Patrick T. Harrison

Two distinct regions of FC gamma RI initiate separate signalling pathways involved in endocytosis and phagocytosis.

Cross‐linking of the high affinity receptor for IgG, Fc gamma RI, can result in both endocytosis of immune complexes and phagocytosis of opsonized particles in myeloid cells, although the cytoplasmic domain of the receptor lacks the tyrosine activation motif which has been implicated in signal transduction triggered by cross‐linking of other Fc receptors. To identify the structural determinants of Fc gamma RI‐mediated ligand internalization, we have expressed Fc gamma RI or truncated versions of Fc gamma RI in COS cells, either alone or in the presence of the Fc epsilon RI gamma subunit (which contains a classical tyrosine activation motif and associates with Fc gamma RI in myeloid cells), and assessed their ability to mediate endocytosis and phagocytosis. We have found that Fc gamma RI alone (in the absence of the gamma subunit) is capable of mediating endocytosis in COS cells and that the process occurs via a novel, tyrosine kinase‐independent signalling pathway. Activation of this pathway following cross‐linking appears to require only the receptor extracellular domain. In contrast, Fc gamma RI phagocytic function in COS cells is dependent on an interaction between the receptor transmembrane domain and the gamma subunit and is mediated by recruitment of tyrosine kinase activity. Our data therefore indicate that distinct domains of the receptor regulate ligand internalization following receptor cross‐linking by either immune complexes (endocytosis) or opsonized particles (phagocytosis) and that these functions are mediated by different intracellular signalling pathways.

C‐reactive protein‐mediated phagocytosis and phospholipase D signalling through the high‐affinity receptor for immunoglobulin G (FcγRI)

C‐reactive protein (CRP) is the prototypic acute‐phase protein in man which performs innate immune functions. CRP‐mediated phagocytosis may be indirect, through activation of complement and complement receptors, or direct, through receptors for the Fc portion of immunoglobulin G (IgG; FcγRs) or even a putative CRP‐specific receptor. No strong evidence has been shown to indicate which receptors may be responsible for phagocytosis or signalling responses. Using BIAcore technology, we confirm that CRP binds directly to the extracellular portion of FcγRI with a threefold higher affinity than IgG (KD = 0·81 × 10−9 m). Binding is Ca2+ dependent and is inhibited by IgG1 but not by phosphorylcholine (PC). CRP opsonization (using CRP concentrations within the normal human serum range) of PC‐conjugated sheep erythrocytes increased phagocytosis of these particles by COS‐7 cells transfected with FcγRI‐II chimaera or FcγRI/γ‐chain. Interferon‐γ‐treated U937 cells, which signal through FcγRI to activate phospholipase D (PLD) in response to cross‐linked IgG, were also activated by CRP without any requirement for further cross‐linking. These studies indicate that CRP is capable of binding to and cross‐linking FcγRI thereby resulting in PLD activation and increased phagocytosis. Uptake by FcγRI has been reported to promote various acquired immune responses suggesting that CRP could act in a similar way.

Viral Vectors in Cancer Immunotherapy: Which Vector for Which Strategy?

Gene therapy involves the transfer of genetic information to a target cell to facilitate the production of therapeutic proteins and is now a realistic prospect as a cancer treatment. Gene transfer may be achieved through the use of both viral and non-viral delivery methods and the role of this method in the gene therapy of cancer has been demonstrated. Viruses represent an attractive vehicle for cancer gene therapy due to their high efficiency of gene delivery. Many viruses can mediate long term gene expression, while some are also capable of infecting both dividing and non-dividing cells. Given the broadly differing capabilities of various viral vectors, it is imperative that the functionality of the virus meets the requirements of the specific treatment. A number of immunogene therapy strategies have been undertaken, utilising a range of viral vectors, and studies carried out in animal models and patients have demonstrated the therapeutic potential of viral vectors to carry genes to cancer cells and induce anti-tumour immune responses. This review critically discusses the advances in the viral vector mediated delivery of immunostimulatory molecules directly to tumour cells, the use of viral vectors to modify tumour cells, the creation of whole cell vaccines and the direct delivery of tumour antigens in animal models and clinical trials, specifically in the context of the suitability of vector types for specific strategies.

Evolution of herpesvirus thymidine kinases from cellular deoxycytidine kinase

The thymidine kinases encoded by herpesviruses of higher vertebrates form a distinct group and are unrelated to the thymidine kinases (TKs) of other organisms. Their evolutionary source has not been identified, but our analysis has revealed a clear relationship with a sequence of human deoxycytidine kinase (dCK) published recently. We report the sequence of the putative TK of channel catfish virus, a herpesvirus of a lower vertebrate, and show that it is also related to dCK. We propose, therefore, that the TKs of herpesviruses of higher and lower vertebrates have evolved, either independently or successively, from a cellular dCK.

FcγRIIa expression with FcγRI results in C-reactive protein- and IgG-mediated phagocytosis

C‐reactive protein (CRP) is a pattern‐recognition molecule, which can bind to phosphorylcholine and certain phosphorylated carbohydrates found on the surface of a number of microorganisms. CRP has been shown recently to bind human Fc receptor for immunoglobulin G (IgG; FcγR)I and mediate phagocytosis and signaling through the γ‐chain. To date, binding of monomeric CRP to FcγRII has been contentious. We demonstrate that erythrocytes opsonized with CRP bind FcγRIIa‐transfected COS‐7 cells. In addition, we demonstrate that FcγRI can use FcγRIIa R131 and H131 to phagocytose erythrocytes coated with IgG or purified or recombinant CRP in the absence of the γ‐chain. COS‐7 cells expressing FcγRIIa or FcγRI alone did not phagocytose opsonized erythrocytes. Such phagocytosis required the cytoplasmic domain of FcγRIIa, as mutation of tyrosine at position 205 and truncation of the cytoplasmic domain from the end of the transmembrane region (position 206), resulting in the loss of the immunoreceptor tyrosine activatory motif, abrogated phagocytosis. FcγRIIa R131 was more efficient than FcγRIIa H131 at mediating CRP‐dependent phagocytosis.

Neuronal-specific and nerve growth factor-inducible expression directed by the preprotachykinin-A promoter delivered by an adeno-associated virus vector.

The ability to manipulate the expression of genes within neurons provides unique opportunities to study the role of individual gene products in nervous system function. Virus vectors are a potentially rapid tool for the experimental manipulation of gene expression in the mammalian nervous system. However, a block to the use of virus vector systems in neurobiology is often the lack of cell-specific expression of the gene within the nervous system, and the immune and inflammatory responses to both the virus vector and the delivered gene. We have generated an adeno-associated virus vector that exploits the restricted expression pattern of the rat preprotachykinin-A promoter to support reporter gene expression. We demonstrate that this virus has a neuronal-specific expression pattern. Moreover, it is shown for the first time that the proximal rat preprotachykinin-A promoter is nerve growth factor inducible. This virus will be a useful tool to (i) modify neuronal phenotype by expressing therapeutic molecules or antisense nucleic acid and (ii) dissect the signal transduction pathways that regulate promoter function in vivo.

Genetic medicines for CF: Hype versus reality

Since identification of the CFTR gene over 25 years ago, gene therapy for cystic fibrosis (CF) has been actively developed. More recently gene therapy has been joined by other forms of “genetic medicines” including mRNA delivery, as well as genome editing and mRNA repair‐based strategies. Proof‐of‐concept that gene therapy can stabilize the progression of CF lung disease has recently been established in a Phase IIb trial. An early phase study to assess the safety and explore efficacy of CFTR mRNA repair is ongoing, while mRNA delivery and genome editing‐based strategies are currently at the pre‐clinical phase of development. This review has been written jointly by some of those involved in the various CF “genetic medicine” fields and will summarize the current state‐of‐the‐art, as well as discuss future developments. Where applicable, it highlights common problems faced by each of the strategies, and also tries to highlight where a specific strategy may have an advantage on the pathway to clinical translation. We hope that this review will contribute to the ongoing discussion about the hype versus reality of genetic medicine‐based treatment approaches in CF. Pediatr Pulmonol. 2016;51:S5–S17.

The interaction between human FCγRI and the γ-chain is mediated solely via the 21 amino acid transmembrane domain of FCγRI

We have established a biological assay to investigate the nature of the non-covalent interaction between two integral type I membrane proteins, Fc gamma RI and gamma-chain. Fc gamma RI, the human high affinity receptor for immunoglobulin G (IgG), is expressed on the surface of macrophages and monocytes and mediates a broad range of important immunological functions. Fc gamma RI relies on a functional interaction with a second integral type I membrane protein, gamma-chain, to mediate many of these functions. For example, Fc gamma RI can only mediate phagocytosis of IgG-coated particles in COS cells when co-expressed with gamma-chain. We have previously shown that the cytoplasmic domain of Fc gamma RI is not necessary for this functional interaction. In this study we have used the phagocytosis assay to investigate the role of the transmembrane region of Fc gamma RI in mediating this functional interaction with gamma-chain by using mutant and chimeric forms of the receptor. Three mutants, which introduce or remove charged residues from a conserved 10 amino acid stretch of amino acids in the proximal transmembrane region of Fc gamma RI, were able to mediate phagocytosis of IgG-coated particles. In contrast, two chimeric receptors, In which 21 of the amino acids in the distal transmembrane region of Fc gamma RI were replaced with the transmembrane region of the related receptors CD2 or LFA3, were expressed but failed to interact functionally with gamma-chain to mediate phagocytosis. Thus, these mutants demonstrate that the interaction between human Fc gamma RI and gamma-chain is mediated solely via these 21 amino acids in the transmembrane domain of Fc gamma RI.

Protein : protein interactions in the lipid bilayer (Review)

At least four different types of interaction between protein transmembrane helices have been described to date. These include the use of charge-pair interactions that can play a positive or negative role in the assembly of multi-subunit complexes such as the T cell receptor, or recruit signal transducing accessory molecules in the case of some Fc receptors. Inter-helix hydrogen bonds have been shown to play an important role in the constitutive activation of certain proto-oncogenes, whereas helix:helix interfaces stabilized solely by van der Waals contacts mediated by non-polar residues also exist. The fourth type of interaction is an inter-chain disulphide linkage which is dependent on a buried charged residue. A role for glycine residues in several of these mechanisms is also suggested. In addition, the use of disulphide mapping to further explore protein:protein interactions within the lipid bilayer is discussed.

E47 retroviral rescue of intrinsic B‐cell defects in senescent mice

In aging, immune responses are dramatically impaired, specifically the ability to produce protective antibodies. We previously showed that with age there is a B‐cell intrinsic decrease in class switch recombination (CSR) because of a decrease in activation‐induced cytidine deaminase (AID). One mechanism we have demonstrated for decreased AID includes increased mRNA degradation of the transcription factor E47, critical for AID transcription. Here, we show by means of a retroviral construct containing the DsRED reporter and the 3′UTR of E47 that the 3′UTR lowers mRNA expression, and particularly in B cells from old mice. This is the first demonstration that the E47 3′UTR directly regulates its degradation. The AID mRNA was not differentially regulated by degradation in aging. Therefore, we have here further established critical components for decreased AID with age. The major aim of this study was to establish conditions for the rescue of the intrinsic defect of aged B cells with retroviral addition of the coding region of E47 in splenic B cells to restore their ability to produce optimal AID and class switch to IgG. In this study, we show that young and old primary B cells overexpressing a stable E47 mRNA up‐regulate E47, AID, and CSR and improve B‐cell immune responses in senescent murine B cells. Our results provide a proof of principle for the rescue of intrinsic B‐cell defects and the humoral immune response in senescence.