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The Journal of Immunology, 2006, 177: 2131-2137.
Copyright © 2006 by The American Association of Immunologists

Type 2 Cytotoxic T Lymphocytes Modulate the Activity of Dendritic Cells Toward Type 2 Immune Responses1

Giandomenica Iezzi2,3, Andrea Boni2,4, Elena Degl’Innocenti, Matteo Grioni, Maria T. S. Bertilaccio and Matteo Bellone5

Cancer Immunotherapy and Gene Therapy Program, Istituto Scientifico H San Raffaele, Milan, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Activated CD8+ T cells can differentiate into type 1 (Tc1) cells, producing mainly IFN-{gamma}, and type 2 (Tc2) cells, producing mostly IL-4, IL-5, and IL-10. Tc1 cells are potent CTL involved in the defense against intracellular pathogens and cancer cells. The role of Tc2 cells in the immune response is largely unknown, although their presence in chronic infections, cancer, and autoimmune diseases is associated with disease severity and progression. Here, we show that mouse Tc2 cells modify, through a cell-to-cell contact mechanism, the function of bone marrow-derived dendritic cells (DC). Indeed, Tc2-conditioned DC displayed a reduced expression of MHC class II and costimulatory molecules, produced IL-10 instead of IL-12, and favored the differentiation of both naive CD4+ and CD8+ T cells toward type 2 cells in the absence of added polarizing cytokines. The novel function for Tc2 cells suggests a type 2 loop in which Tc2 cells modify DC function and favor differentiation of naive T cells to type 2 cells. The type 2 loop may at least in part explain the unexpected high frequency of type 2 cells during a chronic exposure to the Ag.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Similarly to CD4+ T cells (1), CD8+ CTL (Tc)6 can differentiate upon antigenic stimulation into Tc1 cells, which are characterized by high levels of IFN-{gamma} production, or into Tc2 cells, which produce mainly IL-4, IL-5, IL-10, and TGF-beta (2, 3, 4).

Tc1 cells, because of their capacity to efficiently migrate to inflamed tissue (5) where they secrete IFN-{gamma} and kill their targets, are a major defense against tumor and virus-infected cells (5, 6, 7, 8).

Less is known about the function of Tc2 cells, which have been found in chronic human pathologies such as viral infections (4, 9), cancer (10, 11), and neurologic (12) and autoimmune diseases (13). Of relevance, the presence of Tc2 cells has been frequently correlated with disease severity and progression (9, 10, 12). In some circumstances, IL-10-secreting human CD8+ T cells may act as regulatory (Tr) cells (9, 14, 15).

We recently observed (A. Boni, E. Iezzi, E. Degl’Innocenti, M. Grioni, A. Camporeale, and M. Belone, submitted for publication) that bone marrow-derived dendritic cells (DC) exposed to maturation stimuli for 48 h (48h-DC) in the presence of IL-4 favored naive CD8 T cells to differentiate to Tc2 cells that efficiently killed their targets and produced IL-4, IL-5, IL-10, IL-13, IFN-{gamma}, and TGF-beta. Additionally, 48h-DC and not untreated DC or recently activated (8 h) DC were particularly prone to induce IL-10-secreting Tc2 cells. We investigated here the function of 48-DC-induced Tc2 cells.

We found that Tc2 and not Tc1 effectors impaired the capacity of DC to drive Th1 and Tc1 polarization through a cell-to-cell contact mechanism and promoted Th2/Tc2 expansion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and reagents

Eight- to 10-wk-old female C57BL/6 (Charles River Laboratories), C57BL/6-Tg(TcraTcrb)425Cbn/J (OT-1) (16), and C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-2) (17) mice (provided by W. R. Heath, University of Melbourne, Parkville, Victoria, Australia) were housed in a specific pathogen-free animal facility and treated with the approval of the Institutional Ethical Committee of the Istituto Scientifico H San Raffaele (Milan, Italy). Unless specified, chemical reagents were from Sigma-Aldrich, and mAb and ELISA kits were from BD Pharmingen.

Generation of Tc1 and Tc2 cells

Tc1 and Tc2 cells were obtained from lymphoid organs of OT-1 mice as described (A. Boni, E. Iezzi, E. Degl’Innocenti, M. Grioni, A. Camporeale, and M. Belone, submitted for publication). Briefly, naive OT-1 cells (106/ml) were cultured with DC (see below; 5:1 ratio), exposed to LPS (1 µg/ml) for 48 h, and loaded with 100 ng/ml SIINFEKL peptide (18) (Research Genetics) in RPMI 1640 supplemented with penicillin-streptomycin, 10 mM HEPES, 10 mM sodium pyruvate, 50 µM 2-ME, 10% heat-inactivated FCS (19), and IL-12 (3.5 ng/ml) or IL-4 (5 ng/ml; R&D Systems). On day 6 of culture, Tc1 and Tc2 cells were analyzed for surface molecule expression and intracellular cytokine production (ICP) by FACS (19). Cytolytic activity was investigated by a standard 51Cr release assay (19). For enrichment of IL-10-producing cells, Tc2 cells were stimulated for 3 h with anti-CD3 (5 µg/ml) and anti-CD28 (3 µg/ml) mAb and sorted by anti-IL-10 magnetic microbeads (Miltenyi Biotec).

Functional characterization of DC

Bone marrow-derived DC cells were obtained from C57BL/6 mice as reported (19). On day 7 of the in vitro culture, nonadherent and loosely adherent cells were collected and evaluated for mycoplasma contamination by PCR (positive cultures were discarded). DC were also analyzed by FACS after incubation with normal mouse serum for 15 min at 4°C and double staining with the PE-conjugated anti-CD11c mAb and one of the indicated FITC- or biotin-conjugated mAb. Propidium iodide or TOPRO-3 was added immediately before sample acquisition to exclude dead cells. Biotin-conjugated mAb specific for programmed death (PD) 1 and PD ligands (PDL) 1 and 2 were from eBioscience. Alternatively, cells were assessed for ICP (biotin-labeled anti-CD11c followed by CyChrome-labeled streptavidin, PE-conjugated anti-IL12 p70, or FITC-conjugated anti-IL-10 mAb) by FACS after 4 h of stimulation with PMA (60 ng/ml) and ionomycin (1 µg/ml) (19) and by ELISA (IL-12 kit) on culture supernatants.

Tc-DC cocultures

Tc1 and Tc2 cells were cocultured at ratios from 5:1 to 1:1 with 106 DC untreated or exposed overnight to LPS and pulsed or not pulsed with SIINFEKL (10 ng/ml). When needed, neutralizing anti-IL-10 mAb (5 µg/ml; clone JES5-2A5) were added at the beginning of the coculture. After 18 h, cells were analyzed by FACS as described above. Cells were also triple stained by biotin-labeled anti-CD11c, followed by CyChrome streptavidin, FITC-labeled anti-I-Ab, and PE-labeled CaspaTag kit (red caspase (VAD) activity kit; Intergen). Alternatively, T cells were seeded together with Ag-pulsed DC in the upper chamber of a Transwell plate (0.2-µm diameter pore; Corning, Costar), and FACS analyses were performed on unpulsed DC placed in the lower chamber. In other experiments, the cocultured cells were sorted using anti-CD8 magnetic microbeads (Miltenyi Biotec). DC in the negative fraction were either stimulated with PMA/ionomycin and analyzed for ICP (see above) or pulsed with 10 ng/ml SIINFEKL or OVA323–339, irradiated (3000 rad), and seeded at 1:5 or 1:10 ratio with naive OT-1 and OT-2 cells that had been previously stained with CFSE (5 µM) (19). When needed, anti-IL-10 mAb were added as described above. At day 5, cells were analyzed for ICP (IL-4, IL-10, and IFN-{gamma}) as described above.

In vivo experiments

CD45.2+ Tc1 or Tc2 cells, generated as described above, were adoptively transferred i.v. in congenic CD45.1+ hosts (107/mouse). Twelve hours later, equal numbers of CD45.2+ naive OT-2 cells were injected i.v. After one additional day, mice were given a mixture of OVA protein (500 µg) plus SIINFEKL peptide (50 µM) and LPS (10 ng) i.v. Six days later, mice were sacrificed and their splenocytes were either assayed for ICP after 6 h of PMA/ionomycin stimulation or seeded overnight at 106/ml CD45.2+ cells (quantified by flow cytometry) together with OVA323–339 (1 µM) and anti-IL-4R mAbs (5 µg/ml; Genzyme). Cytokine production was measured by ELISA (IL-4 kit) on supernatants.

Statistical analysis

Statistical analyses were performed using the Student t test. Values were considered statically significant for p ≤ 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Ag specific Tc2 cells modulate phenotype and function of Ag-carrying DC

Polarized Tc1 and Tc2 cells were obtained by culturing naive TCR transgenic OT-1 cells (16), with 48h-DC pulsed with SIINFEKL in the presence of exogenous IL-12 or IL-4, respectively (Fig. 1). Tc1 cells (up to ~80%) produced high amounts of IFN-{gamma} but did not contain IL-10- or IL-4-secreting cells. On the contrary, Tc2 cells contained significant proportions of IL-4- and IL-10-producing cells (~15% and 13%, respectively) and a small fraction of cells secreting low IFN-{gamma} levels. Both Tc1 and Tc2 cells exhibited high levels of CD25 and CD44 and intermediate-to-low levels of CD62L (7); when tested in a 51Cr release assay, Tc2 cells were similarly efficient to Tc1 against the specific targets (A. Boni, E. Iezzi, E. Degl’Innocenti, M. Grioni, A. Camporeale, and M. Belone, submitted for publication).


Figure 1
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  		FIGURE 1. In vitro induction of Tc1 and Tc2 cells. Naive OT-1 cells were cocultured with SIINFEKL-pulsed 48h-DC in the absence (Unp) or presence of exogenous IL-12 (Tc1) or IL-4 (Tc2). On day 6 of culture, primed cells were tested for ICP by FACS (FL1-H and FL2-H, fluorescence channels 1 and 2, respectively). The percentage of positive cells is indicated in each quadrant. Data are representative of at least three independent experiments.

 
Because we found that a sizable fraction of Tc2 cells was also producing IL-10 (Fig. 1), we addressed the effects of Tc2 cells on DC during a secondary immune response. Unpulsed or Ag-loaded DC not exposed to LPS were incubated with Tc1 or Tc2 cells for 18 h and analyzed by FACS. Ag-pulsed DC, upon exposure to increasing numbers of Tc2 cells, displayed a strong and dose-dependent reduction in the expression of I-Ab, CD40, CD80, and CD86 molecules (Fig. 2A). In contrast, Ag-loaded DC exposed to Tc1 cells displayed unmodified or even increased levels of costimulatory and especially I-Ab molecules, predominantly at low T:DC ratios (Fig. 2A). Such a phenomenon was already evident after 1 h of coculture with Tc2 and reached a plateau within the first 6 h (Fig. 2B). This phenomenon was also independent of the maturation stage of the DC, because Tc2 cells also caused down-regulation of I-Ab and costimulatory molecules on LPS-treated DC (Fig. 2C), but it was Ag dependent because Tc-DC cocultures in the absence of the nominal Ag altered the DC phenotype only marginally (Fig. 2A). A slight reduction in the expression of Kb, the restriction element for OT-1 cells (16), was present in all cultures irrespective of the presence of the Ag and the Tc population tested (Fig. 2A).


Figure 2
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  		FIGURE 2. Tc2 cells modulate the phenotype of DC. Bone marrow-derived DC were generated by 7 days in vitro culture in the presence of GM-CSF and IL-4 (see Materials and Methods). A, DC not exposed to maturation stimuli were unloaded (empty symbols) or loaded with SIINFEKL (filled symbols), coincubated overnight with Tc1 or Tc2 cells at the indicated T:DC ratio, double stained for CD11c and the indicated surface molecules, and analyzed by FACS. We gated live CD11c+ cells. For each molecule analyzed, MFI value is shown. Expression level on DC cultured alone (Tc2:DC ratio = 0) is shown as control. B, Sixteen, 12, 6, and 1 h before retrieval, DC were left unpulsed (black circles) or pulsed with 10 ng/ml SIINFEKL peptide (white circles) and cocultured with Tc2 cells (ratio 1:2.5). Cells were analyzed by FACS after staining with CD11c, CD8, and TOPRO-3 (to exclude dead cells) and either Kb or CD80. Values are expressed as MFI. C, Forty-eight hours before retrieval, LPS (1 µg/ml) was added to the DC cultures. Then, DC were loaded with SIINFEKL, coincubated overnight with Tc2 cells at 1:2 ratio, double stained for CD11c and the indicated surface molecules, and analyzed by FACS as described above. Histograms illustrate the expression of specific markers on CD11c+ cells alone (white profiles) or cocultured (black profiles) with Tc2 cells. Gray profiles represent isotype controls. Representative examples of at least three independent experiments are shown for each panel.

 
Tc-conditioned DC were also analyzed by FACS for ICP. Approximately 10% of the untreated CD11+ cells produced IL-12, with a few cells (<2%) producing IL-10 (Fig. 3A). These data are not surprising, because it is well known that in vitro manipulated DC may exhibit different levels of spontaneous maturation (20). Strikingly, coculture with Tc2 cells inverted the ratio between IL-12 and IL-10 DC producers. None of the Tc1-exposed DC was IL-10+ or IL-12+. A possible explanation for the apparent lack in IL-12 production by DC after coculture with Tc1 cells was an early cytokine release (21). Indeed, when measured by ELISA, almost 400 pg/ml IL-12 was detected in the DC-Tc1 supernatant, an amount higher than what was found in the supernatant from DC alone (Fig. 3B). As expected, IL-12 was low in the DC-Tc2 coculture (Fig. 3B). To verify whether DC conditioned by Tc cells were still able to release IL-12 at the end of the coculture, CD8+ cells were sorted out by magnetic beads, and equal numbers of the different DC populations were seeded overnight with (1 µg/ml) or without LPS. In the absence of LPS, only DC not precultured with Tc cells produced IL-12 in the range of 100 pg/ml (Fig. 3B). In the presence of LPS, IL-12 production was augmented in all of the wells except the ones containing DC from Tc2 cocultures, therefore suggesting that the Tc2-induced cytokine switch was irreversible.


Figure 3
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  		FIGURE 3. Tc2 cells modulate cytokine production of DC. A, DC loaded with SIINFEKL were cultured overnight alone (none) or together with Tc1 or Tc2 cells (1:2 ratio), double stained for CD11c and the indicated intracellular cytokine, and analyzed by FACS for ICP. Values are expressed as percentage ± SD of triplicates of double-positive cells. B, Supernatants from the cocultures described above (On) were analyzed for the presence IL-12 by ELISA. After coculture, CD8+ cells were sorted out, and equal number of DC were seeded overnight in new wells with or without LPS (1 µg/ml). IL-12 content was measured by ELISA. C, Untreated DC were cocultured overnight with Tc1, Tc2, and IL-10– Tc2 cells (DC:Tc ratio 1:1.5) or IL-10+ Tc2 cells (ratio 1:1) and analyzed by FACS as described above. Values are expressed as the percentage of expression of the indicated marker, where 100% is given to DC coincubated with Tc1 cells. Each panel is representative of at least three independent experiments.

 
To address which Tc2 subpopulation (see Fig. 1) was directly involved in DC conditioning, we enriched Tc2 cells for IL-10+ and IL-10– cells by magnetic beads and tested their ability to induce surface molecule down-regulation on untreated DC. Coculture with Tc1 cells did not alter I-Ab and CD86 expression on DC. These mean fluorescent intensity (MFI) values were considered as the standard. Tc2 cells and IL-10+-enriched Tc2 cells modulated the expression of I-Ab and CD86 on DC, whereas IL-10– Tc2 cells did not (Fig. 3C), therefore suggesting that IL-10+ Tc2 cells were responsible for DC conditioning.

Tc2-induced conditioning of DC requires cell-cell contact, is not IL-10-mediated, and is not related to the cytolytic activity of Tc2 cells

To evaluate whether IL-10 or other potentially inhibitory cytokines produced by Tc2 cells (such as TGF-beta) could play a role in MHC and costimulatory molecule down-regulation, Ag-loaded DC were cultured with Tc1 or Tc2 cells in the presence of neutralizing anti-IL-10 mAb or in a Transwell system to prevent cell-cell interaction. The fact that the dose of anti-IL10 mAb given was enough to block any possible IL-10 activity was confirmed by preliminary experiments in which the same amount of mAb inhibited the capability of IL-10 to prevent the in vitro differentiation of bone marrow precursors to DC and favor the induction of macrophages (data not shown), as previously reported for human monocyte-derived DC (22). Neutralization of IL-10 did not affect Tc2-induced down-regulation of DC surface molecules, whereas the phenomenon was abrogated in Transwell plates (Fig. 4A), thus suggesting that Tc2 cells acted through a cell-to-cell interaction.


Figure 4
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  		FIGURE 4. Mechanism of DC conditioning by Tc2 cells. A, Tc2 cells were coincubated overnight with SIINFEKL-pulsed DC (Tc2:DC ratio = 2.5:1) in the absence or presence of neutralizing anti-IL-10 mAb ({alpha}IL-10), stained, and analyzed as described in the legend to Fig. 1. Alternatively, Tc2 and SIINFEKL-pulsed DC were coincubated in the upper chamber of a Transwell system (tw) in the presence of an equal number of DC in the lower chamber. FACS analysis was performed on the latter DC population. MFI values are shown for each of the molecules analyzed. B, unpulsed (empty symbols) or SIINFEKL-pulsed DC (filled symbols) were cocultured overnight alone (none) or in the presence of Tc1 or Tc2 cells (T:DC ratio = 1:1) and double stained for CD11c and CaspaTag before FACS analysis. Data are expressed as the percentage of double-positive cells. Statistical analyses (Student t test) for SIINFEKL-pulsed DC: none vs Tc1, p < 0.05; Tc1 vs Tc2, p < 0.02; none vs Tc2, not significant. C, DC not exposed to maturation stimuli were loaded with SIINFEKL and cultured overnight alone (black columns) or with Tc2 cells (Tc2:DC ratio = 2.5:1), double stained for CD11c and the indicated surface molecules, and analyzed by FACS. We gated live CD11c+ cells. Values are expressed as percentage of expression of the indicated marker, where 100% is given to DC cultured in the absence of Tc2. A and B are representative examples of at least three independent experiments. C is representative of two independent experiments.

 
Because Tc2 cells did not express TGF-beta on the cell surface (data not shown), we excluded a role for membrane-bound TGF-beta (23) in Tc2-induced DC modulation.

Because both Tc1 and Tc2 cells displayed a strong cytolytic activity against peptide-pulsed targets (A. Boni, E. Iezzi, E. Degl’Innocenti, M. Grioni, A. Camporeale, and M. Belone, submitted for publication), we verified whether down-modulation of surface molecules on DC was due to a selection imposed by Tc cell killing within the DC population. FACS detection of caspase activation, one of the most proximal events in apoptosis, in DC after 18 h of coculture with Tc1 and Tc2 cells showed that DC coincubated with Tc2 cells displayed only slightly higher levels of caspases when compared with DC cultured in the absence of Tc cells (Fig. 4B). Conversely, coculture of peptide-loaded DC with Tc1 cells caused apoptosis in 60% of the DC (Fig. 4B), therefore excluding cytotoxicity as a major mechanism in DC conditioning by Tc2 cells.

Because Tc2-induced conditioning of DC requires cell-cell contact, we extended the FACS analysis to a larger panel of costimulatory molecules expressed on DC that may interact with Tc2 cells. Together with MHC class I and class II, CD80, and CD86 (Fig. 2), untreated DC expressed high levels of PDL-1, PDL-2, and ICAM-1 (data not shown), moderate levels of CD40 (Fig. 2) and OX40 ligand (OX40L), and were negative for VCAM-1 and PD-1 (data not shown). Overnight exposure to LPS caused up-regulation of all the expressed molecules, whereas VCAM-1 and PD-1 remained negative (data not shown). As for the molecules reported in Fig. 2, coculture of untreated DC with Tc2 cells drastically affected the expression of OX40L, PDL-1, PDL-2, and ICAM-1, therefore suggesting that Tc2-conditioning involves several intracellular signaling pathways.

Tc2-conditioned DC polarize naive cells toward a type-2 phenotype both in vitro and in vivo

As a direct consequence of MHC and costimulatory molecule down-modulation and cytokine production alteration, DC might display a decreased APC function. Thus, we tested the ability of untreated, Tc1-exposed, or Tc2-exposed DC to induce proliferation and cytokine production by naive TCR transgenic CD8+ (OT-1) and CD4+ (OT-2) cells in response to the specific antigenic peptide. Naive OT-1 and OT-2 cells were labeled with CFSE and cultured with peptide-loaded untreated, Tc1-exposed, or Tc2-exposed DC. At day 5, cocultures were assessed for proliferation (measured as decrease in the expression of CFSE) and ICP. We did not find any substantial difference in the extent of T cell proliferation among the different cocultures (Fig. 5). In the absence of any added polarizing cytokine, Tc2-exposed DC (at the ratio of 1:10 or 1:5) induced OT-1 cells to produce IL-4 (10 ± 4% cells; Fig. 5A) and IL-10 (20 ± 8%; not shown). Untreated and Tc1-exposed DC induced a higher percentage of cells producing IFN-{gamma} (42 ± 3 and 59 ± 17, respectively; Fig. 5A) than Tc2-exposed DC (19 ± 7%; Fig. 5A) and no or marginal IL-4 (2 ± 1 and 1 ± 1%; Fig. 4A) and IL-10 (2 ± 2 and 1 ± 1%) (DC vs DC plus Tc2: IL-4, p < 0.04; IFN-{gamma}, p < 0.02; and IL-10, p < 0.05; DC plus Tc1 vs DC plus Tc2: IL-4, p < 0.04; IFN-{gamma}, p < 0.01; and IL-10, p < 0.05; and DC vs DC plus Tc1: not statistically significant for all cytokines). Neutralization of IL-10 by mAb did not affect Tc2-conditioned DC function (not shown). OT-2 cells behaved similarly to OT-1 cells (Fig. 5B) except for IL-10, which was never detected. Also in this case, the phenomenon appeared to be independent of the maturation stage of the DC. Indeed, DC exposed overnight to LPS and cocultured with Tc2 cells down-regulated MHC and costimulatory molecules (Fig. 2C) and skewed naive OT-1 cells toward a Tc2 phenotype (Fig. 5C).


Figure 5
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  		FIGURE 5. Tc2-exposed DC induce Tc2/Th2 cell polarization. DC left untreated (A and B) or exposed to LPS overnight (C) after an overnight coculture with Tc1 and Tc2 cells were negatively selected by anti-CD8–-coated beads sorting, pulsed with SIINFEKL (A and C) or OVA223–239 (B), and cocultured with CFSE-labeled naive OT-1 (A and C) or OT-2 (B) cells, respectively. Blasts were analyzed for ICP by FACS at day 6. Dot plots show the level of IFN-{gamma} and IL-4 (A and B) or IL-13 (C) production vs CFSE fluorescence (FL1-H and FL2-H, fluorescence channels 1 and 2, respectively). The percentage of positive cells is indicated in each quadrant. Data are representative of at least three independent experiments. No or only marginal proliferation was detected when OT-1 and OT-2 cells were cultured alone or in the presence of unpulsed DC from any population tested (data not shown).

 
To test the capacity of Tc2 cells to induce DC conditioning in vivo, Tc1 or Tc2 effectors (CD45.2+) from OT-1 mice were adoptively transferred into congenic wild-type mice (CD45.1+) together (12 h apart) with naive OT-2 cells (CD45.2+) and challenged with a mixture of OVA protein, SIINFEKL peptide, and LPS. Animals were killed 6 days after Ag challenge, and the cytokine profile of expanded OT-2 cells was evaluated ex vivo. As expected from the in vitro results (Fig. 5B), ICP analysis showed that Tc2 cells favored a 3-fold enrichment of OT-2 effectors producing IL-4 (Fig. 6B) and slightly reduced numbers of IFN-{gamma}-producing cells (Fig. 6A) as compared with OT-2 effectors from Tc1-transferred mice (IL-4 Tc1 vs Tc2, p < 0.02; IFN-{gamma} Tc1 vs Tc2, p < 0.13; n = 3). The ICP findings were confirmed by an even more striking difference in IL-4 content found by ELISA in the supernatants from splenocytes collected from Tc2- and Tc1-transferred mice and cultured overnight in the presence of OVA323–339 (Fig. 6C). Thus, adoptively transferred Tc2 cells favored polarization of in vivo ongoing immune responses toward a Th2-phenotype.


Figure 6
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  		FIGURE 6. Tc2 effectors promote Th2 cell development in vivo. CD45.2+ Tc1 or Tc2 effectors were adoptively transferred in CD45.1+ wild type recipients together (12 h apart) with naive CD45.2+ OT-2 cells. Mice were immunized with a mixture of OVA protein, SIINFEKL peptide, and LPS i.v. Six days later, splenocytes were tested for ICP after stimulation with PMA/ionomycin (A and B) or seeded in vitro with OVA323–339 (C). A and B, Numbers of IFN-{gamma} and IL-4 producing cells within the fraction of CD4+ CD45.2+ cells. C, IL-4 content in supernatants after a 12-h culture with OVA323–339. Average ± SD is shown. Mice analyzed per group, n = 3.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We show here a previously unrecognized population of Tc2 cells that induce down-modulation of MHC and costimulatory molecules and a cytokine switch in cocultured DC. Tc2-conditioned DC did not block the proliferation of naive T cells but favored the induction of type 2 CD4+ and CD8+ T cells. Tc2-mediated modulation was independent of the DC maturation stage but was strictly dependent on the presence of the nominal Ag on targeted DC. DC conditioning occurred through a cell-to-cell contact mechanism and did not depend on soluble factors or membrane-bound TGF-beta. Nevertheless, IL-10+ Tc2 cells appear to be the main factors responsible for DC conditioning, thus suggesting a strong association between IL-10 production ability and modulating capacity. It is therefore possible that, despite being distinct events, IL-10 secretion and DC conditioning capacities are acquired by Tc2 cells at the same time, perhaps favored by similar microenvironmental factors.

The molecules directly involved in DC modulation by Tc2 cells still need to be identified. OX40 has been described as a costimulatory molecule involved in Th2 polarization (24). OX40 was slightly but consistently up-regulated in Tc2 cells when compared with naive or Tc1 OT-1 cells (A. Boni, E. Iezzi, E. Degl’Innocenti, M. Grioni, A. Camporeale, and M. Belone, submitted for publication). OX40L is marginally expressed on immature and recently activated DC (data not shown), whereas it is strongly up-regulated on 48h-DC (25). As for several other costimulatory molecules, DC-Tc2 cocultures caused down-regulation of OX40L on DC. Hence, it might be possible that OX40-OX40L interaction is actively involved in the generation and maintenance of the Tc2 loop described herein. Also, the role of PD-1-PDL-1/PDL-2 molecules, which have already been reported to be expressed by DC and up-regulated upon activation (26), needs to be explored in the phenomenon described herein. Untreated DC expressed PDL-1 and PDL-2 but not PD-1. PDL molecules were overexpressed upon LPS stimulation (data not shown) and down-regulated upon Tc2 interaction. Because PDL-1/PD-1 interactions may lead to either tolerance (27) or exhaustion (28) of CD8 T cells, and in our model we did not experience either one of the two, we speculate that down-regulation of PDL molecules on Tc2-conditioned DC may have avoided tolerance/exhaustion of T cells and favored the type 2 loop.

CD8+ T cells producing IL-10 are reminiscent of Tr cells. Different subpopulations of CD8+ Tr cells have been described to date that, in humans, may belong to three subtypes (29). Type 1 Tr cells in particular suppress T cells by altering the expression of costimulatory molecules on DC through cell-to-cell contact, therefore resembling the Tc2 population identified here. Nevertheless, the CD8+ T cell population characterized by us did not induce T cell tolerance and should not be considered as a Tr. It has been also reported that vaccination with immature DC favored the induction of IL-10-secreting CD8+ Tr cells (15) whose function required cell-to-cell contact and was largely independent of IL-10 (30). Under our experimental conditions, induction of Tc2 cells was largely facilitated by 48h-DC and exogenous IL-4 (A. Boni, E. Iezzi, E. Degl’Innocenti, M. Grioni, A. Camporeale, and M. Belone, submitted for publication). Hence, depending on their maturation status, DC can favor the induction of Tc2 cells with different effector functions. Our results may suggest that the induction of Tc2 cells with a DC-conditioning potential is favored in the late phases of an immune response (see below).

In mice, an additional population of CD8+ Tr cells has been described that can be induced by vaccination with activated CD4+ T cells and that acts by conventional cytotoxic T cell activity on activated CD4+ T cells expressing the MHC class IB molecule Qa-1 (31). We can exclude the possibility that our Tc2 cells belong to this subtype of CD8+ T cells principally because they did not act through cell lysis. Conversely, Tc1 cells were highly cytolytic against Ag-loaded DC. Nevertheless, DC that survived the Tc1 conditioning were still highly efficient, secreting IL-12 upon LPS stimulation and inducing proliferation and IFN-{gamma} production by naive CD4+ and CD8+ T cells.

Of relevance, upon adoptive transfer into wild-type mice repopulated with naive OT-2 cells, in vitro polarized Tc2 cells were able to skew the phenotype of OT-2 cells toward IL-4-producing cells. Although in vivo detection of IL-4 producing cells is cumbersome (32), we indeed found an at least 3-fold higher expansion of Th2 cells in Tc2-transferred mice when compared with Tc1-transferred animals. These results suggest that DC conditioning may spontaneously occur in vivo.

Recently, Matzinger and collaborators (33) elegantly showed that, after coculture with memory/effector CD4+ T cells from orally tolerized mice, DC preferentially induced naive CD4+ T cells to produce IL-4. DC conditioning required cell-to-cell contact and, at odds with our findings, depended on IL-10. Taken together, our results and their results suggest that both CD4 and CD8 cells producing IL-4 and IL-10 can modify the APC function of DC by favoring type 2 responses. These findings allow us to hypothesize that Tc2 cells, whose induction is favored by DC exposed to maturation stimuli for prolonged periods of time, may exert a role as conditioning cells during the late phase of an ongoing immune response as well as during a chronic exposure to the Ag such as chronic infection, cancer, and autoimmune diseases (4, 9, 10, 11, 12, 13). It would be of particular interest to verify whether the type 2 response found in these pathologic conditions is due at least in part to DC conditioning by Tc2 cells. We are currently investigating this issue in mouse models.

We did not verify whether Tc2 cells directly modulate the function of Tc1 and/or other cell populations. Although this is a likely possibility, we favor the hypothesis that the main targets of conditioning Tc2 cells are professional APCs that orchestrate the immune response.

Besides being effective in the physiologic development of an immune response, modulating CD8+ T cells may be helpful in the clinic. Understanding the molecular mechanisms regulating the generation and action of such type 2-deviating cells may indeed be of interest in developing more effective therapeutic approaches in transplantation and autoimmunity.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Associazione Italiana per la Ricerca sul Cancro, Ministero della Salute, e Ministero dell’Istruzione dell’Università e della Ricerca. Back

2 G.I and A.B. contributed equally to this work. Back

3 Current address: Molecular Biomedicine, Swiss Federal Institute of Technology, Zürich-Schlieren, Switzerland. Back

4 Current address: National Institutes of Health/National Cancer Institute, Building 10, Clinical Research Center, 9000 Rockville Pike, Bethesda, MD 20892-1201. Back

5 Address correspondence and reprint requests to Dr. Matteo Bellone, Cancer Immunotherapy and Gene Therapy Program, Istituto Scientifico H San Raffaele, Via Olgettina 58, 20132 Milan, Italy. E-mail address: bellone.matteo{at}hsr.it Back

6 Abbreviations used in this paper: Tc, CD8+ CTL; Tr, T regulatory; DC, dendritic cell; 48h-DC, DC exposed to maturation stimuli for 48 h; ICP, intracellular cytokine production; MFI, mean fluorescence intensity; OX40L, OX40 ligand; PD, programmed death; PDL, PD ligand. Back

Received for publication October 26, 2005. Accepted for publication May 24, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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