![Figure 1. GM-CSF licensing is a prerequisite to develop suppressor activity in vitro and in vivo. Inhibition of T-cell proliferation was measured using a CFSE dilution assay. (A) Fresh murine BM cells or BM cultured for 1 to 3 days (d) in GM-CSF were added to anti-CD3/CD28–stimulated murine T cells at a ratio 10:1 (T cell/suppressor cell) for 4 days before determining CFSE dilution of T cells by FACS analysis. (B) T-cell suppressor assay where BM cells were used from 3-day cultures with 10 U/mL of GM-CSF or M-CSF, or 100 U/mL of Flt3L or G-CSF. Values correspond to the mean ± SD of 3 independent experiments. Statistics by 1-way analysis of variance (ANOVA) with multiple comparisons and Tukey posttest. *P < .05, ***P < .001. NS, not significant. (C) T-cell suppressor assay where BM cells were used from 3-day cultures with titrated doses GM-CSF (D) C57BL/6 mice were injected daily with GM-CSF or Flt3L with 2 μg/day for 10 days. Then CD11b+ cells from SP and BM were isolated and added to CFSE-labeled, CD3/CD28 antibody-stimulated T cells. Values correspond to the media of 5 independent experiments ± SD. Student t test indicated significance as *P < .05, ***P < .001 when compared with control mice (Ctrl). (E) Clinical autoimmune symptoms in the EAE model with or without preadministration of 4 × 106 L-Mono (day 4 of EAE, IV) in wild-type (WT) mice. Representative of 3 separate experiments. Two-way ANOVA with Bonferroni posttest. *P < .05, **P < .01. (F) L-Mono of WT, Nos2−/−, and Ifngr1−/− mice were tested for suppressive function by titrating them into cultures of CD3/CD28-stimulated T cells. Data represent means ± SD of 2 independent experiments. (G) The T-cell suppressor assay was performed by adding titrated amounts of resting L-Mono alone or L-Mono plus LPS and IFN-γ during the T-cell proliferation assay for 3 days before [3H]-Thymidine was added to the cultures overnight. Extrinsic LPS + IFN-γ activation further boosts suppressor activity of L-Mono by conversion into suppressor monocytes. Statistics by unpaired Student t test by comparing knock-out vs WT controls, ***P < .001.](https://ash.silverchair-cdn.com/ash/content_public/journal/bloodadvances/1/14/10.1182_bloodadvances.2017006858/3/advances006858f1.jpeg?Expires=1724944828&Signature=j9mR-7nMxRKZ9gyWx82y5Iva8thGho4R-W9CIf1vHK~w1NjqaJorC~tvSmizMfvD~zISxHdtKDr-RNobxlOcyg9BT9Q4I0hjiwrsbnxGqny4VUytiqrEri0EP8sawc6t6Yqynl2ebFGRUHDswRf39p9~U6IG8HBKXsbA9NTn~ueXxr6Q9m21GhLy4VK67KsbW1A9PJW1HgnXM4wG2bSiRITQ36dMdn29UrHqP0f0-UTFlR-pUOPpWfqcbkLvbyZHnDMrsAMdXfFu2j9oqtLp~knXvkpYbo4u-1bbk~d0gA7DjkbdYDyZ-prpegJbY8gu1YThF7wqPN9sqAqoqPXSFQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
GM-CSF licensing is a prerequisite to develop suppressor activity in vitro and in vivo. Inhibition of T-cell proliferation was measured using a CFSE dilution assay. (A) Fresh murine BM cells or BM cultured for 1 to 3 days (d) in GM-CSF were added to anti-CD3/CD28–stimulated murine T cells at a ratio 10:1 (T cell/suppressor cell) for 4 days before determining CFSE dilution of T cells by FACS analysis. (B) T-cell suppressor assay where BM cells were used from 3-day cultures with 10 U/mL of GM-CSF or M-CSF, or 100 U/mL of Flt3L or G-CSF. Values correspond to the mean ± SD of 3 independent experiments. Statistics by 1-way analysis of variance (ANOVA) with multiple comparisons and Tukey posttest. *P < .05, ***P < .001. NS, not significant. (C) T-cell suppressor assay where BM cells were used from 3-day cultures with titrated doses GM-CSF (D) C57BL/6 mice were injected daily with GM-CSF or Flt3L with 2 μg/day for 10 days. Then CD11b+ cells from SP and BM were isolated and added to CFSE-labeled, CD3/CD28 antibody-stimulated T cells. Values correspond to the media of 5 independent experiments ± SD. Student t test indicated significance as *P < .05, ***P < .001 when compared with control mice (Ctrl). (E) Clinical autoimmune symptoms in the EAE model with or without preadministration of 4 × 106 L-Mono (day 4 of EAE, IV) in wild-type (WT) mice. Representative of 3 separate experiments. Two-way ANOVA with Bonferroni posttest. *P < .05, **P < .01. (F) L-Mono of WT, Nos2−/−, and Ifngr1−/− mice were tested for suppressive function by titrating them into cultures of CD3/CD28-stimulated T cells. Data represent means ± SD of 2 independent experiments. (G) The T-cell suppressor assay was performed by adding titrated amounts of resting L-Mono alone or L-Mono plus LPS and IFN-γ during the T-cell proliferation assay for 3 days before [3H]-Thymidine was added to the cultures overnight. Extrinsic LPS + IFN-γ activation further boosts suppressor activity of L-Mono by conversion into suppressor monocytes. Statistics by unpaired Student t test by comparing knock-out vs WT controls, ***P < .001.
