FIGURE LEGENDS
Figure 1 The therapeutic response of psoriasis patients treated
with UVB is dictated by a specific cluster of CD4+expressing Treg and TCR-activated markers. (A) UMAP of
CD4+ T cells showing a distinct cluster of
CD25+FOXP3+CD127-cells (cluster 11) by CyTOF analysis. (B) Heatmap showing the
differences among clusters in CD4+ T cells. Each row
represents the median arcsinh-transformed intensities in each cluster,
and each column represents a maker. (C) Receiver operating
characteristic (ROC) curve for the percentage of cluster 11 at baseline
predicting UVB treatment response. Models were fit using logistic
regression, both unadjusted (blue) and adjusted for gender and age
(red). (D) Change in the percentage of cluster 11 CD4+T cells before (pre-UVB) and after (post-UVB) UVB treatment (n = 19).
The P value was calculated using the Wilcoxon signed-rank test. PASI:
Psoriasis Area Severity Index. (E) Left: Biaxial contour plots
illustrating the distribution of HLADR-TIGIT and HLADR-TIM3 expressing
cells within cluster 11, pre- and post-UVB treatment. Numbers indicate
the percentage of cells in each quadrant. Right: Paired Wilcoxon
signed-rank tests were used to compare the frequencies of
HLADRhiTIGIThi and
HLADRhiTIM3hi cells in cluster 11
before and after UVB treatment.
Figure 2 UVB alleviates OVA-induced inflammatory responses in
the skin by increasing OVA-specific Treg cells. (A) Mice were
intraperitoneally injected with OVA/alum on days 0, 7, and 14 and
epicutaneously treated with OVA patches on day 14. The mice were exposed
to UVB every other day three times during the OVA patch application.(B) Images of mouse dorsal skin are shown. (C) Changes
in transepidermal water loss (TEWL). n = 5 for the ctrl and O groups; n
= 3 for O+U groups. (D) Histological changes within the graft
were assessed using hematoxylin and eosin staining. (E-G) Flow cytometry
of cells isolated from skin and draining lymph nodes (LNs). Cells
isolated from the skin were restimulated. IL-4, IL-5, and IL-22 in
CD4+ T cells were analyzed (E).
CD25+Foxp3+ Treg cells and the
markers associated with TCR activation were analyzed in cells isolated
from skin-draining LNs (F). Cells from the skin were incubated with the
OVA323-339 tetramers, followed by the antibodies against
the indicated markers. OVA-specific Treg cells were analyzed (G).
Statistical analyses: one-way ANOVA with Kruskal-Wallis test. Mean ±
SEM. *, p<0.05; **, p<0.01; ***, p<0.001.
Figure 3 UVB prolongs allograft survival by inducing
allograft-specific Treg cells. (A) Schematic depicting the allograft
transplantation experiment. Mice were treated with UVB on alternating
days starting on day 5 (d5) for five treatments post-transplantation. n
= 9 for the No UVB group; n = 8 for the UVB group. (B) Skin
grafts were monitored from d5 post-transplantation. Grafts were
considered rejected when less than 20% viable tissue remained.(C) Histological changes within the graft were assessed using
hematoxylin and eosin staining. (D) Flow cytometry of cells
isolated from the grafts.
CD25+Foxp3+ Treg cells and ICOS,
CTLA-4, and LAG-3 expression were analyzed. (E) Schematic
diagram of the second allograft transplantation. Recipient mice,
previously subjected to a first allograft transplant with or without UVB
treatment, received a second allograft from either C57BL/6 or C3H mice.
(F) Skin graft appearance was monitored until rejection. n = 5 for each
of the groups. Statistical analyses: Gehan-Breskiw-Wilcoxon test. *,
p<0.05; **, p<0.01; ****, p<0.0001.
Figure 4 Antigen-specific Treg cells affect clinical outcomes of
inflammatory skin diseases. (A) Scatter plot illustrating the
correlation between the percentage of cluster 11 in
CD4+ T cells and PASI score in psoriasis patients. The
P value was calculated using Spearman’s correlation. (B) Box-and-whisker
plot comparing the percentage of cluster 11 in CD4+ T
cells in healthy controls (HC) and psoriasis patients with low (PASI ≤
4.5), medium (4.5 < PASI < 9), and high (PASI ≥ 9)
disease severity. PASI: Psoriasis Area Severity Index. (C)
Foxp3DTR-GFP Mice were sensitized with OVA/alum,
challenged by the OVA patch, and treated with UVB.
CD25+GFP+ Treg cells were sorted
from the skin-draining LNs. CD4+GFP-T cells as responder T cells were sorted from the skin-draining LNs of
OTII/Foxp3DTR-GFP mice. Treg and responder T cells
were mixed at a ratio of 1 to 1 and then were administered to the
recipient mice by intravenous injection. The recipient mice were then
sensitized with OVA/alum by intraperitoneal injection and challenged
with the OVA patches. (D) TEWL was measured every other day. n
= 3 for each of the groups. (E) Histological changes within the
graft were assessed using hematoxylin and eosin staining. (F) Cells isolated from the skin were restimulated. IL-5 production on
CD4+ T cells was analyzed. (G) Sorted
CD25+GFP+ Treg cells from mice with
OVA stimulation and UVB treatment were intravenously injected into the
recipient mice. The ears of the mice were then stimulated with 15 mg of
imiquimod-containing cream. (H) Changes in the ear swelling of
mice were shown. n = 4 for each of the groups. (I) Cells
isolated from ears were restimulated. IL-17 and IL-22 on
CD4+ T cells were analyzed. Statistical analyses:
one-way ANOVA with Kruskal-Wallis test. P for trend test was calculated
by ordinary one-way ANOVA. Mean ± SEM. *, p<0.05; **,
p<0.01; ***, p<0.001.
Figure 5 UVB enhances genes involved in cell stability, cell
proliferation, and cellular localization, and suppresses genes
associated with negative regulation of Treg development. (A) Pathway
analysis of upregulated genes in Tregs from skin of mice treated with
UVB compared to those of mice without UVB treatment (n = 2 treated and n
= 2 untreated, pooled samples). GOBP: Gene Ontology Biological Process;
NES: Normalized Enrichment Score. (B) UMAP plot of
CD4+ T cells from patients PBMC samples (2 patients,
with pre- and post-treatment samples). (C) Dot plot of gene markers for
Treg, T memory, T effector, and naive T clusters. (D) Heatmap showing
gene expression changes in CD4+ Tregs from mice and
human PBMC under UVB treatment. (E) Violin plot of negatively-associated
genes (RAB35 , IDH1 , and VAV1 ) with significant gene
expression changes (p<0.05) in human PBMC-derived Treg cells
under UVB treatment. Statistical significance was tested through the
Wilcoxon ranked test.
Figure 6 Treg transition from human CD4+ T
cell lineage and clonal expansion under UVB treatment. (A-D) Analyses of
CD4+ T cells obtained from the periphery of psoriasis
patients treated with UVB and the public dataset generated from healthy
human skin. UMAP of CD4+ T cell clusters with an
overlaid Slingshot trajectory (A). UMAP plot of 9375
CD4+ T cells in the skin with Slingshot trajectory
(B). A chronologically color-coded pseudotime UMAP of
CD4+ T cells from violet (least differentiated) to
yellow (most differentiated) (C). Expression of key genes for pseudotime
trajectory, color coded by the clusters shown in panel A (D). (E) (Left)
UMAP visualization of CD4+ T cell subtypes. (Right)
Clonal expansion overlay on the UMAP, with higher density indicating
greater clonal expansion. (F) Distribution of clonal size across four
CD4+ T cell subtypes under UVB treatment. Numbers
represent cell counts in each condition. (G) Expansion indices were
quantified using the STARTRAC-expa method.