[PMC free article] [PubMed] [Google Scholar] 25. a proinflammatory intestinal Th1/Th2 effector phenotype. These studies demonstrate Treg dysfunction in a spontaneous model of CD-like ileitis. conditions.8,9 Thus, it remains unclear whether Treg dysfunction contributes to the pathogenesis of Crohns disease. Few Anisole Methoxybenzene mechanistic studies have been performed to assess the function of Tregs function of Tregs during spontaneous CD-like ileitis in SAMP mice remains unclear. In this report, we used anti-CD25 antibodies (Ab), a well-established method to deplete natural Tregs depletion of SAMP Tregs significantly increased the severity of spontaneous ileitis; transfer of Treg-depleted SAMP MLN cells significantly increased the severity of adoptively transferred SAMP colitis, 2) a second transfer of non-depleted CD4+CD25+ cells isolated from AKR control mice was able to ameliorate the adoptively transferred SAMP colitis; transfer of non-depleted cells from SAMP mice failed to do so, 3) CD25?Foxp3+ cells, which were significantly expanded in SAMP mice following Treg depletion, do not possess a regulatory function and appear to have a colitogenic phenotype. To our knowledge, these observations provide the first direct evidence suggesting that Tregs are dysfunctional in a spontaneous model of CD-like intestinal inflammation. RESULTS Anti-CD25 Ab treatment depletes CD25+ cells, but not Foxp3+cells, in SAMP mice We first tested the extent of Treg depletion after six weeks of anti-CD25 treatment. In the MLN of SAMP and AKR mice, 99.5% of CD25+ cells were eliminated following anti-CD25 Ab treatment using either Clone PC61 (Figure 1A) or Clone 3C7, a noncompeting anti-CD25 mAb (Supplemental Figure 1). In the spleens, CD25+ cells were also completely eliminated (99.9%, data not shown). In MLN cells of untreated SAMP mice, the proportion of CD4+Foxp3+ cells was significantly higher than in untreated AKR mice (SAMP: 12.50.7% vs. AKR: 9.80.3%, and have lost their regulatory properties. Open in a separate window Figure 5 SAMP CD4+CD25+ cells fail to prevent the development of adoptively transfer-induced colitisCD4+ cells were transferred from anti-CD25 treated SAMP mice into SCID mice (6 wks, n=10) resulting in severe colitis. Six weeks later, 5 colitic SCID recipients per group received a second adoptive transfer of MLN CD4+CD25+ cells (2105) from untreated SAMP or AKR control mice (12 wks, n=3/group). The percentage of FoxP3+ cells was 90.8% and 88.3% in SAMP and AKR mice respectively. (A) Time course of body weight changes after transfer showed significant weight gain in SCID mice treated with AKR versus SAMP CD4+CD25+ cells. (B) Survival analysis of different experimental groups showed 0% survival in SAMP CD4+CD25+ treated SCID mice versus 60% in AKR CD4+CD25+ treated SCID mice. (C) Elevated levels Anisole Methoxybenzene of secreted IL-10 and TGF- measured from 3-day cultures of SAMP (n=4 mice) or AKR (n=6 mice) MLN or spleen CD4+CD25+ T cells (2105 cells/well) stimulated with immobilized anti-CD3 and soluble anti-CD28. (D) TNF-, IFN-, IL-3, IL-4, IL-5, and IL-6 measured from 3-day cultures were elevated in SAMP (n=4 mice) compared to AKR (n=6 mice) MLN or spleen CD4+CD25+ T cells (2105 cells/well) stimulated with immobilized anti-CD3 and soluble anti-CD28. Data are expressed as the mean SEM (*and generated a 10.3% Foxp3+ cells. By comparison, isolation of SAMP CD4+CD25? cells by MACS included only 1 1.4% Foxp3+ cells (Supplemental Figure 3A). Next, we transferred CD4+CD25? enriched cells (5105) into SCID mice to test the function of this cell population or methods significantly lost weight compared to SCID mice that received non-depleted SAMP CD4+ cells (Supplemental Figure 3B). There was no significant difference in body weight between SCID mice transferred with CD25? enriched cells via either method, despite a significant difference in the number of Foxp3+ cells. Rectal prolapse was observed in 60.0% of SCID mice that received CD4+CD25? enriched cells from either or techniques. Histological analysis revealed that SCID mice receiving CD25? enriched cells via either method had significantly more severe colitis, compared to mice receiving SAMP MLN cells treated with control Ab (total inflammatory scores: MACS=14.000.7303 vs. control Ab=7.2502.403, observations regarding Treg function during CD. First, depletion of natural Tregs in SAMP mice by anti-CD25 Ab treatment Anisole Methoxybenzene increases the severity of ileitis and Rabbit Polyclonal to Cytochrome P450 2A6 adoptively transferred SCID colitis. This observation supports a key role for Tregs in the development of spontaneous intestinal inflammation using an model that closely resembles CD. Second, a subsequent transfer of non-depleted CD4+CD25+ cells from AKR Anisole Methoxybenzene control mice is able to ameliorate the induced SCID colitis, whereas non-depleted CD4+CD25+ cells from SAMP mice failed to do so. These results suggest that SAMP Tregs are functionally abnormal and emphasize.