Skin liquid biopsy method for assessing the immune environment of cutaneous T-cell lymphoma lesions


 (179/200 words) Detailed analysis of cells infiltrating lesional skin cannot be performed in skin biopsy specimens by immunohistochemistry or cell separation techniques because small amounts of protein and minor cell populations in the biopsy specimen might be destroyed by enzyme treatment in the isolation step. Here, we describe a skin liquid biopsy method that enables T cell isolation in small amounts of lesional whole blood from patients with cutaneous T-cell lymphoma. Lesional blood, assumed to contain lesional resident cells, cells from capillary vessels, and blood overflowing from capillary vessels in the lesion area, was obtained during regular skin biopsy. The lesional blood showed substantial increases in distinct cell populations, chemokines, and expression of various genes. CD8 + CD45RO + T cells in the lesional blood negatively correlated with the modified severity-weighted assessment tool scores. CD4 + CD45RO + T cells in the lesional blood expressed genes associated with the development of cancer and progression of cutaneous T-cell lymphoma. The skin liquid biopsy technique might provide new insight into the pathogenesis of mycosis fungoides and facilitate evaluation of the treatment efficacy for other skin inflammatory diseases.


Introduction
The tissue environment surrounding skin lesions has an important role in skin disease. Harvesting cells from the lesions can be time-consuming and challenging due to considerable cell and protein loss caused by tissue degradation. Various techniques are used to analyze skin lesion cells and the surrounding environment, including multiphoton excitation microscopy [1], dermal open-ow microperfusion [2], and immersion of skin samples in a medium to extract cells [3]. The cell isolation processes required for these techniques, however, may lead to the loss of critical information. For example, while lymphocytes can be isolated from skin tissue obtained by punch biopsy, the small amount of tissue contains too few cells and thus limited information is provided.
Alternatively, lesional blood samples could provide valuable information regarding the surrounding environment, including the levels and types of cytokines and in ammatory cells, without requiring enzyme treatment. In fact, a previous study successfully used sera from the peripheral blood and from blood obtained from psoriasis lesions to assess the skin lesion environment [4]. Therefore, liquid biopsy of skin lesions might be effective for isolating and analyzing cellular components and serum.
Skin biopsies are regularly obtained to diagnose and assess treatment e cacy for cutaneous T-cell lymphoma (CTCL). Diagnosis of CTCL is relatively di cult [5], however, and effective treatments are not yet clearly established. Better methods that allow for rapid isolation and analysis of resident and systemic pathogenic T cells and effector T cells are necessary to facilitate diagnosis and develop effective treatments. Mycosis fungoides (MF), the most common CTCL, is considered to be a low-grade T-cell lymphoma [6]. The premycotic and mycotic phases can last for several years, but in some cases develop very rapidly [7]. Due to the relatively low awareness and diagnostic di culties of MF, many patients seek dermatologic consultation for the rst time after their condition has already progressed to the mycotic or tumor stage. This delay in diagnosis may lead to tumor formation, ulceration, leukemic transformation, visceral invasion, and death within a few months. The histologic ndings depend on the disease stage. In the erythema stage (stage I), the characteristic features include epidermal hyperplasia, lymphoid exocytosis, and band-like lymphoid in ltration in the super cial dermis. In the plaque stage (stage II), Pautrier's microabscesses are often observed. In the tumor stage (stage III), tumor cells in ltrate the nodular lesions and proliferate with necrosis causing the subsequent development of ulcers in the tumorous lesions [7]. Cells in ltrating MF lesions have an α/β memory T-helper phenotype. In the advanced tumor stage, T-cell markers may be lost and a T-cytotoxic phenotype is observed. Different biopsies of MF lesions obtained from a single patient within a short period of time may reveal several phenotypes [8]. The effector cells that in ltrate MF lesions, however, remain unclear.
A skin liquid biopsy technique could be useful for obtaining more detailed information about the phenotype and immune microenvironment in MF.
In the present study, we performed a skin liquid biopsy, i.e., the collection of a small amount of blood from the lesion site during the skin lesion biopsy to determine the types of T cells induced in MF while concurrently analyzing RNA expression. We were also able to determine the functions of the in ltrating T cells with high accuracy. The development of an effective skin liquid biopsy method may also be useful for evaluating other skin in ammatory diseases, such as atopic dermatitis and psoriasis, as well as their treatment e cacies.

Results
CD4 + /CD8 + T cells are successfully isolated from a small amount of lesional blood.
We obtained 200 to 300 µL of lesional blood from the wound site resulting from the skin biopsy. The cells were separated from the sample using a cell sorter. Approximately 3000 CD4 + T cells and 1000 CD8 + T cells were successfully isolated from 5 µL of peripheral blood (Fig. 1a). The number of cells in lesional blood was slightly lower than that in peripheral blood, but we could collect cells from lesional blood ( Fig. 1b and c). Cytometry by time-of-ight revealed that the lesional blood contained more granulocytes, and fewer monocytes and B cells than the peripheral blood ( Fig. 1d and e). These ndings indicate that the cell population in the lesional blood might differ from that in the peripheral blood. The isolated cells and sera from lesional blood were therefore further analyzed.
Fourteen biopsy specimens were obtained from the lesional skin of MF patients and used for immunohistochemical analysis (Table 1). All MF patients were assessed using the modi ed severity-weighted assessment tool (mSWAT), and skin biopsies were obtained upon admission (Fig. 2a).
While the proportions of CD4 + and CD8 + T cells did not differ signi cantly between lesional blood and peripheral blood in the mass cytometry analysis (Fig. 1e), lesional blood contained signi cantly a greater proportion of CD4 + CD45RO + and CD8 + CD45RO + T cells ( Fig. 2b and c).
Furthermore, the proportion of CD8 + CD45RO + T cells in the lesional and peripheral blood negatively correlated with the mSWAT score (Fig. 2e). The proportion of CD4 + CD45RO + T cells was weakly inversely correlated with the mSWAT score (Fig. 2d). The skin liquid biopsy technique might reveal the phenotypic details of in ltrating cells.  [9]. Therefore, increased levels of CD8 + T cells in CTCL serve as a promising criterion for predicting patient survival and supporting treatment decisions and inclusion of patients in randomized controlled trials [10]. Moreover, the partial activation of CD8 + cytotoxic T cells present in CTCL and their correlation with a better prognosis suggest that they have an important role in the antitumor response [11]. Tissue specimens showed a negative correlation between the mSWAT score and CD8 + CD45RO + T cells with less in ltration of effector CD8 + T cells in advanced cases ( Fig. 2h and i). In contrast, CD4 + CD45RO + T cells in tissue specimens did not correlate with mSWAT ( Fig. 2f and g), consistent with the results from skin liquid biopsy. These in ltrating cells in tissue specimens, however, cannot be easily assessed by only immunohistochemistry.
Chemokine pro les differ between lesional blood and peripheral blood.
In sera simultaneously isolated from peripheral and lesional blood samples, the levels of chemokines such as CCL5, CCL11, CCL17, CCL22, and CXCL11 were signi cantly increased in the lesional blood compared with the peripheral blood (Fig. 2j). The increases in these chemokines are speci c to MF lesions. CCL5, CCL17, and CCL22 are derived from keratinocytes, CCL22 and CXCL11 are derived from endothelial cells, and CCL11 is derived from macrophages in MF lesions [12]. In particular, levels of CCL17 and CCL22, a CCR4 ligand, are upregulated in the epidermis and serum of patients with MF [13][14][15]. Malignant T cells expressing CCR4 are recruited by CCL17 and CCL22 [14]. We found that sera from lesional blood revealed a speci c chemokine environment for MF. The results demonstrated that the chemokine pro le of lesional blood differs from that of peripheral blood, indicating that the skin liquid biopsy technique is feasible for obtaining a detailed chemokine pro le of lesional blood.
CD8 + CD45RO + T cells from lesional and peripheral blood differ in RNA sequence and T-cell receptor repertoire analyses.
On the other hand, CD8 + CD45RO + T cells highly expressed the following representative genes in lesional blood samples: CDKN2B, PTPRT, CREB3L1, CADM4, and INPP5F (Fig. 3e). These genes encode tumor suppressor proteins [26-30] and activate the expression of genes encoding cell cycle inhibitors, including p21 [31]. Gene ontology analysis using 468 genes was performed in CD8 + CD45RO + T cells from MF samples. Among the 10 most enriched biologic processes determined using Metascape in CD8 + CD45RO + T cells isolated from MF samples, the most enriched biologic processes were "negative regulation of intracellular signal transduction", "negative regulation of STAT cascade", and "negative regulation for cellular response to growth factor stimulation" (Fig. 3f). A previous study reported that effector cells (CD8 + CD45RO + T cells) express exhausted phenotypes [32]. Our ndings con rmed that CD8 + CD45RO + T cells from lesional blood negatively regulate cellular responses. On the other hand, genes related to in ammation (S100A12, S100A8, HCK, IL1B and KLF4) were increased in peripheral blood [33][34][35][36]. The TCR repertoire of CD8 + CD45RO + T cells was skewed in lesional blood (erythema area; n = 4) compared with that in peripheral blood ( Fig. 3g and h). These expanded CD8 + CD45RO + T cells might be tumor antigen-speci c but not have the capacity to suppress CTCL cells.
Stage progression and skewed TCR repertoires in CD8 + CD45RO + T cells.
Biopsy specimens and lesional blood were collected from each stage: erythema, plaque, and tumor-stage lesions from the same patient. A certain number of CD8 + CD45RO + cells were found in the erythema areas, but few were found in the plaque and tumor tissue in case 10 ( Fig. 4a, c, e; immuno uorescence staining). We isolated CD8 + CD45RO + T cells from the lesional blood and peripheral blood separately. The isolated cells were subjected to TCR repertoire analysis. The CD8 + CD45RO + T cells in lesional blood showed unique TCR repertoires in lesions of all stages (Fig. 4b, d, f, g). We assume that CD8 + CD45RO + cells in lesional blood would recognize a tumor antigen in tumor and plaque tissue. In previous reports, malignant T-cell clones exhibit heterogeneity in each skin lesion [37]. Our results are consistent with previous ndings that neoplastic T-cell clones vary in skin lesions. Furthermore, different TCR repertoires were present in tumor-stage lesions of the same patient (Fig. 4f), which may result from the generation of different neoplastic T-cell clones for the growth of tumor lesions. In the other patient (case 15), a biopsy was performed from an adjacent lesion, which developed erythema and plaque. CD8 + CD45RO + cells were found in both erythema and plaque lesions ( Fig. 4h and j), and repertoire analysis showed that the same TCRs (TRAV1-2 -TRAJ33) were increased (Fig. 4i, k, l). These results indicate CD8 + T cells with different TCR repertoires are directed for each skin lesion, which is expected given the heterogeneity of malignant T cells in skin lesions.

Discussion
The present study revealed that the composition of lesional blood differs from that of peripheral blood, and lesional blood appears to re ect the condition of the skin's immune environment of lesional areas in MF patients. Many techniques are used to analyze the cells and tissue environments of skin lesions, and the skin liquid biopsy method is a feasible technique for assessing lesion areas. Although the skin liquid biopsy method would not obviate the need for skin biopsy due to the different results, analysis of lesional blood could provide valuable information about in ltrating cells and the separated serum. We assume that lesional blood would contain lesional resident cells, cells from capillary vessels, and blood over owing from capillary vessels, allowing us to identify a unique pattern of cell populations and chemokines in the lesional blood.
Mass cytometry analysis showed differences in the cell populations, and the chemokine assay revealed increases in speci c chemokines in lesional blood. The different types of cells in the lesional blood may be due to the induction of chemokines. Memory T cells, CD45RO + T cells, were frequently obtained in lesional blood, suggesting that these cells are involved in the lesion. The chemokines, which are reported to be associated with MF, were particularly abundant in lesional blood. CCL17 and CCL22 are ligands for CCR4, and these chemokines are strongly associated with the pathogenesis of MF [13,14]. Although we have not yet collected lesional blood from healthy areas, we will be able to clarify the pathogenesis of MF in detail by comparing lesional blood from healthy and lesional areas.
RNA-seq of CD4 + CD45RO + T cells and CD8 + CD45RO + T cells suggested that lesional blood might contain malignant T cells and effector T cells, respectively. This notion was supported by the skewed TCR repertoire in lesional blood ( Fig. 3c and g). In the present study, CD4 + CD45RO + T cells from the lesional blood highly expressed genes relating to cancer progression and CTCL progression. It may thus be possible to isolate lymphocytes that are recruited by chemokines or present in the lesion area from lesional blood. A larger number of differentially expressed genes were detected in the CD4 + CD45RO + cells of the peripheral blood because the cells of lesional blood have individual variations. In addition, there are different types of malignant cells [38], which may be why fewer differentially expressed genes were detected in the CD4 + CD45RO + cells of lesional blood (Fig. 3a).
CD8 + CD45RO + T cells in the lesional blood had a less in ammatory transcriptome than the CD8 + CD45RO + T cells in the peripheral blood. Effector T cells express exhausted phenotypes characterized by the expression of the PD-1, ICOS, TIM-3, LAG-3, and CTLA-4 markers in lesional skin [32]. Although the expression of these genes was not increased, the weak expression of genes of the in ammatory system and cell division suggest that CD8 + CD45RO + T cells in the lesional blood are a type of exhausted T cells [39].
The clones of malignant cells are different in skin lesions in MF [40]. In the present study, we collected CD8 + CD45RO + cells of lesional blood and performed TCR repertoire analysis by skin lesion. Few CD8 + CD45RO + cells, however, were detected in the plaque and tumor tissue in case 10. A characteristic repertoire pattern of CD8 + CD45RO + cells was detected in different skin lesions of case 10.
Case 15 showed a more skewed repertoire of the plaque tissue compared with an area of erythema from the same skin lesion. Although the lesional blood would not be a perfect representation of the cells in this lesion, we could indirectly show that different skin lesions have different clones of malignant cells. We need to further increase the number of cases and investigate the details in the future.
In conclusion, a skin liquid biopsy method for assessing the immune environment of CTCL was successfully developed in this study. Su cient numbers of isolated cells and amounts of sera were successfully obtained from a small amount of lesional blood collected by skin liquid biopsy. The cells and sera isolated from lesional blood can also be used for further gene expression analysis of target cells. Our results might provide new insight into the pathogenesis of MF, a rare cutaneous lymphoma. The technique described in this study could be further applied to evaluate other skin in ammatory diseases, such as atopic dermatitis and psoriasis, as well as for assessing the e cacy of treatments for these diseases.

Methods
Patients. We recruited 19 patients with MF (mean age: 67.10 ± 13.91 years; 9 women, 10 men) from the Department of Dermatology at the Nagoya City University. Exclusion criteria were: 1) age under 20 years, 2) HTLV-1 positive status, and 3) pregnant. The institutional review board of the Nagoya City University Graduate School of Medical Sciences approved the study (approval number: #60-18-0101). Written informed consent was obtained from the patients. All the experimental protocols adhered to relevant ethical guidelines for involving humans. The patients' pro les are described in Table 1. Samples were obtained from the rst 14 patients who visited our department from December 2018 to December 2019 and used for tissue staining, ow cytometry analyses, and chemokine assays. Some samples were subjected to RNA-seq (cases 4 and 12) and TCR repertoire analyses (cases 4, 10, 11, and 14). Samples obtained from cases 15 through 19 were used for RNA-seq or TCR repertoire analyses (Table 1).
Lesional blood collection. Skin biopsies were regularly obtained from the patients for diagnosis and assessment of treatment e cacy. After administering local anesthesia using lidocaine without epinephrine to the skin lesion, a punch biopsy was performed at a depth of 1-to 3-mm to avoid reaching the fatty layer. Oozing blood from the wounded area was collected as quickly as possible into an Eppendorf tube containing anticoagulant to avoid clotting. As anticoagulants, we used 5 µL of 100 U mL − 1 heparin sodium (TERUMO) for ow cytometry analysis, and 3 µL of 0.5 mol L − 1 EDTA (Invitrogen) for the RNA-seq and TCR repertoire analyses. Approximately 200 to 300 µL of blood was collected using a P20 Pipetman (Gilson) from each lesion area. We also collected 30 to 50 µL of lesional blood without anticoagulant to obtain serum. After collecting the lesional blood, we performed another punch biopsy in the same lesion area, again at a su cient depth to obtain a skin sample. Serum was collected and stored at -80°C until analysis. We collected peripheral blood from the patient's arm and treated the peripheral blood in the same manner as the lesional blood.
Mass cytometric immunoassay. The blood cells were stained for mass cytometry after hemolysis using a Maxpar Human Peripheral Blood Phenotyping Panel kit (Fluidigm). Hemolyzed peripheral blood and lesional blood samples were resuspended in 1 mL phosphate-buffered saline and incubated for 5 min at room temperature with 1 mL of Cisplatin-108Pt (Fluidigm). The cells were washed using Maxpar Cell Staining Buffer (Fluidigm) and centrifuged, and the supernatant discarded; the pellets were resuspended in 50 µL of the same buffer. We then added 50 µL of a prepared cocktail of titrated Maxpar metal-conjugated antibodies (Fluidigm). After incubating for 15 min at room temperature, we washed the cells twice and xed them with 2% paraformaldehyde. The stained cells were analyzed by St. Luke's MBL Corp using cytometry by time-of-ight. Mass cytometry data were analyzed using Cytobank (https://www.cytobank.org/).
Cell sorting. CD4 + CD45RO + and CD8 + CD45RO + T cells were sorted using the BD FACSMelody Cell Sorter (BD Bioscience). The sorted T cells were collected for the TCR repertoire and RNA-seq analyses as described below.
RNA-seq analysis. The CD4 + CD45RO + and CD8 + CD45RO + T cells were prepared as described above. The T cells were lysed with TRIzol reagent (Thermo Fisher Scienti c) and stored at -80°C. The lysates were sent to Genewiz Japan Corp for RNA-sEq. RNA-seq and related analyses were completed by Genewiz Japan Corp. In brief, RNA was extracted with chloroform/isopropanol and recovered from the supernatants using RNA Clean and Concentrator-5 columns (ZymoResearch) following the manufacturer's instructions. The RNA purity was assessed with an Agilent 2100 Bioanalyzer. The RNA was subjected to library preparation with the TaKaRa SmartSeq Stranded Kit (Takara Bio) and sequenced with Illumina Hiseq (Illumina). Sequences were mapped to grch38 with HISAT2 (version 2.0.1). Differentially expressed genes were counted using the DESeq2 package in R (version 3.6.3). Up-and downregulated genes were de ned as those (i) differentially expressed in peripheral and lesional blood cells with a Pvalue lower than 0.05, and (ii) having a greater than 2-fold change in the average normalized number of peripheral and lesional blood cells. Gene ontology analysis and enrichment analysis using the Jensen DISEASES dataset of differentially expressed genes was performed using the Enrichr webtool (https://maayanlab.cloud/Enrichr/) and Metascape (https://metascape.org/) [41][42][43].
TCR repertoire analysis. CD4 + CD45RO + and CD8 + CD45RO + T cells were prepared as described above. The T cells were lysed with Isogen-LS (NIPPON GENE) and stored at -80°C. The lysates were sent to Repertoire Genesis Inc. (Ibaraki, Japan) for next-generation sequencing, which was performed as previously described [44]. Brie y, total RNA was converted to complementary DNA (cDNA) with the SuperScript reverse transcriptase (Invitrogen). Double-stranded -cDNA was synthesized and ligated with a 5′ adaptor oligonucleotide, then cut with the SphI restriction enzyme. Next, the double stranded-cDNA was ampli ed through polymerase chain reaction using primers speci c for the adaptor and TCRα constant region. The sequencing was performed with the Illumina MiSeq paired-end platform (2 × 300 bp). Data processing was performed with the repertoire analysis software developed by Repertoire Genesis Inc. TCR sequences were assigned with a dataset of reference sequences from the international ImMunoGeneTics information system database (http://www.imgt.org). The Shannon-Weaver index shows the diversity and is de ned as follows.
Statistics. Statistical analyses were performed using GraphPad Prism 7. All numerical data are summarized using mean ± standard deviation.
Paired or unpaired Student t-tests were used to determine the signi cance of differences between groups, unless otherwise indicated in the gure legend. P-values < 0.05 were considered statistically signi cant.  . Quanti cation of the number of cells per visual eld was performed using the Hybrid Cell Count BZ-H4C analyzer software. Data were statistically analyzed using the Pearson correlation test (2-tailed). j) Results of multiplex chemokine bead assay using sera from peripheral and lesional blood (n = 14). Paired t-test. * P < 0.05, ** P < 0.01.

Supplementary Files
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