Maintaining T lymphocytes in sufficient numbers and at an

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1 NF-κB signaling mediates homeostatic maturation of new T cells Ana Silva, Georgina Cornish 1, Steven C. Ley, and Benedict Seddon 2,3 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 T-cell compartment of the adaptive immune system. IL-7 receptor α ( IL-7Rα) expression is subject to developmental regulation and new T cells induce expression as they leave the thymus, which is essential for their long-term survival. It is not understood how this expression is regulated. Here, we identify a role for the Nuclear Factor κ-b (NF-κB) signaling pathway in controlling expression of IL-7Rα in new T cells. Perturbations to NF-κB signaling, either by deletion of Inhibitor of Kappa-B Kinase-2 (IKK2) or by inhibiting Rel dimer activity, prevented normal IL-7Rα expression in new T cells. Defective IL-7Rα expression resulted in impaired survival and homeostatic cell division responses by T cells that could be attributed to their failure to express IL-7Rα normally. Surprisingly, NF-κB signaling was only required transiently in new T cells to allow their normal expression of IL-7Rα, because IKK2 deletion in mature T cells had no effect on IL-7Rα expression or their normal homeostatic responsiveness. Therefore, we identify a developmental function for NF-κB signaling in the homeostatic maturation of new T cells, by regulating IL-7Rα expression. Maintaining T lymphocytes in sufficient numbers and at an appropriate composition of differentiation states and subtypes is essential for effective immunity. The immune system has evolved a number of homeostatic mechanisms to ensure the size and composition of the T-cell compartment remains remarkably stable over time. The cytokine interleukin (IL)-7 plays a central role in regulating homeostasis of the T-cell compartment. It is essential for normal development of αβ and γδ T cells in the thymus and provides vital survival signals for both naive and memory T cells in the periphery (1). IL-7 is produced by stromal cell components in bone marrow, thymus, and in peripheral lymphoid compartments (2), and there is extensive evidence that production of IL-7 is a key factor that determines and limits the overall size of the peripheral T-cell compartment (3, 4). The receptor for IL-7 is a member of the common-gamma chain (γc) family of cytokine receptors and consists of a heterodimeric complex of IL-7Rα and γc (5). In T cells, IL-7 signaling is primarily regulated at the level of IL-7Rα expression. During T-cell development in the thymus, expression of IL-7Rα by thymocytes is subject to dynamic developmental regulation. IL-7Rα is essential for survival and development of CD4 CD8 double negative (DN) thymocytes (6). Expression is lost at the CD4 CD8 double positive (DP) stage, ensuring that onward development of DP thymocytes is restricted to those that successfully undergo positive selection (7). Immediately following selection, however, IL-7Rα is immediately reexpressed and recent studies suggest that the strength of T-cell receptor (TCR)-mediated positive selection signaling determines the extent of reexpression by SP thymocytes (8). Following egress from the thymus, new T cells continue to mature as recent thymic emigrants (RTE), a process including the further induction of IL-7Rα (9). The identity of the signaling pathways that regulate IL-7Rα expression in new T cells remains unknown. In mature T cells, Fox family transcription factors Foxo1 and Foxp1 are required for expression of IL-7Rα in mature T cells (10, 11). In particular, expression of Foxo1 is constitutively required for IL-7Rα expression (11). Phosphorylation of Foxo1 by PKB/Akt targets its degradation and cytokine receptors that activate PKB/Akt act as negative regulators of IL-7Rα gene expression. As such, both IL-2 and IL-7 negatively regulate IL-7Rα expression (12). Foxo1 binds to an enhancer region upstream of the Il7r gene. Interestingly, the same enhancer also contains conserved binding sites for NF-κB family transcription factors (11). NF-κB transcription factors are heterodimers or homodimers of Rel family members. Dimers are sequestered in cytoplasm of cells by their association with the inhibitory proteins, inhibitors of kappa B (IκB) family, and related proteins p100 and p105. Phosphorylation of IκB by the IκB Kinase (IKK) complex targets IκB for degradation by the proteosome and releases NF-κB dimers to enter the nucleus. Canonical NF-κB signaling is mediated by a trimeric complex of two kinases, IKK1(IKKα) and IKK2(IKKβ), and a third regulatory component, NEMO (IKKγ). Activation of NF-κB by the IKK complex has been implicated in 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 single-positive (SP) thymocytes at the immature HSA hi stage (13). Partial impairment of IKK function by specific ablation of IKK2 impairs development of T reg and CD4 effector/memory cells (13). Peripheral IKK2-deficient T cells also exhibit impairments in homeostatic proliferation responses (14). NF-κB has also been implicated in thymocyte selection because overexpression of dominant-negative IκB affects both double-negative (DN) thymocyte Significance Interleukin (IL)-7 is critical for the maintenance of the peripheral T-cell compartment of the adaptive immune system. Our study identifies a role for the Nuclear Factor κ-b (NF-κB) signalling pathway in the control of IL-7 receptor expression by T cells. Following thymic selection, new T cells specifically upregulate IL-7R even as they leave the thymus, and we reveal that this expression is strictly NF-κB dependent. NF-κB signaling was only required transiently, however, and once fully mature, naive T cells did not require NF-κB signaling to maintain IL-7R expression. Therefore, we reveal a developmental role for NF-κB signaling for the normal maturation and function of new T cells. 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. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Freely available online through the PNAS open access option. 1 Present address: Academic Department of Rheumatology, Center for Molecular and Cellular Biology of Inflammation, King s College London, London SE1 1UL, United Kingdom. 2 To whom correspondence should be addressed. benedict.seddon@ucl.ac.uk. 3 Present address: Institute of Immunity and Transplantation, Division of Infection and Immunity, University College of London, Royal Free Hospital, London NW3 2PF, United Kingdom. This article contains supporting information online at /pnas /-/DCSupplemental. E846 E855 PNAS Published online February 18,

2 survival (15) and reduces development of CD8 lineage thymocytes (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-κB signaling 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 deletion was mediated by CD4 Cre that starts gene excision at the CD4 + CD8 + double-positive (DP) stage and thymocytes were not found to be protein deficient until they had reached CD4 or CD8 single-positive (SP) stage. Because NF-κB signaling has already been implicated in the thymic selection of CD8 T cells (16 18), we also analyzed mice in which Ikk2 was deleted by hucd2 icre that induces gene deletion much earlier, at the DN2 stage of development (19). Phenotype and number of thymocytes from hucd2 icre Ikk2 fx/fx and CD4 Cre Ikk2 fx/fx mice were examined compared with either Cre-negative Ikk2 fx/fx controls or Creexpressing Ikk2 fx/wt mice, in consideration of Cre toxicity. Cre activity was confirmed by measuring YFP expression from a Rosa26R YFP Cre reporter allele (R26R YFP ) (20) also present in these mice (Fig. S1A). Western blot analysis of IKK2 protein abundance in sorted YFP + thymocytes confirmed that YFP expression correlated with deletion of Ikk2 (Fig. S1B). In the absence of Ikk2 expression, number and phenotype of all thymocyte subpopulations was similar to controls, regardless of whether deletion occurred early, using hucd2 icre, or late, via CD4 Cre, in thymic development (Fig. 1 A and B). Examining numbers and phenotype of peripheral T cells that developed in the absence of Ikk2 revealed near-identical defects in naive T- cell numbers regardless of whether Ikk2 was deleted at the DN2 by hucd2 icre or DP thymocyte stages by CD4 Cre (Fig. 1 C and D). Therefore, inducing IKK2 ablation before positive selection of DPs did not further exacerbate the observed reduction in the size of the peripheral naive T-cell compartment. Naive T Cells Express Reduced IL-7Rα in the Absence of Ikk2. Homeostasis of naive T cells depends on the combined activity of TCR signaling induced by self peptide (sp) MHC in peripheral lymphoid organs and IL-7. We therefore assessed the function of homeostatic TCR and IL-7R signaling in Ikk2-deficient mice to investigate the underlying cause of the reduction in naive T cells we observed. Expression levels of CD5 reliably reports tonic homeostatic TCR signaling in peripheral T cells (21, 22). Examining Ikk2-deficient peripheral naive CD4 and CD8 T cells revealed normal CD5 expression (Fig. 2A). In contrast, IL-7Rα levels were substantially reduced on both CD4 and CD8 naive T cells (Fig. 2A).The reduction observed was similar regardless of Fig. 1. Normal thymocyte development in CD4 Cre R26R EYFP Ikk2 fx/fx and hucd2 icre R26R EYFP Ikk2 fx/fx mice. CD4 Cre R26R EYFP Ikk2 fx/fx mice and huc- D2 icre R26R EYFP aged 8 12 wk of age were analyzed by FACS (n = 5 each). CD4 Cre R26R EYFP Ikk2 fx/wt or Ikk2 fx/fx R26R EYFP were used as negative controls (n = 5). (A) Density plots are of CD4 vs. CD8 expression by total live thymocytes from the indicated strain. Numbers indicate percentage of cells in the corresponding gate. (B) Bar charts show total numbers of DP, TCR hi CD4 SP, and TCR hi CD8 SP thymocytes in either IKK2-deficient (gray bars) or control (black bars) mice. (C) Dot plots are of CD4 vs. CD8 expression by TCR hi lymph node cells from the indicated strains. (D) Bar charts show total combined numbers of CD44 lo CD25 naive CD4 or CD44 lo naive CD8 T cells from lymph node and spleen of the indicated strains. Data are representative of five or more experiments. whether Ikk2 was deleted early or late in thymic development (Fig. 2B). To see whether reduced receptor levels were due to differences in Il7r transcription, we measured Il7r mrna. Both CD4 and CD8 naive T cells from CD4 Cre Ikk2 fx/fx R26R EYFP mice had substantially reduced levels of Il7r mrna (Fig. 2C). IL-7 signaling can negatively regulate expression of IL-7Rα (12). Because Ikk2 deletion results in a reduction in total naive T-cell numbers, we sought to confirm that the loss of IL-7Rα expression was cell intrinsic to naive T cells. We therefore generated mixed irradiation chimeras by using bone marrow from CD45.1 WT and CD4 Cre Ikk2 fx/fx R26R EYFP or Ikk2 fx/fx R26R EYFP donors. Only IKK2-deficient naive T cells exhibited reduced IL-7Rα expression in chimeras (Fig. S2A). Furthermore, the relative reduction in IKK2-deficient naive CD8 T cells in mixed chimeras was at least as great as observed in intact mice, and there was evidence that competition with WT cells further reduced numbers of naive CD4 T cells in chimeras (Fig. S2B). Finally, to test the functional impact of reduced IL-7Rα expression in vitro, we measured T cells responses to IL-7 stimulation. Both survival and induction of STAT5 phosphorylation was reduced in Ikk2- deficient naive CD4 and CD8 T cells (Fig. 2 D and E), suggesting that the defect in IL-7Rα expression was functionally significant. IL-7Rα expression is under dynamic regulation during T-cell development. During thymic selection, DP thymocytes lack IL- 7Rα expression. However, IL-7Rα is reexpressed in CD4 SPs and in CD8 lineage DP3 thymocytes, which in both cases depends on IMMUNOLOGY PNAS PLUS Silva et al. PNAS Published online February 18, 2014 E847

3 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 relatively 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 HSA lo thymocytes, before they left the thymus. In Ikk2-deficient thymocytes, induction of IL-7Rα in HSA lo 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 HSA lo 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 HSA hi SP thymocytes and DP3 thymocytes for CD8 lineage cells, but a failure to further induce expression in HSA lo 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 HSA lo 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. Fig. 2. Defective IL-7Rα expression and function in the absence of IKK2 expression. CD4 Cre R26R EYFP Ikk2 fx/fx (fx/fx) and control CD4 Cre R26R EYFP Ikk2 fx/wt (fx/wt) mice (n = 5 each) were analyzed at 8 12 wk of age. (A) Histograms are of CD5 or IL-7Rα expression by CD44 lo CD25 naive CD4 or CD44 lo naive CD8 T cells from lymph nodes from IKK2-deficient (red lines) or control strains (black lines). (B) Bar charts show IL-7Rα MFI, normalized to control, of naive CD4 and naive CD8 T cells in which Ikk2 was deleted by either hucd2 icre (red) or CD4 Cre (blue). (C) Total mrna was purified from sorted CD4 + TCR hi CD44 lo CD25 (CD4) and CD8 + TCR hi CD44 lo (CD8) lymph node cells from CD4 Cre Ikk2 fx/fx R26R EYFP (Ikk2 fx/fx) and control CD4 Cre Ikk2 fx/wt R26R EYFP (Ikk2 fx/wt) mice, and Il7r gene expression was determined by quantitative real-time PCR. Bar charts show expression relative to HPRT by the indicated populations. (D) Total lymph node T cells were cultured in different doses of IL-7 and cell viability assessed by flow cytometry at 48 h. Plots are of percent viable cells for the indicated subset from cultures of IKK2 knockout (Ikk2 fx/fx Cre+) and control (Ikk2 fx/fx Cre-) mice. (E) Total lymph 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 STAT5 stain for the indicated subset from cultures of IKK2 knockout (Ikk2 fx/fx Cre+) and control (Ikk2 fx/wt Cre+) mice. Data and statistically significant differences are representative of three (D and E) ormore(a C) experiments. 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 restricted TCR specific for a peptide of influenza nucleoprotein (24). Our previous studies have shown that successful induction of Il7r gene expression by developing thymocytes depends on the strength of positive selection signaling. Neither DP3 nor CD8SP thymocytes in F5 mice induce Il7r during or following positive selection in the thymus, because it is thought the avidity of their TCR for self peptide-mhc (spmhc) is not sufficiently strong (8). After leaving the thymus, however, F5 T cells do nevertheless induce expression of IL-7Rα and it is not known how this late induction of IL-7Rα is controlled. Because we identified a role for IKK2 in the induction of IL-7Rα late in the maturation of SP thymocytes, we asked whether induction of IL-7Rα in peripheral F5 T cells depended on IKK2 signaling. To test this idea, we generated F5 Rag1 / hucd2 icre R26R EYFP Ikk2 fx/fx mice. Consistent with observations in polyclonal hucd2 icre Ikk2 fx/fx R26R YFP mice, phenotype and number of thymocytes from F5 Rag1 / hucd2 icre R26R EYFP Ikk2 fx/fx mice was similar to controls (Fig. 4A). In contrast, numbers of peripheral F5 T cells were substantially reduced in the absence of IKK2 (Fig. 4B). Significantly, in the absence of IKK2, peripheral F5 T cells almost completely failed to induce IL-7Rα expression (Fig. 4B). There- E848 Silva et al.

4 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 hucd2 icre R26R EYFP Ikk2 fx/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 hucd2 icre R26R EYFP Ikk2 fx/fx mice (gray bars) or Cre -ve littermate controls (black bars) (n = 5 each). CD4 lineage subsets are CD4SP HSA hi thymocytes (HSAhi), CD4SP HSA lo thymocytes (HSAlo) and peripheral CD4 + TCR hi CD44 lo CD25 naive T cells (LN). CD8 lineage subsets analyzed were DP3 thymocytes (DP3) (41), CD8SP HSA hi thymocytes (HSAhi), CD8SP HSA lo thymocytes (HSAlo), and peripheral CD8 + TCR hi CD44 lo 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 TCR hi lymph node T cells from the indicated strain. (D) Histograms are of IL-7Rα by CD4 + TCR hi CD44 lo CD25 naive and CD8 + TCR hi CD44 lo 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 < fore, in F5 mice, IL-7Rα expression by CD8 T cells almost completely depends on the expression of IKK2. IKK2 Is Required for Induction of IL-7Rα by RTE in F5 Mice. To determine more precisely the timing of IL-7Rα expression by F5 T cells, CTV cell dye was injected intrathymically into F5 mice to label a cohort of thymocytes and the phenotype of new F5 T cells assessed as they egressed from thymus to peripheral lymph nodes. CD24 or Heat Stable Antigen (HSA) is expressed at high levels by thymocytes and lost once cells leave the thymus. Consistent with this observation, CTV + F5 T cells were still HSA int 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 HSA int in F5 mice. In light of the changes in HSA expression observed in CTV-labeled F5 T cells, this population likely represents 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 HSA int CD8 T cells in normal F5 hosts (Fig. 5B). That HSA int 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). HSA int F5 T cells were also CD45RB lo (Fig. 5C). Analyzing IL-7Rα expression by HSA int F5 T cells further confirmed that induction of IL-7Rα by F5 T cells occurs among the RTE population. Induction of IL-7Rα by HSA int cells also depends on IKK2 because HSA int 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 HSA int and HSA lo mature naive T cells in F5 Rag1 / Ikk2 fx/fx hucd2 icre R26R YFP mice. Significantly, HSA int 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 HSA lo peripheral F5 T cells were specifically reduced in F5 Rag1 / Ikk2 fx/fx hucd2 icre R26R YFP mice (Fig. 5D), and their under representation accounted for the reduced numbers of peripheral T cells observed in the IKK2-deficient F5 strain. Fig. 4. F5 TCR transgenic T cells fail to induce IL-7Rα in the absence of IKK2. F5 Rag1 / hucd2 icre R26R EYFP Ikk2 fx/fx mice and Cre littermate controls were analyzed at 8 12 wk of age. (A) Density plots are of CD4 vs. CD8 by thymocytes from Cre + and Cre F5 Rag1 / hucd2 icre R26R EYFP Ikk2 fx/fx mice. Bar charts are of total numbers of DP thymocytes (DPs) and CD8 SP thymocytes (SP8) from a litter of Cre + (n = 7) and Cre (n =4)F5Rag1 / hucd2 icre R26R EYFP Ikk2 fx/fx mice. (B) Histograms are of IL-7Rα by CD8 + TCR hi lymph node T cells (solid lines) from F5 Rag1 / hucd2 icre R26R EYFP Ikk2 fx/fx or Cre 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 from lymph nodes and spleen of the mice described in A. Data and statistically significant differences are representative of eight independent experiments. **P < IMMUNOLOGY PNAS PLUS Silva et al. PNAS Published online February 18, 2014 E849

5 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 HSA int (red) and HSA lo (black) T cells. Histograms of CD45RB and IL-7Rα are from gated HSA int (red line) and HSA lo (black lines) F5 T cells from Cre + and Cre F5 Rag1 / hucd2 icre R26R EYFP Ikk2 fx/fx mice. (D) Bar charts show total numbers of HSA int and HSA lo F5 T cells recovered from lymph nodes and spleen of Cre + and Cre F5 Rag1 / hucd2 icre R26R EYFP Ikk2 fx/fx mice. Data and statistically significant differences are representative of four independent experiments. *P < Defective Homeostasis of Ikk2-Deficient F5 T Cells Is Strictly IL-7 Dependent. Because IL-7Rα expression by peripheral CD8 T cells in F5 mice almost completely depends on IKK2 expression, we used this system to investigate the effect of IKK2 deficiency on homeostatic responses of T cells. Homeostatic proliferation by F5 T cells to lymphopenia depends on cytokines IL-7 and, to a lesser extent, IL-15 (25). Following their transfer to lymphopenic Rag1 / hosts, proliferation of YFP+ IKK2-deficient F5 T cells was greatly reduced compared with control F5 T cells, cotransferred to the same hosts (Fig. 6A). The few divisions observed among IKK2-deficient F5 T cells were in part IL-15 dependent, because the same T cells transferred to Rag1 / Il15ra / mice, which have no functional IL-15 (26), failed to proliferate at all (Fig. 6A).There was no proliferation at all in the absence of host IL-7 in Il7 / Rag1 -/ hosts, confirming the nonredundant role of this cytokine in the response. We next assessed survival of IKK2-deficient F5 T cells. First, analyzing cell recoveries from these same experiments revealed substantially reduced recoveries of IKK2-deficient F5 T cells relative to control F5 T cells, which could not be accounted for simply by differences in cell division in lymphopenic Rag1 / hosts (Fig. 6B). Death of IKK2-deficient F5 T cells was greatest in IL-15 deficient hosts, probably because the loss of IL-15 exacerbated the survival defect of IKK2-deficient F5 T cells that already fail to express IL-7Rα. Furthermore, the additive effect of IL-15 deficiency on survival suggested that the requirement for IKK2 was not downstream IL-15 signaling and also that downstream γc cytokine signaling was otherwise intact in IKK2-deficient F5 T cells. To measure survival in replete hosts, IKK2-deficient F5 T cells were transferred to congenic replete F5 hosts. Significantly, IKK2-deficient F5 T cells disappeared more rapidly than IKK2 sufficient control F5 T cells (Fig. 6B). It was possible that the reduced levels of IL-7Rα on IKK2- deficient F5 T cells was alone sufficient to account for the reduced lymphopenia-induced proliferation and survival of F5 T cells. Indeed, IL-15 signaling was able to induce both survival and proliferation of IKK2-deficient F5 T cells, suggesting that downstream γc cytokine signaling was otherwise intact. Analyzing gene expression in IKK2-deficient F5 T cells revealed that other components of the IL-2R family and their associated downstream signaling components were expressed normally in the absence of IKK2 expression (Fig. S3). However, NF-κB signaling is conventionally thought to control cell survival by regulating expression of Bcl2 family members (27 29). Therefore, it was also possible that deletion of IKK2 resulted in other gene expression changes that contributed to the defective survival and proliferation of F5 T cells. To investigate this idea, we first measured expression of the Bcl2 family genes that are expressed in F5 T cells. Significantly, no differences in expression were observed in the absence of IKK2 expression (Fig. 6C). Second, we wanted to determine whether death of IKK2-deficient F5 T cells strictly depended on IL-7 signaling pathway. Others report that ectopic expression of IL-7Rα paradoxically results in cell death (30), because expression is not subject to the same negative regulation of the endogenous Il7r locus (12). Therefore, we instead asked whether IKK2 deficiency had an additive effect on F5 T-cell death in the absence of IL-7, which would indicate an IL-7 independent survival function for IKK2. Significantly, control and IKK2-deficient F5 T cells cotransferred to IL-7 deficient hosts both maintained a similar representation (Fig. 6B) and died with similar kinetics to one another (Fig. 6D). There was therefore no evidence of an additive effect of IKK2 deficiency on cell death in the absence of IL-7. Together, these data suggest that IKK2 dependent up-regulation of IL-7Rα by new T cells is a key function for IKK2 for the normal survival and homeostasis of T cells. Ikk2 Expression Is Required Only Transiently for Normal Peripheral T-cell Homeostasis. Our data showed that IKK2 expression is required for normal T-cell homeostasis and that failure to express IL-7Rα at normal levels is a key underlying mechanism. However, in principle, it was possible that IKK2 expression was also required downstream of TCR and/or IL-7 for the transduction of signals required for homeostatic survival and proliferative responses. Furthermore, previous studies have identified Fox family transcription factors as a critical regulator of IL-7Rα E850 Silva et al.

6 Fig. 6. Defective homeostatic responses of F5 T cells in the absence of IKK2. F5 T cells from F5 Rag1 / hucd2 icre+ R26R EYFP Ikk2 fx/fx (IKK2 ko) and huc- D2 icre- littermates (IKK2 wt) were labeled with CTV cell dye, mixed 1:1, and total cells were transferred to either Rag1 /, Il15ra / Rag1 /, Il7 / Rag1 / or Ly5.1 F5 Rag1 / hosts for 14 d. (A) Histograms are of cell dye labeling in the indicated host by the indicted donor population at d7 and d14 after transfer. (B) Relative cell frequencies of YFP + and YFP donor cells 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 hosts. (C) mrna was isolated from sorted CD8 + TCR hi T cells from F5 Rag1 / Ikk2 fx/fx R26R EYFP hucd2 icre and hucd2 icre -ve littermate. Total mrna was sequenced by a Illumina Genome Analyzer IIx. Following normalization reads were displayed as normalized reads per kilobase of exon per million reads (nrpkm). Bar charts show expression level (nrpkm) of the expression in peripheral T cells (10, 11). They are constitutively 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 R26 CreERT by tamoxifen administration to F5 Rag1 / R26 CreERT R26R EYFP Ikk2 fx/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 intrinsic, we also transferred F5 T cells from F5 Rag1 / R26 CreERT R26R EYFP Ikk2 fx/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 / R26 CreERT R26R EYFP Ikk2 fx/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 transmitting 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 detectable for TNF receptor I and II, TACI (Tnfrsf13b), LIGHTR (Tnfrsf14), GITR (Tnfrsf18), Tnfrsf25, Tnfrsf26, and CD27(Fig. 8A). A similar pattern of expression was also observed by IKK2- deficient F5 T cells (Fig. 8A). F5 CD8 SP thymocytes were stimulated with ligands for these receptors to determine which, if any, could induce expression of IL-7Rα. Culture of thymocytes with APRIL or BAFF (TACI ligands), LIGHT (LIGHTR ligand), TRAIL (ligand for Tnfrsf26), GITRL (GITR ligand), or TL1A (ligand for DR3/Tnfrsf25) failed to induce IL-7Rα expression. Significantly, however, both TNF and soluble CD70 (ligand for indicated genes in control F5 Rag1 / hucd2 icre- R26R EYFP Ikk2 fx/fx samples (black bars; IKK2 WT) vs. F5 Rag1 / hucd2 icre+ R26R EYFP Ikk2 fx/fx samples (red bars; IKK2 KO). Data are mean ± SD RPKM from triplicate biological replicates. (D) Graphs shows cell recovery from spleen, normalized to day 1, of IKK2 WT and IKK2 KO F5 T cells cotransferred to Il7 / Rag1 / hosts. Data are representative of five independent experiments. IMMUNOLOGY PNAS PLUS Silva et al. PNAS Published online February 18, 2014 E851

7 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 HSA hi 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 / R26 CreERT2 R26R EYFP Ikk2 fx/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 + TCR hi T cells from F5 Rag1 / R26 CreERT2 R26R EYFP Ikk2 fx/fx mice 5 d after the final injection with tamoxifen. (B) CD8 + TCR hi 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 / R26 CreERT2 R26R EYFP Ikk2 fx/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 + TCR hi cells. Histogram of YFP expression is by donor CD CD8 + TCR hi 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 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 BI (31) prevented induction of IL-7Rα in control F5 thymocytes (Fig. 8D), whereas IKK2-deficient F5 T cells expressed less IL- 7Rα in response to culture with either ligand (Fig. 8D). Taken together, these data show that homeostatic maturation of new T cells can be induced in vitro by ligation of Tnfrsf members including TNFR and CD27. Discussion Following thymic selection, new T cells induce expression of IL- 7Rα, which is essential for their long-term survival and integration into the peripheral repertoire (8). The mechanisms governing this process are incompletely understood. Here, we identify a role for canonical NF-κB signaling for the homeostatic maturation of new T cells. The requirement for NF-κB signaling was only transient because fully mature peripheral naive T cells were subject to normal homeostasis when IKK2 expression was Fig. 8. Tnfrsf ligands induce IL-7Rα expression on SP thymocytes in vitro. (A) Bar charts show expression level (nrpkm) of the indicated Tnfrsf genes by control (black bars) and IKK2-deficient F5 T cells (red bars). (B) F5Rag1 / thymocytes were cultured for 24 h with either TNF (10 ng/ml), BAFF (100 ng/ 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). Histograms 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 Rag1 / thymocytes were cultured for 24 h with TNF or CD70. Histograms are of IL-7R expression by the indicated subset in the presence of the indicated ligand (red lines) compared with control cultures with no added ligand (gray fills). (D) Thymocytes from hucd2 icre +ve (IKK2 KO) and -ve (WT) F5 Rag1 / R26R EYFP Ikk2 fx/fx donors were cultured for 24 h with TNF or CD70. Cultures of icre -ve control thymocytes were additionally cultured in the presence of IKK2 inhibitor BI (10 μm). Histograms are of IL-7R expression in cultures of TNF or CD70 ligand with vehicle (black lines), ligand and IKK2 inhibitor (BI; red lines) or vehicle alone (gray fills). Data are representative of three independent experiments. E852 Silva et al.

8 thymus that highly depended on NF-κB signaling. This dependence was particularly evident in F5 TCR transgenic mice that fail to induce IL-7Rα immediately following positive selection. In this strain, IL-7Rα was only induced after new T cells had left the thymus. Significantly, IL-7Rα expression in F5 T cells almost completely depended on IKK2 expression. There was also evidence that NF-κB dependent induction of IL-7Rα expression started in the thymus before egress in polyclonal mice because HSA lo CD8 SPs expressed more IL-7Rα protein than the corresponding population in either IKK2-deficient T cells or plck- IκB-PEST expressing thymocytes. Thymocytes that developed in the absence of IKK2 gave rise to mature T cells that failed to make normal homeostatic survival or proliferative responses. In contrast, ablation of IKK2 protein in mature F5 T cells had no impact on their homeostatic survival and proliferative responses. Thus, IKK2 expression was not required for the transmission of either TCR or IL-7R dependent signals known to be essential for inducing both survival and proliferation of naive T cells in response to these homeostatic signals. Rather, the failure to make normal homeostatic responses was associated with a failure of T cells to express normal levels of IL-7Rα when IKK2 gene was deleted before thymic egress. Although Bcl2 family members have been identified as key transcriptional targets of NF-κB (27 29), these genes did not appear to be the targets of NF-κB signaling in new T cells. No differences in expression of any Bcl2 family members were identified in IKK2-deficient T cells. In particular, the survival defect of T cells following thymic deletion of Ikk2 was clearly IL-7 dependent. Control and Ikk2-deficient T cells survived equally in the absence of IL-7 in vivo, and no additive effect of Ikk2 deletion on cell death was observed in the absence of IL-7. In contrast, the absence of IL-15 had additive affects on both survival and LIP of IKK2-deficient F5 T cells, demonstrating that γc signaling was functional downstream of IL-15R in the absence of IKK2. Consistent with this view, γc family receptors, downstream JAK kinases, and STAT transcription factors were expressed normally in IKK2-deficient F5 T cells. The sole exception was Il7r, which was substantially reduced. Therefore, the data strongly support the view that reduced IL- 7Rα expression is the key factor that accounts for the defects in survival and proliferative of Ikk2-deficient T cells. By deleting IKK2 from T cells at different developmental stages, we revealed that NF-κB was required only transiently to permit normal induction of IL-7Rα expression in new T cells. Evidence for this conclusion came from the observation that both IL-7Rα expression and T-cell homeostasis were normal following ablation of IKK2 in fully mature T cells. In addition, T cells in plck IκB-PEST expressing mice also exhibited reduced IL-7Rα expression. Significantly, the dominant negative IκB- PEST is expressed by using proximal Lck promotor that is expressed highly in thymus, but whose expression is greatly reduced in peripheral T cells and such an expression pattern was sufficient to repress induction of IL-7Rα in peripheral T cells. Monitoring IL-7Rα expression in F5 T cells as they left the thymus revealed that induction of IL-7Rα occurred in the first week following thymic egress, timing consistent with the previously reported induction of IL-7Rα by RTE. Therefore, it appears that intact NF-κB signaling is essential for induction of IL-7Rα expression by HSA lo SP thymocytes and RTE. Deletion of IKK2 either early or late in thymic development using hucd2- icre or CD4-Cre, respectively, had an identical outcome on the subsequent expression of IL-7Rα by new T cells. Although it seems likely that NF-κB signaling may be required specifically in RTE for the induction of IL-7Rα, the genetic evidence narrows the possible window in which NF-κB signaling is required to one that starts following positive selection and ends after full maturation of RTE. In future experiments, it will be important to identify the receptor(s) responsible for activating NF-κB dependent induction of IL-7Rα in vivo. However, our data from in vitro culture of F5 thymocytes suggest the Tnfrsf ligands such as TNF and CD70 may be implicated. There is already evidence to suggest these ligands may be present in the thymus because TNF mrna can be detected in dendritic cells ex vivo (32), and CD70 is already implicated in development of regulatory T cells in the thymus (33). Future studies should address whether these ligands alone are sufficient for induction of Il7r expression or whether other NF-κB activating receptors may be involved. The mechanism by which NF-κB regulates Il7r expression appears to be distinct from that of Foxo1, another key regulator of IL-7Rα expression. Foxo1 is constitutively required for normal IL-7Rα expression and provides a mechanism by which cytokine signaling can actively tune peripheral expression of IL-7Rα by T cells. Phosphorylation of Foxo1 by PKB/Akt specifically targets Foxo1 for degradation, resulting in a corresponding loss of IL- 7Rα expression. However, Foxo1 expression was not affected in the absence of IKK2 expression, and we have shown that F5 thymocytes induce Foxo1 expression during thymic development normally (8). The same study showed that TCR signaling in selecting thymocytes normally initiates reexpression of IL-7Rα during thymic development. In F5 thymocytes, however, TCR signaling is not strong enough to activate Il7r gene expression and F5 T cells only induce IL-7Rα in the periphery as RTE in a strictly NF-κB dependent manner. Together these data reveal that Foxo1 expression alone is not sufficient to switch on IL-7Rα in postselection T cells. In contrast, it appears that either TCR or NF-κB signaling are sufficient for independent activation of the Il7r locus. Significantly, in both cases, constitutive activity of these pathways is not required to maintain Il7r expression once it has been initiated (8), whereas constitutive Foxo1 expression is critical for such maintenance in mature T cells (11). Therefore, we speculate that TCR signaling and NF-κB signaling are both capable of opening and initiating expression of the Il7r locus in new T cells and that Foxo1 is required to maintain that expression. The observation that TCR and NF-κB signaling have additive effects on subsequent IL-7Rα expression suggests that they target distinct regulatory regions. Conserved NF-κB binding sites upstream of IL-7Rα have already been noted (11), and it remains to be determined which regulatory regions are specifically targeted by factors downstream of TCR signaling events. However, the observation that TCR signal-induced IL-7Rα expression is normal in IKK2-deficient T cells suggests that factors other than NF-κB are the required downstream of TCR signaling. Taken together, our data suggests that IKK2 expression during development was critical for homeostatic maturation of RTE. The importance of IL-7Rα induction by new T cells for maintenance of the peripheral repertoire was evident in both polyclonal and F5 transgenic mice. The T-cell compartment of IKK2- deficient mice is significantly reduced, particularly in the CD8 compartment. In F5 mice, RTE numbers were largely unaffected in the absence of IKK2, although it should be noted that IL-7Rα expression was still very low in this subset, even in control F5 mice. Therefore, it may not be surprising that survival of control and IKK2-deficient RTE is quite similar. In contrast, the T-cell deficiency in IKK2-deficient F5 mice was accounted for by a specific reduction in the size of the fully mature naive compartment, and most likely reflects the reduced half-life of T cells that fail to express normal IL-7Rα levels in the absence of IKK2. Although up-regulation of IL-7Rα was IKK2 dependent, other RTE markers such as CD45RB and HSA were unaffected by the absence of IKK2. Complete maturation of RTE is suggested to take as long as 3 wk (9). In F5 T cells, maximum up-regulation of IL-7Rα occurred over 6 9 d. Together, these observations suggest that RTE maturation is not a single discrete developmental process but rather involves several distinct regulatory mechanisms, of which NF-κB activation is but one. Other studies have implicated a role for transcriptional repressor NKAP. IMMUNOLOGY PNAS PLUS Silva et al. PNAS Published online February 18, 2014 E853

9 Thymic development is unaffected following CD4 Cre -mediated deletion of NKAP, whereas the peripheral T-cell compartment is profoundly lymphopenic and is comprised almost exclusively of cells with an RTE-like phenotype (34). We found NKAP mrna expression was normal in IKK2-deficient F5 T cells, suggesting that NF-κB activation and NKAP may mediate independent functions during maturation of new T cells. Consistent with this view, IL-7Rα expression and function is reportedly normal in NKAP-deficient mice. In conclusion, our data provide insight into the homeostatic maturation of new T cells that is essential for normal population of peripheral T-cell compartment. Our data show that NF-κB signaling to new T cells is critical for completing the process of homeostatic maturation. Although the receptors that induce NF-κB dependent up-regulation of IL-7Rα have not been reported as yet, others suggest that neither TCR nor IL-7 signaling are involved in maturation of RTE (9). If true, then our data suggest that postselection induction of IL-7Rα occurs in two phases. The first is linked to TCR signaling during positive selection (8), whereas the second starts just as cells leave the thymus and is NF-κB dependent. This second phase of induction may proceed independently of TCR signaling and serve to ensure that all new T cells express a minimal level of IL-7Rα that permits at least some participation in the peripheralt-cellrepertoire. Methods Mice. Mice with conditional alleles of IKK2 (Ikk2 fx/fx ) (35) were intercrossed with mice either expressing transgenic Cre under the control of the human CD2 (hucd2) (19), CD4 expression elements (36), or with mice expressing CreERT from Rosa26 locus (R26 CreERT ) (37). Rosa26 reporter YFP allele (R26R EYFP ) (20) was also bred in, to facilitate identification of cells in which Cre recombinase had been active. The strain combinations were generated as follows: hucd2 Cre R26R EYFP Ikk2 fx/fx, CD4 Cre R26R EYFP Ikk2 fx/fx, and R26 CreERT R26R EYFP Ikk2 fx/fx. Either Cre -ve littermates or Cre +ve Ikk2 fx/wt or Ikk1 fx/wt littermates were used as control. Mice were additionally backcrossed to F5 Rag1 / background to generate F5 Rag1 / hucd2 icre R26R EYFP Ikk2 fx/fx and F5 Rag1 / R26 CreERT R26R EYFP Ikk2 fx/fx. These strains and Il15ra / Rag1 /, Il7 / Rag1 /, Rag1 /,F5Rag1 /, Ly5.1 F5 Rag1 /, Ly5.1 C57Bl6/J, and plck IκB-PEST (15) mice were bred in a conventional colony free of pathogens at the National Institute for Medical Research, London. For analysis of maturation of RTE in vivo, F5 Rag1 / mice were intrathymically injected with 10 μl of10μm CTV dye per thymic lobule. R26 CreERT mice were treated with five consecutive injections of 1 mg of Tamoxifen to induce Cre activity and organ analysis was done no less than 10 d after first tamoxifen injection. Animal experiments were approved by National Institute for Medical Research Ethical Review Panel and under UK Home Office Project License Flow Cytometry. Flow cytometric analysis was performed with thymocytes and lymph node or spleen cells. Cell concentrations of thymocytes, lymph node, and spleen cells were determined with a Scharf Instruments Casy Counter. Cells were incubated with saturating concentrations of antibodies in 100 μl of PBS containing 0.1% BSA and 1 mm azide (PBS-BSA-azide) for 45 min at 4 C followed by two washes in PBS-BSA-azide. Phycoerythrin (PE)-conjugated antibody against IL-7Rα, EF450-conjugated antibodies against CD4, APC-conjugated antibodies against CD5, PE-Cy5 conjugated antibody against TCR (H57-597), APC-Cy7 and APC-conjugated antibody against CD44, pacific orange (PO)-conjugated antibody against CD8, EF450-, and APC-conjugated antibodies against CD45RB, PE-Cy7- conjugated antibody against CD25, streptavidin PE-Cy7 conjugated antibody against HSA/CD24 and Qua2 biotinylated antibodies, PE-conjugated antibody against Ly5.2, and APC-conjugated antibody against Ly5.1 were purchased from ebioscience. Alexa Fluor 647-conjugated antibody against pstat5 (py694) was obtained from BD Biosciences. For detection of pstat5, cells were first stained for surface markers, fixed in 2% (wt/vol) paraformaldehyde for 20 min at room temperature, washed in fluorescence-activated cell sorting (FACS) buffer, and permeabilized in 90% methanol for 30 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 min. Eight-color flow cytometric staining was analyzed on a FACSCanto II (Becton Dickinson) instrument, and data analysis and color compensations were performed with FlowJo V9.5.3 software (TreeStar). Data are displayed on log and biexponential displays. For cell sorting, lymphocytes were incubated with the appropriate antibodies for detection of surface markers and were then purified to >95% purity by high-speed sorting on an Aria flow cytometer (Becton Dickinson). To label cells with CellTrace Violet (CTV) (Invitrogen Molecular Probes), cells were washed and resuspended in 1 PBS at cells per ml. A prewarmed solution of CTV was added to the cell suspension to a final concentration of 5 μm and incubated for 10 min at 37 C. Cells were washed three times in IMDM/2% (wt/vol) BSA (IMDM/BSA). In Vivo Measurements of Survival and Proliferation. LN cells from donor F5 Rag1 / hucd2 icre R26R EYFP Ikk2 fx/fx or F5 Rag1 / R26 CreERT R26R EYFP Ikk2 fx/fx mice were labeled with CTV and i.v. injected in 250 μl of IMDM/BSA into the indicated host mice. hucd2 icre -ve littermates were used as control for T cells from F5 Rag1 / hucd2 icre R26R EYFP Ikk2 fx/fx. CTV-labeled hucd2 icre -ve and hucd2 icre +ve F5 T cells were mixed 1:1 and total T cells transferred. 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) by 2 n. The ratio of these adjusted frequencies was then calculated. To analyze response to influenza A challenge, mice were additionally injected with 100 hemagglutinating (HA) units of virus (A/NT/60 68) i.v. as described (25). Real-Time Quantitative PCR. RNA was isolated from sorted populations of cells with TRIzol (Invitrogen) according to the manufacturer s instructions. CDNA was produced by reverse transcription with SuperScript II (Invitrogen) and standard protocols. Expression of Il7r was determined by real-time PCR with an Applied Biosystems ABI Prism 7900 Sequence Detection System and commercial 6-carboxyfluorescein (FAM) labeled probes (Applied Biosystems). The abundances of mrnas were normalized against that of Hprt1 mrna. In Vitro Culture. Lymphocytes were cultured at 37 C with 5% CO 2 in RPMI medium 1640 (Gibco, Invitrogen Corporation) supplemented with 10% (vol/ vol) FBS (Gibco Invitrogen), 0.1% 2-mercaptoethanol βme (Sigma Aldrich), and 1% penicillin-streptomycin (Gibco Invitrogen) (RPMI-10), with or without different concentrations of mil-7 (Peprotech EC), for 48 h. Cell viability was examined by using LIVE/DEAD cell stain kit (Invitrogen Molecular Probes), following the manufacturer s protocol. For the short-term signaling, lymphocytes were surface stained as described, washed twice with 1 PBS and incubated for 15 min at 37 C with prewarmed 1 PBS alone (unstimulated), with mil-7. Reactions were stopped by placing samples on ice and adding ice-cold 1 PBS. Cells were washed twice with cold 1 PBS and stained for p-stat5. Recombinant TNF, BAFF, LIGHT, APRIL, TRAIL, GITRL, CD70, and TLA1 used to supplement cultures were obtained from R&D. Immunoblotting. Total cell lysates were performed in sorted populations. Cell lysates were immunoblotted by standard methodology, as described (38). Briefly, equal cell numbers ( per sample) were analyzed by NuPage 10% Bis-Tris gel (Invitrogen Novex), transferred onto PVDF membrane (Millipore), and immunoblotted with the antibodies (all diluted 1:1,000) IKK2 (Cell Signaling Technology) and ζ chain (in house) as loading control. Immunodetection was performed by incubation with horseradish peroxidize-conjugated antirabbit (1:10,000) (Southern Biotech) or anti-protein A (1:10,000) (GE Healthcare) and developed by enhanced chemiluminescence (Millipore). RNA Sequencing. Indicated cellular populations were lysed with TRIzol (Invitrogen) andrna was prepared according tomanufacturer s instructions. RNA-seq libraries from F5 Rag1 / hucd2 icre R26R EYFP Ikk2 fx/fx Cre +ve or -ve were prepared for sequencing with the mrna-seq. Eight-sample preparation kit (Illumina) according to the manufacturer s instructions. RNA-seq libraries from cell sorted CD8 + TCR hi lymph node T cells from the two donor strains were prepared using the Illumina duplex-specific nuclease (DSN) protocol (39). Samples were sequenced at the MRC National Institute for Medical Research High Throughput Sequencing Facility by using an Illumina Genome Analyzer IIx, and 36 base-pair single-end reads were obtained using the Illumina pipeline. Reads were aligned to the Mus musculus genome (mm9 assembly) using CLC Genomic Workbench (V5) with standard settings. Aligned reads were mapped to the RefSeq database and were normalized by using the DESeq method (Anders S Huber W 2010 differential expression analysis for sequence count data) using Avadis NGS software V Following normalization reads were displayed as reads per kilobase of exon per million reads (RPKM) (40). Statistics. Differences in quantitative RT-PCR and MFI values were test by unpaired t test (*P < 0.05, **P < 0.01), whereas differences in cell numbers between experimental groups were tested by unpaired t test with Welches Correction (*P < 0.05, **P < 0.01). E854 Silva et al.

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11 Supporting Information Silva et al /pnas Fig. S1. Cre activity and protein ablation by CD4 Cre and hucd2 icre drivers. (A) Histograms are of YFP expression by the indicated thymocyte subset from either hucd2 icre R26R EYFP Ikk2 fx/fx or CD4 Cre R26R EYFP Ikk2 fx/fx mice. (B)YFP + cells were sorted from DP thymocytes of CD4 Cre R26R EYFP Ikk2 fx/fx donors, and DP, total SP, and total lymph node cells are from hucd2 icre R26R EYFP Ikk2 fx/fx donors. The same subsets were sorted from Cre littermates as control, and IKK2 protein levels in cell lysates were determined by Western blot. Tubulin protein was probed as control. Fig. S2. Reduced IL-7Rα expression by IKK2-deficient T cells is cell intrinsic. (A) Mixed irradiation bone marrow chimeras were generated by using 1:1 mix of T- cell depleted bone marrow (10 7 total cells) from CD45.1 C57Bl6/J donors and Ikk2 fx/fx R26R EYFP CD4 Cre (Ly5.1:Cre + )orikk2 fx/fx R26R EYFP as control (Ly5.1:Cre ), transferred into irradiated (500 rads) Rag1 / recipients. (A) After 8 wk, CD4 and CD8 SP thymocytes and peripheral naive CD4 and naive CD8 T cells were analyzed for their expression of IL-7Rα. Histograms show IL-7Rα expression by the indicated subset of Ly5.1 origin (black line) or experimental Ikk2 fx/fx R26R EYFP CD4 Cre + or Cre origin (red lines). (B) Plot shows ratio of naive IKK2-deficient T cells to WT CD4 (diamonds) or CD8 (circles) subsets in mixed chimeras (red symbols), normalized to ratio in DP thymocytes, compared with the ratio of IKK2 deficient to WT naive CD4 and CD8 subsets found in intact mice (black symbols), using data reported in Fig. 1. Data are representative of two independent experiments. Silva et al. 1of2

12 Fig. S3. Cytokine signaling components are normally expressed in the absence of IKK2. RNA sequencing described in Fig. 6C was also analyzed for expression of Fox factors, Il2r family, Jak family, and Stat family members. Fig. S4. IL-7Rα expression by naive T cells does not require constitutive IKK2 expression. R26 CreERT2 R26R EYFP Ikk2 fx/fx mice or R26 CreERT2 R26R EYFP Ikk2 fx/wt controls were injected with tamoxifen for 5 consecutive days. Four weeks later, YFP expression and IL-7Rα levels were determined by flow cytometry. Histograms show YFP expression by total lymph node T cells, and IL-7Rα expression by CD4 + CD44 lo CD25 naive and CD8 + CD44 lo naive T cells from the indicated strain. Data are representative of three experiments. Silva et al. 2of2