Release Of Cytotoxic Granules Research Articles

Fate control engagement augments NK cell responses in LV/hu-IL-12 transduced sarcoma

IntroductionNK cells are an untapped resource for cancer therapy. Sarcomas transduced with lentiviruses to express human IL-12 are only cleared in mice bearing mature human NK cells. However, systemic inflammation limits IL-12 utilization. Fate control a.k.a. “suicide mechanisms” regulate unchecked systemic inflammation caused by cellular immunotherapies. Despite increasing utilization, there remains limited data on immune consequences or tumor-directed effects of fate control. ObjectivesWe sought to engage the mutant thymidylate kinase (mTMPK) metabolic fate control system to regulate systemic inflammation and assess the impact on NK cell effector functions. MethodsPrimary human sarcoma short-passage samples and cell lines were transduced with LV/hu-IL-12_mTMPK engineering expression of IL-12 and an AZT-associated fate control enzyme. We assessed transduced sarcoma responses to AZT engagement and subsequent modulation of NK cell functions as measured by inflammatory cytokine production and cytotoxicity. ResultsAZT administration to transduced (LV/hu-IL-12_mTMPK) short-passage primary human sarcomas and human Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma cell lines, abrogated the robust expression of human IL-12. Fate control activation elicited a specific dose-dependent cytotoxic effect measured by metabolic activity (WST-1) and cell death (Incucyte). NK effector functions of IFN-γ and cytotoxic granule release were significantly augmented despite IL-12 abrogation. This correlated with preferentially induced expression of NK cell activation ligands. ConclusionsmTMPK fate control engagement terminates transduced sarcoma IL-12 production and triggers cell death, but also augments an NK cell-mediated response coinciding with metabolic stress activating surface ligand induction. Fate control engagement could offer a novel immune activation method for NK cell-mediated cancer clearance.

Bortezomib promotes the TRAIL-mediated killing of resistant rhabdomyosarcoma by ErbB2/Her2-targeted CAR-NK-92 cells via DR5 upregulation

Treatment resistance and immune escape are hallmarks of metastatic rhabdomyosarcoma (RMS), underscoring the urgent medical need for therapeutic agents against this disease entity as a key challenge in pediatric oncology. Chimeric antigen receptor (CAR)-based immunotherapies, such as the ErbB2 (Her2)-CAR-engineered natural killer (NK) cell line NK-92/5.28.z, provide antitumor cytotoxicity primarily through CAR-mediated cytotoxic granule release and thereafter - even in cases with low surface antigen expression or tumor escape - by triggering intrinsic NK cell-mediated apoptosis induction via additional ligand/receptors. In this study, we showed that bortezomib increased susceptibility towards apoptosis in clinically relevant RMS cell lines RH30 and RH41, and patient-derived RMS tumor organoid RMS335, by upregulation of the TRAIL receptor DR5 in these metastatic, relapsed/refractory (r/r) RMS tumors. Subsequent administration of NK-92/5.28.z cells significantly enhanced antitumor activity in vitro. Applying recombinant TRAIL instead of NK-92/5.28.z cells confirmed that the synergistic antitumor effects of the combination treatment were mediated via TRAIL. Western blot analyses indicated, that the combination treatment with bortezomib and NK-92/5.28.z cells increased apoptosis by interacting with the NF-κB, JNK and caspase pathways. Overall, bortezomib pretreatment can sensitize r/r RMS tumors to CAR- and by upregulating DR5, to TRAIL-mediated cytotoxicity of NK-92/5.28.z cells.

IL-27 Levels in Bronchoalveolar Lavage Fluid in Children with Post-infectious Bronchiolitis Obliterans.

Pulmonary neutrophils may play a crucial role in the development of bronchiolitis obliterans (BO) following measles virus infection. IL-27 could potentially have a negative regulatory effect on the release of reactive oxygen species and cytotoxic granules in neutrophils. To investigate the levels of IL-27 in the bronchoalveolar lavage fluid (BALF) of children with post-infectious bronchiolitis obliterans (PIBO) and analyze the relationship between IL-27 levels and neutrophil proportions. A total of 24 children with PIBO were recruited for the experimental group, while 23 children with bronchial foreign bodies were included in the control group. Bronchoscopic alveolar lavage was performed in both groups. The levels of IL-27 in BALF were measured using enzyme-linked immunosorbent assay (ELISA). The proportions of neutrophils in BALF were determined by smear staining. The relationship between the levels of IL-27 in BALF and the neutrophil proportions was analyzed by the Pearson test. The levels of IL-27 in BALF were significantly lower in children with PIBO compared to children with bronchial foreign bodies (p<0.05). Additionally, the proportions of neutrophils in BALF were significantly higher in children with PIBO compared to children with bronchial foreign bodies (p<0.05). The levels of IL-27 were negatively correlated with the neutrophil proportions in BALF in children with PIBO (p<0.05), but not in children with bronchial foreign bodies (p>0.05). The present study suggests that a decrease in IL-27 may be associated with an increase in neutrophils in BALF and may contribute to the pathogenesis of PIBO.

PD-1 inhibits T cell actin remodeling at the immunological synapse independently of its signaling motifs.

Engagement of the receptor programmed cell death molecule 1 (PD-1) by its ligands PD-L1 and PD-L2 inhibits T cell-mediated immune responses. Blocking such signaling provides the clinical effects of PD-1-targeted immunotherapy. Here, we investigated the mechanisms underlying PD-1-mediated inhibition. Because dynamic actin remodeling is crucial for T cell functions, we characterized the effects of PD-1 engagement on actin remodeling at the immunological synapse, the interface between a T cell and an antigen-presenting cell (APC) or target cell. We used microscopy to analyze the formation of immunological synapses between PD-1+ Jurkat cells or primary human CD8+ cytotoxic T cells and APCs that presented T cell-activating antibodies and were either positive or negative for PD-L1. PD-1 binding to PD-L1 inhibited T cell spreading induced by antibody-mediated activation, which was characterized by the absence of the F-actin-dense distal lamellipodial network at the immunological synapse and the Arp2/3 complex, which mediates branched actin formation. PD-1-induced inhibition of actin remodeling also prevented the characteristic deformation of T cells that contact APCs and the release of cytotoxic granules. We showed that the effects of PD-1 on actin remodeling did not require its tyrosine-based signaling motifs, which are thought to mediate the co-inhibitory effects of PD-1. Our study highlights a previously unappreciated mechanism of PD-1-mediated suppression of T cell activity, which depends on the regulation of actin cytoskeleton dynamics in a signaling motif-independent manner.

The role of CD8+ T-cell clones in immune thrombocytopenia

AbstractImmune thrombocytopenia (ITP) is traditionally considered an antibody-mediated disease. However, a number of features suggest alternative mechanisms of platelet destruction. In this study, we use a multidimensional approach to explore the role of cytotoxic CD8+ T cells in ITP. We characterized patients with ITP and compared them with age-matched controls using immunophenotyping, next-generation sequencing of T-cell receptor (TCR) genes, single-cell RNA sequencing, and functional T-cell and platelet assays. We found that adults with chronic ITP have increased polyfunctional, terminally differentiated effector memory CD8+ T cells (CD45RA+CD62L−) expressing intracellular interferon gamma, tumor necrosis factor α, and granzyme B, defining them as TEMRA cells. These TEMRA cells expand when the platelet count falls and show no evidence of physiological exhaustion. Deep sequencing of the TCR showed expanded T-cell clones in patients with ITP. T-cell clones persisted over many years, were more prominent in patients with refractory disease, and expanded when the platelet count was low. Combined single-cell RNA and TCR sequencing of CD8+ T cells confirmed that the expanded clones are TEMRA cells. Using in vitro model systems, we show that CD8+ T cells from patients with ITP form aggregates with autologous platelets, release interferon gamma, and trigger platelet activation and apoptosis via the TCR-mediated release of cytotoxic granules. These findings of clonally expanded CD8+ T cells causing platelet activation and apoptosis provide an antibody-independent mechanism of platelet destruction, indicating that targeting specific T-cell clones could be a novel therapeutic approach for patients with refractory ITP.

T cells of colorectal cancer patients’ stimulated by neoantigenic and cryptic peptides better recognize autologous tumor cells

BackgroundPatients with cancers that exhibit extraordinarily high somatic mutation numbers are ideal candidates for immunotherapy and enable identifying tumor-specific peptides through stimulation of tumor-reactive T cells (Tc).MethodsColorectal cancers (CRC) HROC113...

In Vivo Imaging of CD8+ T-Cell‒Mediated Keratinocyte Apoptosis in Graft-Versus-Host Disease‒Like Dermatitis in Involucrin Membrane-Bound Ovalbumin Mice

In the skin lesions of graft-versus-host disease (GVHD), apoptotic keratinocytes (KCs) in the vicinity of CD8+ cytotoxic T lymphocytes (CTLs) or satellite cell necrosis are a characteristic histologic feature ( Rapini, 2012 Rapini R.P. Practical dermatopathology. Elsevier Health Sciences, Edinburgh, Scotland2012 Google Scholar ). Despite sparse lymphocytic exocytosis histologically, CTLs play a major role in KC apoptosis with the release of cytotoxic granules and cytokines, such as granzyme B and IFN-γ respectively, toward the target cells ( Henden and Hill, 2015 Henden A.S. Hill G.R. Cytokines in graft-versus-host disease. J Immunol. 2015; 194: 4604-4612 Crossref PubMed Scopus (120) Google Scholar ). However, the actual in vivo dynamics of CTLs in the process of KC apoptosis in GVHD have largely remained unclear.

3191 – DELINEATING HUMAN NATURAL KILLER CELL DEVELOPMENT THROUGH AN IN VITRO CELL- DIFFERENTIATION MAP AND BARCODE-CLONAL TRACING

Natural Killer (NK) cells represent a unique cytotoxic innate lymphoid lineage that holds great potential for anti-cancer immunotherapy. The process of human NK cell lineage specification and maturation from hematopoietic progenitors is poorly understood. While mouse and human NK cell developmental pathways converged overall in terms of molecular signatures and transcription factor requirements, inherent differences in surface marker repertoire and shared phenotypic features with other innate lymphoid subsets, highlight the need for a platform allowing direct modeling of human NK cell development. Employing an in-vitro differentiation system on OP9 stroma and multi-parameter FACS analysis, we established and characterized a NK cell developmental trajectory that recapitulates multiple diverse maturation stages proposed in a current model of human NK cell development ex-vivo in the bone marrow and tonsils. Differentiation kinetics, expression of transcription factors (Tbet, Eomes) and effector molecules (Perforin, Granzyme-B) confirmed that in-vitro NK cell maturation progressed through this developmental trajectory in a sequential manner. Functional assays including IFNg secretion, release of cytotoxic granules and direct killing of tumor cells supported the increased effector functions as differentiating NK cells progressed from the early to the final maturation stages. We also further characterized NK cell differentiation steps using transcriptomic profiles from single cell RNA-sequencing. Finally, we employ DNA-barcode-clonal tracing to study developmental relationship between different stages in our in vitro NK cell differentiation model. In summary, we established a framework that allows further understanding of human NK cell development and provides a platform for studies on identifying regulatory networks controlling this process. Natural Killer (NK) cells represent a unique cytotoxic innate lymphoid lineage that holds great potential for anti-cancer immunotherapy. The process of human NK cell lineage specification and maturation from hematopoietic progenitors is poorly understood. While mouse and human NK cell developmental pathways converged overall in terms of molecular signatures and transcription factor requirements, inherent differences in surface marker repertoire and shared phenotypic features with other innate lymphoid subsets, highlight the need for a platform allowing direct modeling of human NK cell development. Employing an in-vitro differentiation system on OP9 stroma and multi-parameter FACS analysis, we established and characterized a NK cell developmental trajectory that recapitulates multiple diverse maturation stages proposed in a current model of human NK cell development ex-vivo in the bone marrow and tonsils. Differentiation kinetics, expression of transcription factors (Tbet, Eomes) and effector molecules (Perforin, Granzyme-B) confirmed that in-vitro NK cell maturation progressed through this developmental trajectory in a sequential manner. Functional assays including IFNg secretion, release of cytotoxic granules and direct killing of tumor cells supported the increased effector functions as differentiating NK cells progressed from the early to the final maturation stages. We also further characterized NK cell differentiation steps using transcriptomic profiles from single cell RNA-sequencing. Finally, we employ DNA-barcode-clonal tracing to study developmental relationship between different stages in our in vitro NK cell differentiation model. In summary, we established a framework that allows further understanding of human NK cell development and provides a platform for studies on identifying regulatory networks controlling this process.

NBAS Variants Are Associated with Quantitative and Qualitative NK and B Cell Deficiency

PurposeBiallelic pathogenic NBAS variants manifest as a multisystem disorder with heterogeneous clinical phenotypes such as recurrent acute liver failure, growth retardation, and susceptibility to infections. This study explores how NBAS-associated disease affects cells of the innate and adaptive immune system.MethodsClinical and laboratory parameters were combined with functional multi-parametric immunophenotyping methods in fifteen NBAS-deficient patients to discover possible alterations in their immune system.ResultsOur study revealed reduced absolute numbers of mature CD56dim natural killer (NK) cells. Notably, the residual NK cell population in NBAS-deficient patients exerted a lower potential for activation and degranulation in response to K562 target cells, suggesting an NK cell–intrinsic role for NBAS in the release of cytotoxic granules. NBAS-deficient NK cell activation and degranulation was normalized upon pre-activation by IL-2 in vitro, suggesting that functional impairment was reversible. In addition, we observed a reduced number of naïve B cells in the peripheral blood associated with hypogammaglobulinemia.ConclusionIn summary, we demonstrate that pathogenic biallelic variants in NBAS are associated with dysfunctional NK cells as well as impaired adaptive humoral immunity.

Abstract 1775: Metformin-mediated natural killer cell cytotoxicity in head and neck squamous cell carcinoma

Abstract Head and Neck Squamous Cell Carcinoma (HNSCC) is the 6th most common cancer worldwide. Even with intensive treatment, up to 50% of patients will relapse. Checkpoint inhibitors, including antibodies targeting programmed death 1 (PD-1), have dramatically improved survival outcomes but only elicit a response in 20% of patients. PD-1 is expressed on both T-cells and Natural Killer (NK) cells, and depletion of NK cells has been linked to poor prognosis in several solid tumors. NK cells are essential to the innate immune response, do not require MHC-1 expression for recognition, and can stimulate an anti-cancer response through release of cytotoxic granules and direct killing of cancer cells. Upregulation of several interleukins including IL-6, which induces the JAK/STAT3 pathway and subsequently inhibits cancer cell apoptosis, is correlated with a poor prognosis in HNSCC patients. Importantly, inhibition of phosphorylated STAT3 (pSTAT3) increases NK-specific cancer cytotoxicity, NK cell cytokine release, and anti-tumor cell chemokines. Therefore, we hypothesize that impaired NK cell cytotoxicity results in a poor response to immunotherapy. We have shown that a common anti-diabetes drug with anti-cancer properties, metformin, downregulates NK pSTAT3 with subsequent increase in cytotoxicity of peripheral NK cells in HNSCC patients. Metformin also decreases pSTAT3 pathway related cytokine release from HNSCC cell lines. However, it remains unclear whether metformin-mediated enhanced NK cytotoxicity was through a direct effect on NK cells or indirect through inhibition of pSTAT3 in tumor cells and subsequent decrease in inhibitory cytokine release. In addition, inhibiting pSTAT3 could lead to further activation of innate immunity through activation of NK cell cytotoxicity complementing current PD-1 inhibitor therapy and improved treatment response. Here we report the contribution of metformin-mediated direct and indirect effects on NK cell cytotoxic functions and the combined effect of metformin and pembrolizumab using patient samples from a clinical trial and ex vivo models. Understanding metformin-mediated regulation of pSTAT3 and downstream pathways will provide necessary insight to potentially overcome resistance to PD-1 inhibitors and identifying novel treatment combinations for this disease. Citation Format: McKenzie Crist, Maria Lehn, Trisha Wise-Draper. Metformin-mediated natural killer cell cytotoxicity in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1775.

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.

Systemic and mucosal mobilization of granulocyte subsets during lentiviral infection.

Granulocytes mediate broad immunoprotection through phagocytosis, extracellular traps, release of cytotoxic granules, antibody effector functions and recruitment of other immune cells against pathogens. However, descriptions of granulocytes in HIV infection and mucosal tissues are limited. Our goal was to characterize granulocyte subsets in systemic, mucosal and lymphoid tissues during lentiviral infection using the rhesus macaque (RM) model. Mononuclear cells from jejunum, colon, cervix, vagina, lymph nodes, spleen, liver and whole blood from experimentally naïve and chronically SHIVsf162p3-infected RM were analysed by microscopy and polychromatic flow cytometry. Granulocytes were identified using phenotypes designed specifically for RM: eosinophils-CD45+ CD66+ CD49d+ ; neutrophils-CD45+ CD66+ CD14+ ; and basophils-CD45+ CD123+ FcRε+ . Nuclear visualization with DAPI staining and surface marker images by ImageStream (cytometry/microscopy) further confirmed granulocytic phenotypes. Flow cytometric data showed that all RM granulocytes expressed CD32 (FcRγII) but did not express CD16 (FcRγIII). Additionally, constitutive expression of CD64 (FcRγI) on neutrophils and FcRε on basophils indicates the differential expression of Fc receptors on granulocyte subsets. Granulocytic subsets in naïve whole blood ranged from 25·4% to 81·5% neutrophils, 0·59% to 13·3% eosinophils and 0·059% to 1·8% basophils. Interestingly, elevated frequencies of circulating neutrophils, colorectal neutrophils and colorectal eosinophils were all observed in chronic lentiviral disease. Conversely, circulating basophils, jejunal eosinophils, vaginal neutrophils and vaginal eosinophils of SHIVsf162p3-infected RM declined in frequency. Overall, our data suggest modulation of granulocytes in chronic lentiviral infection, most notably in the gastrointestinal mucosae where a significant inflammation and disruption occurs in lentivirus-induced disease. Furthermore, granulocytes may migrate to inflamed tissues during infection and could serve as targets of immunotherapeutic intervention.

HLA-A2.1-restricted ECM1-derived epitope LA through DC cross-activation priming CD8+ T and NK cells: a novel therapeutic tumour vaccine

BackgroundCD8+ T cell-mediated adaptive cellular immunity and natural killer (NK) cell-mediated innate immunity both play important roles in tumour immunity. This study aimed to develop therapeutic tumour vaccines based on double-activation of CD8+ T and NK cells.MethodsThe immune Epitope database, Molecular Operating Environment software, and enzyme-linked immunosorbent assay were used for epitope identification. Flow cytometry, confocal microscopy, UPLC-QTOF-MS, and RNA-seq were utilized for evaluating immunity of PBMC-derived DCs, CD8+ T or NK cells and related pathways. HLA-A2.1 transgenic mice combined with immunologically reconstituted tumour-bearing mice were used to examine the antitumour effect and safety of epitope vaccines.ResultsWe identified novel HLA-A2.1-restricted extracellular matrix protein 1(ECM1)-derived immunodominant epitopes in which LA induced a potent immune response. We also found that LA-loaded DCs upregulated the frequency of CD3+/CD8+ T cells, CD45RO+/CD69+ activated memory T cells, and CD3−/CD16+/CD56+ NK cells. We demonstrated cytotoxic granule release of LA/DC-CTLs or LA/DC-NK cells and cytotoxicity against tumour cells and microtissue blocks via the predominant IFN-γ/perforin/granzyme B cell death pathway. Further investigating the mechanism of LA-mediated CD8+ T activation, we found that LA could be internalized into DCs through phagocytosis and then formed a LA-MHC-I complex presented onto the DC surface for recognition of the T cell receptor to upregulate Zap70 phosphorylation levels to further activate CD8+ T cells by DC-CTL interactions. In addition, LA-mediated DC-NK crosstalk through stimulation of the TLR4-p38 MAPK pathway increased MICA/B expression on DCs to interact with NKG2D for NK activation. Promisingly, LA could activate CD8+ T cells and NK cells simultaneously via interacting with DCs to suppress tumours in vivo. Moreover, the safety of LA was confirmed.ConclusionsLA-induced immune antitumour activity through DC cross-activation with CD8+ T and NK cells, which demonstrated proof-of-concept evidence for the capability and safety of a novel therapeutic tumour vaccine.

Induction of Unique STAT Heterodimers by IL-21 Provokes IL-1RI Expression on CD8+T Cells, Resulting in Enhanced IL-1β Dependent Effector Function

IL-1β plays critical roles in the priming and effector phases of immune responses such as the differentiation, commitment, and memory formation of T cells. In this context, several reports have suggested that the IL-1β signal is crucial for CTL-mediated immune responses to viral infections and tumors. However, little is known regarding whether IL-1β acts directly on CD8+ T cells and what the molecular mechanisms underlying expression of IL-1 receptors (IL-1Rs) on CD8+ T cells and features of IL-1R+CD8+ T cells are. Here, we provide evidence that the expression of IL-1R type I (IL-1RI), the functional receptor of IL-1β, is preferentially induced by IL-21 on TCR-stimulated CD8+ T cells. Further, IL-1β enhances the effector function of CD8+ T cells expressing IL-21-induced IL-1RI by increasing cytokine production and release of cytotoxic granules containing granzyme B. The IL-21-IL-1RI-IL-1β axis is involved in an augmented effector function through regulation of transcription factors BATF, Blimp-1, and IRF4. Moreover, this axis confers a unique effector function to CD8+ T cells compared to conventional type 1 cytotoxic T cells differentiated with IL-12. Chemical inhibitor and immunoprecipitation assay demonstrated that IL-21 induces a unique pattern of STAT activation with the formation of both STAT1:STAT3 and STAT3:STAT5 heterodimers, which are critical for the induction of IL-1RI on TCR-stimulated CD8+ T cells. Taken together, we propose that induction of a novel subset of IL-1RI-expressing CD8+ T cells by IL-21 may be beneficial to the protective immune response against viral infections and is therefore important to consider for vaccine design.

Cell Density of NK Cells during Ex Vivo Expansion Impacts NK Cell Surface TRAIL Expression

Introduction Natural Killer (NK) cells are an emerging form of cancer immunotherapy currently being tested in clinical trials world-wide. NK cells are innate immune cells that can kill tumor cells via release of cytotoxic granules and via surface expression of the death receptor ligands tumor-related apoptosis-inducing ligand (TRAIL) and Fas ligand. We and others have recently shown that the proteasome inhibitor bortezomib sensitizes tumor cells to NK cell TRAIL-mediated killing by upregulation of death receptor 5. In a recent phase I NK cell dose-escalation study conducted at the NIH (NCT00720785) we have attempted to exploit TRAIL sensitization by administering ex vivo-expanded autologous NK cells to patients with solid tumors or hematologic malignancies that have been pretreated with bortezomib. Ex vivo cultures used to expand clinical grade NK cells for this trial utilize irradiated EBV-LCL feeder cells and IL-2 containing media which upregulates surface expression of TRAIL, substantially augmenting NK cell killing of bortezomib-treated tumors in vitro. Here we characterize the impact of specific expansion conditions used to generate high numbers of NK cells for clinical use on NK cell TRAIL expression. Methods To generate clinical grade ex vivo-expanded NK cells, we first isolated NK cells from patient apheresis products by CD3+ depletion followed by CD56+ selection, and stimulated these enriched NK cells with irradiated EBV-LCL feeder cells at a ratio of 1:10 in X-VIVO 20 supplemented with 10% inactivated human AB serum and recombinant human IL-2 (500 IU/ml). The clinical trial evaluated 8 escalating NK cell dose levels (Figure 1). Cohorts 1-4 received a single infusion of ex vivo-expanded NK cells on day 0 in a dose-escalating fashion (3-6 pts per cohort) and cohorts 5-7 received 1 x 108 NK cells/kg on day 0 and a second escalating dose of NK cells infused on day +5. A "closed bag" Baxter PL732 culture system was used for cohorts 1-7 which was later changed to a GREX500-CS (Wilson Wolf) system in cohorts 7-8. Using flow cytometry, we monitored surface expression of TRAIL on the day NK cells were harvested and infused fresh into patients. We also assessed TRAIL expression on NK cells from a single patient cultured at 6 different cell densities (range: 2.03-16.95 x 106/cm2) using culture conditions mimicking the phase I trial. Results A total of 137 NK cell cultures were harvested and administered fresh to 32 patients. NK cells on the day of harvest expanded a median of 198-fold, 895-fold, and 3637-fold on culture days 14-16, 19-22, and 24-27, respectively. NK cells at harvest contained a median of 99.7% CD3−/CD56+ NK cells, were 68.65% CD16+ and had a median of 88% viability. TRAIL was assessed by mean fluorescence intensity (MFI) with a median surface expression of of 1245 (range 132-4913) at the time of infusion (Figure 1). Expansions for cohort 8 generated 10-14 x109 (1 vessel) and 50-70 x109 NK cells (4-5 vessels) for fresh infusion, enough to support the target dose level of 1x108 (1st harvest) and 5x108 (2nd harvest) NK cells/kg. Remarkably, NK cells grown at higher cell density to reach the target cell numbers for cohort 8 exhibited substantially reduced TRAIL expression (median: 255, range 132-691). Subsequent experiments conducted on NK cells expanded in vitro for 14 days at different cell densities/concentrations showed TRAIL expression (MFI range: 319-1627) inversely correlated with both cell density and concentration (Figure 2). NK cells grown at the highest cell density (16.95 x 106/cm2) and concentration (4.23 x 106/mL) expressed the least amount of TRAIL (MFI 319), in contrast to those cultured at the lowest cell density (2.03 x 106/cm2) and concentration (0.51 x 106/mL), which demonstrated a TRAIL MFI of 1627. Conclusions Although ex vivo cultures using feeder cells make it possible to expand large numbers of NK cells for clinical use in humans, the higher concentrations and density of cells in these cultures reduce NK cell surface expression of TRAIL. In vitro, TRAIL expression appears to inversely correlate with cell density. These data highlight the need to avoid overly concentrating ex vivo expanded NK cells to maximize TRAIL surface expression as a method to potentiate the anticancer effects of adoptively infused NK cells. Disclosures No relevant conflicts of interest to declare.

NKG7 - regulating endosomal pathways?

Ng et al. have identified NKG7 as a regulator of inflammation in response to diverse immunological challenges. While NKG7 was required for the degranulation of cytotoxic cells, additional defects including reduced expansion and trafficking of CD8 T cells, and altered antigen presentation, were noted in NKG7-deficient mice. The precise mechanism by which NKG7 mediates its effects has not been resolved but may involve regulation of endosomal vesicle trafficking.

Mistletoe-Extract Drugs Stimulate Anti-Cancer Vγ9Vδ2 T Cells.

Human phosphoantigen-reactive Vγ9Vδ2 T cells possess several characteristics, including MHC-independent recognition of tumor cells and potent killing potential, that make them attractive candidates for cancer immunotherapeutic approaches. Injectable preparations from the hemi-parasite plant Viscum album L. (European mistletoe) are commonly prescribed as complementary cancer therapy in European countries such as Germany, but their mechanism of action remains poorly understood. Here, we investigated in-depth the in vitro response of human T cells towards mistletoe-extract drugs by analyzing their functional and T-cell-receptor (TCR) response using flow cytometry and high-throughput sequencing respectively. Non-fermented mistletoe-extract drugs (AbnobaViscum), but not their fermented counterparts (Iscador), induced specific expansion of Vγ9Vδ2 T cells among T cells. Furthermore, AbnobaViscum rapidly induced the release of cytotoxic granules and the production of the cytokines IFNγ and TNFα in Vγ9Vδ2 T cells. This stimulation of anti-cancer Vγ9Vδ2 T cells was mediated by the butyrophilin BTN3A, did not depend on the accumulation of endogenous phosphoantigens and involved the same Vγ9Vδ2 TCR repertoire as those of phosphoantigen-reactive Vγ9Vδ2 T cells. These insights highlight Vγ9Vδ2 T cells as a potential target for mistletoe-extract drugs and their role in cancer patients receiving these herbal drugs needs to be investigated.

Abstract B108: Hypoxia induces phenotypic and functional changes in NK cells which negatively impacts antitumor immunity in the solid tumor microenvironment

Abstract Natural killer (NK) cells are key effectors in cancer immunosurveillance, and they can eliminate tumor cells through the release of cytotoxic granules triggered by interactions with natural ligands or through antibody-dependent cellular cytotoxicity (ADCC). NK cell-based treatment has had therapeutic success for hematologic malignancies, but strategies to treat solid tumors have been more limited. One of the biggest barriers to NK cell immunotherapy is immunosuppression within the tumor microenvironment. An important aspect of this immunosuppression is the highly hypoxic environment created by proliferating tumor cells. To further study the effect of hypoxia on NK cell function, our lab has been collaborating with XCell Biosciences to use their novel incubators that precisely control oxygen and pressure levels. We incubated NK cells isolated from peripheral blood mononuclear cells (PBMCs) at three different oxygen (O2) levels: 12% O2 to mimic the peripheral blood, 5% O2 to mimic the bone marrow, and 1% O2 to mimic the solid tumor microenvironment. All comparisons were made against a standard incubator condition. NK cells were incubated for three different time points—24 hours, 72 hours, and 7 days—and activated with low dose IL-15. We conducted a mass cytometry analysis to assess phenotype of PBMCs and enriched NK cells incubated at the different oxygen conditions. NK cells from the 1% O2 condition express fewer activating receptors (NKG2D, Nkp30, Nkp46, DNAM-1) and less perforin and granzyme than NK cells from the higher oxygen conditions. However, the NK cells from 1% O2 conditions express high levels of the activating receptors CD69 and CD16 (which triggers ADCC). This altered activation state is reflected in cell-killing assays using an IncuCyte machine. Here, fluorescently labeled target cells and NK cells are incubated together and images are taken for 48 hours to measure target cell death. We observed that natural cytotoxicity against K562 targets is severely impaired in the NK cells at the lowest oxygen condition but ADCC responses using the antibody rituximab against Raji targets remain intact. We observe a sharp decrease in proliferation, as measured by CellTrace dilution and Ki-67 expression in the NK cells in the 1% O2 condition. We also detect a decrease in glycolytic and mitochondrial metabolism, as measured by a Seahorse Analyzer, as the oxygen levels decrease. Supernatants collected from NK cells in the lower oxygen conditions (12% O2 and 5% O2) contain more inflammatory cytokines (IFNγ, TNFα, GM-CSF) and chemokines (CCL3, CCL4, CCL5) than NK cells in high oxygen conditions, indicating a shift from cytotoxic to a more immunomodulatory state. These results indicate that hypoxia changes the phenotype and function of NK cells. Overall, the insights gained from these experiments can help overcome hypoxia-induced immune suppression in the tumor microenvironment and enhance NK cell-based immunotherapy for solid tumors. Citation Format: Upasana Sunil Arvindam, Pippa Kennedy, Bri Ettestad, Peter Hinderlie, Jeffrey S. Miller, Martin Felices. Hypoxia induces phenotypic and functional changes in NK cells which negatively impacts antitumor immunity in the solid tumor microenvironment [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 B108.

Effects of Combination Hydroxyurea and Chronic Red Blood Cell Transfusion Therapy on the Immune Parameters of Patients with Sickle Cell Disease

Background: Sickle cell disease (SCD) is typically characterized as a red blood cell disorder but our understanding of the effects on the immune system is limited. Patients with sickle cell disease have been shown to have unique inflammatory profiles, immune phenotypes and function. Others have shown that during vaso-occlusive crises patients with SCD have elevated counts of neutrophils, monocytes, and cytokines as well as increased activity of invariant natural killer T cells (iNKT).We have previously shown that hydroxyurea use is associated with a normalization of the increased NK cell number and function. While there are studies that describe on the effects of single therapy, there is little known about combination therapy. Therefore, our study investigated immunological changes in pediatric patients on combination therapy, which was defined as hydroxyurea added to chronic red blood cell transfusion treatment. Methods: Patient data and peripheral blood samples were collected from an ongoing pilot study of combination therapy hydroxyurea and simple chronic transfusion in patients with SCD previously on chronic transfusion for stroke prevention. A total of 11 patients with hemoglobin SS were studied at two time points; baseline (on chronic RBC transfusion only) and 3 months follow up after initiation of hydroxyurea 20 mg/kg/day. Comparisons were performed using paired t-tests with a p-value &lt;0.05 being considered significant. Results: T, B and NK cell percentage was similar between baseline and after 3 months of combination therapy 62.5% (44.3-71.9) vs 67% (17.6-82.7), 16.29%(8.15-30.2) vs. 13.26% (1.07-32.8) and 7.79% (4.16-14.7) vs. 6.88 (1.54-21) (p&gt;0.05). There were no significant differences between markers of NK cell activation between baseline and 3 months as follows: NKG2D 4.89% (0.47-28.4) vs. 24.38% (0.88-63), and NKp30 8.40% (0.81-58.7) vs. 32.42% (0.45-86.9). However there was a significant decrease in the percentage of mature (CD57+) NK cells 33.8% (10.7-67.6) vs. 23.07%(4.23-37), p =0.005. Similar results were also seen when using absolute values of the different lymphocyte subsets. Conclusion: Combination therapy appears to not affect overall percentages of B, T and NK cells but does appear to decrease the percentage of mature CD57+ NK cells that are known to have increased cytolytic activity. We plan to investigate the implications of these findings using NK functional studies such as cytotoxicity assays and cytotoxic granule release to further elucidate if combination therapy can lead to a decrease in NK cell function to normal levels. Additionally we plan to assess the effect on the immune parameters at 1 year as the hydroxyurea effect is likely time-dependent. These findings may have implications for patients on chronic transfusion therapy who plan to undergo bone marrow transplantation where a reduction in the potential for graft rejection by NK cells is desired. Disclosures No relevant conflicts of interest to declare.

Antitumor Efficacy of Human Monocyte-Derived Dendritic Cells: Comparing Effects of two Monocyte Isolation Methods

BackgroundDendritic cells (DCs), which can be used as anti-cancer vaccines, are generally obtained in vitro from isolated CD14+ monocytes (MoDCs). This generates high cell numbers and allows instructing DCs to guarantee effective antitumor responses. However, the impact of the monocyte isolation step in the antitumor effectiveness of the generated MoDCs is still unknown. Here, we compared the most used immunomagnetic technologies for monocyte isolation: magnetic activated cell sorting (MACS) from Miltenyi Biotec and EasySep from STEM CELL.ResultsMACS technology allowed a higher monocyte yield and purity and, by flow cytometry, monocytes displayed higher size and lower granularity. In the resting state, EasySep_MoDCs showed a higher basal expression of HLA-DR, and no significant response to stimulation by LPS and TNF-α. When stimulated with whole tumor cells lysates, both MoDCs expressed similar levels of maturation and co-stimulatory markers. However, when cultured with autologous T cells, MACS_MoDCs induced significantly higher IFN-γ secretion than EasySep_MoDCs, indicating a stronger induction of Th1 cell response profile. Concordantly, T cells induced by MACS_MoDCs also showed a higher release of cytotoxic granules when in contact with tumor cells.ConclusionsOverall, both the MACS and the EasySep isolation immunomagnetic technologies provide monocytes that differentiate into viable and functional MoDCs. In our experimental settings, resting EasySep_MoDCs showed a higher basal level of maturation but show less responsivity to stimuli. On the other hand, MACS_MoDCs, when stimulated with tumor antigens, showed better ability to stimulate Th1 responses and to induce T cell cytotoxicity against tumor cells. Thus, monocyte isolation techniques crucially affect MoDCs’ function and, therefore, should be carefully selected to obtain the desired functionality.