Effective differentiation of double negative thymocytes requires high fidelity replication of mitochondrial DNA in an age dependent manner

One of the most proliferative periods for T cells occurs during their development in the thymus. Increased DNA replication can result in increased DNA mutations in the nuclear genome, but also in mitochondrial genomes. A high frequency of mitochondrial DNA mutations can lead to abnormal mitochondrial function and have negative implications on human health. Furthermore, aging is accompanied by an increase in such mutations through oxidative damage and replication errors. Increased mitochondrial DNA mutations cause loss of mitochondrial protein function, and decrease energy production, substrates, and metabolites. Here we have evaluated the effect of increased mitochondrial DNA mutations on T cell development in the thymus. Using mice carrying a mutant mitochondrial DNA polymerase γ (PolG) that causes increased mitochondrial DNA mutations, we show that high fidelity replication of mitochondrial DNA is pivotal for proper T cell development. Reducing the fidelity of mitochondrial DNA replication results in a premature age-dependent reduction in the total number of CD4/CD8 double negative and double positive thymocytes. Analysis of mitochondrial density in thymocyte subpopulations suggests that this may be due to reduced proliferation in specific double negative stages. Taken together, this work suggests that T cell development is regulated by the ability of mitochondria to faithfully replicate their DNA

EFFECTS OF INCREASED MITOCHONDRIAL DNA MUTATIONS ON T CELL DEVELOPMENT AND FUNCTION

166 pagesMitochondria 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 PolGD257A/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 PolGD257A/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 (PolGD257A/+) 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.2025-09-0

Past, Present, and Future of Rituximab—The World’s First Oncology Monoclonal Antibody Therapy

Rituximab is a chimeric mouse/human monoclonal antibody (mAb) therapy with binding specificity to CD20. It was the first therapeutic antibody approved for oncology patients and was the top-selling oncology drug for nearly a decade with sales reaching $8.58 billion in 2016. Since its initial approval in 1997, it has improved outcomes in all B-cell malignancies, including diffuse large B-cell lymphoma, follicular lymphoma, and chronic lymphocytic leukemia. Despite widespread use, most mechanistic data have been gathered from in vitro studies while the roles of the various response mechanisms in humans are still largely undetermined. Polymorphisms in Fc gamma receptor and complement protein genes have been implicated as potential predictors of differential response to rituximab, but have not yet shown sufficient influence to impact clinical decisions. Unlike most targeted therapies developed today, no known biomarkers to indicate target engagement/tumor response have been identified, aside from reduced tumor burden. The lack of companion biomarkers beyond CD20 itself has made it difficult to predict which patients will respond to any given anti-CD20 antibody. In the past decade, two new anti-CD20 antibodies have been approved: ofatumumab, which binds a distinct epitope of CD20, and obinutuzumab, a mAb derived from rituximab with modifications to the Fc portion and to its glycosylation. Both are fully humanized and have biological activity that is distinct from that of rituximab. In addition to these new anti-CD20 antibodies, another imminent change in targeted lymphoma treatment is the multitude of biosimilars that are becoming available as rituximab’s patent expires. While the widespread use of rituximab itself will likely continue, its biosimilars will increase global access to the therapy. This review discusses current research into mechanisms and potential biomarkers of rituximab response, as well as its biosimilars and the newer CD20 binding mAb therapies. Increased ability to assess the effectiveness of rituximab in an individual patient, along with the availability of alternative anti-CD20 antibodies will likely lead to dramatic changes in how we use CD20 antibodies going forward

TCR Signal Strength and Antigen Affinity Regulate CD8+ Memory T Cells

CD8(+) T cells play a critical role in adaptive immunity, differentiating into CD8(+) memory T cells, which form the basis of protective cellular immunity. Vaccine efficacy is attributed to long-term protective immunity, and understanding the parameters that regulate development of CD8(+) T cells is critical to the design of T-cell mediated vaccines. We show here using mouse models that two distinct parameters: T cell Receptor (TcR) signal strength (regulated by tyrosine kinase ITK) and antigen affinity, play important but separate roles in modulating the development of memory CD8(+) T cells. Unexpectedly, our data reveals that reducing TcR signal strength along with reducing antigen affinity for the TcR leads to enhanced and accelerated development of CD8(+) memory T cells. Additionally, TcR signal strength is able to regulate CD8(+) T cell effector cytokine production independent of TcR antigen affinity. Analysis of RNA-sequencing data reveals that genes for inflammatory cytokines/cytokine receptors are significantly altered upon changes in antigen affinity and TcR signal strength. Furthermore, our findings show that the inflammatory milieu is critical in regulating this TcR signal strength mediated increase in memory development as both CpG treatment or co-transfer of WT and Itk(−/−) T cells eliminates the observed increase in memory cell formation. These findings suggest that TcR signal strength and antigen affinity independently contribute to CD8(+) memory T cell development, which is modulated by inflammation, and suggest that manipulating TcR signal strength, along with antigen affinity, may be used to tune the development of CD8(+) memory T cells during vaccine development

DataSheet_1_Effective differentiation of double negative thymocytes requires high fidelity replication of mitochondrial DNA in an age dependent manner.pdf

One of the most proliferative periods for T cells occurs during their development in the thymus. Increased DNA replication can result in increased DNA mutations in the nuclear genome, but also in mitochondrial genomes. A high frequency of mitochondrial DNA mutations can lead to abnormal mitochondrial function and have negative implications on human health. Furthermore, aging is accompanied by an increase in such mutations through oxidative damage and replication errors. Increased mitochondrial DNA mutations cause loss of mitochondrial protein function, and decrease energy production, substrates, and metabolites. Here we have evaluated the effect of increased mitochondrial DNA mutations on T cell development in the thymus. Using mice carrying a mutant mitochondrial DNA polymerase γ (PolG) that causes increased mitochondrial DNA mutations, we show that high fidelity replication of mitochondrial DNA is pivotal for proper T cell development. Reducing the fidelity of mitochondrial DNA replication results in a premature age-dependent reduction in the total number of CD4/CD8 double negative and double positive thymocytes. Analysis of mitochondrial density in thymocyte subpopulations suggests that this may be due to reduced proliferation in specific double negative stages. Taken together, this work suggests that T cell development is regulated by the ability of mitochondria to faithfully replicate their DNA.</p

Interleukin-2-Inducible T-Cell Kinase Deficiency Impairs Early Pulmonary Protection Against Infection

Interleukin-2 (IL-2) inducible T-cell kinase (ITK) is a non-receptor tyrosine kinase highly expressed in T-cell lineages and regulates multiple aspects of T-cell development and function, mainly through its function downstream of the T-cell receptor. deficiency can lead to CD4 lymphopenia and Epstein-Bar virus (EBV)-associated lymphoproliferation and recurrent pulmonary infections in humans. However, the role of the ITK signaling pathway in pulmonary responses in active tuberculosis due to infection is not known. We show here that human lungs with active tuberculosis exhibit altered T-cell receptor/ITK signaling and that deficiency impaired early protection against in mice, accompanied by defective development of IL-17A-producing γδ T cells in the lungs. These findings have important implications of human genetics associated with susceptibility to due to altered immune responses and molecular signals modulating host immunity that controls activity. Enhancing ITK signaling pathways may be an alternative strategy to target infection, especially in cases with highly virulent strains in which IL-17A plays an essential protective role