Cytokine Cytotoxicity Research Articles

Interleukin-37 promotes colitis-associated carcinogenesis via SIGIRR-mediated cytotoxic T cells dysfunction

Interleukin-37b (hereafter called IL-37) was identified as fundamental inhibitor of natural and acquired immunity. The molecular mechanism and function of IL-37 in colorectal cancer (CRC) has been elusive. Here, we found that IL-37 transgenic (IL-37tg) mice were highly susceptible to colitis-associated colorectal cancer (CAC) and suffered from dramatically increased tumor burdens in colon. Nevertheless, IL-37 is dispensable for intestinal mutagenesis, and CRC cell proliferation, apoptosis, and migration. Notably, IL-37 dampened protective cytotoxic T cell-mediated immunity in CAC and B16-OVA models. CD8+ T cell dysfunction is defined by reduced retention and activation as well as failure to proliferate and produce cytotoxic cytokines in IL-37tg mice, enabling tumor evasion of immune surveillance. The dysfunction led by IL-37 antagonizes IL-18–induced proliferation and effector function of CD8+ T cells, which was dependent on SIGIRR (single immunoglobulin interleukin-1 receptor-related protein). Finally, we observed that IL-37 levels were significantly increased in CRC patients, and positively correlated with serum CRC biomarker CEA levels, but negatively correlated with the CD8+ T cell infiltration in CRC patients. Our findings highlight the role of IL-37 in harnessing antitumor immunity by inactivation of cytotoxic T cells and establish a new defined inhibitory factor IL-37/SIGIRR in cancer-immunity cycle as therapeutic targets in CRC.

Direct comparison of different therapeutic cell types susceptibility to inflammatory cytokines associated with COVID-19 acute lung injury

BackgroundAlthough 90% of infections with the novel coronavirus 2 (COVID-19) are mild, many patients progress to acute respiratory distress syndrome (ARDS) which carries a high risk of mortality. Given that this dysregulated immune response plays a key role in the pathology of COVID-19, several clinical trials are underway to evaluate the effect of immunomodulatory cell therapy on disease progression. However, little is known about the effect of ARDS associated pro-inflammatory mediators on transplanted stem cell function and survival, and any deleterious effects could undermine therapeutic efficacy. As such, we assessed the impact of inflammatory cytokines on the viability, and paracrine profile (extracellular vesicles) of bone marrow-derived mesenchymal stromal cells, heart-derived cells, and umbilical cord-derived mesenchymal stromal cells.MethodsAll cell products were manufactured and characterized to established clinical release standards by an accredited clinical cell manufacturing facility. Cytokines and Extracellular vesicles in the cell conditioned media were profiled using proteomic array and nanoparticle tracking analysis. Using a survey of the clinical literature, 6 cytotoxic cytokines implicated in the progression of COVID-19 ARDS. Flow cytometry was employed to determine receptor expression of these 6 cytokines in three cell products. Based on clinical survey and flow cytometry data, a cytokine cocktail that mimics cytokine storm seen in COVID-19 ARDS patients was designed and the impact on cytokine cocktail on viability and paracrine secretory ability of cell products were assessed using cell viability and nanoparticle tracking analysis.ResultsFlow cytometry revealed the presence of receptors for all cytokines but IL-6, which was subsequently excluded from further experimentation. Despite this widespread expression, exposure of each cell type to individual cytokines at doses tenfold greater than observed clinically or in combination at doses associated with severe ARDS did not alter cell viability or extracellular vesicle character/production in any of the 3 cell products.ConclusionsThe paracrine production and viability of the three leading cell products under clinical evaluation for the treatment of severe COVID-19 ARDS are not altered by inflammatory mediators implicated in disease progression.

Lack of ZnT8 protects pancreatic islets from hypoxia- and cytokine-induced cell death.

Pancreatic β-cells depend on the well-balanced regulation of cytosolic zinc concentrations, providing sufficient zinc ions for the processing and storage of insulin, but avoiding toxic effects. The zinc transporter ZnT8, encoded by SLC30A8,is a key player regarding islet cell zinc homeostasis, and polymorphisms in this gene are associated with altered type 2 diabetes susceptibility in man. The objective of this study was to investigate the role of ZnT8 and zinc in situations of cellular stress as hypoxia or inflammation. Isolated islets of WT and global ZnT8−/− mice were exposed to hypoxia or cytokines and cell death was measured. To explore the role of changing intracellular Zn2+ concentrations, WT islets were exposed to different zinc concentrations using zinc chloride or the zinc chelator N,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN). Hypoxia or cytokine (TNF-α, IFN-γ, IL1-β) treatment induced islet cell death, but to a lesser extent in islets from ZnT8−/− mice, which were shown to have a reduced zinc content. Similarly, chelation of zinc with TPEN reduced cell death in WT islets treated with hypoxia or cytokines, whereas increased zinc concentrations aggravated the effects of these stressors. This study demonstrates a reduced rate of cell death in islets from ZnT8−/− mice as compared to WT islets when exposed to two distinct cellular stressors, hypoxia or cytotoxic cytokines. This protection from cell death is, in part, mediated by a reduced zinc content in islet cells of ZnT8−/− mice. These findings may be relevant for altered diabetes burden in carriers of risk SLC30A8 alleles in man.

Wilful pathogens provoke a gut feeling in Parkinson's disease.

Parkinson’s disease is the second most common neurological disorder marked by characteristic poverty and dysfunction in movement. There are many mechanisms and factors which have been postulated to be associated with the neurodegenerative pathway(s) resulting in distinctive loss of neurons in the substantia nigra. Subsequently, the neuropathology is more widespread and exhibited in other areas of the brain, and enteric nervous system. Aggregates of misfolded α-synuclein or Lewy bodies are the hallmark of the illness and appear to be central in the whole cascade of cell destruction. There are many processes implicated in neuronal destruction including: oxidative stress, excitotoxicity, mitochondrial dysfunction, an imbalance in protein homeostasis and neuroinflammation. Interesting, inflammation induced by pathogens (including, bacteria and viruses) has been associated in the pathogenesis of the disease. Bacteria such as Helicobacter pylori and Helicobacter suis appear to colonise the gut, and elicit systemic immune responses, which is them transmitted via the gut-axis to the brain, where cytotoxic cytokines induce neuroinflammation and cell death. This conforms to the bottom–top hypothesis proposed by Braak. The gut is also implicated in two other theories postulated in the development and progression of the disorder, namely, the top–down and the threshold. There is a possibility that these theories may be inter-linked and operate together to certain degree. Ultimately specific trigger factors or conditions may govern the occurrences of these processes in genetically predisposed individuals. Nevertheless, the importance of pathogen-related gut infections cannot be overlooked, since it can result in dysbiosis of gut microbes, which may orchestrate α-synuclein pathology and eventually cell destruction.

Simultaneous delivery of immune stimulatory gene and checkpoint blocker via targeted nanoparticles to strengthen antitumor immunity

The functions of immune cells are regulated by stimulatory and inhibitory signals. Immune stimulating factors provoke the activation of immune cells and the release of cytotoxic cytokines such as interferon gamma (IFN-γ), which can induce overexpression of programmed cell death protein ligand-1 (PD-L1) on tumor cells, leading to immune evasion. Concurrent blockage of programmed cell death protein-1 (PD-1)/PD-L1 axis will counterbalance the immune suppression attributed to PD-L1 and further augment antitumor responses. Herein, a combined immunotherapy to enhance immune activation and reverse immunosuppression is developed for cancer treatment using a self-assembled nanocarrier-drug system. This study introduces the tumor-targeting RDMCM nanoparticles composed of (cyclo (Arg-Gly-Asp-D-Phe-Lys)-poly(ethylene glycol)-poly(ϵ-caprolactone)) (cRGD-PEG-PCL) copolymer, 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) and methoxy PEG-PCL-methoxy PEG (MPEG-PCL-MPEG) copolymer to deliver IL-12 encoding plasmid (pIL-12) and PD-1/PD-L1 small molecular inhibitor (PD-1/PD-L1i) for cancer therapy. The RDMCM nanoparticles selectively deliver dual drugs to the tumor site by targeting the integrin αvβ3 receptor overexpressed on neoendothelial cells and tumor cells. We disclose that combined treatment can reshape tumor immune micro-environment, modulate lymphocytes, macrophages, natural killer (NK) cells and other immune cells, thus, to boost antitumor activities. Synergistic immunotherapy markedly retards tumor growth without causing significant toxicity or adverse effects. Collectively, the results indicate that the fabricated therapeutic nanoparticle will be a promising candidate for cancer immunotherapy.

Differentiation of a CD4/CD8αβ Double Positive T Cell Population from the CD8 Pool Is Both Predictive and Sufficient to Mediate Graft-Vs-Host Disease

Acute graft-vs-host disease (aGVHD) and cancer relapse remain the primary complications following an allogeneic hematopoietic stem cell transplantation (allo-HSCT) for malignant blood disorders. While post-transplant cyclophosphamide combined with standard GVHD prophylaxis has greatly reduced the overall prevalence and severity of aGVHD, relapse rates remain a concern. There is thus a need to identify the specific human T cell populations mediating GVHD vs GVL activity as a means to develop targeted therapeutics capable of controlling aGVHD without inhibiting GVL activity. In this study, we identify a novel human T cell population that develops after transplant that is predictive and sufficient for GVHD pathology.To determine the role of human T cell populations in aGVHD, we performed xenogeneic transplantation studies using primary human graft tissue from a variety of sources (peripheral blood, G-CSF mobilized peripheral blood, bone marrow and umbilical cord blood) in addition to collecting primary human aGVHD blood samples from our clinic. Using the LD50 dose of human graft tissue, we identified a novel mature CD4 +/CD8αβ + double positive (DP) T cell population that only developed after transplantation. The development of this population was further confirmed in aGVHD patients from our clinic. The presence of DP T cells, irrespective of graft source, was also predictive of lethal GVHD in as early as one week after xenogeneic transplantation.To identify the origin of DP T cells, we transplanted isolated human CD4 or CD8 T cells into mice which showed that DP T cells only arise from the CD8 pool. Furthermore, re-transplantation of flow-sorted CD8 T cells from GVHD mice did not reveal a 2nd wave of DP T cell differentiation. This data, in addition to their highly proliferative state, suggests that DP T cells represent highly activated CD8 T cell clones. The ability of these CD8-derived DP T cells to gain CD4 expression coincides with their co-expression of both RUNX3 and THPOK, the master transcription factors of the CD8 and CD4 lineages respectively, that classically repress each other. Intracellular cytokine staining also revealed that DP T cells are the primary activated T cell population in xenogeneic GVHD, secreting both modulatory and cytotoxic cytokines (e.g. IFNγ, IL-17A, IL-22, perforin and granzyme). Ex vivo re-stimulation or re-transplantation of flow-sorted DP T cells showed that this T cell population is capable of dividing and expanding independent of CD4 and CD8 single positive T cells with the majority of the isolated DP T cells retaining their co-expression of CD4 and CD8.Finally, transplantation of either isolated human peripheral blood CD4 or CD8 T cell populations were capable of causing lethal GVHD. Conversely, re-transplantation of flow-sorted DP, CD8 or CD4 T cells from GVHD mice revealed that DP and CD4 T cells are sufficient to mediate GVHD pathology but re-transplanted CD8 T cell are not. This correlates with the absence of DP T cell differentiation in that re-transplanted CD8 population. The differentiation of DP T cells from chronically activated CD8 T cells represents a novel mechanism of GVHD pathology not previously described. The presence of DP T cells in other chronic inflammatory human diseases also suggests a broader pathology mediated by DP T cells. Further understanding of DP T cell differentiation and pathology may lead to targeted prophylaxis and/or treatment regimens for aGVHD and other human chronic inflammatory diseases. [Display omitted] DisclosuresCapitini: Nektar Therapeutics: Honoraria; Novartis: Honoraria.

CAR Density Influences Antitumoral Efficacy of BCMA CAR-T Cells and Correlates with Clinical Outcome

Background: Chimeric Antigen Receptor-modified T cell (CAR-T) therapies have revolutionized cancer immunotherapy, especially in hematological malignancies. Although great results have been achieved during the last years, long-term efficacy is still compromised in some cases and factors behind CAR-T cell disfunction are not fully understood. Recent studies have shown that the control of CAR expression influences CAR-T fitness and antitumoral efficacy 1. Therefore, we hypothesized that CAR density on the membrane of CAR-T cells could directly affect CAR-T cell function. In this study we perform a functional and genomic analysis of FACS-isolated subpopulations of CAR-T cells with different CAR densities (CAR High and CAR Low).Methodology: Second generation CAR-T cells with 4-1BB costimulatory domain targeting BCMA were generated by lentiviral transduction of αCD3/αCD28 activated T cells that were expanded for 12-14 days in the presence of IL-7/IL-15. Phenotypic analyses were performed by flow cytometry before and after coculture with MM cells. Cytotoxic activity and cytokine production were measured by standard procedures. In vivo antitumoral efficacy was evaluated in xenogeneic tumor models in NSG mice. Transcriptomic (RNA-seq) and epigenetic (ATAC-seq) analysis were performed following stablished protocols 2. Single cell analysis was performed using the Chromium Single Cell Immune Profiling solution from 10x Genomic that allows simultaneous analysis of gene expression and paired T-cell receptors from a single cell. Gene Regulatory Network (GRN) analysis was performed using SimiC, a novel computational method that infers regulatory dissimilarities 3.Results: RNA-seq and ATAC-seq analysis revealed completely different profiles between CAR High- and CAR Low-T cells in both CD4 +and CD8 + cell subsets, with >3500 differentially expressed genes (2086 for CD4 + and 1553 for CD8 +) that were related with increased tonic signaling, T cell activation and proliferation in CAR High-T cells. Functional studies at resting state (before antigen encounter) corroborated that CAR High-T cells presented increased tonic signaling, that lead to a higher basal activation and a more differentiated phenotype with skewed presence of CCR7 +/CD45RA +/CXCR3 + T SCM cells. After antigen-driven activation, increased cytotoxicity and cytokine production was observed in CAR High-T cells, that also presented higher percentage of terminally differentiated effector cells (CCR7 -/CD45RA +), along with increased exhaustion (PD1 +/LAG3 +/TIGIT +). This effect was also observed in the infusion products of CARTBCMA-HCB-01 clinical trial for patients with R/R MM (NCT04309981), where products enriched in CAR High-T cells presented increased cytotoxic activity. Although no significant differences were observed in the antitumoral efficacy in vivo, CAR Low-T cells presented increased persistence, suggesting that higher CAR levels could reduce long-term efficacy.Further characterization of CAR-T cells at single cell level (scRNA-seq) showed enrichment of CAR High-T cells in activated CD4 + and exhausted CD8 + cell clusters. The analysis of regulatory dissimilarities driven by different CAR densities with SimiC revealed an increased activity of the regulon associated to NR4A1 transcription factor (a well-known TF driving T cell exhaustion 4) in CAR High-T cells, providing mechanistic insights of the regulatory networks behind differential functionality of CAR High-T cells.Finally, to evaluate the impact of CAR density in the clinical outcome of CAR-T therapies, we developed a gene signature associated to increased CAR density, that was applied to transcriptomic data available from public studies 5. We score the infusion products of several clinical trials testing CTL019 (NCT01029366, NCT01747486 and NCT02640209) and we observed an enrichment on CAR High signature in the products from non-responder patients.Conclusions: Our data demonstrate that CAR density on the membrane of engineered T cells plays important roles in CAR-T activity with a significant impact on clinical outcome. Moreover, the comprehension of regulatory mechanisms driven by CAR densities at the single cell level offer an important tool for the identification of key regulatory factors that could be modulated for the development of improved therapies. [Display omitted] DisclosuresRodríguez-Otero: Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kite: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Regeneron: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS/Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel and other expenses. Paiva: Bristol-Myers Squibb-Celgene, Janssen, and Sanofi: Consultancy; Adaptive, Amgen, Bristol-Myers Squibb-Celgene, Janssen, Kite Pharma, Sanofi and Takeda: Honoraria; Celgene, EngMab, Roche, Sanofi, Takeda: Research Funding. San-Miguel: AbbVie, Amgen, Bristol-Myers Squibb, Celgene, GlaxoSmithKline, Janssen, Karyopharm, Merck Sharpe & Dohme, Novartis, Regeneron, Roche, Sanofi, SecuraBio, Takeda: Consultancy, Other: Advisory board. Prósper: Oryzon: Honoraria; Janssen: Honoraria; BMS-Celgene: Honoraria, Research Funding.

Deletion of CISH and TGFβR2 in iPSC-Derived NK Cells Promotes High Cytotoxicity and Enhances In Vivo Tumor Killing

Natural killer (NK) cells distinguish tumor from healthy tissue via multiple mechanisms, including recognition of stress ligands and loss of MHC class I expression. Effector function of allogeneic NK cells can be diminished by the lack of functional persistence, as well as tumor-intrinsic immunosuppressive mechanisms, such as production of TGF-β, a pleiotropic cytokine that inhibits immune effector function. Gene editing is the power tool to modify NK cells to potentially overcome these biological limitations. Here, we developed a next-generation iPSC-derived NK cell therapy using CRISPR-AsCas12a gene editing to enhance NK cell function by deleting the CISH and TGFβR2 genes. We hypothesized that knockout of CISH, a negative regulator of IL-2/IL-15 signaling, would improve NK cell effector function, while knockout of the TGF-β receptor gene, TGFβR2, would render NK cells resistant to TGF-β mediated suppression. NK cells are typically isolated from either cord blood or peripheral blood of healthy donors, but recent advances with induced pluripotent stem cells (iPSCs) allows a nearly unlimited supply of iPSC-derived natural killer cells (iNK). In this study, we used CRISPR-Cas12a to generate edited iPSC lines that were differentiated into TGFβ R2-/-/CISH-/- double knockout (DKO) iNK cells.Using flow cytometry-based assays we demonstrate that DKO iNK cells phosphorylated less SMAD2/3 relative to unedited control iNK cells in response to IL-15 and TGF-β, while CISH KO NK cells showed enhanced pSTAT3 upon IL-15 stimulation. Additionally, DKO iNKs produced higher levels of cytotoxic cytokines including IFN-γ and TNF-α in response to PMA/ionomycin stimulation. We next explored the ability of these DKO iNKs in controlling 3D SKOV-3 ovarian tumor spheroids in vitro over 5 days of co-culture. Both freshly generated and cryopreserved DKO iNKs demonstrated significantly better tumor killing as compared to unedited control iNKs. Importantly, there was no difference in tumor killing between freshly generated and cryopreserved DKO iNKs, suggesting that the freeze/thaw process does not impact functional capacity.We utilized the SKOV3-luc IP tumor model to evaluate the in vivo efficacy of cryopreserved iNKs cells. Here, NSG mice with established SKOV3-luc tumors were treated IP with unedited control iNKs or DKO iNKs. DKO iNK cell treatment induced robust anti-tumor efficacy resulting in a significant 7.2- fold and 3.2-fold reduction in tumor burden as compared to vehicle and unedited iNK cell treatment, respectively, at 9 days post-iNK cell dosing.In summary, we demonstrated that TGFβ R2-/-/CISH-/- DKO iPSCs differentiated into iNK cells have potent anti-tumor activity that is maintained after cryopreservation. Together, the increased overall effector function of TGFβ R2-/-/CISH-/- DKO human iNK cells support their development as a potent allogeneic cell-based medicine for cancer. This potential medicine is being investigated with other gene edits for future advancement to clinic. DisclosuresGerew: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Sexton: Editas Medicine: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Wasko: Editas Medicine: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Shearman: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zhang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Chang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Khan: Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

Integrative Characterization of the Role of IL27 In Melanoma Using Bioinformatics Analysis.

Background IL27 has been reported to play dual roles in cancer; however, its effects on the tumor microenvironment (TME), immunotherapy, and prognosis in melanoma remain largely unclear. This study was aimed to uncover the effects of IL27 on TME, immunotherapy and prognosis in patients with melanoma.MethodsRNA-seq data, drug sensitivity data, and clinical data were obtained from TCGA, GEO, CCLE, and CTRP. Log-rank test was used to determine the survival value of IL27. Univariate and multivariate Cox regression analyses were employed to determine the independent predictors of survival outcomes. DAVID and GSEA were used to perform gene set functional annotations. ssGSEA was used to explore the association between IL27 and immune infiltrates. ConsensusClusterPlus was used to classify melanoma tissues into hot tumors or cold tumors.ResultsClinically, IL27 was negatively correlated with Breslow depth (P = 0.00042) and positively associated with response to radiotherapy (P = 0.038). High IL27 expression showed an improved survival outcome (P = 0.00016), and could serve as an independent predictor of survival outcomes (hazard ratio: 0.32 - 0.88, P = 0.015). Functionally, elevated IL27 expression could induce an enhanced immune response and pyroptosis (R = 0.64, P = 1.2e-55), autophagy (R = 0.37, P = 7.1e-17) and apoptosis (R = 0.47, P = 1.1e-27) in patients with melanoma. Mechanistically, elevated IL27 expression was positively correlated with cytotoxic cytokines (including INFG and GZMB), enhanced immune infiltrates, and elevated CD8/Treg ratio (R = 0.14, P = 0.02), possibly driving CD8+ T cell infiltration by suppressing β-catenin signaling in the TME. Furthermore, IL27 was significantly associated with hot tumor state, multiple predictors of response to immunotherapy, and improved drug response in patients with melanoma.Conclusions IL27 was correlated with enriched CD8+ T cells, desirable therapeutic response and improved prognosis. It thus can be utilized as a promising modulator in the development of cytokine-based immunotherapy for melanoma.

Diminished Peripheral CD29hi Cytotoxic CD4+ T Cells Are Associated With Deleterious Effects During SIV Infection.

Cytotoxic CD4+ T cells (CD4+ CTLs) limit HIV pathogenesis, as evidenced in elite controllers (a subset of individuals who suppress the virus without the need for therapy). CD4+ CTLs have also been shown to kill HIV-infected macrophages. However, little is known about their contribution towards HIV persistence, how they are affected following exposure to immune modulators like morphine, and what factors maintain their frequencies and function. Further, the lack of robust markers to identify CD4+ CTLs in various animal models limits understanding of their role in HIV pathogenesis. We utilized various PBMC samples obtained from SIV infected and cART treated rhesus macaques exposed to morphine or saline and subjected to flow cytometry evaluations. Thereafter, we compared and correlated the expression of CD4+ CTL-specific markers to viral load and viral reservoir estimations in total CD4+ T cells. We found that CD29 could be reliably used as a marker to identify CD4+ CTLs in rhesus macaques since CD29hi CD4+ T cells secrete higher cytotoxic and proinflammatory cytokines following PMA/ionomycin or gag stimulation. In addition, this immune cell subset was depleted during untreated SIV infection. Strikingly, we also observed that early initiation of cART reconstitutes depleted CD29hi CD4+ T cells and restores their function. Furthermore, we noted that morphine exposure reduced the secretion of proinflammatory cytokines/cytotoxic molecules in CD29hi CD4+ T cells. Lastly, increased functionality of CD29hi CD4+ T cells as depicted by elevated levels of either IL-21 or granzyme B hi T Bet+ gag specific responses were linked to limiting the size of the replication-competent reservoir during cART treatment. Collectively, our data suggest that CD4+ CTLs are crucial in limiting SIV pathogenesis and persistence.

PD-1, CTLA-4, LAG-3, and TIGIT: The roles of immune checkpoint receptors on the regulation of human NK cell phenotype and functions

The roles of immune checkpoint receptors were defined in many cancers and autoimmune diseases, while there is limited information on their functional roles in the NK cells of healthy individuals. Immune checkpoint receptor expression of NK cell subsets and their association with NK cell functions (cytotoxic capacity and cytokine production) in healthy population were investigated. PD-1, CTLA-4, LAG-3 and TIGIT expression of peripheral blood NK cells, cytokine levels (TNF-α, IFN-γ, IL-10) and cytotoxic functions (granzyme A, perforin, CD107a; with/without K562 target cell stimulation) were evaluated by flow cytometry. CD56dimCD16dim NK cells had the highest expression of TIGIT, while CD56dimCD16− NK cells had highest expression of PD-1, CTLA-4 and LAG-3. PD-1+ NK cells, CTLA-4+ NK cells and LAG-3+ NK cells had increased amount of IL-10 however, reduced IFN-γ and TNF-α levels. Cytotoxic granule expressions (perforin and granzyme A) were reduced in PD-1+ NK cells, CTLA-4+ NK cells and LAG-3+ NK cells. However, TIGIT expression did not alter perforin and granzyme A expressions. Degranulation capacity was reduced in three groups of NK cells (PD-1+ or LAG-3+ or TIGIT+). TIGIT+ NK cells responded strongly to target cell stimulation, while NK cells in the other groups (PD-1+ or CTLA-4+ or LAG-3+) were resistant. PD-1+ NK cells, CTLA-4+ NK cells and LAG-3+ NK cells had a regulatory phenotype, impaired cytotoxic functions, and response to target cell stimulation. In contrast, TIGIT+ NK cells had strong baseline cytotoxic activity that further increased in response to target cell stimulation.

1553TiP A phase III study comparing the efficacy of the combination of doxorubicin and the tumor-targeting human antibody-cytokine fusion protein L19TNF to doxorubicin alone as first-line therapy in patients with advanced or metastatic soft tissue sarcoma

Recombinant human tumor necrosis factor (TNF) is a potent proinflammatory and cytotoxic cytokine that is approved in the EU for the therapy of soft tissue sarcoma (STS) of the limbs, used in combination with melphalan in an isolated-limb perfusion procedure. Although TNF is one of the most potent antitumor cytokines, the considerable therapeutic potential of natural TNF cannot be fully exploited due to severe side effects, which preclude achievement of therapeutically active concentrations in the tumor.

PD-1 Blockade on Tumor Microenvironment-Resident ILC2s Promotes TNF-α Production and Restricts Progression of Metastatic Melanoma.

While pulmonary ILC2s represent one of the major tissue-resident innate lymphoid cell populations at steady state and are key drivers of cytokine secretion in their occupational niche, their role in pulmonary cancer progression remains unclear. As the programmed cell death protein-1 (PD-1) plays a major role in cancer immunotherapy and immunoregulatory properties, here we investigate the specific effect of PD-1 inhibition on ILC2s during pulmonary B16 melanoma cancer metastasis. We demonstrate that PD-1 inhibition on ILC2s suppresses B16 tumor growth. Further, PD-1 inhibition upregulates pulmonary ILC2-derived TNF-α production, a cytotoxic cytokine that directly induces cell death in B16 cells, independent of adaptive immunity. Together, these results highlight the importance of ILC2s and their anti-tumor role in pulmonary B16 cancer progression during PD-1 inhibitory immunotherapy.

Targeted knockdown of Tim3 by short hairpin RNAs improves the function of anti-mesothelin CAR T cells

T-cell immunoglobulin mucin 3 (Tim3) is an immune checkpoint receptor that plays a central role in chimeric antigen receptor (CAR) T cell exhaustion within the tumor microenvironment. This study was aimed to evaluate the effects of targeted-knockdown of Tim3 on the antitumor function of anti-mesothelin (MSLN)-CAR T cells. To knockdown Tim3 expression, three different shRNA sequences specific to different segments of the human Tim3 gene were designed and co-inserted with an anti-MSLN-CAR transgene into lentiviral vectors. To investigate the efficacy of Tim3 targeting in T cells, expression of Tim3 was assessed before and after antigen stimulation. Afterwards, cytotoxic effects, proliferative response and cytokine production of MSLN-CAR T cells and Tim3-targeted MSLN-CAR T cells were analyzed. Our results showed that activation of T cells and MSLN-CAR T cells led to up-regulation of Tim3. Tim3 knockdown significantly decreased its expression in different groups of MSLN-CAR T cells. Tim3 knockdown significantly improved cytotoxic function, cytokine production and proliferation capacity of MSLN-CAR T cells. Our findings indicate that targeted knockdown of Tim3 allows tumor-infiltrating CAR T cells that would otherwise be inactivated to continue to expand and carry out effector functions, thereby altering the tumor microenvironment from immunosuppressive to immunosupportive via mitigated Tim3 signaling.

Pathogenic Characterization of Clostridium perfringens Strains Isolated From Patients With Massive Intravascular Hemolysis.

Sepsis caused by Clostridium perfringens infection is rare but often fatal. The most serious complication leading to poor prognosis is massive intravascular hemolysis (MIH). However, the molecular mechanism underlying this fulminant form of hemolysis is unclear. In the present study, we employed 11 clinical strains isolated from patients with C. perfringens septicemia and subdivided these isolates into groups H and NH: septicemia with (n = 5) or without (n = 6) MIH, respectively. To elucidate the major pathogenic factors of MIH, biological features were compared between these groups. The isolates of two groups did not differ in growth rate, virulence-related gene expression, or phospholipase C (CPA) production. Erythrocyte hemolysis was predominantly observed in culture supernatants of the strains in group H, and the human erythrocyte hemolysis rate was significantly correlated with perfringolysin O (PFO) production. Correlations were also found among PFO production, human peripheral blood mononuclear cell (PBMC) cytotoxicity, and production of interleukin-6 (IL-6) and interleukin-8 (IL-8) by human PBMCs. Analysis of proinflammatory cytokines showed that PFO induced tumor necrosis factor-α (TNF-α), IL-5, IL-6, and IL-8 production more strongly than did CPA. PFO exerted potent cytotoxic and proinflammatory cytokine induction effects on human blood cells. PFO may be a major virulence factor of sepsis with MIH, and potent proinflammatory cytokine production induced by PFO may influence the rapid progression of this fatal disease caused by C. perfringens.

Src/lck inhibitor dasatinib reversibly switches off cytokine release and T cell cytotoxicity following stimulation with T cell bispecific antibodies

BackgroundT cell engagers are bispecific antibodies recognizing, with one moiety, the CD3ε chain of the T cell receptor and, with the other moiety, specific tumor surface antigens. Crosslinking of CD3...

Abstract 1532: EDIT-201: CRISPR-Cas12a gene editing enhances functional metabolism of natural killer cells and enables tumor cell cytolysis in metabolically stressful conditions that inhibit effector cell function

Abstract We have previously described EDIT-201, a healthy donor derived NK cell therapy with CRISPR-Cas12a mediated CISH and TGFBR2 double gene knockout that demonstrated resistance to TGF-β inhibition and increased tumor control in vitro and in vivo. The tumor microenvironment (TME) is known to be nutrient-deprived due to active tumor cell metabolism leading to competition for essential nutrients with infiltrating effector cells, while at the same time being enriched in immunosuppressive metabolites such as lactic acid due to Warburg Metabolism. To explore whether EDIT-201 is functional in such hostile metabolic conditions, we aimed to more accurately model the metabolic microenvironment in a long-term tumor spheroid model. To model this, we generated SK-OV-3 ovarian tumor spheroids in decreasing concentrations of glucose (10-0.5mM) or glutamine (2-0.1mM), two important fuels for NK cell metabolism, as well as increasing concentrations of inhibitory metabolite lactate (0-50mM), or decreasing pH (7.2-6.5). Each of these metabolic conditions are known to suppress effector cell function; we additionally performed spheroid cells co-cultures in the presence of 10ng/mL TGF-β to increase the potential for NK inhibition. We demonstrated that, relative to control NK cells, EDIT-201 demonstrated rapid and sustained tumor killing in all conditions tested including, the absence of critical nutrients. We next created a multifactorial matrix of metabolic conditions where we combined deprivation of multiple nutrients, in the presence of lactate and/or acidic cell culture media. Remarkably, in all combinations tested, we surprisingly found that EDIT-201 continued to demonstrate increased cytotoxicity against SK-OV-3 spheroids relative to control NK cells, suggesting a clear and robust metabolic advantage of EDIT-201 over control NK cells. We additionally observed a corresponding increase in the concentrations of IFN-γ and TNF-α by EDIT-201 in all of these conditions relative to control NK cells. Given that mitochondrial respiration is key to NK cell persistence and function, we next interrogated the mitochondrial function of EDIT-201. We find that EDIT-201 consistently demonstrated greater spare respiratory capacity (SRC) relative to control NK cells, suggesting enhanced mitochondrial reserve as a result of CISHand TGFBR2 knockout. The increase in SRC likely enables EDIT-201 to meet enhanced energy demands necessary to mediate effector function in metabolically challenging conditions, thus sustaining superior cytotoxic capacity and cytokine production. In summary, our data demonstrate enhanced metabolic function of EDIT-201, resulting in superior cytotoxicity in metabolically unfavorable conditions. These data further support the development of EDIT-201 as a novel cell therapy for cancer. Citation Format: Karrie K. Wong, Steven Sexton, Lincy Prem Antony, Kevin Wasko, Jared Nasser, Kelly Donahue, Amanda Pfautz, Shuqi Zhang, Richard A. Morgan, Christopher M. Borges. EDIT-201: CRISPR-Cas12a gene editing enhances functional metabolism of natural killer cells and enables tumor cell cytolysis in metabolically stressful conditions that inhibit effector cell function [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 1532.

Pembrolizumab (anti-PD-1) Treatment Increases Anti-Tumor Activities of XBP1/CD138/CS1-Specific Cytotoxic T Lymphocytes Against Multiple Myeloma

In these studies, we investigated the role of Pembrolizumab (anti-PD-1)to enhance the function of myeloma-specific CD8+ cytotoxic T lymphocytes (CTL) and maintain their antigens-specific CTL activities against multiple myeloma cells. Bone marrow or peripheral blood mononuclear cells (BMMC, PBMC) from healthy donors or MGUS, smoldering multiple myeloma (SMM), newly diagnosed multiple myeloma (MM), relapsed MM, relapsed/refractory MM patients (N=5, respectively) were evaluated for expression of immune checkpoints that regulate antigen-specific CTL development and function. Active MM patients had higher levels of CD138+ tumor cells as compared to MGUS and SMM patients. PD-L1 and PD-L2 expression levels on myeloma cells were also higher in active MM patients than healthy donors. CD4 Treg (CD3+CD4+CD25+FoxP3+), but not CD8 Treg (CD3+CD8+CD25+FoxP3+), frequencies were highest in both BMMC and PBMC from MM patients. PD-L1, but not PD-L2 nor PD-1, was expressed on the CD4 Treg in BMMC and PBMC from MM patients. G-type MDSC (CD11b+CD33+HLA-DRlowCD15+), but not M-type MDSC (CD11b+CD33+HLA-DRlowCD14+), was increased in MM as compared to MGUS, SMM or healthy donors. The MDSC from active MM patients also had high levels of PD-L1 and PD-L2 expression. BMMC and PBMC from newly diagnosed MM patients (N=3) treated in vitro with Pembrolizumab enhanced the CD3+ T cell proliferation as compared to the untreated group. Interestingly, Pembrolizumab treatment induced proliferation of T cells expressing immune agonists (OX40, GITR) beneficial for cell function and immune checkpoint (LAG3) involved in down-regulating T cell activities. XBP1/CD138/CS1 antigens-specific CTL (XBP1/CD138/CS1-CTL) generated in vitro with a cocktail of immunogenic peptides [heteroclitic XBP1 US184-192 (YISPWILAV), heteroclitic XBP1 SP367-375 (YLFPQLISV), native CD138260-268 (GLVGLIFAV), and native CS1239-247 (SLFVLGLFL)] displayed increased proliferation of CD8+ CTL expressing the CD28 co-stimulatory and CD38 activation markers upon treatment with Pembrolizumab. Importantly, the XBP1/CD138/CS1-specific central memory and effector memory CTL subsets increased the cell proliferation, following treatment with the PD-1-specific antibody. In addition, XBP1/CD138/CS1-CTL treated with Pembrolizumab demonstrated increased cytotoxic activity and Th1-specific cytokines production against U266 myeloma cells. Thus, these studies suggest the potential benefit of Pembrolizumab (a-PD-1) therapy in MM patients to enhance XBP1/CD138/CS1 antigens-specific memory CTL proliferation and boost their anti-tumor activities through blockage of the PD-1 specific immune checkpoint specifically expressed on antigens-specific CD8+ CTL. These data provide the framework for an immunotherapeutic strategy encompassing a combination of multipeptide-based cancer vaccine with PD1-specific antibody to enhance the antigens-specific T cells efficacy and promote stronger and long-lasting CD8+ CTL immune responses in myeloma patients. DisclosuresBae:OncoPep: Other: Scientific Founder. Hideshima:C4 Therapeutics: Equity Ownership; Acetylon: Consultancy. Anderson:C4 Therapeutics: Other: scientific founder; Oncopep: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees.

Invariant NKT Are a More Effective and Versatile Platform Than T Cells for CAR Immunotherapy of CD1d-Expressing B Lineage Malignancies: Cellular and Molecular Mechanisms

Anti-CD19 chimeric antigen receptor T cell (CAR19T) immunotherapy has shown promising clinical potential in relapsed/refractory mature B cell malignancies. However, only about half of patients benefit, highlighting the need for more effective CAR-based strategies.iNKT cells are rare but powerful immunoregulatory and cytotoxic T lymphocytes, playing a pivotal anti-tumor role. iNKT cells are restricted by CD1d, a non-polymorphic, glycolipid-presenting HLA I-like molecule. We previously showed that CD1d, as well as on normal B cells, is also expressed on malignant CD19+ B cells in mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL) cells, while in up to 50% of patients with chronic lymphocytic leukemia (CLL) CD1d expression is very low or negative.We tested the hypotheses that a) bi-specific CAR iNKT 19 cells, targeting simultaneously CD19 and CD1d via the CD19-specific CAR and their natural invariant TCR respectively, would be more effective than CART19 cells against CD19+CD1d+ B cell malignancies and b) transcriptional enhancement of CD1d expression would increase the CAR19iNKT cytotoxic effect.We optimized a novel protocol for manufacturing CAR19iNKT cells. Their in vitro reactivity was assessed in cytotoxicity (flow cytometry), cytokine and cytotoxic granule intracellular staining and cytokine release assays (flow cytometry and Luminex technology) as well as real-time Incucyte proliferation assays. In vivo reactivity was assessed in systemic NSG xenograft assays with growth of the C1RCD1d B cells monitored by bioluminescence and magnetic resonance imaging. Transcription factor binding and histone mark enrichment at the promoter of CD1d was assessed by ChiP- and ChIP-re-ChIP-qPCR assays.We manufactured clinical scale 2nd and 3rd generation CAR19iNKT cells using a highly optimized protocol (>75% transduction efficiency, n=8). In vitro validation, using singly- or dual-positive CD1d and CD19 targets, demonstrated that CAR19iNKT cells are CD19-specific, retain their natural CD1d-restricted reactivity and exert additive dual-specific cytotoxicity against CD1d+CD19+ targets. Compared to same donor CAR19T, CAR19iNKT cells display a significantly higher (P<0.0001) expandability and proliferative potential and are equally or more effective in killing CD19+CD1d+ lymphoid cell lines (C1RCD1d and Farage) and consistently more effective against primary MCL, MZL and CLL cells.Since CD1d expression is low/negative in most cases of CLL, we sought to restore CD1d expression on leukemic cells for the purpose of CAR19iNKT cell immunotherapy. We found that surface CD1d expression can be restored by clinically relevant concentrations of ATRA in a time-dependent manner. Mechanistic dissection of CD1d transcriptional regulation in the CD1d-negative B lineage cell line U266 demonstrated the co-existence of bivalent chromatin marks at the promoter of CD1d commensurate with enriched binding and association of EZH2 with RARα. Notably, an EZH2 inhibitor exerted a synergistic effect along with ATRA in restoring CD1d transcription and surface expression and, in line with our hypothesis, 2nd and 3rd generation CAR19iNKT but not CAR19T cells displayed higher cytotoxic activity against ATRA-treated CLL cells.Finally, in an NSG xenograft model of lymphoma, while survival of T and iNKT cell-treated animals was the same as that of untreated animals (n=7, P>0.05), both CAR19T and CAR19iNKT cell-treated animals (n=19) had significantly and comparably improved overall survival (OS; P<0.0001). However, compared to CAR19T, CAR19iNKT cell immunotherapy led to a significantly improved OS (P=0.01) and also better disease control including of CNS lymphoma, with earlier, more profound and sustained complete responses resulting in a significantly improved tumor-free survival (P=0.03).We conclude thatCARiNKT19 are more effective than CART19 cells against CD19+CD1d+ B cell malignancies in vitro and in vivo and ATRA-mediated restoration of CD1d expression enhances the anti-lymphoma effect of CAR19iNKT immunotherapy in vitro . These properties together with the previously demonstrated ability of donor iNKT cells to protect from aGVHD raise the prospect of developing effective ‘off-the-shelf’ CAR19iNKT immunotherapy of lymphomas, which can mitigate against CD19-negative immune escape upon CAR treatment and can be further enhanced by transcriptional and epigenetic treatment. DisclosuresPule:UCL: Patents & Royalties: UCL Business; Autolus Ltd: Employment, Equity Ownership, Research Funding. Maher:Leucid Bio: Other: Chief scientific officer. Karadimitris:GlaxoSmithKline: Research Funding.

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.