Abstract
B lymphocytes (B cells) are widely known to be important players within the context of adaptive immunity and may contribute to immune responses through expression of immunoglobulin (Ig), antigen presentation to CD4+ and CD8+ T cells, and production of effector cytokines. Moreover, in patients with solid tumours, initiation of effective humoral immunity may contribute to tumour growth restriction through specific antigen- directed responses. This Thesis investigates the complex and dynamic roles of B cells in immune responses to solid tumours, with specific focus on immunogenic tumours such as melanoma and subsets of breast cancer. Overall, I aimed to identify novel features and biases among circulating and tumour-infiltrating B lymphocyte (TIL-B) populations, to elucidate their roles and functional significance, and to place these findings within the wider context of the adaptive immune response.In breast cancer, I used flow cytofluorimetric, transcriptomic, fluorescence immunohistochemistry (IHC), single-cell RNA-seq and long-read Ig repertoire studies to evaluate isotype-switched and memory B cell subsets, Ig isotype distribution, and clonal expansion profiles within the circulation and lesions of patients. Isotype-switched and activatory B cell signatures were enhanced among breast TIL-B. Quantitative IHC showed that TIL-B frequently form stromal clusters with T lymphocytes (T cells), and single cell transcriptomic analyses suggested that B and T cells in tumours engage in bidirectional crosstalk. TIL-B-rich tumours demonstrated expansion of IgG isotypes, and IgG isotype-switching was found to be positively associated with survival outcomes, especially in the aggressive triple-negative breast cancer (TNBC) subtypes. Long-read Ig repertoire analysis also indicated that B cell clonal expansion was biased towards IgG, showing expansive clonal families with specific variable region gene combinations and narrow variable region repertoires. Lastly, stronger selection pressure was present in the complementarity determining regions of IgG compared to their clonally related IgA in tumour samples.
In melanoma, I used CyTOF, intracellular cytokine assays, transcriptomic, fluorescence IHC, single-cell RNA-seq and ex vivo co-culture analyses to investigate the phenotype and functional significance of cytokine-expressing B cells in patients with melanoma, including regulatory (IL-10 and/or TGF-β-expressing) and pro-inflammatory (IFN-γ and/or TNF-α-expressing) subsets. Using CyTOF analyses, circulating regulatory B cell populations (IL-10+ plasmablasts and TGF-β+ naïve B cells) were found to be enhanced in melanoma patients compared with a matched healthy volunteer cohort. In concert, intracellular cytokine assay phenotyping revealed a collapse in TNF-α-expressing B cells in melanoma patient compared to healthy volunteer blood. In concordance, TNF-α- expressing B cells were rare among TIL-B. Contrasting to a sparse TNF-α-expressing B cell population, single-cell RNAseq analyses revealed prominent TGF-β-expressing B cell populations in the melanoma tumour microenvironment. CellPhoneDB analyses predicted signaling with Tregs via the immune checkpoint receptor Galectin-9. The less well represented tumour-infiltrating TNF-α-expressing B cells were also predicted to engage in crosstalk with Tregs and may point to potential suppressive activity via TNF-α signaling and the ICOS/ICOSL axis. Lastly, in ex vivo co-cultures, patient-derived B cells enhanced the proliferation of autologous T-helper cells, an effect further enhanced with programmed cell death protein 1 (PD-1) blockade, and allowed T-helper cells to express pro-inflammatory Th1 cytokines (IFN-γ and TNF-α). Patient B cells could also induce FOXP3+ Tregs from conventional T-helper (Tcon) cells in a TGF-β-dependent manner.
In summary, my findings have established contrasting immunostimulatory and immunomodulatory roles of B cells in their response to solid tumours. In breast cancer, and particularly in TNBC patients, B cells and their connected functional signatures were associated with improved clinical outcomes. Isotype-switched B cell lineage traits were prevalent and conferred IgG-biased, clonally expanded, and likely antigen-driven humoral responses. In melanoma, my findings point to a dysregulated cytokine- expressing B cell compartment both in the circulation and in melanoma lesions, favouring regulatory as opposed to pro-inflammatory phenotypes. Within the tumour, cytokine- expressing B cells may engage in crosstalk with and promote Tregs. Furthermore, patient B cells can exert a combination of immunostimulatory and immunomodulatory influences upon autologous T-helper cells in ex vivo culture including expansion of Tregs.
Overall, my findings have unravelled previously unknown features of B cell responses to melanoma and breast cancer, which could help to inform the development of novel therapeutics.
| Date of Award | 1 Sept 2022 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Sophia Karagiannis (Supervisor), Katie E. Lacy (Supervisor) & James Spicer (Supervisor) |