Lenalidomide treatment promotes CD154 expression on CLL cells and enhances production of antibodies by normal B cells through a PI3-kinase-dependent pathway

Abstract

Chronic lymphocytic leukemia (CLL) involves a profound humoral immune defect and tumor-specific humoral tolerance that directly contribute to disease morbidity and mortality. CD154 gene therapy can reverse this immune defect, but attempts to do this pharmacologically have been unsuccessful. The immune-modulatory agent lenalidomide shows clinical activity in CLL, but its mechanism is poorly understood. Here, we demonstrate that lenalidomide induces expression of functional CD154 antigen on CLL cells both in vitro and in vivo. This occurs via enhanced CD154 transcription mediated by a Nuclear Factor of Activated T cells c1 (NFATc1)/Nuclear Factor-kappaB (NF-kappaB) complex and also through phosphoinositide-3 (PI3)-kinase pathway-dependent stabilization of CD154 mRNA. Importantly, CD154-positive CLL cells up-regulate BID, DR5, and p73, become sensitized to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis, and promote costimulatory activation of normal B cells to produce antibodies. In CLL patients receiving lenalidomide, similar evidence of CD154 activation is observed including BID, DR5, and p73 induction and also development of anti-ROR1 tumor-directed antibodies. Our data demonstrate that lenalidomide promotes CD154 expression on CLL cells with subsequent activation phenotype, and may therefore reverse the humoral immune defect observed in this disease. This study is registered at https://ichgcp.net/clinical-trials-registry/NCT00466895" title="See in ClinicalTrials.gov">NCT00466895.

Figures

Figure 1

Case report. (A) White blood cell (WBC) counts were performed by The Ohio State University Clinical Laboratory using Beckman Coulter LH. WBC count is expressed over time as absolute numbers in thousands of cells per microliter (K/μL). (B) IgM, IgA, and IgG production was detected by The Ohio State University Clinical Laboratory using the immunoturbidimetric method (Synchron LX System). Values are expressed as milligrams per deciliter.

Figure 2

Lenalidomide promotes time-dependent CD154 transmembrane protein expression. (A) CD19+ CLL cells were incubated with lenalidomide 0.5μM and vehicle control. CD154 expression was analyzed by flow cytometry after 48 hours of treatment. The graph shows fold change in percentage (%) of CD154+ cells in the lenalidomide-treated group compared with vehicle control (n = 30, P < .001). (B) CD19+ CLL cells were incubated with lenalidomide 0.5μM and vehicle control. CD154 expression was analyzed by flow cytometry after 24, 48, and 72 hours. The graph shows fold change in percentage (%) of CD154+ cells in the lenalidomide-treated group compared with vehicle control (n = 5, P = .006). (C) The panel shows 48-hour results for CD154 expression from a representative experiment. Black line denotes lenalidomide-treated cells and dark gray line denotes vehicle-treated CLL cells. Control IgG1k-PE is shown (light gray line). (D) CLL B cells were treated with vehicle (V) or 0.5μM lenalidomide (L) and after 48 hours, cell-surface proteins were biotinylated, and lysates were prepared. For each condition, 400 μg of protein lysate was subjected to immunoprecipitation using anti-CD154 or anti-IgG1 antibodies. The 39- and 32-kDa bands, corresponding to the 2 transmembrane forms of CD154, are indicated with arrows. Immunoblot shown is representative of 2 patient samples of 5 tested. Error bars represent SD. *Statistical significance.

Figure 3

Lenalidomide increases RNA stability and transcription of CD154 in CLL cells. (A) CD154 mRNA level was measured by real-time reverse-transcription–PCR after 48 hours of treatment with lenalidomide 0.5μM or vehicle control (n = 20, P < .001). (B) After 48 hours with lenalidomide (0.5μM) or vehicle control, transcription was inhibited by the addition of ActD and 107 cells were collected for each time point over a 4.5-hour time course. The percentage of RNA remaining from each time point is plotted. Lenalidomide increases CD154 mRNA stability as demonstrated by significantly diminished decay after ActD treatment of CLL cells compared with vehicle control (n = 6, P = .002). (C) CLL B cells were transfected with CD154 luciferase reporter plasmid along with 1 μg of pCMV-renilla reporter vector or pGL3 empty reporter plasmid (pGL3-basic) as a negative control. After 48 hours with lenalidomide 0.5μM or vehicle control, luciferase activity was determined and corrected for transfection efficiency using renilla activity (n = 13, P = .001 for lenalidomide vs vehicle control). (D) Nuclear extracts from lenalidomide- or vehicle control-treated CLL cells were resolved on SDS–polyacrylamide gel electrophoresis and subjected to Western blotting with anti-NFATc1 or anti–c-Rel antibody. Nuclear protein Brg1 was used as an internal loading control. (E) Lenalidomide treatment of CLL B cells enhanced promoter binding of NFATc1 (n = 3, P < .001) and c-Rel (n = 3, P < .001) to the CD154 promoter by chromatin immunoprecipitation (ChIP). PCR to detect the CD154 promoter region (CD154-κB) was performed on the precipitated DNA (n = 3, P = .004). Error bars represent SD. *Statistical significance.

Figure 4

Lenalidomide-induced CD154 expression on CLL cells is functional. Lenalidomide (L)–treated but not vehicle (VC)–treated CLL B cells induce normal, target B cells to produce antibodies in the presence of PWM. A total of 5 × 104 autologous T cells (AutoTxr) from the healthy target B-cell donor, lenalidomide-treated CLL B cells (L CLLBxr), or vehicle control CLL B cells (VC CLLBxr) were irradiated (20 Gy) and placed in culture with 5 × 104 targets, purified B cells from the healthy donor, in the absence or presence of PWM. Control conditions included 5 × 104 target, unirradiated B cells alone, with and without PWM, and 5 × 104 irradiated and unirradiated CLL B cells, in the absence of target B cells (data not shown). IgM (A) and IgG (B) production was assayed by ELISA from supernatants obtained after 7 days of coculture. (C) A total of 5 × 104 autologous T cells (AutoTxr) from the healthy target B-cell donor, lenalidomide-treated CLL B cells (L CLLBxr), or vehicle control CLL B cells (VC CLLBxr) were irradiated (20 Gy) and placed in culture with 5 × 104 targets, purified B cells from the healthy donor, in the absence or presence of PWM and of a CD40 blocking antibody. Control conditions included 5 × 104 targets, unirradiated B cells alone, with and without PWM, and 5 × 104 irradiated and unirradiated CLL B cells, in the absence of target B cells (data not shown). IgM production was assayed by ELISA from supernatants obtained after 7 days of coculture. Error bars represent SD.

Figure 5

Lenalidomide-induced up-regulation of CD154 is dependent on PI3-kinase. (A) CLL cells were treated with vehicle or 0.5μM lenalidomide for 8 hours. Cell lysates were subjected to immunoblot analysis. (B) IKK-β was immunoprecipitated from cell lysate derived from vehicle or 0.5μM lenalidomide-treated CLL samples and tested for the capability to phosphorylate the GST-IκBα substrate in a kinase buffer containing 20μM ATP and 10 μCi (0.37 MBq) of [γ32P]ATP. The left panel shows a representative experiment of the IKK kinase activity. The graph shows fold change in IKKβ activity in the lenalidomide-treated group compared with vehicle control (n = 8, P = .01). (C) IKK substrate IκB is degraded after lenalidomide treatment. The panel shows 3 representative CLL patients treated with 0.5μM lenalidomide or the vehicle control for 12 hours. (D) After 48 hours with lenalidomide 0.5μM and vehicle control with and without 25μM LY294002, transcription was inhibited by the addition of ActD and 107 cells were collected for each time point over a 4.5-hour time course. The percentage of RNA remaining from each time point is plotted (n = 4, P = .045). (E) A total of 5 × 104 lenalidomide-treated CLL B cells (L) or vehicle control CLL B cells (VC) were irradiated (20 Gy) and placed in culture with 5 × 104 targets, purified B cells from the healthy donor, in the absence or presence of PWM and 25μM LY294002. IgM production was assayed by ELISA from supernatants obtained after 7 days of coculture (n = 8, P = .001). Error bars represent SD.

Figure 6

Lenalidomide promotes CD154 gene therapy phenotype. (A) Whole-cell extracts from 48-hour–treated CLL B cells were analyzed by immunoblot for p73 and BID. Actin was used as internal loading control. (B) Representative histograms show expression of DR5 measured by flow cytometry in CLL B cells cultured without (filled gray) or with (filled black) lenalidomide. The gray- and black-lined open histograms correspond to CLL B cells cultured, respectively, with vehicle or lenalidomide and stained with an isotype monoclonal antibody of irrelevant specificity. (C) Peripheral blood mononuclear cells from before and day 8 of therapy were cultured in the presence of ActD and 107 cells were collected over a 4-hour time course as in Figure 3B (n = 5, P < .05). (D) Whole-cell extracts from patients' peripheral blood mononuclear cells before the dose and on day 8 were analyzed by immunoblot for p73 and BID (representative findings in 6 of 9 patients). (E) Purified recombinant human-ROR1 was run in each lane of an SDS-PAGE gel and then transferred to nitrocellulose membrane. Ponceau red staining was performed to assess both purity and equal loading. The membrane was cut into pieces and probed with rabbit anti–human ROR1 (Cell Signaling), goat anti–human ROR1 (R&D), healthy donor serum (ND), or serum from the index case immediately before lenalidomide (before) and on month 6 of therapy (after). The pieces were incubated with HRP-conjugated anti–rabbit, anti–goat, or anti–human IgG.