Divergent Clonal Differentiation Trajectories of T-Cell Exhaustion

Summary

Scientists characterized Tex (T cell exhaustion) phenotypic diversity by adopting a single-cell multi-omics profiling in mice, which contained two late Tex subtypes: those with terminal exhaustion (Tex^term) and those expressing killer cell lectin-like receptor cytotoxicity (Tex^KLR), respectively. Additionally, the authors reported the three clonal differentiation trajectories of Tex^term-biased, Tex^KLR-biased, and evanescent clones for the aforementioned two phenotypes. In addition, the researchers found that the Tex^KLR differentiation trajectory was associated with low TCR signaling activity, whereas the Tex^term differentiation trajectory was associated with high TCR signaling affinity. In human tumor-infiltrating lymphocytes, the authors observed similar clonal differentiation pathways. These data suggest that T cells exposed to chronic antigens exhibit clonal heterogeneity.

Research Criteria

Using scRNA-seq, scTCR-seq, and scATAC-seq, the authors examine the phenomena of Tex induced by extended antigen exposure.

Fig.1 Experimental design. (Daniel, 2022)

Sample Type

Mouse fresh kidney, liver, and spleen tissues.

Result—Multiomic Map of Tex

To analyze CD8+ T-cell depletion, the authors used mouse models of acute (LCMV Armstrong, Arm) and chronic (LCMV clone 13, Cl13) infection with lymphocytic choroid plexus meningitis virus strains. These two viral strains share immunodominant epitopes that enable direct comparison of antigen-specific T cell responses. The authors performed paired scRNA/TCR-seq as well as scATAC-seq using LCMV glycoprotein tetramer-positive (gp33+) and negative spleen CD8+ T cells from day 8 (D8) and day 21 (D21) post-infection. At day 21 of Cl13 infection, the authors also carried out scRNA/TCR-seq of gp33+ and gp33- phenotypes from two additional organs. Finally, the authors used the defined Tex^prog (PD-1+SLAMF6+CX3CR1-), Tex^int (PD-1+ CX3CR1+SLAMF6-), and Tex^term (PD-1+SLAMF6-CX3CR1-) surface markers for the classification of D21 Cl13 splenic T cells. Overall, the authors obtained 96750 cells by scRNA-seq and detected TCR alpha and beta sequences in 88696 T cells (91.7%), consisting of 5197 T cell clonotypes, in addition to 62731 high-quality cells by scATAC-seq.

Fig.2 Single-cell multiomic atlas of T cell exhaustion during LCMV infection. (Daniel, 2022)

Result—Tex Acquire Organ-Specific Terminal Exhaustion Signatures

The authors re-clustered the scRNA-seq profiles of gp33+ and gp33- CD8+ T cells in the spleen, lung, and liver of D21 Cl13-infected mice and investigated the distribution of Tex subpopulations across organs. Lung T cells displayed an alternative end-stage failure phenotype (Tex^lung) and reduced Tex^prog subpopulations, as well as similar Tex^KLR and Tex^int ratios, compared with spleen T cells, whereas liver T cells were almost exclusively Tex^term, consistent with previous research.

The authors observed a common Tex^term gene signature in all organs despite the tissue-specific differences in Tex. This signature (n=35 genes) contained genes previously associated with depletion, including the immune checkpoint inhibitory receptors Pdcd1 and Tox. Using a previously defined depletion gene signature, the authors ranked the severity of Tex^term loss in each organ. Tex^term derived from the liver scored the highest, followed by Tex^term derived from the spleen and lung. Flow cytometry analysis revealed that Tex^term was more prevalent in the liver (47%) than in the spleen (47%) and lung (51%). In contrast, the spleen had the highest frequency of Tex^prog. Tex^int and Tex^KLR levels were higher in the spleen than in the liver and lung. These findings suggest that T-cell depletion has a common profile of gene expression in multiple organs, but also has microenvironment-specific effects.

Fig.3 Identification of Tex^int, Tex^KLR Expression, and Organ-Specific Tex Subpopulations. (Daniel, 2022)

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Reference

Daniel, B.; et al. Divergent clonal differentiation trajectories of T cell exhaustion. Nature Immunology. 2022, 23(11): 1614-1627.

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