CCR8 Is Expressed by Antigen-Elicited, IL-10-Producing CD4⁺ CD25⁺ T Cells, Which Regulate Th2-Mediated Granuloma Formation in Mice

Abstract

CCR8 was initially described as a Th2 cell-restricted receptor, but this has not been fully tested in vivo. The present study used ex vivo and in vivo approaches to examine the distribution and functional significance of CCR8 among CD4⁺ T cells. Populations of cytokine-secreting CD4⁺ T cells were generated in primed mice with Th1 or Th2 cell-mediated pulmonary granulomas, respectively elicited by i.v. challenge with either Mycobacteria bovis purified protein derivative- or Schistosoma mansoni egg Ag (SEA)-coated beads. Cytokine-producing CD4⁺ T cells were isolated from Ag-stimulated draining lymph node cultures by positive selection. Quantitative analysis of cytokine mRNA indicated enriched populations of IFN-γ-, IL-4-, and IL-10-producing cells. Analysis of chemokine receptor mRNA indicated that IL-10⁺ cells selectively expressed CCR8 in the SEA bead-elicited type 2 response. The IL-10⁺CCR8⁺ populations were CD25⁺ and CD44⁺ but lacked enhanced Foxp3 expression. Adoptive transfer to naive recipients indicated that IL-10⁺ T cells alone could not transfer type 2 inflammation. Analysis of SEA bead-challenged CCR8^−/− mice indicated significantly impaired IL-10 production as well as reductions in granuloma eosinophils. Adoptive transfer of CD4⁺CCR8^+/+ T cells corrected cytokine and inflammation defects, but the granuloma eosinophil recruitment defect persisted when donor cells were depleted of IL-10⁺ cells. Accordingly, local IL-10 production correlated with CCR8 ligand (CCL1) expression and the appearance of CCR8⁺ cells in granulomatous lungs. Thus, IL-10-producing, CCR8⁺CD4⁺CD25⁺CD44⁺ T cells are generated during SEA challenge, which augment the Th2-mediated eosinophil-rich response to the parasite Ags.

Chemokine receptors play an important role in the traf-ficking of immune cells to sites of injury, inflammation, and Ag encounter. These G protein-coupled receptors also regulate multiple cellular functions including growth, differentiation, and programmed cell death (1). Chemokine receptors are differentially expressed by leukocyte subpopulations and their expression is dynamic. T cell chemokine receptor expression changes based upon activation/maturation state, and they are differentially expressed by type 1 (Th1) and type 2 (Th2) Th cells in both human and mouse. Th1 cells are reportedly associated with CXCR3 and CCR5 expression, whereas Th2 cells putatively express CCR3, CCR4, and CCR8 (2–8). Th1 and Th2 cells are distinguished by cytokine production profiles and the nature of the immune response they mediate. Specifically, Th1 cells produce IFN-γ and participate in host cell-mediated responses, whereas Th2 cells are IL-4 and IL-5 producers and are associated with allergic and helminth parasite responses (9). Others and we have shown that Th2-associated cytokines play an important role in mediating the eosinophil-rich granuloma elicited by eggs of the helminth parasite Schistosoma mansoni. We previously reported aberrant Th2 immune responses in vivo in CCR8-deficient (−/−) mice using a model of S. mansoni soluble egg Ag (SEA)³-induced granuloma formation. These mice had no impairment of IL-4-producing cells, yet displayed reduced IL-5 and -13 production, and eosinophil recruitment, and had augmented IFN-γ production in draining lymph node cultures. No impairment was observed when these mice were challenged with Th1-eliciting mycobacterial Ags (10). These results suggested a role for CCR8 in Th2 functional responses in vivo; however, the underlying mechanism was unclear. Because Th2 cells generated in vitro reportedly express CCR8, it was hypothesized that observed defects in CCR8^−/− mice were due to direct effects on Th2 cell function. However, besides being reported on Th2 cells, CCR8 is also expressed by monocytes and macrophages, IL-2-activated NK cells, endothelial cells, and human IL-10-producing T regulatory (Treg) cells (11–14).

In the present study, we used in vivo and ex vivo approaches to test the hypothesis that the aberrant Th2 response observed in CCR8^−/− was due to direct impairment of CD4⁺IL-4⁺ Th2 cell function. Our results dispute this hypothesis and rather suggest that CCR8⁺IL-10⁺ Treg cells are the affected population. Analysis of chemokine receptor transcripts among Ag-elicited CD4⁺ T cells from the lymph nodes of mice undergoing synchronously developing type 1 and type 2 immune responses indicated that IL-10-but not IL-4-secreting cells preferentially expressed transcripts for CCR8 during the type 2 response to SEA. When CD4⁺ T cells were depleted of either CD25⁺ (a Treg marker) cells or CD44⁺ memory cells, there was comparable reduction in IL-10 and CCR8 expression, suggesting the presence of a CD4⁺CD25⁺CD44⁺CCR8⁺ IL-10-producing population. This population did not display Foxp3 expression and thus differed from so-called natural Treg cells (15). Adoptive transfer of the IL-10⁺CD4⁺ T cells to naive recipients was incapable of eliciting an anamnestic Th2 response, indicating they were innately anergic. Examination of CCR8^−/− mice revealed a significant impairment of IL-10 production. Adoptive transfer of CD4⁺ T cells from wild-type mice corrected aberrant type 2 granuloma formation in CCR8^−/− mice but not when IL-10-producing T cells were removed. These findings are consistent with recent reports that helminth infection elicits specialized Treg cells that can support Th2 responses (16, 17). We further show that SEA induce an IL-10-secreting, CD4⁺CD25⁺CD44⁺CCR8⁺ Treg population that appears to be required for full expression of the Th2 response. This population may serve to protect the host from potentially fatal tissue-destructive effects of a Th1-dominated cell-mediated response to tissue-deposited parasite eggs.

Materials and Methods

Animals

Mice lacking CCR8 were generated in the 129Sv × C57BL/6 background as previously described (10). Controls consisted of male CCR8^+/+ 129Sv × C57BL/6 mice obtained from The Jackson Laboratory. Female CBA/J mice were obtained from The Jackson Laboratory. S. mansoni-infected Swiss outbred mice were obtained from Biomedical Research Laboratories. All mice were maintained under specific pathogen-free conditions and provided with food and water ad libitum.

Development of primary and secondary type 1 or type 2 immune response

Secondary type 1 and type 2 lung Ag bead granulomas were generated as previously described (10). Briefly, mice were sensitized with either a s.c. injection of 20 μg of Mycobacteria bovis purified protein derivative (PPD; Department of Agriculture, Veterinary Division, Ames, IA) incorporated into 0.25 ml of CFA (Sigma-Aldrich), or they received an i.p. injection of 3000 S. mansoni eggs in 0.5 ml of PBS. After 14–18 days, mice were challenged by tail vein with 6000 Sepharose 4B beads covalently coupled to either PPD or soluble SEA (World Health Organization, Geneva, Switzerland). To generate primary lung granulomas, naive mice received an i.v. injection of either 6000 PPD beads or 6000 SEA beads. Mice were sacrificed at various time points after bead challenge.

Granuloma and lymph node culture

Following perfusion with cold RPMI 1640, lungs excluding trachea and major bronchi and mediastinal lymph nodes were excised. Lungs were homogenized in a Waring blender. Intact granulomas were collected and digested in RPMI 1640 plus 1000 U/ml type IV collagenase for 25 min at 37°C. Lymph nodes were teased into a single-cell suspension. The cells were cultured in RPMI 1640 plus FBS at 5 × 10⁶ cells/ml in the presence or absence of 5 μg/ml PPD or SEA. Cells were cultured for 24 or 48 h in a 37°C incubator with 5% CO₂. Supernatants were collected by centrifugation and measured by ELISA. Cells were used to isolate cytokine-secreting populations or were lysed for RNA analysis by real-time PCR.

Enrichment and depletion of CD4⁺ T cell subpopulations

To isolate CD4⁺ cytokine-secreting populations, draining lymph node cells were cultured overnight and then labeled with either murine IL-10, IL-4, or IFN-γ catch reagents from MACS cytokine secretion assay kits (Miltenyi Biotec). Cells were incubated for 45 min at 37°C to allow cytokine secretion, enabling the catch reagent to bind to positive, secreting cells. CD3⁺CD4⁺ T cells were negatively selected using a mixture of biotinylated mAbs directed against cell surface Ags on unwanted cells (StemCell Technologies). Cells were incubated with anti-biotin tetrameric Ab complexes and magnetic beads, and passed through the MACS separation column (Miltenyi Biotec). The CD4⁺ cells were labeled with an IL-10, IL-4, or IFN-γ cytokine-specific detection Ab conjugated to PE. Using EasySep positive selection kits (StemCell Technologies), anti-PE Abs and magnetic nanoparticles bind to the PE-labeled cells and allow them to be recovered using the EasySep magnet. CD4⁺ T cells depleted of IL-10 or IL-4 were recovered by collecting those cells that passed through the magnet.

To isolate CD4⁺ cells depleted of CD44 and CD25, biotinylated anti-CD25 or anti-CD44 Abs (BD Pharmingen) were added into the CD4⁺ T cell enrichment mixture. Cells were passed through the MACS separation column as described above.

Cytokine measurement

Murine IL-4, -5, -10, and -13, and IFN-γ were measured from cultured lymph node supernatants by ELISA using commercially available reagents and standards (BD Pharmingen; R&D Systems; PeproTech). Sensitivities fell between 15 and 50 pg/ml.

Morphometry

Individual excised lung lobes were inflated and fixed with 10% buffered formalin for morphometric analysis. Granuloma area was measured in a blinded fashion in H&E-stained sections of paraffin-embedded lungs using computer-assisted morphometry. A minimum of 20 lesions was measured per lung. Only granulomas with full cross-sections of the bead nidus were measured.

Differential analyses of granulomas

After perfusion with cold RPMI 1640, lung lobes were excised, placed in RPMI 1640, and homogenized in a Waring blender. Intact granulomas were collected and digested in RPMI 1640 plus 1000 U/ml type IV collagenase for 25 min at 37°C. After washing, cells were resuspended at 2.5 × 10⁶ cells/ml. A 200-cell differential analysis was performed on duplicate Wright-stained cytospin preparations of dispersed granulomas.

Real-time RT-PCR analysis

Excised lung tissues for RNA isolation were stored in RNA Later (Ambion) at −20°C. Poly(A) pure mRNA was isolated from lung tissue and cultured lymph node cells using poly(A) pure mRNA isolation kits (Ambion). DNA-free DNA removal kit was used to remove any contaminating genomic DNA (Ambion). Each mRNA sample was reverse-transcribed in a 20-μl reaction in a PCR tube using SuperScript II RNase H⁻ Reverse Transcriptase (Invitrogen Life Technologies). Analysis of the transcripts was performed by real-time PCR using the ABI PRISM 7000 Sequence Detection System (Applied Biosystems). For this study, the comparative threshold cycle (CT) method recommended by the manufacturer was adopted. GAPDH acted as the endogenous reference. Primer-probe sets were purchased commercially (Applied Biosystems) except for Foxp3, which was designed according to a previously reported sequence (15). The thermal cycling condition was programmed according to the manufacturer’s instructions. Transcript levels were expressed as arbitrary units and were calculated as previously described (18). Briefly, arbitrary units were calculated from the fluorescence amplification factor as measured by the real-time PCR fluorescent detection unit. The original gene copy number (Co) is related to fluorescence of the generated signal as follows: Co = F × E⁻¹ × I × 2⁻ⁿ, where F is an arbitrary conversion constant, E⁻¹ is amplification efficiency constant (~1 for manufacturer’s real-time primers sets), I is the fluorescent intensity reading, and n is the amplification cycle number. Hence, F × E⁻¹ × I × 2⁻ⁿ constitutes an arbitrary measure of original copy number that is directly related to the fluorescent product and inversely related to cycle number. Because E is roughly equivalent for the various primer sets, the expression levels among genes are comparable at orders of magnitude.

CCR8 knockout (KO) reconstitution protocol

For CCR8 KO reconstitution studies, CD4⁺ T cells, CD4⁺ IL-4⁻ cells, and CD4⁺IL-10⁻ cells were isolated from the spleen of CCR8 wild-type mice. CCR8^+/+ donor mice were sensitized i.p. with 3000 S. mansoni eggs on days 1 and 10. On day 13, spleens were collected, teased into a single-cell suspension, and cultured overnight with Ag. Cells were isolated as described above and were transferred by tail vein injection into CCR8^−/− mice that had been sensitized with S. mansoni eggs 14 days earlier. Recipient CCR8^−/− mice received 1 × 10⁶ cells. Mice were SEA bead challenged the following day and sacrificed 4 days postchallenge.

Adoptive transfer into naive mice

For adoptive transfer studies into naive mice, wild-type mice were challenged with SEA beads 14 days after egg sensitization. Donors were sacrificed 4 days after bead challenge. Draining lymph nodes were collected and cultured overnight with Ag. CD4⁺ cells and CD4⁺IL-10⁺ cells were isolated as described above. A total of 1 × 10⁶ cells was transferred into naive wild-type mice by tail vein injection. The following day, mice were challenged with SEA beads and were sacrificed 4 days later.

Statistical analysis

ANOVA was used for multiple intergroup comparisons with p < 0.05 considered to indicate significance. Pairwise tests were done with Dunnett error protection at 95% confidence interval. The Student’s t test was used for direct comparison to a parallel control group with p < 0.05 considered to indicate significance.

Results

CCR8 transcripts associate with IL-10-producing CD4⁺ T cells

To determine any association between CCR8 and cytokine-producing cells, we initially positively selected IFN-γ, IL-4, and IL-10 cytokine-producing CD4⁺ T cells from the draining mediastinal lymph nodes of mice undergoing anamnestic type 1 or type 2 granulomatous responses, respectively elicited with agarose bead-bound M. bovis PPD or SEA. Because Ag-reactive cytokine-producing cells represent a minute fraction of the CD4⁺ population, cytokine mRNA was measured by quantitative RT-PCR to confirm enrichment of these populations. As shown in Fig. 1A, among the nonenriched CD4⁺ populations, IFN-γ transcripts were more dominant in the type 1 than in the type 2 model consistent with the Th1-dominant nature of the mycobacterial response. Following positive selection of Ag-elicited cytokine-producing cells, there was profound enhancement of corresponding cytokine transcripts in the IFN-γ, IL-4, and IL-10 cytokine-secreting populations (Fig. 1). IL-4- and IL-10-producing cells clearly dominated in the SEA response consistent with Th2 polarization. Interestingly, there was evidence of cytokine-producing subpopulations. IL-10⁺CD4⁺ cells enriched from type 1 nodes also appeared to express low but detectable IFN-γ and IL-4 transcripts, suggesting a Th0-like population as described by others (19), but there was clearly a separate population of cells showing strong IFN-γ expression as evidenced by the 4-fold enrichment among IFN-γ-selected cells. Similarly, there was a 12-fold enhancement of IL-4 mRNA in the IL-4-selected population. Although Th0-like populations were likely also present in type 2 lymph node cultures, most impressive was the 20-fold enrichment of IL-10 mRNA among selected IL-10⁺ cells. This was not associated with a comparable increase in IL-4 or IFN-γ transcripts, suggesting a separate sub-population of IL-10-producing CD4⁺ cells.

FIGURE 1.

Isolation of IFN-γ, IL-4, and IL-10 cytokine-producing CD4⁺ T cells from draining lymph nodes during type 1 (PPD) and type 2 (SEA) granuloma formation. Secondary lung granulomas were induced in CBA/J mice as described in Materials and Methods. On day 3, draining lymph nodes were collected and cultured overnight with Ag. CD4⁺ cytokine-secreting populations were isolated using MACS. The CD4⁺ nonenriched population serves as a control. Relative mRNA transcript levels were measured for IFN-γ (A), IL-4 (B), and IL-10 (C) using real-time PCR. Type 1 (PPD) response (▪); type 2 (SEA) response (▨). Bars are mean arbitrary units ± SD and are derived from three separate experiments.

We next measured chemokine receptor transcripts among these enriched populations to determine any associations with the cytokine-secreting cells. Transcripts for CCR3, CCR4, CCR5, CCR6, CCR7, and CCR8 were measured in each population (Fig. 2). As shown in Fig. 2B, CCR4 transcripts were enriched 2- to 4-fold in IL-4⁺ and IFN-γ ⁺ populations with higher levels noted in the former. Although this finding suggested biased CCR4 expression by Th2 cells, it argues against exclusive CCR4 expression by Th2 cells and supports reports that CCR4 is potentially expressed by both Th1 and Th2 cells (20). CCR3, −5, −6, and −7 showed no association with any of the cytokine-enriched populations. As previously reported, CCR7 transcripts were reduced among Ag-primed compared with naive (Fig. 2E, dashed line) CD4⁺ T cell populations (18). Of the examined receptors, CCR8 displayed the most striking association with IL-10⁺CD4⁺ cells with a 12-fold enhancement of CCR8 transcripts (Fig. 2F). There was no evidence of enriched CCR8 expression among IL-4-secreting cells. The association of CCR8 with IL-10-producing cells was similarly demonstrable in 129 × B6 mice, which had a 5-fold enrichment of CCR8 transcripts among IL-10-positive cells, whereas IL-4 and IFN-γ secretors were <2-fold.

FIGURE 2.

Chemokine receptor transcript association with IL-10-, IL-4-, and IFN-γ-secreting CD4⁺ T cells isolated from draining lymph nodes of CBA/J mice undergoing type 1 or type 2 granulomatous responses. Transcript levels of the chemokine receptors CCR3 (A), CCR4 (B), CCR5 (C), CCR6 (D), CCR7 (E), and CCR8 (F) were measured by real-time PCR. Type 1 (PPD) response (▪); type 2 (SEA) response (▨). E, Dashed line represents transcript levels in naive CD4⁺ T cells. Bars are mean arbitrary units ± SD and are derived from three separate experiments.

Taken together, our results suggest that a variety of cytokine-producing subsets are generated following pathogen Ag challenge, but a strong IL-10-producing CD4⁺ subset with selective CCR8 expression can be identified during the type 2 response to SEA. Furthermore, this population was distinguished from Ag-elicited, CD4⁺, IL-4-producing T cells (classic Th2 cells), which, unlike in vitro-generated Th2 cells, did not appear to selectively express CCR8.

CCR8 and IL-10 transcripts segregate with CD44⁺CD25⁺CD4⁺ T cell populations

In view of previous reports linking CCR8 with human CD25⁺ Treg cells, we investigated whether CD25 was associated with the IL-10⁺CCR8⁺ population that we identified. CD4⁺ T cells derived from draining lymph nodes during type 2 granuloma formation were depleted of either CD25⁺ or CD44⁺ cells, and then transcripts for IL-4, CCR4, IL-10, and CCR8 were measured by real-time PCR. Results are illustrated in Fig. 3. Both CCR4 and IL-4 transcripts were reduced by 64 and 98%, respectively, when CD44⁺ (memory) cells were depleted. Similarly, depletion of CD44⁺ cells eliminated IL-10 and CCR8 transcripts, suggesting an association with memory T cells. When CD25⁺ cells were depleted, there was no effect on CCR4 or IL-4 transcript levels. In contrast, IL-10 and CCR8 transcripts were reduced by 98 and 80% upon CD25⁺ cell depletion. These results suggested the presence of a CD25⁺ T cell subset within the memory population that secretes IL-10 and expresses CCR8 during type 2 pulmonary granuloma formation. Although the production of IL-10 would be consistent with Treg type 1 (Tr1) cells, the expression of CD25 has been associated with so-called “naturally arising” Treg cells (21, 22). The CD25⁺IL-10⁺ population elicited during the type 2 response might represent an overlapping Tr1 population that expresses both IL-10 and CD25 as described by others (23).

FIGURE 3.

Chemokine receptor and cytokine transcript association with CD44⁺ and CD25⁺ activated CD4⁺ T cells isolated from draining lymph nodes of CBA/J mice during type 2 granuloma formation. Following column depletion of either CD44⁺ or CD25⁺ cells, CD4⁺ T cell mRNA transcripts were measured by real-time PCR. Bars are mean arbitrary units ± SD and are derived from two separate experiments. *, p < 0.05 compared with the total CD4⁺ population.

SEA-elicited IL-10-producing cells do not display augmented Foxp3 expression

Naturally arising CD25⁺ Treg cells reportedly express enhanced levels of the transcription factor Foxp3 (15). To determine whether the IL-10-producing populations elicited during SEA granuloma formation selectively expressed this protein, we compared Foxp3 mRNA levels among all CD4⁺ T cells to that of populations of CD25-depleted, IL-10-enriched, and IL-10-depleted CD4⁺ T cells. As shown in Fig. 4, CD25⁺ depletion profoundly reduced Foxp3 transcript levels, suggesting that naturally arising Treg cells were present in the total CD4⁺ population, but these apparently did not produce IL-10 in response to SEA because Foxp3 was not reduced in the IL-10-depleted (IL-10⁻) population. Foxp3 transcript levels were actually reduced among the IL-10-enriched (IL-10⁺) populations, suggesting that these were not “naturally arising” Treg cells. Hence, the CCR8⁺IL-10⁺CD4⁺CD25⁺ T cells were likely generated de novo from naive T cells during the response to SEA.

FIGURE 4.

Foxp3 transcript expression by CD4⁺ T cells and CD4⁺ subpopulations isolated from the draining lymph nodes of mice undergoing type 2 granuloma formation. After overnight culture with Ag, CD4⁺ T cells were either enriched for IL-10 or depleted of CD25 and IL-10. Foxp3 mRNA transcript levels were measured by real-time PCR. Bars are mean arbitrary units ± SD and are derived from two separate experiments. *, p < 0.05 compared with the CD4⁺ control.

Enriched IL-10⁺CD4⁺ T cells fail to adoptively transfer secondary type 2 hypersensitivity granuloma formation

To further characterize the IL-10-secreting CD4⁺ T cell population, they were purified from draining lymph nodes of SEA-sensitized mice and then transferred to naive mice that were subsequently challenged with SEA beads. Control groups received no cells or nonenriched sensitized CD4⁺ T cells. Local type 2 granuloma formation was assessed 3 days after challenge. As shown in Fig. 5, whole CD4⁺ T cell populations effectively transferred an eosinophil-rich, anamnestic type 2 granulomatous response; however, purified IL-10⁺CD4⁺ T cells failed to elicit a full secondary response, although there was a partial recruitment of lesion eosinophils. This finding suggested that, like Treg cells, SEA-elicited CD4⁺IL-10⁺ T cells were anergic or weakly immunogenic when transferred by themselves.

FIGURE 5.

Enriched CD4⁺ IL-10-secreting T cells fail to transfer anamnestic type 2 granulomatous responses to naive mice. Draining lymph nodes were collected from SEA-sensitized and -challenged wild-type (sCCR8^+/+) donors and then cultured overnight with Ag. CD4⁺ and CD4⁺IL-10⁺ enriched cells were isolated from these cultures as described in Materials and Methods, and then 1 × 10⁶ cells were adoptively transferred i.v. into naive wild-type (nCCR8^+/+) syngeneic recipients. Recipient mice were challenged with SEA beads the following day. Granulomas were examined on day 4 after bead challenge. Top panel, Granuloma cross-sectional area; bottom panel, eosinophil content of dispersed granulomas. Data are representative of two separate experiments. Bars are means ± SEM. * p < 0.05 compared with the mice that did not receive cells.

CCR8^−/−mice display impaired IL-10 production in draining lymph node cultures during induction and elicitation phases of the Th2 schistosomal immune response

To help determine the functional relationship of CCR8 and IL-10 production, we examined responses of CCR8 KO (CCR8^−/−) mice. We previously reported that CCR8^−/− mice display partially impaired IL-5 and IL-13 production during type 2 SEA-elicited inflammation but have augmented IFN-γ production (10). To determine whether Th2 cytokine production was affected during the induction or elicitation stages of the immune response, we compared Th2 cytokine profiles in Ag-stimulated draining mediastinal lymph nodes from mice undergoing primary and secondary pulmonary Th2 granulomatous responses to SEA beads. Draining lymph nodes were harvested 4 days after bead challenge. As shown in Fig. 6, IL-5 and IL-13 were 60–70% lower in KO mice during the primary response and 30 – 40% lower upon secondary challenge. IL-10 levels were 80% lower during primary and 70% lower during secondary challenges. As we previously reported, IL-4 was unaffected, suggesting that Th2 cell differentiation was not impaired and that IL-4 was regulated independently of IL-5, IL-10, and IL-13. These findings would also be consistent with the presence of a variety of independent cytokine-producing populations as noted above (Fig. 1).

FIGURE 6.

CCR8 gene knockout is associated with impaired IL-10 production by draining lymph node cultures during primary and secondary type 2 (SEA) granulomatous responses. Primary and secondary lung granulomas were induced in CCR8^+/+ and CCR8^−/− mice as described in Materials and Methods. On day 4, draining lymph nodes were collected and cultured with Ag for 24 or 48 h for secondary or primary responses, respectively. Cytokine levels were determined by ELISA. Bars are means ± SEM of Ag-elicited cytokine levels from a representative experiment. Left sides of panels are primary SEA responses; right sides of panels are secondary SEA responses. CCR8^+/+ mice (▪); CCR8^−/− mice (▨). * p < 0.05 comparing CCR8^+/+ to CCR8^−/− responses.

IL-10⁺CD4⁺CCR8^+/+ T cells reconstitute normal type 2 granuloma formation in CCR8^−/− mice

Our studies of CCR8^−/− mice suggested that CCR8 was required for the full expression of the type 2 response to SEA. We previously demonstrated using Ab-mediated depletion of IL-10 that endogenous IL-10 appears to support the Th2 cytokine production during type 2 pulmonary granuloma formation (24). The source of IL-10 was undetermined at that time and could have been derived from lymphoid and or nonlymphoid sources. Our present findings indicate that schistosomal Ag-elicited CD4⁺CCR8⁺ T cells are a potentially significant source of IL-10. To test whether the aberrant Th2 response observed in CCR8^−/− mice was due to impaired CD4⁺ T cell function, we attempted to reconstitute CCR8^−/− mice by adoptive cell transfer of CCR8^+/+ CD4⁺ T cells.

SEA-sensitized CCR8^−/− recipient mice were i.v. administered purified CD4⁺ T cells from similarly sensitized donor groups of CCR8^+/+ or CCR8^−/− mice. Recipients were challenged with SEA beads, and then inflammatory foci and draining lymph nodes were examined 4 days later. Fig. 7 shows effects on the local inflammatory response and eosinophil content. As shown, adoptive transfer of sensitized CD4⁺CCR8^+/+ T cells did not significantly alter overall lesion size; however, this treatment corrected the impaired eosinophil recruitment observed in CCR8^−/− mice.

FIGURE 7.

Wild-type CCR8^+/+CD4⁺ T cells from SEA-sensitized CCR8 ^+/+ mice reconstitute type 2 granulomatous responses of CCR8^−/− mice. Donor mice were sensitized with 3000 S. mansoni eggs i.p. on days 1 and 10. On day 13, spleens were collected and cultured overnight with Ag. CD4⁺ T cells were isolated using negative selection. A total of 1 × 10⁶ cells was i.v. transferred to SEA-sensitized CCR8^−/− mice. Recipients were challenged with SEA beads the following day. Granulomas were examined on day 4 after bead challenge. Top panel, Granuloma cross-sectional area; bottom panel, eosinophil content of dispersed granulomas. Data are representative of three separate experiments. Bars are means ± SEM.

The above study indicated that wild-type CD4⁺ T cells could correct the defective response in CCR8^−/− mice, but it did not point to a specific CD4⁺ subpopulation in mediating the reconstitution. The effect may have been mediated by populations of classic IL-4⁺ Th2 cells, IL-10⁺ T cells, or another CD4⁺ subset. To distinguish among these possibilities, SEA-sensitized CD4⁺ CCR8^+/+ T cells were depleted of either IL-4- or IL-10-secreting cells before adoptive transfer. Depletion was confirmed by quantitative mRNA analysis for the target cytokines as previously described (18). As shown in Fig. 8, CD4⁺ T cells depleted of IL-4-producing cells restored the normal eosinophil-rich granulomatous response as effectively as the undepleted population and tended to exacerbate the inflammatory reaction. In contrast, when depleted of IL-10-producing cells, the CD4⁺ populations lost the capacity to restore maximum eosinophil recruitment.

FIGURE 8.

Wild-type CCR8⁺CD4⁺ T cells depleted of IL-10-secreting cells fail to reconstitute type 2 granulomatous responses of CCR8^−/− mice. Donor mice were given 3000 S. mansoni eggs i.p. on days 1 and 10. On day 13, spleens were collected and cultured overnight with Ag. CD4⁺ T cells were isolated using negative selection. IL-4- or IL-10-secreting populations were depleted as described in Materials and Methods. A total of 1 × 10⁶ cells was i.v. administered to SEA-sensitized CCR8^−/− mice. Recipients were challenged with SEA beads on the following day. Granulomas were examined on day 4 after bead challenge. Top panel, Granuloma cross-sectional area; bottom panel, eosinophil content of dispersed granulomas. Data are representative of three separate experiments. Bars are means ± SEM.

Surprisingly, assessment of draining lymph node cytokine profiles revealed reconstitution of IL-5 and IL-13 by both IL-4-depleted and IL-10-depleted populations, but there was no reconstitution of IL-10 production (Table I). No significant changes occurred in IL-4 levels. These studies suggested that wild-type CCR8^+/+CD4⁺ T cells harbored non-IL-4- and non-IL-10-producing populations that could migrate to lymph nodes and reconstitute IL-5 and IL-13. In addition, it appeared that IL-10 producers were unable to reach lymph nodes. However, those cells were required to correct the local type 2 inflammatory response.

Table I.

Draining lymph node cytokine levels in CCR8^−/− mice receiving CD4⁺ T cells, CD4⁺ IL-4-depleted cells, and CD4⁺ IL-10-depleted cells isolated from SEA-sensitized CCR8^−/− donors^a

		Cytokine Levels (ng/ml)
Recipients	Donor Cells	IL-4	IL-5	IL-10	IL-13
CCR8+/+	None	0.42 ± 0.10	32.6 ± 1.53	30.7 ± 6.6	44.0 ± 13.7
CCR8−/−	None	1.1 ± 0.43	15.0 ± 10.4	5.2 ± 3.0	13.8 ± 3.5
CCR8−/−	CD4+	1.9 ± 0.45	46.2 ± 9.5*	7.3 ± 2.5	45.4 ± 9.9*
	CCR8+/+
CCR8−/−	CD4+	1.3 ± 0.32	73 ± 13.5*	3.1 ± 1.1	45.7 ± 16.8*
	CCR8+/+
	IL-4 depleted
CCR8−/−	CD4+	1.9 ± 0.52	95 ± 18.9*	5.5 ± 1.6	46.5 ± 5.4*
	CCR8+/+
	IL-10
	depleted

^aCCR8^−/− mice received 1 × 10⁶ cells i.v. as described in Materials and Methods. On day 4 post-bead challenge, draining lymph nodes were collected and cultured overnight with Ag. Cytokine levels were measured by ELISA. Values are means × SEM derived from three separate experiments.

^*, p < 0.05 compared to no-transfer CCR8^−/− group.

CCL1 is induced strongly at sites of schistosomal Ag bead granuloma formation

To determine potential local and regional sites of CCR8⁺CD4⁺IL-10⁺ T cell action, we measured IL-10-producing capacity of cultured granulomas and draining lymph nodes. In addition, transcript levels of CCR8 and its known ligand, CCL1 (TCA-3) were assessed in challenged lungs and draining lymph nodes of wild-type mice. As shown in Fig. 9, IL-10 was detected in granuloma cultures on day 2 and dramatically increased above baseline in lymph node cultures on day 4 after challenge. In challenged lungs, there was a 3500-fold increase in CCL1 transcripts by day 2 followed by the local accumulation of CCR8 transcripts in the lung (Fig. 10). Thus, in lungs, there was a reasonable correlation between CCL1, CCR8, and IL-10 expression. In draining lymph nodes, only marginal levels of CCL1 transcripts were detected, but a 2- to 3-fold increase in CCR8 transcripts occurred on day 4, correlating with IL-10 production. These findings argued that granulomatous lungs were potential sites for the active recruitment of CCR8⁺ cells. The marginal expression of CCL1 in draining lymph nodes suggested that CCR8⁺CD4⁺ T cells were generated de novo in lymph nodes but were likely not actively recruited there by way of CCR8.

FIGURE 9.

IL-10 expression in granulomatous lungs and draining lymph nodes during secondary type 2 (SEA) granulomatous responses. Groups of S. mansoni egg-sensitized CBA/J mice were challenged with SEA beads, and then on days 1, 2, 4, and 8, intact granulomas (1000/ml) and dispersed draining lymph nodes (5 3 10⁶ cells/ml) were cultured, respectively, for 48 and 24 h with 5 μg/ml SEA. IL-10 levels were determined in culture supernatants by ELISA. A, Granuloma cultures; B, lymph node cultures. Three to four mice per point.

FIGURE 10.

CCL1 (TCA-3) (A) and CCR8 (B) transcript induction in lungs and draining lymph nodes during secondary type 2 (SEA) granulomatous responses. Type 2 lung granulomas were induced in groups of S. mansoni egg-sensitized CBA/J mice as described in Materials and Methods. Lungs and draining lymph nodes were collected before bead challenge (day 0) and on days 1, 2, 3, 4, and 8 post-bead challenge. The freshly isolated tissues were used for transcript analysis by real-time PCR. Data are derived from two separate experiments for a total of five to six mice per point.

We next examined CCL1 and IL-10 transcript expression in lungs of CCR8^+/+ and CCR8^−/− mice with day 4 SEA bead granulomas. As shown in Fig. 11A, CCL1 transcripts trended lower in CCR8^−/−, but this did not achieve statistical significance; hence any recruitment defects in CCR8^−/− would be largely due to receptor deletion. In contrast to CCL1, there was a profound reduction of IL-10 transcripts (Fig. 11B), which would be consistent with impaired recruitment or activation of IL-10-producing cells.

FIGURE 11.

CCL1 and IL-10 transcript expression in lungs of CCR8^+/+ wild-type and CCR8^−/− mice during secondary type 2 (SEA) granulomatous responses. Type 2 lung granulomas were induced in groups of S. mansoni egg-sensitized CCR8^+/+ or CCR8^−/− mice as described in Materials and Methods. Lungs were collected on day 3 post-SEA bead challenge. The freshly isolated tissue was used for transcript analysis by real-time PCR. A, CCL1 transcript levels; B, IL-10 transcript levels. Data are derived from four to five individual mice per point.*, p < 0.05 comparing CCR8^+/+ to CCR8^−/− levels.

Discussion

The role of the chemokine receptor CCR8 in immune responses is poorly understood. We previously reported that CCR8^−/− mice had aberrant Th2 immune responses in vivo in a model of helminth, SEA-induced granuloma formation (10). Specifically, CCR8^−/− mice displayed reduced eosinophil recruitment and decreased IL-5 and IL-13 cytokine production, yet had unchanged or augmented IFN-γ production. We also reported that CCR4 and CCR8 transcripts increased among CD4⁺ T cell populations following sensitization with schistosome eggs (18). Others have reported that Th2 cells preferentially express CCR8 (7, 8); thus defects in CCR8^−/− mice may have been due to impaired Th2 cell function. Still other reports have associated CCR8 with human IL-10-producing Treg cells (12, 14). Recently, IL-10-producing Treg cells were identified in mice infected with the helminth S. mansoni and were demonstrated to play a role in promoting or sustaining a Th2-biased response and thereby possibly protecting the host from tissue-destructive Th1-dominant responses (16, 17). In the present study, we used an in vivo approach to investigate whether CCR8 expression was related to Th2 or Treg cell function during the type 2 granulomatous response to SEA.

Our studies demonstrated that CCR8 associated strongly with IL-10 cytokine-producing CD4⁺ T cells isolated from the draining lymph nodes of mice undergoing type 2 granuloma formation. CCR8 expression did not associate with IL-4-producing cells; hence CCR8 is not a marker of classic Th2 cells. In agreement with other reports (25), we detected CCR4 transcript expression in the CD4⁺ IL-4-secreting fractions, but this was not exclusive to Th2 cells as has been purported, because CCR4 mRNA was also detected among IFN-γ-secreting cells elicited with mycobacterial Ag challenge. This has been likewise noted in studies of human T cell chemokine receptor distribution (20).

We found that CCR8⁺IL-10⁺ cells were CD44⁺ and CD25⁺ but were incapable of transferring a secondary inflammatory response. CD25 is constitutively expressed by “naturally arising” anergic Treg cells, and our finding would seem to be in accord with reports demonstrating CCR8 as a marker of human Treg cells (14), but this would likely be a gross oversimplification. Treg cells can be divided into subpopulations including “naturally arising” CD4⁺CD25⁺ Treg, CD25⁻ IL-10-producing Tr1 cells, and TGF-β-producing Treg cells. The former are thymus derived, functionally mature T cells that are characterized by increased expression of the transcription factor Foxp3 (26). These CD4⁺CD25⁺ cells prevent organ-specific autoimmune disease in mice, mediate transplantation tolerance, and may temper pathologic immune responses (27–29). The IL-10-secreting population we observed in our model did not display enhanced Foxp3 expression and thus likely did not represent “naturally arising” Treg cells. Likewise, we could not demonstrate enhanced TGF-β production by this population (data not shown). Tr1 cells are CD4⁺ Ag-specific T cells (22). It has been suggested that induction of Tr1 cells may represent an evasion strategy by pathogens designed to subvert protective Th1 responses. Such a means of immune diversion has been reported in Bordetella pertussis infection (30). Multicellular helminth infections represent a different challenge where Tr1-mediated regulation may be desirable. In schistosomiasis, an early Th1 response switches to a Th2-dominated response after the onset of parasite egg production (31). The Th2-dominant response, although profibrotic, may benefit both host and parasite by preventing massive Th1-mediated bystander organ damage (32, 33). The IL-10-secreting population elicited during SEA challenge appears to most resemble Tr1 cells, but the population was unique in that it appeared to express CD25. CD25 is not a specific marker for Treg cells. It is also a marker for recently activated effector cells. Because our isolation procedures required Ag stimulation, we may have enhanced expression of CD25. Its association with the IL-10-producing population in our model appeared to be selective, because depletion of CD25 had no effect on IL-4 or CCR4 transcripts, but this might reflect the lack of IL-2R (CD25) induction by IL-4-producing cells. It should be also noted that other investigators have reported CD25 expression by IL-10-producing Tr1 cells (34 –36).

In a Th1-dominant mycobacterial PPD-induced granuloma model, we were unable to identify a similar CCR8⁺ IL-10-producing population (Figs. 1C and 2F). This agrees with our report that CCR8^−/− mice had no change in their response to mycobacterial PPD (10). It also raises the possibility that helminth Ags may promote the generation of such populations with the purpose of suppressing underlying Th1-mediated responses. It has been suggested that Th2 cells are less affected by Treg cells than Th1 cells due to their ability to produce and respond to a variety growth factors (37). McKee and Pearce (17) were able to suppress Th1 but not Th2 responses in schistosome-challenged IL-10 KO mice by transferring only 5 × 10⁵ wild-type T cells, suggesting that Th1 cells were sensitive to even low concentrations of IL-10-producing cells.

We directly demonstrated that the aberrant Th2 response observed in CCR8^−/− mice was due to impaired CD4⁺ T cell function, because adoptive transfer of sensitized CCR8^+/+CD4⁺ T cells into CCR8^−/− mice corrected the defects in Th2 cytokine production and eosinophil recruitment. Further characterization of the cell responsible for correcting the CCR8^−/− type 2 granulomatous response pointed to an IL-10⁺CD4⁺ T cell. Specifically, transfer of sensitized CD4⁺ T cells depleted of IL-4 producers restored the eosinophil-rich response; however, the effect was lost when IL-10 producers were removed. Interestingly, removal of IL-4 producers tended to enhance the effect of the IL-10 producers. This may have been an enrichment effect, but potential cross-regulatory mechanisms will need to be investigated.

The finding that both IL-4- and IL-10-depleted populations restored IL-5 and IL-13 cytokine levels in the draining lymph nodes of CCR8^−/− mice was unexpected. We initially predicted that the IL-10-depleted population would fail to correct Th2 cytokine production, yet this was not the case. Our findings argue for independent cytokine-producing populations that have different migratory properties. Specifically, wild-type SEA-elicited IL-5 and IL-13-producing cells appeared to be able to migrate to lymph nodes of CCR8^−/− mice, whereas the IL-10 producers did not. It is tempting to speculate that this was due to differential expression of chemokine receptors and their ligands. Highly sensitive methods to monitor the migration of T cell subsets would be required to directly test this hypothesis.

In contrast to the regional lymph node defect, CCR8^+/+ IL-10-producing CD4⁺ T cells were required to correct the local type 2 response in CCR8^−/− recipients. Our analysis of CCL1 transcript expression indicated that this CCR8 ligand was highly induced in lungs compared with draining lymph nodes. Moreover, its expression corresponded to the appearance of CCR8 transcripts and IL-10 in granulomatous lungs. Potential sources of CCL1 include T cells, mononuclear phagocytes, and endothelial cells, all of which are active components of granulomas. The expression of CCR8 by the IL-10-producing population could permit those cells to be recruited to the lung where they regulate the local inflammatory response by countering cross-inhibitory cytokines such as IL-12 and IFN-γ production, both of which can limit eosinophil recruitment and Th2 cytokine production (38 – 40). Because CCL1 was only minimally expressed in draining lymph nodes, it implies that CCR8⁺CD4⁺ T cells are not actively recruited to nodes via CCR8, but they clearly were generated within lymphoid tissues of sensitized wild-type mice. Because CCR8 is up-regulated in lymphoid tissue following Ag challenge (18), a hypothetical scenario in which CCR8⁺ IL-10-producing T cells are generated within lymph nodes then subsequently migrate to peripheral organs would be reasonable. Thus, CCR8⁺ IL-10-producing cells would have the opportunity to function in both lymphoid and inflammatory sites. Indeed, CCR8⁺ cells appeared to be required for maximum generation of IL-5 and IL-13 production during the induction phase of the T cell response, providing evidence for a lymph node-based function; however, the nature of that induction phase population has yet to be defined.

It should be noted that other groups using OVA-elicited asthma models did not detect impaired Th2 responses in CCR8^−/− mice (41, 42). This is probably related to different experimental conditions. Compared with pathogen Ags, OVA is a very weak inducer of Th2 responses, requiring use of optimal mouse strains, adjuvants, and multiple challenges to elicit an asthmatic response. We have found more consistent results using cockroach Ag asthma and schistosomal egg granuloma models. In contrast to OVA, S. mansoni eggs elicit strong polarized Th2 responses with a single i.p. sensitization of only 500–1000 eggs. Thus, CCR8⁺ IL-10-producing CD4⁺ cells may participate to a greater degree in helminth pathogen Ag-elicited responses, possibly due to underlying innate recognition signals.

In conclusion, we have demonstrated a distinct association of CCR8 with a population of CD44⁺CD25⁺CD4⁺ IL-10-producing T cells generated during the response to egg Ags from the helminth S. mansoni. This T cell population appears to be a Treg cell or other specialized cytokine-producing T cell that supports and/or augments the type 2 granulomatous response likely through IL-10-mediated cross-regulatory mechanisms as demonstrated by others (16, 17, 43). Hence, the ultimate histopathologic features of the type 2 granuloma likely results from the interaction of multiple effector T cell subsets. At present, it is unclear whether this is a host regulatory mechanism co-opted by the parasite, or whether it represents a host adaptation required to mount an appropriate response to a multicellular infectious agent. Additional studies will be needed to define the signals that determine the generation and trafficking of these regulatory cells.