B-cell receptor induced autophagy and phagocytosis in chronic lymphocytic leukaemia

Abstract

Chronic lymphocytic leukaemia (CLL) is a common mature B-cell malignancy with a highly heterogeneous disease course, tightly linked to features of the B-cell receptor (BCR). Higher surface immunoglobulin (sIg)M signalling capacity is associated with poorer outcomes, and inhibitors targeted against BCR-associated kinases induce dramatic clinical responses. In normal B cells, the BCR has a dual capacity to induce intracellular signalling responses, and internalise soluble or particulate antigens for presentation to T-helper cells. These processes are subject to cross-talk, as BCR signalling can be required for antigen internalisation, in addition to regulating antigen presentation machinery expression. There remains a clear need to understand BCR-mediated signalling and antigen internalisation/presentation pathways in CLL cells.
One process with important links to both signalling and antigen presentation is autophagy. Autophagy is tightly regulated by signalling pathways and upregulated in many types of cancer, resulting in breakdown of non-essential, damaged or misfolded proteins/organelles, thereby protecting cells from effects of starvation or stress. However, autophagy also plays specialised roles in the immune system, including antigen processing and presentation, and is known to be activated via BCR signalling in normal B cells.
My initial hypothesis was that BCR stimulation induces autophagy in CLL cells. Using bead immobilised anti-IgM/D, I demonstrated that BCR stimulation increased autophagy markers (LC3B-II, GABARAPL2 p62 and pATG13). Inhibitor studies demonstrated that autophagy induction was dependent on autophagy regulators VPS34 and ULK1/2, and the BCR signalling kinase SYK. Confocal analysis revealed autophagosome turnover following sIgM stimulation (LC3B/LAMP colocalisation), confirming that BCR stimulation induces active autophagy in CLL. An unexpected observation from imaging studies was that anti-IgM coated beads were internalised by CLL cells. I therefore developed a quantitative, flow cytometry assay, to confirm that CLL cells efficiently internalised anti-IgM-coated beads. Internalisation required SYK activity, but not VPS34, and appeared to be mediated by a phagocytic process, requiring actin remodelling but not dynamin. These results demonstrate for the first time that CLL cells can internalise particulate antigen via sIgM.
I performed RNA-seq analysis to probe consequences of sIgM stimulation/autophagy inhibition in CLL cells, demonstrating that anti-IgM treatment results in strong activation of antigen presentation pathways. I therefore investigated the capacity of CLL cells to present antigen from anti-IgM-coated beads, via MHC-II, to T cells. This demonstrated that CLL cells could present particulate-derived antigen to a similar extent as soluble. Both processes appeared independent of autophagy. Presentation from soluble antigen required SYK activity, but SYK inhibition did not affect antigen presentation from particulate antigen, despite inhibiting phagocytosis. Therefore, internalisation of particulate antigen may not be required for presentation, and in fact B cells may secrete proteases to liberate antigen extracellularly.
In summary, these results provide important new insight into the function of the BCR in CLL as a dual mediator of signalling and antigen presentation. Autophagy is a target of signalling, but does not appear to play a key role in either phagocytosis or MHC-II Ag presentation. The ability of CLL cells to present particulate antigen may be particularly important for pathobiology. However, it does not seem to be affected by currently used kinase inhibitors, including ibrutinib, and further analysis of the pathways involved may reveal new targets for therapies aimed at depriving CLL cells of potential T-cell help.

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Identifiers

ORCID for Annabel Rachel Minton: orcid.org/0000-0002-2640-990X

ORCID for Graham Packham: orcid.org/0000-0002-9232-5691

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