CD24 induces changes to the surface receptors of B cell microvesicles with variable effects on their RNA and protein cargo

Abstract

The CD24 cell surface receptor promotes apoptosis in developing B cells, and we recently found that it induces B cells to release plasma membrane-derived, CD24-bearing microvesicles (MVs). Here we have performed a systematic characterization of B cell MVs released from WEHI-231 B lymphoma cells in response to CD24 stimulation. We found that B cells constitutively release MVs of approximately 120 nm, and that CD24 induces an increase in phosphatidylserine-positive MV release. RNA cargo is predominantly comprised of 5S rRNA, regardless of stimulation; however, CD24 causes a decrease in the incorporation of protein coding transcripts. The MV proteome is enriched with mitochondrial and metabolism-related proteins after CD24 stimulation; however, these changes were variable and could not be fully validated by Western blotting. CD24-bearing MVs carry Siglec-2, CD63, IgM, and, unexpectedly, Ter119, but not Siglec-G or MHC-II despite their presence on the cell surface. CD24 stimulation also induces changes in CD63 and IgM expression on MVs that is not mirrored by the changes in cell surface expression. Overall, the composition of these MVs suggests that they may be involved in releasing mitochondrial components in response to pro-apoptotic stress with changes to the surface receptors potentially altering the cell type(s) that interact with the MVs.

Figure 1

The quantification and morphology of MVs released from B cells with and without CD24 stimulation. (A) Representative nanoparticle tracking (NTA) plots of particle sizes and concentrations in supernatants from cells stimulated with either isotype (right) or anti-CD24 (left) Ab after 1 h. Grey lines show ± 1 standard deviation of the mean in measurements. n = 3 biological replicates with 5 technical replicates each. (B) The mean size (left) and concentration (right) ± SEM of particles from supernatants analyzed by NTA, n = 3. Statistical significance was assessed using Students’ paired t-test, ^#p = 0.08. (C) Mean % positive (left) and relative mean fluorescent intensity (MFI; right) of Annexin V-FITC beads used to capture CD24-bearing MVs released from isotype or anti-CD24 stimulated cells for the indicated times, n = 3. Statistics assessed by a two-tailed Student’s paired t-test. **p < 0.01. (D) Representative transmission electron microscopy (TEM) images of Vn96-isolated EVs from cells stimulated with either isotype or anti-CD24 for 1 h. Scale bar = 100 nm.

Figure 2

CD24 stimulation alters the abundance of protein coding transcripts loaded into B cell MVs. (A) Representative RNA size distributions of MVs collected from cells stimulated with either isotype (right) or anti-CD24 (left) Ab after 1 h. (B) The RNA incorporated into MVs from cells after 1 h of either isotype or anti-CD24 Ab stimulations categorized into one of 8 sub-categories containing >0.2% of the total RNA, n = 3 biological replicates. (C) The mean ± SEM percentage of the 8 major non-rRNA transcripts with greater than 2% of RNA abundance from MVs isolated from either isotype (white bars) or anti-CD24 (grey bars) stimulated cells. n = 3, statistics were assessed using a two-tailed Student’s t-test, ^#p = 0.07; *p < 0.05.

Figure 3

Proteomics analysis suggests that CD24 stimulation causes enrichment of proteins from specific functional categories into MVs. Gene Ontology (GO) enrichment analysis was performed on proteins common to MVs from all 6 isotype and anti-CD24 samples in comparison to the proteins enriched in MVs after 1 h anti-CD24 stimulation (Supplemental File 1). Venn diagrams (left panels) show GO terms associated with all MVs or those associated with the proteins enriched in MVs after anti-CD24 stimulation. The numbers indicate the number of unique GO terms associated with the respective protein lists. Enriched GO terms were visualized by Revigo (right panels). (A) CD24-enriched biological process (BP): Cellular amide metabolism (yellow), macromolecular complex assembly (green) and protein localization to nuclear body (blue). (B) CD24-enriched molecular functions (MF): Damaged DNA binding (green), transferase activity (purple) and threonine aldolase activity (blue). (C) CD24-enriched cellular component annotations were primarily grouped as chaperonin-containing T-complex (red). A.A. = amino acid, Synth = synthesis, Carb = carboxylic, Reg = regulation, Pos. = positive, Leng. = length, org = organization, Maint = maintenance, Loc = localization, Est = Establishment, Spm = sperm, Z.P = zona pellucida.

Figure 4

Proteins identified by proteomics analysis of B cell MVs are detectable in both cell lysates and MVs of isotype and anti-CD24 stimulated B cells by Western blot analysis. Cell lysates (5 µg; equivalent to approximately 2.3 × 10⁵ cells) and the corresponding protein from MVs (from 1.0 × 10⁶ cells) were analyzed for expression of SHMT2, EEF1G, GRB2 and HMGB2. HSP90 was used as a loading control after stimulation with isotype (Iso) or anti-CD24 (CD24). n = 5. Two different outcomes were observed for HMGB2, with MVs from CD24 stimulated cells containing high HMGB2 (Upper panels, n = 2) or HMGB2 being low/absent in all vesicles (lower panels n = 3).

Figure 5

CD24 stimulation induces the formation of a unique B cell MV surface phenotype that does not reflect the cells from which they are released. Cells (A,B) and MVs (C,D) were analyzed for their expression of the indicated cell surface markers. Data are shown as mean ± SEM at 1 h or 2 h stimulation with either isotype or anti-CD24 Ab. n = 3–4. Cells or CD24-bearing MVs bound to beads were assessed for the percentage of (A) cells or (C) MVs positive for the individual markers, and (B/D) their relative mean fluorescent intensities (MFI). Significant differences were assessed by two-way ANOVA followed by Tukey post-hoc, if significant. Main effect of stimulation: ^§P < 0.05, ^§§P < 0.01. Significant changes compared to time-matched control: ^#P < 0.1, *P < 0.05, **P < 0.01. Non-statistically significant changes have no indicators.

Figure 6

Summary diagram of the cargo and surface composition of MVs from isotype or CD24 stimulated cells. The differences in surface protein, luminal protein, and RNA transcriptome between basal and CD24 induced MVs are shown. No change indicated by=, absence indicated by X, a significant change indicated by arrows where arrows coloured with a gradient indicate a variable difference in abundance as detected by Western blot.