Immune Tolerance to Self-Major Histocompatability Complex Class II Antigens after Bone Marrow Transplantation: Role of Regulatory T Cells

Suppression of adoptive transfer of syngeneic GVHD. Splenic T cells were harvested from animals after resolution of syngeneic GVHD or from normal animals after intravenous challenge with 60 × 10⁶ effector T cells. Regulatory population was depleted of CD4⁺, CD8⁺, or CD25⁺ cells with immunomagnetic beads. Graded numbers of regulatory cells were adoptively transferred into secondary recipients (n = 6-8) along with 30 × 10⁶ effector T cells harvested from animals with acute syngeneic GVHD (7-10 days after initial onset). Recipients were examined daily for 6 weeks for development of syngeneic GVHD. Presence or absence of syngeneic GVHD was confirmed by biopsy.

Suppression of in vitro cytokine response of autoreactive effector cells to CLIP. Splenic T cells were harvested from normal animals after intravenous challenge with 60 × 10⁶ effector T cells. Regulatory population was depleted of CD4⁺, CD8⁺, or CD25⁺ cells with immunomagnetic beads. Regulatory T cells (1 × 10⁵) were irradiated (1250R) and incubated with equal number of autoreactive T cells harvested from animals with syngeneic GVHD. Cells were stimulated with syngeneic APCs (1 × 10⁴) loaded with CLIP. After 48 hours of incubation, cells were harvested and assessed for levels of IL-2 and IFNγ mRNA transcripts by quantitative PCR. IFNγ and IL-2 mRNA transcripts were not detected in control cultures of irradiated regulatory cells cultured with peptide-loaded APCs. Data were standardized against mRNA transcript levels for housekeeping gene, GAPDH.

Ex vivo analysis of cytokine mRNA transcripts in CLIP-reactive CD4⁺CD25⁺ T-cell subsets. N-CLIP- and CLIP-C-reactive CD4⁺CD25⁺ T cells were isolated flow cytometrically from spleens of normal animals, animals with syngeneic GVHD, and animals after resolution of disease. Cells were assessed for cytokine (IL-2, IL-4, IL-10, and IFNγ) mRNA transcripts by quantitative PCR. Data were standardized against mRNA transcript levels for housekeeping gene, GAPDH. ND = Not detected.

Foxp3 Expression in CLIP-reactive CD4⁺CD25⁺ T-cell subsets. N-CLIP- and CLIP-C-reactive CD4⁺CD25⁺ T cells were isolated flow cytometrically from normal animals, animals with syngeneic GVHD, and animals after resolution of disease. Foxp3 mRNA transcript levels were assessed by quantitative PCR. Data were standardized against GAPD mRNA transcript levels.

Regulation of in vitro cytokine tesponse by N-CLIP- and CLIP-C-reactive T-cell subsets. N-CLIP- and CLIP-C-reactive T cells were isolated flow cytometrically from animals after resolution of syngeneic GVHD. In addition, CLIP-reactive T-cell subsets were depleted of cells expressing CD4, CD8, or CD25 with immunomagnetic beads. Cells (1 × 10⁵) were extensively washed and assessed for their ability to suppress in vitro response of syngeneic GVHD effector lymphocytes (1 × 10⁵) stimulated with CLIP presented by syngeneic APCs (1 × 10⁴). After 48 hours of culture, cells were harvested assessed for levels of IL-2 and IFNγ mRNA transcripts by quantitative PCR. IFNγ and IL-2 mRNA transcripts were not detected in control cultures of irradiated regulatory cells and peptide-loaded APCs. Data were standardized against mRNA transcript levels for housekeeping gene, GAPDH.

Regulatory activity after peptide immunization. Normal Lewis rats were immunized intradermally with syngeneic APCs loaded with either N-CLIP or CLIP-C. Fourteen days later, splenic T cells (1 × 10⁵) were harvested and cocultured with equal number of syngeneic GVHD effector T cells. Spleen cells were stimulated with syngeneic APCs (1 × 10⁴) loaded with CLIP. After 48 hours of culture, cells were harvested assessed for levels of IL-2 and IFNγ mRNA transcripts by quantitative PCR. Data were standardized against mRNA transcript levels for housekeeping gene, GAPDH.

Response of CD4⁺CD25⁺ T cells after immunization with N-CLIP and CLIP-C peptides. Normal Lewis rats were immunized intradermally with N-CLIP and CLIP-C peptides presented by syngeneic APCs. Fourteen days later, CD4⁺CD25⁺ Tcells (1 × 10⁵) were stimulated in vitro with syngeneic APCs (1 × 10⁴) loaded with either N-CLIP or CLIP-C peptide variant. After 48 hours of culture, the cells were harvested assessed for cytokine and Foxp3 mRNA transcript levels by quantitative PCR. Data were standardized against mRNA transcript levels for housekeeping gene, GAPDH. These results are representative of 3 separate experiments.