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CD40 Ligand and CTLA-4 Are Reciprocally Regulated in the Th1 Cell Proliferative Response Sustained by CD8⁺ Dendritic Cells¹

Department of Experimental Medicine, University of Perugia, Perugia, Italy

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Subsets of murine dendritic cells (DCs) from the spleen differ in their ability to induce proliferative responses in both primary and secondary CD4⁺ T cells. Recent evidence indicates that lymphoid-related CD8⁺ DCs fail to provide appropriate signals to freshly isolated secondary CD4⁺ T cells to sustain their proliferation in vitro. In the present study, we examined peptide-pulsed CD8^- and CD8⁺ DCs for ability to stimulate Th1 and Th2 cell clones with the same Ag specificity. Defective ability to induce proliferation was selectively shown by CD8⁺ DCs presenting Ag to the Th1 clone. The deficiency in CD8⁺ DCs was overcome by CD40 triggering before peptide pulsing. When exposed to CD8⁺ DCs in the absence of CD40 activation, the Th1 clone expressed low levels of CD40 ligand and high levels of surface CTLA-4. Neutralization of CTLA-4 during the DC/T cell coculture resulted in increased CD40 ligand expression and proliferation of T cells. Remarkably, the activation of CD40 on DCs under conditions that would increase Th1 cell proliferation, also resulted in down-regulation of surface CTLA-4. These results confirm differential effects of CD8⁺ and CD8^- DCs in the stimulation of Ag-primed Th cells. In addition, they suggest that reciprocal regulation of CD40 ligand and CTLA-4 expression occurs in Th1 cells exposed to CD8⁺ DCs.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Dendritic cells (DCs)³ are unique in their ability to stimulate T cells and initiate adaptive immunity (1). Much is known about the ontogeny of DCs, the factors that influence their maturation and tissue distribution, and the role of these cells as APCs in the induction and regulation of immune responses (2, 3, 4, 5). Recent speculation that distinct subset of DCs may differentially regulate the development of Th1 vs Th2 cells in vivo and that the induction of tolerance vs immunity may also be mediated by different DC subsets are buoyed by the description of phenotypically and functionally distinct DC populations (6, 7).

In the mouse, we have shown that adoptive transfer of Ag-pulsed CD8^- DCs induces a Th1 response, whereas injection of CD8⁺ DCs leads to a state of Ag-specific anergy to a synthetic tumor/self-peptide (8, 9). Although IFN- enhances the tolerogenic activity of CD8⁺ DCs (10), CD40 activation on these cells will abolish their tolerogenic capacity or even trigger the potential for immunogenic presentation of the synthetic peptide (11, 12). Of interest, IL-6 can inhibit the tolerogenic function of CD8⁺ DCs by decreasing IFN-R expression on these cells (13).

These data are consistent with the notion that CD8⁺ DCs are, in general, poor inducers of the production of IL-2 and other cytokines by naive CD4⁺ and especially CD8⁺ T cells and this inhibits subsequent T cell expansion (14, 15, 16). In addition, CD8⁺ DCs may induce Fas-mediated death of many of the naive CD4⁺ T cells they activate (17). Recent evidence indicates that similarly dichotomic effects are mediated by CD8⁺ and CD8^- DCs in the stimulation of secondary CD4⁺ T cells. The differential between the two DC subtypes is even more pronounced than previously observed with primary T cells (18).

A pivotal role in the activation of function of DCs is thought to be played by the CD40-CD40 ligand (CD40L) partnership (19). CD40 activation on DCs increases production of IL-12, which initiates Th1 development (20, 21). Recent evidence indicates that blockade of CD40L in vivo may suppress autoimmunity by down-regulating Th1 differentiation and up-regulating CTLA-4 (22), a surface molecule expressed by T cells that acts as a negative regulator of T cell activation (23, 24, 25). In the present study, by comparatively analyzing CD8^- and CD8⁺ DCs for ability to present Ag to Th1 and Th2 clones with the same Ag specificity, we obtained evidence that the deficiency in the CD8⁺ subset is most pronounced when these cells present Ag peptide to the Th1 clone, which is prevented from expressing significant levels of CD40L. The effect is associated with up-regulation of CTLA-4 and is reversed by activation of CD40 on DCs. Remarkably, mutual regulation of CD40L and CTLA-4 is observed, with blockade of CTLA-4 up-regulating CD40L expression and restoring T cell proliferation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice and reagents

DBA/2J (H-2^d) mice were obtained from Charles River Breeding Laboratories (Calco, Milan, Italy). Female mice were used at the age of 2–4 mo. Source and characteristics of the hamster anti-murine CD40 (HM40-3) mAb used in combination with goat anti-hamster IgG (11, 12, 13) and of the anti-CTLA-4 hamster IgG mAb 4F10 (26) were as described. PE-conjugated anti-CD40L (gp39) mAb MR1, FITC-conjugated anti-CD3 mAb 145-2C11, and PE-conjugated anti-CD28 mAb 37.51 were obtained from BD PharMingen (San Diego, CA). The P815AB peptide (amino acid sequence LPYLGWLVF) was synthesized on solid phase using F-moc for transient NH₂-terminal protection, purified by means of reversed-phase HPLC and characterized by amino acid analysis.

DC preparation and treatments

DCs were prepared and fractionated according to CD11c/CD8 expression using positive selection columns in combination with CD11c and CD8 MicroBeads (Miltenyi Biotec, Bergish Gladbach, Germany), as described previously (13). Briefly, DCs were obtained from collagenase-treated spleens (collagenase type IV; Sigma-Aldrich, St. Louis, MO). Total spleen cells were treated with EDTA to disrupt DC-T cell complexes according to a previously described procedure (27), and EDTA was also present in subsequent steps involving the use of positive selection columns. Cells were resuspended in a 1.080 g/cm³ isoosmotic Nycodenz medium (Sigma-Aldrich), and centrifuged at 3000 rpm for 15 min at 4°C. The low-density fraction at the interface was collected and washed several times. The recovered cells were incubated with CD11c MicroBeads and separated using a positive selection column. Cells were then resuspended in RPMI 1640 medium supplemented with 10% FCS and allowed to adhere for 2 h, and this was followed by an additional 18-h incubation to allow DCs to detach. The recovered cells were routinely 96–98% CD11c⁺ and appeared to consist of 90–95% CD8^- and 5–10% CD8⁺ cells. For preparation of CD8⁺ and CD8^- fractions, the purified DCs were separated using positive selection columns and CD8 MicroBeads. After cell fractionation, the recovered CD8^- cells were 45% CD4⁺ and typically contained <0.5% contaminating CD8⁺ DCs, whereas the CD8⁺ fraction was made up of >95% CD8⁺ DCs. The CD8^- DC fraction consisted of >95% B7-1⁺, >98% B7-2⁺, and >98% CD40⁺ cells, whereas the CD8⁺ fraction consisted of >98% B7-1⁺, >99% B7-2⁺, and >98% CD40⁺ cells. In all CD40 stimulations (11, 12, 13), DCs were incubated on ice for 10 min in PBS plus 10% mouse serum, for 20 min with hamster anti-mouse CD40 mAb (5 µg/ml), and then overnight at 37°C with goat anti-hamster Ab (5 µg/ml) in Iscove’s medium plus 10% FCS. CD40 ligation on DCs routinely involved the use of the second cross-linking Ab, as the latter appears to be necessary for effective DC activation. To check for possible nonspecific effects of anti-CD40 ligation, appropriate controls included incubation of the CD8⁺ DCs in the presence of the second Ab alone, which treatment appeared to be devoid of any functional effect. As an additional control, isotype-matched anti-mouse H-2K^d 31-3-4S, capable of binding to DCs, was also used in place of the primary anti-CD40 reagent (11).

Th cell clones

T cell clones F76 (Th1) and F2 (Th2) were derived by limiting dilution of cultured lines generated from the popliteal lymph nodes of DBA/2 mice immunized with P815AB-pulsed DCs as described elsewhere (28) and were maintained by weekly restimulation of 1 x 10⁵ cells with 5 µM P815AB peptide and 6 x 10⁶ irradiated spleen cells in complete medium containing 40 U/ml human rIL-2.

T cell proliferation assay

Assays were performed in triplicate in flat-bottom 96-well microtiter plates in a total volume of 200 µl. Cultures contained T cell clones (5 x 10⁴ cells/well), purified DCs (at the indicated concentrations), and 5 µM P815AB peptide. Cultures were incubated for 48 h at 37°C. In selected experiments, cultures were prepared in the presence of anti-CTLA-4 mAb at the final concentration of 50 µg/ml as described previously (26). Proliferation of T cells was determined by addition of 1 µCi/well [³H]thymidine 8 h before termination of the culture. Cells were harvested on glass fiber filters and incorporation of radioactive label was measured on a beta counter.

Cytokine determinations in T cell cultures

Cultures were established using 5 x 10⁴ T cells and 5 x 10³ DCs in a 0.2-ml volume in the presence of 5 µM P815AB peptide, and supernatants were harvested at 24 h (for IL-2) or 48 h (for IL-4 and IFN-) for evaluation of cytokine contents by sandwich ELISA, as previously reported (8, 28). Briefly, IL-2 was measured by the use of mAb JES6-1A12 and biotinylated S4B6. IFN- levels were measured using R4-6A2 and biotinylated XMG1.2, whereas IL-4 measurements involved the use of mAb 11B11 and biotinylated BVD6-24G2. Cytokine titers (mean ± SD of replicate samples) were expressed as units per milliliter or nanograms per milliliter, calculated by reference to standard curves.

Cytokine production by DCs

DC culture supernatants were tested for IFN antiviral activity using a cytopathic effect reduction bioassay as previously described (29). DCs (1 x 10⁶) were plated in each well of a 24-well culture plate in a volume of 1 ml in the presence or the absence of anti-CD40 mAb. Cultures were incubated at 37°C in 5% CO₂ for 18 h, after which the supernatant was harvested. Aliquots of each sample (100 µl) were assayed for IFN- biological activity by measuring its ability to confer resistance to vesicular stomatitis virus infection upon L929 cells in the presence of neutralizing anti-mouse IFN- mAb R4-6A2 (29). The activity of the supernatants was determined by comparison to that of rIFN- (ICN Pharmaceuticals, Basingstoke, U.K.). Results are expressed as international IFN units (IU).

The production of IFN- by DCs was assessed by ELISA as indicated above using overnight cultures of fractionated DCs (10⁶) incubated in the presence or absence of anti-CD40 mAb. Aliquots of the same culture supernatants were also assayed for IL-12 p70 contents by ELISA as described elsewhere (11).

Cytofluorometric analysis

T cells (2–4 x 10⁵) cultured in 1 ml with 2 x 10⁴ DCs in the presence of cognate peptide were subjected to cytofluorometric analysis. mAb were used at saturating concentrations to analyze surface expression by two-color direct immunofluorescence. CD3 expression was analyzed by staining with FITC-conjugated 145-2C11 mAb. Gated CD3⁺ cells were analyzed for CD40L, CTLA-4, and CD28 expression. CD40L expression was assessed by means of PE-conjugated MR1 mAb and PE-conjugated hamster IgG as a control. CD28 was determined by staining with PE-conjugated anti-CD28 37.51 mAb and PE-conjugated hamster IgG as a control. Cell surface expression of CTLA-4 was analyzed by standard immunofluorescence on nonpermeabilized cells using PE-conjugated 4F10 mAb. Intracellular CTLA-4 expression was examined by fixing CD3-FITC-labeled cells in 2% paraformaldehyde, permeabilization with 0.5% saponin, and incubation with PE-anti-CTLA-4 or control PE-conjugated hamster IgG. Samples were analyzed on a FACScan (BD Biosciences, San Jose, CA) and data were analyzed using Lysis II software (BD Biosciences). Live cells were selected for analysis using forward vs side scatter gating.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Differential ability of DC subsets to stimulate proliferation of Th1 and Th2 clones

Previous studies have documented differential effects of CD8⁺ and CD8^- DCs in the proliferative response of freshly harvested secondary T cells with specificity for a synthetic influenza virus-related peptide or infectious virus (18). We wanted to assess any possible differential effects of DC subsets on the proliferation of Th1 and Th2 cell clones with specificity for a synthetic tumor/self-peptide, P815AB (28). Cultures were established using Th1 (F76) and Th2 (F2) cells in combination with fractionated CD8^- and CD8⁺ splenic DCs in the presence of cognate peptide. Proliferation of T cells was determined at different times (i.e., 24, 48, 72, and 96 h), showing that maximum proliferation would occur at 48 h (data not shown). As depicted in Fig. 1A, which reports the 48-h data using a range of DC concentrations, the CD8^- and CD8⁺ DCs showed comparable ability to support the growth of Th2 cells. In contrast, significant proliferation of the Th1 clone was only observed when these cells were incubated with CD8^- DCs. When cytokine levels were measured in culture supernatants (28), the Th2 cells appeared to express high levels of IL-4 and IL-2 regardless of the DC subtype added to the coculture as APCs. In contrast, the Th1 cells released significant amounts of IL-2 in addition to IFN- only in the presence of CD8^- DCs (Fig. 1B). It should be noted that the Th1 clone did produce considerable levels of IFN- even when cocultured with CD8⁺ DCs. This finding emphasizes the inability of Th1 cells to proliferate in the presence of CD8⁺ DCs even when producing IFN-.

View larger version (22K): [in this window] [in a new window]   FIGURE 1. Presentation of peptide Ag by CD8- and CD8+ DCs to Th cell clones F76 (Th1) and F2 (Th2). A, Proliferative response of the T cells exposed to different concentrations of P815AB-loaded DCs as APCs. Radioactivity was measured at 48 h and counts are expressed as the mean cpm of replicate samples ± SE. B, Cytokine production in parallel culture supernatants harvested at 24 h (IL-2) or 48 h (IL-4 and IFN-). Data are the means ± SE of replicate determinations in one of three experiments with similar results.

Using an in vivo model of P815AB presentation for induction of CD4⁺ T cell-dependent skin test reactivity, we have previously shown that the failure of peptide-loaded CD8⁺ DCs to initiate T cell reactivity in vivo may be overcome by activation of CD40 in vitro before transfer into recipient hosts (12). We therefore became interested in ascertaining whether CD40 ligation would overcome the inability of CD8⁺ DCs to support the growth of the Th1 clone in vitro in the presence of cognate peptide. Cultures were established as illustrated above using Th1 or Th2 cells incubated with either type of peptide-loaded DCs, which were used either as such or after activation of CD40 in vitro, as previously described (11, 12, 13). Fig. 2A shows that CD40 activation had no effect on the proliferative response of Th2 cells incubated with CD8⁺ or CD8^- DCs. Similarly unchanged was the proliferative response of Th1 cells exposed to CD8^- DCs. In contrast, CD40 activation greatly increased the ability of CD8⁺ DCs to support the growth of the Th1 clone. Remarkably, the proliferation of the latter cells, namely, F76 clone cells exposed to CD40-activated CD8⁺ DCs, was the highest among the different experimental groups in Fig. 2A. In addition, the increase in the proliferative response of the F76 clone was associated with an increased production of IL-2 (from 1.5 ± 0.1 to 6.2 ± 0.3 U/ml) and IFN- (from 5.5 ± 0.27 to 11.4 ± 0.6 ng/ml).

View larger version (20K): [in this window] [in a new window]   FIGURE 2. Effect of CD40 ligation on DC ability to support the growth of Th1 and Th2 cells and to release specific cytokines. A, Purified CD8- and CD8+ DCs, either as such or following CD40 activation, were incubated with Th1 or Th2 cell clones in the presence of cognate peptide. Proliferation of T cells was measured at 48 h. The control treatment consisted of goat anti-hamster Ab in the absence of hamster anti-mouse anti-CD40 mAb. B, Cytokine production was measured by ELISA in culture supernatants of DCs incubated with anti-CD40 mAb for 18 h. Data are the means ± SE of replicate determinations in one of three experiments.

DCs not only stimulate T cells effectively but are also producers of cytokines that have important immune regulatory functions. It has been reported that CD4^-8^- DCs are the main producers of IFN- and that CD8⁺ DCs in contrast produce very large amounts of IL-12 and IFN- (16). To investigate whether a cytokine-mediated pathway may be at work in the defect in Th1 proliferation induced by CD8⁺ DCs, we measured the production of IL-12, IFN-, and IFN- by CD8^- and CD8⁺ DCs in the presence or the absence of CD40 ligation. The baseline production of IFN- was below the detection limit of the assay (i.e., 2 IU/ml) and production remained undetectable after CD40 activation (data not shown). In addition, Fig. 2B shows that the baseline productions of IL-12 and IFN- were also limited. However, following CD40 activation, the levels of IL-12 and IFN- appeared to increase significantly in both DC subsets. Although the apparent lack of IFN- production was somewhat unexpected, it has been reported that optimal release of IFN- by CD8⁺ DC may require a combination of CpG and poly(I:C) (16).

Defective CD40L expression in Th1 cells cultured with CD8⁺ DCs

The ability of CD40 activation to prime CD8⁺ DCs for effective support of Th1 cell growth suggested that defective expression of CD40L may be associated with the poor proliferative response of the latter cells upon coculture with untreated CD8⁺ DCs. As a matter of fact, a crucial role in the activation and function of DCs is considered to be played by the CD40-CD40L interaction (19). CD40L is expressed on activated mature T cells but not on resting T cells. The expression of CD40L on activated T cells is transient and tightly regulated. However, the factors that contribute to the regulation of CD40L expression are not entirely known (30). We wanted to examine CD40L expression in Th1 cells cocultured with CD8^- vs CD8⁺ DCs in the presence of Ag peptide (Fig. 3). We found that CD40L expression could be clearly detected at 24 h of coculture of the Th1 cells with CD8^- DCs, and peak levels were reached at 48 h. In contrast, the limited CD40L expression observed at 24 h of cell incubation with CD8⁺ DCs was totally abrogated at 48 h. When the same cocultures of Th1 and CD8^- or CD8⁺ DCs were examined for CD28 expression, we found that the percentages of CD28⁺ T cells incubated with CD8^- DCs were 45.3 at 3 h, 44.9 at 24 h, and 46.7 at 48 h; on incubation of the T cells with CD8⁺ DCs, the percentages were 46.1 (3 h), 44.5 (24 h), and 45.5 (48 h). Therefore, the poor proliferative response of Th1 cells to peptide-pulsed CD8⁺ DCs appeared to correlate with defective expression of CD40L on the former cells. In parallel experiments, we also found that the Th2 cells were induced to express high levels of CD40L by both CD8^- and CD8⁺ DCs. As an example, the 3-/48-h expressions of the Th2 clone were 5.6/82.0% (for CD8^- DC coculture) and 4.9/80.4% (for CD8⁺ DC coculture).

View larger version (25K): [in this window] [in a new window]   FIGURE 3. CD40 ligand expression in Th1 cells cultured with CD8- or CD8+ DCs. Cultures were established using F76 cells and CD8- or CD8+ DCs in the presence of Ag peptide. At 3, 24, or 48 h of coculture, gated CD3+ cells were examined for expression of CD40L by means of PE-conjugated MR1 mAb (bold lines). Control staining consisted of PE-conjugated hamster IgG (thin lines). Numbers within boxes indicate the percentage of CD3+ cells staining positively for CD40L. Data are from one experiment representative of four.

CTLA-4 present on CD4⁺ T cells acts as a key negative modulator of immune responses by blocking CD28-dependent T cell activation (31). CTLA-4 is also involved in the induction of peripheral T cell tolerance in vivo (32). Therefore, CTLA-4 normally acts as a repressor of T cell activation. Recent evidence suggests that up-regulation of CTLA-4 levels may occur after anti-CD40L treatment, leading to attenuation of Th1 cell activation (22). It appeared therefore of interest to investigate CTLA-4 expression in Th1 cells cultured with CD8^- and CD8⁺ DCs. Although CTLA-4 expression is largely intracellular, TCR ligation induces rapid movement from endosomes to plasma membrane. Nonetheless, CTLA-4 is quickly endocytosed, which precludes significant accumulation on the surface. To examine regulation of the cycling of CTLA-4 between endosomal compartment and the cell surface, the Th1 cells were incubated for different times with either type of DCs before cytofluorometric analysis of expression of surface CTLA-4. Fig. 4A shows that a dramatic increase in CTLA-4 expression was observed at 24–48 h only in Th1 cells exposed to CD8⁺ DCs. When the intracellular levels of CTLA-4 were measured in permeabilized cells, we found that significant and comparable increases were observed in Th1 cells regardless of the DC subtype added to the coculture (Fig. 4B). Therefore, coculture of Th1 cells with CD8⁺ DCs appeared to increase the surface expression of CTLA-4 and to influence the rapid cycling between endosomal compartments and the cell surface. It is interesting to note that, in parallel experiments using Th2 cells, we found that the CD8⁺ DCs would induce a limited expression of surface CTLA-4 (data not shown).

View larger version (21K): [in this window] [in a new window]   FIGURE 4. CTLA-4 expression in Th1 cells cultured with CD8- or CD8+ DCs. Cultures were established using purified DC fractions and F76 cells in the presence of cognate peptide. Gated CD3+ cells were examined for expression of CTLA-4 at 3, 24, or 48 h of coculture. A, Surface staining with anti-CTLA-4-PE (bold lines) or control PE-conjugated hamster IgG (thin lines). Numbers within boxes indicate the percentage of CD3+ cells staining positively for CTLA-4. One experiment representative of three. B, Intracellular staining with anti-CTLA-4-PE or control Ab using permeabilized cells. Data are the means ± SD of three independent experiments.

Blockade of CTLA-4 interactions using neutralizing mAb has been found to augment T cell proliferation in vitro (26, 31), to promote tumor rejection in vivo (33), and to prevent Ag-specific tolerance in vivo (32, 34). We investigated whether the copresence of mAb to CTLA-4 would affect the ability of CD8⁺ DCs to sustain the growth of the Th1 clone. Cultures were established using CD8⁺ DCs and the Th1 clone F76 in the presence of the cognate peptide, with or without the anti-CTLA-4 4F10 mAb. Fig. 5A shows that the poor ability of the CD8⁺ DCs to sustain the growth of the Th1 cells was completely overcome by blockade of CTLA-4. When CD40L expression was examined in the Th1 cells subject to CTLA-4 blockade during coculture with CD8⁺ DCs, a remarkable increase in this expression was found to occur at 24–48 h (Fig. 5B). On examining the production of IL-2 and IFN- by Th1 cell cultures treated with anti-CTLA-4 in the presence of CD8⁺ DCs, we found that blockade of CTLA-4 would result in increased production of IL-2 from 1.0 ± 0.1 to 4.6 ± 0.2 U/ml and IFN- from 5.1 ± 0.2 to 9.4 ± 0.4 ng/ml.

View larger version (19K): [in this window] [in a new window]   FIGURE 5. Blockade of CTLA-4 increases CD40L expression and proliferation in Th1 cells. Cultures were established using peptide-pulsed CD8+ DCs and F76 cells in the presence of anti-CTLA-4 4F10 mAb. A, Proliferation was measured at 48 h in Th1 cells exposed to different concentrations of CD8+ DCs (indicated) in the presence of anti-CTLA-4 mAb, control Ig, or medium alone. Data are the means ± SE in one experiment representative of three. B, Cytofluorometric analysis of Th1 cells exposed for different times to peptide-pulsed CD8+ DCs in the presence of anti-CTLA-4 mAb or medium alone. Gated CD3+ cells were stained with PE-conjugated anti-CD40L MR1 mAb (bold lines) or control Ab (thin lines). Data are from one experiment representative of three.

In chronic experimental myasthenia gravis, the blockade of CD40L in vivo is known to affect both T cell effector and APC functions, with a reduction in B7-2, IL-12, and IFN- levels and increased expression of CTLA-4 (22). We wanted to examine the effect of CD40 activation in vitro in CD8⁺ DCs on the expression of CTLA-4 by Th1 cells. CD8⁺ DCs were subjected to CD40 cross-linking as illustrated above before coculture with Th1 cells in the presence of Ag peptide. Regulation of recycling of CTLA-4 between endosomal compartment and cell surface was investigated. Fig. 6A shows that considerable inhibition of cell surface expression of CTLA-4 was observed at 24–48 h in Th1 cells incubated with CD40-activated CD8⁺ DCs, thus opposing the dramatic increase in CTLA-4 observed in the absence of CD8⁺ DC treatment. In contrast, no major changes were found in the intracellular levels of CTLA-4 under comparable experimental conditions (Fig. 6B). Therefore, it appeared that CD40-mediated changes of CTLA-4 expression by T cells would occur in association with the increased Th1 cell proliferative response observed in Fig. 2.

View larger version (19K): [in this window] [in a new window]   FIGURE 6. CTLA-4 expression in Th1 cells cultured with CD40-activated CD8+ DCs. Cultures were established using F76 cells and peptide-pulsed CD8+ DCs, either as such or after CD40 cross-linking. Gated CD3+ cells were examined for expression of CTLA-4 at 3, 24, or 48 h of coculture. A, Surface expression of CTLA-4 using anti-CTLA-4-PE (bold lines) or hamster IgG-PE (thin lines) in intact cells. Numbers within boxes indicate the percentage of CD3+ cells staining positively for CTLA-4. One experiment representative of three. B, Expression of intracellular CTLA-4 in permeabilized cells reacted with anti-CTLA-4-PE or control Ab. Data (percentage of positive cells) are the means ± SD of three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

CD8⁺ and CD8^- DCs differ in their ability to induce proliferative responses in both primary CD4⁺ and primary CD8⁺ T cells (14, 17). Recent evidence using freshly harvested secondary T cells indicates that CD8^- DCs can activate Ag-primed CD4⁺ T cells and stimulate proliferation. In contrast, interaction with CD8⁺ DCs, though able to induce clustering of the T cells, results in only poor and sometimes barely significant levels of both cytokine production and proliferation (18). Immature DCs may exert immunoregulatory effects also through interaction with T regulatory cells (40). However, a recent study on the regulatory activity of immature and mature CD8⁺ DCs has indicated that neither immune deviation nor induction of regulatory cells may be a significant contributory factor to the modulation in vivo of peripheral T cell function (41).

CD8⁺ DCs were initially described as the major producer of IL-12 p70 (42, 43, 44, 45) and were reported to induce predominantly Th1 responses, whereas CD8^- DCs drive Th2 or mixed Th1/Th2 responses (44, 46). However, recently, the capacity of all CD11c⁺ DCs to produce IL-12 p70, and thus also their ability to induce Th1 responses, has been shown to vary with Ag stimulus (47, 48). In the present study, we made use of highly polarized T cell clones with the same Ag specificity to comparatively analyze the ability of CD8^- and CD8⁺ DCs to stimulate the proliferative response of Th1 vs Th2 cells in the presence of specific Ag.

By using an Ag peptide that is presented differentially by CD8^- and CD8⁺ DCs in a primary response in vivo (8, 9, 10, 11, 12, 13), we made a series of observations that may be relevant to a better understanding of the reciprocal control of Th cell and DC function during the course of a secondary response. We found that defective ability to sustain T cell proliferation was selectively shown by CD8⁺ DCs cultured with Th1 cells (Fig. 1). During the coculture, the latter cells were induced to express considerable amounts of CTLA-4 (Fig. 4), which event was associated with impaired ability to express the CD40L (Fig. 3) and unchanged expression of CD28. Poor expression of CD40L by the T cells likely contributed to suboptimal interaction between DCs and Th1 cells, such that CD40 activation on the DCs fully restored their ability to sustain the growth of the Th1 clone (Fig. 2). Reciprocal regulation appeared to occur between CTLA-4 and CD40L expression, because CTLA-4 neutralization led to increased proliferation and up-regulation of CD40L expression (Fig. 5), and CD40 activation on DCs down-regulated the expression of CTLA-4 (Fig. 6) and up-regulated that of CD40L (data not shown). It is interesting to note that, in line with previous observations (49), CD28 was expressed similarly by the Th1 and Th2 clones used in this study (data not shown).

CTLA-4 is expressed on T cells after activation and shares homology with the CD28 costimulatory receptor. In contrast to CD28, CTLA-4 is considered to be a negative regulator of T cell activation. Cross-linking of CTLA-4 during activation of peripheral T cells reduces IL-2 production and arrests T cells in G₁ (23, 24, 25). In differentiated T cells, CTLA-4 can function to suppress the production of cytokines produced by both Th1 and Th2 cells (49). Several mechanisms have been proposed to explain the inhibitory activity of CTLA-4, including competition for ligand access to CD28, delivery of a signal that antagonizes a CD28 signal, and delivery of a signal that antagonizes a TCR-mediated event (26). Recent evidence indicates that CTLA-4 up-regulation may represent a likely mechanism whereby signaling through CD45 in T cells mediates tolerogenic effects (50). Up-regulation of CTLA-4 has also been shown to result from blockade of CD40L in T cells (22).

Our observation that CTLA-4 is up-regulated in Th1 cells exposed to peptide-pulsed CD8⁺ DCs suggests that these cells may directly regulate the expression of surface CTLA-4 and influence the rapid cycling between endosomal compartments and the cell surface. Although the regulation of CTLA-4 expression is complex and occurs at multiple levels (23, 24), the recent demonstration of a link between CD45 and CTLA-4 that depends on calcineurin-mediated signaling (50) may underscore a role for the activation of such a mechanism by peptide-pulsed CD8⁺ DCs. Interesting in this regard is the observation that CD45 may be differentially expressed on Th1 and Th2 cells (51). Although the data of the present work were generated using a single Th1 clone and a single Th2 clone, we have very recently observed a similar pattern of effects using Th1 and Th2 clones generated in our laboratory to NRP, a synthetic peptide mimotope recognized by diabetogenic T cells in the nonobese diabetic mouse (Ref. 13 and data not shown). In addition, results similar to ours, of a differential effect of CD8⁺ and CD8^- DCs, have been reported for the stimulation of secondary CD4⁺ T cells specific for influenza virus hemagglutinin (18).

Whatever the mechanisms whereby surface CTLA-4 becomes up-regulated in Th1 cells cultured with CD8⁺ DCs, one major observation in the present study is that this event results in impaired expression of the CD40L, such that blocking of CTLA-4 by means of specific Ab will result in up-regulation of CD40L and restore proliferative ability. Again, several mechanisms can be proposed to explain the inhibitory activity of CTLA-4 on CD40L expression, including inhibition of CD28-mediated effects and generation of signals that antagonize or abort TCR-driven events.

Triggering of DCs in vivo through CD40 is a powerful activation stimulus, causing these cells to express the full array of Ag-presenting/costimulatory molecules (30, 20). Moreover, injection of CD40-modulated DCs restores Ag-specific CTL responses in CD4⁺ T cell-depleted mice (36). These data indicate that the function of CD4⁺ Th cells is mediated through CD40-dependent activation of APCs. Recent evidence suggests that the CD40-CD40L pair can act as a switch in vivo, determining whether naive peripheral CTL are primed or tolerized (52) and accounting for the ability of CD40 ligation to convert tumor-specific CD4⁺ T cell tolerance into T cell priming (53). Of interest in this regard may be our current observation that CD40 activation of CD8⁺ DCs will result in impaired expression of CTLA-4 by the Th1 clone cultured with those cells. Although the underlying mechanism remains to be determined, it is possible that an optimally conditioned CD8⁺ DCs may affect the rapid cycling of CTLA-4 between endosomal compartments and cell surface so as to limit the surface expression of CTLA-4.

In conclusion, the poor ability of CD8⁺ DCs to support the growth of a Th1 clone can be overcome by CD40 activation, a maneuver that apparently obviates the poor expression of CD40L by the Th1 cells cultured with CD8⁺ DCs. One possible mechanism via which the T cells are prevented from expressing significant amounts of CD40L may be represented by increased expression of surface CTLA-4, whose neutralization has effects comparable to those of CD40 activation. These data may be among the first to report on mutual regulation between CTLA-4 and CD40L, and may provide insights into the mechanisms through which distinct DC subsets interact with Ag-primed CD4⁺ T cells.

	Acknowledgments

 
We thank Dr. Maria Ferrantini for assistance with the IFN- biological assay.

	Footnotes

 
¹ This work was supported by the Italian Association for Cancer Research and a postdoctoral fellowship from Fondazione Italiana per la Ricerca sul Cancro (to F.F.).

² Address correspondence and reprint requests to Prof. Paolo Puccetti, Department of Experimental Medicine, Pharmacology Section, University of Perugia, Via del Giochetto, I-06126 Perugia, Italy. E-mail address: plopcc{at}tin.it

³ Abbreviations used in this paper: DC, dendritic cell; CD40L, CD40 ligand.

Received for publication February 22, 2002. Accepted for publication May 28, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Steinman, R. M.. 1991. The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. 9:271.[Medline]
2. Heath, W. R., F. R. Carbone. 2001. Cross-presentation, dendritic cells, tolerance, and immunity. Annu. Rev. Immunol. 19:47.[Medline]
3. Pulendran, B., J. Banchereau, E. Maraskovsky, C. Maliszewski. 2001. Modulating the immune response with dendritic cells and their growth factors. Trends Immunol. 22:41.[Medline]
4. Lechler, R., W. F. Ng, R. M. Steinman. 2001. Dendritic cells in transplantation: friend or foe?. Immunity 14:357.[Medline]
5. Shortman, K., W. R. Heath. 2001. Immunity or tolerance? That is the question for dendritic cells. Nat. Immunol. 2:988.[Medline]
6. Fazekas de St. Groth, B.. 1998. The evolution of self-tolerance: a new cell arises to meet the challenge of self-reactivity. Immunol. Today 19:448.[Medline]
7. Moser, M., K. M. Murphy. 2000. Dendritic cell regulation of T_H1-T_H2 development. Nat. Immunol. 1:199.[Medline]
8. Grohmann, U., R. Bianchi, E. Ayroldi, M. L. Belladonna, D. Surace, M. C. Fioretti, P. Puccetti. 1997. A tumor-associated and self antigen peptide presented by dendritic cells may induce T cell anergy in vivo, but IL-12 can prevent or revert the anergic state. J. Immunol. 158:3593.[Abstract]
9. Grohmann, U., R. Bianchi, M. L. Belladonna, C. Vacca, S. Silla, E. Ayroldi, M. C. Fioretti, P. Puccetti. 1999. IL-12 acts selectively on CD8^- dendritic cells to enhance presentation of a tumor peptide in vivo. J. Immunol. 163:3100.[Abstract/Free Full Text]
10. Grohmann, U., R. Bianchi, M. L. Belladonna, S. Silla, F. Fallarino, M. C. Fioretti, P. Puccetti. 2000. IFN- inhibits presentation of a tumor/self peptide by CD8^- dendritic cells via potentiation of the CD8⁺ subset. J. Immunol. 165:1357.[Abstract/Free Full Text]
11. Bianchi, R., U. Grohmann, C. Vacca, M. L. Belladonna, M. C. Fioretti, P. Puccetti. 1999. Autocrine IL-12 is involved in dendritic cell modulation via CD40 ligation. J. Immunol. 163:2517.[Abstract/Free Full Text]
12. Grohmann, U., F. Fallarino, S. Silla, R. Bianchi, M. L. Belladonna, C. Vacca, A. Micheletti, M. C. Fioretti, P. Puccetti. 2001. CD40 ligation ablates the tolerogenic potential of lymphoid dendritic cells. J. Immunol. 166:277.[Abstract/Free Full Text]
13. Grohmann, U., F. Fallarino, R. Bianchi, M. L. Belladonna, C. Vacca, C. Orabona, C. Uyttenhove, M. C. Fioretti, P. Puccetti. 2001. IL-6 inhibits the tolerogenic function of CD8⁺ dendritic cells expressing indoleamine 2,3-dioxygenase. J. Immunol. 167:708.[Abstract/Free Full Text]
14. Winkel, K. D., V. Kronin, M. F. Krummel, K. Shortman. 1997. The nature of the signals regulating CD8 T cell proliferative responses to CD8⁺ or CD8^- dendritic cells. Eur. J. Immunol. 27:3350.[Medline]
15. Kronin, V., K. Winkel, G. Süss, A. Kelso, W. Heath, J. Kirkberg, H. von Boehmer, K. Shortman. 1996. A subclass of dendritic cells regulate the response of naive CD8 T cells by limiting their IL-2 production. J. Immunol. 157:3819.[Abstract]
16. Hochrein, H., K. Shortman, D. Vremec, B. Scott, P. Hertzog, M. O’Keeffe. 2001. Differential production of IL-12, IFN-, and IFN- by mouse dendritic cell subsets. J. Immunol. 166:5448.[Abstract/Free Full Text]
17. Süss, G., K. Shortman. 1996. A subclass of dendritic cells kills CD4 T cells via Fas/Fas-ligand-induced apoptosis. J. Exp. Med. 183:1789.[Abstract/Free Full Text]
18. Kronin, V., C. J. Fitzmaurice, I. Caminschi, K. Shortman, D. C. Jackson, L. E. Brown. 2001. Differential effect of CD8⁺ and CD8^- dendritic cells in the stimulation of secondary CD4⁺ T cells. Int. Immunol. 13:465.[Abstract/Free Full Text]
19. Grewal, I. S., R. A. Flavell. 1998. CD40 and CD154 in cell-mediated immunity. Annu. Rev. Immunol. 16:111.[Medline]
20. Cella, M., D. Scheidegger, K. Palmer-Lehmann, P. Lane, A. Lanzavecchia, G. Alber. 1996. Ligation of CD40 on dendritic cells triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via APC activation. J. Exp. Med. 184:747.[Abstract/Free Full Text]
21. Macatonia, S. E., N. A. Hosken, M. Litton, P. Vieira, C.-S. Hsieh, J. A. Culpepper, M. Wysocka, G. Trinchieri, K. M. Murphy, A. O’Garra. 1995. Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4⁺ T cells. J. Immunol. 154:5071.[Abstract]
22. Im, S. H., D. Barchan, P. K. Maiti, S. Fuchs, M. C. Souroujon. 2001. Blockade of CD40 ligand suppresses chronic experimental myasthenia gravis by down-regulation of Th1 differentiation and up-regulation of CTLA-4. J. Immunol. 166:6893.[Abstract/Free Full Text]
23. Wainas, T. L., D. J. Lenschow, C. Y. Bakker, P. S. Linsley, G. L. Freeman, J. M. Green, C. B. Thompson, J. A. Bluestone. 1994. CTLA-4 can function as a negative regulator of T cell activation. Immunity 1:405.[Medline]
24. Thompson, C. B., J. P. Allison. 1997. The emerging role of CTLA-4 as an immune attenuator. Immunity 7:445.[Medline]
25. Saito, T.. 1998. Negative regulation of T cell activation. Curr. Opin. Immunol. 10:313.[Medline]
26. Fallarino, F., P. E. Fields, T. F. Gajewski. 1998. B7-1 engagement of cytotoxic T lymphocyte antigen 4 inhibits T cell activation in the absence of CD28. J. Exp. Med. 188:205.[Abstract/Free Full Text]
27. Vremec, D., J. Pooley, H. Hochrein, L. Wu, K. Shortman. 2000. CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen. J. Immunol. 164:2978.[Abstract/Free Full Text]
28. Fallarino, F., U. Grohmann, R. Bianchi, C. Vacca, M. C. Fioretti, P. Puccetti. 2000. Th1 and Th2 cell clones to a poorly immunogenic tumor antigen initiate CD8⁺ T cell-dependent tumor eradication in vivo. J. Immunol. 165:5495.[Abstract/Free Full Text]
29. Bianchi, R., M. C. Fioretti, L. Romani, U. Grohmann, E. Cenci, P. Puccetti. 1990. T-cell subsets, IFN- production and efferent specificity in anti-parental tumor immunity induced by mouse sensitization with xenogenized variant cells. Int. J. Cancer 46:653.[Medline]
30. van Kooten, C., J. Banchereau. 2000. CD40-CD40 ligand. J. Leukocyte Biol. 67:2.[Abstract]
31. Krummel, M. F., J. P. Allison. 1995. CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation. J. Exp. Med. 182:459.[Abstract/Free Full Text]
32. Perez, V. L., L. Van Parijs, A. Biuckians, X. X. Zheng, T. B. Strom, A. K. Abbas. 1997. Induction of peripheral T cell tolerance in vivo requires CTLA-4 engagement. Immunity 6:411.[Medline]
33. Leach, D. R., M. F. Krummel, J. P. Allison. 1996. Enhancement of antitumor immunity by CTLA-4 blockade. Science 271:1734.[Abstract]
34. Bluestone, J. A.. 1997. Is CTLA-4 a master switch for peripheral T cell tolerance?. J. Immunol. 158:1989.[Abstract]
35. Bennett, S. R., F. R. Carbone, F. Karamalis, J. F. Miller, W. R. Heath. 1997. Induction of a CD8⁺ cytotoxic T lymphocyte response by cross-priming requires cognate CD4⁺ T-helper help. J. Exp. Med. 186:65.[Abstract/Free Full Text]
36. Ridge, J. P., F. Di Rosa, P. Matzinger. 1998. A conditioned dendritic cell can be a temporal bridge between a CD4⁺ T-helper and a T-killer cell. Nature 393:474.[Medline]
37. Rissoan, M.-C., V. Soumelis, N. Kadowaki, G. Grouard, F. Briere, R. de Waal Malefyt, Y.-J. Liu. 1999. Reciprocal control of T helper cell and dendritic cell differentiation. Science 283:1183.[Abstract/Free Full Text]
38. Shortman, K., C. Caux. 1997. Dendritic cell development: multiple pathways to nature’s adjuvants. Stem Cells 15:409.[Abstract/Free Full Text]
39. Vremec, D., K. Shortman. 1997. Dendritic cell subtypes in mouse lymphoid organs: cross-correlation of surface markers, changes with incubation, and differences among thymus, spleen, and lymph nodes. J. Immunol. 159:565.[Abstract]
40. Roncarolo, M. G., R. Bacchetta, C. Bordignon, S. Narula, M. K. Levings. 2001. Type 1 T regulatory cells. Immunol. Rev. 182:68.[Medline]
41. O’Connell, P. J., L. Wei, Z. Wang, S. M. Specht, A. J. Longar, A. G. Thompson. 2002. Immature and mature CD8⁺ dendritic cells prolong the survival of vascularized heart allografts. J. Immunol. 168:143.[Abstract/Free Full Text]
42. Pulendran, B., J. Lingrappa, M. K. Kennedy, J. Smith, M. Teepe, A. Rudensky, C. R. Maliszewski, E. Maraskovsky. 1997. Developmental pathways of dendritic cells in vivo: distinct function, phenotype, and localization of dendritic cell subsets in Flt3 ligand-treated mice. J. Immunol. 159:2222.[Abstract/Free Full Text]
43. Reis e Sousa, C., S. Hieny, T. Scharton-Kersten, D. Jankovic, H. Charest, R. N. Germain, A. Sher. 1997. In vivo microbial stimulation induces CD40 ligand-independent production of interleukin-12 by dendritic cells and their redistribution to T cell areas. J. Exp. Med. 186:1819.[Abstract/Free Full Text]
44. Maldonado-Lopez, R., T. de Smedt, P. Michel, J. Godfroid, B. Pajak, C. Heirman, K. Thielemans, O. Leo, J. Urbain, M. Moser. 1999. CD8⁺ and CD8^- subclasses of dendritic cells direct the development of distinct T helper cells in vivo. J. Exp. Med. 189:587.[Abstract/Free Full Text]
45. Ohteki, T., T. Fukao, K. Suzue, C. Maki, M. Ito, M. Nakamura, S. Koyasu. 1999. Interleukin 12-dependent interferon- production by CD8⁺ lymphoid dendritic cells. J. Exp. Med. 189:1981.[Abstract/Free Full Text]
46. Pulendran, B., J. L. Smith, G. Caspary, K. Brasel, D. Pettit, E. Maraskovsky, C. R. Maliszewski. 1999. Distinct dendritic cell subsets differentially regulate the class of immune response in vivo. Proc. Natl. Acad. Sci. USA 96:1036.[Abstract/Free Full Text]
47. De Smedt, T., E. Butz, J. Smith, R. Maldonado-Lopez, B. Pajak, M. Moser, C. Maliszewski. 2001. CD8^- and CD8⁺ subclasses of dendritic cells undergo phenotypic and functional maturation in vitro and in vivo. J. Leukocyte Biol. 69:951.[Abstract/Free Full Text]
48. Huang, L. Y., C. Reis e Sousa, Y. Itoh, J. Inman, D. E. Scott. 2001. IL-12 induction by a Th1-inducing adjuvant in vivo: dendritic cell subsets and regulation by IL-10. J. Immunol. 167:1423.[Abstract/Free Full Text]
49. Alegre, M.-L., H. Shiels, C. B. Thompson, T. F. Gajewski. 1998. Expression and function of CTLA-4 in Th1 and Th2 cells. J. Immunol. 161:3347.[Abstract/Free Full Text]
50. Fecteau, S., G. P. Basadonna, A. Freitas, C. Ariyan, M. H. Sayegh, D. M. Rothstein. 2001. CTLA-4 up-regulation plays a role in tolerance mediated by CD45. Nat. Immunol. 2:58.[Medline]
51. Rothstein, D. M., M. F. A. Livak, K. Kishimoto, C. Ariyan, H.-Y. Qian, S. Fecteau, M. Sho, S. Deng, X. X. Zheng, M. H. Sayegh, G. P. Basadonna. 2001. Targeting signal 1 through CD45RB synergizes with CD40 ligand blockade and promotes long-term engraftment and tolerance in stringent transplant models. J. Immunol. 166:322.[Abstract/Free Full Text]
52. Diehl, L., A. Th. Den Boer, S. P. Schoenberger, E. I. H. van der Voort, T. N. M. Schumacher, C. J. M. Melief, R. Offringa, R. E. M. Toes. 1999. CD40 activation in vivo overcomes peptide-induced peripheral cytotoxic T-lymphocyte tolerance and augments anti-tumor vaccine efficacy. Nat. Med. 5:774.[Medline]
53. Sotomayor, E. M., I. Borrello, E. Tubb, F.-M. Rattis, H. Bien, Z. Lu, S. Fein, S. Schoenberger, H. I. Levitsky. 1999. Conversion of tumor-specific CD4⁺ T-cell tolerance to T-cell priming through in vivo ligation of CD40. Nat. Med. 5:780.[Medline]

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