CXCL9, CXCL10, and CCL19 synergistically recruit T-lymphocytes to skin in lichen planus

: Lichen planus (LP) is a chronic, debilitating, inflammatory disease of the skin and mucous membranes that affects 1-2% of Americans. Its molecular pathogenesis remains poorly understood, and there are no FDA-approved treatments. We performed single cell RNA sequencing on paired blood and skin samples (lesional and non-lesional tissue) from 7 LP patients. We discovered that LP keratinocytes and fibroblasts specifically secrete a combination of CXCL9, CXCL10, and CCL19 cytokines. Using an in vitro migration assay with primary human T cells, we demonstrated that CCL19 in combination with either cytokine synergistically enhanced recruitment of CD8 T cells, more than the sum of individual cytokines. Moreover, exhausted T cells in lesional LP skin secreted CXCL13, which along with CCL19 also enhanced recruitment of T cells, suggesting a feed-forward loop in LP. Finally, LP blood revealed decreased circulating naïve CD8 T cells compared to healthy volunteers, consistent with recruitment to skin. Molecular analysis of LP skin and blood samples increased our understanding of disease pathogenesis and identified CCL19 as a new therapeutic target for treatment.


Immune cell landscape in LP skin consists predominantly of CD8 T cells
We collected 4-millimeter skin biopsies from lesional and non-lesional skin from seven LP patients (Figure 1A, Supplemental Table 1).Three male and four female patients were included with a median age of 62 (mean 57).All patients had clinically active disease; five patients were not receiving treatment, and two patients were on oral treatment (hydroxychloroquine and prednisone, respectively).We generated 188,607 high quality single cell RNA sequencing (scRNAseq) profiles (Supplemental Table 2).Unsupervised cell clustering of scRNAseq profiles revealed 29 unique cell populations which were annotated to 10 cell types using marker gene identification and mapping to single cell databases (Figure 1B, Supplemental Figure 1, A-E).The cell types were shared between lesional and non-lesional skin samples, and lymphoid cell populations were consistently enriched by number in all lesional LP skin samples (Supplemental Figure 1, B and C).
Next, we subclustered the lymphoid cell population and identified nine cell types, eight T cell subtypes and one NK cell population (Figure 1, C and D, Supplemental Figure 1, F and G).

Lichen planus skin secretes CXCL9, CXCL10, and CCL19 cytokines
To assess how LP skin may recruit lymphoid cell populations, we subclustered epidermal cell populations and identified eleven unique clusters, which were annotated based on expression of canonical marker genes (Figure 2A, Supplemental Figure 2, A and B).We identified four populations of basal cells along with suprabasal cells, melanocytes and cells of the hair follicle and eccrine glands (18,19).LP is histologically characterized by apoptosis in the basal layer of keratinocytes.Within basal keratinocytes, CXCL9 and CXCL10 were induced as much as 8-fold in lesional skin compared to non-lesional skin, making them two of the highest induced genes (Figure 2B, Supplemental Figure 3A).Melanocytes also markedly expressed CXCL9, CXCL10 and CCL19 (all 2-fold) compared to non-lesional skin (Supplemental Figure 3A).Transcription factor analysis of the keratinocyte populations showed an upregulation of IRF7, ETV7, and STAT2 (Supplemental Figure 3B).
Fibroblasts subclustered into eight unique populations, and four subpopulations exhibited increased expression of CXCL9 (up to 10-fold) and CXCL10 (up to 5-fold) in LP lesional skin compared to non-lesional skin (Figure 2, C and D).Interestingly, all fibroblast populations also substantially induced expression of CCL19 (average 3-fold induction) (Figure 2, D and E).
CCL19 is a chemokine typically expressed in thymus and lymph nodes to regulate immune cell trafficking but does not have an established role in skin inflammatory diseases (20).One population of basal keratinocytes, Basal 1, also induced CCL19 significantly (Figure 2B).
To confirm our scRNAseq findings, we performed immunohistochemistry on LP skin biopsies.CXCL9 and CXCL10 were primarily expressed by keratinocytes in the lower layers of the epidermis and fibroblasts in the superficial dermis (Figure 2E).CCL19 was also strongly expressed in the lower levels of epidermis and in the superficial dermis.We confirmed that CCL19+ dermal staining came from fibroblasts (CD3-, vimentin+, CCL19+) and to a lesser extent tissue-infiltrating T-cells (Supplemental Figure 4A).We additionally assessed skin biopsies of patients with lichen planopilaris (LPP) and psoriasis.LPP is a scalp-restricted clinical variant of lichen planus.Consistent with prior reports, we found increased expression of CXCL10 and CCL19 that localized to the hair follicle epithelium (Supplemental Figure 4B) (33).
We did not observe staining for these chemokines in psoriasis skin samples (Supplemental Figure 4C).Taken together, CXCL9, CXCL10, and CCL19 were the major cytokines induced in keratinocytes and fibroblasts of LP skin.
Next, we compared the chemokine environment in LP to other T cell mediated skin diseases.We analyzed publicly available scRNAseq skin data sets for psoriasis, atopic dermatitis, and vitiligo (21)(22)(23).Within fibroblasts, keratinocytes, and melanocytes, we found that LP lesional skin had the strongest expression of CXCL9, CXCL10, and CCL19 as well as the highest frequency of fibroblasts expressing these chemokines (Figure 2F).Thus, LP skin strongly secretes a unique combination of CXCL9, CXCL10, and CCL19 cytokines.
Cellchat computationally identifies potential ligand-receptor interactions within a population of cells.Global analysis of lesional and non-lesional skin from LP patients revealed upregulation of signaling pathways in the cytotoxic and Th1 lymphocyte response (TNF, IL2, OX40, LT, IFN-II) and chemokine signaling (CCL, CXCL) (Figure 3A and Supplemental Figure 5 A and B).In lesional skin, basal keratinocytes and fibroblasts secreted CXCL9 and CXCL10 which was received by CXCR3 expressed on CD8 Pro T cells, CD8 T1, Treg, and Texh populations in lesional LP skin only (Figure 3B).CCL19 expressed by fibroblasts and basal keratinocytes was received by CCR7 expressed on CD8 Pro T cells, Naïve T cells, CD8 T1, CD8 T2, Tregs and Texh cells (Figure 3C).Taken together, Cellchat analysis suggests that CXCL9, CXCL10, and CCL19 signals converge on T cells in LP skin.

CCL19 works synergistically with CXCL9 and CXCL10 to recruit T cells
To test whether these cytokines recruit T cells, we performed in vitro migration assays with peripheral blood mononuclear cells (PBMCs) from healthy donors (Figure 3D).We quantified migration of CD4 and CD8 T cells by flow cytometry (Supplemental Figure 6A).We tested migration in response to CXCL9, CXCL10, or CCL19, as well as combinations of both CXCL9 and CCL19 and CXCL10 and CCL19 (Figure 3, D-H).Migration index is the ratio of cells that migrated in response to a chemokine stimulus divided by the number of cells that migrated in response to control media.

Compared to vehicle control, CD4 T cells exhibited more migration towards CXCL9
(migration index of 9.5 + 3.6) and CCL19 (8.1 +1.8) alone (Figure 3E, left panel).Combination treatment with CXCL9 and CCL19 induced CD4 T cells to migrate significantly more strongly (36.7+7.3).In fact, these cells migrated more than double the sum of the individual chemokine migration indices, suggesting a synergistic response.Blocking antibodies against CCR7, the receptor for CCL19, significantly ameliorated the combined treatment's effect (12.3+4.5)(Figure 3E, right panel).
CD8 T cells showed an even more pronounced response (Figure 3F).Combination treatment with CXCL9 and CCL19 induced CD8 T cells to migrate significantly more strongly (87.0+21.9)than the sum of individual chemokine migration indices for CXCL9 (13.4+4.9) and CCL19 (15.0+3.1).Blocking antibodies against CCR7 also reduced the combined treatment's effect (29.0+10.8)(Figure 3F, right panel).Taken together, combination treatment of CXCL9 and CCL19 was synergistic for T cell migration and induced almost 3-fold more CD8 T cells migration compared to CD4 T cells.
We repeated migration assays with CXCL10 with and without CCL19.CD4 T cells demonstrated increased migration towards CXCL10 (mean migration index of 3.8+1.0)and CCL19 (17.5+4.0)(Figure 3G).Combination treatment also induced CD4 T cells to migrate more (40.0+9.0)than the sum of the individual cytokines.CCR7 blocking antibodies ameliorated this effect (14.8+4.6)(Figure 3G, right panel).CD8 T cells again showed a more pronounced response (Figure 3H).Combination treatment with CXCL10 and CCL19 induced CD8 T cells to migrate more strongly (68.0+20.3)than the sum of individual chemokine migration indices for CXCL10 (4.7+1.1) and CCL19 (26.8+8.1).CCR7 blocking antibodies also reduced this effect (20.8+5.9),but it did not reach statistical significance (p=0.06; Figure 3H, right panel).In summary, CCL19 worked with CXCL9 or CXCL10 to synergistically amplify the migration of CD4 and CD8 T cells.This effect was greater on CD8 T cells than CD4 T cells.

T cell secreted CXCL13 synergizes with CCL19 to recruit CD8 T lymphocytes
We examined our scRNAseq dataset to identify other cytokines that may recruit CD8 T cells into LP lesional skin.Prior studies demonstrated that CXCL13 is specifically expressed by exhausted T-cells (13,14,24).Indeed, CXCL13 was among the highest induced genes in the Texh (>5-fold induction) and CD8 Pro (>10-fold induction) populations in LP lesional skin compared to non-lesional skin (Figure 4A).Immunohistochemistry of LP skin confirmed that CXCL13 was primarily expressed by infiltrating CD8 T cells (CD3+CXCL13+CD4-) (Figure 4B).CXCL13 canonically signals through the CXCR5 receptor.However, we did not detect expression of CXCR5 in our dataset.CXCL13 has also been shown to bind CXCR3, and this interaction has been functionally demonstrated to induce T-cell migration (25,26).In our dataset, CXCR3 was expressed in CD8 Pro, Treg, CD8 T1, and CD4 CTL populations in lesional LP skin (Figure 4C).Cellchat analysis highlighted that CD8 Pro T cells may signal to other CD8 Pro T cells in lesional LP skin via CXCL13:CXCR3 interactions as well as CD8 T1 and Treg cell populations (Figure 4D).Consistently, Texh cells may also signal to CD8 Pro, CD8 T1 and Treg populations (Figure 4D).
Taken together, Texh and CD8 Pro T cells in lesional LP skin secrete CXCL13 that synergizes with fibroblast-secreted CCL19 to recruit more CD8 T cells.

Circulating levels of naïve CD8 T cells are decreased in LP patients
Tissue-infiltrating lymphocyte populations are generally recruited from peripheral blood.
We assessed if there were any changes in circulating immune cell populations between LP patients and healthy controls.We collected paired PBMC samples from the seven LP patients for scRNAseq (Supplemental Table 1-2).We used publicly available data for three healthy adult controls to create a combined 116,108 cell dataset.After unsupervised clustering, we annotated cell populations using a reference atlas for human PBMCs (Figure 5A, Supplemental Figure 6B) (27,28).We found two lymphoid populations that changed significantly in frequency between LP patients and healthy controls.LP patients exhibited fewer circulating naive CD8 T cells compared to healthy controls (3.3+1.3%versus 10+1.2% in healthy controls, p= 0.02) (Figure 5B).This decrease in circulating naïve CD8 T cells may correspond with the influx of CD8 T cells into LP lesional skin.We also observed that CD4 CTLs were enriched 10-fold in circulating blood of LP patients (2.9+ 0.7% compared to 0.3+0.01% in healthy controls, p=0.04) (Figure 5B).These cells expressed higher levels of granzyme B compared to healthy controls (Supplemental Figure 6C).Notably, none of the circulating cell populations exhibited increased expression of CXCR3 or CCR7 receptors, suggesting that increased recruitment to skin is mediated by changes in cytokine ligand expression in the tissue and not due to changes in receptor expression levels in LP immune cells (Supplemental Figure 6D).
Finally, we performed pseudotime analysis on our LP skin and blood datasets.
Pseudotime rationally defines developmental trajectories of analyzed cells based on single cell transcriptomes which are assumed to be individual variations of developmental states (29).
Unbiased analysis of the naïve CD8 T cell subset from blood and CD8 T cell subsets from lesional and nonlesional LP skin demonstrated naïve peripheral CD8 T cells at the initial state and CD8 T1 and CD8 T2 skin populations at the terminal state (Figure 5C).This trajectory correlated with the upregulation of the genes found on activated CD8 T cells (IFNG, GZMA, GZMK, CCL4, and CCL5) (Figure 5D).Additionally, the pseudotime trajectory correlated with the downregulation of CCR7, which is known to be downregulated after binding to CCL19 (30).
Thus, pseudotime analysis suggests that T cell activation in LP is not a systemic finding and only occurs after local recruitment to the skin.

DISCUSSION:
We showed that infiltrating CD8 T cells in LP are the primary source of IFNg that may trigger keratinocyte apoptosis (5).Moreover, LP lesional skin secreted a unique combination of CXCL9, CXCL10, and CCL19 chemokines.CCL19 in combination with either CXCL9 or CXCL10 synergistically recruited T cells, especially CD8 T cells.We believe this is a dynamic process by which CCL19 bulk-recruits CCR7-expressing naïve T cells to the superficial dermis and that upon activation these cells upregulate CXCR3 which strengthens their retention at the dermal epidermal junction.Notably, exhausted T cells and proliferating CD8 T cells in LP skin specifically secreted CXCL13.CCL19 also synergized with CXCL13 to recruit more CD8 T cells, and this mechanism may establish a feed-forward loop.
Individual roles for CXCL9, CXCL10 and CCL19 have been demonstrated in other skin diseases.CCL19-expressing fibroblasts have been identified in atopic dermatitis and localize to leukocyte-rich areas of the skin (22,31,32).CXCL9 and CXCL10 are expressed in many other skin diseases, and their functional importance was demonstrated for vitiligo, where inhibition of CXCL10 reduced recruitment of T cells and resulted in skin repigmentation (10,11,33).
However, compared to these inflammatory skin diseases, LP skin had the strongest expression of all three cytokines.There are many different clinical variants of LP; patients may have skinrestricted, mucosa-restricted, hair follicle-restricted (known as lichen planopilaris or frontal fibrosing alopecia) or involvement of some combination of those sites.Our samples were only obtained from the skin, and similar increased expression of CXCR3 and CCL19 were noted by bulk sequencing in the hair follicle-restricted variant of LP (33).It may be possible that different chemokines recruit T cells to different body sites; a prior study showed increased CCR5 and CXCR3 expression on CD8 T cells in mucosal LP lesions (7).We did not see CCR5 expression in our skin samples.Taken together, we speculate that this specific combination of CXCL9, CXCL10, and CCL19 is responsible for the robust recruitment of all T cells to skin and possibly hair follicles.More studies are needed to assess CCL19 expression and site-specific lymphocyte migration.
Prior work explored whether CCL19 may amplify recruitment of immune cells in other biological contexts.Sezary syndrome is a subtype of cutaneous T-cell lymphoma that involves both the skin and blood (34).Combination treatment of CCL19 and CXCL13 enhanced the migration of Sezary CD4+ T-cells (35).However, CCL19 was not found in Sezary patient skin samples, which may explain why the T cell infiltrate in Sezary syndrome is not as pronounced as in LP.In cancer biology, local production of CCL19 promotes antitumor responses by recruiting more CD8+ T cells in lung and ovarian cancer models (36,37).Thus, CCL19 may also amplify immune cell recruitment in other biological contexts.The robust lymphocytic infiltrate seen in LP has been histologically termed a "lichenoid band reaction", and this pattern is sometimes seen in patients with allergic drug reactions, lupus erythematosus, and less frequently squamous cell carcinoma.It would be interesting to assess the role of CCL19 in the histologically defined lichenoid band reaction.
What stimulates CCL19 production in LP skin?We found induction of STAT1, STAT2, and IRF7 transcription factors in lesional LP keratinocytes and fibroblasts.Prior studies using bacterial or viral infection models demonstrated that CCL19 expression in dendritic cells is similarly driven by activation of STAT1, STAT2, and IRF7 (38).The identification of an initial trigger for LP has remained elusive, and a viral basis for this disease has been speculated by clinicians for decades (39,40).Future studies are needed to assess the possibility of viralmediated activation of CCL19 in the skin of LP patients.
In recent years, the concept that T cells may become "exhausted" after chronic antigen stimulation in tumors and infections has become established.We are intrigued that CXCL13 secreted by exhausted T cells in LP skin also synergizes with CCL19 to recruit T cells.A functional role for exhausted T cells in autoimmune disease remains unexplored, and we posit this feed-forward mechanism may help to sustain LP skin lesions and warrants further exploration.

Finally, we discovered that LP blood contained fewer naïve CD8 T-cells and more CD4
CTLs.There are no clinical tests available to measure LP disease activity.While cutaneous LP skin activity may be monitored by visual inspection, there are cases of mucosal and esophageal specific lesions that require invasive procedures to assess.A blood test to measure disease activity would benefit patient care by permitting clinicians to assess treatment response.This would help optimize treatment selection and minimize exposure to medication side-effects.
Future studies should explore whether circulating naïve CD8 T cells and/or CD4 CTLs may serve as circulating biomarkers for disease activity.Molecular analysis of skin and blood improved our understanding of disease pathophysiology.Our data suggests that blocking CCL19 may serve as a novel therapeutic strategy for LP.The ability to directly interrogate diseased skin and blood allows this approach to be generalizable to other systemic inflammatory disorders.

Sex as a biological variable
To address sex as a biological variable, we attempted to recruit equal numbers of male and female patients.

Demographic information
Patient data and associated demographics are provided in Supplementary Table 1.Demographic information was provided by the participants with options provided by the investigators.

Human Study Participants
Patients diagnosed with lichen planus were recruited to the study at the Dermatology Clinic of the Perelman Center for Advanced Medicine at the University of Pennsylvania.Diagnosis in each case had been previously confirmed with histology.Patient demographics are provided in Supplementary Table 1.4-5 millimeter (mm) biopsies were taken from lesional and non-lesional skin of patients with active skin disease.All biopsies were transported in saline soaked gauze and processed immediately for optimal cell recovery.Patient whole blood samples were collected in vacutainer tubes with EDTA (Beckton Dickenson) to prevent clotting.Healthy volunteer PBMCs and plasma were obtained from Human Immunology Core (HIC) at the University of Pennsylvania.

Tissue processing and single cell gene RNA-seq library preparation and sequencing
Skin biopsies were suspended in serum-free RPMI 1640 media with DNase (0.2 milligram/milliliter (mg/mL), 12633012, Thermo Fisher Scientific), 20 mM HEPES and 0.25 mg/ml Liberase TM (5401119001, Roche) and minced into <1 mm 3 pieces using sterile Gradle scissors.The suspension was incubated at 37˚C for 90 minutes.The digestion was arrested with FBS and 3 mL of 0.5 M EDTA and the suspension was subsequently filtered through a 70 micrometer cell strainer Fisher Scientific).The cells were then washed twice with PBS containing 1% BSA and resuspended in PBS containing 0.04% BSA and taken for counting on a hemocytometer.Single cell RNA sequencing (scRNA-seq) was performed using 10X Chromium 3 v3.1 kit (1000268, 10X Genomics).The sequencing libraries were prepared per manufacturer's protocol and sequenced 2x100bp paired-end run on the Illumina HiSeq2000/HiSeq2500 platforms at the BGI America.The raw and processed sequencing data details are given in Supplementary Table 2.

PBMC and Plasma Isolation
Patient blood collected in vacutainers was transferred to 50 mL conical tubes and mixed with an equal volume of HBSS (21-023-CV, Corning).The suspension was carefully pipetted over Ficolpaque (17-140-02, GE Healthcare) in a 1:1 ratio by volume.The tubes were centrifuged at 400g, 25 min with 0 acceleration and 0 de-acceleration setting at 10° C. Plasma was carefully aspirated from the top layer while mononuclear cells were retrieved from the interface.Mononuclear cells were washed with PBS and stored in freezing media (10% DMSO in FBS) in 2 mL cryotube vials.The cryovials were frozen in a freezing container at -80 °C for 24 hours before storing in liquid nitrogen.

Figure 1 :
Figure 1: Immune cell landscape in lichen planus.(A) Clinical photograph of lesional and non-lesional skin biopsies from a lichen planus (LP) patient.(B) Identification of cell clusters from lesional and non-lesional LP skin (n=13) (C) UMAP depicts subclustering of lymphoid cells.(D) Analysis of individual subclusters.Marker genes described below title.Density plots demonstrate location of subgroup within UMAP.(E) Bar plots show relative contribution as % of total cells.One-way ANOVA.(F) Dot plot demonstrating levels and percent of cells expressing IFNG, IL17A, and IL4.Mean ± SEM are plotted.*p<0.05.

Figure 2 :
Figure 2: Lichen planus skin secretes CXCL9, CXCL10, and CCL19.(A) UMAP depicting subclustering of epidermal skin cells.(B) Volcano plots of differential gene expression from basal keratinocyte subpopulations from lichen planus (LP) lesional vs nonlesional skin.Expression of CXCL9, CXCL10, and CCL19 are labeled on the plots.A p-value of <0.01 and a two-fold expression change was used for significance.(C) UMAP depicting subclustering of fibroblasts.(D) Volcano plots of differential gene expression from fibroblast subpopulations from LP lesional vs non-lesional skin.Expression of CXCL9, CXCL10, and CCL19 are labeled on the plots.A p-value of <0.01 and a two-fold expression change was used for significance.(E) Representative immunofluorescence images depicting localization of CXCL9 (green), CCL19, (white), and DAPI (blue) in LP skin (n=5 patient samples).The white dotted line depicts the epidermal-dermal junction.Scale bars: 100 micrometer.(F) scRNAseq data from publicly available single-cell data sets for vitiligo, psoriasis and atopic dermatitis were used to analyze skin cells for their expression of chemokines.Dot size corresponds to percent of cells expressing chemokine while color corresponds to level of gene expression.

Figure 3 :
Figure 3: CCL19 synergizes with skin secreted CXCL9 or CXCL10 to recruit T cells.(A) Global analysis of ligand-receptor pathways.Arrows highlight relevant signaling.(B) Analysis of cell-to-cell interactions between epidermal keratinocytes cells and immune cells in lichen planus (LP) lesional skin.Dot color illustrates communication probability, and dot size illustrates p-value.(C) Analysis of cell-to-cell interactions between dermal fibroblasts and immune cells in lesional (salmon color) and non-lesional (blue color) LP skin.Dot color illustrates communication probability, and dot size illustrates p-value.(D) Schematic of migration assay.(E-H) Left panel: migration index (# of migrated cells in response to cytokine/# of migrated cells in response to control) for CD4 and CD8 T cells in response to different conditions of CXCL9, CXCL10, and CCL19 (n=12).Right panel: Combined treatment ± CCR7 blocking antibodies (n=9).Mean ± SEM are plotted.One-way ANOVA was used for migration assays and two-tailed paired Student's t-test was used for CCR7 antibody analysis.* p<0.05; **p<0.01;ns, not significant.

Figure 4 :
Figure 4: CCL19 synergizes with T cell secreted CXCL13 to recruit T cells.(A) Volcano plots of differential gene expression from exhausted (Texh) and CD8 proliferating (CD8 Pro) T cell populations from lesional vs non-lesional lichen planus (LP) skin.Expression of CXCL13, CTLA4, GZMB, and GNLY are labeled.(B) Representative immunofluorescence images of LP skin depicting localization of CD4 (green), CXCL13 (white), CD3 (red), and DAPI (blue) (n=5 patient samples).Scale bars: 100 micrometer.(C) Dot plot demonstrating levels and percent of cells expressing CXCR3 and CXCR5.(D) Analysis of cell-to-cell interactions between immune cells in lesional (salmon color) and non-lesional (blue color) LP skin.(E and F) Left panel: migration index (# of migrated cells in response to cytokine/# of migrated cells in response to control) for CD4 (E) and CD8 T cells (F) in response to different conditions of CXCL13 and CCL19 (n = 9).Right panel: Combined treatment ± CCR7 blocking antibodies (n=7).Mean ± SEM are plotted.One-way ANOVA was used for migration assays and two-tailed paired Student's t-test was used for CCR7 antibody analysis.* p<0.05; ns, not significant.

Figure 5 :
Figure 5: Peripheral naïve CD8 T cells migrate to skin in lichen planus.(A) Identification of cell clusters from lichen planus (LP) blood (n=7) (B) Bar plot shows relative contribution as % of total cells for CD8 naïve and CD4 CTL T cell populations between LP patients and healthy controls (n=3).Mean ± SEM are plotted.Two-tailed unpaired Student's t-test.*p<0.05;ns, not significant.(C) Pseudotime trajectory of naïve CD8 T cells isolated from PBMCs, CD8 T1 and CD8 T2 populations from LP lesional skin.Each dot represents a cell.Top panel shows trajectory through time (blue-scale).Bottom panel shows cells colored according to cell type origin (naïve-salmon; CD8 T1-green; CD8 T2 -blue).(D) Gene expression changes as the cells progress through the pseudotime trajectory (from naïve state in peripheral blood to effector state in tissue).