1. Significance of circulating T-cell clones in Sézary syndrome
by Nicolas Ortonne, Delphine Huet, Caroline Gaudez, Anne Marie-Cardine, Valérie Schiavon, Martine Bagot, Philippe Musette, and Armand Bensussan
Blood
Volume 107(10):
May 15, 2006
©2006 by American Society of Hematology

2. Expression of CD158k and VIM by circulating and cutaneous lymphocytes.
Expression of CD158k and VIM by circulating and cutaneous lymphocytes. (A) Flow cytometry analysis in patient 11 showed CD158kdim and cell-surface VIM positivity before treatment (top row). After treatment with extracorporeal photochemotherapy, there was a dramatic decrease in the CD4+ population associated with a loss of CD158k expression and a significant increase in VIM (bottom row). (B) RT-PCR for CD158k was positive for PBMCs in patients 1, 2, 3, and 4 and for the Sézary cell line Pno, whereas it was negative for CD4+ lymphocytes in a healthy donor. (C) Immunohistochemistry analysis performed in a skin sample from patient 3 showed a predominantly CD3+CD4+ infiltrate along with CD158k+ cells in the dermis and the epidermis. Intraepidermal nests of lymphocytes, or Pautrier microabscesses, can be seen (arrows). Few CD8+ lymphocytes were present in the dermis (arrowheads), whereas only CD4+ T cells are seen in the epidermis. Slides are shown at 200 × original magnification (20 ×/0.50 NA objective), and images were captured with an Axioskop2 microscope (Zeiss, Oberkochen, Germany). Pictures were scanned in with a DP70 camera and software (Olympus, Hamburg, Germany). Hematoxylin and eosin staining is shown on the left, whereas a biotin/avidin system conjugated to alkaline phosphatase was used for CD3, CD4, CD8, and CD158k labeling.
Nicolas Ortonne et al. Blood 2006;107:
©2006 by American Society of Hematology

3. Immunoscopy applied to PBMCs and a skin specimen of patient 2: TCR Vβ spectratyping.
Immunoscopy applied to PBMCs and a skin specimen of patient 2: TCR Vβ spectratyping. (A) GeneScan Analysis of PBMCs in patient 2 revealed a monoclonal signature for Vβ2 (shown to be malignant by 2-color flow cytometry analysis), apparent loss of several families (*) and many contracted profiles (**). (B) Gene-Scan Analysis of a skin sample from the same patient revealed a complete loss of all Vβ families, with a single monoclonal peak of a size identical to that of the malignant T-cell clone identified in the blood, indicating the presence of the malignant T-cell clone in the skin and the complete loss of other T cells.
Nicolas Ortonne et al. Blood 2006;107:
©2006 by American Society of Hematology

4. Identification of malignant and nonmalignant T-cell clones in patients 4, 8, and 10 using TCR Vβ, CD158k, and cell-surface VIM phenotyping.
Identification of malignant and nonmalignant T-cell clones in patients 4, 8, and 10 using TCR Vβ, CD158k, and cell-surface VIM phenotyping. Molecular characteristics of the T-cell clones are shown on the left, and the results of 2-color flow cytometry analyses are shown on the right. (A) A strongly expanded TCR Vβ7-Jβ1.2 T-cell clone was identified in patient 4, expressing CD158k and displaying heterogeneous labeling with SC5 mAb. A minor CD158k– population could be recognized within the Vβ7+ population (arrowhead). (B) A nonmalignant TCR Vβ17-Jβ2.2 T-cell clone was identified in patient 8, accounting for 10% of gated lymphocytes. Two-color flow cytometry showed that the TCR Vβ17+ population displayed heterogeneous expression of cell-surface vimentin (SC5) but was negative for CD158k. (C) A nonmalignant TCR Vβ14-Jβ1.6 T-cell clone was identified in patient 10 during treatment with anti–CD52 mAb (alemtuzumab) that was heavily expanded and did not express CD158k, as seen on the right based on the results of 2-color flow cytometry.
Nicolas Ortonne et al. Blood 2006;107:
©2006 by American Society of Hematology

5. Intraclonal heterogeneity for CD158k and TCR Vβ expression in patients 1 and 13 and cell sorting analysis.
Intraclonal heterogeneity for CD158k and TCR Vβ expression in patients 1 and 13 and cell sorting analysis. (Ai) Patient 13 showed 2 distinct TCRαβdim and TCRαβbright circulating T-cell populations. Two-color flow cytometry showed that CD158k+ cells were present within both T-cell populations. (Aii) GeneScan Analysis applied to sorted populations revealed that the TCRαβdim population only disclosed clonal TCR Vβ5, TCR Vβ15, and TCR Vβ23. In contrast, the TCRαβbright population had a profile similar to that of whole PBMCs, including clonal TCR Vβ5, Vβ23, and Vβ15, suggesting that malignant and nonmalignant T-cell clones were present in the TCRαβbright and TCRαβdim subsets. (Bi) Two-color flow cytometry revealed heterogeneity for CD158k expression within the malignant TCR Vβ17 population in patient 1. A proportion of Vβ17+ cells did not express CD158k. A minor Vβ17dim subpopulation could be recognized within the TCR Vβ17+ CD158k+ population (arrowhead). (Bii) Through RT-PCR, CD158k mRNA was detected at significant levels in the TCR Vβ17+ CD158k+ population; negative results were obtained in the TCR Vβ17+ CD158k– population.
Nicolas Ortonne et al. Blood 2006;107:
©2006 by American Society of Hematology