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-flight revealed that the lesional blood contained more granulocytes, and fewer monocytes and B cells than the peripheral blood (Fig. 1d and e). These findings 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.
CD8 + CD45RO + T-cells in lesional blood negatively correlate with the mSWAT score.
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 modified 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 significantly between lesional blood and peripheral blood in the mass cytometry analysis (Fig. 1e), lesional blood contained significantly 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 infiltrating cells.
Table 1
Pt
|
Age
(yr)
|
Sex
|
Disease
|
Stage
|
mSWAT
|
CD8/45RO
(%, PB)
|
CD8/45RO
(%, LB)
|
Tissue
CD8/45RO
|
CyTOF
|
RNA-seq
|
TCR repertoire
|
Treatment
|
1
|
83
|
M
|
MF
|
IB
|
44
|
10.4
|
11.2
|
45
|
|
|
|
Chemo + PUVA
|
2
|
68
|
M
|
MF
|
IB
|
49
|
0.18
|
0.5
|
45
|
|
|
|
Chemo
|
3
|
61
|
F
|
MF
|
IIIA
|
64
|
3.25
|
3.54
|
44
|
|
|
|
Chemo + PUVA
|
4
|
88
|
F
|
MF
|
IB
|
38
|
7.69
|
9.18
|
89
|
|
CD8/45RO
|
CD8/45RO
|
PUVA
|
5
|
71
|
F
|
MF
|
IB
|
102
|
3.12
|
4.03
|
19
|
○
|
|
|
Chemo + PUVA
|
6
|
82
|
M
|
MF
|
IB
|
40
|
0.65
|
0.72
|
67
|
○
|
|
|
Chemo
|
7
|
56
|
F
|
MF
|
IB
|
36
|
2.22
|
2.33
|
71
|
○
|
|
|
Chemo + PUVA
|
8
|
70
|
M
|
MF
|
IIIA
|
138
|
0.34
|
0.26
|
13
|
|
|
|
Chemo + PUVA
|
9
|
78
|
F
|
MF
|
IB
|
22
|
1.71
|
3.81
|
135
|
○
|
|
|
Chemo + PUVA
|
10
|
71
|
M
|
MF
|
IIB
|
109
|
0.76
|
0.97
|
23
|
○
|
|
CD8/45RO
|
Chemo + PUVA
|
11
|
64
|
F
|
MF
|
IB
|
70
|
2.3
|
2.4
|
82
|
|
|
CD4/45RO
|
Chemo + PUVA
|
12
|
72
|
F
|
MF
|
IB
|
39
|
12.6
|
11.8
|
167
|
|
CD4/45RO
|
|
Chemo + PUVA
|
13
|
78
|
M
|
MF
|
IA
|
15
|
13
|
15
|
178
|
|
|
|
PUVA
|
14
|
72
|
M
|
MF
|
IB
|
22
|
1.5
|
1.9
|
154
|
|
|
CD8/45RO
|
Chemo + PUVA
|
15
|
45
|
F
|
MF
|
IA
|
14
|
1.88
|
2.3
|
|
|
|
CD8/45RO
|
Chemo + PUVA
|
16
|
67
|
M
|
MF
|
IIIA
|
94
|
1.16
|
1.81
|
|
|
CD4/45RO
|
|
Chemo
|
17
|
45
|
M
|
MF
|
IB
|
26
|
1.71
|
1.7
|
|
|
CD4/45RO
|
|
Chemo + PUVA
|
18
|
70
|
M
|
MF
|
IB
|
35
|
3.42
|
3.44
|
|
|
CD8/45RO
|
|
Chemo + PUVA
|
19
|
34
|
F
|
MF
|
IB
|
40
|
1.26
|
1.45
|
|
|
CD8/45RO
|
|
Chemo + PUVA
|
All MF patients are CD4+ MF and underwent skin biopsies on admission treatment. MF: Mycosis Fungoides, Chemo: Chemotherapy (bexarotene), PUVA: Psoralen and Ultraviolet A. |
CD8 + CD45RO + T-cells infiltrate MF lesions and negatively correlate with CTCL pathogenesis. The CD8+ tumor-infiltrating lymphocyte levels in patients with MF correlate with an improved survival rate and exert an antitumor effect [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 infiltration 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 infiltrating cells in tissue specimens, however, cannot be easily assessed by only immunohistochemistry.
Chemokine profiles 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 significantly increased in the lesional blood compared with the peripheral blood (Fig. 2j). The increases in these chemokines are specific 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 [13–15]. Malignant T cells expressing CCR4 are recruited by CCL17 and CCL22 [14]. We found that sera from lesional blood revealed a specific chemokine environment for MF. The results demonstrated that the chemokine profile of lesional blood differs from that of peripheral blood, indicating that the skin liquid biopsy technique is feasible for obtaining a detailed chemokine profile of lesional blood.
CD8 + CD45RO + T cells from lesional and peripheral blood differ in RNA sequence and T-cell receptor repertoire analyses.
We isolated CD4+CD45RO+ and CD8+CD45RO+ T cells from lesional and peripheral blood (n = 3) using a FACS Melody sorter (Becton Dickinson). RNA sequencing (RNA-seq) of the isolated cells was performed to analyze the transcriptome. CD4+CD45RO+ T cells in the lesional blood highly expressed genes relating to cancer progression and CTCL pathogenesis: RGS1, RYR2, DNAH9, ANK2, and TNFRSF21 (Fig. 3a) [16–25]. To further confirm the increases in the 51 highly expressed genes, the genes were enriched in the Jensen Disease library of Enrichr. Enrichr showed that the diseases were related to cancer, including “skin cancer” and “lymphoid leukemia” (Fig. 3b). Although we performed T-cell receptor (TCR) repertoire analysis only for 1 case, CD4+CD45RO+ T cells in the lesional blood exhibited a unique TCR repertoire, showing reduced diversity compared with the TCR repertoire in peripheral blood (Fig. 3c and d).
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 findings confirmed that CD8+CD45RO+ T cells from lesional blood negatively regulate cellular responses. On the other hand, genes related to inflammation (S100A12, S100A8, HCK, IL1B and KLF4) were increased in peripheral blood [33–36]. 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-specific 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; immunofluorescence 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 findings 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.