Benign T cells drive clinical skin inflammation in cutaneous T cell lymphoma

Abstract

Psoralen plus UVA (PUVA) is an effective therapy for mycosis fungoides (MF), the skin-limited variant of cutaneous T cell lymphoma (CTCL). In low-burden patients, PUVA reduced or eradicated malignant T cells and induced clonal expansion of CD8+ T cells associated with malignant T cell depletion. High-burden patients appeared to clinically improve but large numbers of malignant T cells persisted in skin. Clinical improvement was linked to turnover of benign T cell clones but not to malignant T cell reduction. Benign T cells were associated with the Th2-recruiting chemokine CCL18 before therapy and with the Th1-recruiting chemokines CXCL9, CXCL10, and CXCL11 after therapy, suggesting a switch from Th2 to Th1. Inflammation was correlated with OX40L and CD40L gene expression; immunostaining localized these receptors to CCL18-expressing c-Kit+ dendritic cells that clustered together with CD40+OX40+ benign and CD40+CD40L+ malignant T cells, creating a proinflammatory synapse in skin. Our data suggest that visible inflammation in CTCL results from the recruitment and activation of benign T cells by c-Kit+OX40L+CD40L+ dendritic cells and that this activation may provide tumorigenic signals. Targeting c-Kit, OX40, and CD40 signaling may be novel therapeutic avenues for the treatment of MF.

Keywords: Dermatology; Immunology; Lymphomas.

Figure 1. PUVA is an effective therapy for mycosis fungoides that can eradicate malignant T cells in patients with low initial tumor burden.

(A–C) Design of the clinical and translational studies. (A) Fifteen patients were treated with oral 8-methoxypsoralen (8-MOP) and exposed to UVA light. (B) Severity of disease was assessed in a single index lesion using the CAILS scoring system and across the entire body surface area using the mSWAT scoring system. (C) Skin biopsies of a single index lesion were obtained before and after therapy and were studied by HTS, NanoString-based gene expression profiling, and multiplex immunostaining. (D–F) All patients improved clinically with respect to index lesion severity (D) and overall burden of disease (E). Pre-tx, pretreatment. Individual patients (D and E) and aggregate clinical scores (F) are shown. (G) The number of total T cells in skin as assessed by HTS was significantly reduced after therapy but (H) changes in the malignant T cell frequency varied widely among patients. (I–L) Patients with a low initial malignant T cell burden had the best clearance of malignant T cells. (I) There was a wide range of initial malignant T cell frequencies before therapy but patients tended to cluster into either high or low residual malignant T cells groups after therapy. (J–L) Patients with a low initial malignant T cell frequency (<10% or 10%–20%) had the greatest depletion of malignant T cells. The change in the malignant T cell frequency for each patient (J), the mean reduction in malignant T cell frequency in each group (K), and the percentage of patients in each group with the indicated levels of malignant T cell reductions (L) are shown. (M and N) PUVA reduced the absolute number of benign and malignant T cells in skin from low-burden but not high-burden patients. The absolute number of total, malignant, and benign T cells (T cell genomes/100 ng of DNA) in skin for low-burden (M) and high-burden (N) patients are shown. The mean and SEM (error bars) are shown. Differences between 2 sample groups were detected using the 1-tailed Wilcoxon-Mann-Whitney test (α = 0.05). For comparisons of multiple groups, a Kruskal-Wallis 1-way analysis of variance with a Bonferroni-Dunn post hoc test for multiple means testing was used (α = 0.05). All reported P values are adjusted for multiple comparison testing.

Figure 2. Visible inflammation does not reflect malignant T cell burden and reduced inflammation is linked to turnover of benign T cells.

(A) Clinical exam scores in high- and low-burden patients were not significantly different. (B) Two patients are shown in whom visible inflammation (clinical exam scores) improved but the malignant T cell clone remained high after treatment (patient 1, 68%; patient 4, 69%) or even increased (patient 4). (C) Malignant T cell frequency remained high after treatment despite the presence of large numbers of malignant T cells in skin in patient 4, a complete clinical responder. The unique TCR CDR3 sequences of each nonmalignant T cell clone were used to identify which benign T cells persisted after therapy (blue), were eliminated from skin (light green), or were recruited to skin (dark green) after therapy. Persistent benign clones were benign T cell clones that were present in skin both before and after PUVA therapy. (D) Additional patients are shown in whom the malignant T cell burden remained high after therapy despite improvement in clinical inflammation exam scores. (E–H) Improvement in inflammation is correlated with a shift in the benign T cell population but not with depletion of malignant T cells. Improvement in inflammation (mSWAT) did not correlate with reductions in the number of malignant T cells (E), total T cells (F), or benign T cells (G). However, the loss of specific T cell clones from skin and recruitment of a second, distinct T cell population was correlated with reduced inflammation as assessed by mSWAT (H) and CAILS scores (Supplemental Figure 1). Differences between 2 sample groups were detected using the 1-tailed Wilcoxon-Mann-Whitney test (α = 0.05). For correlations, a Pearson’s correlation coefficient with a 2-tailed P value is reported.

Figure 3. Benign T cells have distinct gene association profiles before and after therapy and PUVA induces a shift from Th2 to Th1 chemokine association.

(A) Gene expression before and after therapy was studied by NanoString and genes associated with the benign T cell population were assessed by Pearson’s correlations with CD7 and CD8A (CD8⁺ T cells only). CD7 was strongly correlated with the number of benign T cells as measured by HTS, validating the approach (Supplemental Figure 2). Genes associated with total benign cells had only 1 gene in common and CD8-associated genes before and after therapy were completely distinct. Genes in bold font: r > 0.9, P < 0.001. (B) PUVA induces a shift from Th2- to Th1-associated chemokines. The number of benign T cells, as assessed by CD7 gene expression levels (top 2 panels), correlated strongly with the expression of the Th2-recruiting chemokine CCL18 before but not after therapy and with the Th1-recruiting chemokines CXCL9, -10, and -11 after, but not before, therapy. Similar results were seen when the number of benign T cells was assessed by HTS (bottom panel). For mRNA expression and association studies, Pearson’s correlations were used to identify genes associated with CD7 or CD8A gene counts (as measured by NanoString). Genes with r > 0.8 and P < 0.01 were considered significant. For correlations, a Pearson’s correlation coefficient with a 2-tailed P value is reported.

Figure 4. CCL18 is produced by c-Kit⁺ dendritic cells in mycosis fungoides (MF).

(A) CD163-expressing M2-like macrophages do not produce CCL18 in MF. Coimmunostaining of CCL18 (red) and CD163 (green) demonstrated independent staining. A patient with low-burden stage IA CTCL is shown; a total of 3 stage IA-IB patients showed similar results. (B and C) CCL18 immunostaining colocalized with c-Kit–expressing dermal cells. A stage IB high-burden MF patient is shown; a total of 4 patients showed similar results. (C) Higher-power images of immunostaining of the same stage IA patient studied in A are shown. (D) CCL18-producing c-Kit⁺ cells are CD11c⁺ dendritic cells. A stage IA high-burden patient is shown (first 3 panels) and costaining for all markers in another stage IB high-burden patient is shown (fourth panel). Triple staining appears white. Staining demonstrating that CCL18⁺ cells lacked expression of tryptase are included in Supplemental Figure 4. Similar results were observed in high- and low-burden patients. All lesions are untreated. Results from additional patients are shown in Supplemental Figure 5. A color blind–accessible version of this image is provided in Supplemental Figure 6. Scale bars: 100 μm.

Figure 5. Skin inflammation is not linked to malignant T cell number or frequency but is related to the expression of 2 malignant T cell–associated genes.

(A and B) Skin inflammation (CAILS) was not correlated with the absolute number (A) or relative percentage (B) of malignant T cells in skin in pretreatment samples from 3 clinical cohorts from 3 different institutions. (C) Genes associated with visible inflammation were identified by correlating pretreatment CAILS with genes across the PUVA treatment data set. Nine genes were identified, 3 of which (CD3G, CCR4, and CD5) are widely expressed by skin T cells and 2 of which (OX40L and CD40LG) were strongly associated with the number of malignant T cells in skin as measured by HTS (D). TNFSF4 (OX40L) was strongly associated with malignant T cell numbers (r = 0.959, P = 0.0006) and its receptor TNFRSF4 (OX40) was strongly associated with the number of benign T cells (CD7 gene expression, r = 0.971, P = 0.0003). Genes in bold font: r > 0.9, P < 0.001. CD40 ligand was strongly associated with malignant T cell number (r = 0.932, P = 0.0022) and its ligand CD40 was associated with CD4⁺ T cell and macrophage (CD68, CD163) but not DC markers (CD11c is shown; CCL13, CCL17, CCL22, and HSD11B1 are included in Supplemental Figure 7). For correlations, a Pearson’s correlation coefficient with a 2-tailed P value is reported. Pearson’s correlations were used to identify genes associated with pretreatment CAILS, CD7, CD40 (as measured by NanoString), or malignant T cell counts (as measured by HTS). Genes with r > 0.8 and P < 0.01 were considered significant.

Figure 6. OX40 is expressed by benign T cells and OX40L is expressed by c-Kit⁺ dendritic cells in mycosis fungoides (MF).

(A) Malignant and benign T cells can be discriminated by costaining for CD3 and the TCR Vβ expressed by the malignant T cell clone, as identified by HTS. Malignant T cells costain for CD3 and the malignant T cell TCR Vβ (yellow) and benign T cells stain only for CD3 (green). Stained cells from a patient with high-burden stage IIA are shown; a second field from the same donor is shown in the last panel. (B) OX40 is expressed by T cells in MF. A patient with high-burden stage IIA CTCL is shown. A second field from the same donor is shown in the last panel. Similar results were observed in a total of 3 donors. (C) OX40 is expressed by benign but not malignant T cells. OX40 and malignant T cell TCR Vβ antibodies stained distinct populations of T cells. A patient with high-burden stage IIA MF is shown. A second field from the same donor is shown in the last panel. Similar results were observed in a total of 3 donors. (D) OX40L is not expressed by T cells in MF. Antibodies specific for CD3 and OX40L stained distinct cell populations. A patient with low-burden stage IA MF is shown. The last panel is a higher magnification view of the same staining; similar results were observed in a total of 5 donors. (E) OX40L is expressed by c-Kit⁺ cells in MF. OX40L and c-Kit were colocalized. Patients with high-burden stage IB (first 3 panels) and high-burden stage IA (fourth panel) MF are shown. Similar results were observed in a total of 3 donors. (F) OX40L-expressing cells are CD11c⁺ dendritic cells. A patient with high-burden stage IA (first 3 panels) and a second patient with high-burden stage IB (fourth panel) MF are shown. Staining demonstrating that OX40L⁺ cells lacked expression of tryptase is included in Supplemental Figure 4. All staining was performed on pretreatment skin biopsies. Results from additional patients are shown in Supplemental Figure 8. A color blind–accessible version of this image is provided in Supplemental Figure 9. Scale bars: 100 μm.

Figure 7. CD40/CD40L interactions may participate in an inflammatory synapse created between c-Kit⁺ dendritic cells, malignant T cells, and benign T cells in mycosis fungoides.

(A) CD40L is expressed by malignant T cells. A patient with high-burden stage IIA (left 3 panels) and high-burden stage IB (right panel) are shown. (B) CD40L is also expressed by c-Kit⁺ dendritic cells. A patient with low-burden stage IA (left 3 panels) CTCL is shown; similar results were observed in a total of 3 donors. Costaining of CD40L⁺ cells with the dendritic cell marker CD11c is shown in the right panel. (C) Both benign and malignant T cells express CD40. The left 2 panels show benign T cells (CD3⁺Vβ^–) expressing CD40. The right 2 panels show malignant T cells (CD3⁺Vβ⁺) expressing CD40. Both fields are from the same high-burden stage IIA donor; similar results were observed in a total of 3 donors. (D) An inflammatory synapse is created between dendritic cells, malignant T cells, and benign T cells. Clusters of dendritic cells, benign T cells, and malignant T cells were frequently observed in MF, as illustrated by this costain for OX40L⁺ dendritic cells, CD3, and the malignant TCR Vβ. A patient with high-burden stage IIB is shown; clusters of these cell types were observed in 5 donors. (E) A proposed model for c-Kit⁺ dendritic cell, benign T cell, and malignant T cell interactions in MF. Benign T cells are recruited by c-Kit⁺ dendritic cell–produced CCL18 and activated by OX40/OX40L and CD40/CD40L interactions leading to visible skin inflammation and protumorogenic signals. Dendritic cells may also directly stimulate malignant T cells via CD40/CD40L interactions. All staining was performed on pretreatment skin biopsies. Results from additional patients are shown in Supplemental Figure 13. A color blind–accessible version of this image is provided in Supplemental Figure 14. Scale bars: 50 μm.

Figure 8. Clearance of the malignant T cell clone after PUVA is associated with recruitment of new CD8⁺ T cell clones that express markers of antigen-specific activation, are locally expanded, and may be tumor specific.

(A) Markers of TCR-dependent, antigen-specific activation (CD40L, ITK, LCK, and ZAP-70) are strongly associated with malignant T cells before but not after therapy and are strongly associated with benign and CD8⁺ T cells after, but not before, therapy. Expression of these genes was correlated with malignant T cell number (assessed by HTS), benign T cells (CD7 gene), and CD8⁺ T cells (CD8A gene) before and after therapy. *P < 0.05. (B) The recruitment of new T cell receptors into the tumor is strongly associated with clearance of the malignant T cell clone. The percentage of total T cell clones bearing antigen receptors not seen before therapy is shown on the x axis and the percentage reduction in the malignant T cell clone is shown on the y axis. A Pearson’s correlation coefficient with a 2-tailed P value is reported. (C) Patients who eradicated or greatly reduced malignant T cells had expanded clonal populations of benign T cells after therapy. The number of expanded benign T cell clones making up greater than 1% of total benign T cells after therapy are shown for patients who had a greater than 90% reduction (>90% red), a 50%–90% reduction (50-90% red), or less than a 50% reduction (<50% red) in malignant T cells after PUVA therapy. The mean and SEM are shown; a Kruskal-Wallis 1-way analysis of variance with a Bonferroni-Dunn post hoc test for multiple means testing was used (α = 0.05). The P value is adjusted for multiple comparison testing. (D–F) An illustrative patient (Pt 5) is shown who had marked reductions in clinical inflammation (D), malignant and total benign T cell numbers in skin (E), but an increase in CD8⁺ T cell–associated genes (F and G). Malignant and benign T cell numbers were determined by HTS (E) and expression counts for T cell–associated (CD3 and CD2) and CD8-associated (CD8A, GZMK, KLRK1, and CD6) genes (F and G) were measured by NanoString.