Abstract P138: Controlling For Hypertension Does Not Alter Sex Differences In Renal T Cell Profiles In Doca-Salt Rats.

Hypertension ◽  
2020 ◽  
Vol 76 (Suppl_1) ◽  
Author(s):  
Kasey Belanger ◽  
Jennifer C Sullivan
Keyword(s):  
T Cells ◽  
Sex Differences ◽  
T Cell ◽  
Flow Cytometric Analysis ◽  
Sprague Dawley ◽  
Flow Cytometric ◽  
Acetate Salt ◽  
Sprague Dawley Rats ◽  
To Receive ◽  
Cell Profile

Our lab has shown that there are sex differences in the renal T cell profile in deoxycorticosterone acetate salt (DOCA) hypertension. The current study tested the hypothesis that sex differences in renal T cells in DOCA-salt rats is blood pressure (BP) dependent. At 10 wks of age, male and female Sprague-Dawley rats (N=9-12/group) were anesthetized, a telemeter was implanted for BP measurement, and a right uni-nephrectomy was performed. After one wk, all rats received a subcutaneous slow-release DOCA pellet (200 mg) with 0.9% saline to drink. To prevent DOCA-salt induced increases in BP, a subset of rats were randomized to receive hydrochlorothiazide (HCTZ; 55 mg/kg/day) and reserpine (4.5 mg/kg/day) combined with saline in drinking water. After 3 wks, the remaining kidney was isolated for flow cytometric analysis of T cells. After 3 wks of DOCA-treatment, males had a higher BP than females (P sex =0.0001). Treatment with HCTZ and reserpine prevented DOCA-induced increases in BP in both sexes (P t x t <0.0001; P int =0.307). With DOCA-salt induced hypertension, males had more renal CD4 + T cells (P sex =0.4) and Th17 (P sex =0.02) than females. HCTZ and reserpine decreased both CD4 + cells (P t x t <0.0001) and Th17 cells (P t x t <0.0001) and the decreases were comparable between sexes (CD4 + : P int =0.86; Th17: P int =0.55). With DOCA alone, females had more renal Tregs than males (P sex <0.0001). HCTZ and reserpine did not significantly change renal Tregs in either sex (P t x t =0.11; P int =0.38).Our data suggests that while preventing DOCA-salt induced increases in BP attenuates the increase in renal pro-inflammatory T cells, sex differences in renal T cells is not BP dependent.

scholarly journals Chronic ANG II infusion induces sex-specific increases in renal T cells in Sprague-Dawley rats

2015 ◽  
Vol 308 (7) ◽  
pp. F706-F712 ◽  
Author(s):  
Margaret A. Zimmerman ◽  
Babak Baban ◽  
Ashlee J. Tipton ◽  
Paul M. O'Connor ◽  
Jennifer C. Sullivan
Keyword(s):  
T Cells ◽  
T Cell ◽  
Specific Effect ◽  
Ang Ii ◽  
Sprague Dawley ◽  
Mas Receptor ◽  
Sprague Dawley Rats ◽  
Males And Females ◽  
Anti Inflammatory ◽  
Cell Profile

Recent studies suggest that sex of the animal and T cell impact ANG II hypertension in Rag−/− mice, with females being protected relative to males. This study tested the hypothesis that ANG II results in greater increases in proinflammatory T cells and cytokines in males than in females. Male and female Sprague-Dawley (SD) rats, aged 12 wk, were treated with vehicle or ANG II (200 ng·kg−1·min−1) for 2 wk. Renal CD4+ T cells and Tregs were comparable between vehicle-treated males and females, although males expressed more Th17 and IL-17+ T cells and fewer IL-10+ T cells than females. ANG II resulted in greater increases in CD4+ T cells, Th17 cells, and IL-17+ cells in males; Tregs increased only in females. We previously showed that ANG (1–7) antagonizes ANG II-induced increases in blood pressure in females and ANG (1–7) has been suggested to be anti-inflammatory. Renal ANG (1–7) levels were greater in female SD at baseline and following ANG II infusion. Additional rats were treated with ANG II plus the ANG (1–7)-mas receptor antagonist A-779 (48 μg·kg−1·h−1) to test the hypothesis that greater ANG (1–7) in females results in more Tregs relative to males. Inhibition of ANG (1–7) did not alter renal T cells in either sex. In conclusion, ANG II induces a sex-specific effect on the renal T cell profile. Males have greater increases in proinflammatory T cells, and females have greater increases in anti-inflammatory Tregs; however, sex differences in the renal T cell profile are not mediated by ANG (1–7).

Abstract 254: Chronic Ang II Hypertension Differentially Impacts the Renal T Cell Profile in Males and Females

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Margaret A Zimmerman ◽  
Ashlee J Tipton ◽  
Babak Baban ◽  
Jennifer C Sullivan
Keyword(s):  
T Cells ◽  
Sex Differences ◽  
T Cell ◽  
Th17 Cells ◽  
Female Rats ◽  
Ang Ii ◽  
Sprague Dawley ◽  
Male And Female ◽  
Cell Counts ◽  
Cell Profile

T cells contribute to angiotensin (Ang) II hypertension in male animal models yet less is known in females. We previously showed that hypertensive female rats have more immuno-suppressive T regulatory cells (Tregs) and males have more pro-inflammatory Th17 cells in their kidneys. This study tested the hypothesis that Ang II results in greater expression of renal Tregs in females and Th17 cells in males. 12 wk old male and female Sprague Dawley rats (SD) were treated with Ang II (200 ng/kg/min) or vehicle for 2 wks; blood pressure (BP) was measured via tail cuff and renal T cell profiles were measured via flow cytometry. Ang II infusion increased BP in both sexes (mmHg: males: 130±2 to 152±2; females: 123±1 to 146±1; p<0.05). Females had lower CD3 + T cell counts at baseline vs. males (p<0.0001). Ang II significantly increased CD3 + T cells in both sexes (p<0.0001). Baseline CD4 + T cell counts were comparable between male and female SD. Ang II infusion increased CD4 + T cells in both sexes (p<0.0001), however, expression remained lower in females (p=0.04). Females had greater expression of Tregs (p<0.0001) at baseline. Ang II infusion increased Treg expression in females (p=0.004), with no effect in males. Males expressed more baseline Th17 cells than females (p<0.001). Though Ang II increased levels in both sexes (p<0.001), females maintained significantly reduced Th17 cell expression vs. males (p<0.001). Therefore, Ang II resulted in female SD having greater renal expression of Tregs and males having greater expression of Th17 cells. In conclusion, sex differences in the T cell profile may contribute to observed sex differences in BP control.

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

Blood ◽  
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.

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

Blood ◽  
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.

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

1993 ◽  
Vol 178 (5) ◽  
pp. 1807-1811 ◽  
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.

scholarly journals CD4+ T Cell Fate in Multiple Myeloma

Blood ◽  
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.

scholarly journals Flow Cytometric Analysis Revealed a Significant Accumulation of Terminally Differentiated T cell Subtypes in the Circulating Lymphocytes of Cytomegalovirus (CMV) Positive Follicular Lymphoma Patients

2021 ◽  
pp. 1-9
Author(s):  
Lugos MD ◽  
◽  
Dangana A ◽  
Ntuhun BD ◽  
Oluwatayo BO ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Flow Cytometric Analysis ◽  
B Cell Lymphoma ◽  
T Cell Subsets ◽  
Cmv Infection ◽  
Flow Cytometric ◽  
The Impact

Follicular lymphoma (FL), a non-Hodgkin lymphoma, is an indolent cancer of the B cell lineage that runs a chronic deterioration course that can result in multiple treatment episodes leading to resistance and possible transformation to diffuse large B cell lymphoma. Cytomegalovirus (CMV) reactivation during chemotherapy or after an organ or hematopoietic stem cell transplantation is a major cause of morbidity and mortality. This study tests the hypothesis that some of the heterogeneity of FL might result from chronic infection with Cytomegalovirus (CMV). This research was intended to appraise the impact of CMV infection on the subtypes of T cells in follicular lymphoma patients. We accessed stored peripheral blood mononuclear cells (PMBCs) from patients of known CMV serostatus recruited into an FL clinical trial. We undertook a multicolour flow cytometric analysis of the PBMCs and compared the number of lymphocyte subtypes of CMV-positive and CMV-negative FL patients. Data showed a significant increase in the quantity of terminally differentiated (TEMRA) T cell subsets, including EM3-CD8 (P=0.005), EM3-CD4 (P=0.018), E-CD4 (P=0.029), E-CD8 (P=0.033) and pE2-CD4 (P=0.046) phenotypes, as well as increased NKT cells (P=0.031) among CMV-positive patients compared to the negative group. Our findings support the hypothesis that recurrent infections characterise CMV infection in FL due to accelerated immune senescence and the accumulation of exhausted T cells. Based on the data, a case could be argued for the routine application of CMV screening in FL before treatment with chemo-immunotherapy to implement enhanced infection surveillance in CMV-positive patients. These discoveries can eventually help improve the treatment approaches in the management of FL toward a combinatorial viewpoint for direct cytotoxic and indirect immunomodulatory outlook

Chronic Lymphocytic Leukemia Cells Co-Opt CD200, CD270, CD274 and CD276 to Induce Impaired Actin Polarization At the T Cell Immune Synapse

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 802-802
Author(s):  
Alan G. Ramsay ◽  
Andrew J. Clear ◽  
Alexander Davenport ◽  
Rewas Fatah ◽  
John G. Gribben
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Direct Contact ◽  
Actin Polymerization ◽  
Flow Cytometric Analysis ◽  
Cd8 T Cell ◽  
Lymphocytic Leukemia ◽  
Flow Cytometric ◽  
Cell Synapse

Abstract Abstract 802 We have previously demonstrated that impaired formation of the T cell immunological synapse in response to autologous (auto) antigen-presenting cells (APCs) is a global immunosuppressive mechanism in chronic lymphocytic leukemia (CLL) (J Clin Invest. 2008;118(7):2427-2437). Polymerization of F-actin beneath the area of the T cell:APC contact site generates a structural support for signaling molecules to assemble and regulate appropriate CD4+ T cell activation and cytolytic CD8+ T cell (CTL) effector function. Importantly, direct contact interaction with tumor cells was shown to induce defective actin polarization at the synapse in previously healthy allogeneic (allo) peripheral blood (PB) T cells. Here we have extended our functional screening coculture assays and show that CD200, CD270 (TNF receptor, TNFR-superfamily 14, SF14), CD274 (programmed death ligand 1, PD-L1), and CD276 (B7-H3) are co-opted by primary CLL cells (n=25) to induce impaired actin polymerization at the CD3+ T cell synapse. Antibody neutralizaton of these CLL ligands significantly increased allo T cell synapse actin polymerization with APCs compared to isotype control treated cells (P<.01). Counteracting the combined activity of all four inhibitory proteins on CLL cells showed the largest increase in F-actin synapse polymerization. Importantly, we further demonstrate that direct contact coculture with CLL cells further augmented F-actin polymerization defects in auto PB patient T cells (isolated from low white blood cell count CLL patients), that was prevented by the prior blockade of these CLL inhibitory ligands (P<.01). Next we analyzed the in situ expression of inhibitory ligands and receptors by immunohistochemistry using a CLL lymphoid tissue microarray (TMA). Significantly higher expression of CD200+ CD270+ CD274+ CD276+ CD20+ CLL cells, and CD272+ (B and T lymphocyte attenuator, BTLA) CD279+ (PD-1) CD3+ T cells were detected compared to healthy counterpart cells from reactive control lymph node samples (P<.0001). Notably, higher expression of CD200+ CD274+ CLL cells correlated with poor disease outcome (P<.01). Flow cytometric analysis of peripheral blood patient cells showed that these inhibitory ligands were up-regulated on circulating CLL cells and also their receptors on auto T cells compared to age-matched healthy donor cells (P<.05). Next we investigated the impact of lenalidomide on CLL immunosuppressive signaling interactions with T cells. Both pretreatment of CLL cells with lenalidomide prior to primary coculture and direct addition of drug significantly increased (P <.01) subsequent allo T cell F-actin synapse polarization compared to control treated experiments. Flow cytometric analysis identified that lenalidomide downregulated the expression of these CLL inhibitory ligands and cognate receptors on allo T cells during intercellular contact interactions (P <.01), but not when age-matched healthy B cells were used. We next investigated the effect on cytolytic synapse function and demonstrated that allo CD8+ T cell killing function was significantly enhanced (P <.05) following combinational antibody blockade of CLL inhibitory ligands or lenalidomide treatment compared to control treated leukemic cells. Importantly, lenalidomide treatment blocked further augmented synapse impairment in auto T cells from CLL patients following coculture with CLL tumor cells. As members of the Rho family of GTPases, including RhoA, Rac1 and Cdc42 have been described as key regulators of actin polymerization, we measured their activated GTP-bound state in T cells following direct-contact interaction with CLL tumor cells. We demonstrate decreased active RhoA and Rac1 levels in TCR-stimulated allo T cells on coculture with CLL cells compared with primary coculture with healthy B cells (P <.05). In contrast, combinational antibody blockade of the CLL inhibitory ligands or lenalidomide treatment increased T cell Rho GTPase activity including Cdc42 (P <.05). In conclusion, our findings identify a new mechanism of cancer immunoescape in which CLL tumor cells co-opt multiple inhibitory B7-related molecules that can mediate global immunosuppressive actin defects in both auto and allo T cells. Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

Rapid and Semi-Automated Screening of Multiple Samples for Antigen-Specific T Lymphocytes

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4839-4839
Author(s):  
Anne Richter ◽  
Michaela Niemöller ◽  
Inken Verwohl ◽  
Katrin Lange ◽  
Anna Foerster-Marniok ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Flow Cytometric Analysis ◽  
T Cell Response ◽  
Activation Markers ◽  
Cell Stimulation ◽  
Flow Cytometric ◽  
T Cell Stimulation ◽  
Multiple Samples

Abstract Abstract 4839 Functional characterizations of T lymphocytes are performed to gain understanding on their contribution in certain immunological situations, to monitor the course of diseases, and to track therapeutic interventions. Meanwhile the flow cytometric analysis of antigen-specific T cells examined for intracellular cytokine production and expression of activation markers after a short-term in vitro antigenic challenge is a well-established method for research applications. Despite the advantages of this approach to qualify samples on a single cell level and by multiple parameters, the broad use of this analysis for immune monitoring purposes is hampered. Screening of a lot of samples is time-consuming, requires many manual handling steps, and operator experience in flow cytometric analysis of stimulated T cell samples. To overcome these hurdles, we worked out a complete strategy to rapidly study cytokine and activation marker profiles in antigen-specific T cells of multiple samples by a semi-automated process. For the simultaneous analysis of multiple samples we examined for the T cell stimulation an antigen pre-coated 96-strip-well culture system. This flexible and ready-to-use format provides the opportunity to screen either for a single antigen or in parallel for up to twelve antigen specificities by combining 8-well-strips possessing different antigens. The coated antigens consist of pools of overlapping 15-mer peptides derived from a single viral protein of cytomegalovirus, Epstein-Barr-Virus, or adenovirus. The peptide pools have been designed for activation of the specific CD4+ as well as CD8+ T cells. They are solubilized and thereby accessible for T cell stimulation after addition of a cell sample, e.g. peripheral blood mononuclear cells, suspended in culture medium into the antigen-coated well. After a stimulation period of six hours the induced T cell response is comparable to the activation with a conventional lyophilized and reconstituted peptide pool. To reduce the time and work load for cell harvesting, fixation, permeabilization, and staining, we developed a protocol and reagents to allow a rapid and easy-to-handle intracellular staining procedure. Compared to conventional staining protocols, all steps are executed in the 96-strip-well culture plate, i.e. cell harvesting is dispensable. Without any washing step, cells are fixed and stained with defined reagent cocktails containing antibodies to identify virus-specific CD3+ CD4+ CD154+ and/or CD3+ CD8+ T cells and various Anti-cytokine-fluorochrome conjugates to evaluate the cytokine pattern. With these modifications, we drastically diminished the overall processing time for the staining of up to 96 samples to only 50 minutes. Furthermore, we integrated an automated flow cytometric analysis process. This includes the possibility to measure the samples in the 96-strip-well plates hands-free using pre-defined experiment settings and acquisition templates. We also applied an automated gating strategy for the data analysis. Finally, a report summarizes the results of the T cell response against several viral proteins for all samples tested, e.g. frequencies of cytokine+ CD154+ CD4+ and cytokine+ CD8+ T cell subsets are indicated. Hands-on time for the multi-sample acquisition and analysis is only minimal and the standardized reagents/protocol and sample analysis process decrease inter- and intra-assay variations. In summary, with our newly developed tools and protocols for in vitro T cell stimulation, staining of activation markers as well as intracellular cytokines, and automated flow cytometric analysis we have set up a fast and convenient procedure to routinely monitor antigen-specific T cell responses. Disclosures: Richter: Miltenyi Biotec GmbH: Employment. Niemöller:Miltenyi Biotec GmbH: Employment. Verwohl:Miltenyi Biotec GmbH: Employment. Lange:Miltenyi Biotec GmbH: Employment. Foerster-Marniok:Miltenyi Biotec GmbH: Employment. Brauns:Miltenyi Biotec GmbH: Employment. Kramer:Miltenyi Biotec GmbH: Employment. Höher-Peters:Miltenyi Biotec GmbH: Employment. Büscher:Miltenyi Biotec GmbH: Employment. Assenmacher:Miltenyi Biotec GmbH: Employment. Schmitz:Miltenyi Biotec GmbH: Employment.

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