Down-regulation Of CD8 Research Articles

CRISPR-Cas9 Genome Editing of Primary Chronic Lymphocytic Leukemia Cells

Chronic lymphocytic leukemia (CLL) is a common B-cell neoplasia that exhibits a very heterogeneous course. Over the last decade, multiple molecular studies have described the genomic, epigenomic and transcriptional landscape of the disease. These studies have led to the identification of more than 60 recurrently mutated genes as well as a large number of deregulated genes and regulatory elements. While the prognostic significance of some of these alterations is well described, their specific contributions to the pathogenesis of the disease remains largely unknown. Attempts to address this experimentally have employed genetically engineered mouse models and human cell lines. Although mouse models have been helpful to recapitulate disease aspects that can be only properly studied in vivo, significant limitations exist that prevent extrapolation of data from mice to human. Moreover, cell lines are commonly obtained from patients with end-stage, refractory diseases and are frequently EBV-positive, showing great discrepancies with primary cells, including high proliferation rate and absence of spontaneous apoptosis. In addition, they do not faithfully recapitulate the transcriptional and chromatin landscape of CLL primary cases. Thus, there is a need of better cellular models that recapitulate these disease aspects. An attractive solution to this problem would be the use of primary human CLL cells as a platform for ex vivo genetic manipulation, applying CRISPR-Cas9 genome editing technology. However, this is technically challenging, as (i) CLL cells are critically dependent on stimulatory signals coming from the tumor microenvironment, particularly in the lymph nodes (LN), and (ii) primary cells are resistant to most gene transfer methods. In order to overcome these limitations, we took advantage of a cell culture system that is reminiscent of a LN environment and induces vigorous proliferation of CLL primary cells for several weeks, allowing retroviral gene transfer with high efficiency and minimal toxicity (Mangolini, M. et al., 2022, Nat Commun, in press). To avoid undesired off-target effects induced by the constitutive expression of a CRISPR-Cas9 viral plasmid, we used transient expression systems. As an initial proof of concept, we designed sgRNA targeting the CD19 gene, a pan-B cell marker highly expressed in CLL cells, and assessed the transfection efficiency and cell viability when introducing the Cas9-CD19 sgRNA ribonucleoprotein complex using the Neon Transfection System (Thermo Fisher Scientific). Cryopreserved CLL primary cells (from peripheral blood samples) were expanded for 3 days in vitro and were subsequently electroporated with optimized conditions. Cell viability and electroporation efficiency were assessed 24 hours later and demonstrated a remarkable low percentage of apoptotic cells, with only 10% of cell death compared to non-electroporated cells (measured by flow cytometry using the LIVE/DEAD fixable Aqua dead cell stain, n= 6 cases); while high transfection efficiency was accomplished, with over 80% of cells containing the fluorescent Cas9-ATTO550 sgRNA ribonucleoprotein. Finally, 72 hours after electroporation we observed a downregulation greater than 85% of CD19 levels by flow cytometry (n=6 cases). After successful CD19 downregulation, we aimed at targeting the cell cycle regulatory protein Cyclin D2 (CCND2), which is upregulated by CLL cells at proliferation centers and the most upregulated cyclin in our cell culture system. Two independent sgRNAs were electroporated with the Cas9 protein into 3 CLL primary cases, with a minimum effect on cell viability after 24 hours. CCND2 knock-down (KD) efficiency was of 70% and 60% respectively in all cases studied, assessed by qRT-PCR. Interestingly, despite being exposed to cell culture conditions promoting vigorous proliferation, CCND2 KD blocked cell proliferation, and a cell growth reduction of 50-70% was obtained with both sgRNAs compared with non-targeting controls already at 48 hours. Altogether, these results demonstrate the feasibility to genetically modify primary CLL cells using the CRISPR-Cas9 system. This strategy overcomes the current dependency on cell lines or mouse models and represents a powerful tool to unveil the role of candidate genes involved in CLL pathogenesis.

Genetic and immune crosstalk between severe burns and blunt trauma: A study of transcriptomic data.

Background: Severe burns and blunt trauma can lead to multiple organ dysfunction syndrome, the leading cause of death in intensive care units. In addition to infection, the degree of immune inflammatory response also affects prognosis. However, the characteristics and clinical relevance of the common mechanisms of these major diseases are still underexplored. Methods: In the present study, we performed microarray data analysis to identify immune-related differentially expressed genes (DEGs) involved in both disease progression in burns and blunt trauma. Six analyses were subsequently performed, including gene enrichment analysis, protein‐protein interaction (PPI) network construction, immune cell infiltration analysis, core gene identification, co-expression network analysis, and clinical correlation analysis. Results: A total of 117 common immune-related DEGs was selected for subsequent analyses. Functional analysis emphasizes the important role of Th17 cell differentiation, Th1 and Th2 cell differentiation, Cytokine-cytokine receptor interaction and T cell receptor signaling pathway in these two diseases. Finally, eight core DEGs were identified using cytoHubba, including CD8A, IL10, CCL5, CD28, LCK, CCL4, IL2RB, and STAT1. The correlation analysis showed that the identified core DEGs were more or less significantly associated with simultaneous dysregulation of immune cells in blunt trauma and sepsis patients. Of these, the downregulation of CD8A and CD28 had a worse prognosis. Conclusion: Our analysis lays the groundwork for future studies to elucidate molecular mechanisms shared in burns and blunt trauma. The functional roles of identified core immune-related DEGs and dysregulated immune cell subsets warrant further in-depth study.

Elevated KIR expression and diminished intensity of CD7 on NK cell subsets among treatment naïve HIV infected Ethiopians

Natural killer (NK) cells are crucial effector cells of the innate immune response to viral infections, including HIV, through cytolytic activity and the production of cytokines with anti-HIV activities. We recruited 15 treatment naïve HIV patients and 16 healthy controls (HC) to assess NK cell subsets or expression of multiple markers by flow cytometry. The frequency of circulating CD56brightCD16−ve and CD56dimCD16bright NK cell subsets was significantly lower among the HIV group than in HC. The CD56−veCD16bright subset was higher in HIV patients, but this was only apparent when gated among total NK cells, not total lymphocytes. NK cells among HIV participants also showed a lower and higher frequency of CD8 and HLA-DR expressing cells, respectively. In addition, CD7 median fluorescent intensity and CD2+CD7− frequencies were significantly lower in HIV patients. A distinct population of KIR3DL1/S1 cells was unexpectedly higher among CD56brightCD16−ve NK cells in HIV patients. In conclusion, this study in the Ethiopian setting confirms many previous findings, but the down-regulation of CD7 and enhanced KIR3DL1/S1 within the CD56bright subsets have not been widely reported among HIV patients and merit further research.

Host B cells escape CAR-T immunotherapy by reversible downregulation of CD19.

Anti-CD19-CAR-T cells are a successful clinical immunotherapy for B cell lymphomas, although some lymphomas can escape attack by downregulating surface CD19 levels. An undesirable consequence of this therapy is that it can also eliminate healthy B cells expressing CD19. Therefore, understanding the dynamics of CD19 expression in B cells under CAR-T cell immunotherapy can help mitigate both escape and adverse outcomes. Previous studies suggested that mechanisms responsible for the loss of CD19 expression in lymphomas usually involves genomic deletion or epigenetic modification which permanently removes CD19 as a therapeutic target in these cells. We examined if healthy B cells can use similar processes to lose CD19 expression and escape CAR-T attack. In the presence of CAR-T cells, untransformed B cells both when cultured in vitro or in vivo in non-tumor bearing animals downregulate expression of CD19. We then used adoptive transfer strategies to remove CD19-low B cells from αCD19-CAR-T pressure in vivo. Intriguingly, these B cells systematically recovered surface expression of CD19 comparable to wild-type levels. These data suggest that unlike many cases of lymphomas, healthy B cells downregulate CD19 in a reversible fashion. Taken together, these data suggest a dynamic regulatory process of CD19 surface expression on healthy B cells that could be exploited to modulate the expression of CD19 on cancer cells to improve immunotherapy or minimize the depletion of endogenous B cell compartment during treatment.

Abstract 646: Liquid biopsy transcriptomics identify pathways associated with poor outcomes and immune phenotypes in men with mCRPC

Abstract Androgen receptor signaling inhibitors (ARSi) and taxanes are mainstays for patients with metastatic castration-resistant prostate cancer (mCRPC). However, patient response is heterogeneous, and the molecular underpinnings of treatment resistance are not well elucidated. To identify clinically meaningful mechanisms of treatment resistance, we performed transcriptome analysis of circulating tumor cells (CTCs) isolated from mCRPC patients enrolled in two independent prospective clinical trials: PROPHECY, a clinical study of patients (n=118) treated with abiraterone or enzalutamide followed by docetaxel; and TAXYNERGY where patients were randomized to docetaxel or cabazitaxel treatment. CTCs were obtained at baseline (before treatment), on treatment and at progression and their comprehensive transcriptomic analysis was correlated with clinical outcomes. To uncover potential involvement of the circulating immune macroenvironment (CIME) in treatment resistance, we performed transcriptomic analysis of matching peripheral blood mononuclear cells (PBMCs) using an established, rigorous, blood-derived transcriptional modular framework. In PROPHECY, CTC RNA-seq identified that RB loss concurrently with enhanced E2F signaling networks were associated with intrinsic ARSi resistance. Using single sample GSEA (ssGSEA) score, we identified that the RB/E2F common signature at baseline was associated with short PFS (median PFS=6.5 months) and OS (median OS=24.5 months) (hazard ratio (HR) = 3.5; 95% CI 1.5-8.2) in men with mCRPC. We further developed a BRCA-loss transcriptional signature, and validated it in the SU2C mCRPC patient cohort, expanding the identification of patients with BRCA-loss phenotypes beyond genomic loss. Applying this signature to PROPHECY baseline samples, we showed that men with high BRCA-loss scores experienced shorter OS (HR=2.42; 95% CI=1-5.9). Through the comparison of CTC transcriptomic profiles at progression with baseline, we identified an inflammatory response signature in CTCs which was significantly associated with acquired ARSi resistance. Transcriptomic PBMC analysis further identified enrichment of inflammasome gene signatures at progression, with concurrent downregulation of CD8+ T and NK cells. Furthermore, preliminary data from both clinical trials, showed a significant upregulation of TGF-β1 and corresponding TGFβ-Receptor signaling pathway in CTCs from patients at progression following taxane treatment, suggesting a role for TGFβ pathway in clinical response to taxane chemotherapy. Taken together, these data demonstrate that liquid biopsy transcriptomics of both tumor cells and immune cells can identify molecular pathways associated with treatment resistance paving the way for treatment optimization and the development of novel precision therapies in patients with mCRPC. Citation Format: Jiaren Zhang, Bob Zimmermann, Giuseppe Galletti, Susan Halabi, Ada Gjyrezi, Qian Yang, Santosh Gupta, Akanksha Verma, Andrea Sboner, Monika Anand, Daniel J. George, Simon G. Gregory, Prerna Mahtani, Seunghee Hong, Virginia Pascual, Clio P. Mavragani, Emmanuel S. Antonarakis, David M. Nanus, Scott T. Tagawa, Olivier Elemento, Andrew J. Armstrong, Paraskevi Giannakakou. Liquid biopsy transcriptomics identify pathways associated with poor outcomes and immune phenotypes in men with mCRPC [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 646.

Resveratrol, Modafinil and Caffeine’s modulation of B cell’s CD19 activation receptor

Abstract Resveratrol a natural compound with anti-oxidant properties shown to inhibit cell proliferation and induces cellular apoptosis in Jurkat cell lines. Mechanistically, it was observed that interleukin-2 expression was blocked in T cells, suggesting resveratrol’s anti-inflammatory mechanism may involve downregulation of activation molecules. In this study, we investigated the effects of resveratrol or in combination with Modafinil or caffeine on a B cell line, Nor20. Modafinil is a compound shown to directly act on enzymes in the brain’s free-radical scavenging system and therefore directly reduce free radical levels. Caffeine has been shown to abolish the anti-inflammatory effects of resveratrol in astrocytes. Here we show that Nor20 treated with resveratrol can have up to 47.3% reduction in viability, consistent with previous observations. To determine a mechanism to explain these results, we used flow cytometry to measure the expression of CD19, the dominant receptor for all forms of B cell activation and modulator of B cell activation threshold combined with Modafinil or caffeine. We observed that resveratrol reduced the expression of CD19 up to 51%, Modafinil, inhibited up to 65%, but when resveratrol and Modafinil were combined the effect was not additive remaining at 53% inhibition when compare to the controls. In contrast, caffeine did not affect CD19 expression but when combined with resveratrol, caffeine abolished the downregulation of CD19 by resveratrol returning to the same levels as the untreated controls. Taken together, these results may indicate that resveratrol’s anti-inflammatory effects may initiate at the lymphocyte’s early recognition signaling receptors as a mechanisms to control cell growth. Supported by RevGenetics and Whittier College's Faculty Research Grant.

Increased angiopoietin-like 4 expression ameliorates inflammatory bowel diseases via suppressing CD8+ T cell activities

Angiopoietin-like 4 (ANGPTL4) is involved in inflammation-associated diseases, such as rheumatoid arthritis, type 2 diabetes, atherosclerosis, and chronic obstructive pulmonary disease. The role of ANGPTL4 in the pathogenesis of inflammatory bowel disease (IBD) remains unknown. Here, the plasma ANGPTL4 levels peaked on days 3 and 5, and expression of ANGPTL4 of inflamed colons peaked on days 5 and 7 in mice with dextran sulfate sodium (DSS)-induced colitis. Simultaneously, CD8+T cells in the inflamed colons peaked at day 5 but declined at day 7. However, the ANGPTL4−/− mice treated with DSS exhibited exacerbated colitis with more CD8+T cells and macrophages infiltrating the colons. The exogenous ANGPTL4 protein protected the mice against DSS-induced colitis with less CD8+T cell and macrophage recruitment in the colons. In addition, recombinant ANGPTL4 directly downregulated the IFN-γ and NKG2D expression of CD8+T cells but had no effects on the CD86 expression and TNF-α secretion of macrophages ex vivo. Adding ANGPTL4 protein into ANGPTL4−/− mice almost blocked the onset of DSS-induced colitis. In parallel, the plasma ANGPTL4 levels were elevated in patients with Crohn's disease and ulcerative colitis at mild/moderate stage and restored to normal levels in IBD patients at a severe stage. The higher ANGPTL4 expression in the inflamed colons of patients with IBD was correlated with lower CD8+ cell infiltration, whereas no associations with macrophages. Our results demonstrated the compensatory protective effect of ANGPTL4 on IBD development at least via the downregulation of CD8+T cell activities. Adding the ANGPTL4 protein would have beneficial effects to retard the progression of IBD.

Association of circulating tumor cell RB1 loss RNA signature with outcomes and immune phenotypes in men with mCRPC.

139 Background: Androgen receptor signaling inhibitors (ARSi) are a mainstay for patients with metastatic castration-resistant prostate cancer (mCRPC). However, patient response is heterogeneous and the molecular underpinnings of ARSi resistance are not well elucidated. Methods: We performed transcriptome analysis of circulating tumor cells (CTCs) and peripheral blood mononuclear cells (PBMC) in the context of PROPHECY, a prospective clinical trial of men (n = 118) with mCRPC treated with abiraterone (Abi) or enzalutamide (Enza). We obtained CTCs at baseline (before treatment) and at the time of progression on Abi/Enza, performed a comprehensive transcriptomic analysis of CTC patient samples (n = 40) and correlated with clinical outcomes to identify mechanisms of ARSi resistance. In addition, we also performed a transcriptomic analysis of matching peripheral blood mononuclear cells (PBMCs) in order to uncover potential involvement of the circulating immune macroenvironment (CIME) in ARSi resistance. The proportional hazard model was used to determine the prognostic significance of these signatures in predicting overall survival (OS) and progression-free survival (PFS). Results: CTC RNA-sequencing identified that RB loss concurrently with enhanced E2F signaling transcriptional networks were associated with intrinsic ARSi resistance. Using single sample GSEA (ssGSEA) score, we identified that the RB/E2F common signature at baseline was associated with short PFS (median PFS = 6.5 months) and OS (median OS = 24.5 months) (hazard ratio (HR) = 3.5; 95% CI 1.5-8.2) in men with mCRPC. We further developed a BRCA loss transcriptional signature which we validated in the SU2C mCRPC patient cohort, by showing that BRCA loss transcriptional network reflected BRCA genomic alterations as it was significantly enriched in the SU2C BRCA-altered patients vs unaltered patients. Generating BRCA loss ssGSEA scores in the PROPHECY cohort we observed that patients with high BRCA loss scores at baseline experienced shorter OS (HR = 2.42; 95% CI = 1-5.9). Through the comparison of CTC transcriptomic profiles at progression with baseline, we identified an inflammatory response signature in CTCs which was significantly associated with acquired ARSi resistance. Transcriptomic analysis of matching PBMCs identified enrichment of inflammasome gene signatures indicative of activated innate immunity at progression, with concurrent downregulation of CD8 T and NK cells. Importantly, CTC gene signatures of RB loss/E2F signaling had a significant positive association with this CIME signatures. Conclusions: Taken together, these data demonstrate that liquid biopsy transcriptomics of both tumor cells and immune cells can identify molecular pathways associated with clinical ARSi resistance paving the way for treatment optimization and the development of novel precision therapies in patients with mCRPC.

Characterization of Antigen-Induced CD4+ T-Cell Senescence in Multiple Sclerosis.

Antigen-induced T-cell exhaustion and T-cell senescence are peripheral regulatory mechanisms that control effector T-cell responses. Markers of exhaustion and senescence on T Cells indicate the previous activation by repetitive stimulation with specific antigens. Malignant tumors are accompanied by enhanced T-cell exhaustion and T-cell senescence resulting in immune evasion, while these control mechanisms might be diminished in autoimmune diseases including multiple sclerosis (MS). To better understand the involvement of antigen-induced T-cell senescence in controlling CD4+ T-cell-mediated autoimmune responses in MS, we have analyzed the re-expression of CD45RA and the downregulation of CD28 and CD27 molecules as markers of antigen-induced T-cell senescence in fresh cerebrospinal fluid (CSF)-infiltrating and paired circulating T cells from patients with MS. Patients with different levels of CD4+ T-cell senescence were identified and characterized regarding demographical and clinical features as well as intrathecal markers of neurodegeneration. CD4+ T-cell senescence was also analyzed in control patients to explore a putative deficit of this regulatory mechanism in MS. This study shows heterogeneity of markers of CD4+ T-cell senescence in patients with MS. Patients with high levels of CD4+ T-cell senescence in peripheral blood showed increased frequencies of CSF-infiltrating CD28+ CD27-EM CD4+ T cells with a proinflammatory Th1 functional phenotype. The correlation of these cells with the intrathecal levels of neurofilament light chain, a marker of neurodegeneration, suggests their relevance in disease pathogenesis and the involvement of T-cell senescence in their regulation. Markers of antigen-induced T-senescence, therefore, show promise as a tool to identify pathogenic CD4+ T cells in patients with MS.

Downregulated cytotoxic CD8+ T-cell identifies with the NKG2A-soluble HLA-E axis as a predictive biomarker and potential therapeutic target in keloids.

Keloids are an abnormal fibroproliferative wound-healing disease with a poorly understood pathogenesis, making it difficult to predict and prevent this disease in clinical settings. Identifying disease-specific signatures at the molecular and cellular levels in both the blood circulation and primary lesions is urgently needed to develop novel biomarkers for risk assessment and therapeutic targets for recurrence-free treatment. There is mounting evidence of immune cell dysregulation in keloid scarring. In this study, we aimed to profile keloid scar tissues and blood cells and found that downregulation of cytotoxic CD8+ T cells is a keloid signature in the peripheral blood and keloid lesions. Single-cell RNA sequencing revealed that the NKG2A/CD94 complex was specifically upregulated, which might contribute to the significant reduction in CTLs within the scar tissue boundary. In addition, the NKG2A/CD94 complex was associated with high serum levels of soluble human leukocyte antigen-E (sHLA-E). We subsequently measured sHLA-E in our hospital-based study cohort, consisting of 104 keloid patients, 512 healthy donors, and 100 patients with an interfering disease. The sensitivity and specificity of sHLA-E were 83.69% (87/104) and 92.16% (564/612), respectively, and hypertrophic scars and other unrelated diseases exhibited minimal interference with the test results. Furthermore, intralesional therapy with triamcinolone combined with 5-fluorouracil drastically decreased the sHLA-E levels in keloid patients with better prognostic outcomes, while an incomplete reduction in the sHLA-E levels in patient serum was associated with higher recurrence. sHLA-E may effectively serve as a diagnostic marker for assessing the risk of keloid formation and a prognostic marker for the clinical outcomes of intralesional treatment.

A Multi-Center Retrospective Study of Polatuzumab for Patients with Large B-Cell Lymphoma Relapsed after Standard of Care CAR T-Cell Therapy

A Multi-Center Retrospective Study of Polatuzumab for Patients with Large B-Cell Lymphoma Relapsed after Standard of Care CAR T-Cell Therapy

Semi-Quantitative Analysis of CD19 and CD22 Expression in B-Lymphoblastic Leukemia and Implications for Targerted Immunotherapy

IntroductionAcute lymphoblastic leukemia (ALL) is the most common childhood cancer, accounting for 30% of pediatric cancers and 75% of pediatric leukemias. While overall survival in children with ALL exceeds 85%, survival rates for children who relapse are low. In recent years, targeted immunotherapy has shown significant responses in refractory ALL. CAR-modified T-cells and Blinatumomab are two immunotherapy treatments targeting the CD19 antigen that have shown to be effective in treating patients with relapse refractory ALL. Moxetumomab and Inotuzumab are agents that target CD22 on lymphoblasts. However, some patients do not respond and others relapse with antigen-negative disease after targeted therapy. Expression levels of CD19 and CD22 may predict response to therapy. Hence, it is important to quantify expression levels of CD19 and CD22 in B lymphoblastic leukemias and correlate with response to immunotherapy.MethodsThe flow cytometry archives at the Children's Hospital of Philadelphia from 2002 - 2017 were reviewed. The majority of cases reviewed were de-novo ALL and prior to any targeted immunotherapy. The flow cytometry panels included CD45-PC7, CD19-PE, CD10-PC5, CD9-FITC, CD20-ECD, CD38-FITC, CD34-ECD, and CD22-PE antibodies (all from BD Biosciences). During this time period, the flow cytometry panels were similar, run on similar instruments and analyzed the same way. To quantify the expression of CD19, a 4-point logarithmic scale was used. The scale ranged from 0-3 with 0 representing no expression, 1 representing dim expression, 2 representing normal expression in circulating B cells, and 3 representing bright expression of the surface antigen. CD19 vs CD10 dot plots of CD45dim gated pure populations of blasts were used to accurately quantify the number of CD19-negative events. Other antigens expressed on blasts such as CD34, CD24, CD9, CD38, and HLA-Dr were used if the blasts were CD10-negative. Surface CD22 expression was determined using a 3 point scale since CD22 expression is universally weak compared to CD19. Proportion of CD22 negative cells could not be determined as there was no other blast marker along with CD22 in our panel. Cytogenetics, FISH, and whole genome SNP array (2008-2017) data were obtained on all cases. All cases showed strong expression of CD79a, terminal deoxy transferase (TdT) and lacked strong myeloperoxidase (MPO) or CD3 expression ruling out AML or a mixed phenotype acute leukemia per the current WHO criteria. In parallel, RNA expression data from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) study for Pediatric ALL was analyzed to identify cases with dim CD19 and CD22 expression.Results348 cases of B -ALL were identified, of which 304 were de novo and 45 were relapsed cases. 18% of all B-ALL specimens showed significant (>1%) populations of CD19-negative blasts. 7% of B-ALL specimens were negative for surface CD22. 5% of cases showed downregulation of both CD19 and CD22 antigens suggesting a correlation between various B cell antigens. Of the specimens that showed downregulation of CD19, 3% had the ETV6-RUNX1 translocation, 29% showed hyperdiploidy, and 19% showed rearrangement of the MLL gene. Of the specimens that showed downregulation of CD22, 17% showed the ETV6-RUNX1 translocation, 13% showed hyperdiploidy, and 13% showed rearrangement of the MLL gene. Cases with bright expression of CD19 or CD22 antigen were also determined. 12% of B-ALL showed overexpression of CD19 antigen when compared to normal mature B cells. 11% of B-ALL showed an overexpression of CD22 antigen when compared to normal mature B cells. Of the 157 cases analyzed from the TARGET study, 18% showed dim CD19 RNA expression and 11% showed dim CD22 RNA expression similar to the immunophenotypic findings.ConclusionWe have comprehensively analyzed CD19 and CD22 expression in a large number of B-ALL cases from a single institution. We conclude that a significant number of B-ALL cases have dim CD19 and CD22 that may correlate with response to immunotherapy. Many cases also showed significant populations of CD19-negative cells that may be responsible for antigen negative relapses. We also identified cases with bright CD19 and CD22 that may predict good response to immunotherapy. Future directions include correlation of antigen expression with response to immunotherapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Fas/FasL Signaling Regulates CD8 Expression During Exposure to Self-Antigens.

Activation of self-reactive CD8+ T cells induces a peripheral tolerance mechanism that involves loss of CD8 expression. Because genetic deficiency of Fas and Fasl causes the accumulation of double-negative (DN; CD3+ TCR-αβ+ CD4- CD8-) T cells that have been proposed to derive from CD8+ cells, we decided to explore the role of Fas and FasL in self-antigen-induced CD8 downregulation. To this end, we quantified Fas and FasL induction by different stimuli and analyzed the effects of Fas/FasL deficiency during a protective immune response and after exposure to self-antigens. Our data describes how Fas and FasL upregulation differs depending on the setting of CD8 T cell activation and demonstrates that Fas/FasL signaling maintains CD8 expression during repetitive antigen stimulation and following self-antigen encounter. Together, our results reveal an unexpected role of Fas/FasL signaling and offer a new insight into the role of these molecules in the regulation of immune tolerance.

A new risk factor indicator for papillary thyroid cancer based on immune infiltration

Increasing evidence has indicated a close association between immune infiltration in cancer and clinical outcomes. However, related research in thyroid cancer is still deficient. Our research comprehensively investigated the immune infiltration of thyroid cancer. Data derived from TCGA and GEO databases were analyzed by the CIBERSORT, ESTIMATE, and EPIC algorithms. The CIBERSORT algorithm calculates the proportions of 22 types of immune cells. ESTIMATE algorithm calculates a stromal score to represent all stromal cells in cancer. The EPIC algorithm calculates the proportions of cancer-associated fibroblasts (CAFs) and endothelial cells (ECs), which are the main components of stromal cells. We analyzed the correlation of immune infiltration with clinical characteristics and outcomes of patients. We determined that the infiltration of CD8+ T cells improved the survival of thyroid cancer patients. Overexpression of immune checkpoints was closely related to the development of thyroid cancer. In general, stromal cells were associated with the progression of thyroid cancer. Interestingly, CAFs and ECs had opposite roles in this process. In addition, the BRAFV600E mutation was related to the upregulation of immune checkpoints and CAFs and the downregulation of CD8+ T cells and ECs. Finally, we constructed an immune risk score model to predict the prognosis and development of thyroid cancer. Our research demonstrated a comprehensive panorama of immune infiltration in thyroid cancer, which may provide potential value for immunotherapy.

Downregulation of Lymphoid enhancer-binding factor 1 (LEF-1) expression (by immunohistochemistry and/ flow cytometry) in chronic Lymphocytic Leukemia with atypical immunophenotypic and cytologic features.

Lymphoid enhancer-binding factor 1 (LEF-1) overexpression has been recently remarkably reported in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and has shown utility in distinguishing CLL/SLL from other B-cell lymphomas. CLL has a well-defined immunophenotype, yet, some cases of CLL demonstrate atypical morphology/ phenotype reflected by low Matutes score (atypical CLL). Till date, LEF1 expression has not been systematically studied in cases of CLL with atypical features. In this study, LEF-1 expression was assessed by two different techniques, (immunohistochemistry and flow cytometry), to investigate the expression profile of LEF-1 in cases of CLL/SLL, in comparison with other low-grade B-lymphomas and CLL with atypical features, including atypical immunophenotype and CLL with increased prolymphocytes or morphologically atypical cells. We found that LEF-1 expression is downregulated in CLL with atypical immunophenotype/features compared to classic CLL; Chi-Square P<.0001. The ratio for LEF-1 expression in malignant B-cells/NK (by flow cytometry) in CLL/SLL with classic immunophenotype was higher than atypical CLL and is significantly higher in other small B-cell lymphomas (P<.01). Absence of LEF-1 expression in CLL/SLL is correlated (P<.05) with downregulation of CD5, CD23, CD200, expression of FMC7, brighter expression of CD79b, brighter expression of surface light chain, increased prolymphocytes and lower Matutes score. As downregulation of LEF-1 expression is well correlated with atypical CLL, we suggest adding LEF-1 to Matutes score as a beneficial marker to differentiate classic from atypical CLL LEF-1 could also serve as a potential prognostic indicator for CLL clinical course.

Increased Frequency of TOX+ CD39+ TIGIT+ CD73- CD8+ T Cells in Patients with Newly Diagnosed AML

Increased Frequency of TOX+ CD39+ TIGIT+ CD73- CD8+ T Cells in Patients with Newly Diagnosed AML

Obesity Attenuates T-Cell Function Which Impacts the Efficacy of T-Cell Based Immunotherapies in Acute Lymphoblastic Leukemia

The incidence of obesity continues to rise with over 50% of the world's population predicted to be overweight/obese by the year 2030. The global health impact of this trend is concerning given that obesity is a risk factor for developing cancers of varying etiologies. Alarmingly, the survival outcomes for obese patients with cancer are lower than those observed in lean patients. Obesity is characterized by the accumulation of adipocytes which alters drug dynamics and impacts the function of cancer and immune cells in the tumor microenvironment. Obesity-induced immune defects are troubling given the increasing use of immunotherapy in the treatment of malignancies. Here we show that adipocyte-secreted factors upregulate immunosuppressive mechanisms on human B-cell acute lymphoblastic leukemia (B-ALL) cells, attenuate the function of endogenous T-cells, and compromise the efficacy of T-cell based immunotherapies. To study the impact of adiposity on T-cell function, CD4+ and CD8+ T-cells were purified from the spleens of C57BL/6 mice and activated with PMA/Ionomycin for 72 hours in unconditioned media (UCM), bone marrow stromal-cell conditioned media (SCM), and adipocyte-conditioned media (ACM) followed by flow cytometry analysis for surface marker expression, cytokine production, and the induction of cytolytic mediators. Interestingly, T-cells activated in ACM, but not UCM or SCM, showed an attenuated phenotype highlighted by decreased CD44 and PD-1 expression, diminished cytokine production (IFN-γ/TNF-α) and reduced induction of cytolytic mediators (granzyme B/perforin). These observations were also true in obese, relative to lean, patients with B-ALL where we found that T-cells purified from the peripheral blood mononuclear cells (PBMCs) failed to produce significant levels of TNF-α when stimulated with PMA/Ionomycin. In all, these results demonstrate that adipocyte-secreted factors directly compromise the function of endogenous T-cells, which phenocopies T-cell defects observed in obese relative to lean pediatric patients with B-ALL. We next assessed the impact of adiposity on malignant cells by culturing human B-ALL cell lines in the conditioned mediums described above and performed flow cytometric analysis to assess their surface expression of the B-cell lineage antigen CD19 and proteins that modulate immunity. In addition to being a marker for B-cells, CD19 is the primary target of the T-cell based immunotherapies Blinatumomab and CAR T-cells directed against B-ALL cells. Surprisingly, when human B-ALL cells were co-cultured with adipocytes, every cell line tested (n=6) exhibited lower surface CD19 expression with 5 out 6 reaching statistical significance. Furthermore, adipocyte-secreted factors alone were sufficient to reduce CD19 surface levels on B-ALL cells in 2 of the 6 cell lines tested. Human B-ALL cells cultured in ACM, but not UCM or ACM, also upregulated their surface expression of the immunoinhibitory proteins HVEM, PD-L1, and PD-L2. These results demonstrate that adipocytes directly induce the downregulation of CD19 on human B-ALLs and increase their immune evasive capacity. Given these observations, we hypothesized that adipocyte-secreted factors would compromise T-cell-based immunotherapies targeting CD19-expressing B-ALL cells. To this end, primary human T-cells were engineered to express a CD19-directed chimeric antigen receptor (CAR). CAR T-cells and human B-ALL cells were separately pre-treated for 24 hours in UCM, SCM or ACM followed by co-culture for cytolytic analysis using Annexin-V/PI staining. Adipocyte-secreted factors significantly inhibited CAR T-cell mediated killing of CD19-expressing B-ALL cells at 4 hours. In addition to CAR T-cells, we tested the leukemia killing efficacy of the bispecific T-cell engager, Blinatumomab. After 3 days of culture, we found that Blinatumomab significantly increased the killing capacity of endogenous T-cells with 60-80% of B-ALL cells being killed after 3 days of culture in UCM and SCM. In contrast, we found that ACM significantly compromised the efficacy Blinatumomab with only 30% of B-ALL cells being killed over 3 days when co-cultured with human T-cells. Our pre-clinical data highlights the negative impact of an adipose-rich microenvironment on T-cell function and B-ALL immunogenicity, which subsequently compromises the efficacy of multiple classes of immunotherapies targeting CD19. Disclosures No relevant conflicts of interest to declare.

Abstract 422: IL-10 inhibition accelerates gastric tumorigenesis in mice with either Helicobacter pylori infection or chronic alcohol intake

Abstract Background and Aims: H. pylori is a class I human carcinogen that contributes to the development of gastric cancer through complex interactions between genetic and environmental factors. Alcohol is also classified as a group 1 carcinogen and results in 3.3 million deaths every year. H. pylori infection and alcohol intake are risk factors in gastric carcinogenesis; however, no conclusive model or experimental evidence yet exists showing that a combination of H. pylori and alcohol increases the risk of gastric cancer. Hence, we hypothesized that alcohol and H. pylori infection alone cannot produce gastric cancer in human and mice but combination of both will produce gastric cancer. Here, we provide insight into the molecular mechanisms driving gastric carcinogenesis. Methods: Here, we developed a series of mouse models that progress from chronic gastritis to gastric cancer, induced by infecting H. pylori combined with chronic alcohol consumption and then determining the molecular mechanism of the progression C57-BL/6J-219 and IL-10 knockout mice were infected with the H. pylori (SS1) strain and then fed or not fed alcohol. Results: Here, we show that alcohol consumption in combination with H. pylori infection induce gastric cancer with the IL-10 downregulation through metabolic dysfunction in CD8+ cells. IL-10 knockout accelerates tumor development in mice with either H. pylori infection or alcohol induced gastric cancer or both. Specifically, we show IL-10 inhibits glucose uptake and glycolysis and promotes oxidative phosphorylation with lactate inhibition. Consequently, in the absence of IL-10 signaling, CD8+ cells accumulate damaged mitochondria in a mouse model of gastric cancer induced with the combination of alcohol plus H. pylori infection, and this result in mitochondrial dysfunction and production of IL-1β. Furthermore, downregulation of IL-10 and CD8+ cells switch to stimulate IL1β secretion that facilitates H. pylori colonization to gastric cancer cells and suppress NKX6.3 expression as well as cancer cells apoptosis. Overall, the molecular mechanisms of gastric carcinogenesis include IL-10 inhibition resulting in lowered host immunity via mitochondrial dysfunction of CD8+ lymphocytes; gastric inflammation due to H. pylori infection, alcohol intake, and IL-1β production; and disruption of gastric-specific tumor suppressor NKX6.3 expression, which increases cancer cell survival and proliferation. This is the first study providing evidence that a combination of H. pylori infection and alcohol consumption could lead to the development and progression of gastritis leading to gastric carcinogenesis. Conclusions: This study suggests that anti-inflammatory IL-10 cytokine stimulation could reduce the incidence of gastric cancer, along with lowered alcohol consumption and treatment of H. pylori infection. IL-10 may be a potential therapeutic target and its stimulation may be a novel therapeutic strategy for the treatment of alcohol-associated gastric cancer. Citation Format: Faisal aziz, Ann Bode, Zigang Dong. IL-10 inhibition accelerates gastric tumorigenesis in mice with either Helicobacter pylori infection or chronic alcohol intake [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 422.

Abstract 5516: Immunological effects of SYK inhibition using TAK659 in patients diagnosed with diffuse large B cell lymphoma

Abstract Targeted inhibition of the BCR signaling pathway is a therapeutic option for patients diagnosed with Diffuse Large B-cell Lymphoma (DLBCL) currently under clinical evaluation. Inhibition of the proximal BCR signaling kinase SYK has been shown to have direct cytotoxic effect on malignant B lymphocytes. Moreover, SYK protein also participates in immumomodulatory processes involving the inflammasome, Fcγ receptor and T cell responses in non-malignant settings. Herein we aimed to study the immunological effects of treatment with TAK659 in patients diagnosed with DLBCL. For that we longitudinally studied changes in immune cells subpopulations and cytokines in the peripheral blood of seven patients undergoing treatment with TAK659 (phase I, first in human clinical trial; NCT03123393). Patients received 100 mg of TAK659 orally once a day in 28-day cycles until disease progression, unacceptable toxicities, or withdrawal for other reasons (estimated median treatment duration 6 months). Peripheral blood was collected serially before and during treatment (after 1 week). The relative percentages of CD4 and CD8 T cells were not significantly modified. Interestingly, treatment with TAK659 significantly induced a reduction in regulatory T cells after 1 week. Additionally, treatment with TAK659 induced an increase in the proportion of central memory CD4 T cells after 1 week of treatment while effector memory went down, indicating a shift towards a more effector T cell profile. Regarding the expression of inhibitory receptors we observed a downregulation of PD-1 and LAG-3 in both CD4 and CD8 T cells after 1 week of treatment, as well as downregulation of CD4 and CD8 co-expressing PD-1 with LAG-3. Finally, we observed a reduction in the levels of IFN-γ in the plasma of patients after 1 week of treatment while TNF-α and IL-10 did not change. In order to assess if some of the immunological effects could be due to direct effect of TAK659 in T lymphocytes we treated PBMCs from healthy donors with increasing doses of TAK659 for 30' before 48 hours of TCR stimulation. The in vitro stimulation of T lymphocytes induced an increase in the proportion of regulatory T cells as well as induction of exhaustion markers in both CD4 and CD8 T cells that was counteracted by treatment with TAK659 that did not affect their viability. Overall, these findings provide initial evidence of the role of TAK 659 in potentiating the anti-tumoral immune response during treatment of DLBCL. Therefore, we believe that treatment with TAK659 can help to reinstall immune surveillance by exerting direct immunomodulatory effects which provides rationale for combination therapy not only in B-cell derived malignancies but also in solid tumors. Citation Format: Marta Crespo, Juan C. Nieto, Cristina Hernández, Cecilia del Carmen Carpio, Isabel Jiménez, Carlos Palacio, Júlia Carabia, Pau Abrisqueta, Francesc Bosch. Immunological effects of SYK inhibition using TAK659 in patients diagnosed with diffuse large B cell lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5516.

Abstract 531: AT1412, a patient-derived antibody in development for the treatment of CD9-positive precursor B-acute lymphoblastic leukemia

Abstract Introduction Despite rapid advances in immunotherapeutic options for precursor B-acute lymphoblastic leukemia (BCP-ALL), outcomes remain poor especially for adult ALL and relapsed pediatric ALL. With conventional chemotherapy remission percentages in adult ALL range from 75 to 90%, but relapse rates are high and long-term leukemia-free survival ranges between 35-70% depending on age and risk group. The introduction of CD19-targeting immunotherapy has significantly improved patient outcomes in (relapsed) BCP-ALL. However, tumor escape via downregulation of CD19 occurs in a significant number of patients. An ongoing medical need remains for the identification of alternative immunotherapeutic targets for treatment of ALL. Methods CD9 is expressed in 60-80% of BCP-ALL, is correlated with adverse prognosis and has been proposed as therapeutic target for BCP-ALL. AT1412 is a fully human CD9-targeting antibody, that was identified from a patient cured of metastatic melanoma after adoptive T-cell therapy, using our B-cell immortalization technology (AIMSelect) [Kwakkenbos et al. Nat. Med. 2010]. The antibody was selected based on differential binding to melanoma cells as compared to healthy melanocytes and was shown to be successful in killing melanoma cells in vitro and in vivo. Results Binding of AT1412 to BCP-ALL cell lines SUP-B15, MHH-CALL-2 and CCRF-SB varied as expected based on CD9 levels that we detected using a commercial CD9 antibody. AT1412-induced antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP) correlated with the level of AT1412 binding. No binding was seen to the T-ALL cell line Jurkat. These findings were confirmed in primary ALL samples, obtained prospectively at diagnosis from a cohort of patients with T- or B-ALL (n=38). AT1412 showed binding to the majority of B-ALL samples but not to T-ALL samples. AT1412 induced ADCC of nearly all B-ALL samples it bound to (10/11) and of none of the non-binding B-ALL or T-ALL samples. Cytotoxicity significantly correlated with the level of AT1412 binding. These findings were corroborated by the observation that AT1412 induced specific B-ALL cell death when a bone marrow sample from a newly diagnosed BCP-ALL patient was incubated with AT1412. Remarkably, AT1412 induced cell death in the absence of added effector cells or other (chemo)therapeutic agents, while the sample contained over 80% blasts and as little as 3% lymphocytes. We are currently investigating the in vivo efficacy of the antibody in a humanized immune system mouse model with human BCP-ALL. Conclusion Taken together, the majority of BCP-ALL express CD9 and this is associated with poor prognosis. Our data demonstrate that CD9 can be successfully targeted by the human CD9 antibody AT1412, suggesting that AT1412 has the potential to be developed as a therapeutic antibody for B-ALL. AT1412 is currently being advanced through preclinical development. Citation Format: Greta de Jong, Sophie E. Levie, Remko Schotte, Wouter Pos, Daniel Go, Etsuko Yasuda, Madalina Cercel, Susan E. van Hal, Esmay Frankin, Aniko Szabo, Martijn Kedde, Els Verdegaal, Julien Villaudy, Sjoerd van der Burg, Pauline M. van Helden, Hans van Eenennaam, Hergen Spits, Anita van Rijneveld, Mette D. Hazenberg. AT1412, a patient-derived antibody in development for the treatment of CD9-positive precursor B-acute lymphoblastic leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 531.