Dolores Mahmud

Differentiation stage-specific activation of p38 mitogen-activated protein kinase isoforms in primary human erythroid cells

p38α, p38β, p38γ, and p38δ are four isoforms of p38 mitogen-activated protein (MAP) kinase (MAPK) involved in multiple cellular functions such as cell proliferation, differentiation, apoptosis, and inflammation response. In the present study, we examined the mRNA expression pattern of each of the four isoforms during erythroid differentiation of primary erythroid progenitors. We show that p38α and p38γ transcripts are expressed in early hematopoietic progenitors as well as in late differentiating erythroblasts, whereas p38δ mRNA is only expressed and active during the terminal phase of erythroid differentiation. On the other hand, p38β is minimally expressed in early CD34+ hematopoietic progenitors but not expressed in lineage-committed erythroid progenitors. We also determined the phosphorylation/activation of p38α, MAPK kinase 3/6, and MAPKAP-2 in response to erythropoietin and stem cell factor. We found that phosphorylation of p38α, MAPK kinase kinase 3/6 and MAPKAP-2 occurs only upon growth factor withdrawal in primary erythroid progenitors. Moreover, our data indicate that activation of p38α does not induce apoptosis or promote proliferation of erythroid progenitors. On the other hand, under steady-state culture conditions, both p38α and p38δ isoforms are increasingly phosphorylated activated in the terminal phase of differentiation. This increased phosphorylation/activity was accompanied by up-regulation of heat shock protein 27 phosphorylation. Finally, we demonstrate that tumor necrosis factor α, an inflammatory cytokine that is modulated by p38α, is expressed by differentiating erythroblasts and inhibition of p38α or tumor necrosis factor α results in reduction in differentiation. Taken together, our data demonstrate that both p38α and δ isoforms function to promote the late-stage differentiation of primary erythroid progenitors and are likely to be involved in functions related to erythrocyte membrane remodeling and enucleation.

Phosphorylation of forkhead transcription factors by erythropoietin and stem cell factor prevents acetylation and their interaction with coactivator p300 in erythroid progenitor cells.

The mammalian forkhead transcription factors, FOXO3a (FKHRL1), FOXO1a (FKHR) and FOXO4 (AFX) are negatively regulated by PKB/Akt kinase. In the present study we examined the engagement of forkhead family of transcription factors in erythropoietin (Epo)- and stem cell factor (SCF)-mediated signal transduction. Our data show that all three forkhead family members, FOXO3a, FOXO1a and FOXO4 are phosphorylated in human primary erythroid progenitors. Experiments performed to determine various upstream signaling pathways contributing to phosphorylation of forkhead family members show that only PI-3-kinase pathway is required for inactivation of FOXO3a. Our data also demonstrate that during Epo deprivation FOXO3a interacts with the transcriptional coactivator p300 and such interaction is disrupted by stimulation of cells with Epo. To determine the domains in FOXO3a, mediating its interaction with p300, we performed GST pull-down assays and found that the N-terminus region containing the first 52 amino acids was sufficient for binding p300. Finally, our data demonstrate that FOXO3a and FOXO1a are acetylated during growth factor deprivation and such acetylation is reversed by stimulation with Epo. Thus mammalian forkhead transcription factors are involved in Epo and SCF signaling in primary erythroid progenitors and may play a role in the induction of apoptotic and mitogenic signals.

Regulation of myeloma cell growth through Akt/Gsk3/forkhead signaling pathway.

The interleukin-6 (IL-6) signaling pathway contributes to myeloma cell growth and viability through activation of the PI3/Akt kinase pathway. To understand the downstream signaling elements in the PI3/Akt kinase pathway that are involved in the regulation of myeloma cell growth, we determined the role played by glycogen synthase kinase 3 (Gsk3) and forkhead transcription factors (FH) in the RPMI-8226 myeloma cell line. We demonstrate that both Gsk3 and FH transcription factors FKHRL1 (FOX3a), FKHR (FOXO1a), and AFX (FOXO4) are phosphorylated (inactivated) by IL-6. Further, we show that inhibitors of Gsk3 induce dephosphorylation of FKHRL1 and FKHR at their threonine sites and upregulate the cyclin-dependent kinase inhibitor p27(kip1). Finally, we show that inhibition of Gsk3 activity is sufficient to suppress cell growth and induce apoptosis thus overriding the effects of IL-6 in myeloma cells.

CD11b+GR1+ Myeloid Cells Secrete NGF and Promote Trigeminal Ganglion Neurite Growth: Implications for Corneal Nerve Regeneration

PURPOSE We characterized fluorescent bone marrow cells (YFP(+) BMCs) in the thy1-YFP mouse and determine if they promote trigeminal ganglion (TG) cell neurite growth. METHODS Excimer laser annular keratectomy was performed in thy1-YFP mice, and corneas were imaged. BMCs were harvested from femur and tibia, and the expression of surface markers on YFP(+) BMCs was analyzed by flow cytometry. The immunosuppressive action of BMCs (YFP(+) and YFP(-)) was evaluated in an allogenic mixed lymphocyte reaction (MLR). Neurotrophic action of BMCs (YFP(+) and YFP(-)) was determined in compartmental and transwell cultures of dissociated TG cells. RESULTS Following annular keratectomy, YFP(+) BMCs infiltrated the cornea. YFP(+) BMCs shared surface markers (CD11b+Gr1+Ly6C+Ly6G-F4/80(low)) with monocytic myeloid-derived suppressor cells (MDSCs), had similar morphology, and suppressed T-cell proliferation in allogenic MLR in a dose-dependent manner. YFP(+) BMCs, but not YFP(-) BMCs, significantly increased growth of TG neurites in vitro. When cultured in a transwell with TG neurites, YFP(+) BMCs expressed neurotrophins and secreted nerve growth factor (NGF) in conditioned medium. YFP(+) BMCs that infiltrated the cornea maintained their phenotype and actions (neuronal and immune). CONCLUSIONS YFP(+) BMCs in thy1-YFP mice have immunophenotypic features of MDSCs. They secrete NGF and promote neuroregeneration. Their immunosuppressive and neurotrophic actions are preserved after corneal infiltration. These findings increase our understanding of the beneficial roles played by leukocyte trafficking in the cornea and may lead to therapeutic strategies that use NGF-secreting myeloid cells to repair diseased or injured neurons.

Cord Blood Nucleated Cells Induce Delayed T Cell Alloreactivity

Cord blood (CB) mononuclear cells (MNCs) can be transplanted in HLA mismatched recipients with limited graft rejection or graft-versus-host disease (GVHD). Previous studies have shown that naive T cells and hyporesponsive dendritic cells are largely represented in CB. Data presented here demonstrate that CB MNCs are unable to stimulate allogeneic T cell proliferative or cytotoxic responses in standard in vitro assays. However, a suppressive effect of CB MNCs was ruled out because purified CD34(+) cells or CD14(+) monocytes stimulated T cell responses that were not inhibited by add-back of CB MNCs. The lack of antigen-presenting cell (APC) activity of CB MNCs in primary mixed lymphocyte culture (MLC) did not induce allogeneic T cell anergy. In fact, rechallenge of T cells with CB CD34(+) cells, or immature monocyte-derived dendritic cells (iMo-DCs) in secondary MLC induced potent T cell proliferative responses. A delayed APC activity of CB MNCs was observed after stimulation with irradiated allogeneic T cells for 6 days, likely because of the upregulation of CD86 and HLA-DR on CB cells. Cytotoxic lymphocytes (CTL) were generated after stimulation of blood T cells with CB MNCs for 4 weeks or CB-derived iMo-DCs for 1 week. Concomitant stimulation of T cells with CB iMo-DC obtained from 2 CB units resulted in the generation of CTLs specific for each CB, independently of the CB:CB cell ratio. These data suggest that the APC activity of CB cells possibly increases posttransplant, and may contribute to delayed graft rejection and/or acute and chronic GVHD.

LIGHT elevation enhances immune eradication of colon cancer metastases

The majority of patients with colon cancer will develop advanced disease, with the liver being the most common site of metastatic disease. Patients with increased numbers of tumor-infiltrating lymphocytes in primary colon tumors and liver metastases have improved outcomes. However, the molecular factors that could empower antitumor immune responses in this setting remain to be elucidated. We reported that the immunostimulatory cytokine LIGHT (TNFSF14) in the microenvironment of colon cancer metastases associates with improved patient survival, and here we demonstrate in an immunocompetent murine model that colon tumors expressing LIGHT stimulate lymphocyte proliferation and tumor cell-specific antitumor immune responses. In this model, increasing LIGHT expression in the microenvironment of either primary tumors or liver metastases triggered regression of established tumors and slowed the growth of liver metastases, driven by cytotoxic T-lymphocyte-mediated antitumor immunity. These responses corresponded with significant increases in tumor-infiltrating lymphocytes and increased expression of lymphocyte-homing signals in the metastatic tumors. Furthermore, we demonstrated evidence of durable tumor-specific antitumor immunity. In conclusion, increasing LIGHT expression increased T-cell proliferation, activation, and infiltration, resulting in enhanced tumor-specific immune-mediated tumor regressions in primary tumors and colorectal liver metastases. Mechanisms to increase LIGHT in the colon cancer microenvironment warrant further investigation and hold promise as an immunotherapeutic strategy. Cancer Res; 77(8); 1880-91. ©2017 AACR.

Human CD4+CD25+ Cells in Combination with CD34+ Cells and Thymoglobulin to Prevent Anti-hematopoietic Stem Cell T Cell Alloreactivity

Cotransplantation of human CD34(+) hematopoietic stem cells (HSC) and CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) could prevent anti-HSC alloreactivity and reduce the risk of rejection in HLA mismatched transplants. To pursue this hypothesis we cocultured CD34(+) cells and CD4(+)CD25(+) cells immunomagnetically isolated (Milteny) from human peripheral blood (unmanipulated or granulocyte-colony stimulating factor [G-CSF] mobilized) or cord blood. Enriched Tregs obtained from the same source (autologous) of CD34(+) cells showed greater inhibitory effect on T cell alloreactivity than third-party (allogeneic) Tregs. The immunosuppressive activity of Tregs was maintained after stimulation with allogeneic CD34(+) cells and Tregs did not modify the clonogenic activity of CD34(+) cells in vitro. Cotransplantation of Tregs with CD34(+) cells at 1:1 or 2:1 ratios in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice resulted in normal hematopoietic stem cell engraftment. Incubation with physiologic doses of rabbit antithymocyte globulin (rATG, thymoglobulin) did not affect the number of Tregs in 6-day culture. Upon exposure to thymoglobulin Tregs maintained their suppressive activity, increased expression of CCR7, and released multiple cytokines, primarily interleukin (IL)10. Our findings suggest that human autologous or allogeneic Tregs could be cotransplanted with CD34(+) cells after preparative regimens including thymoglobulin.

A novel L-asparaginase with low L-glutaminase coactivity is highly efficacious against both T and B cell acute lymphoblastic leukemias in vivo

Acute lymphoblastic leukemia (ALL) is the most common type of pediatric cancer, although about 4 of every 10 cases occur in adults. The enzyme drug l-asparaginase serves as a cornerstone of ALL therapy and exploits the asparagine dependency of ALL cells. In addition to hydrolyzing the amino acid l-asparagine, all FDA-approved l-asparaginases also have significant l-glutaminase coactivity. Since several reports suggest that l-glutamine depletion correlates with many of the side effects of these drugs, enzyme variants with reduced l-glutaminase coactivity might be clinically beneficial if their antileukemic activity would be preserved. Here we show that novel low l-glutaminase variants developed on the backbone of the FDA-approved Erwinia chrysanthemi l-asparaginase were highly efficacious against both T- and B-cell ALL, while displaying reduced acute toxicity features. These results support the development of a new generation of safer l-asparaginases without l-glutaminase activity for the treatment of human ALL.Significance: A new l-asparaginase-based therapy is less toxic compared with FDA-approved high l-glutaminase enzymes Cancer Res; 78(6); 1549-60. ©2018 AACR.

T Cell–Mediated Rejection of Human CD34+ Cells Is Prevented by Costimulatory Blockade in a Xenograft Model

A xenograft model of stem cell rejection was developed by co-transplantating human CD34+ and allogeneic CD3+ T cells into NOD-scid ɣ-chainnull mice. T cells caused graft failure when transplanted at any CD34/CD3 ratio between 1:50 and 1:.1. Kinetics experiments showed that 2 weeks after transplantation CD34+ cells engrafted the marrow and T cells expanded in the spleen. Then, at 4 weeks only memory T cells populated both sites and rejected CD34+ cells. Blockade of T cell costimulation was tested by injecting the mice with abatacept (CTLA4-IgG1) from day -1 to +27 (group A), from day -1 to +13 (group B), or from day +14 to +28 (group C). On day +56 groups B and C had rejected the graft, whereas in group A graft failure was completely prevented, although with lower stem cell engraftment than in controls (P = .03). Retransplantation of group A mice with same CD34+ cells obtained a complete reconstitution of human myeloid and B cell lineages and excluded latent alloreactivity. In this first xenograft model of stem cell rejection we showed that transplantation of HLA mismatched CD34+ cells may be facilitated by treatment with abatacept and late stem cell boost.