Site-1 protease function is essential for the generation of antibody secreting cells and reprogramming for secretory activity

Abstract

The unfolded protein response (UPR) and activation of XBP1 is necessary for high secretory efficiency and functional differentiation of antibody secreting cells (ASCs). The UPR additionally includes a branch in which membrane-bound transcription factors, exemplified by ATF6, undergo intramembrane-proteolysis by the sequential action of site-1 (MBTPS1/S1P) and site-2 proteases (MBTPS2/S2P) and release of the cytoplasmic domain as an active transcription factor. Such regulation is shared with a family of CREB3-related transcription factors and sterol regulatory element-binding proteins (SREBPs). Of these, we identify that the CREB3 family member CREB3L2 is strongly induced and activated during the transition from B-cell to plasma cell state. Inhibition of site-1 protease leads to a profound reduction in plasmablast number linked to induction of autophagy. Plasmablasts generated in the presence of site-1 protease inhibitor segregated into CD38^high and CD38^low populations, the latter characterized by a marked reduction in the capacity to secrete IgG. Site-1 protease inhibition is accompanied by a distinctive change in gene expression associated with amino acid, steroid and fatty acid synthesis pathways. These results demonstrate that transcriptional control of metabolic programs necessary for secretory activity can be targeted via site-1 protease inhibition during ASC differentiation.

Figure 1

Expression of CREB3L2 in differentiating primary human B-cells. (A) Schematic of in vitro B-cell differentiation with key stages and phenotypes. (B) Relative mRNA expression levels for IRF4, IRF8, CREB3L2, MBTPS1 and MBTPS2 during in vitro generation of PCs derived from microarray data. (C) Protein expression of CREB3L2 during in vitro differentiation of primary human B-cells. The day of culture is indicated at the top. Intact and dashed arrows show migration of full-length protein and cleaved C-terminus, respectively.

Figure 2

S1P-specific inhibitor PF-429242 blocks CREB3L2 processing. (A) Diagram of CREB3L2 structure with proteolytic cleavage sites. Act domain, Activation Domain. bZIP, Basic Leucine Zipper Domain. TM, Transmembrane Domain. (B) In vitro generated human ASCs were treated with 10 μM PF-429242 or vehicle control (dH2O) on the day that appears first (d6, d8, d10) and protein lysates generated on the day that appears subsequently (d8, d10, d13) were analyzed by Western blotting. Intact and dashed arrows show migration of CREB3L2 full-length protein and cleaved C-terminus, respectively. (C) In vitro differentiated human B-cells were treated with 10 μM PF-429242 or vehicle control (dH2O) at day 6 and analyzed subsequently for phenotype on the indicated days. Live cells were evaluated for expression of B-cell markers CD19 and CD20 and ASC markers CD38 and CD138. Percentages of cells are shown within the quadrant gates. The experiment was performed with 4 donors, a representative donor is shown. (D) Samples treated as in part (B) were assessed for cell number using CountBright beads. (E) In vitro activated human B-cells from 4 donors were treated with indicated amount PF-429242 or vehicle control (dH2O) at day 6 and assessed for cell number at 48 h. Results are displayed as the percentage viable cells relative to control. Supernatants from samples in part (B) were analyzed by ELISA for production of (F) IgM and (G) IgG. Significance was determined by t-test.

Figure 3

Kinetic and dose analysis of S1P-specific inhibitor PF-429242 during the early phase of ASC generation. (A) In vitro activated human B-cells were treated with 10 μM PF-429242 or vehicle control (dH2O) at day 3 and assessed for CREB3L2 processing at 12 h intervals. Representative results are shown from a total of 7 donors. (B) Samples from part (A) were assessed for cell number at the indicated time points. Displayed are the average cell number ± standard deviation derived from 7 donors. (C) In vitro activated human B-cells from 4 donors were treated with indicated amount PF-429242 or vehicle control (dH2O) at day 3 and assessed for cell number at 48 h. Results are displayed as the percentage viable cells relative to control.

Figure 4

S1P inhibition induces autophagy and affects multiple UPR-related TFs. (A) In vitro day 3 B-cells were treated with 10 μM PF-429242 or vehicle control (dH2O) for 72 h and were assessed for cell number using CountBright beads. Shown are the average cell numbers from 7 donors ± standard deviations. Protein lysates generated from day 3 cells treated for 72 h with 10 μM PF-429242 were evaluated for (B) autophagy markers or (C) CREB3L2 and ATF6 by Western blotting. Shown are representative results from 2 donors, a total of 7 donors were evaluated.

Figure 5

S1P inhibition alters phenotypic maturation and prevents antibody secretion. (A) In vitro activated human B-cells were treated with 10 μM PF-429242 at day 3 and analyzed at 12 h intervals. Live cells were evaluated for surface phenotype. (B) The levels of CD38 expression in the lower left quadrant (CD38 low) or upper left quadrant (CD38 high) were determined for 7 donors at the 72 h time point. Data depict the mean ± standard deviation with significance determined by t-test (**P = 0.003, ***P = 0.001). (C) Day 3 human differentiated B-cells were treated with 10 μM PF-429242 or dH2O for 72 h. After the incubation, the cells were sorted based on CD38 (high and low). An equal number of sorted cells (2,000 cells) were used to quantify ASCs using ELISpot for IgM and IgG.

Figure 6

Gene expression analysis identifies distinct gene signature enrichment in PF-429242 treated plasmablasts. (A) Day 3 B-cells from 6 donors were treated with 10 μM PF-429242 or vehicle control (dH2O) and analyzed on day 6 using Illumina bead arrays. (B) Selected genes showing greater than 1.4-fold change in expression are depicted. (C) Enrichment of gene signatures was determined using a hypergeometric test against a curated set of 28,000+ signatures and ontology term associations. Enriched signatures and ontologies were clustered according to genes contributing to enrichment with summary labels shown on the right.