Inflammatory Cytokines Provide a Third Signal for Activation of Naive CD4+ and CD8+ T Cells

Julie M. Curtsinger, Clint S. Schmidt, Anna Mondino, Debra C. Lins, Ross M. Kedl, Marc K. Jenkins and Matthew F. Mescher
J Immunol March 15, 1999, 162 (6) 3256-3262;

Abstract

The effects of inflammatory cytokines on naive T cells have been studied using MHC protein/peptide complexes on microspheres, thus avoiding the use of APCs whose functions may be affected by the cytokines. IL-1, but not IL-12, increased proliferation of CD4+ T cells in response to Ag and IL-2, which is consistent with effects on in vivo priming of CD4+ cells. In contrast, proliferation of CD8+ T cells to Ag and IL-2 required IL-12, and IL-12 replaced adjuvant in stimulating an in vivo response to peptide. These results support a model in which distinct inflammatory cytokines act directly on naive CD4+ and CD8+ T cells to provide a third signal, along with Ag and IL-2, to optimally activate differentiation and clonal expansion.

Initiation of a T cell immune response that leads to full activation rather than tolerance appears to require that the T cell know that “danger” is present 1 , and it has been argued that inflammatory cytokines produced by the innate immune system in response to pathogens provide the “danger signals” to T cells 2 . Potent induction of inflammatory cytokines is likely to account, at least in part, for the adjuvant properties of CFA, LPS, and other adjuvants. Administration of Ag in the absence of adjuvant results in some clonal expansion of CD4+ T cells in draining lymph nodes, but the cells are rendered tolerant to subsequent challenge with Ag. Coadministration of Ag with CFA or LPS increases the extent of clonal expansion and prevents tolerance induction, and these effects can be mimicked by administration of either TNF-α or IL-1 3 . The effects of these may be related, because TNF-α induces IL-1 production 4 . Thus, these inflammatory cytokines are necessary and sufficient to support the response in the absence of other adjuvants.

Where and how the inflammatory cytokines act to promote T cell responses is less clear. Studies of CD4+ T cell lines suggest that IL-1 may contribute to activation through direct interaction with the T cell 5, 6, 7, 8 . Alternatively, the cytokines may act indirectly by up-regulating, on APCs, the expression of ligands that costimulate T cell activation 9 . TNF-α and IL-1 can induce the expression of CD40 on dendritic cells 10 and CD40-dependent signaling can in turn increase B7 expression on APC 11, 12, 13 , thus increasing the level of costimulation available to the T cells. Proliferation and differentiation of CD8+ T cells to generate cytotoxic T lymphocyte responses can also be enhanced by inflammatory cytokines produced by macrophages and/or dendritic cells, cytokines that include IL-1 14 , IL-6 14, 15 , IL-12 16, 17, 18 , and IFN-γ 19 . Like CD4+ T cells, it is unclear whether these cytokines act directly on the CD8+ T cells, or indirectly by enhancing Ag presentation or costimulation by APC.

MHC protein/peptide Ag complexes immobilized on inert microspheres can be used to study T cell activation requirements in the absence of APC 20 , thus making it possible to determine whether inflammatory cytokines have a direct effect on the T cells. Using this approach with highly purified naive T cells from TCR transgenic mice and signal 2 provided in the form of either exogenous IL-2 or coimmobilized B7.1 protein 21 , we found that IL-1 significantly enhanced the response of CD4+ T cells, whereas IL-12 was required for a response by CD8+ T cells. The latter finding predicted that IL-12 may act as an effective adjuvant for in vivo induction of a CD8+ T cell response, and this prediction was confirmed. Many aspects of T cell activation can be accounted for by a two-signal model 22 with signal 1 provided by the TCR and signal 2 by costimulation leading to IL-2 production 23, 24, 25 . The results described in this report suggest that a three signal model may more adequately describe T cell activation requirements, with the third signal being a “danger signal” provided by an inflammatory cytokine, IL-1 in the case of CD4+ T cells and IL-12 in the case of CD8+ T cells, that acts directly on the T cell.

Materials and Methods

T cell purification

Lymph node (LN)3 cells from OT-I mice 26 were harvested and passed over Cellect-plus CD8 enrichment columns (Biotex Laboratories, Edmonton, Alberta, Canada). The cells were then stained with anti-CD44-FITC and anti-CD8-phycoerythrin mAbs and sorted using a FACSvantage flow cytometer (Becton Dickinson, Mansfield, MA) to obtain a population of naive CD8+ CD44low cells (>98% CD44low). LN and spleen cells were harvested from DO11.10 mice 27 , depleted of adherent cells by incubation on plastic petri dishes for 1 h, and passed over Cellect-plus CD4 enrichment columns. The cells were then stained with anti-CD45RB-FITC mAb and sorted to obtain naive CD4+CD45RBhigh cells (>98% CD45RBhigh).

Proliferation and cytotoxicity assays

A total of 5 × 104 purified T cells and 105 Ag-coated latex microspheres were placed in flat-bottom microtiter wells in 200 μl of RPMI 1640 medium supplemented with 10% FCS, 4 mM l-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin and streptomycin, 10 mM HEPES, and 5 μM 2-ME. Where indicated, cultures were supplemented with human rIL-2 (R&D Systems, Minneapolis, MN), mouse rIL-12 (Genetics Institute, Cambridge, MA), or mouse rIL-1β (R&D Systems). Proliferation was measured after 3 days for CD8+ T cells and after 4 days for CD4+ T cells by addition of 1 μCi [3H]TdR per well for the last 8 h of culture. In some experiments, neutralizing antiserum against IL-12 (sheep anti-mouse IL-12; Genetics Institute) or mAb to IL-1R (PharMingen, San Diego, CA) were used. Triplicate determinations were done, and SDs are shown. Cytolytic activity was determined in a standard 4-h 51Cr release assay using E.G7 cells (EL4 cells transfected with OVA) as targets, with EL4 cells included as a control for specificity.

Ags on microspheres

H-2Kb was purified, incorporated onto 5-micron-diameter latex microspheres and pulsed with OVA257–264 as previously described 20 using 3.5 μg H-2Kb per 107 beads for immobilization. Peptide pulsing was done using 2 μM peptide, unless otherwise indicated. IAd/OVA327–339 fusion protein 28 was obtained from L cells transfected with a full-length construct that included the cyoplasmic and transmembrane regions of the class II protein. These cells were a kind gift from Dr. Andrea J. Sant (University of Chicago, Chicago, IL). Cells were solubilized using Triton X-100, and the IAd/OVA327–339 protein was purified by affinity chromatography using M5/114 mAb specific for IAβd. The purified protein was incorporated onto latex microspheres exactly as for H-2Kb 20 , and the extent of immobilization determined by flow cytometry using the M5/114 mAb. Native B7.1 protein was purified by affinity chromatography and immobilized on microspheres as previously described 21, 29 . For all microsphere preparations, the surface density of immobilized B7.1 was determined by flow cytometry to insure that it was in the range previously shown to provide effective costimulation to T cells 21 .

Adoptive transfer and immunization

LN cells were harvested from 2C TCR transgenic mice 30 , labeled with the fluorescent dye PKH26-GL (Sigma, St. Louis, MO) according to the supplier’s protocol and adoptively transferred into C57BL/6 mice (Charles River Laboratory, Wilmington, MA) by suspending 3–4 × 106 CD8+ cells in 0.5 ml PBS and injecting into the tail vein. After transfer, mice were rested for 1 day and then immunized (day 0) by s.c. injection of a total of 0.3 ml Ag in two sites on the back. Ag was SIYRYYGL peptide 31 in either PBS or emulsified in CFA. Cytokines were administered i.p. in 0.1 ml doses on days 0, 1, and 2. IL-12 was administered at 1 μg/day in PBS with 1% mouse serum, and IL-1β was administered at 0.5 μg/day in PBS with 0.1% BSA. On day 3, mice were sacrificed, and cells from draining LN, spleen and peripheral blood were analyzed by flow cytometry to determine the total number of 2C cells and their PKH26-GL fluorescence. 2C cells were identified by staining with 1B2 mAb specific for the TCR (Ref. 32; a gift from Dr. H. Eisen) and anti-CD8 mAb. These methods for identification, enumeration, and phenotypic characterization of adoptively transferred 2C cells, have been described in detail 33 .

Results

IL-12 is needed along with IL-2 and Ag for in vitro stimulation of naive CD8+ T cells

Naive (CD44low) CD8+ T cells were purified by FACS from OT-I mice having a transgenic TCR specific for OVA257–264 peptide and H-2Kb 26 to yield a population having >99% CD8+CD44low T cells (Fig. 1⇓) that are >99% positive for the Vα2 transgenic TCR. The purified cells are Ly-6C−, CD25−, and CD69− and have the forward scatter profile of small resting lymphocytes, consistent with being naive cells (data not shown). Artificial APC were prepared by immobilizing H-2Kb on latex microspheres and pulsing with OVA257–264 to form Ag complexes. Beads prepared in this way stimulate strong Ag-specific proliferation of CD44high memory cells from OT-I mice, provided that exogenous IL-2 is added 20 . In contrast, naive CD44low cells respond only marginally to beads and IL-2 (Ref. 20 and Fig. 2⇓A). However, Ag and IL-2 stimulate vigorous proliferation of naive cells when IL-12 is also added (Fig. 2⇓A). The response depends on all three stimuli (Fig. 2⇓A), and addition of anti-IL-2 mAb to the cultures blocks the response in the presence of IL-12 (data not shown).

FIGURE 1.
FIGURE 1.

Naive CD8+CD44low OT-1 T cell purification by FACS. LN cells from OT-I mice (26) were purified using Cellect-plus CD8 enrichment columns and then stained with anti-CD44-FITC and anti-CD8-phycoerythrin mAbs. The starting population included both CD44low and CD44high expressing cells (···). Cells were separated using a FACSvantage flow cytometer to obtain a population of naive CD8+CD44low cells (——) and a population of CD8+CD44high cells (---).

FIGURE 2.
FIGURE 2.

IL-12 is required to support a maximal proliferative response of naive CD8+ T cells to Ag and IL-2. Purified CD8+CD44low OT-1 T cells (see Fig. 1⇑) were stimulated in microtiter wells with the indicated additions. Purified H-2Kb and Db proteins were immobilized on microspheres and pulsed with 2 μM OVA257–264 peptide. IL-2 was added at 2.5 U/ml and IL-12 at 2 U/ml. A, Proliferation was determined by measuring [3H]TdR incorporation on day 3. (B) Proliferation was determined by measuring [3H]TdR incorporation at the indicated times. B6 spleen stimulator cells were irradiated spleen cells from normal C57BL/6 mice that were pulsed with OVA peptide (B6 spleen + OVA).

Proliferation in response to Ag, IL-2, and IL-12 had essentially the same time course as proliferation in response to C57BL/6 spleen stimulator cells that had been pulsed with OVA257–264 peptide (Fig. 2⇑B). To determine whether the requirement for three stimuli might be resulting from suboptimal amounts of one of the stimuli, the concentration dependence for each was measured. Proliferation as a function of the concentrations of peptide complex (Fig. 3⇓A), IL-12 (Fig. 3⇓B), or IL-2 (Fig. 3⇓C) were determined with the other two stimuli held constant. In all cases, proliferation remained completely dependent on the presence of all three stimuli over the effective concentration ranges of each of the stimuli. In the absence of IL-2, IL-12 alone did not support a response to Ag even at high concentrations (Fig. 3⇓B) and IL-12 did not enhance survival of unstimulated CD8+ T cells (data not shown).

FIGURE 3.
FIGURE 3.

IL-12-dependent enhancement of CD8+CD44low T cell proliferation is concentration-dependent and occurs at all concentrations of peptide Ag and IL-2. Purified CD8+CD44low OT-1 T cells were stimulated in microtiter wells, and proliferation was measured by determining [3H]TdR incorporation on day 3. Ag was H-2Kb immobilized on microspheres and pulsed with varying amounts of OVA257–264 peptide (A) or with 2 μM (B) or 1 μM (C) peptide. IL-2 was added at 2.5 U/ml (A and B) or in varying amounts (C). IL-12 was added at 2 U/ml (A), 3 U/ml (C), or in varying amounts (B).

IL-1, but not IL-12, supports optimal proliferation of naive CD4+ T cells in response to Ag and IL-2

Although IL-12 acts synergistically with Ag and IL-2 in stimulating CD8+ T cells, other inflammatory cytokines including IL-1, IL-6, and TNF-α did not support the response (data not shown). This finding was somewhat surprising because IL-1 can replace adjuvant in supporting Ag-dependent in vivo clonal expansion of CD4+ T cells 3 , whereas IL-12 promotes differentiation to a Th1 phenotype 3, 34 but has little effect on the extent of clonal expansion. These results predicted that CD4+ cells may respond differently than CD8+ cells to inflammatory cytokines in vitro; therefore, experiments were done to compare the two cell types. The response of CD8+ T cells to Ag and IL-2 was not increased by the addition of IL-1β, whereas IL-12 supported vigorous proliferation, and this response was blocked by addition of anti-IL-12 Ab (Fig. 4⇓A). The CD4+ response was examined using naive (CD45Rbhigh) CD4+ T cells from D011.10 TCR transgenic mice 27 and artificial APC made using I-Ad/OVA327–339 fusion protein 28 . Ag alone induced a weak proliferative response that was increased about 2-fold when either IL-2 or IL-1β was added, and addition of both gave the strongest response (Fig. 4⇓B). All three components were required for maximal response (Fig. 4⇓B), and the response was completely blocked by anti-IL-2R mAb (data not shown). In contrast to IL-1, IL-12 had no effect on the CD4+ T cell response to Ag or Ag plus IL-2 (Fig. 4⇓B). Thus, responses of naive CD4+ T cells are enhanced by IL-1 but not IL-12, whereas responses of naive CD8+ T cells require IL-12 but not IL-1.

FIGURE 4.
FIGURE 4.

IL-12 supports proliferation of naive CD8+ T cells, but IL-1 augments proliferation of CD4+ T cells. Naive CD8+ and CD4+ T cells were purified from OT-I and DO11.10 TCR transgenic mice, respectively. Ag were made using H-2Kb immobilized on 5-μm microspheres and pulsed with 2 μM OVA257–264 peptide and I-Ad/OVA323–339 fusion protein on microspheres. In C and D, the Ag-bearing microspheres had purified B7.1 protein coimmobilized on the surfaces. Proliferation was measured by [3H]TdR incorporation after 3 days for CD8+ T cells and 4 days for CD4+ T cells. Cytokines additions were IL-2 at 2.5 U/ml, IL-12 at 2 (C), 2.5 (A) or 3 (B and D) U/ml, and IL-1β at 2.5 (A and C) or 5 (B and D) μg/ml. IL-1 was tested at up to 20 μg/ml on CD8+ T cells and had no effect alone or with Ag and IL-2 (not shown). α12 indicates that neutralizing anti-IL-12 antisera was added to the cultures. α1R indicates that neutralizing anti-IL-1R mAb was added to the cultures.

B7.1 ligand does not eliminate a requirement for inflammatory cytokines for optimal T cell responses

To determine whether the B7.1 costimulatory ligand may alter the requirements for inflammatory cytokines, microspheres having Ag and purified B7.1 coimmobilized on the same surface were prepared and used to stimulate cells. B7.1 was immobilized at a surface density previously shown to yield effective costimulation along with anti-TCR mAb 21 , and this density was confirmed by flow cytometry 21, 29 . The presence of the B7.1 on the same surface as the H-2Kb/OVA257–264 Ag did not eliminate a requirement for IL-12 in stimulating OT-1 cells (Fig. 4⇑C). CD8+ cells make low levels of IL-2, and optimal proliferation required the addition of exogenous IL-2 even when B7.1 was present; biological activity of the B7.1 was confirmed in experiments examining CD4+ cells. When Ag and B7.1 were coimmobilized and used to stimulate DO11.10 cells, B7.1-dependent costimulation caused sufficient IL-2 production to increase the response over that of Ag alone, and IL-1β doubled the response (Fig. 4⇑D). Addition of anti-IL-1R mAb decreased the response to the same level obtained when just Ag/B7.1 beads were used, confirming that the effects are IL-1-dependent. Again, neither IL-12 (Fig. 4⇑D) nor TNF-α (data not shown) enhanced the CD4+ T cell responses. Thus, when signal 2 is provided either in the form of IL-2 or IL-2 and B7.1 ligand, naive CD8+ T cells remain dependent on IL-12 for a strong proliferative response, whereas IL-1β substantially increases the response of CD4+ T cells when either IL-2 or B7.1 ligand are used to provide signal 2.

IL-12 is required for differentiation of naive CD8+ T cells to lytic effector cells

In addition to proliferating in response to Ag-specific stimulation, naive CD8+ T cells develop lytic effector function by day 3. Cytolytic activity of cells cultured with Ag alone or Ag and IL-12 could not be determined because too few viable cells remained to be assayed by day 3. A low level of proliferation of OT-1 cells does occur in response to just Ag and IL-2 ( Figs. 2–4⇑⇑⇑), and sufficient viable cells could be recovered from these cultures on day 3 to determine whether they had acquired lytic activity. Cells stimulated with Ag and IL-2, or with just IL-2 and IL-12 in the absence of Ag, did not develop detectable cytolytic activity (Fig. 5⇓). In contrast, cells stimulated with Ag, IL-2, and IL-12 developed potent Ag-specific cytolytic activity. Thus, in addition to being required to support optimal proliferation, both IL-2 and IL-12 are required along with Ag to support acquisition of lytic effector function by naive cells.

FIGURE 5.
FIGURE 5.

IL-12 is required for development of lytic effector function of CD8+CD44low T cells. Purified naive CD8+CD44low T cells from OT-I mice were placed in culture with Ag and 3 U/ml IL-2. Ag was purified H-2Kb immobilized on 5-micron microspheres and pulsed with 2 μM OVA257–264 peptide. Cytolytic activity was measured on day 3 using EG.7 targets (shown). Release was <7% in all cases for EL-4 target cells examined in parallel. The experiment shown is representative of six of six experiments showing that cytolytic activity is obtained in response to Ag and IL-2 plus IL-12, but not in response to Ag and IL-2 alone (or IL-12 alone).

IL-12 supports in vivo clonal expansion of CD8+ T cells

The in vitro results described above predicted that IL-12 may replace adjuvant in stimulating an in vivo response of CD8+ cells to peptide. This finding was examined by adoptively transferring CD8+ T cells from 2C TCR transgenic mice 30 into C57BL/6 recipients, challenging with SIYRYYGL peptide recognized by the 2C receptor on H-2Kb 31 and assessing the in vivo response by flow cytometry using 1B2 anti-clonotype mAb and anti-CD8 mAb to identify 2C cells 33 . A s.c. injection of just peptide caused minimal clonal expansion in draining lymph nodes or spleen, whereas peptide in CFA caused a large increase in the number of 2C cells in the draining nodes (Fig. 6⇓). IL-12 along with peptide also caused a large increase in 2C cell number, and the increase was seen in both the draining lymph nodes and spleen. It appears likely that the Ag depot effect of the mineral oil in CFA results in the response being limited to the draining lymph nodes, whereas free peptide readily reaches other sites, including the spleen in which clonal expansion can occur if IL-12 is present.

FIGURE 6.
FIGURE 6.

IL-12 replaces adjuvant in stimulating in vivo clonal expansion of CD8+ 2C T cells in response to peptide Ag. Cells from 2C TCR transgenic mice were labeled with PKH26 (see Fig. 7⇓) and adoptively transferred into normal C57BL/6 recipients. SIYRYYGL peptide (50 μg) was injected s.c. in PBS alone (Peptide) or along with IL-12 (Peptide + IL12), IL-1β (Peptide + IL1) or CFA (Peptide + CFA). Controls included mice that received no challenge (Transfer Only) and mice that received just IL-12 (IL-12 only) or just CFA. Cytokines were delivered by i.p. injection on days 0, 1, and 2. On day 3, mice were sacrificed and the numbers of 2C cells in the draining lymph nodes, spleen, and peripheral blood were determined by flow cytometry using 1B2 anti-clonotypic mAb and anti-CD8 mAb to identify the cells. Groups included two mice each, and error bars indicate ranges. Comparable responses to peptide with CFA or IL-12 were obtained in six of six experiments, and nonresponsiveness to peptide and IL-1 in two of two experiments. A, Number of 2C cells in draining lymph nodes on day 3. 2C cells accounted for 0.16% of the total lymphocytes in transfer only LN, and 2.1% in IL-12 + peptide LN. B, Number of 2C cells in the spleens on day 3.

In six experiments, the number of 2C cells in draining lymph nodes on day 3 in mice that received just peptide was increased 2.9 ± 1-fold (average ± SD) over the number present in transfer only mice. In contrast, mice that received peptide and IL-12 had a 42 ± 14-fold increase over the number present in transfer only mice (p < 0.05 for peptide alone vs peptide + IL-12 by Student’s t test). A similar 42 ± 21-fold increase over transfer only occurred when mice received peptide in CFA (p < 0.05 for peptide alone vs peptide + CFA), and there was no significant difference between peptide plus IL-12 vs peptide in CFA (p > 0.9). Consistent with the in vitro results (Fig. 4⇑), IL-1β did not substantially increase the numbers of 2C cells in the draining nodes or spleen in comparison to the numbers present in response to just peptide (Fig. 6⇑). In two independent experiments, peptide and IL-1 caused 6.2- and 7.8-fold increases in the numbers of 2C cells in comparison with transfer only mice, whereas in these same experiments, peptide and IL-12 caused 26- and 40-fold increases. This result is in marked contrast to CD4+ T cells responses, in which IL-1 supports clonal expansion comparable with that obtained using CFA, whereas IL-12 does not 3 .

To ensure that changes in the numbers of 2C cells reflected proliferation, rather than increased migration into the draining nodes or spleen, cells were labeled with PKH26 fluorescent dye before transfer, and PKH26 fluorescence levels were determined on day 3. PKH26 incorporates into the cell membrane, and the fluorescence of the cell decreases as the dye is diluted upon cell division. Peptide alone stimulated some cell division, and administration of peptide with either CFA or IL-12 resulted in more extensive cell division by day 3 (Fig. 7⇓). Less than 10% of 2C cells had undergone more than four divisions in transfer only mice, <30% in peptide only mice, and >65% in peptide + IL-12 or peptide + CFA mice. Although the differences between peptide alone vs peptide along with IL-12 or CFA do not appear great when these profiles are examined, it must be kept in mind that relatively small differences in the number of divisions can result in large differences in the extent of clonal expansion. Furthermore, the extent of clonal expansion will be also be influenced by the rate and extent of cell death. Thus, the results obtained with PKH26 labeling do not provide a useful measure of the relative efficiencies of the different immunizations in expanding the 2C cell population. However, the results do support the conclusion that the majority of 2C cells proliferate in response to Ag, even when this is peptide alone that does not cause large clonal expansion (Fig. 6⇑). Thus, the increased numbers of 2C cells in the draining LN and spleen are not simply due to migration.

FIGURE 7.
FIGURE 7.

Peptide Ag induces in vivo proliferation that is increased by IL-12 administration. Cells recovered on day 3 from mice in the experiment shown in Fig. 6⇑ were examined by flow cytometry to determine the level of PKH26 fluorescence of 1B2+ CD8+ cells. Dotted lines show PKH fluorescence before transfer. Based on dilution of dye, the fraction of cells that have undergone varying extents of division in response to the different stimuli can be determined. For 0 divisions, 2–4 divisions, and ≥5 divisions these are: transfer only 88%, 2%, and 9%; peptide only 1%, 72%, and 28%; peptide + IL-12 1%, 32%, and 67%; peptide + CFA 6%, 12%, and 82%. 2C cells in mice stimulated with peptide only do not exhibit further dilution of dye beyond day 3 (data not shown). Essentially identical results were obtained in two independent experiments.

Discussion

The ability of inflammatory cytokines to enhance T cell responses is becoming increasingly appreciated and is usually interpreted in terms of the ability of these cytokines to increase expression of costimulatory molecules on APC, thus enhancing the level of signal 2 available to the T cells 10, 11, 12, 13 . The results described in this study strongly suggest that instead, or in addition, the inflammatory cytokines can provide an essential third signal that acts directly on the T cell and is necessary to fully activate naive T cells. By using highly purified T cells from TCR transgenic mice and artificial APC in the form of Ag immobilized on microspheres, the potential effects of inflammatory cytokines on APC were eliminated, allowing their direct effects on T cells to be examined. Both CD4 and CD8 T cell responses in vitro were enhanced by inflammatory cytokines when artificial APC were used to present Ag and when signal 2 was provided in the form of either exogenously added IL-2 or coimmobilized B7.1. However, the effective cytokine and the extent to which it is required differ for the two T cell subsets. IL-12 is required along with Ag and IL-2 to stimulate a significant CD8 response ( Figs. 2–5⇑⇑⇑⇑), whereas IL-1 augments a CD4 response to Ag and IL-2 (Fig. 4⇑).

We recently showed that IL-2 and H-2Kb immobilized on microspheres and pulsed with OVA257–264 peptide were sufficient to stimulate proliferation and development of effector function by memory (CD44high) CD8+ T cells from OT-1 mice 20 . In contrast, naive (CD44low) OT-1 cells showed only marginal proliferation and no development of lytic function in response to these stimuli despite the fact that the naive and memory cells expressed the same TCR and had almost identical surface densities of TCR and CD8. Those observations suggested that the signals needed to activate naive and memory CD8+ T cells differed either quantitatively or qualitatively.

It is clear from the present experiments that IL-12 can supply the missing signal to the naive cells, and that this is a direct effect of IL-12 on the T cells ( Figs. 2–5⇑⇑⇑⇑). IL-12 has previously been shown to enhance CD8+ T cell responses 16, 17, 18 , as have numerous other cytokines including IL-1 14 , IL-6 14, 15 , IFN-γ 19 , and others. However, it has been unclear whether these reflect direct effects on the CD8+ T cell or effects on APC that enhance delivery of signal 2 to the T cells. Although IL-12 can supply the missing signal to naive CD8+ T cells in the absence of APC, this was not the case for IL-1 (Fig. 4⇑), TNF-α, or IL-6 (data not shown). It also appears that IL-12 is not acting through its ability to stimulate T cell production of IFN-γ 35 , because the addition of anti-IFN-γ Ab did not block the response to Ag, IL-2, and IL-12 (data not shown). Although only IL-12, of the cytokines tested, was able to support the CD8 T cell response, it is possible that there is some redundancy with other cytokines that are also able to support the response. Redundancy is seen with respect to signal 2 delivery; CD28/B7 seems to be the major pathway but a number of other receptor/ligand pairs have been implicated as providing costimulatory signals. It may also be the case that signal 3 is not always required. Signal 1, or signals 1 and 2, may be sufficient for activation at very high Ag levels or very high TCR affinity for Ag 37, 38 ; quantitative differences in signal 1 may determine whether one, two, or three signals are necessary for full activation. That IL-12 is not always required for CTL responses is suggested by the fact that cells from mice deficient in the IL-12 p40 gene and thus unable to make active IL-12 are still able to generate an allogeneic CTL response 36 . Similarly, we have found that neutralizing anti-IL-12 mAb is unable to block proliferation of CD8+ T cells when peptide pulsed spleen cells are used as APC (data not shown). Experiments are in progress to determine whether quantitative Ag effects, alternative cytokines, or both are involved in overcoming a requirement for IL-12 under some circumstances.

These in vitro results predicted that IL-12 may serve as an effective adjuvant in vivo and were confirmed using an adoptive transfer system to examine clonal expansion of TCR transgenic CD8+ T cells from 2C mice (Figs. 6⇑ and 7⇑). A striking parallel was found between the in vitro and in vivo responses. In the absence of IL-12, a low level of proliferation occurred in vitro in response to Ag and IL-2 (Figs. 2⇑ and 3⇑). In vivo, clonal expansion was minimal in response to just peptide, but the 2C cells had clearly recognized and responded to Ag, as evidenced by that fact that all of the cells recovered from the draining LN had undergone some proliferation (Fig. 7⇑) and a substantial fraction (40–50%) had acquired a CD44high phenotype (data not shown). Addition of IL-12 in vitro resulted in a strong proliferative response, and in vivo administration along with peptide resulted in extensive clonal expansion, as good or better than that obtained using peptide with CFA (Fig. 6⇑). This finding is consistent with previous reports that IL-12 administration can enhance in vivo CTL responses 18, 39 and suggests that IL-12 will provide a means for effectively inducing CD8+ responses when peptide Ags are used for vaccination or therapy.

The in vitro effects of inflammatory cytokines on the CD4+ cells described here also agreed well with in vivo effects that have been previously described 3, 34 . Substantial proliferation occurs in vitro in response to Ag and signal 2 in the form of either coimmobilized B7.1 or exogenous IL-2 and nearly doubles when IL-1 is added (Fig. 4⇑). Similarly, in vivo administration of Ag alone results in substantial B7-dependent clonal expansion 40 , and coadministration of IL-1 approximately doubles the expansion 3 . TNF-α also supports in vivo Ag-specific clonal expansion. However, it does not enhance the in vitro response to Ag and signal 2 (data not shown). It probably acts in vivo by inducing production of IL-1 by APC 4 and is thus inactive in vitro when APC are not present. In contrast to its effects on CD8+ T cells, IL-12 does not significantly enhance either in vitro proliferation or in vivo clonal expansion of CD4+ T cells. However, it does act as an inducer of Th1 differentiation of CD4+ cells 3, 34, 41 .

In vivo administration of just Ag results in significant clonal expansion of CD4+ T cells; the Ag-specific cells are present in expanded numbers but have been rendered tolerant. Coadministration of adjuvants prevents tolerance induction, as does coadministration of IL-1 3, 34 . Tolerance of CD8+ T cells can also result when peptide Ag is administered in the absence of adjuvant 42 ; proliferation is very limited under these conditions (Figs. 6⇑ and 7⇑) and preliminary results suggest that this may lead to clonal deletion (data not shown). Coadministration of IL-12 can reverse this by supporting strong clonal expansion in response to the Ag (Fig. 6⇑). Thus, for both CD4+ and CD8+ T cells, inflammatory cytokines produced by APC in response to pathogens can provide a third signal needed to support optimal proliferation and avoid tolerizing the cells in response to Ag and signal 2. These cytokines may well be the predominant signalers of “danger” that the innate immune system provides to the adaptive immune response 1, 2 . IL-1 will support CD4+ T cell expansion and promote Th2 help for a humoral response. IL-12 will favor a cell-mediated response by promoting Th1 differentiation of CD4 cells 3, 34, 41 and signaling for expansion (Fig. 6⇑) and differentiation (Fig. 5⇑) of CD8+ T cells. Differential effects of pathogens on the production of these two cytokines may have profound effects on the nature of the immune response that can be generated.

Acknowledgments

We thank K. Hogquist, S. Jameson, and D. Mueller for critical reading of the manuscript.

Footnotes

  • ↵1 This work was supported by National Institutes of Health Grants AI 27998 (to M.K.J.), RO1 AI34824 (to M.F.M.), and PO1 AI 35296 (to M.K.J. and M.F.M.). C.S.S. and R.M.K. were supported by National Institutes of Health Training Grants.

  • ↵2 Address correspondence and reprint requests to Dr. Matthew F. Mescher, Center for Immunology, Box 334 Mayo, 420 Delaware St. S.E., Minneapolis, MN 55455. E-mail address: mesch001{at}maroon.tc.umn.edu

  • ↵3 Abbreviation used in this paper: LN, lymph node.

  • Received August 13, 1998.
  • Accepted December 14, 1998.

References

  1. ↵
    Matzinger, P.. 1994. Tolerance, danger, and the extended family. Annu. Rev. Immunol. 12: 991
    OpenUrlCrossRefPubMed
  2. ↵
    Janeway, C. A., C. C. Goodnow, R. Medzhitov. 1996. Immunological tolerance: danger-pathogen on the premises!. Curr. Biol. 6: 519
    OpenUrlCrossRefPubMed
  3. ↵
    Pape, K. A., A. Khoruts, A. Mondino, M. K. Jenkins. 1997. Inflammatory cytokines enhance the in vivo clonal expansion and differentiation of antigen-activated CD4+ T cells. J. Immunol. 159: 591
    OpenUrlAbstract
  4. ↵
    Brouckaert, P., C. Libert, B. Everaerdt, N. Takahashi, A. Cauwels, W. Fiers. 1993. Tumor necrosis factor, its receptors and the connection with interleukin 1 and interleukin 6. Immunobiology 187: 317
    OpenUrlCrossRefPubMed
  5. ↵
    Kurt-Jones, E. A., S. Hamberg, J. Ohara, W. E. Paul, A. K. Abbas. 1987. Heterogeneity of helper/inducer T lymphocytes: lymphokine production and lymphokine response. J. Exp. Med. 166: 1774
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Dinarello, C. A.. 1988. Biology of interleukin 1. FASEB J. 2: 108
    OpenUrlAbstract
  7. ↵
    Weaver, C. T., C. M. Hawrylowicz, E. R. Unanue. 1988. T helper subsets require the expression of distinct costimulatory signals by antigen-presenting cells. Proc. Natl. Acad. Sci. USA 85: 8181
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Rojo, J. M., J. D. Kerner, C. A. J. Janeway. 1989. Selective induction of growth factor production and growth factor receptor expression by different signals to a single T cell. Eur. J. Immunol. 19: 2061
    OpenUrlCrossRefPubMed
  9. ↵
    Liu, Y., C. A. Janeway. 1991. Microbial induction of co-stimulatory activity for CD4 T-cell growth. Int. Immunol. 3: 323
    OpenUrlAbstract/FREE Full Text
  10. ↵
    McLellan, A. D., R. V. Sorg, L. A. Williams, D. N. J. Hart. 1996. Human dendritic cells activate T lymphocytes via a CD40:CD40 ligand-dependent pathway. Eur. J. Immunol. 26: 1204
    OpenUrlCrossRefPubMed
  11. ↵
    Grewal, I. S., J. Xu, R. A. Flavell. 1995. Impairment of antigen-specific T-cell priming in mice lacking cD40 ligand. Nature 378: 617
    OpenUrlCrossRefPubMed
  12. ↵
    Grewal, I. S., H. G. Foellmer, K. D. Grewal, J. Xu, F. Hardardottir, J. L. Baron, C. A. Janeway, R. A. Flavell. 1996. Requirement for CD40 ligand in costimulation induction, T cell activation, and experimental allergic encephalomyelitis. Science 273: 1864
    OpenUrlAbstract/FREE Full Text
  13. ↵
    Yang, Y., J. M. Wilson. 1996. CD40 ligand-dependent T cell activation: requirement of B7-CD28 signaling through CD40. Science 273: 1862
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Renauld, J., A. Vink, J. Van Snick. 1989. Accessory signals in murine cytolytic T cell responses: dual requirement for IL-1 and IL-6. J. Immunol. 143: 1894
    OpenUrlAbstract
  15. ↵
    Okada, M., M. Kitahara, S. Kishimoto, T. Matsuda, T. Hirano, T. Kishimoto. 1988. IL-6/BSF-2 functions as a killer helper factor in the in vitro induction of cytotoxic T cells. J. Immunol. 141: 1543
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Gately, M. K., A. G. Wolitzky, P. M. Quinn, R. Chizzonite. 1992. Regulation of human cytolytic lymphocyte responses by IL-12. Cell. Immunol. 143: 127
    OpenUrlCrossRefPubMed
  17. ↵
    Mehrotra, P., D. Wu, J. Crim, H. Mostowski, J. Siegel. 1993. Effects of IL-12 on the generation of cytotoxic activity in human CD8+ T lymphocytes. J. Immunol. 151: 2444
    OpenUrlAbstract
  18. ↵
    Gately, M. K., R. R. Warrier, S. Honasoge, D. M. Carvajal, D. A. Faherty, S. E. Connaughton, T. D. Anderson, U. Sarmiento, B. R. Hubbard, M. Murphy. 1994. Administration of recombinant IL-12 to normal mice enhances cytolytic T lymphocyte activity and induces production of IFN-γ in vivo. Int. Immunol. 6: 157
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Simon, M., S. Landolfo, T. Diamantstein, U. Hochgeschwender. 1986. Antigen and lectin sensitized cytolytic T lymphocyte precursors require both interleukin 2 and endogenously produced immune γ interferon for their growth and differentiation into effector cells. Curr. Top. Microbiol. Immunol. 126: 173
    OpenUrlPubMed
  20. ↵
    Curtsinger, J. M., D. C. Lins, M. F. Mescher. 1998. CD8+ memory T cells (CD44high, Ly6C+) are more sensitive than naive cells (CD44low, Ly6C−) to TCR/CD8 signaling in response to antigen. J. Immunol. 160: 3236
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Deeths, M. J., M. F. Mescher. 1997. B7-1-dependent costimulation results in qualitatively and quantitatively different responses in CD4+ and CD8+ T cells. Eur. J. Immunol. 27: 598
    OpenUrlCrossRefPubMed
  22. ↵
    Lafferty, K. J., S. J. Prowse, C. J. Simeonovic, H. S. Warren. 1983. Immunobiology of tissue transplantation: a return to the passenger leukocyte concept. Annu. Rev. Immunol. 1: 143
    OpenUrlCrossRefPubMed
  23. ↵
    Jenkins, M. K., J. G. Johnson. 1993. Molecules involved in T-cell costimulation. Curr. Opin. Immunol. 5: 361
    OpenUrlCrossRefPubMed
  24. ↵
    Janeway, C. A., K. Bottomly. 1994. Signals and signs for lymphocyte responses. Cell 76: 275
    OpenUrlCrossRefPubMed
  25. ↵
    Lenschow, D. J., T. L. Walunas, J. A. Bluestone. 1996. CD28/B7 system of T cell costimulation. Annu. Rev. Immunol 14: 233
    OpenUrlCrossRefPubMed
  26. ↵
    Hogquist, K., S. Jameson, W. Heath, J. Howard, M. Bevan, F. Carbone. 1994. T cell receptor antagonist peptides induce positive selection. Cell 76: 17
    OpenUrlCrossRefPubMed
  27. ↵
    Murphy, K., A. Heimberger, D. Loh. 1990. Induction by antigen of intrathymic apoptosis of CD4+ CD8+ TCRlo thymocytes in vivo. Science 250: 1720
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Kozono, H., J. White, J. Clements, P. Marrack, J. Kappler. 1994. Production of soluble MHC class II proteins with covalently bound single peptides. Nature 369: 151
    OpenUrlCrossRefPubMed
  29. ↵
    Curtsinger, J., M. J. Deeths, P. Pease, M. F. Mescher. 1997. Artificial cell surface constructs for studying receptor-ligand contributions to lymphocyte activation. J. Immunol. Methods 209: 47
    OpenUrlCrossRefPubMed
  30. ↵
    Sha, W., C. Nelson, R. Newberry, D. Kranz, J. Russell, D. Loh. 1988. Selective expression of an antigen receptor on CD8-bearing T lymphocytes in transgenic mice. Nature 335: 271
    OpenUrlCrossRefPubMed
  31. ↵
    Udaka, K., K.-H. Wiesmuller, S. Kienle, G. Jung, P. Walden. 1996. Self-MHC-restricted peptides recognized by an alloreactive T lymphocyte clone. J. Immunol. 157: 670
    OpenUrlAbstract
  32. Kranz, D. M., D. H. Sherman, M. V. Sitkovsky, M. S. Pasternack, H. N. Eisen. 1984. Immunoprecipitation of cell surface structures of cloned cytotoxic T lymphocytes by clone specific antisera. Proc. Natl. Acad. Sci. USA 81: 573
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Kedl, R. M., M. F. Mescher. 1997. Migration and activation of antigen-specific CD8+ T cells upon in vivo stimulation with allogeneic tumor. J. Immunol. 159: 650
    OpenUrlAbstract
  34. ↵
    Van Parijs, L., V. L. Perez, A. Biuckians, R. G. Maki, C. A. London, A. K. Abbas. 1997. Role of interleukin 12 and costimulators in T cell anergy in vivo. J. Exp. Med. 186: 1119
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Trinchieri, G.. 1995. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity. Annu. Rev. Immunol. 13: 251
    OpenUrlCrossRefPubMed
  36. ↵
    Magram, J., S. E. Connaughton, R. R. Warrier, D. M. Carvajal, C.-Y. Wu, J. Ferrante, C. Steward, U. Sarmiento, D. A. Faherty, M. A. Gately. 1996. IL-12-deficient mice are defective in IFN-γ production and type 1 cytokine responses. Immunity 4: 471
    OpenUrlCrossRefPubMed
  37. ↵
    Cai, Z., A. Brunmark, M. R. Jackson, D. Loh, P. A. Peterson, J. A. Sprent. 1996. Transfected Drosophila cells as a probe for defining the minimal requirements for stimulating unprimed CD8+ T cells. Proc. Natl. Acad. Sci. USA 93: 14736
    OpenUrlAbstract/FREE Full Text
  38. ↵
    Goldstein, J., H. Mostowsky, J. Tung, H. Hon, M. Brunswick, S. Kozlowski. 1997. Naive alloreactive CD8 T cells are activated by purified major histocompatibility complex class I and antigenic peptide. Eur. J. Immunol. 27: 871
    OpenUrlCrossRefPubMed
  39. ↵
    Noguchi, Y., E. C. Richards, Y.-T. Chen, L. J. Old. 1995. Influence of IL-12 on p53 peptide vaccination against established Meth A sarcoma. Proc. Natl. Acad. Sci. USA 92: 2219
    OpenUrlAbstract/FREE Full Text
  40. ↵
    Khoruts, A., A. Mondino, K. A. Pape, S. L. Reiner, M. Jenkins. 1998. A natural immunological adjuvant enhances T cell clonal expansion through a CD28-dependent, IL-2-independent mechanism. J. Exp. Med. 187: 225
    OpenUrlAbstract/FREE Full Text
  41. ↵
    Abbas, A. K., K. M. Murphy, A. Sher. 1996. Functional diversity of helper T lymphocytes. Nature 383: 787
    OpenUrlCrossRefPubMed
  42. ↵
    Kyburz, D., P. Aichele, D. Speiser, H. Hengartner, R. Zinkernagel, H. Pircher. 1993. T cell immunity after a viral infection versus T cell tolerance induced by soluble viral peptides. Eur. J. Immunol. 23: 1956
    OpenUrlCrossRefPubMed
Back to top

In this issue

The Journal of Immunology: 162 (6)
The Journal of Immunology
Vol. 162, Issue 6
15 Mar 1999
Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section