Engineering Improved CAR T Cell Products with A Multi-Cytokine Particle Platform for Hematologic and Solid Tumors.

Despite the remarkable clinical efficacy of chimeric antigen receptor (CAR) T cells in hematological malignancies, only a subset of patients achieves a durable complete response (dCR). DCR has been correlated with CAR T cell products enriched with T cells memory phenotypes. Therefore, reagents that consistently promote memory phenotypes during the manufacturing of CAR T cells have the potential to significantly improve clinical outcomes. A novel modular multi-cytokine particle (MCP) platform is developed that combines the signals necessary for activation, costimulation, and cytokine support into a single "all-in-one" stimulation reagent for CAR T cell manufacturing. This platform allows for the assembly and screening of compositionally diverse MCP libraries to identify formulations tailored to promote specific phenotypes with a high degree of flexibility. The approach is leveraged to identify unique MCP formulations that manufacture CAR T cell products from diffuse large B cell patients   with increased proportions of memory-like phenotypes MCP-manufactured CAR T cells demonstrate superior anti-tumor efficacy in mouse models of lymphoma and ovarian cancer through enhanced persistence. These findings serve as a proof-of-principle of the powerful utility of the MCP platform to identify "all-in-one" stimulation reagents that can improve the effectiveness of cell therapy products through optimal manufacturing.

Association of race/ethnicity with innate immune tumor microenvironment of children with B-acute lymphoblastic leukemia.

BACKGROUND: Black and Hispanic children with B-acute lymphoblastic leukemia (B-ALL) experience worse outcomes compared with their non-Hispanic white (NHW) counterparts. Immune-based approaches have begun to transform the therapeutic landscape in children with B-ALL. Recent studies identified several alterations in both innate and adaptive immune cells in children with B-ALL that may impact disease risk and outcome. However, the impact of racial/ethnic background on immune microenvironment is less studied, as children of minorities background have to date been severely under-represented in such studies. METHODS: We performed high-dimensional analysis of bone marrow from 85 children with newly diagnosed B-ALL (Hispanic=29, black=18, NHW=38) using mass cytometry with 40 and 38-marker panels. RESULTS: Race/ethnicity-associated differences were most prominent in the innate immune compartment. Hispanic patients had significantly increased proportion of distinct mature CD57 +T-bet+DR+ NK cells compared with other cohorts. These differences were most apparent within standard risk (SR) patients with Hispanic SR patients having greater numbers of CD57 +NK cells compared with other cohorts (43% vs 26% p=0.0049). Hispanic and Black children also had distinct alterations in myeloid cells, with a significant increase in a population of non-classical activated HLA-DR +CD16+myeloid cells, previously implicated in disease progression, compared with NHW counterparts. Racial background also correlated with altered expression of inhibitory checkpoint PD-L1 on myeloid cells. CONCLUSION: There are surprisingly substantial race/ethnicity-based differences in innate immune cells of children with newly diagnosed B-ALL. These differences urge the need to enhance accrual of children from minorities background in immunetherapy trials and may impact their outcome following such therapy.

HLA-DR cancer cells expression correlates with T cell infiltration and is enriched in lung adenocarcinoma with indolent behavior.

Lung adenocarcinoma (ADC) is a heterogeneous group of tumors associated with different survival rates, even when detected at an early stage. Here, we aim to investigate whether CyTOF identifies cellular and molecular predictors of tumor behavior. We developed and validated a CyTOF panel of 34 antibodies in four ADC cell lines and PBMC. We tested our panel in a set of 10 ADCs, classified into long- (LPS) (n = 4) and short-predicted survival (SPS) (n = 6) based on radiomics features. We identified cellular subpopulations of epithelial cancer cells (ECC) and their microenvironment and validated our results by multiplex immunofluorescence (mIF) applied to a tissue microarray (TMA) of LPS and SPS ADCs. The antibody panel captured the phenotypical differences in ADC cell lines and PBMC. LPS ADCs had a higher proportion of immune cells. ECC clusters (ECCc) were identified and uncovered two ADC groups. ECCc with high HLA-DR expression were correlated with CD4+ and CD8+ T cells, with LPS samples being enriched for those clusters. We confirmed a positive correlation between HLA-DR expression on ECC and T cell number by mIF staining on TMA slides. Spatial analysis demonstrated shorter distances from T cells to the nearest ECC in LPS. Our results demonstrate a distinctive cellular profile of ECC and their microenvironment in ADC. We showed that HLA-DR expression in ECC is correlated with T cell infiltration, and that a set of ADCs with high abundance of HLA-DR+ ECCc and T cells is enriched in LPS samples. This suggests new insights into the role of antigen presenting tumor cells in tumorigenesis.

Risk-associated alterations in marrow T cells in pediatric leukemia.

Current management of childhood leukemia is tailored based on disease risk determined by clinical features at presentation. Whether properties of the host immune response impact disease risk and outcome is not known. Here, we combine mass cytometry, single cell genomics, and functional studies to characterize the BM immune environment in children with B cell acute lymphoblastic leukemia and acute myelogenous leukemia at presentation. T cells in leukemia marrow demonstrate evidence of chronic immune activation and exhaustion/dysfunction, with attrition of naive T cells and TCF1+ stem-like memory T cells and accumulation of terminally differentiated effector T cells. Marrow-infiltrating NK cells also exhibit evidence of dysfunction, particularly in myeloid leukemia. Properties of immune cells identified distinct immune phenotype-based clusters correlating with disease risk in acute lymphoblastic leukemia. High-risk immune signatures were associated with expression of stem-like genes on tumor cells. These data provide a comprehensive assessment of the immune landscape of childhood leukemias and identify targets potentially amenable to therapeutic intervention. These studies also suggest that properties of the host response with depletion of naive T cells and accumulation of terminal-effector T cells may contribute to the biologic basis of disease risk. Properties of immune microenvironment identified here may also impact optimal application of immune therapies, including T cell-redirection approaches in childhood leukemia.

Early alterations in stem-like/resident T cells, innate and myeloid cells in the bone marrow in preneoplastic gammopathy.

Preneoplastic lesions carry many of the antigenic targets found in cancer cells but often exhibit prolonged dormancy. Understanding how the host response to premalignancy is maintained and altered during malignant transformation is needed to prevent cancer. In order to understand the immune microenvironment in precursor monoclonal gammopathy of undetermined significance (MGUS) and myeloma, we analyzed bone marrow immune cells from 12 healthy donors and 26 MGUS/myeloma patients by mass cytometry and concurrently profiled transcriptomes of 42,606 single immune cells from these bone marrows. Compared to age-matched healthy donors, memory T cells from both MGUS and myeloma patients exhibit greater terminal-effector differentiation. However, memory T cells in MGUS show greater enrichment of stem-like TCF1/7hi cells. Clusters of T cells with stem-like and tissue-residence genes were also found to be enriched in MGUS by single-cell transcriptome analysis. Early changes in both NK and myeloid cells were also observed in MGUS. Enrichment of stem-like T cells correlated with a distinct genomic profile of myeloid cells and levels of Dickkopf-1 in bone-marrow plasma. These data describe the landscape of changes in both innate and adaptive immunity in premalignancy and suggest that attrition of the bone-marrow-resident T cell compartment due to loss of stem-like cells may underlie loss of immune surveillance in myeloma.

Computational Immune Monitoring Reveals Abnormal Double-Negative T Cells Present across Human Tumor Types.

Advances in single-cell biology have enabled measurements of >40 protein features on millions of immune cells within clinical samples. However, the data analysis steps following cell population identification are susceptible to bias, time-consuming, and challenging to compare across studies. Here, an ensemble of unsupervised tools was developed to evaluate four essential types of immune cell information, incorporate changes over time, and address diverse immune monitoring challenges. The four complementary properties characterized were (i) systemic plasticity, (ii) change in population abundance, (iii) change in signature population features, and (iv) novelty of cellular phenotype. Three systems immune monitoring studies were selected to challenge this ensemble approach. In serial biopsies of melanoma tumors undergoing targeted therapy, the ensemble approach revealed enrichment of double-negative (DN) T cells. Melanoma tumor-resident DN T cells were abnormal and phenotypically distinct from those found in nonmalignant lymphoid tissues, but similar to those found in glioblastoma and renal cell carcinoma. Overall, ensemble systems immune monitoring provided a robust, quantitative view of changes in both the system and cell subsets, allowed for transparent review by human experts, and revealed abnormal immune cells present across multiple human tumor types.

BRAF and MEK inhibitor therapy eliminates Nestin-expressing melanoma cells in human tumors.

Little is known about the in vivo impacts of targeted therapy on melanoma cell abundance and protein expression. Here, 21 antibodies were added to an established melanoma mass cytometry panel to measure 32 cellular features, distinguish malignant cells, and characterize dabrafenib and trametinib responses in BRAFV600mut melanoma. Tumor cells were biopsied before neoadjuvant therapy and compared to cells surgically resected from the same site after 4 weeks of therapy. Approximately 50,000 cells per tumor were characterized by mass cytometry and computational tools t-SNE/viSNE, FlowSOM, and MEM. The resulting single-cell view of melanoma treatment response revealed initially heterogeneous melanoma tumors were consistently cleared of Nestin-expressing melanoma cells. Melanoma cell subsets that persisted to week 4 were heterogeneous but expressed SOX2 or SOX10 proteins and specifically lacked surface expression of MHC I proteins by MEM analysis. Traditional histology imaging of tissue microarrays from the same tumors confirmed mass cytometry results, including persistence of NES- SOX10+ S100ß+ melanoma cells. This quantitative single-cell view of melanoma treatment response revealed protein features of malignant cells that are not eliminated by targeted therapy.

Preparing Viable Single Cells from Human Tissue and Tumors for Cytomic Analysis.

Mass cytometry is a single-cell biology technique that samples >500 cells per second, measures >35 features per cell, and is sensitive across a dynamic range of >104 relative intensity units per feature. This combination of technical assets has powered a series of recent cytomic studies where investigators used mass cytometry to measure protein and phospho-protein expression in millions of cells, characterize rare cell types in healthy and diseased tissues, and reveal novel, unexpected cells. However, these advances largely occurred in studies of blood, lymphoid tissues, and bone marrow, since the cells in these tissues are readily obtained in single-cell suspensions. This unit establishes a primer for single-cell analysis of solid tumors and tissues, and has been tested with mass cytometry. The cells obtained from these protocols can be fixed for study, cryopreserved for long-term storage, or perturbed ex vivo to dissect responses to stimuli and inhibitors. © 2017 by John Wiley & Sons, Inc.

Single cell analysis of human tissues and solid tumors with mass cytometry.

BACKGROUND: Mass cytometry measures 36 or more markers per cell and is an appealing platform for comprehensive phenotyping of cells in human tissue and tumor biopsies. While tissue disaggregation and fluorescence cytometry protocols were pioneered decades ago, it is not known whether established protocols will be effective for mass cytometry and maintain cancer and stromal cell diversity. METHODS: Tissue preparation techniques were systematically compared for gliomas and melanomas, patient derived xenografts of small cell lung cancer, and tonsil tissue as a control. Enzymes assessed included DNase, HyQTase, TrypLE, collagenase (Col) II, Col IV, Col V, and Col XI. Fluorescence and mass cytometry were used to track cell subset abundance following different enzyme combinations and treatment times. RESULTS: Mechanical disaggregation paired with enzymatic dissociation by Col II, Col IV, Col V, or Col XI plus DNase for 1 h produced the highest yield of viable cells per gram of tissue. Longer dissociation times led to increasing cell death and disproportionate loss of cell subsets. Key markers for establishing cell identity included CD45, CD3, CD4, CD8, CD19, CD64, HLA-DR, CD11c, CD56, CD44, GFAP, S100B, SOX2, nestin, vimentin, cytokeratin, and CD31. Mass and fluorescence cytometry identified comparable frequencies of cancer cell subsets, leukocytes, and endothelial cells in glioma (R = 0.97), and tonsil (R = 0.98). CONCLUSIONS: This investigation establishes standard procedures for preparing viable single cell suspensions that preserve the cellular diversity of human tissue microenvironments. © 2016 International Clinical Cytometry Society.

Melanoma-specific MHC-II expression represents a tumour-autonomous phenotype and predicts response to anti-PD-1/PD-L1 therapy.

Anti-PD-1 therapy yields objective clinical responses in 30-40% of advanced melanoma patients. Since most patients do not respond, predictive biomarkers to guide treatment selection are needed. We hypothesize that MHC-I/II expression is required for tumour antigen presentation and may predict anti-PD-1 therapy response. In this study, across 60 melanoma cell lines, we find bimodal expression patterns of MHC-II, while MHC-I expression was ubiquitous. A unique subset of melanomas are capable of expressing MHC-II under basal or IFNγ-stimulated conditions. Using pathway analysis, we show that MHC-II(+) cell lines demonstrate signatures of 'PD-1 signalling', 'allograft rejection' and 'T-cell receptor signalling', among others. In two independent cohorts of anti-PD-1-treated melanoma patients, MHC-II positivity on tumour cells is associated with therapeutic response, progression-free and overall survival, as well as CD4(+) and CD8(+) tumour infiltrate. MHC-II(+) tumours can be identified by melanoma-specific immunohistochemistry using commercially available antibodies for HLA-DR to improve anti-PD-1 patient selection.

High-dimensional single-cell cancer biology.

Cancer cells are distinguished from each other and from healthy cells by features that drive clonal evolution and therapy resistance. New advances in high-dimensional flow cytometry make it possible to systematically measure mechanisms of tumor initiation, progression, and therapy resistance on millions of cells from human tumors. Here we describe flow cytometry techniques that enable a "single-cell " view of cancer. High-dimensional techniques like mass cytometry enable multiplexed single-cell analysis of cell identity, clinical biomarkers, signaling network phospho-proteins, transcription factors, and functional readouts of proliferation, cell cycle status, and apoptosis. This capability pairs well with a signaling profiles approach that dissects mechanism by systematically perturbing and measuring many nodes in a signaling network. Single-cell approaches enable study of cellular heterogeneity of primary tissues and turn cell subsets into experimental controls or opportunities for new discovery. Rare populations of stem cells or therapy-resistant cancer cells can be identified and compared to other types of cells within the same sample. In the long term, these techniques will enable tracking of minimal residual disease (MRD) and disease progression. By better understanding biological systems that control development and cell-cell interactions in healthy and diseased contexts, we can learn to program cells to become therapeutic agents or target malignant signaling events to specifically kill cancer cells. Single-cell approaches that provide deep insight into cell signaling and fate decisions will be critical to optimizing the next generation of cancer treatments combining targeted approaches and immunotherapy.