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. 2011 Jan;60(1):75-85.
doi: 10.1007/s00262-010-0920-3. Epub 2010 Sep 21.

Fludarabine Modulates Composition and Function of the T Cell Pool in Patients With Chronic Lymphocytic Leukaemia

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Free PMC article

Fludarabine Modulates Composition and Function of the T Cell Pool in Patients With Chronic Lymphocytic Leukaemia

Franz Josef Gassner et al. Cancer Immunol Immunother. .
Free PMC article

Abstract

The combination of cytotoxic treatment with strategies for immune activation represents an attractive strategy for tumour therapy. Following reduction of high tumour burden by effective cytotoxic agents, two major immune-stimulating approaches are being pursued. First, innate immunity can be activated by monoclonal antibodies triggering antibody-dependent cellular cytotoxicity. Second, tumour-specific T cell responses can be generated by immunization of patients with peptides derived from tumour antigens and infused in soluble form or loaded onto dendritic cells. The choice of cytotoxic agents for such combinatory regimens is crucial since most substances such as fludarabine are considered immunosuppressive while others such as cyclophosphamide can have immunostimulatory activity. We tested in this study whether fludarabine and/or cyclophosphamide, which represent a very effective treatment regimen for chronic lymphocytic leukaemia, would interfere with a therapeutic strategy of T cell activation. Analysis of peripheral blood samples from patients prior and during fludarabine/cyclophosphamide therapy revealed rapid and sustained reduction of tumour cells but also of CD4(+) and CD8(+) T cells. This correlated with a significant cytotoxic activity of fludarabine/cyclophosphamide on T cells in vitro. Unexpectedly, T cells surviving fludarabine/cyclophosphamide treatment in vitro had a more mature phenotype, while fludarabine-treated T cells were significantly more responsive to mitogenic stimulation than their untreated counterparts and showed a shift towards T(H)1 cytokine secretion. In conclusion, fludarabine/cyclophosphamide therapy though inducing significant and relevant T cell depletion seems to generate a micromilieu suitable for subsequent T cell activation.

Figures

Fig. 1
Fig. 1
PBMCs from 27 untreated CLL patients were incubated for 24 h with complete medium alone, fludarabine (1 μg/ml), mafosfamide (1 μg/ml) or both. The cells were then washed and cultured for subsequent 6 days in complete medium. On day 6, the fraction of CD38-expressing CD4+ (a) or CD8+ (b) T cells was determined. T cell subsets were defined as stated in the Results section and the fraction of memory CD4+ (c) and CD8+ (d) T cells was determined. Values significantly different from untreated controls are indicated by asterisks (*p < .05; **p < .01; ***p < .001)
Fig. 2
Fig. 2
a PBMCs from CLL patients (n = 16) were incubated for 24 h with medium alone, or fludarabine (1 μg/ml). The cells were then washed, stained with CFSE, and cultured for 6 days in the presence of allogeneic DC at a ratio of 10:1. On day 6, the fraction of CFSE-diluted T cells was assessed. The fraction of proliferating T cells in CD38-positive or negative T cell population (b) and in naïve or memory T cell compartment (c) was measured (n = 8). d PBMCs from CLL patients (n = 16) were incubated for 24 h with medium alone, or fludarabine (1 μg/ml). The cells were then washed, stained with CFSE, and cultured for 6 days in the presence of beads coated with anti-CD3/anti-CD28 antibodies at a PBMC/bead ratio of 250:1. On day 6, the fraction of CFSE-diluted CD4+ (d) and CD8+ (e) T cells and the total CD4+ T cell number (f) were assessed. Significance is indicated by asterisks (*p < .05, **p < .01, ***p < .001) (white bars CD4+, grey bars CD8+ T cells)
Fig. 3
Fig. 3
PBMCs from seven untreated CLL patients were incubated with medium alone (co), fludarabine (Flu 1 μg/ml) or mafosfamide (Maf 1 μg/ml) for 24 h. The cells were then washed and incubated for further 6 days in presence of PMA (1 ng/ml) and ionomycin (0.5 μg/ml) at a concentration of 2.5 × 106 cells/ml. Culture supernatants were collected and the concentration of cytokines (ng/ml) was measured by cytokine bead assay (CBA). Values significantly different from untreated controls are indicated by asterisks (*p < .05)
Fig. 4
Fig. 4
a Chemonaïve CLL patients were treated with fludarabine and cyclophosphamide. PBMCs collected before treatment were incubated for 24 h in presence (vitro24) or absence of 1 μg/ml fludarabine. PBMCs were also collected on day 1 (vivo24) and day 3 (vivo72) of the FC treatment. All samples were co-cultured in vitro for 6 days with allogeneic DC and the T cell proliferation rates on day 6 were measured by CFSE dilution. Boxplots indicate the percentage of proliferating CD4+ (white bars) or CD8+ (grey bars) T cells (n = 6). bd Peripheral blood was collected before treatment (0) and after 24 or 72 h of therapy (n = 16). Whole blood staining was performed of each sample and the percentage of (b) CD38+ T cells as well as the T cell subset distribution of (c) CD4+ and (d) CD8+ T cells were determined by flow cytometry (naïve T cells in normal frames, memory T cells in bold frames)
Fig. 5
Fig. 5
a Peripheral blood from five mice was collected 3 days prior to (d-3), during (d3) and after (d7, d11) fludarabine treatment (5 days; 35 mg/kg/day). T cells were stained using specific antibodies and the percentage of CD4+ (a) and CD8+ (b) memory T cells was assessed. c In a separate experiment, the numbers of peripheral blood TH1 and TH2 CD4+ T cells prior to (d0) and after fludarabine treatment (d7, d14) were determined. One representative result of two experiments is shown

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