Differential Responsiveness to IL-2, IL-7, and IL-15 Common Receptor γ Chain Cytokines by Antigen-specific Peripheral Blood Naive or Memory Cytotoxic CD8+ T Cells From Healthy Donors and Melanoma Patients

AbstractMultiple myeloma (MM) is one of the most common hematologic malignancies. Despite extensive therapeutical approaches, cures remain rare exceptions. An important issue for future immunologic treatments is the characterization of appropriate tumor-associated antigens. Recently, a highly glycosylated mucin MUC1 was detected on a majority of multiple myeloma cell lines. We analyzed bone marrow and peripheral blood of 68 patients with HLA-A2–positive myeloma for the presence and functional activity of CD8 T cells specific for the MUC1-derived peptide LLLLTVLTV. Forty-four percent of the patients with MM contained elevated frequencies of MUC1-specific CD8 T cells in freshly isolated samples from peripheral blood (PB) or bone marrow (BM) compared with corresponding samples from healthy donors. BM-residing T cells possessed a higher functional capacity upon specific reactivation than PB-derived T cells with regard to interferon γ (IFN-γ) secretion, perforin production, and cytotoxicity.

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Microarray Analysis Reveals Differences in Gene Expression of Circulating CD8+ T Cells in Melanoma Patients and Healthy Donors

Cancer Research ◽

10.1158/0008-5472.can-03-3396 ◽

2004 ◽

Vol 64 (10) ◽

pp. 3661-3667 ◽

Cited By ~ 19

Author(s):

Tong Xu ◽

Chen-Tsen Shu ◽

Elizabeth Purdom ◽

Demi Dang ◽

Diane Ilsley ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

Microarray Analysis ◽

Cd8 T Cells ◽

Healthy Donors ◽

Melanoma Patients

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P853 Single cell transcriptome analysis identifies unique features in circulating CD8+ T cells that can predict immunotherapy response in melanoma patients

Journal for ImmunoTherapy of Cancer ◽

10.1136/lba2019.7 ◽

2020 ◽

Vol 8 (Suppl 1) ◽

pp. A5.1-A5

Author(s):

Chuan Li ◽

Yee Peng Phoon ◽

Keaton Karlinsey ◽

Ye Tian ◽

Samjhana Thapaliya ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

High Resolution ◽

Single Cell ◽

T Cell Activation ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Cell Activation ◽

Melanoma Patients ◽

Cluster 2

BackgroundImmune checkpoint blockade (ICB) has greatly advanced the treatment of melanoma. A key component of ICB is the stimulation of CD8+ T cells in the tumor. However, ICB therapy only benefits a subset of patients and a reliable prediction method that does not require invasive biopsies is still a major challenge in the field.MethodsWe conducted a set of comprehensive single-cell transcriptomic analyses of CD8+ T cells in the peripheral blood (mPBL) and tumors (mTIL) from 8 patients with metastatic melanoma.ResultsCompared to circulating CD8+ T cells from healthy donors (hPBL), mPBLs contained subsets resembling certain features of mTIL. More importantly, three clusters (2, 6 and 15) were represented in both mPBL and mTIL. Cluster 2 was the major subset of the majority of hPBL, which phenocopied hallmark parameters of resting T cells. Cluster 6 and 15 were uniquely presented in melanoma patients. Cluster 15 had the highest PD-1 levels, with elevated markers of both activation and dysfunction/exhaustion; while Cluster 6 was enriched for ‘dormant’ cells with overall toned-down transcriptional activity except PPAR signaling, a known suppressor for T cell activation. Interestingly, unlike other mTIL clusters that would classically be defined as exhausted, Cluster 15 exhibited the highest metabolic activity (oxidative-phosphorylation and glycolysis). We further analyzed total sc-transcriptomics using cell trajectory algorithms and identified that these three clusters were the most distinct subtypes of CD8 T cells from each other, representing: resting (cluster 2), metabolically active-dysfunctional (cluster 15), and dormant phenotypes (cluster 6). Further, three unique intracellular programs in melanoma drive the transition of resting CD8+ T cells (cluster 2) to both metabolic/dysfunctional (cluster 15) and dormant states (cluster 6) that are unique to tumor bearing conditions. Based on these high-resolution analyses, we developed original algorithms to build a novel ICB response predictive model using immune-blockade co-expression gene patterns. The model was trained and tested using previously published GEO datasets containing CD8 T cells from anti-PD-1 treated patients and presented an AUC of 0.82, with 92% and 89% accuracy of ICB response in the two datasets.ConclusionsWe identified and analyzed unique populations of CD8+ T cells in circulation and tumor using high-resolution single-cell transcriptomics to define the landscape of CD8+ T cell states, revealing critical subsets with shared features in PBLs and TILs. Most importantly, we established an innovative model for ICB response prediction by using peripheral blood lymphocytes.Ethics ApprovalThis study was performed under an IRB approved protocol.

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HLA-A*0201 Restricted Killing Activity of WT1-Specific CTL Generated From the Peripheral Blood Lymphocytes of Normal Healthy Donors.

Blood ◽

10.1182/blood.v114.22.3026.3026 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3026-3026

Author(s):

Deepa Kolaseri Krishnadas ◽

Mindy Stamer ◽

Kim Dunham ◽

Lei Bao ◽

Kenneth Lucas

Keyword(s):

T Cells ◽

Cell Line ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Peripheral Blood Lymphocytes ◽

Target Cells ◽

Cd8 Cells ◽

Blood Lymphocytes ◽

Healthy Donors ◽

Ifn Γ

Abstract Abstract 3026 Poster Board II-1002 The Wilms' tumor antigen (WT1) is over-expressed on several human leukemia and solid tumors, and thus is considered as a potential target for cancer immunotherapy. Combating leukemia by targeting WT1 expressing leukemic cells using in vitro generated WT1-specific CTL is one potential approach, but it is difficult to generate an immune response against WT1 due to low T cell precursor frequency in normal healthy individuals. Earlier studies have shown the generation of WT1-A*0201 peptide specific CTL from CD8+ T cells by cloning. Another study reported the production of IFN- γ by WT-1 specific CD8+ T cells. However, the cytolytic killing ability of these IFN- γ producing cells was not further characterized. Here, we demonstrate the generation of WT1-A*0201 specific CTL from the peripheral blood lymphocytes (PBL) of normal healthy donors using CD137 selection. The PBL were stimulated once with RMFPNAPYL (WT1-A*0201 peptide) pulsed autologous dendritic cells and twice with WT1-A*0201 peptide pulsed irradiated peripheral blood mononuclear cells (PBMC). Following three stimulations, the PBL were selected for CD137+ expression and rapidly expanded with OKT3 and IL-2. The WT1-A*0201 specific CTL showed killing of target cells and production of IFN-γ in an antigen-specific manner. The percent killing of WT1-A*0201 peptide pulsed T2 cells (TAP−, HLA- A2+) and autologous B blast (BB) were significantly higher when compared with their control targets. T2 cells and BB either pulsed with an irrelevant A*0201 peptide or un-pulsed served as the control. We have observed similar results with WT1-A*0201 specific CTL generated from normal donor CD8+ cells. However, the efficiency of WT1-A*0201 CTL generated from PBL to kill target cells and produce IFN- γ was higher than CTL from CD8+ cells. The CTL generated from PBL killed BA25, a WT1 expressing A2+ leukemia cell line but failed to kill Molt-4, a WT1 expressing A2− cell line, clearly indicating HLA-A2 restricted CTL activity. The specificity of the generated CTL were further confirmed by staining with WT1-HLA-A*0201 tetramer. The percentage of WT1-specific CD3+CD8+Tetramer+ cells either remained same or higher in CTL generated from PBL when compared with those generated from CD8+ cells. CD137 selection leads to the generation of significant number of CTL in a shorter time when compared to conventional cloning methods. In addition, generation of WT1-A*0201 specific CTL from PBL avoids CD8+ selection. Currently, we are aiming to generate WT1-specific CTL using an overlapping WT1 peptide-mix in order to widen our ability to treat patients with different HLA types. This study has implications for cellular immunotherapy in leukemia patients who relapse following allogeneic stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

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Circulating T Cells Exhibit Different TIM3/Galectin-9 Expression in Patients with Obesity and Obesity-Related Diabetes

Journal of Diabetes Research ◽

10.1155/2020/2583257 ◽

2020 ◽

Vol 2020 ◽

pp. 1-10

Author(s):

Lili Sun ◽

Shengyi Zou ◽

Sisi Ding ◽

Xuan Du ◽

Yu Shen ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Healthy Controls ◽

Fasting Insulin ◽

Blood Samples ◽

Healthy Donors ◽

Simple Obesity ◽

Circulating T Cells

Aims. Obesity is highly associated with type 2 diabetes mellitus (T2DM). The TIM3/galectin-9 pathway plays an important role in immune tolerance. Herein, we aimed to investigate the expression of TIM3 and galectin-9 in peripheral blood and to evaluate their clinical significance in patients with obesity and obesity-related T2DM. Methods. We performed flow cytometry on peripheral blood samples from healthy donors (HC), patients with simple obesity (OB), and patients with obesity comorbid T2DM (OD). The expression of TIM3 on CD3+, CD4+, and CD8+ T cells was determined. The level of galectin-9 in plasma was detected by ELISA. Results. We demonstrated the enhancement of TIM3 on CD3+, CD4+, and CD8+ T cells in the OB group when compared with healthy controls, while it was decreased significantly in the OD group. The TIM3+CD8+ T cells of the OB group were positively correlated with risk factors including BMI, body fat rate, and hipline. The concentration of galectin-9 of the OD group in plasma was significantly higher than that of healthy donors and the OB group. Moreover, the level of galectin-9 of the OD group was positively correlated with fasting insulin and C-peptide, which were two clinical features that represented pancreatic islet function in T2DM. Conclusions. Our results suggested that TIM3 and galectin-9 may be potential biomarkers related to the pathogenesis of obesity-related T2DM.

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Differential Gene Expression Profile Identifies the Nature of T Cell Defects in AML Patients at Diagnosis.

Blood ◽

10.1182/blood.v112.11.1186.1186 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1186-1186

Author(s):

Rifca Le Dieu ◽

Alan G Ramsay ◽

David Taussig ◽

Richard Mitter ◽

John G. Gribben

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

Gene Expression Profile ◽

Expression Profile ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Tumour Cells ◽

Rhodamine Phalloidin ◽

Healthy Donors

Abstract Contrary to expectation, we have shown that the absolute number of T cells in the peripheral blood of patients presenting with AML is higher than that of healthy individuals (p=0.0006). Previous publications have demonstrated that in vitro the microenvironment in AML is immunosuppressive to healthy T cell function (Buggins et al. 2001). To analyze the molecular nature of T cell defects in patients with AML, we performed global gene expression profiling of peripheral blood CD4 and CD8 T cells from 10 AML patients compared to that of 21 healthy volunteers. The purity of the CD4 T cells was 86% in AML and 94% in healthy donors and 92% for AML and 87% in healthy donors for CD8 T cells. cRNA was hybridised on Affymetrix U133Plus2 chips using standard protocols. Samples were normalised using the MAS5 algorithm and subsequent analysis using Bioconductor software indicated marked differences between the gene expression profiles of CD4 and CD8 T cells from AML patients compared with normal individuals. Using a false discovery rate of 0.01 and fold change greater than 2, 1407 genes were found to be differentially regulated for CD4 and 779 genes for CD8. There was a large overlap in the two gene lists. The microarray data was validated using real-time quantitative PCR. Initial pathway analysis using Ingenuity software indicated many genes involved in cell proliferation, cell death and gene expression. Molecular defects in T cells from AML patients show marked differences from those we have already noted in T cells from patients with CLL. Only 2% of genes for CD4 and 6% of genes for CD8 are differentially regulated in T cells from both AML and CLL patients. These data suggest a different molecular basis for the T cell defects in these disease types. However, 2 genes downregulated in CD8 T cells in AML patients, ACTN1 and FILIP1 suggested impaired actin cytoskeletal activity may contribute to immune dysfunction in AML T cells as we have already demonstrated in CLL. We therefore tested the ability of AML CD4 and CD8 T cells to form intact immunological synapses (IS) with autologous AML blasts in the presence or absence of superantigen. F-actin was visualised with rhodamine phalloidin and recruitment of activated T cell receptor mediated signalling molecules was detected using an anti-phosphotyrosine antibody. Conjugate and synapse formation and phosphotyrosine signalling was assessed by confocal microscopy with at least 50 random conjugates analyzed. Unlike CLL B cells, AML blasts can act as antigen presenting cells (APCs) for conjugate and IS formation with healthy T cells. Again, in contrast to CLL, AML T cells are able to form conjugates with autologous tumour cells but demonstrate impaired IS formation and, in CD8 T cells, decreased phosphotyrosine signalling at the synapse site. This data indicates that T cells in patients presenting with AML are abnormal in terms of their gene expression profile and although they retain the ability to conjugate with autologous blasts, reduced polarisation of F-actin to form immune synapses is seen. We are currently investigating if these changes are induced directly by tumour cells and the functional consequences of the gene expression changes identified. The ability of AML blasts to function as APCs for IS formation is in keeping with the observed GVL effect in this disease. Understanding the nature of T cell defects in patients presenting with AML is fundamental before successful autologous immunotherapeutic strategies can be implemented.

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P862 Clinical benefit potentially evident with immunopharmacodynamic responses in prior-checkpoint failed metastatic melanoma patients treated with imprime PGG and pembrolizumab

Journal for ImmunoTherapy of Cancer ◽

10.1136/lba2019.15 ◽

2020 ◽

Vol 8 (Suppl 1) ◽

pp. A10.1-A10

Author(s):

Anissa Chan ◽

Nandita Bose ◽

Nadine Ottoson ◽

Xiaohong Qiu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Disease Control ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Clinical Benefit ◽

Secondary Resistance ◽

Enhanced Production ◽

Hla Dr ◽

Melanoma Patients

BackgroundCheckpoint inhibitor (CPI) monotherapy has revolutionized the treatment of melanoma, yet most patients are primary nonresponders or develop secondary resistance. Lack of antigen-specific T cell priming and/or immunosuppressive mechanisms leading to T cell exhaustion are critical cancer-extrinsic factors contributing to CPI resistance mechanisms. Immunotherapeutic agents capable of sparking de novo anti-tumor T cell responses or reinvigorating pre-existing exhausted T cell immunity could help reinstate the activity of CPI.MethodsOur Phase 2, multi-center, open label study, NCT02981303 in collaboration with Merck & Co., Inc., is evaluating Imprime PGG (Imprime), a novel yeast derived, Dectin-1 agonist, β-glucan PAMP in combination with pembrolizumab (KEYTRUDA®, pembro) in heavily CPI pre-treated melanoma patients (20 patients; 65% had >2 prior CPI regimens with 17/20 having previously progressed on pembro). Patients received Imprime (4 mg/kg) + pembro (200 mg) intravenously in a 3-week cycle. Here, we present the immunopharmacodynamic (IPD) responses elicited by Imprime and pembro in the peripheral blood of 19 patients.ResultsIn the intent-to-treat population (ITT; N=20), the disease control rate was 45% (1 CR and 8 SD), 6-month and 12-month OS rates were 65% and 45% respectively, and median OS (mOS) was 8.8 months. In the patients showing disease control, a significant increase in CH50, the classical pathway complement function (~0.7-2.6-fold), HLA-DR expression on classical monocytes (~0.61-1.94-fold) and reduction of frequency of PD-1+Tbet-EOMES+ exhausted CD8 T cells (~0.9-4-fold) was observed. Stimulation of peripheral blood mononuclear cells from a subset of patients by CD3/CD28 beads showed enhanced production of IL-2 and IFN-gamma in the CD8 T cells. Some of these IPD responses were also associated with 6-month landmark OS analyses. Additionally, whole blood gene expression analyses showed >2-fold upregulation of several myeloid and T cell activation genes including IFNg, CD83, IP-10, and IL-2RA. Enhanced OS was observed in patients with >1.3 fold increase in CH50 (8/19; HR 0.385; p=0.1) or >1.5-fold reduction in the frequency of exhausted CD8 T cells (8/19; HR 0.102; p=0.001). The IPD responses observed in the ITT population included formation of circulating immune complexes (peak levels ranging from ~4.5-16.1-fold) and production of complement activation protein SC5b9 (~3.4-25.6-fold), and increase in the frequency of HLA-DR+ myeloid cells (~0.43-3.71-fold).ConclusionsOverall, these data, albeit in a small population, demonstrate that Imprime/pembro combination can drive the innate/adaptive IPD responses that are critical for providing clinical benefit to the patients who have progressed through prior CPI treatments.Ethics ApprovalThe study was approved by central and local ethics committees depending on site requirements. The central IRB for the study is Western Institutional Review Board (WIRB), approval number 20162506; all sites received IRB approval before opening the study at the respective sites.

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Immune Responses Against the Tumor-Associated Antigens WT1, MUC-1, PRAME and HER2/Neu in 114 Prospectively Screened Healthy Donors: Effects of Gender and Prior Pregnancy and Implications for Immunotherapy

Blood ◽

10.1182/blood.v120.21.4115.4115 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4115-4115 ◽

Cited By ~ 1

Author(s):

Mathias Lutz ◽

Andrea Worschech ◽

Sabine Gahn ◽

Laura Bernhard ◽

Michael Schwab ◽

...

Keyword(s):

T Cells ◽

Healthy Volunteer ◽

Immune Responses ◽

Cd8 T Cells ◽

Peripheral Blood ◽

High Avidity ◽

Tumor Associated Antigens ◽

Healthy Donors ◽

Significant Difference ◽

Ifn Γ

Abstract Abstract 4115 Background: The graft-versus-leukemia (GvL) effect is a central component of the stem cell allograft's ability to cure hematological malignancies. While it has become evident that donor-derived T lymphocytes targeting tumor-associated antigens (TAAs) contribute significantly to the GvL effect little is known about the frequency and the origin of TAA-directed immune responses in healthy donors. Here we investigate for the first time their frequency against three well-known and transplant-relevant TAAs (WT1, MUC-1, PRAME) and one well-described TAA in solid tumor immunotherapy (HER2/neu) in a prospective manner considering gender and former pregnancy as potential influencing factors. Material and Methods: To detect the very low frequencies of these antigen-specific CD8+ T cells we have used immunodominant peptides of WT1, MUC-1, PRAME and HER2/neu and a quantitative polymerase chain reaction (qPCR) to measure Interferon-gamma (IFN-γ) mRNA production by peptide-pulsed CD8+ T cells from HLA-A*0201-positive healthy volunteers. After obtaining approval by the local ethical committee and written informed consent 114 HLA-A*0201-positive healthy volunteer blood donors were enrolled in this prospective research study including males (median age 40.5 years), nulliparous women (median age 27.5 years) and women with at least one delivery (median age 45.5 years). Each group consisted of 38 individuals as planned in the original study design. Peripheral blood was drawn, peripheral blood mononuclear cells (PBMCs) and CD8+ lymphocytes isolated and stimulated with peptide-loaded (0.1 and 10 μM), irradiated T2 cells. IFN-γ mRNA expression was measured by qPCR. The irrelevant melanoma antigen gp100 was used as a negative control and a stimulation index was calculated accordingly. A two-fold change or more as compared to gp100 was considered a positive result. The polyclonal stimulator PMA and a HLA-A*0201 restricted CMV peptide were used as positive controls. Results: Of the screened 114 healthy volunteer donors 17 (15%) showed immune responses against WT1, 8 (7%) against PRAME, 16 (14%) against MUC-1 and 6 (5%) against HER2/neu with one or both peptide concentrations. Comparing nulli- and multiparous women there was no significant difference regarding the frequency of assessed positive immune responses. However, comparing female (n=76) and male donors (n=36) we found that positive immune responses were more frequently present in men. Using the Mann-Whitney test this difference between men and women was significant for HER2/neu (p=0.0478) and reached borderline significance for PRAME (p=0.0677). This data is presented in Figure 1. Conclusions: In this prospectively screened cohort of healthy volunteer donors we could detect positive responses against WT1, MUC-1, PRAME and HER2/neu containing both low and high avidity immune responses. Prior delivery did not increase the frequency of immune responses. Interestingly, men showed a general tendency towards higher frequencies of immune responses to TAAs, particularly to HER2/neu and PRAME. Thus, the exploitation of donor-derived autoimmunity against selected TAAs by improving donor selection and facilitating adoptive immunotransfer of donor-derived T cells may significantly contribute to the control of the malignant disease after allotransplantation. Disclosures: No relevant conflicts of interest to declare.

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B-cell and T-cell values in peripheral blood in Polish mixed-breed rabbits with addition of blood of meet breeds

Polish Journal of Veterinary Sciences ◽

10.2478/pjvs-2014-0060 ◽

2014 ◽

Vol 17 (3) ◽

pp. 421-426 ◽

Cited By ~ 1

Author(s):

B. Tokarz-Deptuła ◽

P. Niedźwiedzka-Rystwej ◽

B. Hukowska-Szematowicz ◽

M. Adamiak ◽

A. Trzeciak-Ryczek ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Laboratory Animals ◽

Immunological Parameters ◽

Four Seasons ◽

Mixed Breed ◽

The Impact

Abstract In Poland, rabbit is a highly valued animal, due to dietetic and flavour values of its meat, but above all, rabbits tend to be commonly used laboratory animals. The aim of the study was developing standards for counts of B-cells with CD19+ receptor, T-cells with CD5+ receptor, and their subpopulations, namely T-cells with CD4+, CD8+ and CD25+ receptor in the peripheral blood of mixed-breed Polish rabbits with addition of blood of meet breeds, including the assessment of the impact of four seasons of the year and animal sex on the values of the immunological parameters determined. The results showed that the counts of B- and T-cells and their subpopulations in peripheral blood remain within the following ranges: for CD19+ B-cells: 1.05 - 3.05%, for CD5+ T-cells: 34.00 - 43.07%, CD4+ T-cells: 23.52 - 33.23%, CD8+ T-cells: 12.55 - 17.30%, whereas for CD25+ T-cells: 0.72 - 2.81%. As it comes to the season of the year, it was observed that it principally affects the values of CD25+ T-cells, while in the case of rabbit sex, more changes were found in females.

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