Upregulation Of CD107a Research Articles

Upregulation of the NKG2D Ligand ULBP2 by JC Polyomavirus Infection Promotes Immune Recognition by Natural Killer Cells.

JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML), a potentially fatal complication of severe immune suppression with no effective treatment. Natural killer (NK) cells play critical roles in defense against viral infections; however, NK-cell response to JCPyV infection remains unexplored. NK- and T-cell responses against the JCPyV VP1 were compared using intracellular cytokine staining upon stimulation with peptide pools. A novel flow cytometry-based assay was developed to determine NK-cell killing efficiency of JCPyV-infected astrocyte-derived SVG-A cells. Blocking antibodies were used to evaluate the contribution of NK-cell receptors in immune recognition of JCPyV-infected cells. In about 40% of healthy donors, we detected robust CD107a upregulation and IFN-γ production by NK cells, extending beyond T-cell responses. Next, using the NK-cell-mediated killing assay, we showed that coculture of NK cells and JCPyV-infected SVG-A cells leads to a 60% reduction in infection, on average. JCPyV-infected cells had enhanced expression of ULBP2-a ligand for the activating NK-cell receptor NKG2D, and addition of NKG2D blocking antibodies decreased NK-cell degranulation. NKG2D-mediated activation of NK cells plays a key role in controlling JCPyV replication and may be a promising immunotherapeutic target to boost NK-cell anti-JCPyV activity.

Impaired IL-17 and cytotolytic responses to influenza vaccine antigens in aging nonhuman primates

Abstract Despite widespread flu vaccination, influenza remains a major cause of morbidity and mortality in the elderly. The early immune response, including CD4+ helper T cells, and CD8+ CTLs, plays a critical role in efficient control of virus and initiation of adaptive immunity. However, the correlates of protection in the elderly are not well understood. Here, we sought to investigate early cellular responses to re-exposure with seasonal flu vaccine antigens in a cohort of pre-vaccinated young and old rhesus macaques (human age equivalent of 30–45 yrs, and 55–85 yrs). In vitro T cell responses were examined following overnight stimulation of PBMCs with flu vaccine antigens by flow cytometry for Th1-type and Th17-type cytokine responses, and cytolytic functions. Notably, older macaques showed significantly impaired IL-17 production and CD107a upregulation in response to vaccine antigens, along with greater HLA-DR +/PD-1 +CD8 T cells. Further, they produced significantly higher TNF-α than young macaques. These results suggest that circulating flu vaccine-specific T cells in older macaques are deficient in mucosal barrier-protective and cytotolytic functions and possess a dominant proinflammatory function. These findings suggest that re-exposure to flu vaccine antigens in older individuals likely induces a proinflammatory response that is impaired in cytolytic capacity and the ability to repair lung mucosal damage during influenza infection, thereby resulting in more severe disease outcomes. Further studies on in vivo modulation of proinflammatory T cells while enhancing IL-17 functions during vaccination could support the development of more effective influenza vaccine strategies for older adults.

Elucidating the role played by the NKG2 family of receptors in natural killer cell responses to JC polyomavirus

Abstract JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML), a potentially fatal complication of severe immune suppression due to monoclonal antibody treatments for cancer, autoimmune diseases and transplantation, or due to uncontrolled HIV infection. There is currently no effective treatment against PML and novel immunotherapies are urgently needed. Natural killer (NK) cells play critical roles in defense against viral infections, yet NK cell responses to JCPyV remain largely unexplored. Here, we first compared NK and T cell responses against JCPyV VP1 peptide pools by intracellular cytokine staining. In about 40% of healthy donors, we detected robust CD107a upregulation and IFNγ production by NK cells, extending beyond T cell responses. Next, using a novel flow cytometry-based killing assay, we showed that co-culture of NK cells and JCPyV-infected SVG-A cells led to a 65% reduction in infection, on average. To identify receptor-ligand interactions modulating NK cell-mediated responses to JCPyV, we performed co-cultures in the presence of select blocking antibodies. We observed enhanced expression of ligands for the activating NKG2D on infected cells and accordingly, NKG2D blockade resulted in decreased NK cell degranulation. In contrast, blockade of inhibitory NKG2A enhanced NK cell response to JCPyV-infected targets. Moreover, we identified JCPyV-derived peptides that elicit dominant NK cell responses and can stabilize HLA-E, the ligand for NKG2A and the activating NKG2C. Altogether, these findings suggest that modulation of NKG2D and NKG2A/C signaling may play key roles in NK cell-mediated control of JCPyV and may be promising immunotherapeutic targets to boost NK cell anti-JCPyV activity. Supported by grant from NIH (R01 NS116278)

Using the Jurkat reporter T cell line for evaluating the functionality of novel chimeric antigen receptors.

Background: T cells that are genetically modified with chimeric antigen receptor (CAR) hold promise for immunotherapy of cancer. Currently, there are intense efforts to improve the safety and efficacy of CAR T cell therapies against liquid and solid tumors. Earlier we designed a novel CAR backbone (FiCAR) where the spacer is derived from immunoglobulin (Ig) -like domains of the signal-regulatory protein alpha (SIRPα). However, the analysis of novel CAR using primary T cells is slow and laborious. Methods: To explore the versatility of the CAR backbone, we designed a set of variant FiCARs with different spacer lengths and targeting antigens. To expedite the analysis of the novel CARs, we transduced the FiCAR genes using lentiviruses into Jurkat reporter T cells carrying fluorescent reporter genes. The expression of fluorescent markers in response to FiCAR engagement with targets was analyzed by flow cytometry, and cytotoxicity was evaluated using killing assays. Furthermore, the killing mechanisms that are employed by FiCAR-equipped Jurkat T cells were investigated by flow cytometry, and the intracellular pathways involved in signaling by FiCAR were analyzed by phosphoproteomic analysis using mass spectrometry. Results: Seven different CARs were designed and transduced into Jurkat reporter cells. We show that the SIRPα derived FiCARs can be detected by flow cytometry using the SE12B6A4 antibody recognizing SIRPα. Furthermore, FiCAR engagement leads to robust activation of NFκβ and NFAT signaling, as demonstrated by the expression of the fluorescent reporter genes. Interestingly, the Jurkat reporter system also revealed tonic signaling by a HER-2 targeting FiCAR. FiCAR-equipped Jurkat T cells were cytotoxic in cocultures with target cells and target cell engagement lead to an upregulation of CD107a on the Jurkat reporter T cell surface. Phosphoproteomic analyses confirmed signal transduction via the intracellular CD28/CD3ζ sequences upon the interaction of the FiCAR1 with its antigen. In addition, downstream signaling of CD3ζ/ZAP70- SLP-76-PLCγ, PI3K-AKT-NFκB pathways and activation of NFAT and AP-1 were observed. Conclusion: We conclude that the FiCAR backbone can be shortened and lengthened at will by engineering it with one to three SIRPα derived Ig-like domains, and the FiCARs are functional when equipped with different single chain variable fragment target binding domains. The Jurkat reporter system expedites the analysis of novel CARs as to their expression, signaling function, evaluation of tonic signaling issues and cytotoxic activity.

Abstract P191: The combination of CD16A/EGFR bispecific innate cell engager AFM24 with SNK01 NK cells promotes efficacious targeting and killing of EGFR+ tumor cells

Abstract The epidermal growth factor receptor (EGFR) is an overexpressed antigen on several solid cancers resulting in uncontrolled cell proliferation. Current EGFR-targeting agents such as tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mAbs) mainly rely on EGFR signaling inhibition which is challenged by limited efficacy due to dose limiting toxicity, and/or intrinsic or acquired resistance of the tumor by e.g., KRAS or BRAF mutations. To overcome these limitations and to address the high medical need across EGFR-positive cancers, we investigate a unique approach of combining AFM24, a tetravalent bispecific EGFR- and CD16A-binding innate cell engager (ICE), with the patient-specific NK cell product SNK01. AFM24 mediates high affinity targeting and ADCC of EGFR+ tumor cells by NK cells, independent of EGFR signaling inhibition and irrespective of the mutational status of the tumor. AFM24 is currently being tested as monotherapy in a Phase 1/2a study in patients with EGFR-positive tumors. SNK01 is a first-in-kind, autologous non genetically modified NK cell product with significant anti-tumor cytotoxicity and high and homogeneous expression of CD16A, NKG2D, NKp46 and DNAM-1 , that can be consistently produced even from heavily pre-treated cancer patients. SNK01 showed activity against numerous solid tumors in preclinical studies and in the phase I trials. In order to investigate the synergistic potential of this unique combination, SNK01 cells were generated from PBMCs of healthy individuals and their functional activation by AFM24 in response to EGFR-positive A-431 cells was assessed in cytotoxicity assays and flow cytometry-based CD107a (marker for NK cell degranulation) and IFN-γ mobilization assays. AFM24 binding to and CD16A expression on SNK01 cells was measured by flow cytometry. Both, binding of AFM24 and expression of CD16A were detected on nearly 100% of SNK01 cells, indicating saturation of CD16A-positive SNK01 cells with AFM24. In the presence of AFM24, lysis of EGFR-positive A-431 cells by SNK01 cells was significantly enhanced. Likewise, in response to AFM24, upregulation of CD107a and IFN-γ expression by SNK01 cells was significantly increased upon cross-linking to A-431 cells. By contrast, AFM24 alone did not lead to significant NK cell activation in the absence of target cells or vice versa. Hence, activation of SNK01 cells was strictly dependent on AFM24-mediated cross-linking of SNK01 cells with target cells. In conclusion, this combination holds promise to direct the AFM24-augmented anti-tumor activity of SNK01 cells to EGFR-positive tumor cells independent of EGFR signaling pathway mutations thereby providing a potential new treatment option for underserved patient populations. An IND for a Phase I/II combination trial of AFM24 and SNK01 has been cleared and first-patient-in is on track for the second half of 2021. Citation Format: Jens Pahl, Ting-Ying (Hazel) Cheng, Uwe Reusch, Arndt Schottelius, Wolfgang Fischer, Yong Man Kim, Stephen Chen, Joachim Koch. The combination of CD16A/EGFR bispecific innate cell engager AFM24 with SNK01 NK cells promotes efficacious targeting and killing of EGFR+ tumor cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P191.

Engineering Human Induced Pluripotent Stem Cells with Novel Chimeric Antigen Receptors to Generate Natural Killer (NK) Cell Cancer Immunotherapies with Targeted Anti-Tumor Activity

Chimeric antigen receptors (CARs) provide a powerful strategy to direct and enhance anti-tumor activity of immune effector cells. While most studies have evaluated CAR-expression in T cells, here we evaluate and optimize CAR constructs that are specifically designed with natural killer (NK) cell transmembrane and signaling domains. Since NK cell-mediated cytotoxicity does not require self-HLA expression, derivation of NK cells from human induced pluripotent stem cells (iPSCs), combined with the use of NK cell-specific CARs, enables production of a standardized, targeted allogeneic effector cell population. After screening 10 combinations of CARs using an anti-mesothelin (meso) scFv to target ovarian cancer cells, we identified a CAR construct containing the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3ζ signaling domain that mediates a strong increase in intracellular NK cell signaling. iPSC-derived NK cells that express this novel CAR (NKCAR-iPSC-NK cells) have a surface phenotype similar to NK cells isolated from peripheral blood (PB-NK) and unmodified iPSC-derived NK cells. Interestingly, the NKCAR-iPSC-NK cells mediate improved killing of target-expressing targets more than iPSC-derived NK cells expressing a 3rd generation CAR with CD28, CD137, and ζ signaling elements optimized for T cell-mediated activity (TCAR-iPSC-NK cells). We examined intracellular signaling pathways stimulated by our NKG2D-2B4-ζ CAR construct. In NKCAR-iPSC-NK cells, antigen (meso)-expressing targets promoted phosphorylation of Syk and Erk. Site-directed mutagenesis studies demonstrate the requirement for the NKG2D and 2B4 domains to mediate release of cytotoxic granules (CD107a), cytokine (IFN-γ) release and robust tumor cell lysis. Using an ovarian cancer xenograft model, a single dose of NKCAR-iPSC-NK cells markedly inhibited tumor growth, and mediated significantly enhanced survival (84 days) compared to NK cells isolated from peripheral blood (PB-NK cells) (70 day survival), non-CAR-expressing iPSC-NK cells (57 days), and TCAR-iPSC-NK cells (63 days) (p < 0.002 for NKCAR-iPSC-NK cells compared to each of the other 3 NK cell populations). As an additional strategy, we have engineered iPSCs both with this novel CAR using an anti-CD19 scFv and with a high-affinity non-cleavable CD16 receptor (hnCD16 with F158V (high-affinity) and S197P (non-cleavable) substitutions). To prevent antigen loss variants that promote resistance of B cell malignancies to anti-CD19-CAR-T cell-based therapies, this approach now allows us to combine the use of anti-CD19-CAR with anti-CD20 antibody to target B cell malignancies using engineered iPSC-NK cells. This combinatorial targeting of Raji cells (CD19+CD20+ B cell lymphoma) demonstrates enhanced upregulation of CD107a and TNFα with improved elimination of CD19-negative target cells. Together, these strategies provide a comprehensive approach to utilize NKCAR-iPSC-NK cells as a novel strategy to produce “off-the-shelf”, targeted, allogeneic lymphocytes suitable to treat refractory solid tumor and hematologic malignancies. DisclosuresHermanson:Poseida Therapeutics: Employment. Bjordahl:Fate Therapeutics: Employment, Equity Ownership. Mahmood:Fate Therapeutics: Employment. Valamehr:Fate Therapeutics: Employment, Equity Ownership. Kaufman:Fate Therapeutics: Consultancy, Research Funding.

Immunological Aspects of X-Linked Chronic Granulomatous Disease Female Carriers.

X-linked Granulomatous Disease (XL-CGD) carriers were previously thought to be clinically healthy because random X-chromosome inactivation (XCI) allows approximately half of their phagocytes/monocytes to express functional gp91phox protein. This supports the NADPH oxidase activity necessary for the killing of engulfed pathogens. Some XL-CGD carriers suffer from inflammatory and autoimmune manifestations as well as infections, although the skewed-XCI of a mutated allele is reported to be exclusively determinant for infection susceptibility. Indeed, immune dysregulation could be determined by dysfunctional non-phagocytic leukocytes rather than the percentage of functioning neutrophils. Here we investigated in a cohort of 12 X-CGD female carriers at a particular time of their life the gp91phox protein expression/function and how this affects immune cell function. We showed that 50% of carriers have an age-independent skewed-XCI and 65% of them have a misrepresented expression of the wild-type gene. The majority of carriers manifested immune dysregulation and GI manifestations regardless of age and XCI. Immunological investigations revealed an increase in CD19+ B cells, CD56bright-NK cell percentage, a slightly altered CD107a upregulation on CD4+ T cells, and reduced INFγ-production by CD4+ and CD8+ cells. Notably, we demonstrated that the residual level of ROS robustly correlates with INFγ-expressing T cells, suggesting a role in promoting immune dysregulation in carriers.

T cell mediated immunity against influenza H5N1 nucleoprotein, matrix and hemagglutinin derived epitopes in H5N1 survivors and non-H5N1 subjects.

BackgroundProtection against the influenza virus by a specific antibody is relatively strain specific; meanwhile broader immunity may be conferred by cell-mediated immune response to the conserved epitopes across influenza virus subtypes. A universal broad-spectrum influenza vaccine which confronts not only seasonal influenza virus, but also avian influenza H5N1 virus is promising.MethodsThis study determined the specific and cross-reactive T cell responses against the highly pathogenic avian influenza A (H5N1) virus in four survivors and 33 non-H5N1 subjects including 10 H3N2 patients and 23 healthy individuals. Ex vivo IFN-γ ELISpot assay using overlapping peptides spanning the entire nucleoprotein (NP), matrix (M) and hemagglutinin (HA) derived from A/Thailand/1(KAN-1)/2004 (H5N1) virus was employed in adjunct with flow cytometry for determining T cell functions. Microneutralization (microNT) assay was performed to determine the status of previous H5N1 virus infection.ResultsIFN-γ ELISpot assay demonstrated that survivors nos. 1 and 2 had markedly higher T cell responses against H5N1 NP, M and HA epitopes than survivors nos. 3 and 4; and the magnitude of T cell responses against NP were higher than that of M and HA. Durability of the immunoreactivity persisted for as long as four years after disease onset. Upon stimulation by NP in IFN-γ ELISpot assay, 60% of H3N2 patients and 39% of healthy subjects exhibited a cross-reactive T cell response. The higher frequency and magnitude of responses in H3N2 patients may be due to blood collection at the convalescent phase of the patients. In H5N1 survivors, the effector peptide-specific T cells generated from bulk culture PBMCs by in vitro stimulation displayed a polyfunction by simultaneously producing IFN-γ and TNF-α, together with upregulation of CD107a in recognition of the target cells pulsed with peptide or infected with rVac-NP virus as investigated by flow cytometry.ConclusionsThis study provides an insight into the better understanding on the homosubtypic and heterosubtypic T cell-mediated immune responses in H5N1 survivors and non-H5N1 subjects. NP is an immunodominant target of cross-recognition owing to its high conservancy. Therefore, the development of vaccine targeting the conserved NP may be a novel strategy for influenza vaccine design.

Regulation of ROS in myeloid-derived suppressor cells through targeting fatty acid transport protein 2 enhanced anti-PD-L1 tumor immunotherapy

Despite the remarkable success and efficacy of immune checkpoint blockade (ICB) therapy against the PD-1/PD-L1 axis, it induces sustained responses in a sizeable minority of cancer patients due to the activation of immunosuppressive factors such as myeloid-derived suppressor cells (MDSCs). Inhibiting the immunosuppressive function of MDSCs is critical for successful cancer ICB therapy. Interestingly, lipid metabolism is a crucial factor in modulating MDSCs function. Fatty acid transport protein 2 (FATP2) conferred the function of PMN-MDSCs in cancer via the upregulation of arachidonic acid metabolism. However, whether regulating lipid accumulation in MDSCs by targeting FATP2 could block MDSCs reactive oxygen species (ROS) production and enhance PD-L1 blockade-mediated tumor immunotherapy remains unexplored. Here we report that FATP2 regulated lipid accumulation, ROS, and immunosuppressive function of MDSCs in tumor-bearing mice. Tumor cells-derived granulocyte macrophage-colony stimulating factor (GM-CSF) induced FATP2 expression in MDSCs by activation of STAT3 signaling pathway. Pharmaceutical blockade of FATP2 expression in MDSCs by lipofermata decreased lipid accumulation, reduced ROS, blocked immunosuppressive activity, and consequently inhibited tumor growth. More importantly, lipofermata inhibition of FATP2 in MDSCs enhanced anti-PD-L1 tumor immunotherapy via the upregulation of CD107a and reduced PD-L1 expression on tumor-infiltrating CD8+T-cells. Furthermore, the combination therapy blocked MDSC's suppressive role on T- cells thereby enhanced T-cell's ability for the production of IFN-γ. These findings indicate that FATP2 plays a key role in modulating lipid accumulation-induced ROS in MDSCs and targeting FATP2 in MDSCs provides a novel therapeutic approach to enhance anti-PD-L1 cancer immunotherapy.

&lt;p&gt;Drugs for Multiple Sclerosis Activate Natural Killer Cells: Do They Protect Against COVID-19 Infection?&lt;/p&gt;

COVID-19 infection caused by the newly discovered coronavirus severe acute respiratory distress syndrome virus-19 (SARS-CoV-2) has become a pandemic issue across the globe. There are currently many investigations taking place to look for specific, safe and potent anti-viral agents. Upon transmission and entry into the human body, SARS-CoV-2 triggers multiple immune players to be involved in the fight against the viral infection. Amongst these immune cells are NK cells that possess robust antiviral activity, and which do not require prior sensitization. However, NK cell count and activity were found to be impaired in COVID-19 patients and hence, could become a potential therapeutic target for COVID-19. Several drugs, including glatiramer acetate (GA), vitamin D3, dimethyl fumarate (DMF), monomethyl fumarate (MMF), natalizumab, ocrelizumab, and IFN-β, among others have been previously described to increase the biological activities of NK cells especially their cytolytic potential as reported by upregulation of CD107a, and the release of perforin and granzymes. In this review, we propose that such drugs could potentially restore NK cell activity allowing individuals to be more protective against COVID-19 infection and its complications.

Abstract 4519: Photochemically-primed immune cell enrichment and anti-tumor responses to pancreatic cancer

Abstract Pancreatic adenocarcinoma (PDAC) is among the deadliest forms of cancer. Currently, it is the third leading cause of cancer deaths in the United States. At diagnosis, the majority of patients present with advanced primary or metastatic disease that is unresectable. PDAC pathophysiology is challenging for current therapies as immunosuppressive desmoplastic stroma limits responsiveness to treatments including immunotherapy. The latter condition is making a vast proportion of PDAC tumors being immunologically cold as defined by the lack of effective immune infiltration to the tumor. Thus, there is an unmet need for improved treatment strategies. Photodynamic therapy (PDT) is an approved modality that utilizes light, a photo-responsive photosensitizer (PS), and oxygen to generate reactive oxygen species that will kill nearby cells or prime neighboring cells to be more responsive to traditional therapies, and thereby produce a synergistic outcome. We aimed to investigate PDT-induced immunogenicity in PDAC tumors. In this study, we applied PDT in an immunocompetent mouse (C57BL/6 mice), PDAC tumor model and assessed tumour-infiltrating lymphocyte (TIL) infiltration at different time points. Visudyne® used as the PS. Multicolor flow cytometric analysis showed gradual increase of T and B cell infiltration at 1h time point to 5 days after PDT. T cell subset analysis showed a higher infiltration of CD8+ T cells with time. Localization of immune cells using immunofluorescence assays further confirmed latter findings. We observed a presence of proliferating T cells in the T cell zone of Spleen B cell follicles 1h after treating with PDT suggesting an early T cell activation. Activated dendritic cells were localized in the spleens treated with PDT compared to the untreated control. We further evaluated the anti-tumour immune reactivity of CD8+ T cells treated with PDT in an in vitro 3D system consisting of human PDAC cell line, MiaPaCa2, pancreatic fibroblast and human peripheral blood mononuclear cell co-culture. Tumor reactivity by CD8+ T cells was evaluated at baseline, 3 days, 7 days and 2 weeks in co-culture after PDT by staining for the surface expression of degranulation marker CD107a. There was a continuous CD107a upregulation in CD8+ T cells from day 3 to 2 week in co-culture treated with PDT compared to untreated controls demonstrating enhanced PDT priming of T cells, in vitro. In addition, there was an effective tumor cell killing in PDT treated immune-tumour co-cultures. Our data shows a possibility of triggering an immediate and effective immune-response to PDT in PDAC tumors enhancing the immunogenicity of this tumor type. Citation Format: Pushpamali De Silva, Mohammad Ahsan Saad, Assiris P. Camargo, Joseph Swain, Akilan Palanasami, Tayyaba Hasan. Photochemically-primed immune cell enrichment and anti-tumor responses to pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4519.

Abstract A17: Enhanced immune infiltration and antitumor immune reactivity in response to optical priming in pancreatic cancer

Abstract Pancreatic adenocarcinoma (PDAC) is one of the most lethal malignancies with a survival rate of approximately 5% at five years. At diagnosis, the majority of patients present with advanced primary or metastatic disease that is unresectable. PDAC pathophysiology is challenging for current therapies as immunosuppressive desmoplastic stroma limits responsiveness to treatments including immunotherapy. Latter condition is making vast proportion of PDAC tumors immunologically cold as defined by the lack of effective immune infiltration to the tumor. Thus, there is an unmet need for improved treatment strategies. Photodynamic therapy (PDT) is an approved modality that utilizes light, a photoresponsive photosensitizer (PS), and oxygen to generate reactive oxygen species that will kill nearby cells or prime neighboring cells to be more responsive to traditional therapies, and thereby produce a synergistic outcome. We aimed to investigate PDT-induced immunogenicity in PDAC tumors. In this study, we applied PDT in an immunocompetent mouse (C57BL/6 mice), PDAC tumor model and assessed tumor-infiltrating lymphocyte (TIL) infiltration at different time points. Visudyne was used as the PS. Multicolor flow cytometric analysis showed gradual increase of T- and B-cell infiltration at 1h time point to 5 days after PDT. T-cell subset analysis showed a higher infiltration of CD8+ T cells with time. Localization of immune cells using immunofluorescence assays further confirmed latter findings. We observed a presence of proliferating T cells in the T cell zone of spleen B cell follicles 1h after treating with PDT, suggesting an early T-cell activation. Activated dendritic cells were localized in the spleens treated with PDT compared to the untreated control. We further evaluated antitumor immune reactivity of CD8+ T cells treated with PDT in an in vitro 3D system consisting of human PDAC cell line, MiaPaCa2, pancreatic fibroblast, and human peripheral blood mononuclear cell coculture. Tumor reactivity by CD8+ T cells was evaluated at baseline, 3 days, 7 days, and 2 weeks in coculture after PDT by staining for the surface expression of degranulation marker CD107a. There was a continuous CD107a upregulation in CD8+ T cells from day 3 to 2 week in coculture treated with PDT compared to untreated controls, demonstrating enhanced PDT priming of T cells, in vitro. In addition, there was effective tumor cell killing in PDT-treated immune-tumor cocultures. Our data show a possibility of triggering an immediate and effective immune response to PDT in PDAC tumors, enhancing the immunogenicity of this tumor type. Citation Format: Pushpamali De Silva, Mohammad Ahsan Saad, Assiris P. Camargo, Joseph Swain, Akilan Palanasami, Girgis Obaid, Shalin Shetty, Tayyaba Hasan. Enhanced immune infiltration and antitumor immune reactivity in response to optical priming in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A17.

Combined Detection of CD137 and Type 1 Functions Improves Identification and Characterization of the Activated T Lymphocyte Repertoire

Abstract Background CD137 (4-1BB) is widely considered a comprehensive marker of T cell activation, and it is largely used in cancer immunology to determine the proportion of tumor-reactive T cells. However, CD137 detection does not provide specific information regarding the functions of activated T cells. Hence, in this study we tried to determine whether the simultaneous detection of CD137 plus common antitumor immune functions (type 1 functions such as production of cytokines TNFα and IFN-γ, or upregulation of CD107a) was feasible and provided advantages over using both methods separately. Methods The influence of common protein transport inhibitors used to allow cytokine detection, such as Brefeldin A (BFA) or Monensin (MN), on surface and intracellular expression of CD137 was tested in vitro. Activation of tumor infiltrating lymphocytes (TILs) was achieved with unspecific stimuli (PMA and Ionomycin) or naturally presented tumor-antigens (autologous tumor cells). TIL activation was defined as the proportion of CD4+ or CD8+ TILs staining for either CD137 (CD137+ TILs) and/or at least one of TNFα, IFNγ and CD107a (Type 1 function+ TILs) via flow cytometry. Results Our results showed that MN had minimal impact on the ability to detect surface CD137, whereas BFA had a much larger effect regardless of the stimulus employed. Nonetheless, robust results could be obtained when type 1 functions detection was combined with intracellular staining of CD137 in the presence of both BFA and MN. Using this method, we identified three tumor-antigen specific T cell subpopulations, characterized by combined expression of CD137 and type 1 functions or CD137 without type 1 functions and vice-versa. The combined method allowed the identification of a much larger proportion of tumor-reactive T cells, over either of the two methods used alone. Indeed, CD137 alone identified 46% of the total tumor-reactive CD8+ TILs, while 86% of the total tumor-reactive CD8+ TILs were positive for type 1 functions. Conclusion Combined detection of CD137 and common type 1 functions represents a more comprehensive method to identify and characterize the total repertoire of tumor-antigen specific, activated T lymphocytes, regardless of the antigen recognized. Legal entity responsible for the study Herlev and Gentofte Hospital. Funding Research grant A11806 and A12535, The Danish Cancer Society; Research grant R233-2016-3728 and R286-2018-991 Lundbeck Foundation; Clinician-Scientist Grant from the Herlev and Gentofte Hospital Research Council. Disclosure All authors have declared no conflicts of interest.

A Bcma and CD19 Bispecific CAR-T for Relapsed and Refractory Multiple Myeloma

Introduction Chimeric Antigen Receptor T cells (CAR-T) therapy, e.g. B Cell Maturation Antigen (BCMA)-directed CAR-T has provided an encouraging modality for relapsed and refractory management of multiple myeloma (MM). However, a significant portion of patients still relapse with progressive disease after monospecific anti-BCMA CAR-T treatment. It has been demonstrated that CD19-directed CAR-T was effective in certain MM patients, likely due to CD19 expression on subsets of MM cells, and/or undetectable level of CD19 on MM cells. In addition, it has been reported that CD19 could express on the myeloma progenitor cells. To further improve the efficacy and to reduce relapse, we have designed a bispecific CAR-T targeting both BCMA and CD19. In addition to the conventionally-manufactured BCMA-CD19 CAR-T, the bispecific CAR-T was also successfully manufactured in our newly developed FasT CAR-T platform, which shortened the production time to one day. Here we report the results from pre-clinical studies and early results from the first-in-human clinical study. Methods The BCMA-CD19 bispecific CAR was constructed by linking BCMA and CD19 scFv, joined by a CD8 hinge, transmembrane domain, co-stimulatory domain and CD3. CAR-T cells were produced using either the conventional process (GC012) or the FasT CAR-T platform (GC012F). Peripheral blood (PB) mononuclear cells were obtained by leukapheresis either from healthy donors for the pre-clinical study or from patients for the clinical trial. T cells were isolated and used for CAR-T manufacturing. A xenograft mouse model was used to determine the efficacy in vivo. From March 2019 to July 2019, 5 adult relapsed/refractory MM patients (Age 50-59), who had previously received multiple lines of therapies, were enrolled (Table). Among them, 2 had extramedullary diseases. One patient did not receive lymphodepletion, and all other 4 patients received i.v. fludarabine and cyclophosphamide for 3 days. All patients received a single infusion of CAR-T cells, either at dose 1x106/Kg (DL1) (2 patients) or at dose 2x106/Kg (DL2) (3 patients), and the dose escalation is still ongoing. The endpoints of the exploratory trial were to evaluate the safety, feasibility, PK, and clinical efficacy of BCMA-CD19 bispecific CAR-T. Results In pre-clinical study, BCMA-CD19 bispecific CAR-T were very effective in killing CD19+ and/or BCMA + target cells including MM cell lines RPMI8226 and MM.1s (Fig 1). Increased IFN production and CD107a up-regulation were also observed. We demonstrated that BCMA-CD19 CAR-T completely eliminated BCMA+ MM cell line RPMI8226, MM.1s, and CD19+ ALL cell line Nalm6 in in vivo xenograft models. Additionally BCMA-CD19 CAR-T cells were shown to be more cytotoxic than single CAR-T both in vitro and in vivo. BCMA-CD19 CAR-T manufactured in the FasT CAR-T platform was more effective in eliminating MM in a xenograft model (Fig. 1). In the clinical study, the median observation time was 68 days (27-144 days up to 2019/7/30). Five patients were evaluated between 15-59 days post CAR-T infusion. Despite the relatively short disease evaluation time, all 5 patients responded to the treatment: 1 patient achieved sCR, 3 achieved VGPR and 1 achieved PR. Notably, although patient KM001 did not receive any pre-conditioning, however, the patient achieved sCR status on Day 15 and has maintained sCR up to now (129 days). CAR-T PK in the PB was monitored by qPCR and flow cytometry. The CAR-T proliferation peak was reached on Day 10 (D7-D14), and the median peak copy number was 34,039 (12,897-128,775) copies /ug DNA (Fig. 2). Remarkably, despite the encouraging clinical response to the CAR-T treatment, no severe adverse events were encountered during the observation period. Three patients experienced only grade 1 cytokine release syndrome (CRS) and no subject suffered from neurotoxicity of any level (Table). Conclusion Pre-clinical data demonstrated BCMA-CD19 CAR-T cells are effective in eliminating MM tumor cells both in vitro and in vivo. The first-in-human clinical trial also showed extraordinary safety profile and efficacy of BCMA-CD19 bispecific CAR-T in treating R/R MM. The long-term benefit and effect on relapse are being further studied. Bispecific CAR-T manufacturing on the FasT CAR-T platform is successful and has been shown to be more potent. A clinical study to evaluate safety and efficacy of FasT BCMA-CD19 CAR-T is ongoing. Disclosures No relevant conflicts of interest to declare.

A novel simplified method of generating cytomegalovirus-specific cytokine-induced killer cells of high specificity and superior potency with GMP compliance

We describe a method of rendering polyclonal cytokine-induced killer cells (CIK) specific against cytomegalovirus (CMV), focusing on GMP compliance. Peripheral blood mononuclear cells (PBMNC) are stimulated with pooled CMV peptides pp65 and IE-1 for 16–24 h and the reactive T cell subset which up-regulate CD137 is further co-stimulated with anti-CD137, followed by expansion in G-Rex flasks under standard CIK culture condition. This method generates a large number CMV-specific CIK with superior potency compared to published method currently in clinical trials. The cytotoxicity as measured by chromium release assay correlates with the upregulation of CD107a upon peptide re-challenge as measured by flow cytometry. CMV-CIK at maturity consist of mainly late effector memory CD8 T cells and have a skewed TCR repertoire with preferential expansion of a few families. Such CMV-CIK retain their function after freezing and thawing. CMV-CIK thus generated is ready for clinical trial against drug-resistant CMV disease.

Construction of a chimeric antigen receptor bearing a nanobody against prostate a specific membrane antigen in prostate cancer.

Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) is considered to be a novel anticancer therapy. To date, in most cases, single-chain variable fragments (scFvs) of murine origin have been used in CARs. However, this structure has limitations relating to the potential immunogenicity of mouse antigens in humans and the relatively large size of scFvs. For the first time, we used camelid nanobody (VHH) to construct CAR T cells against prostate specific membrane antigen (PSMA). The nanobody against PSMA (NBP) was used to show the feasibility of CAR T cells against prostate cancer cells. T cells were transfected, and then the surface expression of the CAR T cells was confirmed. Then, the functions of VHH-CAR T cell were evaluated upon coculture with prostate cancer cells. At the end, the cytotoxicity potential of NBPII-CAR in T cells was approximated by determining the cell surface expression of CD107a after encountering PSMA. Our data show the specificity of VHH-CAR T cells against PSMA+ cells (LNCaP), not only by increasing the interleukin 2 (IL-2) cytokine (about 400 pg/mL), but also the expression of CD69 by almost 38%. In addition, VHH-CAR T cells were proliferated by nearly 60% when cocultured with LNCaP, as compared with PSMA negative prostate cancer cell (DU-145), which led to the upregulation of CD107a in T cells upto 31%. These results clearly show the possibility of using VHH-based CAR T cells for targeted immunotherapy, which may be developed to target virtually any tumor-associated antigen for adoptive T-cell immunotherapy of solid tumors.

Enhancing the immune stimulatory effects of cetuximab therapy through TLR3 signalling in Epstein-Barr virus (EBV) positive nasopharyngeal carcinoma

ABSTRACT Cetuximab immunotherapy targeting the epidermal growth factor receptor (EGFR) has been used to treat nasopharyngeal cancer (NPC) with some success. Therefore, combining an immune adjuvant to boost the immune microenvironment may improve its clinical efficacy. Herein, we investigate the immune-stimulatory effects of Poly-ICLC (a TLR3 agonist) in enhancing cetuximab-based immunotherapy and correlate these responses with FcɣRIIIa (V158F) or TLR3 single nucleotide polymorphisms (SNPs- L412F and C829T) expressed on immune effector cells. We observed high levels of TLR3 mRNA in NPC cells; and both TLR3 and EGFR expression were unaffected by Poly-ICLC treatment. Cetuximab plus Poly-ICLC significantly enhanced NK-mediated ADCC through up-regulation of CD107a and Granzyme B expression. This effect was independent of FcɣRIIIa-V158F and TLR3-L412F or TLR3-C829T polymorphisms expressed on NK cells. Additionally, IFN-ɣ expression and secretion were doubled following cetuximab plus poly-ICLC treatment; compared to either treatment alone. This effect was independent of TLR3 polymorphisms. Consequentially, adaptive immune responses were also seen with increased DC maturation (CD83), co-stimulatory molecules expression (CD80 and CD86) and increased frequency of EGFR-specific CD8 + T cells following Poly-ICLC treatment. The percentage of CD80+ CD83+ and CD83+ CD86+ DC was highest in the Poly-ICLC plus cetuximab group, compared to either treatment alone. These results demonstrate the effectiveness of Poly-ICLC in enhancing both cetuximab-mediated innate and adaptive anti-tumor immunity against NPC, which is independent of FcɣRIIIa-158, TLR3-L412F or TLR3-C829T polymorphisms. Additionally, Poly-ICLC does not downregulate EGFR expression on NPC cells and hence, will not dampen cetuximab anti-tumor activity.

Decreased NK cell immunity in kidney transplant recipients late post-transplant and increased NK-cell immunity in patients with recurrent miscarriage.

BackgroundThere is evidence that NK-cell reactivity might affect graft outcome in transplant recipients and pregnancy in women.MethodNK-cell subsets were determined in whole blood using eight-colour-fluorescence flow cytometry in patients before and after renal transplantation, patients with recurrent miscarriage (RM) and healthy controls (HC).ResultsPatients late post-transplant (late-Tx) with functioning renal transplants showed abnormally low CD56dimCD16+ NK-cells containing both perforin and granzyme (vs HC p = 0.021) whereas RM patients exhibited abnormally high numbers of these cells (vs HC p = 0.043). CD56dimCD16+perforin+granzyme+ NK-cell counts were strikingly different between the two patient groups (p<0.001). In addition, recipients late-Tx showed abnormally low CD8+ NK-cells (vs HC p<0.001) in contrast to RM patients who showed an abnormal increase (vs HC p = 0.008). CD8+ NK-cell counts were strongly different between the two patient groups (p<0.001). Higher perforin+granzyme+CD56dimCD16+ and CD8+ NK-cells were associated with impaired graft function (p = 0.044, p = 0.032). After in-vitro stimulation, CD56dimCD16+ and CD56brightCD16dim/- NK-cells showed strong upregulation of CD107a and IFNy, whereas the content of perforin decreased dramatically as a consequence of perforin release. Recipients late post-Tx showed less in-vitro perforin release (= less cytotoxicity) than HC (p = 0.037) and lower perforin release was associated with good graft function (r = 0.738, p = 0.037). Notably, we observed strong in-vitro perforin release in 2 of 6 investigated RM patients. When circulating IL10+CD56bright NK-cells were analyzed, female recipients late post-Tx (n = 9) showed significantly higher relative and absolute cell numbers than RM patients (p = 0.002 and p = 0.018, respectively); and high relative and absolute IL10+CD56bright NK-cell numbers in transplant recipients were associated with low serum creatinine (p = 0.004 and p = 0.012) and high glomerular filtration rate (p = 0.011 and p = 0.002, respectively). Female recipients late post-Tx exhibited similar absolute but higher relative numbers of IL10+IFNy- NK-cells than RM patients (p>0.05 and p = 0.016, respectively).ConclusionNK-cells with lower cytotoxicity and immunoregulatory function might contribute to good long-term graft outcome, whereas circulating NK-cells with normal or even increased cytotoxicity and less immunoregulatory capacity are observed in patients with RM.

Perforin and CD107a testing is superior to NK cell function testing for screening patients for genetic HLH

AbstractPrimary hemophagocytic lymphohistiocytosis (HLH) can be caused by biallelic mutations in PRF1, encoding perforin, or UNC13D, STXBP2, STX11, RAB27A, LYST, and AP3B1, encoding proteins involved in cytotoxic lymphocyte degranulation. Natural killer (NK)–cell cytotoxicity assays can quickly screen for all of these genetic diseases, facilitating treatment, but combining NK-cell perforin expression and CD107a upregulation tests can as well. To determine the relative diagnostic accuracies for each approach, we retrospectively reviewed screening test performance in 1614 patients referred for HLH evaluation. For each test, we generated a receiver operating characteristic (ROC) curve, and calculated area under the curve (AUC) and diagnostic parameters at optimal threshold. We generated an AUC for combining perforin and CD107a tests by creating a logistic regression model and applying model-generated coefficients to patient values. Sensitivities of NK-cell function, perforin mean channel fluorescence (MCF), and CD107a MCF to detect biallelic mutations were 59.5%, 96.6%, and 93.8%, with specificities of 72.0%, 99.5%, and 73%. AUCs for NK-cell cytotoxicity, perforin MCF, CD107a MCF, and combined perforin and CD107a MCFs were 0.690, 0.971, 0.860, and 0.838. Perforin and CD107a tests are more sensitive and no less specific compared with NK cytotoxicity testing for screening for genetic HLH and should be considered for addition to current HLH criteria.

Evaluating the repertoire of immune checkpoint markers expressed on peripheral and ascites CD8+ T cells in ovarian cancer.

5571 Background: Understanding the immune checkpoint marker repertoire can facilitate development of therapeutic strategies to improve efficacy of immune-based therapies. We used a novel high-dimensional flow cytometry panel to determine co-expression patterns of immune checkpoint markers and effector function of CD8+ T cells from peripheral blood and ascites of patients newly diagnosed with ovarian cancer. Methods: Peripheral blood and ascites samples were collected from patients with epithelial ovarian cancer (n=8). Cells isolated from peripheral blood and ascites were used for immune profiling by multiparameter flow cytometry of 5 inhibitory receptors (PD-1, LAG-3, TIM-3, TIGIT, and BTLA) on CD8+ T cells, along with 4 functional parameters (production of each of the following: TNF-α, IFN-γ, IL-2, and upregulation of CD107a). A complementary multiplex analysis on plasma and ascites fluid was performed to quantify 14 soluble checkpoint markers. Results: The concentrations of soluble PD-1, TIM-3, LAG-3, CTLA-4, BTLA, IDO, and CD137 were increased in ascites fluid compared to plasma from patients with ovarian cancer. Ascites CD8+ T cells co-express higher levels of inhibitory receptors than peripheral CD8+ T cells. In total, CD8+ T cells in ascites retained the ability to produce effector functions at levels similar to peripheral blood. However, IFN-γ production was retained in PD-1 only expressing CD8+ T cells and decreased in CD8+ T cells co-expressing multiple receptors. Conclusions: High-dimensional flow cytometry allowed for the phenotypic and functional characterization of CD8+ T cells from ovarian cancer patients. The profile of receptor co-expression was distinct in peripheral blood compared to ascites. Collectively, our study suggests that co-expression of factors beyond PD-1 influences CD8+ T cell activity. Thus blocking PD-1 and PD-L1 alone may not be sufficient for CD8+ T cells expressing multiple inhibitory receptors.