Figure 3.

Human CRP enhances the suppressive capacity of mouse MDSCs. Mouse MDSCs were used in co-cultures with mouse T cells to assess their suppressive capacity and the effect of human CRP on MDSC mediated immune suppression. (A) Representative CFSE dilution histograms (normalized to mode as defined in the Materials and Methods) for CD4⁺ T cells cultured without stimulation and in isolation (bottom trace), cultured with stimulating anti-CD3 and anti-CD28 mAbs but in isolation (middle trace; horizontal gate used to demarcate proliferating cells is shown), or co-cultured with BM-MDSCs (10E:1T) in the presence of stimulating anti-CD3 and anti-CD28 mAbs (top trace). Note that in the presence of BM-MDSCs proliferation of T cells is suppressed. See Supplemental Figure 2 for additional details. (B) Pooled results (mean + SEM fraction diluted for n = 2–5 co-cultures each conducted in triplicate) from experiments performed with (i) co-cultures of BM-MDSCs plus CFSE-labeled T cells in the presence of stimulating anti-CD3 and anti-CD28 mAbs (solid bars; experiments done as in the top trace of (A) but with increasing E:T ratios) or with (ii) CFSE-labeled T cells cultured without BM-MDSCs but with stimulating anti-CD3 and anti-CD28 mAbs (cross-hatched bar; experiments done as in the middle trace of A). The asterisks indicate p < 0.0001 for Dunnett's tests compared to the proliferation of the CD3/CD28 stimulated T cells (cross-hatched bar). At E:T ratios of 1:1 or more BM-MDSCs significantly suppressed T cell proliferation. (C) Suppression assays were done using BM-MDSCs plus CD3/CD28 stimulated T cells (5E:1T ratios as in B), and T cell proliferation in the absence of CRP or in the presence of 1–50 μg/ml human CRP was measured. Addition of human CRP dose-dependently enhanced the ability of mouse BM-MDSCs to suppress T cell proliferation. The IC₅₀ and associated 95% confidence interval estimated by non-linear regression analysis is indicated (r² = 0.551, DF = 24 obtained from two separate co-cultures each done in triplicate). (D) Representative CFSE dilution histograms (normalized to mode) for mouse CD4⁺ T cells stimulated with anti-CD3 and anti-CD28 mAbs; from bottom to top the T cells received no further treatment, BM-MDSCs (1E:20T), enriched DCs (immunomagnetically positively-selected; 1E:20T ratio; see Materials and Methods and Supplemental Figure 1), or enriched MDSCs (immunomagnetically negatively-selected; 1E:20T ratio. See Materials and Methods and Supplemental Figure 1). Note that the proliferation of T cells is effectively suppressed by the enriched MDSCs (top trace), whereas it is effectively enhanced by the enriched DC fraction. (E) CFSE dilution histograms (normalized to mode) for mouse CD4⁺ T cells stimulated with anti-CD3 and anti-CD28 mAbs. From bottom to top the T cells received: no further treatment, enriched DCs, enriched DCs plus human CRP, enriched MDSCs, or enriched MDSCs plus CRP. When MDSCs and CRP were added they were added at 1E:20T and 100 μg/ml, respectively. Note that addition of CRP only enhanced the suppressive capacity of enriched MDSCs (top trace). (F) CD4⁺ T cell proliferation indices (normalized to CD3/CD28 stimulated T cells; cross-hatched bar. See the Materials and Methods) for the representative experiment shown in (E). The asterisk indicates significantly more suppression of T cell proliferation (p < 0.05 for Sidak's test) in the presence of enriched MDSCs plus CRP than in the presence of enriched MDSCs without CRP.