A TLR4-HSP70 efferocytic program in thymic macrophages sustains thymic homeostasis and T cell output Free

T cell reconstitution is essential for immune recovery after hematopoietic cell transplantation (HCT) and requires a functional thymus to generate naïve T cells. However, thymic regenerative failure remains poorly understood and lacks therapeutic strategies, leaving patients vulnerable to infections, impaired vaccine responses, and poor immune recovery.

Inspired by historical reports of natural variation in thymic size across inbred mouse strains, we compared multiple inbred strains of mice. We found that C3H/HeJ (HEJ) mice, which harbor a nonfunctional Tlr4 allele, exhibit markedly reduced thymic size, impaired regeneration after injury, and persistent thymic apoptotic debris. In contrast, Tlr4-competent C3H/FeJ (FEJ) mice maintain robust thymic cellularity and regenerative potential. Crossbreeding and genetic mapping confirmed a dominant role for Tlr4 in regulating thymic size. Myeloid-specific deletion of Tlr4 in Csf1r^Cre+Tlr4^flox mice recapitulated the HEJ phenotype, while deletion in endothelial or mesenchymal cells had no effect, indicating a macrophage-intrinsic mechanism.

Mechanistically, Tlr4 signaling in thymic macrophages licensed a p65-dependent efferocytic program involving lysosomal remodeling and metabolic activation. Bulk RNA-seq of sorted thymic macrophages from Tlr4-deficient mice revealed impaired induction of genes regulating phagolysosomal fusion, lipid metabolism, and immune resolution. While ATAC-seq showed minimal changes in global chromatin accessibility, CUT&RUN profiling identified altered p65 binding at enhancers of differentially expressed genes. This suggests that Tlr4 signaling modifies transcription by directing p65 to lysosomal effector gene enhancers.

Functionally, Tlr4-deficient macrophages failed to undergo efferocytic reprogramming, exhibiting impaired apoptotic cell uptake, defective acidification, and reduced lysosomal gene expression as measured by flow cytometry and immunohistochemistry. Live imaging and electron microscopy further revealed intracellular accumulation of undigested apoptotic cargo. This led to increased release of inflammatory mediators from uncleared apoptotic thymocytes, ultimately disrupting the local regenerative niche.

While Tlr4 can bind to microbial products, germ-free and antibiotic-treated mice retained normal thymic size, supporting a non-microbial source of Tlr4 activation. Thus, to identify endogenous Tlr4 ligands, we engineered a Tlr4-TurboID fusion construct and performed proximity-dependent biotin labeling in the presence of apoptotic thymocytes. Mass spectrometry revealed heat shock protein 70 (HSP70) as a Tlr4-interacting protein. Stimulation of macrophages with HSP70 triggered NF-κB activation, suggesting that apoptotic cell–derived HSP70 functions as an endogenous rheostat of macrophage activity.

This axis remains pharmacologically targetable, as treatment with monophosphoryl lipid A (MPLA), a clinically used TRIF-biased Tlr4 agonist, enhanced thymic cellularity, while this did not happen with non-TRIF-biased Tlr4 agonists. MPLA increased efferocytosis in thymic macrophages of UBQ^GFP mice and improved chromatin accessibility in macrophages at loci involved in phagosome acidification and lysosomal metabolism, leading to enhanced output of recent thymic emigrants in Rag2^GFP mice. In contrast, Tlr4-deficient macrophages failed to respond to MPLA, showing no efferocytic or transcriptional activation, confirming that this therapeutic benefit requires intact Tlr4 signaling.

Similarly, human apoptotic thymocytes activated TLR4 in a HEK-Blue TLR4 reporter cell line and human thymic samples had spatially organized apoptotic foci around efferocytotic macrophages in the human thymus during stress. Human thymic macrophages expressed the highest TLR4 expression of all cells by sigle cell sequencing, and CD163+LAMP1 macrophages exhibited active efferocytosis with distinct spatial characteristics in regions of apoptotic thymocyte clearance with active thymopoiesis in samples across lifespan.

Together, our findings define a conserved macrophage-intrinsic Tlr4–HSP70 axis that calibrates thymic regeneration through p65-mediated efferocytic function. This apoptosis-sensing pathway sustains thymic homeostasis and adaptive lymphopoiesis and its therapeutic activation via MPLA represents a promising and translatable strategy to restore thymic function, enhance immune competence, and improve outcomes in patients undergoing cytoreduction, aging, or transplantation.