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IL-1 and TNF- Are Required for IL-12-Induced Development of Th1 Cells Producing High Levels of IFN- in BALB/c But Not C57BL/6 Mice¹

Kazuko Shibuya^*, Douglas Robinson^*, Francesca Zonin^*, Suzanne B. Hartley^*, Steven E. Macatonia^*, Chamorro Somoza^*, Christopher A. Hunter, Kenneth M. Murphy and Anne O’Garra^2,*

^* Department of Immunobiology, DNAX Research Institute of Molecular and Cellular Biology, Inc., Palo Alto, CA 94303; Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110; and Department of Pathobiology, University of Pennsylvania, Philadelphia, PA 19104

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The development of Th1- or Th2-type responses determines the type of immune response that is elicited in response to Ag. Responsiveness to IL-12 is critical for the development of Th1-type CD4⁺ T cells required for cell-mediated immune responses. Addition of IL-12 to primary cultures of CD4⁺ T cells stimulated with OVA and splenocytes or dendritic cells resulted in the development of a Th1 phenotype with the capacity to secrete high levels of IFN- upon restimulation with splenic APC. The present study shows that using dendritic cells to present Ag upon restimulation reveals a requirement for additional cofactors, including IL-1 and TNF-, which were provided by spleen cells but not dendritic cells. Furthermore, these cofactors are required for optimal IL-12-induced Th1 development in BALB/c but not C57BL/6 mice. This differential requirement for such cofactors in IL-12-driven Th1 development may play a role in genetic predisposition to Th1 or Th2 responses to infectious agents.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The class of immune response to a particular infection may be determined by the nature of the T cell help elicited, where characteristic profiles of cytokines produced by distinct Th cells modulate the effector arm of the immune system (1, 2, 3). Th1 cells produce IL-2, IFN-, and lymphotoxin (TNF-ß) and promote cell-mediated effector responses, whereas Th2 cells produce IL-4, IL-5, IL-6, and IL-10, cytokines that can augment humoral responses and stimulate mast cells and eosinophils to promote allergic-type responses (1, 2, 3, 4). Early events in an immune response stimulate the production of cytokines (5, 6, 7, 8, 9, 10, 11), which in turn, direct the subsequent development of Th subsets. These events are strongly dictated by the type of Ag/micro-organism invading a host as well as by the genetic background of the host. It is likely that the dose and route of immunization with Ag may also be determining factors (3, 12, 13, 14).

The most clearly defined factors determining Th1 and Th2 differentiation are cytokines present at the initiation of the immune response, at the stage of activation through the TCR (3, 5, 6, 7, 8, 9, 10, 11, 12, 13). Bacterial infection leads to the activation of macrophages and subsequently NK cells. This innate immune response results in the production of IL-12 and IFN- (8, 15), which then drive the development of Th1-type cells from naive Ag-specific CD4⁺ T cells, leading to the production of high levels of IFN- (8, 10, 11, 15). Conversely, IL-4 induces a Th2 or allergic/humoral-type immune response that can often lead to down-regulation of cell-mediated immunity (5, 6, 7, 9).

Previously, we reported conditions for inducing a Th1 phenotype using the DO11.10 TCR-ß-transgenic mouse (BALB/c genetic background) in which 80% of CD4⁺ T cells express a unique TCR specific for a peptide of OVA (8, 16, 17). Addition of IL-12 to primary cultures of CD4⁺ T cells stimulated with OVA and splenocytes or dendritic cells resulted in the development of a Th1 phenotype with the capacity to secrete high levels of IFN- (8, 17, 18) upon restimulation with splenic APC. The present study shows that additional cofactors, including IL-1 and TNF- are required for optimal IL-12-induced Th1 development in BALB/c but not C57BL/6 mice.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals

Mice transgenic for a TCR-ß recognizing OVA_323–339 (D011.10; BALB/c genetic background) (16) were selected at 6 to 8 wk of age by staining peripheral blood leukocytes with the anti-clonotype mAb KJ1-26 (19). Mice transgenic for a TCR-ß recognizing a peptide of hen egg lysozyme, HEL_74–88,³ were selected at 6 to 8 wk of age by PCR analysis of DNA obtained from ear punches, analyzed for the rearranged transgenic TCR (S. B. Hartley et al., manuscript in preparation). All transgenic mice used in these experiments were heterozygous for the integration of the TCR ß-chains. BALB/c mice between 6 and 10 wk of age were purchased from Simonsen Laboratories (Gilroy, CA). C57BL/6 mice between 6 and 10 wk of age were purchased from The Jackson Laboratory (Bar Harbor, ME).

Culture medium, cytokines, Abs, and Ags

RPMI 1640 (JR Scientific, Inc., Woodland CA) supplemented with 10% FCS (JR Scientific), 2-ME (0.05 mM; Sigma), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), HEPES buffer (10 mM), and sodium pyruvate (1 mM) was used as culture medium. For dendritic cell isolations, RPMI 1640 Dutch modification (Life Technologies, Paisley, Scotland) was used with glutamine, 2-ME (0.05 mM; Sigma), FCS, penicillin, and streptomycin as described above.

Recombinant mouse cytokines were as follows: IFN- (Genzyme, Cambridge, MA), IL-4 (Dr. S. Menon, DNAX), TNF- (Genzyme), and IL-1 (a gift from Peter Lomedico, Roche, Nutley, NJ). Recombinant mouse IL-12 was obtained from PharMingen (San Diego, CA) and was as described previously (20, 21).

Purified rat anti-mouse IL-4, 11B11, (22), anti-TNF- (XT/22), and anti-IFN- (XMG1.2) plus isotype-matched controls were supplied by J. Abrams (23) (DNAX). Anti-mouse IL-1 (ALF 161) and anti-mouse IL-1 receptor mAb (JAMA) along with isotype-matched controls (B122 and L2, respectively) were gifts from Dr. E. Unanue, Washington University (St. Louis, MO) (24, 25, 26). Anti-IL-12 mAbs (C17.8.20) were described by Wysocka and Trinchieri (27). Other Abs used included anti-mouse Mac-1 (M1/70, Caltag, San Francisco, CA); hamster anti-mouse N418 (American Type Culture Collection, Rockville, MD) (28); biotinylated anti-mouse CD8, B220, GR-1, anti-Mac-1, and anti-I-A^d (PharMingen); and anti-mouse CD4 FITC (or phycoerythrin) and anti-L-selectin FITC (PharMingen). mAbs for ELISA, including anti-IL-4 and IFN- reagents, were purified from serum-free hybridoma supernatants as previously described (23).

The antigenic peptides from chicken, OVA_323–339, and HEL_74–88 were synthesized by Biosynthesis (Lewisville, TX).

Preparation of T cells and APCs

CD4⁺ T cells were enriched by negative selection using magnetic activated cell sorting with a mixture of biotinylated anti-CD8, anti-I-A^d, anti-B220, anti-GR-1, and anti-Mac-1 Abs (Miltenyi, Sunnyvale, CA). CD4⁺ T cells, enriched by negative selection using magnetic activated cell sorting were further purified using a FACStar^Plus flow cytometer (Becton Dickinson, Mountain View, CA) to achieve >99% CD4⁺ T cells, demonstrated as naive on the basis of bright L-selectin staining (29). Staining with mAb did not alter the function of the T cells (not shown).

Dendritic cells were enriched from either BALB/c or C57BL/6 spleen cell preparations, as described previously (17, 30), by first removing adherent cells by overnight culture in plastic flasks followed by overlayering onto 2-ml metrizamide gradients (analytical grade; 13.7%; Nycomed Pharma AS, Oslo, Norway) and centrifugation for 10 min at 600 x g to give a population enriched for dendritic cells in the low density fraction. Dendritic cells (N418^highMac-1^low) were further purified to homogeneity by flow cytometry as previously described (17).

Stimulation of transgenic CD4⁺ T cells for cytokine production

Primary stimulations of CD4⁺ T cells (2.5 x 10⁵/well) were conducted using OVA (0.6 µM) or HEL (1 µM) and dendritic cells (1 x 10⁴/well; 1500 rad) or RBC-lysed spleen cells (5 x 10⁶/well; 3000 rad) as APC in a total volume of 2 ml in 24-well plates (Costar). In some cases, T cells were stimulated with cross-linked anti-CD3 (10 µg/ml) plus IL-2 (10 ng/ml). In addition, some cultures received cytokines (IL-4 (10 ng/ml) or IL-12 (10 ng/ml)) or mAbs to block endogenous cytokines (anti-IL-4 (10 µg/ml), anti-IFN- (10 µg/ml), anti-IL-12 (10 µg/ml)). Some cultures also received TNF- (100 U/ml), IL-1 (10 ng/ml), individually or together with IL-12 (10 ng/ml). T cells were expanded threefold into fresh medium at 72 h, or 96 h for anti-CD3-stimulated cultures. Cells were harvested on day 7, washed three times, counted, and restimulated with fresh APC (splenocytes or dendritic cells) and 0.6 µM OVA or HEL at 1 µM. Cultures previously stimulated with anti-CD3 plus IL-2 were restimulated with anti-CD3 plus IL-2. The cell concentrations used were the same as those described for priming. Some cultures also received TNF- (100 U/ml), IL-1 (10 ng/ml), or IL-12 (10 ng/ml), either separately or in combination, during the restimulation. Supernatants were collected at 48 h for the measurement of IL-4 and IFN-. In some cases [³H]thymidine was added to the cultures for the last 4 h of the 72-h incubation to measure DNA synthesis.

Committed Th1 cells and Th1 clones

Committed Th1 cells were derived from FACS-sorted naive TCR transgenic CD4⁺ T cells as previously described (31). In brief, cells were cultured with Ag (OVA), and in this case with dendritic cells in the presence of IL-12 (10 ng/ml) with or without IL-1 and TNF- to induce Th1 development. Cells were harvested at 7 days and restimulated with fresh dendritic cells and Ag in the presence of the above cytokines; this process was repeated two additional times. Committed Th1 populations were restimulated with Ag (OVA, 0.6 µM) and splenic APC. Supernatants were collected at 48 h and assayed for IFN- and IL-4. Proliferation was assessed by addition of 1 µCi of [³H]thymidine/well for 6 h.

The Th1 clone HDK1 is a keyhoe limpet hemocyanin-specific clone in the context of I-A^d (32). The Th1 clone was used in experiments at least 10 days after the last Ag stimulation, after culture in medium with IL-2 alone. Th1 clones were stimulated for 48 h in 96-well plates (at 2 x 10⁴/well) either with cross-linked anti-CD3 (10 µg/ml) with or without IL-2 (10 ng/ml) and anti-CD28 (10 µg/ml), or with PMA and ionomycin (50 and 500 ng/ml, respectively). In addition, Th1 clones were stimulated with Ag (keyhole limpet hemocyanin, 500 µg/ml) and dendritic cells for 48 h. Cytokine production was assessed in supernatants at 48 h, and proliferation was assessed by [³H]thymidine incorporation.

Cytokine assays

IFN- was detected using a two-site sandwich ELISA (33). The sensitivity was 125 pg/ml (1 U/ml = 0.1 ng/ml). The ELISA for IL-4 has been described previously (23), with a level of detectability of 150 pg/ml. Cytokine-secreting cells were assayed on a single cell basis by flow cytometric analysis of intracellular IFN- and IL-4 synthesis as previously described (31).

Statistical analysis

Data from multiple experiments were analyzed by comparison to a defined control value using Dunnett’s test. Analysis was performed using JMP software (SAS Institute, Inc., Cary, NC). In defined cases, pairwise comparison was by Student’s paired t test. p < 0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Capacity of dendritic cells vs spleen cells as APC to drive IL-12-dependent Th1 development

We have previously shown that addition of IL-12 to primary Ag-driven OVA-specific ß-TCR transgenic CD4⁺ T cells stimulated with dendritic cells or spleen cells as APC results in a Th1 phenotype producing high levels of IFN- upon restimulation with splenic APC and OVA (8, 17, 18). We now show that IL-12-driven Th1 development of CD4⁺ T cells primed and restimulated with dendritic cells as APC gave rise to Th1 cells, but these cells produced 10- to 40-fold less IFN- than T cells primed and restimulated using spleen cells as APC (Fig. 1, A and B). However, dendritic cells were 2 logs more potent on a per cell basis in inducing the proliferation of CD4⁺ T cells both in the primary stimulation (data not shown) (17) and during the restimulation (Fig. 1, C and D). This suggested that spleen cells were providing other cofactors required for IL-12-driven Th1 phenotype development.

View larger version (22K): [in this window] [in a new window]   FIGURE 1. IL-12-driven Th1 development upon stimulation with spleen APC results in significantly higher levels of IFN- production by Th1 cells than stimulation with dendritic cells. Naive CD4+ T cells from DO11.10 TCR transgenic mice were cultured in 24-well plates at a density of 2.5 x 105 T cells in 2 ml with irradiated FACS-sorted dendritic cells (1 x 104/well; A and C) or irradiated whole splenic APC (5 x 106/well; B and D) and OVA (0.6 µM) for 7 days in the presence of medium alone (triangles) or with IL-12 (squares). Cells were then harvested, washed, and replated in 24-well plates at 2.5 x 105/ml with Ag presented by dendritic cells (1 x 104/well) or irradiated whole splenic APC (5 x 106/well) and OVA (0.6 µM) for 48 h (top) or in 96-well plates at 2.5 x 104/ml with Ag presented by dendritic cells or irradiated whole splenic APC OVA (0.6 µM), both APC titrated as indicated, for 72 h. IFN- was measured in 48-h supernatants from 24-well plates by ELISA. [3H]thymidine (1 µCi/well) was added to 96-well plates for last 4 h before counting. Results are representative of at least five independent experiments.

IL-1 and TNF- potentiate IL-12-driven Th1 development from naive DO11.10 ß-TCR transgenic CD4⁺ T cells

Based on the apparent similarities between costimulators of IFN- production by T cells and NK cells (8, 11, 17, 34, 35, 36), possible costimulators for IFN- production by developing Th1 cells were TNF- and IL-1. To test this, IL-1 and TNF- were first added to primary cultures of naive CD4⁺ T cells stimulated with dendritic cells as APC, either alone or in the presence of IL-12. Addition of IL-1 or TNF- (either together or individually) in the absence of IL-12 to primary CD4⁺ T cells stimulated with dendritic cells had no effect on the IFN- levels obtained upon restimulation with spleen APC (Fig. 2A). Addition of IL-1 to IL-12, in contrast to TNF-, caused a small, but reproducible, increase in the level of IFN- induced when naive CD4⁺ T cells were stimulated with dendritic cells in primary cultures, but this was still reduced compared with the amounts of IFN- induced using splenic APC. Although TNF- on its own had no effect on IL-12-induced Th1 development, a combination of IL-1 and TNF- with IL-12, in primary cultures stimulated with DC, induced maximal levels of IFN- production equivalent to those induced following primary stimulation of CD4⁺ T cells with splenic APC and Ag (Fig. 2A). The IL-12-induced Th1 development observed in the presence of IL-1 and TNF- was inhibited significantly by addition of anti-IFN- Abs (Fig. 2B). Furthermore, addition of anti-IL-1, anti-IL-1R, and anti-TNF- mAbs to cultures of CD4⁺ T cells undergoing IL-12-driven Th1 development with Ag presented by splenic APC significantly reduced IFN- secretion by these Th1 cells to the level obtained when IL-12-driven Th1 development was in the presence of dendritic cells as APC (Fig. 3, top). Isotype-matched control mAbs had no influence on IFN- secretion (data not shown). In contrast to their effects on IFN- production, these anti-cytokine Abs did not inhibit T cell proliferation (data not shown), suggesting a specific requirement for these costimulators in the induction of IFN-. Although dendritic cells were 100-fold more potent than splenic APC for induction of proliferation of Th1 cells (Fig. 1), demonstrating competence as APC for T cell proliferation, the cofactors required for maximal IFN- production were clearly provided by splenocytes and not dendritic cells.

View larger version (20K): [in this window] [in a new window]   FIGURE 2. A, IL-1 plus TNF- potentiate IL-12-induced Th1 development from BALB/c T cells. Naive CD4+ T cells from DO11.10 TCR transgenic mice were cultured in 24-well plates at a density of 2.5 x 105 T cells in 2 ml with irradiated FACS-sorted dendritic cells (1 x 104/well) and OVA (0.6 µM) for 7 days in the presence of medium alone, IL-12 alone, IL-12 plus IL-1, IL-12 plus TNF-, IL-12 plus IL-1 and TNF-, or IL-4 or with irradiated whole splenic APC (5 x 106/well) and OVA (0.6 µM) for 7 days in the presence of medium alone, IL-12, or IL-4. Cells were then harvested, washed, and replated in 24-well plates at 2.5 x 105/ml with irradiated whole splenic APC (5 x 106/well) and OVA (0.6 µM) for 48 h. IFN- was measured in 48-h supernatants from 24-well plates by ELISA. IFN- production from cells cultured in the presence of dendritic cells (DC) with IL-12, IL-1, and TNF- or in the presence of DC with IL-12 and IL-1 or splenic APC and IL-12 was significantly higher than that from cells cultured with DC and IL-12 by Dunnett’s test (p < 0.05 for each comparison; n = 9). B, Anti-IFN- partially inhibits IL-12-induced Th1 development from BALB/c T cells in the presence of IL-1 and TNF-. Th1 development was performed exactly as described in A; however, cultures containing added anti-IFN- (10 µg/ml) Abs were also included.

View larger version (24K): [in this window] [in a new window]   FIGURE 3. IL-1 plus TNF- potentiate IL-12-induced Th1 development from BALB/c but not C57BL/6 T cells. Naive CD4+ T cells from DO11.10 or TCR7 TCR transgenic mice were cultured in 24-well plates at a density of 2.5 x 105 T cells in 2 ml with irradiated FACS-sorted dendritic cells (1 x 104/well) and OVA (0.6 µM) or HEL (1 µM), respectively, for 7 days in the presence of medium alone, IL-12 alone, IL-12 plus IL-1 and TNF-, or IL-4 or with irradiated whole splenic APC (5 x 106/well) and OVA (0.6 µM) for 7 days in the presence of medium alone; IL-12; IL-12 plus anti-IL-1, anti-IL-1R type I, and anti-TNF- Abs; or IL-4. Cells were then harvested, washed, and replated in 24-well plates at 2.5 x 105/ml with irradiated whole splenic APC (5 x 106/well) and OVA (0.6 µM) or HEL (1 µM), respectively, for 48 h. IFN- was measured in 48-h supernatants from 24-well plates by ELISA. Addition of IL-1 and TNF- to IL-12 in cultures of T cells from BALB/c mice with DC produced a significant increase in IFN- production compared with DC with IL-12 (p < 0.05; n = 6), whereas IL-1 and TNF- had no significant effect on IL-12-induced IFN- production from T cells from C57BL/6 mice cultured with DC (n = 6).

IL-1 and TNF- are required for maximal IL-12-induced Th1 development in mice on a BALB/c but not C57BL/6 genetic background

Since Th1 development has been shown to be impaired in mice on a BALB/c background (37, 38, 39, 40), it was important to determine whether this requirement for IL-1 and TNF- was a general requirement for IL-12-driven Th1 differentiation or was restricted to this genetic background. Figure 3 (bottom) clearly shows that TCR7 mice (C57BL/6), which express an ß-TCR transgene specific for HEL_74–88, show no requirement for IL-1 or TNF- in IL-12-driven Th1 development when Ag was presented in the primary cultures on dendritic cells. Furthermore, IL-12-driven Th1 development on spleen APC was not affected by the addition of Abs directed against IL-1, IL-1 type 1-R, and TNF- (Fig. 3, bottom) in the TCR7 (C57BL/6) mice.

These results suggested that mice on a BALB/c background may not acquire, or conversely may lose, responsiveness to IL-12 during Th1 development (40). To address this, IL-12 induction of CD25, the IL-2R subunit, was measured. IL-12 has been previously shown to up-regulate IL-2 induction of CD25 in Th1 clones (41). IL-12 induced maximal expression of the CD25 on developing Th1 cells in C57BL/6 mice stimulated with dendritic cells and Ag, and IL-1 and TNF- did not increase receptor expression (Fig. 4, bottom). In contrast, CD4⁺ T cells from the DO11.10 mouse, on a BALB/c genetic background, stimulated with dendritic cells and Ag in the presence of IL-12 showed no increased level of CD25 expression above the medium control value unless IL-1 and TNF- were also added (Fig. 4, top). IL-1 and TNF- showed no effect on CD25 expression in the absence of IL-12 (data not shown).

View larger version (39K): [in this window] [in a new window]   FIGURE 4. IL-1 plus TNF- potentiate IL-12-induced IL-2R expression on BALB/c but not C57BL/6 T cells. Naive CD4+ T cells from DO11.10 or TCR7 TCR transgenic mice were cultured in 24-well plates at a density of 2.5 x 105 T cells in 2 ml with irradiated FACS-sorted dendritic cells (1 x 104/well) and OVA (0.6 µM) or HEL (1 µM), respectively, for 5 days in the presence of medium alone, IL-12 alone, or IL-12 plus IL-1 and TNF- (or IL-1 and TNF-, together or alone; data not shown). Cells were harvested, washed, stained with Abs directed against the IL-2R -chain (CD25), and then analyzed by flow cytometry. Results are representative of at least three independent experiments.

View larger version (13K): [in this window] [in a new window]   FIGURE 5. IL-1- plus TNF--potentiated IL-12-induced Th1 development from BALB/c T cells is in part counteracted by neutralization of endogenous IL-4. Naive CD4+ T cells from DO11.10 TCR transgenic mice were cultured in 24-well plates at a density of 2.5 x 105 T cells in 2 ml with irradiated FACS-sorted dendritic cells (1 x 104/well) and OVA (0.6 µM) for 7 days in the presence of medium alone, IL-12 alone, or IL-12 plus IL-1 and TNF-, each in the presence or the absence of anti-IL-4 Abs. Cells were then harvested, washed, and replated in 24-well plates at 2.5 x 105/ml with irradiated whole splenic APC (5 x 106/well) and OVA (0.6 µM) for 48 h. IFN- was measured in 48-h supernatants from 24-well plates by ELISA. Addition of IL-1 and TNF- to IL-12 produced a significant increase in IFN- production compared with the effect of IL-12 alone in the presence or the absence of anti-IL-4 (p < 0.05 compared with controls, by Dunnett’s test; n = 5).

IL-1 and TNF- potentiate IL-12-driven Th1 development in BALB/c but not C57BL/6 mice stimulated in an APC-free system

Our data strongly suggested a requirement for IL-1 and TNF- in IL-12-driven Th1 development in BALB/c but not C57BL/6 mice. However, these TCR-transgenic mice express different TCR transgenes, each specific for a different peptide Ag, OVA_323–339 and HEL_74–88, respectively. Since the affinity of Ag-MHC for the TCR has been proposed to play a role in determining Th subset development (14), this may influence this differential responsiveness to Ag presented on dendritic cells. To rule this out, IL-12-driven Th1 development was examined when CD4⁺ T cells from BALB/c or C57BL/6 mice were stimulated in an APC/Ag-free system with cross-linked anti-CD3 Abs plus IL-2. As shown in Figure 6, IL-12-driven Th1 development was still reduced in CD4⁺ T cells obtained from BALB/c compared with C57BL/6 mice. Furthermore, IL-1 and TNF- potentiated IL-12-driven Th1 development in BALB/c but not C57BL/6 mice. These data also support a direct effect of these cofactors on the T cell as well as possible enhancing effects on APC in previous systems. It is possible that IL-1 and TNF- increase the amounts of IFN- secreted on a per cell basis by Th1 cells induced by IL-12, or that these factors increase the number of IFN--secreting cells within the population. Flow cytometric analysis of intracellular synthesis of IFN- demonstrated that these cofactors indeed increased the number of IFN--secreting Th1 cells resulting from cultures induced with IL-12.

View larger version (24K): [in this window] [in a new window]   FIGURE 6. A, IL-1 plus TNF- potentiate IL-12-induced Th1 development from naive BALB/c but not C57BL/6 T cells stimulated with anti-CD3 in the absence of APC. Naive CD4+ T cells from BALB/c or C57BL/6 mice were cultured for 7 days on anti-CD3-coated 24-well plates with IL-2 in the presence of medium alone, IL-12, or IL-12 plus IL-1 and TNF-. Cells were then restimulated with anti-CD3 and IL-2 for 48 h before supernatants were harvested for assay of IFN- by ELISA. Addition of IL-1 and TNF- produced a significant increase in IL-12-induced IFN- production from T cells from BALB/c mice (p < 0.05; n = 6), but not in those from C57BL/6 mice. B, IL-1 plus TNF- potentiate IL-12-induced Th1 development by increasing the number of IFN--secreting cells developing from naive BALB/c T cells. Naive CD4+ T cells from BALB/c or C57BL/6 mice were cultured for 7 days on anti-CD3-coated 24-well plates with IL-2 in the presence of medium alone, IL-12, or IL-12 plus IL-1 and TNF-. Cells were then restimulated with anti-CD3 and IL-2 for 4 h, with brefeldin A added for the last 2 h, and analyzed by flow cytometry for intracellular synthesis of IFN- and IL-4 as previously described (31). The frequencies of IFN--producing cells are shown on the y-axis, and those of IL-4-producing cells are shown on the x-axis.

IL-1 and TNF- are cofactors for Th1 development but do not completely account for maximal IL-12-driven Th1 development observed when priming and restimulation are induced by spleen APC

IL-12-driven Th1 development observed when CD4⁺ T cells were stimulated with anti-CD3 plus IL-2 during primary and secondary stimulation yielded consistently and significantly lower levels of IFN- upon restimulation than those observed with splenic APC, even in the presence of IL-1 and TNF- (Fig. 6). To address this in more detail, Th1 cells driven by IL-12 plus IL-1 and TNF- were stimulated in the primary cultures with either dendritic cells or spleen cells and were then restimulated with DC or spleen cells in the presence of IL-12 with or without IL-1 and TNF- in an attempt to restore the levels of IFN- to those seen upon restimulation with spleen APC. As observed in Figure 7, although the levels of IFN- seen upon primary and secondary stimulation with DC in the presence of IL-12, plus IL-1 and TNF-, were much enhanced by addition of all three factors upon secondary stimulation with DC, the levels of IFN- achieved were still not equivalent to those achieved upon restimulation with splenic APC plus IL-12.

View larger version (24K): [in this window] [in a new window]   FIGURE 7. Priming and restimulation of CD4+ T cells with Ag and splenic APC yield higher levels of IFN- from IL-12-driven Th1 cells than stimulation with dendritic cells, even in the presence of IL-1 plus TNF-. Naive CD4+ T cells from DO11.10 TCR transgenic mice were cultured in 24-well plates at a density of 2.5 x 105 T cells in 2 ml with irradiated FACS-sorted dendritic cells (1 x 104/well) and OVA (0.6 µM) for 7 days in the presence of medium alone, IL-12 alone, or IL-12 plus IL-1 and TNF- or with irradiated whole splenic APC (5 x 106/well) and OVA (0.6 µM) for 7 days in the presence of medium alone or IL-12. Cells were then harvested, washed, and replated in 24-well plates at 2.5 x 105/ml with irradiated FACS-sorted dendritic cells (DC; 1 x 104/well) and OVA (0.6 µM) in the presence of medium alone, IL-12 alone, or IL-12 plus IL-1 and TNF- for 48 h or with irradiated whole splenic APC (5 x 106/well) and OVA (0.6 µM) for 7 days in the presence of medium alone or IL-12 for 48 h. IFN- was measured in 48-h supernatants from 24-well plates by ELISA. IFN- production in secondary cultures stimulated with splenic APC in the presence of IL-12 was significantly higher than that when cells were restimulated with DC, IL-12, IL-1, and TNF- in cells that had primary stimulation with DC; with IL-12, DC, IL-12, IL-1, and TNF-, or with splenic APC and IL-12 (p < 0.05, by paired t test, comparing splenic APC with IL-12 to DC with IL-12, IL-1, and TNF- in each case; n = 6). Addition of IL-1 and TNF- to DC with IL-12 significantly increased IFN- production in secondary cultures of cells cultured with DC, IL-12, IL-, and TNF- or with splenic APC and IL-12 in the primary stimulation (comparison of DC with IL-12 alone vs DC, IL-12, IL-1, TNF- in secondary culture, p < 0.05).

Committed Th1 cells and Th1 clones lose their responsiveness to IL-1 and TNF-

To determine whether differentiated Th1 cells retain their responsiveness to the cofactors IL-1 and TNF-, two approaches were taken. The first was to repeatedly stimulate CD4⁺ T cells from DO11.10 mice with OVA_323–339 and DC in the presence of IL-12 alone or with IL-12 together with IL-1 and TNF-. As shown in Figure 8, repeated restimulation of the Th1 cells showed that there was no difference in the levels of IFN- produced if they had been stimulated in the presence or the absence of the cofactors, IL-1 and TNF-. This could suggest that inhibitors present early during IL-12-induced development of Th1 cells were lost upon repeated stimulation with the polarizing stimulus. Additionally, it is possible that committed Th1 cells lose responsiveness to these cofactors. This possibility is confirmed by a lack of enhancement of IFN- production or proliferation by Th1 clones by IL-1 and/or TNF- over that seen with IL-12 alone (Fig. 9), in contrast to IL-1 mediated increased proliferation of Th2 clones (data not shown), consistent with prior observations (43).

View larger version (16K): [in this window] [in a new window]   FIGURE 8. IL-1 and TNF- do not synergize with IL-12 to induce proliferation and IFN- production from committed Th1 cells. Naive CD4+ T cells from DO11.10 TCR-transgenic mice were stimulated in 24-well plates at a density of 2.5 x 105 T cells in 2 ml with irradiated FACS-sorted dendritic cells (1 x 104/well) and OVA (0.6 µM) for 7 days in the presence of medium alone, IL-12 alone, IL-12 plus IL-1 and TNF-, or IL-4 or with whole splenic APC (5 x 106/well) and OVA (0.6 µM) for 7 days in the presence of medium alone, IL-12, or IL-4. T cells were harvested and washed after 7 days, and the process was repeated as described above for three rounds of stimulation. After the third stimulation the cells were harvested and restimulated with whole splenic APC (5 x 106/well) and OVA (0.6 µM) for 48 h, and supernatants were removed for assay of IFN- and IL-4 production. Results are representative of three separate experiments.

View larger version (27K): [in this window] [in a new window]   FIGURE 9. IL-1 and TNF- do not potentiate IL-12-induced proliferation and IFN- production from a Th1 clone, HDK 1. Th1 clone cells were rested in IL-2 for at least 10 days from the last Ag stimulation, then cultured for 48 h with either medium alone, IL-12 alone, or IL-12 plus IL-1 and TNF- in the presence of anti-CD3 with or without IL-2, or PMA and ionomycin. IFN- production and proliferation were assessed at 48 h.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Previous studies suggest that APC-derived IL-1 plays an important role as a costimulatory signal for Th2 cells (43, 48). Exogenously supplied IL-1 was shown to costimulate responses of Th2 cells stimulated with mitogens, anti-TCR Abs, or fixed Ag-pulsed APC (43, 46, 48, 49, 50, 51). Furthermore, IL-1 renders Th2 cells sensitive to proliferation induced by IL-4. Some APC, such as monocytes, produce high levels of IL-1 (52) although very potent APC, such as dendritic cells, reportedly do not produce IL-1 (53). Other studies have indicated that certain Th2 clones can produce IL-1 (54, 55) through engagement of CD28 (49), and this leads to increased Th2 IL-4 responsiveness for stimulation of proliferation. In contrast to Th2 cells, there is no evidence to indicate that Th1 cells either produce IL-1 or respond to it (43, 46). In fact, Th1 cells reportedly lack cell surface receptors for IL-1 (43, 48).

Although, Th2 cells clearly respond to IL-1, and Th1 cells lack responsiveness to this factor, we wished to address its possible role as a cofactor in Th1 cell development. Evidence that cofactors are required for IL-12-driven Th1 development is provided in this study. We show that IL-12-driven Th1 development on dendritic cells or anti-CD3 yielded low levels of IFN- from Th1 cells upon restimulation, in contrast to Th1 cells restimulated on spleen APC. Likely candidates as cofactors included IL-1 and TNF-, since the production of IFN- by NK cells stimulated with IL-2 or IL-12 has been shown to be enhanced by IL-1 or TNF- (56, 57, 58), and NK cells have stimulatory requirements similar to those of Th1 cells (8, 34). Furthermore, TNF- has been reported to be an adjuvant for Th1 responses (59) and also to synergize with IL-1 for certain biologic responses (60). Thus, our study lends further evidence for the similarity of developing Th1 cells and NK cells for the production of IFN-.

Szabo et al. (42) and Rogge et al. (61) have recently clarified the molecular basis of the IL-12 unresponsiveness of Th2 cells by showing the down-regulation of IL-12Rß2 by IL-4. They have further demonstrated the requirement for IFN- in Th1 development, which to date has been controversial (9, 10, 17, 18, 42, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71). In addition, IFN- up-regulates IL-12Rß2 and counteracts the inhibitory effects of IL-4. Thus, BALB/c mice, which produce a substantial amount of IL-4 (39, 40), may inhibit IL-12Rß2 expression, imposing the reported requirement for IFN- (42). This may not occur in genetic backgrounds of mice producing low levels of IL-4, such as B10 and C57BL/6. In the present study it is likely that IL-1 and TNF- act either to down-regulate IL-4 or up-regulate IFN- directly, thus increasing the IL-12 responsiveness of BALB/c T cells. Supporting this conclusion, we also noted that anti-IL-4 Abs partially counteracted the requirement for IL-1 and TNF-, but our observation that this was not complete suggests that other inhibitors may also operate to affect IL-12 responsiveness. IL-12-driven Th1 development in the BALB/c mouse in the presence of IL-1 and TNF- is in part dependent on IFN-, because anti-IFN- reduced this cofactor-dependent Th1 development. Thus, it is likely that these cofactors operate by increasing levels of IFN- production from developing Th1 cells, which, as demonstrated by Szabo et al. (42), directly up-regulates the IL-12Rß-2. Since anti-IFN- did not completely reverse the effects of IL-1 and TNF-, it is clear that there is an additional direct effect of these cofactors to increase IL-12 responsiveness.

The results shown in this study may be relevant for understanding responses to intracellular pathogens. For example, susceptibility or resistance to Leishmania major in various strains of mice is complex and probably controlled by several genetic loci (72, 73). Although it is likely to result from the level of IL-12Rß2 expression regulated by IL-4 or IFN-, the immune response may also be affected by other cofactors, such as IL-1 and TNF- and by their interplay with T cells and NK cells (36, 56, 58, 74).

Finally, to address the differential stimulation of Th1 vs Th2 cells by IL-1 published in previous studies, our findings on committed polarized Th1 cells from TCR-transgenic mice and Th1 clones agree with the loss of responsiveness of Th1 clones to IL-1 (43) reported previously.

In summary, we show that IL-1 and TNF- are required for maximal IL-12-driven Th1 development in BALB/c but not C57BL/6 mice, with IL-1 having a dominant effect. Although required for potentiation of IL-12-driven Th1 development in BALB/c mice, these cofactors show no effect on differentiated Th1 cells.

	Acknowledgments

 
We thank John Abrams for advice on statistics, Lewis Lanier for critical review of the manuscript, and Robert Coffman for helpful discussion. We thank Jim Cupp, Dixie Polakoff, Eleni Callas, and Erin Murphy for technical help with flow cytometry, and Maribel Andonian for assistance with graphics.

	Footnotes

 
¹ DNAX is funded by Schering Plough Research Institute.

² Address correspondence and reprint requests to Dr. Anne O’Garra, Department of Immunobiology, DNAX Research Institute of Molecular and Cellular Biology, Inc., 901 California Ave., Palo Alto, CA 94304-1104.

³ Abbreviations used in this paper: HEL, hen egg lysozyme.

Received for publication August 8, 1997. Accepted for publication October 29, 1997.

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