In past studies, researchers have identified a subset of T cells that serve as precursors to exhausted T cells. While these cells show some features of exhaustion, they maintain high proliferative potential and are able to both self-renew and replenish populations of exhausted effector T cells that mediate control in chronic viral infections and tumors. While the T cell precursor subset is known to play an important role in T cell responses, little is known about how or when these cells arise. Recently, Utzschneider and Gabriel et al. investigated this topic and published their findings in Nature Immunology.

To begin, Utzschneider and Gabriel et al. transferred labeled TCR-transgenic CD8⁺ P14 T cells that recognized the gp33-41 CD8⁺ epitope from LCMV into naive mice. Tracking these cells over the course of LCMV Armstrong (acute viral infection) or LCMV Docile (chronic viral infection) infection, the researchers observed that P14 T cells quickly began to separate into either TCF1⁺TIM3^- precursor T (TP; later redefined as ID3⁺TIM3^-) cells or TCF1^-TIM3⁺ effector T (TE; ID3^-TIM3⁺) cell subsets. Interestingly, TP cells were maintained at higher levels during the expansion phase of chronic infection compared to acute; this was found to be the direct result of higher viral loads, and thus higher antigen burden in the chronic infection model.

Next, the researchers compared how TP and TE cells acquired features of exhaustion and found that both subsets upregulated PD-1 and TOX within 3 days of both chronic and acute infection. However, only in chronic infection did TOX and PD-1 expression continue to increase with ongoing cell divisions in both TP and TE cells. TP cells expressed higher levels of TOX and lower levels of IFNγ compared to their TE counterparts, and production of IFNγ by TPs was lower in chronic infection compared to acute. Decreasing the dose of the virus increased IFNγ production by TP cells, suggesting that higher antigen loads led to the generation of TP cells with repressed effector functions.

Next, the researchers performed RNAseq on TP and TE cells at different time points post-infection and found that large numbers of genes were differentially expressed between early TP and early TE cells in both chronic and acute infection. In chronic infection, the difference between TP and TE cells became more pronounced over time, while in acute infection, the difference between the two subtypes became less pronounced as the infection resolved, in line with both subtypes transitioning into resting memory T cells.

Using multidimensional scaling analysis to cluster cells based on  features of T cell exhaustion over time revealed that early (day 5) TP cells in chronic infection resembled established signatures for precursors of exhausted (day 21) T cells, while early TE cells from mice with chronic infection resembled the polyfunctional effector cells that were prominent in acute infection. When the researchers compared exhausted TP and TE cells from chronic infections against corresponding memory cells that developed after acute infection, they were able to identify a core exhaustion signature of 281 genes that included Pdcd1, Lag3, and Tox. Further analysis revealed that early TP cells were highly enriched for genes commonly upregulated in exhausted T cells, while TP cells from acute infection were enriched for genes commonly upregulated in polyfunctional cells. This shows that in the setting of chronic infection, TP cells acquire an exhaustion signature early on. 

Investigating the epigenetic profile of TP cells, Utzschneider and Gabriel et al. found that in chronic infection, exhaustion-related genes like Tox were highly accessible in early TP cells, mature TP cells, and mature TE cells. Analysis of DNA binding motifs in accessible chromatin regions showed that in early TE cells, accessible chromatin regions were enriched for the T-box transcription factors T-BET and EOMES. Meanwhile, in early TP cells, there was a strong enrichment for DNA binding motifs of TCF1, JUN, AP-1, and NFAT family transcription factors, which are commonly activated in response to TCR stimulation. TP cells were also enriched for binding motifs for BACH2, which limits TCR-induced transcriptional changes and promotes the development of memory phenotypes in acute infection. Further, the Bach2 gene itself was highly accessible in early TPs, which corresponded with increased expression of Bach2 in early TPs and even more pronounced expression in mature TPs.

To determine whether BACH2 was required for the generation of TP cells, Utzschneider and Gabriel et al. showed that P14 T cells lacking BACH2 failed to generate TP cell populations in mice with chronic LCMV infection, while overexpression of BACH2 enforced TP cell generation. Deletion of the transcription factor BATF had a similar effect to the overexpression of BACH2, suggesting that BATF and BACH2 regulated transcription in opposing manners, though the effect of BACH2 appeared to be stronger.

Finally, to determine whether exhaustion was imprinted and propagated to progeny, the researchers isolated TP and TE cells from early infections and transferred them to naive hosts which were acutely infected the following day. At day 7 post-infection, all cell populations had expanded, but TPs isolated from the chronic infection setting sustained higher levels of TOX and PD-1. Even memory cells (day 56) derived from this subset maintained high TOX expression, suggesting that exhaustion was imprinted early on. After transfer of TP and TE cells from early infections into chronically infected mice – a setting with persistent antigen stimulation – TE cells failed to proliferate while TP cells expanded robustly, supporting a requirement for TP cells in mounting and maintaining effective T cell responses.

The evidence uncovered by Utzschneider and Gabriel et al. suggests that high amounts of antigen, a harbinger of chronic antigen exposure, initiated early molecular pathways that promoted the generation of TP cells and protected them from undergoing full differentiation, thereby limiting immune pathology. Early on, these precursor T cells acquired features of exhaustion at the epigenetic, transcriptional, and protein expression levels. These features of exhaustion, relevant to both the chronic viral infection and tumor settings, were imprinted early on as a consequence of antigen load, rather than acquired over the course of chronic antigen exposure.

by Lauren Hitchings

Meet the researcher

This week, co-first author Daniel Utzschneider answered our questions.

What prompted you to tackle this research question?
We and others recently identified that exhausted T cell responses to chronic viral infections and in tumors are sustained by a subset of TCF1-expressing cells that retain self-renewal and proliferative potential while also giving rise to TCF1^-TIM3⁺ exhausted effector cells that mediate viral or tumor control. In this study, we were initially interested in determining when and how this dichotomy of TCF1⁺ precursors and TIM3⁺ effector cells is established.

What was the most surprising finding of this study for you?
When comparing CD8⁺ T cells responding to an acute infection (LCMV Armstrong) with those responding to a severe or chronic infection (LCMV Docile), we noted that both T cell responses behaved quite similarly in terms of differentiating into TCF1⁺ precursors and TIM3⁺ effectors. However, early TCF1⁺ precursors found in responses to the chronic LCMV strain already acquired transcriptional and epigenetic features of T cell exhaustion, while TIM3⁺ effector T cells in the same host displayed polyfunctional profiles that resembled the profiles of T cells responding to acute LCMV infections. This discrepancy among virus-specific cell subsets within the same host was quite striking and surprising to us.

What was the coolest thing you’ve learned (about) recently outside of work?
This pandemic has had a crucial impact on our lives. I have been working at home for the majority of the past months, which, fortunately, also allowed me to spend a lot of time with my young family. Thus, I have to admit, the ‘coolest thing’ I have learned recently are the lyrics of ALL Wiggles songs