or cells and the at least one additional therapeutic agent. The administration of the nucleic acid or cells and the at least one additional therapeutic agent can be substantially simultaneous, e.g., the polypeptide can be administered to an individual within about 1 minute to about 24 hours (e.g., within about 1 minute, within about 5 minutes, within about 15 minutes, within about 30 minutes, within about 1 hour, within about 4 hours, within about 8 hours, within about 12 hours, or within about 24 hours) of administration of the at least one additional therapeutic agent. In some cases, the transgene of the present disclosure is administered to an individual who is undergoing treatment with, or who has undergone treatment with, the at least one additional therapeutic agent. The administration of the nucleic acid or cells can occur at different times and / or at different frequencies. In some embodiments, the patient may additionally receive a T cell therapy (e.g., a CAR T cell therapy) or cancer vaccine that targets the tumor. Exemplary T cell therapies and cancer vaccines for GBM may target, e.g., IL13RA2, EGFRvIII, HER2, EphA2, MUC1, EGFR, PD-L1, B7-H3, NKG2D and PDPN, among others (see, e.g., Shraibman et al. Mol Cell Proteomics 2019 18:1255-1268; Wu et al. J Big Data. 20229: 92; Wang et al. Chin Neurosurg J. 20228: 34; SF2024-175-2 Nakagawa et al. Neurooncol Adv. 20235: vdac177; Nehama et al. EBioMedicine. 201947:33- 43; Yang et al. J Immunother Cancer. 20197: 171, Razpotnik et al. Front Immunol. 20178: 1181 and Akhavan et al Immunol Rev. 2019290: 60-84). Other antigens are known for other cancers. Other therapies include immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include inhibitors that target an immune checkpoint polypeptide such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1 and PD-L2. In some cases, the immune checkpoint polypeptide is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR, CD122 and CD137. In some cases, the immune checkpoint polypeptide is an inhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, CD96, TIGIT and VISTA. Further co-therapies include for example, (a) anthracycline therapy (e.g., by administering daunomycin, doxorubicin, or mitoxantrone), (b) alkylating agent therapy (e.g., by administering mechlorethane, cyclophosphamide, ifosfamide, melphalan, cisplatin, carboplatin, nitrosourea, dacarbazine, procarbazine or busulfan), (c) topoisomerase II inhibitor therapy (e.g., by administering etoposide or teniposide), (d) bleomycin therapy, (e) anti-metabolite therapy (e.g., by administering methotrexate, 5-fluorocil, cytarabine, 6-mercaptopurine or 6- thioguanine), (f) vinca alkyloid therapy (e.g., by administering vincristine or vinblastine), (g) steroid therapy (e.g., by administering prednisone or dexamethasone and (h) radiation treatment, etc. Alternative therapies include targeted therapies and non-targeted chemotherapies, where targeted therapy includes treatment with erlotinib (Tarceva), afatinib (Gilotrif), gefitinib (Iressa) or osimertinib (Tagrisso) which may be administered to patients having an activating mutation in EGFR, crizotinib (Xalkori), ceritinib (Zykadia), alectinib (Alecensa) or brigatinib (Alunbrig) which may be administered to patients having an ALK fusion, crizotinib (Xalkori), entrectinib (RXDX-101), lorlatinib (PF-06463922), crizotinib (Xalkori), entrectinib (RXDX-101), lorlatinib (PF-06463922), ropotrectinib (TPX-0005), DS-6051b, ceritinib, ensartinib or cabozantinib which may be administered to patients having an ROS1 fusion, or dabrafenib (Tafinlar) or trametinib (Mekinist) which may be administered to patients having an activating mutation in BRAF. Many other actionable mutations are known. If the patient is going to be switched to a non-targeted chemotherapy, the therapy may be, for example, a platinum-based doublet SF2024-175-2 chemotherapy (in which the platinum-based doublet chemotherapy may comprise a platinum- based agent selected from cisplatin (CDDP), carboplatin (CBDCA), and nedaplatin (CDGP)) and one third-generation agent (selected from docetaxel (DTX), paclitaxel (PTX), vinorelbine (VNR), gemcitabine (GEM), irinotecan (CPT-11), pemetrexed (PEM), and tegafur gimeracil oteracil (S1)). Screening methods In some embodiments, the method may further comprise analyzing the repertoire of peptides presented by MHC proteins on the cell. This may be done by, e.g., mass spectrometry (see, e.g., Bassani-Sternberg et al (MCP 201514: 658-673) or the isotope tagging approach described in Stopfer et al Nature Comm. 202011: 2760), among other methods. This screening method may be done to optimize the modifications that give rise to an altered repertoire of peptides presented by MHC proteins on the cell. Alternative embodiments ERAP2 has a similar structure and function as ERAP1 and, as such, a similarly modified form of ERAP1 is expected to provide similar results. In any embodiment, a modified ERAP1 may be expressed in the cell. These methods may be for increasing the peptide repertoire presented by MHC proteins on a mammalian cell, where the method comprises modifying the cell to express a modified ERAP1 (endoplasmic reticulum aminopeptidase 1) protein, e.g., aminopeptidase-deficient form of ERAP1. In these embodiments, the modified ERAP1 protein may comprise an aminopeptidase domain that has been inactivated by one or more amino acid substitutions, insertions or deletions, or it may lacks an aminopeptidase domain, as described above for ERAP2. The modified ERAP1 protein may be capable of dimerizing with ERAP2 or incapable of dimerizing modified ERAP2. All methods or compositions may be the same except ERAP1 may be employed. The following examples are offered by way of illustration and not by way of limitation. EXPERIMENTALBelow are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. SF2024-175-2 Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for. SUMMARYThis study reports that the ERAP2 haplogroups arose specifically in hominids, with individuals of African ancestry exhibiting additional genetic diversity consisting of two haplotypes of ERAP2-B. The study identifies that nucleic acid sensing induces the expression of ERAP2Iso3in monocyte-derived macrophages. ERAP2Iso3induction is controlled by multiple causal variants on ERAP2-B located within an intronic cis-regulatory element. In vitro, the truncated ERAP2Iso3 protein alters peptide processing cleavage efficiency of full-length ERAP1 and ERAP2. Finally, the first transgenic mice expressing ERAP2Iso1and ERAP2Iso3who do not express these genes endogenously were generated. In vivo, antigen presenting cells from both ERAP2Iso1 and ERAP2Iso3 transgenics stimulate wild type CD8 T cell responses but not in reciprocal. These findings establish that genetic variation at the ERAP2 locus encodes a protein in response to virus that causes differentiated antigen processing which ultimately creates a more diverse peptide-MHC repertoire in vivo. More broadly, this study provides a prototypical example of how an ancient and prevalent disease-associated genetic variation in humans interacts with common environmental exposures to generate distinct and diverse cellular responses. MATERIALS AND METHODS Ancestral Recombination Graph (ARG): Due to recombination, the genealogical history for a sample of DNA sequences may differ at different sites in the genome, and Ancestral Recombination Graph (ARG) is a data structure that stores the information about site-specific genealogies (trees) and how they are related by recombination events. A popular implementation of the ARG data structure is the tree sequence format introduced by Kelleher et al. (2016). This work utilized the genome-wide ARG recently inferred by Wohns et al. (2021) for 3601 modern humans from three whole-genome sequencing projects – the 1000 Genomes Project (1000 Genomes Project Consortium, 2015), the Human Genome Diversity Project (Bergström et al., 2020), and the Simons Genome Diversity Project (Mallick et al., 2016) – as well as 4 archaic hominids (three Neanderthals and a Denisovan) sequenced at high coverages. SF2024-175-2 154 of 3601 modern individuals were included in more than one sequencing project and hence appear in the tree sequence more than once. Duplicated entries were removed so that each individual is represented only once. Mapping rs2548535 (eQTL) and rs1581849348 (VNTR) on the inferred genealogy: rs2548535 (eQTL) is included in the inferred ARG from Wohns et al (2021) and mapped to a unique branch. However, rs1581849348 (VNTR) is a structural variant which was excluded when inferring the ARG. Hence, to map rs1581849348 in the ARG, the following approach was used: first, using the unphased genotype data from gnomAD, the individuals with homozygous VNTR insertions and with genotyping quality score > 90 were determined. The subtree (with 1326 leaves) was then extracted for these individuals from the marginal tree containing the rs1581849348 locus and maximum parsimony was used to infer required mutation events for the locus. It was found that a total of 22 mutation events are needed to explain the data but all of them except for one occur near terminal branches and hence likely correspond to genotyping errors. The remaining mutation occurs on a deep branch, which is taken to be where the structural variant first arose. Dating the variants: Upon mapping a mutation to the inferred ARG, its age can be estimated using the two endpoints of the branch on which the mutation occurred. The branch harboring the mutation for rs2548535 spans 22,200 to 45,780 generations in the past, which translates to 640,000–1,330,000 ya (assuming a generation time of 29 years), while the branch harboring the mutation for rs1581849348 is from 12740 to 16890 generations ago, which translates to 370,000–490,000 ya. Selection analysis of rs1581849348 (VNTR): A tree-based test of positive selection proposed by Speidel et al. (2019) (see page 16 of Supplementary Note, Method Details of their paper) was applied. Intuitively, this method tests for an imbalance in the number of descendant leaves for ancestral branches, and a mutation on a branch is likely to have experienced positive selection if the imbalance for the branch is statistically significant. In the inferred tree for the rs1581849348 locus, it was found that there were at most 14 lineages when the VNTR variant arose and 3650 out of 7200 leaves carry the mutation. The corresponding p-value for the test statistic is 0.00145, indicating a rather strong signal of positive selection. Inference of archaic hominins: In the marginal tree containing rs2548535 and rs1581849348, it was found that all lineages from the three Neanderthal individuals coalesce first SF2024-175-2 amongst themselves and then join the subtree with the A allele at rs2548535 (FIG. 1D). In contrast, the Denisovan (Denisova 5) lineage joined the part of the tree with the G allele at rs2548535 and without the repeat at rs1581849348 (FIG. 1D). In addition to the three high-coverage Neanderthal individuals included in the above inferred ARG, the VCF file (from 1.9X coverage data) was also examined for the Mezmaiskaya 1 Neandertal individual from Prüfer et al. (2017) and it was found that the individual is homozygous for HapA. The Denisova 11 genome (2.6X coverage) from Slon et al. (2018) was also checked; this individual has a Neanderthal mother and Denisovan father. Because Denisova 11 is not included in the inferred ARG, the BAM file provided to us by Kay Prüfer was instead used to determine the ERAP2 genotype of this individual. Specifically, the SNVs that arose on the same branch as rs2548535 (and hence in complete LD with rs2548535) in the inferred ARG were identified and the reads from Denisova 11 covering these variants were examined. Evidence of heterozygosity was observed at these sites, indicating that Denisova 11 contains both HapA and HapB. Phasing and determining haplotype frequencies in different populations: Because there is a lot of missing data in rs1581849348 and the VNTR genotypes are not phased, the inferred marginal tree at this locus was used to impute and phase the VNTR genotype. After imputing and phasing, the frequencies of 2-locus haplotypes (rs2548535, rs1581849348) were calculated in different continental populations, as well as in different sub-populations. Confidence intervals of HapB frequencies: Out of the 8 Neanderthal haplotypes studied, none of them is HapB, which leads to a 95% confidence interval of (0, 0.0063) for the HapB frequency in Neanderthals (ignoring the non-independence of these haplotypes and assuming Hardy-Weinberg equilibrium). Cell Culture and Genotyping: Immune cells from healthy human donors were purchased from Vitalant (San Francisco, USA) as TRIMA residuals. PBMCs were isolated using density centrifugation with Lymphoprep (StemCell, catalog no. 07851) in SepMate tubes (StemCell, catalog no. 85450). RBCs were removed with LCK lysis buffer (ThermoFisher, catalog no. A1049201) and aliquots of PBMCs were made in freezing media composed of 90% FBS (ThermoFisher, catalog no. 10438026) and 10% DMSO (Sigma-Aldrich, catalog no. D8418) and stored in liquid nitrogen. SF2024-175-2 At approximately 18 hours before stimulation, PBMCs were thawed and EasySep magnetic isolation kits by StemCell were used to isolate the different cell types: CD4+ T cells (catalog no. 17952), CD8+ T cells (catalog no. 17953), B cells (catalog no. 17954), NK cells (catalog no. 17955), and monocytes (catalog no. 19359). Isolated cells were cultured in complete RPMI medium consisting of RPMI 1640 Medium with GlutaMAX supplement (ThermoFisher, catalog no. 61870036), 10% FBS, and 100 U / ml Penicillin-Streptomycin (ThermoFisher, catalog no. 15140122). For differentiation to MDMs, monocytes were cultured for 6 days in the above media supplemented with 50 ng / ml rhGM-CSF (R&D Systems, catalog no. 215-GM-010) with fresh media added every 2-3 days. Cells were cultured in 96-well plates (Corning, catalog no. 07- 200-95) at 1-2 million cells per ml. To genotype cells for rs2248372, genomic DNA was extracted from approximately 1-3 million cells of each TRIMA donor via the Wizard SV Genomic DNA Purification System (Promega, catalog no. A2361). Real-time qPCR reactions were set up in triplicates in 384-well plates, each well consisting of genomic DNA, TaqMan Genotyping Master Mix (ThermoFisher, catalog no. 4371353), and the TaqMan rs2248372 genotyping probe (ThermoFisher, Assay ID: C_25649529_10). Reactions were run on the QuantStudio 5 Real-Time PCR System (and rs2248374 A / G genotypes were called using an allelic discrimination plot. U937 cells were purchased from ATCC and cultured according to their guidelines. Stimulation of PBMC Cell Types and ERAP2 isoform quantification: Influenza A virus strain A / PR / 8 / 34 (ATCC, catalog no. VR-95) was added at 50 infectious units per cell. Poly(I:C) HMW (catalog no. tlrl-pic), poly(I:C) (HMW) / LyoVec (catalog no. tlrl-piclv), and ssRNA40 / LyoVec (catalog no. tlrl-lrna40) were purchased from InvivoGen, resuspended in endotoxin-free water, and added to cells at final concentration of 5µg / ml. 3p-hpRNA (InvioGen, catalog no. tlrl-hprna) and G3-YSD (InvivoGen, catalog no. tlrl-ydna) were resuspended in endotoxin-free water, complexed with the LyoVec transfection agent (InvivoGen, catalog no. lyec-12), and added at effective concentration of 5µg / ml to each well. Recombinant human IFN- beta protein (R&D Systems, catalog no, 8499-IF-010) and IFN-gamma protein (R&D Systems, catalog no, 285-IF-100) were resuspended in DPBS solution (ThermoFisher, catalog no. 1419011) and added to cells at 100U / ml. After 18 hours, RNA was extracted from the cells using the Quick-RNA 96 kit (Zymo Research, catalog no. R1052) and reverse-transcribed to cDNA using the SuperScript IV VILO SF2024-175-2 Master Mix (ThermoFisher, catalog no. 11756050) following the manufacturers’ protocols. Triplicate reactions were set up in 384-well plates to measure the expression of genes or isoforms with the following TaqMan probes: full-length ERAP2 (Assay ID: Hs01073632_m1), ERAP2-B short isoforms (Custom Assay ID: APDJ3E7), total ERAP2 (Assay ID: Hs01073624_g1), ISG15 (Assay ID: Hs00192713_m1), and PTMA (Assay ID: Hs02339492_g1). The reaction was carried in the QuantStudio 5 Real-Time PCR System and the incubation protocol was 50 °C for 2 min, 95 °C for 10 min and 40 cycles of 95 °C for 15 s, 60 °C for 1 min. The Ct value of the three technical replicates of each sample were analyzed. If standard deviation greater than 1, the outlier replicate was removed. The Ct values were then averaged for each sample and expression levels of each gene or isoform were normalized to that of PTMA housekeeping gene using this formula: 2^[^^^^^^^ ^^ ^^^^^^^^^^^^^^^^^]. Box plots were generated with ggplot2 in R. Luciferase Assay: Sequences were designed for haplotypes A, B1, and B2 of CRE1, containing 10 genetic variants (with rs2248374). The haplotype sequences were split into inserts of about 320bp centered around each genetic variant (data not shown). All inserts, except rs2548535 B and VNTR B2, were made and cloned in the pNL3.1 luciferase vector (Promega, catalog no. N1031) by Twist Bioscience (San Francisco, USA). Due to the complexity from a repeated sequence in rs2548535 B and VNTR B2 inserts, they were cloned in-house. For each of these inserts, two gBlock gene fragments were ordered (IDT, USA), PCR amplified with the Platinum SuperFi II PCR Master Mix (ThermoFisher, catalog no. 12368010) to add 5’ and 3’ overhangs. Baculovirus protein expression and purification: cDNAs encoding 6X-His tagged human and murine ERAP1 and human ERAP2 isoforms were cloned into pFastBac1 (Invitrogen), confirmed by Sanger sequencing and subsequently used to transform competent DH10Bac Escherichia coli. Recombinant bacmids were isolated and used to transfect Hi-5 adherent insect cells with Cellfectin II (both from Invitrogen) to produce recombinant baculoviruses. Batches of recombinant proteins were produced in nonadherent Hi-5 insect cells grown in Express Five serum-free medium (Invitrogen). After infection with baculovirus, the culture medium containing the secreted ERAP protein was harvested by centrifugation (3000 × g, 30 min, 4 °C). The supernatant was concentrated in a Stirred Ultrafiltration Cell (Amicon, Millipore), adjusted to 50 mm phosphate, 300 mm NaCl, 10 mm imidazole, pH 8.0, and loaded SF2024-175-2 onto a Poly-prep chromatography column (Bio-Rad) pre-loaded with nickel-nitrilotriacetic acid- agarose (Qiagen). The column was washed with the same buffer, containing 20 mm imidazole, and the protein was eluted with a 40–150 mm imidazole gradient. Protein elution was checked by SDS-PAGE. Protein-containing fractions were dialyzed in Vivaspin 500 (Sartorius Stedim Biotech, Goettingen, Germany) against 10 mM Hepes buffer, 100 mM NaCl, pH 7, aliquoted, and stored at −70 °C. X-pNA Cleavage assay: X-pNA substrate (X=designated amino acid) was mixed with 100 nM of full length human ERAP1, ERAP2, or murine ERAP1 (ERAAP), mixed with 0-200 nM of human ERAP2Iso3 in ERAP reaction buffer (50 mM Tris pH 7.2, 150 mM NaCl, 100 µM ZnCl2). Enzymatic cleavage of substrate was monitored by absorbance at OD=410nm over time 0-60min. Cell based antigen processing assay: The β-galactosidase (lacZ)-inducible SHL8 / Kb- specific B3Z hybridomas, H2-Kb expressing APC fibroblasts, and ERAAP / TAP deficient fibroblasts have been previously described. Fibroblasts (200,000 cells) were transfected (FuGENE6, Roche) with plasmids (0.3 µg) containing minigenes encoding ER-targeted and N- terminally extended antigenic peptide (ESS-RK-SIINFEHL, RK-SHL8) along with plasmids (1 µg) encoding ERAP1 and / or ERAP2 isoforms. Total transfected DNA content was normalized across experimental conditions with empty vector. 48 hours after transfection, cells were harvested, peptides were acid extracted (10% formic acid) and filtered through Microcon-10 filters (<10,000 MW cutoff, Amicon), and the filtrate was fractionated by HPLC on a reverse phase C18 column (Vydac) using an Agilent 1260 HPLC system. Fractionated samples were dried by vacuum centrifugation. Fractions were treated with trypsin (7.5 µg / mL) for 3 hours and co-cultured with H2-Kb expressing APCs and B3Z hybridoma cells for 18 hours. LacZ activity induced upon antigen specific T cell activation was measured by conversion of substrate chlorophenolred- β-D-galactopyrannoside to chlorophenol red with measurement of absorbance at 595 nm with 655 nm background subtraction. Identity of peptide fractionation was determined by fractionation of synthetic RK-, K-, and SHL8 peptides (ThermoFisher). Generation of transgenic mouse: A construct expressing ERAP2iso3 under the control of the CAG promoter was introduced into mouse embryos by pronuclear injection and targeted to the safe harbor ROSA26 locus using appropriate sgRNA and homology arms in the targeting vector. PCR was used to detect transgenic founders with appropriate integration into the ROSA26 SF2024-175-2 locus, and transgene expression was measured in tissues using quantitative real-time PCR (PowerUp SYBR Green and QuantStudio 6, ThermoFisher) in purified RNA (Rneasy MicroKit, Qiagen) or by western blotting in tissues lysates (1X RIPA Buffer with protease inhibitor, Cell Signaling Technology). Blots were probed with specific anti-ERAP2 antibodies (AF3830, RND Systems; and HPA034498, Millipore-Sigma). Three founder lines with appropriate insertion were obtained, and germline transmission was confirmed in two founder lines. All experiments shown were performed with progeny of one founder line (#135) with robust transgene expression. T cell immunization and restimulation: Generation and detection of antigen-specific T cells was performed as described with minor modifications. Briefly, T cells that recognize ERAP2iso3-trangene specific peptide-MHC complexes were generated by intraperitoneal immunization of female C57BL / 6J mice (Jackson Labs) with ~20x106splenocytes harvested from male ERAP2iso3-trangenic animals. The spleens from immunized animals were harvested 10 days after immunization. Responding T cells were restimulated (5x106cells per well in 24 well plates) with an equal number of irradiated splenocytes harvested from female ERAP2iso3- trangenic animals in the presence of 50 U / mL recombinant human IL-2 (Peprotech). Responding T cells were passaged and restimulated every 6 days on freshly irradiated transgenic splenocytes or used for assessment of intracellular cytokine production. For this assay, antigen presenting splenocytes from either female wild type or ERAP2iso3-trangenic were depleted of CD4+ and CD8+ cells by magnetic bead depletion (Miltenyi) and stimulated overnight with 500 ng / mL lipopolysaccharide (Sigma), then mixed with responding T cells that had been harvested, enriched for viable cells (Lympholyte-M, Cedar Labs), and rested overnight. Equal numbers (~500,000 cells) of APCs and T cells were cultured for 5 hours in the presence of 10 µg / mL BrefeldinA (Sigma), and then cells were stained for surface marker expression, fixed, permeabilized, and stained for intracellular IFNɣ and TNF⍺ (BD Biosciences). Results ERAP2 haplotypes emerged during human evolution In humans, two highly differentiated ERAP2 haplogroups distinguished by a large number of SNPs in high linkage disequilibrium (LD) have evolved under long-term balancing selection and are associated with immune-mediated diseases (IMIDs) (FIG. 1A and data not SF2024-175-2 shown). In lymphoblastoid cell lines (LCLs), it has been previously described that ERAP2-A encodes a full-length transcript (ERAP2Iso1) while ERAP2-B encodes a transcript degraded by nonsense-mediated decay (NMD) (ERAP2Iso2) due to two premature codons introduced by the usage of an alternate splice site created by rs2248374-G (FIG. 1B). While examining the two ERAP2 haplogroups for differential genetic elements with potential regulatory function, a 100-bp intronic tandem repeat (TR) located 1-bp downstream of the 3’ end of exon 94(rs1581849348) was identified, further segregating ERAP2-B into two haplotypes that either harbored one (ERAP2-B1) or two copies (ERAP2-B2) of the TR (FIGS. 1A-1B). Six additional SNPs are in perfect LD with the copy number of the tandem repeat including rs2927608. Using an inferred ancestral recombination graph5derived from whole-genome sequencing (WGS) data from the 1000 Genomes Project, the Human Genome Diversity Project (HGDP), and the Simons Genome Diversity Project (SGDP), it was estimated that the haplotype frequency is 0.45 for ERAP2-A (containing a single copy of the TR), 0.043 for ERAP2-B1, and 0.51 for ERAP2-B2. There is striking geographic variation in the frequency of ERAP2-B1 and ERAP2-B2 (FIG. 1C); in the Mbuti and Biaka, ERAP2-B1 is the dominant ERAP2-B haplotype. Albeit at lower allele frequencies, ERAP2-B1 is also segregating in populations in South Asia as well as the Caribbean and United States, corresponding to evolutionarily recent population movement and gene flow. In all other geographic locations, the ERAP2-B2 is strongly overrepresented within the ERAP2-B haplogroup. Next, the frequencies of the three ERAP2 haplotypes were examined in ancient and modern human genomes and chimpanzees to assess how they evolved and diverged in hominids. Across 39 chimpanzee genomes, the ERAP2 locus has much lower overall LD than in humans and SNPs are mostly different from humans (data not shown). ERAP2 is present in most mammalian species, except for rodents, where the gene has been lost in the majority of this group during evolution6. Using the aforementioned ancestral recombination graph5, it was estimated that the coalescence time of ERAP2 haplogroups is about 1.28 mya, which agrees with the previous estimate by Andres et al. (1.44 ± 0.55 mya)7but is much younger than the estimate by Cagliani et al. (> 5.08 mya)8(FIG. 1D). Remarkably, only ERAP2-A was detected in 3 high- coverage Neanderthal individuals while only ERAP2-B1 was detected in 1 high-coverage Denisovan individual (FIG. 1D). Furthermore, it was found that Mezmaiskaya 1 Neanderthal individual9is homozygous for ERAP2-A, while Denisova 11 individual10, who has a Neanderthal SF2024-175-2 mother and Denisovan father, has both ERAP2-A and ERAP2-B. These results suggest that the ERAP2-B haplogroup evolved before modern humans split from the Neanderthal / Denisovan supergroup 400 to 800 thousand years ago, but ERAP2-B likely was lost in Neanderthals, while introgression involving this haplogroup occurred between modern humans and Denisovans. In addition, ERAP2-B2 arose even more recently during human evolution. A genealogy-based statistical test for neutrality shows a strong signal of positive selection for ERAP2-B2, consistent with its high frequency in non-African populations and suggests it may be beneficial for adaptation outside of Africa. The ancestral haplogroup (ERAP2-A) has recently been associated with resistance to Yersinia pestis during the European plague in the Middle Ages11and resistance to human immunodeficiency virus-1 (HIV) infection8,12. In contrast, the derived haplogroup (ERAP2-B) has been previously associated with reduced risk for Crohn’s disease, ankylosing spondylitis, and preeclampsia. The association of ERAP2 haplotypes with common human diseases was analyzed by utilizing samples in the 23andme V5 database. Across samples of European ancestry, it was confirmed that ERAP2-B haplogroups are significantly associated with reduced risk for preelampsia and Crohn’s disease (FIG. 1E). Novel ERAP2 isoforms expressed from ERAP2-B in response to nucleic-acid sensing. Recent studies have repeatedly identified ERAP2 as one of the strongest expression quantitative trait loci (eQTLs) in the human genome across various human tissues. Consistent with data in LCLs, these studies have found ERAP2-B to be associated with low transcript abundance. In monocyte-derived dendritic cells (MoDCs), it was found that while ERAP2-B is associated with lower transcript abundance at baseline, it is strikingly associated with the transcription of two previously unknown isoforms (ERAP2Iso3 and ERAP2Iso4 collectively referred to as ERAP2Iso3 / 4), in response to influenza A virus (IAV) infection, but not stimulation with the antiviral cytokine IFN-β13,14. ERAP2Iso3 / 4 differ from the full-length ERAP2Iso1 by the initiation of transcription at exon 9, the alternate splicing of an extended exon 10, and can be distinguished from each other by the alternative splicing between a constitutive and a secondary splice site at exon 15 (FIG. 2A). A reanalysis of RNA-sequencing data across ancestrally diverse samples14confirmed that individuals from African ancestry exclusively harbored ERAP2-B1 and a haplotype-based test showed that ERAP2Iso3 / 4expression was lower in carriers of ERAP2-B1 vs ERAP2-B2 haplotypes (FIG. 2B and data not shown). SF2024-175-2 To validate the IAV-specific transcription of ERAP2Iso3 / 4, qPCR was used to profile myeloid subtypes stimulated with IAV at 10 hours (FIG. 2C). In response to IAV, ERAP2Iso3 / 4 induction was observed in CD14 monocytes (P = 0.002) from ERAP2-B2 individuals, reaching levels beyond the expression of the full-length isoform in controls and infected cells. Interestingly, the expression of ERAP2Iso3 / 4 in monocyte derived macrophages (MDMs) and MoDCs were even higher after IAV infection. Next, inflammatory stimuli that may induce ERAP2Iso3 / 4in MDMs were assessed. IAV is a negative-sense, single-stranded RNA (-ssRNA) virus capable of activating several different pathogen-associated molecular pattern recognition (PAMP) pathways, including endosomal and cytosolic sensors (data not shown). Thus, the requisite stimuli were mapped using synthetic agonists to stimulate each PAMP receptor in MDMs. IAV infection produced the highest induction of ERAP2Iso3 / 4 (FIG. 2D). Treatment with poly(I:C) complexed with transfection reagent (polyIC-LV), which facilitates the entry of double-stranded RNA (dsRNA) mimetic to the cytoplasm leading to stimulation of the cytosolic RNA sensors RIG-I and / or MDA-5, also resulted in significant induction of ERAP2Iso3 / 4 expression (FIG. 2D). Selective stimulation of RIG-I by 5’-triphosphate hairpin RNAs, cGAS-STING by G3-YSD-LyoVec, and endosomal TLR8 by ssRNA40-LyoVec, also upregulated ERAP2Iso3 / 4, suggesting that its induction is a conserved response to nucleic-acid sensing of multiple viral infections beyond IAV. Notably, treatment with poly(I:C) alone, which stimulates endosomal sensing of dsRNA through TLR3, did not significantly induce ERAP2Iso3 / 4. Treatment with type I or type II interferons also significantly induced ERAP2Iso3 / 4– a result not observed in previous work, which may be due to the longer culture time in this study that resulted in the induction of both short and long ERAP2 isoforms. To investigate the possibility that selective upregulation enhancing the ratio of ERAP2Iso3 / 4to ERAP2FLwas a specific response to viral infection and not interferons, allele-specific probes targeting a SNP in exon 11 were utilized (FIG. 2A) to quantify the relative abundance of the ERAP2 isoforms in heterozygous individuals. As expected, cells stimulated with interferons did not exhibit significant differences in allelic ratios relative to unstimulated controls. In cells stimulated with IAV, and to a lesser extent in response to PAMPs, the allelic ratio increased in favor of ERAP2Iso3 / 4expression (FIG. 2E). Altogether, these results suggest that the induction of SF2024-175-2 ERAP2Iso3 / 4is a conserved response to nucleic-acid sensing of multiple viral infections beyond IAV. Causal allele on ERAP2-B consists of two copies of a tandem repeat and a SNP. One of the genomic footprints of long-term balancing selection at the ERAP2 locus is the large region of LD that distinguishes the two haplogroups. While this entire region has been associated with IMIDs, pinpointing the causal variants at the locus is critical for understanding the mechanisms by which disease risk is mediated. Because ERAP2Iso3 / 4share an alternative start site, it was sought to identify the cis-regulatory element(s) (CRE) that would induce their expression in response to IAV. Bulk ATAC-seq on MDMs infected with IAV identified several candidate CREs at the locus, with CRE1 being particularly intriguing for three reasons (FIGS. 3A-3B). First, CRE1 is located downstream but near the predicted transcription start site for ERAP2Iso3 / 4. Second, the ATAC-seq data directly captured additional accessibility at the TR in carriers of ERAP2-B2, suggesting that the TR could function as part of the CRE. Third, QTL analysis of chromatin accessibility in classical monocytes across 350 healthy controls showed that the accessibility of the second ATAC-seq peak within CRE1 (chr5:96896800-968973000) is significantly associated with rs2548535, a SNP tagging ERAP2-B230 bp downstream of the TR located within the peak (FIG. 3C). In addition to the TR and rs2548535, CRE1 contains 4 additional SNPs that distinguish the two haplogroups. To pinpoint the causal alleles within CRE1 that induce ERAP2Iso3 / 4 expression, luciferase reporter assays were performed in U937 cells at rest or after IAV infection at 18 hours (data not shown). 8 constructs covering the two peaks in CRE1 were tested, 6 of which distinguished the three haplotypes within each peak. Two additional constructs for haplotypes A and B1 were padded with random 100 bases to ensure they were the same length as the B2 construct containing two copies of the TR. Of those tested, only the ERAP2-B2 constructs overlapping peak 2 significantly drove luciferase expression at baseline and induced luciferase expression in response to IAV infection (FIG. 3D). To further resolve if individual or multiple causal variants may explain the effects of peak 2, additional reporter assays were performed to test the effects of all possible combinations of the three variants that reside within the peak, including copy number of TR, alleles of rs2548535, and alleles of rs2161657. These experiments first confirmed that ERAP2-B2 differentially affected luciferase expression but not ERAP2-A and ERAP2-B1. The largest effect is driven by the rs2161657-T allele. Strikingly, these analyses also identified an SF2024-175-2 epistatic effect of rs2548535-G allele with rs2161657-T allele to drive expression (FIG. 3E). These results suggest that the effect of rs2548535 is significantly amplified by rs2161657 and that the combination of rs2161657 and rs2548535 allele is the causal cis-regulatory element in the locus that determines the ERAP2Iso3 / 4expression from haplotypes B2. ERAP2Iso3 modifies ERAP1 and ERAP2 peptide cleavage in vitro. To characterize the function of ERAP2Iso3, its activity was studied in vitro. The putative ERAP2Iso3protein isoform induced by IAV infection contains only the C-terminal domain of the full-length protein, lacking the enzymatic domain. However, structural studies have modeled how the C-terminal domain is capable of forming potential interactions with peptide substrates and / or full-length aminopeptidases. Consistent with its lack of aminopeptidase domain, ERAP2Iso3showed no enzymatic activity against a panel of amino acid substrates on its own (FIG. 4A). However, when co-incubated with full-length aminopeptidases, ERAP2Iso3 increased the cleavage efficiency of human and mouse ERAP1 and reduced the cleavage efficiency of human ERAP2 against modified amino acid substrates (X-pNA) (FIG. 4B). These effects were linearly dependent on the ERAP2Iso3 dose (FIG. 4C). When assaying a longer peptide substrate that was poorly trimmed by ERAP2 alone, the addition of ERAP2Iso3 drastically increased the recovery of trimmed peptide intermediates. Similar changes were also observed during the coincubation of ERAP2Iso3 with human or murine ERAP1 with peptide substrates, demonstrating that ERAP2Iso3 modulated the repertoire of peptides generated by full-length aminopeptidases in vitro. Next, it was tested whether ERAP2Iso3functioned similarly in living cells. Using a well- established assay of ER antigen processing, it was found that an extended peptide substrate for ERAP2 was predominantly overtrimmed and thus was eliminated from the antigenic repertoire when expressed with full-length ERAP2 alone (FIG. 4D). However, when ERAP2Iso3was also present, increased amounts of the trimmed intermediate were recovered (FIG. 4D), even though the full-length ERAP2 remained robustly expressed in co-transfected cells. Thus, ERAP2Iso3 also modulates the repertoire of peptides generated by full length aminopeptidases in cells. ERAP2Iso3 alters antigenic peptide repertoires in vivo In Mus musculus, ERAP1 has been extensively characterized and shown to trim peptides in the ER to the optimal lengths for binding and presentation by MHC I molecules. Because ERAP2 is absent in most rodents including mice, comparatively less is known about its function SF2024-175-2 in vivo, and the function of truncated ERAP2 isoforms have been entirely unelucidated. To investigate ERAP2Iso1 and ERAP2Iso3 activity in vivo, two transgenic mouse lines humanized to ubiquitously express ERAP2Iso1 or ERAP2Iso3 on the B6 background were generated (FIG. 5A). These models provide a powerful platform to interrogate if ERAP2Iso1and ERAP2Iso3-induced alterations of peptide-MHC repertoires modulate downstream antigen-specific T cell responses. Transgenic animals expressed ERAP2Iso1 and ERAP2Iso3 in all tissues evaluated, including robust expression in critical immune organs–spleen, lymph nodes, and thymus (FIG. 5B). At baseline, no significant differences in the frequency of immune cell subsets were observed between the transgenic animals and wild type controls (FIG. 5C). In addition, no spontaneous disease pathologies were noted in the transgenic mice (data not shown). Deficiency of ERAP1 has been shown to alter pMHC stability leading to reduced surface expression of MHC. However, surface levels of MHC class I and class II molecules were not altered in ERAP2Iso1 and ERAP2Iso3 transgenic animals (FIG. 5D), suggesting that global stability of pMHC complexes was maintained. It was reasoned that if ERAP2Iso1and ERAP2Iso3nevertheless altered pMHC repertoires in vivo, transgenic animals would maintain self-tolerance against these antigenic targets. However, wild type animals should contain non-tolerized T cells which could recognize this novel pMHC repertoire if immunized with transgenic APCs, an approach previously used to successfully identify the effects of murine ERAP1 deficiency. Consistent with this hypothesis, wild type animals generated robust CD8 T cell responses against APCs from ERAP2Iso1and ERAP2Iso3transgenic mice (FIGS. 5E and 5G). In ERAP2Iso1transgenics, blocking with anti-MHCI antibodies partially reduced CD8 T cell expansion. Note that APCs from ERAP2Iso3 transgenics generated the highest frequency of CD8 T cells (~30%) compared to APCs from ERAP1KO animals (~20%) and ERAP2Iso1 animals (~12%) (FIGS. 5F and 5H). Furthermore, reciprocal immunization experiments show that T cells of ERAP2Iso3transgenic mice maintained tolerance to pMHCs presented by wild type APCs, which was not observed in ERAP1KO animals (FIGS. 5E-5F). Thus, ERAP2Iso1 and ERAP2Iso3 transgene expression did not cause global dysregulation of immune responses, but resulted in the generation of a more diverse immunogenic pMHC repertoire in vivo with antigen-specific functional consequences for the immune system. These results conclusively demonstrate that ERAP2Iso1and, perhaps more importantly, ERAP2Iso3, which does not have a catalytic aminopeptidase domain, impact T cell responses by altering the peptide-MHC repertoire. The SF2024-175-2 selective induction of this mechanism in response to influenza provides a plausible explanation for how balancing selection has maintained ERAP2-B in modern humans in response to selective pressures from pathogens. Similar data is obtained using CD4+T cell (FIG. 6). This data shows that ERAP2 Iso3 also affects CD4+ processing, which is not observed in the ERAAP knockout. This data shows that modulating ERAP2 may affect both class I and class II presentation. Here it is shown that alternative isoforms expressed from the ERAP2-B allele during influenza viral infection are not degraded but are translated into truncated protein isoforms (ERAP2Iso3 and ERAP2Iso4). Using a variety of in vitro and in vivo approaches, it was demonstrated that even though ERAP2Iso3 lacks enzymatic activity, it elicits differentiated antigen processing. Most powerfully, using novel transgenic mice humanized to express ERAP2Iso1and ERAP2Iso3, it was found that alterations in antigen processing generated distinct peptide repertoires that served as specific antigenic targets for T cell responses. Mechanistically, interactions of ERAP2Iso3 with peptide substrates and / or the full-length ERAP1 and ERAP2 proteins likely leads to this differentiated antigen processing. Heterodimers of ERAP1 and ERAP2 have been proposed as the optimal trimming complex of antigenic precursors with the broadest length and residue specificity. ERAP2Iso3 may similarly form functional interactions with the full-length ERAP1 and ERAP2 aminopeptidases, altering the dynamics of their trimming activity and / or their substrate selectivity. Like ERAP1 knockout mice, ERAP2Iso1 and ERAP2Iso3 transgenics do not develop spontaneous autoimmunity or infectious disease. Nevertheless, these animals are a valuable resource for follow up cross breeding experiments to infectious disease, autoimmunity, and cancer immunity models to examine the effects of ERAP2 isoforms in those contexts. REFERENCES 1. Harroud, A. & Hafler, D. A. Common genetic factors among autoimmune diseases. Science 380, 485–490 (2023). 2. Benton, M. L. et al. The influence of evolutionary history on human health and disease. Nat. Rev. Genet. 22, 269–283 (2021). 3. Kerner, G. et al. Genetic adaptation to pathogens and increased risk of inflammatory disorders in post-Neolithic Europe. Cell Genom. 3, 100248 (2023). SF2024-175-2 4. Lu, T.-Y., Human Genome Structural Variation Consortium & Chaisson, M. J. P. Profiling variable-number tandem repeat variation across populations using repeat-pangenome graphs. Nat. Commun. 12, 4250 (2021). 5. Wohns, A. W. et al. A unified genealogy of modern and ancient genomes. Science 375, eabi8264 (2022). 6. Paladini, F., Fiorillo, M. T., Tedeschi, V., Mattorre, B. & Sorrentino, R. The multifaceted nature of aminopeptidases ERAP1, ERAP2, and LNPEP: From evolution to disease. Front. Immunol. 11, 1576 (2020). 7. Andrés, A. M. et al. Balancing selection maintains a form of ERAP2 that undergoes nonsense-mediated decay and affects antigen presentation. PLoS Genet. 6, e1001157 (2010). 8. Cagliani, R. et al. Genetic diversity at endoplasmic reticulum aminopeptidases is maintained by balancing selection and is associated with natural resistance to HIV-1 infection. Hum. Mol. Genet. 19, 4705–4714 (2010). 9. Prüfer, K. et al. A high-coverage Neandertal genome from Vindija Cave in Croatia. Science 358, 655–658 (2017). 10. Slon, V. et al. The genome of the offspring of a Neanderthal mother and a Denisovan father. Nature 561, 113–116 (2018). 11. Klunk, J. et al. Evolution of immune genes is associated with the Black Death. Nature 611, 312–319 (2022). 12. Biasin, M., De Luca, M., Gnudi, F. & Clerici, M. The genetic basis of resistance to HIV infection and disease progression. Expert Rev. Clin. Immunol. 9, 319–334 (2013). 13. Lee, M. N. et al. Common genetic variants modulate pathogen-sensing responses in human dendritic cells. Science 343, 1246980 (2014). 14. Ye, C. J. et al. Genetic analysis of isoform usage in the human anti-viral response reveals influenza-specific regulation of ERAP2 transcripts under balancing selection. Genome Res. 28, 1812–1825 (2018). Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited SF2024-175-2 herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein

Claims

SF2024-175-2 CLAIMS What is claimed is:

1. A method for increasing the peptide repertoire presented by MHC proteins on a mammalian cell, comprising: modifying the cell to express a modified ERAP2 (endoplasmic reticulum aminopeptidase 2) protein.

2. The method of claim 1, wherein the modified ERAP2 protein is aminopeptidase- deficient.

3. The method of claim 1 or 2, wherein the modified ERAP2 protein comprises an aminopeptidase domain that has been inactivated by one or more amino acid substitutions, substitutions or deletions.

4. The method of any of claims 1-3, wherein the modified ERAP2 protein lacks an aminopeptidase domain.

5. The method of any of claims 1-4, wherein the modified ERAP2 is capable of dimerizing with ERAP1.

6. The method of any of claims 1-4, wherein the modified ERAP2 is not capable of dimerizing with ERAP1.

7. The method of any prior claim, wherein the cell is a cancer cell.

8. The method of any of claims 1-4, wherein the cell is not a cancer cell.

9. The method of any prior claim, wherein the cell is a professional antigen presenting cell.SF2024-175-2 10. The method of any prior claim, wherein the MHC proteins are MHC Class I proteins.

11. The method of any of claims 1-9, wherein the MHC proteins are MHC Class II proteins.

12. The method of any prior claim, wherein the modifying is done by modifying the cell to express a nucleic acid encoding the modified ERAP2 protein in the cell.

13. The method of claim 12, wherein modifying is done by introducing an RNA or DNA encoding the modified ERAP2 protein in the cell.

14. The method of claim 13, wherein the RNA or DNA is in a plasmid or viral vector.

15. The method of any of claims 1-11, wherein the modifying is done by directly introducing the aminopeptidase-deficient ERAP2 protein into the cell.

16. The method of any of claims 1-12, wherein the modifying is done by modifying the genome of the cell.

17. The method of any prior claim, wherein the cell is in vitro, in vivo or ex vivo.

18. The method of any prior claim, further comprising analyzing the repertoire of peptides presented by MHC proteins on the cell.

19. A recombinant nucleic acid comprising a coding sequence for a modified ERAP2 protein.

20. The nucleic acid of claim 19, wherein the recombinant nucleic acid is a plasmid or viral vector.

21. The nucleic acid of claim 19 or 20, wherein the coding sequence is operably linked to an inducible, tissue-specific or constitutive promoter.SF2024-175-2 22. A method of increasing the peptide repertoire presented by MHC proteins in a mammalian subject in need thereof, comprising: modifying a cell in the subject to express the modified ERAP2 of any of claims 1-6.

23. The method of claim 22, wherein the subject has cancer or an autoimmune disease.

24. A method of increasing the peptide repertoire presented by MHC proteins, comprising: removing cells from a subject, modifying the cells to express the modified ERAP2 protein of any of claims 1-6.

25. The method of claim, wherein the method further comprises loading the modified cells with antigen.

26. The method of any of claims 24-25, further comprising: re-introducing the cells back into the subject. 27 The method of claim 25 or 26, wherein the antigen is a cancer antigen from the subject.

28. The method of any of claims 24-27, wherein the cells are professional antigen-presenting cells (APCs) or a progenitor thereof.

29. The method of any of claims 24-28, wherein the subject has cancer, an autoimmune disease or an infectious disease.

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