scholarly journals Single-Cell RNA-Seq Reveals Naïve B cells Associated with Better Prognosis of HCC

2019 ◽  
Author(s):  
Jian He ◽  
Yingxin Lin ◽  
Xianbin Su ◽  
Qing Luo ◽  
Shila Ghazanfar ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
B Cells ◽  
Single Cell ◽  
Solid Tumor ◽  
Immune Cells ◽  
High Morbidity ◽  
Malignant Solid Tumor ◽  
Functional States ◽  
Interaction Map ◽  
The Impact

AbstractHepatocellular Carcinoma (HCC) is a type of malignant solid tumor, causing high morbidity and mortality around the world and the major portion of HCC patients is from China. Cancer immunotherapies have shown some clinical responses in treating some types of cancer but did not shown significant efficiency in HCC treatment. This in part due to the impact of immune cells in the tumor microenvironment. It is commonly believed that HCC is a heterogeneous solid tumor and the microenvironment of HCC plays an important role in tumorgenesis and development. Currently, the residents of the microenvironment of HCC is not well-defined and clarification, especially the immune cells, which we believe that paly pivotal roles in tumorgenesis and development. To depict the landscape of the composition, lineage and functional states of the immune cells in HCC, we performed single-cell RNA sequencing on Diethylnitrosamine (DEN)-induced mouse HCC model. We observed heterogeneity within the immune and hepatocytes both in the precancerous condition of tumorigenesis and cancerous condition of HCC. In this study we found that the disease-associated changes appeared early in pathological progression and were highly cell-type specific. Specific subsets of T and B cells preferentially enriched in HCC, and we identified signature genes for each subset. Additionally, we mapped this group of specific cells to the human TCGA database. We found a cluster of naïve B cells characterized by high expression of CD38 associated with better prognosis of human HCC. Our study demonstrates signaling interaction map based on receptor-ligand bonding on the single-cell level could broaden our comprehending of cellular networks in varies status. Our finding provides a new approach for patient stratification and will help further understand the functional states, dynamics and signaling interaction of B cells in hepatocellular carcinoma, and may provide a novel insight and therapeutics for the HCC.

Single Cell Profiling Reveals Unique CXCL13 Positive T Cell Subsets in the Tumor Microenvironment of Lymphocyte Rich Classic Hodgkin Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 32-33
Author(s):  
Tomohiro Aoki ◽  
Lauren C. Chong ◽  
Katsuyoshi Takata ◽  
Katy Milne ◽  
Elizabeth Chavez ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Single Cell ◽  
Immune Cells ◽  
Research Funding ◽  
Immune Cell ◽  
Expression Patterns ◽  
The Other ◽  
Tfh Cells

Introduction: Classic Hodgkin lymphoma (CHL) features a unique crosstalk between malignant cells and different types of normal immune cells in the tumor-microenvironment (TME). On the basis of histomorphologic and immunophenotypic features of the malignant Hodgkin and Reed-Sternberg (HRS) cells and infiltrating immune cells, four histological subtypes of CHL are recognized: Nodular sclerosing (NS), Mixed cellularity, Lymphocyte-rich (LR) and Lymphocyte-depleted CHL. Recently, our group described the high abundance of various types of immunosuppressive CD4+ T cells including LAG3+ and/or CTLA4+ cells in the TME of CHL using single cell RNA sequencing (scRNAseq). However, the TME of LR-CHL has not been well characterized due to the rarity of the disease. In this study, we aimed at characterizing the immune cell profile of LR-CHL at single cell resolution. METHODS: We performed scRNAseq on cell suspensions collected from lymph nodes of 28 primary CHL patients, including 11 NS, 9 MC and 8 LR samples, with 5 reactive lymph nodes (RLN) serving as normal controls. We merged the expression data from all cells (CHL and RLN) and performed batch correction and normalization. We also performed single- and multi-color immunohistochemistry (IHC) on tissue microarray (TMA) slides from the same patients. In addition, an independent validation cohort of 31 pre-treatment LR-CHL samples assembled on a TMA, were also evaluated by IHC. Results: A total of 23 phenotypic cell clusters were identified using unsupervised clustering (PhenoGraph). We assigned each cluster to a cell type based on the expression of genes described in published transcriptome data of sorted immune cells and known canonical markers. While most immune cell phenotypes were present in all pathological subtypes, we observed a lower abundance of regulatory T cells (Tregs) in LR-CHL in comparison to the other CHL subtypes. Conversely, we found that B cells were enriched in LR-CHL when compared to the other subtypes and specifically, all four naïve B-cell clusters were quantitatively dominated by cells derived from the LR-CHL samples. T follicular helper (TFH) cells support antibody response and differentiation of B cells. Our data show the preferential enrichment of TFH in LR-CHL as compared to other CHL subtypes, but TFH cells were still less frequent compared to RLN. Of note, Chemokine C-X-C motif ligand 13 (CXCL13) was identified as the most up-regulated gene in LR compared to RLN. CXCL13, which is a ligand of C-X-C motif receptor 5 (CXCR5) is well known as a B-cell attractant via the CXCR5-CXCL13 axis. Analyzing co-expression patterns on the single cell level revealed that the majority of CXCL13+ T cells co-expressed PD-1 and ICOS, which is known as a universal TFH marker, but co-expression of CXCR5, another common TFH marker, was variable. Notably, classical TFH cells co-expressing CXCR5 and PD-1 were significantly enriched in RLN, whereas PD-1+ CXCL13+ CXCR5- CD4+ T cells were significantly enriched in LR-CHL. These co-expression patterns were validated using flow cytometry. Moreover, the expression of CXCR5 on naïve B cells in the TME was increased in LR-CHL compared to the other CHL subtypes We next sought to understand the spatial relationship between CXCL13+ T cells and malignant HRS cells. IHC of all cases revealed that CXCL13+ T cells were significantly enriched in the LR-CHL TME compared to other subtypes of CHL, and 46% of the LR-CHL cases showed CXCL13+ T cell rosettes closely surrounding HRS cells. Since PD-1+ T cell rosettes are known as a specific feature of LR-CHL, we confirmed co-expression of PD-1 in the rosetting cells by IHC in these cases. Conclusions: Our results reveal a unique TME composition in LR-CHL. LR-CHL seems to be distinctly characterized among the CHL subtypes by enrichment of CXCR5+ naïve B cells and CD4+ CXCL13+ PD-1+ T cells, indicating the importance of the CXCR5-CXCL13 axis in the pathogenesis of LR-CHL. Figure Disclosures Savage: BeiGene: Other: Steering Committee; Merck, BMS, Seattle Genetics, Gilead, AstraZeneca, AbbVie: Honoraria; Roche (institutional): Research Funding; Merck, BMS, Seattle Genetics, Gilead, AstraZeneca, AbbVie, Servier: Consultancy. Scott:Janssen: Consultancy, Research Funding; Celgene: Consultancy; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoString, Research Funding; NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America.; Roche/Genentech: Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy. Steidl:AbbVie: Consultancy; Roche: Consultancy; Curis Inc: Consultancy; Juno Therapeutics: Consultancy; Bayer: Consultancy; Seattle Genetics: Consultancy; Bristol-Myers Squibb: Research Funding.

scholarly journals Identification of Inflammatory Genes, Pathways, and Immune Cells in Necrotizing Enterocolitis of Preterm Infant by Bioinformatics Approaches

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Lili Zhang ◽  
Lizhen Sun ◽  
Mingli Wu ◽  
Jie Huang
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Rna Sequencing ◽  
Necrotizing Enterocolitis ◽  
Immune Cells ◽  
Gastrointestinal Disease ◽  
Gene Set Enrichment Analysis ◽  
High Morbidity ◽  
Expression Levels ◽  
Gene Expression Levels

Background. Necrotizing enterocolitis (NEC) is one of the most serious gastrointestinal disease-causing high morbidity and mortality in premature infants. However, the underlying mechanism of the pathogenesis of NEC is still not fully understood. Methods. RNA sequencing of intestinal specimens from 9 NEC and 5 controls was employed to quantify the gene expression levels. RNA sequencing was employed to quantify the gene expression levels. DESeq2 tool was used to identify the differentially expressed genes. The biological function, pathways, transcription factors, and immune cells dysregulated in NEC were characterized by gene set enrichment analysis. Results. In the present study, we analyzed RNA sequencing data of NECs and controls and revealed that immune-related pathways were highly activated, while some cellular responses to external stimuli-related pathways were inactivated in NEC. Moreover, B cells, macrophages M1, and plasma cells were identified as the major cell types involved in NEC. Furthermore, we also found that inflammation-related transcription factor genes, such as STAT1, STAT2, and IRF2, were significantly activated in NEC, further suggesting that these TFs might play critical roles in NEC pathogenesis. In addition, NEC samples exhibited heterogeneity to some extent. Interestingly, two subgroups in the NEC samples were identified by hierarchical clustering analysis. Notably, B cells, T cells, Th1, and Tregs involved in adaptive immune were predicted to highly infiltrate into subgroup I, while subgroup II was significantly infiltrated by neutrophils. The heterogeneity of immune cells in NEC indicated that both innate and adaptive immunes might induce NEC-related inflammatory response. Conclusions. In summary, we systematically analyzed inflammation-related genes, signaling pathways, and immune cells to characterize the NEC pathogenesis and samples, which greatly improved our understanding of the roles of inflammatory responses in NEC.

scholarly journals Global immune characterization of HBV/HCV-related hepatocellular carcinoma identifies macrophage and T-cell subsets associated with disease progression

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Guohe Song ◽  
Yang Shi ◽  
Meiying Zhang ◽  
Shyamal Goswami ◽  
Saifullah Afridi ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Immune Cells ◽  
Immune Cell ◽  
T Cell Subsets ◽  
M2 Macrophage ◽  
Advanced Hcc ◽  
Immune Cell Subsets

AbstractDiverse immune cells in the tumor microenvironment form a complex ecosystem, but our knowledge of their heterogeneity and dynamics within hepatocellular carcinoma (HCC) still remains limited. To assess the plasticity and phenotypes of immune cells within HBV/HCV-related HCC microenvironment at single-cell level, we performed single-cell RNA sequencing on 41,698 immune cells from seven pairs of HBV/HCV-related HCC tumors and non-tumor liver tissues. We combined bio-informatic analyses, flow cytometry, and multiplex immunohistochemistry to assess the heterogeneity of different immune cell subsets in functional characteristics, transcriptional regulation, phenotypic switching, and interactions. We identified 29 immune cell subsets of myeloid cells, NK cells, and lymphocytes with unique transcriptomic profiles in HCC. A highly complex immunological network was shaped by diverse immune cell subsets that can transit among different states and mutually interact. Notably, we identified a subset of M2 macrophage with high expression of CCL18 and transcription factor CREM that was enriched in advanced HCC patients, and potentially participated in tumor progression. We also detected a new subset of activated CD8+ T cells highly expressing XCL1 that correlated with better patient survival rates. Meanwhile, distinct transcriptomic signatures, cytotoxic phenotypes, and evolution trajectory of effector CD8+ T cells from early-stage to advanced HCC were also identified. Our study provides insight into the immune microenvironment in HBV/HCV-related HCC and highlights novel macrophage and T-cell subsets that could be further exploited in future immunotherapy.

scholarly journals Expression and prognosis analysis of JMJD5 in human cancers

2020 ◽  
Author(s):  
Hui Li ◽  
Qun Li ◽  
Hong Jing ◽  
Jianghai Zhao ◽  
Hui Zhang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
T Cells ◽  
B Cells ◽  
Lung Adenocarcinoma ◽  
Immune Cells ◽  
Prognostic Value ◽  
Normal Tissues ◽  
Human Cancers ◽  
Stomach Adenocarcinoma ◽  
Liver Hepatocellular Carcinoma

Abstract BackgroundJumonjiC (JmjC) domain-containing protein 5 (JMJD5) plays an important role in cancer metabolism. However, the prognostic value of JMJD5 in most human cancers is still unknown. In this study, we aim to investigate the expression and prognostic value of JMJD5, immune cells infiltration, and the correlations among them. MethodsWe performed a detailed cancer vs. normal analysis of JMJD5 mRNA expression via online Tumor Immune Estimation Resource (TIMER). The protein expressions of JMJD5 in various cancers vs. adjacent normal tissues were examined by immunohistochemistry (IHC) of tissue microarray sections (TMAs). Moreover, the Kaplan-Meier Plotter databases were used to evaluate the prognostic values in above cancers. The correlations between JMJD5 expression level and abundances of six immune infiltrating cells (B cells, CD4+ T cells, CD8+ T cells, macrophages, neutrophils and dendritic cells) were explored by TIMER database in breast cancer (BRCA), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC) and stomach adenocarcinoma (STAD). The prognostic values of tumor- infiltrating immune cells were also investigated by TIMER in above four cancers. Finally, the COX proportional hazards model was used to investigate the correlations among clinical outcome, the abundance of immune cell infiltrates and the expression of JMJD5 in above four cancer types.ResultsThe expression of JMJD5 was significantly lower in human breast carcinoma (BRCA), cholangiocarcinoma (CHOL), liver hepatocellular carcinoma (LIHC) and lung cancer (LUC) but higher in prostate adenocarcinoma (PRAD) and stomach adenocarcinoma (STAD) comparing to their respective normal tissues. And high expression of JMJD5 has better prognosis only in BRCA, LIHC, LUC but the opposite effect in STAD. JMJD5 expression is significant correlation with the abundance of six immune cells infiltration in above four cancers. Both the BRCA or lung adenocarcinoma (LUAD) patients with abundance of B cell and the STAD patients with low level of macrophage have a better cumulative survival. ConclusionsWe provided novel evidence of JMJD5 as an essential prognostic biomarker in cancers through analyses the correlation of the JMJD5 expression, tumor-infiltrating B cells and macrophages and prognostic value. This study offers new perspectives therapeutic target in BRCA, LUAD and STAD.

Single-cell RNA-sequencing reveals profound changes in circulating immune cells in patients with heart failure

2020 ◽  
Author(s):  
Wesley T Abplanalp ◽  
David John ◽  
Sebastian Cremer ◽  
Birgit Assmus ◽  
Lena Dorsheimer ◽  
...  
Keyword(s):  
Heart Failure ◽  
Flow Cytometry ◽  
Single Cell ◽  
Rna Sequencing ◽  
Immune Cells ◽  
Healthy Subjects ◽  
Patients With Heart Failure ◽  
Single Cell Rna Sequencing ◽  
The Impact ◽  
Classical Monocytes

Abstract Aims Identification of signatures of immune cells at single-cell level may provide novel insights into changes of immune-related disorders. Therefore, we used single-cell RNA-sequencing to determine the impact of heart failure on circulating immune cells. Methods and results We demonstrate a significant change in monocyte to T-cell ratio in patients with heart failure, compared to healthy subjects, which were validated by flow cytometry analysis. Subclustering of monocytes and stratification of the clusters according to relative CD14 and FCGR3A (CD16) expression allowed annotation of classical, intermediate, and non-classical monocytes. Heart failure had a specific impact on the gene expression patterns in these subpopulations. Metabolically active genes such as FABP5 were highly enriched in classical monocytes of heart failure patients, whereas β-catenin expression was significantly higher in intermediate monocytes. The selective regulation of signatures in the monocyte subpopulations was validated by classical and multifactor dimensionality reduction flow cytometry analyses. Conclusion Together this study shows that circulating cells derived from patients with heart failure have altered phenotypes. These data provide a rich source for identification of signatures of immune cells in heart failure compared to healthy subjects. The observed increase in FABP5 and signatures of Wnt signalling may contribute to enhanced monocyte activation.

scholarly journals Single-cell RNA sequencing shows the immunosuppressive landscape and tumor heterogeneity of HBV-associated hepatocellular carcinoma

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Daniel Wai-Hung Ho ◽  
Yu-Man Tsui ◽  
Lo-Kong Chan ◽  
Karen Man-Fong Sze ◽  
Xin Zhang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Single Cell ◽  
Tumor Cells ◽  
Rna Sequencing ◽  
Immune Cells ◽  
Tumor Heterogeneity ◽  
Sequencing Analysis ◽  
T Cell Infiltration ◽  
Single Cell Rna Sequencing ◽  
Immune Oncology

AbstractInteraction between tumor cells and immune cells in the tumor microenvironment is important in cancer development. Immune cells interact with the tumor cells to shape this process. Here, we use single-cell RNA sequencing analysis to delineate the immune landscape and tumor heterogeneity in a cohort of patients with HBV-associated human hepatocellular carcinoma (HCC). We found that tumor-associated macrophages suppress tumor T cell infiltration and TIGIT-NECTIN2 interaction regulates the immunosuppressive environment. The cell state transition of immune cells towards a more immunosuppressive and exhaustive status exemplifies the overall cancer-promoting immunocellular landscape. Furthermore, the heterogeneity of global molecular profiles reveals co-existence of intra-tumoral and inter-tumoral heterogeneity, but is more apparent in the latter. This analysis of the immunosuppressive landscape and intercellular interactions provides mechanistic information for the design of efficacious immune-oncology treatments in hepatocellular carcinoma.

scholarly journals Age-related sexual dimorphism on the longitudinal progression of blood immune cells in BALB/cByJ mice

2021 ◽  
Author(s):  
Cláudia Serre-Miranda ◽  
Susana Roque ◽  
Palmira Barreira-Silva ◽  
Claudia Nobrega ◽  
Neide Vieira ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
Sexual Dimorphism ◽  
B Cells ◽  
Nk Cells ◽  
Mouse Models ◽  
Immune Cells ◽  
Age Related ◽  
The Impact ◽  
Over Time

Abstract The study of immune system aging is of relevance, considering its myriad of interactions and role in protecting and maintaining body homeostasis. While mouse models have been extensively used to study immune system aging, little is known on how the main immune populations progress over time and what is the impact of sex. To contribute to filling this gap, male and female BALB/cByJ mice were longitudinally evaluated, from 3 to 18 months old, for the main blood populations, assessed by flow cytometry. Using linear mixed-effect models, we observed that the percentages of neutrophils, monocytes, eosinophils and total natural killer (NK) cells increase with aging; while those of B cells, T cells (including CD4 + and CD8 + subsets) and Ly6C + NK cells, decrease. Males present higher percentages of neutrophils and classical monocytes Ly6C high over time, while females present higher percentages of total T cells, both CD4 + and CD8 +, eosinophils and NK cells. Males and females display similar percentages of B cells, even though with opposite accelerated progressions over time. This study revealed that mouse models recapitulate what is observed in humans during aging: an overall proportional decrease in the adaptive and an increase in the innate immune cells. Additionally, it uncovers an age-related sexual dimorphism in the proportion of immune cells in circulation, further strengthening the need to explore the impact of sex when addressing immune system aging using mouse models.

scholarly journals Single-cell analysis of diverse immune phenotypes in malignant pleural effusion

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhong-Yin Huang ◽  
Ming-Ming Shao ◽  
Jian-Chu Zhang ◽  
Feng-Shuang Yi ◽  
Juan Du ◽  
...  
Keyword(s):  
Lung Cancer ◽  
T Cells ◽  
B Cells ◽  
Pleural Effusion ◽  
Small Cell Lung Cancer ◽  
Single Cell ◽  
Immune Cells ◽  
Malignant Pleural Effusion ◽  
Small Cell ◽  
Small Cell Lung

AbstractThe complex interactions among different immune cells have important functions in the development of malignant pleural effusion (MPE). Here we perform single-cell RNA sequencing on 62,382 cells from MPE patients induced by non-small cell lung cancer to describe the composition, lineage, and functional states of infiltrating immune cells in MPE. Immune cells in MPE display a number of transcriptional signatures enriched for regulatory T cells, B cells, macrophages, and dendritic cells compared to corresponding counterparts in blood. Helper T, cytotoxic T, regulatory T, and T follicular helper cells express multiple immune checkpoints or costimulatory molecules. Cell-cell interaction analysis identifies regulatory B cells with more interactions with CD4+ T cells compared to CD8+ T cells. Macrophages are transcriptionally heterogeneous and conform to M2 polarization characteristics. In addition, immune cells in MPE show the general up-regulation of glycolytic pathways associated with the hypoxic microenvironment. These findings show a detailed atlas of immune cells in human MPE and enhance the understanding of potential diagnostic and therapeutic targets in advanced non-small cell lung cancer.

scholarly journals Single-cell RNA-Seq reveals transcriptional heterogeneity and immune subtypes associated with disease activity in human myasthenia gravis

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Wanlin Jin ◽  
Qi Yang ◽  
Yuyao Peng ◽  
Chengkai Yan ◽  
Yi Li ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Myasthenia Gravis ◽  
Disease Activity ◽  
Single Cell ◽  
Immune Cell ◽  
Large Cell ◽  
Cellular Heterogeneity ◽  
T Cell Subsets ◽  
The Impact

AbstractMyasthenia gravis (MG) is a rare autoimmune disease. Although the impact of immune cell disorder in MG has been extensively studied, little is known about the transcriptomes of individual cells. Here, we assessed the transcriptional profiles of 39,243 cells by single-cell sequencing and identified 13 major cell clusters, along with 39 subgroups of cells derived from patients with new-onset myasthenia gravis and healthy controls. We found that B cells, CD4+ T cells, and monocytes exhibited more heterogeneity in MG patients. CD4+ T cells were expanded in MG patients. We reclustered B cells and CD4+ T cells, and predict their essential regulators. Further analyses demonstrated that B cells in MG exhibited higher transcriptional activity towards plasma cell differentiation, CD4+ T cell subsets were unbalanced, and inflammatory pathways of monocytes were highly activated. Notably, we discovered a disease-relevant subgroup, CD180− B cells. Increased CD180− B cells in MG are indicative of a high IgG composition and were associated with disease activity and the anti-AChR antibody. Together, our data further the understanding of the cellular heterogeneity involved in the pathogenesis of MG and provide large cell-type-specific markers for subsequent research.

scholarly journals Dynamic Immune Surveillance in Durable Clinical Response to Combined BTK and BCL2 Inhibition in MCL at Longitudinal Single-Cell Resolution

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1323-1323
Author(s):  
Kevin Wang ◽  
Maurizio Di Liberto ◽  
Yang Hu ◽  
Xiangao Huang ◽  
Rachel M Koldej ◽  
...  
Keyword(s):  
B Cells ◽  
Nk Cells ◽  
Single Cell ◽  
Clinical Response ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Immune Surveillance ◽  
Mantle Cell

Abstract Combined inhibition of BTK with ibrutinib and BCL2 with venetoclax is one of the most promising therapies for B cell malignancies, especially mantle cell lymphoma (MCL), where durable complete remission continued after therapy cessation in some patients (Tam et al, NEJM 2018, Handunnetti ASH 2019). The MCL-intrinsic and extrinsic mechanisms underlying this deep and durable clinical response are unknown, nor have resistance mechanisms been identified. Since BTK is expressed mainly in B lineage cells and venetoclax inhibits BCL2 universally, we hypothesize that BTK inhibition selectively primes MCL cells for vulnerability to BCL2 inhibition while maintaining immune cell homeostasis, leading to differential elimination of MCL cells through immune surveillance. To test this, we undertook integrative longitudinal single-cell RNA-sequencing analysis (scRNA-seq) of PBMCs from sequential tissue and blood specimens (n=32) of 8 MCL patients before and during ibrutinib-venetoclax combination therapy, after therapy cessation or progression, as well as 4 treatment-naïve MCL patients and 4 normal subjects as controls. High dimensional analysis using a unique MCL RNA reference library that we built from bulk RNA-seq data of MCL cells from 57 patients reveals that MCL cells comprise 4 transcriptomically distinct clusters. Cluster 1 (C1) is similar to quiescent normal B cells; C2 resembles hyper-activated B cells enriched for signatures of BCR and cytokine signaling and proinflammatory pathways; C3 represents non-proliferating, long-lived MCL cells that accumulate as disease progresses; and C4 is highly proliferative, expanding with disease progression in untreated patients or on therapy. Integrative analysis of scRNA-seq and CBC with differential showed that homeostasis of all immune cells was maintained throughout ibrutinib-venetoclax therapy and after therapy cessation in 6 MCL patients with a complete response (CR). CD8+T and NK cells were functional, evidenced by the expression of cytotoxic genes such as GNLY, FGFBP2, and GZMH. In contrast, these genes were profoundly suppressed in CD8+T cells that were rapidly depleted on MCL progression after transient response in 2 patients. NK cells were also depleted on progression. In one patient, this was preceded by suppression of cytotoxic genes and loss of MHC-I and MHC-II in MCL cells. Exhaustion did not appear to be the cause. Rather, TSC22D3 upregulation suggests that inhibition of TCR-induced IL2 and IL2R expression and NF-kB activation may underlie the loss of CD8+T and NK cells in ibrutinib-venetoclax resistance. To determine whether BTK inhibition primes MCL cells for subsequent venetoclax killing while sparing other immune cells in a clinical response to ibrutinib-venetoclax, we found that BCL2 expression was higher in MCL cells than in other immune cells pre-therapy. Ibrutinib selectively downregulated the anti-apoptotic MCL1 and upregulated PMAIP1 encoding the pro-apoptotic NOXA in MCL cells, concurrent with TSC22D3 induction and inhibition of the NF-kB signaling pathway. As expected from the lack of BTK expression, CD4+ and CD8+T cells and NK cells expressed MCL1 over PMAIP1 in CR patients, but greater PMAIP1 in patients who developed progressive MCL. Collectively, these data support the hypothesis that BTK inhibition selectively primes MCL cells for venetoclax sensitivity. scRNA-seq analysis further showed that B cells emerging after therapy cessation in CR patients were nearly identical to the bone marrow B cells pre-therapy, as quiescent B cells in cluster 1 and activated B cells in cluster 2. None were detected in clusters 3 or 4. The restoration of B cell immunity confirms CR at the single-cell transcriptome level, speaking to the power of dual BTK and BCL2 inhibition, and supporting the role of immune surveillance in clinical response in targeted therapy. In summary, by unbiased longitudinal scRNA-seq analysis of sequential patient specimens from the ibrutinib-venetoclax clinical trial, we have provided the first evidence that priming of MCL cells for venetoclax vulnerability by BTK inhibition cooperates with immune surveillance to determine the depth and durability in combined targeting of BTK and BCL2 in lymphoma in humans. These findings parallel our longitudinal scRNA-seq analysis of dual BTK and CDK4/6 inhibition, suggesting priming of cancer cells and cooperation with immune surveillance underpin targeted therapy. Disclosures Koldej: CRISPR Therapeutics: Research Funding. Ritchie: CRISPR Therapeutics: Research Funding; Takeda: Research Funding; BMS: Research Funding; Novartis: Honoraria; Amgen Inc: Honoraria, Research Funding; CSL: Honoraria. Martin: ADCT: Consultancy. Elemento: One Three Biotech: Consultancy, Other: Current equity holder; Owkin: Consultancy, Other: Current equity holder; Eli Lilly: Research Funding; AstraZeneca: Research Funding; Johnson and Johnson: Research Funding; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Janssen: Research Funding; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Champions Oncology: Consultancy. Tam: Beigene: Research Funding; Loxo: Honoraria; Beigene: Honoraria; Janssen: Honoraria; Abbvie: Honoraria; Abbvie: Research Funding; Janssen: Research Funding. OffLabel Disclosure: Venetoclax is a BCL2 inhibitor FDA-approved for chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), and acute myeloid leukemia (AML). It was used off-label in combination with ibrutinib in a phase II clinical trial in patients with mantle cell lymphoma.

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