Szymczak-Workman for assistance with histological analysis, S

Szymczak-Workman for assistance with histological analysis, S. survival and in inflammatory sites in the presence of Sema4a-Ig or its isotype control. Results represent the mean of five impartial experiments. *, 0.05, **, 0.01, ***, 0.001 by unpaired t-test. Error bars indicate s.e.m. Nrp1 is usually a homogeneously expressed marker of thymically-derived Tregs, and has also been shown to bind Sema3a, which shares homology with Sema4a, and vascular endothelial growth factor (VEGF) in mediating neural axon Rabbit Polyclonal to PKC zeta (phospho-Thr410) growth and angiogenesis (Supplementary Fig. 3a)10-13. Thus, we hypothesized that Nrp1 functions as a receptor for Sema4a on Tregs. Sema4a could directly and specifically bind to Nrp1, albeit with slightly lower avidity to Sema3a, as demonstrated in an ELISA-based binding assay with recombinant Nrp1 and Sema family proteins and blocking antibodies to Nrp1 or Sema4a (Fig. 1d and Supplementary Fig. 3b-d). Sema4a-Ig also bound to littermate control (deletion was comparable (Supplementary Fig. 6), suggesting that maintenance of immune homeostasis and prevention of autoimmunity may not require Nrp1 signaling. We hypothesized that Nrp1 signaling may regulate Treg function under inflammatory conditions. Tregs are recruited to and are induced by tumor cells, which consequently hamper protective anti-tumor immunity16. in a Sema4a:Nrp1-dependent manner (Supplementary Fig. 7d). Treatment with Sema4a-Ig also resulted in an increased number of intratumoral CD8+ T cells, consistent with observations with expanded Tregs serum starved for 3h, then stimulated as indicated for 6 h prior to IP. d, Transwell suppression assay using melanoma model29, although contrary to their findings we did not observe any differences in Treg prevalence in B16 melanoma tumors (Fig. 2h). However, further studies will be required to delineate in which tumors and under what conditions these two disparate functions, namely regulation of Treg migration and the maintenance of Treg survival and stability, of Nrp1 in Tregs are utilized. Our identification of Nrp1:Sema4a as a pivotal pathway required for intratumoral Treg stability, but dispensable for the maintenance of immune homeostasis, suggests that Sema4a:Nrp1 blockade via antibodies or soluble antagonists may be a viable therapeutic strategy to limit tumor-induced tolerance without evoking autoimmunity. METHODS Antibodies Sema4a staining antibody was purchased from MBL (clone 5E3), and conjugated to biotin or Alexa Fluor 647 in-house. Polyclonal anti-Nrp1 was purchased from R&D Systems (AF566). Monoclonal antibodies were obtained from R&D Systems (Sema4a, 757129; Nrp1, 761704, MAB59941). Most flow cytometric antibodies were purchased from BioLegend. Anti-Foxp3 and anti-Eomes were purchased from eBioscience. KLF2 antibody was purchased from Millipore. Phospho-Akt (S473), phospho-S6K1 (T421/S424), Foxo3a, and pan Akt antibodies were purchased from Cell Signaling Technologies. PTEN-HRP antibody was purchased from Santa Cruz Biotechnology. RNA interference Control siRNA (Catalog # 4390843) and pools of Sema4a (Catalog # 4390771, siRNA # s73547) siRNA were purchased from Life Technologies and resuspended per the manufacturer’s instructions. CD4+ and CD8+ conventional T cells were sorted magnetically by unfavorable selection and transfected by Amaxa (Lonza) with 300 pMol siRNA and 2 g of pMaxGFP control plasmid, rested overnight in Amaxa nucleofector media. Cells were then sorted based on GFP, CD25, and CD45RB expression and cocultured with Treg cells in the top well of a transwell suppression assay. Plasmids Nrp1.mCherry was obtained from Addgene and used as a template to generate retroviral overexpresion constructs. Nrp1WT was generated by adding the native signal sequence and cloned into pMICherry. Nrp1SEA was generated from the WT construct, deleting the terminal SEA motif by mutation of the serine codon to a stop codon. AktWT, AktDN (dominant-negative kinase dead K179M; originally described by Franke et al23), and pBabe empty vector were obtained from D.R. Green. Human T cell populations Human umbilical cord samples were provided by B. Triplett, M. Howard and M. McKenna JAK3-IN-2 at the St. Louis Cord Blood Lender, and were obtained from the umbilical vein JAK3-IN-2 immediately after vaginal delivery with JAK3-IN-2 the informed consent of the mother and approved by St. Louis Cord Blood Lender Institutional Review Board (IRB). Research use approved by the St. Jude IRB. Transwell suppression 1.25 104 Treg purified flow cytometrically purified (CD4+ CD45RBlo and were cloned in-frame to pX-Ig to create a Sema4a- or Nrp1-mouse IgG1.