A Decreased Frequency of Regulatory T Cells in Patients with Common Variable Immunodeficiency

Introduction

Common variable immunodeficiency disorder (CVID) is a heterogeneous syndrome, characterized by deficient antibody production and recurrent bacterial infections in addition abnormalities in T cells. CD4⁺CD25^high regulatory T cells (Treg) are essential modulators of immune responses, including down-modulation of immune response to pathogens, allergens, cancer cells and self-antigens.

Objective

In this study we set out to investigate the frequency of Treg cells in CVID patients and correlate with their immune activation status.

Materials and Methods

Sixteen patients (6 males and 10 females) with CVID who had been treated with regular intravenous immunoglobulin and 14 controls were enrolled. Quantitative analyses of peripheral blood mononuclear cells (PBMC) were performed by multiparametric flow cytometry using the following cell markers: CD38, HLA-DR, CCR5 (immune activation); CD4, CD25, FOXP3, CD127, and OX40 (Treg cells); Ki-67 and IFN-γ (intracellular cytokine).

Results

A significantly lower proportion of CD4⁺CD25^highFOXP3 T cells was observed in CVID patients compared with healthy controls (P<0.05). In addition to a higher proportion of CD8⁺ T cells from CVID patients expressing the activation markers, CD38⁺ and HLA-DR⁺ (P<0.05), we observed no significant correlation between Tregs and immune activation.

Conclusion

Our results demonstrate that a reduction in Treg cells could have impaired immune function in CVID patients.     

Evaluation of STAT3 Signaling in ALDH+ and ALDH+/CD44+/CD24? Subpopulations of Breast Cancer Cells

Background

STAT3 activation is frequently detected in breast cancer and this pathway has emerged as an attractive molecular target for cancer treatment. Recent experimental evidence suggests ALDH-positive (ALDH⁺), or cell surface molecule CD44-positive (CD44⁺) but CD24-negative (CD24⁻) breast cancer cells have cancer stem cell properties. However, the role of STAT3 signaling in ALDH⁺ and ALDH⁺/CD44⁺/CD24⁻ subpopulations of breast cancer cells is unknown.

Methods and Results

We examined STAT3 activation in ALDH⁺ and ALDH⁺/CD44⁺/CD24⁻ subpopulations of breast cancer cells by sorting with flow cytometer. We observed ALDH-positive (ALDH⁺) cells expressed higher levels of phosphorylated STAT3 compared to ALDH-negative (ALDH⁻) cells. There was a significant correlation between the nuclear staining of phosphorylated STAT3 and the expression of ALDH1 in breast cancer tissues. These results suggest that STAT3 is activated in ALDH⁺ subpopulations of breast cancer cells. STAT3 inhibitors Stattic and LLL12 inhibited STAT3 phosphorylation, reduced the ALDH⁺ subpopulation, inhibited breast cancer stem-like cell viability, and retarded tumorisphere-forming capacity in vitro. Similar inhibition of STAT3 phosphorylation, and breast cancer stem cell viability were observed using STAT3 ShRNA. In addition, LLL12 inhibited STAT3 downstream target gene expression and induced apoptosis in ALDH⁺ subpopulations of breast cancer cells. Furthermore, LLL12 inhibited STAT3 phosphorylation and tumor cell proliferation, induced apoptosis, and suppressed tumor growth in xenograft and mammary fat pad mouse models from ALDH⁺ breast cancer cells. Similar in vitro and tumor growth in vivo results were obtained when ALDH⁺ cells were further selected for the stem cell markers CD44⁺ and CD24⁻.

Conclusion

These studies demonstrate an important role for STAT3 signaling in ALDH⁺ and ALDH⁺/CD44⁺/CD24⁻ subpopulations of breast cancer cells which may have cancer stem cell properties and suggest that pharmacologic inhibition of STAT3 represents an effective strategy to selectively target the cancer stem cell-like subpopulation.     

The Ability to Generate Senescent Progeny as a Mechanism Underlying Breast Cancer Cell Heterogeneity

Background

Breast cancer is a remarkably heterogeneous disease. Luminal, basal-like, “normal-like”, and ERBB2+ subgroups were identified and were shown to have different prognoses. The mechanisms underlying this heterogeneity are poorly understood. In our study, we explored the role of cellular differentiation and senescence as a potential cause of heterogeneity.

Methodology/Principal Findings

A panel of breast cancer cell lines, isogenic clones, and breast tumors were used. Based on their ability to generate senescent progeny under low-density clonogenic conditions, we classified breast cancer cell lines as senescent cell progenitor (SCP) and immortal cell progenitor (ICP) subtypes. All SCP cell lines expressed estrogen receptor (ER). Loss of ER expression combined with the accumulation of p21^Cip1 correlated with senescence in these cell lines. p21^Cip1 knockdown, estrogen-mediated ER activation or ectopic ER overexpression protected cells against senescence. In contrast, tamoxifen triggered a robust senescence response. As ER expression has been linked to luminal differentiation, we compared the differentiation status of SCP and ICP cell lines using stem/progenitor, luminal, and myoepithelial markers. The SCP cells produced CD24+ or ER+ luminal-like and ASMA+ myoepithelial-like progeny, in addition to CD44+ stem/progenitor-like cells. In contrast, ICP cell lines acted as differentiation-defective stem/progenitor cells. Some ICP cell lines generated only CD44+/CD24-/ER-/ASMA- progenitor/stem-like cells, and others also produced CD24+/ER- luminal-like, but not ASMA+ myoepithelial-like cells. Furthermore, gene expression profiles clustered SCP cell lines with luminal A and “normal-like” tumors, and ICP cell lines with luminal B and basal-like tumors. The ICP cells displayed higher tumorigenicity in immunodeficient mice.

Conclusions/Significance

Luminal A and “normal-like” breast cancer cell lines were able to generate luminal-like and myoepithelial-like progeny undergoing senescence arrest. In contrast, luminal B/basal-like cell lines acted as stem/progenitor cells with defective differentiation capacities. Our findings suggest that the malignancy of breast tumors is directly correlated with stem/progenitor phenotypes and poor differentiation potential.     

Differential Responses of Human Regulatory T Cells (Treg) and Effector T Cells to Rapamycin

Background

The immunosuppressive drug rapamycin (RAPA) promotes the expansion of CD4⁺ CD25^highFoxp3⁺ regulatory T cells via mechanisms that remain unknown. Here, we studied expansion, IL-2R-γ chain signaling, survival pathways and resistance to apoptosis in human Treg responding to RAPA.

Methodology/Principal Findings

CD4⁺CD25⁺ and CD4⁺CD25^neg T cells were isolated from PBMC of normal controls (n = 21) using AutoMACS. These T cell subsets were cultured in the presence of anti-CD3/CD28 antibodies and 1000 IU/mL IL-2 for 3 to 6 weeks. RAPA (1–100 nM) was added to half of the cultures. After harvest, the cell phenotype, signaling via the PI3K/mTOR and STAT pathways, expression of survival proteins and Annexin V binding were determined and compared to values obtained with freshly-separated CD4⁺CD25^high and CD4⁺CD25^neg T cells. Suppressor function was tested in co-cultures with autologous CFSE-labeled CD4⁺CD25^neg or CD8⁺CD25^neg T-cell responders. The frequency and suppressor activity of Treg were increased after culture of CD4⁺CD25⁺ T cells in the presence of 1–100 nM RAPA (p<0.001). RAPA-expanded Treg were largely CD4⁺CD25^highFoxp3⁺ cells and were resistant to apoptosis, while CD4⁺CD25^neg T cells were sensitive. Only Treg upregulated anti-apoptotic and down-regulated pro-apoptotic proteins. Treg expressed higher levels of the PTEN protein than CD4⁺CD25^neg cells. Activated Treg±RAPA preferentially phosphorylated STAT5 and STAT3 and did not utilize the PI3K/mTOR pathway.

Conclusions/Significance

RAPA favors Treg expansion and survival by differentially regulating signaling, proliferation and sensitivity to apoptosis of human effector T cells and Treg after TCR/IL-2 activation.     

An alternate STAT6-independent pathway promotes eosinophil influx into blood during allergic airway inflammation

Background

Enhanced eosinophil responses have critical roles in the development of allergic diseases. IL-5 regulates the maturation, migration and survival of eosinophils, and IL-5 and eotaxins mediate the trafficking and activation of eosinophils in inflamed tissues. CD4⁺ Th2 cells are the main producers of IL-5 and other cells such as NK also release this cytokine. Although multiple signalling pathways may be involved, STAT6 critically regulates the differentiation and cytokine production of Th2 cells and the expression of eotaxins. Nevertheless, the mechanisms that mediate different parts of the eosinophilic inflammatory process in different tissues in allergic airway diseases remain unclear. Furthermore, the mechanisms at play may vary depending on the context of inflammation and microenvironment of the involved tissues.

Methodology/Principal Findings

We employed a model of allergic airway disease in wild type and STAT6-deficient mice to explore the roles of STAT6 and IL-5 in the development of eosinophilic inflammation in this context. Quantitative PCR and ELISA were used to examine IL-5, eotaxins levels in serum and lungs. Eosinophils in lung, peripheral blood and bone marrow were characterized by morphological properties. CD4⁺ T cell and NK cells were identified by flow cytometry. Antibodies were used to deplete CD4⁺ and NK cells. We showed that STAT6 is indispensible for eosinophilic lung inflammation and the induction of eotaxin-1 and -2 during allergic airway inflammation. In the absence of these chemokines eosinophils are not attracted into lung and accumulate in peripheral blood. We also demonstrate the existence of an alternate STAT6-independent pathway of IL-5 production by CD4⁺ and NK cells that mediates the development of eosinophils in bone marrow and their subsequent movement into the circulation.

Conclusions

These results suggest that different points of eosinophilic inflammatory processes in allergic airway disease may be differentially regulated by the activation of STAT6-dependent and -independent pathways.     

Octa-arginine mediated delivery of wild-type Lnk protein inhibits TPO-induced M-MOK megakaryoblastic leukemic cell growth by promoting apoptosis

Background

Lnk plays a non-redundant role by negatively regulating cytokine signaling of TPO, SCF or EPO. Retroviral expression of Lnk has been shown to suppress hematopoietic leukemic cell proliferation indicating its therapeutic value in cancer therapy. However, retroviral gene delivery carries risks of insertional mutagenesis. To circumvent this undesired consequence, we fused a cell permeable peptide octa-arginine to Lnk and evaluated the efficacy of inhibition of leukemic cell proliferation in vitro.

Methodology/Principal Findings

In this study, proliferation assays, flow cytometry, Western Blot analyses were performed on wild-type (WT), mutant Lnk R8 or BSA treated M-MOK cells. We found that delivered WT, but not mutant Lnk R8 blocked TPO-induced M-MOK megakaryoblastic leukemic cell proliferation. In contrast, WT Lnk R8 showed no growth inhibitive effect on non-hematopoietic HELA or COS-7 cell. Moreover, we demonstrated that TPO-induced M-MOK cell growth inhibition by WT Lnk R8 was dose-dependent. Penetrated WT Lnk R8 induced cell cycle arrest and apoptosis. Immunoprecipitation and Western blots data indicated WT Lnk R8 interacted with endogeneous Jak2 and downregulated Jak-Stat and MAPK phosphorylation level in M-MOK cells after TPO stimulation. Treatment with specific inhibitors (TG101348 and PD98059) indicated Jak-Stat and MAPK pathways were crucial for TPO-induced proliferation of M-MOK cells. Further analyses using TF-1 and HEL leukemic cell-lines showed that WT Lnk R8 inhibited Jak2-dependent cell proliferation. Using cord blood-derived CD34+ stem cells, we found that delivered WT Lnk R8 blocked TPO-induced megakaryopoiesis in vitro.

Conclusions/Significance

Intracellular delivery of WT Lnk R8 fusion protein efficiently inhibited TPO-induced M-MOK leukemic cell growth by promoting apoptosis. WT Lnk R8 protein delivery may provide a safer and more practical approach to inhibit leukemic cell growth worthy of further development.     

Adult Mouse Subventricular Zone Stem and Progenitor Cells Are Sessile and Epidermal Growth Factor Receptor Negatively Regulates Neuroblast Migration

Background

The adult subventricular zone (SVZ) contains stem and progenitor cells that generate neuroblasts throughout life. Although it is well accepted that SVZ neuroblasts are migratory, recent evidence suggests their progenitor cells may also exhibit motility. Since stem and progenitor cells are proliferative and multipotential, if they were also able to move would have important implications for SVZ neurogenesis and its potential for repair.

Methodology/Principal Findings

We studied whether SVZ stem and/or progenitor cells are motile in transgenic GFP+ slices with two photon time lapse microscopy and post hoc immunohistochemistry. We found that stem and progenitor cells; mGFAP-GFP+ cells, bright nestin-GFP+ cells and Mash1+ cells were stationary in the SVZ and rostral migratory stream (RMS). In our search for motile progenitor cells, we uncovered a population of motile βIII-tubulin+ neuroblasts that expressed low levels of epidermal growth factor receptor (EGFr). This was intriguing since EGFr drives proliferation in the SVZ and affects migration in other systems. Thus we examined the potential role of EGFr in modulating SVZ migration. Interestingly, EGFr^low neuroblasts moved slower and in more tortuous patterns than EGFr-negative neuroblasts. We next questioned whether EGFr stimulation affects SVZ cell migration by imaging Gad65-GFP+ neuroblasts in the presence of transforming growth factor alpha (TGF-α), an EGFr-selective agonist. Indeed, acute exposure to TGF-α decreased the percentage of motile cells by approximately 40%.

Conclusions/Significance

In summary, the present study directly shows that SVZ stem and progenitor cells are static, that EGFr is retained on some neuroblasts, and that EGFr stimulation negatively regulates migration. This result suggests an additional role for EGFr signaling in the SVZ.     

Human CD34+/CD90+ ASCs Are Capable of Growing as Sphere Clusters,Producing High Levels of VEGF and Forming Capillaries

Background

Human adult adipose tissue is an abundant source of mesenchymal stem cells (MSCs). Moreover, it is an easily accessible site producing a considerable amount of stem cells.

Methodology/Principal Findings

In this study, we have selected and characterized stem cells within the stromal vascular fraction (SVF) of human adult adipose tissue with the aim of understanding their differentiation capabilities and performance. We have found, within the SVF, different cell populations expressing MSC markers – including CD34, CD90, CD29, CD44, CD105, and CD117 – and endothelial-progenitor-cell markers – including CD34, CD90, CD44, and CD54. Interestingly, CD34⁺/CD90⁺ cells formed sphere clusters, when placed in non-adherent growth conditions. Moreover, they showed a high proliferative capability, a telomerase activity that was significantly higher than that found in differentiated cells, and contained a fraction of cells displaying the phenotype of a side population. When cultured in adipogenic medium, CD34⁺/CD90⁺ quickly differentiated into adipocytes. In addition, they differentiated into endothelial cells (CD31⁺/VEGF⁺/Flk-1⁺) and, when placed in methylcellulose, were capable of forming capillary-like structures producing a high level of VEGF, as substantiated with ELISA tests.

Conclusions/Significance

Our results demonstrate, for the first time, that CD34⁺/CD90⁺ cells of human adipose tissue are capable of forming sphere clusters, when grown in free-floating conditions, and differentiate in endothelial cells that form capillary-like structures in methylcellulose. These cells might be suitable for tissue reconstruction in regenerative medicine, especially when patients need treatments for vascular disease.     

Leukemic Stem Cell Frequency: A Strong Biomarker for Clinical Outcome in Acute Myeloid Leukemia

Introduction

Treatment failure in acute myeloid leukemia is probably caused by the presence of leukemia initiating cells, also referred to as leukemic stem cells, at diagnosis and their persistence after therapy. Specific identification of leukemia stem cells and their discrimination from normal hematopoietic stem cells would greatly contribute to risk stratification and could predict possible relapses.

Results

For identification of leukemic stem cells, we developed flow cytometric methods using leukemic stem cell associated markers and newly-defined (light scatter) aberrancies. The nature of the putative leukemic stem cells and normal hematopoietic stem cells, present in the same patient''s bone marrow, was demonstrated in eight patients by the presence or absence of molecular aberrancies and/or leukemic engraftment in NOD-SCID IL-2Rγ-/- mice. At diagnosis (n = 88), the frequency of the thus defined neoplastic part of CD34+CD38- putative stem cell compartment had a strong prognostic impact, while the neoplastic parts of the CD34+CD38+ and CD34- putative stem cell compartments had no prognostic impact at all. After different courses of therapy, higher percentages of neoplastic CD34+CD38- cells in complete remission strongly correlated with shorter patient survival (n = 91). Moreover, combining neoplastic CD34+CD38- frequencies with frequencies of minimal residual disease cells (n = 91), which reflect the total neoplastic burden, revealed four patient groups with different survival.

Conclusion and Perspective

Discrimination between putative leukemia stem cells and normal hematopoietic stem cells in this large-scale study allowed to demonstrate the clinical importance of putative CD34+CD38- leukemia stem cells in AML. Moreover, it offers new opportunities for the development of therapies directed against leukemia stem cells, that would spare normal hematopoietic stem cells, and, moreover, enables in vivo and ex vivo screening for potential efficacy and toxicity of new therapies.     

Expansion of Cord Blood CD34+ Cells in Presence of zVADfmk and zLLYfmk Improved Their In Vitro Functionality and In Vivo Engraftment in NOD/SCID Mouse

Background

Cord blood (CB) is a promising source for hematopoietic stem cell transplantations. The limitation of cell dose associated with this source has prompted the ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs). However, the expansion procedure is known to exhaust the stem cell pool causing cellular defects that promote apoptosis and disrupt homing to the bone marrow. The role of apoptotic machinery in the regulation of stem cell compartment has been speculated in mouse hematopoietic and embryonic systems. We have consistently observed an increase in apoptosis in the cord blood derived CD34⁺ cells cultured with cytokines compared to their freshly isolated counterpart. The present study was undertaken to assess whether pharmacological inhibition of apoptosis could improve the outcome of expansion.

Methodology/Principal Findings

CB CD34⁺ cells were expanded with cytokines in the presence or absence of cell permeable inhibitors of caspases and calpains; zVADfmk and zLLYfmk respectively. A novel role of apoptotic protease inhibitors was observed in increasing the CD34⁺ cell content of the graft during ex vivo expansion. This was further reflected in improved in vitro functional aspects of the HSPCs; a higher clonogenicity and long term culture initiating potential. These cells sustained superior long term engraftment and an efficient regeneration of major lympho-myeloid lineages in the bone marrow of NOD/SCID mouse compared to the cells expanded with growth factors alone.

Conclusion/Significance

Our data show that, use of either zVADfmk or zLLYfmk in the culture medium improves expansion of CD34⁺ cells. The strategy protects stem cell pool and committed progenitors, and improves their in vitro functionality and in vivo engraftment. This observation may complement the existing protocols used in the manipulation of hematopoietic cells for therapeutic purposes. These findings may have an impact in the CB transplant procedures involving a combined infusion of unmanipulated and expanded grafts.     

Human Cord Blood Stem Cell-Modulated Regulatory T Lymphocytes Reverse the Autoimmune-Caused Type 1 Diabetes in Nonobese Diabetic (NOD) Mice

Background

The deficit of pancreatic islet β cells caused by autoimmune destruction is a crucial issue in type 1 diabetes (T1D). It is essential to fundamentally control the autoimmunity for treatment of T1D. Regulatory T cells (Tregs) play a pivotal role in maintaining self-tolerance through their inhibitory impact on autoreactive effector T cells. An abnormality of Tregs is associated with initiation of progression of T1D.

Methodology/Principal Findings

Here, we report that treatment of established autoimmune-caused diabetes in NOD mice with purified autologous CD4⁺CD62L⁺ Tregs co-cultured with human cord blood stem cells (CB-SC) can eliminate hyperglycemia, promote islet β-cell regeneration to increase β-cell mass and insulin production, and reconstitute islet architecture. Correspondingly, treatment with CB-SC-modulated CD4⁺CD62L⁺ Tregs (mCD4CD62L Tregs) resulted in a marked reduction of insulitis, restored Th1/Th2 cytokine balance in blood, and induced apoptosis of infiltrated leukocytes in pancreatic islets.

Conclusions/Significance

These data demonstrate that treatment with mCD4CD62L Tregs can reverse overt diabetes, providing a novel strategy for the treatment of type 1 diabetes as well as other autoimmune diseases.     

Upregulation of SOCS-3 and PIAS-3 Impairs IL-12-Mediated Interferon-Gamma Response in CD56+ T Cells in HCV-Infected Heroin Users

Background

CD56⁺ T cells are abundant in liver and play an important role in host innate immunity against viral infections, including hepatitis C virus (HCV) infection, a common infection among heroin abusers. We thus investigated the in vivo impact of heroin use or heroin use plus HCV infection on the CD56⁺ T cell frequency and function.

Methodology/Principal Findings

A total of 37 heroin users with (17) or without (20) HCV infection and 17 healthy subjects were included in the study. Although there was no significant difference in CD56⁺ T cell frequency in PBMCs among three study groups, CD56⁺ T cells isolated from the heroin users had significantly lower levels of constitutive interferon-gamma (IFN-γ) expression than those from the normal subjects. In addition, when stimulated by interleukin (IL)-12, CD56⁺ natural T cells from HCV-infected heroin users produced significantly lower levels of IFN-γ than those from the normal subjects. This diminished ability to produce IFN-γ by CD56⁺ T cells was associated with the increased plasma HCV viral loads in the HCV-infected heroin users. Investigation of the mechanisms showed that although heroin use or heroin use plus HCV infection had little impact on the expression of the key positive regulators (IL-12 receptors, STAT-1, 3, 4, 5, JAK-2, and TYK-2) in IL-12 pathway, heroin use or heroin use plus HCV infection induced the expression of suppressor of cytokine signaling protein-3 (SOCS-3) and protein inhibitors of activated STAT-3 (PIAS-3), two key inhibitors of IL-12 pathway.

Conclusion/Significance

These findings provide compelling in vivo evidence that heroin use or heroin use plus HCV infection impairs CD56⁺ T cell-mediated innate immune function, which may account for HCV infection and persistence in liver.     

CD133+ Anaplastic Thyroid Cancer Cells Initiate Tumors in Immunodeficient Mice and Are Regulated by Thyrotropin

Background

Anaplastic thyroid cancer (ATC) is one of the most lethal human malignancies. Its rapid onset and resistance to conventional therapeutics contribute to a mean survival of six months after diagnosis and make the identification of thyroid-cancer-initiating cells increasingly important.

Methodology/Principal Findings

In prior studies of ATC cell lines, CD133⁺ cells exhibited stem-cell-like features such as high proliferation, self-renewal and colony-forming ability in vitro. Here we show that transplantation of CD133⁺ cells, but not CD133⁻ cells, into immunodeficient NOD/SCID mice is sufficient to induce growth of tumors in vivo. We also describe how the proportion of ATC cells that are CD133⁺ increases dramatically over three months of culture, from 7% to more than 80% of the total. This CD133⁺ cell pool can be further separated by flow cytometry into two distinct populations: CD133^+/high and CD133^+/low. Although both subsets are capable of long-term tumorigenesis, the rapidly proliferating CD133^+/high cells are by far the most efficient. They also express high levels of the stem cell antigen Oct4 and the receptor for thyroid stimulating hormone, TSHR. Treating ATC cells with TSH causes a three-fold increase in the numbers of CD133⁺ cells and elicits a dose-dependent up-regulation of the expression of TSHR and Oct4 in these cells. More importantly, immunohistochemical analysis of tissue specimens from ATC patients indicates that CD133 is highly expressed on tumor cells but not on neighboring normal thyroid cells.

Conclusions/Significance

To our knowledge, this is the first report indicating that CD133⁺ ATC cells are solely responsible for tumor growth in immunodeficient mice. Our data also give a unique insight into the regulation of CD133 by TSH. These highly tumorigenic CD133⁺ cells and the activated TSH signaling pathway may be useful targets for future ATC therapies.     

Hypoxia-Induced Mitogenic Factor (HIMF/FIZZ1/RELMα) Recruits Bone Marrow-Derived Cells to the Murine Pulmonary Vasculature

Background

Pulmonary hypertension (PH) is a disease of multiple etiologies with several common pathological features, including inflammation and pulmonary vascular remodeling. Recent evidence has suggested a potential role for the recruitment of bone marrow-derived (BMD) progenitor cells to this remodeling process. We recently demonstrated that hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELMα) is chemotactic to murine bone marrow cells in vitro and involved in pulmonary vascular remodeling in vivo.

Methodology/Principal Findings

We used a mouse bone marrow transplant model in which lethally irradiated mice were rescued with bone marrow transplanted from green fluorescent protein (GFP)⁺ transgenic mice to determine the role of HIMF in recruiting BMD cells to the lung vasculature during PH development. Exposure to chronic hypoxia and pulmonary gene transfer of HIMF were used to induce PH. Both models resulted in markedly increased numbers of BMD cells in and around the pulmonary vasculature; in several neomuscularized small (∼20 µm) capillary-like vessels, an entirely new medial wall was made up of these cells. We found these GFP⁺ BMD cells to be positive for stem cell antigen-1 and c-kit, but negative for CD31 and CD34. Several of the GFP⁺ cells that localized to the pulmonary vasculature were α-smooth muscle actin⁺ and localized to the media layer of the vessels. This finding suggests that these cells are of mesenchymal origin and differentiate toward myofibroblast and vascular smooth muscle. Structural location in the media of small vessels suggests a functional role in the lung vasculature. To examine a potential mechanism for HIMF-dependent recruitment of mesenchymal stem cells to the pulmonary vasculature, we performed a cell migration assay using cultured human mesenchymal stem cells (HMSCs). The addition of recombinant HIMF induced migration of HMSCs in a phosphoinosotide-3-kinase-dependent manner.

Conclusions/Significance

These results demonstrate HIMF-dependent recruitment of BMD mesenchymal-like cells to the remodeling pulmonary vasculature.     

Defects in innate immunity render breast cancer initiating cells permissive to oncolytic adenovirus

Background

Cancer stem cells/initiating cells (CSC/CIC), are thought to exist as a small population in malignant tissues. They are resistant to conventional cancer treatments and possibly underlie post-treatment relapse. The CIC population can be targeted with capsid modified oncolytic adenoviruses.

Methodology/Principal Findings

We studied the mechanisms of innate immunity to oncolytic adenovirus Ad5/3-Delta24 in conventional treatment resistant non-CIC breast cancer cells, breast cancer CD44⁺/CD24^−/low CIC population and normal breast tissue CD44⁺/CD24^−/low stem cells. We compared virus recognition by pattern recognition receptors for adenovirus, Toll-like receptors (TLR) 2 and 9 and virus induced type I interferon (IFN) response regulation in these cell types. We show TLR mediated virus recognition in these non-immune cell types. Normal tissue stem cells have intact type I IFN signaling. Furthermore, TLR9 and TLR2 reside constantly in recognition sites, implying constant activation. In contrast, breast cancer CD44⁺/CD24^−/low CIC have dysregulated innate immune responses featuring dysfunctional virus recognition caused by impaired trafficking of TLR9 and cofactor MyD88 and the absence of TLR2, having a deleterious impact on TLR pattern recognition receptor signaling. Furthermore, the CIC have increased inhibitory signaling via the suppressor of cytokine signaling/Tyro3/Axl/Mer receptor tyrosine kinase (SOCS/TAM) pathway. These defects in contribute to dysfunctional induction of type I IFN response in CIC and therefore permissivity to oncolytic adenovirus.

Conclusions/Significance

CICs may underlie the incurable nature of relapsed or metastatic cancers and are therefore an important target regarding diagnostic and prognostic aspects as well as treatment of the disease. This study addresses the mechanisms of innate infection immunity in stem cells deepening the understanding of stem cell biology and may benefit not only virotherapy but also immunotherapy in general.     

Wnt5a Increases Cardiac Gene Expressions of Cultured Human Circulating Progenitor Cells via a PKC Delta Activation

Background

Wnt signaling controls the balance between stem cell proliferation and differentiation and body patterning throughout development. Previous data demonstrated that non-canonical Wnts (Wnt5a, Wnt11) increased cardiac gene expression of circulating endothelial progenitor cells (EPC) and bone marrow-derived stem cells cultured in vitro. Since previous studies suggested a contribution of the protein kinase C (PKC) family to the Wnt5a-induced signalling, we investigated which PKC isoforms are activated by non-canonical Wnt5a in human EPC.

Methodology/Principal Findings

Immunoblot experiments demonstrated that Wnt5a selectively activated the novel PKC isoform, PKC delta, as evidenced by phosphorylation and translocation. In contrast, the classical Ca²⁺-dependent PKC isoforms, PKC alpha and beta2, and one of the other novel PKC isoforms, PKC epsilon, were not activated by Wnt5a. The PKC delta inhibitor rottlerin significantly blocked co-culture-induced cardiac differentiation in vitro, whereas inhibitors directed against the classical Ca²⁺-dependent PKC isoforms or a PKC epsilon-inhibitory peptide did not block cardiac differentiation. In accordance, EPC derived from PKC delta heterozygous mice exhibited a significant reduction of Wnt5a-induced cardiac gene expression compared to wild type mice derived EPC.

Conclusions/Significance

These data indicate that Wnt5a enhances cardiac gene expressions of EPC via an activation of PKC delta.     

Towards a Clinically Relevant Lentiviral Transduction Protocol for Primary Human CD34+ Hematopoietic Stem/Progenitor Cells

Background

Hematopoietic stem cells (HSC), in particular mobilized peripheral blood stem cells, represent an attractive target for cell and gene therapy. Efficient gene delivery into these target cells without compromising self-renewal and multi-potency is crucial for the success of gene therapy. We investigated factors involved in the ex vivo transduction of CD34⁺ HSCs in order to develop a clinically relevant transduction protocol for gene delivery. Specifically sought was a protocol that allows for efficient transduction with minimal ex vivo manipulation without serum or other reagents of animal origin.

Methodology/Principal Findings

Using commercially available G-CSF mobilized peripheral blood (PB) CD34⁺ cells as the most clinically relevant target, we systematically examined factors including the use of serum, cytokine combinations, pre-stimulation time, multiplicity of infection (MOI), transduction duration and the use of spinoculation and/or retronectin. A self-inactivating lentiviral vector (SIN-LV) carrying enhanced green fluorescent protein (GFP) was used as the gene delivery vehicle. HSCs were monitored for transduction efficiency, surface marker expression and cellular function. We were able to demonstrate that efficient gene transduction can be achieved with minimal ex vivo manipulation while maintaining the cellular function of transduced HSCs without serum or other reagents of animal origin.

Conclusions/Significance

This study helps to better define factors relevant towards developing a standard clinical protocol for the delivery of SIN-LV into CD34⁺ cells.     

Adipocytokines and CD34 progenitor cells in Alzheimer's disease

Background

Alzheimer''s disease (AD) and atherosclerosis share common vascular risk factors such as arterial hypertension and hypercholesterolemia. Adipocytokines and CD34⁺ progenitor cells are associated with the progression and prognosis of atherosclerotic diseases. Their role in AD is not adequately elucidated.

Methods and Findings

In the present study, we measured in 41 patients with early AD and 37 age- and weight-matched healthy controls blood concentrations of adiponectin and leptin by enzyme linked immunoabsorbent assay and of CD34⁺ progenitor cells using flow cytometry. We found significantly lower plasma levels of leptin in AD patients compared with the controls, whereas plasma levels of adiponectin did not show any significant differences (AD vs. control (mean±SD): leptin:8.9±5.6 ng/mL vs.16.3±15.5 ng/mL;P = 0.038; adiponectin:18.5±18.1 µg/mL vs.16.7±8.9 µg/mL;P = 0.641). In contrast, circulating CD34⁺ cells were significantly upregulated in AD patients (mean absolute cell count±SD:253±51 vs. 203±37; P = 0.02) and showed an inverse correlation with plasma levels of leptin (r = −0.248; P = 0.037).In logistic regression analysis, decreased leptin concentration (P = 0.021) and increased number of CD34⁺ cells (P = 0.036) were both significantly associated with the presence of AD. According to multifactorial analysis of covariance, leptin serum levels were a significant independent predictor for the number of CD34⁺ cells (P = 0.002).

Conclusions

Our findings suggest that low plasma levels of leptin and increased numbers of CD34⁺ progenitor cells are both associated with AD. In addition, the results of our study provide first evidence that increased leptin plasma levels are associated with a reduced number of CD34⁺ progenitor cells in AD patients. These findings point towards a combined involvement of leptin and CD34⁺ progenitor cells in the pathogenesis of AD. Thus, plasma levels of leptin and circulating CD34⁺ progenitor cells could represent an important molecular link between atherosclerotic diseases and AD. Further studies should clarify the pathophysiological role of both adipocytokines and progenitor cells in AD and possible diagnostic and therapeutic applications.     

Non-Redundant Role for IL-12 and IL-27 in Modulating Th2 Polarization of Carcinoembryonic Antigen Specific CD4 T Cells from Pancreatic Cancer Patients

Background

Pancreatic cancer is a very aggressive disease with dismal prognosis; peculiar is the tumor microenvironment characterized by an extensive fibrotic stroma, which favors rapid tumor progression. We previously reported that pancreatic cancer patients have a selective Th2 skew in the anti-carcinoembryonic antigen (CEA) CD4⁺ T cell immunity, which correlates with the presence of a predominant GATA-3⁺ tumor lymphoid infiltrate. This has negative effects in both effective anti-tumor immunity and further favoring fibrinogenesis. Aim of this study was to evaluate whether the Th2 polarization of CEA-specific CD4⁺ T cells from pancreatic cancer patients is stable or can be reverted by immunomodulating cytokines.

Methodology/Principal Findings

We first evaluated the influence of IL-12 and IL-27, as single agents and in association, on the polarization of CEA-specific Th2 CD4⁺ T cell clones from a pancreatic cancer patient. We found that only the combination of IL-12 and IL-27 modified the polarization of Th2 effectors by both reduction of IL-5, GM-CSF and IL-13 and induction of IFN-γ production, which lasted after cytokine removal. Second, we evaluated the effect of the combined treatment on polyclonal CEA-specific CD4⁺ T cells in short-time re-stimulation assays. In agreement with the data obtained with the clones, we found that the combined treatment functionally modulated the Th2 polarization of CEA-specific CD4⁺ T cells and enhanced pre-existing Th1 type immunity.

Conclusions/Significance

Collectively, our results demonstrate that tumor antigen specific Th2 CD4⁺ T cells in pancreatic cancer are endowed with functional plasticity. Hence, loco-regional cytokines delivery or targeted therapy based on antibodies or molecules directed to the tumor stroma might improve anti-tumor immunity and ameliorate fibrosis, without systemic toxicity.