Adult T-cell leukemia/lymphoma cells from blood and skin tumors express cytotoxic T lymphocyte-associated antigen-4 and Foxp3 but lack suppressor activity toward autologous CD8+ T cells

Abstract

Adult T cell leukemia/lymphoma (ATL) cells share the CD4(+)CD25(+) phenotype with regulatory T (Treg) cells. However, it is still controversial whether ATL cells are Treg cells. The aim of the present study was to investigate the Treg nature of ATL cells obtained from peripheral blood and skin tumors in terms of their phenotype and function. By flow cytometry and immunohistochemistry, the expression of the Treg-associated molecule cytotoxic T lymphocyte-associated antigen (CTLA)-4 and Foxp3 was examined in freshly isolated circulating and skin-infiltrating tumor cells from 21 ATL patients with skin eruptions. The expression of CTLA-4 on freshly isolated circulating tumor cells was elevated in two of 15 patients, and Foxp3 was expressed intracytoplasmically at high levels in three of nine patients. In five of the patients examined, skin-infiltrating tumor cells bore variously elevated CTLA-4 with high Foxp3 expression. The potentiality of ATL cells as Treg cells was further addressed by stimulating ATL cells with anti-CD3/CD28 monoclonal antibodies and monitoring CTLA-4 expression. With the stimulation, even CTLA-4-low ATL cells expressed higher levels of CTLA-4 than normal CD4(+)CD25(+) cells. To study function, ATL cells isolated from blood and skin tumors were tested for their ability to suppress the proliferation of autologous CD8(+) T cells stimulated with allogeneic lymphocytes. Despite the expression of CTLA-4 and Foxp3, these tumors were incapable of suppressing the proliferation of autologous CD8(+) T cells. ATL cells are phenotypically Treg cells in at least some patients, but lack immunoregulatory functions, at least toward CD8(+) T cells.

Figure 1

Flow cytometric analyses of surface CCR4 or cytotoxic T lymphocyte‐associated antigen (CTLA)‐4 expression in CD4⁺CD25⁺ cells and intracytoplasmic Foxp3 expression in CD4⁺ cells. Peripheral blood mononuclear cells freshly isolated from adult T cell leukemia/lymphoma (ATL) patients and normal volunteers were stained with peridinin chlorophyll protein‐anti‐CD4, fluorescein isothiocyanate‐anti‐CD25, and phycoerythrin‐anti‐CCR4, CTLA‐4 or Foxp3. (a) As represented by case 3, CD4⁺ cells were gated and the percentages of dual‐positive cells for CD25 and CCR4 or CTLA‐4 were counted. (b) In the gated lymphocytes, the percentage of dual‐positive cells for CD4 and Foxp3 was counted. (c) The percentages of cells expressing both CCR4 and CD25 in circulating CD4⁺ cells from ATL patients (n = 15) and normal subjects (n = 8). (d) The percentages of cells expressing both CTLA‐4 and CD25 in circulating CD4⁺ cells from ATL patients (n = 15) and normal subjects (n = 8). (e) The percentages of cells expressing both Foxp3 and CD4 in circulating lymphocytes from ATL patients (n = 9) and normal subjects (n = 3). Welch's t‐test was used for the significance of data comparison. NS, not significant.

Figure 2

Flow cytometric analyses of surface CD4, CCR4, or cytotoxic T lymphocyte‐associated antigen (CTLA)‐4 expression in CD25⁺ cells in skin‐infiltrating tumor cells. Single‐cell suspensions of skin‐infiltrating tumor cells were prepared and stained with the indicated monoclonal antibodies. (a) As represented by case 17, the percentages of dual‐positive cells for CD4 and CD25, CD25 and CCR4, or CD25 and CTLA‐4 were counted in the broad range of small to large cells. (b) The skin‐infiltrating tumor cells in all five patients tested expressed both CCR4 and CTLA‐4 at high levels. (c) The skin tumor‐infiltrating cells were stained with peridinin chlorophyll protein‐anti‐CD4, fluorescein isothiocyanate‐anti‐CD25, and phycoerythrin‐anti‐Foxp3, and CD4⁺CD25^low, CD4⁺CD25^high, or CD4⁻CD25^low populations were identified. CD4⁺CD25^high cells highly expressed Foxp3 compared to CD4⁺CD25^low or CD4⁻CD25^low cells.

Figure 3

Cytotoxic T lymphocyte‐associated antigen (CTLA) expression of adult T cell leukemia/lymphoma (ATL) cells infiltrating the lesional skin. Immunohistochemical staining was carried out using a monoclonal antibody against CTLA‐4 in biopsy specimens of ATL tumors and normal skin. (a–c) Clinical picture of tumorous skin lesions. (d–f) Hematoxylin–eosin staining showing atypical lymphocyte infiltration in the dermis with epidermotropism. (g–j) CTLA‐4 staining showing positivity of ATL tumor cells, infiltrating from the upper dermis to the epidermis. In the normal skin, CTLA‐4 was negative in skin‐homing lymphocytes. (k–n) Control staining with an isotype‐matched IgG2α,κ monoclonal antibody. Original magnifications: ×100 or ×400.

Figure 4

Increased levels of cytotoxic T lymphocyte‐associated antigen (CTLA)‐4 by T‐cell receptor‐mediated stimulation in CD4⁺CD25⁺ cells from adult T cell leukemia/lymphoma (ATL) patients and normal healthy subjects. Peripheral blood mononuclear cells isolated from ATL patients (n = 8) and normal volunteers (n = 3) were cultured for 12, 24, or 48 h with anti‐CD3 and ‐CD28 monoclonal antibodies, and CD25⁺CTLA‐4⁺ cells in CD4⁺ cells were counted by flow cytometry. Welch's t‐test was used for the significance of data comparison.

Figure 5

Lack of regulatory function of circulating adult T cell leukemia/lymphoma (ATL) tumor cells. (a) CD4⁺CD25⁺ T cells were isolated immunomagnetically, and the purity was always higher than 95%, as represented by case 2. A total of 5 × 10⁴ CD8⁺ T cells from ATL patients and a normal volunteer were cultured with autologous CD4⁺CD25⁺ T cells at a ratio of 1:1 under allogeneic peripheral blood mononuclear cell (5 × 10⁴ cells) stimulation for 6 days. On the last day of culture, the cells were tested for [³H]thymidine incorporation. Error bars represent the mean + SD c.p.m. from triplicate cultures. (b) Normal CD4⁺CD25⁺ T cells suppressed the proliferation of allo‐stimulated CD8⁺ T cells. (c) CD4⁺CD25⁺ T cells failed to suppress the proliferation of allo‐stimulated autologous CD8⁺ T cells in four of the ATL patients tested.

Figure 6

Lack of regulatory function of skin‐infiltrating tumor cells against autologous CD8⁺ T cells. (a) Skin‐infiltrating tumor cells isolated from cases 17, 19, and 20 were cultured with autologous CD8⁺ T cells at various ratios under allogeneic peripheral blood mononuclear cell stimulation for 6 days. Student's t‐test was used for the significance of data comparison. (b) CD4⁺CD25⁺ adult T cell leukemia/lymphoma (ATL) tumor cells were isolated immunomagnetically from a lesional skin tumor of case 17, and the purity was as high as 98.8%, as shown in the flow cytometry. ATL tumor cells and autologous CD8⁺ T cells were used for the allo‐mixed lymphocyte reaction system.

Figure 7

Lack of regulatory function of circulating adult T cell leukemia/lymphoma (ATL) tumor cells against autologous CD4⁺CD25⁻ T cells. CD4⁻ cells obtained from peripheral blood mononuclear cells in two ATL patients (case 2 and 21) and a normal healthy control were pretreated with mitomycin C and used as antigen‐presenting cells (APC). Autologous CD4⁺CD25⁻ T cells were also prepared and used as responder cells. A total of 5 × 10⁴ purified CD4⁺CD25⁻ T cells were cultured with the same number of autologous APC and anti‐CD3 monoclonal antibody at 0.5 µg/mL for 3 days with or without purified autologous CD4⁺CD25⁺ T cells at a ratio of 1:1. On the last day of culture, the cells were tested for [³H]thymidine incorporation. Error bars represent the mean + SD cpm from triplicate culture. (a,b) CD4⁺CD25⁺ ATL cells did not suppress the proliferation of autologous CD4⁺CD25⁻ non‐ATL cells. (c) In a normal healthy control, Treg cells suppressed the CD4⁺CD25⁻ T cells.