Presence Of IL-2 Research Articles

IL-7Rαlow CD8+ T Cells from Healthy Individuals Are Anergic with Defective Glycolysis.

Effector memory (EM) CD8+ T cells expressing lower levels of IL-7R α (IL-7Rαlow) from healthy individuals are partly compromised in vitro, but the identity of these cells has remained unclear. In this study, we demonstrate that human IL-7Rαlow EM CD8+ T cells are naturally occurring anergic cells in vivo and impaired in proliferation and IL-2 production but competent in IFN-γ and TNF-α production, a state that can be restored by IL-2 stimulation. IL-7Rαlow EM CD8+ T cells show decreased expression of GATA3 and c-MYC and are defective in metabolic reprogramming toward glycolysis, a process required for the proliferation of T cells. However, IL-7Rαlow EM CD8+ T cells can proliferate with TCR stimulation in the presence of IL-2 and IL-15, suggesting that these cells can be restored to normality or increased activity by inflammatory conditions and may serve as a reservoir for functional immunity.

IL-7 During Antigenic Stimulation Using Allogeneic Dendritic Cells Promotes Expansion of CD45RA-CD62L+CD4+ Invariant NKT Cells With Th-2 Biased Cytokine Production Profile.

Invariant natural killer T (iNKT) cells are innate-like T lymphocytes cells that recognize glycolipid antigens associated with CD1d, non-classical antigen presenting proteins. They can drive either pro-inflammatory (Th-1) or anti-inflammatory (Th-2) immune microenvironment through the production of both Th-1 and Th-2 type cytokines upon activation, thus play a vital role in cancer, infection, and autoimmune diseases. Adoptive cell therapy using ex vivo expanded iNKT cells is a promising approach to enhance anti-tumor immunity or immunosuppression. However, overcoming phenotypic and functional heterogeneity and promoting in vivo persistency of iNKT cells remains to be a challenge. Here, we compared various methods for ex vivo expansion of human iNKT cells and assessed the quality of expansion, phenotype, and cytokine production profile of expanded iNKT cells. While a direct stimulation of iNKT cells in peripheral blood mononuclear cells with agonist glycolipid led to the expansion of iNKT cells in varying degrees, stimulation of enriched iNKT cells by irradiated autologous peripheral blood mononuclear cells or allogeneic dendritic cells resulted in consistent expansion of highly pure iNKT cells. Interestingly, the mode of antigenic stimulation influenced the dominant subtype of expanded iNKT cells. Further, we evaluated whether additional IL-7 or IL-15 during antigenic stimulation with allogeneic dendritic cells can improve the phenotypic heterogeneity and modify cytokine production profile of iNKT cells expanded from 18 consecutive donors. The presence of IL-7 or IL-15 during antigenic stimulation did not affect the fold of expansion or purity of expanded iNKT cells. However, IL-7, but not IL-15, led to a better expansion of CD4+ iNKT cells, enhanced Th-2 type cytokine production of CD4+ iNKT cells, and maintained the expansion of central memory (CD45RA-CD62L+) CD4+ iNKT cells. Our results suggest the addition of IL-7 during antigenic stimulation with allogeneic dendritic cells can promote the expansion of CD62L+Th-2+CD4+ human iNKT cells that can be used as novel immunotherapeutic to control excessive inflammation to treat various autoimmune diseases.

Pro-inflammatory response ensured by LPS and Pam3CSK4 in RAW 264.7 cells did not improve a fungistatic effect on Cryptococcus gattii infection.

BackgroundThe macrophage lineage is characterized by plasticity due to the acquisition of distinct functional phenotypes, and two major subsets are evaluated; classical M1 activation (strong microbicidal activity) and alternative M2 activation (immunoregulatory functions). The M1 subset expresses inducible nitric oxide synthase (iNOS), which is a primary marker to identify these cells, whereas M2 macrophages are characterized by expression of Arginase-1, found in inflammatory zone 1 (Fizz1), chitinase-like molecule (Ym-1), and CD206. The micro-environmental stimuli and signals in tissues are critical in the macrophage polarization. Toll-like receptors (TLR) ligands, such as lipopolysaccharide (LPS), palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4), and ArtinM (mannose-binding lectin) are inductors of M1 subset. The impact of TLR2 and TLR4 signals to fight against Cryptococcus gattii infection is unknown, which is a fungal pathogen that preferentially infects the lung of immunocompetent individuals. The macrophages initiate an immune response to combat the C. gattii, then we evaluated in RAW 264.7 cell the effect of TLR2 and TLR4 agonists on the macrophage polarization dynamic and the impact on the growth of C. gattii.Methods and ResultsWe demonstrated that P3C4, LPS, and ArtinM induced an increase in the levels of iNOS transcripts in RAW 264.7 cells, whereas the relative expression of arginase-1, Ym-1, and Fizz1 was significantly increased in the presence of IL-4 alone. The effects of TLR2 and TLR4 agonists on repolarization from the M2 to M1 subset was evaluated, and the first stimulus was composed of IL-4 and, after 24 h of incubation, the cells were submitted to a second stimulus of P3C4, LPS, ArtinM, or Medium. These TLR agonists induced the production of TNF-α in polarized RAW 264.7 cells to the M2 subset, moreover the measurement of M1/M2 markers using qRT-PCR demonstrated that a second stimulus with LPS for 24 h induced a significant augmentation of levels of iNOS mRNA. This impact of TLR2 and TLR4 agonists in the activation of the RAW 264.7 macrophage was assayed in the presence of C. gattii, the macrophages stimulated with TLR2 and TLR4 agonists for 24 h and co-cultured with C. gattii, as a second stimulus, reached high levels of TNF-α even after incubation with different concentrations of C. gattii. The activation of RAW 264.7 cells induced by TLR2 and TLR4 agonists favored the phagocytosis of C. gattii and inhibited the growth of yeast in the early period of infection. However, RAW 264.7 cells incubated with C. gattii in the presence of TLR2 and TLR4 agonists did not result a significant difference in the colony forming unit (CFU) assay in the early period of C. gattii infection, compared to negative control.ConclusionPolarized RAW 264.7 cells to the M1 subset with TLR2 and TLR4 agonists did not inhibit the growth of C. gattii, whereas robust immunity was identified that could dysregulate host tolerance to this pathogen.

In Vitro Replication of HCV RNA in Peripheral Blood Mononuclear Cells Isolated from Patients Undergoing Treatment for Hepatitis C Virus Infection

Background: Although the liver is the main site for the Hepatitis C Virus (HCV) replication, there is still an essential debate about extrahepatic HCV reservoirs. Objectives: It has been proposed that Peripheral Blood Mononuclear Cells (PBMCs) could be the possible virus replication sites. Therefore, PBMCs may be candidates for recurrent HCV infection after achieving Sustained Virologic Response (SVR). In this study, we designed a lymphocyte culture to explore more about virus replication in PBMCs collected from patients with chronic hepatitis C. Methods: Plasma and PBMC samples were collected from 16 randomly selected seropositive patients for the anti-HCV antibody. Four out of 16 (25%) patients received combination therapy with alpha interferon and ribavirin. PBMCs were isolated from whole blood. Between 106-107 cells were cultured with optimized concentrations of IL-2 (10 mg/ml) and phytohemagglutinin A (5 mg/ml). Total RNA was extracted from the first collected sera and harvested lymphocytes. Constructed plasmids containing the NCR coding region were used to plot the standard curve for the relative quantification of SYBR green real-time PCR. The sensitivity and specificity of the detection were established by using plasmids containing cDNA. Results: With this plasmid containing the NCR coding region, the Limit of Detection (LOD) of in-house-developed real-time RT-PCR sensitivity was 2×101 copies. Using primers for the NCR region, 10 out of 16 (62.5 %) PBMCs were positive for negative-strand HCV RNA. Among the four samples collected from patients with SVR, negative-strand HCV RNA was found in two patient samples. Conclusions: Our results indicated that cultured lymphoid cells from patients with chronic hepatitis, even with SVR, in the presence of IL-2 and PHA, markedly enhanced the detection of HCV RNA replica-tive strands. Therefore, PBMCs may be reservoirs for recurrent hepatitis infection after SVR and antiviral treatment. However, more clinical samples and control groups (lymphocyte culture without mitogen) should be examined to support the data presented in this study.

The Central Role of MAPK-ERK Signaling in IL7-Dependent and IL7-Independent Steroid Resistance Reveals a Broad Application of MEK-Inhibitors Compared to JAK1/2-Inhibition in T-ALL

Introduction Poor prednisolone response in the induction phase of treatment is a risk-stratification marker in the current Dutch DCOG ALL-11 treatment protocol. Physiological or mutational activation of the IL7-receptor (IL7R) provokes steroid resistance in T-ALL. Moreover, relapsed T-ALL patients that harbor IL7R or RAS mutations are considered as an ultra-high-risk group with extremely poor outcome. Aim We studied the contribution of two key IL7R downstream pathways (STAT5B and MAPK-ERK respectively) to steroid resistance in order to optimize future treatment regimens. Results To date, IL7-dependent steroid resistance has been proposed to depend on IL7-induced JAK-STAT signaling and subsequent BCL2 upregulation. Here, we demonstrate that the transforming STATBN642H mutation does not impair steroid sensitivity despite a paradoxical upregulation of BCL2 and BCLXL following steroid treatment. We therefore hypothesized that other pathways contribute to IL7-dependent steroid resistance in T-ALL. By studying T-ALL PDX samples, we observed that physiological IL7-signaling can activate the MAPK-ERK pathway in IL7-dependent and IL7-independent steroid resistant PDX cells. Similar to mutational-driven MAPK-ERK signaling (e.g. by activating IL7R, JAK1 or NRAS mutations), IL7-induced MAPK-ERK signaling leads to the inactivation of the pro-apoptotic protein BIM by inhibitory phosphorylation of both BIM-EL and BIM-L isoforms. By mass spectrometry, we observe that phosphorylated BIM specifically decreases its physical interaction with Aurora kinase A and Bcl-2 family members BMF, BCL2, BCLXL and MCL1, which alters the apoptotic threshold and effectuates steroid resistance. The JAK1/2-inhibitor ruxolitinib and MEK-inhibitor selumetinib are considered for future basket-treatment protocols in IL7R/JAK-STAT-activated or MAPK-ERK-activated leukemias respectively. We demonstrate that both ruxolitinib and selumetinib prevent IL7-induced MAPK-ERK signaling in T-ALL PDX cells. Moreover, ruxolitinib and selumetinib abolish BIM phosphorylation in mutant JAK1-overexpressing SUPT-1 cells, which restores the binding of BIM to BCL2, BCLXL and MCL1. Only selumetinib restores the function of BIM in wild type or mutant NRAS overexpressing SUPT-1 cells, indicating that ruxolitinib only blocks MAPK-ERK signaling in the context of 'upstream' (e.g. mutant IL7R/JAK1 or IL7-induced) pathway activation. Surprisingly, none of our 46 PDX samples respond to ruxolitinib treatment in the absence of IL7, while the majority of these samples demonstrate a robust response towards MEK-inhibition. IL7-dependent steroid resistant PDX samples do respond to ruxolitinib treatment in the presence of IL7, while IL7 exposed IL7-independent steroid resistant PDX samples remain unresponsive to ruxolitinib. We therefore conclude that the therapeutic effect of ruxolitinib is dependent on an IL7-protective effect, limiting its therapeutic application to a selective subset of T-ALL patients. When we combine prednisolone treatment with selumetinib or ruxolitinib, we observe that ruxolitinib only synergizes with prednisolone in IL7-dependent steroid resistant PDX samples in the presence of IL7. Importantly, combination treatment with prednisolone and selumetinib acts highly synergistic in IL7-dependent and IL7-independent steroid resistant samples in the presence and absence of IL7. Conclusion and clinical relevance In addition to described JAK-STAT and PI3K-AKT signaling, our study demonstrates that IL7-induced signaling activates the MAPK-ERK signaling pathway, which actively contributes to IL7-dependent steroid resistance. We demonstrate the central importance of MAPK-ERK signaling in T-ALL, whereas all T-ALL PDX samples tested highly benefit from combined prednisolone and selumetinib treatment. Ruxolitinib only acts synergistically with steroids in the context of IL7-dependent steroid resistance. Moreover, our data proposes that this synergistic effect may (in part) depend on the anti-MAPK-ERK effect downstream of JAK1/2-inhibition. Combined, our study strongly supports the enrollment of T-ALL patients in the current phase I/II SeluDex trial (NCT03705507), and contributes to the optimization and stratification of newly designed T-ALL treatment regimens. Disclosures Bourquin: Servier: Other: Travel Support. Vormoor:AstraZeneca: Research Funding.

Phenotypic and Proliferative Analysis of T Cell and Monocyte Populations in Patients with Intermediate/High/Very High-Risk MDS Compared to Age-Matched Healthy Donors

Background: Myelodysplastic syndromes (MDS) have been associated with alterations in the bone marrow microenvironment and include abnormal innate and adaptive immune responses. The extent to which these changes contribute to the pathogenesis of MDS is unclear. The heterogeneous nature of MDS makes it difficult to distinguish changes in intrinsic cell function for microenvironmental alterations in the mutated myeloid compartment. T cells, which are in the lymphoid compartment and less frequently contain MDS mutations, can potentially be used to evaluate immune dysfunction in the presence of malignant myeloid cells. Here we examine the immunophenotypes and proliferative function of T lymphocyte and monocyte populations from higher-risk MDS patients. Methods: Peripheral blood mononuclear cells (PBMC) from patients with IPSS-R Intermediate/High/Very High-risk MDS (herein referred to collectively as higher-risk MDS) and age-matched healthy donors (n = 15 and n = 12, respectively) were polyclonally stimulated with an antibody against CD3 (OKT3) in the presence of IL-2 (50 U/mL). Phenotypic analysis was performed at baseline and 46 hours after the addition of OKT3 and IL-2. T cell proliferation was measured by staining PBMC with proliferation dye (CellTrace Violet) and measuring fold proliferation after eight days in culture. Proliferation statistical comparisons were made by unpaired t-test. Phenotypic parameters were measured by flow cytometry and statistical analysis performed with the Kruskal-Wallis one-way analysis. Results: As previously reported, higher-risk MDS patient T cells had decreased capacity for proliferation when compared to healthy donor counterparts (Figure 1A p = 0.0096). This difference in proliferative capacity was not associated with either the level of PD-1 on the surface of T cells (r = 0.2956) or the level of PD-L1 on monocytes (r = 0.3859) in stimulated PBMCs. Patients with higher-risk MDS tended to have lower PD-L1 surface expression on monocytes compared to healthy donor counterparts, albeit not significantly (Figure 1C p = 0.1). However, PD-1 expression on T cells was comparable between groups both before and after stimulation (Figure 1B CD4 cells: p = 0.58; CD8 cells p = 0.58). This suggests that T cell proliferation from patients with higher-risk MDS might not be inhibited through the PD-1/PD-L1 axis. Of note, higher-risk MDS patient monocytes expressed less CD80 and HLA-DR both pre- and post-stimulation when compared to healthy donors (Figure 1C CD80: p = 0.0001 and 0.037; DR: p = 0.012 and 0.019, respectively), implying a potential dysfunction in the monocytic stimulatory and antigen presentation capacity. PD-L1 surface expression is low at baseline (an average of <5% for both donors and higher-risk MDS); upon stimulation, there is a high correlation between HLA-DR and PD-L1 in healthy donors (p = 0.0003, r = 0.859). Additionally, there is less upregulation of PD-L1 on the surface of monocytic cells in the higher-risk MDS group and subsequently less correlation between PD-L1/HLA-DR (p = 0.03) in stimulated PBMC samples. Taken together, these results show a decreased baseline expression and/or ability to upregulate monocyte activation markers after stimulation. We hypothesize that these changes might contribute to the observation that higher-risk MDS patient T cells are less capable of proliferation. Conclusion: There was no significant difference in PD-1 and PD-L1 expression between higher-risk MDS and healthy donor patients, despite reduced T cell proliferation among patients with MDS. Additionally, decreased levels of CD80 and HLA-DR on intermediate/high-risk MDS patient monocytes suggests a suppressed ability to upregulate activation markers, possibly leading to decreased T cell proliferation. Targeting the PD-1/PD-L1 axis alone may not be therapeutic in patients with higher-risk MDS, and we hypothesize that the immune evasion observed in higher-risk MDS patients may not be due exclusively to increases in inhibitory interactions like PD-L1 and PD-1, but may result from a decrease in stimulatory receptors, such as CD80, and antigen presentation molecules, such as HLA-DR. Disclosures Fields: PersImmune, Inc.: Ended employment in the past 24 months. Ferrari:PersImmune, Inc.: Ended employment in the past 24 months. Tarke:PersImmune, Inc.: Ended employment in the past 24 months. Luger:Arcturus Therapeutics: Current Employment. Lane:PersImmune, Inc.: Current Employment. Vitiello:PersImmune, Inc.: Current equity holder in private company, Ended employment in the past 24 months. Bejar:Aptose Biosciences, Inc: Current Employment, Current equity holder in publicly-traded company.

GFI1 Is a Downstream Target of EVI1 in Normal Hematopoiesis

Acute myeloid leukemia (AML) with high expression of a transcriptional factor, Ecotropic viral integration site 1 (EVI1), is associated with extremely poor prognosis. EVI1 is, however, also essential for maintaining normal hematopoietic stem cells (HSCs), rendering it potentially difficult to target this molecule. To overcome this therapeutic difficulty, it is important to comprehensively elucidate differentially regulated downstream targets between normal and leukemia cells. In this study, we searched downstream targets of EVI1 in normal hematopoiesis by combining a chromatin immunoprecipitation sequence (ChIP-seq) and RNA-sequence (RNA-seq) analysis using a mouse hematopoietic cell line 32D-cl3 with high EVI1 expression. We deleted Evi1 using CRISPR/Cas9 in 32D-cl3 cells. Evi1 knock-out (KO) 32D-cl3 cells showed comparable cell growth with parental cells in the presence of IL-3, which enables them to proliferate permanently without differentiation. When they are allowed to differentiate by adding G-CSF, the number of KO cells decreased sharply at day 5-6, compared with parental 32D-cl3 cells. Along with the decreased cell number, KO cells also demonstrated higher positive rate of Gr-1 at day 7, a typical marker of differentiation into granulocytes, indicating accelerated differentiation of KO cells. These results indicated that EVI1 is required to maintain undifferentiated status of 32D-cl3 cells in a differentiation-permissive conditions, which can model normal hematopoiesis. We knocked in 3×FLAG tag at the 3' end of the Evi1 gene to perform ChIP-seq using anti-FLAG antibody. By using these knock-in cells, ChIP-seq was performed on day 0 and day 3 of G-CSF treatment, when they had started to differentiate with still maintained EVI1 expression. The peaks observed in undifferentiated day 0 sample were considered to contain a group of genes involved in undifferentiated hematopoietic cells in cooperation with EVI1. Genes associated HDAC class I, RAC1 signaling were enriched in these genes. To investigate the functional implications of the result of ChIP-seq, RNA-seq data using two clones of KO cells and parental cells were combined. We found that 152 genes were significantly up-regulated, and 155 genes were down-regulated in the KO cells, with false discovery rate less than 0.05. Twenty-four genes were identified by extracting common genes between ChIP-seq and RNA-seq; namely, genes which had day 0-specific peaks in ChIP-seq, and whose expression were decreased in the KO cells. In order to further examine the physiological implications of 24 genes in vivo, we referred to the results of RNA-seq using murine bone marrow transplantation model, where murine hematopoietic progenitor cells retrovirally transduced with Evi1 were transplanted into irradiated syngeneic mice, finally leading to AML after a long latency. Samples obtained early after post transplantation and those after AML onset were compared to those of normal hematopoietic progenitor cells. Among the above 24 genes, the expression of 5 genes was increased early after transplantation and decreased after the onset of AML, that is, these genes were up-regulated by EVI1 but don't seem to be involved in AML maintenance. We functionally validated the role of these genes in 32D-cl3 cells. Of the above, CRISPR/Cas9-mediated knock-out of Gfi1(Growth Factor Independent 1 Transcriptional Repressor) and Mfsd2b (Major facilitator superfamily domain containing 2B) in 32D-cl3 cells led to high Gr-1 positivity at day 7 like Evi1-KO cells, suggesting that these genes are involved in the functions of EVI1 in the normal hematopoiesis. The mRNA expression of these genes was compared in LSK (Lineage- Sca1+ c-kit+) cells from the bone marrow of Evi1 conditional knockout (cKO) mice and control mice. The expression of Gfi1 and Mfsd2b was decreased in LSK cells from Evi1 cKO mice. Furthermore, retroviral expression of Gfi1 in LSK cells restored the reduced colony-forming ability of Evi1 cKO cells. These results collectively suggest that GFI1 is regulated by EVI1 and is involved in the function of EVI1 regulating the stemness of hematopoietic stem and progenitor cells in normal hematopoiesis. These findings provide us with the novel insights on EVI1-mediated HSC maintenance as well as on the therapeutic strategy that specifically targets leukemia-specific EVI1 effectors while preserving normal hematopoiesis. Disclosures Kurokawa: Shire Plc: Speakers Bureau; Jansen Pharmaceutical: Speakers Bureau; Ono: Research Funding, Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Eisai: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Teijin: Research Funding; Takeda: Research Funding, Speakers Bureau; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Otsuka: Research Funding, Speakers Bureau; Pfizer: Research Funding; Sanwa-Kagaku: Consultancy; MSD: Consultancy, Research Funding, Speakers Bureau; Chugai: Consultancy, Research Funding, Speakers Bureau; Bioverativ Japan: Consultancy; Celgene: Consultancy, Speakers Bureau; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Nippon Shinyaku: Research Funding, Speakers Bureau.

Preclinical Development of Edit-201, a Multigene Edited Healthy Donor NK Cell with Enhanced Anti-Tumor Function and Superior Serial Killing Activity in an Immunosuppressive Environment

Natural killer (NK) cells distinguish tumor from healthy tissue via multiple mechanisms, including recognition of stress ligands and loss of MHC class I expression. For example, KIR mismatching enables allogenic NK cells to respond to MHC positive tumors in a similar manner to MHC negative tumors, making allogeneic NK cell therapy a promising approach for broad oncology indications. Accordingly, allogenic human HD-NK cells, including gene-modified cells, have demonstrated an impressive safety and efficacy profile when administered to patients with advanced hematologic malignancies. However, 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. To this end, we developed a next-generation allogeneic NK cell therapy using CRISPR-Cas12a gene editing to enhance NK cell function through knockout of the genes CISH and TGFBR2. Both single and simultaneous targeting (DKO) of TGFBR2 and CISH in NK cells using CRISPR-Cas12a produced in/dels at both targets in greater than 80% of NK cells, with greater than 90% of edited NK cells viable at 72 hours post-editing. Importantly, we find that DKO NK cells do not phosphorylate the SMAD2/3 protein downstream of the TGF-b receptor complex and demonstrate increased phosphorylation of pSTAT3 and pSTAT5 upon IL-15 stimulation, consistent with protein level knockout of TGFBR2 and CISH. To determine whether DKO NK cells exhibited superior function relative to control NK cells, we first measured the ability of DKO NK cells to kill Nalm6 cells (adult B cell ALL) relative to unedited control NK cells. We find that in the presence of exogenous TGF-b, DKO NK cells demonstrate improved cytotoxicity against Nalm6 tumor targets by delaying tumor re-growth in comparison to control NK cells. To better characterize the ability of DKO NK cells to kill tumor cells, we developed an in vitro serial killing assay. In this long-duration assay, up to 30 days, control and DKO NK cells (grown in the presence of IL-15) were challenged every 48 hours with a new bolus of Nalm6 tumor targets. Both DKO and unedited NK cells control Nalm6 target cell growth for greater than 18 days (9 additions of new Nalm6 target cells), demonstrating a surprising ability for the same NK cells to serially kill new Nalm6 target cells for a prolonged period of time in vitro. We find that DKO NK cells produce higher levels of IFN-γ and TNF-α relative to control NK cells over the duration of the entire serial killing assay, suggesting that DKO NK cells can continue to produce these inflammatory cytokines even after serial killing. As many tumors, including hematologic malignancies, have high concentrations of TGF-β in their microenvironments, we next tested the ability of DKO NK cells to control the growth of Nalm6 cells in our serial killing assay in the presence of TGF-b. 10ng/mL TGF-β was added at the start of the assay as well as at each addition of new Nalm6 target cells. We observed that control NK cells fail to restrict Nalm6 target cell growth beyond 4 days (after 1 addition of new Nalm6 target cells) whereas DKO NK cells control Nalm6 target cell growth for greater than 18 days (after 9 additions of new Nalm6 target cells). Similar to the serial killing assay without TGF-b, we find that DKO NK cells produce higher concentrations of IFN-γ and TNF-α relative to control NK cells over the duration of the entire serial killing assay. Broadening our repertoire of target cells beyond B cell malignancies is now in progress, including the AML-like cell lines HL-60 and THP-1, the multiple myeloma cell line RPMI 8226, and various solid tumor targets. In summary, using CRISPR-Cas12a we demonstrated highly efficient gene editing of primary human NK cells at two unique targets designed to augment NK cell anti-tumor activity across a variety of malignancies. Most significantly, we demonstrate sustained anti-tumor serial-killing activity in the presence of the potent immunosuppressive cytokine TGF-β. Together, the increased overall effector function of CISH/TGFBR2 DKO primary human NK cells and their ability to serial kill, support their development as a potent allogeneic cell-based medicine for cancer. This potential medicine, termed EDIT-201, is being advanced to clinical study. Disclosures Borges: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wasko:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Nasser:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Donahue:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Pfautz:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Antony:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Leary:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Sexton:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Morgan:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wong:Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

Increasing AMPK Activity in Human T Cells Enhances Memory Subset Formation without Sacrificing in Vitro Expansion

BACKGROUND: While chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of relapsed/refractory acute lymphoblastic leukemia (ALL), treatment failures continue to occur. In studying therapeutic T cell function, it has become clear that achieving a memory-like phenotype is ideal for CAR-T production. This is likely related to the enhanced oxidative metabolic potential of this subset, which allows for improved persistence and enhanced anti-leukemia activity in vivo. However, current expansion protocols drive T cells towards terminal differentiation, decreasing the number of T cells fit for the in vivo environment. Finding methods to improve the yield of memory-like cells without sacrificing T cell expansion has been challenging. AMP-activated protein kinase (AMPK) is a key metabolic regulator responsible for promoting mitochondrial biogenesis and oxidative metabolism, and is more active in memory T cells at baseline. It is similarly induced by TCR ligation, making it unlikely that it would significantly detract from proliferation. These properties make activation of AMPK a potential candidate pathway for improving the yield of more functional T cells for CAR-T cell therapy. METHODS: AMPK is a heterotrimeric protein complex consisting of alpha, beta, and gamma domains. Functionally, the alpha subunit contains the kinase domain, which is activated by phosphorylation. The gamma subunit controls the phosphorylation, and therefore the activity, of the alpha domain. To increase AMPK signaling in T cells, we cloned the gamma subunit into a lentiviral plasmid containing the elongation factor 1a (EF1a) promoter and a green fluorescent protein (GFP) tag. An empty vector, containing GFP only, served as a negative control. Human T cells were isolated from three separate donors, transduced with our lentiviral construct, and expanded in vitro in the presence of IL-2. AMPK activity was assessed by phosphorylation of Thr172 on the AMPKα subunit as well as phosphorylation of S555 on downstream target Unc-51-like autophagy activating kinase (ULK1) using western blot densitometry, normalized to the total protein amounts. Memory marker expression and mitochondrial density (using Mitotracker Red) were analyzed by flow cytometry. Oxidative metabolism and spare respiratory capacity (SRC) were determined using the Seahorse Metabolic Analyzer. Fold changes for in vitro expansion were calculated by adjusting manual cell counts to reflect GFP positivity and CD4+/CD8+ surface staining. RESULTS: The AMPK gamma subunit was efficiently transduced and expressed by human T cells as measured by GFP expression, qRT-PCR, and western blot analysis. Further, AMPK activity increased in GFP+ cells as indicated by the phosphorylation of AMPKα Thr172 (1.93 +/- 0.05 vs 0.6 +/- 0.09, p<0.001) and ULK1 S555 (1.28 +/- 0.11 vs 0.67 +/- 0.08, p<0.01). Cells transduced with AMPK augmented expression of memory markers CD62L, CD27, and CCR7, with an increased yield of stem cell memory-like T cells marked by co-expression of CD45RA and CD62L (Figure 1). In addition, AMPK-transduced T cells showed a statistically significant increase in mitochondrial density along with notable enhancement of SRC and maximal oxygen consumption rates (Figure 2A,B). Furthermore, the rate of expansion of AMPK-transduced T cells did not differ significantly from Empty-transduced controls, and in fact trended towards increased in both CD4+ and CD8+ cells (Figure 3A). Indeed, the improved rate of expansion in AMPK-transduced CD4+ T cells led to a measurable increase in CD4+ T cell percentages by flow cytometry (Figure 3B). DISCUSSION: Here we present an efficient and direct method to increase AMPK activity in human T cells and demonstrate that increased AMPK activity endows T cells with a variety of characteristics ideal for CAR-T cell therapy. These features include increased memory-marker expression, enhanced SRC and oxidative metabolism, equivalent to augmented in vitro expansion, and improved CD4+ T cell yields. Further studies are ongoing to assess the activity and function of AMPK-transduced CAR-T cells both in vitro and in vivo. Disclosures No relevant conflicts of interest to declare.

Super-Enhancer-Driven TOX2 Mediates Oncogenesis in Natural Killer/T Cell Lymphoma

Background: Extranodal natural killer/T-cell lymphoma (NKTL) is an EBV associated, aggressive malignancy. Activation of JAK/STAT pathway, overexpression of EZH2, and alternations in epigenetic program have been implicated in the pathogenesis of NKTL. Combined chemotherapy-radiotherapy is standard treatment for NKTL patients, but often associated with high relapse rate and serious side effects. New drugs, including anti-PD1 antibody pembrolizumab, have been explored. Overall, treatment for NKTL patients remains challenge in clinic. Super-enhancers (SEs) are defined as large clusters of cis-acting enhancers, marked by high level bindings of acetylation of histone H3 lysine 27 (H3K27ac), coactivators and transcription factors. SE associated oncogenes have been demonstrated to play key roles in various types of cancers. In this study, we aim to define the SE landscapes of NKTL for a better understanding of the molecular pathogenesis of NKTL and to identify novel therapeutic targets. Methods: We performed H3K27Ac ChIP-seq and RNA-seq on primary NKTL tumor samples and paired normal tonsil tissues as controls, then the Rank Ordering of Super Enhancers (ROSE) analysis was used to systematically annotate SEs and their associated genes between tumors and controls. A combination of RNA interference (RNAi), CRISPRi (dCas9-KRAB) technologies and overexpression experiments, followed by several functional assays were performed to determine the effects of candidate SE-genes on NKTL cells. Circularized chromatin conformation capture followed by sequencing (4C-seq) experiments which examine the direct contact of SE and its promoter are ongoing. Results: A total of 1266 SEs were identified in more than 2 out of 3 primary NKTL tumors but not in their normal tonsils. RNA-seq revealed overexpression of 1478 genes (false discovery rate <0.001, log2 fold change ≥ 1). We filtered genes that were associated with super-enhancers and also significantly overexpressed in NKTL tumors relative to normal tonsils. Using this rigorous strategy, we pinned down a list of 191 genes with high value for further investigation. Pivotal transcription factors regulating NK cell development and function were highly enriched in the list: TOX2, TBX21, EOMES, RUNX2, ID2, GATA3, GFI1, and MAFB. We then scrutinized the SE constituents of these 3 T-box protein family, including TOX2, TBX21 and EOMES, and found that only TOX2 harbored remarkably high SE peaks specifically in all 3 NKTL primary samples, in contrast, only background signals were presented in normal tonsil tissues. TOX2 expression was significantly higher in the primary NKTL samples and cell lines compared with normal NK cells. Most importantly, high expression of TOX2 was association with worse overall survival (Log rank p value: 0.021, hazard ratio: 2.63), demonstrating its prognostic significance (GSE90784). We used two individual TOX2 specific-shRNAs tagged with GFP to decrease TOX2 expression in NKYS cells. FACS analysis of GFP+ cells revealed a markedly decreased GFP+% cells in TOX2-sh1- and -sh2-treated samples compared with scr-shGFP-transduced cells over 11 days post transduction. These results indicate that silencing TOX2 imposes a strong negative selection pressure on NKYS cell growth. Overexpressed TOX2 resulted in significantly higher cell viability of NKYS-TOX2 cells than NKYS-EV in the culture without IL-2, but not in presence of IL-2. CFU assays also demonstrate that TOX2 was effective in enhancing the clonogenic capacity of NKTL cells. Next, we assessed whether the TOX-SE is functional and causative for the TOX2 dysregulation in NKTL. The repression of the TOX2-SE region significantly decreased TOX2 expression in NKYS cells. Repression of the TOX2-SE region also led to impairment of cell viability and cell growth, consistent with an oncogenic function. Conclusions: Taken together, our integrative approaches defined the landscape of SE and identified high-value SE-associated oncogenes in NKTL. Among them, we demonstrated that TOX2 is a novel tumor driver, which promotes NKTL cell growth and enhances ability of colony formation, as well as protects cell viability under adverse condition. This study not only provides novel insight into the NKTL pathology, but also offer novel, potential therapeutic targets, such as TOX2 for the treatment of NKTL patients. Disclosures Lim: National Cancer Centre Singapore: Current Employment. Chng:Abbvie: Honoraria; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria; Amgen: Honoraria, Research Funding.

Adipose Tissue-Derived Stromal Cells Induce a Highly Trophic Environment While Reducing Maturation of Monocyte-Derived Dendritic Cells.

Allogeneic cell-based therapies using adipose tissue-derived stromal cells (ASCs) offer an off-the-shelf alternative to autologous therapy. An underlying assumption is that ASC can modulate the immune response of the recipient. However, in vitro models are required to explore and identify cell interactions and mechanisms of action, to ensure sufficient and sustained effects, and to document these. In this study, we shed light on the effect of ASC manufactured for clinical use on monocyte-derived dendritic cells and an inflammatory microenvironment. ASCs were isolated from healthy voluntary donors, expanded using a human platelet lysate in bioreactors, and cryopreserved as per clinical use. Monocyte-derived dendritic cells were generated by isolation of monocytes and differentiation with GM-CSF and IL-4. Dendritic cells were cocultured with different ratios of ASC and matured with LPS and IFN-γ. Dexamethasone was included as an immunosuppressive control. Dendritic cells were analyzed by flow cytometry for CD11c, CD40, CD80, CD83, CD86, PD-L1, and HLA-DR, and supernatants were analyzed for FGF2, HGF, IL-10, IL-12p70, LIF, MIF, PDGF, PlGF, and IDO. Reduced expression of maturation markers was observed on ASC-treated dendritic cells, while high levels of PD-L1 were maintained. Interestingly, the expression of CD83 was elevated. Escalating ratios of ASC did not affect the concentration of IL-10 considerably, whereas the presence of IL-12 was reduced in a dose-dependent manner. Besides offsetting the IL-12/IL-10 balance, the concentrations of IDO and MIF were elevated in cocultures. Concentrations of FGF2, HGF, LIF, and PIGF were high in ASC cocultures, whereas PDGF was depleted. In a robust coculture model, the addition of ASC to dendritic cells inhibited the dendritic maturation substantially, while inducing a less inflammatory and more tolerogenic milieu. Despite the exposure to dendritic cells and inflammatory stimuli, ASC resulted in supernatants with trophic factors relevant for regeneration. Thus, ASC can perform immunomodulation while providing a regenerative environment.

MHC/class-II-positive cells inhibit corticosterone of adrenal gland cells in experimental arthritis: a role for IL-1\u03b2, IL-18, and the inflammasome

In experimental arthritis, glucocorticoid secretion is inadequate relative to inflammation. We hypothesized that IL-1 is a key factor for inadequate glucocorticoid secretion in arthritic rats. Collagen type II—induced arthritis (CIA) in DA rats was the model to study effects of IL-1 on adrenal function. In the CIA model, an increase of intraadrenal MHCII-positive cells was observed. MHCII-positive cells or bone marrow-derived dendritic cells inhibited glucocorticoid secretion of adrenal gland cells. IL-1, but also IL-18 and the inflammasome were critical in glucocorticoid inhibition. Arthritic compared to control adrenal gland cells produced higher amounts of CXC chemokines from MHCII+ adrenal cells, particularly CINC-2, which is strongly dependent on presence of IL-1. In CIA, macrophages and/or dendritic cells inhibit glucocorticoid secretion via IL-1 in adrenal glands. These findings show that activated macrophages and/or dendritic cells inhibit glucocorticoid secretion in experimental arthritis and that IL-1β is a decisive factor.

Antigenic competition in the generation of multi-virus-specific cell lines for immunotherapy of human cytomegalovirus, polyomavirus BK, Epstein-Barr virus and adenovirus infection in haematopoietic stem cell transplant recipients

Adoptive transfer of multivirus-specific T cell lines (MVST) is an advanced tool for immunotherapy of virus infections after hematopoietic stem cell transplantation (HSCT). Their preparation includes activation of donor virus-specific T cells by the mixture of oligopeptides derived from immunodominant antigens of several most harmful viruses, i.e. human cytomegalovirus (HCMV), polyomavirus BK (BKV), Epstein-Barr virus (EBV) and adenovirus (ADV). The aim of our study was to find out whether antigenic competition may have an impact on the expansion of virus-specific T cells. MVST from several heathy blood donors were generated using a pulse of overlapping oligopeptides (PepMixes™, derived from the IE1 and pp65 CMV antigens, VP1 and LTAG BKV antigens, BZLF1 and EBNA1 proteins of EBV and hexon protein from ADV) and short time culture in the presence of IL-7 and IL-4. The amount of virus-specific T cells in MVST was measured by ELISPOT and flow cytometry after re-stimulation with individual antigens. To evaluate antigenic competition, MVST were expanded either with a complete set of antigens or with the mixture lacking some of them. MVST expanded with the antigen mixture including CMV antigens contained a lower proportion of the T cells of other antigen specificities. A similar inhibitory effect was not apparent for EBV-derived peptides. The competitive effect of CMV antigens was most pronounced in MVST from CMV-seropositive donors and was mediated by both IE1 and pp65-derived peptides. Antigenic competition did not influence the phenotype of either CMV- or BKV-specific T cells. Both T cell populations had an effector memory phenotype (CD45RO+, CD27-, CCR7-). However, CMV-specific T cells preferentially consist of CD8+ while in BKV-specific T cells, the CD4+ phenotype predominated. Modification of the MVST manufacture protocol to prevent antigenic competition may increase the efficacy of MVST in therapy of BKV infections in HSCT recipients.

Granzyme A-producing T helper cells are critical for acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) can occur after hematopoietic cell transplant in patients undergoing treatment for hematological malignancies or inborn errors. Although CD4+ T helper (Th) cells play a major role in aGVHD, the mechanisms by which they contribute, particularly within the intestines, have remained elusive. We have identified a potentially novel subset of Th cells that accumulated in the intestines and produced the serine protease granzyme A (GrA). GrA+ Th cells were distinct from other Th lineages and exhibited a noncytolytic phenotype. In vitro, GrA+ Th cells differentiated in the presence of IL-4, IL-6, and IL-21 and were transcriptionally unique from cells cultured with either IL-4 or the IL-6/IL-21 combination alone. In vivo, both STAT3 and STAT6 were required for GrA+ Th cell differentiation and played roles in maintenance of the lineage identity. Importantly, GrA+ Th cells promoted aGVHD-associated morbidity and mortality and contributed to crypt destruction within intestines but were not required for the beneficial graft-versus-leukemia effect. Our data indicate that GrA+ Th cells represent a distinct Th subset and are critical mediators of aGVHD.

Interleukin 3-induced GITR promotes the activation of human basophils

Human basophils regulate allergic reactions by secreting histamine, interleukin 4 (IL-4) and IL-13 through key surface receptors FcεRI as well as IL-3R, which are constitutively expressed on basophils. IL-3/IL-3R signaling axis plays key roles in regulating the development and activation of basophils. We and others have shown that IL-3-induced surface receptors e.g. ST2, IL-17RB and IL-2 receptors regulate the biology of basophils. However, the expression and function of IL-3-induced surface proteins on human basophils remain to be elucidated. We in this study aimed to identify new basophil activation regulators by transcriptomic analysis of IL-3-stimulated basophils. Gene expression microarray analysis of IL-3-treated basophils revealed 2050 differentially expressed genes, of which 323 genes encoded surface proteins including GITR. We identified that GITR was preferentially induced by IL-3 rather than anti-IgE, IL-33, fMLP and C5a. IL-3-induced GITR was suppressed by inhibitors targeting JAK2, PI3K and MEK1/2. Stimulation of IL-3-treated basophils by GITR enhanced the expression of IL-4 and IL-13. Moreover, IgE-mediated degranulation was enhanced by GITRL in the presence of IL-3. This transcriptomic analysis of IL-3-activated basophils helps to identify novel activation regulator. IL-3-induced GITR promoted the activation of basophils, adding new evidence supporting GITR as an important player in Th2-associated immune responses.

Plasticity and regulatory mechanisms of human ILC2 functions

Human group 2 innate lymphoid cells (ILC2) represent the innate counterpart of Th2 cells and cooperate with them in helminths protection and in the pathogenesis of allergic diseases. Some reports described ILC2 plasticity and few studies investigated the cellular and molecular mechanisms regulating human ILC2 functions. The aim of this study is to define how immune deviation and immune regulation control human ILC2-mediated immune response. Human circulating ILC2 were expanded in vitro and then cultured in presence of IL-12 or IL-1β plus IL-23 or co-coltured in presence of circulating CD4+CD25highFoxp3+Treg. IL-12 induces IFN-γ production and upregulation of T-bet mRNA level on human circulating ILC2 whereas IL-1β and IL-23 mediate IL-22 production and upregulation of RORC mRNA level. In all these conditions, GATA-3 mRNA level is not reduced and the typical type 2 cytokines are only partially reduced. Moreover, “modulated” ILC2 have reduced ability to induce IgE producing by B cells. ILC2 proliferation, cytokines production and CD154 expression were inhibited by CD4+CD25highFoxp3+ Treg cells. TGF-β reduced CD154 expression on ILC2 stimulated with IL-25/IL-33. This study defines possible cellular and molecular mechanisms responsible for modulation and inhibition of human ILC2 activity. These results may be useful in the development of strategies aimed to dampen ILC2 function in type-2 mediated diseases.

The role of oral supplementation with immunonutrients in the inflammatory response in patients with COVID-19

Introduction: The SARS-CoV-2 related coronavirus has spread to almost every country, causing the COVID-19. The severity of COVID-19 is based on the inflammation caused by the cytokine storm. Inflammatory markers suggest a molecular explanation for the occurrence of severe disease and represent a possible treatment. Objective: This review aims to analyse the plausibility of using oral supplementation with immunonutrients. Discussion: Immunonutrition studies the interactions between nutrition, the immune system, infection, and inflammation of tissues. Arginine is a fundamental amino acid in processes of metabolic stress. Its deficiency implies in the capacity of immune response. ω-3 fatty acids demonstrated improved lung compliance, oxygenation, mechanical ventilation time and intensive care unit stay in patients with acute respiratory distress syndrome. Essential for cell-mediated immunity and T lymphocyte function, dietary nucleotides have been shown to improve the cytotoxicity of natural killer cells. Considering that the inversely proportional relationship between presence of IL-6 and TNF-α and the function of T cells is well established in patients with COVID-19, this reinforces the idea that the release control of these biomarkers can be a way to treat this disease. Conclusion: In this scenario, this review raises the possibility of using immunonutrition to improve immune response of individuals affected by COVID-19.

Abstract 875: Next generation clonal neoantigen targeting T cells, generated using the PELEUSTM bioinformatics platform and the VELOSTM manufacturing method show superior reactivity and phenotypic characteristics than classical TIL products

Abstract Adoptive transfer of tumor infiltrating lymphocytes (TIL) has generated objective clinical responses in patients with advanced metastatic cancers. Therapeutic exploitation of neoantigens as targets can potentially lead to safer and more effective treatment modalities with reduced toxicities. The Achilles Therapeutics trial NCT03517917 enabled the acquisition of matched tumor specimens and peripheral blood samples from patients undergoing routine surgery and facilitated the development of the proprietary VELOSTM manufacturing process, generating a personalized clonal neoantigen specific T cell product. An in-depth characterization of T cells expanded with the VELOSTM process was performed and compared to a standard TIL product. Samples were obtained from patients with primary NSCLC or metastatic melanoma. TIL were expanded from tumor fragments after dissection in the presence of IL-2. Peptide pools corresponding to the clonal mutations that were identified using the PELEUSTM bioinformatics platform were used to pulse dendritic cells (DC) generated from peripheral blood monocytes from each patient. Clonal neoantigen specific T cells (cNeT) were expanded using the VELOSTM process by co-culture of TIL with the peptide-pulsed autologous DC. As a comparison, TIL were expanded with a rapid expansion protocol (REP-TIL) in the presence of allogeneic feeders, anti-CD3 antibody and high-dose IL-2. Intracellular cytokine staining was performed following rechallenge with individual peptide pools encoding the clonal mutations. Single peptide reactivities were identified using ELISPOT and extended flow cytometric analysis of markers associated with T cell fitness or dysfunction was performed to phenotypically characterize the cNeT, TIL and REP-TIL. Analysis of the immune cell composition showed that cNeT, TIL and REP-TIL have similar CD3+ T cell content (median cNeT 90.2%, TIL 87.3%, REP-TIL 95%, n=6) and are composed of CD4+ and CD8+ T cells (median CD4:CD8 ratio- cNeT 11.1, TIL 2.03 and REP-TIL 4.7, n=6). cNeT showed superior clonal neoantigen specificity compared to TIL or REP-TIL. The proportion of CD3+ T cells responding to clonal neoantigen rechallenge was increased in cNeT (median 24.3%) compared to TIL (median 0.6%) and REP-TIL (median 1.8%) (n=5). The VELOSTM process incorporating the PELEUSTM platform for prediction of clonal neoantigens generates T cell products enriched for clonal neoantigen reactivities and superior phenotypic characteristics compared to conventional TIL. The VELOSTM process is currently being used to manufacture cNeT for two first-in-human studies including NSCLC and melanoma patients (NCT04032847, NCT03997474). Ethical approval: The samples for the study were collected under an ethically approved protocol (NCT03517917). Citation Format: Eleni Kotsiou, Tie Zheng Hou, Joseph Robinson, Sonal Varsani, Theres Oakes, Pablo D. Becker, Shreenal Patel, Jennine Mootien, Andrew Craig, Jane Robertson, Edward Samuel, James Reading, Lyra Del Rosario, Andrew Haynes, Samra Turajlic, Farah Islam, David Lawrence, Mariam Jamal-Hanjani, Martin Foster, Sergio A. Quezada, Katy Newton. Next generation clonal neoantigen targeting T cells, generated using the PELEUSTM bioinformatics platform and the VELOSTM manufacturing method show superior reactivity and phenotypic characteristics than classical TIL products [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 875.

Abstract 4482: Monitoring T cell reactivity to allogeneic placental cell products using multiple lymphocyte reaction and TCR sequencing approaches

Abstract Introduction: Allogeneic stem cell-derived therapies offer patients a readily accessible source of off-the-shelf product. However, allogeneic cells may be recognized by the recipient's immune system thus negatively affecting persistence and efficacy. Among people, allelic differences in human leukocyte antigen (HLA) complex genes are the major source of alloantigen which can elicit immunity. While there are established assays for measuring alloantibodies, monitoring T cell alloreactivity is more challenging. Unique clonal populations vary between individuals and functional assays are difficult to scale and standardize if alloreactive populations are rare/infrequent. Here, we evaluate how alloreactive T cells from patients may be identified using multiple lymphocyte reaction (MLR). Next generation sequencing of the TCR locus permits the subsequent identification and monitoring of allogeneic T cell clones in a patient over time. Methods: Placental leukocytes depleted of CD34 cells or peripheral blood mononuclear cells (PBMC) were enriched for monocytes using magnetic-bead isolation followed by dendritic cell (DC) maturation in the presence of GM-CSF and IL-4 for 5 days. DC were activated with lipopolysaccharide for 16 hours then cultured in the presence of CD3+ T cells isolated from healthy 3rd party PBMC for 5 days in the presence of IL-2. Proliferating alloantigen-specific T cells were observed by flow cytometry. TCR locus sequencing was performed on DNA libraries generated from unstimulated and post-MLR cultures using the Illumina platform. Results: In contrast to monocyte derived DCs from PBMC, monocyte derived DCs from placenta required higher levels of cytokine and lipopolysaccharide stimulation to achieve similar level of maturation. Co-culture of placental-derived DCs with unrelated T cells over 5 days resulted in the outgrowth of T cell colonies visible by brightfield microscopy. Flow cytometry confirmed clonal expansion of CD4 and CD8 T cell populations based upon the expression of Ki-67, CD25, and CD69 on cells with a higher forward scatter. To assess feasibility of performing TCR sequencing in unstimulated and post-MLR T cells, one T cell responder was evaluated in response to 3 different DC donors. For each DC donor, a single T cell clone representing 0.35% of the starting repertoire expanded to become the dominant clone at 44%, 61%, and 66% following MLR with the next most frequent clones represented 4%, 3%, and 2% among total T cells, respectively. Our data demonstrate the feasibility of identifying alloreactive T cell clones for translational research monitoring in patients. This approach could inform correlations with cell persistence and help optimize allogeneic cell delivery and dosage strategies in the context of disease state and lymphodepletion. Citation Format: Bhavani Stout, Tanel Mahlakoiv, Robert Hariri, Xiaokui Zhang, William van der Touw. Monitoring T cell reactivity to allogeneic placental cell products using multiple lymphocyte reaction and TCR sequencing approaches [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4482.

Abstract LB-390: Intratumoral electroporation of plasmid-encoded IL-12 and membrane-bound anti-CD3 increases tumor immunogenicity and augments the function of T cell subsets

Abstract Intratumoral (IT) delivery of plasmid IL-12 (tavokinogene telseplasmid; tavo) via electroporation (EP), collectively referred as IT-tavo-EP, generates immunologically-relevant levels of localized IL-12, triggering regression of both treated and distant tumors with minimal toxicity in preclinical and clinical studies. Our previous clinical trial data from melanoma patients treated with IT-tavo-EP identified a treatment-related increase of infiltrating T cells and transcripts related to immune activation, as well as a significant increase in the IFN-γ score of patients with a clinical benefit, suggesting that CD3+ tumor-infiltrating lymphocytes (TIL) may be critical in maximizing the anti-tumor effects of IT-tavo-EP. Furthermore, in-house biomarker data have identified an abundance of non-tumor reactive TIL that, if mobilized could additionally contribute to a clinical response. Accordingly, a plasmid-encoded membrane-bound polyclonal T cell-stimulating anti-CD3 (αCD3) hybrid antibody (scFv) was developed and used in combination with tavo (IT-tavo-αCD3-EP) to broaden the scope and depth of the T cell response. We previously demonstrated that membrane expression of αCD3 on neoplastic and stromal cells could activate CD3+ TIL, driving enhanced proliferation and cytotoxicity in a B16-OVA murine model. Here, using immune profiling of the tumor microenvironment (TME), we have demonstrated that this membrane-bound αCD3 therapeutic can significantly upregulate frequencies of CXCR3+CD8+ T cells and short-lived effector T cells, while reducing PD-1 expression on CD8+ T cells in vivo. Critically, naïve T cells, Treg cells, and exhausted T cells (subsets not typically associated with strong anti-tumor responses) displayed enhanced effector function (IFN-γ and granzyme B release) with engagement of membrane-bound αCD3 and IL-12. Furthermore, we found that this therapeutic approach could equally enhance proliferation of T cells regardless of the affinity for their cognate peptide:MHC, suggesting a TCR independent mechanism. Collectively, these observations demonstrate that IT-tavo-αCD3-EP can mobilize broad subsets of T cells beyond dominant anti-tumor effectors demonstrated. Thus, while enhanced cytolytic function is associated with this therapy, inclusion of additional atypical anti-tumor T cell subsets may also promote reshaping of the TME by production of effector cytokines upon engagement of surface-bound αCD3. Moreover, functional restoration of TIL isolated from a melanoma patient with active clinical progression on anti-PD-1 therapy, was possible with engagement of membrane-bound αCD3 in the presence of IL-12. Collectively, these data continue to support the utility of IT-tavo-αCD3-EP as a promising therapeutic approach for patients with melanoma and other accessible solid tumors. Citation Format: Mia Han, Anandaroop Mukhopadhyay, Bianca Nguyen, Jack Y. Lee, Erica Browning, Jon Salazar, Reneta Hermiz, Lauren Svenson, Chris Baker, Daniel O'Connor, Kellie Malloy, David A. Canton, Christopher G. Twitty. Intratumoral electroporation of plasmid-encoded IL-12 and membrane-bound anti-CD3 increases tumor immunogenicity and augments the function of T cell subsets [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-390.