Early Emergence of Adaptive Mechanisms Sustaining Ig Production: Application to Antibody Therapy

Abstract

Antibody therapy, where artificially-produced immunoglobulins (Ig) are used to treat pathological conditions such as auto-immune diseases and cancers, is a very innovative and competitive field. Although substantial efforts have been made in recent years to obtain specific and efficient antibodies, there is still room for improvement especially when considering a precise tissular targeting or increasing antigen affinity. A better understanding of the cellular and molecular steps of terminal B cell differentiation, in which an antigen-activated B cell becomes an antibody secreting cell, may improve antibody therapy. In this review, we use our recently published data about human B cell differentiation, to show that the mechanisms necessary to adapt a metamorphosing B cell to its new secretory function appear quite early in the differentiation process i.e., at the pre-plasmablast stage. After characterizing the molecular pathways appearing at this stage, we will focus on recent findings about two main processes involved in antibody production: unfolded protein response (UPR) and endoplasmic reticulum (ER) stress. We'll show that many genes coding for factors involved in UPR and ER stress are induced at the pre-plasmablast stage, sustaining our hypothesis. Finally, we propose to use this recently acquired knowledge to improve productivity of industrialized therapeutic antibodies.

Keywords: B cell differentiation; ER stress; RNA-seq; UPR; mAbs.

Figure 1

Schematic representation of the in vitro model of B cells differentiation used in our laboratory. Peripheral NBCs from blood donors are stained with a cell-tracer then stimulated with IL-2, CD40L, CpG and anti-IgM Fab’2. Day-1 activated cells are referred as day-1 ActB. After 4 days, activated B cells that have proliferated (Day-4 ActB) are selected according cell-tracer dilution and stimulated with IL-2, IL-4 and IL-10 to induce their differentiation into plasmablasts. The day after (D5), three populations are detected: (i) CD23+ cells that are stuck in an activated state and unable to differentiate, (ii) CD23- population, containing precursors of plasmablasts (pre-PB) which give rise to (iii) differentiated plasmablasts (PB). The increase in the cytoplasm/nucleus ratio, characteristic of the development of the Ig production machinery in PB, is early detected in the pre-PB stage. RNA-seq data are available for NBC, Day-1 ActB, Day-4 ActB, CD23+, CD23- and PB subsets.

Figure 2

Overview of the UPR signaling pathway under ER stress conditions. Unfolded Protein Response is engaged to respond to increasing amount of proteins in the ER. (1) The ER-resident chaperone BiP binds to unfolded and misfolded proteins and consequently releases the sequestrated IRE1α, PERK and ATF6 ER sensors, leading to their activation following (i) dimerization and auto-phosphorylation of IRE1α and PERK elements or (ii) MBTPS1/S1P and MBTPS2/S2P – mediated cleavage of ATF6α in the Golgi apparatus. Activated ATF6α then translocates into the nucleus and upregulates chaperones expression and factors involved in lipid synthesis and ERAD. Activated PERK phosphorylates eIF2α which represses global protein translation except for ATF4 whose translation is enhanced. ATF4 then upregulates amino acid metabolism and apoptosis. IRE1α, via XBP-1 transcription factor, leads to a broader range of responses with upregulation of many factors including chaperones and those important for lipid synthesis and ERAD. IRE1α regulates as well the factors important for protein folding and secretory functions. Particularly, XBP1 mRNA requires IRE1α to be spliced and efficient as a transcription factor.

Figure 3

Unfolded protein response is temporally regulated during the transition from naive B cells to plasmablasts. (A) Genes belonging to the Gene Ontology – Biological Processes term “Unfolded Protein Response” were selected and their expression in our RNA-seq data was submitted to an unsupervised hierarchical clustering analysis. The resulting heatmap is shown here, delineating four different expression clusters: (1) stable expression, (2) genes upregulated from Day-1, (3) genes upregulated in NBC and (4) genes upregulated in Pre-PB and PB. (B) Means of normalized reads obtained from 3 different experiments at Day-1 ActB, Day-4 ActB, pre-PB and PB stages were compared to the NBC counterpart, whose mean is reduced to 1. Left upper panel shows genes related to the PERK pathway. Right upper panel shows genes related to the IRE1α and ATF6 pathways and lower panels show potential genes which are either implicated (i) in the PERK regulation or (ii) in endoplasmic reticulum modifications related to stress. All of the selected genes are addressed in this review.

Figure 4

PERK is negatively regulated right after B cell activation and maintained under negative control during differentiation. PERK signaling is upregulated in NBCs (light green background). In activated B cells, PERK needs to be dampened by factors limiting PERK activity (light orange background). Our RNA-seq analysis combined with literature emphases on two new potential negative factors: HYOU1 and UFBP1. Then, differentiation (pre-PB/PB) involves important changes including increased production of Ig, which critically increases ER stress and risks of apoptosis; hence, PERK signaling repression needs to be reinforced. Factors potentially involved in this control are part of the ADP/ATP BiP regulation cycle: the DnaJ protein P58^IPK and the nucleotide exchange factors SIL1 and HYOU1. The first wave of negative control is IRE1α/XBP-1/ATF6-independent and the second wave is IRE1α/XBP-1/ATF6-dependent (right gray arrows).

Figure 5

Overview of known or suggested strategies to improve Ig production in CHO cells. In the first steps of NBC activation, a low rate of proteins is processed into the ER leading to no or negligible stress conditions (upper panel). When cells undergo the following steps of differentiation that will result in the generation of highly-producing secreted Ig PBs/PCs, mass of their ER is getting bigger and associated with an important stress. Unfolded Protein Response is then engaged to cope with the increasing amount of proteins in the ER. (1) The ER-resident chaperone BiP binds to unfolded and misfolded proteins and consequently releases the sequestrated IRE1α, PERK and ATF6 ER sensors, leading to their activation following (i) dimerization and auto-phosphorylation of IRE1α and PERK elements or (ii) MBTPS1/S1P and MBTPS2/S2P – mediated cleavage of ATF6α in the Golgi apparatus. (2) DnaJ family members such as P58^IPK (encoded by DNAJC3) bind directly to unfolded and misfolded proteins to guide their transfer onto an ATP-bound BiP. Given the ATPase activity of BiP, an inorganic phosphate (Pi) is instantly released from BiP, leading to a conformational modification which stabilizes protein into the ADP-bound BiP (3). Thereafter, nucleotide-exchange factors (NEFs) such as SIL1 and HYOU1 facilitate the release of ADP from BiP and the rebinding of ATP. 4-5) As a consequence, client is released and then processed to be secreted outside of the cell. The main strategies already proposed to improve Ig production in CHO cells are represented as full orange arrows. The strategies discussed in this review are represented as repressive or permissive dotted orange arrows.