Fig 2: 1a,25(OH)2D3 decreased expression of multiple checkpoint receptors but increased CD28 expression. (A, B) Linear regression analysis between 1a,25(OH)2D3 level and immune checkpoint receptors (PD-1+, Tim-3+, TIGIT+, and CTLA-4+) and costimulatory molecule CD28+ on CD8+, V?9Vd2+ (Vd2+) T cells derived from PBMCs of patients with advanced lung cancer. n=42 for patients with advanced lung cancer (correlation between CTLA-4+ expression and 1a,25(OH)2D3 level, n=23). (C) Overview of study design. Patients with NSCLC received one course of intravenous docetaxel (75 mg/m2) followed with rocaltrol at a dose of 0.5–2.0 µg/day for a total of 3 weeks. (D) Graphs showed the 1a,25(OH)2D3 levels in sera of patients with NSCLC, before and after therapy with rocaltrol (0.5–2.0 µg/day) at the indicated time points. n=53. (E–P), Comparison of PD-1+, Tim-3+, TIGIT+, CTLA-4+, and CD28+ levels on CD8+, V?9Vd2+ T cells, in the peripheral blood of patients, before (pre) and after (post) treatment with rocaltrol (0.5–2.0 µg/day for a total of 3 weeks). n=52–53 (frequency of CTLA-4+ showing CD8+, V?9Vd2+ T cells, n=13). Pearson’s correlations (A, B); unpaired Student’s t-test (D); paired Student’s t-test (F–H); Wilcoxon matched-pairs signed-rank test (E, I–P). Significance was set to p<0.05 and represented as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. n.s., not significant; NSCLC, non-small cell lung cancer; PBMC, peripheral blood mononuclear cell; PD-1, programmed cell death-1; TIGIT, T-cell immunoreceptor with Ig and ITIM domains; Tim-3, T-cell immunoglobulin and mucin-domain containing-3.

Fig 3: VDR is a transcription factor for PD-1, Tim3 and Tigit genes. (A–D) V?9Vd2+ T cells were treated with vitamin D (VD3, 25(OH)D3, 1a,25(OH)2D3) in fresh medium at the indicated concentration for 48 hours. Percentages of PD-1+ (A), Tim-3+ (B), TIGIT+ (C), and CD28+ (D) on V?9Vd2+ T cells are shown. n=3 healthy donors. (E) Protein expression of VDR and PD-1 in vehicle-treated or 1a,25(OH)2D3 (50 nM)-treated human healthy CD8+ and V?9Vd2+ T cells for 48 hours. (F) Confocal imaging of VDR (green) and nucleus (blue) in V?9Vd2+ T cells after treatment with vehicle or 1a,25(OH)2D3 (50 nM) for 12 hours. (G) Cell-surface expression of PD-1+, Tim-3+, TIGIT+, and CD28+ on CD8+ T cells which were selected from the CD8+ T cells of VDR knockout (CRISPR-Cas9-VDR) or control vector (CRISPR-Cas9-NC). n=5–7 healthy donors. (H, I) Effects of VDR overexpression on level of PD-1+, Tim-3+, TIGIT+, and CD28+ on activated CD8+ T cells. Human CD8+ T cells were transduced by PTSB–CMV–GFP–VDR or vector control (PTSB–CMV–GFP–NC) using a lentivirus followed by 1a,25(OH)2D3 (50 nM) treatment two times at 1-day intervals. Statistical analysis for the decline ratio of PD-1+, Tim-3+, and TIGIT+ and raise ratio of CD28+ levels on CD8+ T cells are shown. n=4 healthy donors. (J, K) Graph shows ChIP-quantitative real-time PCR analysis of VDR at the promoter of PD-1, Tim-3, Tigit, and Cd28. The relative enrichment of VDR binding to the promoter of PD-1, Tim-3, Tigit, and Cd28 in vehicle or 1a,25(OH)2D3 (50 nM) pretreated CD8+ T cells for 12 hours is shown (pre site=predicted binding site; CT-1, CT-2=non-binding sites as control). VDR levels were normalized to the input. n=3 healthy donors. Data represent mean±SD, unpaired Student’s t-test (G, I, J). Significance was set to p<0.05 and represented as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001, n.s., not significant; PD-1, programmed cell death-1; TIGIT, T-cell immunoreceptor with Ig and ITIM domains; Tim-3, T-cell immunoglobulin and mucin-domain containing-3; VDR, vitamin D receptor.

Fig 4: In vitro IL-27 stimulation increased expression of CD69 and T-bet in CD4 and CD8 T cellsPBMCs from PLWH (HIV+, n = 17) and healthy controls (HC, n = 17) were cultured in the absence (opened symbols) or presence (closed symbols) of rhIL-27 (100 ng/mL) overnight. Flow cytometry analysis was performed for the expression of: (A) CD69; (B) T-bet, GATA3, and Foxp3+CD25+; (C) TIGIT, Tim3 and LAG3 in CD4 and CD8 T cells. Expressions of the markers are expressed as frequencies of the CD4 and CD8 T cells. The graphs are represented by box and whisker showing the median value with first and third quartiles in the box, with whiskers extending to the minimum and maximum values. Comparisons between the media and IL-27 stimulation were performed by non-parametric Wilcoxon test. Comparisons between the groups were performed by non-parametric Mann–Whitney test. p value <0.05 was considered significant.

Fig 5: Enhanced TIGIT+HIVGag-specific CD8 T cell function is driven by IL-27 dependent upregulation of T-betPBMCs from PLWH (HIV+, n = 9) were cultured in the absence (red opened symbols) or presence (red closed symbols) of rhIL-27 (100 ng/mL) overnight follow by stimulation with HIVGag peptide pool. DMSO was used as control. PBMCs from and healthy controls (HC, n = 9) were cultured in the absence (black opened symbols) or presence (black closed symbols) of rhIL-27 (100 ng/mL) overnight follow by stimulation with were stimulated with CEF (CMV, EBV and Influenza pool of peptides), and DMSO was used as control. IL-27 upregulation of T-bet and CD69 was measured by flow cytometry. (A) Gating strategy for TIGIT+and TIGIT-virus-specific T cells secreting IFN?+TNFa+.(B) Expression of CD69+T-bet+ in cytokine secreting CD8 T cells. TIGIT+CD69+T-bet+ or TIGIT- CD69+T-bet+ cytokine secreting T cells is expressed as frequency of total CD8 T cells. The graph is represented by box and whisker showing the median value with first and third quartiles in the box, with whiskers extending to the minimum and maximum values. Comparisons between culture conditions were performed using non-parametric Wilcoxon test. p value <0.05 was considered significant.