NFκB–Pim-1–Eomesodermin axis is critical for PNAS PLUS maintaining CD8 T-cell 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 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 [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 , 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 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 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). Phosphorylation of p65-NFκB (S536), Eomes expression, and cell viability (A) and IL-7R, CD122, and Bcl-2 expression (B) were determined by flow cytometry after 24 h. Graphs show either fold induction over an isotype control or geo mean fluorescence intensity (gMFI) of OT-I memory population minus the respective isotype control gMFI. (C–G) OVA-stimulated OT-I T cells were transduced with Eomes RV or empty vector (EV) RV and 1 × 106 GFP+ OT-I were transferred into naïve hosts and hosts challenged with 7 × 106 cfu Att-LM-OVA. (C) Frequency of Bcl-2hiEomeshi OT-I GFP+ cells and (D) Eomes and Bcl-2 gMFI at day 25 p.i. (E) At day 42 p.i., memory T cells were harvested and incubated for 24 h ± 50 μMNFκBi. Graph shows frequency of Bcl-2hiEomeshi OT-I GFP+ + cells after treatment. (F) Density plots and graphs show frequency of OT-I GFP cells with differential expression of IL-7R and Eomes at day 45 p.i. (G) Number of RV-Eomes or -EV transduced cells in the lymph nodes 25 d p.i. All graphs show mean ± SD. All data are representative of n ≥ 2 independent experiments, n = 3–6 mice per group. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.001, ****P ≤ 0.0001.

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1608448114 Knudson et al. Downloaded by guest on October 1, 2021 rapamycin did not alter Eomes or T-bet expression. This finding was levels of Bcl-2 (Fig. 1E). Forced expression of Eomes also resulted PNAS PLUS in stark contrast to what happened to effector T cells treated in in an increased frequency of memory cells expressing high levels of parallel (Fig. S3B) (10). These data suggest that, whereas rapamycin- IL-7R (Fig. 1F). Furthermore, overexpression of Eomes enhanced sensitive mTORC1 regulates Eomes expression to program the the generation of memory T cells (Fig. 1G). Because NFκBsig- generation of memory T cells (28), it does not contribute to support naling regulates Eomes and both Eomes and NFκB control Bcl-2 Eomes expression at memory. Interestingly, rapamycin did not in- expression, these data show that NFκB can regulate memory hibit the phosphorylation of mTOR in memory cells (Fig. S3B). survival signals in an Eomes-dependent manner. NFκBhasbeenshowntobindtotheEomespromoterininvitro- We also tested whether Eomes regulates NFκB activity. We generated effector T cells (29), but no studies have addressed found that T cells overexpressing Eomes strongly induce the tran- whether this signaling pathway regulates Eomes during an immune scriptional activity of NFκB compared with their control [empty response or at memory. We observed that NFκB activity was pre- vector (EV)] counterparts as shown by the NFκB reporter luciferase sent in resting memory T cells as shown by the levels of phos- signal detected on Eomes transduced cells (Fig. S4). Thus, both phorylated NFκBatSer-536(Fig.1A) (30, 31). When we treated Eomes and NFκB reciprocally regulate each other in T cells. memory cells with a well-characterized chemical IKKβ/NFκBin- hibitor (NFκBi) (32), we found both a decreased level of phospho- NFκB Signaling Controls Eomes in Activated CD8 T Cells. T-bet and NFκB and a severe loss of Eomes expression in memory T cells. Eomes work together to regulate CD8 T-cell memory (10). Thus, Importantly, this finding was not due to overt toxicity, as cell via- we examined whether NFκB signals were required to regulate bility was similar between control (vehicle treated) and NFκBi- Eomes and T-bet expression in activated T cells. To address this treated memory cells (Fig. 1A). These results indicate that NFκB question, we altered NFκB signaling using gain and loss-of- signals are necessary to maintain Eomes expression in memory cells. function approaches in proliferating T cells. First, we transduced Next, we investigated whether NFκB signaling was involved in CD8 T cells with a construct that encodes constitutive active the regulation of molecules associated with memory survival, an IKKβ (CA-IKKβ) to enhance NFκB signaling (22). CA-IKKβ + aspect of memory quality that has been linked to the level of GFP -transduced cells exhibited lower levels of IκBα (as a Eomes expression (2, 9). Treatment of memory CD8 T cells with consequence of increased proteosomal degradation) than their the NFκB inhibitor did not affect expression of the receptors for EV-transduced counterparts, confirming constitutive NFκB sig- IL-7 or IL-15, discarding a role for NFκB in regulating the input naling (33). Importantly, enhanced IKKβ activity increased of homeostatic signals associated with memory survival and ho- Eomes levels and the percentage of T cells expressing Eomes. By INFLAMMATION

meostasis (Fig. 1B). contrast, T-bet expression was not altered in cells expressing IMMUNOLOGY AND The expression of the antiapoptotic molecule Bcl-2, however, CA-IKKβ (Fig. 2 A–C). was significantly reduced in NFκBi-treated cells (Fig. 1B). Inter- Then, we investigated the direct role of NFκB in the regula- estingly, Eomes-deficient T cells are also impaired in Bcl-2 ex- tion of Eomes and T-bet by overexpressing a dominant negative pression (9). Consistent with this fact, overexpression of Eomes (DN) truncated form of p65-NFκB [DN-p65(trunc)] that selec- led to an increase in the Bcl-2 levels of T cells that were differ- tively inhibits p65-dependent transactivation (34). As expected, entiating to memory (Fig. 1 C and D). We then, addressed we observed lower levels of IκBα (a NFκB target) in DN-p65 + whether NFκB signaling regulated Bcl-2 expression independently (trunc)-GFP transduced T cells (35), indicating NFκB activity of Eomes. We found that memory T cells that overexpressed was effectively inhibited. Transduction of activated T cells with Eomes were refractory to NFκB inhibition and maintained high DN-p65(trunc) also led to a reduction in Eomes expression and a

Fig. 2. NFκB signaling regulates Eomes expression in activated CD8 T cells. OVA-stimulated OT-I T cells were transduced with CA-IKKβ RV (A–C), DN-p65 + + (trunc) RV (D–F), or the respective EV RV. Eomes, T-bet, and IκBα expression were determined by flow cytometry at day 1–2 posttransduction in OT-I GFP cells + expressing similar levels of GFP. Numbers in histograms are geo mean fluorescence intensity (gMFI) (A and D). Graphs show percentage of GFP cells with Eomes, T-bet, or IκBα expression over isotype level (B and E). All graphs show mean ± SD. (C and F) Plots show expression of Eomes, T-bet, and IκBα versus GFP + + level. Arrows show trend of OT-I GFP population. Data are representative of n ≥ 3 independent experiments. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.001.

Knudson et al. PNAS Early Edition | 3of9 Downloaded by guest on October 1, 2021 whereas EV-transduced T cells persisted and generated a mem- ory pool, DN-p65(trunc)-transduced cell frequencies started to decay 2 d posttransfer and were barely detectable at the memory phase (Fig. 3 C and D). This finding was not due to rejection of + donor cells, as the ratio of day 8 to day 100 of EV-GFP OT-I − cells (5.53:1) was similar to the ratio of OT-I GFP cells (5.24:1), + but both were different from the ratio of DN-p65(trunc)–GFP OT-I cells (78.97:1). Importantly, loss of memory cells correlated with a defect in Eomes expression in those cells transduced with DN-p65(trunc) (Fig. 3E). These experiments do not discard a role of NFκB signaling in the generation of effector and memory T cells. Rather, the data presented here indicate that a failure in sustaining NFκB sig- naling after priming, results in impairment in maintaining the survival of CD8 T cells into memory. This conclusion is supported by the fact that control cells reached a plateau after day 30, whereas DN-p65–expressing CD8 T-cell numbers continue de- clining over time. Thus, altogether the data show that NFκB signaling regulates Eomes expression and maintenance of CD8 T-cell memory.

TCR-Dependent NFκB Signaling Is Required for Eomes Expression. TNFR family members can induce NFκB signaling (22, 38). However, TNFR CD27, OX40, TRAIL, and 41BB (which have been linked to memory) were either not expressed at the con- traction phase or did not have a role in regulating Eomes (Figs. S1 and S2). Our published data, on the other hand, suggest that TCR- Fig. 3. NFκB signaling regulates the maintenance of CD8 T-cell memory. (A–E) dependent NFκB signals mediated by PKCθ are required for CD8 The 2 × 105 OT-I T cells were transferred into congenic hosts and challenged T-cell memory (31, 39). Thus, we hypothesized that TCR-dependent with 7 × 106 cfu Att-LM-OVA. Four days p.i., OT-I T cells were isolated and NFκB signaling had a role in the regulation of Eomes. To test this 5 transduced with EV- or DN-p65(trunc) RV with 10 ng/mL IL-7. 1 × 10 EV hypothesis, we transduced T cells with a retroviral vector (RV) + − × 5 + + + + (CD45.2 CD90.1 )and1 10 DN-p65(trunc) (CD45.2 CD90.1 )OT-I GFP cells encodingakinasedeadmutantformofPKCθ (DN-PKCθ)(40), were cotransferred (B) into infection-matched hosts challenged with WT LM + − + which is one of the TCR-proximal intermediates connecting the (CD45.1 CD90.1 ). (C) Kinetics of EV- and p65(trunc)-GFP OT-I T cells were + + κ determined after transfer. Percentage of GFP OT-I cells in the total OT-I CD8 TCR to the IKKc/NF B signalosome (14). The frequency of cells + θ– T-cell pool is shown. Days represent postinfection. (D) Number of GFP OT-I T expressing Eomes, but not T-bet, was diminished in DN-PKC + + + cells at day 100. (E) Expression of Eomes on day 8 in OT-I GFP cells was de- GFP transduced T cells (Fig. 4A), suggesting that PKCθ can termined by flow cytometry. Graph shows fold induction over an isotype regulate Eomes expression in CD8 T cells. control. All graphs show mean ± SD. Data are representative of three inde- We also used an OT-I TCR mutant model impaired in triggering pendent experiments; n = 3–4mice.*P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.001. NFκB signals to unequivocally test the role of TCR-dependent NFκB signaling in Eomes expression. In this model, T cells carrying a point mutation in the TCRβ transmembane domain (βTMDmut significant loss in the frequency of Eomes expressors. Further- or MUT) are exclusively defective in the transduction of NFκB more, there was a direct correlation between GFP expression signals and, importantly, in the generation of memory cells upon κ α – and the levels of Eomes and I B (Fig. 2 D F). Similar to the infection (39). We adoptively transferred WT or βTMDmut OT-I β results obtained with the CA-IKK construct, no change in T-bet naïve T cells into congenic hosts and monitored Eomes and T-bet κ levels occurred when NF B activity was inhibited. These data expression after infection with LM-OVA. We found no differences κ demonstrate that NF B signaling regulates Eomes expression in in T-bet expression between WT and MUT CD8 T cells. However, both activated and memory CD8 T cells. and in agreement with our hypothesis, Eomes expression was im- paired in βTMDmut CD8 T cells. Of note, MUT cells were unable NFκB Signaling Is Required After the Peak of the Immune Response to κ to induce Eomes over naïve levels as they matured to memory, a Maintain CD8 T-Cell Memory. Next, we determined whether NF B feature that has been associated with the competence of T cells to signaling regulates Eomes expression and T-cell survival upon remain in the memory niche (9) (Fig. 4B). Conversely, in conditions κ infection. The role of NF B signaling in the early steps of T-cell where βTMDmut T cells regain NFκB signaling and memory de- activation and proliferation of naïve T cells is well established velopment (weak TCR signal strength provided by infection with κ (36). We reasoned that inhibiting NF B signaling too early in LM-Q4H7) (31), Eomes expression was recovered. As with LM- the response would lead to defects in the activation and pro- OVA challenge, there was no difference in T-bet expression (Fig. liferation of differentiating T cells. These defects would preclude 4C). Hence, these data indicate that TCR-dependent NFκBsig- κ the ability to discern whether NF B has a role later in the re- naling is required for sustained expression of Eomes in T cells that sponse after T-cell priming (37). To circumvent this issue, OT-I transition to memory. T cells were primed in vivo upon Listeria monocytogenes expressing ovalbumin (LM-OVA) infection. We harvested OT-I NFκB and Pim-1 Regulate Each Other in CD8 T Cells. The data in Figs. CD8 T cells 4 d postinfection and transduced them with either 1 and 3 suggest that NFκB signals are required after the peak of DN-p65(trunc) or EV control without additional TCR stimula- the response for persistent Eomes expression and survival of + tion. Then, equal numbers of EV-GFP or DN-p65(trunc)– CD8 T cells in the memory pool. Antigenic and inflammatory + GFP cells were cotransferred into WT-LM (not expressing signals quickly decline after the peak of the response (41) and OVA) infection-matched hosts (Fig. 3 A and B). We observed other extracellular signals such as CD27, IL-7, or IL-15 did not + that both EV- and DN-p65(trunc)–GFP transduced OT-I T affect Eomes expression (Fig. S1). Thus, we hypothesized that cells underwent contraction after adoptive transfer. However, the persistence of NFκB signaling after the peak of the response

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Fig. 4. TCR-dependent NFκB signaling is required for expression of Eomes. (A) OVA-stimulated OT-I cells were transduced with DN-PKCθ RV or EV RV. Eomes + + + and T-bet expression were determined in OT-I GFP cells by flow cytometry at day 1 posttransduction. Graphs show percentage of GFP cells with Eomes or T-bet expression over isotype level. (B and C) The 1 × 103 WT or MUT OT-I T cells were transferred into congenic hosts and challenged with 1 × 103 cfu LM-OVA (B) or LM-Q4H7 (C). Kinetics of Eomes and T-bet expression were determined in the blood or spleen by flow cytometry. Graphs show geo mean fluorescence intensity (gMFI). Dashed line indicates the level of Eomes or T-bet for naïve T cells. All graphs show mean ± SD and all data are representative of two or more independent experiments; n ≥ 3 mice per group. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.001.

and into memory is programmed by cell-intrinsic mechanisms. T cells was similar to WT levels (Fig. 5E) (31). WT and MUT cells Pim-1, a Ser/Thr kinase, is constitutively active once expressed displayed similar levels of Pim-2, suggesting that only Pim-1 is a and has been shown to increase p65-mediated NFκBtrans- target of TCR-dependent NFκB regulation (Fig. S5). INFLAMMATION

activation (42, 43). Most relevant to our studies, Pim-1 is Upon infection, Pim-1 levels in differentiating CD8 T cells at IMMUNOLOGY AND expressed upon TCR stimulation. It is important for T-cell sur- the peak of the LM-OVA response were similar to naïve cells and vival and highly expressed in memory T cells (32, 44, 45). Thus, gradually increased as they developed into memory. This finding we tested whether Pim-1 was involved in maintaining NFκB resembled the profile of Eomes expression at the end of the im- signaling in CD8 T cells. Upon CD3/CD28 stimulation, Pim-1 mune response (Figs. 4B and 5F). MUT T cells, however, failed to slowly accumulated with time (Fig. 5A). In line with the idea that increase Pim-1 expression as they entered the memory phase upon Pim-1 maintains NFκB signals, we observed substantial late LM-OVA but not LM-Q4H7 infection (Fig. 5F). This finding phosphorylation of p65-NFκB [p-p65(S536)] at times where closely matched the defect and recovery of Eomes expression and TCR expression and therefore antigen input is low (39), but Pim-1 memory development in MUT cells observed, depending on TCR expression was maximal (Fig. 5A). In contrast, in CD8 T cells signal strength (Fig. 4 B and C). These results indicate that TCR with inhibited Pim-1 kinase activity, the phosphorylation of p65- signals regulate the increase of Pim-1 levels at the end of the NFκB was ∼50% reduced at all time points. Furthermore, in primary immune response. Furthermore, because MUT cells are control cells, IκBα expression returned to basal levels 3 h post- specifically defective in triggering TCR-dependent NFκB signaling stimulation, whereas in the presence of the Pim-1K inhibitor, (39), these data suggest that antigenic signals use the NFκB IκBα levels continued to decrease due to a reduced input in the pathway to regulate Pim-1 expression in CD8 T cells. NFκB feedback loop that regulates IκBα expression (Fig. 5A) (35). These results were confirmed in T cells retrovirally trans- Pim-1 Kinase Activity Supports Eomes Expression and CD8 T-Cell Memory duced with a vector that encodes a kinase dead form of Pim-1 Fitness. So far, our data show that sustained NFκBsignalsafterthe (DN-Pim-1) (Fig. 5B) (43). Together, these data suggest that peak of the response are important for Eomes expression and Pim-1 regulates the amplitude of NFκB signaling in part by memory quality. They also show that Pim-1 is an NFκB target ca- regulating the expression of IκBα in CD8 T cells. pable of regulating NFκB signals. Thus, we reasoned that if Pim-1 Constitutive activation of NFκB signaling up-regulated Pim-1 supports NFκB, it would have a role in regulating Eomes expression expression, indicating that NFκB signaling also modulates Pim-1 and memory persistence. To test this hypothesis, equal numbers of expression in CD8 T cells (Fig. 5C). This finding agrees with OT-I T cells transduced with DN-Pim-1 or EV (control) were other studies showing that NFκB signaling regulates Pim-1 ex- cotransferred into naïve hosts followed by LM-OVA infection (Fig. pression in B cells (46). Overall, these data show that NFκB 6 A and B). Although T cells containing DN-Pim-1 expanded and Pim-1 regulate each other in CD8 T cells and may function similarly to control T cells, impaired Pim-1 kinase activity gradually to preserve a continuum of NFκB signaling when antigenic/ led to a loss of OT-I responders after the peak of the response (Fig. 6B). This loss was especially evident for KLRG1loIL-7Rhi memory inflammatory signals are low or not present. + precursors (Fig. 6C). As a result, the number of DN-Pim-1–GFP TCR-Dependent NFκB Signaling Regulates Pim-1 Expression. We then memory T cells was severely reduced compared with the number of assessed whether TCR-dependent NFκB signaling was involved in control memory T cells (Fig. 6D and Fig. S6). The defect in memory the regulation of Pim-1. T cells, retrovirally transduced to over- generation was especially marked for memory cells with a TCM express a dead kinase form of PKCθ (DN-PKCθ), were indeed phenotype (Fig. 6 D and E). Importantly, DN-Pim-1–transduced impaired in Pim-1 expression (Fig. 5D). Most importantly, OT-I T cells also exhibited impaired Eomes expression throughout βTMDmut T cells defective in TCR-dependent NFκB signaling the immune response (Fig. 6F). and memory development (39) exhibited lower levels of Pim-1 Pim-1 kinase defective T cells were unable to maintain NFκB than their WT counterparts from the peak of the response into signaling in vivo, as indicated by their inability to induce the memory. Conversely, when TCR-dependent NFκB signaling was expression of the NFκB target IκBα (Fig. 6G). This finding is restored (weak TCR stimulation), Pim-1 expression in βTMDmut consistent with the idea that Pim-1 regulates NFκB late in the

Knudson et al. PNAS Early Edition | 5of9 Downloaded by guest on October 1, 2021 primary immune response. Indeed, at memory, NFκB inhibition (12, 13, 47). Our results confirmed this role and show how NFκB led to a marked defect in Pim-1 expression (Fig. 6H) and, vice versa, signaling regulates the generation of memory T cells in the Pim-1 inhibition resulted in memory T cells exhibiting lower levels context of infection. From our data, we conclude that NFκB of p-NFκB and Eomes expression (Fig. 6I)(9).Tcellswithim- signaling is required after the peak of the response to support paired Pim-1 kinase activity showed a defect in the expression of memory. Because we deliberately impaired NFκB signals after prosurvival factors Bcl-2 and Bcl-xL after day 20 postinfection (Fig. T-cell priming, the loss of memory cells most likely indicates 6J). This finding indicates that Pim-1, similar to NFκB(Figs.1B and that NFκB signals are involved in preserving the survival of T 3),playsaroleinthesurvivalofTcells differentiating into memory. cells that enter the memory pool. Because NFκB regulates + We next evaluated the ability of DN-Pim-1–GFP memory T cells Eomes and Eomes in turn controls Bcl-2 expression, we con- to persist and to respond to reinfection. Memory T cells with de- clude that memory survival is in part due to NFκB-dependent ficient Pim-1 activity showed a much faster decay than their EV- regulation of Bcl-2 expression through Eomes and Pim-1. Im- + GFP control counterparts (Fig. 6K) and were remarkably impaired portantly, our results also indicate that NFκB–Pim-1 activity in their reexpansion and expression of IFN-γ and granzyme B contributes to maintaining high Eomes levels in resting memory upon reinfection (Fig. 6 L and M). We obtained similar results in cells and is required for reexpansion and rapid memory func- analogous experiments performed with memory T cells treated tion upon antigen reencounter. This idea is in agreement with with NFκB inhibitor for 24 h (Fig. S7). Collectively, these data the phenotype of Eomes-deficient CD8 T cells (7, 9). Together, indicate that NFκB and Pim-1 modulate each other in an immune our data strongly suggest that NFκBsignalsplayacritical response and at memory and together contribute to maintain role in maintaining the longevity and the function of memory memory fitness through the regulation of Eomes expression Tcells. (Fig. S8). Signaling pathways mTOR and Wnt/TCF-1 have been shown to regulate Eomes expression and CD8 T-cell memory devel- Discussion opment in a cytokine-dependent manner (10, 48). Unexpectedly, Several reports have previously linked different members of the the data presented here show that TCR signaling uses the NFκB canonical and noncanonical NFκB pathway with T-cell memory pathway to program Eomes expression and CD8 T-cell memory

Fig. 5. TCR-dependent NFκB signaling supports late Pim-1 expression. (A) Naïve OT-I T cells were pretreated with 10 μM Pim-1 kinase inhibitor (Pim-1Ki) or + vehicle (DMSO) for 30 min and stimulated with OVA-tetramer αCD28. Levels of shown were determined by immunoblot. Densitometry intensity shown is relative to unstimulated cells and was corrected for loading. Graphs show fold induction over vehicle nonstimulated (t = 0) cells after normalization to loading control. (B) OVA-stimulated OT-I cells were transduced with DN-Pim-1 or EV RV and restimulated with PMA + ionomycin for the indicated times. Expression of IκBα in OT-I+GFP+ T cells was determined by flow cytometry. (C) Stimulated OT-I T cells were transduced with CA-IKKβ or EV RV. Pim-1 expression + + was determined in OT-I GFP cells by flow cytometry at day 2 posttransduction. Graph shows geo mean fluorescence intensity (gMFI) fold induction over + + isotype level. (D) OVA-stimulated OT-I T cells were transduced with DN-PKCθ RV or control RV. Pim-1 expression was determined in OT-I GFP cells by flow + cytometry at day 2 posttransduction. Graph shows percentage of GFP OT-I cells with Pim-1 over isotype level. (E) Naïve OT-I and MUT CD8 T cells were + stimulated with OVA- or Q4H7-tetramer αCD28. levels shown were determined by immunoblot. Densitometry intensity of Pim-1 relative to unsti- mulated cells was corrected for loading and is shown below the panel. (F) The 5 × 103 (or 104 for LM-Q4H7) OT-I and MUT T cells were transferred into congenic hosts and challenged with 7 × 106 cfu Att-LM-OVA (or 104 cfu LM-Q4H7). Pim-1 expression kinetics (shown as fold induction over a naïve control) were determined in the blood by flow cytometry. All graphs show mean ± SD. Dashed line indicates the level of Pim-1 for naïve T cells. Data are representative of two to three independent experiments; n = 4 mice per group. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.001.

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.1608448114 Knudson et al. Downloaded by guest on October 1, 2021 fitness. It is possible that mTOR, Wnt, and NFκB pathways co- cells via CD27 in an mTOR-independent manner (44), and PNAS PLUS operate, such that a change in the activity of one of them affects CD27–CD70 signals have a role late in the primary response in the Eomes expression. Alternatively, it may be that each of these programming of CD8 T-cell memory (19). However, and without signaling pathways regulates Eomes levels at different times in discarding the role of Pim-1 in survival upon CD27 signaling, our the CD8 T-cell differentiation process. For example, we found data strongly suggest that, at least upon Listeria infection, Pim-1 that rapamycin-sensitive mTOR signaling controls Eomes ex- regulates Eomes in a CD27-independent manner. pression in activated CD8 T cells but not at memory. In contrast, Pim-1 expression can also be regulated by STAT-3 and STAT-5 NFκB signaling regulates Eomes expression in both activated signaling, which would suggest a role for cytokine signaling in and memory CD8 T cells. How these pathways cooperate to the expression of Pim-1 and its downstream target Eomes (49). regulate T-cell memory is an interesting area of study that war- However, we were unable to observe any Eomes dependence on rants further investigation. IL-7 or IL-15 signaling (STAT-5 mediated) at memory. Fur- Our study proposes that the reciprocal regulation of Pim-1 and thermore, MUT cells, which are impaired in Pim-1 and Eomes NFκB maintains the level of NFκB signaling required to enable expression, exhibit normal levels of STAT-5 and STAT-4 activa- Eomes-dependent survival of CD8 T cells as they progress to tion (STAT-4 phosphorylation is enhanced in STAT-3–deficient memory and at memory. This is supported by the fact that inhi- T cells; ref. 50) (31). Rather, the data from the memory-defective bition of either NFκB or Pim-1 leads to defects in the expression βTMDmut model, where TCR-dependent NFκB signaling is im- of the other during the late phase of the primary immune response paired together with late expression of Pim-1 and Eomes, suggest and at memory. What controls the activity of Pim-1 and NFκBlate that TCR signals (although maybe not exclusively) are involved in in the immune response? Pim-1 is involved in the survival of T regulating Pim-1 expression late (after the peak of the response INFLAMMATION IMMUNOLOGY AND

Fig. 6. Pim-1 supports Eomes expression and CD8 T-cell memory fitness. OVA-stimulated OT-I T cells were transduced with DN-Pim-1 or EV RV. The 106 + + congenic EV-GFP and DN-Pim-1–GFP OT-I were transferred into the same (A, B, D, F, and G) or independent (C, E, and J–M) naïve hosts. All hosts were challenged with 7 × 106 cfu Att-LM-OVA. (B) Kinetics of OT-I+GFP+ T cells p.i. were determined in the blood (frequency of total live cells). (C) Kinetics of the lo hi hi lo + + hi hi frequencies of MPECs (KLRG1 IL-7R ) or SLECs (KLRG1 IL-7R )GFP OT-I were determined p.i. (D) Numbers of GFP OT-I T cells and TCM (CD44 CD62L )at + memory. (E) Frequencies of GFP OT-I T cells with a TCM (Left)oraTEM (Right) memory phenotype at memory. (F) Eomes level (fold induction over isotype + + + + control) of OT-I GFP cells in the blood. Dashed line indicates the level of Eomes for naïve T cells. (G) Expression of IκBα in OT-I GFP at day 16 p.i. Graph + shows percentage of GFP OT-I cells expressing IκBα over isotype. (H and I) Memory cells were obtained as in Fig. 1 and treated with 10 ng/mL IL-7 ± 100 μM NFκBi (H)or± 40 μM Pim-1Ki (I). Pim-1 (H) or phospho-NFκB and Eomes (I) levels were determined after 24–48 h. Histograms show geo mean fluorescence intensity (gMFI). All graphs show mean ± SD. (J) Kinetics of expression of Bcl-2, Bcl-xL, Bim, and phospho-Bad are shown in graphs. (K–M) The 2.5 × 105 (K)or 2.5 × 104 (L and M) DN-Pim1 and EV GFP+ OT-I memory T cells were transferred in equal numbers into independent congenic hosts and their frequencies (percentage of donor OT-I T cells (KbOVAtet+CD8+CD45.2+) that are GFP+) were determined over time (K) or upon infection with 2 × 106 pfu VSV-OVA (L). + (M) Graph shows frequencies of OT-I T cells that are GFP that express both granzyme B and IFN-γ on day 4 post–LM-OVA (5 × 105 cfu) infection after ex vivo restimulation with OVA or VSV peptides. All graphs show mean ± SD. Data are representative of two to three independent experiments; n = 3–10 mice. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.001. NS, nonsignificant. (K) Curves were analyzed by nonlinear regression and compared via F test to determine significance.

Knudson et al. PNAS Early Edition | 7of9 Downloaded by guest on October 1, 2021 and at memory) in an NFκB-dependent manner. It is unclear how Bacteria and Viral Infections. Listeria strains were generously provided by NFκB regulates Pim-1 expression. This regulation may happen at M. Bevan, University of Washington, Seattle (LM-OVA and LM-Q4H7) and Sing a biochemical level. Gradual increase of Pim-1 in the memory Sing Way, University of Minnesota, Minneapolis (Att-LM-OVA). Att-LM-OVA strains were grown to an OD600 of 0.1 as in ref. 39, whereas LM-OVA and LM- phase could be related to induced transcription, reduced deg- Q4H7 strains were grown as described in ref. 11. All infections were performed radation, or posttranscriptional mRNA stability (51). Future re- i.v. at least 1 d after adoptive transfer of naïve transgenic T cells. search beyond the scope of this study will be necessary to address OVA-expressing vesicular stomatitis virus (VSV) was provided by L. Lefrançois, these possibilities. University of Connecticut Health Center, Farmington, CT and used as described Eomes expression appears to have a higher impact on TCM previously (53). development, homeostasis, and function than on any other T-cell memory subset (9). Curiously, the absence of NFκB and Pim-1 Ex Vivo T-Cell Function. T cells at different times of the secondary immune signaling has a more profound effect on memory maintenance response were harvested from spleens of host mice and stimulated with cognate (OVA) or control (VSV) null peptides in the presence of Brefeldin A than what would be anticipated by only the specific loss of TCM. (BD Biosciences) as described previously (11, 54). Responding T cells were Thus, it is likely that Eomes has a distinct role beyond the peak identified with a gating strategy that included Kb-OVA tetramers, CD8, and of the response to enable general memory fitness. Such a role is congenic markers. supported by the dramatic loss of memory cells observed after day 45 post-LCMV infection in Eomes-deficient mice (52) and Listeria Titer Assays. Listeria numbers in the spleens of infected mice were by the fact that T cells appear to become Eomes “addicted” for determined as described previously (31). their survival once at memory (7). This role of Eomes at memory does not exclude an additional role of Eomes in programming Retroviral Transduction. Retroviral particles were generated using the Plati- num-E Retroviral Packaging Cell Line (Cell Biolabs) and Genejammer (Agilent TCM development. This role could be related to Pim-1, as both Technologies) according to the manufacturer’s instructions. Spin trans- deficiency in Eomes and Pim-1 activity does not affect the gen- duction was performed two ways. For transduction of in vitro-activated CD8 eration of memory precursor effector cells (MPECs) [or skew the T cells, 24 h after stimulation with 20 nM OVA, OT-I T cells were plated in 96- differentiation toward short lived effector cells (SLEC)] but well plates (5 × 105 cells per well). A total of 100–200 μL viral supernatant rather results in a reduction in the frequency of memory pre- with 8 μg/mL Polybrene (Millipore) and IL-2 was added, and plates were spun × cursors beyond the peak of the response. Interestingly, both at 575 g for 75 min at 32 °C. After a 4-h incubation at 32 °C, cells were Eomes and Pim-1 kinase deficiencies have a more pronounced resuspended in media with 20 nM peptide and IL-2 and incubated at 37 °C overnight. The second spin transduction was performed 24 h later and cells effect in cells of TCM phenotype (9). Nonetheless, our findings were resuspended in IL-2–containing media. For transduction of in vivo- show that maintaining NFκB signals and Pim-1 expression is primed CD8 T cells, OT-I T cells were harvested 4–5 d after challenge. OT-I T critical for supporting T-cell memory quality (maintenance and cells were plated in 96-well plates (5 × 105 per well). Two spin transductions recall potential). These data indicate that, in addition to the local were performed by spinning plates at 1,800 rpm for 75 min at 32 °C after tissue environment signals, cell-intrinsic mechanisms are crucial adding 100–200 μL viral supernatant with 8 μg/mL Polybrene (Invitrogen) to determine the quality of memory T cells. and 10 ng/mL IL-7. After a 4-h incubation at 32 °C, cells were resuspended in media with 10 ng/mL IL-7 and incubated at 37 °C overnight. In both condi- In conclusion, the data presented here show that TCR/antigenic tions, flow cytometry was used to calculate transduction efficiency to ensure κ + signals regulate the induction of a NF B/Pim-1 feedback loop that that equal number of congenically marked OT-I GFP cells transduced with controls expression of Eomes and thereby long-term maintenance the retroviral construct or empty vector were adoptively transferred 24 h of the memory pool. Our study identifies two molecular targets after the last transduction. that can be of aid in the design of better vaccine strategies and For retroviral transduction of Jurkat T cells, the Platinum GP Packaging cell tumor immunotherapies. line was used, and Eomes was cotransduced together with a construct that encodes VSV-G (55). Materials and Methods − − NFκB Activity Luciferase Assay. Retroviral particles containing Eomes or EV RV Mice and Reagents. C57BL/6, C57BL/6 Rag / , B6.SJL, OT-I, and βTMDmut − − − − control and VSV-G were produced using Plat-GP cells (Cell Biolabs) and (MUT) TCR transgenic mice (39), and B6Rag / β2m / were bred and main- Lipofectamine 3000. Jurkat T cells were retrovirally transduced and RV-Eomes tained in accordance with University of Missouri Office of Animal Re- + + GFP or control RV-EV GFP sorted in a BC MoFlow XDP. Next, RV-Eomes or sources Animal Care and Use Committee. Experimental procedures were EV control expressing T cells were transfected with pGL3-NFκB luciferase approved by the University of Missouri Institutional Animal Care and Use reporter (56) construct using Trans-IT Jurkat (Mirus) reagent. Luciferase ex- Committee. OVA, Q4H7, and VSV peptides were from New England Pep- pression was determined by flow cytometry with a biotin-conjugated anti- tides. rmIL-7 and rmIL-15 were from Peprotech. IL-2 (X63-IL-2 hybridoma) luciferase antibody (Rockland) followed by streptavidin-PE staining. was used at 50 units/mL Wedelolactone (NFκB inhibitor); rapamycin, Pim-1 kinase inhibitor IV, and Pim-1/2 kinase inhibitor V were from EMD/ Enrichment of CD8 T Cells. Before transfer into recipient mice, OT-I CD8 T-cell Calbiochem. donors were enriched by positive or negative selection using magnetic beads (Miltenyi Biotec). Antibodies and Flow Cytometry. Anti-CD8α (53-6.7), -CD90.1 (OX-7), –IL-7Rα β γ (A7R34), -CD62L (MEL-14), -CD28 (37.51), -CD122 (TM 1), IFN- (XMG1.2) and Immunoblot Analysis. The 2–5 × 106 T cells were stimulated with OVA- or -Bcl-2 (3F11) were from BD Pharmingen. Anti-CD45.1 (A20), -CD45.2 (I04), Q4H7-tetramer and anti-CD28 subjected to immunoblot as described in ref. -CD27 (LG.7F9 or LG.3A10), -OX40 (OX-86), KLRG1 (2F1), TRAIL and -Eomes 17. Densitometry was determined using Photoshop CC2015. (Dan11Mag) were from eBioscience. Anti–T-bet (4B10) and –Pim-1 (12H8 and κ α – C20) were from Santa Cruz Biotechnology. Anti-I B (5A5 and L35A5), p65- Statistical Analysis. For statistical analysis, two-tailed unpaired Student’s t test NFκB (D14E12), –phospho-p65-NFκB (S536), -Bim (C34C5), –phospho-BAD was applied using GraphPad Prism software. Significance was set at P < 0.05. (40A9) and –phospho-mTOR (S2481) were from Cell Signaling. Anti– *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.001. α-tubulin was from Sigma. Secondary antibodies and anti-granzyme B (GB11) were from Invitrogen. Anti–Bcl-XL (7B2.5) was from Southern Biotech. Flow ACKNOWLEDGMENTS. WethankS.Guerder,M.Croft,S.Reiner,andN.Kimfor cytometry was performed on a Coulter Cyan ADP or a FACSCalibur or LSRII providing retroviral constructs; S. S. Way, M. Bevan, and D. Zehn for providing Fortessa flow cytometer (Becton Dickinson) and analyzed with FlowJo FACS Listeria monocytogenes strains; B. Osborne, S. Jameson, and Sara Hamilton for Analysis Software (Tree Star). critical discussion; R. Kedl for performing experiments with CD70-deficient mice; B. Hahm and M. Vijayan for providing the NFκB reporter construct; and M. Johnson, D. Burke, and M. Lange for providing the VSV-G construct. This × 3– × 4 Adoptive Transfer. Naïve (1 10 5 10 ) or memory CD8 T cells were pu- work was supported by the University of Missouri Mission Enhancement Fund, rified from the lymph nodes of OT-I, MUT, or host mice and transferred i.v. the University of Missouri Life Sciences Fellowship (to K.M.K.), and the National into congenic mice. Institutes of Health Grants R01 AI110420 (to E.T.) and R01 CA35299 (to A.A.).

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