Cyclic AMP Response Element Modulator-α Suppresses PD-1 Expression and Promotes Effector CD4+ T Cells in Psoriasis

Abstract

Effector CD4⁺ T lymphocytes contribute to inflammation and tissue damage in psoriasis, but the underlying molecular mechanisms remain poorly understood. The transcription factor CREMα controls effector T cell function in people with systemic autoimmune diseases. The inhibitory surface coreceptor PD-1 plays a key role in the control of effector T cell function and its therapeutic inhibition in patients with cancer can cause psoriasis. In this study, we show that CD4⁺ T cells from patients with psoriasis and psoriatic arthritis exhibit increased production of IL-17 but decreased expression of IL-2 and PD-1. In genetically modified mice and Jurkat T cells CREMα expression was linked to low PD-1 levels. We demonstrate that CREMα is recruited to the proximal promoter of PDCD1 in which it trans-represses gene expression and corecruits DNMT3a-mediating DNA methylation. As keratinocytes limit inflammation by PD-1 ligand expression and, in this study, reported reduced expression of PD-1 on CD4⁺ T cells is linked to low IL-2 and high IL-17A production, our studies reveal a molecular pathway in T cells from people with psoriasis that can deserve clinical exploitation.

Figure 1:. CD4⁺ T cells from patients with psoriasis exhibit effector cytokine expression profile.

CD4⁺ T cells from patients with psoriasis (N=13), psoriatic arthritis (N=9) and healthy controls (N=13) were cultured for 5 days in the absence or presence of plate-bound CD3 and CD28 antibodies. A) mRNA and protein IL-2 levels; B) mRNA and protein expression of IL-17A (Kruskal Wallis and Dunne’s tests).

Figure 2:. CD4⁺ T cells from patients with psoriasis express increased levels of CREMα.

A) CREM expression (all isoforms) in CD4⁺ T cells from patients with psoriasis (N=13), psoriatic arthritis (N=9) and healthy controls (N=13) after 5 days of simulation with plate-bound CD3 and CD28 antibodies (Mann Whitney U test for unstimulated, NS vs stimulated, ST; Kruskal Wallis between groups). B) Expression of the CREMα isoform in CD4⁺ T cells from patients with psoriasis or psoriatic arthritis and control subjects (Kruskal Wallis and Dunne’s tests).

Figure 3:. PD-1 surface expression on CD4⁺ T cells from patients with psoriasis and control subjects.

A) Left panel: PD-1 expression in CD4⁺ T cells from healthy controls (N=10), psoriasis (N=9) and psoriatic arthritis (N=8) patients after ex vivo isolation and cultured for 5 days (resting: NS; stimulated: ST with plate-bound CD3/CD28). Right panel: Data from the same experiments, emphasizing the proportion of PD-1⁺ CD4⁺ T cells in patients with psoriasis or psoriatic arthritis and controls after 5d of stimulation. B) Left panel: Mean fluorescence intensity (MFI) of PD-1 surface expression on CD4⁺ T cells in stimulated cells from control subjects (N=10) and patients with psoriasis (N=9) or psoriatic arthritis (N=8) after ex vivo isolation and cultured for 5 days (resting: NS; stimulated: ST with plate-bound CD3/CD28). Right panel: Data from the same experiments, emphasizing PD-1 surface density in cells from psoriasis and psoriatic arthritis patients and control subjects (Kruskal Wallis and Dunne’s tests).

Figure 4:. PD-1 mRNA expression and protein shedding in CD4⁺ T cells from patients with psoriasis and control subjects.

A) PD-1 mRNA expression in CD4⁺ T cells from controls (N=13), patients with psoriasis (N=13) or psoriatic arthritis (N=9) (Left panel: PD-1 mRNA expression in resting (NS) and stimulated (for 5 days, ST) T cells; right panel: stimulated T cells from patients with psoriasis, psoriatic arthritis and control subjects; Kruskal Wallis and Dunne’s tests). B) PD-1 levels in cell culture supernatants (Mann Whitney U test).

Figure 5:. CREMα inversely correlates with PD-1 expression in genetically modified cells.

Wild-type, CREMα overexpressing (CREMα TG, left panel), and CREMα-deficient (CREMα Del., right panel) Jurkat T cells were cultured for 24h under resting conditions or in the presence of plate-bound CD3 and CD28 antibodies (N=5 independent experiments). A) PD-1 mRNA levels (logarithmic scale on Y axis). B) Proportion of PD-1 positive cells as assessed by flow cytometry (N=9 independent experiments). C) PD1 mRNA (N=3) and protein levels (N=4) in CD4+ T cells from C57/Bl6 mice overexpressing CREMα in T cells stimulated with plate-bound CD3 and CD28 antibodies for 24h.

Figure 6:. CREMα trans-regulates the PDCD1 proximal promoter.

A) PDCD1 is under the control of a 5’ proximal promoter region. The level of inter-species conservation (human vs mouse) is provided as % conservation over 200 base pairs (Vista Genome browser). The core promoter region an almost complete (7/8, 87.5%) cAMP response element (CRE) -942 base pairs upstream the transcriptional initiation sequence, which is indicated above the graph. B) ChIP with antibodies directed against HA in genetically modified Jurkat T cells overexpressing HA-tagged CREMα, CREMα recruitment to the aforementioned -942 CRE is shown (p values from t tests are shown) (N=4). C) PDCD1 promoter region-driven luciferase activity in wild-type (WT) and genetically modified Jurkat T cells (t tests) (N=6).

Figure 7:. CREMα regulates PD-1 expression through DNMT3a.

A) ChIP assays demonstrating the co-recruitment of DNMT3a and CREMα to the same region within the PDCD1 promoter in Jurkat T cells transfected with HA-CREMα or wild-type WT pLenti vectors (N=4). B) Jurkat T cells transfected with DNMT3a-specific or scrambled miRNAs and stimulated with plate-bound CD3 and CD28 antibodies (Kruskal Wallis and Dunne’s tests) (N=6).

Figure 8:. CREMα expression correlates with PDCD1 methylation.

A) PDCD1 promoter region CpG rich regions CR-B and CR-C. DNA methylation of these regions was tested using bisulfite pyrosequencing (CR-B including 7 CpGs, CR-C1 (3 CpGs), CR-C2 (7 CpGs), CR-C3 (6 CpGs). B) DNA methylation of regions CR-B (CpG 1–3) and CR-C1 (CpG 1–3) in CREMα-deficient Jurkat T cells (blue open triangles) wild-type control cells (Cas9; blue filled triangles) under resting conditions (NS) and in response to stimulation with plate-bound CD3 and CD28 antibodies (ST) (N=4). Only CpGs with variable methylation levels are displayed. pLenti: filled red circles, filled red circles: CREMα overexpressing T cells. C) PDCD1 DNA methylation in CD4⁺ T cells from patients with psoriasis (red, N=11)) and PsA (green, N=8)) and control cells (blue, N=8)). (Mann Whitney U tests; significance levels: **p<0.01; ***p<0.001).

Figure 9:. PD-1 negative T cells display effector T cell cytokine expression profile.

PD-1⁺ and PD-1⁻ Jurkat T cells using wild-type and CREMα-deficient cell. A) PD-1 mRNA expression in PD-1^- WT and CREMα-deficient Jurkat T cells (N=7). B) IL-2 mRNA expression in PD-1⁻ T cells (N=7) and C) mRNA levels of IL-17A (N=7) (Mann Whitney U tests).