CpG-Induced Myeloid CD11b⁺Gr-1⁺ Cells Efficiently Suppress T Cell–Mediated Immunoreactivity and Graft-Versus-Host Disease in a Murine Model of Allogeneic Cell Therapy

Accumulation of CD11b⁺/GR-1⁺ cells in spleens of CpG-treated mice. CpG or CpG+IFA was injected subcutaneously into C57 mice 6 or 10 days, respectively, before flow cytometry analysis. Injections of non-CpG or non-CpG+IFA or IFA alone served as controls and were given in parallel to their relevant opposite (ie, CpG vs non-CpG and CpG+IFA vs non-CpG+IFA). The results shown represent 1 experiment out of 5 experiments conducted.

Characterization of CD11b⁺ cells isolated from spleens of GpG+IFA-treated mice. Flow cytometry was carried out on a CD11b⁺ fraction isolated by magnetic beads from spleens of mice treated with CpG+IFA 10 days earlier. (A) Positive fraction of isolated CD11b cells. (B) Isolated CD11b cells gated for GR-1 cells. (C)-(H) Various phenotypic markers of the CD11b-isolated Gr-1 gated cells. The results shown represent 1 experiment out of 3 experiments conducted.

Distribution of T cells and myeloid cells after CpG treatment. Peripheral blood cells, splenocytes, and lymph node–derived C57 cells were analyzed by flow cytometry to detect T cells (CD3) and/or myeloid cells (CD11b and Gr-1) 10 days after inoculation of CpG+IFA (100 μg). Non-CpG+IFA inoculations served as controls. The results shown represent 1 experiment out of 2 experiments conducted.

In vitro mitogenic responses of splenocytes pretreated with CpG+IFA. Splenocytes derived from C57 mice treated with either CpG+IFA or non-CpG+IFA 10 days earlier were tested for various mitogenic responses (LPS, Con-A, PMA+Ca⁺⁺Iono, and anti-CD3). Results are presented as response percentage in relation to 100% response of naïve nontreated splenocytes. Response percentages were calculated from ³HTdR uptake in proliferation assays of 3 days of mitogenic stimulation and 4 days for the MLR of C57-derived T cells responding to BALB splenocytes. P = .049 and .016 for the comparison of non-CpG +IFA and CpG+IFA treatments in the MLR and in response to Con-A, respectively; P = .105, .12, and .235 for the comparison of non-CpG +IFA and CpG+IFA treatments in response to PMA+Ca⁺⁺Iono, anti-CD3, and LPS, respectively. The results represent the mean ± standard error of 3 separate experiments.

Cytokine secretion after CpG treatment. Splenocytes from C57 mice treated 6 or 10 days earlier with CpG (A) or CpG+IFA (B) were stimulated with Con-A for 48 h. The cytokine levels in the supernatants of the various cultures were measured. The results indicate P = .001, .007, and .36 for the comparison of CpG versus non-CpG for secretion of IL-10, IL-6, and IFN-γ, respectively, and P = .048, .013, and .076 for the comparison of CpG+IFA versus non-CpG+IFA for secretion of IL-10, IL-6, and IFN-γ, respectively.