HLA Ligands Research Articles

CD4 T cells restricted to DRB1*15:01 recognize two Epstein–Barr virus glycoproteins capable of intracellular antigen presentation

Both genetic and environmental factors contribute to multiple sclerosis (MS) risk. Infection with the Epstein-Barr virus (EBV) is the strongest environmental risk factor, and HLA-DR15 is the strongest genetic risk factor for MS. We employed computational methods and in vitro assays for CD4 T cell activation to investigate the DR15-restricted response to EBV. Using a machine learning-based HLA ligand predictor, the EBV glycoprotein B (gB) was predicted to be enriched in epitopes restricted to presentation by DRB1*15:01. In DR15-positive individuals, two epitopes comprised the major CD4 T cell response to gB. Surprisingly, the expression of recombinant gB in a DR15-homozygous B cell line or primary autologous B cells elicited a CD4 T cell response, indicating that intracellular gB was loaded onto HLA class II molecules. By deleting the signal sequence of gB, we determined that this pathway for direct activation of CD4 T cells was dependent on trafficking to the endoplasmic reticulum (ER) within the B cell. We screened seven recombinant EBV antigens from the ER compartment for immune responses in DR15-negative vs. DR15-homozygous individuals. In addition to gB, gH was a key CD4 T cell target in individuals homozygous for DR15. Compared to non-DR15 controls, DR15-homozygotes had significantly higher T cell responses to both gB and gH but not to EBV latent or lytic antigens overall. Responses to gB and gH were slightly elevated in DR15 homozygotes with MS. Our results link MS environmental and genetic risk factors by demonstrating that HLA-DR15 dictates CD4 T cell immunity to EBV antigens.

ImmuneApp for HLA-I epitope prediction and immunopeptidome analysis

Advances in mass spectrometry accelerates the characterization of HLA ligandome, necessitating the development of efficient methods for immunopeptidomics analysis and (neo)antigen prediction. We develop ImmuneApp, an interpretable deep learning framework trained on extensive HLA ligand datasets, which improves the prediction of HLA-I epitopes, prioritizes neoepitopes, and enhances immunopeptidomics deconvolution. ImmuneApp extracts informative embeddings and identifies key residues for pHLA binding. We also present a more accurate model-based deconvolution approach and systematically analyzed 216 multi-allelic immunopeptidomics samples, identifying 835,551 ligands restricted to over 100 HLA-I alleles. Our investigation reveals the effectiveness of the composite model, denoted as ImmuneApp-MA, which integrates mono- and multi-allelic data to enhance predictive performance. Leveraging ImmuneApp-MA as a pre-trained model, we built ImmuneApp-Neo, an immunogenicity predictor that outperforms existing methods for prioritizing immunogenic neoepitope. ImmuneApp demonstrates its utility across various immunopeptidomics datasets, which will promote the discovery of novel neoantigens and the development of new immunotherapies.

Molecular relapse monitoring reveals the domination of impaired NK cell education over impaired inhibition in missing KIR-ligand recognition in patients after unrelated hematopoietic stem cell transplantation for malignant diseases.

Transplantation of HLA and/or KIR mismatched allogeneic hematopoietic stem cells can lead NK cells to different states of activation/inhibition or education/resetting and change anti-tumor immunosurveillance. In this study, we used molecular relapse monitoring to investigate a correlation between either missing ligand recognition or variation of the cognate iKIR-HLA pairs with clinical outcomes in patients with hematological malignancies requiring allogeneic hematopoietic stem cell transplantation (allo-HSCT). Patients (N = 418) with acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome (MDS), or lymphoma receiving T-cell repleted graft from HLA-matched or partly mismatched unrelated donors between 2012 and 2020 in our center were included in this study. Missing-ligand recognition was assessed through the presence or absence of recipients' HLA ligand for a particular inhibitory KIR (iKIR) exhibited by the donor. Inhibitory KIR-HLA pair number variation was defined by loss or gain of a new cognate pair of HLA-KIR within the new HLA environment of the recipient, compared with the donor's one. Considering the results of our research, we drew the following conclusions: (i) loss of iKIR-HLA cognate pair for C1, C2, and/or Bw4 groups led to significant deterioration of disease-free survival (DFS), molecular relapse, overall survival (OS) and non-relapse mortality (NRM) for patients undergoing allo-HSCT in the standard phase of the disease. This phenomenon was not observed in patients who underwent transplantation in advanced hematological cancer. (ii) The missing ligand recognition had no impact if the proportion of HLA mismatches was not considered; however, adjustments of HLA mismatch level in the compared groups highlighted the adverse effect of the missing ligand constellation. (iii) The adverse effect of adjusted missing ligand suggests a predominance of lost NK cell education over lost NK cell inhibition in posttransplant recipients' new HLA environment. Our results suggested that donors with the loss of an iKIR-HLA cognate pair after transplantation should be avoided, and donors who provided an additional iKIR-HLA cognate pair should be preferred in the allo-HSCT donor selection process.

Multi-omics analysis of overexpressed tumor-associated proteins: gene expression, immunopeptide presentation, and antibody response in oropharyngeal squamous cell carcinoma, with a focus on cancer-testis antigens.

The human leukocyte antigen complex (HLA) is essential for inducing specific immune responses to cancer by presenting tumor-associated peptides (TAP) to T cells. Overexpressed tumor associated antigens, mainly cancer-testis antigens (CTA), are outlined as essential targets for immunotherapy in oropharyngeal squamous cell carcinoma (OPSCC). This study assessed the degree to which presentation, gene expression, and antibody response (AR) of TAP, mainly CTA, are correlated in OPSCC patients to evaluate their potential as immunotherapy targets. Snap-frozen tumor (NLigand/RNA=40), healthy mucosa (NRNA=6), and healthy tonsils (NLigand=5) samples were obtained. RNA-Seq was performed using Illumina HiSeq 2500/NovaSeq 6000 and whole exome sequencing (WES) utilizing NextSeq500. HLA ligands were isolated from tumor tissue using immunoaffinity purification, UHPLC, and analyzed by tandem MS. Antibodies were measured in serum (NAb=27) utilizing the KREX™ CT262 protein array. Data analysis focused on 312 proteins (KREX™ CT262 panel + overexpressed self-proteins). 183 and 94 of HLA class I and II TAP were identified by comparative profiling with healthy tonsils. Genes from 26 TAP were overexpressed in tumors compared to healthy mucosa (LFC>1; FDR<0.05). Low concordance (r=0.25; p<0.0001) was found between upregulated mRNA and class I TAP. The specific mode of correlation of TAP was found to be dependent on clinical parameters. A lack of correlation was observed both between mRNA and class II TAP, as well as between class II tumor-unique TAP (TAP-U) presentation and antibody response (AR) levels. This study demonstrates that focusing exclusively on gene transcript levels fails to capture the full extent of TAP presentation in OPSCC. Furthermore, our findings reveal that although CTA are presented at relatively low levels, a few CTA TAP-U show potential as targets for immunotherapy.

The Impact of KIR-HLA Interplay in T-LGL Leukemia

T-Cell large granulocyte lymphocyte leukemia (T-LGLL), characterized by clonal expansions of cytotoxic T cells (CTLs), arises from polyclonal lymphoproliferative responses. Various triggers have been stipulated, ranging from autoimmune, viral or tumor antigens. However, one could also posit that an inherent propensity to overshooting CTL responses contributing to the initial clonal expansions or affecting normal termination of immune response might be at play. For instance, specific HLA risk alleles may be overrepresented or even diagnostic for certain autoimmune conditions. In contrast to the risk allele theory, which enriches haplotypes within the patient population, the concept of HLA evolutionary diversity (HED) posits that the structural divergence between alleles widen the recognition spectrum. HLA alleles also serve as ligands for killer immunoglobulin-like receptors (KIRs). Indeed, the KIR gene family, consisting of 15 genes and 2 pseudogenes, is responsible for regulating the activity of NK and a subset of T cells and thus may play a role in T-LGLL. The main KIR genes are classified as either activating or inhibiting, based on their ability to up or downregulate NK and T cells. As the KIR genes and their HLA ligands genetically assort independently of each other, KIR-HLA “mismatches” are possible. We analyzed HLA alleles in 91 T-LGLL patients vs 130 controls and investigated whether specific presentations of T-LGLL, namely neutropenia and anemia enrich for certain HLA alleles. We found that HLA-A*02 was more frequent in cases with neutropenia (59 vs 47%, p=0.045) and HLA-A*68 in patients with anemia (17 vs 8%, p=0.02) as compared to controls. Interestingly, HLA-A*02/HLA-C*05 was observed typically in STAT3 MT vs WT cases (22 vs 11%). Using the HLA calls, we calculated the HED for HLA class 1/2, but no increased HED could be asserted (consistent with the presence of risk alleles as described above). We then explored whether specific KIR-HLA interactions enhanced an individual's risk for clonal CTL expansion and subsequent T-LGLL occurrence. As KIR gene frequencies can vary based on ethnicity, controls were chosen to match T-LGLL cohort (European ancestry). We applied T1K as a computational method for KIR genotyping and validated the calls by the 4 framework KIR genes (always present): 3DL2, 3DL3, 3DP1, and 2DL4. Along with the framework KIRs, there were no differences in the frequency of KIR genotype between T-LGLL and ethnicity matched controls. However, we found that the frequency of individuals with the 3DL2 gene and either HLA A*03 or HLA A*11 was significantly higher in the T-LGLL compared to controls (26 vs 8%, p = .006). Moreover, we found a higher frequency of 2DL3 combined with HLA C2/C2, (31 vs 14%, p=0.05) in T-LGLL patients with neutropenia. As this KIR-ligand (KIRL) configuration is a mismatch, the normally inhibiting receptor will have no effect on cell activity. Stratified by the presence of anemia, the LGLL cohort had instead a significantly higher proportion of 2DS2 co-expressed with the HLA C1 allele (75.68 vs 35.39%, p=.001), indicating activating genotype. We also found that the number of cases with the co-occurrence of the 3DL2 receptor and absent HLA A*03/A*11 ligands paired with 2DL3 and the HLA C1/C1 allotype was higher in STAT3 MT compared to WT (39.1 vs 16.7% p=0.06). Finally, we attempted to estimate and compare the net KIR activity of T-LGLL patients and controls by creating a formula in which, for each individual, we subtracted the sum of the number of KIR/KIRL inhibitory matches from that of activating KIR/KIRL matches. Mismatches were also included either as activating or inhibiting depending on the KIR/KIRL configuration. Using this formula, we found that the net KIR average value for the control cohort was 0.85 compared to 0.69 for the T-LGLL cohort, indicating that the control cohort had slightly more activating KIR-ligand configurations. Even when mismatches were treated as neutral, the preexcitation balance was not changed in T-LGLL. These findings suggest that other factors may contribute to the baseline preexcitation levels of clonal CTLs in this disease. In conclusion, our study of HLA and KIR-HLA interactions shows modest impact of KIR and KIR-ligand genotype on the propensity for overshooting clonal CTL proliferation and points towards other possible mechanisms such as hypersecretion of high affinity IL6R or other stimulatory factors.

Acylpyrazolone based Square pyramidal Cu(II) complexes: Synthesis, Structural characterization, DFT and antiproliferative properties

σ-donating acylpyarazolone HLb ligand and its Copper complex [Cu(HLb)2(DMF)] were synthesized. [Cu(HLa)2(DMSO)] complex is synthesized from the previously reported HLa ligand. Single crystal analysis revealed the Square Pyramidal geometry of both the complexes with one oxygen fromDMF and DMSO attached at an apical site in the [Cu(HLb)2(DMF)] and [Cu(HLa)2(DMSO)] complexes, respectively and four oxygens of the ligand coordinated to the centralcopper metal at an equatorial position. The electronic spectra of both the complexes have been analyzed using UV–visible spectroscopy. The study investigated the molecular specification utilising the B3LYP/6-31G and B3LYP/LANL2DZ basis set formalism for DFT computational analysis to provide a perfect correlation and comparison with all the experimental values. The Hirshfeld surface area analysis was done to investigate the intermolecular interactions or secondary molecular interaction. The magnetic behaviour of both the synthesized complexes was identified by ESR spectral analysis. Using three different cancer cell lines NCI-H23 (Lung adenocarcinoma cells), HepG2 (Hepatocellular carcinoma) and SH-SY5Y (Neuroblastoma cells), cytotoxicity of both the copper complexes was examined and wound healing assay on [Cu(HLb)2(DMF)] complex was done to check the cell migration.

A Lysine Residue at the C-Terminus of MHC Class I Ligands Correlates with Low C-Terminal Proteasomal Cleavage Probability.

The majority of peptides presented by MHC class I result from proteasomal protein turnover. The specialized immunoproteasome, which is induced during inflammation, plays a major role in antigenic peptide generation. However, other cellular proteases can, either alone or together with the proteasome, contribute peptides to MHC class I loading non-canonically. We used an immunopeptidomics workflow combined with prediction software for proteasomal cleavage probabilities to analyze how inflammatory conditions affect the proteasomal processing of immune epitopes presented by A549 cells. The treatment of A549 cells with IFNγ enhanced the proteasomal cleavage probability of MHC class I ligands for both the constitutive proteasome and the immunoproteasome. Furthermore, IFNγ alters the contribution of the different HLA allotypes to the immunopeptidome. When we calculated the HLA allotype-specific proteasomal cleavage probabilities for MHC class I ligands, the peptides presented by HLA-A*30:01 showed characteristics hinting at a reduced C-terminal proteasomal cleavage probability independently of the type of proteasome. This was confirmed by HLA-A*30:01 ligands from the immune epitope database, which also showed this effect. Furthermore, two additional HLA allotypes, namely, HLA-A*03:01 and HLA-A*11:01, presented peptides with a markedly reduced C-terminal proteasomal cleavage probability. The peptides eluted from all three HLA allotypes shared a peptide binding motif with a C-terminal lysine residue, suggesting that this lysine residue impairs proteasome-dependent HLA ligand production and might, in turn, favor peptide generation by other cellular proteases.

Immunoprecipitation methods impact the peptide repertoire in immunopeptidomics.

Mass spectrometry-based immunopeptidomics is the only unbiased method to identify naturally presented HLA ligands, which is an indispensable prerequisite for characterizing novel tumor antigens for immunotherapeutic approaches. In recent years, improvements based on devices and methodology have been made to optimize sensitivity and throughput in immunopeptidomics. However, developments in ligand isolation, mass spectrometric analysis, and subsequent data processing can have a marked impact on the quality and quantity of immunopeptidomics data. In this work, we compared the immunopeptidome composition in terms of peptide yields, spectra quality, hydrophobicity, retention time, and immunogenicity of two established immunoprecipitation methods (column-based and 96-well-based) using cell lines as well as primary solid and hematological tumor samples. Although, we identified comparable overall peptide yields, large proportions of method-exclusive peptides were detected with significantly higher hydrophobicity for the column-based method with potential implications for the identification of immunogenic tumor antigens. We showed that column preparation does not lose hydrophilic peptides in the hydrophilic washing step. In contrast, an additional 50% acetonitrile elution could partially regain lost hydrophobic peptides during 96-well preparation, suggesting a reduction of the bias towards the column-based method but not completely equalizing it. Together, this work showed how different immunoprecipitation methods and their adaptions can impact the peptide repertoire of immunopeptidomic analysis and therefore the identification of potential tumor-associated antigens.

Large case-control study indicates no association of KIR genotype and risk of developing acute myeloid leukemia.

Immunogenetic association studies may give rise to new hypotheses on the immune surveillance of cancer. We hypothesized that certain combinations of killer immunoglobulin-like receptor (KIR) and HLA genotypes may enhance natural killer (NK) cell immunity against nascent acute myeloid leukemia (AML) and, thereby, lead to a skewed genotype distribution among patients. For this purpose, we analyzed KIR and HLA genotypes of 1767 German patients with AML and compared the results with that of the data of 51 890 German volunteers who had registered with German bone marrow donor file (DKMS). Patient samples were retrieved from the Collaborative Biobank and the biorepository of the Study Alliance Leukemia. All samples were genotyped with high-resolution amplicon-based next-generation sequencing. Because of the large number of controls, this study was very sensitive to detect the impact of KIR genotype. Knowledge on KIRs and their cognate HLA ligands allowed for testing of several hypotheses of NK cell-mediated endogenous leukemia surveillance. We did not find significant differences between the 2 cohorts in regard to the presence or absence of single KIR genes. When grouped based on telomeric or centromeric gene content, the major haplotypes A/A, A/B, and B/B were equally distributed among patients and control subjects. Using information on KIRs and their HLA ligands, we further tested receptor-ligand models and summation models without revealing markedly significant differences between patients and controls, albeit we observed a trend pointing at a minor protective effect of a low number of inhibitory KIR/KIR-ligand pairs. The results suggest that the KIR/KIR-ligand genotype has no effect on the susceptibility for the development of de novo AML.

Killer cell immunoglobulin-like receptor (KIR) genes and their HLA ligands in a Brazilian population.

Killer cell immunoglobulin-like receptors (KIR) exhibit extensive diversity, giving rise to different KIR profiles in populations worldwide. This study aimed to investigate the distribution of KIR genes and HLA ligands in a population from Campinas, southeastern Brazil (n = 292), and to compare their results with other populations. A comprehensive analysis of population-specific genes, genotype, and haplotype frequencies of KIR may facilitate a better understanding of their evolution and role in immunity. The genotyping of 16 KIR genes and HLA class I alleles was performed by the reverse sequence-specific oligonucleotide methodology using the Luminex platform (One Lambda, Inc., Canoga Park, CA). The framework genes were present in all individuals, with the most common non-framework KIR genes detected being KIR2DP1(96.6%), KIR2DL1(95.5%), KIR3DL1(94.5%), KIR2DS4(93.8%) and KIR2DL3(87.3%). KIR2DS1, KIR2DS3, KIR2DS5, and KIR3DS1 presented frequencies below 40%. KIR2DL2, KIR2DL5, and KIR2DS2 showed intermediate frequencies (between53% and 58%). The activating gene KIR2DS5 was the least common in this population (30.8%). Forty-five KIR profiles were found with the commonest being the homozygous A haplotype (27.4%). The distribution of KIR genes in the Brazilian population is similar to Caucasian European and Euro-descendant populations.

Association of KIR Genes Polymorphism and its HLA Ligands in Diffuse Large B-cell Lymphoma.

Non-Hodgkin lymphoma (NHL) is a heterogeneous disease, with each subtype associated with different risk factors. Within this group, diffuse large B-cell lymphoma (DLBCL) can be highlighted, the most common type of NHL.NK cells are key components of the innate immune response and may play an important antitumor role. The objective of the present work was to determine the polymorphism of KIR genes in Brazilian patients with DLBCL. Furthermore, we evaluated the association between the polymorphism of these genes and their ligands with the clinical course of the disease. For the study, 112 patients with DLBCL and 222 voluntary blood and bone marrow donors. The genetic material of these samples were extracted for KIR and HLA typing, determination of HLA ligands, determination of the KIR haplotype and search for the deletion of 22 bp in the KIR2DS4 gene. KIR genotype distributions were made by direct counting using 2 × 2 contingency tables using Fisher's exact test. The magnitude of the association was measured by odds ratio (OR) and 95% confidence interval. P values <.05 were considered significant. Overall survival and progression-free survival were assessed with a Kaplan-Meier estimator. In the present study, an association of HLA-Bw4 and HLA-Bw480I ligand was found with more advanced stages of the disease. Also, an association of the KIR2DL3 gene with a better response to treatment was found. With this, we can conclude that the polymorphism of KIR genes and the association with HLA ligands can influence the prognosis of DLBCL, as well as the response to treatment was found. With this, we can conclude that the polymorphism of KIR genes and the association with HLA ligands can influence the prognosis of DLBCL, as well as the response to treatment.Non-Hodgkin lymphoma (NHL) is a heterogeneous disease, with each subtype associated with different risk factors. Within this group, diffuse large B-cell lymphoma (DLBCL) can be highlighted, the most common type of NHL.NK cells are key components of the innate immune response and may play an important antitumor role. The objective of the present work was to determine the polymorphism of KIR genes in Brazilian patients with DLBCL. Furthermore, we evaluated the association between the polymorphism of these genes and their ligands with the clinical course of the disease. For the study, 112 patients with DLBCL and 222 voluntary blood and bone marrow donors. The genetic material of these samples were extracted for KIR and HLA typing, determination of HLA ligands, determination of the KIR haplotype and search for the deletion of 22 bp in the KIR2DS4 gene. KIR genotype distributions were made by direct counting using 2×2 contingency tables using Fisher's exact test. The magnitude of the association was measured by odds ratio (OR) and 95% confidence interval. P values <.05 were considered significant. Overall survival and progression-free survival were assessed with a Kaplan-Meier estimator. In the present study, an association of HLA-Bw4 and HLA-Bw480I ligand was found with more advanced stages of the disease. Also, an association of the KIR2DL3 gene with a better response to treatment was found. With this, we can conclude that the polymorphism of KIR genes and the association with HLA ligands can influence the prognosis of DLBCL, as well as the response to treatment was found. With this, we can conclude that the polymorphism of KIR genes and the association with HLA ligands can influence the prognosis of DLBCL, as well as the response to treatment.

NK cell defects: implication in acute myeloid leukemia.

Acute Myeloid Leukemia (AML) is a complex disease with rapid progression and poor/unsatisfactory outcomes. In the past few years, the focus has been on developing newer therapies for AML; however, relapse remains a significant problem. Natural Killer cells have strong anti-tumor potential against AML. This NK-mediated cytotoxicity is often restricted by cellular defects caused by disease-associated mechanisms, which can lead to disease progression. A stark feature of AML is the low/no expression of the cognate HLA ligands for the activating KIR receptors, due to which these tumor cells evade NK-mediated lysis. Recently, different Natural Killer cell therapies have been implicated in treating AML, such as the adoptive NK cell transfer, Chimeric antigen receptor-modified NK (CAR-NK) cell therapy, antibodies, cytokine, and drug treatment. However, the data available is scarce, and the outcomes vary between different transplant settings and different types of leukemia. Moreover, remission achieved by some of these therapies is only for a short time. In this mini-review, we will discuss the role of NK cell defects in AML progression, particularly the expression of different cell surface markers, the available NK cell therapies, and the results from various preclinical and clinical trials.

Abstract 2910: NKG2A suppression enhances the function of primary human Natural Killer cells

Abstract In this study we propose inhibition of NKG2A signaling enhances the anti-tumor response of primary human NK cells. One mechanism employed by tumor cells to evade immunosurveillance is through induction of the surface expression of unconventional HLA ligands that agonize inhibitory receptors on immune cells. Specifically on some tumors, HLA-E is either expressed or its expression is known to be induced by IFNγ and is indicative of resistance to immunotherapy. HLA-E agonizes the CD94/NKG2A inhibitory complex on NK cells and some T cells to lessen their cytotoxicity, potentially decreasing the efficacy of cellular therapy with these immune cells. In this study ex vivo-expanded primary human NK cells were shown to have significantly elevated expression of NKG2A. These expanded NK cells also secreted high levels of IFNγ and could induce expression of HLA-E on tumor cells. Suppression of NKG2A by either antibody blockade or gene deletion increased the cytotoxicity of NK cells against the lung cancer cell line A549 stably expressing HLA-E. NKG2A knockout cells had an almost 10-fold decrease in EC50 compared to wild-type NK cells (EC50 0.15 vs 0.94 at 48 h), meaning nearly log-fold fewer NKG2A knockout cells are needed to kill the same number of tumor cells. The effect of NKG2A knockout on cytotoxicity of NK cells against other cancer cell lines stably expressing HLA-E will be presented along with effects of NKG2A suppression on NK cell phenotype, cytokine secretion and degranulation. These initial data suggest that blockade of the CD94/NKG2A inhibitory complex can improve the function of ex vivo expanded human NK cells and could provide an effector population with the potential for enhanced therapeutic efficacy. Citation Format: Tayler J. Croom-Perez, Liza D. Robles-Carrillo, Md Faqrul Hasan, Alicja J. Copik. NKG2A suppression enhances the function of primary human Natural Killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2910.

Abstract 6376: Discovery and preclinical characterization of dual antagonist antibodies targeting both LILRB1 and LILRB2 that enhance innate and adaptive anti-cancer immune responses

Abstract Background: One cause for the failure of checkpoint inhibitors is the immunosuppressive nature of the tumor microenvironment. LILRB1 (ILT2) and LILRB2 (ILT4) are ITIM-containing inhibitory receptors that recognize HLA Class 1 and nonclassical ligands (e.g., HLA-A, HLA-G, etc.). LILRB1 is expressed on myeloid cells and subsets of B, NK, and T cells, while LILRB2 expression is mostly restricted to myeloid cells. Interaction of LILRB1 and LILRB2 receptors with HLA ligands promotes an inhibitory milieu that prevents T cells from attacking cancer cells. The distinct pattern of expression and function of these lymphoid and myeloid checkpoints suggests complementary targeting approaches for cancer immunotherapy. Dual blockade of LILRB1 and LILRB2 receptors by a single antibody that restores both innate and adaptive immune responses is a promising strategy to enhance efficacy of checkpoint inhibitors. Methods: Dual LILRB1 and LILRB2 targeting antibodies were cloned from B cells derived from rabbits immunized with human LILRB2 recombinant protein, and subsequently humanized. Antibodies were evaluated for binding to human LILRB1 and LILRB2 proteins. Dual targeting antibodies were evaluated in a panel of functional and phenotypic assays. Selected antibodies were further tested for efficacy in a humanized NSG-SGM3 tumor model. Results: Dual antibodies were selected based on binding to recombinant human LILRB1 and LILRB2 protein, as well as blocking of HLA-G binding. These antibodies demonstrated binding to cells expressing LILRB1 and LILRB2, with no appreciable binding to other family members. Lead antibodies demonstrated activity in functional cell-based assays modeling LILRB1- or LILRB2-mediated immunosuppression. Dual antibodies also enhanced IFN-γ production by LPS-stimulated human PBMC. Selected clones restored T-cell function from M2c macrophage-mediated suppression in coculture with CD8+ T cell, and enhanced the tumoricidal activity of NK cells. Importantly, the lead antibody demonstrated in vivo efficacy with significant tumor growth inhibition and tumor regression in an SK-MEL-5 tumor model in humanized NSG-SGM3 mice. Conclusions: We have identified dual antagonist antibodies targeting both LILRB1 and LILRB2 antibodies that restore both innate and adaptive immune responses. Additionally, dual antibodies restored CD8+ T cell activation from macrophage-mediated suppression and enhanced NK cell cytotoxic activity. These data provide a strong rationale for further development of dual antibodies as an anti-cancer immunotherapy. Citation Format: Meghan Zuck, Myriam Bouchlaka, Huyen Dinh, Kevin Green, Francisco Zapata, Ramya Chandrasekaran, Tatyana Pisarenko, Lauren Loh, Gajendra S. Naika, Meilyn Sylvestre, Jacob Heit, Raymond Fox, Darbie Whitman, Tom Graddis, Kamal D. Puri, Peter Probst. Discovery and preclinical characterization of dual antagonist antibodies targeting both LILRB1 and LILRB2 that enhance innate and adaptive anti-cancer immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6376.

Abstract 6577: CARMEN: A pan-HLA and pan-cancer proteogenomic database on antigen presentation to support cancer immunotherapy

Abstract Cancer immunotherapy has greatly improved the quality of life of cancer patients and it hinges on the discovery of novel cancer antigens that could be targeted to improve disease outcomes. The creation of databases such as IEDB, SysteMHC, TANTIGEN, caAtlas, HLA Ligand Atlas, Cancer Antigenic Peptide Database, SPENCER and IEAtlas support the immunopeptidomics community in understanding the landscape of antigen presentation. We have developed a pan-cancer, pan-HLA, and pan-tissue database containing immunopeptidomics data mapped to transcriptomic, genomic, immunological and biochemical data. The database was generated from 80 different publicly available immunopeptidomics mass spectrometry datasets collected between 2015-2022 (76 cancer and 4 normal datasets), covering 15 different types of cancers and 152 different HLA-I alleles. The peptides contained in our database were obtained by a combination of closed, open and de novo searches using an in-house developed computational pipeline. Following rigorous false discovery rate estimation at 1% and a second-round search to eliminate any false signals that may not have been detected in the previous round of FDR estimation, we obtained a list of 11.2 million peptide-HLA combinations comprising both coding and non-coding regions of the genome as well as bacterial peptides. These peptides have been mapped to chromosomal coordinates to facilitate adoption by the genomics community of this useful resource on antigen presentation. Pathway/biochemical analysis of each peptide was performed using the rWikiPathways package. Finally, mutations associated with each peptide were annotated using COSMIC and dbSNP resources. Our database includes a FAIR knowledge graph which contextualizes and enriches the data to enable clinicians to take effective therapeutic decisions on the appropriate form of treatment for cancer immunotherapy with the case study of clear cell renal cell carcinoma (ccRCC). We will continue to expand our database with new data over the next two years and expand the scope of its applications to facilitate uptake by the larger scientific community. Citation Format: Ashwin Adrian Kallor, Michał Waleron, Georges Bedran, Patrícia Eugénio, Catia Pesquita, Daniel Faria, Fabio Massimo Zanzotto, Christophe Battail, Ajitha Rajan, Javier Alfaro. CARMEN: A pan-HLA and pan-cancer proteogenomic database on antigen presentation to support cancer immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6577.

The HLA ligandome of oropharyngeal squamous cell carcinomas reveals shared tumour-exclusive peptides for semi-personalised vaccination

BackgroundThe immune peptidome of OPSCC has not previously been studied. Cancer-antigen specific vaccination may improve clinical outcome and efficacy of immune checkpoint inhibitors such as PD1/PD-L1 antibodies.MethodsMapping of the OPSCC HLA ligandome was performed by mass spectrometry (MS) based analysis of naturally presented HLA ligands isolated from tumour tissue samples (n = 40) using immunoaffinity purification. The cohort included 22 HPV-positive (primarily HPV-16) and 18 HPV-negative samples. A benign reference dataset comprised of the HLA ligandomes of benign haematological and tissue datasets was used to identify tumour-associated antigens.ResultsMS analysis led to the identification of naturally HLA-presented peptides in OPSCC tumour tissue. In total, 22,769 peptides from 9485 source proteins were detected on HLA class I. For HLA class II, 15,203 peptides from 4634 source proteins were discovered. By comparative profiling against the benign HLA ligandomic datasets, 29 OPSCC-associated HLA class I ligands covering 11 different HLA allotypes and nine HLA class II ligands were selected to create a peptide warehouse.ConclusionTumour-associated peptides are HLA-presented on the cell surfaces of OPSCCs. The established warehouse of OPSCC-associated peptides can be used for downstream immunogenicity testing and peptide-based immunotherapy in (semi)personalised strategies.

Uranyl(VI) Mixed-ligand complex synthesis and characterization using 4-acylhydrazone-5-pyrazolone and 4-acylpyrazolone: Covalency, crystal assay, DFT study and Hirshfeld analysis

Newly prepared bidentate HLA and tridentate Bz-HLA ligands were used to synthesize the novel uranyl mixed ligand [UO2(HLA)(Bz-HLA)] complex with pentagonal bipyramidal geometry. HLA ligand was prepared via the common acylation method, and the Bz-HLA ligand was prepared using the Schiff base reaction of HLA with a benzohydrazide molecule. FTIR, 1H and 13C NMR, SCXRD, molar conductance, and TG-DTA were mainly implemented to examine the structure of produced compounds. The combined covalency effect of complex due to both ligands has been studied, which was discovered to be influenced by the average bond length values on the pentagonal plane, uranyl axial bond lengths, ligand coordination, and donor ability of various atoms. To demonstrate that solvents have no impact on the covalency of the synthesised complex, Sinha's covalency parameters were computed using UV–vis data. DFT calculations were performed in order to validate experimental findings in comparison to theoretical findings, and theoretical bond characteristics were also investigated through the recognition of global index values. Hirshfeld surface analysis was used to examine the crystal strength, energy characteristics and non-covalent surface interactions. Analysis using cyclic voltammetry(CV) shows that the complex is irreversibly reducing. Understanding the Schiff base of acylpyrazolones and their covalency in uranyl complexes allow for the investigation of a wide range of chemical properties and applications.

Association of KIR2DL5, KIR2DS5, and KIR2DS1 allelic variation and atopic dermatitis

Natural killer cells (NK) have been associated with the pathophysiology of atopic dermatitis (AD). NK function is regulated by killer cell Ig-like receptor family (KIR) receptors that interact with HLA ligands. The study goal was to focus on allelic variation in genes KIR2DL5, KIR2DS5, and KIR2DS1 with respect to AD. This was a case–control study of individuals with (n = 313) and without (n = 176) AD. Associations were estimated using logistic regression. The prevalence of KIR2DL5 was 52.5% (95% CI 48.0,57.0), KIR2DS5 was 33.0% (28.8,37.3), and KIR2DS1 was 33.6% (29.4,38.0). The presence of the KIR2DL5*001:01 increased the odds of having AD by about 86% (odds ratio (OR): 1.86(1.23,2.82) p = 0.003). The risk for individuals homozygous for KIR2DL5*001:01 was even greater (OR: 2.16 (95% CI 1.31,3.53) p = 0.0023). The odds of having AD with KIR2DL5*001:01 was similar in Whites and Blacks. Allelic variation in KIR2DS5 and KIR2DS1 was not associated with AD. There is no known HLA binding ligand for KIR2DL5. The effect of KIR2DL5*001:01 increased in the presence of HLA-B*-21TT leader sequence (2.46(1.37,4.41) p = 0.0025) and the HLA-C2 ligand (2.07 (1.37,4.41, p = 0.000002). Our study shows an independent association of the KIR2DL5*001:01 with AD and is the first study to associate AD with KIR allelic variation.

Exploring the potential of combining IL-2-activated NK cells with an anti-PDL1 monoclonal antibody to target multiple myeloma-associated macrophages

Multiple myeloma (MM) is an incurable disease, characterized by malignant plasma cells in the bone marrow. MM growth is largely dependent on the tumor microenvironment (TME), consisting of complex cellular networks that shape a tumor-permissive environment. Within the TME, tumor-associated cells (TAC) comprise heterogeneous cell populations that collectively support immunosuppression. Reshaping the TME toward an immunostimulatory environment may enhance effectiveness of immunotherapies. Here, we investigated interactions between donor-derived natural killer (NK) cells and TAC, like tumor-associated macrophages (TAM) and M1 macrophages, and assessed whether anti-tumor effector functions of NK cells could be enhanced by an ADCC-triggering antibody targeting macrophages. Monocytes were polarized in vitro toward either M1 or TAM before co-culture with high-dose IL-2-activated NK cells. NK cell responses were assessed by measuring degranulation (CD107a) and IFN-γ production. We found that NK cells degranulated and produced IFN-γ upon interaction with both macrophage types. NK cell responses against PD-L1+ M1 macrophages could be further enhanced by Avelumab, an anti-PD-L1- and ADCC-inducing antibody. Additionally, NK cell responses were influenced by HLA class I, shown by stronger degranulation in NK cell subsets for which the corresponding HLA ligand was absent on the macrophage target cells (KIR-ligand mismatch) compared to degranulation in the presence of the HLA ligand (KIR-ligand match). Our results suggest that NK cells could, next to killing tumor cells, get activated upon interaction with TAC, like M1 macrophages and TAMs, and that NK cells combined with PD-L1 blocking antibodies with ADCC potential could, through IFN-γ secretion, promote a more immune-favorable TME.

Prevalent and immunodominant CD8 T cell epitopes are conserved in SARS-CoV-2 variants

The emergence of SARS-CoV-2 variants of concern (VOC) is driven by mutations that mediate escape from neutralizing antibodies. There is also evidence that mutations can cause loss of Tcell epitopes. However, studies on viral escape from Tcell immunity have been hampered by uncertain estimates of epitope prevalence. Here, we map and quantify CD8 Tcell responses to SARS-CoV-2-specific minimal epitopes in blood drawn from April to June 2020 from 83 COVID-19 convalescents. Among 37 HLA ligands eluted from five prevalent alleles and an additional 86 predicted binders, we identify 29 epitopes with an immunoprevalence ranging from 3% to 100% among individuals expressing the relevant HLA allele. Mutations in VOC are reported in 10.3% of the epitopes, while 20.6% of the non-immunogenic peptides are mutated in VOC. The nine most prevalent epitopes are conserved in VOC. Thus, comprehensive mapping of epitope prevalence does not provide evidence that mutations in VOC are driven by escape of Tcell immunity.