Defining the T cell transcriptional landscape in pediatric liver transplant rejection at single cell resolution.

Acute cellular rejection (ACR) affects >80% of pediatric liver transplant recipients within 5 years, and late ACR is associated with graft failure. Traditional anti-rejection therapy for late ACR is ineffective and has remained unchanged for six decades. Although CD8+ T cells promote late ACR, little has been done to define their specificity and gene expression. Here, we used single-cell sequencing and immune repertoire profiling (10X Genomics) on 30 cryopreserved 16G liver biopsies from 14 patients (5 pre-transplant or with no ACR, 9 with ACR). We identified expanded intragraft CD8+ T cell clonotypes (CD8EXP) and their gene expression profiles in response to anti-rejection treatment. Notably, we found that expanded CD8+ clonotypes (CD8EXP) bore markers of effector and CD56hiCD161- 'NK-like' T cells, retaining their clonotype identity and phenotype in subsequent biopsies from the same patients despite histologic ACR resolution. CD8EXP clonotypes localized to portal infiltrates during active ACR, and persisted in the lobule after histologic ACR resolution. CellPhoneDB analysis revealed differential crosstalk between KC and CD8EXP during late ACR, with activation of the LTB-LTBR pathway and downregulation of TGFß signaling. Therefore, persistently-detected intragraft CD8EXP clones remain active despite ACR treatment and may contribute to long-term allograft fibrosis and failure of operational tolerance.

Adult microglial TGFß1 is required for microglia homeostasis via an autocrine mechanism to maintain cognitive function in mice.

While TGF-ß signaling is essential for microglial function, the cellular source of TGF-ß1 ligand and its spatial regulation remains unclear in the adult CNS. Our data supports that microglia but not astrocytes or neurons are the primary producers of TGF-ß1 ligands needed for microglial homeostasis. Microglia-Tgfb1 KO leads to the activation of microglia featuring a dyshomeostatic transcriptome that resembles disease-associated, injury-associated, and aged microglia, suggesting microglial self-produced TGF-ß1 ligands are important in the adult CNS. Astrocytes in MG-Tgfb1 inducible (i)KO mice show a transcriptome profile that is closely aligned with an LPS-associated astrocyte profile. Additionally, using sparse mosaic single-cell microglia KO of TGF-ß1 ligand we established an autocrine mechanism for signaling. Here we show that MG-Tgfb1 iKO mice present cognitive deficits, supporting that precise spatial regulation of TGF-ß1 ligand derived from microglia is required for the maintenance of brain homeostasis and normal cognitive function in the adult brain.