Figure 7. CnB-deficient Treg are more strongly impaired in maintaining tumor tolerance than CD28-deficient Treg.

(A) Growth rates of subcutaneous MCA-205 tumors CD28 ΔTreg (n=3) or Foxp3^YFP-Cre control mice (n=3) . Means and SEM for one experiment of two are shown. (B) Frequency of Foxp3⁺ Treg in tumor tissue of Foxp3^YFP-Cre and CD28 ΔTreg mice at day 17 or 18 after tumor implantation. Data are representative of 7 (Foxp3^YFP-Cre) and 5 mice (CD28 ΔTreg). (C) Accumulation of CD28-deficient YFP⁺ Treg in tumor tissue is more strongly impaired in competitive (CD28 ΔTreg^het hosts, n=6) than in non-competitive settings (CD28 ΔTreg hosts, n=5). CD28-deficient YFP⁺ Treg were compared with YFP⁺ Treg in Foxp3^YFP-Cre/wt and Foxp3^YFP-Cre hosts, respectively. Bars show means, p-value calculated by Mann-Whitney U test. (D) Growth of MC38 tumors in CD28 ΔTreg (n=4) mice or Foxp3^YFP-Cre control mice (n=6) . Mean and SEM for are shown. (E) Growth of MC38 tumors in mice with inducible Treg-specific deletion of CnB or CD28. Tumor cells were implanted in Foxp3^CreERT2, CnB ΔTreg^ind, and CD28 ΔTreg^ind mice. Arrow indicates start of tamoxifen administration, resulting in the deletion of CnB or CD28. Mean ± SEM and one experiment of two is shown, n=4 per group. (F) Growth of MC38 tumors in mixed chimeras generated in irradiated TCRα^–/– hosts with Foxp3^DTR and either CnB ΔTreg, CD28 ΔTreg or Foxp3^YFP-Cre bone marrow. Upon administration of diphtheria toxin (arrow), mice are acutely converted into functional CnB ΔTreg or CD28 ΔTreg mice without pre-existing lymphoproliferative disease. Foxp3^YFP-Cre + Foxp3^DTR: n=5; CnB ΔTreg + Foxp3^DTR: n=4; CD28 ΔTreg + Foxp3^DTR: n=7. Mean ± SEM is shown.

In A, D, E, and F, * indicates p-values<0.05 against control mice, # indicates p-values<0.05 against CD28 ΔTreg mice, p-values calculated using muliple t-tests with Sidak post-test.