T-cell Surface Research Articles

P233 Environmental and occupational exposures in the development of systemic lupus erythematosus in adults: a narrative review

Abstract Background/Aims Systemic lupus erythematosus (SLE) is a chronic inflammatory disease, of an autoimmune nature, of unknown origin, characterized by loss of tolerance to nuclear antigens and the deposition of immunecomplexes in the tissues leading to inflammation that can affect various organs and systems of the body. This narrative review aims to demonstrate that occupational factors and environmental exposure contribute to the development of SLE in adults. Methods This is a narrative review of literature that seeks to analyse the scientific production on environmental and occupational exposure in the development of systemic lupus erythematosus in adults, published in health journals. A literature search was conducted in the period between November 2020 and July 2021, considering the object of study, initially the articles related to the theme addressed were searched in the PubMed database, in the US National Institutes of Health (NIH) and Scientific Electronic Library Online (SCIELO) portals. Results Exposure to respirable silica dust (i.e. the crystalline form of quartz) is an established risk factor for SLE and other systemic autoimmune diseases. Exposure to toxic components of cigarette smoke (e.g., tar, nicotine, carbon monoxide, polycyclic aromatic hydrocarbons, and free radicals) can induce oxidative stress and directly damage endogenous proteins and DNA, leading to genetic mutations and gene activation, which may be involved in the development of SLE. Smoking stimulates the expression of CD95 on B and CD4 on T-cell surfaces, potentially inducing autoimmunity, and also increases the production of pro- inflammatory cytokines. Exposure to ultraviolet (UV) radiation may exacerbate pre-existing SLE; it is not yet known whether UV exposure plays a role in the pathogenesis of SLE. Thus, UV may result in a redistribution of nuclear antigens to the cell surface or in the production of new forms of autoantigens, effects that may be relevant given the mechanisms believed to be involved in SLE etiology. Several hypotheses have been postulated that air pollution may contribute to autoimmune diseases via oxidative stress, nitrosative stress, and systemic inflammation. Traffic-related air pollutants may cause oxidative stress and and systemic inflammation. The combination of CO with hemoglobin (Hb) to form carboxyhemoglobin (COHb) reduces the oxygen-carrying capacity of the blood and causes tissue hypoxia. Conclusion The articles analysed identified risk factors for exposure to current smoking, silica, ultraviolet radiation, and environmental pollution from CO, NO2, and minute particles. These are caused by oxidative stress, apoptosis inducers, systemic inflammation, genetic mutations, and activation of genes that will lead to immunological changes with increased pro-inflammatory cytokines, activation of genes, and increased autoantigens, leading to predisposition of these exposures in the development of systemic lupus erythematosus. In contrast, alcohol has been shown to be a neutralizing factor in systemic inflammation, causing a decrease in cellular responses and suppressing the synthesis of pro-inflammatory cytokines, showing the association of alcohol with a lower risk of developing SLE. Disclosure B.G. Monteiro: Grants/research support; FUNADESP. C.B. Pizarro: Grants/research support; FUNADESP.

Genotyping An Autoimmune-Associated SIRPg SNP From Archived Oral Biopsy FFPE Tissues

<h3>Introduction</h3> T-cells play critical roles in many autoimmune conditions, such as type I diabetes mellitus (T1D). They are also thought to play a significant role in many immune-mediated disorders affecting the oral soft tissues: lichen planus, graft vs. host disease, Sjogren syndrome. T-cells exert their functions via direct recognition through molecules expressed on the cell surface and secretion of factors that drive or suppress inflammatory responses locally. Signal regulatory protein gamma (SIRPg) is an immunomodulatory protein that is uniquely expressed on the cell surface of human T-cells. Our laboratory has shown that a T1D-linked genetic variant of the gene for SIRPg (rs2281808) correlates with reduced expression of SIRPg on the T-cell surface and that T-cells with less SIRPg surface expression produce more inflammatory molecules. We hypothesize that single nucleotide changes within the gene for SIRPg can lead to a predisposition for the development of immune-mediated pathosis. Our objective for this study was to assess the feasibility of genotyping individuals at single nucleotide polymorphism (SNP) rs2281808 from genomic DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue blocks. <h3>Materials and Methods</h3> Archived, de-identified formalin-fixed paraffin-embedded tissue blocks from oral soft tissue biopsies were used in this study. The tissue blocks ranged from 1-18 years of age. Three 5-mm thick sections per sample were deparaffinized with subsequent genomic DNA extraction using the QIAamp® DNA FFPE Tissue Kit and Deparaffinization Solution. DNA was quantitated using UV spectroscopy at 260 nm. Allelic discrimination PCR for rs2281808 genotyping was done using a TaqMan® assay and probes. <h3>Results</h3> Allelic discrimination PCR for rs2281808 was successful in ∼77% (46/60) of specimens tested. <h3>Conclusions</h3> This pilot study supports the feasibility of our methodology, however, further studies are necessary to assess the accuracy of the genotyping results obtained via this method.

Overcoming biases in causal inference of molecular interactions.

Computer inference of biological mechanisms is increasingly approachable due to dynamically rich data sources such as single-cell genomics. Inferred molecular interactions can prioritize hypotheses for wet-lab experiments to expedite biological discovery. However, complex data often come with unwanted biological or technical variations, exposing biases over marginal distribution and sample size in current methods to favor spurious causal relationships. Considering function direction and strength as evidence for causality, we present an adapted functional chi-squared test (AdpFunChisq) that rewards functional patterns over non-functional or independent patterns. On synthetic and three biology datasets, we demonstrate the advantages of AdpFunChisq over 10 methods on overcoming biases that give rise to wide fluctuations in the performance of alternative approaches. On single-cell multiomics data of multiple phenotype acute leukemia, we found that the T-cell surface glycoprotein CD3 delta chain may causally mediate specific genes in the viral carcinogenesis pathway. Using the causality-by-functionality principle, AdpFunChisq offers a viable option for robust causal inference in dynamical systems. The AdpFunChisq test is implemented in the R package 'FunChisq' (2.5.2 or above) at https://cran.r-project.org/package=FunChisq. All other source code along with pre-processed data is available at Code Ocean https://doi.org/10.24433/CO.2907738.v1. Supplementary materials are available at Bioinformatics online.

Gut Microbiota Modulates the Efficiency of Programmed Cell Death Protein 1 Cancer Immunotherapies.

Program cell death protein 1 (PD1) is considered as an inhibitory molecule that is expressed on the surface of activated T-cells and bound to PD-L1 and PD-L2 ligands. Several types of cancer cells express PD-L1 which can bind to PD1 on the surface of tumor-specific T-cells. PD1/PD-L1 ligation triggers a pathway to protect tumor cells from an effective response of tumor-specific T-cells. Different PD1/PD-L1 blocker antibodies are clinically used to promote the T-cell response against the cancer cells. Current studies suggest that the gut microbiome impacts the efficiency of PD1 blockade therapy in cancer patients. The association of several bacterial species with PD1 responder patients has been determined. The present study reviewed previous reports on the relation between the microbiome and immune checkpoint therapy (ICT). The results of studies were discussed considering adjuvant and molecular mimicry of microbial antigens by tumor-associated antigens and metabolic effects of microbial products on ICT.

Imaging T-cell activation: topography and features of early T-cell contacts

T cells are the drivers of adaptive immunity and detect antigens with unprecedented sensitivity and selectivity. To understand this process, studying the very first interaction of a T cell with an antigen presenting cell (APC) is key. In order to detect an antigen, a close physical contact with the APC needs to be established. This process heavily relies on the interaction of adhesion proteins. Furthermore, the T-cell surface is dominated by microvilli (MV), which are dynamic membrane protrusions involved in antigen search.

Expression and polymorphisms of CD8B gene and its associations with body weight and size traits in sheep

The growth traits are economically important traits in sheep. Improving growth rates will increase the profitability of producers. The aim of this study was to identify alleles of CD8B (encoding T-cell surface glycoprotein CD8 beta chain) that are aberrantly expressed in different tissues and to assess the effects and associations of its different genotypes on weight and size traits in sheep. Using quantitative real-time reverse transcription PCR arrays, expression profiling of CD8B was performed in various organs and tissues. CD8B was ubiquitously expressed, with very high expression in the lung, spleen, lymph, duodenum, and liver. One intronic mutation (chr3:62,718,030 (Oar_rambouillet_v1.0, same below) G > A) was identified using pooled DNA sequencing. Subsequently, the variants (AA, AG, and GG) were genotyped using the KASPar® PCR single nucleotide polymorphism (SNP) genotyping system. The results of association analysis with body weight and body size traits in 1304 sheep showed that increases in multiple phenotypic traits correlated with the AA genotype (body weight, p < 0.05; body length, p < 0.05). Thus, SNP chr3:62,718,030 G > A is a promising molecular marker for marker-assisted selection in sheep breeding.

T-cell surface generation of dual bivalent, bispecific T-cell engaging, RNA duplex cross-linked antibodies (dbBiTERs) for re-directed tumor cell lysis.

Genetic engineered Bispecific T-cell engagers (BiTEs) generate potent cytotoxic effects. Alternately, click chemistry engineered, dual specific bivalent Bispecific T-cell engaging antibodies (dbBiTEs) on T-cell surfaces can be generated from parent monoclonal antibodies. We show the formation of dbBiTEs on the surface of T-cells along with the introduction of complementary 2'-OMe RNA 32-mer oligonucleotides allowing duplex formation between antibodies, designated as dbBiTERs. dbBiTERs generated in solution from anti-CEA and anti-CD3 OKT3 antibodies retained specific binding to CEA positive versus CEA negative cancer cells and to CD3 positive T-cells comparable to dbBiTEs. When T-cells were precoated with dbBiTEs or dbBiTERs and mixed with CEA positive versus CEA negative cancer cells, similar dose dependent and specific cytotoxicity were observed in redirected cell lysis assays. On-cell generated dbBiTERs exerted potent cytotoxic responses against CEA positive targets and were localized at the cell surface by immuno-gold EM. In addition, we demonstrate that target and T-cells, each coated separately with complementary 2'OMe-RNA-linked antibodies can be cross-linked by RNA duplex formation in vitro to generate redirected cell lysis. The facile generation of dbBiTERs with specific cytolytic activity from intact antibodies and their generation on-cell offers a new avenue for antigen specific T-cell therapy.

CD247, a Potential T Cell-Derived Disease Severity and Prognostic Biomarker in Patients With Idiopathic Pulmonary Fibrosis.

BackgroundIdiopathic pulmonary fibrosis (IPF) has high mortality worldwide. The CD247 molecule (CD247, as known as T-cell surface glycoprotein CD3 zeta chain) has been reported as a susceptibility locus in systemic sclerosis, but its correlation with IPF remains unclear.MethodsDatasets were acquired by researching the Gene Expression Omnibus (GEO). CD247 was identified as the hub gene associated with percent predicted diffusion capacity of the lung for carbon monoxide (Dlco% predicted) and prognosis according to Pearson correlation, logistic regression, and survival analysis.ResultsCD247 is significantly downregulated in patients with IPF compared with controls in both blood and lung tissue samples. Moreover, CD247 is significantly positively associated with Dlco% predicted in blood and lung tissue samples. Patients with low-expression CD247 had shorter transplant-free survival (TFS) time and more composite end-point events (CEP, death, or decline in FVC >10% over a 6-month period) compared with patients with high-expression CD247 (blood). Moreover, in the follow-up 1st, 3rd, 6th, and 12th months, low expression of CD247 was still the risk factor of CEP in the GSE93606 dataset (blood). Thirteen genes were found to interact with CD247 according to the protein–protein interaction network, and the 14 genes including CD247 were associated with the functions of T cells and natural killer (NK) cells such as PD-L1 expression and PD-1 checkpoint pathway and NK cell-mediated cytotoxicity. Furthermore, we also found that a low expression of CD247 might be associated with a lower activity of TIL (tumor-infiltrating lymphocytes), checkpoint, and cytolytic activity and a higher activity of macrophages and neutrophils.ConclusionThese results imply that CD247 may be a potential T cell-derived disease severity and prognostic biomarker for IPF.

Epigenetic Priming of Bladder Cancer Cells With Decitabine Increases Cytotoxicity of Human EGFR and CD44v6 CAR Engineered T-Cells.

BackgroundTreatment of B-cell malignancies with CD19-directed chimeric antigen receptor (CAR) T-cells marked a new era in immunotherapy, which yet has to be successfully adopted to solid cancers. Epigenetic inhibitors of DNA methyltransferases (DNMTi) and histone deacetylases (HDACi) can induce broad changes in gene expression of malignant cells, thus making these inhibitors interesting combination partners for immunotherapeutic approaches.MethodsUrothelial carcinoma cell lines (UCC) and benign uroepithelial HBLAK cells pretreated with the DNMTi decitabine or the HDACi romidepsin were co-incubated with CAR T-cells directed against EGFR or CD44v6, and subsequent cytotoxicity assays were performed. Effects on T-cell cytotoxicity and surface antigen expression on UCC were determined by flow cytometry. We also performed next-generation mRNA sequencing of inhibitor-treated UCC and siRNA-mediated knockdown of potential regulators of CAR T-cell killing.ResultsExposure to decitabine but not romidepsin enhanced CAR T-cell cytotoxicity towards all UCC lines, but not towards the benign HBLAK cells. Increased killing could neither be attributed to enhanced target antigen expression (EGFR and CD44v6) nor fully explained by changes in the T-cell ligands PD-L1, PD-L2, ICAM-1, or CD95. Instead, gene expression analysis suggested that regulators of cell survival and apoptosis were differentially induced by the treatment. Decitabine altered the balance between survival and apoptosis factors towards an apoptosis-sensitive state associated with increased CAR T-cell killing, while romidepsin, at least partially, tilted this balance in the opposite direction. Knockdown experiments with siRNA in UCC confirmed BID and BCL2L1/BCLX as two key factors for the altered susceptibility of the UCC.ConclusionOur data suggest that the combination of decitabine with CAR T-cell therapy is an attractive novel therapeutic approach to enhance tumor-specific killing of bladder cancer. Since BID and BCL2L1 are essential determinants for the susceptibility of a wide variety of malignant cells, their targeting might be additionally suitable for combination with immunotherapies, e.g., CAR T-cells or checkpoint inhibitors in other malignancies.

Phase 2 Study of Nivolumab in Epstein-Barr Virus (EBV)-Positive Lymphoproliferative Disorders and EBV-Positive Non-Hodgkin Lymphomas

Introduction: Immune tolerance and evasion plays a significant role in the pathogenesis of EBV+ lymphoproliferative disorders (LPD) and non-Hodgkin lymphomas (NHL). Programmed cell death protein-1 (PD-1) is a signaling molecule on the surface of T-cells that suppresses the cytotoxic effects of T-cells on tumor cells. PD-L1 expression is a marker of poor prognosis in aggressive lymphomas and most EBV+ LPDs demonstrate high levels of PD-L1 expression. Chronic viral infections, such as EBV, also result in T-cell exhaustion that can be reversed by PD-1 blockade. Nivolumab is a fully human IgG4 monoclonal anti-PD-1 antibody which has demonstrated activity and favorable safety in lymphoid malignancies. We hypothesized that PD-1 blockade may reverse the inactivation of tumor-specific effector T-cells and result in anti-tumor responses in EBV+ LPD and NHL.Methods: Relapsed/refractory (R/R) EBV+ LPD and B-cell NHL pts age ≥ 12y with adequate organ function are eligible. Untreated pts are eligible if EBV+ LPD. Exclusions include prior use of PD-1/PD-L1/PD-L2/CD137/CTLA-4 antibodies, prior solid organ transplant and HIV. Pts with immunodeficiency or autoimmune illness are eligible if not requiring steroids or immunosuppression. CNS involvement is permitted if no seizure activity within 4 weeks of study. Nivolumab 480mg IV is given every four weeks for up to 2y. Pts who achieve CR discontinue nivolumab after 1y of treatment. Baseline evaluation includes CT, PET, MRI brain, flow cytometry of peripheral blood and CSF, BM biopsy along with optional tumor biopsy. CT scans are performed after cycles 3, 6, 13 and 19 and end of treatment (EoT). PET is performed after cycles 1, 3 and EoT. Surveillance CT scans are performed q3m for 1y, q6m for yrs 2-5, and annually thereafter.Results: 9 pts, 7 (78%) R/R and 2 (22%) untreated, enrolled between April 2018 and May 2021; 5 (56%) with EBV+ LPD [4 G1-2 lymphomatoid granulomatosis (LYG) and 1 chronic active EBV disease (CAEBV)] and 4 (44%) with EBV+ NHL (all DLBCL, NOS). Median age was 48y (range 30-63) and all pts (100%) had stage III/IV disease. Four pts (44%) had elevated LDH (all DLBCL). Median baseline CD4 and CD8 count (cells/mcL) was 378 (range 99-984) and 86 (range 22-1237), respectively, for LPD and 190 (range 133-255) and 90 (range 9-630), respectively, for NHL. Median EBV VL at baseline (Log10 IU/mL) was 2.55 (range 0-6.78) and 2.53 (range 0-5.33) for LPD and NHL, respectively. Eight (89%) pts had extranodal disease with pulmonary involvement most common in 6 (67%). Median prior therapies were 1 (range 0-1) and 2 (range 1-4) for LPD and NHL pts, respectively. Three (43%) R/R pts were refractory (i.e., <PR) to last therapy.Of 9 pts enrolled, 7 were evaluable for response (1 NHL pt died prior to restaging and 1 NHL pt has not yet been restaged). In 6 measurable pts, tumor reduction was observed in 67% (Fig 1A). ORR and CR rate was 57% (4/7) and 43% (3/7), respectively; 60% (3/5) and 40% (2/5) in LPD and 50% (1/2) and 50% (1/2) in NHL. Median TTR was 3.0m with 3 (75%) of 4 responses ongoing from 6.9m to 35.2m after first response (Fig 1B).Most common adverse events (AEs) (% pts) included maculopapular rash (38%), ALT elevation (25%), AST elevation (25%), CPK elevation (25%) and fatigue (25%). One pt discontinued therapy due to G2 immune-mediated myositis that required prolonged steroid therapy. >G3 AEs included AST elevation in 1 (13%) pt with no G4/G5 or serious adverse events.With a median potential follow up of 12.6m, 12-month PFS and OS was 50.8% (95% CI: 15.7-78.1) and 75.0% (95% CI: 31.5-93.1). In LPD pts, 12-month PFS and OS was 80% (95% CI: 20.4-96.9) and 100%. Three (75%) NHL pts progressed and 2 (50%) died of disease progression. One NHL pt stopped therapy due to apparent disease progression after 2 cycles but later developed CR without further therapy and remains in remission 35.2m after stopping therapy.Conclusion: Nivolumab appears safe in pts with EBV+ LPD and NHL without unexpected toxicities. Preliminary clinical activity, including CRs, is noted in pts with EBV+ LPD and NHL. Additional pts are needed for a better assessment of true activity in these rare entities and correlates of response including PD-1/PD-L1 expression and/or 9p24.1 alterations are ongoing and will be presented at the meeting. [Display omitted] DisclosuresNo relevant conflicts of interest to declare. OffLabel Disclosure:Nivolumab for EBV+ LPD and EBV+ NHL.

A Novel Cotinine-Based System for Switchable Chimeric Antigen Receptor T Cell Immunotherapy

Autologous T cells engineered to express a chimeric antigen receptor (CAR-T cells) have shown impressive outcomes in hematologic malignancies. However, safety concerns derived from the inability to control CAR-T cells in vivo remain a significant challenge to expand the application of CAR-T cell therapy. Switchable CAR-T cells can allow for turning on and off the activation and cytotoxicity of CAR-T cells in vivo, potentially increasing the safety of next-generation CART products. To this goal, CAR constructs targeting non-tumor antigens, e.g., haptens and peptide tags, can be designed and combined with tumor-targeting units that are fused to the hapten or peptide tag. In this study, we developed a novel switchable CAR-T system using cotinine as the hapten and an anti-cotinine CAR-T cell product. Of note, cotinine is physiologically absent in human and pharmacologically inert. A mouse antibody to cotinine was developed and successfully humanized. The anti-cotinine CAR confirmed to be displayed on T-cell surface. We first tested this approach to target epidermal growth factor receptor 2 (HER2) in ovarian cancer. To target HER2, we developed an affibody-based antigen binding domain characterized by high stability, broad spectrum of specificity, and feasibility of large scale chemical synthesis. In in vitro cytotoxicity and cytokine release assays, the activity of CAR-T cells was controlled by cotinine-tagged anti-HER2 affibody in a HER2-specific and dose-dependent manner. We synthesized the cotinine-tagged HER2 affibodies using single isoform cotinine and selected the optimal cotinine-tagged anti-HER2 affibody at the cotinine-based switchable CAR-T system. In an orthotopic ovarian cancer model using NSG mice engrafted with SKOV3, tumor regression was observed only when CAR-T cells were administered in combination with the cotinine-tagged HER2 affibody. In subsequent studies, we demonstrated that the cotinine-based switchable CAR-T system can be applied to other tumor targets such as mesothelin and epidermal growth factor receptor (EGFR). Additional studies are ongoing to determine the kinetics of the on and off CAR-T activation of this system and define the optimal dosing strategy to ensure anti-tumor effect or to interrupt toxicity. In conclusion, we have developed a novel switchable cotinine-based CAR-T system to control CAR-T function in vivo and that could be broadly applicable to wide variety of tumors. DisclosuresKim: AbClon Inc.: Current Employment. Lee: AbClon Inc.: Current Employment. Ruella: viTToria biotherapeutics: Research Funding; Tmunity: Patents & Royalties; AbClon: Consultancy, Research Funding; BMS, BAYER, GSK: Consultancy; Novartis: Patents & Royalties. Chung: AbClon Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Correlative Biological Studies Related to the Response, Peak and Persistence of ARI0002h, an Academic BCMA-Directed CAR-T Cell, with Fractionated Initial Infusion and Booster Dose for Patients with Relapsed and/or Refractory Multiple Myeloma (RRMM)

Correlative Biological Studies Related to the Response, Peak and Persistence of ARI0002h, an Academic BCMA-Directed CAR-T Cell, with Fractionated Initial Infusion and Booster Dose for Patients with Relapsed and/or Refractory Multiple Myeloma (RRMM)

Targeted Proteomics of Pulmonary Hypertension in Sickle Cell Disease

BACKGROUND AND AIMHemolysis, inflammation and coagulopathy associated with sickle cell disease (SCD) can lead to pulmonary hypertension. More than 15% of adult patients with SCD are affected by pulmonary hypertension as measured by a tricuspid regurgitation velocity (TRV) ≥ 2.9 m/sec (Nouraie, M. et al., Validation of a composite vascular high-risk profile for adult patients with sickle cell disease. American Journal of Hematology, 94:E312-E314. 2019). Moreover, sickle cell disease patients with pulmonary hypertension have a much higher mortality risk than those without pulmonary hypertension. There is little knowledge on the proteomic profile of pulmonary hypertension in SCD. Our aim was to conduct a proof-of-concept study to explore targeted serum protein biomarkers that are differentially expressed in SCD patients with elevated tricuspid regurgitation velocities and explore their corresponding pathways. MATERIAL AND METHODSData from the Walk-PHaSST study was used for this study. The Walk-PHaSST was a multicenter study to assess the effect of sildenafil for SCD patients with pulmonary hypertension (Gladwin, M. T. et al., Risk factors for death in 632 patients with sickle cell disease in the United States and United Kingdom. PLoS One, 9:e99489. 2014). In the screening phase of the study, 720 patients with SCD were recruited. Study participants underwent clinical and lab examinations, as well as an echocardiography. We selected two groups of patients: one group of patients with a TRV≤2.6 m/sec and another with TRV≥2.9 m/sec (N =35 in each). The serum concentration of 92 protein biomarkers was measured using an OLINK cardiovascular panel. This panel assess a range of biological process including inflammatory response, angiogenesis, cell adhesion, coagulation, and response to hypoxia. T-tests were performed between the aforementioned groups for each of the serum biomarkers. The Hochberg method was used to calculate the false discovery rate. A volcano plot was created using the Bonferroni correction. Finally, we tested the correlation of the differentially expressed biomarkers with clinical variables measured in blood samples of the participants. These variables include white blood cell (WBC), neutrophil count, thrombospondin-1 (TSP as measure of coagulation), PIGF and VEGF (growth factors involved in angiogenesis), and NT-proBNP (elevated in pulmonary hypertension). RESULTSUsing a false discovery rate of 0.01, we discovered 14 significantly expressed proteins. Six of them passed a Bonferroni corrected overall critical p value < 0.00054 (Figure 1). These included T-cell surface glycoprotein (CD4), lymphotactin (XCL1), SLAM family member 7 (SLAMF7), galectin-9 (GAL9), TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2), and tumor necrosis factor receptor superfamily member 11A (TNFRSF11A). We observed up to a 1.2-fold increase in these 6 protein biomarker levels in the high TRV groups.The correlogram (Figure 2) indicated that there is a slightly positive correlation between WBC and CD4, GAL9, TRAILR2, and TNFRSF11A (r > 0.20). For neutrophil count, we observed significantly negative correlation with selected markers including TNFRSF11A levels (r = -0.51), GAL9 (r = -0.35), XCL1 (r = -0.27), SLAMF7 (r = - 0.21), and CD4 (r < - 0.24). A significantly negative correlation between TSP and GAL9 levels (r = -0.31) was also observed. Finally, we observed a strong, positive correlation between all proteins and serum NT-proBNP levels (r > 0.44). CONCLUSIONCirculatory protein markers of immune response and coagulation are highly expressed in SCD patients with elevated TRV. This provides evidence that these protein biomarkers have potential to be utilized as prognostic markers for pulmonary hypertension in patients with SCD. [Display omitted] DisclosuresLittle: Biochip Labs: Patents & Royalties; Hemex Health, Inc.: Patents & Royalties. Gibbs: Acceleron Pharma: Consultancy, Other: lecture fees; Actelion: Consultancy, Other: lecture fees; Aerovate: Consultancy, Other: lecture fees; Bayer: Consultancy, Other: lecture fees; Compexia: Consultancy, Other: lecture fees; Janssen: Consultancy, Other: lecture fees; MSD: Consultancy, Other: lecture fees ; Pfizer: Consultancy, Other: lecture fees; United Therapeutics: Consultancy, Other: lecture fees. Gordeuk: Modus Therapeutics: Consultancy; Novartis: Research Funding; Incyte: Research Funding; Emmaus: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; CSL Behring: Consultancy. Nouraie: Phoenicia BioScience Inc.: Consultancy.

Haplo-Identical Non-Gene Editing CD7 CAR T-Cells Induced Bone Marrow and Extramedullary Remission in a Pediatric Patient with TP53 Mutated Relapsed and Refractory Early T-Cell Precursor Lymphoblastic Leukemia/Lymphoma after Haploidentical Hematopoietic Stem Cell Transplantation

Patients with relapsed/refractory early T-cell precursor lymphoblastic leukemia/lymphoma (ETP-ALL/LBL) respond poorly to traditional therapy and have dismal prognosis. CD7 expresses in almost all blasts of T-cell lymphoma/leukemia and represents one of the most promising therapeutic targets for T-ALL/LBL by CD7 targeted chimeric antigen receptor modified T cell therapy (CD7-CART). Because of shared CD7 expression in the majority of normal T-cell surfaces, we utilized an non-gene editing strategy by co-transducing CAR-T cells with a CD7 protein expression blocker (PEBL), and successfully overcame the fratricide as well as maintain the proliferation and cytotoxicity of CD7-CART-cells. Here, we presented the efficacy and safety results of CD7-CART therapy in a pediatric patient with TP53 mutated ETP-ALL/LBL. The patient was diagnosed with ETP-ALL/LBL at 2016, achieved and maintained complete remission (CR) for 2 years with traditional chemotherapy. The disease relapsed at a month after discontinuation of chemotherapy. He underwent haploidentical HSCT at the second CR, but suffered relapse again 2 years post haplo-HSCT. TP53 mutation（VAF:96.5%） and extensive extramedullary infiltration was detected at relapse. The patient was resistant to venetoclax combined with decitabine, homoharringtonine, aclarubicin, cytarabine and granulocyte colony stimulating factor (G-CSF), high-dose cytarabine combined with cladribine, G-CSF, chidamide and CD38 CART therapy. Nanobody derived CD7-CART cells were manufactured from lymphocytes of the donor. The CART cells were negative for CD7, CD223 and CD279. 70.5% of blasts in the bone marrow aspirates were observed prior to CAR T-cells infusion. A total of 5×10 6/kg CD7-CART-cells were infused. CR was confirmed at day 30 bone marrow evaluation and maintained at the last followup at day 91. Partial remission was achieved as evaluated by PET-CT scan at day 93. Persistence of CD7-CART-cells can be detected with flowcytometry until day 96 post CAR T-cells infusion. Grade 3 cytokine release syndrome with high fever and hypotension were observed, which was relived by tocilizumab and dexamethosone. No organ dysfuction and immune effector cell-associated neurotoxicity syndrome were observed. In general, we showed for the first time that the nanobody derived CD7-CART with PEBL technology was a potent and safe salvage therapy in a relapsed/refractory ETP-ALL/LBL patient with high tumor burden. DisclosuresNo relevant conflicts of interest to declare.

Divergent Immune Responses to SARS-CoV-2 Vaccines in Immunocompromised Patients.

To The Editor: Understanding the composition and duration of immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination is critical for prevention of infection. The most critical elements of immunity to SARS-CoV-2 are neutralizing antibody and T-cell immunity. Current assessments of immunity and risk for infections largely depended on detection of antibodies to SARS-CoV-2. However, taken alone, this represents an often-unreliable tool due to the evanescent nature of spike immunoglobulin G (IgG) responses.1-3 Here, an important and more durable response involves cytotoxic T-cells that can eliminate virally infected cells and T helper cells, which are critical to coordinating adaptive immunity toward the virus and generating long-lasting immunologic memory. However, T-cell responses are more difficult to assess. These issues become more prescient in determining immunity to SARS-CoV-2 in immunocompromised individuals where a majority show no IgG responses to vaccines and the recent emergence of the Delta variant with reports of >74% vaccine breakthrough cases.4,5 Here, we report on patients demonstrating divergent immune responses to SARS-CoV-2 vaccination. Institutional Review Board approval and informed consent was obtained before performance of assays (Appendix 1, SDC, https://links.lww.com/TP/C290). Seven patients were identified from a cohort of 70 immunocompromised patients, with all demonstrating spike-specific IgG unresponsiveness 2–4 mo postvaccination. All had received treatment with B-cell modifying agents. We subsequently examined CD4+/CD8+ T-cell–spike-specific immune responses in all patients and repeat examination after revaccination in 2 patients (see Appendix 1, SDC, https://links.lww.com/TP/C290, for Methods and Data). CD4+/CD8+ T-cell responses are shown in Figure 1 and demonstrate robust CD4+T-cell (0.94 ± 1.2%, Normal > 0.05%) and CD8+ T-cell (0.89 ± 1.2%, Normal > 0.05%) immune responses to spike peptides. Two patients receiving Johnson & Johnson booster vaccines demonstrated increased T-cell responses but remained spike IgG negative. This suggests that T-cell immune responses to SARS-CoV-2 vaccines are primal and retentive and that B-cell depletion before vaccine exposure prevents the cascade of progression of B-cell activation necessary for SARS-CoV-2 spike IgG production.FIGURE 1.: A, Detectable CD4+/CD8+ T-cell immune responses in vaccinated patients against SARS-CoV-2 spike peptides. Flow cytometry diagrams from 7 patients (P1–P7) assessed after 2 doses of Pfizer or Moderna vaccines. Cells were stimulated by SARS-CoV-2 spike peptides pool (PepMix SARS-CoV-2 [spike glycoprotein, JPT) for 9 h in vitro in the presence of Brefeldin A and anti-CD28/CD49d (BD Bioscience, San Jose, CA). Cells were stained for T-cell surface markers followed by fixation, permeabilization, and intracellular staining of cytokines (BD Bioscience, San Jose, CA). Activated CD4+ T cells (IL-2+TNFα+) and CD8+ T cells (IFNγ+TNFα+) are shown in the upper right-hand corner of each flow box. B, This figure shows immune responses to SARS-CoV-2 spike peptides in 2 patients who obtained external revaccination with the Johnson & Johnson vaccine. As in (A), percentages of activated CD4+ T cells (IL-2+TNFα+) and CD8+ T cells (IFNγ+TNFα+) in blood are shown after first vaccination (2 doses) and after third vaccination (booster). Activated CD4+ T cells (IL-2+TNFα+) and CD8+ T cells (IFNγ+TNFα+) are shown in the upper right-hand corner of each flow box. IFNγ, interferon γ; IL, interleukin; P, patient; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TNFα, tumor necrosis factor α.Absence of IgG responses to SARS-CoV-2 suggests patients be considered “unvaccinated.” However, we show that patients treated with B-cell–modifying agents develop robust T-cell immune responses to SARS-CoV-2 without generating IgG responses. These observations should be considered in light of data presented by Peng et al showing robust CD4+/CD8+ T-cell responses to SARS-CoV-2 after infection. A critical observation was the diversity of T-cell responses that likely extend beyond the persistence of spike antibody. IgG is necessary for sterilizing immunity and T-cells cannot prevent infection as antigen presentation is required, but T-cell immunity can be at the ready for viral elimination. This may provide an inside track for rapid deployment of SARS-CoV-2 immunity, and although not preventing infection, could alter the severity and duration of SARS-CoV-2 disease.6 This is supported by data from Oberhardt et al7 who recently showed that vaccine-induced CD8+ T cells were the main mediators of protection after vaccination since they emerged before detection of neutralizing antibody and expanded after booster vaccination. Thus, detection of T-cell immune responses in patients failing to generate spike-specific IgG may aid in a more comprehensive assessment of immunity to SARS-CoV-2, identifying patients who would no longer be considered “unvaccinated” based on negative spike-specific IgG. This likely has relevance to patients receiving B-cell directed therapies for autoimmune and hematologic diseases. ACKNOWLEDGMENTS The authors would like to express our gratitude to the members of the Transplant Immunotherapy Program and the Transplant Immunology Laboratory at Cedars-Sinai Medical Center for their dedication to improving patient outcomes and safety during the coronavirus disease 2019 pandemic. We would also like to thank the patients who participated in this study by donating their blood for analysis of SARS-CoV-2 reactive T-cells.

PD-1/PD-L1 Checkpoints and Resveratrol: A Controversial New Way for a Therapeutic Strategy.

Simple SummaryOver the last decade, immunotherapies using antibodies targeting the programmed cell death 1 (PD-1) checkpoint or its ligand, programmed death ligand 1 (PD-L1), have emerged as promising therapeutic strategies against cancer. However, some current limitations include a relatively low rate of “responders”, the high cost of the treatment, and a rare risk of hyper-progression. Currently, the main challenge is, therefore, to improve these therapies, for instance, by using combined approaches. Here, we summarize the accumulating evidence that resveratrol (RSV) plays a role in the modulation of the PD-1/PD-L1 axis in cancer cells, suggesting the potential of therapeutic strategies combining RSV with PD-L1 or anti-PD-1 inhibitors. We then discuss the therapeutic potential of polyphenols such as RSV to be used in combination with PD-L1 or PD-1 inhibitors for the management of cancer patients.Immune checkpoints refer to a range of immunoregulatory molecules that modulate the immune response. For example, proteins expressed at the surface of T-cells (including PD-1 and CTLA-4) and their ligands (PD-L1 and B7-1/B7-2, respectively), expressed by cancer cells and antigen-presenting cells, are needed to prevent excessive immune responses. However, they dampen anti-tumor immunity by limiting T-cell activity, making them promising therapeutic targets in cancer. Although immunotherapies using checkpoint blocking/neutralizing antibodies targeting PD-L1 or PD-1 have proven their superiority over conventional chemotherapies or targeted therapies by enhancing T-cell-mediated anti-tumor immunity, some limitations have emerged. These include a relatively low rate of “responders” (<50%; irrespective of cancer type), the high cost of injections, and a rare risk of hyper-progression. For clinicians, the current challenge is thus to improve the existing therapies, potentially through combinatory approaches. Polyphenols such as resveratrol (RSV), a trihydroxystilbene found in various plants and an adjuvant in numerous nutraceuticals, have been proposed as potential therapeutic targets. Beyond its well-known pleiotropic effects, RSV affects PD-L1 and PD-1 expression as well as PD-L1 subcellular localization and post-translational modifications, which we review here. We also summarize the consequences of PD-1/PD-L1 signaling, the modalities of their blockade in the context of cancer, and the current status and limitations of these immunotherapies. Finally, we discuss their potential use in combination with chemotherapies, and, using RSV as a model, we propose polyphenols as adjuvants to enhance the efficacy of anti-PD-1/anti-PD-L1 immunotherapies.

Abstract 1515: Hotspot mutations in KRAS and TP53 targeted by TCR-T cells genetically modified with the Sleeping Beauty transposon/transposase system

Abstract Mutations in critical genes for cell survival and proliferation, e.g., KRAS and TP53, are found as clonal events in multiple tumor types of unrelated people likely due to their importance to the malignant phenotype. T cells recognize products of mutated genes, termed neoantigens, because they are expressed in the tumor but not in the normal tissues; thus, neoantigens are foreign entities from an immunological perspective. T-cell receptors (TCRs) with specificity to the neoantigen in the context of human leukocyte antigen (HLA) on the tumor cell surface can be isolated from the neoantigen-reactive T cell and potentially used for genetically-modified adoptive immunotherapy for any patient with matching mutation and HLA. The purpose of this study was to evaluate the ability of the non-viral Sleeping Beauty transposon/transposase gene transfer system to re-direct the specificity of T cells towards p53 and KRAS neoantigens and to characterize the resultant engineered TCR-T cell populations for specificity and function. Genes encoding the alpha and beta chains of p53 or KRAS neoantigen-specific TCRs were linked by a 2A ribosomal slip site linker and cloned into the clinical Sleeping Beauty transposon. These transposons were co-electroporated into donor peripheral blood leukocytes (PBL) with a DNA plasmid encoding the SB11 transposase and expanded in vitro. Logarithmic proliferation was observed and resulted in large numbers of highly pure TCR-T cells (&amp;gt;80% by introduced TCR expression). The TCR-T cells upregulated 41BB on the T-cell surface and secreted interferon-γ in response to antigen presenting cells with the appropriate HLA molecule pulsed with KRAS or p53 neoantigen peptides but not the cognate wild type peptide, confirming re-directed specificity to mutated KRAS or TP53 genes, respectively. Similarly, TCR-engineered T cells demonstrated cytolysis of tumor cells expressing the neoantigen and appropriate HLA restriction, suggesting that adoptive transfer of these TCR-T cells could mediate anti-tumor responses. In all, we demonstrated that multiple TCRs with unique specificities targeting recurrent p53 and KRAS substitutions in frequent HLA haplotypes could be stably expressed using Sleeping Beauty transposition to re-direct peripheral blood T cells towards tumor cells. Translation of these TCR-T cells into adoptive immunotherapy could result in safe and effective treatments for any cancer patient with matching HLA and KRAS or TP53 hotspot mutations. Citation Format: Ana B. Korngold, Lin-Kin Yong, Ming Zhang, Ugochi Ibekwe, Lenka V. Hurton, Yaoyao Shi, Julissa Simmons, Matthew R. Collinson-Pautz, Eleanor De Groot, Laurence J. Cooper, Drew C. Deniger. Hotspot mutations in KRAS and TP53 targeted by TCR-T cells genetically modified with the Sleeping Beauty transposon/transposase system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1515.

Abstract LB149: A novel cotinine-based system for switchable chimeric antigen receptor T cell immunotherapy

Abstract Autologous T cells engineered to express a chimeric antigen receptor (CAR-T cells) have shown impressive outcomes in hematologic malignancies. However, safety concerns derived from the inability to control CAR-T cells in vivo remain a significant challenge to expand the application of CAR-T cell therapy. Switchable CAR-T cells can allow for turning on and off the activation and cytotoxicity of CAR-T cells in vivo, potentially increasing the safety of next-generation CART products. To this goal, CAR constructs targeting non-tumor antigens, e.g., haptens and peptide tags, can be designed and combined with tumor-targeting units that are fused to the hapten or peptide tag. In this study, we developed a novel switchable CAR-T system using cotinine as the hapten and an anti-cotinine CART cell product. Of note, cotinine is physiologically absent in human and pharmacologically inert. A mouse antibody to cotinine was developed and successfully humanized. The anti-cotinine CAR confirmed to be displayed on T-cell surface. We first tested this approach to target epidermal growth factor receptor 2 (HER2) in ovarian cancer. To target HER2, we developed an affibody-based antigen binding domain characterized by high stability, broad spectrum of specificity, and feasibility of large scale chemical synthesis. In in vitro cytotoxicity and cytokine release assays, the activity of CAR-T cells was controlled by cotinine-tagged anti-HER2 affibody in a HER2-specific and dose-dependent manner. In an orthotopic ovarian cancer model using NSG mice engrafted with SKOV3, tumor regression was observed only when CAR-T cells were administered in combination with the cotinine-tagged HER2 affibody. In subsequent studies, we demonstrated that the cotinine-based switchable CAR-T system can be applied to other tumor targets such as mesothelin and epidermal growth factor receptor (EGFR). Additional studies are ongoing to determine the kinetics of the on and off CART activation of this system and define the optimal dosing strategy to ensure anti-tumor effect or to interrupt toxicity. In conclusion, we have developed a novel switchable cotinine-based CART system to control CART function in vivo and that could be broadly applicable to wide variety of tumors. Citation Format: Ki-Hyun Kim, Soohwan Kim, Ji-Hyeon Choi, Sung-Min Kim, Gae-Baik Kim, Jong-Ho Lee, Hyun-Jong Lee, Sang-Ho Ahn, Guewha Lee, LeiGuang Cui, Seong Yeol Kim, Min Yoon, Ki Hyun Kim, Soohyun Kim, Juwon Lee, Youngjin Han, Youngha Lee, In-Sik Hwang, Bong-Kook Ko, Jong-Seo Lee, Yong Sang Song, Marco Ruella, Junho Chung. A novel cotinine-based system for switchable chimeric antigen receptor T cell immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB149.

GD2-directed bispecific trifunctional antibody outperforms dinutuximab beta in a murine model for aggressive metastasized neuroblastoma

BackgroundNeuroblastoma is the most common extracranial solid tumor of childhood. Patients with high-risk disease undergo extremely aggressive therapy and nonetheless have cure rates below 50%. Treatment with the ch14.18 monoclonal...

CD3G or CD3D Knockdown in Mature, but Not Immature, T Lymphocytes Similarly Cripples the Human TCRαβ Complex.

The human αβ T-cell receptor (TCR) is composed of a variable heterodimer (TCRαβ) and three invariant dimers (CD3γε, CD3δε, and ζζ/CD2472). The role of each invariant chain in the stepwise interactions among TCR chains along the assembly is still not fully understood. Despite the high sequence homology between CD3γ and CD3δ, the clinical consequences of the corresponding immunodeficiencies (ID) in humans are very different (mild and severe, respectively), and mouse models do not recapitulate findings in human ID. To try to understand such disparities, we stably knocked down (KD) CD3D or CD3G expression in the human Jurkat T-cell line and analyzed comparatively their impact on TCRαβ assembly, transport, and surface expression. The results indicated that TCR ensembles were less stable and CD3ε levels were lower when CD3γ, rather than CD3δ, was scarce. However, both defective TCR ensembles were strongly retained in the ER, lacked ζζ/CD2472, and barely reached the T-cell surface (<11% of normal controls) in any of the CD3 KD cells. This is in sharp contrast to human CD3γ ID, whose mature T cells express higher levels of surface TCR (>30% vs. normal controls). CD3 KD of human T-cell progenitors followed by mouse fetal thymus organ cultures showed high plasticity in emerging immature polyclonal T lymphocytes that allowed for the expression of significant TCR levels which may then signal for survival in CD3γ, but not in CD3δ deficiency, and explain the immunological and clinical disparities of such ID cases.