ICOS) Ligand Expression and T Follicular Helper Cell Development

Noncanonical NF-κB regulates inducible costimulator (ICOS) ligand expression and T follicular helper cell development

Hongbo Hua,1, Xuefeng Wua,1,2, Wei Jina,3, Mikyoung Changa, Xuhong Chenga, and Shao-Cong Suna,b,4

aDepartment of Immunology, University of Texas MD Anderson Cancer Center, Houston, TX 77030 and bUniversity of Texas Graduate School of Biomedical Sciences, Houston, TX 77030

Edited by Michael Karin, University of California San Diego School of Medicine, La Jolla, CA, and approved June 27, 2011 (received for review April 12, 2011)

Follicular helper T (Tfh) cells have a central role in mediating a κB enhancer (16, 17). NF-κB is normally sequestered in the humoral immune responses. Generation of Tfh cells depends on cytoplasm by inhibitory proteins (IκBs), and NF-κB activation both T-cell intrinsic factors and the supporting function of B cells, typically involves inducible degradation of IκBα and nuclear but the underlying molecular mechanisms are incompletely un- translocation of p50/RelA and p50/c-Rel NF-κB dimers. The derstood. Here we show that NF-κB–inducing kinase (NIK), a cen- IκBα degradation is in turn triggered through its phosphorylation tral component of the noncanonical NF-κB signaling pathway, is by an IκB kinase (IKK) complex, composed of IKKα and IKKβ κ required for Tfh cell development. Unlike other known Tfh regu- as well as a regulatory subunit, NEMO (NF- B essential mod- κ lators, NIK acts by controlling the supporting function of B cells. ulator) (16, 17). In addition to this canonical pathway of NF- B activation, a noncanonical NF-κB pathway mediates specific NIK and its upstream BAFF receptor regulate B-cell expression of κ inducible costimulator ligand (ICOSL), a molecule required for Tfh functions of NF- B in certain cell types, including B cells (18, 19). This pathway depends on inducible processing of the NF- cell generation. Consistently, injection of a recombinant ICOSL pro- κ tein into NIK-deficient mice largely rescues their defect in Tfh cell B2 precursor protein p100 (20, 21). Because p100 contains an IκB-homologous C-terminal portion, it functions as not only the development. We provide biochemical and genetic evidence indi- precursor of p52, but also an IκB-like molecule that specifically cating that the ICOSL gene is a specific target of the noncanonical κ κ fi κ inhibits a noncanonical NF- B member, RelB. Thus, the in- NF- B. Our ndings suggest that the noncanonical NF- B pathway ducible processing of p100 serves to both generate p52 and in- regulates the development of Tfh cells by mediating ICOSL gene duce the nuclear translocation of the noncanonical NF-κB dimer expression in B cells. p52/RelB. The activated RelB also can function as heterodimers with other NF-κB members, particularly p50. umoral immune responses to protein antigens involve ger- A central signaling component of the noncanonical NF- Hminal center (GC) formation in B-cell follicles of peripheral κB pathway is NF-κB–inducing kinase (NIK), which integrates lymphoid organs and subsequent B-cell differentiation events, noncanonical NF-κB–stimulating signals from a subset of TNF such as antibody isotype switching and selection of high-affinity receptor (TNFR) family members, including CD40, B-cell acti- B-cell clones (1). The successful progress of antibody responses vating factor belonging to TNFR family receptor (BAFFR), and requires cognate help of the antigen-stimulated B cells by a lymphotoxin-β receptor (LTβR) (18, 19, 22). NIK and its down- special CD4 T-cell subset, termed T follicular helper (Tfh) cells stream kinase IKKα stimulate p100 processing by mediating p100 (1, 2). These T cells express the chemokine receptor CXCR5 and phosphorylation and ubiquitination (20, 21). A major function of thus are capable of migrating to the B-cell follicles for efficient the noncanonical NF-κB pathway is regulating humoral immune T cell–B cell interaction. Tfh cells then direct the differentiation responses. Deficiency in NIK or other components of this path- of B cells by secreting cytokines, such as IL-21, and expressing way attenuates GC formation and production of antibodies (18,

surface molecules such as CD40 ligand (CD40L) and pro- 23). However, how precisely NIK and the noncanonical NF-κB IMMUNOLOGY grammed death 1 (PD1) (2). In addition, Tfh cells characteristi- signaling pathway regulate antibody responses is incompletely cally express high levels of inducible costimulator (ICOS), which understood. In this study, we have demonstrated that NIK has delivers a major T-cell costimulatory signal in response to ligation a critical role in antigen-stimulated generation of Tfh cells. This by ICOS ligand (ICOSL; also termed B7h and B7RP-1) (3). function of NIK is not T-cell intrinsic but is mediated through The development of Tfh cells is a multistep process, including regulating the supporting role of B cells. Interestingly, NIK is the initial CD4 T-cell activation by dendritic cells in the T-cell required for maintaining the high-level expression of ICOSL in B zone and the subsequent interaction of Tfh precursor cells with cells. We provide genetic and biochemical evidence that non- – B cells at the T B border of peripheral lymphoid organs (4). In canonical NF-κB members are directly involved in ICOSL gene addition to the T-cell receptor (TCR) and CD28 signals, re- regulation. Thus, our data suggest that the noncanonical NF-κB quired for T-cell activation, the costimulatory signal mediated by – signaling pathway regulates Tfh cell development by controlling ICOS is critical for Tfh cell production (5 8). Because B cells ICOSL gene expression in B cells. constitutively express high levels of ICOSL (9), the T cell–B cell interaction may provide an important mechanism of ICOS costimulation on T cells. Indeed, B cells are known as essential supporting cells in the development of Tfh cells (2, 4). Several Author contributions: H.H. and X.W. designed research; H.H., X.W., W.J., M.C., and X.C. performed research; H.H. and X.W. analyzed data; and S.-C.S. wrote the paper. recent studies suggest that the T cell–Bcell interaction is critical ﬂ for Tfh cell development (10–12), and that this supporting The authors declare no con ict of interest. function of B cells requires their surface expression of ICOSL This article is a PNAS Direct Submission. (7). However, the signaling pathways mediating the homeostatic 1H.H. and X.W. contributed equally to this work. expression of ICOSL and the Tfh-supporting function of B cells 2Present address: Laboratory of Gene Regulation and Signal Transduction, Department of are poorly deﬁned. Pharmacology, School of Medicine, University of California San Diego, La Jolla, CA 92093. The NF-κB signaling pathway has an important role in regu- 3Present address: Howard Hughes Medical Institute and Immunology Program, Memorial lating lymphocyte development and activation (13–15). NF-κB Sloan-Kettering Cancer Center, New York, NY 10065. comprises a family of transcription factors, including RelA, 4To whom correspondence should be addressed. E-mail: [email protected]. RelB, c-Rel, NF-κB1 p50, and NF-κB2 p52, which form different This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. dimeric complexes and transactivate target genes by binding to 1073/pnas.1105774108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1105774108 PNAS | August 2, 2011 | vol. 108 | no. 31 | 12827–12832 Downloaded by guest on September 26, 2021 Results transferred with WT T cells plus NIK KO B cells exhibited se- NIK Regulates Tfh Cell Development in a B-Cell–Dependent Manner. riously defective Tfh cell production (Fig. 1C). Taken together, To understand how the noncanonical NF-κB signaling pathway these results indicate that NIK regulates Tfh cell development by regulates humoral immune responses, we investigated the role of modulating the supporting function of B cells. NIK in Tfh cell development. We immunized WT and NIK KO mice with a strong protein antigen, sheep red blood cells NIK and Its Upstream Receptor BAFFR Regulate ICOSL Expression in B (SRBC), which induces robust GC formation and Tfh cell de- Cells. ICOS/ICOSL interaction is crucial for the development of velopment in the absence of adjuvants (5, 24). We detected Tfh Tfh cells (5–7, 25). In particular, ICOSL is highly expressed on B cells in the spleen of the immunized mice by flow cytometry, cells and is involved in ICOS stimulation during the interaction based on their typical surface markers CXCR5 and PD1. As of B cells and CD4 T cells (7). Given that B cells are constantly κ expected, the spleen of immunized WT mice produced a clear exposed to noncanonical NF- B stimuli, particularly BAFF, we + + κ population of Tfh cells characterized by a CXCR5 PD1 phe- reasoned that the noncanonical NF- B pathway might contribute notype (Fig. 1A). Importantly, the generation of Tfh cells was to the high levels of ICOSL expression in B cells. To test this attenuated in the spleen of NIK KO mice (Fig. 1A). Analyses of hypothesis, we examined the expression of ICOSL on splenic B multiple animals showed that the NIK KO mice had a significantly cells derived from WT and NIK KO mice. As expected, freshly lower percentage of Tfh cells out of total CD4 T cells (Fig. 1B). isolated WT B cells displayed constitutive ICOSL expression The development of Tfh cells requires a complex signaling (Fig. 2A). Moreover, the expression level of ICOSL was sub- program in activated CD4 T cells. B cells also play a critical role stantially reduced, but not completely blocked, in the NIK-de- fi in supporting the differentiation of CD4 T cells to Tfh cells (4). cient B cells (Fig. 2A). To understand how NIK regulates Tfh cell differentiation, we Because homeostatic activation of NIK and noncanonical NF- κ performed lymphocyte adoptive transfer studies to examine B in splenic B cells is mediated primarily by the BAFF/BAFFR whether this function of NIK is in T cells or in B cells. Purified T system (26), we tested whether ICOSL expression is also subject and B cells were transferred to Rag2 KO recipient mice, which to regulation by BAFFR. For these studies, we used the control lack endogenous lymphocytes. After lymphocyte transfer, the A/J mouse and its mutant variant, A/WySnJ, which carries a genetic defect in the BAFFR gene (27). As seen with the NIK KO recipient mice were immunized with SRBC and then subjected fi to Tfh cell analysis. As expected, the mice transferred with WT B cells, the B cells isolated from A/WySnJ mice had a signi - fi cantly lower level of ICOSL expression (Fig. 2B). These results T cells plus WT B cells ef ciently developed Tfh cells after im- κ munization (Fig. 1C). The transfer of NIK KO T cells plus WT B indicate that the noncanonical NF- B signaling pathway, which cells also was associated with the effective development of Tfh is chronically activated in vivo, mediates induction of the high- cells in recipient mice, suggesting that the function of NIK in Tfh level expression of ICOSL in B cells, although other mechanisms cell regulation is not T-cell intrinsic. On the other hand, the mice contribute to this gene expression event as well. NIK Mediates in Vitro ICOSL Induction by Noncanonical NF-κB Inducers. To further assess the role of NIK in mediating the induction of ICOSL expression, we examined whether noncanonical NF-κB inducers stimulate ICOSL expression in vitro, and whether NIK is required for this gene induction event. For these studies, we incubated the B cells in vitro for 48 h to reduce the level of constitutive ICOSL expression. Consistent with the need for BAFFR in maintaining constitutive ICOSL expression in vivo, incubation of WT B cells with BAFF led to potent induction of ICOSL expression in vitro (Fig. 2C). Importantly, the BAFF-stimulated ICOSL expression was dependent on NIK, as demonstrated by its absence in NIK KO B cells. Furthermore, stimulation of CD40, a noncanonical NF-κB inducer mediating T-cell–dependent B-cell activation during an immune response, also led to induction of ICOSL in a NIK-dependent manner (Fig. 2C). In contrast, the canonical NF-κB inducer LPS failed to induce ICOSL expression in both the WT and KO B cells (Fig. 2C). These results indicate a specific role for the noncanonical NF-κB pathway in mediating the induction of ICOSL expression. We also performed studies using the murine M12 B-cell line as a model system, because it had been well characterized for noncanonical NF-κB activation by the B-cell–specific noncanonical NF-κB inducers BAFF and anti-CD40 (28, 29). The surface expression of ICOSL was strongly induced by stimulation with either BAFF or anti-CD40 (Fig. 2D). Consistent with the persistent nature of noncanonical NF-κB signaling, the induction of ICOSL was prolonged, with 48 h as the optimal induction time (Fig. 2D). As seen with primary B cells, the expression of ICOSL in M12 B cells was induced only slightly by the canonical NF-κB inducer LPS (Fig. 2D). These results indicate the involvement of Fig. 1. NIK regulates Tfh cell development in a B-cell–dependent manner. κ +/+ −/− the noncanonical NF- B pathway in the induction of ICOSL (A and B) Age-matched NIK (WT) and NIK (KO) mice were immunized gene expression. This idea was further suggested by parallel real- with SRBC and killed on day 7 after immunization. The frequency of Tfh cells among CD4 T cells was quantified by flow cytometry and presented as time quantitative PCR (qPCR) analyses showing the induction of a representative flow cytometry graph (A) and the mean value of multiple ICOSL mRNA by BAFF and anti-CD40, but not by LPS (Fig. 2E). mice (with each circle or square representing an individual mouse) (B). (C) κ ICOSL Rag2 KO mice were adoptively transferred with a combination of T and Noncanonical NF- B Binds Directly to Promoter and Mediates B cells derived from either WT or NIK KO mice. The recipient mice were the Induction of ICOSL Gene Expression. To examine whether immunized with SRBC antigen and subjected to Tfh cell analyses as described ICOSL serves as a direct target of noncanonical NF-κB, we in A and B. Data are presented as mean value of multiple recipient mice. performed ChIP assays, a technique that detects in vivo binding

12828 | www.pnas.org/cgi/doi/10.1073/pnas.1105774108 Hu et al. Downloaded by guest on September 26, 2021 other hand, we detected very weak binding of the ICOSL promoter by RelA, the core component of the canonical NF-κB complex. These results thus demonstrate that the inducible expression of ICOSL in B cells is associated with binding of noncanonical NF-κB members to the ICOSL promoter. RelB is the core subunit of the noncanonical NF-κB that functions as a heterodimer with either p52 or p50 (22). To functionally examine the requirement of the noncanonical NF- κB in ICOSL gene induction, we performed RNAi-mediated knockdown of RelB. We infected M12 B cells with a control lentiviral vector, pLKO.1, or the same vector encoding a RelB shRNA. Compared with the control cells, the RelB shRNA- infected cells showed markedly lower RelB expression (Fig. 3C). The RelB knockdown moderately reduced the basal level of ICOSL expression and strongly attenuated the BAFF-induced ICOSL expression (Fig. 3D). As an additional approach to conﬁrm the important role of the noncanonical NF-κB pathway in ICOSL gene regulation, we reconstituted the NIK KO B cells with an NIK expression vector via retroviral infection. Expres- sion of exogenous NIK rescued both the basal (Fig. S2) and BAFF-induced (Fig. 3E) ICOSL expression. Taken together with the ChIP assay results, these ﬁndings identify ICOSL as a target gene of the noncanonical NF-κB signaling pathway.

ICOSL Promoter Has a κB Site That Binds Noncanonical NF-κB Members and Mediates ICOSL Promoter Activation. Through DNA sequence analysis, we identified a κB-like element in the promoter region of ICOSL (−347 to −338). This sequence differs slightly from the typical κB consensus sequence, GGGRNTTTCC (30) (Fig. 4A). EMSA revealed binding of this κB-like element by nuclear proteins stimulated by BAFF and anti-CD40 (Fig. 4A). The ICOSL κB-binding complexes also were detected in spleen B cells isolated from WT mice. This κB-binding activity was dependent on NIK, as demonstrated by its absence in B cells derived from the NIK KO mice (Fig. 4A). Parallel antibody supershift assays revealed that the major NF-κB complexes formed with the ICOSL κB site contained the noncanonical NF-κB members Fig. 2. NIK mediates the inducible expression of ICOSL on B cells. (A) Freshly fl p52 and RelB, as well as p50 (Fig. 4B); in contrast, little binding isolated spleen B cells of WT and NIK KO mice were analyzed by ow activity of RelA and c-Rel was detected. cytometry to determine the level of ICOSL expression. Data are represen- To further examine the specificity of the ICOSL in binding to tative of three independent experiments with multiple mice. (B) Spleen B κ fi different NF- B members, we performed EMSA with overex- cells were isolated from control A/J mice or the BAFFR-de cient A/WySnJ κ mice and subjected to flow cytometry analysis of ICOSL surface expression pressed NF- B components. When expressed alone or together, p50 and p52 bound to the ICOSL κB and common κB with level. (C) WT and NIK KO B cells were cultured in vitro for 48 h either in the fi absence (NT) or the presence of BAFF, an agonistic anti-CD40 antibody, or similar ef ciency (Fig. 4C). In contrast, RelA failed to bind to fl the ICOSL κB (Fig. 4C, Upper), despite its efficient binding to LPS. The intensity of ICOSL surface expression was measured by ow IMMUNOLOGY cytometry. (D) M12 B cells were either not treated (NT) or stimulated for the the common κB probe (Fig. 4C, Lower). The RelA/p50 hetero- indicated times with BAFF, anti-CD40, or LPS. The ICOSL surface expression dimer also barely bound to the ICOSL κB (Fig. 4C, Upper). was analyzed by flow cytometry. (E) WT and NIK KO spleen B cells were Furthermore, although RelB alone did not bind ICOSL κBor stimulated in vitro as indicated. For the untreated control (NT), cells were common κB, in line with its inability to form stable homodimers cultured for 12 h without inducers. Total RNA was isolated and subjected to (31, 32), it bound to the ICOSL probe when expressed together real-time qPCR to analyze the level of ICOSL mRNA. Data are presented as with p52 or p52 plus p50 (Fig. 4C, Upper). These results, together fold relative to the NT sample. with those presented in Fig. 4 A and B, suggest that ICOSL contains a κB site that preferentially binds noncanonical NF-κB. To examine whether this intriguing κB site is important for of transcription factors to the regulatory regions of target genes. BAFF-stimulated ICOSL promoter activity, we generated lucif- Analysis of the murine and human ICOSL gene locus revealed erase reporters driven by either WT ICOSL promoter or the two major conserved noncoding sequence (CNS) elements, one same promoter harboring a mutation in the ICOSL κB site. located between −800 and −30 nucleotides and the other located Consistent with the induction of ICOSL expression (Fig. 2), the between −2,300 and −2,100 nucleotides relative to the tran- WT ICOSL promoter was stimulated by anti-CD40 and BAFF, scription start site (Fig. S1). We first performed sequential ChIP but not by LPS (Fig. 4D). Importantly, mutation of the κB site assays to examine which regions were bound by RelB, the core abolished the ICOSL promoter activation (Fig. 4D). These component of the noncanonical NF-κB complex. As expected, in results identify ICOSL κB as a functional DNA element that nonstimulated cells (NT), RelB did not bind to any of the CNS mediates the response to noncanonical NF-κB signals. regions (Fig. 3A). Interestingly, on BAFF stimulation, RelB was bound to the promoter-proximal region, as demonstrated by its Injection of Recombinant ICOSL into NIK KO Mice Largely Rescues pull down of a DNA fragment spanning −434 to −190 (Fig. 3A). Their Tfh Defect. The foregoing results establish ICOSL as a tar- In contrast, the upstream regions (−2,371 to −2,150 and −882 get gene of the noncanonical NF-κB signaling pathway and pro- to −675) did not appreciably bind RelB. Additional ChIP anal- vide a possible molecular mechanism through which this NF-κB yses using both primary spleen B cells and the M12 B-cell line signaling axis regulates Tfh cell development. To further validate demonstrated that in addition to RelB, p50 and p52 also bound the functional significance of the ICOSL gene expression, we to the promoter-proximal region of ICOSL (Fig. 3B). On the tested whether recombinant ICOSL is able to partially or com-

Hu et al. PNAS | August 2, 2011 | vol. 108 | no. 31 | 12829 Downloaded by guest on September 26, 2021 Fig. 3. Noncanonical NF-κB binds to the promoter region of ICOSL gene and is critical for ICOSL induction. (A) Spleen B cells from WT mice were either not treated (NT) or stimulated with BAFF for 24 h. Chromatin IP was performed using either a control Ig (Ig) or anti-RelB antibody, and the precipitated DNA was subjected to PCR using primers that amplify the indicated regions of the ICOSL promoter. Input DNAs also were subjected to PCR to show the efficiency of the primers. (B) WT spleen B cells (Left) or M12 B cells (Right) were stimulated with BAFF for the indicated times. Chromatin IP was performed using either a control Ig or the indicated antibodies, and the precipitated DNA was subjected to PCR using primers that amplify a 300-bp DNA fragment (−490 to −190) of the ICOSL promoter. Data are representative of three independent experiments. (C and D) M12 cells were infected with either the pLKO.1 lentiviral vector or the same vector encoding RelB shRNA. After puromycin selection, the bulk of infected cells were subjected to IB to determine the efficiency of RelB knockdown (C). The control and RelB knockdown cells were either not treated (NT) or stimulated with BAFF for 48 h, and the ICOSL expression level was analyzed by flow cytometry (D). (E)WTorNIK KO splenocytes were infected with ret- roviruses carrying the pCLXSN(GFP) vector (vector), or pCLXSN(GFP)-NIK (NIK). Infected cells were stimulated with BAFF for 48 h, and ICOSL expression on infected B cells was analyzed by flow cytometry (gated on B220+GFP+ cells).

pletely rescue the defect of NIK KO mice in Tfh cell development. Furthermore, we found that injection of a recombinant ICOSL A recombinant ICOSL-Fc fusion protein or a control Fc protein protein into the NIK KO mice largely rescued the defective an- was injected into NIK KO mice on the day of SRBC immunization tigen-stimulated Tfh cell generation. Thus, our data further and at different times after the immunization, followed by flow emphasize the critical role of ICOSL/ICOS interaction in the cytometry analysis of the generation of Tfh cells (Fig. 5A). As induction of Tfh cell differentiation. expected, the control Fc-injected NIK KO mice displayed a sig- B cells are characteristic for their constitutive expression of nificantly reduced level of Tfh cell generation compared with the high levels of ICOSL (9). Although the mechanism mediating WT mice (Fig. 5 B and C). Importantly, injection of recombinant constitutive ICOSL expression in B cells has remained obscure, ICOSL-Fc largely (although not completely) rescued the defect of this gene expression pattern is correlated with constitutive acti- the NIK KO mice in Tfh cell development (Fig. 5 B and C). vation of NF-κB (33). Unlike T cells and many other cell types, Because ICOSL-Fc might have an effect on non-B cells, we B cells are constantly exposed to homeostatic NF-κB stimuli in performed additional ICOSL-Fc rescue experiments using the peripheral lymphoid organs and display chronic NF-κB activity. lymphocyte adoptive transfer model. In brief, Rag2 KO mice One important homeostatic NF-κB–inducing signal is triggered were adoptively transferred with WT T cells plus NIK KO B cells through the binding of BAFF to BAFFR, and this signal pre- and then subjected to ICOSL-Fc rescue studies (Fig. 5 D and E). κ fi dominantly stimulates the noncanonical NF- B signaling path- Under these conditions, ICOSL-Fc again ef ciently rescued the way (26, 29). We have shown that the BAFFR signal is critical defect of the NIK KO B cells in supporting Tfh cell develop- for the constitutive expression of ICOSL in B cells in vivo. ment. Collectively, these results further emphasize the critical Consistently, BAFF stimulated ICOSL expression in vitro in role for NIK-mediated ICOSL expression in regulating Tfh cell both primary B cells and the M12 B-cell line. Our in vitro studies development. also suggest the involvement of the CD40 signal in ICOSL gene Discussion induction. But because CD40 stimulation requires antigen- stimulated T cells, the CD40 signal likely contributes to ICOSL The results presented in this paper identify NIK and its down- induction only during an immune response. stream noncanonical NF-κB as critical factors in the regulation We found strong evidence suggesting an essential role for the of Tfh cell development. Unlike the currently known signaling noncanonical NF-κB in ICOSL gene expression. First, inducers factors, which function in T cells, these factors regulate the κ supporting role of B cells. We obtained biochemical and genetic that trigger the activation of noncanonical NF- B, such as BAFF κ and anti-CD40, effectively induce the expression of ICOSL in B evidence indicating that the noncanonical NF- B pathway reg- κ fi ulates the expression of ICOSL, a costimulatory molecule re- cells. In contrast, canonical NF- B inducer LPS was insuf cient fi to trigger ICOSL expression in B cells. Moreover, RNAi-mediated quired for stimulation of Tfh cell development. These ndings κ identify ICOSL as a target gene of the noncanonical NF-κB knockdown of the core noncanonical NF- B member RelB at- signaling pathway and provide insight into the mechanism by tenuated ICOSL gene induction, providing genetic evidence of κ which this pathway regulates humoral immune responses. the need for noncanonical NF- B in ICOSL gene induction. Some previous studies have demonstrated the requirement for Finally, our ChIP assays revealed the binding of noncanonical ICOSL in the induction of Tfh cells. Genetic deficiency in ICOS NF-κB members to the promoter region of ICOSL, suggesting and ICOSL or blockade of ICOS/ICOSL interactions impairs their direct involvement in ICOSL gene regulation. Of course, Tfh cell development in mice (5–8). Notably, the high-level ex- our data do not exclude the possibility that canonical NF-κBis pression of ICOSL on B cells is particularly important for B-cell– also involved in the regulation of ICOSL gene expression. In mediated supporting function in the generation of Tfh cells (7). particular, BAFF is known to stimulate p50 in addition to the In agreement with these previous studies, we found an associa- noncanonical NF-κB members p52 and RelB (34). We found tion between the attenuated expression of ICOSL in NIK-deficient that p50 also binds to the ICOSL promoter in BAFF-stimulated B cells and reduced production of Tfh cells in immunized NIK B cells. However, because RelA is not a major component of KO mice. This defect is due to the impaired supporting function the NF-κB complex bound to the ICOSL promoter, it is likely of B cells, as demonstrated by adoptive transfer experiments. that p50 may function as a homodimer or a partner of the

12830 | www.pnas.org/cgi/doi/10.1073/pnas.1105774108 Hu et al. Downloaded by guest on September 26, 2021 Fig. 5. Recombinant ICOSL rescues the Tfh cell defect in NIK KO mice. (A) WT and NIK KO mice were immunized with SRBC on day 0 along with injection of a recombinant ICOSL-Fc fusion protein or a control Fc protein. The mice received three additional injections of ICOSL-Fc or control Fc at the indicated days postimmunization and then subjected to Tfh cell analyses. (B and C) The frequency of Tfh cells among CD4 T cells was quantified by Fig. 4. A κB sequence of the ICOSL promoter preferentially binds non- flow cytometry and presented as a representative flow cytometry graph (B) canonical NF-κB members and mediates ICOSL promoter activation. (A) The and mean value of multiple mice (C). (D and E) Rag2 KO mice were adop- sequence of an ICOSL κB was aligned with the consensus κB sequence (Up- tively transferred with a combination of WT T cells and NIK KO B cells. The per). EMSA was performed using the ICOSL κB probe and nuclear extracts recipient mice were immunized with SRBC along with injection with either isolated from nontreated or BAFF- and anti-CD40-stimulated (24 h) M12 cells control Fc or ICOSL-Fc, as described in A. The frequency of Tfh cells among or from freshly purified WT and NIK KO spleen B cells. (B) A supershift assay CD4 T cells was quantified by flow cytometry and presented as a represen- was performed using nuclear extracts of BAFF-stimulated M12 B cells and tative flow cytometry graph (D) and mean values (E). the ICOSL κB probe, in either the absence (none) or the presence of the indicated antibodies or an Ig control. The supershifted bands are indicated by arrows. (C) HEK293 cells were transfected with the indicated NF-κB members, either alone or in combination. Nuclear extracts were subjected to Materials and Methods EMSA using the ICOSL κB and a general κB probe. Immunoblot analysis was Mice. NIK KO mice on a 129Sv/Ev background (38) were provided by Amgen performed to monitor the expression of the different NF-κB proteins. (D) and were maintained in the specific pathogen-free facility of the University +/− M12 cells were infected with pGreenFire lentiviral vectors carrying a lucifer- of Texas MD Anderson Cancer Center. NIK heterozygous mice were bred +/+ −/− ease gene driven by either WT ICOSL promoter (ICOSL-luc) or mutant ICOSL to generate the age-matched NIK (WT) and NIK (KO) mice used in the −/− promoter with mutated κB site (ICOSLΔκB-luc). The cells were either not experiments. Rag2 (Rag2 KO) mice, on a 129Sv/Ev background, were treated (NT) or stimulated for 14 h with LPS, anti-CD40, or BAFF. Luciferase obtained from Taconic. A/J and A/WySnJ mice were obtained from Jackson activity is presented as fold induction compared with the NT pGF cells. Laboratory. All animal experiments were performed in accordance with ’

protocols approved by the University of Texas MD Anderson Cancer Center s IMMUNOLOGY Institutional Animal Care and Use Committee. noncanonical NF-κB member RelB, because RelB is known to form both p52/RelB and p50/RelB heterodimers. Antibodies, Reagents, and Plasmids. Antibodies for p50 (D17), p52 (c-5), RelB The specific involvement of noncanonical NF-κB in ICOSL (C-19), c-Rel (sc-71×), and HSP60 (H-1), as well as control rabbit Ig, were gene induction appears to be due to two different regulatory obtained from Santa Cruz Biotechnology. Fluorescence-labeled antibodies κ for CD4 (L3T4), CD3 (145-2C11), PD-1 (J43), and ICOSL (HK5.3) were pur- mechanisms. First, noncanonical NF- B members are the pre- chased from eBioscience. Anti-mouse CD40 (553721) and fluorescence- dominant components of chronically activated NF-κB complexes labeled antibodies for CD19 (1D3) and CXCR5 (2G8) were purchased from BD in B cells exposed to the homeostatic inducer BAFF. Second, the Biosciences. Other antibodies were as reported previously (28). ICOSL promoter contains a κB element that favors binding by Recombinant ICOSL-Fc (also called B7RP-1-Fc) fusion protein and control Fc the noncanonical NF-κB members. Mutation of this κB element protein were provided by Amgen. Recombinant BAFF protein (PHC1674) was abolished the activation of ICOSL promoter by anti-CD40 and purchased from Biosource. LPS (derived from E. coli 0127:B8) was obtained BAFF. It is important to note, however, that the role of NF-κBin from Sigma-Aldrich, and SRBC was purchased from Cocalico Biologicals. ICOSL gene regulation appears to vary among different cell The pLKO.1-puromycin lentiviral vector and the same vector encoding mouse RelB shRNAs were purchased from Sigma-Aldrich. Three shRNAs types. Previous studies have suggested that canonical NF-κB α targeting different regions of the RelB mRNA were used. To generate the stimuli, such as IL-1 and TNF- , induce the expression of ICOSL luciferase reporter driven by the mouse ICOSL promoter (pGF-ICOSL), a 705- in endothelial cells and fibroblasts (35–37). Our findings suggest bp ICOSL promoter DNA fragment (−570 to +135) was inserted upstream of that LPS is inefficient in the induction of ICOSL expression in the luciferase gene in a lentiviral reporter plasmid, pGreenFire (pGF; System B cells. Whether other canonical NF-κB stimuli induce ICOSL Biosciences). pGF-ICOSLΔκB, a mutant form of pGF-ICOSL that contains point expression in B cells remains to be investigated. Notwithstand- mutations in a κB-like element of the ICOSL promoter (−347 to −338), was fi created using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). ing, our ndings suggest that the NIK-regulated noncanonical ′ κ The following primers were used: sense, 5 -CAGGGACCAGGCCGTTAAC- NF- B signaling pathway plays a predominant role in mediating GTTCTGGGCAGCGTTG-3′; antisense, 5′-CAACGCTGCCCAGAACGTTAACGGC- the high level of ICOSL expression in B cells, a signaling function CTGGTCCCTG-3′. required for the supporting role of B cells in antigen-stimulated The pcDNA expression vectors encoding Flag-tagged p50, p52, and RelB production of Tfh cells. were purchased from Addgene. The pCMV4-p65 was described previously (39).

Hu et al. PNAS | August 2, 2011 | vol. 108 | no. 31 | 12831 Downloaded by guest on September 26, 2021 Cell Culture and shRNA Knockdown. Murine B-cell line M12.4.1 (designated M12 Flow Cytometry. Cell suspensions were subjected to flow cytometry analyses in this paper) was described previously (28). The cells were infected with len- as described previously (41) using a BD Biosciences LSRII flow cytometer. Data tiviruses carrying either the empty pLKO-1 vector or RelB shRNA clones. The were analyzed using FlowJo software. infected cells were then enriched by selection using puromycin (2.0 μg/mL) for 5 d, and the bulk of the infected cells were used in experiments. To produce ChIP Assays. ChIP assays were performed using the Millipore EZ-ChIP Kit the lentiviral particles, the pLKO.1 vectors were transfected into HEK293 cells following the manufacturer’s instructions. In brief, nontreated and treated (using the calcium method) along with packing vectors psPAX2 and pMD2 M12 cells were crosslinked with 1% of formaldehyde (final concentration, (provided by Dr. Xiaofeng Qin, MD Anderson Cancer Center, Houston, TX). vol/vol) for 10 min, lysed in SDS lysis buffer, and sonicated to shear the DNA. fi B cells were puri ed from splenocytes using anti-B220 conjugated mag- The chromatin DNA was subjected to IP using the indicated antibodies or fl netic beads (Miltenyl Biotec) and were either directly subjected to ow a control IgG. After purification, the precipitated DNA was analyzed by PCR cytometry or stimulated in vitro by anti-mouse CD40 (500 ng/mL), BAFF (200 using primers that amplify different regions of the ICOSL promoter. ng/mL), or LPS (100 ng/mL). The primer sequences were as follows: −2371 to −2150, 5′-ACAGGTT- GAGAACCATTCTTCC-3′ and 5′- GAATCCCAGAAAGCCAAATGC-3′; −882 to Mouse Immunization and ICOSL-Fc Injection. For induction of Tfh cells, age- −675, 5′-TAGCCTCAGACTCAAGAGATC-3′ and 5′-CCAGACTTGGCAATCCT- matched WT and NIK KO mice were immunized i.p. with 2 × 109 SRBC (24). GTTC-3′; −434 to −190, 5′-CCAGGTCCGGGCTTTGAACC-3′ and 5′-CATGAGT- In some experiments, the NIK KO mice were injected i.p. with 50 μg of ICOSL- TACAGGTGCCAGGGTG -3′. Fc or 25 μg control Fc (40) on the day of immunization and on days 2, 4, and 6 after immunization. On day 7, spleen cells were isolated for flow cytometry analyses. Statistical Analysis. Two-tailed unpaired t tests were performed using Prism software. A P value < 0.05 was considered significant. Lymphocyte Adoptive Transfer. B220+ B cells and CD90.2+ T cells were isolated from the splenocytes of WT or NIK KO mice using magnetic beads (Miltenyi ACKNOWLEDGMENTS. We thank Amgen for the NIK KO mice and ICOSL-Fc recombinant protein and Xiaofeng Qin for the lentiviral packaging vectors. Biotec). The isolated cells were >95% pure, as determined by flow cytom- We also thank the personnel from the flow cytometry core facility (Karen × 6 etry. WT or NIK KO T cells (5 10 ) were mixed with either WT or NIK KO B Martinez, David He, and Amy Cortez) and the animal facility at MD Ander- × 6 cells (5 10 ) and then injected via a tail vein into Rag2 KO mice. After 16 h, son Cancer Center for technical assistance. This study was supported by the recipient mice were subjected to immunization and ICOSL-Fc injection National Institutes of Health Grants AI057555, AI064639, GM84459-S1, studies as described above. and GM84459.

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