Mitochondria play crucial roles in the development, function, and differentiation of immune cells by providing energy, substrates, and metabolites through mitochondrial proteins. The fidelity of mitochondrial DNA (mtDNA) replication is essential for mitochondria to divide properly. However, as we age, mtDNA accumulates mutations due to multiple rounds of replication, leading to diminished mitochondrial function. One critical protein involved in mtDNA replication is mitochondrial DNA polymerase γ (PolG), which carries a proofreading exonuclease. When the exonuclease function is lacking, mtDNA replication fidelity is reduced, resulting in a 500-fold increase in mtDNA mutations and decreased mitochondrial function in energy-demanding cells. While the dependence of adaptive immune cells on mitochondria at various stages of their lifespan is well-established, the impact of increased mtDNA mutations on T cell biology remains unknown.

In my dissertation, I tested the hypothesis that mice expressing an exonuclease-deficient mutant of PolG, known as PolG^D257A/D257A, prone to elevated mtDNA mutations, would exhibit defects in immune cell development and function. This hypothesis was investigated in various models of T cell development and function, utilizing mice carrying PolG^D257A/D257A. Specifically, we focused on stages of immune cell development where cells are in a highly proliferative state. We chose these stages because we anticipated the most significant differences between the mutant and control groups, considering that mitochondrial replication and mutation load would be highest at these time points. We examined T cell development and the primary and secondary challenge stages during infection conditions, where cells undergo expansion from a naïve to an effector state, followed by contraction and re-expansion. We also considered the age of the mice and grouped them into young, mature, and old categories. We anticipated observing the strongest differences in the mature group, as it is expected to have accumulated more mutations at this time point. Additionally, the study included heterozygous mice (PolG^D257A/+) with one functional and one mutated copy of the PolG gene, resulting in reduced, but not complete, loss of exonuclease function. This allowed for differentiation between the effects of complete loss and reduced exonuclease function, providing a more comprehensive understanding of mtDNA replication fidelity's role in immune cell development and function.

Our findings revealed that reducing mtDNA replication fidelity led to premature age-dependent effects in, 6-8 month old mice, mature mice. In the evaluation of T cell development, we focused on the double negative (DN) stages, which encompass both low and highly proliferative cells. Specifically, DN1 and DN2 represented the low proliferative stages, while DN3 and DN4 were highly proliferative. Intriguingly, we observed an overall reduction in the total number of highly proliferative DN3 cells. Analysis of mitochondria in this thymocyte population suggested that this decrease might be attributed to reduced mitochondrial density. Furthermore, when examining the response of mature CD8⁺ T cells to Listeria monocytogenes infection, our results demonstrated that elevated mtDNA mutations negatively regulated this population. Specifically, low fidelity mtDNA replication resulted in a decrease in the overall number of CD8⁺ T cells capable of mounting an effective infection response, likely due to a decrease in mitochondrial density. Moreover, we observed a preferential increase in the percentage of cells differentiating into memory precursor effector cells compared to short-lived effector cells. Collectively, these findings indicate that mtDNA mutations impair the CD8⁺ T cell response to infection and underscore the importance of mtDNA replication fidelity for optimal T cell effector and memory proliferation.

In summary, our work highlights the importance of faithful replication of mtDNA in the regulation of T cell development and effector function. These findings hold relevance in understanding how CD8⁺ T cells may respond to age-related diseases. Furthermore, since mtDNA mutations accumulate over time, our data provide insights into how CD8⁺ T cells may function in older individuals with an increased mtDNA burden, emphasizing a decline in their functionality.