Non-classical Human Leukocyte Antigen Research Articles

DeepHLAPred: a deep learning-based method for non-classical HLA binder prediction

Human leukocyte antigen (HLA) is closely involved in regulating the human immune system. Despite great advance in detecting classical HLA Class I binders, there are few methods or toolkits for recognizing non-classical HLA Class I binders. To fill in this gap, we have developed a deep learning-based tool called DeepHLAPred. The DeepHLAPred used electron-ion interaction pseudo potential, integer numerical mapping and accumulated amino acid frequency as initial representation of non-classical HLA binder sequence. The deep learning module was used to further refine high-level representations. The deep learning module comprised two parallel convolutional neural networks, each followed by maximum pooling layer, dropout layer, and bi-directional long short-term memory network. The experimental results showed that the DeepHLAPred reached the state-of-the-art performanceson the cross-validation test and the independent test. The extensive test demonstrated the rationality of the DeepHLAPred. We further analyzed sequence pattern of non-classical HLA class I binders by information entropy. The information entropy of non-classical HLA binder sequence implied sequence pattern to a certain extent. In addition, we have developed a user-friendly webserver for convenient use, which is available at http://www.biolscience.cn/DeepHLApred/. The tool and the analysis is helpful to detect non-classical HLA Class I binder. The source code and data is available at https://github.com/tangxingyu0/DeepHLApred.

Perspectives of targeting LILRB1 in innate and adaptive immune checkpoint therapy of cancer.

Immune checkpoint blockade is a compelling approach in tumor immunotherapy. Blocking inhibitory pathways in T cells has demonstrated clinical efficacy in different types of cancer and may hold potential to also stimulate innate immune responses. A novel emerging potential target for immune checkpoint therapy is leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1). LILRB1 belongs to the superfamily of leukocyte immunoglobulin-like receptors and exerts inhibitory functions. The receptor is expressed by a variety of immune cells including macrophages as well as certain cytotoxic lymphocytes and contributes to the regulation of different immune responses by interaction with classical as well as non-classical human leukocyte antigen (HLA) class I molecules. LILRB1 has gained increasing attention as it has been demonstrated to function as a phagocytosis checkpoint on macrophages by recognizing HLA class I, which represents a 'Don't Eat Me!' signal that impairs phagocytic uptake of cancer cells, similar to CD47. The specific blockade of the HLA class I:LILRB1 axis may provide an option to promote phagocytosis by macrophages and also to enhance cytotoxic functions of T cells and natural killer (NK) cells. Currently, LILRB1 specific antibodies are in different stages of pre-clinical and clinical development. In this review, we introduce LILRB1 and highlight the features that make this immune checkpoint a promising target for cancer immunotherapy.

The role of non-classical and chain-related human leukocyte antigen polymorphisms in laryngeal squamous cell carcinoma.

Laryngeal squamous cell carcinoma (LSCC) is the major pathological subtype of laryngeal cancer. It has been shown that alterations of the expression of non-classical human leukocyte antigens (HLA) and the chain-related MIC molecules by malignant cells can lead to escape from the immune system control and certain allele variants may participate in immune editing and therefore be associated with modulation of cancer risk. The aim of the present study was to investigate the role of non-classical HLA class Ib and chain-related MIC polymorphisms, determined at the allelic level by next-generation sequencing (NGS), in patients from the Bulgarian population, diagnosed with LSCC. In the present study DNA samples from 48 patients with LSCC were used. Data was compared to 63 healthy controls analysed in previous studies. HLA genotyping was performed by using the AlloSeq Tx17 early pooling protocol and the library preparation AlloSeq Tx17 kit (CareDx). Sequencing was performed on MiniSeq sequencing platform (Illumina) and HLA genotypes were assigned with the AlloSeq Assign analysis software v1.0.3 (CareDx) and the IPD-IMGT/HLA database 3.45.1.2. The HLA disease association tests revealed a statistically significant predisposing association of HLA-F*01:01:02 (Pc = 0.0103, OR = 24.0194) with LSCC, while HLA-F*01:01:01 (Pc = 8.21e-04, OR = 0.0485) has a possible protective association. Additionally we observed several haplotypes with statistically significant protective and predisposing associations. The strongest association was observed for F*01:01:01-H*01:01:01 (P = 0.0054, haplotype score=-2.7801). Our preliminary study suggests the involvement of HLA class Ib in cancer development and the possible role of the shown alleles as biomarkers of LSCC.

HLA-E-restricted SARS-CoV-2-specific T cells from convalescent COVID-19 patients suppress virus replication despite HLA class Ia down-regulation.

Pathogen-specific CD8+ T cell responses restricted by the nonpolymorphic nonclassical class Ib molecule human leukocyte antigen E (HLA-E) are rarely reported in viral infections. The natural HLA-E ligand is a signal peptide derived from classical class Ia HLA molecules that interact with the NKG2/CD94 receptors to regulate natural killer cell functions, but pathogen-derived peptides can also be presented by HLA-E. Here, we describe five peptides from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that elicited HLA-E-restricted CD8+ T cell responses in convalescent patients with coronavirus disease 2019. These T cell responses were identified in the blood at frequencies similar to those reported for classical HLA-Ia-restricted anti-SARS-CoV-2 CD8+ T cells. HLA-E peptide-specific CD8+ T cell clones, which expressed diverse T cell receptors, suppressed SARS-CoV-2 replication in Calu-3 human lung epithelial cells. SARS-CoV-2 infection markedly down-regulated classical HLA class I expression in Calu-3 cells and primary reconstituted human airway epithelial cells, whereas HLA-E expression was not affected, enabling T cell recognition. Thus, HLA-E-restricted T cells could contribute to the control of SARS-CoV-2 infection alongside classical T cells.

Elevated sHLA-G plasma levels post chemotherapy combined with ILT-2 rs10416697C allele status of the sHLA-G-related receptor predict poorest disease outcome in early triple-negative breast cancer patients.

Triple negative breast cancer (TNBC) shows an aggressive growing and spreading behavior and has limited treatment options, often leading to inferior disease outcome. Therefore, surrogate markers are urgently needed to identify patients at high risk of recurrence and more importantly, to identify additional therapeutic targets enabling further treatment options. Based on the key role of the non-classical human leukocyte antigen G (HLA-G) and its related receptor immunoglobulin-like transcript receptor-2 (ILT-2) in immune evasion mechanisms of tumors, members of this ligand-receptor axis appear to be promising tool for both, defining risk groups and potential therapeutic targets. To follow this, sHLA-G levels before and after chemotherapy (CT), HLA-G 3' UTR haplotypes, and allele variations rs10416697 at the distal gene promoter region of ILT-2 were defined in healthy female controls and early TNBC patients. The results obtained were associated with clinical status, presence of circulating tumor cell (CTC) subtypes, and disease outcome of patients in terms of progression-free or overall survival. sHLA-G plasma levels were increased in TNBC patients post-CT compared to levels of patients pre-CT or controls. High post-CT sHLA-G levels were associated with the development of distant metastases, the presence of ERCC1 or PIK3CA-CTC subtypes post-CT, and poorer disease outcome in uni- or multivariate analysis. HLA-G 3' UTR genotypes did not influence disease outcome but ILT-2 rs10416697C allele was associated with AURKA-positive CTC and with adverse disease outcome by uni- and multivariate analysis. The prognostic value of the combined risk factors (high sHLA-G levels post-CT and ILT-2 rs10416697C allele carrier status) was an even better independent indicator for disease outcome in TNBC than the lymph nodal status pre-CT. This combination allowed the identification of patients with high risk of early progression/death with positive nodal status pre-CT or with non-pathological complete therapy response. The results of this study highlight for the first time that the combination of high levels of sHLA-G post-CT with ILT-2 rs10416697C allele receptor status is a promising tool for the risk assessment of TNBC patients and support the concept to use HLA-G/ILT-2 ligand-receptor axis as therapeutic targets.

DapNet-HLA: Adaptive dual-attention mechanism network based on deep learning to predict non-classical HLA binding sites

Human leukocyte antigen (HLA) plays a vital role in immunomodulatory function. Studies have shown that immunotherapy based on non-classical HLA has essential applications in cancer, COVID-19, and allergic diseases. However, there are few deep learning methods to predict non-classical HLA alleles. In this work, an adaptive dual-attention network named DapNet-HLA is established based on existing datasets. Firstly, amino acid sequences are transformed into digital vectors by looking up the table. To overcome the feature sparsity problem caused by unique one-hot encoding, the fused word embedding method is used to map each amino acid to a low-dimensional word vector optimized with the training of the classifier. Then, we use the GCB (group convolution block), SENet attention (squeeze-and-excitation networks), BiLSTM (bidirectional long short-term memory network), and Bahdanau attention mechanism to construct the classifier. The use of SENet can make the weight of the effective feature map high, so that the model can be trained to achieve better results. Attention mechanism is an Encoder-Decoder model used to improve the effectiveness of RNN, LSTM or GRU (gated recurrent neural network). The ablation experiment shows that DapNet-HLA has the best adaptability for five datasets. On the five test datasets, the ACC index and MCC index of DapNet-HLA are 4.89% and 0.0933 higher than the comparison method, respectively. According to the ROC curve and PR curve verified by the 5-fold cross-validation, the AUC value of each fold has a slight fluctuation, which proves the robustness of the DapNet-HLA. The codes and datasets are accessible at https://github.com/JYY625/DapNet-HLA.

Study of Human Leukocyte Antigen-G gene 14-bp Ins/Del and Codon 93 (CAC/CAT) Polymorphisms Association with Breast Cancer Susceptibility

Human leukocyte antigen-G is a non-classic human leukocyte antigen I molecule. This protein is expressed aberrantly in numerous forms of malignant diseases like glioblastoma multiforme, ovarian cancer, lymphoblastic and breast cancer. Human leukocyte antigen-G polymorphisms association with breast cancer has been extensively studied. One amongst the foremost studied variants was human leukocyte antigen-G 14-base pair insertion/deletion polymorphism. Nevertheless, results are contradictory or inconclusive. We aim to investigate two human leukocyte antigen-G polymorphisms, 14-base pair insertion/deletion 3’ untranslated region and for the first time and codon 93 (CAC/CAT) polymorphisms potentially associated with breast cancer risk in the Tunisian population. This study involved 151 breast cancer patients and 187 healthy controls. Genotyping of the human leukocyte antigen-G codon 93 (CAC/CAT) and 14-base pair insertion/deletion variants was performed respectively by amplification-refractory mutation system and polymerase chain reaction. The analysis of genotype and allele frequencies revealed that human leukocyte antigen-G 14-base pair insertion/deletion polymorphism was associated with breast cancer susceptibility in the Tunisian population (Odds ratio=0.53, 95 % confidence interval=0.33-0.85, p=0.0022) without correlation with clinical parameters. We also observed an association of the human leukocyte antigen-G codon 93 (CAC/CAT) polymorphisms with breast cancer. In terms of analysis of the combined 14-base pair insertion/deletion and codon 93 CT genotype, we detected that the CC insertion/insertion and CC insertion/deletion genotype decrease the risk of breast cancer development (Odds ratio=0.2083; p=0.0097; Odds ratio=0.3571; p=0.0053, respectively). In addition, the haplotype frequency distribution analysis disclosed that the C insertion haplotype has a protective effect against breast cancer development (Odds ratio=0.4749; 95 % confidence interval=0.4425-0.9277; p=0.0153). Taken together, female’s homozygosity for human leukocyte antigen-G 14-base pair insertion and human leukocyte antigen-G codon 93 (CAC) alleles may protect against breast cancer susceptibility.

Targeted capture enrichment and sequencing identifies HLA variants associated with the severity of COVID-19.

Coronavirus disease 2019 (COVID-19) is currently a global pandemic. The pathogenesis of severe COVID-19 has been widely investigated, but it is still unclear. Human leukocyte antigen (HLA) plays a central role in immune response, and its variants might be related to COVID-19 progression and severity. To investigate the hypothesis that individual HLA variations could alter the course of COVID-19 and might be associated with the severity of COVID-19. In this study, we conducted an HLA targeted capture enrichment and sequencing of severe COVID-19 patients matched to mild cases. A total of 16 COVID-19 patients, confirmed by SARS-CoV-2 viral RNA polymerase-chain-reaction (PCR) test and chest computed tomography (CT) scan, were enrolled in this study. The HLA targeted capture enrichment and sequencing were conducted. HLA typing was performed by comparing contigs with IPD-IMGT/HLA Database. In this study, 139 four-digit resolution HLA alleles were acquired. The results showed that HLA-DRB3*01:01 allele was significantly associated with the severity of COVID-19 (odds ratio [OR] = 27.64, 95% confidence interval [CI] = 1.35-560.50, P = 0.0064). And HLA-K*01:01 might be a potential risk factor for COVID-19 severity (OR = 0.11, 95% CI = 0.017-0.66, P = 0.019), but HLA-K*01:02 might be a protective factor (OR = 7.50, 95% CI = 1.48-37.92, P = 0.019). Three non-classical HLA alleles, including HLA-DRB3*01:01, HLA-K*01:01, HLA-K*01:02 were identified to be associated with the severity of COVID-19 by comparing mild and severe patients. The current findings would be helpful for exploring the influence of HLA gene polymorphisms on the development and severity of COVID-19.

Lower HLA-G levels in extravillous trophoblasts of human term placenta in gestational diabetes mellitus than in normal controls

The non-classical human leucocyte antigen (HLA) class I molecule HLA-G is widely known to play a major role in feto-maternal tolerance. We tested the hypothesis that HLA-G expression is altered in placentas of women with gestational diabetes mellitus (GDM) in a specific pattern that depends on fetal sex. HLA-G expression was analysed in a total of 80 placentas (40 placentas from women with GDM and 40 healthy controls) by immunohistochemistry using the semi-quantitative immunoreactive score (IRS). Double immunofluorescence staining identified the cells expressing HLA-G in the decidua and allowed evaluation of the expression pattern. We found a significant (p < 0.001) reduction of HLA-G expression in extravillous cytotrophoblasts (EVTs) in the placentas of women with GDM as compared to the healthy controls and were able to demonstrate that this downregulation was not due to a loss of cell number, but to a loss of expression intensity. A special change in the cell pattern of EVTs was observed, with these cells showing an obvious decrease in HLA-G expression on their cell surface. No significant differences according to fetal sex were found. These data show a possible association between decreased HLA-G expression and presence of GDM and provide new insights into altered placental function in women with GDM.

IMMU-16. SOMATIC MUTATIONS IN HUMAN LEUKOCYTE ANTIGEN GENES AND ANTIGEN PRESENTATION PATHWAY GENES IN MALIGNANT GLIOMA

Abstract Immunotherapeutic approaches in cancer aim to booster T cell mediated immune responses. Tumor-specific T cells recognize target cells via human leukocyte antigen (HLA) molecules before cell specific lysis is initiated. Malignant gliomas are known for their immunoevasive and immunosuppressive properties which impede effective tumor-specific immune responses, including clinical responses to immune checkpoint inhibition. Our aim was to investigate whether somatic mutation in HLA genes and antigen presentation pathway genes are present in malignant gliomas. METHODS We performed next generation sequencing in 138 patients, including matched tumor and blood samples from 100 IDHwt glioblastomas, 25 IDHmut astrocytomas and 13 IDHmut oligodendrogliomas. Sequencing was performed on tumor and peripheral blood for HLA class I (HLA-A, -B, -C), HLA class II (HLA-DR, -DP, -DQ), non-classical HLA molecules (HLA-E, -F, -G, -H), MICA/B, TAP1/2 and beta2 microglobulin (B2M). Identified calls were validated using targeted nanopore sequencing. RESULTS In total, we identified 28 mutations based on Illumina sequencing. Mutations were located in HLA-B (2 mutations), HLA-C (1), B2M (2), HLA-DQA1 (4), HLA-DQB1 (4), HLA-DPB1 (1), HLA-E (1), HLA-F (2), HLA-G (3), HLA-H (1), MICA (3), MICB (2), TAP1 (1) and TAP2 (1). No mutations were found in HLA-A and HLA-DRB1. A frequency cutoff of 5% was applied. Additional mutations (n = 46) below this cutoff were identified. The mutations were found in 30 patients (21.7%). Some patients harbored up to 7-8 mutations in multiple HLA and antigen presenting pathway genes. Most mutations were found in the MICA (MHC class I polypeptide–related sequence A) gene locus, which is engaged by the NKG2D ligand which is broadly expressed on NK cells, gd and ab T cells. CONCLUSION Our study demonstrates that somatic mutations in HLA and antigen presentation pathway genes are frequently present in malignant gliomas.

Induction of pulmonary HLA-G expression by SARS-CoV-2 infection

The non-classical human leukocyte antigen (HLA)-G exerts immune-suppressive properties modulating both NK and T cell responses. While it is physiologically expressed at the maternal–fetal interface and in immune-privileged organs, HLA-G expression is found in tumors and in virus-infected cells. So far, there exists little information about the role of HLA-G and its interplay with immune cells in biopsies, surgical specimen or autopsy tissues of lung, kidney and/or heart muscle from SARS-CoV-2-infected patients compared to control tissues. Heterogeneous, but higher HLA-G protein expression levels were detected in lung alveolar epithelial cells of SARS-CoV-2-infected patients compared to lung epithelial cells from influenza-infected patients, but not in other organs or lung epithelia from non-viral-infected patients, which was not accompanied by high levels of SARS-CoV-2 nucleocapsid antigen and spike protein, but inversely correlated to the HLA-G-specific miRNA expression. High HLA-G expression levels not only in SARS-CoV-2-, but also in influenza-infected lung tissues were associated with a high frequency of tissue-infiltrating immune cells, but low numbers of CD8+ cells and an altered expression of hyperactivation and exhaustion markers in the lung epithelia combined with changes in the spatial distribution of macrophages and T cells. Thus, our data provide evidence for an involvement of HLA-G and HLA-G-specific miRNAs in immune escape and as suitable therapeutic targets for the treatment of SARS-CoV-2 infections.

Human leukocyte antigen HLA-C, HLA-G, HLA-F, and HLA-E placental profiles are altered in early severe preeclampsia and preterm birth with chorioamnionitis

The extravillous trophoblast expresses each of the nonclassical major histocompatibility complex class I antigens-human leukocyte antigens E, F, and G-and a single classical class I antigen, human leukocyte antigen C. We recently demonstrated dynamic expression patterns of human leukocyte antigens C, G, and F during early extravillous trophoblast invasion and placentation. This study aimed to investigate the hypothesis that the immune inflammatory mediated complications of pregnancy such as early preeclampsia and preterm labor may show altered expression profiles of nonclassical human leukocyte antigens. Real-time quantitative polymerase chain reaction, western blot, and immunohistochemistry were performed on placental villous tissues and basal plate sections from term nonlaboring deliveries, preterm deliveries, and severe early-onset preeclampsia, both with and without small-for-gestational-age neonates. Human leukocyte antigen G is strongly and exclusively expressed by the extravillous trophoblast within the placental basal plate, and its levels increase in pregnancies complicated by severe early-onset preeclampsia with small-for-gestational-age neonates relative to those of healthy term controls. Human leukocyte antigen C shows a similar profile in the extravillous trophoblast of preeclamptic pregnancies, but significantly decreases in the villous placenta. Human leukocyte antigen F protein levels are decreased in both extravillous trophoblast and villous placenta of severe early-onset preeclamptic pregnancies, both with and without small-for-gestational-age neonates, compared with those found in term and preterm birth deliveries. Human leukocyte antigen E decreases in blood vessels in placentas from preeclamptic pregnancies relative to its levels in term and preterm birth deliveries. Placental levels of human leukocyte antigens F and C are increased in cases of preterm birth with chorioamnionitis relative to those of cases of idiopathic preterm birth. Dysregulation of placental human leukocyte antigen expression at the maternal-fetal interface may contribute to compromised maternal tolerance in preterm birth with chorioamnionitis and excessive maternal systemic inflammation associated with severe early-onset preeclampsia.

Non-Classical HLA Determinants of the Clinical Response after Autologous Stem Cell Transplantation for Systemic Sclerosis.

Systemic Sclerosis (SSc) is a chronic autoimmune disease with high morbidity and mortality. Autologous Hematopoietic Stem Cell Transplantation (AHSCT) is the best therapeutic option for rapidly progressive SSc, allowing increased survival with regression of skin and lung fibrosis. The immune determinants of the clinical response after AHSCT have yet to be well characterized. In particular, the pivotal role of the Human Leukocyte Antigen (HLA) system is not well understood, including the role of non-classical immuno-modulatory HLA-E and HLA-G molecules in developing tolerance and the role of Natural Killer cells (NK) in the immunomodulation processes. We retrospectively tested whether the genetic and/or circulating expression of the non-classical HLA-E and HLA-G loci, as well as the imputed classical HLA determinants of HLA-E expression, influence the observed clinical response to AHSCT at 12- and 24-month follow-up. In a phenotypically well-defined sample of 46 SSc patients classified as clinical responders or non-responders, we performed HLA genotyping using next-generation sequencing and circulating levels of HLA-G and quantified HLA-E soluble isoforms by ELISA. The -21HLA-B leader peptide dimorphism and the differential expression level of HLA-A and HLA-C alleles were imputed. We observed a strong trend towards better clinical response in HLA-E*01:03 or HLA-G 14bp Del allele carriers, which are known to be associated with high expression of the corresponding molecules. At 12-month post-AHSCT follow-up, higher circulating levels of soluble HLA-E were associated with higher values of modified Rodnan Skin Score (mRSS) (p = 0.0275), a proxy of disease severity. In the non-responder group, the majority of patients carried a double dose of the HLA-B Threonine leader peptide, suggesting a non-efficient inhibitory effect of the HLA-E molecules. We did not find any correlation between the soluble HLA-G levels and the observed clinical response after AHSCT. High imputed expression levels of HLA-C alleles, reflecting more efficient NK cell inhibition, correlated with low values of the mRSS 3 months after AHSCT (p = 0.0087). This first pilot analysis of HLA-E and HLA-G immuno-modulatory molecules suggests that efficient inhibition of NK cells contributes to clinical response after AHSCT for SSc. Further studies are warranted in larger patient cohorts to confirm our results.

Abstract 765: Hybridized-capture sequencing methods bias analysis of antigen presentation genes

Abstract Introduction: Tumor-immune dynamics are often interrogated using hybridized capture-based sequencing approaches which require the use of baits that target the protein-coding regions of the genome. However, the genes encoding the antigen presentation machinery, including human leukocyte antigen (HLA) proteins, are very polymorphic, resulting in thousands of unique genotypes across patient cohorts. Here, using whole exome sequencing (WES) and hybridized capture-based RNA sequencing (RNAseq) from melanoma patients, we show that these genes are not uniformly assessed by common sequencing methods, and require careful analysis for accurate detection of copy number and somatic alterations. Methods: WES and RNAseq data was analyzed from 760 melanoma tumor biopsies, with WES derived from patient-matched normal blood samples. Coverage metrics for 26 antigen presentation genes, including classical and non-classical HLA genes, were evaluated, and the difference in coverage was calculated for heterozygous alleles. Each individual was assessed for heterozygous single nucleotide polymorphisms (SNPs) within each allele, and with respect to the haplotype of the human reference genome. Results: Focusing on the 26 genes assessed, HLA-A, HLA-B, HLA-C, and HLA-DRB1 were the most diverse across our patient cohort; there was an average of 134 unique pairs of HLA alleles in these genes. We compared the SNP profiles of allele combinations to the haplotype of the human reference genome, and observed four patterns: heterozygous alleles are similar to each other and the reference haplotype, heterozygous alleles are different from each other and different to the reference, alleles are very similar to each other and greatly differ from the reference, or one allele is more similar to the reference haplotype. The fourth pattern showed the greatest bias in sequencing, where the allele more similar to the human reference genome had a median 4-fold increase in coverage in normal WES data. Conclusions: Capture-based sequencing shows selection bias, as exome probes have been designed based upon the reported human reference genome, but results in selection bias in hybridized capture sequencing approaches. Alleles that have higher polymorphic differences with respect to the reference are not equally captured, resulting in nonuniform sequencing of heterozygous alleles. Citation Format: Egmidio Medina, Katie Campbell, Antoni Ribas. Hybridized-capture sequencing methods bias analysis of antigen presentation genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 765.

Evaluation of the relationships between HLA-G 14 bp polymorphism and two acute leukemia in a Saudi population

The non-classical Human Leukocyte Antigen G (HLA-G) is an immunomodulatory molecule, with low polymorphism frequency and restricted tissue distribution. Its higher expression was associated with immunoinhibitory properties causing tolerance for cancer progression. The rs371194629 is a 14 bp insertion/deletion polymorphism (Ins/Del) in exon 8 of the 3′ untranslated region (3′-UTR) of the HLA-G gene, has been associated with the stability and the expression level of HLA-G mRNA. In this study, we evaluated the relationship between the HLA-G 14-bp Ins/Del polymorphism and two common blood cancers including acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) in a population from Riyadh in Saudi Arabia. The study groups include 145 patients diagnosed for ALL, 98 patients diagnosed for AML and 115 healthy individuals. For all these individuals, the 14 bp Ins/Del polymorphism was genotyped using PCR methodology. mRNA gene expression of the HLA-G was assessed using quantitative RT-PCR. Logistic regression model analysis for HLA-G 14 bp Ins/Del polymorphism shows no statistically significant correlation with AML and ALL. For the HLA-G mRNA expression, while slightly increased level among healthy individuals compared to patients was observed, no statistically significant differences were obtained. Although our results did not show association between HLA-G with the two leukemia cancer diseases, the role of HLA-G gene awaits further investigations including large number of subjects with more clinical data combinations.

Generation of hypoimmunogenic induced pluripotent stem cells by CRISPR-Cas9 system and detailed evaluation for clinical application

In order to expand the promise of regenerative medicine using allogeneic induced pluripotent stem cells (iPSCs), precise and efficient genome editing of human leukocyte antigen (HLA) genes would be advantageous to minimize the immune rejection caused by mismatches of HLA type. However, clinical-grade genome editing of multiple HLA genes in human iPSC lines remains unexplored. Here, we optimized the protocol for good manufacturing practice (GMP)-compatible CRISPR-Cas9 genome editing to deplete the three gene locus (HLA-A, HLA-B, and CIITA genes) simultaneously in HLA homozygous iPSCs. The use of HLA homozygous iPSCs has one main advantage over heterozygous iPSCs for inducing biallelic knockout by a single gRNA. RNA-seq and flow cytometry analyses confirmed the successful depletion of HLAs, and lineage-specific differentiation into cardiomyocytes was verified. We also confirmed that the pluripotency of genome-edited iPSCs was successfully maintained by the three germ layers of differentiation. Moreover, whole-genome sequencing, karyotyping, and optical genome mapping analyses revealed no evident genomic abnormalities detected in some clones, whereas unexpected copy number losses, chromosomal translocations, and complex genomic rearrangements were observed in other clones. Our results indicate the importance of multidimensional analyses to ensure the safety and quality of the genome-edited cells. The manufacturing and assessment pipelines presented here will be the basis for clinical-grade genome editing of iPSCs.

NKG2C+ NK Cells for Immunotherapy of Glioblastoma Multiforme.

In glioblastoma, non-classical human leucocyte antigen E (HLA-E) and HLA-G are frequently overexpressed. HLA-E loaded with peptides derived from HLA class I and from HLA-G contributes to inhibition of natural killer (NK) cells with expression of the inhibitory receptor CD94/NKG2A. We investigated whether NK cells expressing the activating CD94/NKG2C receptor counterpart were able to exert anti-glioma effects. NKG2C+ subsets were preferentially expanded by a feeder cell line engineered to express an artificial disulfide-stabilized trimeric HLA-E ligand (HLA-E*spG). NK cells expanded by a feeder cell line, which facilitates outgrowth of conventional NKG2A+, and fresh NK cells, were included for comparison. Expansion via the HLA-E*spG feeder cells selectively increased the fraction of NKG2C+ NK cells, which displayed a higher frequency of KIR2DL2/L3/S2 and CD16 when compared to expanded NKG2A+ NK cells. NKG2C+ NK cells exhibited increased cytotoxicity against K562 and KIR:HLA-matched and -mismatched primary glioblastoma multiforme (GBM) cells when compared to NKG2A+ NK cells and corresponding fresh NK cells. Cytotoxic responses of NKG2C+ NK cells were even more pronounced when utilizing target cells engineered with HLA-E*spG. These findings support the notion that NKG2C+ NK cells have potential therapeutic value for treating gliomas.

Human Leukocyte Antigens in Pregnancy and Preeclampsia.

Preeclampsia is a pregnancy-induced hypertensive disorder, the pathophysiology of which includes underlying maternal cardiovascular disease, deficient spiral artery remodeling during placenta development, and inflammatory immune responses at the maternal-fetal interface. Human leukocyte antigens (HLA) are major histocompatibility complex molecules essential for the recognition of foreign antigens that is central to immune defense against pathogens and critical determinants for the immune system discriminating between self and non-self tissues, such as in transplantation. Pregnancy represents a naturally existing “transplantation”, where the maternal immune system must be immunologically tolerant to the developing fetus which is 50% allogeneic. It is then unsurprising that HLA also influence normal pregnancy and pregnancy complications including preeclampsia. Here we review the role of classical and non-classical HLA molecules in influencing normal physiologic function during pregnancy and describe the association of HLA with pathophysiology in preeclampsia.

Mouse and human antibodies bind HLA-E-leader peptide complexes and enhance NK cell cytotoxicity

The non-classical class Ib molecule human leukocyte antigen E (HLA-E) has limited polymorphism and can bind HLA class Ia leader peptides (VL9). HLA-E-VL9 complexes interact with the natural killer (NK) cell receptors NKG2A-C/CD94 and regulate NK cell-mediated cytotoxicity. Here we report the isolation of 3H4, a murine HLA-E-VL9-specific IgM antibody that enhances killing of HLA-E-VL9-expressing cells by an NKG2A+ NK cell line. Structural analysis reveal that 3H4 acts by preventing CD94/NKG2A docking on HLA-E-VL9. Upon in vitro maturation, an affinity-optimized IgG form of 3H4 showes enhanced NK killing of HLA-E-VL9-expressing cells. HLA-E-VL9-specific IgM antibodies similar in function to 3H4 are also isolated from naïve B cells of cytomegalovirus (CMV)-negative, healthy humans. Thus, HLA-E-VL9-targeting mouse and human antibodies isolated from the naïve B cell antibody pool have the capacity to enhance NK cell cytotoxicity.

Abstract P4-04-14: Immunological markers in patients with breast cancer occurring during pregnancy - Results from GBG BCP study

Abstract Background: Breast cancer is one of the most common malignancies during pregnancy. Although breast cancer in pregnancy (BCP) is still a rare event (1 in 3000 to 10000 pregnancies), the incidence is likely to increase as more women tend to delay childbearing into later life and the overall lifetime cancer risk increases with age. Pregnancy presents a complex and unique immunological condition. Pregnant women are widely considered to be in an immunosuppressed state, making them more susceptible to infectious diseases. Recent studies have shown similarities between malignancies and the semi-allogenic fetus in terms of immune evasion strategies, for example upregulation of non-classical human leukocyte antigen G (HLA-G). The loss or downregulation of HLA class I is also a way to escape anti-tumor immunity. The aim of this study was to investigate the tumor biology and immunology of pregnant breast cancer patients and the impact of pregnancy on the immunological characteristics of the breast cancer. Methods: 196 of 2831 patient enrolled in the BCP registry (GBG 29) had available tumor material. After identifying representative tumor regions, tissue microarrays (TMAs) of formalin-fixed paraffin embedded core biopsies or surgical specimens from 126 pregnant breast cancer patients treated with neo-(adjuvant) chemotherapy were constructed. TMAs were stained via immunohistochemistry to assess estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2), Ki-67 (&amp;lt;20% vs ≥20%), and immune markers HLA class I (≤5% vs &amp;gt;5%), HLA-G (≤5% vs &amp;gt;5%), TIGIT and Nectin4 as well as hematoxylin-eosin for the prevalence of tumor-infiltrating lymphocytes (TILs, ≤30% vs 31-60% vs &amp;gt;60%). Results: Median age of the patients was 34 (range 26 - 47) years. At the time of diagnosis 50.8% of patients had cT2 tumor and 36.8% nodal involvement. The pre-dominant histological tumor type was ductal or ductal-lobular-invasive carcinoma (89.5%) with poor differentiation (G3: 68.3%). 78.5% of patients were centrally HER2 negative, 42.1% ER-positive and 53.2% PgR-positive; 53.3% had a high expression of Ki-67 (≥20%). With regards to breast cancer subtypes, most patients had either TNBC (34.7%) or HER2-/HR+ breast cancer (43.8%). HLA class I expression (≤5%) was downregulated in 21.3% of patients while 46.3% showed upregulation of HLA-G expression (&amp;gt;5%). Analyzing HLA-G as continuous variable demonstrated an increased but not significant median expression of HLA-G in T3-4 (N=27) compared to T1-2 (N=99) tumor stage (12.4% vs 3.4%, p=0.226). TILs were detected in 62 out of 126 analyzed patients, of whom 93.5% (N=58) had a low expression of TILs (≤30%). Analysis of TIGIT and Nectin4 is ongoing. Conclusions: The high prevalence of low TILs expression in breast cancer during pregnancy might be a sign of reduced maternal immunity associated with pregnancy. HLA class I downregulation detected in our cohort suggests that it is an independent factor of anti-tumor immunity. Additional immune markers and data from a non-pregnant cohort are currently evaluated to make valid conclusions regarding gestational effects on classical and immunological tumor features. Those data will be presented at the meeting. Citation Format: Kristin Galas, Moritz Gleitsmann, Julia Rey, Christine Solbach, Isabell Witzel, Thomas Karn, Andreas Schneeweiss, Bruno Sinn, Tanja Fehm, Carsten Denkert, Volkmar Müller, Anne-Sophie Litmeyer, Christian Schem, Paul Jank, Frederik Marmé, Jenny Furlanetto, Peter A. Fasching, Elmar Stickeler, Olaf Ortmann, Marion van Mackelenbergh, Valentina Nekljudova, Sibylle Loibl. Immunological markers in patients with breast cancer occurring during pregnancy - Results from GBG BCP study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-04-14.