Donor CD8+ T Cells Facilitate Graft-Versus-Tumor Effect Via Alloantigen Rather Than Tumor-Specific Antigen Recognition Following Murine Myeloablative BMT.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3051-3051
Author(s):  
Asha B. Pillai ◽  
Pearline A. Teo ◽  
Aditi Mukhopadhyay ◽  
Samuel Strober
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Specific Antigen ◽  
Flow Cytometric Analysis ◽  
Antigen Recognition ◽  
Wild Type ◽  
Flow Cytometric ◽  
Tumor Specific Antigen

Abstract Previous data from our group and others has shown that donor CD8+ Tcells mediate graft-versus-tumor (GVT) function in murine myeloablative bone marrow transplantation (BMT) via Perforin/Fas ligand-dependent mechanisms. However, there has to date been no analysis of the mechanism of tumor recognition (i.e allo- versus tumor-specific antigen recognition) by donor CD8+ T cells following myeloablative MHC-mismatched BMT. In order to test the hypothesis that donor CD8 T-cells require allo-antigen recognition to maintain graft-versus-tumor effect, we developed stable full chimeras by transplanting T-cell depleted bone marrow (TCD BM) from C57BL/6 (H-2b) donor mice into myeloablated BALB/c (H-2d) hosts given 800 cGy total body irradiation (TBI) and evaluated the in vivo ability of CD8+ TCR+ splenocytes from these donors to kill the BCL1 tumor (a BALB/c-derived B-cell lymphoma carrying a detectable tumor-specific idiotype which can be monitored via peripheral blood flow cytometric analysis). These chimeras showed complete donor chimerism in myeloid, B- and T-lymphocytic lineages by day +100 following transplantation, and splenocytes from these chimeras exhibited tolerance to host-type but not third-party alloantigens. BALB/c hosts were given 800 cGy TBI on day -1, followed on day 0 by intravenous administration of 500 BCL1 tumor cells and infusion of 0.3x 106 CD8+ T cells of C57 origin (H-2b+) sorted by flow cytometric analysis from the either spleens of the chimeric mice or from the spleens of untreated wild-type C57BL/6 mice. All hosts were given 5 x 106 T cell-depleted wild-type C57 bone marrow cells. All mice were observed for clinical signs of graft-versus-host disease (GVHD) and mortality through day +100. Autopsy was performed at death to assess for sub-clinical target organ involvement with GVHD or tumor. Donor chimerism and BCL1 status was assessed at day +28 and day +100 by 3-color flow cytometric analysis for donor-specific MHC versus T-, B- and myeloid lineage markers as well as tumor-specific idiotype in the peripheral blood (or in the spleen at time of death for animals dying prior to day +100). All animals receiving BCL1 tumor cells and sorted CD8+T cells from wild-type untreated C57 donors cleared tumor idiotype but succumbed to GVHD. All animals receiving tumor cells and sorted chimeric C57 CD8+ T cells remained free of clinical or pathologic evidence of GVHD, but died with tumor progression. Control myeloablated animals given C57 TCD BM alone with BCL1 tumor cells all succumbed to tumor, whereas those receiving C57 whole bone marrow with tumor demonstrated tumor survival without GVHD. The data indicate that chimeric donor peripheral CD8+ T cells, which lose their capacity to induce lethal GVHD in BALB/c hosts, also lose the capacity to eradicate BALB/c-type lymphoma cells. We conclude that CD8+ T cell-induced GVT effect in this model is dependent upon alloantigen rather than tumor-specific antigen recognition.

scholarly journals A reassessment of the role of B7-1 expression in tumor rejection.

1995 ◽  
Vol 182 (5) ◽  
pp. 1415-1421 ◽  
Author(s):  
T C Wu ◽  
A Y Huang ◽  
E M Jaffee ◽  
H I Levitsky ◽  
D M Pardoll
Keyword(s):  
T Cells ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Tumor Rejection ◽  
Type B ◽  
Wild Type ◽  
Systemic Immunity ◽  
Murine Tumor

Introduction of the B7-1 gene into murine tumor cells can result in rejection of the B7-1 transductants and, in some cases, systemic immunity to subsequent challenge with the nontransduced tumor cells. These effects have been largely attributed to the function of B7-1 as a costimulator in directly activating tumor specific, major histocompatibility class I-restricted CD8+ T cells. We examined the role of B7-1 expression in the direct rejection as well as in the induction of systemic immunity to a nonimmunogenic murine tumor. B-16 melanoma cells with high levels of B7-1 expression did not grow in C57BL/6 recipient mice, while wild-type B-16 cells and cells with low B7-1 expression grew progressively within 21 d. In mixing experiments with B7-1hi and wild-type B-16 cells, tumors grew out in vivo even when a minority of cells were B7-1-. Furthermore, the occasional tumors that grew out after injection of 100% B-16 B7-1hi cells showed markedly decreased B7-1 expression. In vivo antibody depletions showed that NK1.1 and CD8+ T cells, but not CD4+ T cells, were essential for the in vivo rejection of tumors. Animals that rejected B-16 B7-1hi tumors did not develop enhanced systemic immunity against challenge with wild-type B-16 cells. These results suggest that a major role of B7-1 expression by tumors is to mediate direct recognition and killing by natural killer cells. With an intrinsically nonimmunogenic tumor, this direct killing does not lead to enhanced systemic immunity.

scholarly journals High frequency of circulating HBcAg-specific CD8 T cells in hepatitis B infection: a flow cytometric analysis

2001 ◽  
Vol 124 (3) ◽  
pp. 435-444 ◽  
Author(s):  
S. Matsumura ◽  
K. Yamamoto ◽  
N. Shimada ◽  
N. Okano ◽  
R. Okamoto ◽  
...  
Keyword(s):  
T Cells ◽  
Hepatitis B ◽  
Cd8 T Cells ◽  
High Frequency ◽  
Flow Cytometric Analysis ◽  
Hepatitis B Infection ◽  
Flow Cytometric

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.

Characterization of NK1.1+ CD8 T Cells in Allogeneic Hematopoietic Cell Transplantation Recipients

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4616-4616
Author(s):  
Yi Wang ◽  
Hui Wang ◽  
Shumei Wang ◽  
Megan Sykes ◽  
Yong-Guang Yang
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Cell Transplantation ◽  
Cd8 T Cells ◽  
Hematopoietic Cell Transplantation ◽  
Flow Cytometric Analysis ◽  
Nkt Cells ◽  
Hematopoietic Cell ◽  
Allogeneic Hematopoietic Cell Transplantation ◽  
Memory Phenotype

Abstract NKT cells from naïve mice are mainly CD4+ or CD4−CD8−. However, it has been reported that CD8+ NKT cells can be expanded in vitro from splenocytes, bone marrow cells and thymocytes of C57BL/6 (B6) mice by stimulation with anti-CD3 mAb and cytokines, and that the expanded CD8+ NKT cells mediate strong graft-vs.-leukemic (GVL) effects without severe GVHD after adoptive transfer into allogeneic mice. We now describe the presence of CD8+NK1.1+ cells in recipient livers (approximately 2–6%), but not in other tissues (spleen, lung, bone marrow, thymus and PBMC), in various allogeneic hematopoietic cell transplantation (allo-HCT) models. The generation of CD8+NK1.1+ cells is likely a consequence of alloresponses, as these cells were not detected in the liver of syngeneic HCT controls. Flow cytometric analysis confirmed that these cells are CD1d-independent, TCRαβ+ T cells with a memory phenotype (CD44+ and CD62L−), and do not express CD49b, Ly-49C/I, Ly-49G2, or Ly-49D. In a sublethally (6 Gy)-irradiated B6-to-B6D2F1 allo-HCT model, NK1.1+ CD8 T cells became detectable by week 2, increased in number until approximately week 8, and gradually declined thereafter but were still detectable in the liver at day 100 after allo-HCT. By using CD45.1 and CD45.2 congeneic donors, we determined that the majority of NK1.1+ CD8 T cells were derived from the donor splenocytes. Furthermore, depletion of CD8+, but not NK1.1+, cells from the donor splenocytes prior to transplantation prevented the generation of NK1.1+ CD8 T cells, indicating that these cells were derived from donor NK1.1−CD8+ splenic T cells. Our data demonstrate that donor CD8 T cells can acquire NK1.1 expression upon activation in allo-HCT recipients, and that these NK1.1+ CD8 T cells maintain a memory phenotype and persist in the recipients with preferential accumulation in the liver. Studies are currently in progress to determine the role of activated donor NK1.1+ CD8 T cells in GVHD and GVL effects.

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.

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

Blood ◽  
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.

Conditional Loss of Dnmt3a Results in Myeloproliferation and Liver-Specific Myeloid Expansion

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 364-364
Author(s):  
Olga A Guryanova ◽  
Yen Lieu ◽  
Kaitlyn R Shank ◽  
Sharon Rivera ◽  
Francine E Garrett-Bakelman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometric Analysis ◽  
Fold Increase ◽  
Extramedullary Hematopoiesis ◽  
Disease Phenotype ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic System ◽  
Flow Cytometric

Abstract Mutations in the DNA methyltransferase 3A (DNMT3A) gene are frequent in normal karyotype de novo acute myeloid leukemia (AML) (20-35%), chronic myelomonocytic leukemia (CMML) (10-20%) and myelodysplastic syndrome (MDS) (8%). Hematopoietic-specific loss of Dnmt3a in a mouse model leads to acquisition of aberrant self-renewal by the HSCs and expansion of the stem/progenitor compartment in bone marrow transplantation studies. Despite these important insights, the impact of hematopoietic deletion of Dnmt3a on disease phenotype in primary, non-transplanted mice has not been described. Mx1-Cre-mediated Dnmt3a ablation in the hematopoietic system in primary mice led to the development of a myeloproliferative neoplasm (MPN) with a 100% penetrance (n=14) and a median age of onset at 47.7 weeks (survival difference between Dnmt3a KO and control animals p<0.0001, Figure 1A). Loss of Dnmt3a in the hematopoietic compartment resulted in thrombocytopenia (platelet counts 250±251.8 K/μl in Dnmt3a KO vs 1260±292.8 K/μl in controls, p<0.002) and overall anemia (hematocrit 25.25±7.48% vs 44.8±5.83%, p<0.006). Marked expansion of the mature Mac1+Gr1+ myeloid cell population in the peripheral blood was evident by flow cytometric analysis (52.3±18.03% in Dnmt3a knock-outs). Myeloproliferation induced by Dnmt3a loss was characterized by marked, progressive hepatomegaly (liver weights 7.25±1.195 g in Dnmt3a-deleted animals vs 1.61±0.266 g in wild-type controls, p<1.75×10^-8, Figure 1B) with moderate splenomegaly (spleen weights 457.5±379.6 mg vs 79.43±21.19 mg, p<0.033). Histopathological analysis revealed massive myeloid infiltration in spleens and livers leading to complete effacement of organ architecture, left shifted myeloid cells, and occasional blasts. In addition, the presence of megakaryocytes in spleens and livers of Dnmt3a-deleted mice was indicative of extramedullary hematopoiesis. The significant myeloid infiltration of liver parenchyma was confirmed by flow cytometric analysis of liver tissue, with Mac1+Gr1+ myeloid cells making up 66.15±11.93% of all viable cells. In line with previous reports, we observed an increased number of immunophenotypically defined stem (Lin-Sca1+cKit+, LSK, 2.013±1.200% in Dnmt3a-ablated mice vs 0.423±0.052% in controls, a 4.76-fold increase, p<0.014) and granulomonocytic progenitor (GMP, Lin-Sca1-cKit+CD34+FcγR+, 2.713±1.593% vs 1.278±0.451%, a 2.12-fold increase, p<0.024) cells in the bone marrow. Consistent with extramedullary hematopoiesis, we were able to detect expanded LSK cell populations in livers and spleens of Dnmt3a-deleted mice. Notably, the myeloid disease phenotype induced by Dnmt3a loss was fully transplantable, including the marked hepatomegaly; these data demonstrate that the liver-specific expansion reflects a cell-autonomous mechanism. To assess relative tropism for different target organs, we next performed homing studies where Dnmt3a-deleted bone marrow cells were competed against wild-type counterparts in lethally irradiated hosts. 48 hours after transplantation, we observed increased tropism of the Dnmt3aΔ/Δ BM cells to the liver and spleen, whereas control cells preferentially localized to the bone marrow (difference between homing to bone marrow and spleen/liver p<0.0115, Figure 1C). These data demonstrate that altered homing and tissue tropism of Dnmt3a KO hematopoietic cells promote extramedullary hematopoiesis and liver involvement. ERRBS and gene expression profiles by RNA-seq in stem and progenitor cell populations demonstrated differential regulation of key biologic pathways, including self-renewal, hematopoietic lineage commitment and differentiation, and heterotypic cell-cell interactions. In conclusion, our studies show that ablation of Dnmt3a in the hematopoietic system leads to myeloid transformation in vivo, with cell autonomous liver tropism and marked extramedullary hematopoiesis. These data demonstrate, in addition to its established role in controlling self-renewal, Dnmt3a serves as an important regulator of the myeloid compartment that limits expansion of myeloid progenitors in vivo. Figure 1 Figure 1. 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.

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.

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