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Proportion of CD4+CD49b+LAG-3+ Type 1 Regulatory T Cells in the Blood of Psoriasis Patients Inversely Correlates with Psoriasis Area and Severity Index
Laboratory for Investigative Dermatology, The Rockefeller University, New York, New York, USADivision of Dermatology, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York, USA
Psoriasis skin lesions are created through chronic T-cell activation and expansion of autoreactive, skin resident αβ T helper type 17 (Th17) cell clones (
), suggesting a defect in normal tolerance mechanisms. A previous study determined that although psoriasis patients have normal numbers of circulating regulatory T (Treg) cells (CD4+CD25+Foxp3+ T cells), psoriatic Treg cells were less effective at suppressing alloreactive T cells compared with Treg cells from healthy individuals (
Dysfunctional blood and target tissue CD4+CD25high regulatory T cells in psoriasis: mechanism underlying unrestrained pathogenic effector T cell proliferation.
). Tr1 cells are IL-10–producing T cells implicated as key regulators of peripheral immune tolerance, and there have been studies speculating the role of Tr1 cells in psoriasis.
reported that IL-10–producing regulatory B cells are impaired in psoriasis and inversely correlate with IL-17– and IFN-γ–producing T cells.
However, it has been challenging to identify psoriasis patients’ Tr1 cells by detecting IL-10 with flow cytometry intracellular staining, because Tr1 cells produce IL-10 only after in vitro re-stimulation (
High levels of interleukin 10 production in vivo are associated with tolerance in SCID patients transplanted with HLA mismatched hematopoietic stem cells.
have shown that surface expression of the markers CD49b and LAG-3 is sufficient to identify Tr1 cells in both murine and human T cells. Gagliani et al. performed genomic profiling and suppressive functional studies of FACS-sorted CD49b+ LAG-3+ T cells in mouse and human, which showed that CD4+CD49b+LAG-3+ T cells secrete large amounts of IL-10 with strong IL-10–dependent suppressive activity, whereas FoxP3 and IL-17 are expressed at low levels (
). Based on Gagliani et al.’s established functional studies, we tested if Tr1 cells, defined by surface markers of CD4+CD49b+LAG-3+, have clinical impacts on psoriasis patients.
We analyzed 12 human blood samples from patients with psoriasis and eight healthy human blood samples to compare Tr1 cell, Treg cell, and activated T cells between patients with psoriasis and healthy individuals (see Supplementary Materials and Methods online). We defined Tr1 cells as CD3+CD4+CD8–CD49b+LAG-3+ T cells, Treg cells as CD3+CD4+CD8–CD25+CD127– cells, and activated T cells as CD3+CD4+CD8–CD69+ cells. We performed flow cytometry analysis of whole blood and measured proportions of these CD3+CD4+CD8– T-cell subsets (Figure 1, and see Supplementary Figures S1 and S2 online). The study protocol was approved by the Rockefeller University institutional review board. Written informed consent was obtained from each patient or healthy participant before enrolment.
Figure 1Flow cytometric gating strategies. CD3+CD4+CD8– circulating T cells are subclassified into Tr1 cells (CD49b+LAG-3+ subset), Treg cells (CD25+CD127–subset), and activated T cells (CD69+ subset). Representative flow cytometry data from healthy control. FSC, forward scatter; Tr1 cell, type 1 regulatory T cell; Treg cell, regulatory T cell.
The proportion of activated (CD69+) T cells in CD3+CD4+ T cells was significantly higher in patients with psoriasis (mean ± standard error of the mean [SEM] = 0.42 ± 0.06%) compared with healthy participants (mean ± SEM = 0.16 ± 0.07%) (P = 0.015) (Figure 2a). In contrast, the proportion of Tr1 (CD49b+LAG-3+) cells in CD3+CD4+ T cells was significantly lower in patients with psoriasis (mean ± SEM = 0.18 ± 0.03%) compared with healthy participants (mean ± SEM = 0.51 ± 0.04%) (P < 0.001). The proportion of Treg (CD25+CD127–) cells tended to be lower in patients with psoriasis (mean ± SEM = 3.01 ± 0.43%) compared with healthy participants (mean ± SEM = 4.09 ± 0.53%), but the difference was not statistically significant (P = 0.132).
Figure 2Tr1 (CD49b+LAG-3+) cells, Treg (CD25+CD127–) cells, and activated (CD69+) T cells in the blood of patients with psoriasis versus healthy participants.(a) Comparison of the subset proportions between patients with psoriasis and healthy participants. (b) Correlations between the subset proportions and Psoriasis Area and Severity Index (PASI) (ρ = Spearman’s rank correlation coefficient). (c) Representative four-color wide-field fluorescence microscopy images of psoriasis nonlesional skin for DAPI (blue), CD4 (green), CD49b (red), and LAG-3 (magenta) staining. Tr1 cell (DAPI+CD4+CD49b+LAG-3+, white) is identified in the dermis of psoriasis nonlesional skin. Scale bar = 100 μm. Tr1 cell, type 1 regulatory T cell; Treg cell, regulatory T cell. DAPI, 4',6-diamidino-2-phenylindole.
The proportion of activated (CD69+) T cells in CD3+CD4+ T cells was positively correlated with Psoriasis Area Severity Index (PASI) (Spearman rank correlation coefficient [ρ] = 0.65, P = 0.002) (Figure 2b). In contrast, the proportion of Tr1 (CD49b+LAG-3+) cells in CD3+CD4+ T cells was inversely correlated with PASI (ρ = –0.63, P = 0.002). The proportion of Treg (CD25+CD127–) cells tended to be inversely correlated with PASI, but the correlation was not statistically significant (ρ = –0.35, P = 0.127).
Next, we studied 20 psoriasis lesional skin, 10 psoriasis nonlesional skin, and 5 normal control skin samples with four-color wide-field fluorescence microscopy and searched for quadruple-stained cells with 4',6-diamidino-2-phenylindole (DAPI) (blue), CD4 (green), CD49b (red), and LAG-3 (magenta). Psoriasis lesional skin showed abundant DAPI+CD4+ T cells in the dermis, but DAPI+CD4+CD49b+LAG-3+ Tr1 cells were not identified (0/20) (see Supplementary Figure S3 online). In contrast, psoriasis nonlesional skin and normal control skin showed only a few DAPI+CD4+ T cells in the dermis. In psoriasis nonlesional skin, DAPI+CD4+CD49b+LAG-3+ Tr1 cells were identified in 6 of 10 samples (Figure 2c). DAPI+CD4+CD49b+LAG-3+ Tr1 cells were not identified in normal skin (0/5) (see Supplementary Figure S4 online).
Our study findings provide preliminary data for further investigation of Tr1 cells in psoriasis with surface markers of CD49b and LAG-3, suggesting a potential role of Tr1 cells in the psoriasis immune mechanism. The proportion of Tr1 cells was decreased in psoriasis patients compared with healthy individuals, and the proportion of Tr1 cells decreased as PASI increased. In addition, Tr1 cells were identified in psoriasis nonlesional skin but not in lesional skin, despite the large increase of T cells in active lesions.
Therefore, we suggest that Tr1 cells, defined by surface markers of CD4+CD49b+LAG-3+, have potential roles in psoriasis pathogenesis. Decreased proportion of Tr1 cells in the blood of psoriasis patients may allow for excess expansion of psoriasis disease-related T cells in either lymph nodes or cutaneous compartments and thus to disease progression. This hypothesis requires further mechanistic studies investigating the cross-talk between Tr1 cells and Th17 cells in the blood and skin of psoriasis human and animal models.
Conflict of Interest
James G. Krueger has been a consultant and has received grants and honoraria from Novartis, Pfizer, Janssen, Lilly, Kadmon, Dermira, Boehringer, BMS, Paraxel, Kineta, Leo Pharma, Amgen, Innovaderm, Vitae, Provectus, Merck, Serono, Biogenldec, Delenex, AbbVie, Sanofi, Baxter, and Xenoport.
Author Contributions
JK designed the study, performed flow cytometry experiments, analyzed bioinformatics data, and co-wrote the paper. JL performed flow cytometry assays and analysis. JG supervised flow cytometry, assays, and analysis. JF-D supervised and performed immunofluorescence studies. SG analyzed bioinformatics data. JGK designed the study and co-wrote the paper.
High levels of interleukin 10 production in vivo are associated with tolerance in SCID patients transplanted with HLA mismatched hematopoietic stem cells.
Dysfunctional blood and target tissue CD4+CD25high regulatory T cells in psoriasis: mechanism underlying unrestrained pathogenic effector T cell proliferation.
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