Kristin Fino

Utilizing regulatory T cells against rheumatoid arthritis

Regulatory T (Treg) cells are essential for normal immune surveillance systems, and their dysfunction leads to development of diseases, such as autoimmune disorders. CD4+CD25+ Treg cells are well-known suppressive cells, which express the transcription factor Foxp3, are indispensable for the maintenance of immune self-tolerance and homeostasis by suppressing aberrant or excessive immune response. Other Foxp3− Treg cells include Tr1, Th3, CD8+CD28−/−, and Qa1-restricted T cells; however, the contribution of these Treg cells to self-tolerance, immune homeostasis as well as preventing autoimmunity is not well defined. Here, we discuss the phenotypes and function of Foxp3+ Treg cells and the potential use of such Treg cells against rheumatoid arthritis (RA). Of note, even though most expanded populations of Foxp3+ Treg cells exhibit suppressive activity, tissue-associated or antigen-specific Treg cells appear superior in suppressing local autoimmune disorders such as RA. In addition, utilizing tissue-associated Foxp3+ Treg cells from stem cells may stable Foxp3 expression and avoid induction of a potentially detrimental systemic immunosuppression.

Downregulation of the CCK-B receptor in pancreatic cancer cells blocks proliferation and promotes apoptosis.

Gastrin stimulates the growth of pancreatic cancer cells through the activation of the cholecystokinin-B receptor (CCK-BR), which has been found to be overexpressed in pancreatic cancer. In this study, we proposed that the CCK-BR drives growth of pancreatic cancer; hence, interruption of CCK-BR activity could potentially be an ideal target for cancer therapeutics. The effect of CCK-BR downregulation in the human pancreatic adenocarcinoma cells was examined by utilizing specific CCK-BR-targeted RNA interference reagents. The CCK-BR receptor expression was both transiently and stably downregulated by transfection with selective CCK-BR small-interfering RNA or short-hairpin RNA, respectively, and the effects on cell growth and apoptosis were assessed. CCK-BR downregulation resulted in reduced cancer cell proliferation, decreased DNA synthesis, and cell cycle arrest as demonstrated by an inhibition of G(1) to S phase progression. Furthermore, CCK-BR downregulation increased caspase-3 activity, TUNEL-positive cells, and decreased X-linked inhibitor of apoptosis protein expression, suggesting apoptotic activity. Pancreatic cancer cell mobility was decreased when the CCK-BR was downregulated, as assessed by a migration assay. These results show the importance of the CCK-BR in regulation of growth and apoptosis in pancreatic cancer. Strategies to decrease the CCK-BR expression and activity may be beneficial for the development of new methods to improve the treatment for patients with pancreatic cancer.

A single nucleotide polymorphism of the cholecystokinin-B receptor predicts risk for pancreatic cancer

There currently are no tests available for early diagnosis or for the identification of patients at risk for development of pancreatic cancer. We report the discovery of single nucleotide polymorphism (SNP) in the cholecystokinin B receptor (CCKBR) gene predicts survival and risk of pancreatic cancer. Growth of human pancreatic cancer is stimulated by gastrin through the CCKBR and an alternatively spliced isoform of the CCKBR gene called CCKCR. One hundred and ten surgically resected benign and malignant pancreatic tissues as well as normal pancreas were prospectively evaluated for CCKBR genotype and protein expression. Analysis demonstrated the expression of the spliced isoform, CCKCR, was associated with a (SNP) (C > A) at position 32 of the intron 4 (IVS 4) of the CCKBR gene. Since the SNP is within an intron, it has not previously been identified in the GWAS studies. Only patients with the A/A or A/C genotypes, exhibited immunoreactivity to a selective CCKCR antibody. Survival among pancreatic cancer patients with the A-SNP was significantly shorter (p = 0.0001, hazard ratio = 3.63) compared with individuals with C/C genotype. Other variables such as surgical margins, lymph node status, histologic grade or adjuvant chemotherapy were not associated with survival. Furthermore, having one or two of the A-alleles was found to increase the risk of pancreatic adenocarcinoma by 174% (p = 0.0192) compared with the C/C wild type. Cancer cells transfected to overexpress the CCKCR demonstrated increased proliferation over controls. Genetic screening for this SNP may aid in early detection of pancreatic cancer in high risk subjects.

Stem cell-derived tissue-associated regulatory T cells ameliorate the development of autoimmunity.

Pluripotent stem cells (PSCs) have the potential to produce almost all of the cells in the body, including regulatory T cells (Tregs). However, the exact conditions required for the development of antigen (Ag)-specific Tregs from PSCs (i.e., PSC-Tregs) are not well delineated. Ag-specific PSC-Tregs can be tissue/organ-associated and migrate to local inflamed tissues/organs to suppress the autoimmune response after adoptive transfer, thereby avoiding potential overall immunosuppression from non-specific Tregs. In this study, we developed a new approach to generate functional Ag-specific Tregs from induced PSCs (iPSCs), i.e., iPSC-Tregs, which had the ability to generate an Ag-specific immunosuppressive response in a murine model of arthritis. We retrovirally transduced murine iPSCs with a construct containing genes of Ag-specific T cell receptor (TCR) and the transcriptional factor FoxP3. We differentiated the iPSCs into Ag-specific iPSC-Tregs using in vitro or in vivo Notch signaling, and demonstrated that adoptive transfer of such Tregs dramatically suppressed autoimmunity in a well-established Ag-induced arthritis model, including the inflammation, joint destruction, cartilage prostaglandin depletion, osteoclast activity, and Th17 production. Our results indicate that PSCs can be used to develop Ag-specific Tregs, which have a therapeutic potential for Treg-based therapies of autoimmune disorders.

C-Myc regulation by costimulatory signals modulates the generation of CD8 + memory T cells during viral infection

The signalling mechanisms of costimulation in the development of memory T cells remain to be clarified. Here, we show that the transcription factor c-Myc in CD8+ T cells is controlled by costimulatory molecules, which modulates the development of memory CD8+ T cells. C-Myc expression was dramatically reduced in Cd28−/− or Ox40−/− memory CD8+ T cells, and c-Myc over-expression substantially reversed the defects in the development of T-cell memory following viral infection. C-Myc regulated the expression of survivin, an inhibitor of apoptosis, which promoted the generation of virus-specific memory CD8+ T cells. Moreover, over-expression of survivin with bcl-xL, a downstream molecule of NF-κB and intracellular target of costimulation that controls survival, in Cd28−/− or Ox40−/− CD8+ T cells, reversed the defects in the generation of memory T cells in response to viral infection. These results identify c-Myc as a key controller of memory CD8+ T cells from costimulatory signals.

Melanoma Immunotherapy in Mice Using Genetically Engineered Pluripotent Stem Cells.

Adoptive cell transfer (ACT) of antigen (Ag)-specific CD8+ cytotoxic T lymphocytes (CTLs) is a highly promising treatment for a variety of diseases. Naive or central memory T-cell-derived effector CTLs are optimal populations for ACT-based immunotherapy because these cells have a high proliferative potential, are less prone to apoptosis than terminally differentiated cells, and have the higher ability to respond to homeostatic cytokines. However, such ACT with T-cell persistence is often not feasible due to difficulties in obtaining sufficient cells from patients. Here we present that in vitro differentiated HSCs of engineered PSCs can develop in vivo into tumor Ag-specific naive CTLs, which efficiently suppress melanoma growth. Mouse-induced PSCs (iPSCs) were retrovirally transduced with a construct encoding chicken ovalbumin (OVA)-specific T-cell receptors (TCRs) and survival-related proteins (i.e., BCL-xL and survivin). The gene-transduced iPSCs were cultured on the delta-like ligand 1-expressing OP9 (OP9-DL1) murine stromal cells in the presence of murine recombinant cytokines (rFlt3L and rIL-7) for a week. These iPSC-derived cells were then intravenously adoptively transferred into recipient mice, followed by intraperitoneal injection with an agonist α-Notch 2 antibody and cytokines (rFlt3L and rIL-7). Two weeks later, naive OVA-specific CD8+ T cells were observed in the mouse peripheral lymphatic system, which were responsive to OVA-specific stimulation. Moreover, the mice were resistant to the challenge of B16-OVA melanoma induction. These results indicate that genetically modified stem cells may be used for ACT-based immunotherapy or serve as potential vaccines.

SH3GLB2/endophilin B2 regulates lung homeostasis and recovery from severe influenza A virus infection

New influenza A viruses that emerge frequently elicit composite inflammatory responses to both infection and structural damage of alveolar-capillary barrier cells that hinders regeneration of respiratory function. The host factors that relinquish restoration of lung health to enduring lung injury are insufficiently understood. Here, we investigated the role of endophilin B2 (B2) in susceptibility to severe influenza infection. WT and B2-deficient mice were infected with H1N1 PR8 by intranasal administration and course of influenza pneumonia, inflammatory, and tissue responses were monitored over time. Disruption of B2 enhanced recovery from severe influenza infection as indicated by swift body weight recovery and significantly better survival of endophilin B2-deficient mice compared to WT mice. Compared to WT mice, the B2-deficient lungs exhibited induction of genes that express surfactant proteins, ABCA3, GM-CSF, podoplanin, and caveolin mRNA after 7 days, temporal induction of CCAAT/enhancer binding protein CEBPα, β, and δ mRNAs 3–14 days after infection, and differences in alveolar extracellular matrix integrity and respiratory mechanics. Flow cytometry and gene expression studies demonstrated robust recovery of alveolar macrophages and recruitment of CD4+ lymphocytes in B2-deficient lungs. Targeting of endophilin B2 alleviates adverse effects of IAV infection on respiratory and immune cells enabling restoration of alveolar homeostasis.

Abstract 2891: Gastrin signaling and Par-4 in pancreatic cancer

Background: The gastrointestinal peptide, gastrin, has been identified as a growth factor in pancreatic cancer. Gastrin stimulates growth of pancreatic cancer through the cholecystokinin (CCK)-2 receptor. Prostate apoptosis response-4 (Par-4) is a tumor-suppressor protein that induces apoptosis in cancer cells but not in normal cells. Par-4 is down-regulated in a variety of cancers, including pancreatic where it is correlated with lower rate of survival. The anti-apoptotic activity of Par-4 is reduced when active Akt1 binds and phosphorylates it. In colon cancer cells, Par-4 also binds to NFκB and repressed NFκB gene transcription. Gastrin stimulation mediated through the CCK-2R regulates NFκB and Akt activity. Hypothesis: Stimulation of human pancreatic cancer cells with gastrin affects Par-4 activity which may be mediated through Akt. Methods: PANC-1 cells were serum starved for 16-hours, pretreated with a CCK-2R specific inhibitor (YM022) or vehicle for 1hr, and then stimulated with 10-8 M gastrin-17 for 20min. Cells were immediately lysed and the whole cell lysate was subjected to immunoprecipitation with magnetic Dynabeads bound to antibodies specific for Par-4 or NFκB(p65). Immunoprecipitation samples were probed for Par-4, and NFκB(p65), and cell lysates were probed for Par-4, NFκB(p65), Akt, phospho-p65 (Ser536), and phospho-Akt (Ser473). PANC-1 cells were transfected with siRNA specific to the CCK-2R or appropriate scrambled control delivered by Lipofectamine 2000. Protein was isolated 72 hours after transfection. Results: Pretreatment of PANC-1 cells with YM022 before gastrin stimulation blocked phosphorylation of Akt while total levels of Akt remained unchanged. siRNA knock-down of CCK-2R also resulted in a decrease of Akt activation. Phosphorylation of NFκB(p65) was increased in cells pretreated with YM022 compared to cells treated with gastrin alone; total levels of NFκB(p65) remained unchanged. Binding of Par-4 to NFκB(p65) was enhanced in cells treated with both gastrin and YM022, compared to cells treated with gastrin alone. Total levels of Par-4 remained unchanged. Conclusion: For the first time we provide evidence supporting that gastrin stimulation of pancreatic cancer cells alters activity of Par-4. Gastrin stimulation decreases binding of Par-4 to NFκB(p65) in PANC-1 cells while pretreatment with YM022 blocks this decrease in binding. The change in Par-4 activity may be mediated through Akt because YM022 and gastrin stimulation alters their activity and they are known binding partners of Par-4. Since Par-4 sensitizes cells to apoptosis induction as exemplified by sensitization of colon cancer cells to the chemotherapeutic agent, 5-fluorouracil, when Par-4 is delivered in vivo, understanding of the mechanism by which Par-4 is down-regulated is important in developing therapies to take advantage of Par-4 induced sensitization to apoptosis. Supported by: R01 CA117926 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2891. doi:10.1158/1538-7445.AM2011-2891

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity.

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

Stem cell-derived tissue- associated regulatory T cells ameliorate the development of autoimmune arthritis

Meeting abstracts Embryonic stem cells have the ability to grow indefinitely while maintaining pluripotency. Under the right circumstance, pluripotent stem cells (PSC) can produce almost all of the cells in the body including regulatory T cell (Tregs). Tregs are essential for normal immune