States of exhaustion

To what extent T cell dysfunction in tumours resembles T cell exhaustion in chronic viral infections, and the mechanisms by which immune checkpoint blockade improves tumour immune surveillance even when T cells are dysfunctional, is poorly understood. In a study published in Nature Immunology, Miller, Sen et al. identify a subpopulation of dysfunctional or exhausted CD8⁺ tumour-infiltrating lymphocytes (TILs) that are polyfunctional and respond to anti-programmed cell death 1 (PD1) therapy. In response to anti-PD1, this subpopulation gives rise to the majority of cytotoxic terminally exhausted TILs.

Credit: Lara Crow/Springer Nature Limited

The authors first compared exhausted CD8⁺ T cells from mice during chronic infection with lymphocytic choriomeningitis virus (LCMV) with CD8⁺ T cells isolated from ovalbumin-expressing B16F10 (B16-OVA) mouse melanoma tumours by single-cell expression analysis. Among exhausted CD8⁺ T cells in LCMV infections, clusters of four subpopulations were found, all of which expressed a T cell exhaustion signature (including Pd1 and Tox). These subpopulations included stem-like or progenitor CD8⁺ T cells (referred to as progenitor exhausted CD8⁺ T cells or T_PE cells from hereon) and terminally exhausted CD8⁺ T cells (T_TE cells). When analysing TILs, signatures derived from LCMV T_PE cells (expressing Tcf7 (which encodes transcription factor 7 (TCF7; also known as TCF1)) and the gene encoding DNA-binding protein inhibitor ID3) and T_TE cells (expressing Tim3 (which encodes T cell membrane protein 3 (TIM3)) were significantly enriched. For isolation of live T cells and flow cytometry analyses, the authors used the cell surface marker SLAMF6 for T_PE cells as it was highly co-expressed with TCF1 in this cell population but not in T_TE cells. Gene expression profiles of the corresponding two subpopulations overlapped significantly between TILs and LCMV T cells. However, the two subpopulations were distinct in their transcriptional and phenotypical state and maintained by distinct epigenetic states: T_PE cells and T_TE cells were distinguishable based on their profiles of chromatin-accessible regions (ChARs), with 13,340 ChARs unique to T_PE cells and 8,085 ChARs unique to T_TE cells in both tumour and LCMV T cells. These ChARs were associated with genes regulating cytokine production, survival and memory in T_PE cells, and cell division, apoptosis and cytotoxicity in T_TE cells. The authors then turned their attention to T_PE and T_TE biology in tumour-bearing mice. In growing tumours, the abundance of T_TE relative to T_PE cells increased. While the T cell receptor (TCR) repertoire was less diverse in T_TE cells than in T_PE cells, it overlapped by 50%. In addition, when SLAMF6⁺TIM3^– T_PE cells were transferred into tumour-bearing congenic mice carrying the differential Ptprc^a pan-leukocyte marker, SLAMF6⁺ as well as TIM3⁺ T cells were recovered 16 days later. TIM3⁺ T cells were more cytotoxic, meaning they produced more interferon-γ and granzyme B in vitro than SLAMF6⁺TIM3^– T_PE cells. This suggests that T_PE cells differentiate into T_TE cells in response to TCR stimulation. Indeed, when SLAMF6⁺TIM3^– T_PE cells were transferred into naive mice, in which no antigen would be present and no TCR stimulation occurred, only SLAMF6⁺TIM3^– T cells could be recovered from the spleens 30–40 days later. Also, whereas transferred T_PE cells persisted without antigen presence, T_TE cells did not.

When naive mice were implanted with B16-OVA tumours 30–40 days after having received T_PE cells, T_PE cells trafficked into the tumour tissue and proliferated there, as indicated by the increased number of T_PE cells recovered from tumour tissue compared with secondary lymphoid organs. Moreover, tumours in mice that received T_PE cells as opposed to T_TE cells grew slower — likely a sign of the improved ability of T_PE cells to proliferate and survive and continuously replenish cytotoxic T_TE cells. In response to anti-PD1 treatment in tumour-bearing congenically marked mice, transferred T_PE cells expanded significantly, whereas T_TE cells did not. T_PE cells also converted into the terminally exhausted phenotype at a higher rate than in control tumours.

In patients with melanoma, CD8⁺ T cell populations expressing TCF1 and PD1, indicative of the T_PE cell type, were present in almost all biopsy samples before immune checkpoint blockade therapy. Also, a higher ratio of TCF1⁺ cells among the total population of PD1⁺CD8⁺ T cells positively correlated with prolonged progression-free survival and overall survival on therapy.

In growing tumours, the abundance of T_TE relative to T_PE cells increased

T cell exhaustion and the heterogeneity of exhausted T cell populations in tumours are a mirror of the T cell exhaustion and heterogeneity appearing in chronic viral infections. These findings can translate into improved strategies for PD1 blockade, in which the expansion of T_PE cells in patients can become a central aim in therapeutic strategies to improve outcomes.