Contrasting the Roles of Costimulation and the Natural Adjuvant Lipopolysaccharide During the Induction of T Cell Immunity

Mice

B10.A mice were purchased from the National Cancer Institute (Frederick, MD). C57BL/6, TNFRI knockout (KO), TNFRII KO, TNFRI/II double KO (29), and B6, D2-TgN(LCK-NFKBIA)5Dwb mice (30) (hereafter referred to as inhibitory protein that dissociates from NF-κB α transgenic (IκB-α Tg) mice) were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were maintained in our animal facility at Oregon State University under specific pathogen-free conditions in accordance with federal guidelines. All mice were between 6 and 12 wk of age.

Reagents, mAbs, and flow cytometry

SEA, staphylococcal enterotoxin B (SEB), LPS, and rat IgG were purchased from Sigma-Aldrich (St. Louis, MO) and administered to mice as i.p. injections in balanced salt solution (BSS) or PBS. The recombinant human IL-1Ra was a kind gift from Amgen (Boulder, CO).

The anti-CD40 mAb-producing hybridoma FGK45.5 was a kind gift from Dr. A. Rolink (Basel Institute, Basel, Switzerland) (31). The anti-IFN-γ-producing hybridoma XMG1.2 (32), the anti-TNF-α-producing hybridoma MP6-XT22 (33), and the anti-IL-6-producing hybridoma MP5-20F3.11 (34) were all obtained from the American Type Culture Collection (Manassas, VA) with anti-IL-6 being obtained with permission from DNAX (Palo Alto, CA). Supernatants from each of the above hybridomas were purified over protein G agarose (Life Technologies, Grand Island, NY) and dialyzed against PBS for injection.

For flow cytometric staining, Abs purchased from BD PharMingen (San Diego, CA) were used: anti-CD4 was conjugated to either PE or APC, anti-CD8 was conjugated to either PE or APC, and anti-TCR Vβ8 was conjugated to FITC. The anti-TCR Vβ3 mAb KJ25-607.7 (35) was purified from hybridoma supernatant over protein G agarose (Life Technologies) and conjugated to FITC as described previously (36). Briefly, purified Ab was dialyzed against 0.1 M NaHCO₃, pH 9.4–9.6. Protein concentration was adjusted to 1 mg/ml and incubated with FITC-Celite (Sigma-Aldrich) for 30 min at room temperature. Free celite was removed by centrifugation and the Ab was dialyzed against PBS for use.

Injection schedule

All injections were i.p. The injection of the SAgs SEA and SEB was considered day 0. Injection of anti-CD40 mAb was done 2 days before SAg injection. LPS was always injected 24 h after the SAgs (day +1). The anti-OX40 mAb was injected 24 and 36 h after SAg injection. For the cytokine blocking experiments in Fig. 5⇓, b and c, we used the neutralizing mAbs anti-TNF-α, anti-IL-6, and anti-IFN-γ, as well as IL-1Ra. These reagents were injected 20 and 30 h after SEA.

Cell processing and flow cytometry

Spleens and peripheral lymph nodes (inguinal, axillary, and bronchial) were teased through nylon mesh sieves (Falcon, BD PharMingen) and RBCs were lysed with ammonium chloride. After washing, cells were counted with a Z1 particle counter (Beckman Coulter, Miami, FL), and spleen cells were further purified over nylon wool as described previously (37). Briefly, 3-ml syringes were filled with 0.12–0.15 g of washed and brushed nylon wool. The columns were prepared with warm BSS 5% FBS, after which the cells were loaded in a 0.5-ml volume and incubated for 30 min at 37°C. After draining 0.5 ml away, the columns were incubated for an additional 30 min, followed by elution with BSS 5% FBS.

For two- and three-color staining, cells were incubated on ice with the primary Abs in the presence of 5% normal mouse serum, culture supernatant from hybridoma cells producing an anti-mouse FcR mAb, 2.4.G2 (38), and 10 μg/ml human γ-globulin (Sigma-Aldrich) to block nonspecific binding. After a 30-min incubation on ice in staining buffer (BSS, 3% FBS, 0.1% sodium azide) with primary Abs, the cells were washed twice and analyzed by flow cytometry, or if a secondary reagent was necessary, the incubation and wash procedures were repeated. Flow cytometry was conducted on a FACSCaliber flow cytometer (BD Biosciences, Mountain View, CA), and the data were analyzed using CellQuest software (BD PharMingen).

Bromodeoxyuridine (BrdU) staining

Mice were injected with 60 μg of SEB, 0.20 mg of anti-CD40, and 10 μg of LPS at the times described above. Additionally, the mice were injected with 1 mg of BrdU (Sigma-Aldrich) dissolved in PBS on days 0, 1, and 2. On day 3, T cells from the peripheral lymph nodes (LNs) and spleens from the treated mice were isolated and stained with biotin-conjugated anti-TCR Vβ8 mAb and then with PE-conjugated streptavidin (BD PharMingen). The cells were then stained with a modified BrdU staining protocol (39). Briefly, the cells were dehydrated and fixed in ice-cold 95% ethanol and then were fixed in BSS containing 1% paraformaldehyde and 0.01% Tween 20. Next, cellular DNA was lightly digested with 50 Kunitz U of DNase I (Sigma-Aldrich), and the cells were stained with anti-BrdU-FITC (BD PharMingen).

In vitro proliferation

Mice were injected with 0.30 μg of SEA, 0.25 mg of anti-CD40 mAb, and 10 μg of LPS as described above. Ten days after SEA injection, spleen cells were isolated for culture. Cells from each in vivo treatment group were plated in triplicate at 5.0 × 10⁵, 2.5 × 10⁵, 1.25 × 10⁵, and 0.63 × 10⁵ cells per well in complete tumor medium (CTM). CTM consists of minimal essential medium with FBS, amino acids, salts, and antibiotics. SEA was added to the wells at a concentration of 1 μg/ml. The cultures were left for 72 h, with 1 μCi of [³H]thymidine (ICN Pharmaceuticals, Costa Mesa, CA) being added for the last 8 h. Incorporation of [³H]thymidine was measured on a 1450 Microbeta Trilux Scintillation Counter (Wallac, Turku, Finland).

RT-PCR

B10.A mice were injected with 0.25 mg of anti-CD40, 0.30 μg of SEA, and 10 μg of LPS as described. At the time points corresponding to 0.5, 1.5, and 6 h after LPS injection (or 24.5, 25.5, and 30 h after SEA injection), peripheral LNs and spleens were removed, and total RNA was prepared from the cell suspensions using RNAwiz according to the manufacturer’s suggested protocol (Ambion, Austin, TX). Synthesis of cDNA was performed with 1 μg of RNA using the Reverse Transcriptase System (Promega, Madison, WI) and accompanying protocol. Aliquots of cDNA were amplified with the GeneAmp PCR System 2400 (Perkin-Elmer, Norwalk, CT). Amplification conditions consisted of a 4-min denaturation step at 94°C, followed by 30 cycles of amplification (95°C for 1 min, 53°C for 1 min, 72°C for 1 min) and a final extension at 72°C for 7 min. PCR products were electrophoretically separated on a 2% agarose gel containing ethidium bromide. The polycompetitor plasmid PQRS (40) was a kind gift from Dr. Reiner (University of Chicago). This plasmid contains cDNA sequences for each cytokine amplified and was used as a positive control. The sequences of the primers are as follows: hypoxanthine phosphoribosyltransferase, 5′-GTT GGA TAC AGG CCA GAC TTT GTT G-3′ and 5′-GAG GGT AGG CTG GCC TAT AGG CT-3′; TNF-α, 5′-GTT CTA TGG CCC AGA CCC TCA CA-3′ and 5′-TAC CAG GGT TTG AGC TCA GC-3′; IL-1β, 5′-AAG CTC TCC ACC TCA ATG GAC AG-3′ and 5′-CTC AAA CTC CAC TTT GCT CTT GA-3′; IL-2, 5′-TCC ACT TCA AGC TCT ACA G-3′ and 5′-GAG TCA AAT CCA GAA CAT GCC-3′; IL-6, 5′-CCT CTG GTC TTC TGG AGT ACC AT-3′ and 5′-GGC ATA ACG CAC TAG GTT TGC CG-3′; IL-15, 5′-ACT GAC AGT GAC TTT CAT CCC A-3′ and 5′-GTG CTG CCT CTG AGC AGC AGG-3′; and IFN-γ, 5′-CAT TGA AAG CCT AGA AAG TCT G-3′ and 5′-CTC ATG AAT GCA TCC TTT TTC G-3′. All primers were made by the Central Services Laboratory at Oregon State University (Corvallis, OR).

Subcellular fractionation

Combined LN and spleen cells from C57BL/6 and IκB-α Tg mice were cultured overnight at a concentration of 10 × 10⁶ cells/ml in CTM in the presence of 2 μg/ml Con A. T cells were purified over nylon wool to between 50 and 90% purity.

Nuclear and cytoplasmic extracts were prepared as described (41). Briefly, cell pellets were resuspended in sucrose buffer (0.32 M sucrose, 3 mM CaCl₂, 0.1 mM EDTA, 10 mM Tris-HCl, pH 8.0, 2 mM Mg acetate, 1 mM DTT, 0.5 mM PMSF) with 0.5% (v/v) IGEPAL by gentle pipetting and were centrifuged. To the cytoplasmic fraction, 0.22 volumes of 5× cytoplasmic extraction buffer (0.15 M HEPES, 0.7 M KCl, 0.015 M MgCl₂) was added. The cytoplasmic fraction was then centrifuged at 15,000 rpm in a microcentrifuge, and the supernatant was transferred to a fresh tube containing 25% (v/v) glycerol and stored at −80°C. The nuclei were washed twice in sucrose buffer without IGEPAL. Nuclei were then resuspended in low salt buffer (20 mM HEPES, 25% glycerol, 1.5 mM MgCl₂, 0.02 M KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF), and then 1 volume of high salt buffer (20 mM HEPES, 25% glycerol, 1.5 mM KCl, 0.2 mM EDTA, 1% IGEPAL, 0.5 mM DTT, 0.5 mM PMSF) was carefully added in 1/4 increments. Nuclei were incubated on ice for 30 min, diluted 1/2.5 with diluent (25 mM HEPES, 25% glycerol, 0.1 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF), and centrifuged at 15,000 rpm in a microcentrifuge at 4°C. Nuclear lysates were stored at −80°C.

DNA binding assay

EMSAs were used to assess sequence-specific binding of DC2.4 nuclear NF-κB/Rel to DNA (41). Briefly, a synthetic 20-bp consensus κB response element probe (upper strand, 5′-GAT CGG CAG GGG AAT TCC CC-3′; and lower strand, 5′-GAT CGG GGA ATT CCC CTG CC-3′) was labeled with [α-³²P]dATP using Klenow fragment (Invitrogen, Carlsbad, CA) and used for DNA binding assays. Nuclear extracts were prepared as described above. Samples (5 μg) were incubated with binding buffer (12 mM HEPES, pH 7.3; 4 mM Tris-HCl, pH 7.5; 100 mM KCl; 1 mM EDTA; 20 mM DTT; 1 mg/ml BSA), 4 μg of poly(dI-dC) (Amersham Pharmacia Biotech, Piscataway, NJ), and 100,000 cpm of ³²P-labeled κB response element for 20 min at room temperature. Anti-RelA, anti-RelB, anti-c-rel, anti-p50, and anti-p52 were added to the reaction mixture according to manufacturer’s instructions (Santa Cruz Biotechnology, Santa Cruz, CA) and incubated for 10 min at room temperature. Samples were analyzed on a 5% polyacrylamide gel in 0.5% Tris-buffered EDTA (44.5 mM Tris, 44.5 mM boric acid, 1 mM EDTA) and visualized by autoradiography.

Immunoblotting

Cytoplasmic extracts were subjected to SDS-PAGE as described (42). Proteins were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA) in 25 mM Tris (pH 8.3), 192 mM glycine, 20% methanol using a Genie Electroblotter (Idea Scientific, Minneapolis, MN). Membranes were blocked overnight at 4°C in TBS (25 mM Tris, pH 7.4, 150 mM NaCl) containing 5% nonfat dry milk. Abs were diluted in TBS containing 1% nonfat dry milk and the membranes were incubated with primary Abs for at least 1.5 h at room temperature. Anti-IκB-α IgG (Santa Cruz Biotechnology) and HRP-conjugated secondary Abs, donkey anti-rabbit IgG (Amersham Pharmacia Biotech) were used according to the manufacturer’s instructions. After each Ab treatment, blots were washed in TBS containing 0.05% Tween 20. Ab complexes were visualized with chemiluminescent reagents (Pierce, Rockford, IL).

LPS and CD40 stimulation synergize to enhance Ag-specific T cell survival

Our previous work with CD40 stimulation found that agonistic anti-CD40 mAb treatment could enhance SEA-mediated T cell expansion in vivo; however, it could not keep the responding T cells alive for very long (7). After 2–3 wk, Ag-specific T cell numbers declined to levels observed in mice treated with SEA alone. This death process was not prevented, only delayed.

A very similar trend was observed during treatment with an anti-OX40 agonistic mAb (8). OX40 stimulation enhanced Ag-specific T cell expansion and, much like CD40 stimulation, could only yield weak long-term survival. However, injection of LPS into mice treated with both Ag and anti-OX40 mAb strongly enhanced T cell survival beyond 2 mo.

With these observations, we hypothesized that LPS injection would prevent the death of T cells from mice treated with SEA and anti-CD40 mAb. Thus, mice were injected with the anti-CD40 agonistic mAb, SEA, and LPS. At various time points after Ag treatment, T cell populations were examined in the lymphoid tissues. Fig. 1⇓ shows a time course of the clonal expansion and survival of splenic CD4 Vβ3 (Fig. 1⇓, a and c) and CD8 Vβ3 (Fig. 1⇓, b and d) T cells stimulated by a combination of SEA, anti-CD40 mAb, and LPS.

In mice treated with SEA alone, CD4 Vβ3 T cell percentages (Fig. 1⇑a) and numbers (Fig. 1⇑c) declined on day 5 below their starting levels. The peak of clonal expansion that normally occurs on day 2–3 was not examined. Co-injection of either anti-CD40 mAb or LPS with SEA produced increased percentages and numbers of T cells on day 5, with LPS yielding more potent T cell expansion and short-term survival than CD40 stimulation. By day 14, however, T cells from these SEA/anti-CD40 or SEA/LPS-treated mice had slightly increased numbers of Vβ3 T cells compared with those mice treated with SEA alone (Fig. 1⇑, c and d). Based on percentages of CD4 Vβ3 T cells on day 14, mice treated with SEA/LPS had 1.5-fold more splenic Ag-specific T cells (3.40 ± 0.49%), and SEA/anti-CD40-treated mice had 2-fold more splenic Ag-specific T cells (4.70 ± 0.25%) than SEA-treated mice (2.31 ± 0.12%). Similar differences were observed when examining T cell numbers (Fig. 1⇑c). Thus, a low level of T cell survival is produced with SEA/anti-CD40 or SEA/LPS treatment.

What is most striking about the results is that a combination of the three signals (SEA, anti-CD40 mAb, and LPS) prevented many more T cells from deleting. This effect is observed by examining both percentages and numbers. Vβ3 T cells from mice stimulated by the three signals accumulated almost 10-fold from their starting populations by day 5 and had declined slightly in number after 2 wk. On day 14, CD4 Vβ3 percentages were increased by 16-fold over SEA alone to 38.46 ± 3.08% (Fig. 1⇑a), whereas the absolute numbers were increased by nearly 13-fold to 40.27 × 10⁵ ± 2.69 (Fig. 1⇑c).

A very similar response to that described above was observed in the LN (data not shown) as well as in the Ag-specific CD8 T cell population (Fig. 1⇑, b and d). In mice injected with SEA, anti-CD40 mAb, and LPS, splenic CD8 Vβ3 T cells accumulated to a much greater level than any other treatment on day 5 and then began to gradually decline in both percentage (Fig. 1⇑b) and number (Fig. 1⇑d). By day 14, there was a 26-fold increase in the percentage of CD8 Vβ3 T cells in three-signal-treated mice (29.90 ± 4.86%) vs SEA-treated mice (1.13 ± 0.02%). In contrast, SEA/LPS- and SEA/anti-CD40-treated mice only produced a 4-fold increase in Ag-specific T cell accumulation. Thus, both Ag-specific CD4 and CD8 T cell populations can be signaled to survive beyond the effector stage by the combined action of signal 1 (SAg), signal 2 (costimulation), and signal 3 (LPS).

We next tested whether these rescued T cells were functional by stimulating them in vitro with recall Ag using SEA. As shown in Fig. 2⇓, mice treated with three signals in vivo for 10 days proliferated effectively when restimulated in vitro. This proliferation was less on a per cell basis than other weaker in vivo treatments yielded in vitro, which was likely due to the depleted resources and energetic costs of undergoing such a potent response. These data show that the T cells that are rescued from death by the combination of SAg, costimulation, and LPS are not anergic.

Multiple costimulatory signals cannot substitute for LPS

Stimulation of APCs through CD40 causes an increase in the expression of both B7 molecules as well as MHC class II (7, 43). In addition, LPS stimulation can also enhance B7 expression and prolong OX40 expression on activated T cells (8, 44). We hypothesized that because LPS can synergize with both CD40 and OX40 stimulation to promote Ag-specific T cell survival, its mechanism of action may involve combined signaling via CD28 and OX40 on the SAg-specific cells.

To test this idea, we injected mice with a triple combination of SEA, an agonistic anti-CD40 mAb, and an agonistic anti-OX40 mAb. The time course of this experiment is shown in Fig. 3⇓, a and b, and the same scale as in Fig. 1⇑ is used to allow a direct comparison between the two figures.

The percentages of both CD4 (Fig. 3⇑a) and CD8 (Fig. 3⇑b) T cells expressing Vβ3 behaved in a similar manner. Treatment of mice with SEA and anti-OX40 mAb enhanced the percentage of Ag-specific T cells in the spleen above that observed with SEA alone on day 3, but on subsequent days, the difference between the two treatments was minimal. SEA and anti-CD40 mAb treatment enhanced the CD4 Vβ3 and CD8 Vβ3 percentages in the spleen at the peak of expansion by 4- to 5-fold above that observed with SEA alone. By days 7 and 12, however, there was less than a 2-fold increase in the percentage of T cells remaining. When SEA, anti-CD40 mAb, and anti-OX40 mAb were injected in combination, the observed response was very similar to that from injecting SEA and anti-CD40 mAb. Ag-specific T cell expansion was enhanced 4- to 5-fold above starting levels on day 3, but after day 3, little survival was observed. There was a slightly larger percentage of Ag-specific T cells remaining on day 12 after the combined treatment (∼4-fold above SEA alone), but this was a small fraction of that observed with LPS (Fig. 1⇑). Thus, costimulation via CD40 and OX40 can yield small levels of T cell survival, but when combined with signals produced upon LPS injection, the survival effect is greatly potentiated, suggesting that LPS does not rescue Ag-specific T cells merely by stimulating through OX40 or CD40.

Because the survival response observed during three-signal stimulation does not appear to be a sum of multiple costimulatory signals being delivered, the next focus of study was whether the LPS response could be enhanced further. Because LPS can synergize with either CD40 or OX40 stimulation to improve Ag-specific T cell survival, it was necessary to examine whether combining all of these signals could enhance the amount of surviving T cells.

Mice were injected with a combination of SEA, LPS, anti-CD40, and anti-OX40. After 10 days, the T cell populations were examined. In mice injected with SEA, anti-CD40, and LPS, 18.96 ± 2.86% of the CD4 T cells (Fig. 3⇑c) and 8.54 ± 1.49% of the CD8 T cells (Fig. 3⇑d) were SEA specific. A slightly larger percentage was observed in mice injected with SEA, anti-OX40, and LPS: 19.50 ± 3.81% CD4 Vβ3 and 9.26 ± 2.23% CD8 Vβ3 T cells. In mice treated with both mAbs, SEA and LPS, little enhancement of T cell survival was observed, increasing to 24.29 ± 4.27% in the CD4 Vβ3 population and 11.56 ± 3.17% in the CD8 Vβ3 population. In these experiments, we used 2.5 times less anti-CD40 mAb to conserve on reagent, and thus the magnitude of the response is lessened compared with Fig. 1⇑. Increasing the LPS dose to 15 μg did not further enhance T cell survival, yielding slightly lower percentages and numbers of SEA-specific T cells than observed with 10 μg (data not shown). Again, the numbers of SEA-specific T cells as well as the LN cells both behaved in a similar fashion (data not shown). Thus, costimulation of T cells via either CD40 or OX40 stimulation in the presence of LPS can produce a substantial population of surviving Ag-specific T cells that cannot be significantly augmented by additional costimulation through these receptors. This again suggests that single or multiple costimulatory signals are not the major factor determining T cell survival beyond the effector phase in vivo.

Proinflammatory cytokines are not necessary for T cell survival in the presence of anti-CD40 stimulation and LPS

LPS stimulation of APCs can trigger the production of many proinflammatory cytokines. To begin to understand how LPS promoted survival in our model, mRNA expression of various cytokines was examined at early time points after LPS injection by RT-PCR (Fig. 4⇓). The goal was to identify candidate cytokines that may be contributing to the survival response.

Expression of many of the important proinflammatory cytokines examined in Fig. 4⇑ were enhanced upon LPS treatment in the LN and spleen. All reactions were normalized to hypoxanthine phosphoribosyltransferase, which allowed for semiquantitative comparisons to be made. TNF-α, IL-1β, IL-6, IL-15, and IFN-γ mRNA were all up-regulated to varying degrees during the first 6 h after LPS treatment compared with mice that received no LPS but were examined at the exact same time. IL-2 was also examined, but it was not observed in any treated or untreated mouse at the times tested (data not shown).

Previous work with mice injected with SEA and LPS showed that TNF-α can substitute to a small degree for LPS in keeping Ag-specific T cells alive (19). The survival effect was not nearly as potent as that observed with LPS injection, but it was significant. This result, coupled with the fact that TNF-α message expression was increased by LPS injection in the three-signal model system, suggested that TNF-α might be at least partially responsible for long-term T cell survival in the model described in this report.

To investigate the importance of this cytokine in the three-signal model system, mice deficient in either or both TNFRs were used (29). Because the knockout mice are on a C57BL/6 background, we used SEB instead of SEA to avoid the effect of endogenous mouse mammary tumor viruses on Vβ3 T cells. Each mouse strain was injected with SEB and anti-CD40 mAb with or without LPS, and lymphoid tissues were examined on day 10 for the presence of SEB-specific T cells (TCR Vβ8). Fig. 5⇓a shows the percentage of CD4 Vβ8 T cells in the spleens of the treated mice 10 days after SEB injection.

In C57BL/6 mice treated with SEB and anti-CD40 stimulation, CD4 Vβ8 T cells declined to an average percentage of 18.00 ± 0.92% after 10 days. When those mice were given LPS, the percentage of SEB-specific T cells rose to 32.69 ± 2.85%; an 85% increase. Mice deficient in TNFRI had 22.30 ± 1.18% splenic CD4 Vβ8 T cells in the absence of LPS treatment, but a 63% increase to 36.34 ± 2.13% was observed when LPS was co-injected. T cells from TNFRII knockout mice increased by 69% upon LPS treatment, rising from 18.35 ± 0.79% CD4 Vβ8 T cells without LPS to 31.13 ± 3.30% with LPS.

In the absence of one TNFR, there remained the possibility that the other receptor was still delivering a survival signal due to redundancy of function between the two receptors. Thus, mice deficient in both TNFRs were treated with three signals as in the other strains. Mice receiving SEB and anti-CD40 mAb had a CD4 Vβ8 T cell percentage on day 10 of 20.03 ± 1.00%, but when injected with LPS, the percentage rose 63% to 32.65 ± 1.79%, similar to that observed in the other mice. Overall, these data suggest that signaling through TNFRI and TNFRII is dispensable for long-term T cell survival when CD40 stimulation is used in combination with LPS.

The observation that T cell survival can still occur in the absence of TNFR signaling does not mean that such signaling cannot contribute to survival. Other proinflammatory cytokines may be contributing a survival signal and thus may be substituting for TNFR signaling. To get at the issue of whether other cytokines were delivering the signals necessary to keep activated T cells alive, a combination of cytokines was blocked in vivo during SEA, anti-CD40, and LPS stimulation.

B10.A mice were injected with SEA, anti-CD40, and LPS, but before and after LPS injection, IL-1Ra, anti-TNF-α mAb and anti-IL-6 mAb were all injected to try to neutralize as much activity of these three cytokines as possible. Because the RT-PCR data showed that IFN-γ expression was also increased after LPS injection (Fig. 4⇑), another treatment group was set up in which IFN-γ was blocked with a mAb to investigate what effect this cytokine had on survival. The percentages (Fig. 5⇑b) and numbers (Fig. 5⇑c) of CD4 Vβ3 T cells 10 days after SEA injection are shown.

SEA treatment led to the expected decline in T cell percentages, decreasing from a 6% CD4 Vβ3 T cell population in uninjected mice to a little over 2% by day 10. SEA/anti-CD40 mAb and SEA/LPS treatments both produced small increases in T cell percentages in comparison to SEA alone, again showing that two signals can generate some T cell survival post effector stage. Mice treated with SEA/anti-CD40/LPS and the control IgG exhibited a large increase in percentage of SEA-specific T cells to 22.12 ± 4.32%. What was most interesting, however, was that when IL-1β, TNF-α, and IL-6 were all neutralized, the percentage of surviving T cells actually increased slightly to 27.42 ± 5.34%. Even blocking IFN-γ did not decrease survival, yielding 23.25 ± 6.56% CD4 Vβ3 T cells after 10 days in vivo.

A much better enhancement of T cell survival after cytokine neutralization was observed based on CD4 Vβ3 T cell numbers (Fig. 5⇑c). Mice receiving three-signal stimulation and the control rat IgG had 23.28 × 10⁵ ± 6.31 SEA-specific T cells remaining after 10 days. Blocking TNF-α, IL-1β, and IL-6 increased the numbers of surviving T cells to 42.40 × 10⁵ ± 11.2, whereas IFN-γ inhibition increased the numbers to 35.83 × 10⁵ ± 14.62.

CD8 Vβ3 T cells responded a little differently to the cytokine blocking studies than the CD4 Vβ3 T cells did (data not shown). Both percentages and numbers of CD8 T cells expressing Vβ3 in the spleen were increased poorly, if at all, by inhibition of proinflammatory cytokines. This could represent a difference in activation/survival requirements between CD4 and CD8 T cells. However, what is identical between both kinds of T cells is that blocking TNF-α, IL-1β, IL-6, and IFN-γ did not adversely affect survival and, perhaps surprisingly, enhanced T cell survival to varying degrees.

To verify that the cytokines were inhibited by the neutralizing mAbs, serum samples were taken from the injected mice 1 day after the LPS and Ab injections. ELISAs were performed using anti-rat IgG to detect the neutralizing Abs in the serum using a sandwich ELISA. In the control rat IgG group, quantities of rat IgG were detected in the serum that exceeded the maximum detectable amount (data not shown). In the anti-TNF-α-, anti-IL-6-, and IL-1Ra-treated groups there was slightly less Ab detected, but still large amounts remained in the serum (data not shown). The anti-IFN-γ-treated mice also had detectable levels of rat IgG in their serum (data not shown). The ELISA data show that there were large amounts of rat IgG Abs present in the serum over 24 h after LPS treatment. This suggests that the cytokines were neutralized soon after they were secreted, and thus this data is consistent with the data collected from the TNFR KO mice. Furthermore, attempts to substitute LPS with TNF-α, IL-1β, and IL-6 to induce SAg-specific T cell survival failed, even though large doses of the cytokines were used (data not shown). Collectively, these data suggest that CD40 stimulation circumvents a role for proinflammatory cytokines in long-term T cell survival.

Similar results were observed when IL-1β and IL-6 were inhibited in TNFR double-knockout mice (data not shown). No adverse effects on long-term T cell survival were observed in this model system, and some slight increases in surviving T cell numbers were observed. Thus, from these data it appears that the proinflammatory cytokines TNF-α, IL-1β, IL-6, and IFN-γ are not necessary for long-term T cell survival and in fact may actually be somewhat detrimental, possibly playing a role in attenuating an Ag-specific CD4 T cell response to Ag, strong costimulation, and LPS treatment. This is consistent with previous reports showing that TNF-α and IFN-γ readily exert cytotoxic effects on lymphocytes (40, 45, 46). IL-1β and IL-6, however, appear to be more protective (47, 48), although IL-β does play a role in the death of β-cells in diabetes (49).

A mixed role for NF-κB during T cell survival

The transcription factor NF-κB is very important for inflammatory responses as well as T cell proliferation and survival (16, 17, 50, 51, 52). Many inflammatory cytokines, as well as LPS, activate transcriptional activity of this molecule (53, 54, 55, 56). To examine the role of NF-κB in T cell survival in vivo, mice Tg for a mutant IκB-α molecule that cannot be degraded were used (30). IκB-α is a repressor of NF-κB that prevents the translocation of NF-κB into the nucleus (57). When IκB-α is phosphorylated, it is targeted for ubiquitination (58, 59). Subsequent degradation of IκB-α releases NF-κB and allows it to translocate to the nucleus and initiate transcription. Thus, these mutant IκB-α Tg mice have reduced NF-κB binding to DNA response elements. Additionally, this transgene is under the control of the lck promoter, thus limiting its expression, and ultimately NF-κB inhibition, to the T cell population, allowing an examination of how effective T cell survival is in the presence of reduced NF-κB activity.

As shown in Fig. 6⇓a, the percentage of splenic CD4 Vβ8 T cells in C57BL/6 mice given SEB and anti-CD40 mAb without LPS was 20.91 ± 0.83% and rose to 33.26 ± 3.35% when LPS was injected. A very similar increase was observed in the IκB-α Tg mice whose T cell populations increased from 22.97 ± 1.12% without LPS to 33.90 ± 3.03% with LPS (Fig. 6⇓b). These numbers correspond to a 59% difference between treatments in C57BL/6 mice and a 48% difference in the Tg mice. A similar trend was observed in the absolute numbers of CD4 Vβ8 T cells, which increased by 32% in the C57BL/6 mice and by 31% in the Tg mice that received LPS (data not shown). A few individual mice had much higher levels of CD4 Vβ8 T cells than observed in control strains. One Tg mouse in particular had over 70% of its CD4 T cells bearing Vβ8 in the spleen 10 days after SEB injection. This is in contrast to C57BL/6 mice, which never rose above 50%.

Expression of IκB-α by Western blot and EMSAs assessing NF-κB activity were performed using T cells from mice shown in Fig. 6⇑b. The percentages shown in the lanes of the Western and EMSA blots represent the percentage of CD4 Vβ8 T cells in SEB/anti-CD40/LPS-treated mice used in Fig. 6⇑b. Thus, the 39% lane represents cells from a mouse that had 39% CD4 Vβ8 T cells in Fig. 6⇑b. The data show little variation in transgene expression regardless of the amount of rescue (Fig. 6⇑c). Furthermore, data measuring NF-κB activity showed profound inhibition of NF-κB in the cells taken from the transgenic mice as expected (Fig. 6⇑b). Therefore, these controls support our hypothesis that survival of Ag-activated T cells beyond the effector stage is not dependent on NF-κB.

LPS can circumvent proliferation defects in NF-κB-deficient T cells

Because LPS-induced long-term T cell survival can still occur in NF-κB-deficient T cells, it was important to investigate whether T cell responses were deficient and whether LPS could somehow overcome that deficiency. Thus, IκB-α Tg or C57BL/6 mice were injected with different combinations of SEB, anti-CD40 mAb, and LPS and were given BrdU injections for 3 days. After this treatment, the percentages and numbers of Ag-specific T cells incorporating BrdU were evaluated (Fig. 7⇓).

Both C57BL/6 and IκB-α Tg mice had similar percentages of Vβ8 T cells incorporating BrdU in the LN on day 3 (Fig. 7⇑, a and c). Based on these percentages of BrdU incorporation, no major proliferation defect was apparent. SEB treatment mediated ∼40% of the responding T cells to take up BrdU in both normal and IκB-α Tg mice. Three-signal treatment only slightly enhanced the incorporation of BrdU to ∼50% of the Vβ8 population.

When the absolute numbers of Vβ8 T cells taking up BrdU was examined, however, a major defect was observed (Fig. 7⇑, b and d). C57BL/6 mice had similar numbers of Vβ8 T cells incorporating BrdU regardless of whether SEB alone or three signals were delivered. The number of NF-κB-deficient T cells taking up BrdU was strongly reduced in SEB-treated mice, strongly supporting the notion that NF-κB activity promotes survival during clonal expansion. However, co-injection of anti-CD40 and LPS with SEB increased the numbers by 4.5-fold, suggesting that such stimulation enhances short-term survival, as measured by accumulation of Ag-specific T cells, in an NF-κB-independent manner.