Figure 1.
During colitis, T cells accumulate in the inflamed regions of the colon.
Mice were treated with DSS for 6 days and sacrificed on day 7. The mice displayed signs of colitis including (A) an increased Disease Activity Index (DAI) and (B) shortened colons. C) Immunohistochemical staining of CD3+ cells in colons obtained from both control (left panes) and DSS-treated (right panes) mice. Top panes are 200x (bar: 5 µm) and bottom panes are 400x magnification (bar: 1 µm). Increased CD3+ cells are observed in inflamed colons. D) Naïve (CD4+ CD62L+ CD44-), central memory (TCM, CD4+ CD62L+ CD44+) and effector memory (TEM, CD4+ CD62L-CD44+) T cells were measured in the mLNs and colon mononuclear cell suspensions using flow cytometry. Results are expressed as mean + SEM, N = 4-6 mice per group. *** P < 0.001; **** P < 0.0001.
Figure 2.
Th17 cells are detected in the spleen after colitis resolution.
IFNγ and IL-17A producing CD4+ T cells were detected in the spleens and mLNs, 14 days after the start of DSS using intracellular cytokine staining. A) Percentages depicted are the populations of cytokine expressing CD4+ cells within the total CD4+ population. Bars indicate the mean, N = 8 mice per group. ** P < 0.01. B) Representative FACS contour dot plots for spleen and mLN showing intracellular staining of IL-17A and IFNγ within the gated CD4+ T cell population. Percentages within CD4+ T cell population are shown.
Figure 3.
Oral antigens are presented in the draining lymph nodes of both healthy and DSS treated mice.
A) OVA presentation in the gastrointestinal tract was visualized by the proliferation of adoptively transferred, CFSE labeled, OTII T cells. Representative FACS dot plots displaying OTII T cell proliferation within the mLNs of both healthy and DSS-treated mice after oral gavage of saline, “no antigen” or oral gavage with OVA, “ovalbumin”. The loss of CFSE intensity is an indication of dividing T cells. B) Percent proliferated OTII cells found within isolated mLNs. C) Percent proliferated OTII cells in the non-local, axillary lymph nodes. Results for (B) and (C) are expressed as mean + SEM, N = 4 mice per group, pooled from two independent experiments. * P < 0.05; ** P < 0.01.
Figure 4.
DSS and OVA treated mice have the same clinical phenotype as mice treated with DSS alone.
Mice were treated with both DSS and OVA for 6 days, and several clinical parameters were measured during disease progression and after sacrifice. A) Percent weight gain relative to starting weight was measured in each individual mouse for 14 days. B) DAI was calculated on day 6 and day 13 for all groups as described in the materials and methods. N = 10-15 mice before day 7, N = 5-10 after day 7, mice are pooled from two independent experiments. Mesenteric LNs C) and spleens D) were obtained from mice sacrificed on day 14. Cell suspensions were prepared and total cells counted in each organ. Samples (N = 5) were pooled from two independent experiments. E) SAA was measured in the serum from mice sacrificed on day 14. Samples (N = 5) were pooled from two independent experiments. F) Several colons collected from mice on day 14 were examined and scored for histological damage parameters as described in the materials and methods (N = 3, from a single experiment). G) Representative photos are shown for colonic sections from control, DSS-treated, OVA treated controls and OVA and DSS-treated mice. Bar is 5 µm. All graphical results are expressed as mean + SEM in the bar graphs. * P < 0.05; ** P < 0.01.
Figure 5.
DSS and OVA treated mice have aspecific T cell responses similar to mice treated with DSS alone.
Mice were either treated with DSS alone or with DSS and OVA. Spleens and mLNs were removed, stimulated and examined by flow cytometry. A) Percentages of CD4+ T cells were determined within cultures after stimulation with anti-CD3 for 48 hours. N = 4-9 mice per group. B) The ratios of the percentages of Foxp3- and Foxp3+ cells (Foxp3-/Foxp3+) were determined within the CD4+ T cell population. N = 4-9 mice per group. Using intracellular cytokine staining, percentages of IL-17A+ (C) and IFNγ+ cells (D) within the CD4+ cell population were determined after stimulation. N = 3 mice per group. All graphical results are expressed as mean + SEM in the bar graphs.
Figure 6.
OVA-directed, CD4+ Foxp3-T cells are detected only in mice treated with DSS and OVA.
Both mLN and spleen cell suspensions were prepared 14 days after the start of the DSS and OVA treatment for all groups. Cells were stimulated with OVA, and CD69 expression was measured using flow cytometry. A) Representative FACS dot plots of the CD69 expression in CD4+ Foxp3- (Tconv) and CD4+ Foxp3+ (Treg) T cells in the spleen. Percentages shown are the % CD69+ cells in the specific gated Tconv or Treg populations. B) The mean percentage CD69 expression was calculated for Tconv and Treg in the spleen cells of each group. C) Representative FACS plots of the CD69 expression in Tconv and Treg cells in the mLN. D) The mean percentage CD69 expression was calculated for Tconv and Treg cells in the mLN. Results are expressed as mean + SEM, N = 6-9 per group. *** P < 0.001; **** P < 0.0001.