Abstract

Natural Killer (NK) cells are cytotoxic innate lymphoid cells that play an important role in the surveillance and elimination of virally-infected and malignantly-transformed cells. These primary effector functions are achieved by producing cytokines and chemokines which activate and recruit additional immune effectors, as well as direct cytotoxic killing via perforin and granzymes. NK cell function is tightly regulated by a balance of germ-line encoded activating, inhibitory, co-stimulatory, and cytokine receptors expressed at the cell surface. Through these activating and inhibitory receptors, NK cells recognize and kill targets without prior sensitization through the loss of self-identifying molecules such as major histocompatibility complex (MHC) class I that bind to inhibitory receptors on NK cells (detection of ‘missing self’) or through the upregulation of ligands recognized by activating receptors on NK cells that can overcome inhibitory signals. The ability of NK cells to eliminate malignant cells has been established in the hematopoietic cell transplantation (HCT) setting, where a ‘graft vs leukemia’ effect can be exploited clinically through allogenic HCT and through adoptive NK cell therapy to treat hematologic malignancies such as acute myeloid leukemia (AML). NK cells briefly stimulated with the pro-inflammatory cytokines IL-12/15/18 become long-lived, memory-like (ML) NK cells with the ability to respond robustly upon reactivation with cytokines, activating receptors, or tumor target engagement, and further clinical trials have demonstrated their safety and efficacy as cellular therapy for AML. However, many of the factors that affect response versus treatment failure after ML NK cell therapy remain unknown. Unexpectedly, multidimensional immune correlatives revealed a novel, direct correlation between CD8α expression on donor ML NK cells and treatment failure. However, the impact of CD8α on human NK cell subsets, phenotype, and function are poorly understood. While CD8αβ has been extensively characterized on human and mouse T cells as a co-receptor for the T cell receptor (TCR), CD8α is not expressed on murine NK cells. Approximately 40% of human NK cells express a homodimeric CD8αα chain, and a much smaller fraction of NK cells (1-2%) express CD8αα. CD8αα contains an extracellular region that can bind to the conserved α3 region of HLA class I, a transmembrane domain, and a short cytoplasmic tail that interacts with the Src tyrosine kinase Lck. There are few and conflicting reports on the function of CD8α on the biology of human NK cells. Given the paucity of data on CD8α and human NK cells, and the striking negative association between CD8α expression and treatment response in our recent data, my thesis aimed to comprehensively elucidate the functional role of CD8α on conventional NK cells. Here, we show that CD8α marks a spectrum of functionality on human NK cells, and plays a functional, inhibitory role. We demonstrated that sorted CD8α- NK cells had superior tumor control of K562-engrafted NSG mice, and that this was likely mediated by an enhanced capacity for proliferation and survival of CD8α- NK cells. We observed that CD8α expression was dynamic and induced by IL-15, likely mediated by the transcription factor RUNX3. A subset of sorted CD8α- NK cells induced CD8α expression (‘induced’ or ‘iCD8α+’), while the remainder persisted as CD8α- (‘persistent CD8α- ‘), and those sorted CD8α+ sustained CD8α expression (‘sustained CD8α+’). Notably, NK cells with induced CD8α expression were the most proliferative in vitro and in vivo in NSG models. Mechanistically, we found that iCD8α NK cells had greater expression of IL-15Rb and gc components, which resulted in a greater induction of downstream signaling following IL-15 stimulation. Interestingly, we found that the differences in IL-15R expression were due to a preferential expansion and upregulation of CD8α in cells with greater pre-existing IL-15Rb expression. We demonstrated that these enhanced IL-15 signals corresponded to greater expression of nutrient receptors, glucose uptake, and overall metabolic activity. Further, we showed that this also translated to enhanced responses to tumor and cytokine stimulation in iCD8α+ NK cells, and this hyperfunctionality persisted even after three weeks in vivo. We also interrogated the functional role of CD8α using CRISPR-Cas9 deletion of CD8A in primary human NK cells, and did not identify a role in proliferation, survival, or responses to IL-15 signaling. However, we found that CD8 KO led to greater cytokine production and degranulation following activating receptor ligation, particularly through NKp30. This was likely mediated by CD8α enhancing the inhibitory function of KIR3DL1, rather than by abrogating activating receptor signaling. In summary, this thesis highlights a role for induced CD8α expression in marking a time-dependent functional capacity, in addition to identifying a novel role for CD8α in inhibiting NK activation. These findings highlight the importance of interrogating the dynamics of NK cell marker acquisition as they relate to functionality, particularly in the context of understanding NK cell biology and improving cellular therapies.

Committee Chair

Todd Fehniger

Committee Members

Anthony French; Jacqueline Payton; Marco Colonna; Megan Cooper

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Immunology)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

5-8-2025

Language

English (en)

Author's ORCID

https://orcid.org/0000-0003-0037-9324

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