Expansion and Characterization of Iovance Marrow Infiltrating Lymphocytes: A Potential Novel Therapeutic Strategy for the Treatment of Acute Myeloid Leukemia

Abstract Background: Acute myeloid leukemia (AML) is a poor-prognosis malignancy arising from hematopoietic stem/progenitor cells. To date, novel immunotherapies such as checkpoint inhibitors, vaccines and adoptive cell therapy (ACT) using CAR T cells have demonstrated only modest success for the treatment of patients who are ineligible for marrow transplantation and have minimal residual disease; additional approaches are warranted (Beyar-Katz O and Gill S, Clin Cancer Res 2018). ACT with tumor infiltrating lymphocytes (TIL) has emerged as an effective treatment for patients with metastatic melanoma (Goff SL et al, J Clin Oncol 2016), likely owing to the heterogeneous population of tumor-reactive T cells that comprises the TIL products. As demonstrated for solid cancers, such tumor-reactive T cells are preferentially found in the tumor microenvironment (Gros A et al JCI 2014; Thommen DS et al Nat Med 2018). By avoiding the highly immunosuppressive tumor microenvironment, ex vivo activation of those cells rescues them from tolerance and anergic status. We hypothesized that, in the case of AML for which the bone marrow represents the tumor microenvironment, tumor antigen-specific T cells could be recovered from the patient bone marrow to produce a highly effective therapeutic product that is cytotoxic to AML tumor cells. We present findings related to the ex vivo expansion of Iovance marrow infiltrating lymphocytes (MIL) for the treatment of AML patients. Methods: Immune cell and non-immune cell fractions were sorted from bone marrow mononuclear cells. Immune cell fractions loaded with sonicated non-immune cell fractions were expanded for 14 days in the presence of αCD3/αCD28 beads and interleukin-2 (IL-2) to generate MIL products. Phenotypic and functional characteristics of the cells were determined by flow cytometry and enzyme-linked immunospot assay (ELISpot). Results: MIL were generated from isolated bone marrow mononuclear cells (n=2) with a mean expansion fold of 86 (range 78-93). Equal percentage of CD4+ and CD8+ T cell subsets constituted the MIL products. Phenotypic analysis of the cells showed that the majority of T cell subsets are effector memory and CD28 positive. Low percentages of the T cell subsets were positive for immunosuppressive markers PD-1 and LAG3. ELISpot analysis demonstrated that MIL were readily activatable and produced normal levels of IFNγ in response to CD3/CD28 stimulation. Antigen specificity of MIL is being investigated. Conclusion: We demonstrated the feasibility of MIL expansion from bone marrow mononuclear cells from AML patients. MIL are functionally active and mostly comprised of effector memory T cells. Confirmation of tumor cell antigen specificity will determine whether MIL may deploy a robust anti-tumor activity in vivo. Disclosures Karyampudi: Iovance Biotherapeutics: Employment, Equity Ownership. Frank:Iovance Biotherapeutics: Employment, Equity Ownership. Blaskovich:Iovance Biotherapeutics: Equity Ownership. Chartier:Iovance Biotherapeutics: Equity Ownership.

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The differential immune responses to COVID-19 in peripheral and lung revealed by single-cell RNA sequencing

10.1101/2020.08.15.20175638 ◽

2020 ◽

Author(s):

Gang Xu ◽

Furong Qi ◽

Hanjie Li ◽

Qianting Yang ◽

Haiyan Wang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Mononuclear Cells ◽

Immune Cell ◽

Close Association ◽

Suppressor Cells ◽

T Cell Subsets ◽

Peripheral Blood Mononuclear ◽

Peripheral T Cells

Understanding the mechanism that leads to immune dysfunction induced by SARS-CoV2 virus is crucial to develop treatment for severe COVID-19. Here, using single cell RNA-seq, we characterized the peripheral blood mononuclear cells (PBMC) from uninfected controls and COVID-19 patients, and cells in paired broncho-alveolar lavage fluid (BALF). We found a close association of decreased dendritic cells (DC) and increased monocytes resembling myeloid-derived suppressor cells (MDSC) which correlated with lymphopenia and inflammation in the blood of severe COVID-19 patients. Those MDSC-like monocytes were immune-paralyzed. In contrast, monocyte-macrophages in BALFs of COVID-19 patients produced massive amounts of cytokines and chemokines, but secreted little interferons. The frequencies of peripheral T cells and NK cells were significantly decreased in severe COVID-19 patients, especially for innate-like T and various CD8+ T cell subsets, compared to health controls. In contrast, the proportions of various activated CD4+ T cell subsets, including Th1, Th2 and Th17-like cells were increased and more clonally expanded in severe COVID-19 patients. Patients' peripheral T cells showed no sign of exhaustion or augmented cell death, whereas T cells in BALFs produced higher levels of IFNG, TNF, CCL4 and CCL5 etc. Paired TCR tracking indicated abundant recruitment of peripheral T cells to the patients' lung. Together, this study comprehensively depicts how the immune cell landscape is perturbed in severe COVID-19.

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IL-12 Gene Electrotransfer Triggers a Change in Immune Response within Mouse Tumors

Cancers ◽

10.3390/cancers10120498 ◽

2018 ◽

Vol 10 (12) ◽

pp. 498 ◽

Cited By ~ 13

Author(s):

Guilan Shi ◽

Chelsea Edelblute ◽

Sezgi Arpag ◽

Cathryn Lundberg ◽

Richard Heller

Keyword(s):

Immune Response ◽

T Cells ◽

Tumor Microenvironment ◽

Mononuclear Cells ◽

Immune Cell ◽

Immune Memory ◽

Control Group ◽

Gene Electrotransfer ◽

Cell Content ◽

Peripheral Blood Mononuclear

Metastatic melanoma is an aggressive skin cancer with a relatively low survival rate. Immune-based therapies have shown promise in the treatment of melanoma, but overall complete response rates are still low. Previous studies have demonstrated the potential of plasmid IL-12 (pIL-12) delivered by gene electrotransfer (GET) to be an effective immunotherapy for melanoma. However, events occurring in the tumor microenvironment following delivery have not been delineated. Therefore, utilizing a B16F10 mouse melanoma model, we evaluated changes in the tumor microenvironment following delivery of pIL-12 using different GET parameters or injection of plasmid alone. The results revealed a unique immune cell composition after intratumoral injection of pIL-12 GET. The number of immune memory cells was markedly increased in pIL-12 GET melanoma groups compared to control group. This was validated using flow cytometry to analyze peripheral blood mononuclear cells as well as delineating immune cell content using immunohistochemistry. Significant differences in multiple cell types were observed, including CD8+ T cells, regulatory T cells and myeloid cells, which were induced to mount a CD8+PD1− T cells immune response. Taken together, these findings suggest a basic understanding of the sequence of immune activity following pIL-12 GET and also illuminates that adjuvant immunotherapy can have a positive influence on the host immune response to cancer.

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Immunological Profiles of The Breast Cancer Microenvironment Represented by Tumor-Infiltrating Lymphocytes and PD-L1 Expression

10.21203/rs.3.rs-1109400/v1 ◽

2021 ◽

Author(s):

Toru Hanamura ◽

Shigehisa Kitano ◽

Hiroshi Kagamu ◽

Makiko Yamashita ◽

Mayako Terao ◽

...

Keyword(s):

Breast Cancer ◽

T Cells ◽

B Cell ◽

Natural Killer ◽

Immune Cell ◽

Tumor Infiltrating Lymphocytes ◽

Leukocyte Infiltration ◽

Cell Fractions ◽

Antigen Presenting ◽

Infiltrating Lymphocytes

Abstract Purpose: Histologically assessed tumor-infiltrating lymphocytes and programmed cell death 1 ligand 1 (hPD-L1) are established prognostic or predictive biomarkers in certain subsets of breast cancer. However, the association with immune response complexity is not fully understood. In this study, the immune cell fractions in breast cancer tissue and blood were evaluated to analyze their association with histologically assessed tumor-infiltrating lymphocytes and PD-L1. Methods: Forty-five tumor and 18 blood samples were collected from patients with breast cancer. Total leukocyte counts, proportions of 11 types of immune cells, and PD-L1 expression in each cell fraction were evaluated using multicolor flow cytometry. Histologically assessed tumor-infiltrating lymphocytes and PD-L1 were evaluated using hematoxylin and eosin staining and immunohistochemistry, respectively. Results: The immune cell composition of blood was partly correlated with that of tumor tissue but the abundance ratio of each fraction was different between them. A higher histologically assessed tumor-infiltrating lymphocyte proportion was associated with increased leukocyte infiltration, a higher proportion of CD4+ and CD8+ T cells, and a lower proportion of natural killer cells and natural killer T cells. PD-L1 was highly expressed in the non-B-cell antigen-presenting cell fractions (monocyte/macrophage, nonclassical monocyte, myeloid-derived suppressor, dendritic, and myeloid dendritic cell) in tumors. Histologically assessed PD-L1 positivity reflected PD-L1 expression well in these fractions, as well as increased leukocyte infiltration in tumors. Conclusion: Our results indicate that histologically assessed tumor-infiltrating lymphocytes reflect differences in immune responses in the tumor microenvironment. Non-B-cell antigen-presenting cell fractions are primarily involved in the PD-L1 pathway in breast cancer microenvironments.

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Identification of Human Immune Cell Subtypes Most Vulnerable to IL-1β-induced Inflammatory Signaling Using Mass Cytometry

10.1101/2020.04.19.047274 ◽

2020 ◽

Author(s):

Hema Kothari ◽

Corey M. Williams ◽

Chantel McSkimming ◽

Mythili Vigneshwar ◽

Eli R. Zunder ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mononuclear Cells ◽

Immune Cell ◽

T Cell Subsets ◽

Effector Memory ◽

High Morbidity ◽

Cytokine Storm ◽

Mass Cytometry ◽

Immune Cell Subsets

ABSTRACTIL-1β has emerged as a key mediator of the cytokine storm linked to high morbidity and mortality from COVID-19 and blockade of the IL-1 receptor (IL-1R) with Anakinra has entered clinical trials in COVID-19 subjects. Yet, knowledge of the specific immune cell subsets targeted by IL-1β and IL-1β-induced signaling pathways in humans is limited. Utilizing mass cytometry (CyTOF) of human peripheral blood mononuclear cells, we identified effector memory CD4 T cells and CD4−CD8low/-CD161+ T cells as the circulating immune subtypes with the greatest expression of p-NF-κB in response to IL-1β stimulation. Notably, CCR6 distinctly identified T cells most responsive to IL-1β. Other subsets including CD11c myeloid dendritic cells (mDCs), classical monocytes (CM), two subsets of natural killer cells (CD16−CD56brightCD161− and CD16−CD56dimCD161+) and a population of lineage−(Lin-) cells expressing CD161 and CD25 also showed IL-1β-induced expression of p-NF-kB. The IL-1R antagonist, Anakinra significantly inhibited IL-1β-induced p-NF-kB in the CCR6+ T cells and CD11c mDCs with a trending inhibition in CD14 monocytes and Lin−CD161+CD25+ cells. IL-1β also induced a rapid but much less robust increase in p-p38 expression as compared to p-NF-kB in the majority of these same immune cell subsets. Prolonged IL-1β stimulation greatly increased p-STAT3 and to a much lesser extent p-STAT1 and p-STAT5 in T cell subsets, monocytes, DCs and the Lin−CD161+CD25+ cells suggesting IL-1β-induced production of downstream STAT-activating cytokines, consistent with its role in cytokine storm. Interindividual heterogeneity and inhibition of this activation by Anakinra raises the intriguing possibility that assays to measure IL-1β-induced p-NF-kB in CCR6+ T cell subtypes could identify those at higher risk of cytokine storm and those most likely to benefit from Anakinra therapy.

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Bronchoalveolar Lavage Fluid IFN-γ+Th17 Cells and Regulatory T Cells in Pulmonary Sarcoidosis

Mediators of Inflammation ◽

10.1155/2014/438070 ◽

2014 ◽

Vol 2014 ◽

pp. 1-9 ◽

Cited By ~ 17

Author(s):

Anders Tøndell ◽

Torolf Moen ◽

Magne Børset ◽

Øyvind Salvesen ◽

Anne Dorthea Rø ◽

...

Keyword(s):

T Cells ◽

Bronchoalveolar Lavage ◽

Th17 Cells ◽

Bronchoalveolar Lavage Fluid ◽

Immune Cell ◽

Lavage Fluid ◽

Pulmonary Sarcoidosis ◽

T Cell Subsets ◽

Diffuse Parenchymal Lung Diseases ◽

Cell Fractions

In sarcoidosis, increased Th17 cell fractions have been reported in bronchoalveolar lavage fluid, and elevated numbers of Th17 cells producing IFN-γhave been observed in peripheral blood. The balance between Th1, Th17, and FoxP3+CD4+T cell subsets in sarcoidosis remains unclear. Bronchoalveolar lavage fluid cells, from 30 patients with sarcoidosis, 18 patients with other diffuse parenchymal lung diseases, and 15 healthy controls, were investigated with flow cytometry for intracellular expression of FoxP3. In a subset of the patients, expression of the cytokines IL17A and IFN-γwas investigated. The fractions of FoxP3+CD4+T cells and Th17 cells were both lower in sarcoidosis compared to controls (P=0.017andP=0.011, resp.). The proportion of Th17 cells positive for IFN-γwas greater in sarcoidosis than controls (median 72.4% versus 31%,P=0.0005) and increased with radiologic stage (N=23,rho=0.45, andP=0.03). IFN-γ+Th17 cells were highly correlated with Th1 cells (N=23,rho=0.64, andP=0.001), and the ratio of IFN-γ+Th17/FoxP3+CD4+T cells was prominently increased in sarcoidosis. IFN-γ+Th17 cells may represent a pathogenic subset of Th17 cells, yet their expression of IFN-γcould be a consequence of a Th1-polarized cytokine milieu. Our results indicate a possible immune cell imbalance in sarcoidosis.

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Integrated Immunological Analysis of the Bone Marrow Tumor Microenvironment in Myeloproliferative Neoplasms to Determine Potential Efficacy of Immune Checkpoint Blockade

Blood ◽

10.1182/blood.v126.23.2766.2766 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2766-2766

Author(s):

Robert Orlowski ◽

Alexander Huang ◽

Mercy Gohil ◽

James Mangan ◽

Marissa Vignali ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Tumor Microenvironment ◽

Cd8 T Cells ◽

Immune Checkpoint ◽

Employment Equity ◽

Healthy Donors ◽

Significant Difference ◽

Equity Ownership

Abstract BACKGROUND: Immune checkpoint blockadewith anti-PD-1/PD-L1 therapyhas demonstrated remarkable efficacy in multiple tumor types. Biomarker candidates for predicting likelihood of response to targeted immunotherapy are being actively investigated including inhibitory or activating receptors on CD8+ lymphocytes, corresponding ligands on tumor or antigen-presenting cells (APCs), T-cell functionality, and the T-cell receptor (TCR) repertoire found within a tumor microenvironment. Myelofibrosis (MF) and Chronic Myeloid Leukemia (CML) are tumors responsive to immunotherapy, most notably allogeneic transplantation (alloSCT), and donor lymphocyte infusion. Although tyrosine kinase inhibitors can improve patient outcomes, a potentially curative therapeutic option other than alloSCT is needed. PURPOSE: To determine the immune profile of the bone marrow tumor microenvironment in patients with CML and MF compared to healthy donors in order to assess the rationale and potential efficacy of novel immune checkpoint therapies. METHODS: Cryopreserved bone marrow aspirate mononuclear cells (MNCs) from healthy donors (HDs) (n=11), untreated CML (n=9) or MF (n= 12) were analyzed by flow cytometry. CD3+ CD8+ lymphocytes were divided into naïve, central memory (CM), effector memory (EM), and terminal effector (TEMRA) subsets for analysis. Expression of immune checkpoint receptors including PD-1, 4-1BB, TIM3, LAG3, and TIGIT were evaluated on each population. Known corresponding ligands including PD-L1 and PD-L2 were assessed in CML samples on blasts, plasmacytoid dendritic cells (pDCs), myeloid dendritic cells (mDCs), and monocytes. T-cell function was evaluated by cytokine production, cytotoxicity, and proliferation in CD3+ CD8+ PD1+ or PD1- populations. To assess the TCR repertoire found within the tumor microenvironment, non-naive CD8+ T-cells were sorted into PD-1+ and PD-1- populations, and then CDR3 region of the TCRB gene, together with sufficient flanking sequence to identify most V, D, and J genes was sequenced using the immunoSEQ platform from Adaptive Biotechnologies. RESULTS: There was a significant difference in the CML CD3+ CD8+ subset distribution compared to HDs with EM% increased at 60.01% vs. 41.25% (p =0.0137), and TEMRA 44.51% vs. 20.64% (p=0.0004). CM% trended downwards (32.15% to 21.58%, p=0.118) while naïve% was equivalent in CML and HDs (22.13% vs. 20.87%). The percentage of PD-1+ non-naïve CD8+ T-cells (EM, TEMRA, CM combined) was significantly increased in CML samples at 55.14% (range 31-69%) compared to HDs at 38.98% (range 34.8% to 55.5%; p=0.0050). PD-1 expression was consistently increased across all subgroups in CML (CM: 67.06% vs 53.22%, EM: 60.01% vs. 41.25%, TEMRA: 44.51% vs 20.64% p <0.05 for all). There was no statistically significant difference in CML compared to HDs for secondary receptors including TIGIT, TIM3, LAG3, or 4-1BB. Fewer than 5% of CML blasts were positive for the PD-L1 or PD-L2 ligands, however PD-L1 expression was increased on mDCs compared to HD samples (53.08% vs 24.63%; p=0.0015). In contrast to these findings in CML there was no significant proportional difference in CD8+ subsets, PD-1 status, or other receptors between MF and HDs. Anti-CD3/28 stimulation did not induce differential IFN-γ/TNF-alpha production, granzyme production, or proliferation (Ki67+) among the CD8+ PD-1+ or PD-1- T-cells from CML samples. To begin to estimate T cell clonality in the bone marrow tumor microenvironment, TCRβ sequencing of sorted non-naïve CD8- T-cells showed several clones markedly overrepresented in the diseased PD-1+ compartment. Conclusions: The CML tumor microenvironment is enriched in CD8+ T-cells expressing the inhibitory receptor PD-1 while APC subsets express increased PD-L1. This represents a potential axis of tumor driven immunosuppression amenable to immune checkpoint blockade. This is in contrast to MF, where the immunoprofile was not detectably different from healthy donors. These findings may reflect differences in tumor immunogenicity, cytokine mileu, or the APC types present. In-vivo testing using murine models for both diseases is underway to gain a better understanding of the role of immune checkpoint therapies. Disclosures Mangan: Incyte Corporation: Membership on an entity's Board of Directors or advisory committees. Vignali:Adaptive Biotechnologies: Employment, Equity Ownership. Emerson:Adaptive Biotechnologies: Employment, Equity Ownership. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Yusko:Adaptive Biotechnologies: Employment, Equity Ownership.

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Immunophenotyping reveals longitudinal changes in circulating immune cells during radium-223 therapy in patients with metastatic castration-resistant prostate cancer

10.1101/2020.11.11.20229831 ◽

2020 ◽

Author(s):

J.H.A. Creemers ◽

M.J. van der Doelen ◽

S. van Wilpe ◽

R. Hermsen ◽

T. Duiveman-de Boer ◽

...

Keyword(s):

Prostate Cancer ◽

T Cells ◽

Mononuclear Cells ◽

Immune Cell ◽

T Cell Subsets ◽

Castration Resistant Prostate Cancer ◽

Linear Regression Models ◽

Castration Resistant ◽

Radium 223 ◽

Immunological Mechanisms

ABSTRACTPurposeRadium-223 improves overall survival (OS) in men with bone metastatic castration-resistant prostate cancer (mCRPC). While the exact mechanism behind this survival benefit remains unclear, radium-induced immunological mechanisms might contribute to the OS advantage. We performed a comprehensive evaluation of the immunological changes in mCRPC patients by phenotyping the peripheral blood mononuclear cells (PBMCs) during radium-223 therapy.Experimental DesignIn this prospective, single-arm, exploratory study, PBMCs of 30 mCRPC patients were collected before, during, and after treatment with radium-223. Lymphocyte and monocyte counts were analyzed to get insight into general immune cell trends. Next, we analyzed changes in T cell subsets, myeloid-derived suppressor cells (MDSCs), and immune checkpoint expression using linear regression models. Per subset, the 6-month change (% of baseline) was determined. Bootstrapped 95% confidence intervals were used to measure the degree of uncertainty of our findings.ResultsWe observed a substantial decrease in absolute lymphocyte counts (−0.12 * 10^9 cells/L per injection, 95% CI: -0.143 - -0.102). Simultaneously, an increase was observed in the proportion of T cells that expressed costimulatory (ICOS) or inhibitory (TIM-3, PD-L1, and PD-1) checkpoint molecules. Moreover, the fraction of two immunosuppressive subsets – the regulatory T cells and the monocytic MDSCs – increased throughout treatment. These findings were not more pronounced in patients with an ALP response during therapy.ConclusionImmune cell subsets in patients with mCRPC changed during radium-223 therapy, which warrants further research into the possible immunological consequences of these changes.

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The differential immune responses to COVID-19 in peripheral and lung revealed by single-cell RNA sequencing

Cell Discovery ◽

10.1038/s41421-020-00225-2 ◽

2020 ◽

Vol 6 (1) ◽

Cited By ~ 3

Author(s):

Gang Xu ◽

Furong Qi ◽

Hanjie Li ◽

Qianting Yang ◽

Haiyan Wang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Rna Sequencing ◽

Mononuclear Cells ◽

Immune Cell ◽

Close Association ◽

T Cell Subsets ◽

Peripheral T Cells ◽

Single Cell Rna Sequencing

Abstract Understanding the mechanism that leads to immune dysfunction in severe coronavirus disease 2019 (COVID-19) is crucial for the development of effective treatment. Here, using single-cell RNA sequencing, we characterized the peripheral blood mononuclear cells (PBMCs) from uninfected controls and COVID-19 patients and cells in paired broncho-alveolar lavage fluid (BALF). We found a close association of decreased dendritic cells (DCs) and increased monocytes resembling myeloid-derived suppressor cells (MDSCs), which correlated with lymphopenia and inflammation in the blood of severe COVID-19 patients. Those MDSC-like monocytes were immune-paralyzed. In contrast, monocyte-macrophages in BALFs of COVID-19 patients produced massive amounts of cytokines and chemokines, but secreted little interferons. The frequencies of peripheral T cells and NK cells were significantly decreased in severe COVID-19 patients, especially for innate-like T and various CD8+ T cell subsets, compared to healthy controls. In contrast, the proportions of various activated CD4+ T cell subsets among the T cell compartment, including Th1, Th2, and Th17-like cells were increased and more clonally expanded in severe COVID-19 patients. Patients’ peripheral T cells showed no sign of exhaustion or augmented cell death, whereas T cells in BALFs produced higher levels of IFNG, TNF, CCL4, CCL5, etc. Paired TCR tracking indicated abundant recruitment of peripheral T cells to the severe patients’ lung. Together, this study comprehensively depicts how the immune cell landscape is perturbed in severe COVID-19.

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Immunophenotyping Reveals Longitudinal Changes in Circulating Immune Cells During Radium-223 Therapy in Patients With Metastatic Castration-Resistant Prostate Cancer

Frontiers in Oncology ◽

10.3389/fonc.2021.667658 ◽

2021 ◽

Vol 11 ◽

Author(s):

Jeroen H. A. Creemers ◽

Maarten J. van der Doelen ◽

Sandra van Wilpe ◽

Rick Hermsen ◽

Tjitske Duiveman-de Boer ◽

...

Keyword(s):

Prostate Cancer ◽

T Cells ◽

Mononuclear Cells ◽

Immune Cell ◽

T Cell Subsets ◽

Castration Resistant Prostate Cancer ◽

Linear Regression Models ◽

Castration Resistant ◽

Radium 223 ◽

Immunological Mechanisms

BackgroundRadium-223 improves overall survival (OS) in men with bone metastatic castration-resistant prostate cancer (mCRPC). While the exact mechanism behind this survival benefit remains unclear, radium-induced immunological mechanisms might contribute to the OS advantage. We performed a comprehensive evaluation of the immunological changes in mCRPC patients by phenotyping the peripheral blood mononuclear cells (PBMCs) during radium-223 therapy.Materials and MethodsIn this prospective, single-arm, exploratory study, PBMCs of 30 mCRPC patients were collected before, during, and after treatment with radium-223. Lymphocyte and monocyte counts were analyzed to get insight into general immune cell trends. Next, we analyzed changes in T cell subsets, myeloid-derived suppressor cells (MDSCs), and immune checkpoint expression using linear regression models. Per subset, the 6-month change (% of baseline) was determined. Bootstrapped 95% confidence intervals were used to measure the degree of uncertainty of our findings.ResultsWe observed a substantial decrease in absolute lymphocyte counts (-0.12 * 10^9 cells/L per injection, 95% CI: -0.143 - -0.102). Simultaneously, an increase was observed in the proportion of T cells that expressed costimulatory (ICOS) or inhibitory (TIM-3, PD-L1, and PD-1) checkpoint molecules. Moreover, the fraction of two immunosuppressive subsets – the regulatory T cells and the monocytic MDSCs – increased throughout treatment. These findings were not more pronounced in patients with an alkaline phosphatase response during therapy.ConclusionImmune cell subsets in patients with mCRPC changed during radium-223 therapy, which warrants further research into the possible immunological consequences of these changes.

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CD8+HLA-DR+ T Cells Are Increased in Patients with Severe Aplastic Anemia

Blood ◽

10.1182/blood.v120.21.4395.4395 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4395-4395

Author(s):

Zonghong Shao ◽

Chunyan Liu ◽

Xiao Liu ◽

Le Feng ◽

Rong Fu ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Normal Control ◽

Mononuclear Cells ◽

Granzyme B ◽

Bone Marrow Mononuclear Cells ◽

Hla Dr ◽

Group 2 ◽

Normal Controls ◽

Group 1

Abstract Abstract 4395 OBJECTIVES: To investigate the number and function of CD8+HLA-DR+ cells, which were considered to be activated CTL, in peripheral blood to further explore the pathogenesis of SAA. METHODS: The proportion of CD8+HLA-DR+T cells was analyzed by flow cytometry in peripheral blood of 38 SAA (26 untreated and 12 recovered) patients and 23 normal controls. Also the expression of perforin, granzyme B, TNF-β and FasL in CD8+HLA-DR+T cells was analyzed by flow cytometry and RT-PCR. Moreover, the apoptosis of CD3- bone marrow cells from normal persons after coculture with CD8+HLA-DR+T cells from untreated SAA patients was detected by staining with AnnexinV. The level of lactate dehydrogenase (LDH) in the supernatant were determined by automatic biochemistry analyzer. RESULTS: The ratios of CD8+HLA-DR+T cells to CD8+T cells and to CD3+T cells were 39.3 ± 8.1% and 27.8±7.1% in SAA patients, and were significantly higher than the corresponding ratios in controls (18.3 ± 6.7% and 8.5 ± 2.3%) (P < 0.05). The expression values for perforin, granzyme B, TNF-β and FasL in CD8+HLA-DR+T cells in the untreated SAA group were 8.5%, 96.1%, 94.3% and 72.1%, respectively, which were higher than the corresponding values in the control group (1.8%, 82.0%, 32.9%, 15.6%). The mRNA expression values for perforin, granzyme B, TNF-β and FasL in CD8+HLA-DR+T cells in the untreated SAA group were 0.66±0.25, 0.56±0.26, 0.61±0.16, 0.77±0.24, respectively, which were significantly higher than those in the control group (0.53±0.14, 0.40±0.13, 0.46±0.15, 0.58±0.16). (P < 0.05). The apoptosis values in SAA group 1 (CD8+HLA-DR+ T cells from untreated SAA patients and CD3- bone marrow mononuclear cells from remission patients), SAA group 2 (CD8+HLA-DR+ T cells from untreated SAA patients and CD3- bone marrow mononuclear cells from normal controls), the remission group (CD8+HLA-DR+ T cells and CD3- bone marrow mononuclear cells from remission patients) and the normal control (CD8+HLA-DR+ T cells and CD3- bone marrow mononuclear cells from normal controls) were 41.12 ± 24.84%, 45.81 ± 20.47%, 35.03 ± 22.09%, 20.95 ± 13.82%. There were no significant differences between SAA group 1, SAA group 2, and the remission group (P > 0.05). However, the apoptosis in each of these groups was higher than in the normal control (P < 0.05). The LDH levels in SAA group 1, SAA group 2, the remission group and normal control were 74.56 ± 49.13 U/L, 62.61 ± 31.76 U/L, 61.06 ± 28.41 U/L, and 28.60 ± 8.91 U/L. There were no significant differences between SAA group 1, SAA group 2, and the remission group (P > 0.05). However, the level of LDH in these groups was significantly higher than in the normal control (P < 0.05). CONCLUSION: CD8+HLA-DR+T cells might contribute to bone marrow failure in SAA. Immunosuppressive therapy dramatically reduced the quantity and function of CD8+HLA-DR+T cells, and inhibitors of CD8+HLA-DR+T cells or other factors involved in this pathway will be attractive therapeutic targets in SAA. Disclosures: No relevant conflicts of interest to declare.

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