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The immunosuppressive drug rapamycin (RAPA) promotes the expansion of CD4(+) CD25(high)Foxp3(+) regulatory T cells via mechanisms that remain unknown. Here, we studied expansion, IL-2R-gamma chain signaling, survival pathways and resistance to apoptosis in human Treg responding to RAPA. 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(high)Foxp3(+) 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. 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.

Introduction: Adaptive regulatory T cells (Tr1) are induced in the periphery by environmental stimuli. CD73 expression and adenosine (ADO) production by tumor cells may influence Tr1 generation and their immunosuppressive activity. Material and Methods: Tr1 were generated in co-cultures of CD4+CD25neg T cells, autologous immature dendritic cells (iDC), and irradiated ADO-producing CD73+ or non-producing CD73neg breast cancer (BrCa) cell lines (TU). The expression of ectonucleotidases and other surface markers on Tr1 was determined by flow cytometry. Tr1-mediated suppression of proliferation was evaluated in CFSE-based assays. Luciferase-based ATP detection assays and mass spectrometry were used to measure ATP hydrolysis and ADO levels. Cytokine levels were measured by ELISA or Luminex. CD73 expression on tumor cells or T cells in TU tissues was assessed by immunofluorescence. Results: CD73+ TU induced higher numbers of Tr1 cells (p < 0.01) than CD73neg TU. Tr1TU73+ hydrolyzed more exogenous ATP, produced more ADO, and mediated higher suppression than Tr1TU73neg (p < 0.05 for all). ARL67156, an ectonucleotidase inhibitor, and ZM241385, A2A receptor antagonist, reduced suppression of proliferation mediated by Tr1TU73+ cells (p < 0.01). Basal-like primary BrCa cells expressed higher levels of ectonucleotidases and induced more Tr1 than less aggressive primary luminal-like BrCa. Conclusion: BrCa producing ADO (CD73+ TU) favor the induction of Tr1, which expresses CD39 and CD73, hydrolyzes ATP to ADO, and effectively suppresses anti-tumor immunity.

The immunosuppressive drug rapamycin (RAPA) promotes the expansion of CD4.sup.+ CD25.sup.high Foxp3.sup.+ regulatory T cells via mechanisms that remain unknown. Here, we studied expansion, IL-2R-[gamma] chain signaling, survival pathways and resistance to apoptosis in human Treg responding to RAPA. CD4.sup.+ CD25.sup.+ and CD4.sup.+ CD25.sup.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.sup.+ CD25.sup.high and CD4.sup.+ CD25.sup.neg T cells. Suppressor function was tested in co-cultures with autologous CFSE-labeled CD4.sup.+ CD25.sup.neg or CD8.sup.+ CD25.sup.neg T-cell responders. The frequency and suppressor activity of Treg were increased after culture of CD4.sup.+ CD25.sup.+ T cells in the presence of 1-100 nM RAPA (p<0.001). RAPA-expanded Treg were largely CD4.sup.+ CD25.sup.high Foxp3.sup.+ cells and were resistant to apoptosis, while CD4.sup.+ CD25.sup.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.sup.+ CD25.sup.neg cells. Activated Treg±RAPA preferentially phosphorylated STAT5 and STAT3 and did not utilize the PI3K/mTOR pathway. 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.

Toll-like receptors (TLRs) expressed on immune cells trigger inflammatory responses. TLRs are also expressed on ovarian cancer (OvCa) cells, but the consequences of signaling via the TLR4/MyD88 pathway in these cells are unclear. Here, TLR4 and MyD88 expression in OvCa tissues (n=20) and cell lines (OVCAR3, SKOV3, AD10, A2780 and CP70) was evaluated by RT-PCR, Western blots and immunohistochemistry. Cell growth, apoptosis, NF-κB translocation, IRAK4 and TRIF expression and cJun phosphorylation were measured following tumor cell exposure to the TLR4 ligands, lipopolysacharide (LPS) or Paclitaxel (PTX). Culture supernatants were tested for cytokine levels. TLR4 was expressed in all tumors, tumor cell lines and normal epithelium. MyD88 was detectable in tumor tissues and in 3/5 OvCa lines but not in normal cells. In MyD88+ SCOV3 cells, LPS or PTX binding to TLR4 induced IRAK4 activation and cJun phosphorylation, activated the NF-κB pathway and promoted IL-8, IL-6, VEGF and MCP-1 production and resistance to drug-induced apoptosis. Silencing of TLR4 in SCOV3 cells with siRNA resulted in p-cJun downregulation and a loss of PTX resistance. In PTX sensitive, MyD88neg A2780 cells, TLR4 stimulation upregulated TRIF, and TLR4 silencing eliminated this effect. Thus, TLR4/MyD88 signaling supports OvCa progression and chemoresistance, promoting immune escape.