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Clinical Trial
. 2007 Dec;150(3):523-30.
doi: 10.1111/j.1365-2249.2007.03521.x. Epub 2007 Oct 22.

Increase of CD4+ CD25+ regulatory T cells in the peripheral blood of patients with metastatic carcinoma: a Phase I clinical trial using cyclophosphamide and immunotherapy to eliminate CD4+ CD25+ T lymphocytes

S Audia  1 , A Nicolas, D Cathelin, N Larmonier, C Ferrand, P Foucher, A Fanton, E Bergoin, M Maynadie, L Arnould, A Bateman, B Lorcerie, E Solary, B Chauffert, B Bonnotte
Affiliations
Clinical Trial

Increase of CD4+ CD25+ regulatory T cells in the peripheral blood of patients with metastatic carcinoma: a Phase I clinical trial using cyclophosphamide and immunotherapy to eliminate CD4+ CD25+ T lymphocytes

S Audia et al. Clin Exp Immunol. 2007 Dec.

Abstract

We determined the number and functional status of CD4+ CD25(high) regulatory T cells (Treg) in blood samples from patients with metastatic carcinoma, and evaluated their sensitivity to a single intravenous infusion of cyclophosphamide. Treg numbers were significantly higher in 49 patients with metastatic cancer (9.2% of CD4+ T cells) compared to 24 healthy donors (7.1%). These cells expressed the transcription factor forkhead box P3 (FoxP3), glucocorticoid-induced tumour necrosis factor receptor family-related protein (GITR) and intracellular CD152, and demonstrated a suppressive activity in vitro against CD4+ CD25- autologous proliferation. At a single intravenous infusion, cyclophosphamide failed, in association with a non-specific immunotherapy by intratumoral bacille Calmette-Guérin (BCG), to modulate significantly Treg numbers or function. Metastatic cancer is associated with an expansion of peripheral blood CD4+ CD25(high) FoxP3+ GITR+ CD152+ Treg cells whose immunosuppressive properties do not differ from those of healthy subjects. Moreover, cyclophosphamide administration may not represent an optimal therapy to eliminate Treg, which further underlines the need to identify specific agents that would selectively deplete these cells.

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Figures

Fig. 1
Fig. 1
Increase of circulating CD4+CD25high T lymphocytes in cancer patients. Representative flow cytometry analysis of CD4+CD25+ T cells from a healthy donor (a) or a patient with metastatic cancer (b). (c) Percentage of circulating CD4+CD25high cells from patients with metastatic cancer (n = 49) and healthy donors (n = 24). CD4+CD25high cells are presented as a percentage of total CD4+ cells. Each plot represents an individual patient. P < 0·01 using Student's t-test.
Fig. 2
Fig. 2
Phenotypic analysis of CD4+CD25high T cells and forkhead box P3 (FoxP3) expression. (a) CD4+ cells were sorted in CD4+CD25high and CD4+CD25− and then stained with anti-CD25, anti-glucocorticoid-induced tumour necrosis factor receptor family-related protein (GITR) and anti-CD152 antibodies. CD4+CD25+ and CD4+CD25− cell populations were analysed separately. (b) cDNA obtained from purified populations of CD4+CD25− and CD4+CD25high cells from 49 cancer patients and 24 healthy donors were subjected to quantitative real-time polymerase chain reaction (PCR) analysis using primers specific for FoxP3 and Abelson (Abl), as described in Materials and Methods.
Fig. 3
Fig. 3
CD4+CD25high T cells from cancer patients and healthy donors exhibit a similar suppressive potential. (a) Sorted CD4+CD25high cells from nine healthy donors and 12 patients with metastatic cancer were cultured with autologous CD4+CD25− stimulated with microbeads-conjugated anti-CD3 and anti-CD28 antibodies. T cell proliferation was evaluated using [3H]-thymidine incorporation. Proliferation was determined in triplicate cultures. Inhibition rate of proliferation is expressed as a percentage of co-culture proliferations of 100 000 CD4+CD25− with CD4+CD25+ at a CD4+CD25−/CD4+CD25+ ratio of 2 : 1, compared to proliferation of CD4+CD25− alone. (b) Sorted CD4+CD25high cells from four representative cancer patients (kidney cancer: two, lung cancer: two) were cultured with autologous CD4+CD25− stimulated with anti-CD3 and anti-CD28 microbeads. Similar assay as described in (a).
Fig. 4
Fig. 4
T regulatory (Treg) evolution following a single cyclophosphamide injection. (a) Determination of the percentage of CD4+CD25+ Treg to total CD4+ by flow cytometry at different time points after administration of the indicated doses of cyclophosphamide. The therapeutic protocol is described in Material and methods. (b) Evaluation of forkhead box P3 (FoxP3) expression in peripheral CD4+ T cells by real time reverse transcription–polymerase chain reaction (RT–PCR) at different time-points after administration of the indicated doses of cyclophosphamide. (c) Immunohistochemical analyses of metastatic tumours were performed at days 0 and at 28 of the therapeutic protocol on serial sections using monocloal antibodies: anti-CD68 that labels monocytes, anti-CD11c for myeloid dendritic cells, anti-CD8 and anti-CD25. Representative immunohistochemistry analyses of two patients (1 and 5) are shown.
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