Cutaneous T-cell Lymphoma Skin Research Articles

LAIR1 prevents excess inflammatory tissue damage in S. aureus skin infection and Cutaneous T-cell Lymphoma.

Patients with cutaneous T cell lymphoma (CTCL) experience high morbidity and mortality due to S. aureus skin infections and sepsis, but the causative immune defect is unclear. We previously identified high levels of LAIR2, a decoy protein for the inhibitory receptor LAIR1, in advanced CTCL. Mice do not have a LAIR2 homolog, so we used Lair1 knock-out (KO) mice to model LAIR2 overexpression. In a model of subcutaneous S. aureus skin infection, Lair1 KO mice had significantly larger abscesses and areas of dermonecrosis compared to WT. Lair1 KO exhibited a pattern of increased inflammatory responses in infection and sterile immune stimulation, including increased production of proinflammatory cytokines and myeloid chemokines, neutrophil ROS, and collagen/ECM remodeling pathways. Notably, Lair1 KO infected skin had a similar bacterial burden and neutrophils and monocytes had equivalent S. aureus phagocytosis compared to WT. These findings support a model in which lack of LAIR1 signaling causes an excessive inflammatory response that does not improve infection control. CTCL skin lesions harbored similar patterns of increased expression in cytokine and collagen/ECM remodeling pathways, suggesting that high levels of LAIR2 in CTCL recapitulates Lair1 KO, causing inflammatory tissue damage and compromising host defense against S. aureus infection.

Immunosequencing applications in cutaneous T-cell lymphoma.

Immunosequencing has emerged as a newer clinical test for assessment of T-cell clonality in the blood and skin of cutaneous T-cell lymphoma (CTCL) patients. Utilization of immunosequencing, also known as high-throughput sequencing of the T-cell receptor (HTS-TCR), enables identification and quantification of the precise genetic signature of dominant T-cell clones. Although immunosequencing is more sensitive than commonly used methods such as polymerase chain reaction (PCR) paired with capillary electrophoresis or flow cytometry, it remains underutilized for CTCL management. Nonetheless, incorporation of HTS-TCR in clinical practice offers distinct advantages compared to other molecular analyses that may improve diagnostic evaluation, prognostication, and disease monitoring in CTCL. The objective of this comprehensive review is to provide a thorough explanation of the application of immunosequencing in the context of CTCL. We describe the significance of T-cell clonality and the methods used to detect it, including a detailed comparison between PCR paired with capillary electrophoresis and HTS-TCR. The utilization of immunosequencing in the blood and skin of CTCL patients is discussed in depth, specifically outlining how HTS-TCR can assist in diagnosing CTCL, predicting outcomes, and tracking disease progression. Finally, we address the potential applications of immunosequencing in clinical management and research as well as the novel challenges it presents.

Gemcitabine and Liposomal Doxorubicin (GemDox) for the Treatment of Relapsed and Refractory T-Cell Lymphomas

Introduction: Aggressive T-cell lymphomas (TCL) account for 10-15% of non-Hodgkin's lymphomas and have lower rates of complete response (CR), overall response (OR), and shorter duration of response (DOR) to chemotherapy than other types of NHL, particularly in the relapsed setting. Cutaneous T-Cell lymphoma (CTCL) is treated initially with skin directed therapies, but systemic therapy or cytotoxic chemotherapy may become necessary to control disease. Second-line chemotherapy benefits only a small minority of TCL patients and more effective treatment regimens are needed. Gemcitabine and liposomal doxorubicin have both shown single agent activity in TCL and have been used in combination in patients with relapsed B-cell lymphomas (BCL) and Hodgkin lymphomas (HL), though responses are typically not durable. Therefore, we evaluated a combination of gemcitabine and liposomal doxorubicin (GemDox) for patients with relapsed/refractory (r/r) TCL. Methods: In this single-center, retrospective study, we evaluated the safety, efficacy, and survival (PFS/OS using Kaplan-Meier method) of GemDox in TCL. Patients either received gemcitabine 1000 mg/m 2 and liposomal doxorubicin 15 mg/m 2 either on days 1 and 8 every 21 days (21-day regimen) or gemcitabine 1000 mg/m 2 and liposomal doxorubicin 15 mg/m 2 on days 1 and 15 every 28 days (28-day regimen). Results: 18 patients (13 CTCL, 5 PTCL) with r/r aggressive TCL were treated with GemDox, with 16 evaluable for treatment response. Patients were heavily pre-treated with a median number of 4 prior therapies (range 2-7). This included 62% of CTCL patients with nodal involvement. The overall response rate (ORR) was 50% (19% CR and 31% PR) with a median PFS and OS of 5 and 15 months respectively. In patients that had a response to treatment, the median PFS was 11 months while median OS was not reached. The clinical benefit rate [CR/Partial response (PR)/stable disease (SD)] for CTCL and PTCL was 82% and 40% respectively. The median PFS and OS for CTCL patients was 6 months and 14 months respectively, with an ORR of 55% (18% CR and 36% PR). For PTCL , the median PFS and OS were 4 and 21 months respectively, with an ORR of 40% (20% CR and 20% PR). The clinical benefit (CR/PR/SD) rate for the 21-day vs 28-day regimen was 71% and 67%, respectively, with similar PFS and OS, and no significant difference in toxicities. The median duration of response (DOR) for patients that obtained a response (CR/PR) in evaluable patients was 13 months; 17 months for CTCL, and 5 months for PTCL. We observed a substantial decrease in CTCL skin manifestations, as patients that had a CR/PR had a mean decrease of 76% in mSWAT BSA of 70%. Further, six patients (38%) (5 CTCL, 1 PTCL) were able to proceed to allogeneic stem cell transplant (allo-HSCT). Interestingly, 4 CD30+ CTCL patients were previously treated with brentuximab vedotin (BV). Of those, three (75%) had a response to GemDox, while only one achieved a response (PR) to BV. This demonstrates that GemDox is a potentially effective regimen in heavily refractory CD30+ CTCL patients that fail treatment with BV. 11 patients (61%) experienced any grade 3/4 toxicity and hematologic toxicities were the most common including: anemia (33%), neutropenia (22%), lymphopenia (17%) and thrombocytopenia (17%)., GemDox was very well tolerated with the most common grade 3/4 non-hematologic toxicity being fatigue (17%). 11% of patients discontinued therapy due to adverse events. Conclusion: GemDox produced an excellent ORR of 50%, with an impressive ORR of 82% in CTCL, suggesting this is an effective regimen for heavily pre-treated TCL patients. Additionally, a large number of patients with CTCL with heavy skin burden, including those refractory to BV, were able to achieve remarkable skin responses. Importantly, in this study, 75% of patients who responded to GemDox were able to proceed to allo-HSCT, with 66% experiencing a sustained response. Based on these findings, GemDox should be further investigated as a therapeutic option in relapsed aggressive TCL, particularly in CTCL and considered as a potential bridging therapy patients with a treatment goal of obtaining allo-HSCT and providing a long-term benefit.

Skin infiltrating NK cells in cutaneous T-cell lymphoma are increased in number and display phenotypic alterations partially driven by the tumor.

Cutaneous T-cell lymphomas (CTCL) are characterized by focal infiltration of malignant T cell clones in solitary skin lesions. Many CTCL patients experience an indolent disease, but some progress to advanced disease with high fatality. We hypothesized that natural killer (NK) cells participate in local control of tumor growth in CTCL skin. Immunohistochemistry and flow cytometry analysis of the density, localization, phenotype and function of NK cells in twenty-nine fresh or formalin-fixed skin biopsies from twenty-four CTCL patients and twenty-three biopsies from twenty healthy controls highlighted higher numbers of CD56+CD3- NK cells in CTCL skin. A reduced fraction of CTCL skin NK cells expressed the maturation marker CD57, the cytotoxic protein granzyme B and the activation marker CD69, indicating reduced tumor-killing abilities of the NK cells. Retained expression of immune checkpoint proteins or inhibitory proteins including PD1, TIM3, LAG3, CD73 and NKG2A and the activating receptors CD16 and NKp46 indicated maintained effector functions. Indeed, the capacity of NK cells to produce anti-tumor acting IFNγ upon PMA+ionomycin stimulation was similar in cells from CTCL and healthy skin. Co-cultures of primary human NK cells or the NK cell line NKL with CTCL cells resulted in reduced levels of granzyme B and CD69, indicating that close cellular interactions with CTCL cells induced the impaired functional NK cell phenotype. In conclusion, increased numbers of NK cells in CTCL skin exhibit a partially impaired phenotype in terms of activity. Enhancing NK cell activity with NK cell activating cytokines such as IL-15 or immune checkpoint blockade therefore represents a potential immunotherapeutic approach in CTCL.

PD-1 Overexpression in Sézary Syndrome Is Epigenetically Regulated

PD-1 Overexpression in Sézary Syndrome Is Epigenetically Regulated

Endolysin Inhibits Skin Colonization by Patient-Derived Staphylococcus Aureus and Malignant T-Cell Activation in Cutaneous T-Cell Lymphoma

Staphylococcus aureus is suspected to fuel disease activity in cutaneous T-cell lymphomas. In this study, we investigate the effect of a recombinant, antibacterial protein, endolysin (XZ.700), on S.aureus skin colonization and malignant T-cell activation. We show that endolysin strongly inhibits the proliferation of S.aureus isolated from cutaneous T-cell lymphoma skin and significantly decreases S.aureus bacterial cell counts in a dose-dependent manner. Likewise, exvivo colonization of both healthy and lesional skin by S.aureus is profoundly inhibited by endolysin. Moreover, endolysin inhibits the patient-derived S.aureus induction of IFNγ and the IFNγ-inducible chemokine CXCL10 in healthy skin. Whereas patient-derived S.aureus stimulates activation and proliferation of malignant T cells invitro through an indirect mechanism involving nonmalignant T cells, endolysin strongly inhibits the effects of S.aureus on activation (reduced CD25 and signal transducer and activator of transcription 5 phosphorylation) and proliferation (reduced Ki-67) of malignant T cells and cell lines in the presence of nonmalignant T cells. Taken together, we provide evidence that endolysin XZ.700 inhibits skin colonization, chemokine expression, and proliferation of pathogenic S.aureus and blocks their potential tumor-promoting effects on malignant T cells.

CD25 expression could be a prognostic marker of bexarotene monotherapy for cutaneous T-cell lymphomas.

Bexarotene is often administered to phototherapy-resistant early cutaneous T-cell lymphoma (CTCL) patients as one of the first-line therapies in real-world practice. Since bexarotene reduces the expression of CCR4 in CTCL cells and CCL22 to decrease serum CCL22 levels, bexarotene inhibits the migration of CTCL cells, as well as other CCR4+ cells, such as cytotoxic T cells and regulatory T cells, in the lesional skin of CTCL. In this report, the efficacy of bexarotene in 28 cases of CTCL, as well as its correlations with immunohistochemical profiles of tumour-infiltrating leucocytes (TILs), was retrospectively investigated. The overall response rate at 1 and 4months for the total cohort was 70.8% (95% CI, 50.6%-86.3%) and 47.8% (95% CI, 29.2%-67.0%), respectively. The disease control rate for the total cohort at 4months was 65.2% (95% CI, 44.8%-81.3%). The mean event-free survival for all patients was 4.1months (0.3-68.5months). In addition, the immunoreactive cells were calculated using digital microscopy, suggesting that the ratio of CD25+ cells among TILs was significantly increased in patients who responded to bexarotene (p=0.0209), whereas there were no significant differences in the ratios of CD8+ cells, granulysin+ cells, and Foxp3+ cells among TILs between responder and non-responder patients. Collectively, the ratio of CD25 expression among TILs might be a predictive biomarker for the efficacy of bexarotene.

464 CCR8 is a new therapeutic target in cutaneous T-cell lymphomas

Cutaneous T-cell lymphomas (CTCL) are rare non-Hodgkin lymphomas of skin-tropic T cells. Advanced-stage CTCL are frequently refractory to treatment and early relapses are common. There is a clear unmet need for new treatments with long-term efficacy in CTCL. CCR8 is a chemokine receptor expressed by skin resident memory T cells which are suspected to be the tumor cells of origin in mycosis fungoides. CCR8 is also strongly expressed by tumor-infiltrating Treg involved in immune escape. We combined blood flow cytometry in 13 patients with CTCL and healthy controls, in vitro stimulation of CTCL patients cells, Nanostring gene expression analysis, and single cell RNA sequencing analysis on human CTCL skin to study CCR8 expression and function in CTCL.

Receptor Targeted Delivery of the p38γ Inhibitor PIK-75 By Organic-Core Templated Lipid Nanoparticles in Cutaneous T Cell Lymphoma

Introduction: Patients with relapse/refractory cutaneous T cell lymphomas (CTCL) have limited therapeutic options. p38γ, part of the mitogen-activated protein kinases (MAPK) pathway, has been identified as a critical driver of CTCL. PIK-75, a known p110α inhibitor, was found to inhibit p38γ and induced cell death in CTCL cells in vitro and in vivo tumor xenograft models. However, PIK-75 is very hydrophobic and difficult to systemically deliver and achieve clinical efficacy. Therefore, designing a carrier to formulate and enable targeted cellular delivery of PIK-75 is a logical next step to facilitate translation. We, and others, have shown that scavenger receptor type B1 (SR-B1) is often overexpressed in hematologic malignancies and can be targeted by synthetic lipid nanoparticles for efficient drug delivery. Our group pioneered the use of inorganic and organic core (oc) "templates” to synthesize lipid nanoparticles (LNP) that target SR-B1. The ocLNPs are an ideal construct for the delivery of hydrophobic drugs to malignant cells. Therefore, we hypothesized that ocLNPs could be adapted to formulate PIK-75 and deliver this drug via SR-B1 to CTCL cells to induce cell death. Methods: A suite of organic core molecules were synthesized and tested for their ability to support the synthesis of PIK-75 loaded ocLNPs. The PIK-75 ocLNPs were synthesized by combining an organic core with PIK-75, phospholipids, and apolipoprotein A-I. The drug loaded ocLNPs were purified by dialysis and filtration. PIK-75 ocLNPs were characterized for size (size exclusion chromatography, dynamic light scattering and electron microscopy), surface charge (zeta potential) and drug loading. SR-B1 expression in primary CTCL samples was assessed by RNA sequencing. SR-B1 positive CTCL cell lines (HH and HuT78), and SR-B1 positive normal cells, HepG2 (hepatocytes) and THP-1 (macrophages), as well as SR-B1 negative PIK-75-sensitive U266B1 (myeloma) cells were used for these studies. Expression of SR-B1 in all cell lines was confirmed by western blot and flow cytometry. Cell viability was measured using the MTS assay. An SR-B1 blocking antibody was used to demonstrate the necessity of SR-B1 for PIK-75 ocLNP targeting and drug delivery. Results: Nineteen out of 49 primary patient lesional CTCL skin samples (38.8%) demonstrated SR-B1 over-expression, defined as an expression level greater than two standard deviations above the mean expression measured in normal, CD3+ peripheral T cells using RNAseq data. Fourteen of the 19 samples (74%) were from patients with advanced disease (stage III and IV). PIK-75 was successfully incorporated into ocLNPs. Data showed that ~16 PIK-75 molecules were loaded per ocLNP. PIK-75 ocLNPs induced significant cell death in SR-B1 positive CTCL cell lines HH (IC50 = 3.95 nM) and HuT 78 (IC50 = 6.33 nM), while treatment of an PIK-75 sensitive, SR-B1 negative myeloma cell line (U266B1) with PIK-75 ocLNPs resulted in minimal cell death. Treatment of HH cells with PIK-75 ocLNPs for a 2 hour "pulse” followed by 72 hours of additional culture was completed to test active SR-B1 targeting. Data reveal significant cell death, which was significantly reduced by the addition of an SR-B1 blocking antibody (10 nM PIK-75 ocLNPs, viability 31.6% ± 4.3% w/o blocking antibody vs viability 83.6% ± 8.4% w/ blocking antibody). Treatment of hepatocytes and macrophages, both known to express SR-B1, with PIK-75 ocLNPs revealed no cell death even after 72 hours of culture. Conclusions: ocLNPs can be successfully loaded with drug cargo, in this case hydrophobic PIK-75, enabling active delivery via SR-B1 in CTCL. PIK-75 ocLNPs potently induced cell death in CTCL cells while having minimal negative effects on hepatocytes or macrophages. These results provide proof-of-principle that ocLNPs can be successfully leveraged to deliver hydrophobic small molecule drugs by actively targeting SR-B1.

STAT3 Is a Potential Therapeutic Target for Histone Deacetylase Inhibitor-Resistant Cutaneous T-Cell Lymphoma

Background: Histone deacetylase inhibitors (HDACis), such as vorinostat and romidepsin, are effective treatment agents for various T-cell lymphomas, including cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma, and adult T-cell lymphoma/leukemia. Despite the promising anti-lymphoma activity of HDACis, drug resistance remains a significant clinical problem. Therefore, novel therapeutic strategies are required to overcome HDACi resistance. In this study, we generated HDACi-resistant CTCL cell lines and investigated the mechanisms of HDACi resistance. Aims: This study aimed to identify the mechanism of resistance to HDACis in CTCLs and to explore new therapeutic targets. Methods: The CTCL cell lines MyLa, MJ, and Hut78 were used in this study. Vorinostat-resistant cell lines, namely MyLa resistant, HUT78 resistant, and MJ resistant, were prepared by repeatedly exposing cells to increasing concentrations of vorinostat. To maintain drug resistance, the cells were continuously cultured in a medium containing 2.5 μM vorinostat. Following this, we conducted a comprehensive gene expression analysis using a microarray platform to investigate the deregulated genes in the resistant cell lines. Results: CTCL cell lines resistant to vorinostat were also resistant to romidepsin, suggesting that vorinostat-resistant cell lines showed cross-resistance to other HDACis. Next, we selected genes whose expression was upregulated more than 1.5 times in the three resistant cell lines and performed pathway analysis. Pathway analysis revealed that the upregulated genes were significantly enriched in the cytokine-mediated signaling pathways. Among the upregulated genes, we focused on interleukin-6 signal transducer (IL6ST)/glycoprotein (gp) 130, which forms a heterogeneous complex with IL6 and IL6 receptors, and activates the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway. Furthermore, flow cytometric analysis revealed that surface gp130 expression was significantly upregulated in the resistant cell lines. Moreover, the MyLa-resistant and MJ-resistant cell lines showed increased sensitivity to a gp130 inhibitor (SC144). Gp130 activation could activate the JAK/STAT pathway; therefore, we examined the expression level of the activated forms of STAT3 and p-STAT3 and found that p-STAT3 was significantly upregulated in the resistant cell lines. To investigate whether p-STAT3 upregulation is involved in the development of vorinostat resistance, we performed STAT3 knockdown using siRNA. We confirmed that STAT3 knockdown significantly decreased the survival of the resistant cell lines. Moreover, resistant cell lines showed increased sensitivity to the STAT3-selective inhibitor C188-9. However, compared to the controls, the resistant cell lines showed no difference in their sensitivity towards the JAK1/2 inhibitor ruxolitinib. This suggests that the STAT3 activation occurs not only through the JAK/STAT pathway but also through a pathway that is not mediated by JAK. We also examined the expression level of p-STAT5; however, it was downregulated in the resistant cell lines, which was in contrast to that of p-STAT3. These findings suggest that STAT3 activation, which is mediated by HDAC inhibition, could contribute to the resistance of CTCL to HDACis. Finally, we examined the gp130 and p-STAT3 protein expression in CTCL skin samples, which were obtained from the same patients before and after vorinostat treatment. We found that the gp130 and p-STAT3 expression was significantly increased in the specimens during relapse after vorinostat treatment. Conclusion: Activation of STAT3 in HDACi-resistant CTCL cells was enhanced by gp130, which contributed to their resistance. These results suggest that STAT3 could be the new therapeutic target in HDACi-resistant CTCL and that gp130/STAT3 inhibition could be an important therapeutic strategy. To further elucidate the detailed mechanism of HDACi resistance, analysis of the pathways activating STAT3 without JAK and the target proteins of STAT3 is warranted.

110 Non-invasive molecular analysis for CTCL detection and differentiation from other skin conditions with adhesive smart sticker skin sample collection

Cutaneous T cell lymphoma (CTCL) is a complex and heterogenous disease with a range of pathogenic, diagnostic, prognostic and therapeutic courses. Broadly, CTCL is characterized by cutaneous infiltrates of aberrant monocolonal T-lymphocytes. Clinically and histopathologically, CTCL often mimics psoriasis, eczema and other benign dermatoses. Diagnosis of CTCL is often delayed by 4-6 years from the time lesions initially appear, and typically requires multiple skin biopsies and blood tests. In this study, we employed non-invasive adhesive patches to collect samples from lesional skin of CTCL (n=12) and psoriasis (n=10) patients as well as non-lesional skin from healthy (n=12) volunteers.

The Robust Tumoricidal Effects of Combined BET/HDAC Inhibition in Cutaneous T-Cell Lymphoma Can Be Reproduced by ΔNp73 Depletion

Combined BET inhibitor/histone deacetylase inhibitor treatment induces marked apoptosis of cutaneous T-cell lymphoma (CTCL) with minimal normal T-cell toxicity. At 96 hours when apoptosis was extensive, a majority of CTCL lines showed ≥2-fold suppression of T-cell survival factors (e.g., AKT1, BCL2 antiapoptotic factors, BIRC5, CD40, CD70, GADD45A, PRKCA, TNFRSF1B, ΔNp73) and ≥2-fold upregulation of proapoptotic factors and tumor suppressors (e.g., ATM, BAK, BIM, multiple caspases, FHIT, HIC1, MGMT, NOD1) (P < 0.05). The largest alterations were in TP73 isoform expression, resulting in increased TAp73/ΔNp73 ratios in CTCL lines and leukemic Sézary cells. Targeted ΔNp73 inhibition by small interfering RNA knockdown resulted in robust CTCL apoptosis comparable with that induced by BET inhibitor/histone deacetylase inhibitor with minimal normal T-cell toxicity. Chromatin immunoprecipitation analysis showed that BET inhibitor/histone deacetylase inhibitor treatment reduced RNA polymerase II binding to ΔNp73, MYC, and AKT1 while increasing its binding to TAp73. CTCL skin lesions expressed both TAp73 and ΔNp73 isoforms in situ. In aggregate, these findings implicate TAp73/ΔNp73 balance as a major factor governing CTCL survival, show that the expression of p73 isoforms can be altered by molecular biological and pharmaceutical means, show that p73 isoforms are expressed across the entire CTCL clinical spectrum, and identify the p73 pathway as a potential target for therapeutics.

IL-32 Supports the Survival of Malignant T Cells in Cutaneous T-cell Lymphoma

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous collection of non-Hodgkin’s lymphomas derived from T cells that are tropic for the skin. Our understanding of this disease has been hampered by the fact that malignant T cells survive poorly in culture and do not survive well even in sophisticated xenograft models (Townsend et al., 2016). Isolation and culture of T cells from CTCL skin lesions lead to an overgrowth of benign infiltrating T cells and loss of the malignant T-cell clone. It is an inescapable irony that malignant T cells capable of killing patients die in even the most supportive in vitro and in vivo environments.

Increased Chlormethine-Induced DNA Double-Stranded Breaks in Malignant T Cells from Mycosis Fungoides Skin Lesions

Mycosis fungoides (MF) is a type of cutaneous T-cell lymphoma. Chlormethine (CL) is recommended as first-line therapy for MF, with a major purpose to kill tumor cells through DNA alkylation. To study the extent of treatment susceptibility and tumor specificity, we investigated the gene expression of different DNA repair pathways, DNA double-stranded breaks, and tumor cell proliferation of clonal TCR Vβ+ tumor cell populations in cutaneous T-cell lymphoma skin cells on direct exposure to CL. Healthy human T cells were less susceptible to CL exposure than two T-lymphoma cell lines, resulting in higher proportions of viable cells. Interestingly, in T cells from MF lesions, we observed a downregulation of several important DNA repair pathways, even complete silencing of RAD51AP1, FANC1, and BRCA2 involved in homologous recombination repair. In the presence of CL, the double-stranded DNA breaks in malignant MF skin T cells increased significantly as well as the expression of the apoptotic gene CASP3. These data point toward an important effect of targeting CL on MF skin tumor T cells, which support CL use as an early cutaneous lymphoma treatment and can be of synergistic use, especially beneficial in the setting of combination skin-directed therapies for cutaneous T-cell lymphoma.

685 Reprogramming of tumor-associated macrophages with anti-PD-L1 & lenalidomide in cutaneous T cell lymphoma

M2-like tumor-associated macrophages (TAMs) promote the growth of cutaneous T cell lymphoma (CTCL) by inducing immunosuppression. Little is known about the underlying mechanisms of PD1+ M2-like TAM phenotypes in CTCL. Understanding the mechanisms of macrophage reprogramming will identify novel therapeutic targets to induce anti-tumor responses. We used multiplex immunofluorescence on CTCL skin samples demonstrating co-localization of PD1 on CD163+ M2 macrophages. RNA-seq analysis on tissue sections from same CTCL specimens revealed an up-regulation of the TLR/NF-κB and JAK/STAT signaling pathways.

Ganglioside GD3 May Suppress the Functional Activities of Benign Skin T Cells in Cutaneous T-Cell Lymphoma.

In cutaneous T-cell lymphoma (CTCL), which arises from skin-tropic memory T cells, malignant T cells and benign T cells are confined in the same skin lesions. It is thus difficult to evaluate the phenotypic characteristics and functional activities of benign T cells in CTCL. Disialoganglioside with three glycosyl groups (GD3) is increasingly expressed on the surface of solid malignant tumor cells and takes part in tumor progression and suppression of tumor immunity. However, the role of GD3 in CTCL is not well-understood. In this study, the malignant and benign T cells in CTCL skin lesions were distinguished by flow cytometry and their phenotypic characteristics were compared with those of T cells from control skin specimens. In CTCL skin lesions, the benign T cells included limited resident memory T cells (TRM), which are sessile in skin and known to exert strong antitumor function. The benign T cells showed diminished Th17 property, and the expression of GD3 was high in the malignant T cells. The expression of GD3 in the malignant T cells inversely correlated with IL-17A production from the benign CD4 T cells. GD3 from the malignant T cells was implied to be involved in suppressing the Th17 activity of the benign T cells independent of the regulation of TRM differentiation in CTCL. Revealing the role of GD3 in inhibiting the production of IL-17A in CTCL would aid the understanding of the suppressive mechanism of the antitumor activity by malignant tumor cells.

Cutaneous T-Cell Lymphoma Skin Microbiome Is Characterized by Shifts in Certain Commensal Bacteria but not Viruses when Compared with Healthy Controls

Cutaneous T-Cell Lymphoma Skin Microbiome Is Characterized by Shifts in Certain Commensal Bacteria but not Viruses when Compared with Healthy Controls

Expression Profiles of Genes Encoding Cornified Envelope Proteins in Atopic Dermatitis and Cutaneous T-Cell Lymphomas.

The skin barrier defect in cutaneous T-cell lymphomas (CTCL) was recently confirmed to be similar to the one observed in atopic dermatitis (AD). We have examined the expression level of cornified envelope (CE) proteins in CTCL, AD and healthy skin, to search for the differences and their relation to the courses of both diseases. The levels of FLG, FLG2, RPTN, HRNR, SPRR1A, SPRR1B, SPRR3 and LELP-1 mRNA were determined by qRT-PCR, while protein levels were examined using the ELISA method in skin samples. We have found that mRNA levels of FLG, FLG2, LOR, CRNN and SPRR3v1 were decreased (p ≤ 0.04), whereas mRNA levels of RPTN, HRNR and SPRR1Av1 were increased in lesional and nonlesional AD skin compared to the healthy control group (p ≤ 0.04). The levels of FLG, FLG2, CRNN, SPRR3v1 mRNA increased (p ≤ 0.02) and RPTN, HRNR and SPRR1Av1 mRNA decreased (p ≤ 0.005) in CTCL skin compared to the lesional AD skin. There was a strong correlation between the stage of CTCL and increased SPRR1Av1 gene expression at both mRNA (R = 0.89; p ≤ 0.05) and protein levels (R = 0.94; p ≤ 0.05). FLG, FLG2, RPTN, HRNR and SPRR1A seem to play a key role in skin barrier dysfunction in CTCL and could be considered a biomarker for differential diagnosis of AD and CTCL. SPRR1Av1 transcript levels seem to be a possible marker of CTCL stage, however, further studies on a larger study group are needed to confirm our findings.

Interleukin-31 is overexpressed in skin and serum in cutaneous T-cell lymphomas but does not correlate to pruritus.

IntroductionCutaneous T-cell lymphomas (CTCL) are malignant lymphoproliferative disorders accompanied by persistent pruritus. Pruritogenic role of interleukin-31 (IL-31) has been studied extensively and was proven in atopic dermatitis (AD), while its role in CTCL is still rather vague.AimTo investigate IL-31 serum level along with IL-31, IL-31 receptor α (IL-31RA) and oncostatin M receptor β (OSMR) skin expression in CTCL and compare it to controls: AD and healthy volunteers.Material and methodsThe level of IL-31 in serum was measured using ELISA, while IL-31 and receptors’ expression in the skin were measured using immunohistochemistry and correlated with the stage of disease and pruritus severity.ResultsExpression of IL-31 and IL-31 receptor in serum and skin were significantly higher in CTCL and AD in comparison to healthy controls. No significant correlation between the IL-31 serum level and pruritus severity in CTCL patients was found. There was also no correlation between IL-31/IL-31RA/OSMR expression in the skin and CTCL pruritus, while IL-31 and IL-31RA in CTCL skin negatively correlated with the stage of disease.ConclusionsOur data indicate that IL-31 does not play a crucial role in pruritus in CTCL but it is rather involved in the pathogenesis of the disease. It seems that IL-31 plays an essential role in the pruritus pathomechanism that is unique to AD.

Dysregulated Expression of Antimicrobial Peptides in Skin Lesions of Patients with Cutaneous T-cell Lymphoma.

Mycosis fungoides and Sezary syndrome belong to the group of primary cutaneous T-cell lymphomas. Because of the inflammatory appearance of the skin lesions, we hypothesized that antimicrobial peptides might be dysregulated in these conditions, similar to in inflammatory skin conditions. Samples from 20 patients with cutaneous T-cell lymphoma were analysed using immunohistochemistry and enzyme-linked immunoassay (ELISA) of skin washing fluids of hBD-2, hBD-3, RNase 7 and psoriasin. Immunohistochemistry results were compared with previous analyses of healthy and psoriatic skin. ELISA and immunohistochemistry revealed a higher expression of psoriasin in lesional cutaneous T-cell lymphoma compared with non-lesional and healthy samples. Immunohistochemistry showed an increase in hBD-2 in lesional cutaneous T-cell lymphoma skin compared with healthy skin. The expression profile of antimicrobial peptides in cutaneous T-cell lymphoma appears to be dysregulated, indicating a potential role of antimicrobial peptides in cutaneous T-cell lymphoma. A larger prospective study and functional studies are needed to improve our understanding of the role of antimicrobial peptides in cutaneous T-cell lymphoma.