Analysis of membrane exchange between lymphocytes

Abstract

Membrane transfer is the cell-to-cell contact dependent exchange of plasma membrane and surface molecules between cells. It has been described for a wide range of immune cells, nevertheless the molecular mechanisms mediating this exchange remain unclear. The studies outlined in this Thesis provide new insights and a better understanding of the transfer process. Previous studies in my supervisor’s laboratory showed that activated antigen- specific B cells and T cells readily donate their antigen-specific receptors to neighbouring lymphocytes of an unrelated specificity via plasma membrane exchange. Results obtained in this Thesis confirmed and further characterised several properties of antigen receptor transfer as well as suggesting new potential mediators of membrane exchange. The results in Chapter 3 detail important properties of membrane exchange between B cells, and based on these unique features propose a molecular mechanism for this process. It was confirmed that B cells require appropriate activation conditions in order to donate membranes and cell surface proteins, with naïve B cells being much less capable of transfer. Additionally, it was confirmed that activated B cells are capable of transferring membranes and surface proteins at 4oC, thus excluding many of the currently proposed energy dependent mechanisms. Chapter 3 describes the investigation of an energy independent mechanism of exchange involving cell penetrating peptide (CPP) motifs. The results obtained demonstrate that analogous to transfer between B cells, CPPs are up to 40-fold more efficiently taken up by activated, rather than naïve, B cells and that this uptake occurs at both 37oC and 4oC. These data suggest that receptors involved in the initiation of membrane transfer may contain CPP motifs important in facilitating this process. Chapter 4 describes attempts to identify the molecular basis of membrane transfer via a comparative gene expression analysis. This study took advantage of the finding that B cells require appropriate activation conditions to achieve efficient levels of membrane transfer. In an effort to unravel the proteins involved in this process, transcriptomes of LPS stimulated B cells, which are known to efficiently transfer membranes, were compared with the transcriptomes of CpG stimulated and unstimulated B cells which are less efficient at transferring membranes. Bioinformatics analysis identified five proteins containing CPP motifs that may play a role in B cell membrane transfer, namely ALCAM, AMIGO2, CTLA-4, Slp3 and TIGIT. Monoclonal antibodies (mAbs) specific for ALCAM, CTLA-4, TIGIT and the TIGIT ligand, PVR, demonstrated increased expression of all these proteins on the cell surface of LPS activated B cells. Furthermore, incubation of B cells with ALCAM, TIGIT and TIGIT + PVR mAbs resulted in significantly enhanced membrane transfer, whereas the CTLA-4 mAb, known to block CTLA-4 binding, had no effect. Based on these data and other published findings it is hypothesised that mAbs specific for ALCAM and TIGIT/PVR may crosslink these receptors, creating a patching effect, similar to a lipid raft, in which there are areas of localised membrane destabilisation and, consequently, more accessible areas for CPP-mediated membrane fusion. Based on the current understanding of membrane transfer it should be possible to harness this phenomenon to enhance antigen specific CTL responses in the adoptive immunotherapy of established tumours. Thus, experiments described in Chapter 5 used an adoptive T cell immunotherapy model to investigate whether TCR sharing can be harnessed to control tumour growth. The model utilised the ovalbumin (OVA)-expressing EG7 (EG7-OVA) thymoma cell line to establish tumours in mice. These tumours are susceptible to killing by OVA-specific TCR transgenic (OT-I) CTL, thus resulting in a reduction in EG7-OVA tumour volume. To establish the influence of antigen receptor transfer in the control of tumour growth, perforin deficient OT-I CTLs, which have been shown previously to be less effective at bringing about tumour rejection due to their lack of perforin, and CTLs of an unrelated specificity (B6.SJL.TCRP14 CTLs) were used. Transferred alone each of these CTL populations should be incapable of inducing tumour regression. However, when adoptively transferred together if TCR transfer occurs the B6.SJL.TCRP14 CTL should acquire OVA-specific TCR from the perforin deficient OT-I CTLs and gain the capacity to recognise and eliminate the EG7- OVA tumour cells and thereby mediate tumour regression. The results obtained, however, demonstrated that perforin deficient OT-I CTLs, but not B6.SJL.TCRP14 CTLs, were as efficient as wild type OT-I CTLs at eliminating established EG7-OVA tumours in vivo. This finding indicated that the CTLs used in this model do not require perforin to control tumour growth, with subsequent in vitro studies suggesting involvement of the Fas/FasL pathway. Furthermore, results described in this Chapter demonstrate that extrodinarily small numbers of CTLs within tumours can induce tumour regression, suggesting that in vivo many other mechanisms work in concert to control EG7-OVA tumour growth, NK cells being the most obvious. In addition, variations in the results obtained with the EG7-OVA cell line in different laboratories may be due to genetic drift of the cell line, thus highlighting the importance of stringent cell line authentication. In conclusion, this Thesis identified several candidate proteins required for efficient membrane transfer between B cells as well as providing evidence for a potential membrane exchange mechanism involving proteins that contain CPP motifs that facilitate membrane fusion and thus the transfer of membranes and associated cell surface proteins. Show more