Lenalidomide treatment of chronic lymphocytic leukaemia patients reduces regulatory T cells and induces Th17 T helper cells

Chronic lymphocytic leukaemia (CLL) remains an incurable disease, and the development of novel treatment options is therefore imperative. The immunomodulatory drug lenalidomide has shown promise in early clinical trials (Chanan-Khan & Porter, 2006; Chanan-Khan & Cheson, 2008; Ferrajoli et al, 2008). However, the exact mode of action of lenalidomide is unclear, and development of a ‘tumour flare reaction’ (TFR) is a critical side effect (Andritsos et al, 2008). In order to understand the mode of action of lenalidomide in CLL, we performed an analysis of intra-patient follow-up samples to assess the potential in vivo target cell population that contributes to effects and side effects. We initially investigated whether correlations that were previously reported (Andritsos et al, 2008) between in vitro changes of surface markers on CLL cells and development of TFR could also be found in vivo. We analysed peripheral blood lymphocytes (PBL) from three patients (Table SI) that were treated with lenalidomide (5 mg/d starting dose, escalated by 5 mg/d every 28 d). Two of the patients developed TFR upon therapy (Fig 1). However, the most pronounced upregulation of CD86 and CD40 in vivo was detected in Patient 2 who did not experience a TFR (Fig 2A), while Patient 1 (with a TFR) showed no upregulation of CD40. These findings would suggest that there is no strict correlation of development of a TFR with in vivo induction of CD40 and/or CD86 on CLL cells. This is in contrast to effects of lenalidomide in vitro, which have also been reported previously (Andritsos et al, 2008): treatment resulted in upregulation of CD40 and CD86 in the majority of samples (9/12 and 11/12, respectively, Figure S1). Interestingly this effect was also observed directly on pure CD19 sorted cells (8/12 and 11/12), excluding indirect induction by non-malignant bystander cells (Figure S1). In contrast, only 30–58% of patients experience a TFR after treatment in vivo, depending on lenalidomide dosage (Chanan-Khan & Porter, 2006; Ferrajoli et al, 2008). This makes it unlikely that in vitro upregulation of CD40 and CD86 predicts TFR. Also, in contrast to previous in vitro analyses (Lapalombella et al, 2008), in vivo only one of the patients presented with more CD38 cells during treatment (Patient 1, Fig 2A). A lymph node (LN) aspirate obtained from Patient 3 during TFR however showed twofold more CD38 CLL cells than cells drawn from PBL (data not shown), but no LN aspirate from a timepoint before initiation of treatment was available for comparison. Thus, our findings uncovered interesting differences between the in vitro and in vivo effects of lenalidomide on CLL cells in a small number of patients. Similar to thalidomide, lenalidomide is postulated to impact not only on CLL cells, but also activate and induce proliferation of T cells and natural killer (NK) cells in a mouse model (Hernandez-Ilizaliturri et al, 2005), in healthy volunteers and in patients suffering from multiple myeloma (Chang et al, 2006) and activate them for antibody-dependent cellular cytotoxicity (ADCC) against CLL cells in vitro (Lapalombella et al, 2008). Similarly, we found induction of CD16CD56NK cell numbers in 2/3 CLL patients after treatment with lenalidomide in vivo (Fig 2B). At the same time, the activation status of NK cells decreased as measured by CD69 and CD25 levels. However, it has to be kept in mind that these are early