Keyvan Keyvanfar

Bmi1 regulates mitochondrial function and the DNA damage response pathway

Mice deficient in the Polycomb repressor Bmi1 develop numerous abnormalities including a severe defect in stem cell self-renewal, alterations in thymocyte maturation and a shortened lifespan. Previous work has implicated de-repression of the Ink4a/Arf (also known as Cdkn2a) locus as mediating many of the aspects of the Bmi1-/- phenotype. Here we demonstrate that cells derived from Bmi1-/- mice also have impaired mitochondrial function, a marked increase in the intracellular levels of reactive oxygen species and subsequent engagement of the DNA damage response pathway. Furthermore, many of the deficiencies normally observed in Bmi1-/- mice improve after either pharmacological treatment with the antioxidant N-acetylcysteine or genetic disruption of the DNA damage response pathway by Chk2 (also known as Chek2) deletion. These results demonstrate that Bmi1 has an unexpected role in maintaining mitochondrial function and redox homeostasis and indicate that the Polycomb family of proteins can coordinately regulate cellular metabolism with stem and progenitor cell function.

Rabbit ATG but not horse ATG promotes expansion of functional CD4+CD25highFOXP3+ regulatory T cells in vitro.

Regulatory T cells (Treg) play important roles in suppressing immune responses and maintaining tolerance. Rabbit antithymocyte globulin (rATG) and horse ATG (hATG) are widely used in the treatment of immune-mediated syndromes, but their effects on Treg are unknown. We show here that in vitro culture of normal human peripheral blood mononuclear cells (PBMCs) with a low-dose rATG resulted in marked expansion of functional Treg by converting CD4+CD25- T cells to CD4+CD25+ T cells. hATG did not expand but rather decreased Treg. Immuno-blot showed increased expression of FOXP3 and NFAT1 in CD4+CD25- and CD4+CD25+ T cells exposed to rATG. PBMCs treated with rATG displayed increased interleukin-10 in culture supernatants than those treated with hATG. Furthermore, rATG and hATG showed differences in their potential to stimulate CD4+ T cells as examined using different activation markers. Microarray revealed that rATG induced markedly different gene-expression patterns in PBMCs, compared with hATG-treated or untreated PBMCs. Our findings indicate that rATG expanded Treg, probably through transcriptional regulation by enhanced NFAT1 expression, in turn conferring CD4+CD25- T cell FOXP3 expression and regulatory activity. The therapeutic effects of rATG may occur not only because of lymphocyte depletion but also enhanced Treg cell number and function.

Th17/Treg ratio in human graft-versus-host disease

Th17 cells have never been explored in human graft-versus-host disease (GVHD). We studied the correlation between the presence of Th17 cells with histologic and clinical parameters. We first analyzed a cohort of 40 patients with GVHD of the gastrointestinal tract. Tumor necrosis factor (TNF), TNF receptors, and Fas expression, and apoptotic cells, CD4(+)IL-17(+) cells (Th17), and CD4(+)Foxp3(+) cells (Treg) were quantified. A Th17/Treg ratio less than 1 correlated both with the clinical diagnosis (P < .001) and more than 2 pathologic grades (P < .001). A Th17/Treg ratio less than 1 also correlated with the intensity of apoptosis of epithelial cells (P = .03), Fas expression in the cellular infiltrate (P = .003), TNF, and TNF receptor expression (P < .001). We then assessed Th17/Treg ratio in 2 other independent cohorts; a second cohort of 30 patients and confirmed that Th17/Treg ratio less than 1 correlated with the pathologic grade of GI GVHD. Finally, 15 patients with skin GVHD and 11 patients with skin rash but without pathologic GVHD were studied. Results in this third cohort of patients with skin disease confirmed those found in patients with GI GVHD. These analyses in 96 patients suggest that Th17/Treg ratio could be a sensitive and specific pathologic in situ biomarker of GVHD.

Ex vivo characterization of polyclonal memory CD8 + T-cell responses to PRAME-specific peptides in patients with acute lymphoblastic leukemia and acute and chronic myeloid leukemia

Preferentially expressed antigen of melanoma (PRAME) is aberrantly expressed in hematologic malignancies and may be a useful target for immunotherapy in leukemia. To determine whether PRAME is naturally immunogenic, we studied CD8(+) T-cell responses to 4 HLA-A*0201-restricted PRAME-derived epitopes (PRA100, PRA142, PRA300, PRA425) in HLA-A*0201-positive patients with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and healthy donors. CD8(+) T cells recognizing PRAME peptides could be detected ex vivo in 4 of 10 ALL, 6 of 10 AML, 3 of 10 CML patients, and 3 of 10 donors by HLA-A2 tetramer analysis and flow cytometry for intracellular interferon-gamma. The frequency of PRAME-specific CD8(+) T cells was greater in patients with AML, CML, and ALL than healthy controls. All peptides were immunogenic in patients, while responses were only detected to PRA300 in donors. High PRAME expression in patient peripheral blood mononuclear cells was associated with responses to greater than or equal to 2 PRAME epitopes compared with low PRAME expression levels (4/7 vs 0/23, P = .001), suggesting a PRAME-driven T-cell response. PRAME-specific T cells were readily expanded in short-term cultures in donors and patients. These results provide evidence for spontaneous T cell reactivity against multiple epitopes of PRAME in ALL, AML, and CML. The potential for developing PRAME as a target for immunotherapy in leukemia deserves further exploration.

Th17 immune responses contribute to the pathophysiology of aplastic anemia

T helper type 17 (Th17) cells have been characterized based on production of interleukin-17 (IL-17) and association with autoimmune diseases. We studied the role of Th17 cells in aplastic anemia (AA) by isolating Th17 cells from patients blood (n = 41) and bone marrow (BM) mononuclear cells (n = 7). The frequency and total number of CD3(+)CD4(+)IL-17-producing T cells were increased in AA patients at presentation compared with healthy controls (P = .0007 and .02, respectively) and correlated with disease activity. There was an inverse relationship between the numbers of Th17 cells and CD4(+)CD25(high)FoxP3(+) regulatory T cells (Tregs) in the blood of AA patients. Concomitant with the classical Th1 response, we detected the presence of CD4(+) and CD8(+) IL-17-producing T cells in a mouse model of lymph node infusion-induced BM failure. Although anti-IL-17 treatment did not abrogate BM failure, early treatment with the anti-IL-17 antibody reduced the severity of BM failure with significantly higher platelet (P < .01) and total BM cell (P < .05) counts at day 10. Recipients that received anti-IL-17 treatment had significantly fewer Th1 cells (P < .01) and more Treg cells (P < .05) at day 10 after lymph node infusion. Th17 immune responses contribute to AA pathophysiology, especially at the early stage during disease progression.

Lenalidomide-induced upregulation of CD80 on tumor cells correlates with T-cell activation, the rapid onset of a cytokine release syndrome and leukemic cell clearance in chronic lymphocytic leukemia

In chronic lymphocytic leukemia (CLL) lenalidomide causes striking immune activation, possibly leading to clearance of tumor cells. This study shows that upregulation of CD80 on tumor cells and T-cell activation, which appears to be dispensable for the drug’s anti-tumor effects, correlate with unique toxicities of lenalidomide in CLL. This provides a rationale for combinations of lenalidomide with immunosuppressive agents. See related perspective article on page 1198. Background In chronic lymphocytic leukemia lenalidomide causes striking immune activation, possibly leading to clearance of tumor cells. We conducted this study to investigate the mechanism of action of lenalidomide and the basis for its unique toxicities in chronic lymphocytic leukemia. Design and Methods Patients with relapsed chronic lymphocytic leukemia were treated with lenalidomide 20 mg (n=10) or 10 mg (n=8) daily for 3 weeks on a 6-week cycle. Correlative studies assessed expression of co-stimulatory molecules on tumor cells, T-cell activation, cytokine levels, and changes in lymphocyte subsets. Results Lenalidomide upregulated the co-stimulatory molecule CD80 on chronic lymphocytic leukemia and mantle cell lymphoma cells but not on normal peripheral blood B cells in vitro. T-cell activation was apparent in chronic lymphocytic leukemia, weak in mantle cell lymphoma, but absent in normal peripheral blood mononuclear cells and correlated with the upregulation of CD80 on B cells. Strong CD80 upregulation and T-cell activation predicted more severe side effects, manifesting in 83% of patients as a cytokine release syndrome within 8–72 h after the first dose of lenalidomide. Serum levels of various cytokines, including tumor necrosis factor-α, increased during treatment. CD80 upregulation on tumor cells correlated with rapid clearance of leukemic cells from the peripheral blood. In contrast, neither the severity of the cytokine release syndrome nor the degree of T-cell activation in vitro correlated with clinical response. Conclusions Upregulation of CD80 on tumor cells and T-cell activation correlate with unique toxicities of lenalidomide in chronic lymphocytic leukemia. However, T-cell activation appears to be dispensable for the drug’s anti-tumor effects. This provides a rationale for combinations of lenalidomide with fludarabine or alemtuzumab.

Enrichment of hematopoietic stem cells with SLAM and LSK markers for the detection of hematopoietic stem cell function in normal and Trp53 null mice

OBJECTIVE To test function of hematopoietic stem cells (HSCs) in vivo in C57BL/6 (B6) and Trp53-deficient (Trp53 null) mice by using two HSC enrichment schemes. MATERIALS AND METHODS Bone marrow (BM) Lin-CD41-CD48-CD150+ (signaling lymphocyte activation molecules [SLAM]), Lin-CD41-CD48-CD150- (SLAM-) and Lin-Sca1+CD117+ (LSK) cells were defined by fluorescence-activated cell staining (FACS). Cellular reactive oxygen species (ROS) level was also analyzed by FACS. Sorted SLAM, SLAM-, and LSK cells were tested in vivo in the competitive repopulation (CR) and serial transplantation assays. RESULTS SLAM cell fraction was 0.0078%+/-0.0010% and 0.0135%+/-0.0010% of total BM cells in B6 and Trp53 null mice, and was highly correlated (R2=0.7116) with LSK cells. CD150+ BM cells also contained more ROSlow cells than did CD150- cells. B6 SLAM cells repopulated recipients much better than B6 SLAM- cells, showing high HSC enrichment. B6 SLAM cells also engrafted recipients better than Trp53 null SLAM cells in the CR and the follow-up serial transplantation assays. Similarly, LSK cells from B6 donors also had higher repopulating ability than those from Trp53 null donors. However, whole BM cells from the same B6 and Trp53 null donors showed the opposite functional trend in recipient engraftment. CONCLUSION Both SLAM and LSK marker sets can enrich HSCs from B6 and Trp53 mice. Deficiency of Trp53 upregulates HSC self-renewal but causes no gain of HSC function.

Mitochondrial Membrane Potential Identifies Cells with Enhanced Stemness for Cellular Therapy.

Long-term survival and antitumor immunity of adoptively transferred CD8(+) T cells is dependent on their metabolic fitness, but approaches to isolate therapeutic T cells based on metabolic features are not well established. Here we utilized a lipophilic cationic dye tetramethylrhodamine methyl ester (TMRM) to identify and isolate metabolically robust T cells based on their mitochondrial membrane potential (ΔΨm). Comprehensive metabolomic and gene expression profiling demonstrated global features of improved metabolic fitness in low-ΔΨm-sorted CD8(+) T cells. Transfer of these low-ΔΨm T cells was associated with superior long-term in vivo persistence and an enhanced capacity to eradicate established tumors compared with high-ΔΨm cells. Use of ΔΨm-based sorting to enrich for cells with superior metabolic features was observed in CD8(+), CD4(+) T cell subsets, and long-term hematopoietic stem cells. This metabolism-based approach to cell selection may be broadly applicable to therapies involving the transfer of HSC or lymphocytes for the treatment of viral-associated illnesses and cancer.

Activation of CD44, a receptor for extracellular matrix components, protects chronic lymphocytic leukemia cells from spontaneous and drug induced apoptosis through MCL-1

Survival of chronic lymphocytic leukemia (CLL) cells in vivo is supported by the tissue microenvironment, which includes components of the extracellular matrix. Interactions between tumor cells and the extracellular matrix are in part mediated by CD44, whose principal ligand is hyaluronic acid. Here, we show that CD44 is more highly expressed on CLL cells of the clinically more progressive immunglobulin heavy chain variable gene (IGHV)-unmutated subtype than on cells of the IGHV-mutated type. Engagement of CD44 activated the phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen activated protein kinase (MAPK)/ERK pathways and increased myeloid cell leukemia sequence 1 (MCL-1) protein expression. Consistent with the induction of these anti-apoptotic mechanisms, CD44 protected CLL cells from spontaneous and fludarabine-induced apoptosis. Obatoclax, an antagonist of MCL-1, blocked the pro-survival effect of CD44. In addition, obatoclax synergized with fludarabine to induce apoptosis of CLL cells. In conclusion, components of the extracellular matrix may provide survival signals to CLL cells through engagement of CD44. Inhibition of MCL-1 is a promising strategy to reduce the anti-apoptotic effect of the microenvironment on CLL cells.

Ex Vivo-Generated CD36+ Erythroid Progenitors Are Highly Permissive to Human Parvovirus B19 Replication

ABSTRACT The pathogenic parvovirus B19 (B19V) has an extreme tropism for human erythroid progenitor cells. In vitro, only a few erythroid leukemic cell lines (JK-1 and KU812Ep6) or megakaryoblastoid cell lines (UT7/Epo and UT7/Epo-S1) with erythroid characteristics support B19V replication, but these cells are only semipermissive. By using recent advances in generating large numbers of human erythroid progenitor cells (EPCs) ex vivo from hematopoietic stem cells (HSCs), we produced a pure population of CD36+ EPCs expanded and differentiated from CD34+ HSCs and assessed the CD36+ EPCs for their permissiveness to B19V infection. Over more than 3 weeks, cells grown in serum-free medium expanded more than 800,000-fold, and 87 to 96% of the CD36+ EPCs were positive for globoside, the cellular receptor for B19V. Immunofluorescence (IF) staining showed that about 77% of the CD36+ EPCs were positive for B19V infection, while about 9% of UT7/Epo-S1 cells were B19V positive. Viral DNA detected by real-time PCR increased by more than 3 logs in CD36+ EPCs; the increase was 1 log in UT7/Epo-S1 cells. Due to the extensive permissivity of CD36+ EPCs, we significantly improved the sensitivity of detection of infectious B19V by real-time reverse transcription-PCR and IF staining 100- and 1,000-fold, respectively, which is greater than the sensitivity of UT7/Epo-S1 cell-based methods. This is the first description of an ex vivo method to produce large numbers of EPCs that are highly permissive to B19V infection and replication, offering a cellular system that mimics in vivo infection with this pathogenic human virus.