Weekly Digests
‹ Back to April

Potassium determines the fate of T cells

April 17, 2019

In a paper recently published in and featured on the cover of Science, Vodnala and Eil et al. investigated why some patients have dysfunctional tumor-infiltrating lymphocytes (TILs), while others have T cells that behave like stem cells in their capacity to self-renew, proliferate, and destroy large tumors. They found that this difference is driven by the levels of potassium within the tumor microenvironment (TME).

Previous research has shown that T cell differentiation toward an effector state is metabolism-dependent, and that high levels of extracellular potassium cation (K+) – which is released by necrotic tumor cells – within the tumor interstitial fluid in the TME suppress T cell activation and effector function after TCR engagement, independent of PD-1 and CTLA-4. To uncover the mechanism behind this suppression, Vodnala and Eil et al. analyzed the metabolites of T cells conditioned in media with either elevated or normal levels of K+. Elevated extracellular K+ led to reduced nutrient uptake by the T cells despite unaltered extracellular nutrient availability. This functional caloric restriction did not affect T cell survival or proliferative potential.

The metabolite analysis, together with RNAseq and fluorescent confocal microscopy, also indicated that elevated extracellular K+ induced autophagosome formation and maturation, and maintained ongoing autophagy within T cells. Activity of the nutrient-sensing signaling cascades (Akt-mTOR and AMPK) showed that autophagy was induced by a starvation response, triggered by the low nutrient uptake.

In addition to ongoing autophagy, the T cells had enhanced mitochondrial function, which prompted the researchers to take a look at acetyl-coenzyme A (AcCoA). Mitochondrial AcCoA is involved in the citric acid cycle for oxidative phosphorylation, while the nucleocytosolic AcCoA plays a role in histone and non-histone protein acetylation. In the context of elevated extracellular K+, the total cellular AcCoA (a surrogate marker for mitochondrial AcCoA) was elevated, consistent with mitochondria-driven metabolism; however, the nucleocytosolic AcCoA and its precursor citrate were reduced.

Examining the impact of reduced levels of nucleocytosolic AcCoA revealed a crucial role in determining the phenotype and function of the T cell. Decreased nucleocytosolic AcCoA led to decreased histone acetylation and reduced the activation of T cell effector and exhaustion genes, including Ifng, Pdcd1 (PD-1), Cd244 (2B4), Havcr2 (Tim-3), and Klrg1. In contrast to reducing the expression of genes associated with effector functions, inhibition, and suppression of cytolysis, elevated extracellular K+-induced starvation response preserved the expression of genes associated with CD8+ T cell stemness, including the transcription factor Tcf7, the lymphoid homing marker CD62L, and the costimulatory marker CD27. Similar delay in the acquisition of effector functions was observed in CD4+ T cells. To demonstrate a causal link between nucleocytosolic AcCoA and these functional consequences, Vodnala and Eil et al. supplemented the media with AcCoA or its precursor acetate and observed a functional reversal of T cell autophagy, reduction in stem-like properties, and acquisition of effector functions even in the context of elevated K+ levels. In addition, they found that autophagy was required for retaining stemness in the context of elevated K+.

While high potassium levels limit the anticancer activity of T cells in the TME, inducing stemness properties in T cells ex vivo could enhance the effectiveness of T cell-based immunotherapies. In mice with established B16 melanoma, adoptively transferred pmel T cells (which respond to the gp100 antigen) that had been activated in elevated K+ conditions showed improved persistence in the tumor and secondary lymphoid organs; remained in a less differentiated, more multipotent state within the tumor and spleen; and significantly enhanced tumor regression and survival. Secondary transfer of tumor TILs from mice that had received elevated K+-conditioned T cells showed that the TILs had an increased capacity for recall response to antigen, demonstrating that the stem-like state was maintained. Nucleocytosolic AcCoA could be suppressed not only by elevated extracellular K+ levels but also by 2-hydroxycitrate, an inhibitor of a key enzyme involved in the conversion of citrate to AcCoA. Treatment of pmel T cells with 2-hydroxycitrate prior to adoptive transfer affected T cell function in a manner similar to extracellular K+ and led to increased tumor regression and survival in mice with established subcutaneous B16 tumors and pulmonary metastases. Thus, reduction in nucleocytosolic AcCoA, either by a dysregulatory TME or by specific manipulation, preserved T cell stemness.

To determine clinical relevance, the researchers examined neoantigen-specific, tumor-infiltrating T cells from patient tumors (including melanoma and cancers of the colon, lung, and ovary), and surprisingly discovered that exposure to elevated K+ levels led to an increase in the portion of cells expressing CD62L, despite previous chronic exposure to antigen.

Overall, Vodnala and Eil et al. elucidated a mechanism in which elevated extracellular potassium levels within the TME reduced the nutrient uptake by T cells, triggering a starvation response. This caloric restriction resulted in metabolic reprogramming: increased and ongoing autophagy, mitochondria-driven energy production, and depletion of nucleocytosolic AcCoA. Decreased nucleocytosolic AcCoA led to reduced histone acetylation and epigenetic reprogramming, which resulted in reduced acquisition of effector functions and preservation of T cell stemness. The results of this study suggest that ex vivo reprogramming of the metabolic state of T cells could improve the persistence and effectiveness of T cell-based immunotherapies.

by Anna Scherer

Meet the Researcher

We asked Suman Vodnala, one of the first authors of this study, to tell us a bit more about his research and about himself.

Suman Vodnala, with lead author Nicholas Restifo, celebrating the publication of their paper.


What prompted you to tackle this research question?
From our previous studies we observed that immune cells have differential expression of monovalent transporters and channels to maintain the electrochemical gradients of the cells. We observed that naïve “young”-like T cells have higher expression of these voltage-gated channels and are differentially regulated during their progressive differentiation. This prompted us to study the importance of these channels and the role of monovalent ion’s impact on immune cells.

What was the most surprising finding of this study for you?
The most surprising thing in this study is that a simple ion (K+) can regulate and preserve the essential functions of the cell through epigenetics.

Finally, outside of the lab, what was your recent most exciting “discovery”?
Outside the lab, I custom-design quadcopters and fly them during my leisure time.

References:

Vodnala S.K., Eil R., Kishton R.J., Sukumar M., Yamamoto T.N., Ha N.H., Lee P.H., Shin M., Patel S.J., Yu Z., Palmer D.C., Kruhlak M.J., Liu X., Locasale J.W., Huang J., Roychoudhuri R., Finkel T., Klebanoff C.A., Restifo N.P. T cell stemness and dysfunction in tumors are triggered by a common mechanism. Science. 2019 Mar 29.

In the Spotlight...

GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells

Having demonstrated that GPRC5D, normally found only in the immune-privileged tissue of hair follicles, is expressed on multiple myeloma (MM) cells, independent of BCMA (B cell maturation antigen) expression, Smith et al. utilized a human B cell-derived scFv phage display library and Jurkat Nur77 reporter T cells in vitro and MM xenografts in vivo to identify and optimize a GPRC5D-specific CAR construct. Anti-GPRC5D CAR T cells exhibited cytotoxic activity similar to anti-BCMA CAR T cells, eradicated MM even in case of BCMA antigen loss relapse, and promoted long-term survival in mice. No fur loss or other toxicities were observed in mice or monkeys.

Skin cancer precursor immunotherapy for squamous cell carcinoma prevention

In a prospective analysis of 86 patients with actinic keratosis (a precursor to squamous cell carcinoma [SCC]), a 4-day course of calcipotriol + 5-fluorouracil (5-FU) decreased the risk of developing SCC on face and scalp within 3 years of treatment compared with Vaseline + 5-fluorouracil. Mechanistically, calcipotriol induced the expression of thymic stromal lymphopoietin in keratinocytes and, together with 5-FU, led to a robust epidermal tissue-resident memory response consisting mostly of CD4+ T cells. No protection was observed against developing basal cell carcinoma, which was found to have a cold and immunosuppressive TME.

Neoantigen-directed immune escape in lung cancer evolution

Integrating genomic, transcriptomic, epigenomic, and pathologic data, Rosenthal et al. analyzed 258 regions from 88 untreated, early-stage NSCLC tumors. Immune infiltration varied significantly and was inversely correlated with subclonal mutation diversity both between and within patients. Neoantigens were silenced or eliminated under immune pressure via multiple mechanisms, including subclone loss, loss of heterozygosity in neoantigenic or antigen presentation genes, and hypermethylation (epigenetic silencing) of genes harboring neoantigenic mutations. Tumor capacity for immune evasion was prognostic.

Immune signature drives leukemia escape and relapse after hematopoietic cell transplantation

Analyzing the genome and gene expression profiles of acute myeloid leukemia (AML) samples taken from 40 patients at diagnosis and at relapse after allogeneic hematopoietic cell transplantation, Toffalori et al. found changes that mediate immune escape and prevent donor-derived T cells from recognizing AML. AML blasts downregulated costimulatory ligands and adhesion molecules (e.g. CD11A) and upregulated inhibitory molecules (e.g. PD-L1, B7-H3), while donor T cells upregulated PD-1. AML blasts also downregulated CIITA (regulator of HLA-II transcription) and the expression of HLA-II molecules.

STING-mediated disruption of calcium homeostasis chronically activates ER stress and primes T cell death

Wu et al. elucidate the mechanisms underlying T cell death in the context of gain-of-function STING mutation (STINGN153S/+). STINGN153S/+ mice had reduced numbers of T cells, which displayed elevated apoptosis and unfolded protein response (UPR) and died following CD3/CD28 stimulation ex vivo, a phenomenon associated with aberrant STING localization and ER calcium signaling. A “UPR motif” identified in the cGAMP-binding domain of STING was found to be essential for UPR induction and NFκB signaling following TCR activation, linking chronic STING activation, UPR, and calcium homeostasis in TCR stimulation-mediated T cell death.

Contributed by Alex Najibi

Deciphering CD4+ T cell specificity using novel MHC-TCR chimeric receptors

To improve identification of CD4+ T cell-specific epitopes, Kisielow et al. engineered TCR- T cell lines to express chimeric receptors (extracellular MHC-II domains linked with intracellular TCR signaling machinery; MCR) that were loaded with peptides from a peptide library. CD4+ T cell recognition of cognate peptide presented on MCR triggered a fluorescent-NFAT reporter in MCR cells. This system discovered novel viral and tumor neoantigen epitopes in mouse models of influenza and E0771 breast cancer with high sensitivity, and vaccination with an identified neoantigen restricted tumor growth. Human MCRs effectively detected CD4+ T cell epitopes in influenza.

Contributed by Alex Najibi

Tumor-Specific Delivery of Immune Checkpoint Inhibitors by Engineered AAV Vectors

Reul and Frisch et al. used a HER2-targeted adeno-associated viral (AAV) vector to deliver genes encoding immune checkpoint inhibitors to tumors. In HER2+ tumor-bearing mice, i.v. treatment with either HER2-AAV or untargeted AAV induced anti-PD-1 expression in the tumor, but HER2-AAV caused less accumulation of anti-PD-1 in the liver and serum. When combined with chemotherapy, HER2-AAV-anti-PD-1 marginally reduced tumor progression. The researchers also tested HER2-AAV delivery of genes for human nivolumab, which targeted tumors, but nivolumab was expressed at low levels compared to the murine anti-PD-1 construct.

REVIEW: Immunomodulatory Bonds of the Partnership between Dendritic Cells and T Cells

Bourque and Hawiger review DC subsets and the major pathways involved in cross-talk with T cells. Molecular markers, cytokines, and functions of conventional DCs (cDC1s and cDC2s) and plasmacytoid DCs are discussed, emphasizing unique, context-specific functions of each subset in driving T cell phenotypes (Th1, Th2, Th17, pTreg). Key pathways including CTLA-4-CD80/86, TGFβ, BTLA-HVEM, PD-1-PD-L1/L2, CD27-CD70, and IL-12 family of cytokines (IL-12, IL-23, IL-27) are highlighted for their bidirectional nature, in which both DCs and T cells influence the other to maintain homeostasis or to modulate tolerogenic or stimulatory immune responses.

Contributed by Alex Najibi

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.