Intestinal Lamina Propria Retaining CD4⁺CD25⁺ Regulatory T Cells Is A Suppressive Site of Intestinal Inflammation

Murine intestinal LP CD4⁺CD25⁺ T_REG cells as well as splenic CD4⁺CD25⁺ T_REG cells inhibit the development of colitis induced by adoptive transfer of CD4⁺CD45RB^high T cells into SCID mice. Seven SCID mice in each group were injected i.p. with the following T cell subpopulations: 1) splenic CD4⁺CD45RB^high T cells alone (3 × 10⁵ cells); 2) splenic CD4⁺CD45RB^high T cells (3 × 10⁵ cells) + splenic CD4⁺CD25⁺ T cells (1 × 10⁵ cells); or 3) splenic CD4⁺CD45RB^high T cells (3 × 10⁵ cells) + LP CD4⁺CD25⁺ T cells (1 × 10⁵ cells). A, Body weight during 7 wk after transfer. ∗, p < 0.05 compared with mice transferred with CD45RB^high T cells alone at 7 wk after transfer. B, Gross appearance of the colon, MLNs, and spleen from SCID mice transferred with splenic CD4⁺CD45RB^high T cells alone (upper), splenic CD4⁺CD45RB^high T cells + splenic CD4⁺CD25⁺ T cells (middle), or splenic CD4⁺CD45RB^high T cells + LP CD4⁺CD25⁺ T cells (lower) at 7 wk after transfer. C, Clinical score at 7 wk after transfer. ∗, p < 0.05 compared with mice transferred with CD45RB^high T cells alone. D, Histopathology of distal colon at 7 wk after transfer. Original magnification, ×40. E, Histological score at 7 wk after transfer. ∗, p < 0.05 compared with mice transferred with CD45RB^high T cells alone.

Cotransfer of intestinal LP CD4⁺CD25⁺ T_REG cells inhibits the expansion of LP CD4⁺ T cells and Th1 cytokine production in SCID mice transferred with CD4⁺CD45RB^high T cells. Transfer protocol is described in Fig. 2. A, Recovered LP CD4⁺ T cells at 7 wk after transfer. Data are indicated as the mean ± SD of seven mice in each group. ∗, p < 0.05 compared with mice transferred with CD45RB^high T cells alone. B, Cytokine production by LP CD4⁺ T cells. LP CD4⁺ T cells were stimulated with plate-coated anti-CD3 mAb and soluble anti-CD28 mAb for 72 h. Cytokines in the supernatants were measured by ELISA. Data are indicated as the mean ± SD of seven mice in each group. ∗, p < 0.05 compared with mice transferred with CD45RB^high T cells alone.

Splenic CD4⁺CD25⁺ T_REG cells suppress the development of colitis in LTα^−/− × Rag2^−/− mice transferred with CD45RB^high T cells. CD4⁺CD45RB^high T cells (3 × 10⁵ cells) from Ly5.2-C57BL/6 congenic mice were injected into Ly5.2 background LTα^+/+ × Rag2^−/− and LTα^−/− × Rag2^−/− mice with or without the cotransfer of 1 × 10⁵ splenic CD4⁺CD25⁺ T_REG cells derived from Ly5.1-C57BL/6 mice (n = 7 mice per each group). A, Disease activity index during 10 wk after transfer. ∗, p < 0.05, LTα^+/+ × Rag2^−/− mice vs LTα^−/− × Rag2^−/− mice, transferred with splenic CD4⁺CD45RB^high T cells and splenic CD4⁺CD25⁺ T_REG cells. B, The lack of MLNs in LTα^−/− × Rag2^−/− mice. The abdominal MLN area was dissected and examined for the presence or absence of MLNs in LTα^−/− × Rag2^−/− mice and LTα^+/+ × Rag2^−/− mice after adoptive transfer. C, Histopathology of distal colon at 10 wk after transfer. Original magnification, ×20 (top) and ×100 (bottom). D, Histological score at 10 wk after transfer. ∗, p < 0.05 compared with the paired LTα^+/+ × Rag2^−/− or LTα^−/− × Rag2^−/− mice transferred with splenic CD4⁺CD45RB^high T cells alone. NS, Not significant.

Splenic CD4⁺CD25⁺ T_REG cells suppress the production of Th1 cytokines in LTα^−/− × Rag2^−/− mice transferred with CD45RB^high T cells. CD4⁺CD45RB^high T cells (3 × 10⁵ cells) from Ly5.2-C57BL/6 congenic mice were injected into Ly5.2 background LTα^+/+ × Rag2^−/− and LTα^−/− × Rag2^−/− mice with or without the cotransfer of 1 × 10⁵ splenic CD4⁺CD25⁺ T_REG cells derived from Ly5.1-C57BL/6 mice (n = 7 mice per each group) as described in Fig. 4. Cytokine production by LP CD4⁺ T cells was measured by specific ELISA. LP CD4⁺ T cells were stimulated with plate-coated anti-CD3 mAb and soluble anti-CD28 mAb for 72 h. Cytokines in the supernatants were measured by ELISA. Data are indicated as the mean ± SD of seven mice in each group. ∗, p < 0.05 compared with the paired LTα^+/+ × Rag2^−/− or LTα^−/− × Rag2^−/− mice transferred with splenic CD4⁺CD45RB^high T cells alone. NS, Not significant.

Splenic CD4⁺CD25⁺ T_REG cells suppress the expansion of pathogenic LP CD4⁺ T cells in LTα^−/− × Rag2^−/− mice transferred with CD45RB^high T cells. CD4⁺CD45RB^high T cells (3 × 10⁵ cells) from Ly5.2-C57BL/6 congenic mice were injected into Ly5.2 background LTα^+/+ × Rag2^−/− and LTα^−/− × Rag2^−/− mice with or without the cotransfer of 1 × 10⁵ splenic CD4⁺CD25⁺ T_REG cells derived from Ly5.1-C57BL/6 mice (n = 7 mice per each group) as described in Fig. 4. A, Recovered LP CD4⁺ T cells at 10 wk after transfer. Data are indicated as the mean ± SD of seven mice in each group. ∗, p < 0.05 compared with the paired LTα^+/+ × Rag2^−/− or LTα^−/− × Rag2^−/− mice transferred with splenic CD4⁺CD45RB^high T cells alone. B, Distribution of CD11c⁺ dendritic cells (green) and CD4⁺ T cells (red) in the colon after adoptive transfer. Original magnification, ×100 (left) and ×400 (right).

Splenic CD4⁺CD25⁺ T_REG cells migrate into the colonic LP and are sustained in the LP in LTα^−/− × Rag2^−/− mice transferred with CD45RB^high T cells. A, The ratio of CD4⁺CD25⁺ T_REG (Ly5.1⁺) cells to total CD4⁺ T cells (Ly5.1⁺ + Ly5.2⁺) at 10 wk after transfer was analyzed by gating Ly5.1 or Ly5.2 on CD4⁺ cells. Results shown are from seven mice per group. NS, Not significant. B, Spleen (SP) and LP cells were collected and labeled for Ly5.1, Ly5.2, CD4, and intracellular Foxp3. Ly5.2⁺ and Ly5.1⁺ CD4⁺ cells were gated and analyzed for the presence of converted Ly5.2⁺CD4⁺Foxp3⁺ cells and Ly5.1⁺CD4⁺Foxp3⁺ cells, respectively. Number in upper right quadrant represents the percentage of inducible CD4⁺Foxp3⁺ cells per CD4⁺ cells.