T Cell-Intrinsic Requirement for NF-κB Induction in Postdifferentiation IFN-γ Production and Clonal Expansion in a Th1 Response

Radiah A. Corn, Mark A. Aronica, Fuping Zhang, Yingkai Tong, Sarah A. Stanley, Se Ryoung Agnes Kim, Linda Stephenson, Ben Enerson, Susan McCarthy, Ana Mora and Mark Boothby
J Immunol August 15, 2003, 171 (4) 1816-1824; DOI: https://doi.org/10.4049/jimmunol.171.4.1816

Abstract

NF-κB/Rel transcription factors are linked to innate immune responses and APC activation. Whether and how the induction of NF-κB signaling in normal CD4+ T cells regulates effector function are not well-understood. The liberation of NF-κB dimers from inhibitors of κB (IκBs) constitutes a central checkpoint for physiologic regulation of most forms of NF-κB. To investigate the role of NF-κB induction in effector T cell responses, we targeted inhibition of the NF-κB/Rel pathway specifically to T cells. The Th1 response in vivo is dramatically weakened when T cells defective in their NF-κB induction (referred to as IκBα(ΔN) transgenic cells) are activated by a normal APC population. Analyses in vivo, and IL-12-supplemented T cell cultures in vitro, reveal that the mechanism underlying this T cell-intrinsic requirement for NF-κB involves activation of the IFN-γ gene in addition to clonal expansion efficiency. The role of NF-κB in IFN-γ gene expression includes a modest decrease in Stat4 activation, T box expressed in T cell levels, and differentiation efficiency along with a more prominent postdifferentiation step. Further, induced expression of Bcl-3, a trans-activating IκB-like protein, is decreased in T cells as a consequence of NF-κB inhibition. Together, these findings indicate that NF-κB induction in T cells regulates efficient clonal expansion, Th1 differentiation, and IFN-γ production by Th1 lymphocytes at a control point downstream from differentiation.

Understanding the regulation of immunity and autoimmunity requires dissecting how the amounts of effector cytokine released in response to Ag are controlled. To this end, much attention has focused on the differentiation step at which proliferating Th precursor cells activate previously silent cytokine genes and become committed to a new state such as the Th1 phenotype (1, 2). However, T cell contributions to acquired immune responses also require lymphocyte proliferation, clonal expansion, the survival of activated cells, and reinduction of gene expression in differentiated effectors in addition to the acquisition of effector function by naive precursors. Thus, the outcome of an Ag-specific immune response may be modulated by changing the efficiency with which lymphocyte clones expand, the frequency of differentiation into effector cells, cytokine gene expression levels within the activated effector population, or combinations of these processes. The magnitude of gene expression during a response in vivo might be regulated by controlling either the frequency with which naive precursors differentiate into IFN-γ-producing Th1 cells or by regulating gene expression in each cycling, committed effector. In support of this overall model, i.v. administration of soluble OVA peptide without adjuvant did not prevent the differentiation of TCR-transgenic cells into efficient Th1 cells; instead, the overall response was diminished because clonal expansion was inefficient as compared to Ag administered in a potent adjuvant (3).

Differentiation and postdifferentiation gene expression are regulated by overlapping sets of transcription factors. Stat4 activated by IL-12, and IFN regulatory factor (IRF)-1,7 a transcription factor originally described as an IFN-regulated protein, make important contributions in vivo during the regulated differentiation of Th1 cells (4, 5, 6). IRF-1 induction is downstream of signaling by Stat1 and apparently Stat4, but not Stat6 (7, 8). The need for IRF-1 in generating a type 1 T cell-dependent response reflects a cooperation between IRF-1 actions in APCs, which are the most important, and a modest T cell-intrinsic role (6). Along this same branch, a Stat4-dependent transcription factor designated Ets-related molecule influences effector T cell development in vitro but its role in vivo remains to be determined (9). These regulatory proteins can be considered as serving an IFN-γ/IL-12-dependent limb of Th1 development. Ultimately, these and other proteins may serve to fix the Th1 phenotype by regulating a transcription factor termed T box expressed in T cells (T-bet) (10, 11) and IL-12Rβ2 expression (12). Expression of T-bet is diminished in Stat4-deficient mice and becomes restricted to developing Th1 effectors in vitro through selective increases under conditions promoting Th1 differentiation and reciprocal decreases in activated, uncommitted T cells cultured under Th2 conditions (11, 12, 13).

In addition to the transcription factors noted above, TCR signaling leads to the nuclear induction of members of the NF-κB/Rel transcription factors. These proteins comprise a family whose evolutionary roots are tightly linked to innate host defenses against pathogens, and are prototypic links in inflammatory responses to microbes (14, 15). Consistent with these roots, important roles in inflammation and innate immunity in mice have been identified for individual genes encoding NF-κB/Rel proteins (14, 15, 16). However, it has not been clear whether the NF-κB pathway plays a role in regulating the development or effector function of the type 1 effector T cells essential for resolving infections by intracellular pathogens. Some work using mice deficient in one subunit that is part of the prototypic NF-κB has suggested that there is no role for NF-κB induction in T cells in regulating Th1 differentiation or IFN-γ production (17, 18). Thus, the weak Th1-mediated inflammation observed in vivo when the NF-κB subunit p50/NF-κB1 is absent may be due exclusively to a lack of APC-derived IL-12 (19, 20).

Physiologic induction of the NF-κB/Rel pathway is regulated by the controlled release of dimeric complexes from cytosolic retention molecules termed inhibitors of κB (IκBs) (21, 22). Cell surface receptor engagement activates an enzymatic complex, the IκB kinases (IKKs), leading to regulated serine phosphorylation, ubiquitination, and degradation of IκBs such as IκBα (14, 21, 22). Because of this convergence on IκBs of most pathways regulating NF-κB induction, a mutant IκBα molecule serves as a trans-dominant inhibitor of NF-κB induction and provides a dominant phenocopy of the inhibition of the physiologic checkpoint for regulation (23, 24). In transgenic mouse lines, IκBα molecules mutated at their N termini block NF-κB induction by a range of physiologic stimuli including TCR/CD28 and TNF-α (25, 26, 27). A transgene consisting of a cDNA encoding an N-terminal deletion of IκBα (IκBα(ΔN)) was expressed at high levels in mature T cells when under the control of the lck proximal promoter and linked to a locus control region derived from the human CD2 gene (25). Experiments in which delayed-type hypersensitivity (DTH) or allergic lung disease were elicited in these transgenic mice revealed that the type 1 T cell-dependent response was dramatically inhibited, whereas type 2 T cell responses in vivo were decreased little if at all (28). As noted above, however, the contribution of NF-κB to type 1 (Th1) responses has been ascribed to its role in IL-12 expression rather than a T cell-intrinsic function (17, 18). Moreover, it was possible that there was an indirect contribution of other types of T cell to the forms of response studied in vivo, since IκBα(ΔN) mice are strikingly deficient in conventional CD8αβ T cells (25, 29) which might influence the strength of the type 1 response.

In the present study, we have used in vitro and in vivo approaches to investigate the T cell-intrinsic contributions of NF-κB transcription factors to a Th1 response. In particular, we have determined whether such responses are attenuated by inhibition of NF-κB under conditions where the T cells have access to normal APC populations or to sufficient IL-12, and measured the induction of gene products implicated in the IFN-γ response in vivo. In analyzing the role of NF-κB induction, we have dissected the contributions to a type 1 response of the fundamental mechanisms outlined above: clonal expansion/proliferation, differentiation into Th1 cells, and regulation of IFN-γ expression after expansion and differentiation. Together, the data establish that induction of NF-κB within T cells regulates normal IFN-γ production in vivo and in vitro in the presence of normal APCs and exogenous IL-12. An effect on Th1 development efficiency was observed, but the predominant roles of NF-κB induction were to promote clonal expansion during the Th1 response and to control the amount of IFN-γ produced from a differentiated Th1 population.

Materials and Methods

Reagents and mice

Cytokines, mAbs, and immunofluorescent detection reagents were purchased from BD PharMingen (San Diego, CA) except for purified cytokines recombinant human (rhu) IL-2 (BRMP program; National Cancer Institute, Frederick, MD), IL-12 and -18 (Leinco Technologies, St. Louis, MO) and murine IFN-γ (R&D Systems, Minneapolis, MN). For immunoblotting, anti-Bcl-3 and its control peptide for confirmation of specificity were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), anti-Stat4 and activation-specific anti-phospho-Stat4 were obtained from Zymed Laboratories (San Francisco, CA), anti-cyclophilin B from Alexis (Carlsbad, CA) and anti-T-bet was a generous gift of L. Glimcher (Harvard University, Boston, MA). Custom-synthesized peptides (OVA323–339; IKK inhibitor (30)) were obtained (Research Genetics, Birmingham, AL) at >70% purity mass spectrometry. Pyrrolidine dithiocarbamate (PDTC) was purchased from Sigma-Aldrich (St. Louis, MO). DO-11.10 mice (BALB/c) transgenic for a TCR recognizing OVA323–339 peptide (31), and H-2d lck-IκBα(ΔN) transgenic mice (25) backcrossed to BALB/c (n > 7) were bred in the Vanderbilt University Medical College mouse facility (Nashville, TN) in specific pathogen-free conditions using microisolator cages and were used in accordance with applicable regulations after institutional approval. Breeding stock and BALB/c transfer recipients (4- to 8-wk-old females) were from The Jackson Laboratory (Bar Harbor, ME).

Cell cultures and generation of effector T cell populations

CD4+ T cells from spleen and lymph nodes of TCR-transgenic mice were isolated by negative selection using anti-CD8 and anti-MHC II microbeads (Miltenyi Biotec, Auburn, CA) according to manufacturer’s instructions. Naive L-selectin (CD62L)-high cells were then positively selected using bead-conjugated anti-CD62L mAb at an Ab:buffer ratio of 1:50. The resultant cells were >95% CD4+, CD44low, and >90% CD4+, CD62Lhigh. Cells were maintained in supplemented medium containing 10% heat-inactivated FBS as described (25). These naive cells were cultured at 5 × 106 cells/ml together with APCs from BALB/c nontransgenic mice obtained by panning and were plated at a 1:1 ratio with T cells and OVA323–339 (1 μg/ml; Research Genetics). Alternatively, nontransgenic T cells were activated with plate-bound Abs against CD3ε and CD28 as described (25, 32). For development of Th1-polarized populations, IL-12 (1 or 10 ng/ml), anti-IL-4, (1 μg/ml), and rIL-2 (10 U/ml) were added (day 1; day 3). For Th2-polarized cells, rIL-4 (10 ng/ml), anti-IL-12 (2 μg/ml), anti-IFN-γ (1 μg/ml), and rhuIL-2 (10 U/ml) were used. Cells were harvested for restimulation at day 6, centrifuged on a Ficoll step gradient, rinsed, and restimulated. To determine the polarization of each population, a portion was restimulated using BALB/c APCs and 1 μg/ml OVA323–339 peptide (33), followed by ELISA to measure IL-4 and IFN-γ in the culture supernatants. For retroviral transduction experiments, IκBα(ΔN) cDNA was inserted into the bicistronic vector MSCV2.2-IRES-GFP (GFP-RV) (34). Single cell suspensions of naive CD4+ T cells were prepared from spleen and lymph nodes of DO11.10 TCR-transgenic mice and were peptide-activated as above. Retrovirus-containing supernatants were collected 48 h after transfection of the ecotropic ΦNX packaging cell line with retrovector plasmids, and centrifuged (1 h, 10,000 × g) with peptide-activated T cells (34, 35) which were then cultured under Th1-polarizing conditions. After 6 days of culture, cells were washed, sorted for green fluorescent protein (GFP+) cells, and restimulated (48 h) using plate-bound anti-CD3 (10 μg/ml) and anti-CD28 (2.5 μg/ml), IL-12 plus IL-18 (10 ng/ml each), or with peptide-pulsed APCs. Cytokine production was determined by ELISA of these culture supernatants (28); the frequency of cytokine-producing cells was measured by restimulation of Th2-polarized cells (PMA plus ionomycin) and intracellular staining as described above. For pharmacological inhibition of NF-κB in T cells, lymphocytes (as above) were preincubated 90 min in culture medium supplemented with PDTC (50, 25, and 5 μM) (36, 37, 38) or vehicle (PBS), then stimulated with plate-bound anti-CD3/anti-CD28, and cultured under Th1- or Th2-polarizing conditions (as above).

Adoptive transfers, flow cytometric analyses, and cytokine production assays

Equal numbers of naive, CD4+, KJ1-26+D011.10 T cells from spleen and lymph nodes of donor mice of the indicated genotypes were transferred into syngeneic recipients by i.v. injection. The next day, mice were immunized with OVA323–339 emulsified 1:1 with CFA as described (33). After 6 days, single cell suspensions from draining lymph nodes were used for enumeration of donor-derived (CD4+, KJ1-26+) T cells and restimulation with the indicated concentrations of peptide. Cytokine measurements were performed as described (32, 33). IL-18Rα expression was quantitated by three-step indirect immunofluorescent staining with anti-IL-18Rα (R&D Systems), biotinylated anti-IgG goat, and streptavidin-PerCP. For FACS analysis of intracellular cytokines, cells were cultured 6 h in the presence of Golgi Stop (BD PharMingen) starting 2 h after restimulation (either APCs with 1 μg/ml OVA323–339 peptide, or 10 ng/ml PMA and 1 μM ionomycin (Sigma-Aldrich), for which results were equivalent). Immunofluorescent staining was performed using allophycocyanin anti-CD4 and biotinylated anti-clonotypic KJ1-26 mAb with streptavidin-PerCP to detect transferred DO-11.10 T cells. Stained cells were then washed, fixed (30 min, 4% paraformaldehyde in PBS), permeabilized with 0.1% saponin, and further stained with PE-anti-IFN-γ plus FITC-anti-IL-4. Induction and quantitation of DTH in response to OVA was performed as described (28). Samples were analyzed using a FACSCalibur flow cytometer and CellQuest software (BD Biosciences, San Jose, CA), gating on forward-scatter and side-scatter properties of viable lymphoid cells.

Cell extraction, mobility shift assays, and quantitation of RNAs and proteins

Whole cell and nuclear protein extracts were prepared from cultured cells and assayed by electrophoretic mobility shift or immunoblotting with the indicated Ab preparations as described (10, 25) or according to manufacturer’s recommendations. Total RNA was obtained using TRIzol for use in Northern blot analyses as described (32).

Results

A T cell-intrinsic role of NF-κB in IFN-γ production by Th1 cells in vivo

Previous work indicated that the type 1 T cell-dependent response was substantially decreased in transgenic mice expressing a dominant inhibitor of the nuclear induction of NF-κB/Rel complexes in T lineage cells, whereas the release of IL-4 in response to Ag increased (28). Expression of this transgene, termed IκBα(ΔN), was targeted to T lineage cells using well-characterized T cell-specific control elements, and expression had not been detected in cells outside of the T lineage. However, it was possible that the inhibitory protein is expressed in a vital cellular subset (e.g., dendritic cells) below the limits of biochemical detection and such expression influenced IL-12 production and the outcome of responses in vivo (39, 40). In this regard, a defect of type 1 responses in p50/NF-κB1-deficient mice may be attributable to the dependence of Th1 development on NF-κB-regulated IL-12 production from DCs rather than reflecting any T cell-intrinsic function (17, 18, 19). Further, the type 1 response in vivo is determined by contributions from CD8 and NKT cells in addition to the Th1 subset. Therefore, we sought to determine whether the block to IFN-γ production stemming from inhibition of the IκB/NF-κB/Rel pathway was a T cell-intrinsic process, and investigated whether exogenous IL-12 corrected the defect.

The inhibitory IκBα(ΔN) transgene was backcrossed onto a BALB/c background expressing the DO11.10 TCR transgene (31). DO11.10 T cells expressing the mutated IκBα were transferred into BALB/c recipients to provide an ample pool of normal APCs and T cells, while control mice received cells from DO11.10 littermates with normal NF-κB signaling in their T cells. After immunization with OVA323–339 peptide, the production of IFN-γ upon peptide restimulation of draining lymph node cells from recipients of IκBα(ΔN) T cells was dramatically decreased (Fig. 1⇓A). It was possible that we obtained this result because the inhibition of NF-κB in T cells led to a failure to localize to the draining lymph nodes, survive after activation, or undergo any clonal expansion. Enumeration of DO11.10 CD4+ draining lymph node cells with the KJ1-26 mAb revealed that viable donor T cells were present, albeit at reduced numbers (Fig. 1⇓B). Overall, donor T cell numbers in immunized mice receiving wild-type T cells were 5- to 6-fold greater than those that received T cells subjected to NF-κB inhibition. Accordingly, impaired clonal expansion is one mechanism by which T cell-intrinsic NF-κB induction regulates the Th1 response in vivo, independent of any effects on APCs.

FIGURE 1.
FIGURE 1.

T cell-specific inhibition of NF-κB leads to decreased Th1 response in vivo. T cells were prepared from BALB/c-DO11.10 TCR transgenic mice expressing IκBα(ΔN), or littermates with normal NF-κB signaling. Equal numbers of CD4+ T cells, as determined by flow cytometry, were transferred into syngeneic mice (2 × 106 cells per recipient) which were then analyzed (unimmunized controls) or immunized with OVA323–339 peptide in CFA. A, Draining lymph node cells from mice immunized with peptide were harvested 6 days later, restimulated in vitro with the indicated concentrations of OVA323–339, and culture supernatants were assayed for IFN-γ. Under these assay conditions, no IL-4 production was detectable and no IFN-γ was produced by unimmunized controls (data not shown). B and C, An attenuation of clonal expansion which is insufficient to account for the decrease in IFN-γ production. The frequency of donor-derived T cells was determined by staining with the anti-clonotypic mAb KJ1-26 and CD4 (B), allowing calculation of the numbers of donor-derived cells from wild-type and IκBα(ΔN) donors (∗∗∗, p < 0.001 relative to wild-type controls). Using these numbers, the production of IFN-γ per donor-derived cell was calculated for each sample, averaged, and plotted (C).

However, the diminution in donor cell multiplication was insufficient to account fully for the dramatic decrease in Th1 response (Fig. 1⇑A). Calculation of the amount of IFN-γ released per viable CD4+KJ1-26+ T cell showed that the presence of the IκBα(ΔN) transgene led to an ∼95% reduction in the IFN-γ production per Ag-specific T cell (Fig. 1⇑C). Because the APC population and all other cells in the recipient mice are normal, we conclude that a Th1 response was potently inhibited in vivo when the induction of the NF-κB/Rel pathway was inhibited in T cells. Further, although the results showed that clonal expansion decreased, there was an even more dramatic impairment in IFN-γ production per donor-derived T cell.

A T cell-intrinsic defect of the Th1 response in vitro under conditions of NF-κB inhibition

The adoptive transfer data indicate that induction of NF-κB/Rel proteins within T cells (as distinct from APCs, which were normal in these experiments) was of critical importance for the Th1 response in vivo. We further investigated the role of NF-κB in effector T cell responses using in vitro activation, differentiation, and restimulation of T cells. This experimental approach provided the opportunity to measure the Th1 response under conditions where activated T cells were exposed to equal supplies of exogenous IL-2 and the Th1 differentiative cytokine IL-12, while also neutralizing negative effects which might derive from increased production of IL-4 (1, 2, 28). T cells from IκBα(ΔN) mice and wild-type littermates were activated either by an antigenic peptide or by polyclonal stimulation and grown in the presence of added IL-2 and IL-12. Consistent with the findings of experiments performed in vivo, the multiplication of T cells under Th1 conditions was decreased when NF-κB was inhibited (5.5 ± 1.1 vs 3.3 ± 0.9, p = 0.03). Importantly, IFN-γ production by equal numbers of replated IκBα(ΔN) cells was significantly diminished compared to controls when viable activated T cells were restimulated (Fig. 2⇓, A and B). Addition of IL-18 along with IL-2 and IL-12 was also unable to reverse the defect of IκBα(ΔN) T cells. Similar results were obtained when a population of normal APCs was provided to the T cells at the time of their initial activation and differentiation as well as upon restimulation (Fig. 2⇓C). These findings show that a T cell-intrinsic defect in IFN-γ production results from NF-κB inhibition even with IL-12 and normal APCs present.

FIGURE 2.
FIGURE 2.

Transgenic inhibition of NF-κB leads to decreased Th1 response in vitro under Th1-polarizing and nonpolarizing conditions. A, Equal numbers of CD4+ non-TCR-transgenic T cells of the indicated genotype were activated with plate-bound anti-CD3/anti-CD28 in vitro and cultured in IL-2 under Th1- or Th2-polarizing conditions, or without polarization, as indicated. After 6 days, equal numbers of rinsed cells were restimulated with anti-CD3/anti-CD28 and resultant cytokines in culture supernatants were quantitated by ELISA. Data shown are from one experiment representative of four with similar results. B, As in A except that DO11.10 TCR-transgenic cells were activated with OVA323–339 peptide (1 μg/ml), in the presence or absence of exogenous IL-18 as indicated, and restimulated with wild-type BALB/c APCs plus peptide (1 μg/ml). Shown are mean (±SEM) data from three independent experiments. Th1 low, 1 ng/ml IL-12; Th1 high, 10 ng/ml IL-12. C, As in B except that the initial activation was performed using a 1/1 mixture with wild-type (WT) APCs.

We also tested the effect of inhibiting NF-κB/Rel transcription factors using transgene-independent approaches. The agent PDTC serves as a selective inhibitor of NF-κB induction (36, 37), and control mobility shift experiments showed that it inhibited NF-κB but not AP-1 mobility shift activities at concentrations of 5–50 μM (Fig. 3⇓A). When the effect of PDTC treatment on T cell responses was determined, this NF-κB inhibitor dramatically decreased IFN-γ production by equal numbers of restimulated cells after priming and culture in Th1-polarizing conditions (Fig. 3⇓B). Consistent with the findings obtained using the transgenic system, the multiplication of T cells in PDTC-treated cultures was reduced as well (data not shown). Thus, inhibition of IFN-γ production was observed using a second independent approach that bypasses any potential abnormality of the naive T cell repertoire that might be imposed by expression of the IκBα(ΔN) transgene prior to stimulation and Th1 differentiation. A retrovector encoding the IκBα(ΔN) protein generated sufficient levels of protein to inhibit NF-κB induction (90–95%) (Fig. 3⇓C, lower panel), a degree similar to that of the transgene (25). When DO11.10 T cells were activated, transduced with this retrovector or the empty-vector control (GFP-RV), differentiated under Th1 conditions, and restimulated, IFN-γ production was substantially inhibited (Fig. 3⇓D). IFN-γ production reactivated by a distinct IL-12/IL-18-induced pathway (41) was also diminished (data not shown). We conclude that transgene-independent inhibition of NF-κB decreases the IFN-γ production capacity of a developing Th1 population, congruent with the data with using transgenic T cells in vivo and in vitro. Further, these findings suggest that regulation of IFN-γ gene expression after differentiation of the activated Th precursor might be one component of the requirement for NF-κB induction in the Th1 response.

FIGURE 3.
FIGURE 3.

Decreased IFN-γ production after nontransgenic inhibition of NF-κB. A, Selective inhibition of NF-κB by PDTC. NF-κB and AP-1 mobility shift assays were performed using extracts from splenocytes pretreated with vehicle alone or the indicated concentrations of PDTC and then activated 18 h with plate-bound anti-CD3 plus anti-CD28. As a loading control, immunoblots of the extracts were probed with anti-cyclophilin B. B, PDTC inhibition of the Th1 response in vitro. Equal numbers of splenocytes were pretreated with PDTC or vehicle and activated as in A. After culture under Th1- or Th2-polarizing conditions as indicated, equal numbers of rinsed cells were restimulated with plate-bound anti-CD3/anti-CD28, and cytokines in the culture supernatants were measured by ELISA. C, Inhibition of NF-κB by retrovector-mediated expression of IκBα(ΔN) after activation of nontransgenic T cells. BALB/c splenocytes activated with anti-CD3/anti-CD28 2 days previously were infected with retrovirions packaging an IκBα(ΔN)-IRES-GFP (IκBα(ΔN)) or empty IRES-GFP retrovector (GFP-RV). After preparative sorting of GFP-positive cells and 6 days of expansion in IL-2, extracts were prepared from equal numbers of rinsed or restimulated cells. Upper panel, Anti-FLAG immunoprecipitates from equal amounts of extract were subjected to Western blotting using anti-IκBα Abs and compared to immunoprecipitates (IP) of the same amount of extract protein from thymocytes of IκBα(ΔN) transgenic mice as a reference standard. Lower panel, EMSA of equal amounts of extract protein from transduced cells restimulated with PMA/ionomycin were performed with a radiolabeled κB oligonucleotide and compared to extracts from cells which were not restimulated. D, Naive, CD4+ DO11.10 T cells were activated with peptide under Th1 conditions and infected with retrovirions as in C. After preparative sorting, GFP+ cells were expanded under Th1 conditions. Equal numbers of rinsed cells were restimulated with anti-CD3/anti-CD28, followed by ELISA to measure IFN-γ in the culture supernatants.

NF-κB is essential for both postdifferentiation IFN-γ production and full Th1 development

A transcription factor may regulate the production of effector cytokines by regulating initiation of the Th1 or Th2 program of cytokine genes (42), subsequent to such differentiation, or, in a case such as GATA-3, at both steps (43, 44, 45). If a factor acted exclusively after the differentiation step, then the frequency of cells scoring as positive for effector cytokine expression by intracellular staining would be normal despite diminished expression of the transcriptional protein and impaired effector response. To dissect the T cell-intrinsic contributions of NF-κB to regulation of the Th1 response, CD4 T cells from wild-type and IκBα(ΔN) mice were differentiated under Th1 conditions in vitro and the frequency of CD4+ T cells which scored as IFN-γ-positive after restimulation was measured (Fig. 4⇓A). This analysis showed that the inhibition of NF-κB led to an impairment in Th1 development (the frequency of IFN-γ-positive cells), albeit one less dramatic than the decrease in per cell IFN-γ production observed in the adoptive transfer experiments in vivo (Fig. 1⇑). We next determined whether similar results would be observed with a polyclonal population of T cells whose developmental history was independent of any perturbation of signaling. Nontransgenic T cells cultured under continuous Th1 conditions following activation with anti-CD3 and -CD28 were infected with replication-defective retrovectors, expanded, and analyzed. The transduced CD4+ T cells which expressed IκBα(ΔN) developed a lower frequency of IFN-γ+ cells than the population infected with control GFP-RV (Fig. 4⇓B). These data indicate that inhibition of NF-κB in T cells reduced the efficiency of Th1 development, but not to a degree which quantitatively accounts for the overall inhibition of Th1 responses. To test the possibility that IFN-γ production after differentiation represents a major point of regulation by NF-κB, a polarized Th1 population was generated from TCR-transgenic cells and treated with PDTC just prior to restimulation through the Ag receptor. IFN-γ release was markedly decreased compared to untreated cells (Fig. 4⇓C), indicating that postdifferentiation production rates were inhibited. Because the magnitude of decrease in per cell IFN-γ production (Fig. 1–3⇑⇑⇑) was 5- to 10-fold greater than the decrease in frequency of Th1 cells in these populations (Fig. 4⇓A), we conclude that a major role of NF-κB is in regulating rates of IFN-γ production by differentiated Th1 cells at the time of their restimulation. Inhibition of the differentiation process itself appears also to contribute, but to a quantitatively lesser extent.

FIGURE 4.
FIGURE 4.

Role of NF-κB inducibility in Th1 differentiation efficiency and postdifferentiation IFN-γ production. A, Wild-type (WT) and IκBα(ΔN) T cells, each on a BALB/c DO11.10 TCR transgenic background, were stimulated with OVA323–339 peptide and syngeneic APCs and cultured 6 days in neutral or polarizing conditions (Th1, IL-12, and anti-IL-4; Th2, IL-4, and anti-IL-12 plus anti-IFN-γ) as detailed in Materials and Methods. Equal numbers of rinsed cells were restimulated and stained for CD4 and the indicated intracellular cytokines (IL-4 and IFN-γ). Shown are data gated on the CD4+ cells in the restimulated population. B, As in A, except that the cells represent the GFP+ cells after retroviral transductions using empty vector (GFP-RV) or IκBα(ΔN)-encoding vector, as described in Fig. 3⇑ (C and D). C, DO11.10 T cells were activated and cultured under Th1-polarizing conditions as in Fig. 3⇑. Equal numbers of rinsed cells were then pretreated with vehicle or the indicated concentration of PDTC and restimulated.

T-bet and Bcl-3 levels depend on NF-κB/Rel induction in primary T cells

The data above establish a T cell-intrinsic role for NF-κB induction in the clonal expansion needed for a Th1 response and in the expression of IFN-γ in Th1 cells. To investigate molecular mechanisms by which the NF-κB/Rel transcription factors regulate IFN-γ production, we focused on several inducible gene products that have been implicated in regulating the Th1 response. IL-12 and IL-18 program the Th1 response of uncommitted, activated CD4+ T cells, with IL-12 signaling Th1 differentiation and IL-18Rα expression through the induction of Stat4 (1, 2, 4, 12, 46). After activation and short-term culture, CD4 T cells from IκBα(ΔN) mice exhibited decreases in Stat4 protein levels and in the quantitative levels of activated (tyrosine-phosphorylated) Stat4 compared to wild-type controls (Fig. 5⇓A). Notwithstanding this decrease, IL-12 receptors were clearly able to signal. Consistent with this, the induction and level of cell surface IL-18 receptor expression on T cells with inhibition of NF-κB were slightly increased compared to controls (Fig. 5⇓B). The T-box transcription factor T-bet is critical for the emergence of a differentiated Th1 population in vitro and in vivo, and induction of this protein is promoted by Stat4 (10, 11). In addition to a role in differentiation, T-bet may control postdifferentiation gene expression since it can trans-activate a human IFN-γ minigene in transient transfection assays (10). After culture under conditions polarizing toward Th1 differentiation followed by restimulation, the level of T-bet mRNA and protein were less than half of control levels when NF-κB was inhibited (Fig. 5⇓, C and D). No difference in the induction of IRF-1 mRNA was detected (data not shown). Thus, T-bet levels were diminished as a consequence of decreased NF-κB induction. As noted above, however, the levels of these regulators of Th1 differentiation in T cells from IκBα(ΔN) mice appeared unlikely to account entirely for the severity of the IFN-γ production defect.

FIGURE 5.
FIGURE 5.

Decreased induction of Stat4 and T-bet as a consequence of NF-κB inhibition. A, Stat4 protein levels and IL-12 induction of phospho-Stat4 with inhibition of NF-κB. CD4+ T cells were purified from lymphoid organs of DO11.10 TCR-transgenic mice bearing or lacking the IκBα(ΔN) transgene. After activation with anti-CD3 (2.5 μg/ml) and anti-CD28 (1 μg/ml) and growth in the presence of IL-2 and anti-IL-4, rinsed cells were divided, cultured in medium alone (6 h), followed by medium with anti-IL12 or IL-12 (30 min). Extracts of these cultures were then subjected to immunoblot analyses using anti-phospho-Stat4, total Stat4, and cyclophilin B as a loading control. B, Expression and IL-12 induction of IL-18Rα despite inhibition of NF-κB. T cells from DO11.10 mice bearing or lacking the IκBα(ΔN) transgene were either stained for KJ1-26, CD4, and IL-18Rα, or activated with OVA323–339 peptide and syngeneic APCs, cultured under Th1-inducing conditions (2 days), and then stained. Shown are fluorescence histograms of staining detected on CD4+, clonotype+ cells in the viable lymphoid gate. C and D, Diminished T-bet expression in IκBα(ΔN) effector T cells. Wild-type and IκBα(ΔN) T cells, each on a BALB/c DO11.10 TCR transgenic background, were stimulated with OVA323–339 peptide and syngeneic APCs and cultured 6 days in polarizing conditions (Th1, wild-type, and transgenic; Th2, wild-type only). Whole cell RNA (C) or protein (D) extracts from equal numbers of rinsed cells were analyzed in Northern (C) and Western (D) blots using probes specific for T-bet or the indicated loading controls. WT, wild type.

Recently, it has been proposed that TNF-α induction of Bcl-3, an atypical IκB-like molecule which can trans-activate target genes through its interaction with p50/NF-κB1 or p52/NF-κB2 homodimers, is regulated by NF-κB in nonlymphoid cells (47, 48, 49). Further, mice rendered deficient in Bcl-3 expression by gene targeting have defects in B cell number, germinal center formation, and impaired IFN-γ production in response to an intracellular parasitic infection (50, 51). Measurements of Bcl-3 protein by immunoblotting after TCR stimulation revealed a significant reduction in the level of Bcl-3 in T cells expressing IκBα(ΔN) or subjected to inhibition by the NF-κB inhibitor PDTC, as compared to controls (Fig. 6⇓, A and B). This decrease in Bcl-3 levels was also observed in experiments performed using a specific IKK inhibitor peptide (30) (200 μM) vs DMSO vehicle, data not shown). Other evidence suggests that the AP-1 transcription factors may regulate Bcl-3 levels (52). However, the inhibition of NF-κB did not cause a global defect in induction of transcription factors linked to TCR signaling, inasmuch as AP-1 could be induced normally under conditions where NF-κB was not (Fig. 6⇓C). We conclude that levels of Bcl-3 after T cell activation are influenced by NF-κB induction, suggesting that a defect in this induction step may contribute to the mechanism by which conventional NF-κB/Rel dimers regulate the strength of the Th1 effector response in vivo.

FIGURE 6.
FIGURE 6.

Decreased Bcl-3 as a consequence of NF-κB inhibition. A, NF-κB-dependent induction of Bcl-3. Extracts were prepared from purified CD4+ T cells of wild-type (WT) and IκBα(ΔN) mice after activation with anti-CD3/28 (1 and 3 days) and subjected to immunoblotting using anti-Bcl-3. B, Extracts were prepared from CD4 T cells activated with anti-CD3/anti-CD28 after preincubation with PDTC where indicated. Immunoblot measurements were performed using anti-Bcl-3 and anti-cyclophilin Abs as indicated. C, Normal AP-1 induction in IκBα(ΔN) T cells. Extracts of naive CD4+ T cells isolated by magnetic sorting and activated as indicated were subjected to mobility shift assays using radiolabeled dsDNA probes containing an NF-κB or AP-1 binding site. Immunoblot measurements were performed using anti-cyclophilin Abs as controls for similarity of the protein extract in each sample.

Discussion

TCR engagement, costimulatory receptors, and cytokines such as IL-12 and IL-4 regulate signaling pathways leading to the activation of transcription factors which determine effector function. A limited set of the evoked proteins are helper subset-specific and necessary for the reinforcement or amplification of the Th1 or Th2 pathway (2). Other transcriptional regulators appear to be subset nonspecific, and a fundamental question is how these transcription factors control subset-specific responses. Thus, members of the NF-κB/Rel family of transcription factors are activated after TCR ligation, particularly in Th1 more than Th2 cells (53), but it has been unclear whether the inducibility of NF-κB has a T-cell intrinsic or preferential role in the regulation of Th1 effector functions. Previously, we reported that T lineage-specific expression of a mutated form of the IκBαprotein (designated IκBα(ΔN)) blocked DTH and Th1-specific cognate help to Ab production (28, 54). In contrast, Ag-evoked IL-4 production in draining lymph nodes and Th2-dependent help to Ag-specific IgE were increased in the IκBα(ΔN) transgenic mice (28). These data indicated that NF-κB has a preferential role in type 1 as opposed to type 2 responses but did not dissect the relative contributions of clonal expansion, Th1 differentiation, and postdifferentiation production of IFN-γ. In this study, we report that NF-κB induction within T cells is crucial for the Th1 response, independent of the role for this transcription factor in APC production of IL-12. Further, NF-κB induction is required not only for clonal expansion but also for full differentiation and substantial IFN-γ production by each committed Th1 cell.

The magnitude of an immune response is influenced by both the capacity of an effector cell to produce cytokines such as IFN-γ, as well as the number of Ag-specific effector cells involved in the response. Our data indicate that the reduction in IFN-γ production after inhibition of NF-κB in vivo or in vitro is due in part to decreased multiplication of Ag-specific T cells after activation. Specifically, fewer IκBα(ΔN) than wild-type KJ1-26+ donor cells were recovered from the draining lymph nodes of immunized BALB/c recipients. Further, when cultured under Th1-polarizing conditions with exogenous IL-2, IκBα(ΔN) cells did not expand as well as wild-type cells although, intriguingly, each population grew equally well under Th2 conditions. This contribution of NF-κB to clonal expansion and thereby to an effective Th1 response may be related to findings on clonal anergy induction by soluble protein Ag in the absence of adjuvant (3, 33). Thus, it has been suggested that this form of anergy in CD4+ T cells primarily inhibits clonal expansion (3), and it is intriguing to note that the inducibility of NF-κB may be diminished in other forms of anergy induction (55, 56, 57). This particular type of clonally anergic cell in lymphoid tissues developed a normal Th1 effector capacity as compared to controls immunized with peptide in adjuvant (3). In considering which receptors and signal transducers participate in the NF-κB induction which we show to be involved in Th1 responses in vivo, it is intriguing to note that IκBα degradation and NF-κB induction were reduced after anti-CD3 stimulation of receptor-interacting protein (RIP)-2 deficient T-cells (58, 59). Of note, induction of other transcription factors also was affected by the lack of this signaling intermediate. Consistent with our data, these cells exhibited reduced proliferative responses in vitro as well as a selective decrease in production of IFN-γ but not IL-4 by polarized Th1 and Th2 cells, respectively (58, 59). Thus, it seems likely that the ability of RIP-2 to connect a set of cell surface receptors to the IKK/IκB/NF-κB axis is a key feature of the mechanism by which the RIP adapter protein contributes to the T cell-intrinsic regulation of an adaptive Th1 response.

In terms of the decrease in clonal expansion, previous work suggests that NF-κB regulation of T cell survival (25) is likely to be a significant contributory mechanism and that several independent target genes downstream from NF-κB may be involved. We found that the level of Bcl-3 protein was reduced when NF-κB was inhibited during T cell activation. Perhaps consistent with this observation, studies in the HepG2 tissue culture cell line indicated that Bcl-3 has two high-affinity κB sites in its promoter and is induced by TNF-α in a NF-κB-dependent manner (47). Further, a study of the mechanisms by which adjuvants are able to promote the survival of superantigen-activated T cells found that expression of Bcl-3 was increased 10-fold when T cells were activated along with endotoxin or vaccinia virus and T cell survival was improved when Bcl-3 was overexpressed in activated T cells by retroviral transduction (60). Thus, the attenuated ability of IκBα(ΔN) T-cells to up-regulate Bcl-3 expression may contribute to their inefficient clonal expansion and weak Th1 effector response. However, the mechanisms by which NF-κB induction in T cells mediates clonal expansion are likely to be multifaceted and involve a number of survival factors. Bcl-xL is a key antiapoptotic gene that also contains functional κB binding sites in its promoter (61, 62), and we have observed a decrease in Bcl-xL levels in primary CD4+ T cells after activation when NF-κB is inhibited.8 Interestingly, constitutive expression of Bcl-xL in IκBα(ΔN) transgenic T cells increased their ability to produce IFN-γ and mount DTH responses in vivo, although the levels of IFN-γ production remained significantly below those of wild-type cells (M. A. Aronica and M. Boothby, unpublished observations). This finding suggests that enhanced protection of IκBα(ΔN) T cells from death during growth and Th1 cell differentiation may improve their function. Further, the link between inhibition of clonal expansion and the preferential role of NF-κB in the type 1 response (Fig. 1⇑ and Ref. 28) may relate to a report that Th1 cells exhibit preferential susceptibility to activation-induced cell death (63). However, in addition to Bcl-3 and Bcl-2 it is possible that still other factors, such as growth arrest and DNA damage protein (GADD) 45β or γ, are also involved downstream from NF-κB (64, 65).

A key finding is that IκBα(ΔN) T cells, in addition to their inefficient multiplication, produced less IFN-γ on a per cell basis than wild-type cells. Rather than being due exclusively to a defect in differentiation into Th1 cells, intracellular cytokine staining showed that NF-κB induction was required for normal levels of Th1 differentiation but the principal defect in quantitative terms was in the production of IFN-γ after restimulation. Consistent with the conclusion that NF-κB regulates rates of IFN-γ gene expression at a postdifferentiative step, a functional κB binding site in the closely related human IFN-γ promoter region and its cooperation with an intronic κB site have been reported (66). Although it is unlikely that the lineage-specific transcription factor T-bet is the only factor regulating the efficiency of Th1 differentiation, the magnitude in the reduction of T-bet in IκBα(ΔN) cells is consistent with a principal role for NF-κB after differentiation as well as with the modest decrease in IL-12-induced phospho-Stat4. Further, NF-κB inducibility appears critical for both a cytokine-activated, TCR-independent mechanism of evoking a burst of IFN-γ gene expression in differentiated Th1 effectors (41) and also for the conventional Ag receptor signaling pathway (Fig. 3⇑). Interestingly, an increase in the efficiency of Th2 differentiation was also noted. This finding suggests that a modest block to postdifferentiation of IL-4 caused at high concentrations of the inhibitor PDTC may be counterbalanced by an increase in the frequency of Th2 cells. Further, the result suggests that the previously reported (28) increase Ag-specific IL-4 elicited from draining lymph node cells in allergen-immunized IκBα(ΔN) mice may be due both to a decrease in local IFN-γ production and to an intrinsic increase in the probability of Th2 differentiation among activated naive CD4 T cells. In addition to the mechanistic findings reported in this study, still other factors may contribute to the defects of clonal expansion and IFN-γ production in vivo. We have previously shown that Stat5A induction by IL-2 or IL-4 receptors is decreased in IκBα(ΔN) T cells despite normal expression of IL-2R chains, whereas the efficiency of Stat6 induction is normal (67). First, these results show that some types of cytokine receptor signaling to Janus kinase-Stat pathways are attenuated in the IκBα(ΔN) transgenic T cells while others are unimpaired. Second, the inefficient signaling to Stat5 might represent another contribution to the defective Th1 response. Clonal expansion and IFN-γ production were decreased for DO11.10 T cells which lacked expression of the IL-2Rα chain (68), but the efficiency of neither Stat5 induction nor differentiation was quantitated so that the relationship to findings when NF-κB is inhibited is not clear.

The decreases in clonal expansion and postdifferentiation IFN-γ production which result when NF-κB is inhibited in T cells appear to exert powerful physiologic consequences in vivo. Compared to wild-type littermates, IκBα(ΔN) transgenic mice exhibit a significant reduction in the incidence and severity of collagen-induced arthritis (54), an immunopathology on the DBA/1 (H-2q) background in which Th1 cells are thought to play a role. In addition, ongoing studies show that this T lineage-restricted inhibition of NF-κB leads to nearly complete protection against autoimmune diabetes in NOD mice (S.-Y. A. Kim, S. A. Stanley, B. Enerson, A. L. Mora, and M. Boothby, unpublished observations). Prior studies have suggested that individual NF-κB/Rel proteins are important for the development of Th1 responses in infection or autoimmunity, but a key role of these proteins is in regulating the production of IL-12 by APCs. Whether there also is a T cell-intrinsic function of NF-κB in type 1 responses has been unclear. Mice deficient in RIP-2, a serine/threonine kinase essential for linking Toll-like receptors and TCRs to the activation of NF-κB and AP-1, were unable to defend against infection with the intracellular pathogen Listeria monocytogenes, a phenomenon attributable to defects in both macrophage and T cell populations (59). NF-κB2 (p52)-deficient mice of a background normally resistant to Leishmania major infection failed to mount a Th1 response and developed chronic nonhealing lesions after challenge with this intracellular pathogen (69). However, NF-κB2-null T cells were able to acquire a Th1 phenotype and provide protection against disease progression in lymphocyte-deficient recipient mice (69), suggesting that under these conditions, disease susceptibility was due to defects extrinsic to T cells. In contrast, susceptibility of NF-κB2-deficient mice to Toxoplasma gondii infection was attributed to decreased IFN-γ production as a consequence of a loss of CD4+ and CD8+ T cells (70).

An effective immune response involves not only regulating the acquisition of Th1 and Th2 cell differentiated phenotypes, but also requires that the activated T cell populations survive, clonally expand, and that differentiated effectors express cytokine genes at levels adequate for clearing infection. In aggregate, our data establish that the inducibility of NF-κB/Rel transcription factors within T cells makes critical contributions to the Th1 immune response both during and after the differentiation process. Further, we have uncovered evidence that these actions of conventional NF-κB factors can be correlated with a decrease in the induction of the atypical IκB-like protein Bcl-3. In quantitative terms, the two most substantial roles are in clonal expansion and in the postdifferentiation regulation of IFN-γ gene expression, while a less marked contribution to the frequency of IFN-γ-positive T cells also was detected. Because patients subject to IFN-γ-mediated autoimmune disease will already bear differentiated Th1 effectors specific for autoantigens, these findings have the important implication that T cell-specific inhibition of NF-κB may effectively decrease the function of these pathogenic inflammatory T cells.

Acknowledgments

We gratefully acknowledge the helpful discussions and critical review of the manuscript by G. Miller and S. Joyce, as well as generous gifts of essential reagents by L. H. Glimcher.

Footnotes

  • ↵1 This work was supported by National Institutes of Health Grants AI36997 and AI49460, as well as by core facilities of the Vanderbilt-Ingram Cancer Center (Comprehensive Cancer Center Grant CA68485) and Diabetes Center Grant P60 DK20593. R.A.C. was supported by U.S. Department of Education Graduate Assistance in Areas of National Need Grant P200A010123, National Institutes of Health Award R25 GM62459 to the Meharry-Vanderbilt Alliance, and then Grant AI-49460-02S1.

  • ↵2 R.A.C. and M.A.A. were co-first authors.

  • ↵3 Current address: Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195.

  • ↵4 F.Z., Y.T., and S.A.S. made equal contributions.

  • ↵5 Current address: Department of Medicine, Emory University, Atlanta, GA 30303.

  • ↵6 Address correspondence and reprint requests to Dr. Mark Boothby, Department of Microbiology and Immunology, Vanderbilt University Medical School, Nashville, TN 37232-2363. E-mail address: mark.boothby{at}vanderbilt.edu

  • ↵7 Abbreviations used in this paper: IRF, IFN regulatory factor; T-bet, T box expressed in T cells; IκB, inhibitor of κB; IKK, IκB kinase; DTH, delayed-type hypersensitivity; PDTC, pyrrolidine dithiocarbamate; rhu, recombinant human; GFP, green fluorescent protein; RIP, receptor-interacting protein.

  • ↵8 A. Mora, S. Goenka, M. Aronica, S. Stanley, D. W. Ballard, and M. Boothby. NF-κB regulates cell cycling, survival, and the homeostatic expansion of T cells in vivo. Submitted for publication.

  • Received February 14, 2003.
  • Accepted June 12, 2003.

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