Figure 6. CD28 optimizes the differentiation of tumor-infiltrating eTreg.

(A) Ex vivo analysis of NFAT1 and NFAT2 nuclear localization in control or CD28-deficient Foxp3⁺ Treg immediately upon isolation from LNs and spleen of CD28 ΔTreg or Foxp3^YFP-Cre control mice and processed as described for Fig. 5A. One experiment out of two is shown, p-values calculated through Mann-Whitney U test. (B) Analysis of IκB down-regulation in YFP⁺ Treg from CD28 ΔTreg^het and Foxp3^YFP-Cre/wt control mice. Mean and SD of normalized average of triplicate measures are shown. One experiment representative of two is shown. (C) Frequencies of CD44^low CD62L^hi cTreg and CD44^hi CD62L^neg eTreg among total YFP⁺ Treg in tdLNs in CD28 ΔTreg^het or Foxp3^YFP-Cre/wt control mice. Means and SEM of one experiment (4 mice per group) out of 3 is shown. (D, E) Expression of CXCR3, CCR4 and CCR5 (D) and ICOS, CTLA-4 and Ki67 (E) in YFP⁺ Treg in tdLNs of CD28 ΔTreg^het (‘CD28-def.’) and Foxp3^YFP-Cre/wt control mice (‘WT’) 11 days after MCA-205 tumor implantation. In both (D) and (E) quantification of one experiment (n=3–4) representative of 3 performed is shown. Bars represent medians, p-values calculated by Mann-Whitney U test. (F, G) Expression of CXCR3, CCR4 and CCR5 (F) and ICOS, CTLA-4 and Ki67 expression (G) in tumor tissue of CD28 ΔTreg^het (‘CD28-def’) and Foxp3^YFP-Cre/wt control (‘WT’) mice 11 days after MCA-205 tumor implantation. Data are pooled from 3 independent experiments (n=5–7). MFIs were normalized to the average MFI of WT Treg within each experiment, p-values calculated by Mann-Whitney U test.