Elucidating the Role of Novel Proteins in Plasma Cell Differentiation and Antibody Secretion

Antibodies play an essential role in the humoral component of the immune system. They are produced by Antibody Secreting Cells (ASCs) which are capable of secreting thousands of antibody molecules per second. To facilitate this function, ASCs undergo a dramatic upregulation of their biosynthetic pathway, and therefore represent a unique physiological model of constitutive secretion. In a previous study performed in the lab, a large number of poorly characterised genes were identified as highly upregulated in ASCs compared to naïve B-cells, suggesting that they may be novel factors involved in intracellular trafficking and antibody secretion. To investigate the role of these genes in ASC physiology we have first established a model of B cell differentiation and antibody secretion based on I.29 cells. These cells respond to LPS treatment, expand their biosynthetic capacity, and secrete antibodies. Importantly, they also upregulate the expression of many genes which were identified in the previous proteogenomic work (e.g., FNDC3B, SEC24D, CREB3L2, RRBP1, CRELD2 and TMEM214). To determine the function of these genes, we have developed a CRISPR/Cas9 based platform in I.29 cells and have demonstrated the feasibility of this approach by disrupting the expression of several genes including the R-SNARE VAMP3, the transcription factor CREB3L2 and the coat protein SEC24D. SEC24D, a component of the COPII coat, is one of the most differentially expressed genes in ASC and its expression is thought to be regulated by CREB3L2. Loss of SEC24D did not impact IgM secretion two days post LPS induction. However, its loss leads to a significant reduction in the levels of other COPII components. Surprisingly, the levels of FNDC3B are also perturbed in these cells. FNDC3B is an ER localised protein recently shown to have a role in ER proteostasis suggesting that ER proteostasis in cells with disrupted SEC24D function may be perturbed. Loss of CREB3L2 did not impact the production of antibodies or the expression levels of SEC24D suggesting that other members of this transcription factor family may regulate the levels of SEC24D expression. Finally, we investigated the role of the OASIS family of transcription factors including CREB3L2 in regulating the expression of SEC24D. We have observed that these transcription factors resulted in increased expression of SEC24D, increased cell size and an expanded staining of SEC24D structures in HeLa cells, making them interesting targets for secretory pathway engineering in non-professional secretory cells.