FIG 3.

CD11b⁺ GR1^low/int cells from CD11c/Nef Tg mice show T cell suppressive activity mediated by iNOS. Sorted purified CD11b⁺ Gr1⁺ cells (10⁵) from Tg or non-Tg mice were cocultured for 3 days with CFSE-labeled normal CD4⁺ or CD8⁺ T cells (10⁵) purified by cell sorting and stimulated with anti-CD3 and anti-CD28 to induce their proliferation. (A and B) Representative FACS profiles (A) and quantification (B) of CFSE dilution in non-Tg CD4⁺ and CD8⁺ T cells cocultured (+) or not (−) with purified CD11b⁺ Gr1^low/int cells from Tg or non-Tg mice. In panel B, data are expressed as a ratio of the percentage of stimulated T cells reaching 4 and 5 divisions (m4 + m5) in the presence (+) of CD11b⁺ Gr1^low/int cells over those cultured in their absence (−) (normalized at 1). Results from nine experiments are shown. (C) CFSE dilution of purified CD4⁺ T cells from AD10 TcR Tg mice stimulated with Pcc and cocultured (+) or not (−) with CD11b⁺ Gr1^low/int cells from non-Tg or Tg mice. Values are from three experiments and expressed as in panel B. (D) In vivo adoptive transfer. CFSE-labeled CD4⁺ T cells (3 × 10⁶ to 4 × 10⁶) from AD10 TcR Tg mice were transferred by i.v. tail vein inoculation into normal recipient B10BR mice; 48 h later, mice were injected i.v. with non-Tg or Tg purified CD11b⁺ Gr1^low/int cells (2 × 10⁶ to 3× 10⁶) and, within 1 h, inoculated s.c. with Pcc-loaded DC (3 × 10⁶). Values normalized at 1 in the absence of transfer are from 3 independent experiments (n = 5 Tg and 5 non-Tg). (E) Quantification of CFSE dilutions of non-Tg CD4⁺ (left) or CD8⁺ (right) T cells cocultured with Tg CD11b⁺ Gr1^low/int cells, as described for panel B, in the presence (+) or not (−) of l-NMMA. Values are from 3 independent experiments. (F and G) Representative FACS profile (F) and quantification (G) of CFSE dilution of purified non-Tg CD4⁺ and CD8⁺ T cells cocultured with purified CD11b⁺ Gr1^low/int cells from wild-type (+/+) or iNOS-deficient heterozygous (+/−) or homozygous (−/−) CD11c/Nef Tg mice. Values are expressed as in panel B. Data are from five experiments. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.