Figure 2.

PDPN⁺ LNSCs inhibit CD4⁺ T cell proliferation in vitro. (A) CellTrace-labeled CD4⁺CD25⁻CD69⁻CD11c⁻ (15 × 10⁴) T cells sorted from splenocytes of naive C57BL/6 mice were activated with aCD3 (0.2 μg/mL) and IL-2 (20 ng/mL) for 3.5 d in the presence or absence of PDPN⁺ LNSCs (5 × 10⁴) sorted from tumors and tdLNs on day 14 after tumor inoculation. (B) T cells isolated and activated as in A were co-cultured with PDPN⁺ LNSCs sorted from naive mice. (C) PDPN⁺ LNSCs were separated from T cells using a 0.4 μm transwell insert. Proliferation was assessed by flow cytometry based on CellTrace dilution. Graphs denote the mean percentages and standard deviations of CD4⁺ T cells in each proliferation generation and of dividing CD4⁺ T cells. (D) Mean and standard deviation of the percentage of CD4⁺ T cells per generation of proliferation in cultures of CellTrace-labeled CD4⁺CD25⁻CD69⁻CD11c⁻ (15 × 10⁴) T cells with aCD3 (0.2 μg/mL) and IL-2 (20 ng/mL) in the absence (w/o sup) or presence of 3.5 d supernatants from co-cultures of PDPN⁺ LNSCs with CD4⁺ T cells (PDPN sup) or from cultures of CD4⁺ T cells alone (T cell sup) are presented. Means have been calculated from 15 to 19 independent experiments. Numbers on FACS plots denote frequency of gated population or proliferation index (p.i.). **p < 0.005, ***p < 0.0005.