Figure 4. Treatment of relapsing melanoma requires both T_reg depletion and blockade of PD-L1.

(A) Treatment of relapsing melanoma requires combination T_reg depletion and anti-PD-L1 therapy. C57BL/6 lymphopenic RAG^−/− mice (5–10/group) were inoculated with B16.F10 melanoma (2 × 10⁵ cells). Tumor-bearing mice were treated with 2 × 10⁵ naïve TRP-1 Foxp3-DTR CD4⁺ T cells by intravenous tail vein injection on day 7–10 after tumor inoculation. Tumors were followed until recurrence of melanoma. At recurrence, DT (diphtheria toxin) was injected i.p. at the prescribed concentration of 50 mg/Kg every other day for three doses total. At recurrence, anti-PD-L1 was given as a bolus injection i.p. at 500 μg for the first dose and subsequently given every three days thereafter at 200 μg/injection for 5 doses. For combination therapy, both were given at the same time as described above. PBS controls had no effect (not shown). Data shown is representative of more than 5 experiments. (B) Blockade of PD-L1 and depletion of tumor-specific T_reg cells reinvigorates tumor-specific CD4⁺ T cells by reducing inhibitory receptor expression of PD-1 and LAG-3, and increases IL-7R and CXCR3. Flow cytometry shows four groups (No recurrence, DT only, DT plus anti-PD-L1 antibody, and anti-PD-L1 antibody only). Gray filled histograms represent no recurrence, blue solid line represents DT therapy only, red solid line represents anti-PD-L1 only, and green solid line represents therapy with DT and anti-PD-L1 together. All therapies were given at time of recurrence. Flow cytometry is performed on each group 3–5 days after the last dose of therapy. Shown is representative of 5 experiments. (C) Tumor-specific CD4⁺ T cells regain effector function with dual therapy as defined by re-expression of IFN-γ and TNF-α when compared to relapsing groups with single therapies (anti-PD-L1 (7.3%), DT (3.04%), +DT and anti-PD-L1 (17.4%), no recurrence (18.6%)). Experiment repeated three times. (D) Gross depiction of tumor regression after combination therapy. Days indicate day after therapy was given. All tumors depicted are relapsing tumors that were previously treated as a primary tumor. (E) IFN-γ inducible chemokines (CXCL9 and CXCL10) are highly increased during recurrence and return to treatment levels with combination therapy. Each dot represents and individual mouse. (F) Total number of tumor-specific CD4⁺ T cells in the spleen stabilizes to non-relapsing levels with combination therapy. Absolute number of TRP-1 CD4⁺ T cells during different treatments. Each bar graph represents 5–10 individual mice.