Combinations of the highly polymorphic KIR and HLA-I genes are associated with numerous human diseases. Interpreting these associations requires a molecular understanding of the multiple killer-cell immunoglobulin-like receptor (KIR)–human leukocyte antigen-1 (HLA-I) receptor–ligand interactions on natural killer (NK) cells and identifying the salient features that underlie disease risk. We hypothesize that a critical discriminating factor in KIR–HLA-I interactions is the selective detection of HLA-I-bound peptides by KIRs. We propose a ‘peptide selectivity model’, where high-avidity KIR–HLA-I interactions reflect low selectivity for peptides conferring consistent NK cell inhibition across different tissue immunopeptidomes. Conversely, lower-avidity interactions (including those with activating KIRs) are more dependent on HLA-I-bound peptide sequence, requiring an appreciation of how HLA-I immunopeptidomes influence KIR binding and regulate NK cell function. Relevant to understanding NK cell function and pathology, we interpret known KIR–HLA-I combinations and their associations with certain human diseases in the context of this ‘peptide selectivity model’.

Mutations in KRAS are some of the most common across multiple cancer types and are thus attractive targets for therapy. Recent studies demonstrated that mutant KRAS generates immunogenic neoantigens that are targetable by adoptive T-cell therapy in metastatic diseases. To expand mutant KRAS-specific immunotherapies, it is critical to identify additional HLA-I allotypes that can present KRAS neoantigens and their cognate T-cell receptors (TCR). Here, we identified a murine TCR specific to a KRAS-G12V neoantigen (7VVVGAVGVGK16) using a vaccination approach with transgenic mice expressing HLA-A*03:01 (HLA-A3). This TCR demonstrated exquisite specificity for mutant G12V and not WT KRAS peptides. To investigate the molecular basis for neoantigen recognition by this TCR, we determined its structure in complex with HLA-A3(G12V). G12V-TCR CDR3β and CDR1β formed a hydrophobic pocket to interact with p6 Val of the G12V but not the WT KRAS peptide. To improve the tumor sensitivity of this TCR, we designed rational substitutions to improve TCR:HLA-A3 contacts. Two substitutions exhibited modest improvements in TCR binding avidity to HLA-A3 (G12V) but did not sufficiently improve T-cell sensitivity for further clinical development. Our study provides mechanistic insight into how TCRs detect neoantigens and reveals the challenges in targeting KRAS-G12V mutations.

preprint by Fast et al. describes TAPIR, a computational tool for predicting TCR–pMHC interactions.

Genetic studies associate killer cell immunoglobulin-like receptors (KIRs) and their HLA class I ligands with a variety of human diseases. The basis for these associations and the relative contribution of inhibitory and activating KIR to NK cell responses are unclear. Because KIR binding to HLA-I is peptide dependent, we performed systematic screens, which totaled more than 3500 specific interactions, to determine the specificity of five KIR for peptides presented by four HLA-C ligands. Inhibitory KIR2DL1 was largely peptide sequence agnostic and could bind ~60% of hundreds of HLA-peptide complexes tested. Inhibitory KIR2DL2, KIR2DL3, and activating KIR2DS1 and KIR2DS4 bound only 10% and down to 1% of HLA-peptide complexes tested, respectively. Activating KIR2DS1, previously described as weak, had high binding affinity for HLA-C, with high peptide sequence specificity. Our data revealed MHC-restricted peptide recognition by germline-encoded NK receptors and suggest that NK cell responses can be shaped by HLA-I-bound immunopeptidomes in the context of disease or infection.

In cancer, non-synonymous DNA base changes alter protein sequence and produce neoantigens that are detected by the immune system. For immune detection, neoantigens must first be presented on class I or II human leukocyte antigens (HLA) followed by recognition by peptide-specific receptors, exemplified by the T-cell receptor (TCR). Detection of neoantigens represents a unique challenge to the immune system due to their high similarity with endogenous 'self' proteins. Here, we review insights into how TCRs detect neoantigens from structural studies and delineate two broad mechanistic categories: 1) recognition of mutated 'self' peptides and 2) recognition of novel 'non-self' peptides generated through anchor residue modifications. While mutated 'self' peptides differ only by a single amino acid from an existing 'self' epitope, mutations that form anchor residues generate an entirely new epitope, hitherto unknown to the immune system. We review recent structural studies that highlight these structurally distinct mechanisms and discuss how they may lead to differential anti-tumor immune responses. We discuss how T cells specific for neoantigens derived from anchor mutations can be of high affinity and provide insights to their use in adoptive T cell transfer-based immunotherapy.

Dimorphic amino acids at positions 77 and 80 delineate HLA-C allotypes into two groups, C1 and C2, which associate with disease through interactions with C1 and C2-specific natural killer cell receptors. How the C1/C2 dimorphism affects T cell recognition is unknown. Using HLA-C allotypes that differ only by the C1/C2-defining residues, we found that KRAS-G12D neoantigen-specific T cell receptors (TCRs) discriminated between C1 and C2 presenting the same KRAS-G12D peptides. Structural and functional experiments, and immunopeptidomics analysis revealed that Ser77 in C1 and Asn77 in C2 influence amino acid preference near the peptide C-terminus (pΩ), including the pΩ-1 position, in which C1 favors small and C2 prefers large residues. This resulted in weaker TCR affinity for KRAS-G12D-bound C2-HLA-C despite conserved TCR contacts. Thus, the C1/C2 dimorphism on its own impacts peptide presentation and HLA-C-restricted T cell responses, with implications in disease, including adoptive T cell therapy targeting KRAS-G12D-induced cancers.

Complete cancer regression occurs in a subset of patients following adoptive T cell therapy (ACT) of ex vivo expanded tumor-infiltrating lymphocytes (TILs). However, the low success rate presents a great challenge to broader clinical application. To provide insight into TIL-based immunotherapy, we studied a successful case of ACT where regression was observed against tumors carrying the hotspot mutation G12D in the KRAS oncogene. Four T cell receptors (TCRs) made up the TIL infusion and recognized two KRAS-G12D neoantigens, a nonamer and a decamer, all restricted by human leukocyte antigen (HLA) C*08:02. Three of them (TCR9a, 9b, and 9c) were nonamer-specific, while one was decamer-specific (TCR10). We show that only mutant G12D but not the wild-type peptides stabilized HLA-C*08:02 due to the formation of a critical anchor salt bridge to HLA-C. Therapeutic TCRs exhibited high affinities, ranging from nanomolar to low micromolar. Intriguingly, TCR binding affinities to HLA-C inversely correlated with their persistence in vivo, suggesting the importance of antigenic affinity in the function of therapeutic T cells. Crystal structures of TCR–HLA-C complexes revealed that TCR9a to 9c recognized G12D nonamer with multiple conserved contacts through shared CDR2β and CDR3α. This allowed CDR3β variation to confer different affinities via a variable HLA-C contact, generating an oligoclonal response. TCR10 recognized an induced and distinct G12D decamer conformation. Thus, this successful case of ACT included oligoclonal TCRs of high affinity recognizing distinct conformations of neoantigens. Our study revealed the potential of a structural approach to inform clinical efforts in targeting KRAS-G12D tumors by immunotherapy and has general implications for T cell-based immunotherapies.

Natural killer (NK) cells have an important role in immune defense against viruses and cancer. Activation of human NK cell cytotoxicity toward infected or tumor cells is regulated by killer cell immunoglobulin-like receptors (KIRs) that bind to human leukocyte antigen class I (HLA-I). Combinations of KIR with HLA-I are genetically associated with susceptibility to disease. KIR2DS4, an activating member of the KIR family with poorly defined ligands, is a receptor of unknown function. Here, we show that KIR2DS4 has a strong preference for rare peptides carrying a Trp at position 8 (p8) of 9-mer peptides bound to HLA-C*05:01. The complex of a peptide bound to HLA-C*05: 01 with a Trp at p8 was sufficient for activation of primary KIR2DS4(+) NK cells, independent of activation by other receptors and of prior NK cell licensing. HLA-C*05:01(+) cells that expressed the peptide epitope triggered KIR2DS4(+) NK cell degranulation. We show an inverse correlation of the worldwide allele frequency of functional KIR2DS4 with that of HLA-C*05: 01, indicative of functional interaction and balancing selection. We found a highly conserved peptide sequence motif for HLA-C*05: 01-restricted activation of human KIR2DS4(+) NK cells in bacterial recombinase A (RecA). KIR2DS4(+) NK cells were stimulated by RecA epitopes from multiple human pathogens, including Helicobacter, Chlamydia, Brucella, and Campylobacter. We predict that over 1,000 bacterial species could activate NK cells through KIR2DS4, and propose that human NK cells also contribute to immune defense against bacteria through recognition of a conserved RecA epitope presented by HLA-C*05:01.

The killer-cell Ig-like receptor (KIR) family, expressed mainly in natural killer (NK) cells, includes an activation receptor of unknown function, KIR2DS4. Here we show that KIR2DS4 is restricted by HLA-C*05:01 with a strong preference for tryptophan at position 8 of 9-mer peptides. ‘Self’ peptides with Trp8 eluted from HLA-C*05:01 are rare and only one out of 12 bound KIR2DS4. An HLA-C*05:01-peptide complex that bound KIR2DS4 was sufficient for strong activation of primary KIR2DS4 + NK cells, independently of coactivation by other receptors and of prior NK cell licensing. A highly conserved sequence in bacterial recombinase A, which is essential for DNA repair and survival, includes an epitope that bound to HLA-C*05:01 and activated KIR2DS4 + NK cells. Thus, in addition to their established role in defense against viruses and cancer, NK cells may have also evolved to detect and respond to hundreds of bacterial species through recognition of a conserved RecA epitope.

IL-8-dependent inflammation is a hallmark of host lung innate immunity to bacterial pathogens, yet in many human lung diseases, including chronic obstructive pulmonary disease, bronchiectasis, and pulmonary fibrosis, there are progressive, irreversible, pathological changes associated with elevated levels of IL-8 in the lung. To better understand the duality of IL-8-dependent host immunity to bacterial infection and lung pathology, we expressed human IL-8 transgenically in murine bronchial epithelium, and investigated the impact of overexpression on lung bacterial clearance, host immunity, and lung pathology and function. Persistent IL-8 expression in bronchial epithelium resulted in neutrophilia, neutrophil maturation and activation, and chemotaxis. There was enhanced protection against challenge with Pseudomonas aeruginosa, and significant changes in baseline expression of innate and adaptive immunity transcripts for Ccl5, Tlr6, IL-2, and Tlr1. There was increased expression of Tbet and Foxp3 in response to the Pseudomonas antigen OprF, indicating a regulatory T-cell phenotype. However, this enhanced bacterial immunity came at a high price of progressive lung remodeling, with increased inflammation, mucus hypersecretion, and fibrosis. There was increased expression of Ccl3 and reduced expression of Claudin 18 and F11r, with damage to epithelial organization leading to leaky tight junctions, all of which resulted in impaired lung function with reduced compliance, increased resistance, and bronchial hyperreactivity as measured by whole-body plethysmography. These results show that IL-8 overexpression in the bronchial epithelium benefits lung immunity to bacterial infection, but specifically drives lung damage through persistent inflammation, lung remodeling, and damaged tight junctions, leading to impaired lung function.

HLA-F, a non-classical MHC molecule, is not known to present peptides. Dulberger et al. (2017) show that HLA-F contains a distinct peptide-binding groove and can present a diverse array of peptides. LIR1, however, recognized HLA-F away from bound peptide, leaving open whether peptide-HLA-F-specific T and NK receptors exist.

Background Human natural killer (NK) cell activity is regulated by a family of killer-cell Ig-like receptors (KIR) that bind human leucocyte antigen (HLA) class I. Combinations of KIR and HLA genotypes are associated with disease, including susceptibility to viral infection and disorders of pregnancy. KIR2DL1 binds HLA-C alleles of group C2 (Lys 80 ) and KIR2DL2 and KIR2DL3 bind HLA-C alleles of group C1 (Asn 80 ). However, this model does not capture allelic diversity in HLA-C or the impact of HLA-bound peptides. The goal of this study was to determine the extent to which the endogenous HLA-C peptide repertoire can influence the specific binding of inhibitory KIR to HLA-C allotypes. Results The impact of HLA-C bound peptide on inhibitory KIR binding was investigated taking advantage of the fact that HLA-C*05:01 (HLA-C group 2, C2) and HLA-C*08:02 (HLA-C group 1, C1) have identical sequences apart from the key KIR specificity determining epitope at residues 77 and 80. Endogenous peptides were eluted from HLA-C*05:01 and used to test the peptide dependence of KIR2DL1 and KIR2DL2/3 binding to HLA-C*05:01 and HLA-C*08:02 and subsequent impact on NK cell function. Specific binding of KIR2DL1 to the C2 allotype occurred with the majority of peptides tested. In contrast, KIR2DL2/3 binding to the C1 allotype occurred with only a subset of peptides. Cross-reactive binding of KIR2DL2/3 with the C2 allotype was restricted to even fewer peptides. Unexpectedly, two peptides promoted binding of the C2 allotype-specific KIR2DL1 to the C1 allotype. We showed that presentation of endogenous peptides, or predicted HIV Gag peptides, by HLA-C can promote KIR cross-reactive binding. Conclusions KIR2DL2/3 binding to C1 is more peptide selective than that of KIR2DL1 binding to C2, which provides an explanation for why KIR2DL3–C1 interactions appear weaker than KIR2DL1–C2. In addition, cross-reactive binding of KIR is characterized by even higher peptide selectivity. We demonstrate a hierarchy of functional peptide selectivity of KIR–HLA-C interactions with relevance to NK cell biology and human disease associations. This selective peptide sequence-driven binding of KIR provides a potential mechanism for pathogen as well as self-peptide to modulate NK cell activation through altering levels of inhibition.

Background: Human natural killer (NK) cell activity is regulated by a family of killer cell immunoglobulin-like receptors (KIRs) that bind human leukocyte antigen (HLA) class I. Combinations of KIR and HLA genotypes are associated with disease, including susceptibility to viral infection and disorders of pregnancy. KIR2DL1 binds HLA-C alleles of group C2 (Lys(80)). KIR2DL2 and KIR2DL3 bind HLA-C alleles of group C1 (Asn(80)). However, this model cannot explain HLA-C allelic effects in disease or the impact of HLA-bound peptides. The goal of this study was to determine the extent to which the endogenous HLA-C peptide repertoire can influence the specific binding of inhibitory KIR to HLA-C allotypes.Results: The impact of HLA-C bound peptide on inhibitory KIR binding was investigated taking advantage of the fact that HLA-C*05:01 (HLA-C group 2,C2) and HLA-C*08:02 (HLA-C group 1, C1) have identical sequences apart from the key KIR specificity determining epitope at residues 77 and 80. Endogenous peptides were eluted from HLAC*05:01 and used to test the peptide dependence of KIR2DL1 and KIR2DL2/3 binding to HLA-C*05:01 and HLA-C*08:02 and subsequent impact on NK cell function. Specific binding of KIR2DL1 to the C2 allotype occurred with the majority of peptides tested. In contrast, KIR2DL2/3 binding to the C1 allotype occurred with only a subset of peptides. Cross-reactive binding of KIR2DL2/3 with the C2 allotype was restricted to even fewer peptides. Unexpectedly, two peptides promoted binding of the C2 allotype-specific KIR2DL1 to the C1 allotype. We showed that presentation of endogenous peptides or HIV Gag peptides by HLA-C can promote KIR cross- reactive binding.conclusion: KIR2DL2/3 binding to C1 is more peptide selective than that of KIR2DL1 binding to C2, providing an explanation for KIR2DL3-C1 interactions appearing weaker than KIR2DL1-C2. In addition, cross-reactive binding of KIR is characterized by even higher peptide selectivity. We demonstrate a hierarchy of functional peptide selectivity of KIR-HLA-C interactions with relevance to NK cell biology and human disease associations. This selective peptide sequence-driven binding of KIR provides a potential mechanism for pathogen as well as self-peptide to modulate NK cell activation through altering levels of inhibition.

Killer cell immunoglobulin-like receptor/HLA class I (KIR/HLA-I) combinations are associated with disease risk, implicating functional roles for NK cells (NKCs) or KIR(+) T cells. KIR/HLA-I interactions can act through inhibition of NKC activation by target cells and NKC licensing for greater intrinsic responsiveness. We compared licensing conferred by the weaker, HLA-C group 1/KIR2DL3, and the stronger, HLA-C group 2/KIR2DL1, inhibitory combinations. The "rheostat model" predicts weaker licensing by HLA-C1/KIR2DL3 interactions than HLA-C2/KIR2DL1. We analyzed degranulation in NKC subsets expressing single and multiple receptors for HLA-I. NKG2A had the strongest licensing impact, while KIR2DL3, KIR2DL1, and KIR3DL1 were weaker, and not significantly different to each other. Presence of one or two matched HLA-C allotypes did not alter licensing of KIR2DL3(+) and KIR2DL1(+) NKC. Coexpression of activating KIR2DS1 disarmed KIR2DL3(+) and KIR2DL1(+) NKC to a similar extent. KIR3DL1 and NKG2A combined for more enhanced licensing of double-positive NKC than the combination of KIR2DL3 and KIR2DL1. Thus, KIR2DL3 and KIR2DL1 have similar capacity to license NKC, suggesting that inhibitory signal strength and amount of available HLA-C ligands do not correlate with NKC licensing. Altogether, our results show that the basis for disease associations of HLA-C and KIR2DL likely encompasses factors other than licensing.

Background Multiple sclerosis is generally considered an autoimmune disease resulting from interaction between predisposing genes and environmental factors, together allowing immunological self-tolerance to be compromised. The precise nature of the environmental inputs has been elusive, infectious agents having received considerable attention. A recent study generated an algorithm predicting naturally occurring T cell receptor (TCR) ligands from the proteome database. Taking the example of a multiple sclerosis patient-derived anti-myelin TCR, the study identified a number of stimulatory, cross-reactive peptide sequences from environmental and human antigens. Having previously generated a spontaneous multiple sclerosis (MS) model through expression of this TCR, we asked whether any of these could indeed function in vivo to trigger CNS disease by cross-reactive activation. Findings A number of myelin epitope cross-reactive epitopes could stimulate T cell immunity in this MS anti-myelin TCR transgenic model. Two of the most stimulatory of these ‘environmental’ epitopes, from Dictyostyelium slime mold and from Emiliania huxleyi , were tested for the ability to induce MS-like disease in the transgenics. We found that immunization with cross-reactive peptide from Dictyostyelium slime mold (but not from E. huxleyi ) induces severe disease. Conclusions These specific environmental epitopes are unlikely to be common triggers of MS, but this study suggests that our search for the cross-reactivity triggers of autoimmune activation leading to MS should encompass epitopes not just from the ‘infectome’ but also from the full environmental ‘exposome.’

Signaling by immunoreceptors is often initiated by phosphorylation of cytosolic tyrosines, which then recruit effector molecules. In the case of MHC class I-specific inhibitory receptors, phosphorylation of cytosolic tyrosine residues within ITIMs results in recruitment of a protein tyrosine phosphatase that blocks activation signals. Recent work showed that signaling by an HLA-C-specific killer cell Ig-like receptor (KIR) is independent of signaling by activation receptors. It is not known how ITIM phosphorylation is initiated and regulated. In this article, we show that substitution of His-36 in the first Ig domain of KIR2DL1 with alanine (KIR2DL1-H36A) resulted in constitutive KIR2DL1 self-association and phosphorylation, as well as recruitment of tyrosine phosphatase SHP-1. Furthermore, substitution of His-36 with a similar bulky amino acid, phenylalanine, maintained the receptor in its unphosphorylated state, suggesting that steric hindrance by the His-36 side chain prevents constitutive KIR2DL1 self-association and ITIM phosphorylation. The equally strong phosphorylation of KIR2DL1 and KIR2DL1-H36A after inhibition of tyrosine phosphatase by pervanadate suggested that KIR2DL1-H36A is selectively protected from dephosphorylation. We propose that KIR phosphorylation is controlled by the accessibility of ITIM to tyrosine phosphatases and that KIR binding to HLA-C must override the hindrance that His-36 puts on KIR2DL1 self-association. Expression of KIR2DL1-H36A on NK cells led to stronger inhibition of lysis of HLA-C+ target cells than did expression of wild-type KIR2DL1. These results revealed that ITIM phosphorylation is controlled by self-association of KIR and that His-36 serves as a gatekeeper to prevent unregulated signaling through KIR2DL1.

Background Pathogenic or regulatory effects of natural killer (NK) cells are implicated in many autoimmune diseases, but evidence in multiple sclerosis (MS) and its murine models remains equivocal. In an effort to illuminate this, we have here analysed expression of the prototypic NK cell marker, NCR1 (natural cytotoxicity triggering receptor; NKp46; CD335), an activating receptor expressed by virtually all NK cells and therefore considered a pan-marker for NK cells. The only definitive ligand of NCR1 is influenza haemagglutinin, though there are believed to be others. In this study, we investigated whether there were differences in NCR1 + cells in the peripheral blood of MS patients and whether NCR1 + cells are present in white matter lesions. Results We first investigated the expression of NCR1 on peripheral blood mononuclear cells and found no significant difference between healthy controls and MS patients. We then investigated mRNA levels in central nervous system (CNS) tissue from MS patients: NCR1 transcripts were increased more than 5 times in active disease lesions. However when we performed immunohistochemical staining of this tissue, few NCR1 + NK cells were identified. Rather, the major part of NCR1 expression was localised to astrocytes, and was considerably more pronounced in MS patients than controls. In order to further validate de novo expression of NCR1 in astrocytes, we used an in vitro staining of the human astrocytoma U251 cell line grown to model whether cell stress could be associated with expression of NCR1. We found up-regulation of NCR1 expression in U251 cells at both the mRNA and protein levels. Conclusions The data presented here show very limited expression of NCR1 + NK cells in MS lesions, the majority of NCR1 expression being accounted for by expression on astrocytes. This is compatible with a role of this cell-type and NCR1 ligand/receptor interactions in the innate immune response in the CNS in MS patients. This is the first report of NCR1 expression on astrocytes in MS tissue: it will now be important to unravel the nature of cellular interactions and signalling mediated through innate receptor expression on astrocytes.

The molecular mechanisms underpinning central nervous system damage in multiple sclerosis (MS) are complex and it is widely accepted that there is an autoimmune component. Both adaptive and innate immune effector mechanisms are believed to contribute to tissue disease aetiology. HLA-E is a non-classical MHC class Ib molecule that acts as the ligand for the NKG2A inhibitory receptor present on natural killer (NK) and CD8+ cells. Peptide binding and stabilization of HLA-E is often considered to signal infection or cell stress. Here we examine the up-regulation of HLA-E in MS brain tissue. Expression is significantly increased in white matter lesions in the brain of MS patients compared with white matter of neurologically healthy controls. Furthermore, using quantitative immunohistochemistry and confocal microscopy, we show increased HLA-E protein expression in endothelial cells of active MS lesions. Non-inflammatory chronic lesions express significantly less HLA-E protein, comparable to levels found in white matter from controls. Increased HLA-E protein levels were associated with higher scores of inflammation. These results suggest the potential for an effect in central nervous system pathogenesis from HLA-E modulation in stressed tissue. Co-localization with infiltrating CD8+ cells implicates a possible role for HLA-E-restricted regulatory CD8+ cells, as has been proposed in other autoimmune diseases.