The aggregation of proteins as a result of intrinsic or environmental stress may be cytoprotective, but is also linked to pathophysiological states and cellular ageing. We analysed the principles of aggregate formation and the cellular strategies to cope with aggregates in Escherichia coli using fluorescence microscopy of thermolabile reporters, EM tomography and mathematical modelling. Misfolded proteins deposited at the cell poles lead to selective re-localization of the DnaK/DnaJ/ClpB disaggregating chaperones, but not of GroEL and Lon to these sites. Polar aggregation of cytosolic proteins is mainly driven by nucleoid occlusion and not by an active targeting mechanism. Accordingly, cytosolic aggregation can be efficiently re-targeted to alternative sites such as the inner membrane in the presence of site-specific aggregation seeds. Polar positioning of aggregates allows for asymmetric inheritance of damaged proteins, resulting in higher growth rates of damage-free daughter cells. In contrast, symmetric damage inheritance of randomly distributed aggregates at the inner membrane abrogates this rejuvenation process, indicating that asymmetric deposition of protein aggregates is important for increasing the fitness of bacterial cell populations.
The improved antibody responses of class-switched memory B cells depend on enhanced signaling from their B cell antigen receptors (BCRs). However, BCRs on both naive and antigen-experienced B cells use the canonical immunoglobulin-associated alpha and beta-protein signaling subunits. Here we identified a BCR isotype-specific signal-amplification mechanism. Whereas immunoglobulin M (IgM)-containing BCRs initiated intracellular signals exclusively through immunoglobulin-associated alpha- and beta-proteins, IgG- and IgE-containing BCRs also used a conserved tyrosine residue in the cytoplasmic segments of immunoglobulin heavy chains. When phosphorylated, this tyrosine recruited the adaptor Grb2, resulting in sustained protein kinase activation and prolonged generation of second messengers, which together culminated in enhanced B cell proliferation. Hence, membrane-bound IgG and IgE exert antigen recognition as well as costimulatory functions, thereby rendering memory B cells less dependent on T cell help.
The vigorous response of IgG-switched memory B cells to recurring pathogens involves enhanced signalling from their B-cell antigen receptors (BCRs). However, the molecular signal amplification mechanisms of memory-type BCRs remained unclear. Here, we identify the immunoglobulin tail tyrosine (ITT) motif in the cytoplasmic segments of membrane-bound IgGs (mIgGs) as the principle signal amplification device of memory-type BCRs in higher vertebrates and decipher its signalling microanatomy. We show that different families of protein tyrosine kinases act upstream and downstream of the ITT. Spleen tyrosine kinase (Syk) activity is required for ITT phosphorylation followed by recruitment of the adaptor protein Grb2 into the mIgG-BCR signalosome. Grb2 in turn recruits Bruton’s tyrosine kinase (Btk) to amplify BCR-induced Ca2+ mobilization. This molecular interplay of kinases and adaptors increases the antigen sensitivity of memory-type BCRs, which provides a cell-intrinsic trigger mechanism for the rapid reactivation of IgG-switched memory B cells on antigen recall.
The efficacy of adoptive cell therapy for solid tumours is hampered by the poor accumulation of the transferred T cells in tumour tissue. Here, we show that the forced expression of the C-X-C chemokine receptor type 6 (CXCR6, whose ligand is highly expressed by human and murine pancreatic cancer cells and by tumour-infiltrating immune cells) in antigen-specific T cells enhanced the recognition and lysis of pancreatic cancer cells and the efficacy of adoptive cell therapy for pancreatic cancer. In mice with subcutaneous pancreatic tumours treated with T cells with either a transgenic T-cell receptor or a murine chimeric antigen receptor targeting the tumour-associated antigen epithelial cell-adhesion molecule, and in mice with orthotopic pancreatic tumours or patient-derived xenografts treated with T cells expressing a chimeric antigen receptor targeting mesothelin, the T cells exhibited enhanced intratumoral accumulation, exerted sustained antitumoral activity and prolonged animal survival only when co-expressing CXCR6. Arming tumour-specific T cells with tumour-specific chemokine receptors may represent a promising strategy for the realization of adoptive cell therapy for solid tumours.
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