High-scatter T cells: a reliable biomarker for malignant T cells in cutaneous T-cell lymphoma

Abstract

In early-stage cutaneous T-cell lymphoma (CTCL), malignant T cells are confined to skin and are difficult to isolate and discriminate from benign reactive cells. We found that T cells from CTCL skin lesions contained a population of large, high-scatter, activated skin homing T cells not observed in other inflammatory skin diseases. High-scatter T (T(HS)) cells were CD4(+) in CD4(+) mycosis fungoides (MF), CD8(+) in CD8(+) MF, and contained only clonal T cells in patients with identifiable malignant Vβ clones. T(HS) cells were present in the blood of patients with leukemic CTCL, absent in patients without blood involvement, and contained only clonal malignant T cells. The presence of clonal T(HS) cells correlated with skin disease in patients followed longitudinally. Clonal T(HS) cells underwent apoptosis in patients clearing on extracorporeal photopheresis but persisted in nonresponsive patients. Benign clonal T-cell proliferations mapped to the normal low-scatter T-cell population. Thus, the malignant T cells in both MF and leukemic CTCL can be conclusively identified by a unique scatter profile. This observation will allow selective study of malignant T cells, can be used to discriminate patients with MF from patients with other inflammatory skin diseases, to detect peripheral blood involvement, and to monitor responses to therapy.

Figure 1

CTCL skin lesions contain a high-scatter population of T cells not seen in normal skin or lesional skin from 3 other T cell–mediated inflammatory skin disorders. (A) T cells were isolated from normal human skin (12 patients) or lesional skin from patients with atopic dermatitis (6 patients), contact dermatitis (1 patient), or psoriasis (6 patients) with the use of short-term explant cultures. All samples tested contained a single population of CD3⁺ T cells. (B) T cells were then isolated from CTCL skin lesions with the use of short-term explant cultures. Patients with all stages of CTCL exhibited 2 clear populations of T cells. The lower-scatter population had a forward scatter (reflecting size) and a side scatter (reflecting complexity) similar to that of T cells from normal skin, whereas the high-scatter population consisted of larger and more complex cells. Eight representative patients are shown, similar results were obtained in 30 additional patients. SSC-A indicates side-scatter area; FSC-H, forward-scatter height; PerCP, peridin chlorophyll protein; APC, allophycocyanin.

Figure 2

High-scatter T cells from CTCL lesional skin are highly activated CD4⁺ skin homing T cells with elevated FOXP3 expression. (A) Comparison of the phenotype of high- and low-scatter T cells isolated from MF lesional skin. T_HS cells were uniformly CD4⁺ and expressed high levels of the activation markers CD69 and CD25 and the skin-homing addressins CCR4 and CLA. In contrast, low-scatter T cells contained a mixed CD4 and CD8 T cells with variable expression of activation markers and skin-homing addressins. Similar results were observed in 6 additional patients with stage I-III CTCL. (B) MF lesional skin contains a unique population of T cells with intermediate CD4 and FOXP3 expression. MF skin lesions contained both FOXP3^high regulatory T cells (Tregs), also found in normal skin, and a novel population of T cells expressing intermediate levels of both CD4 and FOXP3. (C) Selective gating on this novel population of T cells (CD4^intFOXP3^int) showed that it corresponded closely to the T_HS cell population. Reversing the gating confirmed that the high-scatter T-cell population was comprised entirely of these CD4^intFOXP3^int T cells. A second patient with stage IB disease is also shown. Similar findings were observed in 4 additional patients. (D) In a patient with CD8⁺ MF diagnosed by histopathologic studies, a clonal population of CD8⁺ T cells expressing TCR Vβ 13.2 existed in lesional skin. T_HS cells in this patient were CD8⁺. SSC-A indicates side-scatter area; FSC-H, forward-scatter height. In gated histograms, the % total cells in each quadrant are shown.

Figure 3

T_HS cells from the lesional skin of L-CTCL and advanced MF are clonal and malignant. (A) Spectratype analysis was used to identify clonal populations of T cells in the blood of patients with L-CTCL. The number of individual peaks within each Vβ subfamily is reflective of the T-cell receptor diversity within that subfamily. Patient 151 had a clonal T-cell population expressing TCR Vβ 11.1. (B) Flow cytometric studies confirmed the presence of an expanded clonal population of CD4⁺ T cells in the blood that expressed TCR Vβ 11.1. This population had high expression of CCR4 and variable CLA expression. (C) T cells isolated from the involved skin of this patient contained 2 clear T-cell populations. The T_HS cell population was composed entirely of clonal and malignant T cells. Similar findings were observed in 3 additional patients with L-CTCL. (D-E) High-scatter lesional skin T cells from patients with MF with identifiable T-cell clones were uniformly clonal and malignant. T cells were isolated from lesional skin and TCR diversity was analyzed with the use of flow cytometry. In a subset of patients, an identifiable clone was found. Selectively gating on the high-scatter population of lesional skin T cells showed that the T_HS cell population was uniformly composed of clonal malignant T cells. SSC indicates side scatter. In gated histograms, the % total cells in each quadrant are shown.

Figure 4

T_HS cells in early-stage MF are enriched for clonal or oligoclonal T-cell populations. (A) T_HS and low-scatter T cells were isolated from a panel of patients with stage IA-stage IB MF. (B) The preferential presence of T cells expressing particular TCR Vβ subfamilies was analyzed by comparing the fold change in the frequency of each TCR Vβ subfamily in the high- versus low-scatter population (percentage of TCR Vβ high-scatter/percentage of low-scatter). There was a trend toward oligoclonality or clonality in the high-scatter population. The 3 patients shown had identifiable T-cell clones on TCRγ PCR. SSC-H indicates side-scatter height; FSC-H, forward scatter-height.

Figure 5

Clonal and malignant T_HS cells are present in the blood of patients with L-CTCL. (A) T_HS cells were present in the blood of patients with CTCL with known blood involvement but absent in patients without evidence of peripheral blood disease. Representative patients are shown; a T_HS cell population has been confirmed in 16 patients with L-CTCL. (B) Blood T_HS cells were obscured by monocytes in some patients. Staining for CD3 in addition to CD4 was required to show these cells. (C) T_HS cells were uniformly clonal and malignant in patients with L-CTCL and identifiable malignant T-cell clones. Shown are 8 patients with L-CTCL with identifiable T-cell clones. T_HS cell populations (red) were present in all patients. Selective gating on T_HS cells showed that most were clonal malignant T cells. SSC-A indicates side-scatter area; FSC-H, forward-scatter height.

Figure 6

T_HS cells from the blood of patients with L-CTCL were clonal proliferative CD4⁺ CCR4⁺ memory T cells expressing central memory T-cell markers. The phenotype of high-scatter (red) and low-scatter (black) T cells of 2 patients with L-CTCL are shown. In both patients, T_HS cells virtually all clonal CD4⁺ cells expressing high levels of CCR4 and variable levels of CLA. Most T_HS cells coexpressed the T_CM cell markers L-selectin and CCR7 and retained CD27 expression. Two representative patients are shown; similar results were found in 6 additional patients with L-CTCL. In gated histograms, the % total cells in each quadrant are shown.

Figure 7

The presence of T_HS cells in blood correlates with skin disease severity. (A) Peripheral blood T cells were drawn from patient 099 (stage IV L-CTCL) over a 4-year period during which the extent of her skin disease fluctuated. The presence of T_HS cells (red) correlated with the severity of skin disease. At all time points, T_HS cells were clonal CD4⁺ T cells expressing the previously identified malignant TCR Vβ17 clonotype. The absolute CD4 T-cell count and CD4/CD8 ratio are also shown. (B-D) Successful clearing of skin lesions on ECP is associated with loss of the high-scatter malignant T-cell clone in patients with L-CTCL. (B) Three T-cell populations were typically evident in patients with CTCL with blood involvement on ECP. The lowest-scatter T-cell population, shown in green, had high levels of caspase 6 activation, consistent with apoptosis (data not shown). Two patients with stage IV L-CTCL with identifiable malignant clones and extensive skin involvement were studied. (C) Patient 099 had worsening of skin disease on ECP and had large numbers of clonal T_HS cells demonstrable in the blood. (D) Patient 119 experienced complete clearing of skin lesions on ECP, and study of her blood at the time of skin clearing showed very few high-scatter clonal T cells. Both patients also had some cells expressing the malignant Vβ subunit in the low-scatter T-cell population. SSC-A indicates side-scatter area; FSC-H, forward-scatter height; PE, phycoerythrin; APC, allophycocyanin; and FITC, fluorescein isothiocyanate. In gated histograms, the % total cells in each quadrant are shown.

Figure 8

Benign clonally expanded T cells are found in the low-scatter T-cell population and can be discriminated from malignant T-cell clones. (A) Spectratype analysis of blood T cells showed a clonal T-cell population expressing TCR Vβ16.1 in a patient with long-standing stable stage IB MF and no other evidence of peripheral blood involvement. (B) Spectratype analysis of T cells isolated from skin lesions showed a diverse T-cell population expressing TCR Vβ16.1. (C) Further analysis of peripheral blood showed a lack of T_HS cells and showed that the expanded TCR Vβ16.1⁺ T-cell population was CD8⁺, lacked expression of CCR4, and produced effector cytokines, including interferon γ (IFNγ) and tumor necrosis factor-α (TNFα), suggesting that these cells represented a benign expanded CD8 clonal T-cell population. SSC-A indicates side-scatter area; FSC-H, forward-scatter height; and IL-4, interleukin-4.