T-cell responses to influenza virus in pigs

The tools and techniques for the study of porcine T-cells lag behind what is currently attainable in human T-cells, so this thesis was initially focused on improvements in this field. This study established long-term culture of porcine T-cells, T-cell clone procurement and relevant T-cell assays. These techniques were then used to investigate cytotoxic T-cell responses to Influenza A virus (IAV) in pigs. IAV is highly mutative and novel strains can be generated following reassortment between different viral strains. IAV is endemic in the global pig population and in some circumstances the virus can pass between humans and pigs and other animals. Pigs can therefore, potentially be a source for the generation of new and possibly pandemic influenza strains. The risk this poses to global human health, together with the negative effects of IAV infection within pig herds, highlights the need to improve our knowledge of IAV in pigs. This study identified four new MHC class I restricted IAV epitopes, derived from the viral nucleoprotein. Cytotoxic T-cells recognising these IAV epitopes were detected at high numbers ex vivo in samples from vaccinated pigs. The structures of these IAV epitopes in complex with their respective MHC class I molecules were resolved and revealed the primary anchor positions within the peptides. This enabled peptide binding motifs to be defined for two porcine MHC-I alleles. These peptide binding motifs can be utilised for efficient epitope prediction. This study also identified super-agonist ligands for two of the MHC-I restricted IAV epitopes. Overall, this work has opened up the study of porcine T-cells to a level previously unattainable and has contributed to our knowledge of IAV in pigs. It has paved the way for further experiments investigating IAV in pigs, other porcine diseases and for using pigs as an animal model for human disease

Reprogramming of various cell types to a beta-like state by Pdx1, Ngn3 and MafA

The three transcription factors, PDX1, NGN3 and MAFA, are very important in pancreatic development. Overexpression of these three factors can reprogram both pancreatic exocrine cells and SOX9-positive cells of the liver into cells resembling pancreatic beta cells. In this study we investigate whether other cell types can be reprogrammed. Eight cell types are compared and the results are consistent with the idea that reprogramming occurs to a greater degree for developmentally related cells (pancreas, liver) than for other types, such as fibroblasts. Using a line of mouse hepatocyte-derived cells we screened 13 compounds for the ability to increase the yield of reprogrammed cells. Three are active and when used in combination they can increase the yield of insulin-immunopositive cells by a factor of six. These results should contribute to the eventual ability to develop a new cure for diabetes based on the ability to reprogram other cells in the body to a beta cell phenotype

Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR–CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR–CD3 complexes. We found that only TCR–CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR–pMHC affinity determined the density of TCR–CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR–CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination

Characterizing and correcting immune dysfunction in non-tuberculous mycobacterial disease

Non-tuberculous mycobacterial pulmonary disease (NTM-PD) is a chronic, progressive, and growing worldwide health burden associated with mounting morbidity, mortality, and economic costs. Improvements in NTM-PD management are urgently needed, which requires a better understanding of fundamental immunopathology. Here, we examine temporal dynamics of the immune compartment during NTM-PD caused by Mycobacterium avium complex (MAC) and Mycobactereoides abscessus complex (MABS). We show that active MAC infection is characterized by elevated T cell immunoglobulin and mucin-domain containing-3 expression across multiple T cell subsets. In contrast, active MABS infection was characterized by increased expression of cytotoxic T-lymphocyte-associated protein 4. Patients who failed therapy closely mirrored the healthy individual immune phenotype, with circulating immune network appearing to ‘ignore’ infection in the lung. Interestingly, immune biosignatures were identified that could inform disease stage and infecting species with high accuracy. Additionally, programmed cell death protein 1 blockade rescued antigen-specific IFN-γ secretion in all disease stages except persistent infection, suggesting the potential to redeploy checkpoint blockade inhibitors for NTM-PD. Collectively, our results provide new insight into species-specific ‘immune chatter’ occurring during NTM-PD and provide new targets, processes and pathways for diagnostics, prognostics, and treatments needed for this emerging and difficult to treat disease

Comparison of peptide-major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells

Fluorochrome-conjugated peptide–major histocompatibility complex (pMHC) multimers are widely used for flow cytometric visualization of antigen-specific T cells. The most common multimers, streptavidin–biotin-based ‘tetramers’, can be manufactured readily in the laboratory. Unfortunately, there are large differences between the threshold of T cell receptor (TCR) affinity required to capture pMHC tetramers from solution and that which is required for T cell activation. This disparity means that tetramers sometimes fail to stain antigen-specific T cells within a sample, an issue that is particularly problematic when staining tumour-specific, autoimmune or MHC class II-restricted T cells, which often display TCRs of low affinity for pMHC. Here, we compared optimized staining with tetramers and dextramers (dextran-based multimers), with the latter carrying greater numbers of both pMHC and fluorochrome per molecule. Most notably, we find that: (i) dextramers stain more brightly than tetramers; (ii) dextramers outperform tetramers when TCR–pMHC affinity is low; (iii) dextramers outperform tetramers with pMHC class II reagents where there is an absence of co-receptor stabilization; and (iv) dextramer sensitivity is enhanced further by specific protein kinase inhibition. Dextramers are compatible with current state-of-the-art flow cytometry platforms and will probably find particular utility in the fields of autoimmunity and cancer immunology

EdU labeling index in five cell types.

<p>Those which generated insulin-immunopositive cells showed a reduction in EdU label. Errors are standard errors, n=3.</p

Effects of the small molecule combination.

<p>A. ASH cells transduced with <i>Ad-PNM</i>, without and with the DNB combination. PDX1 is immunostained green and insulin red. Scale bar 100µm. B. Percentage of PDX1-positive cells that are also insulin-positive. Comparisons are by one way ANOVA with Games-Howell post hoc test, * indicates significant enhancement. Errors are standard errors, n=6.</p

Expression of various beta cell genes provoked by transduction with <i>Ad-PNM</i>.

<p>A-H. Qualitative RT-PCRs showing the effect of <i>Ad-PNM</i> on the expression of a panel of beta cell genes. 30 cycles were used unless otherwise shown. The misaligned bands in the first two lanes of mouse fibroblast <i>Mnx1</i> are primer dimers, not RNA.</p

Insulin-positive cells generated from different cell types.

<div><p>A,B,C Expression of PDX1, NGN3, MAFA in ASH cells. The proteins are shown in green, DAPI in blue.</p> <p>D. Histogram of the percentage PDX1-positive cells which are insulin-positive in different cell types. </p> <p>E,E’ Rat hepatocytes, some PDX1-positive cells are insulin-positive. </p> <p>F,F’ Rat hepatocytes, insulin-positive cells are also C-peptide-positive. </p> <p>G Mouse ASH cells, some PDX1-positive cells are insulin-positive. </p> <p>H. Mouse ASH cells, showing cytoplasmic location of insulin. Inset, high magnification showing insulin-containing granules.</p> <p>I,I’ Mouse ASH cells showing co-expression of C-peptide with insulin. </p> <p>J Rat AR42j-B13 cells showing a high proportion of insulin-positive cells. </p> <p>K,K’ Rat AR42j-B13 cells showing co-expression of C-peptide with insulin. </p> <p>L. Rat AR42j-B13 showing insulin-containing granules.</p> <p>Scale bars 100µm, except H inset and L which are 25 µm. Blue color is DAPI throughout.</p></div

Synthetic hookworm-derived peptides are potent modulators of primary human immune cell function that protect against experimental colitis in vivo

The prevalence of autoimmune diseases is on the rise globally. Currently, autoimmunity presents in over 100 different forms and affects around 9% of the world’s population. Current treatments available for autoimmune diseases are inadequate, expensive, and tend to focus on symptom management rather than cure. Clinical trials have shown that live helminthic therapy can decrease chronic inflammation associated with inflammatory bowel disease and other gastrointestinal autoimmune inflammatory conditions. As an alternative and better controlled approach to live infection, we have identified and characterized two peptides, Acan1 and Nak1, from the excretory/secretory component of parasitic hookworms for their therapeutic activity on experimental colitis. We synthesized Acan1 and Nak1 peptides from the Ancylostoma caninum and Necator americanus hookworms and assessed their structures and protective properties in human cell–based assays and in a mouse model of acute colitis. Acan1 and Nak1 displayed anticolitic properties via significantly reducing weight loss and colon atrophy, edema, ulceration, and necrosis in 2,4,6-trinitrobenzene sulfonic acid–exposed mice. These hookworm peptides prevented mucosal loss of goblet cells and preserved intestinal architecture. Acan1 upregulated genes responsible for the repair and restitution of ulcerated epithelium, whereas Nak1 downregulated genes responsible for epithelial cell migration and apoptotic cell signaling within the colon. These peptides were nontoxic and displayed key immunomodulatory functions in human peripheral blood mononuclear cells by suppressing CD4+ T cell proliferation and inhibiting IL-2 and TNF production. We conclude that Acan1 and Nak1 warrant further development as therapeutics for the treatment of autoimmunity, particularly gastrointestinal inflammatory conditions.</p