TGF-β1 levels were Selleck Sotrastaurin also higher in TDLNs draining TGF-β1-expressing tumors than tumors not expressing TGF-β1. B, Serum TGF-β1 levels measured in the same mice as in panel A. Serum TGF-β1 levels did not differ among the groups. *P < 0.05. n = 5 in each group. To begin assessing DC-mediated immunity in this model, we used flow cytometry to determine the

numbers and phenotypes of DCs within the TDLNs and non-TDLNs from wild SCCVII tumor-bearing mice on day 14 after tumor implantation. Figure 3A shows that TDLNs from these mice contained approximately 1.5 to 5 times as many CD11c+ DCs as non-TDLNs. Numbers of CD11c+CD86+ mature DCs were also increased 1.5 to 5 times within TDLNs, as compared to non-TDLNs (Figure 3B). Clearly, the PF-01367338 cost immune response to tumor antigen was higher in TDLNs than in non-TDLNs. ARS-1620 cell line Figure 3 Increases in the number and biological activity of DCs within TDLNs in wild SCCVII tumor-bearing mice. A, Numbers of CD11c+ DCs in TDLNs and non-TDLNs on day 14 after tumor inoculation. B, Numbers of CD11c+CD86+ mature DCs in TDLNs and non-TDLNs. The immune response of DCs to tumor antigen was higher in TDLNs than non-TDLNs. *P < 0.05. n = 10 in each group. To assess the inhibition of DC migration into TDLNs by tumor-derived TGF-β1, we used flow cytometry to count the numbers of DCs within TDLNs and non-TLDNs. We found that migration of DCs into TDLNs was

inhibited in mice inoculated with the three TGF-β1-expressing clones, resulting in a significant reduction in the numbers of CD11c+ DCs within TDLNs (Figure 4A). By contrast, there was no significant difference between the numbers of CD11+ DCs

in non-TDLNs from mice inoculated with mock or TGF-β1 transfectants. To identify the maturation status of the DCs within TDLNs, we also counted the numbers of CD11c+ and CD86+ DCs. We found that the TDLN/non-TDLN ratio for both CD11c+ cells and CD86+CD11c+ mature DCs was reduced in mice PLEK2 inoculated with TGF-β1-expressing clones (Figure 4B, C). Figure 4 Tumor-derived TGF-β1 reduces the number of DCs within TDLNs. A, Numbers of CD11c+ DCs in TDLNs and non-TDNLs from mice inoculated with TGF-β1-tranfected or mock-transfected tumor cells. B, TDLN/non-TDLN ratios for CD11c+ DCs in mice inoculated with TGF-β1-transfected or mock-transfected cells. C, To determine the maturation status of DCs within TDLNs, numbers of CD11c+ and CD86+ DCs were counted, after which the TDLN/non-TDLN ratio for CD11c+CD86+ DCs was calculated. * P < 0.05. To further clarify the mechanism underlying the reduction in the numbers of DCs within TDLNs, we injected the tumors with CFSE-labeled bmDCs and then counted the numbers of labeled cells within the TDLNs. With this method, we were able to distinguish migrated CFSE-labeled bmDCs from autologous DCs within TDLNs. Flow cytometric analysis of the TDLNs showed that significantly fewer immature (no added LPS) CFSE+ bmDCs migrated from TGF-β1-expressing tumors than from mock-transfected tumors (Figure 5A).