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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 naïve unfractionated, CD4⁺ T cells and CD4⁺ T cell subsets in mixed lymphocyte culture (MLC) to self-DA, and allogeneic stimulators Piebald Virol Glaxo rat strain (PVG) and Lewis. Proliferation of unfractionated and CD4⁺, CD4⁺CD25⁻, and CD4⁺CD25⁺ T cells from naïve DA rats in MLC was assessed. The response to fully allogeneic PVG stimulator cells was compared to self-DA (A,B) and other fully allogeneic Lewis stimulator cells (B). Significant differences are shown as *p < 0.05, **p < 0.01, and ***p < 0.001. (A) Time course with unfractionated cells and CD4⁺ T cells showed peak proliferation to PVG (●) at day 4 and maintained at day 5, after which it waned. There was significant proliferation to DA (○), peaking at day 5. With CD4⁺CD25⁻ T cells, proliferation was greatest at day 4 and day 5; however, there was little difference in response to PVG and self-DA. Proliferation of CD4⁺CD25⁻ T cells was greater than unfractionated and enriched CD4⁺ T cells. CD4⁺CD25⁺ T cells proliferation was much less than that of CD4⁺ or CD4⁺CD25⁻ T cells. There was a detectable response to PVG at days 3 and 4 that waned by day 5 and 6. In subsequent experiments, proliferation of CD4⁺CD25⁺ T cells was assayed at day 4, and of CD4⁺ and CD4⁺CD25⁻ at day 4 or 5. (B) Comparison of proliferation in MLC of CD4⁺ T cells and subsets to self-DA, PVG, and Lewis stimulator cells. All populations had similar responses to PVG () and Lewis (◼) that were greater than proliferation to self-DA (◻) (***p < 0.001). (C) Comparison of responses of serially diluted naïve DA CD4⁺ T cells (△) and CD4⁺CD25⁻ T cells (▲) in MLC stimulated by a constant number of PVG cells. CD4⁺CD25⁻ T cells had significantly higher proliferation to PVG compared to CD4⁺ T cells indicating that removal of naïve CD4⁺CD25⁺ T cells significantly enhanced proliferation.

Inline graphic — Comparison of proliferation of naïve unfractionated, CD4⁺ T cells and CD4⁺ T cell subsets in mixed lymphocyte culture (MLC) to self-DA, and allogeneic stimulators Piebald Virol Glaxo rat strain (PVG) and Lewis. Proliferation of unfractionated and CD4⁺, CD4⁺CD25⁻, and CD4⁺CD25⁺ T cells from naïve DA rats in MLC was assessed. The response to fully allogeneic PVG stimulator cells was compared to self-DA (A,B) and other fully allogeneic Lewis stimulator cells (B). Significant differences are shown as *p < 0.05, **p < 0.01, and ***p < 0.001. (A) Time course with unfractionated cells and CD4⁺ T cells showed peak proliferation to PVG (●) at day 4 and maintained at day 5, after which it waned. There was significant proliferation to DA (○), peaking at day 5. With CD4⁺CD25⁻ T cells, proliferation was greatest at day 4 and day 5; however, there was little difference in response to PVG and self-DA. Proliferation of CD4⁺CD25⁻ T cells was greater than unfractionated and enriched CD4⁺ T cells. CD4⁺CD25⁺ T cells proliferation was much less than that of CD4⁺ or CD4⁺CD25⁻ T cells. There was a detectable response to PVG at days 3 and 4 that waned by day 5 and 6. In subsequent experiments, proliferation of CD4⁺CD25⁺ T cells was assayed at day 4, and of CD4⁺ and CD4⁺CD25⁻ at day 4 or 5. (B) Comparison of proliferation in MLC of CD4⁺ T cells and subsets to self-DA, PVG, and Lewis stimulator cells. All populations had similar responses to PVG () and Lewis (◼) that were greater than proliferation to self-DA (◻) (***p < 0.001). (C) Comparison of responses of serially diluted naïve DA CD4⁺ T cells (△) and CD4⁺CD25⁻ T cells (▲) in MLC stimulated by a constant number of PVG cells. CD4⁺CD25⁻ T cells had significantly higher proliferation to PVG compared to CD4⁺ T cells indicating that removal of naïve CD4⁺CD25⁺ T cells significantly enhanced proliferation.