Stereotypical chronic lymphocytic leukemia B-cell receptors recognize survival promoting antigens on stromal cells

Abstract

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world. Survival of CLL cells depends on their close contact with stromal cells in lymphatic tissues, bone marrow and blood. This microenvironmental regulation of CLL cell survival involves the stromal secretion of chemo- and cytokines as well as the expression of adhesion molecules. Since CLL survival may also be driven by antigenic stimulation through the B-cell antigen receptor (BCR), we explored the hypothesis that these processes may be linked to each other. We tested if stromal cells could serve as an antigen reservoir for CLL cells, thus promoting CLL cell survival by stimulation through the BCR. As a proof of principle, we found that two CLL BCRs with a common stereotyped heavy chain complementarity-determining region 3 (previously characterized as "subset 1") recognize antigens highly expressed in stromal cells--vimentin and calreticulin. Both antigens are well-documented targets of autoantibodies in autoimmune disorders. We demonstrated that vimentin is displayed on the surface of viable stromal cells and that it is present and bound by the stereotyped CLL BCR in CLL-stroma co-culture supernatant. Blocking the vimentin antigen by recombinant soluble CLL BCR under CLL-stromal cell co-culture conditions reduces stroma-mediated anti-apoptotic effects by 20-45%. We therefore conclude that CLL BCR stimulation by stroma-derived antigens can contribute to the protective effect that the stroma exerts on CLL cells. This finding sheds a new light on the understanding of the pathobiology of this so far mostly incurable disease.

Figure 1. Subset 1 CLL B-cell receptors Ig014 and Ig044 recognize proteins highly expressed in nurse-like cell extracts.

A: Two-dimensional gel electrophoresis of nurse-like cell protein extracts. Protein samples were prepared and electrophoresis was performed. The gel was subjected to Coomassie blue staining. pI  =  isoelectric pH value, M.Wt.  =  molecular weight. The rectangles indicate the areas magnified in C. B: CLL B-cell receptors Ig014 and Ig044 recognize partially overlapping protein spots in two-dimensionally separated nurse-like cell protein extracts. Gels run in parallel to the ones shown in Figure 1A were subjected to Western blotting using Ig014 and Ig044 as primary antibodies, followed by secondary detection with a horseradish peroxidase-conjugated antibody. A set of protein spots ranging from 45 to 57 kDa and a pI between 4.5 and 5.5 were detected by both Ig014 and Ig044. Another highly expressed protein spot (no. 27) was detected by Ig044, only. The rectangles indicate the areas magnified in C. C: Overlay of spots detected by Ig014 and Ig044, respectively, and the corresponding regions of the nurse-like cell protein map in Figure 1A. Delta 2D software (Decodon) was used for analysis. The magnified regions correspond to the highlighted regions from figure 1A and B. Overlapping spots are depicted in brown/black and marked by black arrows whereas spots which were only present on the Coomassie stained protein maps from Figure 1A are depicted in blue.

Figure 2. CLL B-cell receptor Ig014 recognizes and immunoprecipitates vimentin from stromal cell protein extracts and culture supernatants.

A: Vimentin is recognized in various stromal cell protein extracts by recombinant CLL BCR Ig014 and a commercially available vimentin-antibody. Protein extracts were generated from human cell lines expressing no vimentin (MCF-7) or low levels of vimentin (HEK293T, HeLa) as well as protein extracts from the vimentin-expressing mouse stromal cell line M210B4 and from NLCs of three patients (I, II, III) with CLL. Protein extracts were separated by SDS-PAGE and subjected to Western blotting using Ig014 or a commercially available vimentin antibody for detection. A loading control was performed by staining with an anti-β-actin antibody. B: Vimentin can be immunoprecipitated from nurse-like cell protein extracts and supernatant. Immunoprecipitations were performed from a nurse-like cell extract (NLC) and supernatant (SN) using Ig014 and sepharose-G beads as described in the materials and methods section. A HeLa cell extract and HeLa supernatant was used as a negative control. Precipitates were separated by SDS-PAGE and subjected to Western blotting using Ig014 or a commercial anti-vimentin antibody as primary antibodies. Note that in the left panel the light (LC) and heavy chains (HC) of the Ig014 immunoglobulin used for immunoprecipitation is also detected because an anti-human immunoglobulin secondary antibody was used when Western blot staining was done with Ig014 as the primary antibody.

Figure 3. Stromal cells express high amounts of vimentin which can be immunostained with the CLL B-cell receptor Ig014.

A: Visualization of vimentin expression in M210B4 stromal cells and nurse-like cells (NLC) by immunofluorescence confocal microscopy. M210B4 cells and NLCs were cultured on coverslips, fixed by paraformaldehyde and permeabilized by Triton-X-100. Vimentin was visualized using a FITC-conjugated anti-vimentin antibody (green). Cell membranes and cytoplasm were counterstained with Alexa Fluor 594 phalloidin (red). The right panel shows an overlay of both stainings. Stromal cells are shown at low (upper panel) and high magnification (intermediate panel). B: Visualization of Ig014 staining of M210B4 stromal cells by immunofluorescence confocal microscopy. Stainings were performed using Ig014 as primary antibody, followed by secondary detection with an anti-human FITC-conjugated antibody (anti-hu. FITC; green). Cell membranes and cytoplasm were counterstained with Alexa Fluor 594 phalloidin (red).

Figure 4. Viable stromal cells actively display vimentin on their cell surface.

A: Vimentin is displayed on the surface of non-permeabilized M210B4 stromal cells as visualized by confocal microscopy. Cells were cultured and fixed with paraformaldehyde as above but omitting the permeabilization step. A FITC-conjugated Vimentin antibody stained the outer surface of the cells (green "corona") as shown in the left panel. Intracellular counterstaining could not be performed due to the non-permeabilized state of the cells. The right image highlights the cell margins by a dotted grey line. B: Stromal cells (as used for immunofluorescence stainings, Figure 4A) display vimentin independently of apoptotic events. Cell viability was assessed before the vimentin staining ("0 h"), as well as 24 and 48 hours later ("24 h" and "48 h"). Less than 1% of cells underwent apoptosis at time point "0 h" and "24 h" and less than 3% at time point "48 h" as demonstrated by staining with the apoptosis marker 7-AAD (interspersed purple cells, highlighted by white arrows). Nuclei were counterstained with DAPI (blue). Pictures were taken using conventional fluorescence microscopy.

Figure 5. Stromal cell displayed or secreted antigen protects CLL 014 cells from spontaneous apoptosis.

A and B: Soluble recombinant B-cell receptor Ig014 competes stromal cell-mediated CLL 014 cell protection from spontaneous apoptosis. CLL 014 cells were cultured alone or in co-culture with the stromal cell line M210B4. B-cell receptor-mediated stroma-CLL cell interactions were blocked by the recombinant B-cell receptor Ig014 or the control immunoglobulin IgGr. After six days of culture, CLL viability was assessed either by trypan blue staining (A) or by FACS analysis using 7-AAD (B). The diagrams show the percentage of viable CLL 014 cells at this time point (data are means of triplicates [A] or duplicates [B] +/− SEM). C: Time course of CLL 014 apoptosis on stroma blocked with the recombinant B-cell receptor Ig014 and with the control IgGr. Under the experimental conditions described in panel A and B of this figure, CLL 014 apoptosis was measured at different time points. To detect early and late phases of apoptosis, the measurement included staining with annexin V and 7-AAD followed by FACS analysis (Data are means of duplicates +/− SEM). D: Stromal cell-mediated CLL 014 cell protection can be competed by a known anti-vimentin antibody. CLL 014 cells were cultured alone, in co-culture with the stromal cell line M210B4 or with stroma cells blocked with either control B-cell receptor IgGr, CLL B-cell receptor Ig014, or a commercially available rabbit anti-mouse/human vimentin antibody. Apoptosis was measured after 12 hours of culture by staining with annexin V and PI followed by FACS analysis. CLL 014 cell viability reductions under blocked stroma conditions were calculated as relative values with unblocked stroma support (set to 100%). Data are means of duplicate experiments +/− SEM. E: A vimentin secreting "feeder layer" exerts a protective effect on CLL 014 cells. CLL 014 cells were cultured on a layer of HEK 293T cells transfected with a vimentin encoding secretory eukaryotic expression vector or an empty control vector. The viability of CLL 014 cells was determined at different time points of the co-culture experiment by FACS analysis using 7-AAD (data means of duplicates +/− SEM).