The inhibitory receptor Siglec-G controls the severity of chronic lymphocytic leukemia

Abstract

Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in adults in the Western world. B cell receptor (BCR) signaling is known to be crucial for the pathogenesis and maintenance of CLL cells which develop from mature CD5⁺ B cells. BCR signaling is regulated by the inhibitory co-receptor Siglec-G and Siglec-G-deficient mice have an enlarged CD5⁺ B1a cell population. Here, we determine how Siglec-G expression influences the severity of CLL. Our results show that Siglec-G deficiency leads to earlier onset and more severe course of the CLL-like disease in the murine Eμ-TCL1 model. In contrast, mice overexpressing Siglec-G on the B cell surface are almost completely protected from developing CLL-like disease. Furthermore, we observe a downmodulation of the human ortholog Siglec-10 from the surface of human CLL cells. These results demonstrate a critical role for Siglec-G in disease progression in mice, and suggest that a similar mechanism for Siglec-10 in human CLL may exist.

Keywords: BCR signaling; CLL; Siglec-10; Siglec-G overexpressing mice; Siglecs.

Figure 1. Earlier and stronger expansion of CLL‐like cells in the blood of TCL1 × Siglecg ^−/− mice

To monitor the progression of the CLL‐like population, blood was collected from the animals every 4 weeks up to an age of 60 weeks and analyzed by flow cytometry.

1. Representative FACS plots show examples of stainings of B220^low CD5⁺ B cells. (CLL‐like cells) and the selected gate for (B).

2. The diagram displays the percentage of CLL‐like cells in relation to all lymphocytes over a period of 60 weeks. Shown are mean values with ± SD. Significant differences between groups were determined by one‐way ANOVA with Kruskal–Wallis test (no normal distribution) and corrected for multiple comparison with Dunn's test, **P < 0.01, ***P < 0.001. Summarized data from more than 40 independent experiments for individual time points, comprising n = 4–75 animals per time point and genotype. X = no surviving TCL1 × Siglecg ^−/− animals at the age of 60 weeks. + indicates that in these groups only 4 animals were used for the analysis and therefore no calculation of significance was possible.

Source data are available online for this figure.

Figure EV1. Earlier expansion of the CLL‐like cell population in TCL1 × Siglecg^−/− mice and later expansion in TCL1 × Siglecg‐R26^ki/ki mb1^cre mice, shown for individual mice over time

1. A, B

   Shown are percentages of B220^low CD5⁺ cells in the blood of individual mice over time in (A) comparison of TCL1 and TCL1 × Siglecg^−/− mice and in (B) TCL1 × Siglecg‐R26^ki/ki and TCL1 × Siglecg‐R26^ki/ki mb1^cre control mice. To distinguish different mice different symbols were used to represent individual mice that are connected by lines. N = 15 TCL1; n = 15 TCL1 × Siglecg ^−/−; n = 22 TCL1 × Siglecg‐R26^ki/ki; n = 17 TCL1 × Siglecg‐R26^ki/ki mb1^cre.

Figure EV2. Earlier signs of leukocytosis and lymphocytosis in TCL1 × Siglecg ^−/− mice

1. A–D

   For hematological analysis of blood cells (A) the leukocyte count (B) the lymphocyte count (C) the platelet count and (D) the red blood cell count were determined with an Advia 120 hematology analysis machine. The mean values are shown with SD. Significant differences between groups were determined by one‐way ANOVA with Kruskal–Wallis and corrected for multiple comparison with Dunn's test, *P < 0.05, **P < 0.01, ***P < 0.001. n = 5–16 animals per time point and genotype, summarized from at least five independent experiments.

Figure 2. Earlier splenomegaly, earlier expansion, and infiltration of CLL‐like cells in spleen and liver and lower survival of TCL1 × Siglecg ^−/− mice

1. Depicted are representative photographs of the spleen on graph paper of the different genotypes at 36 and 48 weeks of age. The spleen weight was determined and the mean values with ± SD are shown for every time point. Significant differences between groups were tested by one‐way ANOVA with Kruskal–Wallis test and corrected for multiple comparison with Dunn's test.

2. The absolute cell numbers of B220^low CD5⁺ lymphocytes at different time points in the spleen are shown as mean values with ± SD. Significant differences between groups were tested by one‐way ANOVA with Kruskal–Wallis test and corrected for multiple comparison with Dunn's test. The different time points were tested separately. Alongside representative dot plots show selection and percentages of B220^low CD5⁺ cells in the spleen pre‐gated on single cells, living cells and lymphocytes.

3. The percentage of B220^low CD5⁺ leukocytes in the liver at different time points is illustrated. Besides representative dot plots show the selection of B220^low CD5⁺ cells in the liver. Cells were pre‐gated on single cells, living cells and CD45⁺ leukocytes. Shown are mean values with ± SD. Significant differences between groups were tested by ordinary one‐way ANOVA with Šídák's post‐hoc test.

4. The survival of TCL1 × Siglecg ^−/− mice compared to TCL1 mice is illustrated with the Kaplan–Meier survival curve (n = 81 for TCL1, n = 101 for TCL1 × Siglecg ^−/−). The groups were compared for significant differences via log‐rank test.

Data information: (A–C) Data are from at least 10 independent experiments for individual time points, comprising n = 5–12 animals per time point and genotype. X = no surviving TCL1 × Siglecg ^−/− animals at the age of 60 weeks. *P < 0.05, **P < 0.01, ****P < 0.0001. Source data are available online for this figure.

Figure 3. TCL1 × Siglecg ^−/− mice develop leukemic clones earlier than TCL1 controls

Analysis of the IgV_H repertoire of CD19⁺ sorted blood cells by NGS. Circos plots depicting the frequencies of V_HJ_H usage from three individual mice for the different time points are shown. The number of productive sequences is indicated under the respective plot. The V_H family of prominent clones is highlighted. The cross indicates that the animals died before reaching the time point and therefore no analysis could be performed. n.d. indicates no data available. Data are from one experiment, comprising n = 3 mice per genotype.

Figure 4. Overexpressed Siglec‐G prevents development of CLL‐like cells in the blood of TCL1 mice almost completely

To monitor the progression of the CLL‐like population, blood was collected from the animals every 4 weeks up to an age of 48 weeks and analyzed by flow cytometry. Genotypes of the mice are: controls (Siglecg‐R26^ki/ki), Siglec‐G overexpressing mice (Siglecg‐R26^ki/ki mb1^cre), transgenic TCL1 controls (TCL1 × Siglecg‐R26^ki/ki) and transgenic TCL1 Siglec‐G overexpressing mice (TCL1 × Siglecg‐R26^ki/ki mb1^cre).

1. Representative FACS plots show examples of stainings of B220^low CD5⁺ B cells. (CLL‐like cells) and the selected gate for (B).

2. The diagram displays the percentage of B220^low CD5⁺ cells in respect to lymphocytes over a time period of 48 weeks. Shown are mean values with ± SD. Significant differences between groups were determined by one‐way ANOVA with Kruskal–Wallis test (no normal distribution) and corrected for multiple comparison with Dunn's test. All time points were tested separately. *P < 0.05, **P < 0.01, ***P < 0.001. Summarized data from more than 20 independent experiments with n = 5–20 animals per time point and genotype are shown.

Source data are available online for this figure.

Figure EV3. Generation of Siglec‐G overexpressing mice

1. For the generation of Siglec‐G overexpressing mice the cDNA of Siglec‐G was cloned into the target vector, between the short (SA) and the long arm (LA) of the ROSA26 (R26) locus. Overexpression was facilitated by the CAG enhancer cassette, which contains the chicken ß‐actin promoter and the early enhancer element of cytomegalovirus. A transcriptional stop cassette flanked by two loxP sites ensured B cell‐specific expression by mating with the mb1cre mouse strain. The cre recombinase specifically removes the stop cassette so that the enhancer cassette is brought in front of the cDNA and transcription of the Siglec‐G cDNA occurs.

2. Siglecg‐R26^ki/ki mb1^cre mice showed a five to eightfold overexpression of Siglec‐G on the surface of mature B cells in the spleen and peritoneal cavity, respectively. The histograms represent the expression of Siglec‐G on conventional B2 cells of the spleen.

3. In the peritoneal cavity a reduction of B1a cells was observed in Siglecg‐R26^ki/ki mb1^cre mice in contrast to the enlargement of this population in Siglecg^−/− mice. Shown are the mean values of the absolute cell counts. Cells were pre‐gated for single, living lymphocytes subsequently, B1a cells were identified as B220^low and CD5⁺. Significant differences between groups were determined with Kruskal–Wallis and corrected for multiple comparison with Dunn's test, *P < 0.05. n = 6–14 animals per genotype; every dot represents a mouse. Data are summarized from 5 independent experiments.

Figure EV4. No leukocytosis or lymphocytosis in TCL1 × Siglecg‐R26^ki/ki × mb1^cre mice

1. A–D

   For hematological analysis of blood cells (A) the leukocyte count (B) the lymphocyte count (C) the platelet count and (D) the red blood cell count were determined with an Advia 120 hematology analysis machine. The mean values are shown with ± SD. Significant differences between groups were determined by one‐way ANOVA with Kruskal–Wallis and corrected for multiple comparison with Dunn's test, *P < 0.05, **P < 0.01. n = 5 animals per time point and genotype, summarized from at least 5 independent experiments.

Figure 5. No splenomegaly, hardly any expansion of CLL‐like cells in spleen and liver and better survival of Siglec‐G overexpressing TCL1 mice

1. Depicted are representative photographs of the spleen on graph paper of the different genotypes at 36 and 48 weeks of age.

2. The spleen weight in g was determined and the mean values with ± SD are shown for every time point. Significant differences between groups were tested by one‐way ANOVA with Kruskal–Wallis test and corrected for multiple comparison with Dunn's test. n = 8–15 animals per time point and genotype.

3. Absolute cell numbers of B220^low CD5⁺ lymphocytes at 36 and 48 weeks of age in the spleen are shown as mean values with ± SD. Significant differences between groups were tested by one‐way ANOVA with Kruskal–Wallis test and corrected for multiple comparison with Dunn's test. The different time points were tested separately. n = 8–13 animals per time point and genotype.

4. Representative dot plots show selection and percentages of B220^low CD5⁺ B cells in the spleen pre‐gated on single cells, living cells and lymphocytes.

5. The percentage of B220^low CD5⁺ lymphocytes in the liver at 36 and 48 weeks of age is illustrated. Shown are mean values with ± SD. Significant differences between groups were tested by ordinary one‐way ANOVA with Šídák's post‐hoc test. n = 7–14 animals per time point and genotype.

6. Representative dot plots for the selection of B220^low CD5⁺ cells in the liver. Cells were pre‐gated on single cells, living cells and CD45⁺ leukocytes.

7. The differences in survival are shown as Kaplan–Meier plots and log‐rank test n = 59 for TCL1 × Siglecg‐R26^ki/ki, n = 57 for TCL1 × Siglecg‐R26^ki/ki mb1^cre.

Data information: (B), (C), (E). Data are from at least 10 independent experiments for individual time points. *P < 0.05, **P < 0.01, ***P < 0.001. n = 48 for TCL1 × Siglecg‐R26^ki/ki, n = 52 for TCL1 × Siglecg‐R26^ki/ki mb1^cre. Source data are available online for this figure.

Figure 6. No indication of leukemic clone development in Siglec‐G overexpressing TCL1 mice

Analysis of the IgV_H repertoire of CD19⁺ sorted blood cells by NGS. Circos plots depicting the frequencies of V_HJ_H usage from three individual mice for the different time points are shown. The number of productive sequences is indicated under the respective plot. When less than 500 productive sequences were obtained, these were marked with an asterisk. The V_H family of prominent clones is highlighted. n.d. indicates no data available. Data are from one experiment, comprising n = 3 mice per genotype.

Figure 7. Higher BCR‐induced calcium response in CLL‐like cells of TCL1 × Siglecg ^−/− mice and increased intracellular signaling responses in TCL1 and TCL1 × Siglecg ^−/− mice, but not in Siglec‐G overexpressing TCL1 mice

1. Shown is a representative dot plot for the selection strategy for CLL‐like B cells from the spleen of TCL1 × Siglecg^−/− mice. For the analysis of all B cells (B220+ and B220^low cells), conventional B2 cells plus the B220^low CD5⁺ population was used. Cells were pre‐gated for single cells and lymphocytes.

2. To measure calcium influx cells were loaded with Indo‐1. Shown is the mean calcium concentration as ratio of bound to unbound Indo‐1 versus time in s. First the basal level was monitored for 50 s in Krebs–Ringer solution. Subsequently, the BCR was stimulated with 13 μg/ml anti‐IgM F(ab)₂ (shown by the black arrow). Shown is one representative result of four independent experiments. Mice were between 36 and 48 weeks of age. To measure intracellular signaling splenic cells were pre‐gated on single, living cells and lymphocytes, then B220⁺ cells, including B220^low CD5⁺ lymphocytes were selected.

3. TCL1 and TCL1 × Siglecg ^−/− B cells were analyzed by intracellular staining with phopho‐specific antibodies (or total protein detecting antibody Bcl2).

4. TCL1 × Siglecg‐R26^ki/ki or TCL1 × Siglecg‐R26^ki/ki mb1‐cre B cells were analyzed by intracellular staining with phopho‐specific antibodies (or total protein detecting antibody Bcl2).

Data information: Shown are geometric mean values normalized to the WT with ± SD. Significant differences between groups were tested either by ordinary one‐way ANOVA with Šídák's post‐hoc test if there was a normal distribution, or by one‐way ANOVA with Kruskal–Wallis test and corrected for multiple comparison with Dunn's test if there was no overall normal distribution. *P < 0.05, **P < 0.01. Data are from at least five independent experiments, summarized for n = 7–11 animals. Mice were between 36 and 48 weeks of age. Source data are available online for this figure.

Figure 8. Downmodulation of human Siglec‐10 surface expression on CLL cells compared to normal residual B cells

1. Peripheral blood B cells of CLL patients are pre‐gated on single, living B lymphocytes (CD19⁺). The mean fluorescence intensity (MFI) of surface Siglec‐10 is given from 35 IgV‐mutated (left) and 18 IgV‐unmutated CLL cases (right), always including tumor cells (CD20^lowCD5^high) and normal residual B cells (CD20^highCD5⁻) as paired samples.

2. The transcript expression (normalized counts from bulk mRNA sequencing) of SIGLEC10 is given from five healthy donors, discriminating naïve CD5⁻ (5 donors), mature CD5⁺ (86 donors), and CD27⁺ memory B‐cell (8 donors) subsets, from CLL tumor cells (n = 9) and from five paired normal residual B (NRB) cells (n = 5).

Data information: Wilcoxon rank sum test, **P < 0.01, ***P < 0.001. Median as central band, box encompassed from first to third quartile, whiskers are the smallest or largest value no further than 1.5 * IQR (range from first to third quartiles) from the hinge. Source data are available online for this figure.

Figure EV5. Downmodulation of human Siglec‐10 surface expression on CLL cells compared to normal residual B cells

1. Peripheral blood B cells, pre‐gated on single, living B lymphocytes (CD19⁺). One representative plot for the gating of CLL cells (CD20^lowCD5^high) and normal residual (NRB) cells (CD20^highCD5⁻) is given.

2. Data as in Fig 8A, separated by IgV‐mutation status (mCLL and uCLL, top and bottom row, respectively) and Binet A versus Binet B or C stage of the patient (left versus right column).

Data information: Wilcoxon signed‐rank test, *P < 0.05, ***P < 0.001. Samples are paired biological replicates. Median as central band, box encompassed from first to third quartile, whiskers are the smallest or largest value no further than 1.5 * IQR (range from first to third quartiles) from the hinge.