Regulatory CD4+CD25+ cells reverse imbalances in the T cell pool of bone marrow transplanted TGepsilon26 mice leading to the prevention of colitis

Abstract

Background and aims: Erroneous thymic selection of developing T lymphocytes may be responsible for the expansion of self reactive T cells or may contribute to the absence of regulatory T cells important in controlling peripheral inflammatory processes. Colitis in bone marrow (BM) transplanted Tgepsilon26 mice is induced by abnormally activated T cells developing in an aberrant thymic microenvironment. We investigated the protective role of regulatory CD4+CD25+ T cells in this model.

Methods: BM from (C57BL/6 x CBA/J) F1 mice was transplanted into specific pathogen free Tgepsilon26 mice (BM-->Tgepsilon26). Transplanted mice received no cells (control), sorted CD4+CD25+, or CD4+CD25- cells from mesenteric lymph nodes (MLN) of normal mice. MLN cell subsets were analysed using membrane markers. Cytokine secretion of MLN cells was measured using intracellular cytokine staining and cytokine secretion in anti-CD3 stimulated cell cultures. Colitis was measured by histological scores.

Results: CD4+CD25+ cells were reduced in the MLNs of BM-->Tgepsilon26 mice. Transfer of regulatory CD4CD4+CD25+ but not of CD4+CD25- cells reduced the number of MLN CD4+ T cells in BM-->Tgepsilon26 recipients and increased the number of MLN CD8+ cells, thereby normalising the CD4+/CD8+ ratio. CD4+CD25+ but not CD4+CD25- cell transfer into BM-->Tgepsilon26 mice reduced the number of tumour necrosis factor alpha+ CD4+ cells and increased the secretion of transforming growth factor beta by MLN cells. Transfer of 3 x 10(5) CD4+CD25+ cells after BM transplantation into Tgepsilon26 mice prevented colitis whereas CD4+CD25- cells had no protective effect.

Conclusions: These results suggest that defective selection or induction of regulatory T cells in the abnormal thymus is responsible for the development of colitis in BM-->Tgepsilon26 mice. Transfer of CD4+CD25+ cells can control intestinal inflammation in BM-->Tgepsilon26 mice by normalising the number and function of the MLN T cell pool.

Figure 1

Flow cytometric analysis of expression of CD4⁺CD25⁺ in mesenteric lymph node (MLN) cells of Tgɛ26 mice transplanted with wild-type bone marrow (BM⇒Tgɛ26) (B) and of syngenic wild-type mice (A). Dot plots showing representative proportions of CD4⁺CD25⁺ MLN cells in Tgɛ26 mice 4–6 weeks after bone marrow (BM) transplantation and in syngenic wild-type (WT) mice.

Figure 2

Intracytoplasmic staining of tumour necrosis factor α (TNF-α) (A) and production of TNF-α (B) and interferon γ (IFN-γ) (C) measured by ELISA. (A) Summation of intracytoplasmic staining of TNF-α is shown on gated CD4⁺ mesenteric lymph node (MLN) cells from wild-type (WT) mice and from Tgɛ26 mice transplanted with wild-type bone marrow (BM⇒Tgɛ26) that received 3×10⁵ CD4⁺CD25⁺ cells, 1.5×10⁵ CD4⁺CD25⁺ cells, 3×10⁵ CD4⁺CD25⁻ cells, or no T cells, analysed by flow cytometry five hours after stimulation with immobilised anti-CD3 antibody. Mean (SEM) percentage of cytokine positive cells are shown. *p<0.05 versus BM⇒Tgɛ26 mice that received 1.5×10⁵ CD4⁺CD25⁺ cells, versus BM⇒Tgɛ26 mice that received 3×10⁵ CD4⁺CD25⁻ cells, and versus BM⇒Tgɛ26 mice. (B) TNF-α and (C) IFN-γ in supernatants of MLN cell cultures three days after stimulation with anti-CD3 antibody. Values represent means (SEM) in supernatants of MLN cell cultures from BM⇒Tgɛ26 mice that received 3×10⁵ CD4⁺CD25⁺ cells or 3×10⁵ CD4⁺CD25⁻ cells.

Figure 3

Weight loss. Body weight of Tgɛ26 mice transplanted with wild-type bone marrow (BM⇒Tgɛ26) that received 3×10⁵ CD4⁺CD25⁺ cells, 3×10⁵ CD4⁺CD25⁻ cells, or no cells (control group) was measured twice per week and was divided by starting body weight (on the day of bone marrow transplantation) to calculate the percentage of body weight at each time point. Body weights were plotted as mean (SEM).

Figure 4

Blinded histological scores of the colon of Tgɛ26 mice. Tgɛ26 recipients were transplanted with bone marrow from normal mice and one week later injected with 3×10⁵ CD4⁺CD25⁺ cells, 1.5×10⁵ CD4⁺CD25⁺ cells, or 3×10⁵ CD4⁺CD25⁻ cells or did not receive T cells. Mice were killed 5–6 weeks later. Inflammation was scored on a scale of 0–4 (see materials and methods) and results are expressed as mean (SEM). *p<0.05 versus all other groups.

Figure 5

Photomicrographs of the colons of Tgɛ26 mice five weeks after bone marrow (BM) transplant or after transplantation of BM plus transfer of different T cell subsets. Representative haematoxylin-eosin stained sections from the distal colon are shown (magnification 200×). (A) Normal colon from a non-transplanted specific pathogen free (SPF) Tgɛ26 mouse. (B) Severe colitis in a SPF BM transplanted Tgɛ26 mouse demonstrating crypt hyperplasia, decreased goblet cells, pronounced lamina propria lymphocytic infiltration, and crypt abscesses. (C) No signs of inflammation in a Tgɛ26 mouse that received BM plus 3×10⁵ CD4⁺CD25⁺ cells. (D) Active colitis in a BM transplanted Tgɛ26 mouse which was reconstituted with only 1.5×10⁵ CD4⁺CD25⁺ cells. (E) Severe colitis in a SPF Tgɛ26 mouse that was injected with BM plus 3×10⁵ CD4⁺CD25⁻ cells. Extensive lamina propria cellular infiltration, crypt hyperplasia, and decreased goblet cells are evident.

Figure 6

Cytokine production detected in supernatants of mesenteric lymph node (MLN) cell cultures three days after stimulation with anti-CD3 antibody for transforming growth factor β (TGF-β) (A) and anti-CD28 antibody for interleukin 10 (IL-10) (B). TGF-β and IL-10 were measured by ELISA. Values represent means (SEM) per CD4⁺ MLN cells in supernatants of MLN cell cultures from Tgɛ26 mice reconstituted with bone marrow (BM) plus 3×10⁵ CD4⁺CD25⁺ cells or BM plus 3×10⁵ CD4⁺CD25⁻ cells. *p<0.05 versus Tgɛ26 mice reconstituted with BM plus CD4⁺CD25⁻ cells.