NFκB–Pim-1–Eomesodermin axis is critical for PNAS PLUS maintaining CD8 T-cell memory quality Karin M. Knudsona,b,1, Curtis J. Pritzla,1, Vikas Saxenaa, Amnon Altmanc, Mark A. Danielsa, and Emma Teixeiroa,2 aDepartment of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO 65212; bLaboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20814; and cDivision of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121 Edited by Stephen P. Schoenberger, La Jolla Institute for Allergy and Immunology, La Jolla, CA, and accepted by Editorial Board Member Philippa Marrack January 13, 2017 (received for review May 26, 2016) T-cell memory is critical for long-term immunity. However, the factors TCR stimulation leads to induction of several signaling path- involved in maintaining the persistence, function, and phenotype of the ways key for T-cell activation. One of these is NFκB. NFκB memory pool are undefined. Eomesodermin (Eomes) is required for the signaling has been previously implicated in the maintenance of establishment of the memory pool. Here, we show that in T cells naïve T cells and generation of memory T cells (12, 13), although transitioning to memory, the expression of high levels of Eomes is not the molecular mechanisms regarding how NFκB regulates these constitutive but rather requires a continuum of cell-intrinsic NFκB signal- processes remain undefined. NFκB signaling is coupled to the ing. Failure to maintain NFκB signals after the peak of the response led TCR through PKCθ (14). PKCθ mediates the activation of the to impaired Eomes expression and a defect in the maintenance of CD8 IKK complex or IKKc (composed of IKKα and IKKβ and IKKγ/ T-cell memory. Strikingly, we found that antigen receptor [T-cell receptor NEMO), which phosphorylates IκBα, a negative regulator of (TCR)] signaling regulates this process through expression of the NFκB- NFκB signaling, and NFκB. Phosphorylated IκBα is ubiquiti- dependent kinase proviral integration site for Moloney murine leukemia nated and degraded by the proteosome. This phenomenon frees virus-1 (PIM-1), which in turn regulates NFκB and Eomes. T cells defective NFκB subunits to translocate to the nucleus and mediate tran- in TCR-dependent NFκB signaling were impaired in late expression of scription of different genes, including IκBα (15). Pim-1, Eomes, and CD8 memory. These defects were rescued when TCR- Here, we investigated how Eomes is regulated during infection dependent NFκB signaling was restored. We also found that NFκB–Pim-1 κ and at memory. We found that the NF B pathway regulates the INFLAMMATION signals were required at memory to maintain memory CD8 T-cell lon- expression of Eomes and is critical for long-term maintenance of IMMUNOLOGY AND gevity, effector function, and Eomes expression. Hence, an NFκB–Pim-1– memory T cells and their responses. Our data indicate that this Eomes axis regulates Eomes levels to maintain memory fitness. process is cell intrinsic and regulated by proviral integration site for Moloney murine leukemia virus-1 (PIM-1) and TCR signals. CD8 T-cell memory | NFkB | Pim-1 | Eomesodermin Results emory CD8 T cells provide long-term protection against Maintenance of Eomes Expression at Memory Is Not Regulated by Mintracellular pathogens and tumors. T-cell receptor (TCR), IL-15, IL-7, CD27, or OX40. Memory T cells express high levels of costimulatory, and inflammatory signals are required early dur- Eomes, but it is unclear whether this elevated expression is the ing infection for efficient memory CD8 T-cell differentiation (1). result of extrinsic or intrinsic cell mechanisms. To address this In addition, IL-7 and IL-15 signals support survival and self- issue, we investigated the role of survival and homeostatic renewal of the memory pool when antigen and inflammatory signals signals that could induce Eomes expression during the memory have ceased (2). Interestingly, the memory T-cell pool is hetero- phase. Self-peptide–MHC–TCR (self-pMHC) interaction is not geneous and contains distinct T-cell subsets [effector memory (TEM), central memory (TCM), stem cell memory (TSCM), and res- Significance ident memory (TRM)] that differ in phenotype, longevity, location, and recall capacity (3). It is currently unknown whether functional Mice and humans whose T cells are deficient in NFκB signaling differences in the memory pool are maintained by cell-intrinsic lack memory T cells, but the mechanism behind this is unclear. mechanisms or by local tissue environmental signals, as described We found that NFκB signaling is required during the resolution recently for TRM cells (4, 5). Likewise, whether the transcription phase of the immune response to maintain long-term CD8 factors and signaling pathways driving memory differentiation are memory. NFκB signaling is necessary for preserving expression also involved in the maintenance of memory quality remains un- of Eomesodermin and prosurvival Bcl-2 in memory T cells, in a explored. The T-box transcription factors T-bet and Eomesodermin cell-intrinsic process where T-cell receptor (TCR) signals and (Eomes) are major regulators of effector function and memory Pim-1 kinase are involved. Our study defines an unexpected programming. During the immune response, the Eomes/T-bet ratio role of NFκB and Pim-1 signaling in the maintenance of T-cell changes, gradually increasing as T cells transition to memory (6). memory quality. Furthermore, it identifies targets and specific Eomes is highly expressed in memory T cells and is considered times of intervention where protective T-cell memory could be crucial for the maintenance of TCM quality (7, 8). As such, failure to reinforced in vaccines and cancer immunotherapies by manip- express Eomes leads to poor development of TCM function and an ulation of the NFκB–Pim-1–Eomesodermin axis. inability of memory cells to survive, homeostatically proliferate, and reexpand upon rechallange (7, 9). Upon infection, high levels of Author contributions: K.M.K., C.J.P., M.A.D., and E.T. designed research; K.M.K., C.J.P., inflammation repress Eomes and increase T-bet expression in a and V.S. performed research; A.A. contributed new reagents/analytic tools; K.M.K., C.J.P., process that is dependent on the mechanistic target of rapamycin V.S., M.A.D., and E.T. analyzed data; and K.M.K., M.A.D., and E.T. wrote the paper. (mTOR) signaling pathway (10). On the other hand, we have de- The authors declare no conflict of interest. scribed that weak TCR signals induce strong expression of Eomes This article is a PNAS Direct Submission. S.P.S. is a Guest Editor invited by the Editorial and favor TCM differentiation (11). Together, these results suggest Board. that both TCR and inflammatory signals play an important role in 1K.M.K. and C.J.P. contributed equally to this work. regulating Eomes expression. However, how the TCR regulates 2To whom correspondence should be addressed. Email: [email protected]. Eomes to generate memory T cells and maintain the quality of the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. memory pool has not been addressed. 1073/pnas.1608448114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1608448114 PNAS Early Edition | 1of9 Downloaded by guest on October 1, 2021 required for memory CD8 T-cell maintenance (16); however, it is We also evaluated the role of IL-7 and IL-15 in the induction unclear whether tonic signals via the TCR are required for Eomes of Eomes expression because both are key regulators of memory expression at memory. Thus, we adoptively transferred memory T survival and homeostasis (2). Neither blockade (24, 25) nor ad- cells into MHC class I sufficient or deficient lymphopenic hosts dition of exogenous IL-7 and IL-15 to memory cells (26, 27) (17) and determined Eomes levels. At 28 d posttransfer, we ob- changed Eomes levels (Fig. S1C). Unexpectedly, these results served no change in Eomes expression regardless of the presence indicate that the maintenance of Eomes expression in resting of self-pMHC (Fig. S1A). memory T cells is not dependent on self-pMHC or TNFR or Costimulatory molecules of the TNFR family can regulate homeostatic cytokines (9). memory generation and secondary responses (18–20). One of them, NFκB Signaling Is Necessary for Maintaining High Eomes and Bcl-2 CD27, has been previously linked to Eomes expression (21). Expression in Memory CD8 T Cells. mTOR signaling regulates both However, we observed that neither activation of CD27 nor OX40 Eomes and T-bet expression upon infection (10). Thus, we tested signaling with agonist antibodies (22, 23) altered Eomes expression whether this pathway was involved in maintenance of Eomes levels in memory T cells (Fig. S1B). In addition, we were unable to detect in memory T cells. For this testing, we used rapamycin, a chemical expression of other TNFR family members previously associated inhibitor of mTOR signaling, because memory T cells have a slow with memory quality, such as 4-1BB and TRAIL, in memory cells cell division rate and are not amenable to retroviral transduction. (reviewed in ref. 20), suggesting they are most likely not involved in mTOR activity was higher in memory cells than in naïve T cells the regulation of Eomes once a T cell is at memory (Fig. S2). (Fig. S3A). Nonetheless, treatment of memory CD8 T cells with Fig. 1. NFκB signaling is required at memory to maintain Eomes and Bcl-2 expression. (A and B) The 2 × 105 naive OT-I cells were transferred into congenic hosts and challenged with 1 × 104 cfu LM-OVA. At ≥28 d postinfection (p.i.) memory cells were harvested and treated with 0.1 ng/mL IL-7 ± 40–100 μMNFκB inhibitor (NFκBi).