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

Abstract

Follicular helper T (Tfh) cells have a central role in mediating humoral immune responses. Generation of Tfh cells depends on both T-cell intrinsic factors and the supporting function of B cells, but the underlying molecular mechanisms are incompletely understood. Here we show that NF-κB–inducing kinase (NIK), a central component of the noncanonical NF-κB signaling pathway, is required for Tfh cell development. Unlike other known Tfh regulators, NIK acts by controlling the supporting function of B cells. NIK and its upstream BAFF receptor regulate B-cell expression of inducible costimulator ligand (ICOSL), a molecule required for Tfh cell generation. Consistently, injection of a recombinant ICOSL protein into NIK-deficient mice largely rescues their defect in Tfh cell development. We provide biochemical and genetic evidence indicating that the ICOSL gene is a specific target of the noncanonical NF-κB. Our findings suggest that the noncanonical NF-κB pathway regulates the development of Tfh cells by mediating ICOSL gene expression in B cells.

Humoral immune responses to protein antigens involve germinal center (GC) formation in B-cell follicles of peripheral lymphoid organs and subsequent B-cell differentiation events, such as antibody isotype switching and selection of high-affinity B-cell clones (1). The successful progress of antibody responses requires cognate help of the antigen-stimulated B cells by a special CD4 T-cell subset, termed T follicular helper (Tfh) cells (1, 2). These T cells express the chemokine receptor CXCR5 and thus are capable of migrating to the B-cell follicles for efficient T cell–B cell interaction. Tfh cells then direct the differentiation of B cells by secreting cytokines, such as IL-21, and expressing surface molecules such as CD40 ligand (CD40L) and programmed death 1 (PD1) (2). In addition, Tfh cells characteristically express high levels of inducible costimulator (ICOS), which delivers a major T-cell costimulatory signal in response to ligation by ICOS ligand (ICOSL; also termed B7h and B7RP-1) (3).

The development of Tfh cells is a multistep process, including the initial CD4 T-cell activation by dendritic cells in the T-cell zone and the subsequent interaction of Tfh precursor cells with B cells at the T–B border of peripheral lymphoid organs (4). In addition to the T-cell receptor (TCR) and CD28 signals, required for T-cell activation, the costimulatory signal mediated by ICOS is critical for Tfh cell production (5–8). Because 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 recent studies suggest that the T cell–Bcell interaction is critical for Tfh cell development (10–12), and that this supporting function of B cells requires their surface expression of ICOSL (7). However, the signaling pathways mediating the homeostatic expression of ICOSL and the Tfh-supporting function of B cells are poorly defined.

The NF-κB signaling pathway has an important role in regulating lymphocyte development and activation (13–15). NF-κB comprises a family of transcription factors, including RelA, RelB, c-Rel, NF-κB1 p50, and NF-κB2 p52, which form different dimeric complexes and transactivate target genes by binding to a κB enhancer (16, 17). NF-κB is normally sequestered in the cytoplasm by inhibitory proteins (IκBs), and NF-κB activation typically involves inducible degradation of IκBα and nuclear translocation of p50/RelA and p50/c-Rel NF-κB dimers. The IκBα degradation is in turn triggered through its phosphorylation by an IκB kinase (IKK) complex, composed of IKKα and IKKβ as well as a regulatory subunit, NEMO (NF-κB essential modulator) (16, 17). In addition to this canonical pathway of NF-κB activation, a noncanonical NF-κB pathway mediates specific functions of NF-κB in certain cell types, including B cells (18, 19). This pathway depends on inducible processing of the NF-κB2 precursor protein p100 (20, 21). Because p100 contains an IκB-homologous C-terminal portion, it functions as not only the precursor of p52, but also an IκB-like molecule that specifically inhibits a noncanonical NF-κB member, RelB. Thus, the inducible processing of p100 serves to both generate p52 and induce the nuclear translocation of the noncanonical NF-κB dimer p52/RelB. The activated RelB also can function as heterodimers with other NF-κB members, particularly p50.

A central signaling component of the noncanonical NF-κB pathway is NF-κB–inducing kinase (NIK), which integrates noncanonical NF-κB–stimulating signals from a subset of TNF receptor (TNFR) family members, including CD40, B-cell activating factor belonging to TNFR family receptor (BAFFR), and lymphotoxin-β receptor (LTβR) (18, 19, 22). NIK and its downstream kinase IKKα stimulate p100 processing by mediating p100 phosphorylation and ubiquitination (20, 21). A major function of the noncanonical NF-κB pathway is regulating humoral immune responses. Deficiency in NIK or other components of this pathway attenuates GC formation and production of antibodies (18, 23). However, how precisely NIK and the noncanonical NF-κB signaling pathway regulate antibody responses is incompletely understood. In this study, we have demonstrated that NIK has a critical role in antigen-stimulated generation of Tfh cells. This function of NIK is not T-cell intrinsic but is mediated through regulating the supporting role of B cells. Interestingly, NIK is required for maintaining the high-level expression of ICOSL in B cells. We provide genetic and biochemical evidence that noncanonical NF-κB members are directly involved in ICOSL gene regulation. Thus, our data suggest that the noncanonical NF-κB signaling pathway regulates Tfh cell development by controlling ICOSL gene expression in B cells.

Results

NIK Regulates Tfh Cell Development in a B-Cell–Dependent Manner.

To understand how the noncanonical NF-κB signaling pathway regulates humoral immune responses, we investigated the role of NIK in Tfh cell development. We immunized WT and NIK KO mice with a strong protein antigen, sheep red blood cells (SRBC), which induces robust GC formation and Tfh cell development in the absence of adjuvants (5, 24). We detected Tfh cells in the spleen of the immunized mice by flow cytometry, based on their typical surface markers CXCR5 and PD1. As expected, the spleen of immunized WT mice produced a clear population of Tfh cells characterized by a CXCR5⁺PD1⁺ phenotype (Fig. 1A). Importantly, the generation of Tfh cells was attenuated in the spleen of NIK KO mice (Fig. 1A). Analyses of multiple animals showed that the NIK KO mice had a significantly lower percentage of Tfh cells out of total CD4 T cells (Fig. 1B).

Fig. 1.

NIK regulates Tfh cell development in a B-cell–dependent manner. (A and B) Age-matched NIK^+/+ (WT) and NIK^−/− (KO) mice were immunized 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 a representative flow cytometry graph (A) and the mean value of multiple mice (with each circle or square representing an individual mouse) (B). (C) Rag2 KO mice were adoptively transferred with a combination of T and B cells derived from either WT or NIK KO mice. The recipient mice were immunized with SRBC antigen and subjected to Tfh cell analyses as described in A and B. Data are presented as mean value of multiple recipient mice.

The development of Tfh cells requires a complex signaling program in activated CD4 T cells. B cells also play a critical role in supporting the differentiation of CD4 T cells to Tfh cells (4). To understand how NIK regulates Tfh cell differentiation, we performed lymphocyte adoptive transfer studies to examine whether this function of NIK is in T cells or in B cells. Purified T and B cells were transferred to Rag2 KO recipient mice, which lack endogenous lymphocytes. After lymphocyte transfer, the recipient mice were immunized with SRBC and then subjected to Tfh cell analysis. As expected, the mice transferred with WT T cells plus WT B cells efficiently developed Tfh cells after immunization (Fig. 1C). The transfer of NIK KO T cells plus WT B cells also was associated with the effective development of Tfh cells in recipient mice, suggesting that the function of NIK in Tfh cell regulation is not T-cell intrinsic. On the other hand, the mice transferred with WT T cells plus NIK KO B cells exhibited seriously defective Tfh cell production (Fig. 1C). Taken together, these results indicate that NIK regulates Tfh cell development by modulating the supporting function of B cells.

NIK and Its Upstream Receptor BAFFR Regulate ICOSL Expression in B Cells.

ICOS/ICOSL interaction is crucial for the development of Tfh cells (5–7, 25). In particular, ICOSL is highly expressed on B cells and is involved in ICOS stimulation during the interaction of B cells and CD4 T cells (7). Given that B cells are constantly exposed to noncanonical NF-κB stimuli, particularly BAFF, we reasoned that the noncanonical NF-κB pathway might contribute to the high levels of ICOSL expression in B cells. To test this hypothesis, we examined the expression of ICOSL on splenic B cells derived from WT and NIK KO mice. As expected, freshly isolated WT B cells displayed constitutive ICOSL expression (Fig. 2A). Moreover, the expression level of ICOSL was substantially reduced, but not completely blocked, in the NIK-deficient B cells (Fig. 2A).

Fig. 2.

NIK mediates the inducible expression of ICOSL on B cells. (A) Freshly isolated spleen B cells of WT and NIK KO mice were analyzed by flow cytometry to determine the level of ICOSL expression. Data are representative of three independent experiments with multiple mice. (B) Spleen B cells were isolated from control A/J mice or the BAFFR-deficient A/WySnJ mice and subjected to flow cytometry analysis of ICOSL surface expression level. (C) WT and NIK KO B cells were cultured in vitro for 48 h either in the absence (NT) or the presence of BAFF, an agonistic anti-CD40 antibody, or LPS. The intensity of ICOSL surface expression was measured by flow cytometry. (D) M12 B cells were either not treated (NT) or stimulated for the indicated times with BAFF, anti-CD40, or LPS. The ICOSL surface expression was analyzed by flow cytometry. (E) WT and NIK KO spleen B cells were stimulated in vitro as indicated. For the untreated control (NT), cells were cultured for 12 h without inducers. Total RNA was isolated and subjected to real-time qPCR to analyze the level of ICOSL mRNA. Data are presented as fold relative to the NT sample.

Because homeostatic activation of NIK and noncanonical NF-κB in splenic B cells is mediated primarily by the BAFF/BAFFR system (26), we tested whether ICOSL expression is also subject to regulation by BAFFR. For these studies, we used the control 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 B cells, the B cells isolated from A/WySnJ mice had a significantly lower level of ICOSL expression (Fig. 2B). These results indicate that the noncanonical NF-κB signaling pathway, which is chronically activated in vivo, mediates induction of the high-level expression of ICOSL in B cells, although other mechanisms 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 the noncanonical NF-κB pathway in the induction of ICOSL gene expression. This idea was further suggested by parallel real-time quantitative PCR (qPCR) analyses showing the induction of ICOSL mRNA by BAFF and anti-CD40, but not by LPS (Fig. 2E).

Noncanonical NF-κB Binds Directly to ICOSL Promoter and Mediates the Induction of ICOSL Gene Expression.

To examine whether ICOSL serves as a direct target of noncanonical NF-κB, we performed ChIP assays, a technique that detects in vivo binding of transcription factors to the regulatory regions of target genes. Analysis of the murine and human ICOSL gene locus revealed two major conserved noncoding sequence (CNS) elements, one located between −800 and −30 nucleotides and the other located between −2,300 and −2,100 nucleotides relative to the transcription start site (Fig. S1). We first performed sequential ChIP assays to examine which regions were bound by RelB, the core component of the noncanonical NF-κB complex. As expected, in nonstimulated cells (NT), RelB did not bind to any of the CNS regions (Fig. 3A). Interestingly, on BAFF stimulation, RelB was bound to the promoter-proximal region, as demonstrated by its pull down of a DNA fragment spanning −434 to −190 (Fig. 3A). In contrast, the upstream regions (−2,371 to −2,150 and −882 to −675) did not appreciably bind RelB. Additional ChIP analyses using both primary spleen B cells and the M12 B-cell line demonstrated that in addition to RelB, p50 and p52 also bound to the promoter-proximal region of ICOSL (Fig. 3B). On the 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.

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) WT or NIK KO splenocytes were infected with retroviruses 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).

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. Expression 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 p52 and RelB, as well as p50 (Fig. 4B); in contrast, little binding activity of RelA and c-Rel was detected.

Fig. 4.

A κB sequence of the ICOSL promoter preferentially binds noncanonical NF-κB members and mediates ICOSL promoter activation. (A) The sequence of an ICOSL κB was aligned with the consensus κB sequence (Upper). EMSA was performed using the ICOSL κB probe and nuclear extracts isolated from nontreated or BAFF- and anti-CD40-stimulated (24 h) M12 cells or from freshly purified WT and NIK KO spleen B cells. (B) A supershift assay was performed using nuclear extracts of BAFF-stimulated M12 B cells and 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 EMSA using the ICOSL κB and a general κB probe. Immunoblot analysis was performed to monitor the expression of the different NF-κB proteins. (D) M12 cells were infected with pGreenFire lentiviral vectors carrying a luciferease gene driven by either WT ICOSL promoter (ICOSL-luc) or mutant ICOSL promoter with mutated κB site (ICOSLΔκB-luc). The cells were either not treated (NT) or stimulated for 14 h with LPS, anti-CD40, or BAFF. Luciferase activity is presented as fold induction compared with the NT pGF cells.

To further examine the specificity of the ICOSL in binding to different NF-κB members, we performed EMSA with overexpressed NF-κB components. When expressed alone or together, p50 and p52 bound to the ICOSL κB and common κB with similar efficiency (Fig. 4C). In contrast, RelA failed to bind to the ICOSL κB (Fig. 4C, Upper), despite its efficient binding to the common κB probe (Fig. 4C, Lower). The RelA/p50 heterodimer also barely bound to the ICOSL κB (Fig. 4C, Upper). Furthermore, although RelB alone did not bind ICOSL κB or common κB, in line with its inability to form stable homodimers (31, 32), it bound to the ICOSL probe when expressed together with p52 or p52 plus p50 (Fig. 4C, Upper). These results, together 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 BAFF-stimulated ICOSL promoter activity, we generated luciferase reporters driven by either WT ICOSL promoter or the same promoter harboring a mutation in the ICOSL κB site. Consistent with the induction of ICOSL expression (Fig. 2), the WT ICOSL promoter was stimulated by anti-CD40 and BAFF, but not by LPS (Fig. 4D). Importantly, mutation of the κB site abolished the ICOSL promoter activation (Fig. 4D). These results identify ICOSL κB as a functional DNA element that mediates the response to noncanonical NF-κB signals.

Injection of Recombinant ICOSL into NIK KO Mice Largely Rescues Their Tfh Defect.

The foregoing results establish ICOSL as a target gene of the noncanonical NF-κB signaling pathway and provide a possible molecular mechanism through which this NF-κB signaling axis regulates Tfh cell development. To further validate the functional significance of the ICOSL gene expression, we tested whether recombinant ICOSL is able to partially or completely rescue the defect of NIK KO mice in Tfh cell development. A recombinant ICOSL-Fc fusion protein or a control Fc protein was injected into NIK KO mice on the day of SRBC immunization and at different times after the immunization, followed by flow cytometry analysis of the generation of Tfh cells (Fig. 5A). As expected, the control Fc-injected NIK KO mice displayed a significantly reduced level of Tfh cell generation compared with the WT mice (Fig. 5 B and C). Importantly, injection of recombinant ICOSL-Fc largely (although not completely) rescued the defect of the NIK KO mice in Tfh cell development (Fig. 5 B and C).

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 flow cytometry and presented as a representative flow cytometry graph (B) and mean value of multiple mice (C). (D and E) Rag2 KO mice were adoptively transferred with a combination of WT T cells and NIK KO B cells. The recipient mice were immunized with SRBC along with injection with either control Fc or ICOSL-Fc, as described in A. The frequency of Tfh cells among CD4 T cells was quantified by flow cytometry and presented as a representative flow cytometry graph (D) and mean values (E).

Because ICOSL-Fc might have an effect on non-B cells, we performed additional ICOSL-Fc rescue experiments using the lymphocyte adoptive transfer model. In brief, Rag2 KO mice were adoptively transferred with WT T cells plus NIK KO B cells and then subjected to ICOSL-Fc rescue studies (Fig. 5 D and E). Under these conditions, ICOSL-Fc again efficiently rescued the defect of the NIK KO B cells in supporting Tfh cell development. Collectively, these results further emphasize the critical role for NIK-mediated ICOSL expression in regulating Tfh cell development.

Discussion

The results presented in this paper identify NIK and its downstream noncanonical NF-κB as critical factors in the regulation of Tfh cell development. Unlike the currently known signaling factors, which function in T cells, these factors regulate the supporting role of B cells. We obtained biochemical and genetic evidence indicating that the noncanonical NF-κB pathway regulates the expression of ICOSL, a costimulatory molecule required for stimulation of Tfh cell development. These findings identify ICOSL as a target gene of the noncanonical NF-κB signaling pathway and provide insight into the mechanism by which this pathway regulates humoral immune responses.

Some previous studies have demonstrated the requirement for ICOSL in the induction of Tfh cells. Genetic deficiency in ICOS and ICOSL or blockade of ICOS/ICOSL interactions impairs Tfh cell development in mice (5–8). Notably, the high-level expression of ICOSL on B cells is particularly important for B-cell–mediated supporting function in the generation of Tfh cells (7). In agreement with these previous studies, we found an association between the attenuated expression of ICOSL in NIK-deficient B cells and reduced production of Tfh cells in immunized NIK KO mice. This defect is due to the impaired supporting function of B cells, as demonstrated by adoptive transfer experiments. Furthermore, we found that injection of a recombinant ICOSL protein into the NIK KO mice largely rescued the defective antigen-stimulated Tfh cell generation. Thus, our data further emphasize the critical role of ICOSL/ICOS interaction in the induction of Tfh cell differentiation.

B cells are characteristic for their constitutive expression of high levels of ICOSL (9). Although the mechanism mediating constitutive ICOSL expression in B cells has remained obscure, this gene expression pattern is correlated with constitutive activation of NF-κB (33). Unlike T cells and many other cell types, B cells are constantly exposed to homeostatic NF-κB stimuli in peripheral lymphoid organs and display chronic NF-κB activity. One important homeostatic NF-κB–inducing signal is triggered through the binding of BAFF to BAFFR, and this signal predominantly stimulates the noncanonical NF-κB signaling pathway (26, 29). We have shown that the BAFFR signal is critical for the constitutive expression of ICOSL in B cells in vivo. Consistently, BAFF stimulated ICOSL expression in vitro in both primary B cells and the M12 B-cell line. Our in vitro studies also suggest the involvement of the CD40 signal in ICOSL gene induction. But because CD40 stimulation requires antigen-stimulated T cells, the CD40 signal likely contributes to ICOSL induction only during an immune response.

We found strong evidence suggesting an essential role for the noncanonical NF-κB in ICOSL gene expression. First, inducers that trigger the activation of noncanonical NF-κB, such as BAFF and anti-CD40, effectively induce the expression of ICOSL in B cells. In contrast, canonical NF-κB inducer LPS was insufficient to trigger ICOSL expression in B cells. Moreover, RNAi-mediated knockdown of the core noncanonical NF-κB member RelB attenuated ICOSL gene induction, providing genetic evidence of the need for noncanonical NF-κB in ICOSL gene induction. Finally, our ChIP assays revealed the binding of noncanonical NF-κB members to the promoter region of ICOSL, suggesting their direct involvement in ICOSL gene regulation. Of course, our data do not exclude the possibility that canonical NF-κB is also involved in the regulation of ICOSL gene expression. In particular, BAFF is known to stimulate p50 in addition to the noncanonical NF-κB members p52 and RelB (34). We found that p50 also binds to the ICOSL promoter in BAFF-stimulated B cells. However, because RelA is not a major component of the NF-κB complex bound to the ICOSL promoter, it is likely that p50 may function as a homodimer or a partner of the noncanonical NF-κB member RelB, because RelB is known to form both p52/RelB and p50/RelB heterodimers.

The specific involvement of noncanonical NF-κB in ICOSL gene induction appears to be due to two different regulatory mechanisms. First, noncanonical NF-κB members are the predominant components of chronically activated NF-κB complexes in B cells exposed to the homeostatic inducer BAFF. Second, the ICOSL promoter contains a κB element that favors binding by the noncanonical NF-κB members. Mutation of this κB element abolished the activation of ICOSL promoter by anti-CD40 and BAFF. It is important to note, however, that the role of NF-κB in ICOSL gene regulation appears to vary among different cell types. Previous studies have suggested that canonical NF-κB stimuli, such as IL-1 and TNF-α, induce the expression of ICOSL in endothelial cells and fibroblasts (35–37). Our findings suggest that LPS is inefficient in the induction of ICOSL expression in B cells. Whether other canonical NF-κB stimuli induce ICOSL expression in B cells remains to be investigated. Notwithstanding, our findings suggest that the NIK-regulated noncanonical NF-κB signaling pathway plays a predominant role in mediating the high level of ICOSL expression in B cells, a signaling function required for the supporting role of B cells in antigen-stimulated production of Tfh cells.

Materials and Methods

Mice.

NIK KO mice on a 129Sv/Ev background (38) were provided by Amgen and were maintained in the specific pathogen-free facility of the University of Texas MD Anderson Cancer Center. NIK^+/− heterozygous mice were bred to generate the age-matched NIK^+/+ (WT) and NIK^−/− (KO) mice used in the experiments. Rag2^−/− (Rag2 KO) mice, on a 129Sv/Ev background, were obtained from Taconic. A/J and A/WySnJ mice were obtained from Jackson Laboratory. All animal experiments were performed in accordance with protocols approved by the University of Texas MD Anderson Cancer Center's Institutional Animal Care and Use Committee.

Antibodies, Reagents, and Plasmids.

Antibodies for p50 (D17), p52 (c-5), RelB (C-19), c-Rel (sc-71×), and HSP60 (H-1), as well as control rabbit Ig, were obtained from Santa Cruz Biotechnology. Fluorescence-labeled antibodies for CD4 (L3T4), CD3 (145-2C11), PD-1 (J43), and ICOSL (HK5.3) were purchased from eBioscience. Anti-mouse CD40 (553721) and fluorescence-labeled antibodies for CD19 (1D3) and CXCR5 (2G8) were purchased from BD Biosciences. Other antibodies were as reported previously (28).

Recombinant ICOSL-Fc (also called B7RP-1-Fc) fusion protein and control Fc protein were provided by Amgen. Recombinant BAFF protein (PHC1674) was purchased from Biosource. LPS (derived from E. coli 0127:B8) was obtained from Sigma-Aldrich, and SRBC was purchased from Cocalico Biologicals.

The pLKO.1-puromycin lentiviral vector and the same vector encoding mouse RelB shRNAs were purchased from Sigma-Aldrich. Three shRNAs targeting different regions of the RelB mRNA were used. To generate the luciferase reporter driven by the mouse ICOSL promoter (pGF-ICOSL), a 705-bp ICOSL promoter DNA fragment (−570 to +135) was inserted upstream of the luciferase gene in a lentiviral reporter plasmid, pGreenFire (pGF; System Biosciences). pGF-ICOSLΔκB, a mutant form of pGF-ICOSL that contains point mutations in a κB-like element of the ICOSL promoter (−347 to −338), was created using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The following primers were used: sense, 5′-CAGGGACCAGGCCGTTAACGTTCTGGGCAGCGTTG-3′; antisense, 5′-CAACGCTGCCCAGAACGTTAACGGCCTGGTCCCTG-3′.

The pcDNA expression vectors encoding Flag-tagged p50, p52, and RelB were purchased from Addgene. The pCMV4-p65 was described previously (39).

Cell Culture and shRNA Knockdown.

Murine B-cell line M12.4.1 (designated M12 in this paper) was described previously (28). The cells were infected with lentiviruses carrying either the empty pLKO-1 vector or RelB shRNA clones. The 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 the lentiviral particles, the pLKO.1 vectors were transfected into HEK293 cells (using the calcium method) along with packing vectors psPAX2 and pMD2 (provided by Dr. Xiaofeng Qin, MD Anderson Cancer Center, Houston, TX).

B cells were purified from splenocytes using anti-B220 conjugated magnetic beads (Miltenyl Biotec) and were either directly subjected to flow cytometry or stimulated in vitro by anti-mouse CD40 (500 ng/mL), BAFF (200 ng/mL), or LPS (100 ng/mL).

Mouse Immunization and ICOSL-Fc Injection.

For induction of Tfh cells, age-matched WT and NIK KO mice were immunized i.p. with 2 × 10⁹ SRBC (24). In some experiments, the NIK KO mice were injected i.p. with 50 μg of ICOSL-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.

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 Biotec). The isolated cells were >95% pure, as determined by flow cytometry. WT or NIK KO T cells (5 × 10⁶) were mixed with either WT or NIK KO B cells (5 × 10⁶) and then injected via a tail vein into Rag2 KO mice. After 16 h, the recipient mice were subjected to immunization and ICOSL-Fc injection studies as described above.

Flow Cytometry.

Cell suspensions were subjected to flow cytometry analyses as described previously (41) using a BD Biosciences LSRII flow cytometer. Data were analyzed using FlowJo software.

ChIP Assays.

ChIP assays were performed using the Millipore EZ-ChIP Kit following the manufacturer's instructions. In brief, nontreated and treated M12 cells were crosslinked with 1% of formaldehyde (final concentration, vol/vol) for 10 min, lysed in SDS lysis buffer, and sonicated to shear the DNA. The chromatin DNA was subjected to IP using the indicated antibodies or a control IgG. After purification, the precipitated DNA was analyzed by PCR using primers that amplify different regions of the ICOSL promoter.

The primer sequences were as follows: −2371 to −2150, 5′-ACAGGTTGAGAACCATTCTTCC-3′ and 5′- GAATCCCAGAAAGCCAAATGC-3′; −882 to −675, 5′-TAGCCTCAGACTCAAGAGATC-3′ and 5′-CCAGACTTGGCAATCCTGTTC-3′; −434 to −190, 5′-CCAGGTCCGGGCTTTGAACC-3′ and 5′-CATGAGTTACAGGTGCCAGGGTG -3′.

Statistical Analysis.

Two-tailed unpaired t tests were performed using Prism software. A P value < 0.05 was considered significant.