Figure 4. MDSC mediate anti-proliferative effects of peg-Arg I in mice.
(A) Proliferation, as tested by BrdU, in CD45.1+/OT-1 T-cells adoptively transferred into CD45.2+ mice that received peg-Arg I or peg-BSA (1 mg/mouse), immunization with siinfekl, and treatments with anti-Gr-1 antibody or IgG, as described in the Methods (n=10 for each group). (B) Mice (n=10) were injected i.p. with peg-Arg I, peg-BSA, or PBS every 2 days for 7 days. Then, percentages of CD11b+ Gr-1+ were tested in spleens by flow cytometry. (C) Proliferation of MDSC was determined by BrdU, as described in the Methods. (D) Samples from B (0.5 mg/mouse) were tested for G-MDSC (CD11b+ Ly6G+ Ly6Clow) and M-MDSC (CD11b+ Ly6Gneg Ly6Chigh). (E) Proliferation of CFSE-labeled T-cells was measured 72 hours after co-culture with different ratios of splenic CD11b+ Gr-1+ cells from peg-Arg I, peg-BSA or PBS-treated mice. (F) Arginase I and iNOS in splenic CD11b+ Gr-1+ cells from mice treated with peg-Arg I, peg-BSA or PBS. (G) Proliferation of CFSE-labeled T-cells co-cultured with splenic peg-Arg I-induced MDSC (1:1/2 ratio) after addition of NN (200 μM), L-NMMA (500 μM) or L-Arg (2 mM). (H) 1×106 3LL cells were injected s.c. into mice (n=5), followed by peg-Arg I, peg-BSA or PBS injections i.p. (1 mg) every 3 days. (I) CD11b+ Gr-1+ within spleens of peg-Arg I, peg-BSA or PBS-treated 3LL-bearing mice (17 days). Results are expressed as mean +/- SEM from 3 experiments. Non-statistical significant differences (ns): p > 0.05;** p<0.01; *** p < 0.001.