# Notch Signaling Exacerbates Pulmonary Fibrosis by Regulating the Differentiation of CD4+ Tissue-Resident Memory T Cells

## TL;DR

CD4+ tissue-resident memory T cells worsen lung fibrosis by promoting inflammation, and their differentiation is regulated by the Notch signaling pathway.

## Contribution

Identifies CD4+ TRM cells as pathogenic drivers in pulmonary fibrosis and reveals Notch signaling as a key regulator of their differentiation.

## Key findings

- CD4+ TRM cells are present in higher numbers in fibrotic lungs and correlate with disease severity.
- Depletion of CD4+ TRM cells reduces fibrosis in mouse models.
- Notch signaling inhibition suppresses pro-fibrotic functions of CD4+ TRM cells.

## Abstract

The involvement of the immune system in pulmonary fibrosis is established, the precise contributions of tissue-resident memory T (TRM) cells are still poorly defined. This study sought to define the contribution of CD4+ TRM cells to pulmonary fibrosis, their origin, and regulatory mechanisms. We combined bioinformatic analysis of human fibrotic lung single-cell RNA-sequencing data with experiments in a bleomycin-induced C57BL/6 mouse model. Flow cytometry, targeted in vivo depletion, lymphocyte trafficking blockade, cell co-culture, and pharmacological inhibition were employed. CD4+ TRM cells were observed at higher frequencies within fibrotic lung tissue. Their presence correlated with disease severity, and they were found to exhibit a pro-inflammatory and pro-fibrotic phenotype. Their specific depletion alleviated fibrosis. These cells primarily originated from recruited circulating lymphocytes, as blocking this recruitment reduced TRM accumulation and attenuated disease. Furthermore, the Notch signaling pathway was activated in fibrotic lung CD4+ TRM cells, and its inhibition suppressed their differentiation and impaired their pro-fibrotic function. We conclude that CD4+ TRM cells are pathogenic drivers in pulmonary fibrosis, originating from circulating precursors and being regulated by Notch signaling, underscoring their relevance for therapeutic intervention.