The Vitamin D Analog, TX527, Promotes a Human CD4⁺CD25^highCD127^low Regulatory T Cell Profile and Induces a Migratory Signature Specific for Homing to Sites of Inflammation

TX527 reduces the proliferative capacity of CD4⁺ and CD8⁺ T cells. Purified human CD3⁺ T cells were activated with anti-CD3/anti-CD28 and repeatedly treated with vehicle (ctr) or TX527 (10⁻⁸ M) every second day as described before. On day 8, cells were harvested, CFSE-labeled, and seeded out again in the presence or absence of TX527 (10⁻⁸ M). The cultures were supplemented with IL-2 (12.5 ng/ml) on the usual 2-d intervals to maintain T cell growth. On day 10, T cells were harvested and stained with mAbs directed against CD4 and CD8 before flow cytometric analysis was performed. The proliferating cell fraction was identified by a decrease of CFSE signal. Dead cells were excluded from analysis using 7-AAD staining. Mean values ± SEM of the percentages of proliferating cell fractions from one representative experiment are shown. A total of three independent donors were tested. *p < 0.05; **p < 0.005 versus ctr.

Cytokine responses in CD4⁺ and CD8-enriched T cell fractions are differentially regulated by TX527. Human CD3⁺ T cells, activated by anti-CD3/anti-CD28 and treated with vehicle (ctr) or TX527 (10⁻⁸ M) as described earlier, were further separated into a CD4⁺ and a CD8-enriched T cell populations following the 10-d culture period. The relative mRNA levels of IFN-γ, IL-17, IL-4, and IL-10 in the different T cell fractions were analyzed by quantitative real-time RT-PCR. The values depicted are the mean ± SEM of one representative experiment out of two to four independently tested donors. *p < 0.05 versus ctr. ND, not detected.

TX527 modulates surface expression of T cell activation and Treg markers. Expression of activation and Treg markers by human T cells, activated by anti-CD3/anti-CD28 and cultured for 10 d in the presence of the vehicle (solid lines) or TX527 (dashed lines), was analyzed by flow cytometry. A, Histogram overlays, gated on CD4⁺ or CD8⁺ T cell subsets, represent surface expression of T cell activation markers CD69 and CD40L. B, The upper panel shows histogram overlays, gated on CD4⁺ T cells, depicting surface expression of Treg markers CD25 and CD127. The lower panel shows contour graphs, depicting coexpression of CD25^high and CD127^low on the CD4⁺ T cell population. The results are representative of three to eight independent replicates performed on different donors.

TX527 drives de novo conversion of naive T cells into functional Tregs. A, TX527-mediated induction of CD4⁺CD25^highCD127^low Tregs from nTreg-depleted CD3⁺ T cells. The upper panel depicts the composition of the starting T cell population as determined by FACS, either depleted (right panel) or not (left panel) for nTregs. The lower panel shows the percentage of end-stage CD4⁺CD25^highCD127^low Tregs induced following anti-CD3/anti-CD28 stimulation and treatment with TX527 or vehicle within the corresponding starting populations. B and C, The functional ability of TX527-induced Tregs to counteract activation and proliferation of autologous CD4⁺CD25^- T responder cells was analyzed. For this, CD4⁺CD25⁺ T cells, isolated from 10-d cultured TX527-treated (TX527 suppressors) or vehicle-treated T cells (ctr suppressors), were cocultured with Cell Proliferation Dye labeled-CD4⁺CD25⁻ responder T cells in the presence of accessory cells and soluble anti-CD3. Activation and proliferation of responder T cells were measured by surface CD69 expression (12 h) (B) and by Cell Proliferation Dye dilution (72 h) (C), respectively. Different responder-to-suppressor ratios were implemented (1:2, 1:1, and 1:0.25). To control for crowding, a condition with identical numbers of unlabeled CD4⁺CD25⁻ responders instead of suppressors was included. The upper panels (B, C) depict histogram plots of the above-mentioned parameters for responder T cells alone or cocultured with TX527 suppressors. The lower panels show the percent suppression of responder T cell activation and proliferation for all the experimental conditions tested (B, C). The data represent one experiment out of three with identical outcome. D, TX527 triggers the induction of IL-10 within the CD4⁺CD25^highCD127^low T cell population. Data shown illustrate relative IL-10 mRNA levels (mean ± SEM) of FACS-sorted CD4⁺CD25^highCD127^low TX527-treated (TX527-Treg) and vehicle-treated (ctr-Treg) cells. E, Expression of Treg-associated markers by human TX527-treated T cells. CD3⁺ T cells, activated by anti-CD3/anti-CD28 and cultured in the presence of the vehicle (solid lines) or TX527 (dashed lines), were analyzed by FACS for surface expression of OX40, CTLA-4, GITR, FOXP3, and ICOS. The data are shown by histogram overlays, gated on CD4⁺ T cells, of one representative experiment out of four independent donors tested.