CD107a Expression Research Articles

Transcription factor TOX maintains the expression of Mst1 in controlling the early mouse NK cell development.

Rationale: TOX is a DNA-binding factor required for the development of multiple immune cells and the formation of lymph nodes. However, the temporal regulation mode of TOX on NK cell development and function needs to be further explored. Methods: To investigate the role of TOX in NK cells at distinct developmental phases, we deleted TOX at the hematopoietic stem cell stage (Vav-Cre), NK cell precursor (CD122-Cre) stage and late NK cell developmental stage (Ncr1-Cre), respectively. Flow cytometry was used to detect the development and functional changes of NK cell when deletion of TOX. RNA-seq was used to assess the differences in transcriptional expression profile of WT and TOX-deficient NK cells. Published Chip-seq data was exploited to search for the proteins directly interact with TOX in NK cells. Results: The deficiency of TOX at the hematopoietic stem cell stage severely retarded NK cell development. To a less extent, TOX also played an essential role in the physiological process of NKp cells differentiation into mature NK cells. Furthermore, the deletion of TOX at NKp stage severely impaired the immune surveillance function of NK cells, accompanied by down-regulation of IFN-γ and CD107a expression. However, TOX is dispensable for mature NK cell development and function. Mechanistically, by combining RNA-seq data with published TOX ChIP-seq data, we found that the inactivation of TOX at NKp stage directly repressed the expression of Mst1, an important intermediate kinase in Hippo signaling pathway. Mst1 deficient at NKp stage gained the similar phenotype with Toxfl/flCD122Cre mice. Conclusion: In our study, we conclude that TOX coordinates the early mouse NK cell development at NKp stage by maintaining the expression of Mst1. Moreover, we clarify the different dependence of the transcription factor TOX in NK cells biology.

Extracellular Vesicles Expressing CD19 Antigen Improve Expansion and Efficacy of CD19-Targeted CAR-T Cells

CAR-T cell therapy is effective in the treatment of certain hematological malignancies, and the expansion and functional persistence of CAR-T cells in vivo are crucial to clinical efficacy. The aim of this study was to investigate the potential of extracellular vesicles (EVs) modified with the CAR antigen to promote the efficacy of CAR-T cells in vivo. We generated HEK293T-derived EVs to present the CD19 antigen as the CAR target. In vitro, EVs expressing CD19 antigen (CD19 EVs) were co-incubated with anti-CD19 CAR-T cells. Then, proliferation, cytokine secretion, CD107a expression, tumor killing, subsets, and immune checkpoint expression were measured to assess CAR-T cell function. After infusion of CD19 EVs pretreated CAR-T cells into a lymphoma xenograft mouse model, flow cytometry and digital PCR were used to measure the expansion of CAR-T cells, and tumor volumes were continuously monitored to assess the anti-tumor efficacy of CAR-T cells in vivo. Another mouse model was created to investigate the effect of in vivo injection of CD19 EVs on the functional persistence of CAR-T cells, and safety was determined by histopathology of the main organs. CD19 EVs activated CAR-T cells in an antigen-specific and dose-dependent manner and promoted the selective expansion and cytokine secretion of co-cultured CAR-T cells. Specifically, CD19 EVs preferably increased the expansion of the CAR-T subpopulation with a high surface CD19-CAR density and consequently enhanced the anti-tumor activity of CAR-T cells. Futhermore, CD19-EVs-primed CAR-T cells achieved superior proliferation and anti-tumor effects in a mouse model with lymphoma xenograft. In vivo administration of CD19 EVs promoted the functional persistence of CAR-T cells in the xenograft mouse model. Our findings indicate that antigen-expressing EVs can be utilized as a boost to improve CAR-T cell efficacy in vitro and in vivo.

The Immunoregulatory Effect of Aconite Treatment on H22 Tumor-Bearing Mice via Modulating Adaptive Immunity and Natural Killer-Related Immunity.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and, in its advanced stages, has a 5-year survival rate of only 3% to 5%. Despite novel mechanisms and treatments being uncovered over the past few years, effective strategies for HCC are currently limited. Previous studies have proven that aconite can suppress tumor growth and progression and prevent the recurrence and metastasis of multiple cancers, but the underlying molecular mechanisms are largely unknown. In this study, different doses of aconite were applied to mice bearing subcutaneous HCC tumors. It was found that aconite had a therapeutic effect on H22 tumor-bearing mice in a dose-dependent manner by reducing tumor volumes and prolonging survival times, which could be attributed to the immunoregulatory effect of aconite. Furthermore, results showed that high-dose administration of aconite could enhance adaptive immunity and natural killer (NK) cell-mediated immunity by regulating the secretion of interferon-γ, upregulating T cells and NK cells, and modulating the expression of the NK cytotoxicity biomarker CD107a and the inhibitory receptor TIGIT. This study revealed a novel mechanism through which aconite exerts antitumor effects, not merely through apoptosis induction pathways, providing more sound evidence that aconite has the potential to be developed into an effective anti-HCC agent.

Development of FAP-Targeted Chimeric Antigen Receptor NK-92 Cells for Non-Small Cell Lung Cancer.

Over the past two decades, great progress has been made in advancing the early detection and multimodal treatment of non-small cell lung cancer (NSCLC). However, overall cure rates and survival rates of NSCLC are still not satisfactory, and research into new therapies is needed. This study attempted to construct human Fibroblast Activation Protein-Chimeric Antigen Receptor Natural killer (NK)-92 cells (hFAP-CAR-NK-92 cells) and explore their potential therapeutic effects in NSCLC. Immunohistochemistry analysis was carried out to examine fibroblast activation protein (FAP) and Gasdermin E (GSDME) expression in clinical specimens of lung adenocarcinoma and squamous cell carcinoma tissue. Then the engineered hFAP-CAR-NK-92 cells efficiency was determined in vitro with lactate dehydrogenase (LDH) cytotoxicity assay and the cell morphology of A549, H226, and cancer-related fibroblast (CAF) was observed by electron microscopy. After the co-culture of target cells and effect cells, flow cytometry was employed for examining the CD107a expression in the effect cells, and western blotting was conducted for the cleavage levels of Caspase 3 and GSDME proteins in the target cells. The safety and efficacy of hFAP-CAR-NK-92 cells adoptive transfer immunotherapy in a tumor-bearing mouse were evaluated. Clinical studies have shown FAP positivity in patients with NSCLC. Compared with A549 or H226 cells alone, FAP expression was notably raised in A549+CAF cells or H226+CAF cells in nude mice, respectively (p < 0.05). The killing efficiency of K562 cells was not significantly different between hFAP-CAR-NK-92 and NK-92 cells (p > 0.05). The hFAP-CAR-NK-92 cells presented a higher killing efficiency against the hFAP-target (A549-hFAP, H226-hFAP and CAF-hFAP) cells than the NK-92 cells (p < 0.05). The degranulation of CD107a and cleavage levels of GSDME and Caspase 3 protein in the hFAP-CAR-NK-92 group were higher than those in the NK-92 group (p < 0.05). The 300 nM Granzyme B also induced pyroptosis in hFAP- or GSDME-positive cells (p < 0.05). In vivo experiments revealed that hFAP-CAR-NK-92 cells inhibited tumor progression of hFAP-positive NSCLC (p < 0.05). In this study, we successfully constructed hFAP-CAR-NK-92 cells and confirmed that hFAP-CAR-NK-92 cells could target hFAP-positive NSCLC to inhibit the progression of NSCLC by activating the Caspase-3/GSDME pyroptosis pathway.

Reduced Number and Immune Dysfunction of CD4+ T Cells in Obesity Accelerate Colorectal Cancer Progression

Obesity, a known risk factor for various types of cancer, reduces the number and function of cytotoxic immune cells in the tumor immune microenvironment (TIME). However, the impact of obesity on CD4+ T cells remains unclear. Therefore, this study aimed to clarify the impact of obesity on CD4+ T cells in the TIME. A tumor-bearing obese mouse model was established by feeding with 45% high-fat diet (HFD), followed by inoculation with a colon cancer cell line MC38. Tumor growth was significantly accelerated compared to that in mice fed a control diet. Tumor CD4+ T cells showed a significant reduction in number and an increased expression of programmed death-1 (PD-1), and decreased CD107a expression and cytokine such as IFN-γ and TNF-α production, indicating dysfunction. We further established CD4+ T cell-depleted HFD-fed model mice, which showed reduced tumor infiltration, increased PD-1 expression in CD8+ T cells, and obesity-induced acceleration of tumor growth in a CD4+ T cell-dependent manner. These findings suggest that the reduced number and dysfunction of CD4+ T cells due to obesity led to a decreased anti-tumor response of both CD4+ and CD8+ T cells to ultimately accelerate the progression of colorectal cancer. Our findings may elucidate the pathogenesis for poor outcomes of colorectal cancer associated with obesity.

IL-10‒Producing Potency from Blood B Cells Correlates with the Prognosis of Alopecia Areata

IL-10‒Producing Potency from Blood B Cells Correlates with the Prognosis of Alopecia Areata

Neutrophil phenotypes implicated in the pathophysiology of post-traumatic sepsis.

The disruption of immune homeostasis after trauma is a major cause of post-traumatic organ dysfunction and/or sepsis. Recently, a variety of neutrophil phenotypes with distinct functions have been identified and suggested as involved in various clinical conditions. The association between neutrophil phenotypes and post-traumatic immunodeficiency has also been reported, yet the specific neutrophil phenotypes and their functional significance in post-traumatic sepsis have not been fully clarified. Therefore, we sought to investigate neutrophil phenotypic changes in a murine model, as these may hold prognostic value in post-traumatic sepsis. Third-degree burns affecting 25% of the body surface area were used to establish trauma model, and sepsis was induced 24 h later through cecal ligation and puncture (CLP). The Burn/CLP post-traumatic sepsis model and the Sham/CLP control model were established to assess the immunological status after trauma. Histopathological evaluation was performed on the spleen, liver, kidneys, and lung tissues. Immunological evaluation included the assessment of neutrophil markers using mass cytometry as well as cytokine measurements in serum and ascitic fluid through multiplex analysis using LUMINEX®. The Burn/CLP group had a lower survival rate than the Sham/CLP group. Histopathological examination revealed an impaired immune response and more advanced organ damage in the Burn/CLP group. Furthermore, the Burn/CLP group exhibited higher levels of transforming growth factor-beta 1 in the blood and generally lower levels of cytokines than the Sham/CLP group. CD11b, which is involved in neutrophil adhesion and migration, was highly expressed on neutrophils in the Burn/CLP group. The expression of CD172a, which is related to the inhibition of phagocytosis, was also upregulated on neutrophils in the Burn/CLP group. The expression of sialic acid-binding lg-like lectin F and CD68 also differed between the two groups. Different neutrophil phenotypes were observed between Burn/CLP and Sham/CLP groups, suggesting that neutrophils are implicated in the immune imbalance following trauma. However, further studies are needed to prove the causal relationships between neutrophil phenotypes and outcomes, including survival rate and organ dysfunction.

Cord blood derived NK cells activated in counter with tumor cells.

NK cells are initially known for their ability to kill tumor cells with no prior sensitization. Production of mature and long lasting NK cells from Umbilical Cord Blood (UCB) by using cytokines could be a promising method for immunotherapy. NK cells were generated from cord blood cells using IL2, IL7, and IL15 cytokines and measured expression of CD57 and NKp46 markers. Afterward, their capacity in the elimination of malignant cells (Reh cell line) was evaluated by assessment of interferon-γ (as cytokine production sign) and CD107-a expression (as cytotoxic function symptom) using flow cytometry. Our results showed efficient NKp46 + , and CD57 + NK cells generated on day 14. Also, expression of CD107-a and IFN-γ following co-culture with Reh cell lines significantly increased in comparison to the control. Taken together, we have reported one of the best culture conditions for the generation of CD57 + NK cells with on feeder cells and showed appropriate capacity in counter reh cell lines as a target.

Identification of the CD16low CD56low CD38pos Natural Killer Cell as a Key Subset in Patients with Multiple Myeloma.

Natural killer (NK) cells are classified as innate immune cells which can directly recognize and kill tumor cells without antigen sensitization. NK cell-based adoptive immunotherapy for blood malignancies has attracted more attention in recent years. To analyze different NK cell subsets in the peripheral blood and bone marrow (BM) of patients with multiple myeloma (MM). Using flow cytometry we analyzed: (i) the distribution of distinct NK cell subpopulations (i.e. CD16low CD56low, CD16pos CD56high, CD16neg CD56high, CD16high CD56low, CD16neg CD56low, CD16low CD56low CD38pos) in the BM from MM patients at distinct disease stages. (ii) the expression of NKG2D, DNAM-1 and NKp30, and (iii) the expression of CD107a in CD16low CD56low CD38pos and CD16low CD56low CD38neg NK cells subsets. CD16low CD56low CD38pos was the dominant subset in BM from patients with MM at the CR stage with a decreased expression of NKp30. CD16low CD56low CD38pos subset showed a higher proportion of CD107a expression compared to CD16low CD56low CD38neg cells. In vitro experiments indicated that the CD16low CD56low CD38pos NK cell subset possesses more cytotoxicity than CD16low CD56low CD38neg NK cells. Our data suggest that CD16low CD56low CD38pos NK cells may reflect as an effector population with the potential therapeutic target in patients with MM. This group of cells may be useful for adoptive immunotherapy in MM in the future.

Transcriptional upregulation of CXCL13 is correlated with a favorable response to immune checkpoint inhibitors in lung adenocarcinoma.

The chemokine CXCL13 is known to influence local anti-tumor immunity by recruiting immune cells and forming tertiary lymphoid structures (TLS). It has been hypothesized that TLS, led by the expression of CXCL13, could be a predictive or prognostic biomarker for immunotherapy. We investigated the predictive value of CXCL13 to immune checkpoint inhibitors (ICI) in lung adenocarcinoma. We constructed an exploratory dataset (n=63) and a validation dataset (n=57) in metastatic lung adenocarcinoma patients treated with ICI. Based on the clinical response, the difference in gene expression profile, including CXCL13, was evaluated. From the exploratory dataset, CXCL13 expression was significantly upregulated in the ICI responders (p=0.002). Survival analysis using a cut-off value of the median expression value of CXCL13 showed prolonged progression-free survival (PFS) (p=0.004) and overall survival (OS) (p=0.007). CXCL13 expression was correlated with other immune response genes, such as GZMA, CD8A, IFNG, PRF1, TLS-related gene sets and its receptor, CXCR5. Notably, subgroup analyses based on CXCL13 expression and CD8A showed that CXCL13-upregulated patients demonstrated comparably prolonged survival regardless of CD8A expression. In the validation dataset, CXCL13 upregulation also demonstrated a significant prolongation of both PFS (p=0.050) and OS (p=0.026). We observed that CXCL13 upregulation is correlated to better ICI response in lung adenocarcinoma. Our results support that CXCL13 could be an important chemokine in shaping the immunoactive tumor microenvironment which affects the anti-tumor effect of ICI.

The clinicopathological significance and relapse predictive role of tumor microenvironment of intrahepatic cholangiocarcinoma after radical surgery.

This study attempts to detect the expression of FoxP3, CD68, CD8α, and PD-L1 in the tumor microenvironment (TME) of intrahepatic cholangiocarcinoma (ICC), and analyze the relationship between the corresponding cells and clinicopathological characteristics as well as prognosis of ICC. RNA sequencing (RNA-seq) provided the general landscape of the TME in ICC. A total of 99 ICC patients and the corresponding specimens were used for multiplex immunofluorescence and relapse-free survival (RFS) was analyzed. Flow cytometry further validated the effect of regulatory T (Treg) cells on ICC relapse. RNA-seq data showed that the infiltration of Treg cells, CD8+ T cells, and macrophages were likely associated with ICC relapse. The survival analysis based on multiplex immunofluorescence showed that the high FoxP3(+) Treg cells ratio and low CD68(+) macrophages ratio in mesenchyme were associated with higher RFS rate, respectively. Low FoxP3(+) Τreg cells ratio was associated with more perineural invasion, and high CD68(+) macrophages ratio was correlated with more lymph node metastasis. Cox regression analysis revealed that FoxP3(+) Treg cells ratio was an independent predictive factor for ICC relapse. Flow cytometry showed that TregIII was the predominant Treg cell subtype in both tumor tissue and peripheral blood of ICC patients, and high TregIII abundance in peripheral blood was significantly associated with longer RFS of ICC patients. High FoxP3(+) Treg cells ratio in the mesenchyme of ICC tumor tissue predicted longer RFS and was an independent favorable prognostic factor for ICC patients. Among all Treg cell subtypes, TregIII in peripheral blood was correlated with the RFS of ICC patients.

614 Modulation of NK cell activation by calcium from alginate dressings in vitro

Early after skin wounds, innate immune cells, including natural killer (NK) cells, infiltrate the wound bed within the first 24 hours. The application of widely used calcium alginate dressings facilitates cutaneous healing by releasing Ca2+ ions into the exudate and locally enriching its level. Since Ca2+ ions have been identified as one of the main activators of NK functions, the effects of two alginate dressings, Algosteril® (Algo, pure Ca2+ alginate) and Biatain® Alginate (BIA, Ca2+ alginate with CMC), and CaCl2 solution were compared on NK cell cytotoxicity by analyzing K562 lysis and CD107a expression as well as on their IFN-γ and TNF-α production.

DR-01, a Non-Fucosylated Anti-CD94 Antibody, Depletes Leukemic Cells in Ex Vivo and In Vivo Models of Large Granular Lymphocyte Leukemia

Large granular lymphocyte leukemia (LGLL) is a rare leukemia more frequently occurring in the elderly population with three major subtypes: T-LGLL, chronic lymphoproliferative disorder of NK cells (CLPD-NK) and aggressive NK cell leukemia (ANKL). Over 50% of patients develop complications including severe neutropenia, thrombocytopenia and anemia, thus predisposing patients to opportunistic infections. In addition, treatments are limited to standard immunosuppressive therapy such as methotrexate, cyclosporine or cyclophosphamide, with complete response rates of only 50% or less for each agent. Thus, novel and effective therapies for LGLL patients are critically needed. Dren Bio is developing DR-01, a non-ligand blocking, non-fucosylated monoclonal antibody targeting CD94. CD94 is a type II transmembrane receptor identified on mature, cytotoxic lymphocytes such as healthy NK and terminally differentiated CD8 T cells. In the context of disease, CD94 is known to be upregulated on LGLL cells. DR-01 functions through depletion of target cells via antibody-mediated cellular cytotoxicity (ADCC) by means of fratricide, a method in which the same cell type induces ADCC on each other. Through characterization of DR-01 using various ELISA and cell-based assays, DR-01 was determined to bind with nanomolar affinity to CD16a (EC50: 0.04µg/mL) and to CD94 on healthy human donor NK cells, cynomolgus monkey NK cells and LGLL cells (EC50: 0.5-1nM). DR-01 induces potent, dose-dependent depletion of CD94+ cells in ex vivo ADCC assays (IC50: 1ng/mL) of peripheral blood mononuclear cells (PBMCs) from healthy and diseased individuals. Furthermore, DR-01 has been shown to be well tolerated in a 4-week dose-range finding and Good Laboratory Practice (GLP) study in cynomolgus monkeys from 5-100mg/kg on a weekly dosing regimen. A 12-color flow cytometry panel was applied to a cohort of patient LGLL PBMCs to profile CD94 expression and other cytotoxic markers associated with the disease, including CD16, CD57 and functional binding partners of CD94 such as NKG2A and NKG2C. CD94 was identified on 97% (37/38) of patient samples screened, ranging from 400 to 725,000 receptors per cell. All patient leukemic cells expressed variable levels of CD16 and CD57. NKG2A and NKG2C were present in over 5% of total leukemic cells in 37% (14/38) and 21% (8/38) of patient samples, respectively. In addition, DR-01 was able to deplete LGL leukemic cells with high potency and in a dose-dependent manner in a functional ADCC assay (IC50:1-10ng/mL). The mechanism of fratricide was demonstrated through degranulation of the leukemic population via expression of CD107a. Cytokine profiling results are pending. To demonstrate in vivo efficacy of DR-01 in a disease model of LGLL, humanized NSG-Tg(hIL-15) mice engrafted with patient LGLL splenocytes were utilized. Optimal splenocyte engraftment (5-20% human CD45+ cells in blood) was achieved within 3 weeks post-inoculation with 10 million PBMCs per pre-sublethally irradiated mouse. The engrafted leukemic population partially preserved the CD94 expression profile from the patient leukemic cells and was successfully depleted (>90%) in blood, bone marrow, spleen and liver 1-week post-treatment with a single dose of DR-01 at 5mg/kg. Overall, DR-01 is a non-fucosylated anti-CD94 antibody with high affinity and potent leukemic cell depleting ability ex vivo and in vivo. The favorable toxicity profile of DR-01 in cynomolgus monkeys precede clinical studies in LGLL patients. Additional profiling and evaluation of DR-01 in LGLL patient samples ex vivo as part of a pre-phase I study is ongoing. A phase I/II trial has been initiated to assess the safety and efficacy of DR-01 in previously treated LGLL patients.

Heme Oxygenase-1 Inhibition the Cytotoxicity of NK Cells to AML By Downregulating HLA-C

Backgroud and objective: Acute myeloid leukemia (AML) is a malignant clonal disease of hematopoietic stem cells with abnormal differentiation of myeloid primordial naive cells, which has the highest incidence in adult Acute leukemia with a low cure rate . Clinically, less than one-third of adult AML patients can achieve sustained remission after cytarabine combined with anthracycline chemotherapy, and over 50% relapse after remission. Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is still the only way to cure leukemia clinically. However, due to age limitation, infection, graft-versus-host disease, and other complications, it is not suitable for all AML patients In recent years, numerous novel drugs have provided more options for the treatment of relapsed and refractory AML while the overall response rate is unsatisfactory and there is no significant advantage in improving overall survival Exploring the mechanism affecting AML relapse and refractory treatment, formulating effective coping strategies are the key to overcome the treatment bottleneck of AML Methods: Bone marrow nucleated cells were obtained from 15 newly diagnosed AML patients, 15 recurrent AML patients and 15 normal controls. The expressions of HO-1 and HLA-C were detected by quantitative reverse transcription PCR (RT-PCR) and Western blot. In vitro cell experiments, we constructed SiHO-1 lentiviral vector to transfect AML cell lines, cocultured NK cells with it, measured the change of CD107a expression on NK cell surface, and silenced HLA-C expression with small interfering RNA. The silenced AML cell lines were co-cultured with NK cells, and CD107a secretion on NK cells was measured by flow cytometry. Results: HO-1 expression in AML patients was higher than that in healthy donors (P<0.05), while HLA-C expression was lower than that in healthy donors (P<0.01). Additionally, the plasma HO-1 levels gradually increased, while the HLA-C levels gradually decreased from healthy donors to patients with relapsed AML. In addition, Pearman correlation analysis indicated that HLA-C expression was significantly correlated with HO-1 expression (r=0.635, P<0.001). In AML cell lines, HO-1 silencing enhanced the cytotoxicity of NK cells to AML cells (P＜0.01), while HO-1 overexpression weakened the cytotoxicity of NK cells to AML cells (P＜0.05). The expression of CD107a in NK cells after HLA-C silencing was lower than the control group in U937 and THP-1 cell line. (P＜0.05). Conclusion: decreased HLA-C expression is associated with increased HO-1 expression during the progression of AML. Besides, the inhibition of HO-1 promotes HLA-C expression, which can therefore promote cytotoxicity of NK cells to AML cells.

Detection, Monitoring and Cell Kinetics of Circulating CAR-T Cells in B-Cell Malignancies

Introduction CAR-T cell therapy has proven to be a disruptive treatment in the hematology field. Tisagenlecleucel and Axicabtagene ciloleucel showed impressive efficacy in relapsed/refractory B-cell lymphoma; however, less than 50% of patients maintain a long-term response and predictors of outcome are still inconsistently defined. Tisa-cel and Axi-cel kinetics have been poorly investigated in real life settings so it becomes necessary to evaluate possible biomarkers that predict response or toxicity. In clinical trials, the efficacy of the treatment is determined by the in vivo expansion and persistence of CAR-T cells. Real life evaluations assessing the immunophenotypic composition of CAR-T cell infusion products and analyzing how these specific features affect CAR-T cell expansion and persistence in vivo and thus outcome, are scarce. Here, we aimed to optimize the detection of CD19 CAR-T cells in blood and to identify phenotypic features associated with post-infusion expansion and outcomes. Methods CD19 CAR-T cells were monitored by flow cytometry from peripheral blood in 48 patients with diffuse large B-cell lymphoma, transformed follicular lymphoma, primary mediastinal B cell lymphoma or B-cell acute lymphoblastic leukemia. Flow cytometry approach was performed for determining cell kinetics (n=48) at days 5, 7, 11, 14, 17 and 20 (±1) after CAR-T cells infusion. In parallel, immunophenotype characterization of CAR- T lymphocytes was conducted at the day of CAR-T expansion in blood. Results After testing different commercially available antibodies for the detection of CD19 CAR-T cells, CD19 protein conjugated with biotin and identified by streptavidin showed the highest resolution in contrast to Antigen CD19 labeled FITC (stain index of 22.19 vs 4.64, p=0.0286). The median day of peak expansion of CAR-T cells was 7 days (range of 5-14 days) after infusion, reaching a median of 119 CAR+ cells/uL (range of 2.4.-1019 CAR+ cells/uL) in blood. Analysis of immune-phenotypic profile of overall circulating T lymphocytes displayed no significant differences in the distribution of naive/memory/effector from CD4+ and/or CD8+ subpopulations between non-CAR T (normal T lymphocytes) and CAR-T cells. However, CAR-T cells showed a significantly higher expression levels of activation (CD69+), proliferation (Ki67+), cytotoxicity (CD107a+) and exhaustion (PD1+TIM3+, PD1+LAG3+) markers in comparison to T lymphocytes (p<0.05). Overall, 31.6% (6/19) patients reached complete remission while 47.4% (9/19) achieved partial response at the first month after CAR-T therapy with event free survival (EFS) of 73% at 1 year (median follow-up of 1 year). Remarkably, cases with lower expression levels of PD-1 and LAG-3 (i.e <5.2%) in CD4+CAR+ T cells at the day of peak expansion had a worse outcome (EFS at 1 year 44% vs 90% for patients with low vs high expression of the above exhaustion markers, p=0.04) (Figure 1A). Similarly, patients with a highly cytotoxicity pattern (i.e CD107a expression >6%) within CD8+CAR+ T cells had a shorter EFS (50%) as compared to those with <6% of CD8+CAR+CD107a+ (89%) (p=0.06) (Figure 1B). Finally, patients with >3.2% CD4-CD8- T cells within the nonCAR T lymphocytes showed superior outcome as compared to those patients who displayed lower levels of double-negative T cells (100% vs 50% of EFS rates, respectively) (p=0.03). Conclusions In our cohort, the median day of CD19 CAR-T cell peak expansion as assessed by flow cytometry was 7 days after infusion. Markers of exhaustion within the CAR+ population and percentage of double-negative T lymphocytes allows an early identification of prognostic groups in order to guide therapeutic actions. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Investigating ITP Plasma for Effect on Platelet Production Using the Autologous Peripheral Blood Based Megakaryopoiesis Assay

Investigating ITP plasma for effect on platelet production using the autologous peripheral blood based megakaryopoiesis assay Nikola Ivetic, Ishac Nazy, Rumi Claire, Angela Huynh John G. Kelton, Donald M. Arnold. Background: Studies focused on megakaryocyte number and function are needed to understand platelet underproduction as a potential mechanism of immune thrombocytopenia (ITP). Most experimental studies have relied on animal models, specialized cell lines, or exogenous and non-autologous sources of hematopoietic stem and progenitor cells (HSPCs) as a source of megakaryocytic cells and platelet production. We used HSPCs isolated from ITP patients themselves to examine the effects their plasma has megakaryocyte growth and platelet production. Study Design and Methods: Megakaryocytes were produced from peripheral blood CD34+ HSCs directly from patients with ITP and healthy controls. HSPC (CD45+ CD34+) were isolated, expanded for 4 days and then cultured with thrombopoietin (TPO) and stem cell factor (SCF) for 8 days. Megakaryocyte numbers were evaluated by flow cytometry using CD41a expression for megakaryopoiesis and CD42b expression for maturation. Platelet production was analyzed using CD41a expression and platelet forward and side scatter profile sizes. The addition of autologous ITP plasma and plasma depleted of IgG were also tested to determine the effects of antibodies on megakaryopoiesis and thrombopoiesis. Results: Peripheral blood derived megakaryocytes were cultured from HSPCs from ITP patients (N=10), and normal controls (N=9). Approximately 2,200±1,100 hematopoietic stem and progenitor cells were isolated per ml of peripheral blood and a 5-fold increase in stem cells was obtained after an expansion of the isolated ITP patient HSPC population. Following an additional 8 days of culture with TPO, 67±12% of the cultured cells were CD41a+ indicating they were undergoing megakaryopoiesis and 54±15% of the CD41a+ population expressed the CD42b marker for mature megakaryocytes. Of the 10 ITP cultures grown, four grew significantly more mature megakaryocytes in comparison to healthy controls. The addition of ITP plasma did not inhibit megakaryocyte growth or maturity, but platelet production was reduced with two samples demonstrating a 50% reduction in platelet levels in comparison to the . Relative to the number of mature megakaryocytes cultured there was a lower number of platelets detected in ITP cultures (3.34±1.9 platelets per mature megakaryocyte) in comparison to the healthy control group (8.1±1.4 platelets per mature megakaryocyte). Conclusions: The addition of patient plasma had no inhibiting effect on megakaryocyte counts in an autologous assay but a significant decrease in platelet number (thrombopoiesis) was detected. A subset of ITP HSPC generated more megakaryocytes when compared to healthy donors, indicating these cells are generating more megakaryocytes. This is to our knowledge the first autologous investigation of ITP plasma on patient HSPC undergoing megakaryopoiesis.

Two Sides of the Same Transdifferentiated Coin: A Case Vignette and Review of the Literature Exploring the Relationship between B Cell Non-Hodgkin Lymphoma, Histiocytic Sarcoma and Interdigitating Dendritic Cell Sarcoma

WHO classifies Histiocytic Sarcoma (HS) and Interdigitating Dendritic Cell Sarcoma (IDCS) as separate, unrelated, entities under histiocytic/dendritic cell neoplasms. We do not fully understand the pathogenesis of HS and IDCS, yet both have been shown to transdifferentiate from B cell non-Hodgkin lymphomas. Diagnosis is challenging and requires application of a wide variety of immunohistochemical studies, flow cytometry and molecular analysis. We present a case of HS which evolved from CLL/SLL and presented as a diagnostic challenge due to unusual features overlapping with what is typically seen in IDCS. An 82-year-old male with a history of CLL/SLL presented with right infiltrative tonsillar mass and cervical lymphadenopathy. Biopsy of the lymph node revealed two abnormal populations of cells : Twenty percent of cells were consistent with CD20+ kappa surface light chain restricted CLL/SLL cells. Eighty percent of cells were histiocytic cells with an abnormal immunophenotype showing expression of CD4, CD43, fascin, HLA-DR, PU.1, (weak) S100 and CD1a without CD163, lysozyme, CD34, CD117, CD21, CD23 or CD14. Expert consultation favored the diagnosis of HS with transdifferentiation from CLL/SLL. PCR for IgH gene rearrangement, performed on separate HS-rich and CLL-rich areas of the biopsy detected identical clonal peaks. Next generation sequencing detected pathogenic KRAS mutations (p.G12D, p.A146P) and likely pathogenic BCOR (p.K839fs*17) and FBXW7 (p.R465S) mutations. While HS was the favored diagnosis, some immunophenotypic features were unusual, including diffuse fascin immunoreactivity, weak S100, and CD1a immunoreactivity which raised the possibility of so-called IDCS. While clonal evolution of HS and IDCS from B cell Non-Hodgkin lymphomas has been reported, there is a paucity of literature on these entities. We suggest that there may be a pathogenic relationship between these two neoplasms that has yet to be elucidated due to their rarity and diagnostic challenge. Few clinical laboratories can perform the breadth of markers required for diagnosis. Both neoplasms have varied clinical presentations with nodal or extranodal involvement and portend poor prognosis. Review of the literature shows significant overlap in immunophenotypic and genomic characteristics of IDCS and HS, suggesting they may be related manifestations of the same neoplasm. A case report has described the initial presentation of IDCS that subsequently developed into HS, although sampling may have been an issue. We identified 11 cases, including our case in the literature presenting with positive histiocytic and dendritic cell markers referred as hybrid dendritic-histiocytic sarcomas or histiocytic sarcoma with interdigitating dendritic cell differentiation. Immunohistochemical analysis of these cases showed positivity rates of S100 (100%), lysozyme (82%) and CD1a (18%). One case in the literature described fascin immunoreactivity, as was observed in our case. Four of six cases had CD163 immunoreactivity. A hybrid neoplasm with overlapping features of follicular and interdigitating dendritic cells has also been reported, further confounding our understanding of these neoplasms. Large studies analyzing genomic features of these neoplasms have shown recurrent mutations in RAS-MAPK pathway, as was seen in our case. Further studies of the relationship between HS, IDCS should include gene expression profiling of these entities which may also yield therapy targets. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract PR019: Mutant KRAS-specific T cell receptors directed against prevalent G12D and G12V variants exhibit potent cytotoxic activity and CD8 co-receptor independence

Abstract Background: KRAS mutations drive tumorigenesis in &amp;gt;90% of pancreatic ductal adenocarcinomas (PDAC). Recent clinical evidence demonstrates potent and durable anti-tumor activity of adoptively transferred KRAS-specific T cells in select patients with metastatic KRAS-mutated solid tumors including PDAC. We have defined mutant KRAS (mKRAS) epitopes restricted to high prevalence HLA class I alleles and validated this oncoprotein as an immunological target amenable to T cell receptor (TCR)-based therapeutic approaches. TCR therapies utilizing HLA class I-restricted TCRs often fail to harness CD4+ T cell activity vital for sustained anti-tumor immune responses. Here, we characterize HLA class I-restricted TCRs isolated from healthy donors and vaccinated patients targeting KRAS G12D or G12V, the most prevalent mKRAS variants observed in PDAC tumors. Methods: mKRAS-specific TCR sequences were identified from healthy donors or vaccinated PDAC patients enrolled on IRB-approved clinical studies at the University of Pennsylvania. Transgenic expression of synthetic TCRs was attained via lentiviral transduction of reporter cell lines or gene-edited (TCRabnull) primary CD8+ or CD4+ T cells. Limiting peptide dilution and combinatorial peptide library assays were used to assess antigen specificity, affinity and cross-reactivity. TCR reactivity to mKRAS cell lines was assessed by FACS analysis, in vitro cytotoxicity assays (4h 51Cr-release, xCELLigence Real Time Cell Analysis), and mouse xenograft tumor models. Results: TCRs exhibit specificity to the cognate KRAS G12D or G12V epitope without cross-reactivity to wild-type KRAS. TCRs are of high affinity and promote cytolysis of HLA-matched tumor cell lines harboring endogenous KRAS mutations irrespective of histologic origin. The adoptive transfer of gene-edited (TCRabnull) primary CD8+ T cells expressing synthetic TCRs exhibit potent in vivo antitumor activity in mouse xenograft tumor models of metastatic cancer. Select TCRs retain functional avidity independent of the CD8 co-receptor as determined by multimer binding and limiting peptide dilution assays. Disruption of the CD8:HLA binding interface via the introduction of D227A and T228A mutations within the HLA a3 domain has limited to no effect on TCR-mediated CD8+ T cell cytolytic activity. Transgenic expression of co-receptor independent TCRs by CD4+ T cells confers cytolytic activity against KRAS-mutated cell lines in both short- and long-term cytotoxicity assays, and flow cytometric analysis confirms antigen-specific expression of CD107a, IFN-g, TNF-a and IL-2 in a subset of CD4+ T cells. Conclusion: This study identifies TCRs with high therapeutic potency for the clinical development of TCR therapy trials targeting mKRAS in PDAC patients. Select TCRs exhibit CD8 co-receptor independence. CD4+ T cells redirected with co-receptor independent TCRs exhibit cytotoxic activity and express Th1 cytokines which may serve to enhance the persistence and anti-tumor activity of adoptive TCR therapy targeting mKRAS. Citation Format: Adham S. Bear, Rebecca B. Nadler, John Scholler, Robert H. Vonderheide, Gerald P. Linette, Beatriz M. Carreno. Mutant KRAS-specific T cell receptors directed against prevalent G12D and G12V variants exhibit potent cytotoxic activity and CD8 co-receptor independence [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR019.

CD8+ TEMRA Clones Cause Platelet Lysis in Immune Thrombocytopenia

Background Immune thrombocytopenia (ITP) is traditionally thought of as an antibody-mediated disease. However, there are a number of features to suggest alternative mechanisms of platelet destruction. In this study we use a multi-dimensional approach to explore the role of terminally differentiated effector memory CD8+ T cells (TEMRA). Methods We characterised 83 adults with chronic ITP and 30 age-matched controls using immunophenotyping, next generation sequencing of T cell receptor (TCR), single cell (sc) RNA sequencing and functional assays. Next generation DNA sequencing of TCRβ was carried out using the Illumina MiSeq platform. Analysis of the raw TCR sequences was performed using MiXCR. Single cell immune profiling (Gene expression and TCR) was done using Chromium Single Cell V(D)J and 5' Library kits from 10x Genomics. The constructed library was sequenced on the HiSeq platform (Illumina). Seurat v.2.3.4 was used for gene expression analysis. Interactions between CD8+ T cells and platelets were measured by co-culturing CD8+ T cells with autologous platelets overnight and by perfusing them on the autologous platelet covered VenaFluoro8+ microchip channel. Results We found an expansion of CD8+ CD45RA+ CD62L- TEMRA cells expressing IFN gamma, TNF alpha and granzyme B, with no evidence of physiological exhaustion (failure to upregulate TIM-3 or PD-1 expression) in ITP patients which correlated with disease activity. DNA TCR sequencing revealed that expansion of TEMRA cells in ITP was associated with reduced T cell repertoire diversity (p ≤ 0.0001) and lower Simpson's diversity index (p ≤ 0.05). Patients with ITP had a higher number of T cell clones occupying more than 5% of the T cell repertoire when compared to age matched controls (p ≤ 0.05). Expanded clones were not shared across ITP patients and displayed no known antigen specificities when compared to available databases of TCR sequences, providing evidence for the lack of virally driven clonal expansion. Clonally expanded CD8+ T cells persisted over many years in ITP patients and characterised disease severity. Longitudinal samples from 9 patients showed expanded clones and reduced T cell repertoire diversity during active disease (platelet count of less than 30 x 109/L) compared to stable disease (platelet count greater or equal to 30 x 109/L) (p ≤ 0.01; r = 0.56 - Figure 1 shows an example patient). To better characterise CD8+ T cells, we performed paired scRNA and scTCR sequencing on isolated whole CD8+ T cells from patients with active and stable disease. Paired sequencing enables transcriptomic characterisation of T cell clones. ScRNA sequencing revealed four distinct clusters in CD8+ T cells: naïve, TEMRA, central memory (CM) and natural killer T cells (NKT). By mapping scTCR CD8+ sequencing to single cell gene expression, we found that the largest expanded clones showed an aggregative distribution, indicating transcriptional homogeneity and were almost exclusively comprised of TEMRA cells (Figure 2). Consistent with immunophenotyping and DNA TCR sequencing, expanded TEMRA cell clones were expanded in active disease compared to stable disease. To assess the impact of these CD8+ T cells on platelets in ITP, PBMCs from patients with ITP and HC were passed across a platelet layer at flow rates consistent with venous blood flow. CD8+ T cells in ITP patients interacted with platelets 4x more frequently than HCs. Isolated CD8+ T cells were cultured over night with autologous platelets to further explore these interactions: CD8+ T cell:platelet aggregates were identified more frequently in the co-cultures from patients with ITP (p ≤ 0.05) and were inhibited by blocking MHC Class I (p ≤ 0.01). CD8+ T cells within the aggregates had increased CD107a expression compared to non-aggregated CD8+ T cells (p ≤ 0.01), indicating cytolytic activity and release of Granzyme and Perforin. Correspondingly, platelets in the aggregates showed increased CD62P expression, a marker of activation and impending death (p ≤ 0.001). Conclusions Here we demonstrate clonal expansion of disease-associated TEMRA CD8+ T cells in ITP. These cells bind to platelets and cause TCR-mediated platelet activation and death providing an antibody-independent mechanism of platelet destruction. This could serve as biomarker to direct treatment in patients with refractory ITP and targeting specific T-cell clones could be a novel therapeutic option in refractory ITP. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

HSV1716 Infects Multiple Myeloma Cells and Leads to CD138 Downregulation

Multiple myeloma (MM) is a plasma cell malignancy that accounts for approximately 10% of all hematological cancers. MM is classified as incurable due to a high relapse rate after the initial therapy and failure to achieve long-term minimal residual disease (MRD). Therefore, new therapies and combinations are needed. Oncolytic viruses (OVs) are genetically modified viruses that preferentially infect and lyse malignantly transformed cells. HSV1716 is a replication-restricted herpes-simplex virus type I (HSV-1)-based OV (HSV-OV) which has been evaluated in several clinical trials. HSV-OVs have been shown to infect and kill MM cell lines and primary malignant plasma cells. Furthermore, HSV-OV mediated target cell killing was demonstrated to be enhanced by natural killer (NK) cells, an immune cell subset that naturally recognizes malignantly transformed or virally infected cells. The underlying mechanisms behind HSV-OV-enhanced NK-tumor cell killing, however, have not yet been elucidated. Here, we aim to monitor phenotypic changes of MM cells upon HSV1716 infection and thus explore the basis for NK cell recognition of HSV-OV infected target MM cells. The MM cell lines RPMI8226 and MM1S were infected with HSV1716 at various multiplicities of infection (MOIs) over a three-day timecourse. Target cell death and expression of viral glycoproteins were measured at different timepoints by flow cytometry. HSV1716 caused oncolysis of 60% of RPMI8226, and 70% of MM1S cells 48h post-infection (p.i.) at an MOI of 0.5. Under these conditions, the expression of viral glycoprotein E on live cells reached 74% on RPMI8226 cells and 37% on MM1S cells. Primary NK (pNK) cells were isolated from healthy donor peripheral blood mononuclear cells (PBMCs) and co-cultured with infected and uninfected MM cells for 4 hours. CD107a as a marker for NK cell degranulation, as well as the release of IFNγ and TNF were measured on CD3-CD56+ pNK cells by flow cytometry. CD107a expression increased from 15.6% (+/- 0.9%) to 56.2% (+/- 2.4%) following infection of RPMI8226 cells and from 15.6% (+/- 3.4%) to 59.2% (+/- 4.5%) following infection of MM1S cells. Similarly, the expression of IFNγ and TNF increased by four- and three-fold, respectively, as compared to uninfected target cells. To monitor changes in the expression of NK cell ligands on infected MM cells, a 16-color flow cytometry panel was designed. RPMI8226 and MM1S cells were infected at different MOIs, and ligand expression was analyzed at 24h, 48h, and 72h p.i.. Most significantly, an early and progressive downregulation of CD138 expression from 96% on uninfected cells to 79 % (24h MOI 0.5), 70% (48h MOI 0.5), and 22% (72h MOI 0.5) on live RPMI8226 cells and from 99% on uninfected cells to 82% (24h MOI 0.5), 65% (48h MOI 0.5) and 23% (72h MOI 0.5) on live MM1S cells was observed. Of note, the expression of CD38 remained stable at each of these timepoints. While downregulation of HLA class I molecules was observed, the expression of other NK cells ligands on RPMI8226 or MM1S cells did not notably differ between infected and uninfected cells. Together, our data confirm that HSV-OV enhances NK cell-mediated killing of MM cells. While expression of ligands for activating NK cell receptors was not altered on infected versus uninfected RPMI8226 and MM1S cells, a strong downregulation of CD138 with maintained CD38 expression was observed upon HSV1716 infection. The implementation of oncolytic virotherapy as an immunotherapeutic platform for cancer treatment adds a new and promising therapeutic tool for combination therapies to the treatment options of MM.