Modulation of the IL-6/STAT3 Signaling Axis in CD4+ T Cells As a Potential Immune Mechanism of Action of Azacytidine in High-Risk Myelodysplastic Syndromes

Azacytidine (AZA), the mainstay of therapy in high risk Myelodysplastic syndromes (HR-MDS), affects CD4+ T-cell polarization and function, but the effect of these changes on tumor immunity is unclear. Signal transducer and activator of transcription (STAT) proteins are key regulators of differentiation and polarization of CD4+ T-cells in both health and cancer, but the STAT signaling architecture of CD4+ T-cell subsets in HR-MDS and its modulation by AZA are currently unknown. We applied single-cell phosphospecific flow cytometry in peripheral blood mononuclear cells from 67 HR-MDS patients at various time-points during AZA therapy. Unsupervised clustering of pretreatment STAT signaling profiles (SPs) of CD4+ T-cells revealed three signaling clusters (SCs), mainly differing in the potentiated responses of STAT3 to IL-6 stimulation (IL-6/STAT3 node). Compared to SC#1 and SC#3, patients in SC#2 displayed higher IL-6/STAT3 levels, higher levels of naïve (TN, p=0.05) and lower levels of PD1+ (p=0.04) and central memory ( TCM, p=0.04) CD4+ T-cells, and longer median overall survival (mOS, p=0.028, fig 1A). Moreover, comparisons of single signaling nodes revealed that the IL-6/STAT3 node correlated inversely with PD1+ (p=0.02) and IL-4+ (p=0.04) and positively with naïve CD4+ (p=0.04) and IFNγ+CD8+ T-cells (p=0.01). No other differences in clinicobiologic parameters, CD4+ and CD8+ T-cell subpopulations (FOXP3, IFNγ, IL-4, IL-17, Perforin and Helios) were noted among the 3 SCs and all other single nodes. To assess the effect of AZA on STAT signaling, we clustered the fold fold-change of pre- versus 6-month post-AZA SPs in CD4+ T-cells. Again the IL6/STAT3 node was the only differentiator among the clusters, and, by single node analysis, downregulation of IL6/STAT3 at 6th cycle (n=26) was associated with better response to AZA (p=0.02) and longer mOS (p=0.03), compared to upregulation of the same node (n=22); the latter also accompanied by an increase of IFNγ+CD8+ cells after AZA, (p=0.02, fig 1B). Further supporting a direct and beneficial modulation of the IL-6/STAT3 axis in CD4+ T-cells by AZA, the kinetics of IL-6/STAT3 during AZA therapy revealed a marked downregulation of the former node both at day15 (p=0.04) and cycle 6 after AZA (p=0.04) in responders (n=5), while no changes were observed in non-responders (n=7). We further compared the transcriptional profiles of isolated bone marrow CD4+ T-cells between responders (n=4) and non-responders to AZA (n=4) by RNA-seq, both prior and after AZA. No significant differences in pretreatment gene expression were identified. By contrast, 105 genes were differentially expressed at cycle 6 compared to pretreatment in responders (FDR<0.2) and 145 genes in non-responders. Gene set enrichment (GSEA) revealed a significant downregulation of the IL-6/STAT3 pathway and the overall inflammatory response after AZA in responders, but a marked upregulation in non-responders, confirming the flow cytometry results (fig 1C). To trace the molecular background associated with the differential regulation of the IL-6/STAT3 pathway we constructed mutational profiles by targeted DNA sequencing of 156 genes in blood mononuclear cells. No associations were found between pre or post-treatment IL-6/STAT3 node and mutational burden. By contrast, mutations in RNA splicing and STAG2 correlated with lower (p=0.02) and higher (p=0.017) pretreatment levels of IL6/STAT3, respectively. Notably, all 5 patients with NPM1/DNMT3A double mutation upregulated significantly IL6/STAT3 after AZA (p=0.03, fig 1D). Collectively, our results reveal for the first time that downregulation of the IL-6/STAT3 signaling axis in CD4+ T cells may represent an immune-mediated mechanism of action of AZA. However, the antileukemic activity of IL-6/STAT3LowCD4+ T-cells appears to be independent from modulation of common metrics of tumor immunity, as, paradoxically, the detrimental IL-6/STAT3 upregulation was linked with an expansion of antitumor T cell subsets and decrease of the immunosuppressive ones. The IL-6/STAT3 axis is notoriously pro-tumorigenic and pharmacologic inhibition of various individual modules of this pathway in cancer is under development. Our findings may serve as a guidepost for the ongoing investigation of IL-6/STAT3 axis inhibition as a therapeutic strategy to overcome azacytidine resistance in HR-MDS. Figure 1 Disclosures Vassilakopoulos: Celgene / GenesisPharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; WinMedica: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Pappa:Gilead: Honoraria, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene / GenesisPharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Research Funding. Papaemmanuil:Celgene: Research Funding. Kotsianidis:Celgene: Research Funding.

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Intra-Tumoral CD8+ T-Cells in Follicular Lymphoma Contain Large Clonal Expansions That Are Amenable to Dual-Checkpoint Blockade

Blood ◽

10.1182/blood-2019-130050 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2793-2793 ◽

Cited By ~ 1

Author(s):

Karthik Nath ◽

Soi C. Law ◽

Muhammed B. Sabdia ◽

Lilia Merida De Long ◽

Mohamed Shanavas ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd4 T Cells ◽

Research Funding ◽

Checkpoint Blockade ◽

T Cell Subsets ◽

Advisory Committees ◽

Cell Clones ◽

Tcr Repertoire

Introduction. Intra-tumoral T-cell infiltration is associated with R-CHOP responsiveness in aggressive B-cell lymphoma (Keane, Lancet Haem 2015). These patients also have a broad (i.e. diverse) intra-tumoral T-cell receptor (TCR) repertoire with a ~20% superior survival compared to those with a narrow (i.e. clonal) repertoire after R-CHOP therapy. Here, the major contributor to the TCR clonal expansion were CD8+ T cells (Keane, CCR 2017). Paradoxically, our recent results in Follicular Lymphoma (FL) (Tobin, JCO in press) found that clonal T-cell expansions were markedly enriched in those patients that experienced progression of disease within 24 months (POD24). Given that FL is a histological subtype associated with a tumor microenvironment distinct from DLBCL including numerous CD4+ T-follicular helper cells (TFH), we now expand upon these findings by comparing TCR repertoires across histological subtypes. We then established whether the TCR repertoire in FL is related to differential TCR clonal expansions between different T-cell subsets and immune checkpoints. Finally, the overlap between tissue and blood TCR repertoires was investigated. Methods. Firstly, unbiased, high-throughput TCRβ sequencing (ImmunoSEQ, Adaptive Biotechnologies) was compared in 164 FFPE tissues (12 healthy nodes, 40 FL, 88 DLBCL, and as a comparator tumor known to be sensitive to checkpoint blockade and to have a high neoantigen burden, 24 melanoma tissues). Next, to determine the contribution of individual T-cell subsets to overall clonality, a further 21 fresh de-aggregated/cryopreserved FL tumor samples were FACS sorted into four T-cell groupings: CD8+ cytotoxic T-lymphocytes (CTLs), CD4+ TFH, CD4+ regulatory T-cells (TREGs) and 'other' (non-TFH/TREG) CD4+ T-cells. Flow cytometry quantified the expression of the checkpoints LAG3, TIM3 and PD1. Then, 5 FL paired tissue/blood samples were tested for shared TCR clones. Results. FL exhibited strikingly reduced TCR repertoire clonality (higher diversity) compared to DLBCL, melanoma and healthy lymph nodes (Fig 1A). Analysis of de-aggregated sorted nodal T-cells revealed a more complex TCR repertoire. The outcome measure was median clonality index (CIx ranging from '0' or minimal, to '1' or maximal clonality). Large T-cell clones in FL (CIx=0.12) predominantly resided within the CTL subset (34% all T-cells). By contrast, there was marked T-cell diversity in TFH (CIx=0.04; 27% all T-cells), TREG (CIx=0.02; 7% all T-cells) and 'other' CD4+ T-cells (CIx=0.02; 32% all T-cells) (Fig 1B). The CTL population had a bimodal expression for PD1 (+51%/-49%), a marker in FL that has been shown to remain functionally active unless co-expressed with LAG3 and/or TIM3 (Yang, Oncotarget 2017). These dual-checkpoint expressing CTLs have reduced capacity to produce cytokines or lytic granules (i.e. they are 'exhausted'). Notably, 54% of the PD1+ CTLs co-expressed either LAG3 or TIM3. Put together, these results are consistent with expanded CTL clones that are frequently functionally exhausted. In contrast, TFH, TREG and 'other' CD4+ T-cells had a low expression of LAG3 and TIM3, although PD1 was frequently found (as expected, particularly in the TFH cells). Finally, in paired tissue/blood samples, there was weak overlap between the circulating and intra-tumoral TCR repertoire in CTLs and TFH T-cells. Conclusion. Although FL has a markedly less clonal TCR repertoire compared to DLBCL, melanoma and even healthy nodes, this result is misleading. Detailed analysis on sorted intra-tumoral T-cell subsets in FL revealed large clonal expansions in CTLs, with approximately half of these classified as functionally exhausted (dual-positive for PD1 and LAG3 and/or TIM3), a state potentially amenable to reversal by dual-checkpoint blockade. The explanation for TCR repertoire diversity lies in CD4+ T-cells (representing approximately two-thirds of T-cells, including the large TFH subset). T-cells in blood did not reflect FL tissue T-cell clones, further highlighting the need for sorted intra-tumoral nodal tissues to accurately assess TCR repertoires in FL. Further characterization of the neo-antigenic targets that CTL clones potentially recognize is required. These results have implications for therapeutic vaccine design and selective recruitment of patients for immune checkpoint blockade. Disclosures Keane: MSD: Consultancy; Gilead: Consultancy; Celgene: Consultancy; Roche: Consultancy, Other: Travel Grant; BMS: Research Funding. Gandhi:Roche: Honoraria, Other: Travel Support; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Research Funding.

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Pre-Existing T Cell Subsets Determine Anti-PD1 Blockade Response in Richter's Transformation

Blood ◽

10.1182/blood-2020-138601 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 42-43

Author(s):

Prajish Iyer ◽

Lu Yang ◽

Zhi-Zhang Yang ◽

Charla R. Secreto ◽

Sutapa Sinha ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Gene Signature ◽

T Cell Subsets ◽

Rna Seq ◽

Advisory Committees

Despite recent developments in the therapy of chronic lymphocytic leukemia (CLL), Richter's transformation (RT), an aggressive lymphoma, remains a clinical challenge. Immune checkpoint inhibitor (ICI) therapy has shown promise in selective lymphoma types, however, only 30-40% RT patients respond to anti-PD1 pembrolizumab; while the underlying CLL failed to respond and 10% CLL patients progress rapidly within 2 months of treatment. Studies indicate pre-existing T cells in tumor biopsies are associated with a greater anti-PD1 response, hence we hypothesized that pre-existing T cell subset characteristics and regulation in anti-PD1 responders differed from those who progressed in CLL. We used mass cytometry (CyTOF) to analyze T cell subsets isolated from peripheral blood mononuclear cells (PBMCs) from 19 patients with who received pembrolizumab as a single agent. PBMCs were obtained baseline(pre-therapy) and within 3 months of therapy initiation. Among this cohort, 3 patients had complete or partial response (responders), 2 patients had rapid disease progression (progressors) (Fig. A), and 14 had stable disease (non-responders) within the first 3 months of therapy. CyTOF analysis revealed that Treg subsets in responders as compared with progressors or non-responders (MFI -55 vs.30, p=0.001) at both baseline and post-therapy were increased (Fig. B). This quantitative analysis indicated an existing difference in Tregs and distinct molecular dynamic changes in response to pembrolizumab between responders and progressors. To delineate the T cell characteristics in progressors and responders, we performed single-cell RNA-seq (SC-RNA-seq; 10X Genomics platform) using T (CD3+) cells enriched from PBMCs derived from three patients (1 responder: RS2; 2 progressors: CLL14, CLL17) before and after treatment. A total of ~10000 cells were captured and an average of 1215 genes was detected per cell. Using a clustering approach (Seurat V3.1.5), we identified 7 T cell clusters based on transcriptional signature (Fig.C). Responders had a larger fraction of Tregs (Cluster 5) as compared with progressors (p=0.03, Fig. D), and these Tregs showed an IFN-related gene signature (Fig. E). To determine any changes in the cellular circuitry in Tregs between responders and progressors, we used FOXP3, CD25, and CD127 as markers for Tregs in our SC-RNA-seq data. We saw a greater expression of FOXP3, CD25, CD127, in RS2 in comparison to CLL17 and CLL14. Gene set enrichment analysis (GSEA) revealed the upregulation of genes involved in lymphocyte activation and FOXP3-regulated Treg development-related pathways in the responder's Tregs (Fig.F). Together, the greater expression of genes involved in Treg activation may reduce the suppressive functions of Tregs, which led to the response to anti-PD1 treatment seen in RS2 consistent with Tregs in melanoma. To delineate any state changes in T cells between progressors and responder, we performed trajectory analysis using Monocle (R package tool) and identified enrichment of MYC/TNF/IFNG gene signature in state 1 and an effector T signature in state 3 For RS2 after treatment (p=0.003), indicating pembrolizumab induced proliferative and functional T cell signatures in the responder only. Further, our single-cell results were supported by the T cell receptor (TCR beta) repertoire analysis (Adaptive Biotechnology). As an inverse measure of TCR diversity, productive TCR clonality in CLL14 and CLL17 samples was 0.638 and 0.408 at baseline, respectively. Fifty percent of all peripheral blood T cells were represented by one large TCR clone in CLL14(progressor) suggesting tumor related T-cell clone expansion. In contrast, RS2(responder) contained a profile of diverse T cell clones with a clonality of 0.027 (Fig. H). Pembrolizumab therapy did not change the clonality of the three patients during the treatment course (data not shown). In summary, we identified enriched Treg signatures delineating responders from progressors on pembrolizumab treatment, paradoxical to the current understanding of T cell subsets in solid tumors. However, these data are consistent with the recent observation that the presence of Tregs suggests a better prognosis in Hodgkin lymphoma, Follicular lymphoma, and other hematological malignancies. Figure 1 Disclosures Kay: Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding; Sunesis: Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; MEI Pharma: Research Funding. Ansell:AI Therapeutics: Research Funding; Takeda: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Bristol Myers Squibb: Research Funding; Regeneron: Research Funding; Seattle Genetics: Research Funding; ADC Therapeutics: Research Funding. Ding:Astra Zeneca: Research Funding; Abbvie: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; DTRM: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: pembrolizumab

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CD4+ T Cell Fate in Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-117674 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4494-4494

Author(s):

Rachel Elizabeth Cooke ◽

Jessica Chung ◽

Sarah Gabriel ◽

Hang Quach ◽

Simon J. Harrison ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Research Funding ◽

Flow Cytometric Analysis ◽

Advisory Committees ◽

Mitochondrial Mass ◽

Flow Cytometric

Abstract The average incidence of multiple myeloma (MM) is in the 7th decade that coincides with the development of immunosenescence and thymic atrophy, meaning that lymphocyte recovery after lymphopenia-inducing therapies (most notably autologous stem cell transplant, ASCT) is largely reliant on homeostatic proliferation of peripheral T cells rather than replenishing the T cell pool with new thymic emigrants. We have previously shown that there is a significant reduction in circulating naïve T cells with a reciprocal expansion of antigen-experienced cells from newly diagnosed MM (NDMM) to relapsed/refractory disease (RRMM). This results in a reduced TCR repertoire and the accumulation of senescence-associated secretory phenotype cytotoxic T cells, which maintain the ability to produce IFNγ but lose proliferative potential. A reduction in CD4:8 ratio is also a characteristic finding in MM with disease progression, which can be explained by high IL-15 levels in lymphopenic states that preferentially drive expansion of CD8+ memory T cells. We wanted to further evaluate what changes were occurring in the CD4+ T cell population with disease progression in MM. We analyzed paired peripheral blood (PB) samples from patients with NDMM and RRMM, and compared with age-matched normal donors (ND). In the NDMM cohort, we examined T cells from PB samples at baseline, after 4 cycles of lenalidomide and dexamethasone (len/dex), and after ASCT; and in the RRMM cohort samples from baseline and after 6 cycles of len/dex. We firstly confirmed in flow cytometric analysis of T cells at serial intervals in NDMM patients that the reduction in circulating naïve T cells and in CD4:8 ratio occurs post ASCT and does not recover by time of last follow-up. We next utilised RNA-seq to analyse differences in CD4+ T cells from NDMM, RRMM and ND. CD4+ T cells from RRMM showed downregulation of cytosolic ribosomal activity but maintenance of mitochondrial ribosomal activity and significant upregulation of pathways involved with calcium signalling. To this end, we evaluated mitochondrial biogenesis and metabolic pathways involved with mitochondrial respiration. Flow cytometric analysis of mitochondrial mass showed a marked increase in RRMM compared with ND, in keeping with a shift towards memory phenotype. Key rate-limiting enzymes in fatty acid β-oxidation (CPT1-A, ACAA2 and ACADVL) were all significantly increased in RRMM compared with ND. To analyse whether these cells were metabolically active, we also measured mitochondrial membrane potential and reactive oxygen species (ROS), gating on cells with high mitochondrial mass. Mitochondrial membrane potential was significantly increased in RRMM compared with ND, although ROS was reduced. The significance of this is not clear, as ROS are not only implicated in cell senescence and activation-induced cell death, but are also positively involved in tyrosine kinase and PI3K-signalling pathways. PD-1 has been shown to play a role in transitioning activated CD4+ T cells from glycolysis to FAO metabolism, and elevating ROS in activated CD8+ T cells. We analysed PD-1 expression on T cells in RRMM and at treatment intervals in NDMM (as described earlier). The proportion of CD4+ and CD8+ T cells expressing PD-1 was increased 4-6 months post-ASCT and remained elevated in CD4+ T cells 9-12 months post-ASCT, but normalised to baseline levels in CD8+ T cells. Increased PD-1 expressing CD4+ T cells was also evident in RRMM patient samples. This may suggest that in the lymphopenic state, PD-1 expression enhances longevity in a subset of CD4+ T cells by promoting reliance on mitochondrial respiration; however, their ability to undergo homeostatic proliferation is impaired. In CD8+ T cells, high PD-1 expression may lead to cell death via ROS accumulation, and these cells do not persist. ASCT remains a backbone of myeloma treatment in medically fit patients. However, this leads to significant permanent defects in the T cell repertoire, which may have unintended adverse outcomes. Additionally, T cells post-ASCT may not be metabolically adequate for the production of CAR-T cells, nor respond to checkpoint blockade therapies. Disclosures Quach: Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Sanofi Genzyme: Research Funding; Janssen Cilag: Consultancy. Harrison:Janssen-Cilag: Other: Scientific advisory board. Prince:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Transcriptomic Analysis of CD4+ T Cell Dysfunction during Gvhd: Evidence for Profound Reprograming of T Cell Signaling during Acute Gvhd That Is Controlled during CD28:CD80/86 Costimulation Blockade with Abatacept

Blood ◽

10.1182/blood-2019-131955 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 596-596

Author(s):

Benjamin Watkins ◽

Yvonne Suessmuth ◽

Kayla Betz ◽

Alison Yu ◽

Brandi Bratrude ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

T Cell Activation ◽

Cd4 T Cells ◽

Research Funding ◽

Costimulation Blockade ◽

Research Support ◽

Advisory Committees ◽

Highly Correlated

Although acute graft-versus-host-disease (AGVHD) is one of the major causes of non-relapse mortality after hematopoietic stem cell transplant (HCT), we are still unable to predict which patients will develop the most severe form of this disease, or which molecular pathways are dysregulated in the T cells that cause disease. Thus, understanding the molecular features of AGVHD is a critical unmet need. To address this, we have performed a companion mechanistic study as a part of our completed Phase 2 trial of abatacept, a CD28:CD80/86 costimulation blockade agent, for severe AGVHD prevention (Clinicaltrials.gov # NCT01743131, 'ABA2'). ABA2 has demonstrated significant improvement in AGVHD in patients prophylaxed with abatacept in addition to calcineurin inhibition (CNI) + Methotrexate (MTX) compared to controls receiving CNI/MTX alone. To begin to uncover mechanisms responsible for the control of AGVHD with abatacept, and given that CD4+ T cells have been consistently documented to be the main therapeutic target of this drug, we interrogated the transcriptome of CD4+ T cells reconstituting in patients prophylaxed with abatacept compared to CNI/MTX. To perform this analysis, we flow cytometrically sorted CD4+ T cells on Days 21-28 post-transplant from all patients on ABA2, as well as a cohort of 12 untransplanted healthy controls, and subsequently performed mRNA-sequencing on these cells. Weighted Gene Correlation Network Analysis (WGCNA) was performed on the top 6000 most variant transcripts from the resulting sequencing data. Hierarchical clustering of the WGCNA co-expression matrix enabled the identification of self-assembling modules (SAMs) that met a threshold of coexpression (Figure 1A). For the ABA2 dataset, we considered the following variables in the WGCNA model: patient cohort (7/8 patients, 8/8 patients, healthy controls), +/- prophylaxis with abatacept, CMV reactivation, EBV reactivation, Grade of GVHD (0-4), relapse, non-relapse mortality, and all-cause mortality. The WGCNA clustering analysis resulted in the identification of 4 discrete SAMs, which were highly correlated with clinical variable metamodules. This analysis revealed a strong positive correlation of a 476-gene SAM (the Turquoise module) in patients prophylaxed with CNI/MTX + placebo and anti-correlation of this module in patients prophylaxed with CNI/MTX + abatacept, as demonstrated in both the WGCNA heatmap and through Gene Set Enrichment Analysis (Figure 1 A-B). These opposing correlations suggested that interrogation of this module would reveal mechanistic correlates with standard prophylaxis that were decoupled by abatacept. Pathway analysis using the Reactome database (Figure 1C) revealed the turquoise SAM to be dominated by four types of pathways: (1) Those that define canonical cell-cycle pathways (2) Those involved in T cell metabolism (3) Those involved in apoptosis and (4) Those involved in T cell activation, consistent with upregulation of these transcripts in placebo versus abatacept patients. In addition to being highly correlated with patients receiving placebo, the expression of a subset of the transcripts in the Turquoise module were also directly correlated with the severity of AGVHD in these patients. Thus, linear regression analysis of the 476 transcripts in this module identified a subset of 93 genes for which transcript expression level was increased both in placebo compared to abatacept, and for which expression level also positively correlated with Grade of AGVHD. As with the Turquoise module as a whole, this subset of genes also formed a highly correlated network, linking transcripts involved in T cell proliferation, apoptosis, activation, metabolism as well as the T cell checkpoint (Figure 1D). This analysis represents the first comprehensive interrogation of the transcriptomic correlates of AGVHD. It identifies a novel set of transcripts which positively associate with the severity of AGVHD, and which costimulation blockade with abatacept down-regulates and de-couples from AGVHD severity. These results suggest a profound reprograming of T cell activation with abatacept that is correlated with the control of AGVHD. Disclosures Qayed: Bristol-Myers Squibb: Honoraria. Langston:Astellas Pharma: Other: Research Support; Incyte: Other: Research Support; Jazz Pharmaceuticals: Other: Research Support; Chimerix: Other: Research Support; Takeda: Other: Research Support; Kadmon Corporation: Other: Research Support; Novartis: Other: Research Support; Bristol Myers Squibb: Other: Research Support. Blazar:Fate Therapeutics, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees. Kean:HiFiBio: Consultancy; BlueBirdBio: Research Funding; Gilead: Research Funding; Regeneron: Research Funding; EMDSerono: Consultancy; FortySeven: Consultancy; Magenta: Research Funding; Kymab: Consultancy; Jazz: Research Funding; Bristol Meyers Squibb: Patents & Royalties, Research Funding. OffLabel Disclosure: Abatacept: Approved for Rheumatoid Arthritis; used in this trial for prevention of GVHD.

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Increased TACE (Tumor necrosis factor-alpha±-converting enzyme; ADAM17) Activity Associates with Decreased CD62L Expression, Increased Soluble CD62L Plasma Levels and Predicts Molecular Response to Nilotinib Therapy in Patients with Early Chronic Phase Chronic Myelogenous Leukemia (CML-CP): Results from an ENEST1st Substudy

Blood ◽

10.1182/blood.v126.23.4033.4033 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4033-4033 ◽

Cited By ~ 1

Author(s):

Sieghart Sopper ◽

Satu Mustjoki ◽

Angelica Loskog ◽

Bjorn T. Gjertsen ◽

Guenther A. Gastl ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Immune Cell ◽

T Cell Subsets ◽

Molecular Response ◽

Advisory Committees ◽

Expression Levels

Abstract Background and Aim: Tyrosine kinase inhibitors (TKI) imatinib and dasatinib modulate immune responses in vitro and in vivo. Immunological surveillance in the MRD-situation might be of particular relevance for long-term control or even elimination of CML-repopulating stem cells. Moreover, baseline immunological characteristics may be associated with response to TKI therapy. Little is known about potential immune-modulatory effects of nilotinib in vivo. The ENEST1st study (NCT01061177) evaluated the role of first-line nilotinib therapy in CML-CP. The primary endpoint was the MR4 rate at 18 months. A comprehensive immunological monitoring program within this ENEST1st substudy characterized baseline and therapy-induced immunological variables to correlate them with biological disease characteritics and clinical response parameters. Methods: Peripheral blood was taken prior to treatment initiation and after 6 and12 months (mo) from 52 patients. Samples were analyzed by nine colour flow cytometry employing six panels of optimized antibodies to determine various leukocyte populations (e.g. T cell subpopulations including Treg and NKT cells, NK cells, B cells, monocytes, MDSC, dendritic cell subsets). Plasma concentrations of soluble CD62L (sCD62L) and TACE (tumor necrosis factor-α-converting enzyme; ADAM17, CD156b), the metalloproteinase inducing proteolytic cleavage of CD62L from the cell surface, were either measured by ELISA or (in case of the enzymatic activity of TACE) using a fluorogenic assay. Changes in immune cell parameters were correlated to biological disease features and clinical endpoints. Results: The most striking finding of this study is the drastic loss of the lymph-node homing marker CD62L on immune cells (T cell subsets and granulocytes) at baseline (basCD62L), which increased back to normal levels during nilotinib therapy. The proportion of basCD62L+ cells among both CD4+ and CD8+ T cell subsets significantly correlated with Sokal score (both as continous and categorial variable, i.e. high vs. low/int). Low basCD62L expression levels on both T cell subsets correlate with increased spleen size, higher BM and PB blast and WBC counts as well as it correlates to higher BCR-ABL copy numbers at almost all time points during treatment. Similarly, lower basCD62L on either CD4+ or CD8+ T cells is linked to a longer duration to reach the respective molecular endpoint. Patients reaching MR4 at 18 months (primary study endpoint) had significantly higher levels of basCD62L on both CD4+ (p=0.02) and CD8+ (p=0.008) T cells. Consequently, MR4 at 18 months was attained in a significantly higher percentage of patients in the basCD62hi compared to the CD62lo patients (63% vs. 13.0%). Vice versa, patients who reached MR4 at 18 months had significantly higher proportions of basCD62L expressing cells among both CD4+ and CD8+ T cells. Moreover, as depicted by a cumulative response rate, patients with high proportions of basCD62Lhi T cells, achieved MMR and MR4 significantly earlier and in a higher proportion throughout the observation period. A detailed characterization of other T cell differentiation marker (CD45RA, CD45R0, CD28, CD27, and CD95) did not reveal significant baseline T cell subset alterations as explanation for altered CD62L expression. In contrast to low basCD62L surface expression levels, its shed form sCD62L is significantly increased at diagnosis but subsequently drops back during nilotinib therapy. Similar to surface CD62L expression, also sCD62L associates with biological disease features and molecular response to nilotinib. Finally, low CD62L surface expression was associated with elevated sCD62L levels and increased proteolytic activity but not total amount of TACE. Conclusion: At baseline, increased proteolytic activity of TACE sheds CD62L from the immune cell surface. During nilotinib therapy, TACE activity gets normalized leading to re-expression of CD62L on T cells and vice versa a drop of sCD62L. Low baseline T cell expression levels of CD62L and increased sCD62L levels correlate to a more aggressive CML phenotype and are linked to inferior molecular response to nilotinib in early CML-CP. Larger prospective studies including also other TKIs are needed to confirm the prognostic relevance of sCD62L/CD62L expression as response-prediction marker, as this marker is easy to measure by ELISA in plasma samples or flow-cytometry. Disclosures Mustjoki: Finnish Cancer Institute: Research Funding; Sigrid Juselius Foundation: Research Funding; Academy of Finland: Research Funding; the Finnish Cancer Societies: Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Signe and Ane Gyllenberg Foundation: Research Funding. Loskog:RePos Pharma AB: Membership on an entity's Board of Directors or advisory committees; Vivolux AB: Membership on an entity's Board of Directors or advisory committees; Lokon Pharma AB: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; NEXTTOBE AB: Membership on an entity's Board of Directors or advisory committees; Alligator Bioscience AB: Patents & Royalties. Gjertsen:Haukeland University Hospital: Research Funding. Giles:Novartis: Consultancy, Honoraria, Research Funding. Ossenkoppele:Pfizer: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Porkka:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria.

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Galectin-3 Signaling in Donor T Cells Regulates Acute Graft Versus Host Disease (aGvHD) after Allogenic Transplantation

Blood ◽

10.1182/blood-2021-150777 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2765-2765

Author(s):

Hemn Mohammadpour ◽

Takemasa Tsuji ◽

Cameron R. MacDonald ◽

Joseph L. Sarow ◽

Jingxin Qiu ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd4 T Cells ◽

Research Funding ◽

T Cell Proliferation ◽

Advisory Committees ◽

Human T Cells ◽

Donor T Cells

Abstract Galectin-3 (Gal-3) is a unique member of the galectin family of lectins. Gal-3 possesses immune-regulatory functions depending on the immune cell and the immunologic situation. There are no studies that specifically delineate the role of Gal-3 in the setting of acute GvHD but mounting research suggests that dysregulation of pathways involving the galectin family may contribute to the pathogenesis of other immune disorders. Gal-3 is expressed by many types of immune cells, including T-cells. It suppresses signaling downstream of the TCR, decreases effector T-cell cytokine production, but increases the development and differentiation of memory T cells, myeloid cells, and macrophages. We investigated the mechanisms and downstream events of Gal-3 signaling in donor T cells after Allo-HCT, using Gal-3 knockout (Gal-3 -/-) mice. We further studied the effect of Gal-3 in controlling aGvHD incidence and severity while preserving the Graft-versus Leukemia (GvL) effect by overexpressing Gal-3 in human T cells. We utilized both a major MHC-mismatch (C57B/6 (H-2 b) into BALB/c (H-2 k) model and a MHC-matched, multiple minor histocompatibility antigen (miHA) mismatched B6 (H-2 b) into C3H/SW (H-2 b) model. Lethally irradiated recipient BALB/c and C3H/SW WT animals were injected with T cell depleted bone marrow alone (3 ×10 6) or with splenic T cells derived from allogeneic WT or Gal-3 -/- B6 donors (0.7 × 10 6 T cells in B6 → BALB/c and 1.5 × 10 6 in B6 → C3H/SW). We found that donor T cells express Gal-3 after Allo-HCT and that Gal-3 expression in WT T cells plays an important role in controlling GvHD, as evidenced by less severe weight loss, decreased clinical GvHD scores, and longer survival when compared to mice receiving Gal-3 -/- donor T cells (Figure 1A). We studied the mechanisms by which Gal-3 signaling controls the severity of aGvHD. Using flow cytometry analysis, we determined that Gal-3 plays a critical role in T cell proliferation and exhaustion. Gal-3 -/- T cells have a cytotoxic T phenotype with increased IFN-ℽ and GM-CSF production in T cells from the spleen and liver tissues on days 7 and 14 after Allo-HCT when compared to WT T cells (Figure 1B). There was a significant increase in T cell proliferation in Gal-3 -/- CD4 +T cells with a significantly higher level of IFN- ℽ mediated activation induced cell death (AICD) when compared to WT T cells. Gal-3 expression in T cells significantly increased the expression of exhaustion markers evidenced by a higher percentage of Slamf6 + Tim-3 + in WT T cells when compared to Gal-3 -/- T cells (Figure 1B). Gal-3 induced T cell exhaustion by through overactivation of NFAT signaling (data not shown). We sought to determine whether overexpression of Gal-3 in human T cells could control GvHD without affecting GVL. Gal-3 was overexpressed in human T cells using retrovirus containing Gal-3, vector alone and control T cells: Gal-3 T cells (T RV-Gal-3), GFP T cells (T RV-GFP) and control T cells were injected in irradiated NSG-HLA-A2 mice. All human cells expressed HLA-A2. Gal-3 overexpression in T cells effectively controlled the severity and mortality of GvHD after Allo-HCT in this humanized murine model of GvHD, evidenced by decreased body weight loss and decreased GvHD clinical scores in recipients transplanted with Gal-3 T cells when compared to control or GFP T cells (Figure 1C). Gal-3 overexpression did not impair the GvL effect when T cells cultured with Raji and THP-1 cell lines in vitro (data not shown). Gal-3 overexpression in T cells increased the frequencies of exhausted CD4 + T cells, and central memory CD4 + T cells while decreasing the percentage of effector CD4 T cell and INF-ℽ + CD4 + T cells. Clinical GI colon biopsies from patients undergoing allo-HCT were evaluated for Gal-3 expression in T cells using the multi-color Vectra 3 Automated Quantitative Pathology Imaging System. T cells in the colon biopsies expressed Gal-3. There was a significant correlation between Gal-3 MFI in CD4+ T cells, and GI histopathology score when analyzing Gal-3 intensity on Gal-3-expressing T cells. The Gal-3 MFI in CD4+ T cells was significantly lower in biopsies with higher colon GI histopathology scores (III-IV) compared to with lower colon GI histopathology scores I-II. In conclusion, these data reveal how Gal-3 can influence donor T cell proliferation and function in preclinical aGvHD models and point to the feasibility of manipulation of Gal-3 signaling to ameliorate aGvHD in the clinical setting. Figure 1 Figure 1. Disclosures Blazar: Rheos Medicines: Research Funding; Carisma Therapeutics, Inc: Research Funding; Equilibre Pharmaceuticals Corp: Research Funding; Tmunity Therapeutics: Other: Co-founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees. McCarthy: Magenta Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bluebird: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Juno: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Intravital Imaging of Bright Cyan-Fluorescent AML Model Reveals Impact of CXCR4 Inhibitor on AML and Immune Cell Dynamics

Blood ◽

10.1182/blood-2019-131945 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2674-2674

Author(s):

Tomasz Zal ◽

Mateusz Rytelewski ◽

Rodrigo Jacamo ◽

Malgorzata Anna Zal ◽

Meenakshi Shanmugasundaram ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Immune Surveillance ◽

T Cell Subsets ◽

Amoeboid Movement ◽

Perivascular Spaces ◽

Advisory Committees ◽

Equity Ownership

INTRODUCTION: CXCR4 chemokine receptor inhibitors such as BL-8040 (BioLineRx) have been investigated by us and others as possible anti-leukemic drugs due to their ability to "mobilize" leukemia cells out of the BM and into the circulation, where they are more sensitive to chemotherapy. However, the exact mode of cell relocation remains unclear. CXCR4/CXCL12 signaling pathway also participates in BM homing of immune cells, including both central memory T cells and immunosuppressive CD4+FoxP3+ T-regulatory cells (T-reg). Therefore, CXCR4 inhibition has the potential to either counteract or enhance the process of AML immune surveillance. Therefore, we sought to develop a syngeneic AML model for intravital 2-photon microscopy (TPM) compatible with existing immune reporter mouse strains, which typically occupy the green, yellow and red fluorescence channels. HYPOTHESIS: CXCR4 inhibition decreases AML and T cell BM cellularity by increasing the rate of intravascular cell entry and/or decreasing the rate of circulating cell homing back to BM. MODEL: The cyan-colored fluorescent protein mTurquoise2 was lentivirally introduced into C57BL6-origin AML cells containing the MLL, ENL-FLT3, ITD, and p53-/- mutations, termed AML1-mTurq2. Syngeneic FoxP3-GFP/CD11c-YFP/hCD2-DsRed reporter mice were generated by inter-breeding of the corresponding strains, respectively highlighting T-reg, myeloid antigen presenting cells, and all T cells. After intravenous infusion of 1E5 AML1-mTurq2 cells, 1-2% blasts appeared in peripheral blood on day 9, increasing to 70% on day 15-20 when animals had to be euthanized. TREATMENT: Mice with >1% blasts were given BL-8040 I.P. in two daily 400 µg doses followed by imaging 24 h later, or intravenously during imaging 10 µg and 50 µg one hour later. ANALYSIS: Disease progression was characterized by blood flow cytometry, symptom scoring and thick-mount organ tissue fluorescence microscopy. Intravital TPM of the calvarial bone marrow (BM) was performed through intact bone under general anesthesia. By interline multiplexing dual femtosecond lasers with four-sensor detection for 8 distinct channels, mTurquoise2 and SHG were recorded by the same sensor at, respectively, 860 and 990 nm excitation, along with GFP, YFP, DsRed and dextran-TRITC (blood tracer). AML and T cell subsets were 3-D tracked using Imaris software. RESULTS: AML1-mTurq2 cells stably and uniformly expressed bright cyan fluorescence, suitable for intravital TPM with low incident laser powers and fast imaging rates in deep tissue locations. In C57BL6 mice, sparse AML cell clusters were found in BM perivascular spaces on day 1 after cell infusion. AML cells were slowly motile (~4 um/min) and highly proliferative, gradually filling BM spaces and emerging in other organs. T cells and CD11c dendritic cells were present in leukemic BM, and the vasculature appeared largely intact and well perfused. T cells interacted with AML cells and the stroma, migrating with high average velocities (~10 µm/min) and slowing down to ~3 µm/min in late-stage disease. After 2 days of BL-8040 treatment, disease symptom scores improved from 3 to 1 while the untreated controls progressed from 3 to 4 (range 0-6). TPM revealed a 4-fold reduction of AML cellularity in BM. Cellular velocities of both AML and T cells were unchanged by BL-8040 treatment. After acute drug administration, a fraction of stromal AML cells begun entering capillary vessel lumens by amoeboid movement. The intravasated AML cells adhered to vessel wall for 1-2 minutes before rapid detachment. Some cells remained tethered while already loose in the blood stream. CONCLUSIONS: A novel, brightly cyan-fluorescent syngeneic AML1-mTurq2 AML model is advantageous for 6-color intravital microscopy of cell trafficking and immune surveillance in optimal compatibility with green, yellow and red reporters of cell lineages and tissue architecture. Using this model, we show that CXCR4 inhibitor BL-8040 decreases AML BM cellularity by increasing the frequency of intravasation without increasing AML migratory velocity. Disclosures Zal: Daiichi-Sankyo: Research Funding; NIH-CTEP: Research Funding; BioLineRx: Research Funding; VueBio.com: Equity Ownership; NIH/NCI: Research Funding; CPRIT: Research Funding; Moleculin Biotech, Inc.: Research Funding. Andreeff:BiolineRx: Membership on an entity's Board of Directors or advisory committees; Aptose: Equity Ownership; Eutropics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy.

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Pharmacologic Inhibition of SUMO-Activating Enzyme Potentiates Interferon Response and T Cell-Mediated Anti-Tumor Immunity in Chronic Lymphocytic Leukemia (CLL) and Lymphoma Models

Blood ◽

10.1182/blood-2021-146347 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3719-3719

Author(s):

Vi Lam ◽

Xiaoguang Wang ◽

Scott R Best ◽

Nur Bruss ◽

Tingting Liu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd4 T Cells ◽

Research Funding ◽

Foxp3 Expression ◽

Tcr Signaling ◽

Advisory Committees

Abstract Introduction: CLL is characterized by deficient immunity which clinically manifests as increased predisposition towards malignancies and infectious complications. T-cells from patients with CLL exhibit a skewed repertoire with predominance of Tregs as well as impaired immune synapse formation and cytotoxic function. Small ubiquitin-like modifier (SUMO) family proteins regulate a variety of cellular processes, including nuclear trafficking, gene transcription and cell cycle progression, via post-translational modification of target proteins. Sumoylation regulates NFκB signaling, IFN response and NFAT activation, processes indispensable in immune cell activation. Despite this, the role of sumoylation in T cell biology in context of cancer is not known. TAK-981 is a small molecule inhibitor of the SUMO-activating enzyme (SAE) that forms a covalent adduct with an activated SUMO protein, thereby preventing its transfer to the SUMO-conjugating enzyme (Ubc9). Here, we investigated the immunomodulatory effects of TAK-981 in CLL. Methods: T cells from patients with CLL were purified using Dynabeads. For polarization assays, FACS-sorted naïve CD4+ T cells were cultured for 7 days in control or differentiation media. For gene expression profiling (GEP; Clariom S), RNA was harvested after 3 and 24 hours of TCR engagement from FACS-sorted naïve CD4+ T cells. For in vivo immunization experiments, CD4+KJ1-26+ cells were inoculated IV into BALB/cJ mice. Mice received 100 µg IV ovalbumin ± R848 followed by TAK-981 7.5 mg/kg or vehicle control IV twice weekly for 10 days prior to spleen collection. Both recipient and transplanted splenocytes were analyzed. For analysis of tumor-infiltrating lymphocytes (TILs), BALB/c mice were injected with 1x10 6 A20 lymphoma cells and treated as above. TAK-981 was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA). Results: T cells from patients with CLL demonstrated high baseline protein sumoylation that slightly increased following TCR engagement (αCD3/CD28). Treatment with TAK-981 significantly downregulated SUMO1 and SUMO2/3-modified protein levels yet did not disrupt early TCR signaling as evidenced by sustained ZAP70, p65/NFκB and NFAT activation detected by immunoblotting, immunocytochemistry and GEP. Treatment with TAK-981 resulted in dose-dependent upregulation of the early activation marker CD69 in CD4 + T cells following 72 and 96 hours of TCR stimulation vs. control. Meanwhile, expression of CD25, HLA-DR and CD40L was delayed in the presence of TAK-981. Interestingly, CD38, an IFN response target, was induced two-fold in TAK-981-treated cells after 24 hours and persisted at high levels at subsequent timepoints. T cell proliferation was reduced in the presence of high (1 μM) but not low/intermediate concentrations of TAK-981, accompanied by reduced S phase entry and decreased synthesis of IL-2. However, T cells did not undergo apoptosis under those conditions. Targeting SAE in either control or Th1/Treg polarizing conditions facilitated an increase in IFNγ and loss of FoxP3 expression (accompanied by decreased IL-2/STAT5), suggesting a shift towards Th1 and away from Treg phenotype, respectively. GEP (Reactome, GSEA) confirmed a dramatically upregulated IFN response in TAK-981-treated CD4 + naïve T cells. Furthermore, targeting SAE enhanced degranulation (CD107a), IFNγ and perforin secretion in cytotoxic CD8+ T cells and potentiated T cell cytotoxicity in allogeneic assays with lymphoma cells (OCI-LY3, U2932) as targets. Consistent with our in vitro data, OVA-stimulated transplanted transgenic KJ1-26+ splenocytes, as well as total CD4+ T cells from recipient mice treated with TAK-981 in vivo exhibited a significant reduction in expression of FoxP3 and an increased production of IFNγ (Figure 1). In the A20 syngeneic model, treatment with TAK-981 similarly downregulated FoxP3 expression in CD4+ TILs and induced IFNγ secretion in CD8+ TILs. Conclusion. Using a combination of in vitro and in vivo experiments, we demonstrate that pharmacologic targeting of sumoylation with TAK-981 does not impair proximal TCR signaling in T cells obtained from patients with CLL, but leads to rebalancing toward healthy immune T cell subsets via induction of IFN response and downmodulation of Tregs. These data provide a strong rationale for continued investigation of TAK-981 in CLL and lymphoid malignancies. Figure 1 Figure 1. Disclosures Siddiqi: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; TG Therapeutics: Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncternal: Research Funding; Janssen: Speakers Bureau; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Danilov: SecuraBio: Research Funding; Bayer Oncology: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Takeda Oncology: Research Funding; TG Therapeutics: Consultancy, Research Funding; Rigel Pharm: Honoraria; Abbvie: Consultancy, Honoraria; Beigene: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; Gilead Sciences: Research Funding; Bristol-Meyers-Squibb: Honoraria, Research Funding; Astra Zeneca: Consultancy, Honoraria, Research Funding.

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Characteristics and Prognosis of AML Patients with or without a History of Clonal Hematopoiesis

Blood ◽

10.1182/blood.v126.23.224.224 ◽

2015 ◽

Vol 126 (23) ◽

pp. 224-224

Author(s):

Felicitas Thol ◽

Larissa Köhler ◽

Sabrina Klesse ◽

Razif Gabdoulline ◽

Arnold Kloos ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Mononuclear Cells ◽

Intensive Therapy ◽

Control Group ◽

Advisory Committees ◽

Clonal Hematopoiesis ◽

Travel Grant

Abstract Background: Clonal hematopoiesis of indeterminate potential (CHIP) is defined by the detection of mutations in genes like DNA methyltransferase 3A (DNMT3A) and has recently been described to occur in healthy people and to predispose them to myeloid malignancies. DNMT3A is frequently mutated in acute myeloid leukemia (AML) and mutations have been detected in CD3 positive T-cells of some AML patients. In these patients DNMT3A mutations are early events that are likely to arise from CHIP. It is unknown how a history (hx) of CHIP influences the characteristics of AML patients and their response to therapy. We studied this question on the basis of a large cohort of DNMT3A mutated AML patients. Patients and Methods: 171 DNMT3A mutated AML patients (aged 18-87 years) were included in our study. 127 patients were treated intensively in trials of the AMLSHG and AMLSG. 34 patients received non-intensive therapy and for 10 patients the therapy is unknown. 148 patients carried a mutation at arginine R882. At the time of diagnosis and relapse samples were further sequenced for 54 genes involved in leukemia with next generation sequencing (NGS) on the Illumina platform. Library preparation of diagnostic samples was performed with the TruSight Myeloid sequencing panel (Illumina). T-cells (CD3+ CD11b- CD14- CD33-) were purified by flow cytometry from AML samples at the time of diagnosis. DNMT3A mutational analysis of T-cell samples and of mononuclear cells during remission or at relapse was performed also with ultra-deep sequencing using customized DNMT3A NGS primers. Presence of a DNMT3A mutation in sorted T cell populations was used as an indicator of a hx of CHIP. Results: A total of 40 patients (23%) were found to have the DNMT3A mutation in mononuclear cells and T-cells (hx of CHIP), while 131 patients (77%) had a DNMT3A mutation in mononuclear cells, but not T-cells (control cohort). Comparing these two patient cohorts revealed that significantly more patients in the hx of CHIP cohort had secondary AML (p=0.009), were older (p=0.005) and less likely to receive intensive treatment (p=0.047) while other clinical parameters did not significantly differ. Analysing the mutational profile of 54 genes revealed that the number of mutations per patient between these 2 groups was similar (median 5 vs 4 mutations, p=0.39). Patients with a hx of CHIP were significantly more likely to harbour mutations in TET2 (p=0.006), RUNX1 (p=0.004), SF3B1 (p=0.049), U2AF1 (p=0.015) but less likely to be NPM1 mutated (p=0.005). There was no significant difference in the allelic burden of DNMT3A in the CHIP hx (mean 43.6) vs control group (mean 44.5). The mean variant allele frequencies of DNMT3A, RUNX1 and NPM1 were highest (44, 45 and 43 respectively) as compared to other mutated genes like IDH1, IDH2 and FLT3 (32, 37 and 34). In relapse samples (n=11), the identical DNMT3A mutation could always be identified. However, patients with a hx of CHIP (n=2) had comparable allelic frequencies compared to diagnosis of mutated DNMT3A (<10% difference), but not NPM1 (> 10% difference), while 7 out of 9 patients in the control group had a change in the allelic frequency at the time of relapse (mostly reduction). In all remission samples DNMT3A mutations could be identified with ultra-deep NGS but with variable allelic frequencies (0.13-50.01% in the control group, 0.25-70.14% in the hx of CHIP group). In the cohort of patients with intensive therapy there was no difference in CR rates between hx of CHIP and control groups (82 vs 90%, p=0.31). Overall survival (OS) was not influenced by a hx of CHIP (whole cohort: HR 1.09; 95%CI 0.67-1.79; P=.73; intensively treated cohort: HR 0.72; 95%CI 0.34-1.51; P=.38). Relapse-free survival (RFS) was also not different in the hx of CHIP vs the control group (HR 1.06; 95%CI 0.58-1.93; P=.85; intensively treated cohort only HR 0.91; 95%CI 0.46-1.78; P=.78). However, when looking at the influence of allogeneic stem cell transplantations (HSCT) on outcome in intensively treated patients, patients with a hx of CHIP showed abenefit from HSCT (HR 0.082; 95%CI 0.009-0.75; P= 0.027 Figure 1A) as compared to the control group (HR 0.68; 95%CI 0.39-1.21; P= 0.19, Figure 1B). Conclusion: AML patients with a hx of CHIP, as defined by mutated DNMT3A in T-cells, show a distinct clinical and molecular profile and may benefit from HSCT. Figure 1A. Figure 1A. Figure 1B. Figure 1B. Disclosures Bug: TEVA Oncology, Astellas: Other: Travel Grant; NordMedica, Boehringer Ingelheim, Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene, Novartis: Research Funding. Fiedler:Pfizer, Amgen, Kolltan: Research Funding; Teva, Amgen, Astellas: Other: Travel Grant; Karyopharm: Research Funding. Schlenk:Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Research Funding; Arog: Honoraria, Research Funding; Teva: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Research Funding.

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Mechanisms of CD4 T Cell Tumor Immunity in a Preclinical Model of Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-117129 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1947-1947

Author(s):

Selma Bekri ◽

Reunet Rodney-Sandy ◽

Diana Gruenstein ◽

Bjarne Bogen ◽

Daniel Levey ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Tumor Immunity ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Research Funding ◽

Cd8 T Cell ◽

Cd4 T Cell ◽

Advisory Committees

Abstract The majority of immune therapies for cancer have focused on generating tumor-specific CD8+ T cells that directly engage and kill tumor cells. However, recent data demonstrate a critical role of CD4 T cells in tumor immunity. Animal models showed that MHC class II epitopes derived from mutated tumor-associated proteins (neo-antigens, neoAg) conferred protection against different mouse tumor types. Clinical studies of neoAg vaccines demonstrated anti-tumor activity, with the majority of neoAg-specific immune responses being CD4 T cells. These tumor Ag-specific CD4 T cells could confer protective anti-tumor immunity even in the absence of CD8 T cells directed against the same antigen. These findings are even more striking because most tumor cells do not express MHC class II molecules and CD4 T cells cannot directly kill tumor cells. In order to understand how CD4 T cells eliminate tumors in the absence of direct cytolytic activity and vaccine specific CD8 T cells, we used an animal model of multiple myeloma, MOPC315.BM (MOPC) cell line derived from BalB/C background, that mimics many of the features of the human disease. MOPC cells do not express MHCII and produce an IgA with a unique mutated neoAg in the l2 light chain called Idiotype l2.315 (Id). This Id antigen is well characterized and known to activate CD4 T cells, however, when used as a peptide vaccine alone, it is weakly immunogenic and fails to elicit protective immune responses. We used an Id peptide sequence fused with a high affinity HSP70 binding site that elicited strong Id-specific CD4 T cell IFNg responses that protect against MOPC tumor growth. No Id-specific CD8 T cell or B cell responses were detected, and Id-specific CD4 cells did not directly lyse MOPC cells in vitro. These CD4 responses were specific to the neoAg, as they did not cross react with the wild type peptide fused to the same HSP70 binding sequence. These findings lead to the hypothesis that Id-specific CD4 T cells confer protective immunity by promoting cross-priming of CD8 T cells against non-Id, MOPC-associated antigens. To investigate this hypothesis, splenocytes and bone marrow cells were harvested from mice either challenged with MOPC cells only (No vax + MOPC), vaccinated with the Id peptide only (Id vax+No MOPC) or Id vaccinated and challenged with MOPC cells (Id vax + MOPC). CD4 and CD8 T cell activation was assessed by measuring IFNg and TNFa production by intracellular cytokine staining (ICS) after eight days of in vitro restimulation with Id peptide and irradiated MOPC cells. As shown in fig.1, in the absence of Id vaccination, no or very little immune activation was observed in the spleen and bone marrow. Id vaccination alone induced CD4 T cells in both compartments. In mice that received both Id vax and MOPC challenge, there was induction of CD8 T cell, with a notable trend towards high levels of CD8 activity in the tumor microenvironment (bone marrow). These CD8 T cells were not detected when cells from the same animals were restimulated with the Id peptide alone, indicating that these CD8 responses were against non-Id, MOPC-associated Ags (data not shown). All together these data support a model in which Id-specific CD4 T cell provide helper activity to promote cross-priming of CD8 T cell against MOPC-associated Ags only in the presence of tumor challenge. These responses are present both systemically and in the tumor site. We are currently studying the specificity of these CD8 T cells and the potential role of innate immune cells such as NK cells and macrophages in the anti-tumor immune response initiated by Id-specific CD4 T cells. These findings will help better understand the mechanisms of CD4 mediated anti-tumor immunity and promote the development of combination immunotherapy strategies to improve the efficacy of vaccines targeting CD4 antigens. Disclosures Bekri: Agenus Inc: Research Funding. Levey:Agenus Inc: Employment, Equity Ownership. Cho:BMS: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Agenus Inc.: Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; J & J: Consultancy; Genentech Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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