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Biology subjects

Thomas, M. M.

Publications and source records attributed to Thomas, M. M..

2 recordsLinked to original sources

Dnmt3a-mediated de novo methylation balances Th1 and Tfh memory cell plasticity and functionality

Following acute viral infection, naive CD4+ T cells differentiate into T follicular helper (Tfh) and T helper 1 (Th1) cells that generate long-lived memory cells. However, it is unclear how memory Tfh and Th1 cells maintain their lineage commitment. We demonstrate that Tfh and Th1 lineages acquire distinct Dnmt3a-dependent de novo DNA methylation programs that are preserved into memory. Dnmt3a deletion impairs lineage commitment and functionality of memory Th1 and Tfh cells, resulting in aberrant Runx1 upregulation that represses germinal center Tfh cell differentiation. In contrast, transient pharmacological DNA methyltransferase inhibition during priming impairs repression of Tfh-associated genes while properly silencing Runx1, and results in enhanced Tfh cell functionality in primary and secondary responses to viral infections. Together, these findings demonstrate that Dnmt3a-mediated epigenetic programing is required to enforce T helper lineage commitment and preserve Tfh and Th1-specific functions during the recall response to infection, and reveal novel strategies to improve long-lived adaptive immunity against infectious diseases. SUMMARYThis article demonstrates that Dnmt3a-dependent epigenetic programing regulates functionality and plasticity of Th1 and Tfh memory cells. Furthermore, early pharmacological inhibition of such programing enhances GC Tfh cell differentiation, suggesting novel strategies for modulating the immune response to viral infections.

immunology↗

Influenza A Viral Burst Size from Thousands of Infected Single Cells Using Droplet Quantitative PCR (dqPCR)

An important aspect of how viruses spread and infect is the viral burst size, or the number of new viruses produced by each infected cell. Surprisingly, this value remains poorly characterized for influenza A virus (IAV), commonly known as the flu. In this study, we screened tens of thousands of cells using a microfluidic method called droplet quantitative PCR (dqPCR). The high-throughput capability of dqPCR enabled the measurement of a large population of infected cells producing progeny virus. By measuring the fully assembled and successfully released viruses from these infected cells, we discover that the viral burst sizes for both the seasonal H3N2 and the 2009 pandemic H1N1 strains vary significantly, with H3N2 ranging from 101 to 104 viruses per cell, and H1N1 ranging from 101 to 103 viruses per cell. Some infected cells produce average numbers of new viruses, while others generate extensive number of viruses. In fact, we find that only 10% of the single-cell infections are responsible for creating a significant portion of all the viruses. This small fraction produced approximately 60% of new viruses for H3N2 and 40% for H1N1. On average, each infected cell of the H3N2 flu strain produced 709 new viruses, whereas for H1N1, each infected cell produced 358 viruses. This novel method reveals insights into the flu virus and can lead to improved strategies for managing and preventing the spread of viruses. Author summaryViruses infect and exploit host cells to reproduce and spread. The viral burst size, or the number of viral particles released from an infected cell, plays a critical role in understanding infection dynamics and overall viral fitness. However, accurately determining burst size for many single cells using conventional laboratory methods can be challenging. Here, we introduce dqPCR, a droplet microfluidic method for the rapid measurement of influenza virus numbers produced by thousands of individual cells. Our findings revealed that only a small proportion of infected cells are responsible for producing a significant portion of the total viral population. By utilizing this method in future studies, we can gain a deeper understanding of the role of diversity in rapidly evolving viruses.

bioengineering↗