Interrogating hypoxia-mediated CD8\(^{+}\) T cell dysfunction in multiple myeloma

Fulton-Ward, Taylor ORCID: 0000-0001-8407-7184 (2024). Interrogating hypoxia-mediated CD8\(^{+}\) T cell dysfunction in multiple myeloma. University of Birmingham. Ph.D.

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Abstract

Introduction: Multiple myeloma (MM) is an incurable plasma cell malignancy that develops in the bone marrow (BM). The BM is understood to be immunosuppressive and hypoxic, which may limit therapeutic responses to immune-directed therapies. This thesis interrogates whether the hypoxic nature of the BM environment drives CD8\(^{+}\) T cell dysfunction.

Methods: CD8\(^{+}\) T cells are isolated from healthy human peripheral blood and incubated overnight in normoxia (21% oxygen) or hypoxia (1% oxygen) to allow oxygen level equilibration. Cells are activated by anti-CD3/anti-CD28 antibodies and analysed for immune function, metabolism and signalling. MM patient samples, including blood and BM mononuclear cells, are analysed ex vivo by flow cytometry.

Results: CD8\(^{+}\) T cells stimulated in hypoxia demonstrate impaired proliferation, CD25 expression and IFN-gamma production. However, TNF-alpha production, granzyme-B expression and CD107a externalisation are unaffected by hypoxia. A defect in T cell signalling lies at the level of mTOR, with impact on downstream targets. Stable isotope-based metabolic tracing identifies a reduction of activation-induced glycolysis, glucose oxidation and glutaminolysis in hypoxia. Bulk RNA sequencing and flow cytometric analysis identify BNIP3 as a marker of hypoxia and potential mechanisms driving mTOR suppression in hypoxia are explored. Stimulation of CD8\(^{+}\) T cells with a BCMAxCD3 bispecific antibody and BCMA-expressing MM cell lines bring these findings into a therapeutic context. CD8\(^{+}\) T cells from the BM of MM patients demonstrate impaired proliferation and expression of c-Myc compared to those from the PB.

Conclusion: Hypoxia impairs CD8\(^{+}\) T cell signalling, activation and specific effector functions alongside activation-induced metabolic reprogramming. These intrinsic effects correlate with reduced function of MM patient CD8\(^{+}\) T cells found in the BM compared to the PB. This work could aid understanding the limitations of response with immune-directed therapies acting within a hypoxic environment.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):