MYXV impairs activation-induced proliferation of human effector T lymphocytes. To determine whether MYXV can impair the postactivation functions of T lymphocytes, the levels of cell proliferation of stimulated T lymphocytes were assessed using flow cytometry. T cells were preloaded with the tracking dye CTV at 37°C for 20 minutes and then either mock-treated or incubated with ± anti-CD3/CD28 microbeads as described in “Methods.” T lymphocytes were then incubated in a humidified chamber at 37°C, and 5% CO₂ for 72 hours or 96 hours to allow for the proliferation of stimulated T cells. At the indicated time points, cells were stained for CD3, CD4, CD8, CD25, and CD69. FCS-Express version 4 was used to analyze the characteristic subpopulations of dividing lymphocytes, and to determine the percentage of proliferation, the proliferation index (PI) and the division index (DI) (see supplemental Methods for a detailed description). (A) Histograms showing populations of mock-treated T lymphocytes (black outline) and MYXV-treated T lymphocytes (blue diagonal) in unstimulated conditions at 72 and 96 hours. The histograms reveal no CTV shift to the left, indicating low numbers in proliferation, and complete overlap when treated with MYXV, indicating no effect of MYXV on lymphocyte proliferation of unstimulated T cells. (B) In stimulated conditions, the mock-treated T lymphocytes (red) proliferate as evidence by leftward CTV stain shifting of the population. However, the MYXV-treated T cells (green diagonal) remains unchanged, indicating full suppression of T cell proliferation. This case is representative of a full responder donor. (C) Control treatments, in unstimulated conditions, showing lack of T-cell proliferation in a partial responder donor. (D) Under stimulated conditions with a partial responder donor, the mock-treated T lymphocytes (red) proliferate as evidence by leftward shifting of the CTV-stained population. However, the MYXV-treated T cells (green diagonal) exhibit an intermediate CTV shifted pattern, indicating partial suppression proliferation by MYXV. (E) To determine whether live virus is needed to suppress the proliferation of T cells, T lymphocytes were incubated with live vMyx-GFP, heat-inactivated vMyx-GFP, or UV-inactivated vMyx-GFP at an equivalent MOI = 10 and ± anti-CD3/CD28 beads. After 72 hours, the proliferation of CTV-tagged T cells was evaluated using flow cytometry. Data indicate that proliferation of T cells is suppressed only in the presence of live MYXV. In contrast, inactivated MYXV did not affect the activation-induced proliferation of T cells. (F) To investigate whether MYXV can affect Tregs, proliferation of nTregs was evaluated using flow cytometry. (i) Histograms of 1 representative donor, showing the proliferation patterns of CTV-tagged CD4⁺CD25⁺FoxP3⁺ (left panels) and CD4⁺CD25⁺Helios⁺ (right panels). (ii-iii) Summaries of the percentage of proliferation of CTV-tagged CD4⁺CD25⁺FoxP3⁺ and CD4⁺CD25⁺Helios⁺, respectively, of different donors (N = 4).