Follicular lymphoma (FL) is the most common form of indolent lymphoma in northern Europe, with approximately 2000 cases diagnosed each year in the UK. It is a malignancy derived from germinal centre (GC) B-lymphocytes and is critically dependent on non-malignant immune cells within the tumour microenvironment (TME) for growth and survival. FL retains a follicular structure similar to reactive GCs and, like its non-malignant counterparts, is infiltrated by T follicular helper cells (TFH). TFH are a specialised subset of CD4+ T-cells that are essential for supporting proliferation, affinity maturation and differentiation of healthy GC B-cells. In FL, TFH appear to create a tumour-permissive environment that supports FL growth, although the mechanisms by which FL B-cells and TFH provide mutual support are not known. The aim of this thesis is to investigate the hypothesis that TFH play a key role in driving FL growth and progression. Here, the nature of interactions between TFH and FL B-cells, both in in vitro culture and in archival FL tissue, are explored using novel imaging techniques. Firstly, it is shown that TFH are identifiable within FL lymph node (LN) tissue and fine needle aspirate, comprising just over a quarter (28.6%) of all CD4+ T-cells. Secondly, it is demonstrated that these FL cell suspensions can be used to explore the mutual role of TFH and FL B-cells on cell survival and activation in vitro. Attempts were made to overcome the limited availability of LN tissue by generating TFH from peripheral blood T-cells. However, it was not possible to successfully obtain cells with a true TFH phenotype from either CXCR5+CD4+ T-cells or naïve T-cells. In culture studies, expression of the activation markers CD86 and HLA-DR on FL B-cells was enhanced by co-culture with TFH, but not by other, non-TFH CD4+ T-cells. FL B-cell survival was also higher in the presence of FL T-cells, including TFH. Conversely, ICOS-L expression was lower on FL B-cells that were cultured with TFH, compared to without, indicating the formation of active ICOS/ICOS-L interactions. ICOS stimulation is known to be critical for TFH survival. Accordingly, co-culture with FL B-cells increased survival of TFH and supported persistence of the TFH phenotype in vitro. FL B-cells also increased expression of the activation marker CD69 on TFH. These results demonstrate dynamic, mutually supportive interactions between TFH and FL B-cells. In FL tissue, there was a close spatial correlation between TFH and expression of MYC in FL B-cells by confocal immunofluorescence microscopy. However, accurate characterisation of complex cell populations in FL tissue requires highly multiplexed imaging techniques. Use of imaging mass cytometry (IMC) was explored as a novel method to assess spatial interactions of TFH within the FL TME. This technique allowed assessment of 20 antigens within the same tissue section and could identify TFH in archival FL tissue. It was possible to replicate previously published findings with confocal imaging by demonstrating a very close correlation between TFH and FL proliferation. This novel imaging technique holds the ability to greatly enhance our knowledge of spatial interactions within FL tissue and potentially identify prognostic networks within the FL TME in future. This thesis provides insights into the mechanisms by which TFH and FL B-cells co-operate to provide mutual support and promote FL growth. TFH therefore represent an exciting potential therapeutic target in FL. Given the critical role that the TME plays in supporting FL growth, better understanding of these interactions will assist in developing novel therapeutic strategies to improve patient outcomes.
An investigation into the role of T follicular helper cells in follicular lymphoma
Phillips, B. (Author). 1 Mar 2020
Student thesis: Doctoral Thesis › Doctor of Medicine by Research