Expression Of HLA Class Research Articles

Direct conversion of human umbilical cord mesenchymal stem cells into dopaminergic neurons for Parkinson's disease treatment

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor deficits due to the depletion of nigrostriatal dopamine. Stem cell differentiation therapy emerges as a promising treatment option for sustained symptom relief. In this study, we successfully developed a one-step differentiation system using the YFBP cocktail (Y27632, Forskolin, SB431542, and SP600125) to effectively convert human umbilical cord mesenchymal stem cells (hUCMSCs) into dopaminergic neurons without genetic modification. This approach addresses the challenge of rapidly and safely generating functional neurons on a large scale. After a 7-day induction period, over 80 % of the cells were double-positive for TUBB3 and NEUN. Transcriptome analysis revealed the dual roles of the cocktail in inducing fate erasure in mesenchymal stem cells and activating the neuronal program. Notably, these chemically induced cells (CiNs) did not express HLA class II genes, preserving their immune-privileged status. Further study indicated that YFBP significantly downregulated p53 signaling and accelerated the differentiation process when Pifithrin-α, a p53 signaling inhibitor, was applied. Additionally, Wnt/β-catenin signaling was transiently activated within one day, but the prolonged activation hindered the neuronal differentiation of hUCMSCs. Upon transplantation into the striatum of mice, CiNs survived well and tested positive for dopaminergic neuron markers. They exhibited typical action potentials and sodium and potassium ion channel activity, demonstrating neuronal electrophysiological activity. Furthermore, CiNs treatment significantly increased the number of tyrosine hydroxylase-positive cells and the concentration of dopamine in the striatum, effectively ameliorating movement disorders in PD mice. Overall, our study provides a secure and reliable framework for cell replacement therapy for Parkinson's disease.

IL-10 and TGF-β, but Not IL-17A or IFN-γ, Potentiate the IL-15-Induced Proliferation of Human T Cells: Association with a Decrease in the Expression of β2m-Free HLA Class I Molecules Induced by IL-15.

IL-15 is a homeostatic cytokine for human T and NK cells. However, whether other cytokines influence the effect of IL-15 is not known. We studied the impact that IL-10, TGF-β, IL-17A, and IFN-γ have on the IL-15-induced proliferation of human T cells and the expression of HLA class I (HLA-I) molecules. Peripheral blood lymphocytes (PBLs) were labeled with CFSE and stimulated for 12 days with IL-15 in the absence or presence of the other cytokines. The proportion of proliferating T cells and the expression of cell surface HLA-I molecules were analyzed using flow cytometry. The IL-15-induced proliferation of T cells was paralleled by an increase in the expression of HC-10-reactive HLA-I molecules, namely on T cells that underwent ≥5-6 cycles of cell division. It is noteworthy that the IL-15-induced proliferation of T cells was potentiated by IL-10 and TGF-β but not by IL-17 or IFN-γ and was associated with a decrease in the expression of HC-10-reactive molecules. The cytokines IL-10 and TGF-β potentiate the proliferative capacity that IL-15 has on human T cells in vitro, an effect that is associated with a reduction in the amount of HC-10 reactive HLA class I molecules induced by IL-15.

Assessment of cancer cell-expressed HLA class I molecules and their immunopathological implications.

Immunotherapy using immune checkpoint inhibitors (ICIs) has shown superior efficacy compared with conventional chemotherapy in certain cancer types, establishing immunotherapy as the fourth standard treatment alongside surgical intervention, chemotherapy, and radiotherapy. In cancer immunotherapy employing ICIs, CD8-positive cytotoxic T lymphocytes are recognized as the primary effector cells. For effective clinical outcomes, it is essential that the targeted cancer cells express HLA class I molecules to present antigenic peptides derived from the tumor. However, cancer cells utilize various mechanisms to downregulate or lose HLA class I molecules from their surface, resulting in evasion from immune surveillance. Correlations between prognosis and the integrity of HLA class I molecules expressed by cancer cells have been consistently found across different types of cancer. This paper provides an overview of the regulatory mechanisms of HLA class I molecules and their role in cancer immunotherapy, with a particular emphasis on the significance of utilizing pathological tissues to evaluate HLA class I molecules expressed in cancer cells.

Prognostic significance of immunohistochemical classification utilizing biopsy specimens in patients with extensive-disease small-cell lung cancer treated with first-line chemotherapy and immune checkpoint inhibitors

PurposeAlthough immune checkpoint inhibitors (ICIs), together with cytotoxic chemotherapy (chemoimmunotherapy), have been adapted for the initial treatment of extensive-disease small-cell lung cancer (ED-SCLC), they have achieved limited success. In ED-SCLC, a subtype of SCLC, the expression of immune-related molecules and clinical data are not well understood in relation to ICI treatment efficiency.MethodsWe examined lung biopsy specimens from patients diagnosed with ED-SCLC treated with chemoimmunotherapy or chemotherapy. SCLC subtype, expression of HLA class I, and infiltration of CD8-positive cells were examined using immunohistochemistry (IHC). Subsequently, the association between clinical factors, IHC results, and progression-free survival or overall survival was assessed.ResultsMost of the cases showed the achaete-scute homolog 1 (ASCL1) subtype. Among the 75 SCLC cases, 29 expressed high levels of HLA class I, while 46 showed low levels or a negative result; 33 patients were characterized as CD8-high, whereas 42 were CD8-low. In the chemoimmunotherapy cohort, multivariate analysis revealed a correlation between CD8-high and improved survival. Specifically, patients in the CD8-high group of the chemoimmunotherapy cohort experienced enhanced survival compared to those in the chemotherapy cohort, which was attributed to ICI addition. IHC subtype analysis demonstrated a survival advantage in the SCLC-I and SCLC-A groups when ICI was combined with chemotherapy compared to chemotherapy alone.ConclusionOur study highlights the predictive value of IHC-classified subtypes and CD8-positive cell infiltration in estimating outcomes for patients with ED-SCLC treated with chemoimmunotherapy as a first-line therapy. These findings have practical implications for daily clinical assessments and treatment decisions.

Characteristics of adaptive immune effectors involved in the secondary xenogeneic immune response to human melanoma cells

Introduction. Current approaches are being developed for adoptive cancer therapy using T-cells genetically modified with T-cell receptors (TCRs) with specificity for tumor antigens. The complexities of identifying antigen-specific TCRs in a patient’s repertoire and selecting therapeutic receptors necessitate the development of experimental strategies for generating tumor-specific T cells. One of such approaches could be the xenogeneic immunization of mice with human tumor cells. It seems plausible that the T cell repertoire stimulated by xenogeneic vaccination could be a source of TCRs suitable for adoptive cancer immunotherapy.Aim. To assess the prospects for using xenogeneic immunizations to generate tumor-specific memory T cells and identify their TCRs suitable for adoptive immunotherapy, we studied the dynamics of the secondary xenogeneic response in a model of induction of an immune response in mice to human melanoma cells.Materials and methods. Mice were immunized with human melanoma cells, and 45 days later, they were re-challenged with the immunizing tumor. The dynamics of the development of the secondary immune response in vivo and the composition of the involved effectors of adaptive immunity were analyzed by flow cytometry. The proliferation of lymphocytes from immune mice in response to human melanoma cells was evaluated in in vitro culture.Results. The secondary xenogeneic response was characterized by a more intense accumulation of T cells and the rapid development of the effector phase at the injection site of human melanoma. This correlated with an enhanced in vitro proliferative response of lymphocytes from immune animals to xenoantigens of the immunizing tumor. CD4+ and CD8+ memory T cells contributed equally to the development of a secondary response to human melanoma cells expressing HLA class I and II molecules. When only HLA class I was expressed on the cells of the immunizing xenogeneic tumor, CD8+ memory cells were formed, which dominated the secondary immune response.Conclusion. Our findings confirmed the formation of a specific immunological memory for xenoantigens during xenogeneic immunization. This suggests the possibility of generating xenogeneic TCRs specific for human tumor antigens, which opens up opportunities to developing approaches for screening among them for receptor variants suited for adoptive immunotherapy of human cancers.

Post-Transplant Nivolumab Plus Unselected Autologous Lymphocytes in Truly Refractory Hodgkin Lymphoma Patients Is Highly Effective and Responses Are Mediated By NK Cells

Background In the relapsed/refractory Hodgkin Lymphoma (HL) setting (RHL), the achievement of complete remission (CR) is fundamental in order to proceed with allogeneic stem cell transplantation (HSCT) consolidation. Immune checkpoint inhibitors (CI) have demonstrated clinical activity in patients relapsing after ASCT, although only 20% CR rate is observed. In order to improve the efficacy of CI in RHL, we previously reported a salvage treatment based on early CI therapy following post-autologous stem cell transplantation (ASCT) with the reinfusion of unselected autologous lymphocytes infusions (ALI). The rationale of this approach is based on the achievement of a disease debulking through ASCT conditioning. Early administration of CI (nivolumab) is performed on a minimal disease burden, while reinfusions of ALI reduce post ASCT immune depression eliciting CI efficacy. Aims The aim of this study was to evaluate the efficacy of the procedure in terms of CR rates and overall survival (OS) after an extended follow up. Biological endpoint was to investigate the lymphocyte subpopulations involved in the mechanism of response and investigate the role of NK-cells mediating anti tumor responses in HL. Methods Patient were eligible to this study after failure to respond to first line treatment (i.e. positive PET2 or PET6 scan) and underwent autologous lymphocyte apheresis upon enrollment. All patients received conventional 2nd line chemo followed by 3rd line chemo-immunotherapy with Brentuximab-Vedotin (BV) in non-responding patients. Patient failing to achieve CR after BV were enrolled in the treatment arm and proceeded to ASCT + CI and ALI, whereas patients responding to 2nd- or 3rd treatment received ASCT followed by ALI alone, as a control cohort (figure 1A). NK cell degranulation assay was evaluated by staining for CD107a (LAMP-1) expression on healthy-donors derived NK cells exposed to HL cell lines (L428 and L250), in the presence or absence of monoclonal antibodies targeting inhibitory or activating receptors. Surface and intracellular staining was performed prior to flow cytometry and analysis was performed on the CD3-/CD56+ gated population. Results Twenty-one patients with RHL (median age 32 years; range 18-65) underwent lymphocyte apheresis after failure of 1st line chemo and then proceeded to salvage therapy. Thirteen patients failed to respond to 2nd and 3rd line therapy and thus received early post-transplant CI supported with four ALI. Eight patients responded to 2nd line chemo (n=6) or 3rd line BV (n=2) and received ASCT followed by ALI alone. No adverse events were recorded, specifically, no immune-related toxicity was observed. All patients receiving ALI + CI (treated patients) achieved negative PET scan CR. Eight of them received HSCT consolidation. One patient refusing HSCT after achieving CR in this study relapsed and responded to CI re treatment followed by HSCT. All patients in the treatment arm are alive and disease-free after a median follow-up of 32 months (95% CI 26.4-51.0). Median disease-free survival (DFS) was not reached in treatment arm (Fig 1B). Four of the patients in the control arm relapsed (50%). Three of them responded to 3rd line therapy and proceeded to HSCT. One patient achieved CR with conventional CI treatment but refused HSCT and the other relapsing patient died because of progressive lymphoma after failure to achieve CR with conventional single agent CI. Median DFS in the control arm was 22 months (Fig 1B). NK cells showed higher expansion and a faster maturation in the treatment arm, compared to the control arm (p<0.05). As expected, HL cells lines do not express HLA class I and II, but express activating NK cells receptors (aNKG2D, aNKp30, aNKp46, aDNAM-1). In vitro, the activation of each of those receptors was able to trigger NK cells degranulation. Similarly, PD-L1 and PD-L2 blockade on HL cell lines significantly increased NK cell degranulation (p<0.05). Taken together, those observations support the role of NK cells in this setting. Conclusions Our data show very high anti-tumor activity of ALI + CI for RHL, allowing most patients to proceed to HSCT. RHL patients in the treatment arm had an excellent outcome if compared to the control arm, which included patient who did respond to conventional treatment. Biological data suggests that this approach may accelerate NK cell development/maturation and suggest that NK cells may mediate response to CI in HL.

Tryptophan metabolism promotes immune evasion in human pancreatic β cells

To resist the autoimmune attack characteristic of type 1 diabetes, insulin producing pancreatic β cells need to evade T-cell recognition. Such escape mechanisms may be conferred by low HLA class I (HLA-I) expression and upregulation of immune inhibitory molecules such as Programmed cell Death Ligand 1 (PD-L1). The expression of PD-L1, HLA-I and CXCL10 was evaluated in the human β cell line, ECN90, and in primary human and mouse pancreatic islets. Most genes were determined by real-time RT-PCR, flow cytometry and Western blot. Activator and inhibitor of the AKT signaling were used to modulate PD-L1 induction. Key results were validated by monitoring activity of CD8+ Jurkat T cells presenting β cell specific T-cell receptor and transduced with reporter genes in contact culture with the human β cell line, ECN90. In this study, we identify tryptophan (TRP) as an agonist of PD-L1 induction through the AKT signaling pathway. TRP also synergistically enhanced PD-L1 expression on β cells exposed to interferon-γ. Conversely, interferon-γ-mediated induction of HLA-I and CXCL10 genes was down-regulated upon TRP treatment. Finally, TRP and its derivatives inhibited the activation of islet-reactive CD8+ T cells by β cells. Collectively, our findings indicate that TRP could induce immune tolerance to β cells by promoting their immune evasion through HLA-I downregulation and PD-L1 upregulation. Dutch Diabetes Research Foundation, DON Foundation, the Laboratoire d'Excellence consortium Revive (ANR-10-LABX-0073), Agence Nationale de la Recherche (ANR-19-CE15-0014-01), Fondation pour la Recherche Médicale (EQ U201903007793-EQU20193007831), Innovative Medicines InitiativeINNODIA and INNODIA HARVEST, Aides aux Jeunes Diabetiques (AJD) and Juvenile Diabetes Research Foundation Ltd (JDRF).

Abstract P3-07-12: Allogeneic, Antigen-Presenting, GM-CSF-secreting, SV-BR-1-GM Whole Cell Therapeutic Vaccine in Advanced Metastatic Breast Cancer

Abstract Background: SV-BR-1-GM is an irradiated allogeneic cell line derived from a hormone receptor-negative (HR-) HER2-positive (HER2+) breast cancer (BC), now engineered to express HLA class I and II antigens, secrete GM-CSF and function as an antigen-presenting cell. Here we report post-hoc exploratory data for metastatic BC (MBC) patients treated with the SV-BR-1-GM regimen (SV) alone (NCT03066947) and in combination (CO) with immune checkpoint inhibitors (ICIs) (NCT03328026). Methods: SV includes cyclophosphamide 300 mg/m2 i.v. 48-72 hours prior to SV-BR-1-GM (20 × 106 cells) intradermally followed by interferon-alpha-2b at the SV-BR-1-GM inoculation sites on days 2 and 4. SV was given every 2 weeks for 3 cycles and then monthly in NCT03066947, and every 3 weeks in combination with PD-1 inhibitors in NCT03328026. Treatment was continued until disease progression or unacceptable toxicities. Results: A total of 34 refractory MBC patients (pts) were treated, including 24 with SV, and 14 with CO, including 4 who crossed over from SV to CO plus one who restarted the SV after a protocol specified administrative interruption in treatment. Median prior regimens were 5; 55% of pts had hormone receptor-positive (HR+) BC, 42% had HER2+ BC, and 16% were triple-negative BC (TNBC). Objective response rates (ORR) were 4% for monotherapy and 7% for combination. Clinical benefit rate (CBR) was 21% for monotherapy and 29% for combination. Responses and clinical benefits were seen across most BC subtypes (see table). Notably, CBR among 10 pts with HR+ BC (any HER2) who received the combination was 50% (5/10). The duration of response (DOR) among 4 patients treated with monotherapy was 49-223 days and DOR among 6 patients treated with combination was 72-292 days. Median PFS was 2.8 months for monotherapy and 4.2 months for combination. Median OS was 7.0 months for monotherapy, and 12.0 months for combination. Of 28 pts with available prior treatment data, 12 (43%) had PFS on study treatment exceeded PFS on prior treatment, of these 8/12 had >1 HLA match and 5/8 had ≥ 2 loci HLA-matched with SV-BR-1-GM. PFS ratio improvement was independent of the prior number of lines of therapy or BC subtypes. The SV-BR-1GM regimen was well tolerated with the most common treatment-related adverse events (AEs) being injection site reaction in 67%. There was no dose-limiting toxicity, special interest AEs such as cytokine storm, and no grade 5 observed. There were 2 non-treatment-related grade 4 AEs: worsening pleural effusion and altered mental status. Significant improvement in PFS was observed in patients with matched HLA and in association with the delayed-type hypersensitivity skin test, peripheral blood circulating tumor cells, and cancer-associated macrophages. Conclusions: SV-BR-1-GM demonstrated promising activity in patients with MBC. Treatment was well tolerated with no concerning AEs. The PFS ratio compares favorably with prior penultimate standards of care, more notably in patients with matched HLA. Clinical benefits were observed across multiple subtypes of BC, particularly in patients with HR+ disease receiving combination therapy. Phase II clinical trial is currently ongoing to evaluate the efficacy of SV-BR-1-GM in combination with ICIs. Clinical benefit and PFS on SV-BR-1-GM SV-BR-1-GM Whole Cell Therapeutic Vaccine in Heavily Pre-Treated Metastatic Breast Cancer Results Stratified by HER2 and HLA Matching Citation Format: Saranya Chumsri, William Williams, Mingjin Chang, Miguel Lopez-Lago, Charles Wiseman, Jarrod Holmes, Chaitali Nangia, Karim Mohammed, Minal Barve, Shaker Dakhil, Bonnie Guerin, Giuseppe Del Priore, Carmen Calfa. Allogeneic, Antigen-Presenting, GM-CSF-secreting, SV-BR-1-GM Whole Cell Therapeutic Vaccine in Advanced Metastatic Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-07-12.

Tumor cell HLA class I expression and pathologic response following neoadjuvant immunotherapy for newly diagnosed head and neck cancer

ObjectivesBiomarkers are needed to identify patients likely to respond to neoadjuvant immunotherapy (NIT) prior to receiving definitive treatment. Materials and MethodsWe hypothesized that expression of tumor cell HLA class I would correlate with pathologic response (PR) following NIT for primary untreated head and neck cancer. Multispectral immunofluorescence of pre- and post-treatment biopsy specimens from a neoadjuvant study of bintrafusp alfa, a dual TGF-β and PD-L1 inhibitor, was performed. ResultsDiscordant expression of tumor cell HLA class I and PD-L1 measured by multispectral immunofluorescence was observed with most positive tumor cells expressing HLA class I or PD-L1 but not both. Spatial analysis revealed colocalization between tumor parenchyma T cells and HLA class I positive tumors cells, but no clear colocalization between T cells and PD-L1 positive tumor cells. Greater pre-treatment tumor cell HLA class I expression, but not PD-L1 expression or tumor T cell infiltration, correlated with the development of a PR. Additionally, increased tumor cell HLA class I expression after NIT compared to before NIT correlated with development of a PR, whereas inconsistent changes in PD-L1 and T cell infiltration were observed after treatment in all patients. ConclusionsThese data provide the rationale for the study of tumor cell HLA class I expression in larger prospective studies powered to determine the performance of biomarkers of PR in newly diagnosed HNSCC patients receiving NIT.

Co-expression of HLA-E and HLA-G in Porcine Endothelial Cells Attenuates Human Natural Killer Cell-Mediated Cytotoxicity

INTRODUCTION: Pig-to-human xenotransplantation (XTx) is a promising solution to the organ shortage. Genetically-modified (GM) pigs lacking major xenoantigens have reduced hyperacute rejection and prolonged xenograft survival. Despite these advancements, acute rejection remains a barrier to clinical XTx. Natural killer cells (NK) play a crucial role in transplantation. In the context of XTx, suppressing human NK activity by expressing HLA class I molecules in pig organs may protect xenografts from immune attack. Therefore, we aimed to co-express HLA class I molecules in GM pig endothelial cell line null for 5 key xenoantigens (5GKO) and to evaluate human NK response to 5GKO+HLA class I+ cells. METHODS: DNA sequences encoding HLA-E and HLA-G were designed, synthesized, and cloned into expression vectors. Three GM porcine cell lines (5GKO.HLA-E, 5GKO.HLA-G, 5GKO.HLA-E+G) were generated by transfection. IL-2–activated human peripheral blood mononuclear cells (PBMCs) from 5 donors were co-cultured with GM porcine cell lines to initiate NK cell–mediated immune responses. NK activation (CD107a expression) was evaluated by flow cytometry. Three experimental replicates were averaged and analyzed by one-way ANOVA and a post hoc Tukey test. RESULTS: We successfully co-expressed HLA-G, HLA-E, and HLA-E+G in 5GKO cells. Co-expression of HLA-E+G in 5GKO cells significantly reduced human NK activation compared with 5GKO, 5GKO.HLA-E, and 5GKO.HLA-G. CONCLUSION: Co-expression of HLA-E and HLA-G in porcine cells significantly reduces NK cell–mediated cytotoxicity and shows promise for incorporation into GM pigs for pig-to-human xenotransplantation.

Reversible epigenetic alterations regulate class I HLA loss in prostate cancer

Downregulation of HLA class I (HLA-I) impairs immune recognition and surveillance in prostate cancer and may underlie the ineffectiveness of checkpoint blockade. However, the molecular mechanisms regulating HLA-I loss in prostate cancer have not been fully explored. Here, we conducted a comprehensive analysis of HLA-I genomic, epigenomic and gene expression alterations in primary and metastatic human prostate cancer. Loss of HLA-I gene expression was associated with repressive chromatin states including DNA methylation, histone H3 tri-methylation at lysine 27, and reduced chromatin accessibility. Pharmacological DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibition decreased DNA methylation and increased H3 lysine 27 acetylation and resulted in re-expression of HLA-I on the surface of tumor cells. Re-expression of HLA-I on LNCaP cells by DNMT and HDAC inhibition increased activation of co-cultured prostate specific membrane antigen (PSMA)27-38-specific CD8+ T-cells. HLA-I expression is epigenetically regulated by functionally reversible DNA methylation and chromatin modifications in human prostate cancer. Methylated HLA-I was detected in HLA-Ilow circulating tumor cells (CTCs), which may serve as a minimally invasive biomarker for identifying patients who would benefit from epigenetic targeted therapies.

Advances in the study of HLA class Ib in maternal-fetal immune tolerance.

The HLA class Ib molecule is an alloantigen that causes transplant rejection on behalf of individual human and plays an important role in maternal-fetal immune tolerance. Early studies on HLA class Ib focused on the mechanism of HLA-G-induced immune escape, but in recent years, studies on the mechanism of HLA-G have deepened and gradually explored the mechanism of HLA-E and HLA-F, which are also HLA class Ib molecules. In the maternal-fetal interface, trophoblast cells express HLA class Ib molecules to protect the fetus from maternal immune cells by binding to inhibitory receptors of decidual immune cells (DICs) and shifting Th1/Th2 balance toward Th2 bias. Further studies on the molecular mechanism of HLA class Ib molecules provide a reference for its application in the field of clinical assisted reproduction.

Immune Evasion of Hepatoma Cancer Stem-Like Cells from Natural Killer Cells

Poor prognosis in liver cancer is due to its high frequency of intrahepatic metastasis. Cancer stem-like cells (CSLCs), which possess the properties of stemness, tumor initiation capability, and resistance to therapy, also exhibit metastatic potential. Immune surveillance plays an important role in the accomplishment of metastasis. Herein, the property of immune evasion in CSLCs was investigated. Sphere cells were induced as CSLCs using a sphere induction medium containing neural survival factor-1. The expression of genes involved in immune evasion was determined using RNA-sequencing for sphere and parental cells followed by validation using flow cytometric analysis and ELISA. Susceptibility to natural killer (NK) cell-mediated cytotoxicity was examined by a chromium release assay. A xenograft model using BALB/c nu/nu mice was used to assess tumor growth. Gene set enrichment analysis was performed for interpreting RNA sequencing. The cell surface expressions of PD-L1, PD-L2, and CEACAM1 were upregulated and those of ULBP1 and MICA/MICB were downregulated in SK-sphere, CSLCs derived from SK-HEP-1, compared with that in parental cells. Levels of soluble MICA were elevated in conditioned medium from SK-sphere. Expression of HLA class I was not downregulated in SK-sphere. The susceptibilities to NK cell-mediated killing and secreted perforin were significantly lower in both CSLCs derived from SK-HEP-1 and HLE than in parental cells. Tumors formed upon inoculation of SK-sphere in immunodeficient mice harboring NK cells were larger than those formed upon inoculation of parental cells. Human hepatoma cell line-derived CSLCs may possess immune evasion properties, especially from NK cell-mediated immunity.

Abstract 2812: CSPG4-specific CAR.CIK lymphocyte-based immunotherapy to eliminate HLA class I-defective melanoma tumors

Abstract Purpose of this study is to explore the preclinical efficacy of Chondroitin Sulfate Proteoglycan 4 (CSPG4)-specific CAR-engineered Cytokine-Induced Killer lymphocytes (CIK) to eradicate melanoma cells with defective HLA class I (HLA-I). The latter abnormality plays a major role in clinical resistance to Checkpoint Inhibitors (CI). CSPG4 was selected as the target because of its high expression on melanoma and its restricted distribution in normal tissues. Our approach is based on CAR.CIK redirected against CSPG4. CIK, ex vivo expanded T-NK lymphocytes endowed with intrinsic HLA-independent antitumor activity, were used as effectors. Experimental procedure. CAR.CIK were generated by retroviral transduction of patient derived PBMC with a vector encoding a 2nd generation CSPG4-CAR with 4-1BB co-stimulation. Surface HLA-I expression was evaluated by flow cytometry on melanoma cell lines (Mel), derived by surgical biopsies. These cells also served as targets for CSPG4 CAR.CIK. The activity of CSPG4 CAR.CIK was analyzed in vitro and in immunodeficient mice grafted with a patient-derived HLA-defective melanoma. Mice were treated intravenously with 3x106 CAR.CIK (5 cells infusions in total). Results. CAR.CIK were efficiently generated from 4 melanoma patients. Mean expression of CSPG4-CAR was 48±8%, the rate of ex vivo expansion (84 fold) and phenotypic characteristics (CD3+CD8+=71±13%, CD3+ CD56+=22.5±13%, NKG2D+= 68±32.3%) were comparable with unmodified controls. Membrane HLA-I molecules were detected in 23/24 Mel samples, with a variable membrane density per cell (median 21232, range 787-49871). Sample Mel17 did not express HLA-I because of a start lost mutation (p.Met1Ile) in β2microglobulin. CSPG4 was intensely expressed by all Mel (78±5%), with no correlation to HLA-I levels. CSPG4 CAR.CIK efficiently killed 10 Mel samples in vitro, including Mel17 (HLA-I negative) and 2 Mel with the lowest HLA-I density (Mel71, Mel72). Mean Mel-specific killing by CSPG4 CAR.CIK was significantly higher compared with unmodified CIK especially at low effector/target (E/T) ratios (85% vs 40% at E/T=1:1; 46% vs 9% at E/T=1:8, p<0.0001). In vivo, CSPG4 CAR.CIK caused a significant inhibition of the HLA-negative Mel17 tumor growth (p<0.0001) as compared to controls. Antitumor activity was confirmed by the reduction of tumor weight and metabolic activity (by fluorescent glucose uptake) measured on the residual explanted tumors. Antitumor response persisted up to 2 weeks after end of the treatment. Conclusions. We reported the activity of CSPG4 CAR.CIK against melanoma, including those with low or defective HLA-I expression. CIK may provide a valid platform for CAR-based strategies against melanoma and solid tumors in general. Our data provide the rationale to implement clinical studies exploring the proposed strategy in melanoma patients not responding or relapsing after immunotherapy with CI. Citation Format: Giulia Cattaneo, Lidia Giraudo, Loretta Gammaitoni, Ilenia Iaia, Fabrizio Carnevale-Schianca, Alberto Pisacane, Enrico Berrino, Caterina Marchiò, Luca Paruzzo, Andrea Michela Biolato, Chiara Donini, Marco Basiricò, Elisa Landoni, Soldano Ferrone, Valeria Leuci, Massimo Aglietta, Gianpietro Dotti, Dario Sangiolo. CSPG4-specific CAR.CIK lymphocyte-based immunotherapy to eliminate HLA class I-defective melanoma tumors [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 2812.

Abstract LB563: Integrated multiomic profiling identifies the epigenetic regulator PRC2 as a therapeutic target to counteract leukemia immune escape and relapse

Abstract Background: Evasion from immune control represents one of the main drivers of acute myeloid leukemia (AML) relapse after allogeneic hematopoietic cell transplantation (allo-HCT). In particular, up to 40% of AML relapses display complete loss of surface expression of HLA class II molecules without any genetic lesion explaining this phenotype (Christopher et al, N Engl J Med, 2018; Toffalori et al, Nat Med, 2019). This led us to investigate the links between epigenetic changes, immune evasion and post-transplantation relapse. Methods: Starting from primary AML samples pairwise collected from five patients at diagnosis and relapse with non-genomic loss of HLA class II expression, we generated Patients-Derived Xenografts (PDXs) into NOD-SCID γ-chain null mice. Leukemic cells expanded in the mice and their original human counterparts were characterized for changes in gene expression (by RNA-Seq), DNA methylation profile (by RRBS), and chromatin accessibility (by ATAC-Seq). The results obtained by all these approaches and in the different patients were integrated by Multi-Omics Factor Analysis (MOFA) and Gene Set Enrichment Analysis (GSEA). Finally, we tested the immunological effects of epigenetic drugs on AML cells and on their recognition by T cells in ex-vivo short-term cultures and in PDXs. Results: We verified that PDXs faithfully recapitulate immune-related differences between AML diagnosis and post-transplantation relapse, including loss of expression of HLA class II molecules. Differences between diagnosis and post-transplantation relapse samples were mostly explained by changes in chromatin accessibility, and largely unrelated to the DNA methylation profile. In particular, in all five patients analyzed, we documented genomewide chromatin compaction at time of relapse, that was particularly evident for HLA class II genes and their master regulator CIITA, and not detected in relapses after sole chemotherapy. Integration of all the high-throughput technologies by MOFA, and of results from different patients by GSEA, pointed to the Polycomb Repressive Complex 2 (PRC2) as the main candidate mediator of HLA class II silencing.Pharmacological inhibition of PRC2 subunits rescued HLA class II expression in AML relapses ex vivo and in vivo, with consequent recovery of leukemia recognition by CD4+ T cells. Conclusions: Our results uncover a novel link between epigenetics and leukemia immune escape, which may rapidly translate into innovative strategies to cure or prevent AML post-transplantation relapse. Citation Format: Valentina Gambacorta, Stefano Beretta, Martina Ciccimarra, Laura Zito, Kety Giannetti, Angela Andrisani, Daniela Gnani, Lucia Zanotti, Giacomo Oliveira, Matteo G. Carrabba, Davide Cittaro, Ivan Merelli, Fabio Ciceri, Raffaella Di Micco, Luca Vago. Integrated multiomic profiling identifies the epigenetic regulator PRC2 as a therapeutic target to counteract leukemia immune escape and relapse [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 LB563.

193-LB: Differentiation of PTPN2 Knockout Pluripotent Stem Cells into Stem-Cell–Derived ß-Like Cells Leads to Increased Stimulation of Diabetogenic TCR T Cell Transductants

Type 1 diabetes (T1D) is an autoimmune disease marked by permanent loss of insulin-producing pancreatic β cells. Genome wide association studies have identified protein tyrosine non receptor type 2 (PTPN2) as a high-risk T1D gene. PTPN2 has been classically implicated in immune cell function, but is expressed in T cells, B cells, and in pancreatic β cells. Mice with a β cell specific knock out (KO) of ptpn2 exhibit impaired function and increased susceptibility to stress induced β cell death. We hypothesized that altered PTPN2 expression in pancreatic beta cells may contribute to T1D disease development. In order to study PTPN2 in the human context we generated CRISPR/Cas9 mediated clonal PTPN2 knock out (KO) hPSCs, abolishing the functional domain of PTPN2. PTPN2 KO and WT hPSCs were differentiated into functional stem cell derived beta-like cells (sBCs) using our published protocol without significant differences in key developmental markers or insulin content. Compared to WT sBC, PTPN2 KO sBCs at steady state and under T1D stress conditions show increased expression of HLA Class I molecules important in T cell recognition. To investigate the interaction of KO or WT sBCs with diabetogenic T cells we employed an HLA-peptide-TCR matched coculture system we recently established. Using this system, we observed increased stimulation of diabetogenic TCR T cell transductants by KO sBC compared to WT. Our data provide evidence for a potential mechanism by which PTPN2, a known T1D high-risk gene may contribute to an immunogenic phenotype of β cells. Our findings suggest that dysregulation of PTPN2 expression contributes to an autoimmune response towards human β cells thus contributing to the pathogenesis of type 1 diabetes. Disclosure J. Q. Matuschek: None. H. A. Russ: Consultant; Eli Lilly and Company, Sigilon Therapeutics, Inc. T. M. Triolo: None. A. W. Michels: Employee; ImmunoMolecular Therapeutics, Stock/Shareholder; ImmunoMolecular Therapeutics. K. Mcdaniel: None. M. S. Hansen: None. S. Williams: None. R. Castro-gutierrez: None. A. Shilleh: None. Funding NIH: K12DK094712

B2M gene knockout in HEK293T cells by non-viral delivery of CRISPR-Cas9 system for the generation of universal cells

BackgroundAllogeneic stem cells are the most potent sources for replacing cell, tissue, and organ malfunctions. The clinical use of these stem cells has been limited due to the risk of immune system rejection due to the incompatibility of human leukocyte (HLA) antigens between donors and recipients. To overcome this limitation, we used the CRISPR/Cas9 system to eliminate the β2 microglobulin (B2M) gene, which plays a vital role in the expression of HLA class I.ResultsNon-viral transfer of two gRNAs targeting the first exon and intron in the B2M gene results in large deletions in the target region. In addition, the results of this study showed that 11.11% and 22.22% of cells received genomic changes as homozygous and heterozygous, respectively.ConclusionIn conclusion, we have shown that the dual guide RNA strategy is a simple and efficient method for modifying genes. As a result, these cells can be proposed as universal cells that are not detectable in the cell therapy system and transplantation by the receptor immune system.

Abstract P2-14-02: Overall survival following treatment with a modified whole tumor cell targeted immunotherapy in patients with advanced breast cancer

Abstract Background: SV-BR-1-GM is a GM-CSF secreting breast cancer cell line derived from a Grade II (moderately differentiated) breast tumor that also expresses HLA class I & II antigens and is able to function as an antigen-presenting cell. Irradiated SV-BR-1-GM is used in a regimen including pre-dose low-dose cyclophosphamide and post-dose injection of IFNα2b into the inoculation sites. The SV-BR-1-GM regimen has been used alone (“monotherapy”, ClinicalTrials.gov NCT03066947 - study completed) and in combination with checkpoint inhibitors (“combination”, ClinicalTrials.gov NCT03328026 - study ongoing). Here we report survival data for patients with advanced metastatic breast cancer (aMBC) treated with the SV-BR-1-GM regimen. Methods: 27 patients with refractory aMBC were treated with the SV-BR-1-GM regimen as monotherapy (cycles every 2 weeks x3 and then monthly). The combination study uses the SV-BR-1-GM regimen with PD-1 inhibitors (PD-1i) pembrolizumab or retifanlimab with cycles every 3 weeks (12 patients dosed to date). Here we report progression free survival (PFS) and overall survival (OS) for patients where that data was collected. Results: A total of 35 patients received the SV-BR-1-GM regimen. The SV-BR-1-GM regimen alone (monotherapy) was given to 27 and 12 received the regimen with a PD-1i checkpoint inhibitor (combination therapy): 4 subjects crossed over from monotherapy. Patients had been heavily pre-treated, median prior regimens = 5. Most patients were estrogen receptor and/or progesterone receptor positive, 18% were Her2/neu positive and 33% were triple negative. The treatment was generally safe and well tolerated. The disease control rate was 30% for the SV-BR-1-GM regimen alone and 33% for the combination with a PD-1i. Several patients had objective complete regression of selected metastases. Median progression free survival was 2.8 months for the SV-BR-1-GM regimen alone and 4.2 months for the PD-1i combination. Median overall survival was 7.0 months for the SV-BR-1-GM regimen alone (data available on 9 patients), and 12.0 months for the PD-1i combination (data available on 7 patients). Conclusions: The median OS compares favorably with published data regarding survival in third line trials (Kazmi Breast Cancer Res Treat. 2020 Aug 17). The protracted OS seen in some subjects suggests some patient subpopulations are more likely to derive clinical benefit. The SV-BR-1-GM regimen alone or in combination with a PD-1i, when administered to heavily pre-treated patients with aMBC, may have elicited effective immune responses in some patients. TablePatients by StudyCharacteristicSV-BR-1-GM Regimen Alone (n=27)SV-BR-1-GM Regimen + PD-1i (n=12)All Patients* (n=35)Age60 ± 1063 ± 1060 ± 10Mean Prior Systemic Regimens5 (range 0-12)6 (range 1-10)5 (range 0-12)% ER/PR +52%75%58%% Her2/neu +15%17%18%% Triple Negative36%25%33%Delayed-type Hypersensitivity81%91%82%Disease Control Rate30%33%29%Median (Range) Progression Free Survival (months)2.8 (0.4-7.4) (n=27)4.2 (0.8-9.4) (n=11)2.8 (0.4-9.4) (n=34)Median (Range) Overall Survival (months)7.0 (1-41) (n=9)12.0 (5.1-21.4) (n=7)10.2 (1-41) (n=14)• Note that 4 patients crossed over from the monotherapy study to the combination therapy study. Citation Format: William Williams, Shaker R Dakhil, Carmen Calfa, Jarrod P Holmes, Saveri Bhattacharya, Jason Lukas, Elizabeth Tan-Chui, George E Peoples, Vivek G Sunkari, Markus D Lacher, Charles L Wiseman. Overall survival following treatment with a modified whole tumor cell targeted immunotherapy in patients with advanced breast cancer [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 P2-14-02.

A rational approach to assess off-target reactivity of a dual-signal integrator for T cell therapy

Cell therapy is an emerging therapeutic modality with the power to exploit new cancer targets and potentially achieve positive outcomes for patients with few other options. Like all synthetic treatments, cell therapy has the risk of toxicity via unpredicted off-target behavior. We describe an empirical method to model off-tumor, off-target reactivity of receptors used for investigational T cell therapies. This approach utilizes an optimal panel of diverse human cell-lines to capture the large majority of protein-coding gene expression in adult human tissues. We apply this cell-line set to test Jurkat and primary T cells engineered with a dual-signal integrator, called TmodTM, that contains an activating receptor (activator) and a separate inhibitory receptor (blocker). In proof-of-concept experiments, we use CD19 as the activating antigen and HLA-A*02 as the blocker antigen. This specific Tmod system, which employs a blocker targeting a ubiquitously expressed HLA class I antigen to inhibit CAR activation, has an inherent mechanism for selectivity/safety, designed to activate only when a specific HLA class I antigen is lost. Nonetheless, it is important to test off-target reactivity in functional assays, especially given the disconnect between ligand-binding and function among T cell receptors (TCRs) and chimeric antigen receptors (CARs). We show these cell-based assays yield consistent results with high sensitivity and specificity. The general strategy is likely applicable to more traditional single-receptor CAR- and TCR-T therapeutics.

Prognostic Value of HLA Class I in Patients with Hepatocellular Carcinoma.

Downregulation of HLA class I molecules is a major tumor escape mechanism from immune attack. However, its prognostic impact for patients with hepatocellular carcinoma is still unclear. This study aimed to investigate whether HLA class I has prognostic significance in patients with hepatocellular carcinoma. A cohort of 132 patients with hepatocellular carcinoma was enrolled. HLA class I expression was detected by immunohistochemistry. Levels of HLA class I expression were dichotomized as low and high according to staining intensity or staining percentage of positive tumor cells, respectively. Association of HLA class I expression with clinical characteristics and survival was analyzed. None of the clinical characteristics, including gender, age, virus infection, cirrhosis, AFP, vascular invasion, tumor size and number, was significantly associated with staining percentage of HLA class I or staining intensity (p > 0.05). Low staining percentage of HLA class I was significantly associated with a worse survival (p = 0.011), which was further confirmed by Cox regression hazards model in multivariate analysis (HR 0.416, 95% CI 0.204 - 0.849, p = 0.016). Staining intensity of HLA class I was not significantly associated with survival (p > 0.05). Expression of HLA class I might be a significant prognostic factor in hepatocellular carcinoma, and downregulation of HLA class I was significantly associated with a worse survival in terms of expression percentage of HLA class I.