An integral T cell pathway helps regulate female gene expression

The well-established existence of sex differences in immune responses, including immune responses driven by T cells, cuts both ways. Females tend to have a stronger immune response to pathogens, yet most patients who have autoimmune diseases involving dysregulated T cells are female.

In the School of Veterinary Medicine, Montserrat Angueraand her lab have been interested in how the X chromosome is involved in these processes. In certain female mammals, gene expression is regulated through X chromosome inactivation, where one of the two X chromosomes is silenced. This process is initiated and maintained by expression of Xist RNA, a long noncoding RNA molecule, from the inactive X chromosome.

Previous work from the Anguera Labshowed that unstimulated T cells lack Xist RNA and other modifications on the inactive X chromosome, but when T cells are activated with an antigen the Xist RNA and epigenetic marks return. She and collaborators wondered: What signals are necessary for the Xist RNA to return? And how much of the inactive X chromosome is silenced in T cells?

Researchers have now found the answers. The maintenance of X chromosome inactivation depends on nuclear factor kappa B (NF-κB), a transcription factor, and some X-linked genes that are specific to T cells can escape transcriptional silencing. This is the first study linking NF-κB signaling pathways, which are critical in T cell development, embryonic development, and many cell types, with X chromosome inactivation maintenance in T cells in females. Their findings are published in the journal Science Immunology.

"X chromosome inactivation maintenance is critical for female cell viability, health, and proper function, and the T cell is a really important immune cell, so it's exciting that we've been able to connect this cell activation pathway that people think about a lot in T cells with this fundamental female process," says Katherine Forsyth, a postdoctoral fellow in the Anguera Lab. She is co-first author along with Natalie Toothacre, a genetics and epigenetics Ph.D. student in the Perelman School of Medicine.

"NF-κB activity in T cells is critical for protection during immune challenge," the authors write, and so "understanding the molecular mechanisms between NF-κB signaling and the regulation of X chromosome inactivation maintenance will shed light on understanding the genetic and epigenetic contributions underlying sex-biased immune responses during infection."

Anguera says elucidating what happens in a healthy state lays the groundwork and creates a road map for researchers to understand X-linked gene expression and T cell modifications in the context of autoimmune diseases. Forsyth adds that some disorders are related to NF-κB signaling, but a lot remains unknown about sex biases and relationships to the X chromosome.

The new study demonstrated the role of the NF-κB pathway in regulating X inactivation maintenance in mouse and human T cells, using genetic deletion, chemical inhibitors, and patients with immunodeficiencies.

To profile the status of the future inactive chromosome in both unstimulated and activated T cells, the researchers used allele-specific RNA sequencing and Cleavage Under Targets & Release Using Nuclease (CUT&RUN) techniques.

Toothacre explains that CUT&RUN is a way of determining where a specific protein is bound to DNA, and in this case researchers were interested in heterochromatic histone modifications known to be enriched on the inactive X chromosome. They were able to isolate and sequence the DNA to see where this mark is enriched on the X chromosome and how stimulation changes enrichment.

Forsyth explains that the researchers became interested in NF-κB in the first place because they knew that NF-κB signaling occurs downstream of T cell receptor engagement, which is necessary for T cell activation. But Anguera notes that her lab had not previously done work related to NF-κB.

They teamed with Penn Vet's Michael May, an expert on NF-κB signaling pathways, and with Neil Rombergfrom the Children's Hospital of Philadelphia, who saw a presentation Forsyth gave and asked if she had looked at patients with NF-κB deficiencies. Forsyth explained that Romberg had extra cells of a patient with combined variable immunodeficiency disease, which is sometimes caused by NF-κB mutations, from a previous study and was able to get more vials of blood samples from collaborators to use in this study.

"It just really highlights how important collaboration is across the Penn community and having these awesome people that we can collaborate with to really push our science and to elevate it to this level," Anguera says.

Montserrat C. Anguera is an associate professor in the Department of Biomedical Sciences at the University of Pennsylvania School of Veterinary Medicine.

Katherine S. Forsyth is a postdoctoral fellow in the Anguera Lab at Penn Vet.

Natalie E. Toothacre is a genetics and epigenetics doctoral candidate in the Cell and Molecular Biology Graduate Group at Penn.

The other co-authors are Amanda M. Driscoll, Lindsey A. Shallberg, Daniel P. Beiting, and Michael J. May of Penn Vet; Nikhil Jiwrajka of Penn Vet and the Perelman School of Medicine; Neil Romberg of the Perelman School of Medicine and Children's Hospital of Philadelphia;

Charlotte Cunningham-Rundles of the Icahn School of Medicine at Mount Sinai; Sara Barmettler and Jocelyn Farmer of Massachusetts General Hospital; and James Verbsky and John Routes of the Medical College of Wisconsin.

This research was supported by the Lupus Research Alliance (Target in Lupus); National Institutes of Health (R01 AI134834, AI168047, T32 DK-07780, T32-AR076951-01, R01 AI146026, R01 AR066567, R21 AI173679, and K23AI163350); H. Ralph Schumacher Rheumatology Research Fund; Benjamin and Mary Siddons Measey Foundation; Scleroderma Research Foundation; and Jeffrey Model Foundation.