NF-Κb Signaling Mediates Homeostatic Maturation of New T Cells

NF-κB signaling mediates homeostatic maturation of new T cells

Ana Silva, Georgina Cornish1, Steven C. Ley, and Benedict Seddon2,3

Division of Immune Cell Biology, Medical Research Council National Institute for Medical Research, London, NW7 1AA, United Kingdom

Edited by Rafi Ahmed, Emory University, Atlanta, GA, and approved January 24, 2014 (received for review October 15, 2013) Interleukin (IL)-7 is critical for the maintenance of the peripheral pression (11). Phosphorylation of Foxo1 by PKB/Akt targets its T-cell compartment of the adaptive immune system. IL-7 receptor degradation and cytokine receptors that activate PKB/Akt act as α ( IL-7Rα) expression is subject to developmental regulation and negative regulators of IL-7Rα gene expression. As such, both IL-2 new T cells induce expression as they leave the thymus, which is and IL-7 negatively regulate IL-7Rα expression (12). Foxo1 binds essential for their long-term survival. It is not understood how this to an enhancer region upstream of the Il7r gene. Interestingly, the expression is regulated. Here, we identify a role for the Nuclear same enhancer also contains conserved binding sites for NF-κB Factor κ-B (NF-κB) signaling pathway in controlling expression of family transcription factors (11). IL-7Rα in new T cells. Perturbations to NF-κB signaling, either by NF-κB transcription factors are heterodimers or homodimers deletion of Inhibitor of Kappa-B Kinase-2 (IKK2) or by inhibiting of Rel family members. Dimers are sequestered in cytoplasm of α Rel dimer activity, prevented normal IL-7R expression in new T cells by their association with the inhibitory proteins, inhibitors α cells. Defective IL-7R expression resulted in impaired survival and of kappa B (IκB) family, and related proteins p100 and p105. homeostatic cell division responses by T cells that could be attrib- Phosphorylation of IκB by the IκB Kinase (IKK) complex targets α κ uted to their failure to express IL-7R normally. Surprisingly, NF- B IκB for degradation by the proteosome and releases NF-κB signaling was only required transiently in new T cells to allow their dimers to enter the nucleus. Canonical NF-κB signaling is me- normal expression of IL-7Rα, because IKK2 deletion in mature T diated by a trimeric complex of two kinases, IKK1(IKKα) and cells had no effect on IL-7Rα expression or their normal homeo- IKK2(IKKβ), and a third regulatory component, NEMO (IKKγ). static responsiveness. Therefore, we identify a developmental κ function for NF-κB signaling in the homeostatic maturation of Activation of NF- B by the IKK complex has been implicated in new T cells, by regulating IL-7Rα expression. regulating T-cell development, homeostasis, and function at various check points. The complete block in NF-κB signaling that results from ablation of NEMO causes a developmental arrest in aintaining T lymphocytes in sufficient numbers and at an single-positive (SP) thymocytes at the immature HSAhi stage Mappropriate composition of differentiation states and sub- (13). Partial impairment of IKK function by specific ablation of types is essential for effective immunity. The immune system has IKK2 impairs development of T and CD4 effector/memory cells evolved a number of homeostatic mechanisms to ensure the size reg (13). Peripheral IKK2-deficient T cells also exhibit impairments in and composition of the T-cell compartment remains remarkably homeostatic proliferation responses (14). NF-κB has also been stable over time. The cytokine interleukin (IL)-7 plays a central implicated in thymocyte selection because overexpression of dom- role in regulating homeostasis of the T-cell compartment. It is κ essential for normal development of αβ and γδ T cells in the inant-negative I B affects both double-negative (DN) thymocyte thymus and provides vital survival signals for both naive and memory T cells in the periphery (1). IL-7 is produced by stromal Significance cell components in bone marrow, thymus, and in peripheral lymphoid compartments (2), and there is extensive evidence that Interleukin (IL)-7 is critical for the maintenance of the periph- production of IL-7 is a key factor that determines and limits the eral T-cell compartment of the adaptive immune system. Our overall size of the peripheral T-cell compartment (3, 4). The study identifies a role for the Nuclear Factor κ-B (NF-κB) sig- receptor for IL-7 is a member of the common-gamma chain (γc) nalling pathway in the control of IL-7 receptor expression by family of cytokine receptors and consists of a heterodimeric T cells. Following thymic selection, new T cells specifically up- complex of IL-7Rα and γc (5). regulate IL-7R even as they leave the thymus, and we reveal In T cells, IL-7 signaling is primarily regulated at the level of that this expression is strictly NF-κB dependent. NF-κB signal- IL-7Rα expression. During T-cell development in the thymus, ing was only required transiently, however, and once fully expression of IL-7Rα by thymocytes is subject to dynamic de- mature, naive T cells did not require NF-κB signaling to main- velopmental regulation. IL-7Rα is essential for survival and de- tain IL-7R expression. Therefore, we reveal a developmental velopment of CD4 CD8 double negative (DN) thymocytes (6). role for NF-κB signaling for the normal maturation and func- Expression is lost at the CD4 CD8 double positive (DP) stage, tion of new T cells. ensuring that onward development of DP thymocytes is re- stricted to those that successfully undergo positive selection (7). Author contributions: A.S., S.C.L., and B.S. designed research; A.S. and G.C. performed research; A.S. and B.S. analyzed data; and A.S. and B.S. wrote the paper. Immediately following selection, however, IL-7Rα is immediately reexpressed and recent studies suggest that the strength of The authors declare no conflict of interest. T-cell receptor (TCR)-mediated positive selection signaling This article is a PNAS Direct Submission. determines the extent of reexpression by SP thymocytes (8). Freely available online through the PNAS open access option. Following egress from the thymus, new T cells continue to ma- 1Present address: Academic Department of Rheumatology, Center for Molecular and Cellular Biology of Inflammation, King’s College London, London SE1 1UL, ture as recent thymic emigrants (RTE), a process including the United Kingdom. further induction of IL-7Rα (9). 2To whom correspondence should be addressed. E-mail: [email protected]. The identity of the signaling pathways that regulate IL-7Rα 3Present address: Institute of Immunity and Transplantation, Division of Infection and expression in new T cells remains unknown. In mature T cells, Immunity, University College of London, Royal Free Hospital, London NW3 2PF, United Fox family transcription factors Foxo1 and Foxp1 are required Kingdom. α for expression of IL-7R in mature T cells (10, 11). In particular, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. expression of Foxo1 is constitutively required for IL-7Rα ex- 1073/pnas.1319397111/-/DCSupplemental.

E846–E855 | PNAS | Published online February 18, 2014 www.pnas.org/cgi/doi/10.1073/pnas.1319397111 Downloaded by guest on October 1, 2021 survival (15) and reduces development of CD8 lineage thymo- PNAS PLUS cytes (16, 17), whereas gain-of-function IKK2 mutants enhance CD8 development (18). Because NF-κB signaling has multiple roles in T-cell development and regulatory elements upstream of the Il7r gene contain NF-κB binding sites, we considered the possibility that NF-κBsig- naling may also regulate IL-7Rα expression during development. We tested this idea by ablating IKK2 expression at different stages of T-cell development and then assessing IL-7Rα expression and T- cell homeostatic responses. We found that deletion of Ikk2 in the thymus resulted in generation of T cells with profound survival and homeostatic defects that could be directly attributed to a specific defect in IL-7Rα expression. Significantly, deleting Ikk2 in mature T cells had little or no impact on their homeostasis, suggesting that a key requirement for IKK2 expression in T cells was for the homeostatic maturation of new T cells and was not a direct downstream target of homeostatic signaling. We identify control of IL- 7Rα expression as the key mechanism of the NF-κB–dependent homeostatic maturation process. Results IKK2 Signaling Is Redundant for Normal Thymocyte Development. To investigate whether NF-κB signaling regulates expression of IL- 7Rα in T cells, we analyzed mice in which the IKK2 kinase component of the IKK complex was conditionally deleted in T lineage. IKK2-deficient mice have been reported to have normal thymic development but reduced numbers of peripheral T cells,

in particular the CD8 subset (13). In this study, however, Ikk2 Cre EYFP fx/fx Cre Fig. 1. Normal thymocyte development in CD4 R26R Ikk2 and deletion was mediated by CD4 that starts gene excision at the iCre EYFP fx/fx Cre EYFP fx/fx + + huCD2 R26R Ikk2 mice. CD4 R26R Ikk2 mice and huC- CD4 CD8 double-positive (DP) stage and thymocytes were D2iCre R26REYFP aged 8–12 wk of age were analyzed by FACS (n = 5 each). not found to be protein deficient until they had reached CD4 CD4Cre R26REYFP Ikk2fx/WT or Ikk2fx/fx R26REYFP were used as negative controls or CD8 single-positive (SP) stage. Because NF-κB signaling has (n = 5). (A) Density plots are of CD4 vs. CD8 expression by total live thymo- already been implicated in the thymic selection of CD8 T cells cytes from the indicated strain. Numbers indicate percentage of cells in the (16–18), we also analyzed mice in which Ikk2 was deleted by corresponding gate. (B) Bar charts show total numbers of DP, TCRhi CD4 SP, huCD2iCre that induces gene deletion much earlier, at the DN2 and TCRhi CD8 SP thymocytes in either IKK2-deficient (gray bars) or control hi stage of development (19). Phenotype and number of thymocytes (black bars) mice. (C) Dot plots are of CD4 vs. CD8 expression by TCR lymph iCre fx/fx Cre fx/fx node cells from the indicated strains. (D) Bar charts show total combined from huCD2 Ikk2 and CD4 Ikk2 mice were exam- − numbers of CD44loCD25 naive CD4 or CD44lo naive CD8 T cells from lymph ined compared with either Cre-negative Ikk2fx/fx controls or Cre- fx/WT node and spleen of the indicated strains. Data are representative of five or expressing Ikk2 mice, in consideration of Cre toxicity. Cre more experiments. activity was confirmed by measuring YFP expression from a Rosa26RYFP Cre reporter allele (R26RYFP) (20) also present in these mice (Fig. S1A). Western blot analysis of IKK2 protein whether Ikk2 was deleted early or late in thymic development + abundance in sorted YFP thymocytes confirmed that YFP ex- (Fig. 2B). To see whether reduced receptor levels were due to pression correlated with deletion of Ikk2 (Fig. S1B). In the ab- differences in Il7r transcription, we measured Il7r mRNA. Both sence of Ikk2 expression, number and phenotype of all CD4 and CD8 naive T cells from CD4Cre Ikk2fx/fx R26REYFP mice thymocyte subpopulations was similar to controls, regardless of had substantially reduced levels of Il7r mRNA (Fig. 2C). IL-7 whether deletion occurred early, using huCD2iCre, or late, via α Cre signaling can negatively regulate expression of IL-7R (12). CD4 , in thymic development (Fig. 1 A and B). Examining Because Ikk2 deletion results in a reduction in total naive T-cell numbers and phenotype of peripheral T cells that developed in numbers, we sought to confirm that the loss of IL-7Rα expres- the absence of Ikk2 revealed near-identical defects in naive T- sion was cell intrinsic to naive T cells. We therefore generated cell numbers regardless of whether Ikk2 was deleted at the DN2 iCre Cre mixed irradiation chimeras by using bone marrow from CD45.1 C Cre fx/fx EYFP fx/fx EYFP by huCD2 or DP thymocyte stages by CD4 (Fig. 1 and WT and CD4 Ikk2 R26R or Ikk2 R26R donors. D). Therefore, inducing IKK2 ablation before positive selection Only IKK2-deficient naive T cells exhibited reduced IL-7Rα of DPs did not further exacerbate the observed reduction in the expression in chimeras (Fig. S2A). Furthermore, the relative size of the peripheral naive T-cell compartment. reduction in IKK2-deficient naive CD8 T cells in mixed chimeras Naive T Cells Express Reduced IL-7Rα in the Absence of Ikk2. Ho- was at least as great as observed in intact mice, and there was meostasis of naive T cells depends on the combined activity of evidence that competition with WT cells further reduced num- TCR signaling induced by self peptide (sp) MHC in peripheral bers of naive CD4 T cells in chimeras (Fig. S2B). Finally, to test lymphoid organs and IL-7. We therefore assessed the function of the functional impact of reduced IL-7Rα expression in vitro, we homeostatic TCR and IL-7R signaling in Ikk2-deficient mice to measured T cells responses to IL-7 stimulation. Both survival investigate the underlying cause of the reduction in naive T cells and induction of STAT5 phosphorylation was reduced in Ikk2- we observed. Expression levels of CD5 reliably reports tonic deficient naive CD4 and CD8 T cells (Fig. 2 D and E), suggesting homeostatic TCR signaling in peripheral T cells (21, 22). Ex- that the defect in IL-7Rα expression was functionally significant. amining Ikk2-deficient peripheral naive CD4 and CD8 T cells IL-7Rα expression is under dynamic regulation during T-cell revealed normal CD5 expression (Fig. 2A). In contrast, IL-7Rα development. During thymic selection, DP thymocytes lack IL-

levels were substantially reduced on both CD4 and CD8 naive T 7Rα expression. However, IL-7Rα is reexpressed in CD4 SPs and IMMUNOLOGY cells (Fig. 2A).The reduction observed was similar regardless of in CD8 lineage DP3 thymocytes, which in both cases depends on

Silva et al. PNAS | Published online February 18, 2014 | E847 Downloaded by guest on October 1, 2021 TCR signaling during selection (8). New T cells are also reported to up-regulate IL-7Rα again once they leave the thymus, as recent thymic emigrants (9). To determine whether Ikk2 deletion was affecting one or other of these points of regulation, we analyzed IL-7Rα expression in different thymocyte subsets. Initial induction of IL-7Rα by DP3 and SP thymocytes appeared rela- tively normal in Ikk2-deficient mice (Fig. 3A). Closer analysis of CD8 lineage thymocytes revealed that following initial induction of IL-7Rα in DP3 thymocytes, expression was further induced among the most mature HSAlo thymocytes, before they left the thymus. In Ikk2-deficient thymocytes, induction of IL-7Rα in HSAlo thymocytes and peripheral naive T cells was substantially reduced compared with controls (Fig. 3B). Significantly, both naive CD4 and CD8 T cells expressed IL-7Rα at similar levels to the most mature HSAlo thymic precursor, suggesting that new T cells fail to induce IL-7Rα expression in the absence of IKK2 expression as they leave the thymus and enter the peripheral compartment.

Normal IL-7Rα Expression by Peripheral T Cells Depends on Canonical NF-κB Signaling. Functions for IKK2 other than NF-κB activation have been described in other cell types (23). We therefore wished to assess whether IKK2 was regulating IL-7Rα expression in peripheral T cells by activating NF-κB signaling. We therefore analyzed pLck-IκB-PEST mice (15) in which T cells express a dominant negative inhibitor of κB transgene. IκB- PEST is proteolytically resistant following its phosphorylation by the IKK complex and, therefore, inhibits Rel dimer release to the nucleus. Significantly, IL-7Rα expression was reduced in both CD4 and CD8 naive T cells of pLck-IκB-PEST mice (Fig. 3C). Comparing IL-7Rα expression by SP and peripheral subsets in pLck-IκB-PEST mice revealed normal induction of IL-7Rα in postselection HSAhi SP thymocytes and DP3 thymocytes for CD8 lineage cells, but a failure to further induce expression in HSAlo CD8 SP thymocytes and peripheral naive T cells (Fig. 3E). Among CD4 lineage cells, there was also evidence that expression was already reduced in pLck-IκB-PEST mice at the HSAlo CD4 SP stage. Therefore, these data suggest that normal induction of IL-7Rα in new T cells depends on activation of canonical NF-κB signaling by IKK2.

Peripheral Expression of IL-7Rα in F5 TCR Transgenic Mice Depends on IKK2 Expression. To investigate whether the failure to up-regulate IL-7Rα expression in Ikk2-deficient peripheral T cells was a specific defect in new T cells as they left the thymus, we analyzed F5 TCR transgenic mice. T cells in F5 mice express a class I re- stricted TCR specific for a peptide of influenza nucleoprotein (24). Our previous studies have shown that successful induction Fig. 2. Defective IL-7Rα expression and function in the absence of IKK2 of Il7r gene expression by developing thymocytes depends on the expression. CD4Cre R26REYFP Ikk2fx/fx (fx/fx) and control CD4Cre R26REYFP strength of positive selection signaling. Neither DP3 nor CD8SP Ikk2fx/WT(fx/WT) mice (n = 5 each) were analyzed at 8–12 wk of age. (A) thymocytes in F5 mice induce Il7r during or following positive − Histograms are of CD5 or IL-7Rα expression by CD44loCD25 naive CD4 or selection in the thymus, because it is thought the avidity of their CD44lo naive CD8 T cells from lymph nodes from IKK2-deficient (red lines) or TCR for self peptide-MHC (spMHC) is not sufficiently strong control strains (black lines). (B) Bar charts show IL-7Rα MFI, normalized to (8). After leaving the thymus, however, F5 T cells do neverthe- control, of naive CD4 and naive CD8 T cells in which Ikk2 was deleted by less induce expression of IL-7Rα and it is not known how this either huCD2iCre (red) or CD4Cre (blue). (C) Total mRNA was purified from + hi lo − + hi lo late induction of IL-7Rα is controlled. Because we identified sorted CD4 TCR CD44 CD25 (CD4) and CD8 TCR CD44 (CD8) lymph α node cells from CD4Cre Ikk2fx/fx R26REYFP (Ikk2 fx/fx) and control CD4Cre Ikk2fx/WT a role for IKK2 in the induction of IL-7R late in the maturation R26REYFP(Ikk2 fx/WT) mice, and Il7r gene expression was determined by of SP thymocytes, we asked whether induction of IL-7Rα in quantitative real-time PCR. Bar charts show expression relative to HPRT by peripheral F5 T cells depended on IKK2 signaling. To test this − − the indicated populations. (D) Total lymph node T cells were cultured in idea, we generated F5 Rag1 / huCD2iCre R26REYFP Ikk2fx/fx different doses of IL-7 and cell viability assessed by flow cytometry at 48 h. mice. Consistent with observations in polyclonal huCD2iCre Plots are of percent viable cells for the indicated subset from cultures of IKK2 Ikk2fx/fx R26RYFP mice, phenotype and number of thymocytes − − knockout (Ikk2 fx/fx Cre+) and control (Ikk2 fx/fx Cre-) mice. (E) Total lymph from F5 Rag1 / huCD2iCre R26REYFP Ikk2fx/fx mice was similar to node T cells were cultured in different doses of IL-7 for 15 min, and phospho- STAT5 levels were assessed by flow cytometry. Plots show MFI phospho controls (Fig. 4A). In contrast, numbers of peripheral F5 T cells STAT5 stain for the indicated subset from cultures of IKK2 knockout (Ikk2 fx/fx were substantially reduced in the absence of IKK2 (Fig. 4B). Sig- Cre+) and control (Ikk2 fx/WT Cre+) mice. Data and statistically significant nificantly, in the absence of IKK2, peripheral F5 T cells almost differences are representative of three (D and E)ormore(A–C)experiments. completely failed to induce IL-7Rα expression (Fig. 4B). There-

E848 | www.pnas.org/cgi/doi/10.1073/pnas.1319397111 Silva et al. Downloaded by guest on October 1, 2021 sistent with this observation, CTV+ F5 T cells were still HSAint PNAS PLUS by day 3 following dye injection and also largely IL-7Rα negative (Fig. 5A). CTV+ cells induced IL-7Rα and lost HSA over the following 6 d. A discrete population of peripheral T cells are HSAint in F5 mice. In light of the changes in HSA expression observed in CTV-labeled F5 T cells, this population likely rep- resents RTE up to 6 d after thymic egress. Consistent with this view, expression of HSA and IL-7Rα by CTV+ F5 T cells at day 3 after intrathymic dye injection was comparable to HSAint CD8 T cells in normal F5 hosts (Fig. 5B). That HSAint F5 T cells are RTE was further confirmed by measuring expression of CD45RB, another marker of RTE that is up-regulated during their maturation (9). HSAint F5 T cells were also CD45RBlo (Fig. 5C). Analyzing IL-7Rα expression by HSAint F5 T cells further confirmed that induction of IL-7Rα by F5 T cells occurs among the RTE population. Induction of IL-7Rα by HSAint cells also depends on IKK2 because HSAint F5 T cells from IKK2-deficient mice failed to express IL-7Rα. IKK2-deficient F5 mice have reduced numbers of peripheral naive T cells. To determine which peripheral compartments were reduced, we counted − − numbers of HSAint and HSAlo mature naive T cells in F5 Rag1 / Ikk2fx/fx huCD2iCre R26RYFP mice. Significantly, HSAint F5 T cells were present in similar numbers to controls in the absence of IKK2, suggesting that RTE in F5 mice were not reduced in the absence in IKK2 and also suggesting that thymic output was similar to controls in these mice. In contrast, mature HSAlo − − peripheral F5 T cells were specifically reduced in F5 Rag1 / Ikk2fx/fx huCD2iCre R26RYFP mice (Fig. 5D), and their under representation accounted for the reduced numbers of peripheral T cells observed in the IKK2-deficient F5 strain.

Fig. 3. Up-regulation of IL-7Rα by peripheral T cells is defective in the absence of normal NF-κB signaling. (A) Histograms are of IL-7Rα by the indicated thymocyte subset from huCD2iCre R26REYFP Ikk2fx/fx mice (red lines) or Cre -ve littermate controls (black lines). (B) Bar charts are of IL-7Rα MFI by CD4 and CD8 lineage cells from huCD2iCre R26REYFP Ikk2fx/fx mice (gray bars) or Cre -ve littermate controls (black bars) (n = 5 each). CD4 lineage subsets are CD4SP HSAhi thymocytes (HSAhi), CD4SP HSAlo thymocytes (HSAlo) and – peripheral CD4+ TCRhi CD44lo CD25 naive T cells (LN). CD8 lineage subsets analyzed were DP3 thymocytes (DP3) (41), CD8SP HSAhi thymocytes (HSAhi), CD8SP HSAlo thymocytes (HSAlo), and peripheral CD8+ TCRhi CD44lo naive T cells (LN). (C–E) pLck IκB-PEST and WT littermate controls were analyzed at 8–12 wk of age. (C) Density plots are of CD4 vs. CD8 by TCRhi lymph node T cells from the indicated strain. (D) Histograms are of IL-7Rα by CD4+ TCRhi − CD44lo CD25 naive and CD8+ TCRhi CD44lo naive lymph node T cells from WT (black lines) and pLck IκB-PEST (red line) mice. IL-7Rα by DP thymocytes are shown as negative control (gray bars). (E) Bar charts are of IL-7Rα MFI by CD4 and CD8 lineage cells from the indicated strains. Data and statistically significant differences are representative of three or more experiments. *P < 0.05, **P < 0.01. Fig. 4. F5 TCR transgenic T cells fail to induce IL-7Rα in the absence of IKK2. −/− iCre EYFP fx/fx – α F5 Rag1 huCD2 R26R Ikk2 mice and Cre littermate controls fore, in F5 mice, IL-7R expression by CD8 T cells almost com- were analyzed at 8–12 wk of age. (A) Density plots are of CD4 vs. CD8 by – − − pletely depends on the expression of IKK2. thymocytes from Cre+ and Cre F5 Rag1 / huCD2iCre R26REYFP Ikk2fx/fx mice. Bar charts are of total numbers of DP thymocytes (DPs) and CD8 SP thymo- IKK2 Is Required for Induction of IL-7Rα by RTE in F5 Mice. To de- cytes (SP8) from a litter of Cre+ (n = 7) and Cre– (n =4)F5Rag1−/− huCD2iCre termine more precisely the timing of IL-7Rα expression by F5 T R26REYFP Ikk2fx/fx mice. (B) Histograms are of IL-7Rα by CD8+ TCRhi lymph −/− iCre EYFP fx/fx – cells, CTV cell dye was injected intrathymically into F5 mice to node T cells (solid lines) from F5 Rag1 huCD2 R26R Ikk2 or Cre label a cohort of thymocytes and the phenotype of new F5 T cells controls, compared with DP thymocytes (gray fill) or CD8 SP (broken lines) thymocytes from the same donor. Bar chart shows total numbers of F5 T cells assessed as they egressed from thymus to peripheral lymph from lymph nodes and spleen of the mice described in A. Data and sta- nodes. CD24 or Heat Stable Antigen (HSA) is expressed at high tistically significant differences are representative of eight independent IMMUNOLOGY levels by thymocytes and lost once cells leave the thymus. Con- experiments. **P < 0.01.

Silva et al. PNAS | Published online February 18, 2014 | E849 Downloaded by guest on October 1, 2021 Fig. 5. Expression of IL-7Rα by RTE in F5 mice depends on IKK2. (A) CTV cell dye was injected intrathymically into F5 Rag1−/− mice, and CTV+ cells were analyzed in peripheral lymph nodes days (d)3, 6, and 9 after injection. Dot plot shows CD8 vs. CTV labeling by lymph node T cells at d3. Histograms are of HSA − (Upper) and IL-7Rα (Lower) by CTV+ (red lines) and CTV (black lines) F5 T cells at the days indicated, compared with F5 DP thymocytes (gray fill). (B) Dot and − density plots are of IL-7Rα vs. HSA by CTV and CTV+ F5 T cells at d3 after intrathymic dye injection. (C) Histograms are of HSA expression by lymph node F5 T cells (solid black lines) compared with DP (gray fill) and CD8 SP (broken line) F5 thymocytes. Bars indicate the gates used to identify HSAint (red) and HSAlo (black) T cells. Histograms of CD45RB and IL-7Rα are from gated HSAint (red line) and HSAlo (black lines) F5 T cells from Cre+ and Cre– F5 Rag1−/− huCD2iCre R26REYFP Ikk2fx/fx mice. (D) Bar charts show total numbers of HSAint and HSAlo F5 T cells recovered from lymph nodes and spleen of Cre+ and Cre– F5 Rag1−/− huCD2iCre R26REYFP Ikk2fx/fx mice. Data and statistically significant differences are representative of four independent experiments. *P < 0.01.

Defective Homeostasis of Ikk2-Deficient F5 T Cells Is Strictly IL-7 ing gene expression in IKK2-deficient F5 T cells revealed that Dependent. Because IL-7Rα expression by peripheral CD8 T other components of the IL-2R family and their associated cells in F5 mice almost completely depends on IKK2 expression, downstream signaling components were expressed normally in we used this system to investigate the effect of IKK2 deficiency the absence of IKK2 expression (Fig. S3). However, NF-κB on homeostatic responses of T cells. Homeostatic proliferation signaling is conventionally thought to control cell survival by by F5 T cells to lymphopenia depends on cytokines IL-7 and, to regulating expression of Bcl2 family members (27–29). There- a lesser extent, IL-15 (25). Following their transfer to lympho- fore, it was also possible that deletion of IKK2 resulted in other − − penic Rag1 / hosts, proliferation of YFP+ IKK2-deficient F5 T gene expression changes that contributed to the defective sur- cells was greatly reduced compared with control F5 T cells, vival and proliferation of F5 T cells. To investigate this idea, we cotransferred to the same hosts (Fig. 6A). The few divisions first measured expression of the Bcl2 family genes that are observed among IKK2-deficient F5 T cells were in part IL-15 expressed in F5 T cells. Significantly, no differences in expression − − dependent, because the same T cells transferred to Rag1 / were observed in the absence of IKK2 expression (Fig. 6C). − − Il15ra / mice, which have no functional IL-15 (26), failed to Second, we wanted to determine whether death of IKK2-deficient proliferate at all (Fig. 6A).There was no proliferation at all in the F5 T cells strictly depended on IL-7 signaling pathway. Others − − − absence of host IL-7 in Il7 / Rag1-/ hosts, confirming the non- report that ectopic expression of IL-7Rα paradoxically results in redundant role of this cytokine in the response. We next assessed cell death (30), because expression is not subject to the same survival of IKK2-deficient F5 T cells. First, analyzing cell negative regulation of the endogenous Il7r locus (12). Therefore, recoveries from these same experiments revealed substantially we instead asked whether IKK2 deficiency had an additive effect reduced recoveries of IKK2-deficient F5 T cells relative to on F5 T-cell death in the absence of IL-7, which would indicate control F5 T cells, which could not be accounted for simply by an IL-7 independent survival function for IKK2. Significantly, − − differences in cell division in lymphopenic Rag1 / hosts (Fig. control and IKK2-deficient F5 T cells cotransferred to IL-7– 6B). Death of IKK2-deficient F5 T cells was greatest in IL-15– deficient hosts both maintained a similar representation (Fig. deficient hosts, probably because the loss of IL-15 exacerbated 6B) and died with similar kinetics to one another (Fig. 6D). the survival defect of IKK2-deficient F5 T cells that already fail There was therefore no evidence of an additive effect of IKK2 to express IL-7Rα. Furthermore, the additive effect of IL-15 deficiency on cell death in the absence of IL-7. Together, these deficiency on survival suggested that the requirement for IKK2 data suggest that IKK2 dependent up-regulation of IL-7Rα by was not downstream IL-15 signaling and also that downstream γc new T cells is a key function for IKK2 for the normal survival and cytokine signaling was otherwise intact in IKK2-deficient F5 T homeostasis of T cells. cells. To measure survival in replete hosts, IKK2-deficient F5 T cells were transferred to congenic replete F5 hosts. Significantly, Ikk2 Expression Is Required Only Transiently for Normal Peripheral IKK2-deficient F5 T cells disappeared more rapidly than IKK2 T-cell Homeostasis. Our data showed that IKK2 expression is re- sufficient control F5 T cells (Fig. 6B). quired for normal T-cell homeostasis and that failure to express It was possible that the reduced levels of IL-7Rα on IKK2- IL-7Rα at normal levels is a key underlying mechanism. How- deficient F5 T cells was alone sufficient to account for the re- ever, in principle, it was possible that IKK2 expression was also duced lymphopenia-induced proliferation and survival of F5 T required downstream of TCR and/or IL-7 for the transduction cells. Indeed, IL-15 signaling was able to induce both survival of signals required for homeostatic survival and proliferative and proliferation of IKK2-deficient F5 T cells, suggesting that responses. Furthermore, previous studies have identified Fox downstream γc cytokine signaling was otherwise intact. Analyz- family transcription factors as a critical regulator of IL-7Rα

E850 | www.pnas.org/cgi/doi/10.1073/pnas.1319397111 Silva et al. Downloaded by guest on October 1, 2021 expression in peripheral T cells (10, 11). They are constitutively PNAS PLUS required for normal Il7r gene expression in T cells, because their conditional deletion results in immediate perturbations to IL-7Rα expression. Therefore, it was important to determine whether IKK2 expression was also a constitutive requirement for normal IL-7Rα expression. We analyzed IL-7Rα expression and homeostasis of F5 T cells in which Ikk2 was deleted specifically in mature peripheral T cells that had already undergone normal thymic and postthymic maturation. Induction of R26CreERT by − − tamoxifen administration to F5 Rag1 / R26CreERT R26REYFP Ikk2fx/fx mice induced Ikk2 deletion in ∼40–50% of mature peripheral F5 T cells, identified by their expression of EYFP reporter (Fig. 7A) and confirmed by Western blot (Fig. 7B). Analyzing IL-7Rα expression in these populations revealed that Ikk2 deletion in mature F5 T cells had no effect on IL-7Rα expression levels (Fig. 7C). To confirm this behavior was cell in- − − trinsic, we also transferred F5 T cells from F5 Rag1 / R26CreERT R26REYFP Ikk2fx/fx donors to congenic F5 hosts, followed by induction of gene deletion by tamoxifen administration to these − hosts. IL-7Rα expression in YFP+ and YFP donor populations matched each other and that of congenic host F5 T cells (Fig. 7D). These findings were also confirmed in polyclonal mice 4 wk after deletion of IKK2 (Fig. S4). These data show that IKK2 expression was not required to maintain IL-7Rα expression in fully mature peripheral T cells. Finally, we wanted to determine whether IKK2 was necessary for the transduction of signals required for survival and lymphopenia induced proliferation (LIP). − − Total F5 T cells from F5 Rag1 / R26CreERT R26REYFP Ikk2fx/fx mice were transferred to either congenically labeled F5 hosts or − − lymphopenic Rag1 / hosts following induction of gene deletion by tamoxifen. Remarkably, both LIP (Fig. 7E) and survival (Fig. 7F) were unaffected by loss of IKK2 expression. Therefore, IKK2 expression was not required for transmission of the signals required for normal mature naive T-cell homeostasis. Taken together, our data indicate that IKK2 was only required transiently during development to allow normal induction of Il7r expression in new T cells.

Signaling from Tnfrsf Receptors Induces IL-7Rα Expression by Thymocytes in Vitro. The surface receptors responsible for trans- mitting signals required for homeostatic maturation of new T cells have not been described. Because we identified a central role for the canonical NF-κB signaling in control of Il7r expression, we reasoned that receptors capable of activating this pathway should be involved. Others have excluded roles for TCR signaling and IL-7 in the maturation of RTE (9). We therefore examined the role of TNF receptor superfamily (Tnfrsf) members because many of these receptors are potent activators of NF-κB. Analyzing mRNA expression by control F5 T cells revealed which Tnfrsf members were expressed. mRNA was de- tectable for TNF receptor I and II, TACI (Tnfrsf13b), LIGHTR (Tnfrsf14), GITR (Tnfrsf18), Tnfrsf25, Tnfrsf26,andCD27(Fig. 8A). A similar pattern of expression was also observed by IKK2- deficient F5 T cells (Fig. 8A). F5 CD8 SP thymocytes were stim- Fig. 6. Defective homeostatic responses of F5 T cells in the absence of IKK2. ulated with ligands for these receptors to determine which, if any, − − F5 T cells from F5 Rag1 / huCD2iCre+ R26REYFP Ikk2fx/fx (IKK2 ko) and huC- could induce expression of IL-7Rα. Culture of thymocytes with iCre- D2 littermates (IKK2 wt) were labeled with CTV cell dye, mixed ∼1:1, and APRIL or BAFF (TACI ligands), LIGHT (LIGHTR ligand), 2 × 106 total cells were transferred to either Rag1−/−, Il15ra−/− Rag1−/−, Il7−/− −/− −/− TRAIL (ligand for Tnfrsf26), GITRL (GITR ligand), or TL1A Rag1 or Ly5.1 F5 Rag1 hosts for 14 d. (A) Histograms are of cell dye α labeling in the indicated host by the indicted donor population at d7 and (ligand for DR3/Tnfrsf25) failed to induce IL-7R expression. − d14 after transfer. (B) Relative cell frequencies of YFP+ and YFP donor cells Significantly, however, both TNF and soluble CD70 (ligand for were determined by flow cytometry and frequencies adjusted to exclude the predicted expansive effects of cell division (Methods). Graphs show ratio of − − − YFP+:YFP F5 T cells, normalized to input ratio at day 1 after transfer in the indicated genes in control F5 Rag1 / huCD2iCre- R26REYFP Ikk2fx/fx samples − − indicated hosts. (C) mRNA was isolated from sorted CD8+ TCRhi T cells from (black bars; IKK2 WT) vs. F5 Rag1 / huCD2iCre+ R26REYFP Ikk2fx/fx samples (red − − F5 Rag1 / Ikk2fx/fx R26REYFP huCD2iCre and huCD2iCre -ve littermate. Total bars; IKK2 KO). Data are mean ± SD RPKM from triplicate biological repli- mRNA was sequenced by a Illumina Genome Analyzer IIx. Following nor- cates. (D) Graphs shows cell recovery from spleen, normalized to day 1, of malization reads were displayed as normalized reads per kilobase of exon IKK2 WT and IKK2 KO F5 T cells cotransferred to Il7−/− Rag1−/− hosts. Data IMMUNOLOGY per million reads (nRPKM). Bar charts show expression level (nRPKM) of the are representative of five independent experiments.

Silva et al. PNAS | Published online February 18, 2014 | E851 Downloaded by guest on October 1, 2021 ablated. Induction of IL-7Rα expression in new T cells was identified as a key mechanism of the maturation process, and a failure to up-regulate IL-7Rα normally resulted in a reduction in the size of the naive T-cell compartment. The present study found evidence that NF-κB signaling played a nonredundant role for the normal induction of IL-7Rα by new T cells as they left the thymus. Perturbation of NF-κB signaling either at the level of the IKK complex, by specific ablation of IKK2, or inhibition of Rel-dimer nuclear translocation by the expression of a dominant negative inhibitor of κB protein, both resulted in a failure of new T cells to induce IL-7Rα expression to normal levels. SP thymocytes induce IL-7Rα expression immediately following selection and depend on TCR-mediated positive selection signaling (8). Interestingly, this initial induction of IL-7Rα immediately after positive selection occurred independently of NF-κB signaling because HSAhi SP thymocytes expressed normal IL-7Rα regardless of how NF-κB activity was perturbed. In contrast, we identified a second phase of IL-7Rα induction that occurred in new T cells as they egressed the

Fig. 7. Normal homeostasis of F5 T cells following peripheral deletion of − − Ikk2. Ikk2 deletion was induced in peripheral F5 T cells by treating F5 Rag1 / R26CreERT2 R26REYFP Ikk2fx/fx mice with five daily injections of tamoxifen and mice analyzed five or more days after the final injection. (A) Histogram − − shows YFP expression by CD8+ TCRhi T cells from F5 Rag1 / R26CreERT2 R26REYFP Ikk2fx/fx mice 5 d after the final injection with tamoxifen. (B) CD8+ TCRhi lymph node T cells were sorted into YFP+ and YFP– fractions, and IKK2 protein was measured in by Western blot. CD3ζ was probed as control. (C) − Histograms are of IL-7Rα expression by YFP+ (red), YFP (black) cells from A, compared with DP thymocytes of the same donor as negative control (gray − − fill). (D) T cells from F5 Rag1 / R26CreERT2 R26REYFP Ikk2fx/fx mice were − − transferred to CD45.1 F5 Rag1 / hosts, which were then received five injections of tamoxifen. Ten days later, phenotype of donor populations was assessed by FACS. Density plot shows CD45.2 v CD45.1 in recipient mice by CD8+TCRhi cells. Histogram of YFP expression is by donor CD45.2+ CD8+TCRhi − cells. Histograms of IL-7Rα expression are of YFP (black) and YFP+ (red) donor populations (solid lines) compared with DP thymocytes as negative control (gray) and host CD8 peripheral T cells (broken black lines) in both cases. (E and F) F5 T cells from A were labeled with cell dye and transferred at 2 × 106 per mouse to either Rag1−/−, Il15ra−/− Rag1−/−, Il7−/− Rag1−/−,or Ly5.1 F5 Rag1−/− hosts (n > 4 each) for 14 d. (E) Bar charts show mean division − index of YFP+ and YFP cells in the indicated hosts. (F) Graphs show the ratio − of YFP+:YFP F5 T cells from A, in the indicated hosts at d1 and d14 after transfer, normalized to ratio at d1. Data are representative of two (D)or four or more (A–C, E,andF) experiments.

CD27) induced strong up-regulation of IL-7Rα protein (Fig. 8B). TNF and CD70 induced expression on SP but not DP thymocytes (Fig. 8C). IL-7Rα expression depended on IKK2 signaling because pharmacological inhibition of IKK2 activity with BI605906 (31) prevented induction of IL-7Rα in control F5 thymocytes Fig. 8. Tnfrsf ligands induce IL-7Rα expression on SP thymocytes in vitro. (A) (Fig. 8D), whereas IKK2-deficient F5 T cells expressed less IL- Bar charts show expression level (nRPKM) of the indicated Tnfrsf genes by − − 7Rα in response to culture with either ligand (Fig. 8D). Taken control (black bars) and IKK2-deficient F5 T cells (red bars). (B)F5Rag1 / together, these data show that homeostatic maturation of new T thymocytes were cultured for 24 h with either TNF (10 ng/mL), BAFF (100 ng/ cells can be induced in vitro by ligation of Tnfrsf members in- mL), LIGHT (100 ng/mL), APRIL (100 ng/mL), TRAIL (100 ng/mL), GITRL (100 ng/mL), CD70 (100 ng/mL), TLA1 (100 ng/mL) or IL-7 (10 ng/mL). Histo- cluding TNFR and CD27. grams are of IL-7R expression by CD8 SP thymocytes in stimulated cultures (black lines) compared with cells cultured alone as control (gray fills). (C)F5 − − Discussion Rag1 / thymocytes were cultured for 24 h with TNF or CD70. Histograms are Following thymic selection, new T cells induce expression of IL- of IL-7R expression by the indicated subset in the presence of the indicated 7Rα, which is essential for their long-term survival and in- ligand (red lines) compared with control cultures with no added ligand (gray − − tegration into the peripheral repertoire (8). The mechanisms fills). (D) Thymocytes from huCD2iCre +ve (IKK2 KO) and -ve (WT) F5 Rag1 / EYFP fx/fx governing this process are incompletely understood. Here, we R26R Ikk2 donors were cultured for 24 h with TNF or CD70. Cultures κ of iCre -ve control thymocytes were additionally cultured in the presence of identify a role for canonical NF- B signaling for the homeostatic μ κ IKK2 inhibitor BI605906 (10 M). Histograms are of IL-7R expression in cul- maturation of new T cells. The requirement for NF- B signaling tures of TNF or CD70 ligand with vehicle (black lines), ligand and IKK2 in- was only transient because fully mature peripheral naive T cells hibitor (BI; red lines) or vehicle alone (gray fills). Data are representative of were subject to normal homeostasis when IKK2 expression was three independent experiments.

E852 | www.pnas.org/cgi/doi/10.1073/pnas.1319397111 Silva et al. Downloaded by guest on October 1, 2021 thymus that highly depended on NF-κB signaling. This de- duction of IL-7Rα in vivo. However, our data from in vitro PNAS PLUS pendence was particularly evident in F5 TCR transgenic mice culture of F5 thymocytes suggest the Tnfrsf ligands such as TNF that fail to induce IL-7Rα immediately following positive selec- and CD70 may be implicated. There is already evidence to tion. In this strain, IL-7Rα was only induced after new T cells had suggest these ligands may be present in the thymus because TNF left the thymus. Significantly, IL-7Rα expression in F5 T cells mRNA can be detected in dendritic cells ex vivo (32), and CD70 almost completely depended on IKK2 expression. There was also is already implicated in development of regulatory T cells in the evidence that NF-κB–dependent induction of IL-7Rα expression thymus (33). Future studies should address whether these ligands started in the thymus before egress in polyclonal mice because alone are sufficient for induction of Il7r expression or whether HSAlo CD8 SPs expressed more IL-7Rα protein than the cor- other NF-κB–activating receptors may be involved. responding population in either IKK2-deficient T cells or pLck- The mechanism by which NF-κB regulates Il7r expression IκB-PEST–expressing thymocytes. appears to be distinct from that of Foxo1, another key regulator Thymocytes that developed in the absence of IKK2 gave rise of IL-7Rα expression. Foxo1 is constitutively required for normal to mature T cells that failed to make normal homeostatic survival IL-7Rα expression and provides a mechanism by which cytokine or proliferative responses. In contrast, ablation of IKK2 protein signaling can actively tune peripheral expression of IL-7Rα by T in mature F5 T cells had no impact on their homeostatic survival cells. Phosphorylation of Foxo1 by PKB/Akt specifically targets and proliferative responses. Thus, IKK2 expression was not re- Foxo1 for degradation, resulting in a corresponding loss of IL- quired for the transmission of either TCR or IL-7R dependent 7Rα expression. However, Foxo1 expression was not affected in signals known to be essential for inducing both survival and the absence of IKK2 expression, and we have shown that F5 proliferation of naive T cells in response to these homeostatic thymocytes induce Foxo1 expression during thymic development signals. Rather, the failure to make normal homeostatic normally (8). The same study showed that TCR signaling in responses was associated with a failure of T cells to express selecting thymocytes normally initiates reexpression of IL-7Rα normal levels of IL-7Rα when IKK2 gene was deleted before during thymic development. In F5 thymocytes, however, TCR thymic egress. Although Bcl2 family members have been iden- signaling is not strong enough to activate Il7r gene expression tified as key transcriptional targets of NF-κB (27–29), these and F5 T cells only induce IL-7Rα in the periphery as RTE in genes did not appear to be the targets of NF-κB signaling in new a strictly NF-κB–dependent manner. Together these data reveal T cells. No differences in expression of any Bcl2 family members that Foxo1 expression alone is not sufficient to switch on IL-7Rα were identified in IKK2-deficient T cells. In particular, the sur- in postselection T cells. In contrast, it appears that either TCR or vival defect of T cells following thymic deletion of Ikk2 was NF-κB signaling are sufficient for independent activation of the clearly IL-7 dependent. Control and Ikk2-deficient T cells sur- Il7r locus. Significantly, in both cases, constitutive activity of vived equally in the absence of IL-7 in vivo, and no additive ef- these pathways is not required to maintain Il7r expression once it fect of Ikk2 deletion on cell death was observed in the absence of has been initiated (8), whereas constitutive Foxo1 expression is IL-7. In contrast, the absence of IL-15 had additive affects on critical for such maintenance in mature T cells (11). Therefore, both survival and LIP of IKK2-deficient F5 T cells, demon- we speculate that TCR signaling and NF-κB signaling are both strating that γc signaling was functional downstream of IL-15R capable of opening and initiating expression of the Il7r locus in in the absence of IKK2. Consistent with this view, γc family new T cells and that Foxo1 is required to maintain that expres- receptors, downstream JAK kinases, and STAT transcription sion. The observation that TCR and NF-κB signaling have ad- factors were expressed normally in IKK2-deficient F5 T cells. ditive effects on subsequent IL-7Rα expression suggests that they The sole exception was Il7r, which was substantially reduced. target distinct regulatory regions. Conserved NF-κB binding sites Therefore, the data strongly support the view that reduced IL- upstream of IL-7Rα have already been noted (11), and it remains 7Rα expression is the key factor that accounts for the defects in to be determined which regulatory regions are specifically tar- survival and proliferative of Ikk2-deficient T cells. geted by factors downstream of TCR signaling events. However, By deleting IKK2 from T cells at different developmental the observation that TCR signal-induced IL-7Rα expression is stages, we revealed that NF-κB was required only transiently to normal in IKK2-deficient T cells suggests that factors other than permit normal induction of IL-7Rα expression in new T cells. NF-κB are the required downstream of TCR signaling. Evidence for this conclusion came from the observation that Taken together, our data suggests that IKK2 expression during both IL-7Rα expression and T-cell homeostasis were normal development was critical for homeostatic maturation of RTE. following ablation of IKK2 in fully mature T cells. In addition, T The importance of IL-7Rα induction by new T cells for main- cells in pLck IκB-PEST–expressing mice also exhibited reduced tenance of the peripheral repertoire was evident in both poly- IL-7Rα expression. Significantly, the dominant negative IκB- clonal and F5 transgenic mice. The T-cell compartment of IKK2- PEST is expressed by using proximal Lck promotor that is deficient mice is significantly reduced, particularly in the CD8 expressed highly in thymus, but whose expression is greatly re- compartment. In F5 mice, RTE numbers were largely unaffected duced in peripheral T cells and such an expression pattern was in the absence of IKK2, although it should be noted that IL-7Rα sufficient to repress induction of IL-7Rα in peripheral T cells. expression was still very low in this subset, even in control F5 Monitoring IL-7Rα expression in F5 T cells as they left the mice. Therefore, it may not be surprising that survival of control thymus revealed that induction of IL-7Rα occurred in the first and IKK2-deficient RTE is quite similar. In contrast, the T-cell week following thymic egress, timing consistent with the pre- deficiency in IKK2-deficient F5 mice was accounted for by a viously reported induction of IL-7Rα by RTE. Therefore, it specific reduction in the size of the fully mature naive com- appears that intact NF-κB signaling is essential for induction of partment, and most likely reflects the reduced half-life of T cells IL-7Rα expression by HSAlo SP thymocytes and RTE. Deletion that fail to express normal IL-7Rα levels in the absence of IKK2. of IKK2 either early or late in thymic development using huCD2- Although up-regulation of IL-7Rα was IKK2 dependent, other iCre or CD4-Cre, respectively, had an identical outcome on the RTE markers such as CD45RB and HSA were unaffected by the subsequent expression of IL-7Rα by new T cells. Although it absence of IKK2. Complete maturation of RTE is suggested to seems likely that NF-κB signaling may be required specifically in take as long as 3 wk (9). In F5 T cells, maximum up-regulation of RTE for the induction of IL-7Rα, the genetic evidence narrows IL-7Rα occurred over ∼6–9 d. Together, these observations the possible window in which NF-κB signaling is required to one suggest that RTE maturation is not a single discrete develop- that starts following positive selection and ends after full matu- mental process but rather involves several distinct regulatory

ration of RTE. In future experiments, it will be important to identify mechanisms, of which NF-κB activation is but one. Other studies IMMUNOLOGY the receptor(s) responsible for activating NF-κB–dependent in- have implicated a role for transcriptional repressor NKAP.

Silva et al. PNAS | Published online February 18, 2014 | E853 Downloaded by guest on October 1, 2021 Thymic development is unaffected following CD4Cre-mediated on log and biexponential displays. For cell sorting, lymphocytes were in- deletion of NKAP, whereas the peripheral T-cell compartment is cubated with the appropriate antibodies for detection of surface markers profoundly lymphopenic and is comprised almost exclusively of and were then purified to >95% purity by high-speed sorting on an Aria cells with an RTE-like phenotype (34). We found NKAP mRNA flow cytometer (Becton Dickinson). To label cells with CellTrace Violet (CTV) (Invitrogen Molecular Probes), cells were washed and resuspended in 1× PBS expression was normal in IKK2-deficient F5 T cells, suggesting × 6 κ at 10 10 cells per mL. A prewarmed solution of CTV was added to the cell that NF- B activation and NKAP may mediate independent suspension to a final concentration of 5 μM and incubated for 10 min at functions during maturation of new T cells. Consistent with this 37 °C. Cells were washed three times in IMDM/2% (wt/vol) BSA (IMDM/BSA). view, IL-7Rα expression and function is reportedly normal in NKAP-deficient mice. In Vivo Measurements of Survival and Proliferation. LN cells from donor F5 In conclusion, our data provide insight into the homeostatic Rag1−/− huCD2iCre R26REYFP Ikk2fx/fx or F5 Rag1−/− R26CreERT R26REYFP Ikk2fx/fx maturation of new T cells that is essential for normal population of mice were labeled with CTV and i.v. injected in 250 μL of IMDM/BSA into the peripheral T-cell compartment. Our data show that NF-κBsignal- indicated host mice. huCD2iCre -ve littermates were used as control for T cells −/− iCre EYFP fx/fx iCre ing to new T cells is critical for completing the process of homeo- from F5 Rag1 huCD2 R26R Ikk2 . CTV-labeled huCD2 -ve and κ – huCD2iCre +ve F5 T cells were mixed 1:1 and 2 × 106 total T cells transferred. static maturation. Although the receptors that induce NF- B + − dependent up-regulation of IL-7Rα have not been reported as yet, To assess relative survival of YFP and YFP F5 T cells, expansive effects of proliferation were excluded by dividing frequency of cells in each division (n) others suggest that neither TCR nor IL-7 signaling are involved in by 2n. The ratio of these adjusted frequencies was then calculated. To ana- maturation of RTE (9). If true, then our data suggest that post- lyze response to influenza A challenge, mice were additionally injected with selection induction of IL-7Rα occurs in two phases. The first is 100 hemagglutinating (HA) units of virus (A/NT/60–68) i.v. as described (25). linked to TCR signaling during positive selection (8), whereas the second starts just as cells leave the thymus and is NF-κB dependent. Real-Time Quantitative PCR. RNA was isolated from sorted populations of cells This second phase of induction may proceed independently of TCR with TRIzol (Invitrogen) according to the manufacturer’s instructions. CDNA signaling and serve to ensure that all new T cells express a minimal was produced by reverse transcription with SuperScript II (Invitrogen) and level of IL-7Rα that permits at least some participation in the pe- standard protocols. Expression of Il7r was determined by real-time PCR with ripheralT-cellrepertoire. an Applied Biosystems ABI Prism 7900 Sequence Detection System and com- mercial 6-carboxyfluorescein (FAM)–labeled probes (Applied Biosystems). The Methods abundances of mRNAs were normalized against that of Hprt1 mRNA. Mice. Mice with conditional alleles of IKK2 (Ikk2fx/fx) (35) were intercrossed In Vitro Culture. Lymphocytes were cultured at 37 °C with 5% CO in RPMI with mice either expressing transgenic Cre under the control of the human 2 medium 1640 (Gibco, Invitrogen Corporation) supplemented with 10% (vol/ CD2 (huCD2) (19), CD4 expression elements (36), or with mice expressing vol) FBS (Gibco Invitrogen), 0.1% 2-mercaptoethanol βME (Sigma Aldrich), CreERT from Rosa26 locus (R26CreERT) (37). Rosa26 reporter YFP allele and 1% penicillin-streptomycin (Gibco Invitrogen) (RPMI-10), with or with- (R26REYFP) (20) was also bred in, to facilitate identification of cells in which out different concentrations of mIL-7 (Peprotech EC), for 48 h. Cell viability Cre recombinase had been active. The strain combinations were generated was examined by using LIVE/DEAD cell stain kit (Invitrogen Molecular as follows: huCD2Cre R26REYFP Ikk2fx/fx,CD4Cre R26REYFP Ikk2fx/fx,and CreERT EYFP fx/fx fx/WT Probes), following the manufacturer’s protocol. For the short-term signaling, R26 R26R Ikk2 . Either Cre -ve littermates or Cre +ve Ikk2 or fx/WT lymphocytes were surface stained as described, washed twice with 1× PBS Ikk1 littermates were used as control. Mice were additionally back- −/− −/− iCre EYFP and incubated for 15 min at 37 °C with prewarmed 1× PBS alone (unsti- crossed to F5 Rag1 background to generate F5 Rag1 huCD2 R26R Ikk2fx/fx and F5 Rag1−/− R26CreERT R26REYFP Ikk2fx/fx . These strains and Il15ra−/− mulated), with mIL-7. Reactions were stopped by placing samples on ice and − − − − − − − − − − − − × × Rag1 / , Il7 / Rag1 / , Rag1 / ,F5Rag1 / , Ly5.1 F5 Rag1 / , Ly5.1 C57Bl6/J, adding ice-cold 1 PBS. Cells were washed twice with cold 1 PBS and and pLck IκB-PEST (15) mice were bred in a conventional colony free of stained for p-STAT5. Recombinant TNF, BAFF, LIGHT, APRIL, TRAIL, GITRL, pathogens at the National Institute for Medical Research, London. For CD70, and TLA1 used to supplement cultures were obtained from R&D. analysis of maturation of RTE in vivo, F5 Rag1−/− mice were intrathymically injected with 10 μLof10μM CTV dye per thymic lobule. R26CreERT mice were Immunoblotting. Total cell lysates were performed in sorted populations. Cell treated with five consecutive injections of 1 mg of Tamoxifen to induce Cre lysates were immunoblotted by standard methodology, as described (38). × 5 activity and organ analysis was done no less than 10 d after first tamoxifen Briefly, equal cell numbers (2 10 per sample) were analyzed by NuPage 10% injection. Animal experiments were approved by National Institute for Bis-Tris gel (Invitrogen Novex), transferred onto PVDF membrane (Millipore), Medical Research Ethical Review Panel and under UK Home Office Project and immunoblotted with the antibodies (all diluted 1:1,000) IKK2 (Cell Signal- ζ− License 80.2506. ing Technology) and chain (in house) as loading control. Immunodetection was performed by incubation with horseradish peroxidize-conjugated anti- Flow Cytometry. Flow cytometric analysis was performed with 2–5 × 106 rabbit (1:10,000) (Southern Biotech) or anti-protein A (1:10,000) (GE Healthcare) thymocytes and 1–5 × 106 lymph node or spleen cells. Cell concentrations of and developed by enhanced chemiluminescence (Millipore). thymocytes, lymph node, and spleen cells were determined with a Scharf Instruments Casy Counter. Cells were incubated with saturating concen- RNA Sequencing. Indicated cellular populations were lysed with TRIzol trations of antibodies in 100 μL of PBS containing 0.1% BSA and 1 mM azide (Invitrogen) and RNA was prepared according to manufacturer’s instructions. −/− iCre EYFP fx/fx (PBS-BSA-azide) for 45 min at 4 °C followed by two washes in PBS-BSA-azide. RNA-seq libraries from F5 Rag1 huCD2 R26R Ikk2 Cre +ve or -ve Phycoerythrin (PE)-conjugated antibody against IL-7Rα, EF450-conjugated were prepared for sequencing with the mRNA-sEq. Eight-sample prepara- antibodies against CD4, APC-conjugated antibodies against CD5, PE-Cy5– tion kit (Illumina) according to the manufacturer’s instructions. RNA-seq li- + hi conjugated antibody against TCR (H57-597), APC-Cy7– and APC-conjugated braries from cell sorted CD8 TCR lymph node T cells from the two donor antibody against CD44, pacific orange (PO)-conjugated antibody against strains were prepared using the Illumina duplex-specific nuclease (DSN) pro- CD8, EF450-, and APC-conjugated antibodies against CD45RB, PE-Cy7- con- tocol (39). Samples were sequenced at the MRC National Institute for Medical jugated antibody against CD25, streptavidin PE-Cy7–conjugated antibody Research High Throughput Sequencing Facility by using an Illumina Genome against HSA/CD24 and Qua2 biotinylated antibodies, PE-conjugated anti- Analyzer IIx, and 36 base-pair single-end reads were obtained using the Illumina body against Ly5.2, and APC-conjugated antibody against Ly5.1 were pur- pipeline. Reads were aligned to the Mus musculus genome (mm9 assembly) chased from eBioscience. Alexa Fluor 647-conjugated antibody against using CLC Genomic Workbench (V5) with standard settings. Aligned reads were pSTAT5 (pY694) was obtained from BD Biosciences. For detection of pSTAT5, mapped to the RefSeq database and were normalized by using the DESeq cells were first stained for surface markers, fixed in 2% (wt/vol) para- method (Anders S Huber W 2010 differential expression analysis for sequence formaldehyde for 20 min at room temperature, washed in fluorescence-account data) using Avadis NGS software V1.3.1. Following normalization reads tivated cell sorting (FACS) buffer, and permeabilized in 90% methanol for 30 were displayed as reads per kilobase of exon per million reads (RPKM) (40). min on ice. Cells were then washed in FACS buffer and incubated with Alexa Fluor 647-conjugated antibody against pSTAT5 at room temperature for 30 Statistics. Differences in quantitative RT-PCR and MFI values were test by min. Eight-color flow cytometric staining was analyzed on a FACSCanto II unpaired t test (*P < 0.05, **P < 0.01), whereas differences in cell numbers (Becton Dickinson) instrument, and data analysis and color compensations between experimental groups were tested by unpaired t test with Welches were performed with FlowJo V9.5.3 software (TreeStar). Data are displayed Correction (*P < 0.05, **P < 0.01).

E854 | www.pnas.org/cgi/doi/10.1073/pnas.1319397111 Silva et al. Downloaded by guest on October 1, 2021 ACKNOWLEDGMENTS. We thank Prof. Michael Karin for the use of Ikk2fx/fx and Prof. Philip Cohen for kindly providing IKK2 inhibitor BI605906. This PNAS PLUS mice, Prof. Reinhard Voll for the provision and use of pLck-IκB-PEST mice, work is supported by MRC Programme U117573801.

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