Weekly Digests
‹ Back to December

Stat5 battles Tox for epigenetic control of T cell exhaustion

December 20, 2023

In cancer immunotherapy, reversing or staving off T cell exhaustion is highly useful, and while current checkpoint blockades show substantial efficacy, they don’t appear to rewire the epigenetics that drive exhaustion in the first place. Investigating the epigenetic programming of exhaustion and potential ways to mitigate it, Beltra et al. identified Stat5 as an antagonist to Tox-mediated exhaustion programming, and interrogated ways to utilize this pathway to improve immunity.

T cell exhaustion occurs in a distinct lineage of T cells (Tex), which progress from progenitor (Texprog) to intermediate (Texint) to terminally (Texterm) exhausted states. Based on previous research showing that expression of the transcription factor Tox, which mediates exhaustion programming, is lower and some effector functions are higher in Texint cells, Beltra et al. investigated whether Tox may have an antagonist in this setting. Looking at several sets of gene expression data, the researchers identified the Stat5a transcription factor as being positively correlated with effector and memory T cell functions, and inversely correlated with Tox and exhaustion-related genes.

To better understand the role of Stat5a in T cell exhaustion, the researchers evaluated viral antigen-specific CD8+ T cells transduced with constitutively active Stat5a (STAT5CA) in chronic LCMV-infected mice. At day 8, the T cells had differentiated into either effector-like (Ly108-Tim3+) T cells or Tex precursors, (Ly108+Tim3-). Compared to control cells, a higher portion of STAT5CA cells differentiated into effector-like cells, forming fewer Tex precursors. STAT5CA cells also showed lower levels of Tox per cell and had increased expression of effector-related molecules.

Next, the researchers evaluated the role of Stat5a through genetic deletion of Stat5a-b. In mice treated with these cells, then infected with chronic LCMV, the researchers found that a higher proportion of Stat5a-b-deleted cells were Tex precursor cells, with very few becoming effector-like cells. Further, these cells expressed higher levels of Tox, and were significantly reduced in number compared to control cells, suggesting that Stat5 plays a role in effector differentiation.

Returning to STAT5CA model, the researchers performed ATACseq on cells taken at day 8 post-infection, and found that compared to control cells, the effector-like and Tex precursor-like cell subsets in STAT5CA showed unique profiles, and were more distinct from one another, suggesting that constitutive expression of Stat5a drives diversion. Analysis of differentially accessible peaks (DAPs) showed that the majority of changes in STAT5CA cells reflected reduced exhaustion and a shift towards a more effector-like state. Further, analysis of open chromatin regions showed that a large portion of genes where accessibility varied between STAT5CA cells and control cells contained one or more Stat5 binding sites. The Tox locus itself contained some of the highest numbers of direct Stat5-binding sites, and both Tox and Tox-dependent regions were less accessible.

Looking at the longer-term impacts of constitutive Stat5a expression, the researchers evaluated cells at d27 and found that STAT5CA cells outnumbered co-adoptively transferred control cells, reflecting better maintenance of Tcf1- compartment. Looking at cells expressing Cx3cr1, the researchers identified a population Texint cells, which were mostly derived from controls, and a similar, but distinct population of effector-NK-like cells, which was mostly composed of STAT5CA cells. These results suggested that Stat5a activation drove virus-specific CD8+ T cells into a distinct effector-NK-like state, rather than towards the typical exhaustion phenotypes.

Based on the enhanced accumulation and effector-NK-like features of STAT5CA cells, Beltra et al. evaluated their therapeutic potential in mice with established B16 tumors expressing the viral target antigen. While control cells only slightly delayed tumor growth, STAT5CA cells substantially decreased tumor burdens, resulting in enhanced survival in mice.

Evaluating the role of Stat5 in endogenous T cell responses, the researchers evaluated mice treated with Stat5iKO cells. After one month in a chronic infection setting, these cells were reduced compared to controls, and while they expressed PD-1 and Tox, most remained Texprog cells and failed to express molecules associated with Texint or Texterm states. This could not be rescued with PD-L1 blockade, suggesting that Stat5 is essential in the transition from the Texprog to Texint cell state. Similar results were observed with inducible deletion of Stat5a-b in mature Texprog.

Using CITEseq, the researchers were able to distinguish between Texprog1 and Texprog2 (quiescent, lymphoid tissue resident and proliferating, migrating progenitors, respectively), and found that Stat5a-b-deficient Texprog2 cells retained higher expression of progenitor-associated molecules that would typically be reduced during the transition from Texprog1. Further, the few Texint and Texterm cells that developed from Stata-b-deficient cells lacked expression of effector genes and killer lectin-like receptors, suggesting impaired re-entry into the cell cycle from quiescence and a failure to synthesize new proteins, resulting in defective differentiation.

Given the seemingly beneficial features associated with Stat5a expression, the researchers developed a strategy to take advantage of the Stat5a axis in a therapeutic setting though an orthogonal IL-2:IL2Rb system that allowed for selective targeting of Tex cells. In mice carrying adoptively transferred T cells transduced with the orthoIL2Rb-chain, treatment with orthoIL-2 specifically expanded those cells into Texprog2 and Texint cells, reducing the frequency of Texprog1 and Texterm cells in a dose- and Stat5a-b-dependent manner. This effect was enhanced when anti-PD-L1 was administered at the same time as orthoIL-2. Interestingly, this effect was more evident than when PD-L1 blockade was administered in the setting of Stat5a constitutive expression, suggesting that there is better synergy when the Stat5 activation and PD-1 blockade occur simultaneously.

Investigating whether the orthoIL-2:IL2Rb system could rewire established Tex cells, the team found that after 27 days in a chronic infection model, ortho-IL2Rb cells transferred into naive mice and treated with orthoIL-2 showed reduced Tox expression, robust expansion, with increased effector functions and polyfunctionality, especially in combination with anti-PD-1. ATACseq and analysis of transcription factor binding sites showed that these cells had distinct chromatin landscapes in which Teff and Tmem-like patterns were enriched, but epigenetic scars of exhaustion still remained.

Overall, these results show that while Tox programs exhaustion, Stat5a antagonizes Tox, playing a role in the transition of Texprog1 cells towards more differentiated exhaustion states. Enforced activation of Stat5a can directly inhibit Tox and subsequent exhaustion programming, instead inducing epigenetic remodeling that drives cells towards a durable effector NK-like state that is therapeutically useful, especially in combination with PD-1 axis blockade.

Write-up and image by Lauren Hitchings

Meet the researcher

This week, first author Jean-Christophe Beltra answered our questions.

What was the most surprising finding of this study for you?
We were very thrilled to see that manipulating Stat5 not only triggered a durable resistance of antigen-specific CD8+ T cells to exhaustion (even in the most stringent conditions), but also partially rescued cells that were already fully committed to the exhaustion lineage. Indeed, exhausted CD8+ T cells are epigenetically stable and, until now, few approaches capable of reversing this process have been identified.

The rescued CD8+ T cells were rewired towards a hybrid epigenetic state, displaying diminished exhaustion traits, partial restoration of effector/memory marks, and the acquisition of epigenetic marks unique to enhanced Stat5 activity. These findings present an exciting prospect: the creation of hybrid cell-types that, through precise manipulation of key signals, would incorporate the best attributes of each of the known developmental states (exhaustion, effector, memory), and would also foster unconventional features to achieve new cell states ideally equipped to eliminate cancer cells.

What is the outlook?
I consider this study along with other recent publications a pivotal proof of concept, affirming that the developmental trajectory of exhausted CD8+ T cells CAN be redirected by targeting specific signals (as demonstrated in our case for Stat5). While cytokine-based immunotherapies have proven efficient in the past, the use of these approaches has been constrained by severe off-target effects. However, the current blooming of sophisticated synthetic approaches and engineered tools now present an opportunity to direct cytokine-based therapy specifically to the desired cell type, potentially overcoming prior therapeutic limitations.

Empowering these new approaches with key fundamental discoveries on CD8+ T cell exhaustion holds promises for compelling translational applications. Of course, additional research is imperative to pinpoint the most efficient signals or, more likely, combinations of signals capable of reversing CD8+ T cell exhaustion and reigniting the full antitumor potential of these cells.

What was the coolest thing you’ve learned (about) recently outside of work?
Over the past few years, I have been traveling quite a lot, primarily for my research activities, often taking the opportunity to bring along my entire family. It is truly amazing to witness the impact those journeys have had on my children – how they have absorbed diverse cultures and cultivated a heightened sense of curiosity and reflection. I am also happy to have sparked in them a budding interest for adventures!


Beltra JC, Abdel-Hakeem MS, Manne S, Zhang Z, Huang H, Kurachi M, Su L, Picton L, Ngiow SF, Muroyama Y, Casella V, Huang YJ, Giles JR, Mathew D, Belman J, Klapholz M, Decaluwe H, Huang AC, Berger SL, Garcia KC, Wherry EJ. Stat5 opposes the transcription factor Tox and rewires exhausted CD8+ T cells toward durable effector-like states during chronic antigen exposure. Immunity. 2023 Dec 12.

In the Spotlight...

Innate TCRβ-chain engagement drives human T cells toward distinct memory-like effector phenotypes with immunotherapeutic potentials

Taking a cue from the non-clonotypic activation and positive selection of γδT cells via natural ligands binding to the germline CDR2/HV4 region of the TCRγδ, Vantourout et al. demonstrated that similar targeted antibodies produce highly proliferative αβT cells. Detailed flow, single-cell (RNAseq and CITEseq), and pathway analysis demonstrated that both naive and memory cells were activated and characterized by a unique set of up- and downregulated genes and transcription factors, generating a novel “memory-like effector” (TMLE) state with high effector potential and resident memory characteristics, distinct from cells induced with canonical anti-CD3ε and super antigen-stimulated cells.

Contributed by Ed Fritsch

Structural and physical features that distinguish tumor-controlling from inactive cancer neoepitopes

To improve identification of protective neoepitopes, Custodio et al. analyzed multiple structural features of a strongly, a moderately, and a non-protective neoepitope (and their WT counterparts) from a murine model. All three epitopes contained 2o anchor mutations, which enhanced MHC I affinity, and there was an inverse relationship between protection and affinity of the cognate WT peptide. Structurally, the peptide backbone did not differ much from WT for any of the neoepitopes, but the most protective was more rigid in the MHC groove and presented an exposed aromatic side chain. An AI-assisted structural modeling program (based on AlphaFold) was best at predicting the observed structure.

Contributed by Ed Fritsch

X-CHIME enables combinatorial, inducible, lineage-specific and sequential knockout of genes in the immune system

LaFleur et al. developed and validated four novel CHimeric IMmune Editing (CHIME) CRISPR-Cas9 bone marrow delivery systems for rapid combinatorial (C), inducible (I), lineage-specific (L), and sequential (S) in vivo KO of single genes in hematopoietic cells of adult mice, enabling the study of genes in distinct contexts. C-CHIME KO of the highly homologous PTPN1 and PTPN2 non-receptor tyrosine phosphatases – which impact T cell immunity and for which embryonic KO is lethal – during immune development resulted in lethality associated with bone marrow hypoplasia, and after immune development, (S-CHIME) led to enteritis and lethality.

Contributed by Paula Hochman

Acquisition of suppressive function by conventional T cells limits antitumor immunity upon T(reg) depletion

Whiteside et al. investigated mechanisms underlying the limited clinical success of Treg cell-depleting therapies. Using a mouse model of Treg-targeted immunotherapy, depletion of Foxp3+ Tregs drove expansion and conversion of CD4+Foxp3- conventional T (Tconv) cells within tumors to acquire a Treg-like transcriptional profile with suppressive activity ex vivo. Treg ablation resulted in activation and expansion of CCR8+ Tconv cells that suppressed tumor immunity via IL-10 production. Inhibition of IL-10 by conditional deletion of T cell Il10 or by Ab blockade of IL-10 signaling was synergistic with Treg depletion, and prevented treatment resistance.

Contributed by Katherine Turner

Comprehensive single-cell analysis demonstrates radiotherapy-induced infiltration of macrophages expressing immunosuppressive genes into tumor in esophageal squamous cell carcinoma

Integrating scRNAseq, CODEX, and VISIUM data from esophageal squamous cell carcinoma patient tissues, Oyoshi and Du et al. substantiated increased immune cell infiltration into tumors, with dynamic changes in immunostimulatory and immunosuppressive gene expression during and following radiation therapy (RT), particularly among myeloid cells. A subgroup of myeloid cells with PD-L1 expression exhibited upregulation of coinhibitory and protumor genes, such as SIRPA, IDO1, CCL3, and IL-6, suggesting novel combinations with anti-PD-L1. Interestingly, some PD-L1+ myeloid cells also upregulated antitumor pathways.

Contributed by Shishir Pant

Allogeneic NK cells induce monocyte-to-dendritic cell conversion, control tumor growth, and trigger a pro-inflammatory shift in patient-derived cultures of primary and metastatic colorectal cancer

Toffoli et al. demonstrated that an off-the-shelf, clinical-stage allogeneic NK cell product (derived from ex vivo-expanded and -differentiated umbilical cord blood CD34+ hematopoietic stem cells) induced the differentiation of infiltrating monocytic cells to an activated DC-like phenotype, triggered tumor cell lysis, and controlled tumor growth efficiently in both primary and metastatic colorectal cancer single-cell suspensions. The combination of NK cells with a toll-like receptor 7/8 agonist (R848) activated CD8+ and CD4+ TlLs, reduced activated Tregs, and induced a pro-inflammatory (IFNγ, IL-2, IL-12p70, and IFNα) cytokine and chemokine release profile.

Contributed by Shishir Pant

Everything New this Week In...

Close Modal

Small change for you. Big change for us!

This Thanksgiving season, show your support for cancer research by donating your change.

In less than a minute, link your credit card with our partner RoundUp App.

Every purchase you make with that card will be rounded up and the change will be donated to ACIR.

All transactions are securely made through Stripe.