Flow cytometric measurement of STAT1 and STAT3 phosphorylation in CD4 + and CD8 + T cells—clinical applications in primary immunodeficiency diagnostics

BackgroundOverexpression of TAM receptors, including MERTK, in some cancers are integral for chemoresistance, proliferation and metastasis.1 Our group has previously demonstrated that T cells also express MERTK and engagement of MERTK signaling is responsible for increased proliferation, functional capacity and metabolic fitness.2 It is therefore important to further study the effect of MERTK inhibition on T cell function in the context of cancer treatments where MERTK inhibitors may play a role. Here we provide evidence that MERTK inhibition impacts greatly on T cell proliferation, specifically reducing phosphorylated mTOR. We have also demonstrated that MERTK expression is increased on CD8 central memory subsets during longterm expansion providing evidence that this signaling pathway may be important for sustaining T memory responses.Materials and MethodsFlow cytometric analysis was used to investigate the effect of titration of MERTK small molecule inhibitor UNC2025 on healthy donor T cells activated with CD3/CD28 dynabeads. Cell trace dye was used to track proliferation of CD4 and CD8 T cells along with markers of memory differentiation (CCR7 and CD45RO), activation (CD137) and function (IFNy, Tnfa and IL-2). MERTK signaling was assessed using phospho flow cytometric methodology of phosphorylated mTOR, AKT, ERK1/2, p38-MAPK and STAT5. Long term cultures of donor T cells of up to 28 days were investigated for MERTK expression alongside memory differentiation.ResultsWe demonstrated that moderate concentrations of MERTK inhibitor reduced proliferation of activated T cells. Despite inhibition of cell division, cell size still increased 2 fold compared to resting cells and cell viability remained unchanged. Additionally, the proportion of central memory to effector memory populations and intracellular cytokine production was not impacted. Analysis of molecules involved in MERTK signaling revealed that phosphorylated mTOR was significantly modulated following the addition of MERTK inhibitor. Long term culture of CD8 T cells demonstrated MERTK was significantly increased following early and late re-stimulation, and expression of MERTK was strongly associated with central memory subsets.ConclusionsOur results demonstrate that inhibition of MERTK signaling on T cells reduces cell division where mTOR is significantly impacted. Despite this, other functional aspects, such as intracellular cytokine production remain unchanged. Therefore, interruption of MERTK signaling on T cells has a specific effect on cell division rather than cytotoxic function on a cell by cell basis. This has potential ramifications on the use of MERTK inhibitors to treat tumors where the ability to form substantial cytotoxic T cell populations might be reduced. In addition, increased MERTK expression on central memory subsets during long term culture suggests this signaling pathway could be critical for generating memory pools of T cells and provide new avenues for the improvement of adoptive T cell therapy protocols.ReferencesCummings CT, Deryckere D, Earp HS, Graham DK. Molecular pathways: MERTK signaling in cancer. Clin Cancer Res 2013;19(19):5275–5280.Peeters MJW, Dulkeviciute D, Draghi A, et al. MERTK Acts as a Costimulatory Receptor on Human CD8+T Cells. Cancer Immunol Res 2019;7(9):1472–1484.Disclosure InformationR.M. Powell: None. M.J.W. Peeters: None. A. Rachbech: None. P.T. Straten: None.

Download Full-text

T Cell Receptor Repertoire of Patients with Chronic Graft-Versus Host Disease.

Blood ◽

10.1182/blood.v108.11.5174.5174 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5174-5174

Author(s):

Olga Y. Azhipa ◽

Scott D. Rowley ◽

Michele L. Donato ◽

Robert Korngold ◽

Thea M. Friedman

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Flow Cytometric Analysis ◽

T Cell Responses ◽

T Cell Repertoire ◽

Cell Repertoire ◽

Cell Responses ◽

Flow Cytometric ◽

Complexity Index

Abstract Chronic GVHD (cGVHD) is a major risk factor in patients receiving allogeneic hematopoietic cell transplantation (HCT), and is a complicated syndrome with a combination of autoimmune-like features and a range of multiorgan manifestations. Currently, efforts are being made to standardize the criteria for diagnosis and staging of cGVHD, but there is little understanding of the pathogenesis of the disease, associated biomarkers, and the immune perturbations that may result. Reconstitution of the T cell repertoire after allo-HCT often takes several months to a year, and may be significantly impaired or skewed in patients who develop cGVHD. We thus sought to assess the immune T cell status of cGVHD patients by TCR Vβ CDR3-size spectratype analysis. A cohort of 9 patients who underwent allo-HCT (PBMC n=7; BM n=2) were enrolled in the study. The underlying diseases in these patients were CML (n=1), AML (n=4), ALL (n=1), CLL (n=1), and MM (n=2). Patients received either reduced intensity or myeloablative conditioning before transplantation, and 8 of the 9 had a previous history of acute GVHD. Furthermore, the patients did not have evidence of infectious disease. PBMC was collected from each patient at one time point ranging from 2 wk to 3 yr from the time they were diagnosed with cGVHD. The onset of cGVHD ranged from 100 d to 3 yr post-HCT (median of 5 mo). Flow cytometric analysis was performed on peripheral blood lymphocytes from 7 of the 9 patients to analyze recovery of different subpopulations. PCR amplification of the CDR3 region of 21 TCR Vβ genes was used to analyze the diversity of the T cell repertoire. The PCR products were run on a sizing gel to separate the CDR3-lengths, and further analyzed by ABI GeneMapper software. Flow cytometric analysis revealed diverse percentages of CD4+ and CD8+ T cells among the 7 patients tested, which were correlated with the post-HCT period. Two patients who received HCT, 4 and 9 months before blood sampling, had only 3% and 4% CD4+ and 3% and 9% CD8+ T cells in their PBMC sample, respectively. On the other hand, the remaining 5 patients, who were all at later time points post-HCT, had CD4+ and CD8+ T cell percentages within normal range. One patient had a ratio close to the normal 2:1 CD4/CD8 ratio, two patients had a 1:1 ratio, and four had inverse CD4/CD8 ratios. Based on CDR3-size spectratype analysis, we determined the recipient TCR-Vβ complexity index within each resoluble family, which represented the percentage of the number of peaks found for each Vβ relative to that found in the average corresponding Vβ family of 10 healthy donors. We considered Vβ to be fully complex if the complexity index exceeded 85%. The results indicated that 41 to 88% of resolved Vβ in all 9 patients were fully complex, with the lower range corresponding to those patients sampled early post-HCT. Vβ 1, 2, 4, 6, 8, 12, and 13 families revealed the best recovery in all patients, even in patients after 4-mo post-HCT. Importantly, extensive skewing of the repertoire within most of the TCR Vβ families were found in all 9 recipients, suggesting that there were active heterogenous T cell responses in those patients with cGVHD. As to what these T cell responses were directed to remains to be seen, and could theoretically involve autoantigens, alloantigens, tumor antigens, or sub-detectable infectious agents. In any case, the presence of a wide-ranging T cell response in these patients may serve as an important new diagnostic indicator for cGVHD.

Download Full-text

Prevention of Acute GvHD During MHC Haploidentical BMT: Evaluating the Efficacy of T-Cell Costimulation Blockade Using a Novel Rhesus Macaque Transplant Model.

Blood ◽

10.1182/blood.v114.22.2455.2455 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2455-2455

Author(s):

Weston Miller ◽

Caleb E. Wheeler ◽

Angela Panoskaltsis-Mortari ◽

Allan D Kirk ◽

Christian P Larsen ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Rhesus Macaque ◽

Cd8 T Cells ◽

Flow Cytometric Analysis ◽

Costimulation Blockade ◽

Flow Cytometric ◽

Gvhd Prophylaxis ◽

High Level ◽

Cd127 Expression

Abstract Abstract 2455 Poster Board II-432 Introduction: While hematopoietic stem cell transplantation (HSCT) offers a cure for many hematologic diseases, it remains plagued by often fatal graft-versus-host disease (GvHD). Despite the inadequacy of current GvHD prevention strategies, especially for MHC-mismatched HSCT, the pace of the clinical introduction of novel therapeutics has been slow, likely due to the lack of a suitable translational model to rigorously test the immunologic and clinical impact of novel biologic therapies. Among the most promising of these therapies include those that block T cell costimulation blockade. While they have been used for both autoimmune disease and to prevent rejection of solid organ transplants, costimulation blockade reagents have not yet been evaluated for efficacy in preventing clinical GvHD. Here we describe a novel primate model of MHC-mismatched GvHD, that has allowed us, for the first time, to evaluate the mechanisms controlling GvHD in a primate translational system, and to evaluate the efficacy of costimulation blockade for the prevention of primate GvHD, even across haplo-MHC barriers. Methods: Using DNA microsatellite-based pedigree analysis and MHC haplotype determination, we have developed the first MHC-defined Rhesus macaque HSCT system. MHC haplo-identical transplant pairs were chosen, and recipients prepared for transplant with TBI (8 Gy, as a single dose, with lung shielding to 6 Gy). Animals were either treated with no immunosuppression post-transplant (controls) or with a costimulation blockade-based regimen which included CD28/B7 blockade with abatacept (20mg/kg every 7 days), CD40/CD154 blockade with the 3A8 anti-CD40 monoclonal antibody (maintenance dosing at 5mg/kg twice weekly) as well as sirolimus to maintain serum trough levels between 5-10 ng/mL. Either leukopheresis-derived peripheral blood stem cells or bone marrow was used for transplant (average total nucleated cell dose = 9.3 +/-2.7×108/kg; average CD3+ cell dose = 1.1 +/- 0.88 ×108/kg) Donor engraftment was measured by microsatellite analysis, and GvHD was graded clinically using standard scales. The immune phenotype after transplant was determined by multicolor cell- and serum-based flow cytometric analyses. Results: Seven haploidentical transplants have been completed. Three controls received no immunosuppression. These animals demonstrated rapid and complete donor engraftment, with donor T cell activation and proliferation occurring within one week of transplant, coincident with the onset of severe clinical GvHD, which predominantly targeted the GI tract. Flow cytometric analysis showed loss of CD127 expression on both CD4+ and CD8+ T cells, consistent with their rapid clonal expansion and differentiation. Multiplexed luminex cytokine analysis demonstrated high-level secretion of the inflammatory cytokines IFNγ, and IL18, as well as the counter-regulatory cytokine IL-1RA. Importantly, no rise in TNF, IL-1b, nor IL17 was measured despite severe GvHD. In contrast, four treated animals received a haplo-identical BMT in the setting of abatacept/anti-CD40 and sirolimus for GvHD prophylaxis. All of these recipients demonstrated rapid donor engraftment, but, unlike the controls, they were protected against clinical GvHD—they displayed neither the skin rash nor the profuse diarrhea noted in the control animals. Flow cytometric analysis demonstrated maintenance of CD127 expression on both CD4+ and CD8+ T cells. Furthermore, luminex analysis revealed that expression of IFNγ, IL18 and IL-1RA were all normal in the setting of GvHD prophylaxis with costimulation blockade and sirolimus. Conclusions: We have established a robust model of haplo-identical HSCT and GvHD using an MHC-defined Rhesus macaque colony. This model has allowed us to begin to determine the mechanisms underlying GvHD during primate haplo-identical BMT and to assess the efficacy of novel regimens to prevent this disease. We find that unprotected primate GvHD is characterized by rapid T cell proliferation, with concomitant loss of expression of CD127 on both CD4+ and CD8+ T cells. In addition, it is associated with a cytokine storm, including high level secretion of IFNγ, IL18 and IL-1RA into the serum. Finally, we find that CD28/CD40-directed costimulation blockade in combination with sirolimus can effectively inhibit both the clinical and cellular hallmarks of GvHD during haplo-identical BMT, and thus may deserve close clinical scrutiny as a possible prophylaxis strategy during these high risk transplants. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Preclinical Evaluation Of Engineered T Cells In Multiple Myeloma: Uncovering a Mechanism Of Immune Escape

Blood ◽

10.1182/blood.v122.21.4205.4205 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4205-4205

Author(s):

Zandra K. Klippel ◽

Jeffrey Chou ◽

Andrea M. H. Towlerton ◽

Paul F Robbins ◽

Lilien Voong ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

Tumor Cells ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Immune Escape ◽

Adoptive Therapy ◽

Myeloma Cells ◽

Nsg Mice ◽

Flow Cytometric

Abstract Introduction Adoptive immunotherapy is an increasingly effective modality of cancer therapy. The ability to redirect the antigenic specificity of patient-derived T cells toward autologous tumor cells through introduction of T-cell receptors (TCRs) or chimeric antigen receptors (CARs) enables reproducible manufacturing of tumor-reactive T cell products even in patients who carry few, if any, tumor-reactive T cells in their peripheral blood repertoire. We present the results of our pre-clinical studies of adoptive therapy with T cells transduced with a retroviral vector that encodes an enhanced-affinity (a95:LY) variant of the HLA-A*02:01-restricted, NY-ESO-1157-165-specific 1G4 TCR to redirect CD8+ T cells from HLA-A*02:01+ multiple myeloma patients to HLA-A*02:01+, NY-ESO-1-expressing myeloma cells. Methods CD3-stimulated peripheral blood mononuclear cells from HLA-A*02:01+ multiple myeloma patients were retrovirally transduced with the NY-ESO-1157-165-specific 1G4 a95:LY TCR. CD8+ TCR-transduced cells were isolated by flow cytometric sorting with a NY-ESO-1157-165/HLA-A*02:01 tetramer. The cytolytic activity of CD8+tetramer+ cells was evaluated by 51Cr release assay using as target cells the multiple myeloma cell lines U266 (HLA-A*02:01+ NY-ESO-1+) and UM-9 (HLA-A*02:01- NY-ESO-1+), and T2 cells with or without exogenous NY-ESO-1157-165 peptide. The U266 cell line was stably transduced with luciferase-containing retrovirus and used to develop a xenograft model of diffuse myeloma in NOD/scid/IL-2Rg-null (NSG) mice in order to evaluate the anti-myeloma activity of adoptive therapy with CD8+ TCR-transduced T cells. Mice that received TCR-transduced CD8+cells and developed disease were sacrificed, and human CD138+ cells were harvested from marrow and other sites for evaluation by flow cytometry, HLA-A typing, NY-ESO-1 expression, and loss of heterozygosity (LOH) analysis of the Major Histocompatibility Complex (MHC) on chromosome 6 with short tandem repeat (STR) probes to determine the mechanism of immune escape. Results CD8+ TCR-transduced cells were specifically cytolytic against HLA-A*02:01+, NY-ESO-1+ tumor cells. Intravenous injection of luciferase-transduced U266/Luc in sub-lethally irradiated NSG mice led to the development of a multiple myeloma-like disease. Mice that received U266/Luc without T cells (control) developed progressive disease within 2 weeks, and met criteria for euthanasia by week 9. Mice that received U266/Luc with sham-transduced cells developed myeloma more slowly, yet all met criteria for euthanasia by week 18 after U266/Luc injection. Of the 6 mice that received U266/Luc and NY-ESO-1-specific TCR-transduced CD8+ T cells, 4 did not have any evidence of myeloma by bioluminescence at the end of study (week 18), and 2 had low burden disease at that point. Kaplan-Meier survival analysis demonstrated significant improvement of overall survival in the mice that received TCR-transduced T cells (Log-rank test p< 0.0001). Flow cytometric analysis of human CD138+ cells isolated from the 2 mice that developed myeloma despite adoptive therapy with NY-ESO-1-specific T cells demonstrated selective loss of surface HLA-A*02 expression, with preserved expression of other MHC class I molecules. Real-time PCR analysis also confirmed preserved expression of HLA-A, B2M, and NY-ESO-1. Low resolution HLA-A typing of genomic DNA from myeloma cells from these 2 mice revealed loss of HLA-A*02, but retention of HLA-A*03. LOH analysis using 7 STR markers mapping to the MHC on chromosome 6p21.3 and 2 markers on chromosome 15 (control) demonstrated LOH in the MHC involving the HLA-A locus in myeloma cells from both of the mice that developed disease despite TCR-transduced T cells. The extent of LOH in the myeloma cells from the 2 mice was distinct. Conclusions LOH in the MHC as a mechanism of immune scape has been described in allogeneic transplantation for AML, but has not been described in multiple myeloma. We identified LOH affecting the HLA-A allele targeted by adoptively transferred TCR-transduced T cells. Given that NY-ESO-1-specific TCR-transduced cells have recently entered clinical testing, this mechanism of immune escape should be evaluated in patients that fail therapy despite persistence of adoptively transferred T cells. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Expression of two alpha chains on the surface of T cells in T cell receptor transgenic mice.

Journal of Experimental Medicine ◽

10.1084/jem.178.5.1807 ◽

1993 ◽

Vol 178 (5) ◽

pp. 1807-1811 ◽

Cited By ~ 90

Author(s):

W R Heath ◽

J F Miller

Keyword(s):

T Cells ◽

T Cell ◽

Transgenic Mice ◽

T Cell Receptor ◽

Cd8 T Cells ◽

Flow Cytometric Analysis ◽

Cell Receptor ◽

Specific Monoclonal Antibody ◽

Beta Chain ◽

Flow Cytometric

CD8+ T cells taken directly from mice expressing a Kb-specific T cell receptor (TCR) transgene expressed the transgenic TCR in a bimodal profile as detected by flow cytometric analysis using a clonotype-specific monoclonal antibody. Those cells expressing the lower density of the transgenic TCR expressed the transgenic beta chain and two different alpha chains on their surface. One alpha chain was the product of the alpha transgene, whereas the other was derived by endogenous rearrangement. This report provides the first demonstration that T cells isolated directly from mice may express two different TCR clonotypes on their surface. The potential consequences of this finding for studies using TCR transgenic mice and for the induction of autoimmunity are discussed.

Download Full-text

Comparison of two flow cytometric methods enumerating CD4 T cells and CD8 T cells

Cytometry ◽

10.1002/(sici)1097-0320(19980901)33:1<76::aid-cyto9>3.0.co;2-9 ◽

1998 ◽

Vol 33 (1) ◽

pp. 76-82 ◽

Cited By ~ 11

Author(s):

Jan Storek ◽

Monja A. Dawson ◽

David G. Maloney

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Flow Cytometric

Download Full-text

CD4+ T Cell Fate in Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-117674 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4494-4494

Author(s):

Rachel Elizabeth Cooke ◽

Jessica Chung ◽

Sarah Gabriel ◽

Hang Quach ◽

Simon J. Harrison ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Research Funding ◽

Flow Cytometric Analysis ◽

Advisory Committees ◽

Mitochondrial Mass ◽

Flow Cytometric

Abstract The average incidence of multiple myeloma (MM) is in the 7th decade that coincides with the development of immunosenescence and thymic atrophy, meaning that lymphocyte recovery after lymphopenia-inducing therapies (most notably autologous stem cell transplant, ASCT) is largely reliant on homeostatic proliferation of peripheral T cells rather than replenishing the T cell pool with new thymic emigrants. We have previously shown that there is a significant reduction in circulating naïve T cells with a reciprocal expansion of antigen-experienced cells from newly diagnosed MM (NDMM) to relapsed/refractory disease (RRMM). This results in a reduced TCR repertoire and the accumulation of senescence-associated secretory phenotype cytotoxic T cells, which maintain the ability to produce IFNγ but lose proliferative potential. A reduction in CD4:8 ratio is also a characteristic finding in MM with disease progression, which can be explained by high IL-15 levels in lymphopenic states that preferentially drive expansion of CD8+ memory T cells. We wanted to further evaluate what changes were occurring in the CD4+ T cell population with disease progression in MM. We analyzed paired peripheral blood (PB) samples from patients with NDMM and RRMM, and compared with age-matched normal donors (ND). In the NDMM cohort, we examined T cells from PB samples at baseline, after 4 cycles of lenalidomide and dexamethasone (len/dex), and after ASCT; and in the RRMM cohort samples from baseline and after 6 cycles of len/dex. We firstly confirmed in flow cytometric analysis of T cells at serial intervals in NDMM patients that the reduction in circulating naïve T cells and in CD4:8 ratio occurs post ASCT and does not recover by time of last follow-up. We next utilised RNA-seq to analyse differences in CD4+ T cells from NDMM, RRMM and ND. CD4+ T cells from RRMM showed downregulation of cytosolic ribosomal activity but maintenance of mitochondrial ribosomal activity and significant upregulation of pathways involved with calcium signalling. To this end, we evaluated mitochondrial biogenesis and metabolic pathways involved with mitochondrial respiration. Flow cytometric analysis of mitochondrial mass showed a marked increase in RRMM compared with ND, in keeping with a shift towards memory phenotype. Key rate-limiting enzymes in fatty acid β-oxidation (CPT1-A, ACAA2 and ACADVL) were all significantly increased in RRMM compared with ND. To analyse whether these cells were metabolically active, we also measured mitochondrial membrane potential and reactive oxygen species (ROS), gating on cells with high mitochondrial mass. Mitochondrial membrane potential was significantly increased in RRMM compared with ND, although ROS was reduced. The significance of this is not clear, as ROS are not only implicated in cell senescence and activation-induced cell death, but are also positively involved in tyrosine kinase and PI3K-signalling pathways. PD-1 has been shown to play a role in transitioning activated CD4+ T cells from glycolysis to FAO metabolism, and elevating ROS in activated CD8+ T cells. We analysed PD-1 expression on T cells in RRMM and at treatment intervals in NDMM (as described earlier). The proportion of CD4+ and CD8+ T cells expressing PD-1 was increased 4-6 months post-ASCT and remained elevated in CD4+ T cells 9-12 months post-ASCT, but normalised to baseline levels in CD8+ T cells. Increased PD-1 expressing CD4+ T cells was also evident in RRMM patient samples. This may suggest that in the lymphopenic state, PD-1 expression enhances longevity in a subset of CD4+ T cells by promoting reliance on mitochondrial respiration; however, their ability to undergo homeostatic proliferation is impaired. In CD8+ T cells, high PD-1 expression may lead to cell death via ROS accumulation, and these cells do not persist. ASCT remains a backbone of myeloma treatment in medically fit patients. However, this leads to significant permanent defects in the T cell repertoire, which may have unintended adverse outcomes. Additionally, T cells post-ASCT may not be metabolically adequate for the production of CAR-T cells, nor respond to checkpoint blockade therapies. Disclosures Quach: Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Sanofi Genzyme: Research Funding; Janssen Cilag: Consultancy. Harrison:Janssen-Cilag: Other: Scientific advisory board. Prince:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Download Full-text