Single-Cell Mass Cytometry Identifies Mechanisms of Resistance to Immunotherapy in AML

Background: The mechanisms of resistance to immunotherapies in patients with acute myeloid leukemia (AML) are not well characterized and biomarkers for improved immunotherapeutic strategies are critical. Multiple phase II/III clinical trials combining hypomethylating agents (HMA), azacitidine (AZA) and decitabine, with immune checkpoint inhibitors are now underway for patients with newly diagnosed and relapsed/refractory (R/R) AML and MDS based on data suggesting that 1) hypomethylating agents increase tumor expression of PD-L1 and PD-1 in myeloid malignancies(Yang H et al. Leukemia 2014); 2) blocking the PD-1/PD-L1 signaling axis has profound anti-leukemic response (Zhou et al. Blood 2011); and 3) clinical responses to Azacitidine/PD1 inhibitor (nivolumab) in relapsed AML (Daver et al., Cancer Discovery). Here, we characterize the baseline immune landscape and potential mechanisms of resistance using single-cell mass cytometry (CyTOF) profiling of serially collected samples from R/R AML patients undergoing therapy with AZA and PD-L1 inhibitor avelumab [NCT02953561]. Methods: Bone marrow (BM) and peripheral blood (PB) samples were collected from 9 patients prior to treatment with AZA and avelumab and after cycles 1, 3, and 5 (as available). To interrogate immune profiling in these samples, we have developed and optimized a novel CyTOF antibody panel that includes immunophenotypic markers to distinguish AML stem cells and blasts from adjacent immune cell subsets (T, B, NK cells, and monocytes), as well as known checkpoint ligands and receptors. Data was analyzed using the uniform manifold approximation and projection (UMAP) algorithm (McInnes et al. arXiv 2018) implemented through Cytofkit (Chen et al. PLoS Comput Biol 2016). Results: We first used the multi-parameter immunophenotyping to characterize the immune microenvironment in normal and leukemic BM prior to therapy. At baseline, the composition of AML BM CD4 and CD8 T cells contained a significantly smaller fraction of naïve T-cells when compared to healthy bone marrow controls (both p<0.001). The proportion of terminally differentiated CD8+ cells was also significantly less in AML BM (p<0.001). Conversely, effector memory CD4 and CD8 cells comprised the major fraction of T-cells in AML BMs (Fig 1D). Due to the small number of patients we investigated response to therapy on a cycle-by-cycle basis rather than overall patients' outcomes. By this approach, we found that the relative ratio of T cells to blasts did not significantly predict response, however, patients with blast reduction or stable disease after 1 cycle of treatment had significantly lower CD4:CD8 ratios (i.e. more CD8 cells per CD4 cell) than those who progressed (p=0.04). In fact, CD4:CD8 ratio at the beginning of the cycle was significantly correlated with relative blast reduction in the bone marrow (R2=0.25, p=0.035) (Fig 1E). This is consistent with what has been seen in other cancer types where low CD4:CD8 has been associated with favorable prognosis (Sato et al. PNAS 2005). Baseline PD-L1 expression levels in these patient samples was low and did not predict response to therapy. While 4 of the 9 patients experienced and initial blast reduction, 7 developed disease progression while on the trial. Serial measurements from the same patients allowed us to track both resistant and newly emerging clones over the course of therapy (representative patient; Fig 1A-C). While PD-L1 levels were consistently low, we did observe high PD-L2 protein expression in AML cells resistant (the 7 with progressive disease) to HMA/PD-L1 inhibition, and PD-L2 was also frequently expressed in the emerging clones not present at baseline. We profiled the AML clones from each patient present at disease progression for the expression of other known checkpoint ligands and receptors (Fig 1F). Expression of PD-L2, OX40, and TIM-3 was detected in the majority of these resistant clones (100%, 86%, and 71%, respectively). Conclusions: Single-cell characterization of R/R AML reveals that the immune landscape of these patients at baseline is significantly altered when compared to healthy bone marrow. The ratio of CD:/CD8 and composition of residual T cells appear to be the most important predictors of response to HMA/PD-L1 inhibition. Finally, AML cells express a variety of other immune checkpoints, particularly PD-L2 but also OX40 and TIM3, that should be considered for future combination therapy. Disclosures Short: AstraZeneca: Consultancy; Amgen: Honoraria; Takeda Oncology: Consultancy, Research Funding. Konopleva:Astra Zeneca: Research Funding; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Genentech: Honoraria, Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Calithera: Research Funding; Agios: Research Funding.

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Dissecting alterations in human CD8+ T cells with aging by high-dimensional single cell mass cytometry

Clinical Immunology ◽

10.1016/j.clim.2019.01.005 ◽

2019 ◽

Vol 200 ◽

pp. 24-30 ◽

Cited By ~ 1

Author(s):

Min Sun Shin ◽

Kristina Yim ◽

Kevin Moon ◽

Hong-Jai Park ◽

Subhasis Mohanty ◽

...

Keyword(s):

T Cells ◽

Single Cell ◽

Cd8 T Cells ◽

Cell Mass ◽

High Dimensional ◽

Mass Cytometry

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Proliferation tracing with single-cell mass cytometry optimizes generation of stem cell memory-like T cells

Nature Biotechnology ◽

10.1038/s41587-019-0033-2 ◽

2019 ◽

Vol 37 (3) ◽

pp. 259-266 ◽

Cited By ~ 21

Author(s):

Zinaida Good ◽

Luciene Borges ◽

Nora Vivanco Gonzalez ◽

Bita Sahaf ◽

Nikolay Samusik ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

Single Cell ◽

Cell Mass ◽

Mass Cytometry ◽

Cell Memory

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Multiple Inhibitory Receptors Are Expressed on Central Memory and Memory Stem T Cells Infiltrating the Bone Marrow of AML Patients Relapsing after Allo-HSCT

Blood ◽

10.1182/blood.v128.22.4564.4564 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4564-4564 ◽

Cited By ~ 2

Author(s):

Maddalena Noviello ◽

Francesco Manfredi ◽

Tommaso Perini ◽

Giacomo Oliveira ◽

Filippo Cortesi ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Research Funding ◽

Central Memory ◽

Cd8 Cells ◽

Effector Functions ◽

Cell Dysfunction ◽

T Cell Dysfunction ◽

Cell Effector

Abstract Background:Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is the only cure for high-risk acute myeloid leukemia (AML). Unfortunately, relapse still remains the major cause of death after HSCT. We investigated if T-cell dysfunction is associated to post-transplant relapse. Patients and Methods: To this,we longitudinally analyzed the T-cell dynamics in bone marrow (BM) and peripheral blood (PB) of 32 AML patients receiving HSCT from HLA identical (HLAid, 20 pts) or HLA haploidentical (haplo, 12 pts) donors. Samples were analysed by multi-parametric flow cytometry to investigate the expression of inhibitory receptors (IRs) on CD4 and CD8 T-cell subsets defined by CD45RA, CD62L and CD95 expression, and to assess the proportion of regulatory T cells (Tregs; CD4+CD25+FoxP3+). Results were also analyzed with the BH-SNE algorithm, an unbiased computational method for the analysis of FACS data. To evaluate T-cell effector functions, the CD107a degranulation assay was performed and the production of cytokines (IL-2, IFNg and TNFa) was measured by intracellular staining. BM and PB were collected 60 days after HSCT and at relapse (median 237 days; 16 pts) or, when complete remission was maintained (CR; 16 pts), at 1 year. Samples from 8 healthy donors (HD) were used as controls. Results:After transplant, BM and PB T cells showed a lower CD4/CD8 ratio (p<0.01) and a preferential late differentiation profile (p<0.05) when compared to HD. A higher proportion of BM Tregs was documented at relapse (p<0.01), independently from the donor source. We next investigated the expression of several IRs as T-cell exhaustion markers. After haplo-HSCT, PD-1, CTLA-4, 2B4 and Tim-3 were significantly upregulated in BM and PB T cells at all time-points, compared to HD and independently from the clinical outcome. Conversely, after HLAid-HSCT, at the late time-point, patients who relapsed, displayed a higher frequency of BM infiltrating T cells expressing PD-1, CTLA-4 and Tim-3 than CR pts (p<0.05) and HD (p<0.01). We then investigated the profile of each T-cell subset in our cohort. In the BM of HD the IR expression was confined to effector memory and effectors. While a similar IR distribution was observed in CR, at relapse, PD-1, 2B4 and Tim-3 were also upregulated in BM infiltrating central memory (p<0.01) and memory stem T cells (p<0.05). Interestingly, at relapse, leukemia expressed PD-L1 (9/9 cases) and Galectin-9 (6/9). The levels of Tim-3 on BM CD8 cells associates with that of Galectin-9 on autologous blasts (p<0.05), suggesting a preferential role for this immunomodulatory axis after HSCT. Based on phenotype similarities, the BH-SNE algorithm positioned HD samples separately from transplanted pts in bi-dimensional maps. 93 significant clusters were identified. Clusters associated with relapse after HLA-id (5) and after haplo (15) were composed of T cells expressing multiple IRs, while CR-specific clusters were diminished in IR fluorescence. To verify whether the T-cell exhaustion phenotypic profile at relapse associates with functional impairment, we evaluated T-cell effector functions upon polyclonal stimulation. Strikingly, we observed a lower degranulation ability of CD8 cells at relapse when compared to CR (p<0.05). In two patients, selected based on samples availability, we isolated and expanded by rapid expansion protocol (REP) T cells expressing one or more IRs (IR+) or no IR (IR-). Expansion rates were high and similar in IR+ and IR- T cells (mean fold increase 624 and 781, respectively at day 21). The degranulation ability measured ex-vivo in those patients (mean 4.4% on CD8 cells) was dramatically increased upon REP expansion (95% and 88.9% for IR+ and IR-, respectively). Similarly, the frequency of IFN-g producing CD8 cells increased in IR+ and IR- cells upon REP, indicating that the T-cell dysfunction observed at relapse can be efficiently reversed. We next challenged IR+ and IR- T cells against autologous blasts. Preliminary results suggest that IR+ T cells are enriched in leukemia specificity (elimination index of 66% and 44% in IR+ and IR- cells respectively at an E/T ratio of 100:1). Conclusions: After HSCT, the molecular signature of exhausted CD8 cells in relapsing pts includes PD-1, CTLA-4, 2B4 and Tim-3. The expression of IRs on early differentiated central memory and memory stem T cells at relapse suggests a wide, though reversible, immunological dysfunction mediated by AML relapsing blasts. Disclosures Bondanza: TxCell: Research Funding; MolMed SpA: Research Funding; Formula Pharmaceuticals: Honoraria. Ciceri:MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: 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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Single Cell RNA-Seq Reveals Deranged Developmental Hierarchy with Coexisting Oncogenic States and Immune Evasion Programs in ETP T-ALL

Blood ◽

10.1182/blood-2019-130482 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3953-3953

Author(s):

Amy Guillaumet-Adkins ◽

Praveen Anand ◽

Huiyoung Yun ◽

Yotam Drier ◽

Anna Rogers ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Single Cell ◽

Cd8 T Cells ◽

Research Funding ◽

Single Cells ◽

Pearson Correlation ◽

Normal Donor ◽

Developmental Hierarchy

Introduction: Early T-cell precursor acute lymphoblastic leukemia (ETP T-ALL) is a distinct subtype of T-ALL characterized by higher rates of relapse and induction failure. Large-scale genetic sequencing studies have identified frequently mutated oncogenes and gene fusions in ETP T-ALL, while bulk transcriptome analyses have revealed expression features resembling myeloid precursors and myeloid malignancies. However, the contributions of intra-tumoral functional heterogeneity and microenvironment to tumor biology and treatment failure remain unknown. Methods: We performed full-length single-cell RNA-sequencing of 5,077 malignant and normal immune cells from bone marrow or blood from five patients with relapsed/refractory ETP T-ALL (based on immunophenotyping, all with NOTCH1 mutations), before and after targeted therapy against NOTCH1. These patients were enrolled on a phase I trial with the γ-secretase inhibitor (GSI) BMS-906024 (NCT01363817). Expression of selected genes was validated by RT-PCR, flow cytometry and immunohistochemistry. Results: Single cell transcriptome analyses revealed a deranged developmental hierarchy characterized by co-expression of stemness programs in multiple malignant cells implying ineffectual commitment to either lymphoid or myeloid lineage. Most ETP T-ALL cells co-expressed HSC (hematopoietic stem cell), CMP (common myeloid progenitor) and CLP (common lymphoid progenitor) signatures simultaneously (Pearson correlation: CLP-CMP: R= 0.41, p < 2.2e-16; HSC-CLP: R= 0.53; p < 2.2e-16; HSC-CMP: R = 0.39, p <2.2e-16). Only a fraction of cells (less than 15%) demonstrated mutually exclusive CLP or HSC signatures. In contrast, CLP, CMP and HSC signatures were not co-expressed and always negatively correlated in normal bone marrow cells (CLP-CMP: R= -0.11, p < 2.2e-16; HSC-CLP: R= -0.38; p < 2.2e-16; HSC-CMP: R = -0.67, p <2.2e-16). Direct targeting of NOTCH1 as the driving oncogene has shown disappointing results in the clinical setting due to the rapid development of resistance. PI3K activation has been shown as a genetic mechanism of Notch resistance, however it is unclear if transcriptional rewiring can give rise to PI3K dependent cells after Notch inhibition. To address this question, we predicted the activity of signaling pathways in single cells after Notch inhibitor treatment using PROGENy. Most single cells demonstrated loss of Notch signaling. PI3K signaling activity was the most anti-correlated signaling pathway to Notch signaling (Pearson correlation: R= -0.51, p < 2.2e-16). Of note, this population preexisted at a frequency of ~30% in the untreated population, coexisting with cells with high Notch activation. Analysis of the immune microenvironment revealed an oligoclonal T-cell population in ETP T-ALL compared to normal donor T-cells. CD8+ T-cells from ETP patients expressed markers of T-cell exhaustion (PDCD1, TIGIT, LAG3, HAVCR2). Analyses of expression levels of the respective ligands on leukemic blasts and the predicted interaction with their receptors on endogenous CD8+ T-cells demonstrated the highest interaction score between HAVCR2 and its ligand LGALS9. LGALS9 was universally expressed in all leukemic cells, which was confirmed by flow cytometry staining in leukemic blasts and IHC staining in bone marrow of 8 patients with ETP T-ALL and 7 patients with T-ALL. T-ALL supernatant increased expression levels of the exhaustion markers HAVCR2,TIGIT and decreased effector marker GZMB in polyclonal activated normal donor CD8+ T-cells (RT-PCR). This effect was abrogated by neutralizing LGALS9 and could be rescued with recombinant LGALS9. Conclusion: We identified deranged developmental hierarchy characterized by co-expression of stemness programs in multiple malignant cell states and ineffectual commitment to either lymphoid or myeloid lineage in ETP T-ALL. Leukemic blasts demonstrate preexisting heterogeneity of diverse oncogenic states as evidenced by opposing PI3K and Notch activity, suggesting possible novel combination therapies. Notch inhibition abolishes the Notch high state without effecting the PI3K active state. Finally, we demonstrate a possible role for HAVCR2-LGALS9 interactions in causing CD8+ T-cell dysfunction in ETP T-ALL patients, which may provide a novel therapeutic strategy in this disease. Disclosures Silverman: Takeda: Consultancy; Servier: Consultancy, Research Funding. Lane:AbbVie: Research Funding; Stemline Therapeutics: Research Funding; N-of-One: Consultancy. DeAngelo:Glycomimetics: Research Funding; Amgen, Autolus, Celgene, Forty-seven, Incyte, Jazzs, Pfizer, Shire, Takeda: Consultancy; Blueprint: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Abbvie: Research Funding. Lohr:Celgene: Research Funding; T2 Biosystems: Honoraria.

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Potent Anti-Tumor Activity of Bcma CAR-T Therapy Against Heavily Treated Multiple Myeloma and Dynamics of Immune Cell Subsets Using Single-Cell Mass Cytometry

Blood ◽

10.1182/blood-2019-130341 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1859-1859 ◽

Cited By ~ 2

Author(s):

Yongxian Hu ◽

Zhang Yanlei ◽

Guoqing Wei ◽

Chang alex Hong ◽

He Huang

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Immune Cell ◽

Cell Mass ◽

Mass Cytometry ◽

Car T Cells ◽

Car T Cell ◽

Immune Cell Subsets ◽

Car T

Background BCMA CAR-T cells have demonstrated substantial clinical activity against relapsed/refractory multiple myeloma (RRMM). In different clinical trials, the overall response rate (ORR) varied from 50% to 100%. Complete remission (CR) rate varied from 20% to 80%. Here we developed a BCMA CAR-T cell product manufactured via lentiviral vector-mediated transduction of activated T cells to express a second-generation CAR with 4-1BB costimulatory domain and evaluated the efficacy and safety, moreover, dynamics of immune cell subsets using single-cell mass cytometry during treatment were analyzed. Methods Our trial (ChiCTR1800017404) is a phase 1, single-arm, open-label single center study to evaluate the safety and efficacy of autologous BCMA CAR-T treatment for RRMM. Patients were subjected to a lymphodepleting regimen with Flu and Cy prior to CAR-T infusion. BCMA CAR-T cells were administered as a single infusion at a median dose of 3.5 (1 to 6) ×106/kg. MM response assessment was conducted according to the International Uniform Response Criteria. Cytokine-release syndrome (CRS) was graded as Lee DW et al described (Blood.2014;124(2):188-195). Phenotypic analysis of peripheral blood mononuclear cells (PBMCs), frozen BCMA CAR-T aliquots, phenotype and in vivo kinetics of immune cell subsets after CAR-T infusion were performed by single-cell mass cytometry. Results As of the data cut-off date (August 1st, 2019), 33 patients, median age 62.5 (49 to 75) years old were infused with BCMA CAR-T cells. The median observation period is 8.0 (0.7 to 18) months. ORR was 100% (The patient who died of infection at 20 days after CAR-T infusion were excluded). All the 32 patients achieved MRD negative in bone marrow by flow cytometry in 2 weeks after CAR-T infusion. Partial response (4 PR, 12.1%), VGPR (7 VGPR, 21.2%), and complete response (21 CR, 63.6%) within 12 weeks post CAR-T infusion were achieved. Durable responses from 4 weeks towards the data cut-off date were found in 28/33 patients (84.8%) (Figure 1a). All patients had detectable CAR-T expansion by flow cytometry from Day 3 post CAR-T cell infusion. The peak CAR-T cell expansion in CD3+ lymphocytes of peripheral blood (PB) varied from 35% to 95% with a median percentage of 82.9%. CRS was reported in all the 33 patients, including 4 with Grade 1, 13 with Grade 2 and 16 with Grade 3. During follow-up, 1-year progression-free survival (PFS) was 70.7% (Figure 1b) and overall survival (OS) was 71.7% (Figure 1c). Multivariate analysis of patients with PR and patients with CR+VGPR revealed that factors including extramedullary infiltration, age>60 years old, high-risk cytogenetics, late stage and CAR-T cell dose were not associated with clinical response (P>0.05). Single-cell mass cytometry revealed that the frequency of total T cells, CD8+ T cells, NK cells and CD3+CD56+ NKT cells in PB was not associated with BCM CAR-T expansion or clinical response. CD8+ Granzyme B+ Ki-67+ CAR-T cells expanded prominently in CRS period. As serum cytokines increased during CRS, non-CAR-T immune cell subsets including PD1+ NK cells, CD8+ Ki-67+ ICOS+ T cells expanded dominantly implying that non-CAR-T cells were also activated after CAR-T treatment. After CRS, stem cell like memory CAR-T cells (CD45RO+ CCR7- CD28- CD95+) remain the main subtype of CAR-T cells (Figure 1d). Conclusions Our data showed BCMA CAR-T treatment is safe with prominent efficacy which can overcome the traditional high-risk factors. We also observed high expansion level and long-term persistence of BCMA CAR-T cells contribute to potent anti-myeloma activity. Stem cell like memory CAR-T cells might be associated with long-term persistence of BCMA CAR-T cells. These initial data provide strong evidence to support the further development of this anti-myeloma cellular immunotherapy. Figure 1. Disclosures No relevant conflicts of interest to declare.

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Cytokine-Induced Memory-like NK Cells Have a Distinct Single Cell Transcriptional Profile and Persist for Months in Adult and Pediatric Leukemia Patients after Adoptive Transfer

Blood ◽

10.1182/blood-2021-147370 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3825-3825

Author(s):

Jennifer A. Foltz ◽

Melissa M. Berrien-Elliott ◽

David A. Russler-Germain ◽

Carly C. Neal ◽

Jennifer Tran ◽

...

Keyword(s):

Clinical Trial ◽

T Cells ◽

Transcription Factors ◽

Nk Cells ◽

Single Cell ◽

Adoptive Transfer ◽

Research Funding ◽

Nk Cell ◽

Mass Cytometry

Abstract Natural killer (NK) cells are innate lymphoid cells that mediate anti-tumor responses and exhibit innate memory following stimulation with IL-12, IL-15, and IL-18, thereby differentiating into cytokine-induced memory-like (ML) NK cells. ML NK cells have well-described enhanced anti-tumor properties; however, the molecular mechanisms underlying their enhanced functionality are not well-understood. Initial reports of allogeneic donor ML NK cellular therapy for relapsed/refractory (rel/ref) acute myeloid leukemia (AML) demonstrated safety and a 47% CR/CRi rate (PMID32826231). In this setting, allogeneic ML NK cells are rejected after 3 weeks by recipient T cells, which precludes long-term evaluation of their biology. To address this limitation, we conducted a clinical trial for rel/ref AML patients that added adoptive transfer of same-donor ML NK cells on day +7 of a reduced-intensity conditioning (RIC) MHC-haploidentical HCT, followed by 4 doses of IL-15 (N-803) over 2 weeks (NCT02782546). Since the ML NK cells are from the HCT donor, they are not rejected, but remain MHC-haploidentical to the patient leukemia. Using samples from these patients, we profiled the single cell transcriptomes of NK cells using multidimensional CITE-seq, combining scRNAseq with a custom NK panel of antibodies. To identify donor ML NK cells in an unbiased fashion, we developed a CITE-seq ML NK classifier from in vitro differentiated paired conventional NK (cNK) and ML NK cells. This classifier was applied via transfer learning to CITE-seq analyzed samples from the donor (cNK cells) and patients at days +28 and +60. This approach identified 28-40% of NK cells as ML at Day +28 post-HCT. Only 1-6% of donor peripheral blood NK cells and 4-7% of NK cells in comparator leukemia patients at day +28 after conventional haplo-HCT alone were identified as ML NK cells (Fig 1A). These ML NK cells had a cell surface receptor profile analogous to a previously reported mass cytometry phenotype. Within the CITE-seq data, ML NK cells expressed a transcriptional profile consistent with enhanced functionality (GZMK, GZMA, GNLY), secreted proteins (LTB, CKLF), a distinct adhesome, and evidence of prior activation (MHC Class II and interferon-inducible genes). ML NK cells had a unique NK receptor repertoire including increased KIR2DL4, KLRC1(NKG2A), CD300A, NCAM1(CD56) , and CD2 with decreased expression of the inhibitory receptor KLRB1(CD161). Furthermore, ML NK cells upregulated HOPX, a transcription factor implicated in memory T cells and murine CMV adaptive NK cells. Additionally, ML NK cells downregulated transcription factors related to terminal maturation (ZEB2) and exhaustion (NR4A2). We next sought to identify changes during ML differentiation in patients post-HCT from day +28 to +60 post-HCT. Trajectory analysis identified a ML NK cell state distinct from cNK cells that was present at least 60 days post-HCT (Fig 1B). The ML transcriptional phenotype continued to modulate during late differentiation, including downregulation of GZMK and NCAM1, and upregulation of maturation related transcription factors, while maintaining high expression of HOPX. ML NK cells retained their enhanced functionality during in vivo differentiation, as patient ML NK cells had significantly increased IFNγ production compared to cNK cells after restimulation with leukemia targets or cytokines using mass cytometry (Fig. 2). Subsequently, we confirmed the ML CITE-seq profile in an independent clinical trial treating pediatric AML relapsed after allogenic HCT with same-donor ML NK cells (NCT03068819). In this setting, ML NK cells expressed a similar transcriptional signature and persisted for at least 2 months in the absence of exogenous cytokine support. Thus, ML NK cells possess a distinct transcriptional and surface proteomic profile and undergo in vivo differentiation while persisting within patients for at least 2 months. These findings reveal novel and unique aspects of the ML NK cell molecular program, as well as their prolonged functional persistence in vivo in patients, assisting in future clinical trial design. Figure 1 Figure 1. Disclosures Foltz: Kiadis: Patents & Royalties: TGFbeta expanded NK cells; EMD Millipore: Other: canine antibody licensing fees. Berrien-Elliott: Wugen: Consultancy, Patents & Royalties: 017001-PRO1, Research Funding. Bednarski: Horizon Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Fehniger: Wugen: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties: related to memory like NK cells, Research Funding; ImmunityBio: Research Funding; Kiadis: Other; Affimed: Research Funding; Compass Therapeutics: Research Funding; HCW Biologics: Research Funding; OrcaBio: Other; Indapta: Other.

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Expression of S100A9 in Bone Marrow Cells Differentiates Refractory Cytopenia with Multilineage Dysplasia (RCMD) from Refractory Anemia with Excess Blasts (RAEB) and Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood.v128.22.4303.4303 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4303-4303 ◽

Cited By ~ 1

Author(s):

Paul Brent Ferrell ◽

Caroline R. Maier ◽

Mikael Roussel ◽

Michael R. Savona ◽

Jonathan Michael Irish

Keyword(s):

Bone Marrow ◽

T Cells ◽

Board Of Directors ◽

Research Funding ◽

Bone Marrow Cells ◽

Myeloid Cells ◽

Mass Cytometry ◽

Immune Microenvironment ◽

Advisory Committees ◽

Marrow Cells

Abstract Introduction: Myelodysplastic syndrome (MDS) is a heterogeneous group of bone marrow disorders with a yearly incidence of approximately 13,000 in the United States. It has been observed that both genetic mutations within stem and progenitor cells and a disordered immune microenvironment are present early in MDS. Abnormal levels of inflammatory cytokines as well increased numbers of suppressive cell types, such as regulatory T cells and myeloid derived suppressor cells (MDSC) have been noted in MDS bone marrow. MDSC are recently discovered subset of myeloid cells with specific immune regulatory functions, such as T cells suppression, seen in pathological conditions, such as cancer. Recent data suggest MDSC may play a critical role in MDS pathogenesis, and that S100A9, a danger-associated molecular pattern (DAMP) produced by some myeloid cells, including neutrophils, monocytes and MDSC, is a key signal for bone marrow immune dysregulation. Here, we report a systems immunology approach to cell type discovery within MDS bone marrow using high dimensional mass cytometry. Methods: Bone marrow aspirate samples with informed consent from MDS (n=19) and AML (n=4) patients were collected and cryopreserved following red blood cell lysis for storage by the Vanderbilt Hematology Tissue Repository, a tissue repository approved by the local Institutional Review Board (IRB). Samples were acquired for the study and stained with a 35-marker panel of metal tagged mass cytometry antibodies and analyzed with a mass cytometer (CyTOF). Cellular populations were then characterized using biaxial gating as well as viSNE, SPADE and hierarchical clustering as has been previously reported (Diggins et al. Methods 2015, Ferrell et al. PLoS One, 2016). Results: Unsupervised viSNE analysis of 35-markers per cell revealed distinct cellular subsets within each sample. Interestingly, one of the strongest marker signals was expression of S100A9, which was seen in multiple cells types including phenotypic MDSC. Further analysis revealed that as a percentage of bone marrow cells, S100A9 expression was significantly more common in RCMD vs. RAEB and AML (30.0% (n=10) vs. 10.9% (n=9) and 2.4% (n=4), respectively, p<0.05 for each comparison) (Figure 1A). Additionally, three paired RCMD/AML samples were available for analysis. Within these patients, the percentage of S100A9+ cells dropped from a mean of 41.7% in RCMD to a mean of 1.84% in AML bone marrow (Figure 1B&C). Conclusion: S100A9 is both a distinguishing feature of RCMD and of disease progression within MDS. Because of its important role inflammation and cellular recruitment, S100A9 may correlate with bone marrow cellular inflammation and could represent a viable target in treatment of the disordered immune microenvironment present in MDS, especially RCMD. Disclosures Savona: Celgene: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Membership on an entity's Board of Directors or advisory committees; Amgen Inc.: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; Takeda: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees. Irish:Incyte: Research Funding; Janssen: Research Funding; Cytobank, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Low-Dose Interleukin-2 Therapy Activates Circulating T Follicular Regulatory Cells and Suppresses Circulating T Follicular Helper Cells in Patients with Chronic Gvhd

Blood ◽

10.1182/blood.v130.suppl_1.74.74 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 74-74

Author(s):

Yusuke Kamihara ◽

Edouard Forcade ◽

John Koreth ◽

Hongye Liu ◽

Tomohiro Kubo ◽

...

Keyword(s):

B Cell ◽

Single Cell ◽

Board Of Directors ◽

Research Funding ◽

Low Dose ◽

Cell Mass ◽

Mass Cytometry ◽

Advisory Committees ◽

Healthy Donors

Abstract Introduction: T follicular helper (TFH) and T follicular regulatory (TFR) cells play important roles in the regulation of B-cell immunity. While TFH promote B cell functions in the germinal center (GC), TFR function as negative regulators of the GC response. Previous studies in murine models established that TFH and GC B cells are required for the development of chronic graft-versus-host disease (cGVHD). We previously reported that circulating TFH (cTFH) were more functionally activated in patients with active cGVHD compared with patients with no cGVHD. Low-dose IL-2 therapy has been shown to selectively expand CD4Treg and improve cGVHD symptoms. In the current study, we examined the effects of IL-2 therapy on cTFH and circulating TFR (cTFR) in patients with steroid resistant cGVHD. Methods: Single cell mass cytomtery (CyTOF) was performed on cryopreserved peripheral blood mononuclear cells (PBMC) from healthy donors and 17 adult patients with active cGVHD receiving daily low-dose IL-2 therapy (Koreth et al. Blood 2016). A panel of 35 metal-tagged monoclonal antibodies was used to simultaneously examine the phenotypic and functional effects of low-dose IL-2 on lymphocyte populations in vitro and in vivo. The analytic panel included 22 cell surface markers to identify distinct lymphocyte subsets and 13 intracellular markers to measure functional status and activation of specific signaling pathways. Before staining for surface and intracellular antigens, serial samples from individual patients were barcoded to ensure uniformity of analysis. viSNE was used to visualize of high-dimensional data on a two-dimensional map and quantify single cell mass cytometry data. Results: In PBMC from healthy donors, expression of CD25 (IL-2Rα), CD95, CTLA-4, BLIMP-1 and GITR was higher in cTFR compared with cTFH. To examine the response to IL-2 in vitro, PBMC from healthy donors were stimulated with IL-2 for 15 minutes (Figure 1A). At low IL-2 concentrations (1 to 10 IU/mL), phospho-STAT5 (p-STAT5) was selectively activated in cTFR compared with cTFH. At high IL-2 concentrations (100 to 1,000 IU/mL), p-STAT5 was activated in both cTFR and cTFH. To examine the response to IL-2 in vivo, we used mass cytometry to examine serial PBMC samples from cGVHD patients receiving daily low dose IL-2 therapy (1x106 IU/M2/day). Selective expansion of cTFR was noted after 1 week of treatment and cTFR expansion remained stable for the 12 week duration of therapy. Expanded cTFR increased expression of p-STAT5, FoxP3, BCL6, HLA-DR (Figure 1B) and CD25, CD95, CTLA-4, ICOS, Ki67 and Helios 1 week after starting IL-2. cTFR:cTFH ratio increased rapidly after starting low dose IL-2 and paralleled the increased Treg:Tcon ratio (Figure 1C). Activated TFH and TFR can be identified by expression of ICOS and PD-1. The expansion of ICOS+PD-1+ cTFR was evident after 1 week of IL-2 and remained elevated at the end of therapy. In contrast, ICOS+PD-1+ cTFH increased 1 week after starting IL-2 therapy but subsequently decreased and fell below baseline 6 and 12 weeks after starting IL-2 (Figure 1D). Activated ICOS+PD-1+ cTFR expressed higher levels of p-STAT5, BCL-6, FoxP3, HLA-DR and CD25 during low dose IL-2 therapy. In contrast, these functional markers were not increased in ICOS+PD-1+ cTFH during IL-2 therapy (Figure 1B). Conclusion: Single cell mass cytometry analysis revealed that daily low dose IL-2 therapy induces selective activation and increased expression of functional proteins in ICOS+PD-1+ cTFR. In contrast, activated ICOS+PD-1+ cTFH were suppressed during IL-2 therapy. The selective activation of cTFR and suppression of cTFH provide a mechanism whereby low dose IL-2 therapy can promote B cell tolerance as well as T cell tolerance in patients with cGVHD. Disclosures Forcade: Neovii: Other: Travel grant. Koreth: Amgen Inc.: Consultancy; Prometheus Labs: Research Funding; Kadmon Corp: Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals: Research Funding; Takeda Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Nikiforow: Kite Therapeutics: Membership on an entity's Board of Directors or advisory committees. Armand: Infinity: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Otsuka: Research Funding; Tensha: Research Funding; Sequenta/Adaptive: Research Funding; Genmab: Consultancy; Affimed: Research Funding; Sigma Tau: Research Funding; Merck & Co., Inc.: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Roche: Research Funding. Cutler: Bristol-Myers Squibb: Consultancy; Pfizer: Consultancy; Kite: Consultancy; Pharmacyclics: Consultancy; Incyte: Consultancy; Astellas: Consultancy.

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Single Cell Signaling Responses Stratify Immune and Leukemia Cell Populations in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2019-129602 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2680-2680

Author(s):

Kathryn I Sunthankar ◽

Benjamin J Reisman ◽

Candace H Cote ◽

Paul Brent Ferrell

Keyword(s):

Bone Marrow ◽

T Cells ◽

Cd8 T Cells ◽

Intracellular Signaling ◽

Myeloid Leukemia ◽

Research Funding ◽

Leukemia Cell ◽

Mass Cytometry ◽

Acute Myeloid ◽

Mass Tag

In healthy hematopoiesis, cell identity and signaling response are tightly linked, with predictable cell type-specific responses to cytokines and growth factors. However, this correlation is often disrupted in myeloid malignancies, including acute myeloid leukemia (AML), wherein signaling responses may be driven directly by kinase mutational activation or cell state changes due to epigenetic alterations, for instance. In order to resolve ligand-driven signaling pathways in bone marrow, tools that allow simultaneous phenotypic characterization and functional cellular responses at single cell resolution are needed. Here, we present the use of a high content mass cytometry panel combined with mass-tag cell barcoding in order to characterize cell identity and signaling responses in bone marrow hematopoietic cells from healthy donors and leukemic patients. We created a phospho-specific mass cytometry panel comprising 24 surface phenotyping markers to resolve the predominant cellular subsets within bone marrow and blood. We perturbed cellular signaling with nine growth factors, cytokines, and chemicals and measured immediate (15 minute) responses at 10 intracellular signaling markers (pSTAT1, pSTAT3, pSTAT5, pSYK, pp38, pERK1/2, pS6, pNFkB, IkBa, & pAKT). To improve robustness of the signaling response analysis, we used mass-tag cell barcoding with palladium prior to surface and intracellular antibody staining, followed by computational debarcoding. Downstream analysis was performed with Cytobank and R. The data set included thirty-five AML patient samples and seven healthy controls with greater than 300,000 cells collected over the 10 barcoded conditions (unstimulated and 9 stimulation conditions). Dimensionality reduction with uniform manifold approximation and projection (UMAP) combined with topological clustering (HDBSCAN) enabled initial data analysis and was followed by expert identification of resultant clusters via surface marker expression. Density-based clustering of the common UMAP embedding of all samples identified known subsets of hematopoietic cells (B cells, CD4 (CD25+ and CD25-) and CD8 T cells, double negative (DN) T cells, NK cells (three subsets), erythroblasts, several subsets of myeloid and leukemia cells, and hematopoietic stem cells (HSCs)). Mass-tag cell barcoding provided stable UMAP embeddings for each sample over the 10 stimulation conditions. High dimensional signaling response was calculated per cell and per each major cell subset for the 90 nodes (9 conditions by 10 markers) and hierarchical clustering stratified samples based upon signaling signatures. Signaling responses varied across non-leukemia and leukemia cell populations in AML samples, whereas cellular phenotypes were more well correlated with signaling phenotypes in healthy samples. Heterogeneity in signaling response was driven by variability seen in several "stimulation:response" pairs. The most impactful pairs to clustering of AML blasts were IFNγ:pSTAT1, GM-CSF:pSTAT5, IL-3:pSTAT5, PMA:pS6, and IL-6:pSTAT3. Favorable risk samples (by European LeukemiaNet risk stratification) were found to have significantly larger pSTAT5 increases to IL-3 and GM-CSF than both intermediate and adverse risk subgroups. In CD8 T cells, responsiveness to PMA and IL-10 drove clustering, and, in particular, samples with ELN adverse risk showed reduced PMA:pS6 and PMA:pERK responses. We present a robust evaluation of intracellular signaling responses in the bone marrow cellular environment of AML. These data provide rationale for ongoing investigation aimed at targeting both leukemia and non-leukemia cell signaling pathways in the treatment of AML. Disclosures Ferrell: Incyte: Research Funding; Forma Therapeutics: Research Funding; Agios: Consultancy; Astex Pharmaceuticals: Research Funding.

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