Investigating the role of target cell availability in the pathogenesis of feline immunodeficiency virus infection

Feline immunodeficiency virus (FIV) is a naturally occurring lentivirus of domestic cats, which shares many similarities with its human counterpart, human immunodeficiency virus (HIV). FIV infects its main target cell, the CD4+ T lymphocyte, via interactions with its primary receptor CD134 (an activation marker expressed on activated CD4+ T lymphocytes), and, the chemokine receptor CXCR4. According to the different ways in which FIV isolates interact with CD134, FIV may be categorised into two groups. The first group contains strains that tend to dominate during the earlier phase of infection, such as GL8 and CPG41. These strains are characterized by their requirement for an additional interaction with the second cysteine rich domain (CRD2) of the CD134 molecule and are classified as “CRD2-dependent” strains. The second group, on the other hand, contains either laboratory-adapted isolates or isolates that emerge after several years of infection, such as PPR or the GL8 variants that emerged in cats 6 years post experimental infection and were studied in this thesis. These isolates are designated “CRD2-independent” as they can infect target cells without interacting with CRD2 of the CD134 molecule. This study provides the first evidence that FIV compartmentalisation is related to FIV-CD134 usage and the tissue availability of CD134+ target cells. In tissue compartments containing high levels of CD134+ cells such as peripheral blood and lymph nodes, CRD2-dependent viruses predominated, whereas CRD2-independent viruses predominated in compartments with fewer CD134+ cells, such as the thymus. The dynamics of CD4+CD134+ T lymphocytes at different stages of FIV infection were also described. The levels of CD4+CD134+ T lymphocytes, which were very high in the early phase, gradually decreased in the later phase of infection. The dynamics of CD4+CD134+ T lymphocyte numbers appeared to correlate with FIV tropism switching, as more CRD2-independent viruses were isolated from cats in the late phase of infection. Moreover, it was observed that pseudotypes bearing Envs of CRD2-dependent variants infected CD134+ target cells more efficiently than pseudotypes bearing Envs of CRD2-independent variants, confirming the selective advantage of CRD2-dependent variants in environments with high levels of CD134+ target cells. In conclusion, this study demonstrated that target cell types and numbers, as well as their dynamics, play important roles in the selection and expansion of FIV variants within the viral quasispecies. Improved understanding of the roles of target cells in FIV transmission and pathogenesis will provide important information required for the development of an improved, more successful protective FIV vaccine and will provide insight into the development of effective vaccines against other lentiviral infections such as HIV.

Macrophages and CD4 T-cells in rheumatoid arthritis and their modulation by JAK inhibitors

Background: Rheumatoid arthritis (RA) is a chronic inflammatory arthritis that causes significant morbidity and mortality and has no cure. Although early treatment strategies and biologic therapies such as TNFα blocking antibodies have revolutionised treatment, there still remains considerable unmet need. JAK kinase inhibitors, which target multiple inflammatory cytokines, have shown efficacy in treating RA although their exact mechanism of action remains to be determined. Stratified medicine promises to deliver the right drug to the right patient at the right time by using predictive ‘omic biomarkers discovered using bioinformatic and “Big Data” techniques. Therefore, knowledge across the realms of clinical rheumatology, applied immunology, bioinformatics and data science is required to realise this goal. Aim: To use bioinformatic tools to analyse the transcriptome of CD14 macrophages derived from patients with inflammatory arthritis and define a JAK/STAT signature. Thereafter to investigate the role of JAK inhibition on inflammatory cytokine production in a macrophage cell contact activation assay. Finally, to investigate JAK inhibition, following RA synovial fluid stimulation of monocytes. Methods and Results: Using bioinformatic software such as limma from the Bioconductor repository, I determined that there was a JAK/STAT signature in synovial CD14 macrophages from patients with RA and this differed from psoriatic arthritis samples. JAK inhibition using a JAK1/3 inhibitor tofacitinib reduced TNFα production when macrophages were cell contact activated by cytokine stimulated CD4 T-cells. Other pro-inflammatory cytokines such as IL-6 and chemokines such as IP-10 were also reduced. RA synovial fluid failed to stimulate monocytes to phosphorylate STAT1, 3 or 6 but CD4 T-cells activated STAT3 with this stimulus. RNA sequencing of synovial fluid stimulated CD4 T-cells showed an upregulation of SOCS3, BCL6 and SBNO2, a gene associated with RA but with unknown function and tofacitinib reversed this. Conclusion: These studies demonstrate that tofacitinib is effective at reducing inflammatory mediator production in a macrophage cell contact assay and also affects soluble factor mediated stimulation of CD4 T-cells. This suggests that the effectiveness of JAK inhibition is due to inhibition of multiple cytokine pathways such as IL-6, IL-15 and interferon. RNA sequencing is a useful tool to identify non-coding RNA transcripts that are associated with synovial fluid stimulation and JAK inhibition but these require further validation. SBNO2, a gene that is associated with RA, may be biomarker of tofacitinib treatment but requires further investigation and validation in wider disease cohorts.

Investigating the CD4+ T cell response to infection with Trypanosoma brucei

African trypanosomes are a protozoan parasite with the ability to cause disease states in both humans and animals; rendering them an important and relevant subject of study. These diseases have a vast socioeconomic impact upon the African continent and are perpetuated in part by the parasite's ability to evade the adaptive immune response. This thesis describes CD4+ T cell response to trypanosome infection, through the use of murine infection models and in vitro assays.

An investigation of the relationship between the components of tumour microenvironment, signal transduction pathways and outcome in breast cancer

Breast cancer, the most commonly diagnosed type of cancer in women, is a major cause of morbidity and mortality in the western world. Well-established risk factors of breast cancer are mostly related to women’s reproductive history, such as early menarche, late first pregnancy and late menopause. Survival rates have improved due to a combination of factors, including better health education, early detection with large-scale use of screening mammogram, improved surgical techniques, as well as widespread use of adjuvant therapy. At initial presentation, clinicopathological features of breast cancer such as age, nodal status, tumour size, tumour grade, and hormonal receptor status are considered to be the standard prognostic and predictive markers of patient survival, and are used to guide appropriate treatment strategies. Lymphovascular invasion (LBVI), including lymphatic (LVI) and blood (BVI) vessel invasion, has been reported to be prognostic and merit accurate evaluation, particularly in patients with node negative tumours who might benefit from adjuvant chemotherapy. There is a lack of standard assessment and agreement on distinguishing LVI from BVI despite the major challenges in the field. A systematic review of the literatures, examining methods of detection and the prognostic significance of LBVI, LVI and BVI, was carried out. The majority of studies used haematoxylin and eosin (H&E) and classical histochemistry to identify LVI and BVI. Only few recent studies used immunohistochemistry (IHC) staining of the endothelium lining lymphatic and blood vessels, and were able to show clear differences between LVI and BVI. The prognostic significance of LBVI and LVI was well-documented and strongly associated with aggressive features of breast tumours, while the prognostic value and the optimal detection method of BVI were unclear. Assessment and prognostic value of LBVI on H&E sections (LBVIH&E) was examined and compared to that of LVI and BVI detected using IHC with D2-40 for LVI (LVID2–40) and Factor VIII for BVI (BVIFVIII) in patients with breast cancer including node negative and triple negative patients (n=360). LBVIH&E, LVID2–40 and BVIFVIII were present in 102 (28%), 127 (35%) and 59 (16%) patients respectively. In node negative patients (206), LBVIH&E, LVID2–40 and BVIFVIII were present in 41 (20%), 53 (26%) and 21 (10%) respectively. In triple negative patients (102), LBVIH&E, LVID2–40 and BVIFVIII were present in 35 (29%), 36 (35%) and 14 (14%) respectively. LBVIH&E, LVID2–40 and BVIFVIII were all significantly associated with tumour recurrence in all cohorts. On multivariate survival analysis, only LVID2–40 and BVIFVIII were independent predictors of cancer specific survival (CSS) in the whole cohort (P=0.022 and P<0.001 respectively), node negative (P=0.008 and P=0.001 respectively) and triple negative patients (P=0.014 and P<0.001 respectively). Assessment of LVI and BVI by IHC, using D2-40 and Factor VIII, improves prediction of outcome in patients with node negative and triple negative breast cancer and was superior to the conventional detection method. Breast cancer is recognised as a complex molecular disease and histologically identical tumours may have highly variable outcomes, including different responses to therapy. Therefore, there is a compelling need for new prognostic and predictive markers helpful of selecting patients at risk and patients with aggressive diseases who might benefit from adjuvant and targeted therapy. It is increasingly recognised that the development and progression of human breast cancer is not only determined by genetically abnormal cells, but also dependent on complex interactions between malignant cells and the surrounding microenvironment. This has led to reconsider the features of tumour microenvironment as potential predictive and prognostic markers. Among these markers, tumour stroma percentage (TSP) and tumour budding, as well as local tumour inflammatory infiltrate have received recent attention. In particular, the local environment of cytokines, proteases, angiogenic and growth factors secreted by inflammatory cells and stromal fibroblasts has identified crucial roles in facilitating tumour growth, and metastasis of cancer cells through lymphatic and/or blood vessel invasion. This might help understand the underlying process promoting tumour invasion into these vessels. An increase in the proportion of tumour stroma and an increase in the dissociation of tumour cells have been associated with poorer survival in a number of solid tumours, including breast cancer. However, the interrelationship between these variables and other features of the tumour microenvironment in different subgroups of breast cancer are not clear. Also, whether their prognostic values are independent of other components of the tumour microenvironment have yet to be identified. Therefore, the relationship between TSP, clinicopathological characteristics and outcome in patients with invasive ductal breast cancer, in particular node negative and triple negative disease was examined in patients with invasive ductal breast cancer (n=361). The TSP was assessed on the haematoxylin and eosin-stained tissue sections. With a cut-off value of 50% TSP, patients with ≤50% stroma were classified as the low-TSP group and those with >50% stroma were classified as the high-TSP group. A total of 109 (30%) patients had high TSP. Patients with high TSP were old age (P=0.035), had involved lymph node (P=0.049), Her-2 positive tumours (P=0.029), low-grade peri-tumoural inflammatory infiltrate (P=0.034), low CD68+ macrophage infiltrate (P<0.001), low CD4+ (P=0.023) and low CD8+ T-lymphocytes infiltrate (P=0.017), tumour recurrence (P=0.015) and shorter CSS (P<0.001). In node negative patients (n=207), high TSP was associated with low CD68+ macrophage infiltrate (P=0.001), low CD4+ (P=0.040) and low CD8+ T-lymphocytes infiltrate (P=0.016) and shorter CSS (P=0.005). In triple negative patients (n=103), high TSP was associated with increased tumour size (P=0.017) high tumour grade (P=0.014), low CD8+ T-lymphocytes infiltrate (P=0.048) and shorter CSS (P=0.041). The 15-year cancer specific survival rate was 79% vs 21% in the low-TSP group vs high-TSP group. On multivariate survival analysis, a high TSP was associated with reduced CSS in the whole cohort (P=0.007), node negative patients (P=0.005) and those who received systemic adjuvant therapy (P=0.016), independent of other pathological characteristics including local host inflammatory responses. Therefore, a high TSP in invasive ductal breast cancer was associated with recurrence and poorer long-term survival. The inverse relation with the tumour inflammatory infiltrate highlights the importance of the amount of tumour stroma on immunological response in patients with invasive ductal breast cancer. Implementing this simple and reproducible parameter in routine pathological examination may help optimise risk stratification in patients with breast cancer. Similarly, the relationship between tumour budding, clinicopathological characteristics and outcome was examined in patients with invasive ductal breast cancer (n=474), using routine pathological sections. Tumour budding was associated with several adverse pathological characteristics, including positive lymph node (P=0.009), presence of LVI (P<0.001), and high TSP (P=0.001) and low-grade general peri-tumural inflammatory infiltrative (P=0.002). In node negative patients, a high tumour budding was associated with presence of LVI (P<0.001) and low-grade general peri-tumural inflammatory infiltrative (P=0.038). On multivariate survival analysis, tumour budding was associated with reduced CSS (P=0.001), independent of nodal status, tumour necrosis, CD8+ and CD138+ inflammatory cells infiltrate, LVI, BVI and TSP. Furthermore, tumour budding was independently associated with reduced CSS in node negative patients (P=0.004) and in those who have low TSP (P=0.003) and high-grade peri-tumoural inflammatory infiltrative (P=0.012). A high tumour budding was significantly associated with shorter CSS in luminal B and triple negative breast cancer subtypes (all P<0.001). Therefore, tumour budding was a significant predictor of poor survival in patients with invasive ductal breast cancer, independent of adverse pathological characteristics and components of tumour microenvironment. These results suggest that tumour budding may promote disease progression through a direct effect on local and distant invasion into lymph nodes and lymphatic vessels. Therefore, detection of tumour buds at the stroma invasive front might therefore represent a morphologic link between tumour progression, lymphatic invasion, spread of tumour cells to regional lymph nodes, and the establishment of metastatic dissemination. Given the potential importance of the tumour microenvironment, the characterisation of intracellular signalling pathways is important in the tumour microenvironment and is of considerable interest. One plausible signalling molecule that links tumour stroma, inflammatory cell infiltrate and tumour budding is the signal transducer and activator of transcription (STAT). The relationship between total and phosphorylated STAT1 (ph-STAT1), and total and ph-STAT3 tumour cell expression, components of tumour microenvironment and survival in patients with invasive ductal breast cancer was examined. IHC of total and ph-STAT1/STAT3 was performed on tissue microarray of 384 breast cancer specimens. Cellular STAT1 and cellular STAT3 expression at both cytoplasmic and nuclear locations were combined and identified as STAT1/STAT3 tumour cell expression. These results were then related to CSS and phenotypic features of the tumour and host. A high ph-STAT1 and a high ph-STAT3 tumour cell expression was associated with increased ER (P=0.001 and P<0.001 respectively) and PR (all P<0.05), reduced tumour grade (P=0.015 and P<0.001 respectively) and necrosis (all P=0.001). Ph-STAT1 was associated with increased general peri-tumoural inflammatory infiltrate (P=0.007) and ph-STAT3 was associated with lower CD4+ T-lymphocyte infiltrate (P=0.024). On multivariate survival analysis, including both ph-STAT1 and ph-STAT3 tumour cell expression, only high ph-STAT3 tumour cell expression was significantly associated with improved CSS (P=0.010) independent of other tumour and host-based factors. In patients with high necrosis grade, high ph-STAT3 tumour cell expression was independent predictor of improved CSS (P=0.021). Ph-STAT1 and ph-STAT3 were also significantly associated with improved cancer specific survival in luminal A and B subtypes. STAT1 and STAT3 tumour cell expression appeared to be an important determinant of favourable outcome in patients with invasive ductal breast cancer. The present results suggest that STATs may affect disease outcome through direct impact on tumour cells, and the surrounding microenvironment. The above observations of the present thesis point to the importance of the tumour microenvironment in promoting tumour budding, LVI and BVI. The observations from STATs work may suggest that an important driving mechanism for the above associations is the presence of tumour necrosis, probably secondary to hypoxia. Further work is needed to examine the interaction of other molecular pathways involved in the tumour microenvironment, such as HIF and NFkB in patients with invasive ductal breast cancer.