CD4+ and CD8+ T Cell Priming for Contact Hypersensitivity Occurs Independently of CD40-CD154 Interactions

Anton V. Gorbachev, Peter S. Heeger and Robert L. Fairchild
J Immunol February 15, 2001, 166 (4) 2323-2332; DOI: https://doi.org/10.4049/jimmunol.166.4.2323

Abstract

The primary effector cells of contact hypersensitivity (CHS) responses to dintrofluorobenzene (DNFB) are IFN-γ-producing CD8+ T cells, whereas CD4+ T cells regulate the magnitude and duration of the response. The requirement for CD40-CD154 engagement during CD8+ and CD4+ T cell priming by hapten-presenting Langerhans cells (hpLC) is undefined and was tested in the current study. Similar CHS responses to DNFB were elicited in wild-type and CD154−/− animals. DNFB sensitization of CD154−/− mice primed IFN-γ-producing CD8+ T cells and IL-4-producing CD4+ T cells. However, anti-CD154 mAb MR1 given during hapten sensitization inhibited hapten-specific CD8+, but not CD4+, T cell development and the CHS response to challenge. F(ab′)2 of MR1 failed to inhibit CD8+ T cell development and the CHS response suggesting that the mechanism of inhibition is distinct from that of CD40-CD154 blockade. Furthermore, anti-CD154 mAb did not inhibit CD8+ T cell development and CHS responses in mice depleted of CD4+ T cells or in CD4−/− mice. During in vitro proliferation assays, hpLC from mice treated with anti-CD154 mAb during DNFB sensitization were less stimulatory for hapten-primed T cells than hpLC from either control mice or mice depleted of CD4+ T cells before anti-CD154 mAb administration. These results demonstrate that development of IFN-γ-producing CD8+ T cells and the CHS response are not dependent on CD40-CD154 interactions. This study proposes a novel mechanism of anti-CD154 mAb-mediated inhibition of CD8+ T cell development where anti-CD154 mAb acts indirectly through CD4+ T cells to impair the ability of hpLC to prime CD8+ T cells.

Contact hypersensitivity (CHS)3 is a T cell-mediated immune response of the skin in sensitized individuals to subsequent contact with the sensitizing hapten (1, 2). During sensitization for CHS, hapten-presenting Langerhans cells (hpLC) migrate to the skin-draining lymph nodes and prime the hapten-specific T cells, which mediate the response to hapten challenge (3, 4, 5). Results from both clinical studies and experimental models of CHS have supported a role for CD4+ and CD8+ T cells as the effector cells of the response (6, 7, 8, 9). Studies using either Ab-mediated depletion of CD4+ vs CD8+ T cells or mice with targeted deletions of class II MHC genes have implicated CD8+ T cells as the primary effector cells in CHS responses to the model haptens dinitrofluorobenzene (DNFB) and oxazolone (9, 10, 11). These results are supported by studies from this laboratory demonstrating that most of the IFN-γ-producing cells induced by epicutaneous sensitization with DNFB or oxazolone are hapten-specific CD8+ T cells (12, 13). In contrast, most of the hapten-specific CD4+ T cells develop to type 2 cytokine (e.g., IL-4, IL-5, and IL-10)-producing cells. CD4+ T cells also regulate the magnitude and duration of the CHS response, although the mechanism of this regulation remains undefined. In addition to recognition of specific hapten/MHC complexes, development of the CD8+ effector and the CD4+ regulatory T cell populations requires engagement of B7-2 costimulatory molecules expressed by hpLC (14). The requirement for other molecular interactions between LC and hapten-specific T cells during priming for CHS remains largely unknown.

T cell engagement of the CD40 costimulatory molecule expressed by APCs is required for the development of many T cell responses. The ligand for CD40, CD154, is rapidly expressed on CD4+ T cells during cellular activation (15, 16). The use of CD154−/− mice has indicated that these interactions play a crucial role in the induction of CD4+ T cell-mediated immune responses and CD4+ T cell-dependent humoral responses (17, 18, 19). The requirement for CD40-CD154 interactions during the development of CD8+ T cell-mediated responses is not as clear as for CD4+ T cell-mediated responses. Whereas some studies have indicated the development of CD8+ T cell responses independently of CD40-mediated costimulation (20, 21, 22, 23), other studies have indicated that CD40-CD154 interactions are required for development of certain CD8+ T cell responses (24, 25).

Several recent studies have suggested that CD40-CD154 interactions might be required during the development of T cells mediating CHS responses (26, 27). This requirement has not been tested directly. In the present report we have tested the development of hapten-specific CD4+ and CD8+ T cell populations and the elicitation of CHS responses following DNFB sensitization of CD154−/− mice and in wild-type mice treated with anti-CD154 mAb during sensitization. The results indicate that CD8+ and CD4+ T cell development in CHS is not dependent on CD40-CD154 interactions. However, treatment with anti-CD154 mAb during sensitization inhibits the development of hapten-specific CD8+ T cells and the CHS response indirectly through CD4+ T cell-mediated effects. The results indicate a novel mechanism for the ability of therapeutic strategies using anti-CD154 mAb to inhibit CD8+ T cell responses.

Materials and Methods

Mice

BALB/c and C57BL/6 mice were obtained through Dr. Clarence Reeder (National Cancer Institute, Frederick, MD). CD4−/− and CD154−/− mice on the C57BL/6 genetic background and IL4−/− mice on the BALB/c background were purchased from The Jackson Laboratory (Bar Harbor, ME). Adult female mice, 8–10 wk old, were used throughout these studies.

Abs and cytokines

The following hybridomas were obtained from American Type Culture Collection (Manassas, VA): GK1.5 (anti-mouse CD4), 2.43 (anti-mouse CD8), and 145.2C11 (anti-mouse CD3). mAb from the hybridomas YTS191.1.2 (anti-mouse CD4) (28), KJ23a (anti-Vβ17a) (29), and MR1 (anti-mouse CD154) (30) were also used in these studies. mAb from the culture supernatant of the IgG-producing hybridomas was purified by protein G chromatography. PE-labeled mAbs specific for CD4 and CD8; capture and detection mAbs for IL-4-, IL-10-, and IFN-γ-specific ELISA; and recombinant IFN-γ, IL-4, and IL-10 were purchased from PharMingen (San Diego, CA). Polyclonal hamster IgG was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). F(ab′)2 of MR1 were prepared using the ImmunoPure Fab′ Preparation Kit (Pierce, Rockford, IL). The digestion was performed according to the manufacturer’s instructions, and the purity of F(ab′)2 was evaluated by SDS-PAGE. The activity of F(ab′)2 was tested by the ability to inhibit allo-reactive responses of mouse lymph node cells (LNC) in vitro. Purified MR1 was FITC labeled using the QuickTag FITC conjugation kit according to the manufacturer’s instructions (Roche, Indianapolis, IN).

Hapten sensitization and elicitation of CHS

Mice were sensitized to DNFB by painting the shaved abdomen with 25 μl of 0.25% DNFB (Sigma, St. Louis, MO) and with 5 μl on each footpad on days 0 and +1. On day +5 hapten-sensitized and control unsensitized mice were challenged with 10 μl of 0.2% DNFB on both sides of each ear. The increase in ear swelling was measured at 24-h intervals after challenge using an engineer’s micrometer (Mitutoyo, Elk Grove Village, IL) and expressed in units of 10−4 in. as previously described (12). The ear swelling response is presented as the mean increase of each group of four sensitized or nonsensitized mice (i.e., eight ears) ± SEM.

CD4+ and CD8+ T cell depletion

For in vivo depletion of CD4+ T cells mice were injected with 200 μg of each anti-CD4 mAb, YTS 191 and GK1.5, i.p. on 3 consecutive days before hapten sensitization on days 0 and +1 as previously described (11, 12, 28). For in vivo depletion of CD8+ T cells 200 μg of anti-CD8 mAb 2.43 was injected on 3 consecutive days. In each experiment treated sentinel mice were used to evaluate the efficiency of CD4+ or CD8+ T cell depletion by Ab staining and flow cytometric analysis of spleen and LNC and was always >95% compared with cells from control, rat IgG-treated mice. Treated mice were rested 1–3 days before sensitization with DNFB. For in vitro depletion of CD4+ or CD8+ T cells, LNC from hapten-sensitized mice were incubated with specific Ab-coated magnetic beads (Dynabeads, Dynal, Oslo, Norway). The efficiency of this depletion was >95% for the target T cell population.

Isolation of hpLC

The isolation of hpLC was performed using the method described by Bigby and coworkers (31). Mice were painted with 0.25% DNFB as described above, and LNC were prepared 24 after the final painting (i.e., day +2 postsensitization). The cells were washed with RPMI 1640, resuspended at 107 cells/ml in RPMI 1640 supplemented with 5% FCS, layered over 3 ml of 14.5% metrizamide (Sigma) in PBS, and centrifuged at 600 × g for 15 min. The interface cells were collected and washed twice with complete medium. Microscopic and flow cytometry examination of cells from the interface indicated 60–80% surface Ig−/class II MHC+ cells with dendritic cell (DC) morphology.

Cell culture

Cells from skin draining lymph nodes from DNFB-sensitized and, as a negative control, from unsensitized mice were prepared on day +4 and stimulated to produce cytokines by culture on anti-CD3 mAb-coated wells. The 96-well U-bottom tissue culture plates were precoated with 30 μl/well anti-CD3 mAb 145.2C11 (25 μg/ml) for 90 min at 37°C. As a negative control, wells were coated with an Ab to a Vβ not expressed by BALB/c or C57BL/6 T cells, anti-Vβ17a mAb KJ 23a. The wells were washed, and 2 × 105 LNC were delivered to each well in 200 μl of complete RPMI medium and cultured in a 7% CO2 humidified incubator at 37°C. After 48 h the culture supernatants were harvested and assayed for cytokine production by ELISA.

T cell allo-reactivity was assayed by standard MLR. As stimulator cells, BALB/c spleen cells were treated with 50 μg/ml of mitomycin C (Sigma) for 30 min at 37°C and washed twice in RPMI 1640, then 106 cells in 100 μl of complete RPMI 1640 medium were delivered to wells of a 96-well tissue culture plate. Responder LNC from naive C57BL/6 mice were added in 100 μl of complete RPMI 1640 medium at 2 × 105 cells/well (E:T cell ratio, 1:5). BALB/c LNC cultured with the BALB/c stimulator spleen cells were used as a negative control. Control hamster IgG, anti-CD154 mAb MR1, or F(ab′)2 of MR1 was added to cell cultures at concentrations of 0.5, 5, and 50 μg/ml. Cells were cultured for 4 days in complete RPMI 1640 and were pulsed with 1 μCi/well [3H]thymidine. Cells were harvested 24 h later onto fiber filter mats, and 3H incorporation was determined by liquid scintillation counting.

In experiments testing the proliferation of DNFB-immune T cells, cocultures of 2 × 105 CD4+ or CD8+ T cells and 2 × 104 hpLC given 2000 rad of gamma irradiation were established in triplicate in the wells of 96-well U-bottom culture plates. After 48 h cultures were pulsed with 1 μCi/well [3H]thymidine and harvested 18–20 h later, and 3H incorporation was determined by liquid scintillation counting.

Cytokine-specific ELISA

Polyvinylchloride ELISA plates were coated with capture anti-IFN-γ, anti-IL-4, or IL-10 mAb in 0.1 M bicarbonate buffer, pH 8.6, overnight at 4°C and then blocked with 5% FCS/0.5% gelatin in PBS. Duplicate aliquots of all supernatants were tested undiluted and in at least two dilutions. As a positive control, each plate also included recombinant cytokine titrated to obtain a standard curve for quantitation. Following incubation overnight at 4°C, each plate was washed, and the biotin-labeled anti-cytokine mAb was added. The plate was incubated for 2 h at 37°C and washed, and alkaline phosphatase-conjugated streptavidin (Fisher Scientific, Pittsburgh, PA) was added. Following a final incubation for 1 h at 37°C the plate was washed, and the assay was developed by addition of the substrate p-dinitrophenylphosphate (Sigma). Results were read at 405 nm, and mean values were calculated. The concentration of cytokine in each test supernatant was calculated using the standard curve on each plate.

Enzyme-linked immunospot (ELISPOT) assays for enumeration of hapten-specific IFN-γ- and IL-4-producing cells

ELISPOT assays were performed as previously described (32, 33). Briefly, ELISPOT plates (Unifilter 350, Polyfiltronics, Rockland, MA) were coated with 4 μg/ml IFN-γ- or IL-4-specific mAb and incubated overnight at 4°C. The plates were blocked with 1% BSA in PBS and then washed four times with PBS. LNC from unsensitized or DNFB-sensitized mice treated with control IgG or anti-CD154 mAb were prepared on day +5 postsensitization and used as responder cells. Syngeneic spleen cells from naive mice were treated with 50 μg/ml mitomycin C and then labeled with 100 μg/ml DNBS before use as stimulator cells as previously described (33, 34). Responder and stimulator cells were cultured in serum-free HL-1 medium (BioWhittaker, Walkersville, MD) supplemented with 1 mM l-glutamine. Stimulator cells were plated at 5 × 105 cells/well with 2 × 105 responder cells/well. Responder cells plated with unlabeled splenocytes were used as a negative (hapten-specificity) control. After 24 h of cell culture at 37°C in 5% CO2, cells were removed from the plate by extensive washing with PBS/0.05% Tween 20. Biotinylated anti-IFN-γ or anti-IL-4 mAb (4 μg/ml) was added, and the plate was incubated overnight at 4°C. The following day the plate was washed three times with PBS/0.05% Tween 20, and conjugated streptavidin-alkaline phosphatase for IFN-γ or streptavidin-HRP for IL-4 was added to each well. After 2 h at room temperature the plates were washed with PBS/0.05% Tween-20, nitro blue tetrazolium/5-bromo-4-chloro-30-indolyl substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added for detection of IFN-γ, and 3-amino-9-ethylcarbazole (Pierce) was added for detection of IL-4. The resulting spots were counted with an ELISPOT image analyzer (developed at Cellular Technology, Cleveland, OH) that was designed to detect ELISA spots with predetermined criteria for size, shape, and colorimetric density.

Stimulation and flow cytometric analysis of CD154 expression

LNC were prepared from naive mice or from hapten-sensitized mice 24, 48, or 72 h after sensitization and CD4+- and CD8+-enriched cell populations were prepared using Dynabeads. For anti-CD3 mAb-mediated activation, the cells were washed with RPMI 1640, and 2 × 105-cell aliquots were cultured in 96-well plates coated with anti-CD3 mAb 145.2C11 or, as a negative control, anti-Vβ17a mAb KJ23 for 16 h at 37°C. Following the culture, the cells were washed twice with staining buffer (Dulbecco’s PBS with 2% FCS/0.2% NaN3), and 5 × 105-cell aliquots were incubated on ice in 100 μl of rat serum (Rockland, Gilbertsville, PA) diluted 1/1000 in the staining buffer. After 30 min the cells were washed twice and stained with FITC-labeled anti-mouse CD154 mAb and PE-labeled anti-mouse CD4 or CD8 mAb. After 30 min on ice the cells were washed five times, resuspended in staining buffer, and analyzed by two-color flow cytometry using a FACScan and CellQuest software (Becton Dickinson, San Jose, CA). Sample data were collected on 10,000 ungated cells. For analysis of CD154 expression, the entire CD4+ or CD8+ T cell population was gated, and the percentage of CD154-expressing T cells was calculated by dividing the amount of positively stained gated cells by the total number of gated cells.

Results

CHS responses and development of IFN-γ-producing CD8+ and IL-4-producing CD4+ T cells to hapten sensitization in CD154−/− mice

To begin to examine the requirement for CD40-CD154 interactions for the induction and elicitation of CHS responses, wild-type C57BL/6 and CD154−/− mice were sensitized with DNFB, and the ear swelling responses to hapten challenge were compared. The magnitudes of CHS to DNFB in sensitized CD154−/− and wild-type mice were similar (Fig. 1⇓). Ear swelling in unsensitized mice challenged with DNFB was low for both sets of animals, indicating that sensitization was required for the response. Depletion of CD8+ T cells before hapten sensitization abrogated the response to hapten challenge in each group. Depletion of CD4+ T cells before DNFB sensitization resulted in slightly elevated ear swelling responses at 24 h postchallenge in each group. These results indicated that CD8+, and not CD4+, T cells were required for CHS responses to DNFB in wild-type and CD154−/− mice.

FIGURE 1.
FIGURE 1.

CHS responses to DNFB in wild-type and CD154−/− mice. Groups of four C57BL/6 wild-type and CD154−/− mice were treated with 200 μg of rat IgG, anti-CD4 mAb, or anti-CD8 mAb on 3 consecutive days before sensitization with 0.25% DNFB on days 0 and +1. On day +5, sensitized mice and a group of naive mice were ear challenged with 0.2% DNFB, and the thickness of the challenged ears was measured 24 h later. The mean increase in ear thickness following DNFB challenge is shown in units of 10−4 in. ± SEM. The results are representative of three individual experiments.

The induction of cytokine-producing CD4+ and CD8+ T cells following DNFB sensitization of wild-type and CD154−/− mice was compared. CD4+ and CD8+ T cell-enriched populations were prepared from the skin-draining lymph nodes of hapten-sensitized mice, and aliquots were cultured on anti-CD3 or, as a control, anti-Vβ17a mAb-coated culture wells. After 48 h the supernatants were collected and tested for IFN-γ and IL-4 (Fig. 2⇓). During in vitro stimulation with anti-CD3 mAb, immune CD8+ T cells from CD154−/− mice produced readily detectable levels of IFN-γ, although this production was consistently lower (e.g., by ∼30%) compared with production by immune CD8+ T cells from wild-type mice. Although low levels of IFN-γ were produced by immune CD4+ T cells from wild-type mice, this was not observed by CD4+ T cells from CD154−/− mice. Similar levels of IL-4 were produced by anti-CD3 mAb-stimulated CD4+ T cells from sensitized CD154−/− and wild-type mice. As previously reported (12, 13, 14), culture of naive T cells on anti-CD3 mAb coated wells produced low to undetectable levels of IFN-γ and IL-4 (Fig. 2⇓), and culture of immune T cells from C57BL/6 mice on anti-Vβ17a mAb coated wells produced undetectable levels of the cytokines (data not shown). These results indicated that CD40-CD154 interactions were not required for, but may augment, the development of IFN-γ-producing CD8+ and IL-4-producing CD4+ T cells during sensitization for CHS.

FIGURE 2.
FIGURE 2.

Cytokine production by CD4+ and CD8+ T cells from wild-type and CD154−/− mice sensitized with DNFB. Groups of C57BL/6 wild-type mice or CD154−/− mice were sensitized with 0.25% DNFB. On day +4, CD4+-enriched (□) and CD8+-enriched (▪) cell populations were prepared from lymph nodes of the sensitized and naive mice, and 2 × 105-cell aliquots were cultured on anti-CD3 or anti-Vβ17a mAb-coated wells for 48 h. The supernatants were collected and analyzed by ELISA for production of IFN-γ and IL-4. Cell culture on control anti-Vβ17a mAb-coated wells did not stimulate detectable cytokine production by cells from any of the tested groups. Results are representative of two independent experiments. ND, Not detectable.

Anti-CD154 mAb inhibits effector CD8+ T cell development to DNFB sensitization

The results presented above indicated the ability of DNFB sensitization to induce effector CD8+ T cells and CHS responses in CD154−/− mice, but anti-CD154 mAb has been reported to inhibit CHS responses (26). To begin to investigate the effect of anti-CD154 mAb on the induction and elicitation of CHS in our hands, groups of BALB/c mice were injected with different doses of anti-CD154 mAb MR1 or 500 μg of polyclonal hamster IgG at the time of DNFB sensitization, and ear swelling responses were measured 24 h after hapten challenge. Administration of anti-CD154 mAb MR1 during hapten sensitization inhibited CHS responses in a dose-dependent manner. Greater than 70% inhibition of CHS was achieved in BALB/c mice with 250 μg of MR1 (Fig. 3⇓) and in C57BL/6 mice given 500 μg of MR1 (data not shown).

FIGURE 3.
FIGURE 3.

Dose-dependent inhibition of CHS by anti-CD154 mAb. On days 0 and +1 groups of four BALB/c mice were sensitized with 0.25% DNFB and injected with the indicated doses of anti-CD154 mAb or with 500 μg of hamster IgG. On day +5 sensitized and nonsensitized mice were ear challenged with 0.2% DNFB, and the change in ear thickness was measured 24 h later. The mean increase in ear thickness following DNFB challenge is shown in units of 10−4 in. ± SEM. ∗, p < 0.005 compared with response in group 1.

To test the effect of MR1 on the development of the CD4+ and CD8+ T cell populations in CHS, IFN-γ and IL-4 production by immune CD4+ and CD8+ T cells from control and MR1-treated mice was compared. CD4+ and CD8+ T cell-enriched populations from LNC of mice treated with control Ig or MR1 during DNFB sensitization were cultured on anti-CD3 mAb-coated wells, and after 48 h the supernatants were collected and tested for IFN-γ, IL-4, and IL-10. CD8+ T cells from mice treated with control Ig during DNFB sensitization were stimulated to produce IFN-γ by culture on anti-CD3 mAb-coated wells (Fig. 4⇓). CD8+ T cells from mice treated with MR1 during DNFB sensitization produced much lower levels of IFN-γ, correlating with the inhibition of the CHS response by MR1. Immune, but not naive, CD4+ T cells produced lower levels of IFN-γ than the CD8+ T cells, and this production was also inhibited by MR1 treatment.

FIGURE 4.
FIGURE 4.

Effect of anti-CD154 mAb on cytokine production by hapten-primed CD8+ and CD4+ T cells. On days 0 and +1 BALB/c mice were given 300 μg of anti-CD154 mAb or control hamster IgG i.p. and sensitized with 0.25% DNFB. On day +4 CD4+ (□) and CD8+ (▪) enriched cell populations were prepared from lymph nodes of sensitized and naive mice, and 2 × 105-cell aliquots were cultured on anti-CD3 mAb-coated wells for 48 h. The supernatants were collected and analyzed by ELISA for production of IFN-γ, IL-4, and IL-10. Cell culture on control anti-Vβ17a mAb-coated wells did not stimulate detectable cytokine production. Results are representative of three independent experiments. ∗, p < 0.005; ∗∗, p < 0.01 (compared with levels in control mice). ND, Not detectable.

In contrast to IFN-γ production, CD4+ T cells from mice treated with control Ig or MR1 during DNFB sensitization produced IL-4 and IL-10 in anti-CD3 mAb-coated wells. It was noteworthy that IL-4, but not IL-10, production by CD4+ T cells from MR1-treated mice was significantly increased compared with production by immune CD4+ T cells from control Ig-treated mice (Fig. 4⇑). T cells from naive mice produced low to undetectable levels of IFN-γ, IL-4, and IL-10. Culture on control anti-Vβ17a mAb-coated wells did not stimulate detectable cytokine production by immune CD4+ or CD8+ T cells (not shown).

To further test the effects of anti-CD154 mAb treatment on the development of CD8+ and CD4+ T cell populations during hapten sensitization for CHS, ELISPOT assays were performed on T cells from naive or DNFB-sensitized mice treated with either control IgG or anti-CD154 mAb. Enriched CD4+ or CD8+ T cells were used as responder cells, and DNBS-labeled syngeneic splenocytes were used as stimulator cells. After 24 h of culture, the cells were removed from the plates, and the numbers of IFN-γ- and IL-4-producing cells in each group were quantified using an ELISPOT assay. In contrast to cells from naive mice, cells from DNFB-sensitized mice contained many IFN-γ-producing CD8+ T cells and IL-4-producing CD4+ T cells when cultured with DNBS-labeled stimulator cells (Fig. 5⇓). Background numbers of spots were observed when the immune CD4+ and CD8+ T cell populations were cultured with unlabeled splenocytes (not shown). Consistent with the results observed in the cytokine ELISA experiments, the number of hapten-specific IFN-γ-producing CD8+ T cells was reduced to background levels, and the number of IL-4-producing CD4+ T cells was increased by treatment with anti-CD154 mAb during DNFB sensitization. Collectively, these data indicated that MR1 treatment during DNFB sensitization inhibited the development of IFN-γ-producing CD8+ and CD4+ T cells, but had an enhancing effect on the development of the IL-4-producing CD4+ T cell population.

FIGURE 5.
FIGURE 5.

Effect of anti-CD154 mAb on the development of IFN-γ-producing CD8+ and IL-4-producing CD4+ T cells in DNFB-sensitized mice. On days 0 and +1 BALB/c mice were given 300 μg of anti-CD154 mAb or control hamster IgG i.p. and sensitized with 0.25% DNFB. On day +5, CD4+-enriched (□) and CD8+-enriched (▪) cell populations were prepared from lymph nodes of sensitized and naive mice, and 2 × 105-cell aliquots were cultured with 5 × 105 DNBS-labeled spleen cells on ELISPOT plates coated with anti-IFN-γ or anti-IL-4 mAb. After 24 h the cells were removed, and the ELISPOT assay was developed. The number of IFN-γ- and IL-4-producing cells for each group represents the mean number detected in triplicate wells using an immunospot image analyzer after subtraction of spots from control wells containing T cells with unlabeled spleen cells (less than five spots per well for both IFN-γ and IL-4). The results are representative of two experiments. ∗, p < 0.001; ∗∗, p < 0.05 (compared with numbers in the control, hamster IgG-treated group).

Anti-CD154 mAb-mediated inhibition of CHS is not due to blockade of CD40-CD154 interactions

The augmentation of hapten-specific CD4+ T cells responses by MR1 treatment during DNFB sensitization suggested that anti-CD154 mAb might not be blocking CD40-CD154 interactions during T cell priming for CHS. To examine this more closely, F(ab′)2 of MR1 were prepared and tested. The function of these fragments was first demonstrated by the ability to inhibit the induction of alloreactive T cell proliferation in mixed lymphocyte cultures (Fig. 6⇓). Groups of mice were given 500 μg of control Ig, MR1, or the F(ab′)2 during sensitization with DNFB. In contrast to MR1, administration of the F(ab′)2 of MR1 during DNFB sensitization did not inhibit ear swelling responses or the induction of IFN-γ-producing CD8+ T cells (Fig. 7⇓). These results suggested that anti-CD154 mAb did not mediate inhibition of CHS through blockade of CD40-CD154 interactions.

FIGURE 6.
FIGURE 6.

Inhibition of alloreactive T cell responses by F(ab′)2 of anti-CD154 mAb MR1. Responder spleen cells from C57BL/6 or BALB/c mice (2 × 105/well) were cocultured with 106 mitomycin C-treated BALB/c spleen cells. At the initiation of culture, 0.5 μg/ml of control hamster IgG, anti-CD154 mAb MR1, or F(ab′)2 of MR1 were added to the indicated cultures. After 4 days cultures were pulsed with 1 μCi of [3H]thymidine for 18 h, and 3H incorporation was determined by liquid scintillation counting. The data represent the mean 3H incorporation of triplicate cultures ± SEM. ∗, p < 0.005.

FIGURE 7.
FIGURE 7.

F(ab′)2 of anti-CD154 mAb MR1 do not inhibit CHS or the development of IFN-γ-producing CD8+ T cells to DNFB sensitization. Groups of C57BL/6 mice were treated with 500 μg of control hamster IgG, anti-CD154 mAb MR1, or F(ab′)2 of MR1 at the time of sensitization with DNFB on days 0 and +1. A, On day +5 mice were challenged with 0.2% DNFB, and the thickness of the challenged ears was measured 24 h later. B, LNC suspensions from each group of sensitized mice and a group of naive mice were prepared on day +4, and CD8+ T cell-enriched populations were prepared. Cell aliquots (2 × 105/well) were cultured on anti-CD3 or anti-Vβ17a mAb-coated wells. The supernatants were collected 48 h later and analyzed by ELISA for the production of IFN-γ by the CD8+ T cells. Culture on anti-Vβ17a did not stimulate IFN-γ by cells from any group. The results shown are representative of three individual experiments. ∗, p < 0.005.

Low to undetectable expression of CD154 by DNFB-primed CD8+ T cells following in vitro stimulation

The results indicating that anti-CD154 mAb inhibited the development of CD8+ T cells in CHS suggested that hapten-primed CD8+ T cells might express CD154 during hapten priming. T cell expression of CD154 was tested following anti-CD3 mAb or PMA/ionomycin stimulation of CD4+ and CD8+ T cell-enriched populations from naive or DNFB-primed mice. In addition, several different times of culture from 6–16 h were tested at which optimal expression of CD154 on CD4+ T cells had been previously reported (15, 16). Both naive and hapten-primed CD4+ T cells taken 24 h after sensitization expressed easily detectable levels of CD154 following stimulation by anti-CD3 mAb (Fig. 8⇓, activated cells). Culture on anti-Vβ17a mAb-coated wells did not stimulate CD4+ T cells to express CD154 (Fig. 8⇓, resting cells). In contrast to CD4+ T cells, expression of CD154 on CD8+ T cells following culture of naive or hapten-primed T cells on anti-CD3 mAb-coated wells for 6–16 h was minimal to undetectable. Detectable expression of CD154 following a 6-h culture with PMA/ionomycin was also observed on naive and immune CD4+, but not CD8+, T cells (data not shown).

FIGURE 8.
FIGURE 8.

CD154 expression on naive and DNFB-primed CD4+ and CD8+ T cells following culture on anti-CD3 mAb-coated wells. On days 0 and +1 BALB/c mice were sensitized with DNFB, and 24 h later (day +2) CD4+ and CD8+ T cell populations were prepared from the lymph nodes of the sensitized and naive mice, and 2 × 105-cell aliquots were cultured on anti-CD3 mAb-coated wells. After 16 h the cells were collected and stained with PE-labeled anti-CD4 or anti-CD8 mAb and FITC-labeled anti-CD154 mAb, MR1. The number in the upper right corner indicates the percentage of CD154+ cells in the CD4+ and CD8+ T cell populations. The results shown are representative of five individual experiments.

Anti-CD154 mAb-mediated inhibition of CHS is dependent on CD4+ T cells

The expression of CD154 on activated CD4+, but not CD8+, T cells suggested that the inhibitory effect of MR1 on the development of IFN-γ-producing CD8+ T cells in CHS might be mediated indirectly through CD4+ T cells. Two approaches were used to test this possibility. First, groups of mice were treated with control rat IgG or anti-CD4 mAb on days −3 through −1 before hapten sensitization and MR1 treatment on days 0 and +1. The efficiency of CD4+ T cell depletion in anti-CD4 mAb-treated mice was >95% as monitored by flow cytometry (not shown). Consistent with our previous observations (12, 13), depletion of CD4+ T cells before hapten sensitization increased the magnitude and duration of CHS (Fig. 9⇓A). Although MR1 treatment during DNFB sensitization inhibited CHS in control IgG-treated mice, this treatment did not inhibit the CHS response in CD4-depleted mice.

FIGURE 9.
FIGURE 9.

Anti-CD154 mAb during DNFB sensitization of CD4+ T cell-depleted wild-type or CD4−/− mice does not inhibit CHS. A, On days −4, −3, and −2 groups of four C57BL/6 mice were treated with 200 μg of control rat IgG (□ and ▴) or anti-CD4 mAb (• and ⋄) and 2 days later (i.e., days 0 and +1) were sensitized with 0.25% DNFB. Groups of control and CD4+ T cell-depleted mice were given 500 μg of anti-CD154 mAb MR1 (▴ and •) on each day of sensitization. B, Groups of four CD4−/− mice were sensitized with 0.25% DNFB and were given 500 μg of control IgG (□) or MR1 (▴) on days 0 and +1. On day +5 DNFB-sensitized and groups of unsensitized (+) mice were ear challenged with 0.2% DNFB, and the thickness of the challenged ears was measured at 24-h intervals. The mean increase in ear thickness following DNFB challenge is shown in units of 10−4 in. ± SEM. The results shown are representative of three individual experiments.

To further examine the CD4+ T cell requirement for MR1-mediated inhibition of CHS, CD4−/− mice were treated with MR1 during DNFB sensitization. In contrast to wild-type mice, the CHS response in CD4−/− mice was not inhibited by MR1 treatment (Fig. 9⇑B). Production of IFN-γ by CD8+ T cells from CD4 mAb-depleted (Fig. 10⇓) or CD4−/− (not shown) mice was also not inhibited by MR1 treatment during DNFB sensitization. These data indicated that MR1-mediated inhibition of CD8+ T cell development and the CHS response required CD4+ T cells.

FIGURE 10.
FIGURE 10.

Anti-CD154 mAb during DNFB sensitization of CD4+ T cell-depleted wild-type mice does not inhibit IFN-γ-producing CD8+ T cell development. On days −4, −3, and −2 groups of C57BL/6 mice were treated with 200 μg of control rat IgG or anti-CD4 mAb and 2 days later (i.e., days 0 and +1) were sensitized with 0.25% DNFB. Groups of the control and CD4+ T cell-depleted mice were given 500 μg of anti-CD154 mAb MR1 on each day of sensitization. On day +4 CD8+-enriched cell populations were prepared from lymph nodes of sensitized and naive mice, and 2 × 105-cell aliquots were cultured on anti-CD3 mAb-coated wells for 48 h. The supernatants were collected and analyzed by ELISA for production of IFN-γ. Cell culture on control anti-Vβ17a mAb-coated wells did not stimulate detectable cytokine production. Results are representative of two independent experiments. ∗, p < 0.001 compared with levels in controls.

Reduced stimulation of immune CD8+T cell proliferation by hpLC from anti-CD154 mAb-treated mice

Preliminary experiments indicated that MR1-mediated inhibition of CHS and CD8+ T cell development was not abrogated by anti-IL-4 mAb or in IL-4−/− mice (data not shown). Because these results indicated that the increased CD4+ T cell production of IL-4 induced by MR1 was not the mechanism by which the anti-CD154 mAb inhibited the induction of CHS, we then asked whether the effect might be mediated during the initial stage of T cell priming with hapten. To begin to examine this, we tested the ability of hpLC isolated from mice treated with control IgG or with anti-CD154 mAb during DNFB sensitization to stimulate immune CD8+ T cell proliferation. As previously observed (12, 13), hpLC obtained from control IgG-treated mice induced DNFB immune, but not naive, CD8+ T cells to proliferate (Fig. 11⇓A, group 1 vs group 5). Culture of immune CD8+ T cells without hpLC resulted in low levels of proliferation similar to naive cells (not shown). Consistent with the inhibited development of CD8+ T cell development in animals treated with anti-CD154 mAb during DNFB sensitization, coculture of these CD8+ T cells and hpLC from control IgG-treated mice resulted in low levels of T cell proliferation (group 2). In contrast to immune CD8+ T cell proliferation in cocultures with hpLC from control IgG-treated mice, reduced proliferation of immune CD8+ T cells was observed in cultures with hpLC from mice treated with anti-CD154 mAb during DNFB sensitization (group 3 vs group 1). Coculture of CD8+ T cells from mice treated with anti-CD154 mAb during DNFB sensitization and hpLC from mice treated with anti-CD154 mAb during sensitization also resulted in low levels of T cell proliferation (group 4). Whereas hpLC from wild-type mice treated with anti-CD154 mAb had reduced capacity to stimulate the proliferation of immune CD8+ T cells, this reduced stimulation was not observed in cocultures of immune CD8+ T cells and hpLC isolated from the lymph nodes of CD154−/− mice treated with anti-CD154 mAb (Fig. 11⇓B).

FIGURE 11.
FIGURE 11.

Reduced proliferation of immune CD8+ T cells during culture with hpLC from anti-CD154 mAb-treated mice. A, On days 0 and +1 BALB/c mice were sensitized with 0.25% DNFB and injected with 300 μg of hamster or anti-CD154 mAb MR1. To isolate hpLC, LNC suspensions were prepared on day +2 and centrifuged through a 14.5% metrizamide gradient. The interface cells (hpLC) were collected and given 2000 rad of gamma irradiation. On day +4 CD8+-enriched cells were prepared from lymph nodes of DNFB-sensitized and naive mice, and 2 × 105-cell aliquots were cultured with 2 × 104 hpLC from control or MR1-treated mice. After 48 h cultures were pulsed with 1 μCi of [3H]thymidine for 18 h, and 3H incorporation was determined by liquid scintillation counting. The data represent the mean 3H incorporation of triplicate cultures ± SEM. B, C57BL/6 wild-type and CD154−/− mice were sensitized with DNFB and treated with 500 μg of control hamster Ig or anti-CD154 mAb. The hpLC were isolated and cultured with CD8+ T cells from sensitized or naive wild-type mice as described in A. The data represent the mean 3H incorporation of triplicate cultures ± SEM. C, BALB/c mice were sensitized and treated with control Ig or MR1 as described above. In addition, a group of the mice was depleted of CD4+ T cells before DNFB sensitization. The hpLC were isolated from each group and cultured with CD8+ T cells from sensitized or naive mice as described above. The data represent the mean incorporation of triplicate cultures ± SEM. The results are representative of three individual experiments. ∗, p < 0.05 compared with proliferation of control immune CD8+ T cells cultured with hpLC from control IgG-treated mice.

Finally, the role of CD4+ T cells in the reduced hpLC stimulation of immune CD8+ T cell proliferation was tested. As observed above, hpLC from MR1-treated/DNFB-sensitized mice stimulated lower proliferation of immune CD8+ T cells than hpLC from control Ig-treated,/DNFB-sensitized mice (Fig. 11⇑C). However, hpLC isolated from mice depleted of CD4+ T cells before MR1 treatment and DNFB sensitization induced higher proliferation of immune CD8+ T cells than hpLC from mice treated with control Ig during sensitization.

Discussion

The goal of the current study was to test whether development of the effector CD8+ T cell and regulatory CD4+ T cell compartments during hapten sensitization for CHS are dependent on CD40-CD154 interactions. Administration of anti-CD154 mAb to allograft recipients has been effective in inhibiting the induction of alloreactive responses and results in prolonged allograft survival in transplant models (35, 36, 37, 38). These results have suggested that the Ab inhibits both CD4+ and CD8+ T cells responses to alloantigens. However, a recent study reported that anti-CD154 mAb prevented CD4+, but not CD8+, T cell-mediated rejection of cardiac allografts (39). The generation of primary CD8+ T cell responses to many viruses and parasites is not dependent on CD40-CD154 interactions (20, 21, 22). Alternatively, a number of studies have indicated that these interactions are required for some CD8+ T cell responses. In many of these studies anti-CD154 mAb inhibition of CD8+ T cells was mediated indirectly by blocking the priming and/or function of cells providing help for the generation of the CD8+ T cell response. In a model of graft-vs-host disease, anti-CD154 mAb inhibited the development of minor histocompatibility Ag-specific CD4+ T cells that produced IL-2 required for effector CD8+ T cell expansion (24). The induction of hapten-specific CD8+ T cells in response to skin sensitization with haptens, however, is not dependent on CD4+ T cell-derived growth factors or accessory signals (8, 9, 10, 11, 12, 13, 14).

As shown in the current report, CD40-CD154 interactions are not required for the development of the effector CD8+ or the regulatory CD4+ T cells during DNFB sensitization for CHS. The development of IFN-γ-producing CD8+ T cells and IL-4-producing CD4+ T cells and the magnitude of the CHS response were similar in CD154−/− and wild-type animals. Furthermore, CHS responses in CD154−/− mice depleted of CD4+ T cells before sensitization were of greater magnitude than responses in control CD154−/− mice, indicating the presence of regulatory CD4+ T cell activity during CHS in CD154−/− mice. A low level of IFN-γ-producing CD4+ T cells was observed in sensitized wild-type, but not CD154−/−, mice. Other laboratories have also reported that the induction of IFN-γ-producing, but not IL-4-producing, CD4+ T cells requires CD40-CD154 interactions (40, 41). In contrast to our results, Moodycliffe and coworkers (27) recently reported the absence of CHS responses to FITC sensitization and challenge in CD154−/− mice. The results also indicated the inability of LC to migrate from the skin sensitization site to the draining lymph nodes, accounting for the absence of responses in these mice. At this time it is difficult to reconcile these results with those presented in the current report. A possible explanation may lie in the different haptens tested in each study. In support of this, we have previously observed that responses to FITC are considerably weaker than responses to DNFB (33). Although the results in CD154−/− animals may be dependent upon the sensitizing hapten under study, the results in the current report clearly demonstrate that CD154 is not required for the induction of T cells and CHS responses to at least some haptens.

Despite the induction of IFN-γ-producing CD8+ T cells and CHS responses in DNFB-sensitized CD154−/− mice, administration of anti-CD154 mAb during sensitization inhibited the development of IFN-γ-producing CD8+ T cells and the CHS response to hapten challenge. Inhibition of CHS responses to DNFB by anti-CD154 mAb was previously reported by Tang and coworkers (26). During these studies decreased IFN-γ production by in vitro stimulated LNC was also observed in mice treated with MR1 during DNFB sensitization, but the effect of Ab treatment on the development of the individual CD4+ and CD8+ T cell populations in CHS was not tested. This and many other studies demonstrating the inhibitory effects of anti-CD154 mAb on T cell priming have assumed that the inhibitory mechanism was mediated through blockade of the CD40-CD154 interactions required for T cell priming (26, 35, 36, 37, 38, 39). In the current report we have demonstrated that MR1-mediated inhibition of CHS is not due to direct blockade of CD40-CD154 interactions during CD8+ T cell priming for CHS, but is due to an active process requiring CD4+ T cells. First, CD8+ T cell development and the CHS response were not inhibited when either CD4−/− mice or wild-type mice depleted of CD4+ T cells were treated with anti-CD154 mAb during hapten sensitization. Because CD4+ T cells are not required for the development of the effector CD8+ T cells in CHS, the anti-CD154 mAb-mediated inhibition observed in the presence of CD4+ T cells is not due to the blockade of CD4+ T cell development. Furthermore, administration of anti-CD154 mAb augmented the development of hapten-specific (i.e., IL-4-producing) CD4+ T cells. Second, administration of F(ab′)2 during sensitization did not inhibit CD8+ T cell priming or CHS responses. Although these fragments were active in inhibiting alloreactive T cell responses in vitro, it is difficult to assess their activity in vivo. Administration of the fragments, however, did result in extended CHS responses compared with those in control Ig-treated mice (A. Gorbachev, unpublished observations). The reasons for this enhancing effect are under investigation.

Several recent studies have indicated that CD40-CD154 engagement is required for CD4+ T cells to deliver signals to DC, which then enable the DC to prime functional cytolytic CD8+ T cells responses (42, 43, 44). The ability of agonist anti-CD40 mAbs to replace CD4+ T cells in this maturation suggests the absence or lack of sufficient CD154 expression by CD8+ T cells to direct DC maturation during this priming. Low level CD154 expression on CD8+ T cells following in vitro stimulation by various methods has been previously reported, although this expression may be restricted to CD8+ T cells producing type 2 cytokines and having the ability to provide helper signals to B cells for Ig production (45, 46). Lefrancois and coworkers (25) recently reported the CD154 blockade-mediated inhibition of mucosal CD8+ T cell expansion in response to OVA and suggested that these T cells may express CD154 during Ag priming. Thus, in contrast to CHS, CD40-CD154 interactions appear to play a crucial role in the development of CD8+ T cell-mediated mucosal immune responses. Consistent with reports from other laboratories (15, 16), CD154 expression was easily detectable on DNFB-primed CD4+ T cells following TCR-mediated stimulation in vitro. In conjunction with the lack of observed effects of anti-CD154 mAb directly on CD8+ T cells during priming for CHS, the results of the current study indicate that CD8+ T cells do not express CD154 during the development of this immune response.

The mechanism by which anti-CD154 mAb treatment promotes immunoregulatory CD4+ T cell activity and inhibits the development of effector CD8+ T cells is not entirely clear at this time. It is also not clear whether the anti-CD154 mAb enhances natural CD4+ T cell immunoregulation in the response or generates an entirely different form of CD4+ T cell immunoregulation. IL-4-producing, but not IL-10-producing, CD4+ T cell development was amplified by anti-CD154 mAb treatment during sensitization. In contrast, Blair and coworkers (47) demonstrated increased production of IL-10 by human CD4+ T cells stimulated in vitro with both anti-CD3 and anti-CD154 mAb. Initially, the increased CD4+ T cell production of IL-4 appeared to be an obvious potential mechanism for the immunoregulation induced by anti-CD154 mAb. A regulatory role for IL-4 in CHS has been reported in one, but not another, study (48, 49). However, the inhibition of IFN-γ-producing CD8+ T cell development also occurred in IL-4−/− mice treated with MR1 during sensitization (A. Gorbachev, unpublished observations). In conjunction with the absence of increased CD4+ T cell production of IL-10, these results suggested that inhibition of CHS by anti-CD154 mAb was not mediated through up-regulated CD4+ T cell production of immunoregulatory cytokines.

These results led us to consider the effects of anti-CD154 mAb directly on the CD4+ T cells during interaction with the hapten-presenting LC, rather than blocking these interactions. As discussed above, CD4+ T cells deliver positive signals through CD154 to DC, which then render the DC competent to generate CD8+ cytolytic responses (42, 43, 44). It is conceivable that Ab ligation of CD154 expressed by CD4+ T cells during these interactions may induce the delivery of negative signals to the DC, which undermine DC function during CD8+ T cell priming. In support of this, in vitro studies have indicated that ligation of CD154 on CD4+ T cells delivers signals affecting cytokine production and other immune functions (47, 50, 51). On this basis, we postulated that Ab ligation of CD154 expressed by CD4+ T cells during priming by hpLC may result in the delivery of negative signaling to the hpLC, and this signaling renders the hpLC incompetent for stimulation of hapten-specific CD8+ T cell maturation, resulting in inhibition of the CHS response (Fig. 12⇓). This model is supported by the observations in this report. The inhibitory activity of the anti-CD154 mAb is dependent on CD4+ T cells and apparently requires Ab Fc-Fc receptor ligation. Although wild-type CD4+ T cells express CD154 during priming, the CD4+, but not the CD8+, T cell population develops in wild-type mice treated with anti-CD154, indicating that the Ab does not block CD4+ T cell priming. The hpLC isolated from the anti-CD154 mAb treated animals have reduced capacity to stimulate immune CD8+ T cell proliferation in vitro, and this reduced hpLC function requires CD4+ T cells during anti-CD154 mAb administration. Other consequences of anti-CD154 mAb treatment on the hpLC have not yet been identified. However, there is no apparent decrease in the number of hpLC in the skin-draining lymph nodes of mice treated with control Ig and anti-CD154 mAb during hapten sensitization, and hpLC isolated from mice treated with MR1 during hapten sensitization express levels of class II MHC and B7-2 equivalent to those of hpLC from control treated mice (A. Gorbachev, unpublished observations).

FIGURE 12.
FIGURE 12.

Proposed model of CD4+ T cell-mediated inhibition of effector CD8+ T cell development by anti-CD154 mAb treatment during hapten sensitization for CHS. A, Recent results from several laboratories have suggested that CD154-CD40 engagement during interaction of CD4+ T cells and DC “conditions” the DC with the ability to generate Ag-specific CD8+ T cells responses (42 ,43 ,44 ). B, Neither CD4+ T cell interaction with hpLC nor CD154-CD40 engagement is required for the development of IFN-γ-producing CD8+ T cells during priming for CHS. CD154 is expressed on CD4+ T cells during interaction with hpLC, however, and Ab ligation of CD154 on the CD154 during this interaction results in hpLC that are unable to generate the hapten-specific CD8+ T cell response.

In summary, the current report indicates for the first time that the development of effector and regulatory T cells participating in responses to a cutaneous hapten application does not require CD40-CD154 interactions. The inhibition of T cell development in these responses by anti-CD154 mAb treatment is not achieved through direct blockade of CD40-CD154 interactions, but through a mechanism mediated by anti-CD154 binding to CD4+ T cells during priming. Anti-CD154 mAb actively promotes a CD4+ T cell-mediated mechanism that regulates or restricts the ability of hpLC to prime the effector CD8+ T cell population. These findings have potential practical value, considering the proposed use of anti-CD154 mAb for therapy of many clinical problems.

Acknowledgments

We thank Tara Engeman for help with preparing the figures.

Footnotes

  • ↵1 This work was supported by National Institutes of Health Grant AR44673 (to R.L.F.).

  • ↵2 Address correspondence and reprint requests to Dr. Robert L. Fairchild, NB3-79, Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195-0001. E-mail address: fairchr{at}ccf.org

  • ↵3 Abbreviations used in this paper: CHS, contact hypersensitivity; DC, dendritic cells; DNFB, 2,4-dinitrofluorobenzene; LNC, lymph node cells; hpLC, hapten-presenting Langerhans cells; ELISPOT, enzyme-linked immunospot.

  • Received August 16, 2000.
  • Accepted November 28, 2000.

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