Chikara Takahashi - Academia.edu (original) (raw)

Papers by Chikara Takahashi

Research paper thumbnail of Abstract PO084: Patterns of T cell clonal expansion in cancer patients associate with response to immunotherapy

Poster Presentations - Proffered Abstracts, 2021

Upon encountering their cognate antigens, T cells can undergo clonal expansion to produce multipl... more Upon encountering their cognate antigens, T cells can undergo clonal expansion to produce multiple copies of a cell with a shared T cell receptor (TCR). Despite the fundamental role of clonal expansion in cancer immunity, little is known about its relationship with T cell subpopulations or antitumor responses in cancer patients. Here we have performed single-cell RNA sequencing (scRNA-seq) and deep analysis of TCR clonotypes (scTCR-seq) in cancer patients across several indications, assessing the profiles of TCRs in the various populations of T cells in tumors, normal adjacent tissue (NAT), and peripheral blood. We found that, although most clonotypes were represented by a single cell, the remaining clonal lineages showed expansion in either NAT or tumor exclusively, or dual-residence with expansion in both compartments. In a subset of patients, we find clear evidence of clonotypic expansion of T effector- and effector memory-like cells not only within the tumor but also in NAT. Importantly, expanded clonotypes found in the tumor and NAT can also typically be detected in peripheral blood, suggesting a continuously replenishment from sites outside of the tumor with fresh, non-exhausted replacement cells. Our data further suggests a continued activity of the cancer immunity cycle in these patients, the acceleration of which may be associated with clinical response. Citation Format: Shravan Madireddi, Thomas D. Wu, Patricia E. de Almeida, Romain Banchereau, Ying-Jiun J. Chen, Avantika S. Chitre, Hina Iftikhar, William E. O9Gorman, Amelia Au-Yeung, Chikara Takahashi, Leonard D. Goldstein, Chungkee Poon, Shilpa Keerthivasan, Sanjeev Mariathasan, Meghna Das Thakur, Mahrukh A. Huseni, Marcus Ballinger, Ivette Estay, Patrick Caplazi, Zora Modrusan, Lelia Delamarre, Ira Mellman, Richard Bourgon, Jane L. Grogan. Patterns of T cell clonal expansion in cancer patients associate with response to immunotherapy [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO084.

Research paper thumbnail of Mechanistic convergence of the TIGIT and PD-1 inhibitory pathways necessitates co-blockade to optimize anti-tumor CD8+ T cell responses

Research paper thumbnail of Intratumoral plasma cells predict outcomes to PD-L1 blockade in non-small cell lung cancer

Cancer Cell, 2022

Inhibitors of the programmed cell death-1 (PD-1/PD-L1) signaling axis are approved to treat non-s... more Inhibitors of the programmed cell death-1 (PD-1/PD-L1) signaling axis are approved to treat non-small cell lung cancer (NSCLC) patients, based on their significant overall survival (OS) benefit. Using transcriptomic analysis of 891 NSCLC tumors from patients treated with either the PD-L1 inhibitor atezolizumab or chemotherapy from two large randomized clinical trials, we find a significant B cell association with extended OS with PD-L1 blockade, independent of CD8+ T cell signals. We then derive gene signatures corresponding to the dominant B cell subsets present in NSCLC from single-cell RNA sequencing (RNA-seq) data. Importantly, we find increased plasma cell signatures to be predictive of OS in patients treated with atezolizumab, but not chemotherapy. B and plasma cells are also associated with the presence of tertiary lymphoid structures and organized lymphoid aggregates. Our results suggest an important contribution of B and plasma cells to the efficacy of PD-L1 blockade in NSCLC.

Research paper thumbnail of Transcriptomic profiling of adjuvant colorectal cancer identifies three key prognostic biological processes and a disease specific role for granzyme B

PLOS ONE, 2021

Background Colorectal cancer (CRC) is a leading cause of cancer-related deaths, with a 5% 5-year ... more Background Colorectal cancer (CRC) is a leading cause of cancer-related deaths, with a 5% 5-year survival rate for metastatic disease, yet with limited therapeutic advancements due to insufficient understanding of and inability to accurately capture high-risk CRC patients who are most likely to recur. We aimed to improve high-risk classification by identifying biological pathways associated with outcome in adjuvant stage II/III CRC. Methods and findings We included 1062 patients with stage III or high-risk stage II colon carcinoma from the prospective three-arm randomized phase 3 AVANT trial, and performed expression profiling to identify a prognostic signature. Data from validation cohort GSE39582, The Cancer Genome Atlas, and cell lines were used to further validate the prognostic biology. Our retrospective analysis of the adjuvant AVANT trial uncovered a prognostic signature capturing three biological functions—stromal, proliferative and immune—that outperformed the Consensus Mol...

Research paper thumbnail of Visualization of Mass Cytometry Signal Background to Enable Optimal Core Panel Customization and Signal Threshold Gating

Methods in molecular biology, 2019

Signal interference or overlap in mass cytometry is minimal compared to flow cytometry but must s... more Signal interference or overlap in mass cytometry is minimal compared to flow cytometry but must still be considered for optimal panel design and assay sensitivity. Here we describe a procedure for evaluating signal interference dynamics in the context of a 25-parameter core immunophenotyping panel. Specifically, a mass-minus-many (MMM) approach was used to assess background signals in "empty" or "blank" channels intended for further customization. Through this approach cell type-specific variability in signal background is revealed. Further panel customization can thus be performed with an understanding of cell type and channel-specific background levels to enable rational panel design and the objective delineation of gating thresholds during analysis.

Research paper thumbnail of Cell Survival Signal Cutting Edge : 4-1 BB Is a Bona Fide CD 8 T

Research paper thumbnail of Intratumoral CD103+ CD8+ T cells predict response to PD-L1 blockade

Journal for ImmunoTherapy of Cancer, 2021

BackgroundCD8+ tissue-resident memory T (TRM) cells, marked by CD103 (ITGAE) expression, are thou... more BackgroundCD8+ tissue-resident memory T (TRM) cells, marked by CD103 (ITGAE) expression, are thought to actively suppress cancer progression, leading to the hypothesis that their presence in tumors may predict response to immunotherapy.MethodsHere, we test this by combining high-dimensional single-cell modalities with bulk tumor transcriptomics from 1868 patients enrolled in lung and bladder cancer clinical trials of atezolizumab (anti-programmed cell death ligand 1 (PD-L1)).ResultsITGAE was identified as the most significantly upregulated gene in inflamed tumors. Tumor CD103+ CD8+ TRM cells exhibited a complex phenotype defined by the expression of checkpoint regulators, cytotoxic proteins, and increased clonal expansion.ConclusionsOur analyses indeed demonstrate that the presence of CD103+ CD8+ TRM cells, quantified by tracking intratumoral CD103 expression, can predict treatment outcome, suggesting that patients who respond to PD-1/PD-L1 blockade are those who exhibit an ongoing ...

Research paper thumbnail of Peripheral T cell expansion predicts tumour infiltration and clinical response

Nature, 2020

Despite the resounding clinical success in cancer treatment of antibodies that block the interact... more Despite the resounding clinical success in cancer treatment of antibodies that block the interaction of PD1 with its ligand PDL1 1 , the mechanisms involved remain unknown. A major limitation to understanding the origin and fate of T cells in tumour immunity is the lack of quantitative information on the distribution of individual clonotypes of T cells in patients with cancer. Here, by performing deep single-cell sequencing of RNA and T cell receptors in patients with different types of cancer, we survey the profiles of various populations of T cells and T cell receptors in tumours, normal adjacent tissue, and peripheral blood. We find clear evidence of clonotypic expansion of effector-like T cells not only within the tumour but also in normal adjacent tissue. Patients with gene signatures of such clonotypic expansion respond best to anti-PDL1 therapy. Notably, expanded clonotypes found in the tumour and normal adjacent tissue can also typically be detected in peripheral blood, which suggests a convenient approach to patient identification. Analyses of our data together with several external datasets suggest that intratumoural T cells, especially in responsive patients, are replenished with fresh, non-exhausted replacement cells from sites outside the tumour, suggesting continued activity of the cancer immunity cycle in these patients, the acceleration of which may be associated with clinical response. Large-scale single-cell sequencing of RNA and T cell receptors in samples from patients with cancer shows clonotypic expansion of effector-like T cells not only in tumour tissue but also in normal adjacent tissues and peripheral blood, which associates with clinical response to cancer immunotherapy.

Research paper thumbnail of Quantitative Comparison of Conventional and t-SNE-guided Gating Analyses

Frontiers in Immunology, 2019

Dimensionality reduction using the t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm ... more Dimensionality reduction using the t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm has emerged as a popular tool for visualizing high-parameter single-cell data. While this approach has obvious potential for data visualization it remains unclear how t-SNE analysis compares to conventional manual hand-gating in stratifying and quantitating the frequency of diverse immune cell populations. We applied a comprehensive 38-parameter mass cytometry panel to human blood and compared the frequencies of 28 immune cell subsets using both conventional bivariate and t-SNE-guided manual gating. t-SNE analysis was capable of stratifying every general cellular lineage and most sub-lineages with high correlation between conventional and t-SNE-guided cell frequency calculations. However, specific immune cell subsets delineated by the manual gating of continuous variables were not fully separated in t-SNE space thus causing discrepancies in subset identification and quantification between these analytical approaches. Overall, these studies highlight the consistency between t-SNE and conventional hand-gating in stratifying general immune cell lineages while demonstrating that particular cell subsets defined by conventional manual gating may be intermingled in t-SNE space.

Research paper thumbnail of Deep Profiling of Integrin β7 Throughout the Peripheral Immune System in Healthy Human Subjects

Research paper thumbnail of Mass cytometry panel optimization through the designed distribution of signal interference

Cytometry Part A, 2016

Mass cytometry is capable of measuring more than 40 distinct proteins on individual cells making ... more Mass cytometry is capable of measuring more than 40 distinct proteins on individual cells making it a promising technology for innovating biomarker discovery. However, in order for this potential to be fully realized, best practices in panel design need to be further defined in order to achieve consistency and reproducibility in data analysis. Of particular importance are controls that reveal, and panel design principles that mitigate the effects of signal interference or overlap. We observed a disparity between the staining profiles of two noncompeting anti-integrin b7 mAbs and hypothesized that signal interference was responsible. A mass-minus-one (MMO) control was applied and demonstrated that signal overlap caused the perceived interclonal discrepancy in b7 expression. Panel redesign in consideration of mass-cytometry specific interference dynamics dramatically improved concordance between both mAbs by redistributing background signals caused by overlap. These studies visualize how signal overlap can complicate mass cytometry data interpretation and demonstrate how the rational distribution of interference can greatly improve panel design and data quality. V C 2016 International Society for Advancement of Cytometry Key terms mass cytometry; panel optimization; CyTOF; signal interference MASS cytometry is rapidly becoming an established technology for the high dimensional analysis of single cells. In mass cytometry, antibodies tagged with elemental isotopes are used to achieve levels of dimensionality (401 parameters) that are currently unattainable with fluorescent molecules and flow cytometry (1-3). Recent mass cytometry-based studies have identified biomarkers of systemic lupus erythematosus pathogenesis (4) and susceptibility to viral infection (5) thus highlighting the utility of this technology in clinical studies. As flow cytometry evolved into a mature tool, concerted efforts were made to standardize data analysis and interpretation, especially for clinical assays (6-9). Similar efforts are now required for the reproducible application of mass cytometry (10,11). Specifically, control methods are needed to objectively gate cell populations into subsets based on the expression levels of particular parameters. In some situations, obviously bimodal markers (e.g., CD4 & CD8 on T cells) provide sufficient resolution to unambiguously define thresholds in the absence of controls. However, many cytometric markers (e.g., CD25) display distributions that are continuous with background signal and thus do not produce two clearly resolved populations. In these circumstances it is critical to distinguish between background and true signal. This leads to the question: "What are the sources of background in mass cytometry data?" While mass cytometry is not affected by autofluorescence, other sources of background exist (12,13), and thus gates cannot be set at 108 without incurring false

Research paper thumbnail of Cutting edge: 4-1BB is a bona fide CD8 T cell survival signal

Journal of immunology (Baltimore, Md. : 1950), 1999

After recognition of Ag/MHC and ligation of a costimulatory molecule, resting T cells will clonal... more After recognition of Ag/MHC and ligation of a costimulatory molecule, resting T cells will clonally expand and then delete to very low levels. Previously, it was shown that deletion can be prevented by coinjection of cytokines or proinflammatory agents such as adjuvants. Here, we demonstrate that ligation of 4-1BB blocks deletion of superantigen-activated T cells in the absence of adjuvant or additional cytokine treatment. Nearly 10 times as many staphylococcal enterotoxin A-specific T cells were detected in the spleens of mice injected 21 days previously with staphylococcal enterotoxin A and an agonist anti-4-1BB Ab compared with mice given staphylococcal enterotoxin A and a control IgG. Even though both CD4- and CD8-activated T cells expressed 4-1BB, a higher proportion of CD8 T cells were rescued compared CD4 T cells. These data suggest that although 4-1BB provides costimulation, it may also promote long-term T cell survival.

Research paper thumbnail of Effector CD8 T cells possess suppressor function after 4-1BB and Toll-like receptor triggering

Proceedings of the National Academy of Sciences, 2003

To better understand how innate and adaptive immune responses interact with each other, we combin... more To better understand how innate and adaptive immune responses interact with each other, we combined 4-1BB T cell costimulation with specific adjuvants to determine whether these treatments would influence specific T cell expansion and function in vivo . In the presence of 4-1BB ligation and Toll-like receptor 3 (TLR)3 and/or TLR4 triggering, CD8 T cell clonal expansion and survival was augmented profoundly. Specific T cells primed in vivo with TLR ligands responded normally to in vitro recall stimulus, but, surprisingly, copriming with 4-1BB costimulation significantly impaired the recall response even though many more specific effector T cells were rescued in vivo . Here, we demonstrate that the rescued CD8 T cells suppressed CD4 T cell proliferation via a type β transforming growth factor-dependent mechanism. Thus, 4-1BB and TLR ligands induce survival of specific effector CD8 T cells with suppressive recall potential, which may explain the dual role that 4-1BB activation plays in...

Research paper thumbnail of Development of a robust flow cytometry-based pharmacodynamic assay to detect phospho-protein signals for phosphatidylinositol 3-kinase inhibitors in multiple myeloma

Journal of Translational Medicine, 2013

Background The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple m... more Background The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients. Methods We conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and ...

Research paper thumbnail of 703 Gut-Homing CD4+ Lymphocytes Are Specifically Targeted in Cynomolgus Monkeys Dosed with Anti-Beta7 Antibodies

Gastroenterology, 2008

role of Ucns and CRF receptors in GI diseases is unclear. Aim: To evaluate the contribution of CR... more role of Ucns and CRF receptors in GI diseases is unclear. Aim: To evaluate the contribution of CRFR2 to inflammation, using a murine model of Crohn's colitis, and to test whether Ucn1 exerts its anti-inflammatory effects via CRFR2. Methods: An intracolonic enema of trinitrobenzene sulfonic acid (TNBS) was used to induce colitis in male C57 mice. An enema of vehicle was given to control group. First, the effect of TNBS was evaluated on the mortality rate and severity of colitis in wild type mice (Wt), CRFR2-/-(KO), and CRFR2+/-(Ht) mice. Second, Ucn1 was given (ip) when TNBS colitis was induced, to test whether Ucn1 could still exert its anti-inflammatory effects and ameliorate colitis in CRFR2 KO mice. Results: At a 5 mg dose of TNBS, resultant colitis in Wt C57 mice caused a~20% mortality rate 2 days after injection of TNBS. Surprisingly, 5 mg of TNBS did not cause any mortality in CRFR2-/-KO mice. In contrast, TNBS resulted in over 60% mortality rate within 3 days of TNBS injection in CRFR2+/-micE. colitis resulted in marked shortening of colon length in from 8.4±0.16 cm (controls) to 6.1±0.27 cm, p<0.05 (Wt), and the length was further shortened in KO and Ht mice to 5.6±0.2 cm. Adrenals were hypertrophied in mice with colitis, weighing twice as much as control adrenals. Colitis significantly increased histologic damage seen in H&E stained colon tissue of Wt and CRFR2 KO or Ht mice. Ucn 1 injection did not alter the mortality rate in Wt or KO mice with colitis, whereas Ucn1 dramatically rescued CRFR2+/-mice with colitis; none died until the experiment ended. This increase in survival rate was accompanied by significant improvement in colon histology as seen in H&E stained sections from CRFR2+/-mice. MPO levels were significantly decreased in Wt and Ht mice treated with Ucn1+TNBS (1.53±0.3 and 2.4±0.9 mU/mg) compared with KO Ucn1+TNBS mice (7.9±1.9 mU/mg, p<0.05). Interestingly, adrenal weights of CRFR2+/mice treated with Ucn1 were comparable to control levels, whereas adrenal weights remained elevated in Wt and KO mice treated with Ucn1. Conclusions: Perturbations in the CRFR2 receptor expression levels dramatically alter mice' ability to handle acute inflammatory stress. Complete lack of CRFR2 probably results in major developmental compensatory changes that enable the KO mice to better survive acute inflammatory stress. Supported in part by Hellman Foundation award to AB.

Research paper thumbnail of Sa.117. Gut-Homing CD4+ Lymphocytes are Specifically Targeted in Cynomolgus Monkeys Dosed with Anti-Beta7 Antibody

Clinical Immunology, 2008

Research paper thumbnail of OX40 Ligation Enhances Cell Cycle Turnover of Ag-Activated CD4 T Cells in Vivo

Cellular Immunology, 2001

OX40 costimulates T cells, increases activated T cell longevity, and promotes memory acquisition.... more OX40 costimulates T cells, increases activated T cell longevity, and promotes memory acquisition. T cells activated in vivo with agonist anti-OX40 and ovalbumin have a unique pattern of survival and cell division compared to control cells, but are able to respond to recall Ag equally well. BrdU incorporation shows that early cellular division rates of the anti-OX40-treated and the control groups are similar. Nevertheless, more BrdU(+) Ag-specific T cells accumulate in lymphoid tissue upon anti-OX40 administration. Thus, OX40 ligation does not necessarily lead to increased cell cycle entry, but promotes the accumulation of dividing cells. However, CFSE staining shows that OX40 ligation allows cells to progress through more cellular division cycles, while control cells stall or die. Moreover, OX40 ligation leads to a proportional decrease in apoptotic Ag-specific T cells. Thus, OX40 ligation boosts immunity by promoting an increase in the number cell cycles completed, thereby increasing the life span of Ag-activated CD4 T cells.

Research paper thumbnail of Differential clonal expansion of CD4 and CD8 T cells in response to 4-1BB ligation: contribution of 4-1BB during inflammatory responses

Immunology Letters, 2001

Cell surface proteins of the tumor necrosis factor (TNF) family of receptors have been intimately... more Cell surface proteins of the tumor necrosis factor (TNF) family of receptors have been intimately involved in inducing T cell death. A feature of these family members that is less well studied is their ability to rescue T cells from apoptosis. One such member is 4-1BB; an activation induced surface receptor on CD4 and CD8 T cells. This study demonstrates that the costimulatory effects of 4-1BB, which was found to enhance clonal expansion, required cross-linking of the receptor. The survival of the activated CD8 T cells following expansion was not associated with an increase in Bcl-2 expression. Provided that 4-1BB signaling was present, the amplification of activated CD8 T cell growth in vivo was independent of CD28 ligation. In vivo clonal expansion of activated CD4 T cells, however, was not as responsive to 4-1BB cross-linking. Moreover, 4-1BB-induced expansion was comparable to that mediated by LPS which can incite multiple costimulatory signals. Furthermore, LPS-mediated growth and survival of superantigen (SAg) stimulated T cells appeared to be partially dependent on interactions between 4-1BB and 4-1BB ligand (4-1BBL).

Research paper thumbnail of 703 Gut-Homing CD4+ Lymphocytes Are Specifically Targeted in Cynomolgus Monkeys Dosed with Anti-Beta7 Antibodies

Gastroenterology, 2008

No abstract is available. To read the body of this article, please view the PDF online. ... © 200... more No abstract is available. To read the body of this article, please view the PDF online. ... © 2008 AGA Institute. Published by Elsevier Inc. All rights reserved. ... Visit SciVerse ScienceDirect to see if you have access via your institution. ... Advertisements on this site do not constitute ...

Research paper thumbnail of Abstract PO084: Patterns of T cell clonal expansion in cancer patients associate with response to immunotherapy

Poster Presentations - Proffered Abstracts, 2021

Upon encountering their cognate antigens, T cells can undergo clonal expansion to produce multipl... more Upon encountering their cognate antigens, T cells can undergo clonal expansion to produce multiple copies of a cell with a shared T cell receptor (TCR). Despite the fundamental role of clonal expansion in cancer immunity, little is known about its relationship with T cell subpopulations or antitumor responses in cancer patients. Here we have performed single-cell RNA sequencing (scRNA-seq) and deep analysis of TCR clonotypes (scTCR-seq) in cancer patients across several indications, assessing the profiles of TCRs in the various populations of T cells in tumors, normal adjacent tissue (NAT), and peripheral blood. We found that, although most clonotypes were represented by a single cell, the remaining clonal lineages showed expansion in either NAT or tumor exclusively, or dual-residence with expansion in both compartments. In a subset of patients, we find clear evidence of clonotypic expansion of T effector- and effector memory-like cells not only within the tumor but also in NAT. Importantly, expanded clonotypes found in the tumor and NAT can also typically be detected in peripheral blood, suggesting a continuously replenishment from sites outside of the tumor with fresh, non-exhausted replacement cells. Our data further suggests a continued activity of the cancer immunity cycle in these patients, the acceleration of which may be associated with clinical response. Citation Format: Shravan Madireddi, Thomas D. Wu, Patricia E. de Almeida, Romain Banchereau, Ying-Jiun J. Chen, Avantika S. Chitre, Hina Iftikhar, William E. O9Gorman, Amelia Au-Yeung, Chikara Takahashi, Leonard D. Goldstein, Chungkee Poon, Shilpa Keerthivasan, Sanjeev Mariathasan, Meghna Das Thakur, Mahrukh A. Huseni, Marcus Ballinger, Ivette Estay, Patrick Caplazi, Zora Modrusan, Lelia Delamarre, Ira Mellman, Richard Bourgon, Jane L. Grogan. Patterns of T cell clonal expansion in cancer patients associate with response to immunotherapy [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO084.

Research paper thumbnail of Mechanistic convergence of the TIGIT and PD-1 inhibitory pathways necessitates co-blockade to optimize anti-tumor CD8+ T cell responses

Research paper thumbnail of Intratumoral plasma cells predict outcomes to PD-L1 blockade in non-small cell lung cancer

Cancer Cell, 2022

Inhibitors of the programmed cell death-1 (PD-1/PD-L1) signaling axis are approved to treat non-s... more Inhibitors of the programmed cell death-1 (PD-1/PD-L1) signaling axis are approved to treat non-small cell lung cancer (NSCLC) patients, based on their significant overall survival (OS) benefit. Using transcriptomic analysis of 891 NSCLC tumors from patients treated with either the PD-L1 inhibitor atezolizumab or chemotherapy from two large randomized clinical trials, we find a significant B cell association with extended OS with PD-L1 blockade, independent of CD8+ T cell signals. We then derive gene signatures corresponding to the dominant B cell subsets present in NSCLC from single-cell RNA sequencing (RNA-seq) data. Importantly, we find increased plasma cell signatures to be predictive of OS in patients treated with atezolizumab, but not chemotherapy. B and plasma cells are also associated with the presence of tertiary lymphoid structures and organized lymphoid aggregates. Our results suggest an important contribution of B and plasma cells to the efficacy of PD-L1 blockade in NSCLC.

Research paper thumbnail of Transcriptomic profiling of adjuvant colorectal cancer identifies three key prognostic biological processes and a disease specific role for granzyme B

PLOS ONE, 2021

Background Colorectal cancer (CRC) is a leading cause of cancer-related deaths, with a 5% 5-year ... more Background Colorectal cancer (CRC) is a leading cause of cancer-related deaths, with a 5% 5-year survival rate for metastatic disease, yet with limited therapeutic advancements due to insufficient understanding of and inability to accurately capture high-risk CRC patients who are most likely to recur. We aimed to improve high-risk classification by identifying biological pathways associated with outcome in adjuvant stage II/III CRC. Methods and findings We included 1062 patients with stage III or high-risk stage II colon carcinoma from the prospective three-arm randomized phase 3 AVANT trial, and performed expression profiling to identify a prognostic signature. Data from validation cohort GSE39582, The Cancer Genome Atlas, and cell lines were used to further validate the prognostic biology. Our retrospective analysis of the adjuvant AVANT trial uncovered a prognostic signature capturing three biological functions—stromal, proliferative and immune—that outperformed the Consensus Mol...

Research paper thumbnail of Visualization of Mass Cytometry Signal Background to Enable Optimal Core Panel Customization and Signal Threshold Gating

Methods in molecular biology, 2019

Signal interference or overlap in mass cytometry is minimal compared to flow cytometry but must s... more Signal interference or overlap in mass cytometry is minimal compared to flow cytometry but must still be considered for optimal panel design and assay sensitivity. Here we describe a procedure for evaluating signal interference dynamics in the context of a 25-parameter core immunophenotyping panel. Specifically, a mass-minus-many (MMM) approach was used to assess background signals in "empty" or "blank" channels intended for further customization. Through this approach cell type-specific variability in signal background is revealed. Further panel customization can thus be performed with an understanding of cell type and channel-specific background levels to enable rational panel design and the objective delineation of gating thresholds during analysis.

Research paper thumbnail of Cell Survival Signal Cutting Edge : 4-1 BB Is a Bona Fide CD 8 T

Research paper thumbnail of Intratumoral CD103+ CD8+ T cells predict response to PD-L1 blockade

Journal for ImmunoTherapy of Cancer, 2021

BackgroundCD8+ tissue-resident memory T (TRM) cells, marked by CD103 (ITGAE) expression, are thou... more BackgroundCD8+ tissue-resident memory T (TRM) cells, marked by CD103 (ITGAE) expression, are thought to actively suppress cancer progression, leading to the hypothesis that their presence in tumors may predict response to immunotherapy.MethodsHere, we test this by combining high-dimensional single-cell modalities with bulk tumor transcriptomics from 1868 patients enrolled in lung and bladder cancer clinical trials of atezolizumab (anti-programmed cell death ligand 1 (PD-L1)).ResultsITGAE was identified as the most significantly upregulated gene in inflamed tumors. Tumor CD103+ CD8+ TRM cells exhibited a complex phenotype defined by the expression of checkpoint regulators, cytotoxic proteins, and increased clonal expansion.ConclusionsOur analyses indeed demonstrate that the presence of CD103+ CD8+ TRM cells, quantified by tracking intratumoral CD103 expression, can predict treatment outcome, suggesting that patients who respond to PD-1/PD-L1 blockade are those who exhibit an ongoing ...

Research paper thumbnail of Peripheral T cell expansion predicts tumour infiltration and clinical response

Nature, 2020

Despite the resounding clinical success in cancer treatment of antibodies that block the interact... more Despite the resounding clinical success in cancer treatment of antibodies that block the interaction of PD1 with its ligand PDL1 1 , the mechanisms involved remain unknown. A major limitation to understanding the origin and fate of T cells in tumour immunity is the lack of quantitative information on the distribution of individual clonotypes of T cells in patients with cancer. Here, by performing deep single-cell sequencing of RNA and T cell receptors in patients with different types of cancer, we survey the profiles of various populations of T cells and T cell receptors in tumours, normal adjacent tissue, and peripheral blood. We find clear evidence of clonotypic expansion of effector-like T cells not only within the tumour but also in normal adjacent tissue. Patients with gene signatures of such clonotypic expansion respond best to anti-PDL1 therapy. Notably, expanded clonotypes found in the tumour and normal adjacent tissue can also typically be detected in peripheral blood, which suggests a convenient approach to patient identification. Analyses of our data together with several external datasets suggest that intratumoural T cells, especially in responsive patients, are replenished with fresh, non-exhausted replacement cells from sites outside the tumour, suggesting continued activity of the cancer immunity cycle in these patients, the acceleration of which may be associated with clinical response. Large-scale single-cell sequencing of RNA and T cell receptors in samples from patients with cancer shows clonotypic expansion of effector-like T cells not only in tumour tissue but also in normal adjacent tissues and peripheral blood, which associates with clinical response to cancer immunotherapy.

Research paper thumbnail of Quantitative Comparison of Conventional and t-SNE-guided Gating Analyses

Frontiers in Immunology, 2019

Dimensionality reduction using the t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm ... more Dimensionality reduction using the t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm has emerged as a popular tool for visualizing high-parameter single-cell data. While this approach has obvious potential for data visualization it remains unclear how t-SNE analysis compares to conventional manual hand-gating in stratifying and quantitating the frequency of diverse immune cell populations. We applied a comprehensive 38-parameter mass cytometry panel to human blood and compared the frequencies of 28 immune cell subsets using both conventional bivariate and t-SNE-guided manual gating. t-SNE analysis was capable of stratifying every general cellular lineage and most sub-lineages with high correlation between conventional and t-SNE-guided cell frequency calculations. However, specific immune cell subsets delineated by the manual gating of continuous variables were not fully separated in t-SNE space thus causing discrepancies in subset identification and quantification between these analytical approaches. Overall, these studies highlight the consistency between t-SNE and conventional hand-gating in stratifying general immune cell lineages while demonstrating that particular cell subsets defined by conventional manual gating may be intermingled in t-SNE space.

Research paper thumbnail of Deep Profiling of Integrin β7 Throughout the Peripheral Immune System in Healthy Human Subjects

Research paper thumbnail of Mass cytometry panel optimization through the designed distribution of signal interference

Cytometry Part A, 2016

Mass cytometry is capable of measuring more than 40 distinct proteins on individual cells making ... more Mass cytometry is capable of measuring more than 40 distinct proteins on individual cells making it a promising technology for innovating biomarker discovery. However, in order for this potential to be fully realized, best practices in panel design need to be further defined in order to achieve consistency and reproducibility in data analysis. Of particular importance are controls that reveal, and panel design principles that mitigate the effects of signal interference or overlap. We observed a disparity between the staining profiles of two noncompeting anti-integrin b7 mAbs and hypothesized that signal interference was responsible. A mass-minus-one (MMO) control was applied and demonstrated that signal overlap caused the perceived interclonal discrepancy in b7 expression. Panel redesign in consideration of mass-cytometry specific interference dynamics dramatically improved concordance between both mAbs by redistributing background signals caused by overlap. These studies visualize how signal overlap can complicate mass cytometry data interpretation and demonstrate how the rational distribution of interference can greatly improve panel design and data quality. V C 2016 International Society for Advancement of Cytometry Key terms mass cytometry; panel optimization; CyTOF; signal interference MASS cytometry is rapidly becoming an established technology for the high dimensional analysis of single cells. In mass cytometry, antibodies tagged with elemental isotopes are used to achieve levels of dimensionality (401 parameters) that are currently unattainable with fluorescent molecules and flow cytometry (1-3). Recent mass cytometry-based studies have identified biomarkers of systemic lupus erythematosus pathogenesis (4) and susceptibility to viral infection (5) thus highlighting the utility of this technology in clinical studies. As flow cytometry evolved into a mature tool, concerted efforts were made to standardize data analysis and interpretation, especially for clinical assays (6-9). Similar efforts are now required for the reproducible application of mass cytometry (10,11). Specifically, control methods are needed to objectively gate cell populations into subsets based on the expression levels of particular parameters. In some situations, obviously bimodal markers (e.g., CD4 & CD8 on T cells) provide sufficient resolution to unambiguously define thresholds in the absence of controls. However, many cytometric markers (e.g., CD25) display distributions that are continuous with background signal and thus do not produce two clearly resolved populations. In these circumstances it is critical to distinguish between background and true signal. This leads to the question: "What are the sources of background in mass cytometry data?" While mass cytometry is not affected by autofluorescence, other sources of background exist (12,13), and thus gates cannot be set at 108 without incurring false

Research paper thumbnail of Cutting edge: 4-1BB is a bona fide CD8 T cell survival signal

Journal of immunology (Baltimore, Md. : 1950), 1999

After recognition of Ag/MHC and ligation of a costimulatory molecule, resting T cells will clonal... more After recognition of Ag/MHC and ligation of a costimulatory molecule, resting T cells will clonally expand and then delete to very low levels. Previously, it was shown that deletion can be prevented by coinjection of cytokines or proinflammatory agents such as adjuvants. Here, we demonstrate that ligation of 4-1BB blocks deletion of superantigen-activated T cells in the absence of adjuvant or additional cytokine treatment. Nearly 10 times as many staphylococcal enterotoxin A-specific T cells were detected in the spleens of mice injected 21 days previously with staphylococcal enterotoxin A and an agonist anti-4-1BB Ab compared with mice given staphylococcal enterotoxin A and a control IgG. Even though both CD4- and CD8-activated T cells expressed 4-1BB, a higher proportion of CD8 T cells were rescued compared CD4 T cells. These data suggest that although 4-1BB provides costimulation, it may also promote long-term T cell survival.

Research paper thumbnail of Effector CD8 T cells possess suppressor function after 4-1BB and Toll-like receptor triggering

Proceedings of the National Academy of Sciences, 2003

To better understand how innate and adaptive immune responses interact with each other, we combin... more To better understand how innate and adaptive immune responses interact with each other, we combined 4-1BB T cell costimulation with specific adjuvants to determine whether these treatments would influence specific T cell expansion and function in vivo . In the presence of 4-1BB ligation and Toll-like receptor 3 (TLR)3 and/or TLR4 triggering, CD8 T cell clonal expansion and survival was augmented profoundly. Specific T cells primed in vivo with TLR ligands responded normally to in vitro recall stimulus, but, surprisingly, copriming with 4-1BB costimulation significantly impaired the recall response even though many more specific effector T cells were rescued in vivo . Here, we demonstrate that the rescued CD8 T cells suppressed CD4 T cell proliferation via a type β transforming growth factor-dependent mechanism. Thus, 4-1BB and TLR ligands induce survival of specific effector CD8 T cells with suppressive recall potential, which may explain the dual role that 4-1BB activation plays in...

Research paper thumbnail of Development of a robust flow cytometry-based pharmacodynamic assay to detect phospho-protein signals for phosphatidylinositol 3-kinase inhibitors in multiple myeloma

Journal of Translational Medicine, 2013

Background The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple m... more Background The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients. Methods We conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and ...

Research paper thumbnail of 703 Gut-Homing CD4+ Lymphocytes Are Specifically Targeted in Cynomolgus Monkeys Dosed with Anti-Beta7 Antibodies

Gastroenterology, 2008

role of Ucns and CRF receptors in GI diseases is unclear. Aim: To evaluate the contribution of CR... more role of Ucns and CRF receptors in GI diseases is unclear. Aim: To evaluate the contribution of CRFR2 to inflammation, using a murine model of Crohn's colitis, and to test whether Ucn1 exerts its anti-inflammatory effects via CRFR2. Methods: An intracolonic enema of trinitrobenzene sulfonic acid (TNBS) was used to induce colitis in male C57 mice. An enema of vehicle was given to control group. First, the effect of TNBS was evaluated on the mortality rate and severity of colitis in wild type mice (Wt), CRFR2-/-(KO), and CRFR2+/-(Ht) mice. Second, Ucn1 was given (ip) when TNBS colitis was induced, to test whether Ucn1 could still exert its anti-inflammatory effects and ameliorate colitis in CRFR2 KO mice. Results: At a 5 mg dose of TNBS, resultant colitis in Wt C57 mice caused a~20% mortality rate 2 days after injection of TNBS. Surprisingly, 5 mg of TNBS did not cause any mortality in CRFR2-/-KO mice. In contrast, TNBS resulted in over 60% mortality rate within 3 days of TNBS injection in CRFR2+/-micE. colitis resulted in marked shortening of colon length in from 8.4±0.16 cm (controls) to 6.1±0.27 cm, p<0.05 (Wt), and the length was further shortened in KO and Ht mice to 5.6±0.2 cm. Adrenals were hypertrophied in mice with colitis, weighing twice as much as control adrenals. Colitis significantly increased histologic damage seen in H&E stained colon tissue of Wt and CRFR2 KO or Ht mice. Ucn 1 injection did not alter the mortality rate in Wt or KO mice with colitis, whereas Ucn1 dramatically rescued CRFR2+/-mice with colitis; none died until the experiment ended. This increase in survival rate was accompanied by significant improvement in colon histology as seen in H&E stained sections from CRFR2+/-mice. MPO levels were significantly decreased in Wt and Ht mice treated with Ucn1+TNBS (1.53±0.3 and 2.4±0.9 mU/mg) compared with KO Ucn1+TNBS mice (7.9±1.9 mU/mg, p<0.05). Interestingly, adrenal weights of CRFR2+/mice treated with Ucn1 were comparable to control levels, whereas adrenal weights remained elevated in Wt and KO mice treated with Ucn1. Conclusions: Perturbations in the CRFR2 receptor expression levels dramatically alter mice' ability to handle acute inflammatory stress. Complete lack of CRFR2 probably results in major developmental compensatory changes that enable the KO mice to better survive acute inflammatory stress. Supported in part by Hellman Foundation award to AB.

Research paper thumbnail of Sa.117. Gut-Homing CD4+ Lymphocytes are Specifically Targeted in Cynomolgus Monkeys Dosed with Anti-Beta7 Antibody

Clinical Immunology, 2008

Research paper thumbnail of OX40 Ligation Enhances Cell Cycle Turnover of Ag-Activated CD4 T Cells in Vivo

Cellular Immunology, 2001

OX40 costimulates T cells, increases activated T cell longevity, and promotes memory acquisition.... more OX40 costimulates T cells, increases activated T cell longevity, and promotes memory acquisition. T cells activated in vivo with agonist anti-OX40 and ovalbumin have a unique pattern of survival and cell division compared to control cells, but are able to respond to recall Ag equally well. BrdU incorporation shows that early cellular division rates of the anti-OX40-treated and the control groups are similar. Nevertheless, more BrdU(+) Ag-specific T cells accumulate in lymphoid tissue upon anti-OX40 administration. Thus, OX40 ligation does not necessarily lead to increased cell cycle entry, but promotes the accumulation of dividing cells. However, CFSE staining shows that OX40 ligation allows cells to progress through more cellular division cycles, while control cells stall or die. Moreover, OX40 ligation leads to a proportional decrease in apoptotic Ag-specific T cells. Thus, OX40 ligation boosts immunity by promoting an increase in the number cell cycles completed, thereby increasing the life span of Ag-activated CD4 T cells.

Research paper thumbnail of Differential clonal expansion of CD4 and CD8 T cells in response to 4-1BB ligation: contribution of 4-1BB during inflammatory responses

Immunology Letters, 2001

Cell surface proteins of the tumor necrosis factor (TNF) family of receptors have been intimately... more Cell surface proteins of the tumor necrosis factor (TNF) family of receptors have been intimately involved in inducing T cell death. A feature of these family members that is less well studied is their ability to rescue T cells from apoptosis. One such member is 4-1BB; an activation induced surface receptor on CD4 and CD8 T cells. This study demonstrates that the costimulatory effects of 4-1BB, which was found to enhance clonal expansion, required cross-linking of the receptor. The survival of the activated CD8 T cells following expansion was not associated with an increase in Bcl-2 expression. Provided that 4-1BB signaling was present, the amplification of activated CD8 T cell growth in vivo was independent of CD28 ligation. In vivo clonal expansion of activated CD4 T cells, however, was not as responsive to 4-1BB cross-linking. Moreover, 4-1BB-induced expansion was comparable to that mediated by LPS which can incite multiple costimulatory signals. Furthermore, LPS-mediated growth and survival of superantigen (SAg) stimulated T cells appeared to be partially dependent on interactions between 4-1BB and 4-1BB ligand (4-1BBL).

Research paper thumbnail of 703 Gut-Homing CD4+ Lymphocytes Are Specifically Targeted in Cynomolgus Monkeys Dosed with Anti-Beta7 Antibodies

Gastroenterology, 2008

No abstract is available. To read the body of this article, please view the PDF online. ... © 200... more No abstract is available. To read the body of this article, please view the PDF online. ... © 2008 AGA Institute. Published by Elsevier Inc. All rights reserved. ... Visit SciVerse ScienceDirect to see if you have access via your institution. ... Advertisements on this site do not constitute ...