CD164 identifies CD4+ T cells highly expressing genes associated with malignancy in Sézary syndrome: the Sézary signature genes, FCRL3, Tox, and miR-214

Abstract

Sézary syndrome (SS), a leukemic variant of cutaneous T-cell lymphoma (CTCL), is associated with a significantly shorter life expectancy compared to skin-restricted mycosis fungoides. Early diagnosis of SS is, therefore, key to achieving enhanced therapeutic responses. However, the lack of a biomarker(s) highly specific for malignant CD4⁺ T cells in SS patients has been a serious obstacle in making an early diagnosis. We recently demonstrated the high expression of CD164 on CD4⁺ T cells from Sézary syndrome patients with a wide range of circulating tumor burdens. To further characterize CD164 as a potential biomarker for malignant CD4⁺ T cells, CD164⁺ and CD164^-CD4⁺ T cells isolated from patients with high-circulating tumor burden, B2 stage, and medium/low tumor burden, B1-B0 stage, were assessed for the expression of genes reported to differentiate SS from normal controls, and associated with malignancy and poor prognosis. The expression of Sézary signature genes: T plastin, GATA-3, along with FCRL3, Tox, and miR-214, was significantly higher, whereas STAT-4 was lower, in CD164⁺ compared with CD164^-CD4⁺ T cells. While Tox was highly expressed in both B2 and B1-B0 patients, the expression of Sézary signature genes, FCRL3, and miR-214 was associated predominantly with advanced B2 disease. High expression of CD164 mRNA and protein was also detected in skin from CTCL patients. CD164 was co-expressed with KIR3DL2 on circulating CD4⁺ T cells from high tumor burden SS patients, further providing strong support for CD164 as a disease relevant surface biomarker.

Keywords: CD164; FCRL3; KIR3DL2; Sézary syndrome; Tox; miR-214.

Fig. 1. CD164⁺CD4⁺ T cells from Sézary syndrome patients are the predominant population expressing the Sézary signature phenotype and significantly upregulated expression of FCRL3 and Tox

PBMC from high tumor burden SS patients diagnosed as B2, upper panel (with ≥50% of CD26⁻CD164⁺CD4⁺ T cells, n=15, left graph), and PBMC from medium-to low tumor burden patients diagnosed as B1-B0, lower panel (with ≤50% CD26⁻CD164⁺CD4⁺ T cells, n=9, left graph), were sorted into CD164⁺/CD164⁻ CD4 ⁺ T cells. Each population was assessed for the expression of mRNA for T plastin, GATA-3, STAT-4, FCRL3 and Tox by qRT-PCR. CD4⁺T-cells isolated from PBMC of healthy donors (n=9, pooled) were used as controls. Graphs show relative expression of given mRNA in CD164⁺ and in CD164⁻ CD4⁺ T cells. Error bars represent mean +/− SEM. The nonparametric Wilcoxon signed-rank test was used to assess differences between groups. P values: *** <0.0005; **<0.005; * <0.05.

Fig. 2. CD164 mRNA is expressed in skin from Sézary syndrome and Mycosis Fungoides patients

Skin samples from 7 Sézary syndrome patients (SS), 9 mycosis fungoides patients (MF), 4 atopic dermatitis patients (AD), 3 psoriasis patients (PS) and 9 healthy controls (HC) were examined for the expression of the genes by qRT-PCR. Fold difference for CD164 was calculated versus expression levels in skin from healthy controls. Error bars represent mean +/− SEM. Unpaired t- test used to assess differences. P values: *** <0.0005; **<0.005; * <0.05.

Fig. 3. CD164 is expressed on the cell surface of skin-resident CD4⁺ T cells from Sézary syndrome patients

Fig. 3a demonstrates percentage of CD164⁺ CD4⁺ T cells isolated from skin of 5 Sézary syndrome patients (SS) and 11 healthy controls (HC) analyzed by flow cytometry. Error bars represent mean +/− SEM. P value: * <0.05. Fig. 3b shows a representative histogram illustrating CD164 expression on CD4⁺ T cells isolated from skin of SS patient (left panel) and healthy control (right panel).

Fig. 4. MicroRNA-214 is mainly expressed in CD164⁺CD4⁺ T cells from Sézary syndrome patients

CD4⁺T cells from B2 patients (Fig. 4a, n=17), and from B1-B0 patients (Fig. 4b, n=8) were assessed for the expression of miR-214 by qRT-PCR. The expression of miR-214 was subsequently assessed in sorted CD164⁺/CD164⁻CD4⁺ T cells from B2 patients (Fig. 4c, n=15). Error bars represent mean +/− SEM. P values: **<0.005;

Fig. 5. CD164 and KIR3DL2 are co-expressed on CD4⁺ T cells from Sézary syndrome patients

PBMC from patients diagnosed as B2 (Fig. 5a, n=31) and B1-B0 (Fig. 5b, n=10) were gated on CD3⁺/CD4⁺ T cells and analyzed for the expression of KIR3DL2, CD164 and CD26 by flow cytometry. Graphs show percentage of CD4⁺ T cells from each patient expressing these molecules. Fig. 5c shows that CD164 and KIR3DL2 are co-expressed on CD4⁺T cells from SS patient diagnosed with advanced B2 disease. Error bars represent mean +/− SEM.