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. Author manuscript; available in PMC: 2013 Feb 15.

Published in final edited form as: Transplantation. 2012 Feb 15;93(3):272–282. doi: 10.1097/TP.0b013e31823ffd39

The islet grafts, D-LN, spleen and blood were collected from the recipients of islet alone, islet/DC or islet/MDSC on post operative day (POD) 3, 10, or >90 (LT) for immunohistochemical and flow analyses. (A) Cotransplantation with MDSC reduces CD8⁺ T cells. Left panels: The number of CD4⁺ and CD8⁺ T cells in islet grafts was calculated based on flow analysis on POD 3, 10 and >90, and expressed as mean/graft±1SD. Right panels: Sections of islet allograft on POD 10 were stained with anti-CD4 (red) and anti-CD8 (green) mAb. (B and C) Cotransplantation with MDSC enhances Treg cells. (B) Cotransplantation with MDSC enhances Foxp3⁺ Treg cells. Isolated cells (POD 10) from indicated compartments were stained for CD4, CD25 and Foxp3, and analyzed by flow cytometry gated on CD4⁺ cells. The number is percentage of positive cells. (C) left panels: The frequencies of Foxp3⁺ cells in islet grafts and D-LN kinetically analyzed by flow cytometry, and expressed as mean±1SD. Right panels: Sections of islet grafts and D-LN (POD 10) were stained for CD4 (red) and Foxp3 (green). (D) CD4⁺CD25⁺ cells contribute to T cell hyporesponsiveness. T cells isolated from recipient D-LN (POD 10) were stimulated by graded number of irradiated BALB/c splenocytes for 3 days. T cells from islet/MDSC recipients showed markedly lower proliferative response, which was restored to the levels similar to islet/DC group when the CD4⁺CD25⁺ T cells were deleted (Treg cell deleted) by magnetic bead (Militenyi Biotech) sorting. (E) Suppressive activity of CD4⁺CD25⁺ cells is Ag-specific. T cells isolated from D-LN or spleen in islet/MDSC recipients (POD 10) were separated (using the CD4⁺CD25⁺ cell isolation kit) into Treg (CD4⁺CD25⁺) and non-Treg (CD25^-) portions. The function of these cell populations were studied in a one-way MLR culture. To test suppressive activity, graded numbers of CD4⁺CD25⁺ cells (Treg) were added at beginning into an MLR culture, in which CD4⁺CD25^- T-cells (Non-Treg) were stimulated by irradiated BALB/c or C3H splenocytes for 5 days. T cell proliferation was determine by thymidine uptake. The data are representative of three separate experiments.

The islet grafts, D-LN, spleen and blood were collected from the recipients of islet alone, islet/DC or islet/MDSC on post operative day (POD) 3, 10, or >90 (LT) for immunohistochemical and flow analyses. (A) Cotransplantation with MDSC reduces CD8⁺ T cells. Left panels: The number of CD4⁺ and CD8⁺ T cells in islet grafts was calculated based on flow analysis on POD 3, 10 and >90, and expressed as mean/graft±1SD. Right panels: Sections of islet allograft on POD 10 were stained with anti-CD4 (red) and anti-CD8 (green) mAb. (B and C) Cotransplantation with MDSC enhances Treg cells. (B) Cotransplantation with MDSC enhances Foxp3⁺ Treg cells. Isolated cells (POD 10) from indicated compartments were stained for CD4, CD25 and Foxp3, and analyzed by flow cytometry gated on CD4⁺ cells. The number is percentage of positive cells. (C) left panels: The frequencies of Foxp3⁺ cells in islet grafts and D-LN kinetically analyzed by flow cytometry, and expressed as mean±1SD. Right panels: Sections of islet grafts and D-LN (POD 10) were stained for CD4 (red) and Foxp3 (green). (D) CD4⁺CD25⁺ cells contribute to T cell hyporesponsiveness. T cells isolated from recipient D-LN (POD 10) were stimulated by graded number of irradiated BALB/c splenocytes for 3 days. T cells from islet/MDSC recipients showed markedly lower proliferative response, which was restored to the levels similar to islet/DC group when the CD4⁺CD25⁺ T cells were deleted (Treg cell deleted) by magnetic bead (Militenyi Biotech) sorting. (E) Suppressive activity of CD4⁺CD25⁺ cells is Ag-specific. T cells isolated from D-LN or spleen in islet/MDSC recipients (POD 10) were separated (using the CD4⁺CD25⁺ cell isolation kit) into Treg (CD4⁺CD25⁺) and non-Treg (CD25^-) portions. The function of these cell populations were studied in a one-way MLR culture. To test suppressive activity, graded numbers of CD4⁺CD25⁺ cells (Treg) were added at beginning into an MLR culture, in which CD4⁺CD25^- T-cells (Non-Treg) were stimulated by irradiated BALB/c or C3H splenocytes for 5 days. T cell proliferation was determine by thymidine uptake. The data are representative of three separate experiments.

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