Expression Of Human Leukocyte Antigen Class Research Articles

Human leukocyte antigen (HLA) class I expression on Hodgkin-Reed-Sternberg cells is an EBV-independent major determinant of microenvironment composition in classic Hodgkin lymphoma.

Hodgkin-Reed-Sternberg cells (HRSCs) in classic Hodgkin Lymphoma (HL) frequently lack expression of human leukocyte antigen class I (HLA-I), considered to hamper activation of cytotoxic T cells in the tumor microenvironment (TME). Here, we demonstrate HLA-I expression on HRSCs to be a strong determinant of TME composition whereas expression of HLA-II was associated with only minor differential gene expression in the TME. In HLA-I-positive HL the HRSC content and expression of CCL17/TARC in HRSCs are low, independent of the presence of Epstein-Barr virus in HRSCs. Additionally, HLA-I-positive HL shows a high content of CD8+ cytotoxic T cells. However, an increased expression of the inhibitory immune checkpoint LAG3 on CD8+ T cells in close proximity to HRSCs is observed. Suggesting interference with cytotoxic activity, we observed an absence of clonally expanded T cells in the TME. While HLA-I-positive HL is not associated with an unfavorable clinical course in our cohorts, they share features with the recently described H2 subtype of HL. Given the major differences in TME composition, immune checkpoint inhibitors may differ in their mechanism of action in HLA-I-positive compared to HLA-I-negative HL.

Mesenchymal Stromal Cells Increase the Natural Killer Resistance of Circulating Tumor Cells via Intercellular Signaling of cGAS-STING-IFNβ-HLA.

Circulating tumor cells (CTCs) shed from primary tumors must overcome the cytotoxicity of immune cells, particularly natural killer (NK) cells, to cause metastasis. The tumor microenvironment (TME) protects tumor cells from the cytotoxicity of immune cells, which is partially executed by cancer-associated mesenchymal stromal cells (MSCs). However, the mechanisms by which MSCs influence the NK resistance of CTCs remain poorly understood. This study demonstrates that MSCs enhance the NK resistance of cancer cells in a gap junction-dependent manner, thereby promoting the survival and metastatic seeding of CTCs in immunocompromised mice. Tumor cells crosstalk with MSCs through an intercellular cGAS-cGAMP-STING signaling loop, leading to increased production of interferon-β (IFNβ) by MSCs. IFNβ reversely enhances the type I IFN (IFN-I) signaling in tumor cells and hence the expression of human leukocyte antigen class I (HLA-I) on the cell surface, protecting the tumor cells from NK cytotoxicity. Disruption of this loop reverses NK sensitivity in tumor cells and decreases tumor metastasis. Moreover, there are positive correlations between IFN-I signaling, HLA-I expression, and NK tolerance in human tumor samples. Thus, the NK-resistant signaling loop between tumor cells and MSCs may serve as a novel therapeutic target.

The HLA-I landscape confers prognosis and antitumor immunity in breast cancer.

Breast cancer is a highly heterogeneous disease with varied subtypes, prognoses and therapeutic responsiveness. Human leukocyte antigen class I (HLA-I) shapes the immunity and thereby influences the outcome of breast cancer. However, the implications of HLA-I variations in breast cancer remain poorly understood. In this study, we established a multiomics cohort of 1156 Chinese breast cancer patients for HLA-I investigation. We calculated four important HLA-I indicators in each individual, including HLA-I expression level, somatic HLA-I loss of heterozygosity (LOH), HLA-I evolutionary divergence (HED) and peptide-binding promiscuity (Pr). Then, we evaluated their distribution and prognostic significance in breast cancer subtypes. We found that the four breast cancer subtypes had distinct features of HLA-I indicators. Increased expression of HLA-I and LOH were enriched in triple-negative breast cancer (TNBC), while Pr was relatively higher in hot tumors within TNBCs. In particular, a higher Pr indicated a better prognosis in TNBCs by regulating the infiltration of immune cells and the expression of immune molecules. Using the matched genomic and transcriptomic data, we found that mismatch repair deficiency-related mutational signature and pathways were enriched in low-Pr TNBCs, suggesting that targeting mismatch repair deficiency for synthetic lethality might be promising therapy for these patients. In conclusion, we presented an overview of HLA-I indicators in breast cancer and provided hints for precision treatment for low-Pr TNBCs.

Prognostic impact of the bone marrow tumor microenvironment, HLA-I and HLA-Ib expression in MDS and CMML progression to sAML

ABSTRACT Genetic aberrations and immune escape are fundamental in MDS and CMML initiation and progression to sAML. Therefore, quantitative and spatial immune cell organization, expression of immune checkpoints (ICP), classical human leukocyte antigen class I (HLA-I) and the non-classical HLA-Ib antigens were analyzed in 274 neoplastic and 50 non-neoplastic bone marrow (BM) biopsies using conventional and multiplex immunohistochemistry and correlated to publicly available dataset. Higher numbers of tissue infiltrating lymphocytes (TILs) were found in MDS/CMML (8.8%) compared to sAML (7.5%) and non-neoplastic BM (5.3%). Higher T cell abundance, including the CD8+ T cell subset, inversely correlated with the number of pathogenic mutations and was associated with blast BM counts, ICP expression, spatial T cell distribution and improved patients’ survival in MDS and CMML. In MDS/CMML, higher PD-1/PD-L1/PD-L2 and HLA-I, but lower HLA-G expression correlated with a significantly better patients’ outcome. Moreover, a closer spatial proximity of T cell subpopulations and their proximity to myeloid blasts showed a stronger prognostic impact when compared to TIL numbers. In sAML – the continuum of MDS and CMML – the number of TILs had no impact on prognosis, but higher CD28 and HLA-I expression correlated with a better outcome of sAML patients. This study underlines the independent prognostic value of the tumor microenvironment in MDS/CMML progression to sAML, which shows the most pronounced immune escape. Moreover, new prognostic markers, like HLA-G expression and spatial T cell distribution, were described for the first time, which might also serve as therapeutic targets.

CSPG4 CAR-redirected Cytokine Induced Killer lymphocytes (CIK) as effective cellular immunotherapy for HLA class I defective melanoma

BackgroundEven acknowledging the game-changing results achieved in the treatment of metastatic melanoma with the use of immune checkpoint inhibitors (ICI), a large proportion of patients (40–60%) still fail to respond or relapse due to the development of resistance. Alterations in the expression of Human Leukocyte Antigen class I (HLA-I) molecules are considered to play a major role in clinical resistance to ICI. Cellular immunotherapy with HLA-independent CAR-redirected lymphocytes is a promising alternative in this challenging setting and dedicated translational models are needed.MethodsIn this study, we propose an HLA-independent therapeutic strategy with Cytokine Induced Killer lymphocytes (CIK) genetically engineered with a Chimeric Antigen Receptor (CAR) targeting the tumor antigen CSPG4 as effector mechanism. We investigated the preclinical antitumor activity of CSPG4-CAR.CIK in vitro and in a xenograft murine model focusing on patient-derived melanoma cell lines (Mel) with defective expression of HLA-I molecules.ResultsWe successfully generated CSPG4-CAR.CIK from patients with metastatic melanoma and reported their intense activity in vitro against a panel of CSPG4-expressing patient-derived Mel. The melanoma killing activity was intense, even at very low effector to target ratios, and not influenced by the expression level (high, low, defective) of HLA-I molecules on target cells. Furthermore, CAR.CIK conditioned medium was capable of upregulating the expression of HLA-I molecules on melanoma cells. A comparable immunomodulatory effect was replicated by treatment of Mel cells with exogenous IFN-γ and IFN-α. The antimelanoma activity of CSPG4-CAR.CIK was successfully confirmed in vivo, obtaining a significant tumor growth inhibition of an HLA-defective Mel xenograft in immunodeficient mice.ConclusionsIn this study we reported the intense preclinical activity of CSPG4-CAR.CIK against melanoma, including those with low or defective HLA-I expression. Our findings support CSPG4 as a valuable CAR target in melanoma and provide translational rationale for clinical studies exploring CAR-CIK cellular immunotherapies within the challenging setting of patients not responsive or relapsing to immune checkpoint inhibitors.

T-Cell Mediated Immune Rejection of Beta-2-Microglobulin Knockout Induced Pluripotent Stem Cell-Derived Kidney Organoids.

Immune evasive induced pluripotent stem cell (iPSC)-derived kidney organoids, known as "stealth" organoids, hold promise for clinical transplantation. To address immune rejection, we investigated the impact of genetically modifying human leukocyte antigen (HLA) class I in kidney organoids prior to transplantation. By using CRISPR-Cas9, we successfully knocked out beta-2-microglobulin (B2M), resulting in iPSCs devoid of HLA class I surface expression. In vitro, the B2M knockout protected kidney organoids derived from these iPSCs against T-cell rejection. To assess in vivo protection, unmodified (control) and B2M-/- kidney organoids were transplanted into humanized mice engrafted with human peripheral blood mononuclear cells (PBMCs). Successful engraftment of human PBMCs was confirmed, and after 4 weeks, we observed no discernible difference in the infiltration rate, proliferation, or cytotoxicity of CD4+ and CD8+ T cells between control and B2M-/- organoids. Both groups of organoids showed compromised tissue integrity, displaying tubulitis and loss of tubule integrity. Notably, while B2M-/- organoids failed to express HLA class I on their cell surface, there was preexisting expression of HLA class II in both control and B2M-/- organoids transplanted into mice with human PBMCs. HLA class II expression was not limited to antigen-presenting cells but also evident in epithelial cells of the kidney organoid, posing an additional immunological challenge to its transplantation. Consequently, we conclude that B2M knockout alone is insufficient to protect iPSC-derived kidney organoids from T-cell-mediated immune rejection. Additionally, our findings suggest that modulating HLA class II signaling will be necessary to prevent rejection following transplantation.

The role of the interferon/JAK-STAT axis in driving islet HLA-I hyperexpression in type 1 diabetes.

The hyperexpression of human leukocyte antigen class I (HLA-I) molecules on pancreatic beta-cells is widely accepted as a hallmark feature of type 1 diabetes pathogenesis. This response is important clinically since it may increase the visibility of beta-cells to autoreactive CD8+ T-cells, thereby accelerating disease progression. In this review, key factors which drive HLA-I hyperexpression will be explored, and their clinical significance examined. It is established that the presence of residual beta-cells is essential for HLA-I hyperexpression by islet cells at all stages of the disease. We suggest that the most likely drivers of this process are interferons released from beta-cells (type I or III interferon; possibly in response to viral infection) or those elaborated from influent, autoreactive immune cells (type II interferon). In both cases, Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathways will be activated to induce the downstream expression of interferon stimulated genes. A variety of models have highlighted that HLA-I expression is enhanced in beta-cells in response to interferons, and that STAT1, STAT2 and interferon regulatory factor 9 (IRF9) play key roles in mediating these effects (depending on the species of interferon involved). Importantly, STAT1 expression is elevated in the beta-cells of donors with recent-onset type I diabetes, and this correlates with HLA-I hyperexpression on an islet-by-islet basis. These responses can be replicated in vitro, and we consider that chronically elevated STAT1 may have a role in maintaining HLA-I hyperexpression. However, other data have highlighted that STAT2-IRF9 may also be critical to this process. Thus, a better understanding of how these factors regulate HLA-I under chronically stimulated conditions needs to be gathered. Finally, JAK inhibitors can target interferon signaling pathways to diminish HLA-I expression in mouse models. It seems probable that these agents may also be effective in patients; diminishing HLA-I hyperexpression on islets, reducing the visibility of beta-cells to the immune system and ultimately slowing disease progression. The first clinical trials of selective JAK inhibitors are underway, and the outcomes should have important implications for type 1 diabetes clinical management.

HLA Class I Expression Is Associated with DNA Damage and Immune Cell Infiltration into Dysplastic and Neoplastic Lesions in Ulcerative Colitis.

Human leukocyte antigen class I (HLA-I) is considered a genetic pathogen for ulcerative colitis (UC). This study aimed to investigate the significance of DNA damage and HLA-I expression in infiltrating immune cells and immune checkpoint protein PD-L1 expression in dysplasia/colitic cancer (CC) and sporadic colorectal cancer (SCRC). We performed immunohistochemical staining for HLA-I, PD-L1, γH2AX (DNA damage marker), and immune cell markers such as CD8, FOXP3, CD68, and CD163 (in surgically resected specimens from 17 SCRC patients with 12 adjacent normal mucosa (NM) and 9 UC patients with 18 dysplasia/CC tumors. The ratio of membrane HLA-I-positive epithelial cells in UC and dysplasia/CC tissues was significantly higher than that in NM and SCRC. High HLA-I expression in dysplasia/CC was associated with high positivity of γH2AX and PD-L1 expression compared to SCRC. The infiltration of CD8-positive T cells and CD68-positive macrophages in HLA-I-high dysplasia/CC was significantly higher than in UC and SCRC. Dysplasia/CC specimens with DNA damage exhibited high levels of HLA-I-positive epithelial cells with high CD8- and CD68-positive immune cell infiltration compared to UC and SCRC specimens. Targeting DNA damage in UC may regulate immune cell infiltration, immune checkpoint proteins, and carcinogenesis by modulating DNA damage-induced HLA-I antigen presentation.

136-OR: The MDA5627X Variant Is Associated with Type 1 Diabetes Protection via Attenuated Inflammatory Antiviral Responses

Type 1 Diabetes (T1D) is an autoimmune disease characterized by destruction of pancreatic β cells. Genetics and environmental factors play crucial roles in T1D development. Four single nucleotide polymorphisms in IFIH1, encoding the viral sensor MDA5 have been linked to T1D. MDA5 promotes anti-viral responses and may link genetics with environmental factors to promote or suppress T1D. A stop-gain mutation in IFIH1 (rs35744605) results in a non-functional MDA5 protein (MDA5627X). We hypothesized that MDA5627X protects against T1D by dampening inflammatory anti-viral responses resulting in reduced autoimmunity. We gene edited human induced pluripotent stem cells at rs35744605 using a CRISPR/Cas9 strategy to generate iPSC encoding the stop-gain variant (MDA5627X). Macrophages (Mφ) have been demonstrated to be indispensable for T1D-initiation and regulate anti-viral responses. We differentiated iPSCs into Mφ and studies responses to high molecular weight Poly I:C to mimic viral infection. The outcome measures were Type 1 Interferon (IFN1) production, antigen presentation, and immune activation markers. Treatment of MDA5 intact Mφ with Poly I:C resulted in 60-fold increase in IFN1 production and a 3-fold elevation in human leukocyte antigen Class I (HLA-I) expression. In contrast, treated MDA5627X Mφ failed to increase IFN1 or HLA-I at any Poly I:C dose. While Poly I:C did not increase HLA-I expression, untreated MDA5627X Mφ had elevated HLA-1 compared to MDA5 intact Mφ (P=0.0306). Also, we assessed the expression of CD80, CD86, CD40, and MHC-II, however there were no differences in the expression between Mφ with intact MDA5 or MDA5627X. In contrast, IFNg and LPS treatment elevated expression of CD80, CD86, CD40, and MHC-II in both Mφ populations. In conclusion, our data point to a protective effect of the MDA5627X variant in T1D through decreased inflammatory IFN1 levels and T cell activation by Mφ. Disclosure O.Iwaloye: None. L.H.Armitage: None. A.M.Meacham: None. C.E.Mathews: None. Funding National Institutes of Health (R01DK127497, P01AI042288, UH3DK122638)

Immunomodulatory effect of human dedifferentiated fat cells: comparison with adipose-derived stem cells.

Dedifferentiated fat cells (DFATs), which are originated by the dedifferentiation of adipocytes, display surface markers of mesenchymal stem cells and are able to differentiate into different cell types, thus, yielding a huge therapeutic potential in repairing damaged tissues and organs. The use of allogeneic stem cells from healthy donors constitutes the basis of a new strategy for cell therapy in the field of transplantation and the first requirement for allografts is determining their immunological properties. In this study, human DFATs and ADSCs were passaged as in vitro models to investigate their immunomodulatory effects. Phenotypic analysis of cell surface markers and three-line differentiation protocols were used to identify stem cells. The immunogenic phenotypes of DFATs and ADSCs were analyzed by flow cytometry and a mixed lymphocyte reaction was used to assess their immune function. The characteristics of stem cells were confirmed by phenotypic identification of cell surface markers and three-line differentiation. Flow cytometry analysis showed that P3 generation DFATs and ADSCs contained human leukocyte antigen (HLA) class I molecules, but did not express HLA class II molecules and costimulatory molecules CD40, CD80 and CD86. Moreover, allogeneic DFATs and ADSCs could not induce the proliferation of peripheral blood mononuclear cells (PBMCs). In addition, both populations were shown to inhibit the Concanavalin A-stimulated proliferation of PBMCs and act as third-party cells responsible for inhibiting the mixed lymphocyte response. DFATs have immunosuppressive properties similar to ADSCs. Based on this, allogeneic DFATs have potential applications in tissue repair or cell therapy.

Combination of Expanded Allogeneic NK Cells and T Cell-Based Immunotherapy Exert Enhanced Antitumor Effects.

Immunotherapies based on immune checkpoint blockade, neoantigen-reactive tumor-infiltrating lymphocytes and T cell receptor-engineered T cells (TCR-T) have achieved favorable clinical outcomes in tumor treatment. However, sustained immune response and tumor regression have been observed only in a few patients due to immune escape. Natural killer (NK) cells can mediate direct tumor lysis and target cancer cells with low or no expression of human leukocyte antigen class I (HLA-I) that are no longer recognized by T cells during immune escape. Therefore, the combination of T cell-based immunotherapy and NK cell therapy is a promising strategy for improving antitumor response and response rate. However, allogeneic NK cells for adoptive cell therapy have been limited by both the required cell number and quality. Here, we developed an efficient manufacturing system that relies on genetically modified K562 cells for the expansion of high-quality NK cells derived from peripheral blood mononuclear cells. NK cells with the optimal expansion and activity were identified by comparing the different culture systems. Furthermore, we demonstrated that the cooperation of NK cells with tumor-reactive T cells or with NY-ESO-1-specific TCR-T cells further enhanced tumors lysis, especially against tumors with downregulated HLA-I expression. The advantages of HLA-mismatch and non-rejection by other allogeneic immune cells demonstrated the potential of "off-the-shelf" NK cells with the capacity to target tumors for immunotherapy. Our results indicate that the combination strategy based on T cell and allogeneic NK cell immunotherapy might have potential for overcoming the barrier of immune incompetence caused by HLA-I downregulation.

Immunohistochemical Analysis of Common Cancer Antigens in Head and Neck Squamous Cell Carcinoma

The prognosis of recurring and metastatic head and neck squamous cell carcinoma (HNSCC) is poor. Although immune checkpoint inhibitors have expanded the treatment options for HNSCC, the response rates are low. Alternatively, cancer vaccines and T-cell therapies are being developed. Identification of useful common cancer antigens and confirmation of human leukocyte antigen (HLA) class I expression are required. Immunohistochemistry analyses were performed for 10 antigens (FOXM1, TGFBI, SPARC, HSP105α, WT1, AFP, GPC3, PP-RP, KIF20A, KM-HN-1) and HLA class I using specimens of 56 surgical cases. Staining intensity, percentage of stain-positive areas, and localization of staining in the tumor cells and normal tissue were evaluated. Staining of FOXM1, TGFBI, SPARC, and HSP105α was more predominant in tumor cells than that in normal cells. The expression rates of these antigens in tumor cells were 60.7%, 58.9%, 73.2%, and 50.0%, respectively. Regarding sites, the expression rates of these antigens in oral cancer were high at 57.1%, 71.4%, 81.0%, and 66.7%, respectively. Furthermore, the expression of HLA class I was 83.9% in all cases. Of these, 68.1% showed expression on the plasma membrane. FOXM1, TGFBI, SPARC, and HSP105α could be useful common cancer antigens, and HLA class I is expressed on the plasma membrane of cancer cells in many cases. The results suggest that cancer vaccines and T-cell therapy may be clinically viable options for HNSCC treatment.

Expression of HLA class I is associated with immune cell infiltration and patient outcome in breast cancer

Downregulation of human leukocyte antigen (HLA) class I is one mechanism of escaping anti-tumor immunity by tumor cells. This study was conducted to compare HLA class I expression in ductal carcinoma in situ (DCIS) and invasive breast carcinoma (IBC) and to evaluate its association with immune cell infiltration of the tumors and clinical outcome of the patients. A total of 830 cases comprising 288 DCIS and 542 IBC were included in this study. Immunohistochemistry for HLA class I expression was performed using HLA-ABC in tissue microarrays and was analyzed in relation to clinicopathologic characteristics of tumors and infiltration of CD4+, CD8+, and FOXP3+ tumor-infiltrating lymphocyte (TIL) subsets and PD-L1+ immune cells. As a whole, there was no difference in HLA class I expression between DCIS and IBC when dichotomized into high or low expression. However, in the HR-negative group, a high level of HLA class I expression was more frequent in IBC than DCIS. On the contrary, in the HR-positive group, a complete loss of HLA class I expression was more frequently observed in IBC than DCIS. High HLA class I expression level was generally associated with aggressive clinicopathologic features of IBC and was associated with high CD4+, CD8+, and FOXP3+ TIL and PD-L1+ immune cell infiltration in both DCIS and IBC. In survival analyses, HLA class I expression was not associated with clinical outcome in DCIS and IBC as a whole; however, low HLA class I expression was associated with poor clinical outcome in HR-negative IBC, especially in triple-negative subtype. In conclusion, this study showed that HLA class I expression increased in association with increased immune cell infiltration during in situ to invasive transition of HR-negative breast cancer, and HLA class I down-regulation had a prognostic value in HR-negative breast cancer.

Effects of tumor-infiltrating CD8+ T cells, PD1/PD-L1 axis, and expression patterns of HLA class I on the prognosis of patients with malignant pleural mesothelioma who underwent extra-pleural pneumonectomy.

Programmed cell death protein-1 (PD1), PD1 ligand 1 (PD-L1), and human leukocyte antigen (HLA) class I molecule play pivotal roles in T cell-induced anti-tumor immunity; however, the clinical impact of these parameters in resected malignant pleural mesothelioma (MPM) cases is unknown. We immunohistochemically evaluated the tumor infiltrated lymphocytes (TILs), PD1/PD-L1 axis, and expression of HLA class I in resected specimens from 58 patients with MPM who underwent extra-pleural pneumonectomy (EPP). Higher infiltration of CD3-TIL, CD8-TIL, and PD1-TIL, loss of HLA class I, and overexpression of PD-L1 by tumor cells (PD-L1 TC) or immune cells (PD-L1 IC) were observed in 34 (58.6%), 27 (46.6%), 41 (70.7%), 45 (77.6%), 29 (50.0%), and 33 (56.4%) of 58 cases, respectively. Interestingly, the CD3-TIL score positively correlated with PD-L1 TC and PD1-TIL scores. HLA class I expression level was inversely correlated with the expression levels of PD-L1 TC and PD-L1 IC. Multivariate analysis showed that age, histology, and node metastasis were independent prognostic factors for 5-year overall survival (OS) and loss of HLA class I coincided with a positive prognosis (p = 0.011). The concomitant lack of infiltrating CD8+ T cells with no loss of HLA class I predicted worse 5-year OS (p = 0.007). Moreover, cluster classifications among multiple immunoparameters showed that categories among CD3/PD-L1 TC/HLA class I (p = 0.043), CD8/PD1/HLA class I (p = 0.032), CD8/PD-L1 TC/HLA class I (p = 0.011), and PD1/PD-L1 TC/HLA class I (p = 0.032) predicted 5-year OS in EPP cases for MPM. These immunoparameters could guide surgical indications for patients with MPM.

Regulation of HLA class I expression by non-coding gene variations.

The Human Leukocyte Antigen (HLA) is a critical genetic system for different outcomes after solid organ and hematopoietic cell transplantation. Its polymorphism is usually determined by molecular technologies at the DNA level. A potential role of HLA allelic expression remains under investigation in the context of the allogenic immune response between donors and recipients. In this study, we quantified the allelic expression of all three HLA class I loci (HLA-A, B and C) by RNA sequencing and conducted an analysis of expression quantitative traits loci (eQTL) to investigate whether HLA expression regulation could be associated with non-coding gene variations. HLA-B alleles exhibited the highest expression levels followed by HLA-C and HLA-A alleles. The max fold expression variation was observed for HLA-C alleles. The expression of HLA class I loci of distinct individuals demonstrated a coordinated and paired expression of both alleles of the same locus. Expression of conserved HLA-A~B~C haplotypes differed in distinct PBMC’s suggesting an individual regulated expression of both HLA class I alleles and haplotypes. Cytokines TNFα /IFNβ, which induced a very similar upregulation of HLA class I RNA and cell surface expression across alleles did not modify the individually coordinated expression at the three HLA class I loci. By identifying cis eQTLs for the HLA class I genes, we show that the non-coding eQTLs explain 29%, 13%, and 31% of the respective HLA-A, B, C expression variance in unstimulated cells, and 9%, 23%, and 50% of the variance in cytokine-stimulated cells. The eQTLs have significantly higher effect sizes in stimulated cells compared to unstimulated cells for HLA-B and HLA-C genes expression. Our data also suggest that the identified eQTLs are independent from the coding variation which defines HLA alleles and thus may be influential on intra-allele expression variability although they might not represent the causal eQTLs.

Abstract P071: Gemcitabine augments HLA class I expression in pancreatic cancer cells through alterations in transcript production and surface stability

Abstract Background: Pancreatic adenocarcinoma, or PDAC, is the fourth leading cause of cancer-related deaths in the United States. Gemcitabine, a nucleoside analog, is a primary standard of care in pancreatic cancer. In addition to its normative cytotoxic function, evidence suggests that this chemotherapy drug also harnesses immunomodulatory capabilities in the form of increasing human leukocyte antigen (HLA) class I expression in lung, breast, colon, and cholangiocarcinoma cells. HLA class I is a complex (alpha heavy chain and beta 2-microglobulin light chain) located at the surface of nearly all cells where it presents peptides, including cancer-associated peptides, to cytotoxic T cells. Recognition of these peptides as atypical leads to T-cell mediated lysis of the presenting cell. Subsequently, understanding the ability of gemcitabine and the mechanisms by which it influences the HLA class I complex are of great importance. Methods: To investigate the effect of gemcitabine treatment on HLA class I expression in pancreatic cancer, alterations in HLA class I protein levels were monitored via western blot analysis, flow cytometry, and quantitative polymerase chain reaction (qPCR) in three pancreatic cancer cell lines. Changes in surface stability of the HLA class I complex were evaluated through brefeldin A (BFA) assays at the 72-hour time point. For western blot and flow cytometry experiments, impacts on the alpha heavy chain were further assessed for all three types of alpha heavy chains (HLA-A, HLA-B/C) at 72 and 96 hours. In qPCR experiments, alterations were analyzed for the HLA-A, HLA-B, and beta 2-microglobulin transcripts after a 48-hour gemcitabine exposure period. Results: Administration of gemcitabine to pancreatic cancer cell lines (S2-013, Capan-1, PANC-1) increased total protein levels of both HLA class I constituents (alpha heavy chain and beta-2-microglobulin light chain). All PDAC cell lines evaluated demonstrated enhanced surface expression of HLA-A2 and HLA-B/C with maximal increases of 3 and 2.5-fold respectively, as indicated by flow cytometry. Our qPCR analysis of the Capan-1 and S2-013 cell lines revealed increases in the levels of HLA-A, HLA-B, and beta 2-microglobulin transcripts (3-12-fold). BFA assays suggested that gemcitabine treatment also enhances stability of the surface HLA class I in the S2-013 and PANC-1 cell lines. Conclusion: In summary, gemcitabine exhibits an immunomodulatory ability to stimulate expression of HLA class I in pancreatic cancer cells. We have demonstrated that this increase in HLA class I is seen at the mRNA, total protein, surface, and stability level. Characterizing the ability of gemcitabine to influence the HLA class I complex and defining the mechanisms by which it does so will increase the potential for identification of suitable immunotherapies, including novel peptide-based cancer vaccines with enhanced treatment efficacy for PDAC patients. Citation Format: Alaina C. Larson, Shelby M. Knoche, Joyce C. Solheim. Gemcitabine augments HLA class I expression in pancreatic cancer cells through alterations in transcript production and surface stability [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P071.

Human Leukocyte Antigen Class I Deficiency in Gastric Carcinoma: An Adaptive Immune Evasion Strategy Most Common in Microsatellite Instable Tumors.

Immune checkpoint inhibitor therapy is effective only for a subset of patients with gastric cancer. Impaired neoantigen presentation caused by deficiency of human leukocyte antigen class I (HLA-I) has been reported as a common mechanism of immune evasion which is associated with resistance to immune checkpoint blockade. To elucidate the significance of HLA-I deficiency in gastric cancer with special focus on microsatellite instable (MSI) and Epstein-Barr virus (EBV)-positive tumors, we examined HLA-I expression on tumor cells and correlated the results with clinicopathologic features, programmed death-ligand 1 (PD-L1) expression, and degree of tumor-infiltrating immune cells. This study included 58 MSI, 44 EBV-positive, and 107 non-EBV non-MSI tumors for comparison. The frequency of HLA-I deficiency (≥1% tumor cells) was significantly higher in MSI tumors (52%) compared with EBV-positive tumors (23%) and the other tumors (28%). In contrast, PD-L1 expression levels were highest in EBV-positive tumors, followed by MSI tumors, with the lowest prevalence in the other tumors in both Tumor Proportion Score and Combined Positive Score. HLA-I deficiency was significantly more frequent in advanced tumors (pT2-4) than in early tumors (pT1) in MSI and non-EBV non-MSI subtypes. In addition, the degree of CD8-positive cells infiltration was significantly reduced in HLA-I deficient tumor areas compared with HLA-I preserved tumor area within a tumor. Based on our observations, HLA-I, as well as PD-L1, should be considered as a common mechanism of immune escape especially in the MSI subtype, and therefore could be a biomarker predicting response to immune checkpoint inhibitor therapy in gastric cancer.

Abstract 1658: Gemcitabine impacts expression of antigen presentation proteins by pancreatic cancer cells

Abstract Background: Pancreatic adenocarcinoma is one of the deadliest type of cancers with a 5-year overall survival rate of 10%. A standard chemotherapy approach for pancreatic cancer is treatment with gemcitabine. Gemcitabine is also considered a potential immunomodulatory agent, capable of increasing human leukocyte antigen (HLA) class I. HLA class I is a heterodimer of a highly polymorphic alpha heavy chain (HLA-A, -B, or -C) and the light chain beta-2-microglobulin (β2m) that is loaded with an endogenous antigen in the endoplasmic reticulum. The pool of antigens available for HLA class I loading is selected by the transporter associated with antigen processing protein (TAP). However, HLA class I peptide loading can occur independently of TAP, causing the HLA class I molecules to present a different antigen pool to cytotoxic T cells. Because many cancers downregulate HLA class I and prevent intracellular tumor antigens from being presented to cytotoxic T cells, understanding more about gemcitabine's capability to increase HLA class I expression is of great importance. Methods: Alterations in HLA class I and TAP protein levels were monitored via western blot analysis in a pancreatic cancer cell line (S2-013) in both a time and dose-dependent manner. The time points and dosages which solicited the largest increase in HLA class I expression were also examined by flow cytometry. Results: Administration of gemcitabine to S2-013 pancreatic cancer cells increased total HLA class I α heavy chain protein, with maximal increases of 141% (HLA-A) and 323% HLA-B/C. Flow cytometry revealed a 3-fold increase in HLA-A2 surface expression post-gemcitabine treatment and a maximal 2-fold increase in HLA-B/C expression at the corresponding gemcitabine concentration, but differing time point. Both western blot and flow cytometry analyses indicate HLA-B/C generally has its largest increase in expression at an earlier time point than HLA-A. In addition, high concentrations of gemcitabine that promoted HLA-A,B,C surface expression were associated with a large decrease in TAP total protein. Conclusion: In summary, gemcitabine demonstrates an ability to stimulate expression of HLA class I in pancreatic cancer cells, with the rate dependent on the HLA class I allele. Because gemcitabine treatment increases HLA class I surface expression with a simultaneous decrease in TAP expression, our findings indicate that gemcitabine may induce TAP-independent peptide loading of HLA class I. These results suggest the potential for gemcitabine treatment as a priming mechanism to enhance immunotherapy efficacy and potentially alter vaccination strategies in pancreatic cancer. Citation Format: Alaina C. Larson, Shelby M. Knoche, Joyce C. Solheim. Gemcitabine impacts expression of antigen presentation proteins by pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1658.

Expression of HLA class I and class II genes in patients with multiple skin warts

Human leukocyte antigens (HLAs), which are genetic markers that have critical roles in the immune response against pathogens, vary greatly among individuals. The aim of the study is to investigate the frequency of HLA class I (HLA-A, HLA-B and HLAC) and class II (HLA-DRB1, HLA-DQB1 and HLA-DQA1) genes in patients with multiple skin warts and to elucidate the role of these genes in the genetic susceptibility to skin warts. Peripheral venous blood samples were collected from 100 patients with multiple skin warts and 300 healthy individuals (controls). HLA typing was performed after DNA isolation from the blood samples. The HLA-A*02 (odds ratio [OR]: 0.12; p=0.0019), HLA-DQA1*03:01 (OR: 0.45; p=0.0017) and DQA1*05:01 (OR: 0.17; p<0.0001) genes were significantly more prevalent in the patients than in the healthy individuals and were thus identified as risk genes. The HLA-DQA1*01:01 (OR: 0.17; p<0.0001), HLA-DQA1*01:02 (OR: 0.17; p<0.0001), HLA-DQA1*01:03 (OR: 0.11; p<0.0001), HLA-DQA1*02:01 (OR:027; p<0.0001) and HLA-DQA1*05:05 (OR:0.16; p<0.0001) genes were classified as protective genes because of their low frequencies in the patients. The limitation of the study is that Human papillomavirus typing could not be performed while investigating the relationship between skin warts and HLA class I and class II genes. Our data suggest the role of HLA genes in the development of skin warts. However, other components of the major histocompatibility complex system and acquired factors of the immune system could also be involved and should be further investigated.

Spatial Analysis and Clinical Significance of HLA Class-I and Class-II Subunit Expression in Non-Small Cell Lung Cancer.

To analyze the distribution, associated immune contexture, and clinical significance of human leukocyte antigen (HLA) class-I and HLA class-II subunits in non-small cell lung cancer (NSCLC). Using spatially resolved and quantitative multiplexed immunofluorescence we studied the tumor/stromal tissue distribution, cancer cell-specific defects, and clinicopathologic/survival associations of β2 microglobulin (β2M), HLA-A, and HLA-B,-C heavy chains, as well as HLA class-II β chain in >700 immunotherapy-naïve NSCLCs from four independent cohorts. Genomic analysis of HLA genes in NSCLC was performed using two publicly available cohorts. Cancer cell-specific downregulation of HLA markers was identified in 30.4% of cases. β2M was downregulated in 9.8% (70/714), HLA-A in 9% (65/722), HLA-B,-C in 12.1% (87/719), and HLA class-II in 17.7% (127/717) of evaluable samples. Concurrent downregulation of β2M, HLA-B,-C, and HLA class-II was commonly identified. Deleterious mutations in HLA genes were detected in <5% of lung malignancies. Tumors with cancer cell-specific β2M downregulation displayed reduced T cells and increased natural killer (NK)-cell infiltration. Samples with cancer cell HLA-A downregulation displayed modest increase in CD8+ T cells and NK-cell infiltration. Samples with cancer cell-selective HLA-B,-C or HLA class-II downregulation displayed reduced T cells and NK-cell infiltration. There was limited association of the markers with clinicopathologic variables and KRAS/EGFR mutations. Cancer cell-selective downregulation of the HLA subunits was associated with shorter overall survival. Our results reveal frequent and differential defects in HLA class-I and HLA class-II protein subunit expression in immunotherapy-naïve NSCLCs associated with distinct tumor microenvironment composition and patient survival.