Biology subjects

Long, E. O.

Publications and source records attributed to Long, E. O..

9 recordsLinked to original sources

bioRxiv2026

Using peptide-exchange systems to interrogate peptide-specific KIR binding to HLA Class -I

The killer-cell immunoglobulin-like receptors (KIR) are a family of activating and inhibitory HLA class I (HLA-I) binding receptors expressed on natural killer (NK) cells and subsets of T cells. The KIR detect HLA-I molecules in a peptide-dependent manner, with some KIR displaying exquisite peptide-specificity. Studying peptide recognition by KIR often uses TAP-deficient cell lines expressing single HLA-I alleles, which are heterogenous and time consuming to generate. Here, we established an alternative approach using peptide-exchange technologies hitherto developed for studying T cell recognition of HLA-I. We tested two methods; dipeptide-mediated peptide exchange and open-HLA-I, HLA-I molecules consisting of heavy chain-{beta}2m disulphide bonded dimers. We combined peptide-exchange technologies with SpyTag-SpyCatcher chemistry to allow rapid detection of KIR binding via HLA-I displayed on plates or cells. We demonstrated the fidelity of this system with peptides of known KIR specificity bound to HLA-C*05:01. We then screened a peptide library to identify novel strong KIR2DS4 binding peptides presented by HLA-C*04:01. Peptide-exchanged HLA-C was functionally competent, promoting activation of KIR2DS4+ NK cells and inhibiting activation of KIR2DL1+ NK cells. Together, we show that peptide-exchangeable HLA-I molecules are ligands for KIR, presenting a flexible, efficient system for examining the peptide-sequence dependent recognition of HLA-I by KIR.

immunology↗

bioRxiv2024

Identification and Structural Characterization of a mutant KRAS-G12V specific TCR restricted by HLA-A3

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 can be targeted in 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 the common HLA-I allotype, 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 CDR3{beta} and CDR1 {beta} formed a hydrophobic pocket to interact with p6 Val of the G12V but not 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 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. [203]

immunology↗

bioRxiv2023

NK cell-induced damage to P. falciparum-infected erythrocytes requires ligand-specific recognition and releases parasitophorous vacuoles that are phagocytosed by monocytes in the presence of immune IgG

Natural killer (NK) cells lyse virus-infected cells and transformed cells through polarized delivery of lytic effector molecules into target cells. We have shown that NK cells lyse Plasmodium falciparum-infected red blood cells (iRBC) via antibody-dependent cellular cytotoxicity (ADCC). A high frequency of adaptive NK cells, with elevated intrinsic ADCC activity, in people chronically exposed to malaria transmission is associated with reduced parasitemia and resistance to disease. How NK cells bind to iRBC and the outcome of iRBC lysis by NK cells has not been investigated. We applied gene ablation in inducible erythrocyte precursors and antibody-blocking experiments with iRBC to demonstrate a central role of CD58 and ICAM-4 as ligands for adhesion by NK cells via CD2 and integrin M{beta}2, respectively. Adhesion was dependent on opsonization of iRBC by IgG. Live imaging and quantitative flow cytometry of NK-mediated ADCC toward iRBC revealed that damage to the iRBC plasma membrane preceded damage to P. falciparum within parasitophorous vacuoles (PV). PV were identified and tracked with a P.falciparum strain that expresses the PV membrane-associated protein EXP2 tagged with GFP. After NK-mediated ADCC, PV were either found inside iRBC ghosts or released intact and devoid of RBC plasma membrane. Electron microscopy images of ADCC cultures revealed tight NK-iRBC synapses and free GFP+ vesicles similar to GFP+ PV isolated from iRBC lysates by cell sorting. The titer of IgG in plasma of malaria-exposed individuals that bound PV was two orders of magnitude higher than IgG that bound iRBC. This immune IgG stimulated efficient phagocytosis of PV by primary monocytes. The selective NK-mediated damage to iRBC, resulting in release of PV, and subsequent phagocytosis of PV by monocytes may combine for efficient killing and removal of intra-erythrocytic P.falciparum parasite. This mechanism may mitigate the inflammation and malaria symptoms during blood-stage P. falciparum infection.

immunology↗

bioRxiv2023

Functional genomics identifies extension of complex N-glycans as a mechanism to evade lysis by natural killer cells

Somatic mutations can lead to the transformation of healthy cells into malignant cells and allow their evasion from immune surveillance. To uncover genes that play a role in the detection and lysis of tumor cells by natural killer (NK) cells, a B lymphoblastoid cell line was subjected to a genome-wide CRISPR screen. Among the top hits that facilitated NK evasion was SPPL3, which encodes an intramembrane protease that cleaves transmembrane glycosyltransferases in the Golgi apparatus. SPPL3-deficient cells accumulated glycosyltransferases, such as acetylglucosaminyltransferase 5 (MGAT5), and displayed increased N-glycosylation. Binding of NK receptors NKG2D and CD2 to their corresponding ligands MICB and CD58, and binding of rituximab to CD20, was disrupted by SPPL3-deletion. Inhibition of N-glycan maturation restored receptor binding and sensitivity to NK cells. To elucidate the mechanism of this resistant phenotype, a secondary CRISPR screen was performed in SPPL3-deficient cells. This screen identified glycosyltransferases that catalyze the formation of highly branched N-glycans and N-acetyl-lactosamine (LacNAc) extensions as key regulators that prevent killing. A significant enrichment of poly-LacNAc-containing tetra-antennary species was confirmed by glycoproteomic analysis. These findings provide mechanistic insight into how SPPL3 deletions have been linked to cancer.

immunology↗

bioRxiv2023

Innate receptors with high specificity for HLA class I-peptide complexes

Genetic studies associate killer-cell immunoglobulin-like receptors (KIR) 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. As KIR binding to HLA-I is peptide-dependent, we performed systematic screens totaling over 3,500 specific interactions to determine the specificity of five KIR for peptides presented by four HLA-C ligands. Inhibitory KIR2DL1 was largely peptide sequence agnostic, binding approximately 60% of hundreds of HLA-peptide complexes tested. Inhibitory KIR2DL2, KIR2DL3, and activating KIR2DS1 and KIR2DS4 bound only 10%, 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 germ-line encoded NK receptors and imply that NK cell responses can be shaped by HLA-I bound immunopeptidomes in the context of disease or infection.

immunology↗

bioRxiv2021

T cells discriminate between groups C1 and C2 HLA-C

Dimorphic residues 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 (TCR) discriminated groups C1 and C2 HLA-C, due to effects on peptide presentation and TCR affinity. Structural and functional experiments combined with immunopeptidomics analysis revealed that C1-HLA-C favors smaller amino acids at the peptide C-terminus minus-1 position (p{Omega}-1), and that larger p{Omega}-1 residues diminished TCR recognition of C1-HLA-C. After controlling for peptide presentation, TCRs exhibited weaker affinities for C2-HLA-C despite conserved TCR contacts. Thus, the C1/C2 dimorphism impacts peptide presentation and HLA-C restricted T cell responses, with implications in multiple disease contexts including adoptive T cell therapy targeting KRAS-G12D-induced cancers.

immunology↗

bioRxiv2019

HLA-C-restricted presentation of a conserved bacterial epitope to an innate NK cell receptor

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.

immunology↗

bioRxiv2018

NK cells inhibit Plasmodium falciparum growth in red blood cells via antibody dependent cellular cytotoxicity

Antibodies acquired naturally through repeated exposure to Plasmodium falciparum are essential in the control of blood-stage malaria. Antibody-dependent functions may include neutralization of parasite-host interactions, complement activation, and activation of Fc receptor functions. A role of antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells in protection from malaria has not been established. Here we show that IgG isolated from adults living in a malaria-endemic region activated ADCC by primary human NK cells, which lysed infected red blood cells (RBCs) and inhibited parasite growth in an in vitro assay for ADCC-dependent growth inhibition. RBC lysis by NK cells was highly selective for infected RBCs in a mixed culture with uninfected RBCs. Human antibodies to P. falciparum antigens PfEMP1 and RIFIN were sufficient to promote NK-dependent growth inhibition. As these results implicate acquired immunity through NK-mediated ADCC, antibody-based vaccines that target bloodstream parasites should consider this new mechanism of action.

immunology↗

bioRxiv2016

Canonical and cross-reactive binding of NK cell inhibitory receptors to HLA-C allotypes is dictated by peptides bound to HLA-C

BackgroundHuman 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 (Lys80) and KIR2DL2 and KIR2DL3 bind HLA-C alleles of group C1 (Asn80). 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.\n\nResultsThe 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.\n\nConclusionsKIR2DL2/3 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.

immunology↗