scholarly journals Circulating mucosal-associated invariant T cells identify patients responding to anti-PD-1 therapy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sara De Biasi ◽  
Lara Gibellini ◽  
Domenico Lo Tartaro ◽  
Simone Puccio ◽  
Claudio Rabacchi ◽  
...  
Keyword(s):  
T Cells ◽  
Metastatic Melanoma ◽  
Checkpoint Inhibitors ◽  
Granzyme B ◽  
In Silico Analysis ◽  
Response To Treatment ◽  
Multiparameter Flow Cytometry ◽  
Mait Cells ◽  
Single Cell Rna Sequencing ◽  
Silico Analysis

AbstractImmune checkpoint inhibitors are used for treating patients with metastatic melanoma. Since the response to treatment is variable, biomarkers are urgently needed to identify patients who may benefit from such therapy. Here, we combine single-cell RNA-sequencing and multiparameter flow cytometry to assess changes in circulating CD8+ T cells in 28 patients with metastatic melanoma starting anti-PD-1 therapy, followed for 6 months: 17 responded to therapy, whilst 11 did not. Proportions of activated and proliferating CD8+ T cells and of mucosal-associated invariant T (MAIT) cells are significantly higher in responders, prior to and throughout therapy duration. MAIT cells from responders express higher level of CXCR4 and produce more granzyme B. In silico analysis support MAIT presence in the tumor microenvironment. Finally, patients with >1.7% of MAIT among peripheral CD8+ population show a better response to treatment. Our results thus suggest that MAIT cells may be considered a biomarker for patients responding to anti-PD-1 therapy.

scholarly journals Circulating mucosal associated invariant T cells identify patients responding to anti-PD1 therapy

2020 ◽  
Author(s):  
Sara De Biasi ◽  
Lara Gibellini ◽  
Domenico Lo Tartaro ◽  
Emilia Mazza ◽  
Simone Puccio ◽  
...  
Keyword(s):  
T Cells ◽  
Metastatic Melanoma ◽  
Cd8 T Cells ◽  
Checkpoint Inhibitors ◽  
In Silico Analysis ◽  
T Cell Response ◽  
Response To Treatment ◽  
Multiparameter Flow Cytometry ◽  
Mait Cells ◽  
Silico Analysis

Abstract Immune checkpoint inhibitors that maintain anti-tumor T cell response are used for treating patients with metastatic melanoma. Since the response to treatment is extremely variable, biomarkers are urgently needed to identify patients who could benefit from such therapy. We combined single-cell RNA-sequencing and multiparameter flow cytometry to determine changes in circulating CD8+ T cells in patients with metastatic melanoma. A total of 28 patients starting anti-PD1 therapy were enrolled and followed for 6 months: 17 responded to therapy, whilst 11 did not. The proportion of activated and proliferating CD8+ T cells and of mucosal associated invariant T (MAIT) cells was significantly higher in responders before starting therapy and was maintained over time. MAIT cells expressed higher level of CXCR4 and produced more granzyme B; in silico analysis revealed that they are present in the tumor microenvironment. Finally, patients with higher levels of MAIT showed a better response to treatment.

scholarly journals 281 Measuring immune checkpoint inhibitor efficacy using primary patient-derived 3D spheroids

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A305-A305
Author(s):  
Kathryn Appleton ◽  
Katy Lassahn ◽  
Ashley Elrod ◽  
Tessa DesRochers
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Checkpoint Inhibitors ◽  
Clinical Success ◽  
Single Cells ◽  
Granzyme B ◽  
Immune Checkpoints ◽  
Dependent Manner ◽  
Luminex Technology

BackgroundCancerous cells can utilize immune checkpoints to escape T-cell-mediated cytotoxicity. Agents that target PD-1, PD-L1 and CTLA4 are collectively deemed immune checkpoint inhibitors (ICIs), and many have been approved for treatment of non-small cell lung cancer (NSCLC) and melanoma. Unfortunately, many patients do not respond to these therapies and often experience disease progression. Immunohistochemistry assays to predict response to ICIs have been inconsistent in their readouts and often patients with low expression levels respond to ICIs. Understanding the determinants of ICI response in individual patients is critical for improving the clinical success of this drug class. Using patient-derived spheroids from NSCLC and melanoma primary tissue, we developed a multi-plexed assay for detecting ICI efficacy.MethodsNine NSCLC and 11 melanoma primary tumor samples were dissociated to single cells, classified for immune checkpoint expression and cell content by flow cytometry, and seeded for spheroid formation. Spheroids were treated with pembrolizumab, nivolumab, atezolizumab, ipilimumab or durvalumab across a range of concentrations and monitored for cytotoxicity at 24-hours and viability at 72-hours by multiplexing CellTox™ Green Cytotoxicity Assay and CellTiter-Glo® 3D Cell Viability Assay. IFNγ and granzyme B secretion was assessed using Luminex technology. ICI response was evaluated by determining the concentration-response relationship for all three read-outs.ResultsIncreased IFNγ and granzyme B were detected for every ICI in one or more patient samples. ICI-induced IFNγ secretion inversely correlated with PD-1+ immune cells. Durvalumab was significantly more cytotoxic for both NSCLC and melanoma spheroids compared to the other ICIs and significantly reduced spheroid viability with mean spheroid survival decreasing to 19.5% for NSCLC and 58.2% for melanoma. We evaluated if there was an association between durvalumab response and cell composition and found that percent spheroid survival significantly correlated with CD8+ T-cells for both NSCLC (r=-0.7920, p=0.0191) and melanoma (r=-0.6918, p=0.0390). Furthermore, CD8+ T-cells correlated with durvalumab-induced granzyme B secretion for NSCLC (r=-0.7645, p=0.0271) and melanoma (r=-0.7419, p=0.0221).ConclusionsIn this study we show ICI-specific increases in immune-related analytes in a concentration-dependent manner for NSCLC and melanoma patient-derived spheroids. We detected spheroid cytotoxicity following short term ICI treatment which closely mirrored decreased spheroid viability at a later timepoint. Finally, we can decipher response mechanisms as exemplified by durvalumab-induced granzyme B secretion correlating with the presence of CD8+ T-cells which results in reduced spheroid viability for both tested cancer indications.

Peripheral immunological response to treatment with checkpoint inhibitor in recurrent/metastatic nasopharyngeal carcinoma.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e18519-e18519
Author(s):  
Dora Lai Wan Kwong ◽  
Ngar-Woon Kam ◽  
Ho-Yin Luk ◽  
Tae Yang Desmond Hung ◽  
Man Kim Yim ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Checkpoint Inhibitors ◽  
Immunological Response ◽  
Blood Analysis ◽  
Response To Treatment ◽  
Chemotherapy Response ◽  
Group 2 ◽  
Drug Responsiveness ◽  
Cellular Phenotypes

e18519 Background: A better understanding of peripheral cellular phenotypes in association with checkpoint inhibitors (CPI) responsiveness in recurrent/metastatic NPC could profoundly impact our knowledge of NPC immunopathology. Methods: Blood were collected from 11 patients with recurrent/metastatic NPC who received CPI (pembrolizumab) after failing second-line chemotherapy. Response to pembrolizumab was assessed by imaging and EBV DNA. Patients who achieved CR/PR were considered responsive and those with SD/PD were considered non-responsive. Four patients in the responsive group also had baseline blood before pembrolizumab. PBMC were freshly isolated and stored until analysis. For surface staining, cells were rested overnight at 37°C before resuspended in BD Brilliant stain with 2.5ng/µl Fc block for 15mins. Cells were then co-stained: 7-AAD, CD19-BV510, CD3-Alexa Fluo 700, CD4-BV510, CD279(PD-1)-BB515, CD197(CCR7)-BV421, CD45RO-APC, CD45RA-Pe-Cy5, CD8-APC-H7, CD27-PE, CD95-PE-Cy7, incubated on ice for 30mins. Samples were washed and acquired on NovoCyte Quanteon. Immune phenotypes were correlated with clinical response. Results: We found that: 1) %CD3 was upregulated in responsive group; 2) CD4/CD8 ratio did not directly stratify drug responsiveness; 3) frequency of PD1-expressing CD8+ T cells was significantly reduced in responsive group; 4) lower frequency of CCR7+PD1+CD8+ T cells in responsive group, suggesting that these may be highly differentiated and have the ability to move into peripheral sites in response to inflammatory chemokines; 5) frequency of naïve and TEMRA CD8+ T cells, but not Tscm was upregulated in responsive group; 6) finally, an interesting finding of sustained CD19+ subsets was observed in non-responsive group. Conclusions: Our results suggest that peripheral blood analysis may provide valuable insights into NPC patients’ responses to PD-1-targeted therapies. [Table: see text]

scholarly journals Deregulated Expression of Immune Checkpoints on Circulating CD4 T Cells May Complicate Clinical Outcome and Response to Treatment with Checkpoint Inhibitors in Multiple Myeloma Patients

2021 ◽  
Vol 22 (17) ◽  
pp. 9298
Author(s):  
Anna Kulikowska de Nałęcz ◽  
Lidia Ciszak ◽  
Lidia Usnarska-Zubkiewicz ◽  
Irena Frydecka ◽  
Edyta Pawlak ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Checkpoint Inhibitors ◽  
Effector Function ◽  
Response To Treatment ◽  
Immune Checkpoints ◽  
Effective Control ◽  
Small Subset

Unlike solid-tumor patients, a disappointingly small subset of multiple myeloma (MM) patients treated with checkpoint inhibitors derive clinical benefits, suggesting differential participation of inhibitory receptors involved in the development of T-cell-mediated immunosuppression. In fact, T cells in MM patients have recently been shown to display features of immunosenescence and exhaustion involved in immune response inhibition. Therefore, we aimed to identify the dominant inhibitory pathway in MM patients to achieve its effective control by therapeutic interventions. By flow cytometry, we examined peripheral blood (PB) CD4 T cell characteristics assigned to senescence or exhaustion, considering PD-1, CTLA-4, and BTLA checkpoint expression, as well as secretory effector function, i.e., capacity for IFN-γ and IL-17 secretion. Analyses were performed in a total of 40 active myeloma patients (newly diagnosed and treated) and 20 healthy controls. At the single-cell level, we found a loss of studied checkpoints’ expression on MM CD4 T cells (both effector (Teff) and regulatory (Treg) cells) primarily at diagnosis; the checkpoint deficit in MM relapse was not significant. Nonetheless, PD-1 was the only checkpoint distributed on an increased proportion of T cells in all MM patients irrespective of disease phase, and its expression on CD4 Teff cells correlated with adverse clinical courses. Among patients, the relative defect in secretory effector function of CD4 T cells was more pronounced at myeloma relapse (as seen in declined Th1/Treg and Th17/Treg cell rates). Although the contribution of PD-1 to MM clinical outcomes is suggestive, our study clearly indicated that the inappropriate expression of immune checkpoints (associated with dysfunctionality of CD4 T cells and disease clinical phase) might be responsible for the sub-optimal clinical response to therapeutic checkpoint inhibitors in MM.

scholarly journals Cellular analysis of bronchoalveolar lavage fluid to narrow differential diagnosis of checkpoint inhibitor-related pneumonitis in metastatic melanoma

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Sabino Strippoli ◽  
Livia Fucci ◽  
Antonio Negri ◽  
Daniela Putignano ◽  
Marco Luigi Cisternino ◽  
...  
Keyword(s):  
T Cells ◽  
Bronchoalveolar Lavage ◽  
Metastatic Melanoma ◽  
Cancer Progression ◽  
Checkpoint Inhibitors ◽  
Complete Response ◽  
Lavage Fluid ◽  
Blood Parameters ◽  
Cryptogenic Organizing Pneumonia ◽  
Cellular Analysis

Abstract Background The diagnosis of check-point inhibitor-related pneumonitis (CIP) relies on radiological and clinical patterns which are not specific and can mimic other conditions (cancer progression, infectious diseases or interstitial pneumonitis). Cell pattern analysis of bronchoalveolar lavage (BAL) is well-known to support the diagnosis of interstitial lung disease; nevertheless, this analysis is somewhat performed and not required by immune-toxicity management guidelines for CIP. Methods We performed BAL analysis in 5 metastatic melanoma (MM) patients who developed CIP among 112 patients treated with checkpoint inhibitors. We also correlated the BAL features with the computed tomography (CT) scan patterns and with various peripheral blood parameters to better define the profile of this patient population. Results BAL flow cytometer and cytopathology analyses showed typical and homogeneous features with increased lymphoid population, prevalent CD8 + T cells and inversion of the CD4/CD8 ratio. Moreover, the extent of activated CD3 + HLA-DR + T cells was related to the grading of adverse events. Blood leucocytosis, hypoxemia, normal values for procalcitonin and lactate dehydrogenase were also found together with a cryptogenic organizing pneumonia-like radiologic pattern. In all our patients, CIP was associated with partial or complete response. Conclusions Identification of a specific BAL cellular pattern allows clinicians to place this investigation in the appropriate position of CIP diagnosis and management to avoid misdiagnosis or considering this condition as progressive disease and delaying proper treatment.

scholarly journals Clinical Development of Immune Checkpoint Inhibitors

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Ayumu Ito ◽  
Shunsuke Kondo ◽  
Kohei Tada ◽  
Shigehisa Kitano
Keyword(s):  
Clinical Trials ◽  
T Cells ◽  
Metastatic Melanoma ◽  
Tumor Cells ◽  
Immune Checkpoint ◽  
Immune Checkpoint Inhibitors ◽  
Checkpoint Inhibitors ◽  
Clinical Development ◽  
Phase Iii ◽  
Immune Checkpoint Molecules

Recent progress in cancer immunotherapy has been remarkable. Most striking are the clinical development and approval of immunomodulators, also known as immune checkpoint inhibitors. These monoclonal antibodies (mAb) are directed to immune checkpoint molecules, which are expressed on immune cells and mediate signals to attenuate excessive immune reactions. Although mAbs targeting tumor associated antigens, such as anti-CD20 mAb and anti-Her2 mAb, directly recognize tumor cells and induce cell death, immune checkpoint inhibitors restore and augment the antitumor immune activities of cytotoxic T cells by blocking immune checkpoint molecules on T cells or their ligands on antigen presenting and tumor cells. Based on preclinical data, many clinical trials have demonstrated the acceptable safety profiles and efficacies of immune checkpoint inhibitors in a variety of cancers. The first in class approved immune checkpoint inhibitor is ipilimumab, an anti-CTLA-4 (cytotoxic T lymphocyte antigen-4) mAb. Two pivotal phase III randomized controlled trials demonstrated a survival benefit in patients with metastatic melanoma. In 2011, the US Food and Drug Administration (FDA) approved ipilimumab for metastatic melanoma. Several clinical trials have since investigated new agents, alone and in combination, for various cancers. In this review, we discuss the current development status of and future challenges in utilizing immune checkpoint inhibitors.

scholarly journals Immunomodulatory Molecules On Lung Cancer Stem Cells From Lymph Nodes Aspirates

Cancers ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 838
Author(s):  
Agata Raniszewska ◽  
Iwona Kwiecień ◽  
Rafał Sokołowski ◽  
Elżbieta Rutkowska ◽  
Joanna Domagała-Kulawik
Keyword(s):  
Lung Cancer ◽  
Stem Cells ◽  
T Cells ◽  
Cancer Stem Cells ◽  
Lymph Nodes ◽  
Cd8 T Cells ◽  
Checkpoint Inhibitors ◽  
Lymphocyte Subpopulation ◽  
Endobronchial Ultrasound ◽  
Multiparameter Flow Cytometry

Over the past decade, immune checkpoint inhibitors have revolutionized the treatment of non-small cell lung cancer (NSCLC). Unfortunately, not all patients benefit from PD-(L)1 blockade, yet, the PD-L1 tumor cell expression is the only approved biomarker, and other biomarkers have been investigated. In the present study, we analyzed the presence of immunomodulatory molecules: PD-L1, CD47, CD73, Fas, and FasL on mature tumor cells (MTCs) and cancer stem cells (CSCs) in lymph nodes (LNs) aspirates and refer it to the lymphocyte subpopulation in peripheral blood (PB). PB samples and LNs aspirates obtained during the endobronchial ultrasound-guided transbronchial needle aspiration (EBUS/TBNA) procedure of 20 patients at different stages of NSCLC. The cells were analyzed by multiparameter flow cytometry. We reported the higher frequency of MTCs and CSCs expressing the investigated immunomodulating molecules in metastatic LNs than in nonmetastatic. The expression of CD47 and PD-L1 was significantly higher on CSCs than on MTCs. Among the lymphocyte subpopulation in PB, we observed a higher frequency of PD-1+ CD8 T cells and Fas+ CD8 T cells in patients with confirmed metastases than in nonmetastatic. Next, we found that the percentage of FasL+ MTCs correlated with the frequency of Fas+ CD3 T cells in LNs aspirates and Fas+ CD8 T cells in PB. Finally, we found that patients with metastatic disease had a significantly higher FasL+/Fas+ MTCs ratio than patients with nonmetastatic disease. Both MTCs and CSCs express different immunomodulatory molecules on their surface. The frequency of FasL+ MTCs associates with altered distribution of Fas+ lymphocyte subpopulations in LNs and PB.

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