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. 2017 Aug 22;8:994. doi: 10.3389/fimmu.2017.00994

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Copyright © 2017 Hall, Robinson, Plain, Verma, Tran, Nomura, Carter, Boyd and Hodgkinson.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Comparison of proliferation of CD4⁺, CD4⁺CD25⁻, and CD4⁺CD25⁺ T cells from tolerant hosts in mixed lymphocyte culture (MLC) to self-DA, specific-donor Piebald Virol Glaxo rat strain (PVG), and to third-party Lewis. Proliferation of CD4⁺, CD4⁺CD25⁻, and CD4⁺CD25⁺ T cells subjected to MLC with either self (DA), specific-donor (PVG), or third party (Lewis) at day 4, assessed by ³H thymidine incorporation and expressed as cpm. (A) Proliferation of cell subsets to donor antigens in MLC. CD4⁺ T cells from tolerant hosts (top left panel) had a similar response to specific-donor PVG () and third-party Lewis (◼) that was much greater than to self-DA (◻). Two other experiments had similar results. For CD4⁺CD25⁻ T cells from tolerant hosts (top right panel), the response to specific-donor PVG was less than to third-party Lewis. This response of CD4⁺CD25⁻ T cells was not greater than that of CD4⁺ T cells. CD4⁺CD25⁺ T cells from DA rats tolerant to PVG graft did not respond to specific-donor PVG, and this response was not greater than to self-DA (top right panel). CD4⁺CD25⁺ T cells from tolerant hosts had significantly higher proliferation to third-party Lewis compared to specific-donor PVG or self-DA. Bottom row panel shows 4 of the 8 replicate experiments that confirmed CD4⁺CD25⁺ T cells from tolerant hosts did not respond or had low response to specific-donor strain PVG compared to their response to third-party Lewis. Significant differences in responses are marked as * if p < 0.05; ** if p < 0.01, and *** if p < 0.001. (B) Limiting dilution assay of cells from tolerant hosts in MLC. The response of unfractionated cells (top panel) and CD4⁺ T cells (middle panel) was similar to specific-donor PVG () and third party Lewis (●) at all points, and was greater than that to self-DA (○). CD4⁺CD25⁻ T cells from tolerant hosts (bottom panel), in contrast to CD4⁺ T cells (middle panel), had a marked increased response to third-party Lewis but not to specific-donor PVG. Significant at three dilutions. Collectively, these findings suggest that CD4⁺CD25⁺ T cells from tolerant animals paradoxically did not suppress the response of CD4⁺CD25⁻ T cells from tolerant hosts to specific-donor alloantigen, but retain the capacity to suppress responses to self-DA cells and third-party Lewis alloantigen. Symbols for significant differences are: ⋄ PVG vs DA; ☆ Lewis vs DA; ★ Lew vs PVG; 1 symbol if p < 0.05; 2 symbols if p < 0.01, and 3 symbols if p < 0.001.

Inline graphic — Comparison of proliferation of CD4⁺, CD4⁺CD25⁻, and CD4⁺CD25⁺ T cells from tolerant hosts in mixed lymphocyte culture (MLC) to self-DA, specific-donor Piebald Virol Glaxo rat strain (PVG), and to third-party Lewis. Proliferation of CD4⁺, CD4⁺CD25⁻, and CD4⁺CD25⁺ T cells subjected to MLC with either self (DA), specific-donor (PVG), or third party (Lewis) at day 4, assessed by ³H thymidine incorporation and expressed as cpm. (A) Proliferation of cell subsets to donor antigens in MLC. CD4⁺ T cells from tolerant hosts (top left panel) had a similar response to specific-donor PVG () and third-party Lewis (◼) that was much greater than to self-DA (◻). Two other experiments had similar results. For CD4⁺CD25⁻ T cells from tolerant hosts (top right panel), the response to specific-donor PVG was less than to third-party Lewis. This response of CD4⁺CD25⁻ T cells was not greater than that of CD4⁺ T cells. CD4⁺CD25⁺ T cells from DA rats tolerant to PVG graft did not respond to specific-donor PVG, and this response was not greater than to self-DA (top right panel). CD4⁺CD25⁺ T cells from tolerant hosts had significantly higher proliferation to third-party Lewis compared to specific-donor PVG or self-DA. Bottom row panel shows 4 of the 8 replicate experiments that confirmed CD4⁺CD25⁺ T cells from tolerant hosts did not respond or had low response to specific-donor strain PVG compared to their response to third-party Lewis. Significant differences in responses are marked as * if p < 0.05; ** if p < 0.01, and *** if p < 0.001. (B) Limiting dilution assay of cells from tolerant hosts in MLC. The response of unfractionated cells (top panel) and CD4⁺ T cells (middle panel) was similar to specific-donor PVG () and third party Lewis (●) at all points, and was greater than that to self-DA (○). CD4⁺CD25⁻ T cells from tolerant hosts (bottom panel), in contrast to CD4⁺ T cells (middle panel), had a marked increased response to third-party Lewis but not to specific-donor PVG. Significant at three dilutions. Collectively, these findings suggest that CD4⁺CD25⁺ T cells from tolerant animals paradoxically did not suppress the response of CD4⁺CD25⁻ T cells from tolerant hosts to specific-donor alloantigen, but retain the capacity to suppress responses to self-DA cells and third-party Lewis alloantigen. Symbols for significant differences are: ⋄ PVG vs DA; ☆ Lewis vs DA; ★ Lew vs PVG; 1 symbol if p < 0.05; 2 symbols if p < 0.01, and 3 symbols if p < 0.001.