Fig 1.
Soluble factors promote MAdCAM-1 costimulated naïve CD4+ T cells to express TRM cell surface markers.(A) The frequency (%) of CD45RO- CD4+ T cells on day 0 (x-axis) and the frequency of CD69+/CD103+ cells on day 7 in multiple independent donors (y-axis) following costimulation of bulk CD4+ T cells with MAdCAM-1 (upper), VCAM-1 (middle) or CD28 mAb (lower) in the presence of RA and TGF-β (closed circles, n = 43). The solid black line and equation represent a linear regression of that includes only bulk cultures. The dashed line represents a quadratic polynomial regression that includes both bulk and purified CD45RO- cells. Linear correlation coefficients are provided for each panel. (B) CD69/CD103 coexpression from a representative donor following MAdCAM-1 costimulation of CD45RO-/CD45RO+ CD4+ T cell cultures reconstituted at different ratios. Left panels indicate the approximate ratio of CD45RO- to CD45RO+ cells on day 0. Right panels indicate the coexpression of CD69 (y-axis) and CD103 (x-axis) on day 7, with the frequency of double positive cells indicated. (C) Frequency of CD69+/CD103+ cells following MAdCAM-1, VCAM-1, and CD28 mAb costimulation of matched bulk (B) and naïve (N) CD4+ T cell cultures (n = 8) harvested at day 7. Y-axis indicates % double positive cells. (D) Coexpression of CD69 and CD103 following MAdCAM-1 costimulation of purified CD45RO- cells in the absence (UL panel) or presence of VCAM-1 (UR), MAdCAM-1 (LL) or CD28 mAb (LR) bulk culture supernatants, from a representative donor. (E) Treatment of purified CD45RO- cells as in panel D (n = 11) harvested at day 7. Error bars indicate standard deviation (*: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001).
Fig 2.
Naïve and memory CD4+ T cell proliferation in response to MAdCAM-1, VCAM-1, and CD28 mAb costimulation. (A) Flow cytometric dot plots of CD4+ T cell proliferation by dye dilution, from a representative donor, costimulated with MAdCAM-1, VCAM-1 or CD28 mAb without (upper) and with (lower) TGF-β. Reconstituted cultures included naïve cells (CD45RO-) labelled with CFSE (y-axis), and memory cells labelled with Far Red (x-axis). Division index (DI) for each population is indicated. (B and C) DI of naïve and memory cells from 4 donors (y-axis), costimulated as in panel A with and without TGF-β, as indicated (*: p < 0.05). (D) CD4+ T cells costimulated as in panel A. Average frequency of CD103, CD69 and CCR5 expression on naïve cells in each division cycle, in the presence or absence of TGF-β as indicated (n = 3). d0 represents non-divided cell populations with each subsequent dn + 1 representing an additional division. Comparisons between MAdCAM-1 + RA and MAdCAM-1 + RA + TGF-β are provided. Error bars indicate standard deviation (p < 0.0001).
Fig 3.
IL-6 promotes MAdCAM-1 mediated TRM differentiation of naïve CD4+ T cells.(A) Flow cytometric analysis comparing CD69+/CD103+ upregulation on naïve CD4+ T cells costimulated with MAdCAM-1, RA and TGF-β in the absence vs presence of exogenous IL-6, IL-10, TNF-α, and IFN-γ, as indicated. Treatment with day 4 culture supernatant included for comparison (n = 6). (B) Coexpression of CD69 and CD103 following MAdCAM-1 costimulation (with RA and TGF-β) of bulk CD4+ T cells in the absence or presence of tocilizumab (added on day 0 and day 4) as indicated (n = 6). (C) Coexpression of CD69 and CD103 following VCAM-1 or CD28 mAb costimulation in the absence or presence of exogenous IL-6 (added on day 0 and day 4). MAdCAM-1 costimulation included for comparison (n = 8). (D) Average CD69/CD103 coexpression on MAdCAM-1 costimulated cells treated with IL-6 on day 4 vs day 0 and day 4 (n = 12). Tocilizumab addition included as a control. (E) Flow cytometric time course (days 0-5) (x-axis) of α4β7, CCR9 and CCR5 expression on purified naïve CD4+ T cells costimulated with MAdCAM-1 in the absence or presence of IL-6, RA, or IL-6 + RA. Average % positive cells (n = 3) is reported (y-axis). Error bars indicate standard deviation (*: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001).
Fig 4.
JAK/STAT signaling and TRM CD4+ T cell formation.(A) Schematic of JAK/STAT signaling pathways. (B) Western blot analysis of lysates from naïve CD4+ T cell cultures costimulated with MAdCAM-1 with the addition of day 4 bulk CD4+ T cell supernatants (left) or exogenous IL-6 (right). Lysates harvested from 0-60 min post treatment were reacted with anti phospho -STAT1, -STAT2 and -STAT3 mAbs and total anti -STAT1 and -STAT3 mAbs. Actin mAb staining included as a control. (C) Flow cytometric analysis of naïve CD4+ T cells (n = 5) costimulated with MAdCAM-1 (+ RA and TGF-β) in the absence or presence of either IFN-β or IL-6, as indicated. Cytokines added on both day 0 and day 4. (D) Flow cytometric analysis of CD69/CD103 coexpression of bulk CD4+ T cells (n = 11) following MAdCAM-1, VCAM-1 or CD28 mAb costimulation (+ RA and TGF-β) in the absence or presence of ruxolitinib. Ruxolitinib added on day 0 or day 0 and 4, as indicated. (E) Flow cytometric analysis of CD69/CD103 coexpression of bulk CD4+ T cells (n = 6) following MAdCAM-1, VCAM-1 or CD28 mAb costimulation (+ RA and TGF-β) in the absence or presence of AZD1480. AZD1480 added on day 0 or day 0 and 4, as indicated. Error bars indicate standard deviation (*: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001).