B-CLL cells acquire APC- and CTL-like phenotypic characteristics after stimulation with CpG ODN and IL-21

Abstract

CpG oligodeoxynucleotides (CpG) and IL-21 are two promising agents for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). Recently, we reported that the combination of CpG and IL-21 (CpG/IL-21) can induce granzyme B (GrB)-dependent apoptosis in B-CLL cells. Here, we demonstrate that treatment of B-CLL cells with CpG and IL-21 results in the development of antigen-presenting cell (APC)-like cells with cytotoxic features. These properties eventually give rise to B-CLL cell apoptosis, independently of their cytogenetic phenotype, whereas normal B-cell survival is not negatively affected by CpG/IL-21. APC- and CTL-typical molecules found to be up-regulated in CpG/IL-21-stimulated B-CLL cells include GrB, perforin, T-bet, monokine-induced by IFN-γ and IFN-γ-inducible protein 10 (IP-10), as well as molecules important for cell adhesion, antigen cross-presentation and costimulation. Also induced are molecules involved in GrB induction, trafficking and processing, whereas the GrB inhibitor Serpin B9 [formerly proteinase inhibitor-9 (PI-9)] is down-modulated by CpG/IL-21. In conclusion, CpG/IL-21-stimulated B-CLL cells acquire features that are reminiscent of killer dendritic cells, and which result in enhanced immunogenicity, cytotoxicity and apoptosis. Our results provide novel insights into the aberrant immune state of B-CLL cells and may establish a basis for the development of an innovative cellular vaccination approach in B-CLL.

Keywords: cross-presentation; cytotoxic B cells; immunogenization; leukemia vaccine; toll-like receptor agonist.

Fig. 1.

CpG and IL-21 induce apoptosis in CD5⁺ B-CLL cells, but not in CD5⁻ B cells. (A) PBMC from five subjects with CD5⁺ B-CLL (left panel), two subjects with CD5⁻ B-CLL (middle panel) and four healthy subjects (right panel) were isolated and cultured for 4 days in the presence of CpG, IL-21 or both. Cells were harvested, stained with antibodies to CD19, CD5, annexin V and propidium iodide (PI) and analyzed by flow cytometry. Shown are mean percentages of viable B cells determined by annexin V/PI staining. Error bars indicate SEM for left and right panels and standard deviation for middle panel. (B and C) B-CLL cells from three different donors with high cellularity (>80% CD19⁺CD5⁺ B-CLL cells) were incubated with IL-21, CpG or a combination of both, in the presence of increasing concentrations of a monoclonal anti-CD5 antibody. Viability of B-CLL cells was determined by FACS, using annexin V/PI. Line graphs (B) and bar graphs (C) show absolute numbers of viable B-CLL cells on day 4. Error bars indicate SEM from three independent experiments.

Fig. 2.

GrB expression is higher in normal B cells compared with B-CLL cells. (A and B) Healthy B cells or B-CLL cells were isolated from peripheral blood and either immediately stained for GrB (0h) or cultured for 16h in the presence of IL-21, CpG, anti-BCR and combinations. Brefeldin A was added for four more hours, cells were stained for intracellular GrB and analyzed by FACS. (A) Bar graphs show average percentages of GrB-expressing healthy B cells (n = 10) compared with B-CLL cells (n = 4). Error bars indicate SEM. (B) In a second experiment with a different B-CLL cohort, we directly compared normal B cells and B-CLL cells. Bar graphs show average percentages of GrB-producing healthy B cells (n = 10) and B-CLL cells (n = 6). Error bars indicate SEM, dots indicate individual donors.

Fig. 3.

GrB production in B cells requires activation of members of the JAK/STAT pathway. (A) Purified B cells from healthy donors or B-CLL patients (>99.5%) were cultured for 15min with IL-21, anti-BCR or CpG. Analysis of pJAK1, pJAK3, pSTAT1 and pSTAT3 was performed by western immunoblotting, each membrane was controlled for correct loading by stripping and restaining the membrane for β-actin (n = 3). (B) Purified healthy B cells and B-CLL cells were cultured for 20min with IL-21 and anti-BCR in the presence or absence of the JAK inhibitor P6. Analysis of pSTAT1 and pSTAT3 was performed by western immunoblotting (n = 3). (C and D) Purified normal B cells and B-CLL cells were cultured for 16h with IL-21 and anti-BCR in the presence or absence of P6. After incubation, cells were stained for intracellular GrB. Shown is GrB production of CD5⁺CD19⁺ B-CLL cells and CD19⁺ B cells as determined by flow cytometry. Panels in (C) illustrate representative dot plots out of five donors (healthy B cells) or four patients (B-CLL). Panels in (D) indicate percentages of GrB⁺ B cells with each line indicating an individual donor.

Fig. 3.

GrB production in B cells requires activation of members of the JAK/STAT pathway. (A) Purified B cells from healthy donors or B-CLL patients (>99.5%) were cultured for 15min with IL-21, anti-BCR or CpG. Analysis of pJAK1, pJAK3, pSTAT1 and pSTAT3 was performed by western immunoblotting, each membrane was controlled for correct loading by stripping and restaining the membrane for β-actin (n = 3). (B) Purified healthy B cells and B-CLL cells were cultured for 20min with IL-21 and anti-BCR in the presence or absence of the JAK inhibitor P6. Analysis of pSTAT1 and pSTAT3 was performed by western immunoblotting (n = 3). (C and D) Purified normal B cells and B-CLL cells were cultured for 16h with IL-21 and anti-BCR in the presence or absence of P6. After incubation, cells were stained for intracellular GrB. Shown is GrB production of CD5⁺CD19⁺ B-CLL cells and CD19⁺ B cells as determined by flow cytometry. Panels in (C) illustrate representative dot plots out of five donors (healthy B cells) or four patients (B-CLL). Panels in (D) indicate percentages of GrB⁺ B cells with each line indicating an individual donor.

Fig. 4.

IL-21 and CpG induce a CTL-like transcriptional profile in B-CLL cells and up-regulate molecules involved in GrB uptake and inhibition. Purified healthy B cells or B-CLL cells were cultured with IL-21, CpG or both. (A) After 16h, cells were harvested, mRNA prepared and RT–PCR for indicated molecules performed. Data are representative for three independent experiments. (B) On day 4, B cells were analyzed by flow cytometry for the expression of CD222. Error bars indicate SEM (healthy B cells n = 3, B-CLL cells n = 9). (C) Cell lysates were prepared after 16h and expression of Serpin B9 and β-actin determined by western immunoblot. Shown is one representative blot out of three with similar results.

Fig. 5.

CpG and IL-21 synergistically induce MIG in B-CLL, but not in normal B cells. Purified healthy B cells or B-CLL cells (>99.5%) were incubated in the presence of IL-2, IL-21, IFN-γ and CpG as indicated. (A) Total RNA was isolated after 16h and RT–PCR for MIG and IP-10 mRNA performed (n = 3). (B) 1×10⁵ B cells were cultured as indicated on a 96-well ELISpot plate coated with specific antibodies to MIG. After 16h, plates were developed and MIG-secreting cells counted. (C) Bar graphs illustrate average dot numbers out of three independent experiments. Error bars show SEM. (D) On day 4, B-CLL cells were analyzed by flow cytometry for the expression of the chemokine receptor CXCR3. Bar graphs represent means of three independent experiments and error bars show SEM.

Fig. 6.

IL-21 and CpG induce B-CLL cell up-regulation of molecules required for cellular cross-talk. PBMC from B-CLL patients were cultured in the presence or absence of IL-21 and CpG for 4 days. Then they were harvested, stained for the markers indicated and analyzed by FACS. Error bars indicate SEM from three independent experiments for each marker.

Fig. 7.

B-CLL cells activated with IL-21 and CpG are superior to B-CLL cells stimulated with CpG alone to induce allogeneic CD8⁺ T-cell proliferation. B-CLL cells were purified and cocultured with carboxyfluorescein diacetate succinimidyl ester-stained allogeneic T cells at a ratio of 2:1 for 6 days in the presence of CpG, IL-21 or both reagents as indicated. Then cells were harvested, stained for CD3 and CD8 and analyzed by FACS. (A) Dot plots show CD3⁺ T cells from one representative experiment out of four with similar results. T cells cultured in the absence of B-CLL cells did not proliferate (top row). (B) Bar graphs show average CD8⁺ T-cell proliferation from four individual experiments. Error bars indicate SEM.