Innate Immune Sensing in Anti-Tumor Immunity and Cancer Immunotherapy

Abstract Most cancers express tumor antigens that can be recognized by T cells of the host. The fact that cancers that become clinically relevant grow, nonetheless implies that immune escape must occur to allow cancer outgrowth. We have observed two major subsets of human melanoma metastases based on gene expression profiling and confirmatory assays. One subgroup of patients has a T cell-inflamed phenotype that includes expression of chemokines, T cell markers, and a type I interferon (IFN) signature. In contrast, the other major subset lacks this phenotype and appears to display immune "exclusion". The mechanisms of immune escape are likely distinct in these two phenotypes, and therefore the optimal immunotherapeutic interventions necessary to promote clinical responses may be different. The T cell-inflamed tumor microenvironment subset shows the highest expression of negative regulatory factors, including PD-L1, IDO, and FoxP3+ Tregs, and evidence for T cell-intrinsic anergy has also emerged aided by a recently defined functional role of EGR2. In addition, the mechanism of induction of these inhibitory mechanisms has been elucidated-PD-L1 and IDO are induced by IFN-g, and Tregs are largely recruited by the chemokine CCL22, both being produced by activated CD8+ effector T cells. Preclinical experiments have confirmed a critical role for each of these mechanisms in limiting anti-tumor T cell efficacy in vivo, giving candidate treatment strategies for translation back into the clinic. These include anti-PD-1/PD-L1 mAbs, IDO inhibitors, and approaches to deplete CD25+ Tregs and/or reverse anergy. The presence of multiple inhibitory mechanisms in the same tumor microenvironment argues that combination therapies may be advantageous. Preclinical data have indicated synergy between anti-CTLA-4 +/- anti-PD-L1 +/- IDO inhibition. Clinical translation of multiple combination immunotherapies is promising and ongoing. In contrast to the T cell-inflamed melanomas, a new paradigm may be needed to promote de novo inflammation in cases of the non-T cell-infiltrated tumor microenvironment. Natural innate immune sensing of tumors appears to occur via the host STING pathway, type I IFN production, and cross-priming of T cells via CD8a+ dendritic cells. New strategies are being developed to engage or mimic this pathway as a therapeutic endeavor, including STING agonists. A phase I study of intratumoral injection of the first STING agonist is ongoing. As an environmental variable, recent work has implicated the commensal microbiota as a regulator of DC activation status and systemic anti-tumor immunity, and clinical analysis of microbiota sequencing in the context of checkpoint blockade is ongoing. Disclosures Gajewski: Evelo: Patents & Royalties: Patent application; Jounce: Consultancy; Merck: Consultancy, Research Funding; Aduro: Patents & Royalties: Patent application; Incyte: Consultancy, Research Funding; Celldex: Consultancy, Research Funding; BMS: Research Funding; Roche/Genentech: Consultancy, Research Funding; Bayer: Consultancy; Abbvie: Consultancy.

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T Cell Exhaustion and Downregulation of Cytotoxic NK Cells – an Immune Escape Mechanism in Adult Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v124.21.3781.3781 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3781-3781

Author(s):

Eolia Brissot ◽

Sawa Ito ◽

Kit Lu ◽

Carly Cantilena ◽

B. Douglas Smith ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Nk Cells ◽

Research Funding ◽

Immune Escape ◽

Nk Cell ◽

T Cell Exhaustion ◽

Cell Exhaustion ◽

Healthy Donors

Abstract Adult acute lymphoblastic leukemia (ALL) remains a therapeutic challenge with less than 40% long term survival. There is growing evidence that malignant diseases exert an immune editing effect which blocks antitumor immunity and permits tumor growth through immune evasion. Such tumor escape represents an obstacle for anticancer immunotherapy. In ALL such immune escape mechanisms are not well characterized. We therefore profiled cellular immunity in ALL, by characterizing the subsets of T cells, regulatory T cells (Treg), natural killers (NK) cells and γd T cells, using various functional markers including T cell exhaustion and NK cell activating or inhibitory molecules. Forty ALL patients were included in the study. The median age was 39 y (range, 18-75). Thirty-six presented with B-lineage ALL and 4 with T-lineage ALL. Mononuclear cells were isolated from blood (n=19) or bone marrow (n=21) at the onset of leukemia or at relapse. The median infiltration of blasts was 85% (range 24-96%). Healthy donor peripheral blood (n=12) and bone marrow (n=9), from age and gender matched population, were simultaneously analyzed as controls. Extra-and intra cellular staining were performed using using antibodies directed against CD3, CD4, CD8, CD45, CD45, CD45RA, CD45RO, CCR7, CD95, CD27, CD19, CD14, CD127, CD25, Foxp3, Helios, αβTCR, HLA-DR, CD117, CD20, CD10, CD22, CD34, LAG3, PD1, PDL1, CD56, NKG2A, NKG2C, NKG2D, KIR2DL1, KIR2DL3, CD57, CD33, CD11b, CD15, CD38 and CD24. Data were acquired on a BD LSRFORTESSA flow cytometer. The expression of programmed cell death 1 (PD-1, CD279) receptor on CD8+T cells was significantly increased in blood and bone marrow of ALL patients compared to healthy donors (p<0.0001 and p=0.004, respectively) (Fig. 1). Focusing on the different subsets, CD8+ effector memory T cells significantly over-expressed PD-1 in blood and bone marrow of ALL patients compared to healthy donors (p=0.008 and p=0.04, respectively). Moreover, there was a significant positive correlation between PD-1 expression on CD8+ effector memory T cells and blast infiltration (R2=0.23, 95%CI 0.026-0.76, p=0.04). Expression of the co-inhibitory receptor lymphocyte-activation gene 3 (LAG-3, CD223) was similar in ALL patients compared to healthy donors. A significantly higher frequency of T regulators (CD25+, CD127 low, Foxp3+) was found in bone marrow microenvironment in ALL patients (4.3% versus 1.6%, p=0.02). Concerning γd T cells, frequency was similar in blood and bone marrow of ALL patients compared with healthy donors. There was a significantly lower frequency of CD56dimNKG2A+KIR-CD57- (p=0.02) in the bone marrow of ALL patients indicating a maturation arrest. Interestingly, expression of the activating receptor NKG2D which plays an important role in triggering the NK cell–mediated tumor cell lysis was significantly reduced in NK cells of ALL patients while no difference in NK cell expression of NKG2C was found(Fig. 2). Adult patients with ALL show evidence of immune-editing of T cells and NK cells. This global immunosuppressive mechanism may contribute to the eventual escape of ALL from immune control. PD-1, overexpression, described in acute myeloid leukemia and chronic myeloid leukemia has been implicated in T-cell exhaustion and subsequent tumor immune evasion. Our data suggests similar immune escape mechanisms pertain in ALL. Effective antileukemia immunotherapy will require targeting one or more of these immunosuppressive pathways to achieve optimum results. Disclosures Fathi: Seattle Genetics, Inc.: Consultancy, Research Funding; Takeda pharmaceuticals International Co.: Research Funding; Exelixis: Research Funding; Ariad: Consultancy.

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Single-Cell Multi-Omic Analysis Uncovers Comprised Immune Function and Primary Resistance Mechanism in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2021-149022 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 378-378

Author(s):

Jianbiao Zhou ◽

Jonathan Adam Scolnick ◽

Stacy Xu ◽

Melissa Ooi ◽

Priscella Shirley Chia ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Nk Cells ◽

Single Cell ◽

Research Funding ◽

Immune Escape ◽

Nk Cell ◽

Primary Resistance ◽

Bm Niche

Abstract Background: Approximately 20% of AML patients do not respond to induction chemotherapy (primary resistance) and 40-60% of patients develop secondary resistance, eventually leading to relapse followed by refractory disease (RR-AML). Diversified molecular mechanisms have been proposed for drug resistance and RR phenotype. However, we still cannot predict when relapse will occur, nor which patients will become resistant to therapy. Single-cell multi-omic (ScMo) profiling may provide new insights into our understanding of hematopoietic stem cell (HSC) differentiation trajectories, tumor heterogeneity and clonal evolution. Here we applied ScMo to profile bone marrow (BM) from AML patients and healthy controls. Methods: AML samples were collected at diagnosis with institutional IRB approval. Cells were stained with a panel of 62 DNA barcoded antibodies and 10x Genomics Single Cell 3' Library Kit v3 was used to generate ScMo data. After normalization, clusters were identified using Uniform Manifold Approximation and Projection (UMAP) and annotated using MapCell (Koh and Hoon, 2019). We analyzed 23,933 cells from 4 adult AML BM samples, and 39,522 cells from 2 healthy adults and 3 sorted CD34+ normal BM samples. Gene set enrichment analysis (GSEA) and Enrichr program were used to examine underlying pathways among differentially expressed genes between healthy and AML samples. Results: We identified 16 cell types between the AML and normal samples (Fig 1a) amongst 45 clusters in the UMAP projection (Fig 1b). Comparative analysis of the T cell clusters in AML samples with healthy BM cells identified an "AML T-cell signature" with over-expression of genes such as granzymes, NK/T cell markers, chemokine and cytokine, proteinase and proteinase inhibitor (Fig 2a). Among them, IL32 is known to be involved in activation-induced cell death in T cells and has immunosuppressive role, while CD8+ GZMB+ and CD8+ GZMK+ cells are considered as dysfunctional or pre-dysfunctional T cells. Indeed, Enrichr analysis showed the top rank of phenotype term - "decreased cytotoxic T cell cytolysis". We next examined whether NK cells, are similarly dysfunctional in the AML ecosystem. The "AML NK cell signature" includes Fc Fragment family, IFN-stimulated genes (ISGs), the effector protein-encoding genes and other genes when compared to normal NK cells (Fig 2b). GSEA analysis revealed "PD-1 signalling" among the top 5 ranked pathways in AML-NK cells, though no increase in PD-1 protein nor PDCD1 gene were identified in these cells. Inhibitory receptor CD160 was expressed higher in AML samples along with exhaustion (dysfunction) associated genes TIGIT, PRF1 and GZMB (Fig 2c). Enrichr analysis uncovered enrichment of "abnormal NK cell physiology and "impaired natural killer cell mediated cytotoxicity". Similarly, the "AML monocyte signature" was significantly enriched with genes in "Tumor Infiltrating Macrophages in Cancer Progression and Immune Escape" and "Myeloid Derived Suppressor Cells in Cancer Immune Escape". We also analyzed HSPC component in one pair of cytogenetically matched, untreated complete remission (CR) /RR AML pair (Fig 2d). Notably, half of the 10 genes overexpressed in RR-AML, CXCR4, LGALS1, S100A8, S100A9, SRGN (Serglycin), regulate cell-matrix interaction and play pivotal roles in leukemic cells homing bone marrow niche. The first 4 of these genes have been demonstrated as prognostic indicators of poor survival and associated with chemo-resistance and anti-apoptotic function. Furthermore, single-cell trajectory analysis of this CR/RR pair illustrated a change in differentiation pattern of HSPCs in CR-AML to monocytes in RR-AML. We are currently analyzing more AML samples to validate these findings. Conclusions: Our ScMo analysis demonstrates that the immune cells are systematically reprogrammed and functionally comprised in the AML ecosystem. Upregulation of BM niche factors could be the underlying mechanism for RR-AML. Thus, reversing the inhibited immune system is an important strategy for AML therapy and targeting leukemic cell-BM niche interaction should be considered for cases with high expression of these molecules on AML HSPCs. Note: J.Z. and J.A.S. share co-first authorship. Figure 1 Figure 1. Disclosures Scolnick: Proteona Pte Ltd: Current holder of individual stocks in a privately-held company. Xu: Proteona Pte Ltd: Current Employment. Ooi: Jansen: Honoraria; Teva Pharmaceuticals: Honoraria; GSK: Honoraria; Abbvie: Honoraria; Amgen: Honoraria. Lovci: Proteona Pte Ltd: Current Employment. Chng: Aslan: Research Funding; Takeda: Honoraria; Johnson & Johnson: Honoraria, Research Funding; BMS/Celgene: Honoraria, Research Funding; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Antengene: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; AbbVie: Honoraria.

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Phase I Experience with a Bi-Specific CAR Targeting CD19 and CD22 in Adults with B-Cell Malignancies

Blood ◽

10.1182/blood-2018-99-110142 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 490-490 ◽

Cited By ~ 24

Author(s):

Nasheed Hossain ◽

Bita Sahaf ◽

Matthew Abramian ◽

Jay Y. Spiegel ◽

Katie Kong ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Phase I ◽

B Cell ◽

Single Cell ◽

Research Funding ◽

Immune Escape ◽

Car T Cells ◽

Cytokine Analysis ◽

Car T

Abstract Autologous CD19 directed CAR T-cell therapy has response rates of >70% in adult acute lymphoblastic leukemia (ALL) and >40% in adult diffuse large B cell lymphoma (DLBCL). Large trials (ZUMA-1/JULIET/TRANSCEND) have highlighted that many patients fail to achieve durable responses. Several groups have reported on the phenomenon of CD19 immune escape as a cause (Grupp et al, NEJM 2013, Neelapu et al, NEJM 2017) and the NIH Pediatric Oncology Branch has shown CD22 as an alternative target (Fry et al, Nat Med. 2018). We developed a bi-specific CAR construct targeting CD19 & CD22 with intracellular signaling domains incorporating 4-1BB and CD3ζ (CD19/CD22.BB.z) to overcome CD19 immune escape. Here, we present our Phase I experience with this bi-specific CAR in adults. This is a single institution phase I dose escalation study enrolling patients Age ≥ 18 years with relapsed/refractory B-ALL or DLBCL after standard therapies. Primary aim is to determine feasibility of manufacturing the bi-specific CAR and safety at three dose levels (1 x 106 CAR T cells/kg, 3 x 106 CAR T cells/kg, 1 x 107 CAR T cells/kg). Clinical response was evaluated as a secondary endpoint utilizing standard response criteria for ALL and DLBCL. All patients underwent lymphodepletion with cyclophosphamide (500mg/m2 daily x3 doses) and fludarabine (30mg/m2 daily x 3 doses) followed by CAR infusion two days later. Patients were assessed at pre-defined time-points (Day 28, Month 3, 6, 9, 12 then every 6-12 months) with correlative assessments including immunophenotyping, single cell RNAseq, CAR qtPCR, serum and single cell cytokine analysis. Seven adult patients (5 DLBCL, 2 ALL), aged 35 - 75 years have been enrolled and 6 treated, all at dose Level 1 [Table 1]. The first 3 patients received freshly harvested cells and the remaining received cryopreserved cells (1 patient treated twice received initial fresh then cryopreserved product). None received systemic bridging therapy before CAR T infusion. Six patients developed reversible cytokine release syndrome (CRS,4 with Grade 1, 2 with Grade 2), onset between Day 1 to 13, and 2 patients received tocilizumab & systemic steroids. Three patients developed neurotoxicity (1 with grade 2, Day 8-11 and the others grade 1) with grade 2 neurotoxicity managed with steroids. Four patients required growth factor support beyond Day 28 and all treated patients show persistent B-cell aplasia. Two patients achieved CR: an ALL patient with disease in bone marrow/blood/CNS was MRD negative at day 28 & 60; a 75yo DLBCL patient achieved PR at day 28 and CR at month 3. Three others have ongoing PR and one died of progressive disease after initial PR at Day 28. A patient with PD at Day 28 subsequently treated with radiation and 2-months of revlimid/rituximab, now has no detectable disease 6 months post CAR-T. One patient with initial 6-month PR received a second infusion due to PD, did not develop CRS or CRES with 2nd infusion and has SD at Day 28 Notably, the patient experienced a lack of CAR-T expansion with the second infusion, raising the possibility of an immunogenic response to the CAR-T cell infusion. Flow analysis of all patients' peripheral blood showed CAR expansion peaked at median Day 13 (range Day 10-20) and CARs remained detectable [Figure 1]. Multi-parametric CyTOF phenotyping of the CAR19-22 products showed significant numbers of transduced CAR-T memory stem cells (phenotype: CD3+CD8+CD45RA+CD127+CD27+CCR7+). Single cell cytokine secretion analysis (Isoplexis,Rossi et al Blood 2018) revealed high polyfunctional strength index (PSI) in both CD4+ and CD8+ cell subsets in each patient's pre-infusion CAR product that reflected phenotypic expansion in patients. Additional correlative studies, including cytokine analysis, qtPCR based CAR quantification and CyTOF phenotypic analysis of the CAR-T cells will be presented. This first adult phase I trial of bi-specific CAR targeting CD19 & CD22 shows low toxicity with promising efficacy including achievement of CR in adult DLBCL and ALL patients. We have escalated dose to 3x 106 CAR T cells/kg and an expansion study of 60 patients will follow. CAR-T cells expanded within the first 20 days and continue to be detectable through 6 months. Disclosures Muffly: Shire Pharmaceuticals: Research Funding; Adaptive Biotechnologies: Research Funding. Miklos:Janssen: Consultancy, Research Funding; Genentech: Research Funding; Pharmacyclics - Abbot: Consultancy, Research Funding; Kite - Gilead: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

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Eliciting Anti-Tumor T Cell Immunity in Chronic Lymphocytic Leukemia (CLL) with PD-L1/PD-1 Blockade Is Enhanced By Avadomide Immunotherapy through the Triggering of Immunogenic Interferon Signaling

Blood ◽

10.1182/blood-2018-99-111605 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 237-237 ◽

Cited By ~ 1

Author(s):

Nikolaos Ioannou ◽

Patrick R. Hagner ◽

Fadi Towfic ◽

Anita K. Gandhi ◽

Kostas Stamatopoulos ◽

...

Keyword(s):

T Cells ◽

Cell Migration ◽

T Cell ◽

Research Funding ◽

Type I ◽

Combination Immunotherapy ◽

Employment Equity ◽

Cell Immunity ◽

Treatment Naïve ◽

Equity Ownership

Abstract Immune checkpoint blockade has demonstrated potential to reactivate anti-tumor immunity and regress tumors. However, response rates in B-cell non-Hodgkin lymphoma patients have been lower compared to Hodgkin lymphoma, with no activity reported in a recent trial of anti-PD-1 immunotherapy in relapsed CLL. This suggests that certain lymphoma subtypes may harbor non-immunogenic tumor microenvironments (TME). Our preliminary studies revealed that avadomide (CC-122), a cereblon modulator, can enhance T cell immune synapse signaling with autologous CLL cells, resulting in a concomitant increase in PD-1/PD-L1 expression at repaired synapses, indicating that avadomide may represent a complementary treatment partner for checkpoint inhibition. Here, we have extended our pre-clinical studies to investigate how these immunotherapy drugs alter the function and gene signatures of previously exhausted T cells from treatment naïve CLL patients (representing disease heterogeneity). Cytotoxicity assays revealed that treating primary T cells and autologous CLL cells with avadomide (1 μM, 48h) activated anti-tumor T cell killing function (P<.01, n=16), while combining avadomide with anti-PD-1 or anti-PD-L1 treatment significantly enhanced (P<.01) T cell killing function compared to these immunotherapies alone. Notably, anti-PD-1 monotherapy induced comparatively modest improvements of effector function. We next performed RNA sequencing on highly purified T cells from treatment naïve CLL patients, representing extremes of prognosis (n=6 good and n=6 poor including disease harboring TP53 abnormalities), following 18 h treatment with avadomide (100 nM) or anti-PD-1 (nivolumab) or PD-L1 (durvalumab) alone (10 μg/ml) or in combinations. Differential expression pathway analysis revealed that the top functional gene categories common for all the avadomide and combination treated samples (independent of anti-PD-1 or anti-PD-L1 monotherapy) were related to the response to type I and II IFN signaling, as well as inflammatory/stimulatory cytokine TNF-α, proliferative IL-6/JAK/STAT3, and IL-2/STAT5 responses. Type I IFN drives expression of chemokines CXCL10 and CXCL9, which are linked to enhanced tumor-infiltrated lymphocyte recruitment, and these chemoattractant genes were significantly upregulated in the avadomide and combination treated patient samples. In addition to their immunostimulatory roles, type II IFN (IFN-γ) and chronic type I IFN signaling have been linked to T cell resistance/exhaustion. We detected upregulated PD-L1 transcript in avadomide and combination treated T cells - supporting blockade of this inhibitory ligand. To investigate the ability of avadomide to modulate T cell migration, we performed comparative quantitative time-lapse microscopy analysis of pretreated patient T cells (n=12). These assays revealed that avadomide, as well as anti-PD-1 or anti-PD-L1 monotherapies, significantly enhanced (P<.01) T cell migration, and this response was significantly augmented by combination immunotherapy. Luminex protein analysis and cell migration assays confirmed the transcriptome data, highlighting the ability of avadomide and combination immunotherapy to significantly (P<.01) induce the release of CXCL10 (as well as IFN-γ, IL-2, GM-CSF and IL-8) from patient T cells, thereby attracting autologous T cells (CXCR3+). Avadomide as well as its pairing with anti-PD-L1 were also found to increase the proliferation of patient T cells (P<.01, n=6), particularly CD8+ T cells. Patient-derived xenograft models demonstrate that the therapeutic treatment of established tumors (3 weeks post-engraftment) with avadomide treatment (0.5 mg/kg) can activate anti-tumor T cells and significantly reduce disease volume (P<.01) that was associated with increased infiltration of PD-L1+ CD8+ T cells. Pairing avadomide with anti-PD-L1 durvalumab led to maximal activation of anti-tumor T cell immunity. In conclusion, our findings support the concept that PD-L1/PD-1 blockade in CLL could be enhanced when combined with avadomide through the promotion of immunogenic IFN signaling that enhances T cell infiltration and function. We believe these data support the rationale for combination immunotherapy that could convert a non-immunogenic TME into a 'hot' T cell inflamed TME which would be sensitive to checkpoint blockade. Disclosures Hagner: Celgene Corporation: Employment, Equity Ownership. Towfic:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corporation: Employment, Equity Ownership. Stamatopoulos:Abbvie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Patten:L Hoffman La Roche: Honoraria, Research Funding; Gilead Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel, Research Funding; AbbVie Inc: Honoraria, Other: travel; Janssen: Honoraria, Other: travel. Vardi:Gilead: Research Funding; Janssen: Honoraria. Ramsay:MedImmune: Research Funding; Roche Glycart AG: Research Funding; Celgene Corporation: Research Funding.

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Pharmacologic Inhibition of SUMO-Activating Enzyme (SAE) with TAK-981 Augments Interferon Signaling and Regulates T Cell Differentiation in Ex Vivo studies of Chronic Lymphocytic Leukemia (CLL)

Blood ◽

10.1182/blood-2019-127539 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1760-1760 ◽

Cited By ~ 1

Author(s):

Vi Lam ◽

Scott R Best ◽

Nur Bruss ◽

Tingting Liu ◽

Taylor Hashiguchi Rowland ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Biology ◽

Research Funding ◽

Ex Vivo ◽

Lymphocytic Leukemia ◽

Interferon Response ◽

Type I ◽

Lymphoid Malignancies ◽

T Cell Biology

Introduction: Novel therapies widely used in treatment of CLL and lymphoma, e.g. Bruton tyrosine kinase and phoshpoinotiside-3 kinase inhibitors, have complex immunomodulatory effects. Detailed understanding of the immune-modulatory effects of novel agents will help battle toxicities and inform the development of combination approaches in CLL and other lymphoid malignancies. Small ubiquitin-like modifier (SUMO) family proteins regulate target protein function by post-translational modification. Sumoylation regulates a variety of cellular processes, including nuclear import/export, transcriptional regulation, protein stability and cell cycle progression. While sumoylation has been shown to be deregulated in cancer and may contribute to carcinogenesis, it has also been implicated in T cell biology and function. Importantly, sumoylation may regulate NFκB signaling and PLCγ1-mediated NFAT activation, both indispensable for T-cell activation. Despite this, the role of sumoylation in broad aspects of T cell biology remain largely understudied. TAK-981 is a small molecule SAE inhibitor that forms an irreversible covalent adduct with SUMO molecules, thereby preventing transfer of SUMO from the E1 (SAE) to the E2 (Ubc9) enzyme, leading to a decrease in SUMO-conjugated proteins. Here, we investigated the immunomodulatory effects of TAK-981 in CLL ex vivo. Methods: Peripheral blood mononuclear cells were isolated from patients with CLL and T cells were purified using Dynabeads. TAK-981 was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA). For gene expression analysis, FACS-sorted naïve CD4+ T cells were pre-treated with TAK-981 for 1 hour and then subjected to concurrent T-cell receptor (TCR; αCD3/CD28) stimulation; RNA was harvested 3 or 24 hours after stimulation and analyzed on a Clariom S microarray chip. For polarization assays, FACS-sorted naïve CD4+ T cells were cultured for up to 7 days under Th1/2/17/Treg-polarizing conditions. Results: Protein sumoylation was induced within 15 minutes (SUMO1) and 24 hours (SUMO2/3) of TCR stimulation. Treatment with TAK-981 depleted nearly all polySUMO2/3-modified proteins but had less effect on SUMO1 conjugation in T cells at 24 hours. GSEA and Reactome pathway analysis of gene expression microarray data from TAK-981-treated CD4+ naïve TCR-stimulated T cells demonstrated minimal changes in NFκB- or NFAT-regulated genes. Significantly upregulated genes included those involved in transcriptional initiation/elongation (3 h), type I interferon response genes and PI3K/AKT signaling (24 h). Meanwhile, genes regulating cell cycle transition and DNA damage responses were downregulated. Activation (CD69, CD25, CD40L and HLA-DR) and survival of CD4+ andCD8+ T cell subpopulations was unimpeded by SAE inhibition within the first 24 hours of treatment, but was modestly reduced at 48 and 96 hours. T cell proliferation (CFSE, Edu) was reduced in a dose and time-dependent manner following exposure to TAK-981 (by 27.8% and 60.8% following treatment with 0.05 and 1.0 μM TAK-981 for 72 hours, respectively). Allogeneic T-cell cytotoxicity (using OCI-LY19 and OCI-LY3 lymphoma cells as target cells) was not disrupted by SAE inhibition. Remarkably, TCR-activated CD4+ naïve T cells treated with TAK-981 exhibited increased expression of CD38, a type I/II interferon response molecule [1]. Furthermore, sorted CD4+ naïve T cells showed enhanced IFNγ production, a type II IFN (as analyzed by flow cytometry), and increased TH1 differentiation in both TH1-polarized and non-polarized conditions. By contrast, differentiation of both TH17 and inducible regulatory T cells (iTregs) was reduced under the respective polarizing conditions. This was accompanied by diminished IL-2expression within the CD4+ T cell population. Conclusions: Our data suggest that targeting SAE may shift the T cell balance toward healthy immune cell subsets in CLL via induction of type I/II interferon response. TH1 polarization was accompanied by a reduction of immunosuppressive Treg phenotype. These data provide a strong rationale for continued investigation of TAK-981 in CLL and lymphoid malignancies. Bürgler, S., et al., Chronic Lymphocytic Leukemia Cells Express CD38 in Response to Th1 Cell-Derived IFN-γ by a T-bet-Dependent Mechanism. The Journal of Immunology, 2015. 194(2): p. 827-835. Disclosures Huszar: Takeda Pharmaceuticals: Employment, Equity Ownership. Danilov:Verastem Oncology: Consultancy, Other: Travel Reimbursement , Research Funding; Celgene: Consultancy; Aptose Biosciences: Research Funding; Seattle Genetics: Consultancy; Takeda Oncology: Research Funding; MEI: Research Funding; Pharmacyclics: Consultancy; AstraZeneca: Consultancy, Research Funding; Bristol-Meyers Squibb: Research Funding; Abbvie: Consultancy; Genentech: Consultancy, Research Funding; Bayer Oncology: Consultancy, Research Funding; Janssen: Consultancy; Gilead Sciences: Consultancy, Research Funding; TG Therapeutics: Consultancy; Curis: Consultancy.

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Duobody-CD3xCD20 Induces Potent Anti-Tumor Activity in Malignant Lymph Node B Cells from Patients with DLBCL, FL and MCL Ex Vivo, Irrespective of Prior Treatment with CD20 Monoclonal Antibodies

Blood ◽

10.1182/blood-2019-125765 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4066-4066

Author(s):

Hilma J Van Der Horst ◽

A. Vera de Jonge ◽

Ida H Hiemstra ◽

Anne T Gelderloos ◽

Daniella RAI Berry ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Microenvironment ◽

Tumor Cell ◽

Cytotoxic Activity ◽

Board Of Directors ◽

Tumor Cells ◽

T Cell Activation ◽

Research Funding ◽

Advisory Committees

DuoBody-CD3xCD20 (GEN3013) is a novel clinical-stage CD3 bispecific antibody (bsAb) targeting CD20-positive tumor cells. GEN3013 was previously shown to induce potent T cell-mediated cytotoxicity towards B cell Non-Hodgkin lymphoma (B-NHL) cell lines in vitro and in vivo. Here, we investigated the cytotoxic activity of GEN3013 in tumor cells obtained from lymph node (LN) biopsies of B-NHL patients, who were newly diagnosed (ND) or relapsed from/refractory to (RR) treatment regimens containing CD20 monoclonal antibodies. Moreover, we explored whether specific tumor microenvironment characteristics could be associated with sensitivity to GEN3013. To test the intrinsic susceptibility of B-NHL cells to GEN3013, independent of interpatient variation in tumor T cell frequency or activation status, single cell suspensions obtained from LN of B-NHL patients were incubated with GEN3013 in the presence of allogeneic PBMC from a single donor, at an effector to target (E:T) ratio 10:1. GEN3013 (30 ng/mL) induced median tumor cell lysis of 64% in Diffuse Large B Cell Lymphoma (DLBCL, n=14), 69% in Follicular Lymphoma (FL, n=14) and 84% in Mantle Cell Lymphoma (MCL, n=8) samples, with EC50 values ranging from 0.01-3.9 ng/ml. Importantly, cytotoxic activity of GEN3013 was comparable in ND (n=24) and RR (n=12) patients (Figure 1). In these assays considerable heterogeneity in T cell activation, as assessed by expression of CD25, CD69 and granzyme B release, was observed. Furthermore, high expression of T cell activation markers was not always associated with high levels of GEN3013 cytotoxic activity, suggesting tumor-intrinsic resistance mechanisms. In parallel, in all B-NHL samples GEN3013-mediated cytotoxicity was assessed without the addition of allogeneic PBMCs, thus purely relying on T cells present in the LN biopsy. In this setting, median tumor cell lysis was lower; 18% in DLBCL (range 0-46%), 17% in FL (range 0-46%) and 0% in MCL (range 0-11%), but strongly correlated with the number of T cells present in the single cell suspensions. Analysis of the tumor microenvironment by 7 color immunohistopathology of matched FFPE-embedded tumor biopsies (n=24), confirmed that the T cell frequency in the tumor biopsies was the major determinant of GEN3013 cytotoxic activity in DLBCL, FL and MCL. Moreover, experiments using (MACS) purified T cells from 4 DLBCL and 5 FL LN biopsies demonstrated that the intrinsic capacity of tumor LN T cells to induce GEN3013 mediated cytotoxicity was comparable to healthy donor T cells. Detailed tumor microenvironment analysis based on 7 color immunohistopathology staining, including relative frequency and spatial distribution of CD4 and CD8 T cells and macrophages, as well as the T cell activation status, in relation to sensitivity to GEN3013 mediated tumor cell lysis is ongoing and results will be presented. In conclusion, GEN3013 induced potent cytotoxicity in tumor cells of DLBCL, FL and MCL patients ex vivo, irrespective of prior treatment with CD20 monoclonal antibodies. Autologous T-cells at the tumor site were able to mediate GEN3013-induced cytotoxicity, and cytotoxic activity was enhanced in presence of PBMCs suggesting that optimal tumor cell kill by GEN3013 is dependent on T-cells in the tumor microenvironment. The cytotoxic capacity of B-NHL patient T cells within the tumor microenvironment was comparable to healthy donor peripheral blood T cells, emphasizing the therapeutic potential of CD3 bsAb in B-NHL. A First-in-Human trial to assess the safety and preliminary efficacy of GEN3013 in B-NHL patients is currently ongoing (NCT03625037). Figure 1 Cytotoxic activity induced by GEN3013 compared to CD3xcontrol bsAb (both 30ng/ml) towards tumor cells obtained from lymph node (LN) biopsies of newly diagnosed (ND) versus relapse or refractory (RR) DLBCL, FL and MCL patients. GEN3013 achieved comparable lysis in ND versus RR patients (Mann-Whitney U test; not significant). Error bars represent median ± interquartile range. Figure 1 Disclosures Van Der Horst: Genmab: Other: Financial Support. Hiemstra:Genmab: Employment, Equity Ownership, Other: Warrants. de Jong:Genmab: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Chamuleau:Genmab: Research Funding. Zweegman:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding. Breij:Genmab: Employment, Other: Warrants. Roemer:Genmab: Research Funding. Mutis:Celgene: Research Funding; Janssen Research and Development: Research Funding; Onkimmune: Research Funding; Genmab: Research Funding.

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Integrative Immunogenomic Characterization of Diffuse Large B-Cell Lymphoma (DLBCL) Identifies Four Molecular Subtypes with Distinct Immune Landscapes

Blood ◽

10.1182/blood-2019-131562 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 924-924

Author(s):

Michael Leukam ◽

James Godfrey ◽

Sravya Tumuluru ◽

Girish Venkataraman ◽

Sonali M. Smith ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Research Funding ◽

Immune Escape ◽

M2 Macrophages ◽

T Helper ◽

Gene Set ◽

M1 And M2 Macrophages

Background: Effective treatment of relapsed/refractory (r/r) DLBCL remains a major unmet need. Checkpoint blockade therapy (CBT) leads to durable responses in a small subset of r/r DLBCL patients, but limited understanding of predictive biomarkers and characteristics of host immune responses have slowed development of DLBCL immunotherapy. We previously described a subset of "T-cell inflamed" DLBCLs marked by PD-L1 gene alterations and increased likelihood of response to CBT. In this study, we aimed to identify and group gene expression patterns associated with immune features in a large number of DLBCL cases available in published datasets. We investigate the immunogenomic features of each group and corroborate findings in primary DLBCLs. Methods: Gene sets reflecting a broad array of activation states or subtypes of tumor-infiltrating immune cells were selected from previous studies (n = 143). Expression by case was scored for each set by applying gene set variation analysis (GSVA) to previously published DLBCL bulk RNAseq profiles (n = 1189). The resulting score matrix was reduced with principal component analysis (PCA); the first 10 components were used to hierarchically cluster each case into related groups. Immune cell fractions were estimated from RNAseq counts via deconvolution analysis. Differentially expressed genes (DEG) for each cluster were identified by false discovery rate (FDR) < 0.05 and log2 fold change of > 1.5. The cytolytic gene expression (CYT) score, associated with T-cell immunity against solid tumors, was computed for each case. Protein-coding mutations found in ≥ 5% of cases by whole exome sequencing analysis were filtered for driver mutations (MutSig2CV q-value < 0.1). PD-L1 gene amplification status was determined where copy number array data were available (n = 471). RNAseq (Illumina HiSeq 2000 platform) was performed on 24 fixed and embedded treatment-naive DLBCL tumors from an institutional biorepository. GSVA scores were projected onto the PCA and clusters assigned. CD4+ and CD8+ T-cells per high-power field (HPF) were assessed by immunohistochemistry (IHC). Results: Four clusters of immune gene set expression were identified in existing DLBCL datasets, termed "inflamed", "intermediate-M", "intermediate-T" and "cold". There is no association of any cluster with a difference in overall survival or enrichment in a cell of origin subtype. The inflamed cluster has the highest mean CYT score (Fig 1A, p < 0.001) and highest deconvolution-estimated fraction of CD8+ T-cells, M1 and M2 macrophages, dendritic cells, T-helper 1, and T-helper 2 cells (Fig 1C-E, p < 0.001). Significantly upregulated DEGs include CXCL9, CXCL10, CCL8, and CXCR6, which have been associated with a T-cell inflamed phenotype in solid tumors. Immune escape mechanisms in the inflamed cluster are suggested by upregulated DEGs of VSIG4 and IDO1, and significant enrichment for PD-L1 gene amplifications compared to the cold cluster (Fig 1B, 8.6% vs 1%, p = 0.01). The cold cluster has the lowest mean CYT score (p < 0.001), and lowest deconvolution scores for CD8+ T-cells, M1 and M2 macrophages, and dendritic cells (p < 0.001). The cold cluster harbors more mutations in MYD88 (27%),TMSB4X (11.6%), and FOXO1 (8%) and fewer SOCS1 (7%) mutations than other clusters (FDR < 0.25). Intermediate-T and intermediate-M clusters share mid-range values of estimated CD8+ T cells and CYT scores, but intermediate-M contained more frequent PD-L1 amplifications (Fig 1B, 9% vs 0.9%, p = 0.02), lower estimated Th1 fraction (p < 0.001), and higher estimated total macrophages (p < 0.001) than intermediate-T. Representatives of each cluster were identified in primary DLBCL tumors (n = 24). Mean CD4+ T-cell count per HPF was higher in inflamed cluster DLBCLs compared to cold (45.1 vs 10.1, p = 0.032). A non-significant increase in CD8+ cells was also seen (21.3 vs 14.2 per HPF). Conclusion: In this first comprehensive immunogenomic study of DLBCL, we define differences in host immune response by gene set expression, associate oncogenic mutations with immune exclusion, and discover expression of a number of immune escape genes in inflamed cases. Primary samples analyzed to date support the immune response patterns found by computational analysis. A greater understanding of heterogeneity in host response to DLBCL may help identify subsets of DLBCLs with inherent vulnerability to CBT and other immunotherapies. Disclosures Smith: Portola Pharmaceuticals: Research Funding. Kline:Merck: Honoraria; Merck: Research Funding.

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Chromatin State and Immunotherapy

Blood ◽

10.1182/blood-2018-99-109432 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. SCI-16-SCI-16

Author(s):

W. Nicholas Haining

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Microenvironment ◽

Viral Infection ◽

Cd8 T Cells ◽

Research Funding ◽

Checkpoint Blockade ◽

Chronic Viral Infection ◽

T Cell Exhaustion ◽

Cell Exhaustion

Abstract The functional impairment of T cell-mediated immunity within the tumor microenvironment (TME) is a defining feature of many cancers. Checkpoint blockade therapy seeks to reinvigorate T cell responses by targeting inhibitory receptors such as PD-1, which are upregulated by dysfunctional TILs. However, the fundamental mechanisms underlying T cell dysfunction in the TME remain poorly understood, as are the mechanisms by which checkpoint blockade overcomes this dysfunction. Initial studies of dysfunctional CD8+ T cells in both human and mouse tumors suggested that they share features of T cell exhaustion, including co-inhibitory receptor upregulation and defects in cytokine production. However, more recent studies have suggested that TIL dysfunction is a unique state that is distinct from T cell exhaustion. Here we show that anti-PD-1 therapy acts on a specific subpopulation of CD8+ tumor-infiltrating lymphocytes (TILs) in melanoma mouse models as well as patients with melanoma. We find that dysfunctional CD8+ TILs possess canonical epigenetic and transcriptional features of T cell exhaustion, mirroring those seen in chronic viral infection. Similar to chronic viral infection, exhausted CD8+ TILs contain a subpopulation of "stem-like exhausted" T cells that have a distinct regulatory state. Stem-like exhausted TILs also have critical functional attributes that are not shared by the majority "terminally exhausted" TILs: they retain more polyfunctionality, persist following transfer into tumor-bearing mice, and differentiate to repopulate terminally exhausted TILs in the TME. As a result, stem-like exhausted CD8+ TILs are better able to control tumor growth than terminally exhausted cells. Stem-like exhausted, but not terminally exhausted, CD8+ TILs can respond to anti-PD-1 therapy without reversion of their exhausted epigenetic state. CD8+ T cells with a stem-like exhausted phenotype can be found in human melanoma samples and patients with a higher fraction of this subpopulation in their tumors have a significantly longer duration of response to combination checkpoint blockade therapy. Responsiveness to checkpoint blockade is therefore restricted to a subpopulation of exhausted TILs that retain specific functional properties which enable them to control tumors. Approaches to expand stem-like exhausted CD8+ T cells in the tumor microenvironment may be an important component of improving checkpoint blockade response. Disclosures Haining: Rheos Medicines: Consultancy; Iomx Therapeutics: Consultancy; Third Rock Ventures: Consultancy; Roche: Research Funding; Calico: Research Funding; Novartis: Research Funding; Tango Therapeutics: Consultancy, Equity Ownership.

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Chromatin Accessibility Profiling to Increase Diagnostic Accuracy and Refine Cell-of-Origin Classification of Mature T-Cell Lymphomas

Blood ◽

10.1182/blood-2021-150034 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 809-809

Author(s):

Edith Julia ◽

Sylvain Mareschal ◽

Amel Chebel ◽

Camille Golfier ◽

Tony Andreas Müller ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Research Funding ◽

Single Cells ◽

Patent Application ◽

Chromatin Accessibility ◽

Open Chromatin ◽

Cell Of Origin ◽

T Cell Lymphomas ◽

Origin Identification

Abstract Background: Mature T-cell lymphomas and leukemias (MTCL) are heterogeneous diseases with dismal prognosis. Differentiating between the numerous entities requires specialized pathology expertise and studies show up to 20% change in diagnosis after expert review of cases (Laurent, JCO, 2017). Assay for transposase accessible chromatin sequencing (ATAC-seq) is a simple technique to profile open chromatin regions (OCR) proven to be highly discriminant for cell-of-origin identification regardless of cell activation status (Shih, Cell, 2016). We applied ATAC-seq to MTCL in order to explore the epigenetic landscape of these diverse entities, compared them to normal T-cell subtypes and built a predictive model to help diagnosis. Method: Ten-thousand FACS-sorted single cells from primary MTCL samples and 50µm section of frozen tumoral tissue from the TENOMIC French T-cell Lymphoma Consortium were processed according to the previously published FAST-ATAC and OMNI-ATAC protocols respectively (Corces, Nat Genetics, 2016 & Nat. Methods, 2017). Concurrently we applied FAST ATAC to different normal T- and NK-cell subsets sorted from healthy donor PBMC or lymph node suspensions. Sequencing data were processed by an adapted version of ENCODE ATAC-seq pipeline. Matrix of insertion events in peaks by sample was obtained, normalized and most variant peaks were selected for UMAP projection. Results: In total, 678 normal and tumoral samples were sequenced to provide a comprehensive landscape of chromatin accessibility in MTCL. Epigenetic profiling by ATAC-seq of FACS-sorted tumoral samples resulted in a complete segregation of the known MTCL entities (AITL, TFH-PTCL, ALK+ and ALK- ALCL, HSTL, CTCL, ATLL, LGL and T-PLL). Most PTCL-NOS (13/17) clustered with a pre-defined MTCL subtype (mainly AITL/TFH-phenotype PTCL, CTCL and lymphomas exhibiting cytotoxic features). All but one discordant diagnosis between pathology and ATAC-seq (1/11) led to revised diagnosis after pathology review. Unsupervised clustering of normal NK- and T-cell subtypes (N=49) and sorted tumoral lymphoma cells (N=104) confirmed that AITL derive from TFH cells. HSTL and LGL closely segregated with NK- and gamma-delta T cells, in line with their known innate-like phenotype. Surprisingly, the cell-of-origin of T-PLL seems to be naïve T cells despite the known expression of central memory markers on leukemic cells. Beyond epigenetic classification, background reads from ATAC-seq profiles were used to detect copy number variation (CNV), such as isochromosome 7q in HSTL. In addition, HTLV1 and EBV viral sequence detection in ATAC-seq reads strengthened identification of ATLL and NKTCL cases. Finally, using unsupervised deconvolution approaches, we were able to discriminate different MTCL subtypes from 223 processed bulk frozen samples. All known MTCL subtypes were differentiated (AITL/PTCL-TFH, HSTL, NKTCL, ATLL, ALK- and ALK+ ALCL, MEITL, EATL). A subgroup of PTCL-NOS harboring GATA3 OCRs and a distinctly high CNV number was isolated that might correspond to previously described PTCL-GATA3 subtypes (Iqbal, Blood, 2019). A random forest model was trained to predict diagnosis based on chromatin-accessibility clusters defined in the discovery cohort of patients. The model showed accurate prediction performance by cross-validation. External validation on 172 samples collected from 5 tertiary care centers will be presented at the meeting. Conclusion: ATAC-seq is a fast and cost-effective technique to help and refine MTCL pathological classification and allows for putative cell-of-origin identification in lymphoma. Training of a machine learning model to predict MTCL entity diagnosis based on ATAC-seq analysis of fresh or frozen samples shows promising results. Figure 1 Figure 1. Disclosures Sibon: Janssen: Consultancy; Abbvie: Consultancy; iQone: Consultancy; Takeda: Consultancy; Roche: Consultancy. Drieux: Genexpath: Patents & Royalties: The author is a potential inventor on a patent application for the LymphoSign, which has been licensed for by Genexpath Patents & Royalties.. Ruminy: Genexpath: Patents & Royalties: The author is a potential inventor on a patent application for the LymphoSign, which has been licensed for by Genexpath Patents & Royalties. . Salles: Takeda: Consultancy; Velosbio: Consultancy; Ipsen: Consultancy; Allogene: Consultancy; Miltneiy: Consultancy; Genentech/Roche: Consultancy; Genmab: Consultancy; Janssen: Consultancy; Loxo: Consultancy; Kite/Gilead: Consultancy; Regeneron: Consultancy, Honoraria; Morphosys: Consultancy, Honoraria; Novartis: Consultancy; Incyte: Consultancy; Rapt: Consultancy; Epizyme: Consultancy, Honoraria; Debiopharm: Consultancy; BMS/Celgene: Consultancy; Beigene: Consultancy; Abbvie: Consultancy, Honoraria; Bayer: Honoraria. Gaulard: Alderaan: Research Funding; Sanofi: Research Funding; Innate Pharma: Research Funding; Gilead: Consultancy; Takeda: Consultancy, Honoraria. Bachy: Kite, a Gilead Company: Honoraria; Novartis: Honoraria; Daiishi: Research Funding; Roche: Consultancy; Takeda: Consultancy; Incyte: Consultancy.

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