HLA-DP Expression Research Articles

Pervasive loss of antigen presentation in solid tumors driven by loss of HLA class I expression.

e14654 Background: Human Leukocyte Antigens (HLA) play a key role in inducing responses to immune checkpoint inhibitor (ICI) therapy. HLA class I (HLA-I) expression is essential for antigen presentation in cancer cells and its loss leads to immune evasion. Expression of HLA-II shows presence of adaptive immune response via antigen presenting cells. To investigate the complex interplay of HLA class I/II in solid tumors, we study the landscape and association of several HLA phenotypes with biomarkers of tumor response and resistance to ICIs in a large pan-cancer cohort. Methods: A real-world cohort of 24,186 solid tumors was assessed by an immune expression profiling assay of 395 genes that were background subtracted, normalized, and ranked yielding a percentile rank value [0-100]. HLA Class I and class II expressions were estimated as average expression of HLA-A, HLA-B, HLA-C genes, and HLA HLA-DP, HLA-DQ, HLA-DR genes, respectively. Both HLA classes were labelled as high (≥66), moderate (≥33 & <66) or low (<33). We studied the distribution of HLA I/II phenotype defined as Ihi/IIhi comprised hot tumors with no loss of HLA-I and Ilo/IIhi defines immune evasion via loss of HLA-I in hot tumors. Ihi/IIlo phenotype comprises less inflamed tumors with high HLA-I expression and Ilo/IIlo comprises of cold tumors. We also performed differential expression of ICI targets including PD-1, PD-L1, LAG-3, TIM3 and expression-based signatures of inflammation and cell proliferation. Results: Loss of HLA-I expression was observed in 44.1% (n= 10669) of all solid tumors with most frequent loss observed in prostate (55%, n=450) cancer. Similarly, loss of HLA-II expression was observed in 34.7% (n=8396) of solid tumors with highest loss observed in neuroendocrine (59%; n=345) cancers. 49% of all tumors presented with four HLA-phenotypes, where 17.9% (n=4332) were characterized by Ilo/IIlo phenotype. Ihi/IIlo was present in 13% (n=3127) of all tumors. Both Ihi/IIhi and Ilo/IIhi phenotypes were significantly (p<0.001) associated with increased tumor inflammation. Other ICI targets such as PD-L1, TIM3, CTLA4 and LAG3 were significantly (p<0.001) overexpressed in this immune evasion phenotype compared to other phenotypes. Conversely, Ilo/IIlo phenotype was significantly associated with cold tumors (p<0.001), hinting at lack of adaptive immune response in these tumors. Conclusions: This study offers insight into pervasive loss of antigen presentation via HLA expression. Concurrent loss of HLA Class I expression with increased class II expression and additional ICI target co-expression is a potential immune escape mechanism in immunologically active solid tumors. Distinct and significant association of these MHC phenotypes with tumor inflammation, checkpoint markers and PD-L1 highlight the potential use of comprehensive immune profiling for further studying combination strategies to correct soft loss of HLA-I and ICI therapies.

Novel HLA-Ilo/HLA-IIhi phenotype and immune evasion in triple-negative breast cancer.

1095 Background: Immune checkpoint inhibitors (ICIs) are a therapeutic option for patients with triple-negative breast cancer (TNBC), however resistance and non-response remain an unmet clinical need. Human leukocyte antigens (HLAs) play an important role in induction and regulation of host immune responses. Downregulation of HLA class I expression is one mechanism by which cancer cells evade the immune control. HLA typing and understanding the role of HLAs can provide valuable insight into optimizing immunotherapy efficacy in TNBC. In this study, we evaluate the association of HLA I/II phenotypes with biomarkers of tumor immune interaction and overall survival (OS) in a real-world breast cancer cohort. Methods: A targeted RNA-seq assay was performed on 142 breast cancer samples. HLA I and HLA II expressions were estimated as average expression of HLA-A, HLA-B, HLA-C and HLA-DP, HLA-DQ, HLA-DR genes, respectively, by a percentile rank values [0-100]. Cohen D index was used to estimate ratio of HLA-I and II in each specimen. We particularly focused on the HLA-Ilow(lo)/HLA-IIhigh(hi) ratio given a proposed mechanism of immune escape by tumor cells (low expression of HLA-I) in response to a highly immune infiltrated state where there is increased expression of HLA-II by the immune cells. We studied the co-expression of HLA-Ilo/HLA-IIhi with other checkpoint targets using Spearman correlation(rs). The impact of HLA phenotypes on OS were analyzed using Kaplan-Meier analysis and Chi-squared assessed associations. Results: The cohort consisted of 106 non-TNBC (ER+/HER2- or HER2+) and 36 TNBC tumors. Most samples originated from metastatic visceral sites (96, 68%) followed by primary breast (33, 23%), and lymph nodes (13, 9%). Among TNBC, HLA-Ilo/HLA-IIhi phenotype displayed a significantly higher co-expression of immune checkpoint targets such as PD-1, PD-L1, TIM3 and LAG-3 than HLA-Ihi/HLA-IIlo phenotype (p<0.001). However, this difference was not observed between HLA-Ilo/HLA-IIhi and HLA-Ihi/HLA-IIlo phenotypes in non-TNBC tumors. In TNBC, there was no significant difference in OS between HLA-Ilo/HLA-IIhi when compared to HLA-Ihi/HLA-IIlo (43 vs. 37 months, p=0.21). Similarly, in non-TNBC, there were no significant differences in OS between HLA-Ilo/HLA-IIhi when compared to HLA-Ihi/HLA-IIlo (107 vs. 66 months, p=0.5). Conclusions: Concurrent loss of HLA I with increased HLA II expression was associated with co-expression of immune checkpoint molecules indicative of immune escape mechanism but not survival outcomes in TNBC. This same effect was not seen in non-TNBC with a more immunologically silent environment. Characterization of HLA phenotypes by immune profiling provides insight into the complex interplay of tumor immune microenvironment and offers an opportunity for developing novel approaches to overcome the immune evasion with cancer vaccines and adoptive cell therapies in TNBC.

An EBV-related CD4 TCR immunotherapy inhibits tumor growth in an HLA-DP5+ nasopharyngeal cancer mouse model.

Adoptive transfer of T cell receptor-engineered T cells (TCR-T) is a promising strategy for immunotherapy against solid tumors. However, the potential of CD4+ T cells in mediating tumor regression has been neglected. Nasopharyngeal cancer is consistently associated with EBV. Here, to evaluate the therapeutic potential of CD4 TCR-T in nasopharyngeal cancer, we screened for CD4 TCRs recognizing EBV nuclear antigen 1 (EBNA1) presented by HLA-DP5. Using mass spectrometry, we identified EBNA1567-581, a peptide naturally processed and presented by HLA-DP5. We isolated TCR135, a CD4 TCR with high functional avidity, that can function in both CD4+ and CD8+ T cells and recognizes HLA-DP5-restricted EBNA1567-581. TCR135-transduced T cells functioned in two ways: directly killing HLA-DP5+EBNA1+ tumor cells after recognizing EBNA1 presented by tumor cells and indirectly killing HLA-DP5-negative tumor cells after recognizing EBNA1 presented by antigen-presenting cells. TCR135-transduced T cells preferentially infiltrated into the tumor microenvironment and significantly inhibited tumor growth in xenograft nasopharyngeal tumor models. Additionally, we found that 62% of nasopharyngeal cancer patients showed 50%-100% expression of HLA-DP on tumor cells, indicating that nasopharyngeal cancer is well suited for CD4 TCR-T therapy. These findings suggest that TCR135 may provide a new strategy for EBV-related nasopharyngeal cancer immunotherapy in HLA-DP5+ patients.

Actives from the Micro-Immunotherapy Medicine 2LMIREG® Reduce the Expression of Cytokines and Immune-Related Markers Including Interleukin-2 and HLA-II While Modulating Oxidative Stress and Mitochondrial Function.

Micro-immunotherapy (MI) is a therapeutic option employing low doses (LD) and ultra-low doses (ULD) of cytokines and immune factors to help the organism at modulating the immune responses. In an overpowering inflammatory context, this strategy may support the restoration of the body's homeostasis, as the active ingredients of MI medicines' (MIM) could boost or slow down the physiological functions of the immune cells. The aim of the study is to evaluate for the first time the in vitro anti-inflammatory properties of some actives employed by the MIM of interest in several human immune cell models. In the first part of the study, the effects of the actives from the MIM of interest were assessed from a molecular standpoint: the expression of HLA-II, interleukin (IL)-2, and the secretion of several other cytokines were evaluated. In addition, as mitochondrial metabolism is also involved in the inflammatory processes, the second part of the study aimed at assessing the effects of these actives on the mitochondrial reactive oxygen species (ROS) production and on the mitochondrial membrane potential. We showed that the tested actives decreased the expression of HLA-DR and HLA-DP in IFN-γ-stimulated endothelial cells and in LPS-treated-M1-macrophages. The tested MIM slightly reduced the intracellular expression of IL-2 in CD4+ and CD8+ T-cells isolated from PMA/Iono-stimulated human PBMCs. Additionally, while the secretion of IL-2, IL-10, and IFN-γ was diminished, the treatment increased IL-6, IL-9, and IL-17A, which may correspond to a "Th17-like" secretory pattern. Interestingly, in PMA/Iono-treated PBMCs, we reported that the treatment reduced the ROS production in B-cells. Finally, in PMA/Iono-treated human macrophages, we showed that the treatment slightly protected the cells from early cell death/apoptosis. Overall, these results provide data about the molecular and functional anti-inflammatory effects of several actives contained in the tested MIM in immune-related cells, and their impact on two mitochondria-related processes.

D-2-hydroxyglutarate supports a tolerogenic phenotype with lowered major histocompatibility class II expression in non-malignant dendritic cells and acute myeloid leukemia cells.

D-2-hydroxyglutarate (D-2-HG) accumulates in patients with acute myeloid leukemia (AML) with mutated isocitrate dehydrogenase (IDH) and in other malignancies. D-2-HG suppresses antitumor T-cell immunity but little is known about potential effects on non-malignant myeloid cells. Here we show that D-2-HG impairs human but not murine dendritic cell differentiation, resulting in a tolerogenic phenotype with low major histocompatibility class II expression. In line with this, IDH-mutated AML blasts exhibited lower expression of HLA-DP and were less susceptible to lysis by HLA-DP-specific T cells. Interestingly, besides its expected impact on DNA demethylation, D-2-HG reprogrammed metabolism towards increased lactate production in dendritic cells and AML. Vitamin C accelerated DNA demethylation, but only the combination of vitamin C and glycolytic inhibition lowered lactate levels and supported major histocompatibility complex class II expression. Our results indicate an unexpected link between the immunosuppressive metabolites 2-HG and lactic acid and suggest a potentially novel therapeutic strategy with combinations of anti-glycolytic drugs and epigenetic modulators (hypomethylating agents) or other therapeutics for the treatment of AML.

HLA-DPB1 genotype variants predict DP molecule cell surface expression and DP donor specific antibody binding capacity.

The contribution of alloresponses to mismatched HLA-DP in solid organ transplantation and hematopoietic stem cell transplantation (HCT) has been well documented. Exploring the regulatory mechanisms of DPB1 alleles has become an important question to be answered. In this study, our initial investigation focused on examining the correlation between the rs9277534G/A SNP and DPB1 mRNA expression. The result showed that there was a significant increase in DPB1 mRNA expression in B lymphoblastoid cell lines (BLCLs) with the rs9277534GG genotype compared to rs9277534AA genotype. In addition, B cells with the rs9277534GG exhibited significantly higher DP protein expression than those carrying the rs9277534AA genotype in primary B cells. Furthermore, we observed a significant upregulation of DP expression in B cells following treatment with Interleukin 13 (IL-13) compared to untreated B cells carrying rs9277534GG-linked DPB1 alleles. Fluorescence in situ hybridization (FISH) analysis of DPB1 in BLCL demonstrated significant differences in both the cytoplasmic (p=0.0003) and nuclear (p=0.0001) localization of DP mRNA expression comparing DPB1*04:01 (rs9277534AA) and DPB1*05:01 (rs9277534GG) homozygous cells. The study of the correlation between differential DPB1 expression and long non-coding RNAs (lncRNAs) showed that lnc-HLA-DPB1-13:1 is strongly associated with DP expression (r=0.85), suggesting the potential involvement of lncRNA in regulating DP expression. The correlation of DP donor specific antibody (DSA) with B cell flow crossmatch (B-FCXM) results showed a better linear correlation of DP DSA against GG and AG donor cells (R2 = 0.4243, p=0.0025 and R2 = 0.6172, p=0.0003, respectively), compared to DSA against AA donor cells (R2 = 0.0649, p=0.4244). This explained why strong DP DSA with a low expression DP leads to negative B-FCXM. In conclusion, this study provides evidence supporting the involvement of lncRNA in modulating HLA-DP expression, shedding lights on the intricate regulatory mechanisms of DP, particularly under inflammatory conditions in transplantation.

T cell receptor-engineered T cells derived from target human leukocyte antigen-DPB1-specific T cell can be a potential tool for therapy against leukemia relapse following allogeneic hematopoietic cell transplantation.

Human leukocyte antigen (HLA)-DPB1 antigens are mismatched in approximately 70% of allogeneic hematopoietic stem cell transplantations (allo-HSCT) from HLA 10/10 matched unrelated donors. HLA-DP-mismatched transplantation was shown to be associated with an increase in acute graft-versus-host disease (GVHD) and a decreased risk of leukemia relapse due to the graft-versus-leukemia (GVL) effect. Immunotherapy targeting mismatched HLA-DP is considered reasonable to treat leukemia following allo-HCT if performed under non-inflammatory conditions. Therefore, we isolated CD4+ T cell clones that recognize mismatched HLA-DPB1 from healthy volunteer donors and generated T cell receptor (TCR)-gene-modified T cells for future clinical applications. Detailed analysis of TCR-T cells expressing TCR from candidate clone #17 demonstrated specificity to myeloid and monocytic leukemia cell lines that even expressed low levels of targeted HLA-DP. However, they did not react to non-hematopoietic cell lines with a substantial level of targeted HLA-DP expression, suggesting that the TCR recognized antigenic peptide is only present in some hematopoietic cells. This study demonstrated that induction of T cells specific for HLA-DP, consisting of hematopoietic cell lineage-derived peptide and redirection of T cells with cloned TCR cDNA by gene transfer, is feasible when using careful specificity analysis.

HLA-DPA1*02:01~B1*01:01 is a risk haplotype for primary sclerosing cholangitis mediating activation of NKp44+ NK cells

ObjectivePrimary sclerosing cholangitis (PSC) is characterised by bile duct strictures and progressive liver disease, eventually requiring liver transplantation. Although the pathogenesis of PSC remains incompletely understood, strong associations with HLA-class...

Specificity of HLA monoclonal antibodies and their use to determine HLA expression on lymphocytes and peripheral blood stem cells.

HLA Class I and II expression are known to differ locus-to-locus, however, HLA expression on the cell-surface is frequently reported as the total amount of HLA Class I or II antigens. This is despite evidence that indicates the differential expression of HLA can influence patient outcomes post-transplantation. Although numerous commercially available HLA monoclonal antibodies (mAbs) exist to characterize HLA expression, there is currently a lack of detailed information regarding their reactivities to HLA specificities. The specificities of locus-specific HLA mAbs (nine Class I and four Class II mAbs) were evaluated by two solid-phase Luminex single antigen bead assays. The reactivity patterns of these mAbs were then confirmed by flow cytometry using lymphocytes and PBSCs (peripheral blood stem cells). Out of the 13 HLA mAbs tested, only four (one Class I and three Class II mAbs) displayed intra-locus reactivity without also reacting to inter-locus specificities. Epitope analysis revealed the presence of shared epitopes across numerous HLA loci, explaining much of the observed inter-locus reactivity. The specificity of the HLA mAbs seen in solid-phase assays was confirmed against PBSCs and lymphocytes by flow cytometry. Using this method, we observed differences in the cell surface expression of HLA-C, HLA-DR, HLA-DQ, and HLA-DP between PBSCs and lymphocytes. Our results emphasize the need to characterize the reactivity patterns of HLA mAbs using solid-phase assays before their use on cells. Through understanding the reactivity of these HLA mAbs, the cellular expression of HLA can be more accurately assessed in downstream assays.

HLA-DP on Epithelial Cells Enables Tissue Damage by NKp44+ Natural Killer Cells in Ulcerative Colitis

Ulcerative colitis (UC) is characterized by severe inflammation and destruction of the intestinal epithelium, and is associated with specific risk single nucleotide polymorphisms in HLA class II. Given the recently discovered interactions between subsets of HLA-DP molecules and the activating natural killer (NK) cell receptor NKp44, genetic associations of UC and HLA-DP haplotypes and their functional implications were investigated. HLA-DP haplotype and UC risk association analyses were performed (UC: n= 13,927; control: n= 26,764). Expression levels of HLA-DP on intestinal epithelial cells (IECs) in individuals with and without UC were quantified. Human intestinal 3-dimensional (3D) organoid cocultures with human NK cells were used to determine functional consequences of interactions between HLA-DP and NKp44. These studies identified HLA-DPA1∗01:03-DPB1∗04:01 (HLA-DP401) as a risk haplotype and HLA-DPA1∗01:03-DPB1∗03:01 (HLA-DP301) as a protective haplotype for UC in European populations. HLA-DP expression was significantly higher on IECs of individuals with UC compared with controls. IECs in human intestinal 3D organoids derived from HLA-DP401pos individuals showed significantly stronger binding of NKp44 compared with HLA-DP301pos IECs. HLA-DP401pos IECs in organoids triggered increased degranulation and tumor necrosis factor production by NKp44+ NK cells in cocultures, resulting in enhanced epithelial cell death compared with HLA-DP301pos organoids. Blocking of HLA-DP401-NKp44 interactions (anti-NKp44) abrogated NK cell activity in cocultures. We identified an UC risk HLA-DP haplotype that engages NKp44 and activates NKp44+ NK cells, mediating damage to intestinal epithelial cells in an HLA-DP haplotype-dependent manner. The molecular interaction between NKp44 and HLA-DP401 in UC can be targeted by therapeutic interventions to reduce NKp44+ NK cell-mediated destruction of the intestinal epithelium in UC.

Development of TCR-T cell therapy targeting mismatched HLA-DPB1 for relapsed leukemia after allogeneic transplantation.

Relapsed leukemia after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a significant challenge, with the re-emergence of the primary disease being the most frequent cause of death. Human leukocyte antigen (HLA)-DPB1 mismatch occurs in approximately 70% of unrelated allo-HSCT cases, and targeting mismatched HLA-DPB1 is considered reasonable for treating relapsed leukemia following allo-HSCT if performed under proper conditions. In this study, we established several clones restricted to HLA-DPB1*02:01, -DPB1*04:02, and -DPB1*09:01 from three patients who underwent HLA-DPB1 mismatched allo-HSCT using donor-derived alloreactive T cells primed to mismatched HLA-DPB1 in the recipient's body after transplantation. A detailed analysis of the DPB1*09:01-restricted clone 2A9 showed reactivity against various leukemia cell lines and primary myeloid leukemia blasts, even with low HLA-DP expression. T cell receptor (TCR)-T cells derived from clone 2A9 retained the ability to trigger HLA-DPB1*09:01-restricted recognition and lysis of various leukemia cell lines in vitro. Our study demonstrated that the induction of mismatched HLA-DPB1 specific T cell clones from physiologically primed post-allo-HSCT alloreactive CD4+ T cells and the redirection of T cells with cloned TCR cDNA by gene transfer are feasible as techniques for future adoptive immunotherapy.

Inhibited MHC Class I and MHC Class II antigen processing and presentation upon Sars-CoV-2 infection

Abstract Major histocompatibility (MHC) bound viral peptides are the immune signatures to initiate the selective and cross-reactive T cell responses. Identifying the antigenic targets of adaptive immunity to SARS-CoV-2 is a key towards understanding the pathogenesis of Sars-CoV-2 and the rapidly evolving mutants. We isolated the immunopeptidomes to identify naturally processed and presented MHC-II (HLA-DR, HLA-DP) and MHC-I (HLA-ABC) bound canonical and out-of-frame viral peptides. We identified 5 HLA-DR canonical epitopes (13 peptides) from spike protein, 2 HLA-DP canonical epitopes (4 peptides) from spike and membrane proteins, 1 canonical MHC-Class I peptides and 5 out-of-frame MHC-I peptides from different ORF proteins. The viral peptides were presented in lower abundances as compared to host peptides obscuring the identification of the MHC bound viral peptides. Some of these peptides were shown to recall T cell responses in COVID-19 donors in other reported studies. We observed the downregulation of surface and total HLA-ABC, HLA-DR and HLA-DP expression upon Sars-CoV-2 infection possibly restricting the presentation of viral peptides and therefore delaying the immune response. The mechanisms by which SARS-CoV-2 evades adaptive immune responses mediated by MHC-I are studied in detail by different groups. Hence, we further investigating the modulators of the MHC-II pathway using proteomics-based approach. We observed significant downregulation of CD74 protein, CIITA protein and cathepsins in infected cells affecting the processing and presentation of viral peptides. These results may help in understanding significantly altered antigen presentation in Sars-CoV-2 infected cells.

HLA-DR, -DP, -DQ expression status of parathyroid tissue as a potential parathyroid donor selection criteria and review of literature

BackgroundBasic and clinical studies about parathyroid allotransplantation have to be utilized with more definitive criteria for longer graft survival. Several reports demonstrated different isolation and cultivation methods for parathyroid cells to minimize their immunogenicity. In this study, we aim to compare and evaluate the clinical characteristics and the status of HLA class II expression changes in parathyroid tissue. MethodsA total of 22 parathyroid hyperplasia tissue donors was included in this study. Clinical characteristics were evaluated and compared with the HLA-DR, -DP, -DQ mRNA, and protein expression levels which were determined by qRT-PCR and Western blot. ResultsWe have compared the clinical characteristics (age, dialysis duration, frequency, recurrency of hyperparathyroidism and, calcimimetic usage) and HLA class II expression. HLA class II mRNA and protein levels showed varied expression patterns between tissues. Only, the HLA-DP has high mRNA expression levels without affecting the protein level when compared with the ages of the tissue donors. In addition, the HLA-DR, HLA-DP, and HLA-DQα1 protein expression levels showed a permanent and varied expression rate between tissues. ConclusionExpression of HLA class II molecules in parathyroid cells appears to constitute a decisive factor. Despite the lack of clinical outcomes, present data proposes new insight with a detailed understanding of parathyroid immunogenicity. In the future, randomized controlled clinical trials are needed for the accurate assessment of the effect of the varied HLA class II expression profiles in parathyroid tissue.

MP66-19 SURVEY OF B AND PLASMA CELLS IN BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology III (MP66)1 Sep 2021MP66-19 SURVEY OF B AND PLASMA CELLS IN BLADDER CANCER Yuanshuo Wang, Jorge Daza, Giuliana Magri, Emilie Grasset, Kristin Beaumont, Adam Farkas, Li Wang, Rachel Brody, Reza Mehrazin, Peter Wiklund, Robert Sebra, Jun Zhu, Matthew Galsky, Nina Bhardwaj, John Sfakianos, and Amir Horowitz Yuanshuo WangYuanshuo Wang More articles by this author , Jorge DazaJorge Daza More articles by this author , Giuliana MagriGiuliana Magri More articles by this author , Emilie GrassetEmilie Grasset More articles by this author , Kristin BeaumontKristin Beaumont More articles by this author , Adam FarkasAdam Farkas More articles by this author , Li WangLi Wang More articles by this author , Rachel BrodyRachel Brody More articles by this author , Reza MehrazinReza Mehrazin More articles by this author , Peter WiklundPeter Wiklund More articles by this author , Robert SebraRobert Sebra More articles by this author , Jun ZhuJun Zhu More articles by this author , Matthew GalskyMatthew Galsky More articles by this author , Nina BhardwajNina Bhardwaj More articles by this author , John SfakianosJohn Sfakianos More articles by this author , and Amir HorowitzAmir Horowitz More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002106.19AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Tumor infiltrating B cells in bladder cancer (BCa) have been associated with cancer invasion, but little is understood regarding their role in the tumor microenvironment. In this study, we analyzed single cell sequencing data from tumor (n=19) and PBMC (n=20) samples from 26 BCa patients to profile the heterogeneity of B cells and plasma cells (PCs). METHODS: Single cell data was imported into R (v4.03) using the SingleCellExperiment package, and outliers in mitochondrial gene percentage, library size, or detected genes were discarded. Normalization, deconvolution, and library size adjustment were done using the scran and batchelor R packages. Trajectory analysis was done using the Monocle R package. RESULTS: We identified 3,158 CD45+ naïve, non-switched memory (NSM), and switched memory (SM) B cells and PCs according to their marker gene expression (Fig 1). The majority of naïve and memory B cells were from PBMC, whereas plasma cells were primarily from tumor. To understand the heterogeneity of PCs, we performed clustering and trajectory analysis on 1,411 PCs and identified eight clusters (Fig 2A). Though most clusters consisted mainly of tumor cells, clusters 7 is almost exclusively from PBMC and is at the beginning of the pseudotime trajectory (Fig 2A). Its lower expression of PC markers such as MZB1, XBP1, and immunoglobulin genes suggest that it’s at an earlier stage of PC maturation (Fig 2A). Clusters 1, 6, and 8 contain PCs likely representative of mature plasmablasts, as they are at the end of the pseudotime trajectory and express high levels PC markers and immunoglobulin genes (Fig 2A). Lastly, cluster 4 upregulates expression of HLA-DP, HLA-DQ, and HLA-DR genes further on the pseudotime trajectory (Fig 2B). CONCLUSIONS: From our survey of B cells and PCs from a large single cell BCa cohort, we observed the majority of PCs to be found in the tumor. Tumor PCs demonstrate a high degree of heterogeneity, encompassing at least an early stage subset, a more mature plasmablast-like subset, and another subset with upregulated HLA expression with potential for T-cell stimulatory function. Source of Funding: institutional funds © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e1137-e1137 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yuanshuo Wang More articles by this author Jorge Daza More articles by this author Giuliana Magri More articles by this author Emilie Grasset More articles by this author Kristin Beaumont More articles by this author Adam Farkas More articles by this author Li Wang More articles by this author Rachel Brody More articles by this author Reza Mehrazin More articles by this author Peter Wiklund More articles by this author Robert Sebra More articles by this author Jun Zhu More articles by this author Matthew Galsky More articles by this author Nina Bhardwaj More articles by this author John Sfakianos More articles by this author Amir Horowitz More articles by this author Expand All Advertisement Loading ...

Rapid generation of monocyte-derived antigen-presenting cells with dendritic cell-like properties.

One of the major challenges in cellular therapy is the establishment and validation of simple and fast production protocols meeting good manufacturing practice (GMP) requirements. Dendritic cells (DCs) are of particular therapeutic interest, due to their critical role in T cell response initiation and regulation. Conventional wisdom states that DC generation from monocytes is a time-consuming protocol, taking up to 7-9 days. This study systematically screened and validated numerous culture components and conditions to identify the minimal requirements, which can give rise to functional monocyte-derived antigen-presenting cells (MoAPCs) in less than 48 h (36 h MoAPC). A total of 36 h MoAPCs were evaluated in terms of surface marker expression, endocytic capability, and induction of antigen-specific T cell expansion via flow cytometry. Screening of media compositions, glucose concentrations, and surface marker kinetics, particularly DC-SIGN as a DC-specific marker, allowed the generation of DC-like APCs in 36 h (36 h MoAPCs). A total of 36 h MoAPCs displayed a similar phenotype to 48 h MoAPC and standard 7 d MoDCs in terms of HLA-DP,DQ,DR, CD83, and DC-SIGN expression, while CD1a was preferentially expressed in standard MoDCs. Functional evaluation revealed that 36 h MoAPCs displayed reduced endocytosis capabilities and IL-12p70 production. However, 36 h MoAPCs were able to induce T cell expansion both in an allogenic and antigen-specific setting. Our results indicate that mature 36 h MoAPCs possess DC-like capabilities by inducing antigen-specific T cell responses. This study has important implications for the generation of DC-based cellular therapies, allowing a more cost and time-efficient generation of APCs.

Role of HLA-DP Expression in Graft-Versus-Host Disease After Unrelated Donor Transplantation.

The main aim of this study was to evaluate the significance of HLA-DPB1 expression in acute graft-versus-host disease (GVHD) after hematopoietic cell transplantation (HCT) from HLA-A, -B, -C, -DRB1, -DQB1-matched and -mismatched unrelated donors. Between January 1, 2017, and January 10, 2019, we assessed 19,136 patients who received HCT from an HLA-A, -B, -C, -DRB1, -DQB1-matched or -mismatched unrelated donor performed in Australia, the European Union, Japan, North America, and the United Kingdom between 1988 and 2016. Among transplant recipients with one HLA-DPB1 mismatch, the patient's mismatched HLA-DPB1 allotype was defined as low or high expression. Multivariable regression models were used to assess risks of GVHD associated with high expression relative to low expression HLA-DPB1 mismatches. The effect of increasing numbers of HLA-DPB1 mismatches on clinical outcome was assessed in HLA-mismatched transplant recipients. In HLA-A, -B, -C, -DRB1,-DQB1-matched transplant recipients, donor mismatching against one high-expression patient HLA-DPB1 increased moderate (odds ratio [OR], 1.36; P = .001) and severe acute GVHD (OR, 1.32; P = .0016) relative to low-expression patient mismatches, regardless of the expression level of the donor's mismatched HLA-DPB1. Among transplant recipients with one HLA-A, -B, -C, -DRB1, or -DQB1 mismatch, the odds of acute GVHD increased with increasing numbers of HLA-DPB1 mismatches (OR, 1.23 for one; OR, 1.40 for two mismatches relative to zero mismatches for moderate GVHD; OR, 1.19 for one; OR, 1.40 for two mismatches relative to zero for severe GVHD), but not with the level of expression of the patient's mismatched HLA-DPB1 allotype. The level of expression of patient HLA-DPB1 mismatches informs the risk of GVHD after HLA-A, -B, -C, -DRB1, -DQB1-matched unrelated HCT, and the total number of HLA-DPB1 mismatches informs the risk of GVHD after HLA-mismatched unrelated HCT. Prospective consideration of HLA-DPB1 may help to lower GVHD risks after transplantation.

Expression of HLA-DP in patients with neuromyelitis optica spectrum disorders

Objective: To investigate the effect of HLA-DP gene expression on the susceptibility and disease status of neuromyelitis optica spectrum disorders (NMOSD). Methods: A total of 86 NMOSD patients (52 in acute phase and 34 in remission phase), 52 multiple sclerosis (MS) patients (20 in acute phase and 32 in remission phase) diagnosed in Department of Neurology, the Third Affiliated Hospital of Sun Yat-sen University and 29 healthy controls were enrolled prospectively. Genotyping of HLA-DP was performed. The expression levels of HLA-DP molecules in peripheral blood B cells and monocytes were measured by flow cytometry. The transcription levels of HLA-DPB1 mRNA in peripheral blood mononuclear cells (PBMC) were measured by real time-PCR. The results were compared among different groups Results: There was no statistically significant difference of the distributions of HLA-DPB1*0501/HLA-DPB1*0501, HLA-DPB1*0501/X and X/X genotypes and the frequencies of allele of HLA-DPB1*0501 among NMOSD, MS patients and healthy controls (P=0.96 and 0.71, respectively). The expression levels of HLA-DP on the surface of B cells in NMOSD patients, especially in remission phase patients, were significantly higher than those in healthy controls(212±328 and 374±394 vs 55±57, P=0.049 and 0.002, respectively). The expression levels of HLA-DP on the surface of monocytes in NMOSD patients in acute phase were significantly higher than those in healthy controls(158±175 vs 65±90, P=0.025). The transcription levels of PMBC HLA-DPB1 mRNA in acute phase and remission phase of NMOSD patients were significantly higher than those in healthy controls (3.0±1.4 and 2.9±1.3 vs 1.5±1.4, P=0.000 and 0.003, respectively). The expression levels of HLA-DP molecules on the surface of peripheral blood B cells and monocytes and the transcription levels of PMBC HLA-DPB1 mRNA in MS patients at the acute and remission stages were not significantly different from those in healthy controls. The expression levels of HLA-DP molecules on the surface of B cells in patients with HLA-DPB1*0501/HLA-DPB1*0501, HLA-DPB1*0501/X and X/X genotypes were statistically different (P=0.017). Conclusion: HLA-DP gene transcription and molecular expression levels in antigen presenting cells may affect the susceptibility and disease status of NMOSD patients, while HLA-DPB1*0501 allele may affect the transcription and molecular expression levels of HLA-DP gene in antigen presenting cells.

Genome-Wide Association Study Identifies an Immune-Related Etiology for Severe Aplastic Anemia

Introduction. Acquired severe aplastic anemia (SAA) is a life-threatening disorder characterized by severe progressive pancytopenia and hypocellular bone marrow. The etiology of acquired SAA is not understood but believed to be related to abnormal immune responses to environmental exposures. We conducted a genome-wide association study (GWAS) to identify common germline variants associated with SAA. Methods. We identified 895 patients with SAA who underwent related or unrelated hematopoietic cell transplant (HCT) with clinical data and pre-HCT blood samples available in the Center for International Blood and Marrow Transplant Research (CIBMTR) database and biorepository. Pre-HCT DNA was extracted from blood of patients with SAA and genome-wide genotyping was conducted using the Illumina OmniExpress array. We excluded 93 inherited bone marrow failure cases. The SAA cases were grouped cases into discovery and validation sets based on time of batch sample receipt. Analyses were limited to patients of European ancestry based on principal component analyses to minimize the potential effect of population stratification. Controls were genomically matched and selected from previously scanned cancer-free subjects at the Cancer Genomics Research Laboratory, Division of Cancer Epidemiology and Genetics, NCI. The final analysis included 534 acquired SAA cases (359 in the discovery set and 175 in the validation set), and 2,455 controls (1,396 in the discovery set, and 1,059 in the validation set). Results. Patients with SAA in this study received HCT between 1989-2015 at a median age of 21 years, 56% were male, and the median time between SAA diagnosis and HCT was 11 months. Strong genome-wide association signals were identified across the human leukocyte antigen (HLA) genes encoded at the major histocompatibility complex (MHC) on chromosome 6p21 (Figure 1). The top SNP was located in the P4 binding pocket of the HLA class II gene HLA-DPB1(rs1042151A>G, p.Met105Val, pooled-odds ratio [OR]=1.75, 95% confidence interval [CI]=1.50-2.03, p=1.94x10-13). The expression of HLA-DP in CD19+ cells from 175 healthy donors was significantly different by rs1042151 A>G genotype (p=2.04x10-6) (Figure 2). A second SNP near HLA-B, rs28367832G>A, also reached genome-wide significance (pooled-OR=1.49, 95% CI=1.22-1.78, p=7.27x10-9). Copy-number variant analysis and next generation sequencing also identified somatic, clonal copy-neutral loss-of-heterozygosity affecting class I HLA genes in 8.6% of the SAA cases and none of the controls. Conclusion. This SAA GWAS identified strong association signals between common germline genetic variants in HLA class I and II genes and SAA. The main SNP is associated with changes in HLA-DP expression suggesting a key role for this locus in SAA etiology. This study adds further evidence to the connection between SAA and immune dysregulation. Disclosures Cerhan: Janssen: Membership on an entity's Board of Directors or advisory committees; NanoString: Research Funding; Celgene: Research Funding. Lee:Incyte: Research Funding; Syndax: Research Funding; Amgen: Research Funding; Novartis: Research Funding; Takeda: Research Funding; Kadmon: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; AstraZeneca: Research Funding.

Characterization of Magnitude and Antigen Specificity of HLA-DP, DQ, and DRB3/4/5 Restricted DENV-Specific CD4+ T Cell Responses.

Background: Dengue Virus (DENV) associated disease is a major public health problem. Assessment of HLA class II restricted DENV-specific responses is relevant for immunopathology and definition of correlates of protection. While previous studies characterized responses restricted by the HLA-DRB1 locus, the responses associated with other class II loci have not been characterized to date. Accordingly, we mapped HLA-DP, DQ, and DRB3/4/5 restricted DENV-specific CD4 T cell epitopes in PBMCs derived from the DENV endemic region Sri Lanka.Methods: We studied 12 DP, DQ, and DRB3/4/5 alleles that are commonly expressed and provide worldwide coverage >82% for each of the loci analyzed and >99% when combined. CD4+ T cells purified by negative selection were stimulated with pools of HLA-predicted binders for 2 weeks with autologous APC. Epitope reactive T cells were enumerated using IFNγ ELISPOT assay. This strategy was previously applied to identify DRB1 restricted epitopes. In parallel, membrane expression levels of HLA-DR, DP, and DQ proteins was assessed using flow cytometry.Results: Epitopes were identified for all DP, DQ, and DRB3/4/5 allelic variants albeit with magnitudes significantly lower than the ones previously observed for the DRB1 locus. This was in line with lower membrane expression of HLA-DP and DQ molecules on the PBMCs tested, as compared to HLA-DR. Significant differences between loci were observed in antigen immunodominance. Capsid responses were dominant for DRB1/3/4/5 and DP alleles but negligible for the DQ alleles. NS3 responses were dominant in the case of DRB1/3/4/5 and DQ but absent in the case of DP. NS1 responses were prominent in the case of the DP alleles, but negligible in the case of DR and DQ. In terms of epitope specificity, repertoire was largely overlapping between DRB1 and DRB3/4/5, while DP and DQ loci recognized largely distinct epitope sets.Conclusion: The HLA-DP, DQ, and DRB3/4/5 loci mediate DENV-CD4 specific immune responses of lower magnitude as compared to HLA-DRB1, consistent with their lower levels of expression. The responses are associated with distinct and characteristic patterns of immunodominance, and variable epitope overlap across loci.

Identification of the rs9277534 HLA-DP expression marker by next generation sequencing for the selection of unrelated donors for hematopoietic cell transplantation

Mismatching of an unrelated donor against a high-expression HLA-DPB1 recipient allele is associated with a high risk of graft-versus-host disease and mortality. The Seattle Cancer Care Alliance (SCCA) and Fred Hutchinson Cancer Research Center transplant program employs an algorithm to match for HLA-A, B, C, DRB1, DQB1 and DPB1 alleles (12/12) and to avoid, whenever possible, donor mismatching against a recipient high-expression HLA-DPB1 allele. HLA-DPB1 expression is associated with the rs9277534 A/G polymorphism located in the 3′UTR of the HLA-DPB1 gene. Next generation sequencing of HLA-DPB1 using the Illumina TruSight HLA V2 Sequencing Panel and Conexio Assign software analyses provides information on rs9277534 variants without the need for any additional SNP testing. Here we present the molecular location of rs9277534 in NGS data and discuss the challenges to resolve HLA-DPB1 ambiguities.