Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2013 Aug 1.
Published in final edited form as: J Immunol. 2012 Jun 27;189(3):1220–1227. doi: 10.4049/jimmunol.1103033

Cell-cell interaction with APC, not IL-23, is required for naive CD4 cells to acquire pathogenicity during Th17 lineage commitment1

Guangpu Shi *, Jenna D Lovaas *, Cuiyan Tan *, Barbara P Vistica *, Eric F Wawrousek ‡, Mehak K Aziz *, Rachael C Rigden *, Rachel R Caspi *, Igal Gery *,2
PMCID: PMC3401365  NIHMSID: NIHMS382475  PMID: 22745380

Abstract

Subpopulations of pathogenic or nonpathogenic Th17 cells were reported to develop when pre-sensitized CD4 cells were activated with their target Ag during polarization by either IL-23 or IL-6 and TGF-β, respectively. Here, we generated two Th17 subpopulations by using a system in which naïve CD4 cells from TCR transgenic mice specific to hen egg lysozyme (HEL) are polarized with IL-6/TGF-β and, concurrently, are activated either with HEL presented by APC, or with anti-CD3/CD28 Abs. Only the former cells were pathogenic, inducing inflammation in eyes expressing HEL. Naïve CD4 cells activated by the anti-CD3/CD28 Abs acquired pathogenicity, however, when co-cultured with HEL/APC. Importantly, however, the naïve CD4 cells did not acquire pathogenicity when co-cultured with APC stimulated with LPS, or when separated from the HEL presenting cells by a semi-permeable membrane. Unlike with pre-sensitized Th17, soluble IL-23 does not participate in pathogenicity acquisition by naïve CD4 cells: no pathogenicity was induced by adding IL-23 to cultures activated with anti-CD3/CD28 Abs. Furthermore, Abs against IL-23 or IL-23R did not inhibit acquisition of pathogenicity in cultures of naïve CD4 cells activated by HEL/APC. Our data thus show that, unlike pre-sensitized CD4 cells, naïve CD4 polarized toward Th17 phenotype acquire pathogenicity only by direct interaction with APC presenting the Ag, with no apparent involvement of soluble IL-23. We suggest that the Th17 lymphocytes derived from naive CD4 cells participate in pathogenic and other immune processes, along with the IL-23 dependent Th17 cells.

Keywords: T cells, Autoimmunity, Inflammation, Transgenic/Knockout mice

Introduction

Numerous studies in recent years have established the importance of the Th17 population in the immune system. Th17 cells are crucial in the protection against bacteria, parasites and fungi, in particular in the gut and lungs and play a critical role in the majority of immune-mediated inflammatory conditions in humans and experimental animals (1-5).

The process of Th17 generation had been controversial in early studies, in particular with regard to the role of IL-23 in the process (2, 6). More recent studies have clarified the issue, however, by providing evidence to show that Th17 cells are normally generated by the polarizing activity of two cytokines, IL-6 and TGF-β, whereas IL-23 is crucial for the maturity and pathogenic effectiveness of these cells (4, 7). In addition, IL-1 and IL-21 contribute to a vigorous generation of Th17 cells (8, 9).

Unlike the phenotype stability of Th1, Th17 cells were found to exhibit a high level of plasticity, readily acquiring non-Th17 phenotypes when exposed to other cytokine environments (10-12). Thus, culturing Th17 cells in media containing polarizing cytokines specific for Th1 or Treg resulted in the majority of Th17 expressing the corresponding new phenotype within 2-3 days. Importantly, the acquisition of new phenotypes by Th17 cells was also observed in vivo (10, 13).

Another unique feature of the Th17 population is its heterogeneity; Th17 lines generated by different procedures were found to exhibit different immunological features (14). Ghoreschi et al. (15) reported on a Th17 subpopulation generated by polarization of naïve precursors by IL-6, IL-23 and IL-1β, but without TGF-β. These Th17 cells were pathogenic and capable of inducing EAE. More recently, Kim et al. (16) discovered the existence of a subpopulation of “natural Th17” which are generated in the mouse thymus, before antigen exposure. Of particular interest to the present study is the early observation made by McGeachy et al (17), of two Th17 subsets, one highly pathogenic and the other completely lacking this capacity. The two subsets originated from presensitized T-cells, reactivated in culture with the Ag in the presence of either IL-23, or IL-6 and TGF-β. Only the previous subset developed pathogenicity, monitored by their capacity to induce immune-mediated inflammation.

We have previously reported on the activities of Th17 cells generated from naïve CD4 cells with transgenic (Tg) TCR specific to hen egg lysozyme (HEL). When activated with HEL, presented by APC, in the presence of IL-6 and TGF-β, these Th17 cells induced ocular inflammation in recipient mice expressing HEL in their lens (10, 18, 19). Here, we analyzed the phenotype of these pathogenic cells and compared it with that of another Th17 subpopulation, which is non-pathogenic, that we generated by activating the naïve CD4 cells with anti-CD3/CD28 Abs, along with the same polarizing cytokines, IL-6 and TGF-β. Our data indicate that the major factor determining pathogenicity of these Th17 cells, or lack thereof, is the availability of cell-cell contact with APC presenting HEL, rather than soluble IL-23.

Materials and Methods

Mice

All studies were carried out with (B10.BR × FVB/N) F1 mice, generated as detailed elsewhere (10, 18). The lines used here included Tg mice expressing HEL in their lens (“HEL-Tg”), or HEL-specific TCR on the majority of their T-lymphocytes (designated “3A9”) (10, 18), or syngeneic wild type mice. HEL is expressed exclusively in eyes of the HEL-Tg mice; this neo-self Ag cannot be detected in the circulation or any other tissue of these mice (20). All manipulations were performed in compliance with the NIH Resolution on the Use of Animals in Research.

Generation of Th17 lines

Naïve T cells were enriched from splenocytes and lymph node cells of 3A9 mice, using T cell columns (R&D). CD4 cells expressing the Tg TCR were then sorted by FACSAria II (BD Biosciences), using the clonotypic mAb “1G12” (10, 18). The naïve CD4 cells were incubated for 4 days, in 12-well plates, at 0.5 × 106 in 2 ml of Th17 polarizing medium (10, 18). The polarizing cytokines, IL-6 and TGF-β, were added at 100 and 10 ng/ml respectively, similarly to the study by McGeachy et al. (17). Activation of the naïve CD4 cells, concurrently with the polarization, was carried out by either HEL, at 2 μg/ml, presented by irradiated wild type splenocytes, or by spleen DC, as indicated (“HEL/APC”), or by anti-CD3/CD28 Abs, bound to the plate walls by incubation for 1 h, at 2 μg/well (“PbAb”) (21).

In certain experiments, additional agents and Abs were added to the culture medium, alone, or in combination, as indicated. These included: IL-23 (e-Bioscience), at 20 ng/ml on day 1 or 2 and at 10 ng/ml on day 3 of culture; LPS (DIFCO), at 100 ng/ml; anti-IL-23-p40 Ab (a gift from Giorgio Trinchieri, National Cancer Institute, NIH), at 50 μg/ml, or anti-IL-23R Ab (R&D System), at 10 μg/ml.

To generate Th1 lines, naïve CD4 cells were incubated for 4 days, in 12-well plates, at 0.5 × 106 in 2 ml of Th1 polarizing medium, as detailed elsewhere (10, 18).

DC isolation

DC were isolated from naïve syngeneic wild-type mouse spleens, using the method described by Tang et al. (22). Briefly, spleens were minced into small fragments and digested with collagenase D (Roche) and DNase I (Sigma-Aldrich) for 45 min at 37°C. Cells were collected after passing through a 70-μM nylon cell strainer (BD Falcon) and treated with EDTA for 5 min. Following red cell lysis and Fc receptor blocking, cells were incubated with anti-CD11c-conjugated magnetic beads (Miltenyi Biotec) at 4°C for 15 min. The positive population was purified using autoMACS (Miltenyi Biotec). The isolated preparation consisted of ~90% cells positive for CD11c.

HEL/APC and PbAb Th17 cells generated in transwell cultures

For HEL/APC and PbAb Th17 generated in transwell cultures, the sorted CD4 cells (0.5×106) were cultured with APC at the top compartment in a volume of 0.5 ml containing HEL and Th17 polarizing cocktail (10, 18, 19), while the same number of sorted CD4 cells, in a volume of 1.5 ml, containing HEL and the polarizing cocktail, but with no APC, were cultured in the bottom compartments that were coated with anti-CD3/CD28 Abs, as described above. Th1 were similarly cultured, for comparison. The system is also demonstrated in Fig. 6A.

Figure 6.

Figure 6

Open in a new tab

Cell-cell interaction is required for acquisition of pathogenicity by Th17 cells, but not by Th1 cells. Transwell cultures (A) were set-up with HEL-specific naïve CD4 cells activated with either APC in the top compartment, or anti-CD3/CD28 Abs in the bottom compartment, along with HEL and polarizing cytokines for either Th17 or Th1 in both compartments. Following 4 days of incubation, the cells collected from each compartment were injected into HEL-Tg recipients. (B and C) Summaries of three repeated experiments, recording the severity levels of individual eyes of recipient mice injected with Th17 or Th1 cells, collected from the top or bottom compartments and scored as detailed in Kim et al. (23). (D and E) Sections of the anterior and posterior eye segments of representative recipient eyes collected 5 days post cell transfer. Severe changes were induced by Th1 collected from both culture compartments (E), but only from the top compartment of Th17 cultures (D). C, cornea; I, iris; R, retina; V, vitreous; L, lens; O, optic nerve.

Quantitative PCR Analysis

Total RNA was extracted from cultured cells with TRIzol (Invitrogen-Life Technologies). RNA (5 μg), SuperScript III Reverse Transcriptase (Invitrogen Life Technologies), and oligo(dT)12–16 were used for first-strand cDNA synthesis. Primer-probe sets for qPCR quantifying expression of mouse IL-23R, CD40L, ICOS, AHR, CCL2, CCL5, CCL20, CCL22, CXCL2, CXCL10, CCR2, CCR6, CXCR3 and GAPDH or β-actin (internal control) were purchased from Applied Biosystems. Primers used for ROR-γt and ROR-α were as follows: ROR-γt, 5′-CCGCTGAGAGGGCTTCAC-3′ and 5′-TGCAGGAGTAGGCCACATTACA-3′ and ROR-α, 5′-CGTGTCCATGGCAGAACTAGAA-3′ and 5′-GCAAGTACTGGCAGGTTTCCA-3′. Fluorescence-labeled probes used are: ROR-γt, 5′-AAGGGCTTCTTCCGCCGCAGCCAGCAG-BHQ-1-3′ and ROR-α, 5′-CCTTGCCCAGAACATATCCAAATCCCA-BHQ-1-3′. PCR parameters were as recommended for the TaqMan Universal PCR Master Mix kit (Applied Biosystems).

Measurement of cytokines released into Th17 cell culture supernatant

Cell culture supernatants were sampled on days 2, 3 or 4 of culture. The supernatants were tested for the levels of IL-10, IL-17, IL-22 and GM-CSF, using ELISA kits from R&D System, and for the levels of IL-21 and IL-17F, using ELISA kits from BioLegend, according to the manufacturers’ instructions.

Flow cytometric analysis of surface, nuclear and intracellular molecules

Conventional methods were used for analysis of surface molecule expression.

For Foxp3 staining, Th17 cells were fixed and permeabilized with the Fixation/Permeabilization buffer for 1 h at 4°C before intracellular staining with allophycocyanin-conjugated anti-Foxp3, following the procedure recommended by the manufacturer (eBioscience).

For intracellular cytokine staining, polarized Th17 cell cultures were stimulated with 20 ng/ml PMA and 1 μM ionomycin (Sigma-Aldrich), plus Golgi-Stop (BD Biosciences), for 5 h. The stimulated cells were then stained with the corresponding Abs for surface and intracellular molecules.

Data were acquired using a FACSCalibur (BD Biosciences) and analyzed using FlowJo (Tree Star).

Adoptive transfer of Th17 line cells

After 4 day incubation, PbAb or HEL/APC Th17 cells were injected, at 4×106, via the tail vein, into groups of naïve HEL-Tg mice. Recipient eyes were collected on day 5 post-cell transfer and processed for histology by conventional H&E staining. Severity of the inflammation was scored according to histological changes, as described elsewhere (23).

Measuring cell division rate by CFSE dilution

PbAb or HEL/APC Th17 cells (107 cells/ml) were labeled with CFSE (10 μM; Molecular Probes) for adoptive transfer, as described elsewhere (19, 24). CFSE-labeled cells were washed, re-suspended in RPMI and injected i.v. into recipient mice (4×106 cells). Recipient mice were euthanized on the indicated days post-cell transfer, and splenocytes were collected and stained with anti-CD4-PE. Data were acquired using a FACSCalibur. CFSE dye dilution was analyzed using FlowJo.

Statistical analysis

Unpaired, two-tail t test was performed for comparison of transcript level in Th17 or cytokine level in supernatant, as well as for comparison of the frequency of transferred cells recovered from recipient spleen. p ≤ 0.05 was defined as statistically significant.

Results

Th17 cells generated by activation with HEL/APC are pathogenic, whereas those generated by activation with anti-CD3/CD28 Abs are not

We generated two subpopulations of Th17 cells by activation of sorted naïve CD4 cells specific to HEL with either the target Ag, HEL, presented by APC (“HEL/APC”), or with plate-bound anti-CD3/CD28 Abs (“PbAb”), in the presence of the same polarizing cytokines, IL-6 and TGF-β. To test for the pathogenic capacity of the two Th17 subpopulations, we adoptively transferred the cells into syngeneic recipients expressing HEL in their eyes (10, 18, 19). Fig. 1A depicts representative sections of eyes from recipients injected with cells of the two subpopulations. Severe ocular inflammatory changes are seen in the recipient of HEL/APC Th17 cells, whereas no inflammatory changes could be detected in eyes of the recipient of PbAb Th17 cells.

Figure 1.

Figure 1

Open in a new tab

Unlike HEL/APC Th17, PbAb Th17 cells are non-pathogenic. (A) Four million cells of the two subpopulations were adoptively transferred into HEL-Tg mice and 5 days later, eyes of recipients were examined histologically for inflammatory changes. Sections of representative eyes show no pathological changes in the recipient of PbAb Th17 cells, but severe panuveitic changes in the recipient of HEL/APC Th17 cells. The changes mainly include edema and cellular infiltration in the cornea, cellular and proteinaceous exudates in the anterior chamber and the vitreous, and intense infiltration in the retina. (B) Transferred PbAb Th17 cells are alive and proliferate in the recipient spleen when exposed to HEL. CSFE division analysis of transferred cells in recipients injected with HEL (“PbAb Th17 + HEL”) and their controls in recipients with no HEL injection (“PbAb Th17”). The data are shown by flow cytometry results of a representative experiment (upper panel) and as mean percentage ± SEM of 1G12+ cells among total CD4 cells in recipient spleens, on day 3 post-cell injection, in three independent experiments (lower panel). (C) Low proportions of FoxP3+ cells among both PbAb and HEL/APC Th17 subpopulations, measured by flow cytometry. A representative experiment; similar results were obtained in two additional experiments.

The lack of pathogenicity by PbAb-activated Th17 cells could be attributed to their low viability in vivo. To examine this possibility, we used the CFSE division method, to test the capacity of the transferred cells to proliferate when exposed to their target Ag. We and others (25, 26) have reported that adoptively transferred autopathogenic T-cells initially migrate to the host spleen, where they proliferate before invading the target tissue. Fig. 1B shows a representative experiment. Adoptively transferred CFSE-labeled PbAb Th17 cells proliferated poorly in the spleen of recipient mice, but vigorous proliferation was seen in recipient mice that were also injected with HEL. This observation thus provides evidence that the injected PbAb-activated cells are viable and fully capable of responding to their target Ag. Importantly, the PbAb-activated Th17 cells in this system retained their non-pathogenicity despite their re-activation by the Ag in vivo (data not shown). The possibility that the non-pathogenic activity of PbAb-stimulated Th17 cells was due to high levels of T-regulatory cells was ruled out by the finding that the proportion of cells expressing FoxP3 in PbAb-activated cells was similar to that of the pathogenic HEL/APC Th17 cells (Fig. 1C).

Molecular analysis of the two Th17 subpopulations

To define the two Th17 subpopulations molecularly, we compared their capacity to produce several molecules characteristic for the Th17 lineage. Both subpopulations expressed transcripts of the two Th17 specific transcription factors, RORα and RORγt, with higher levels seen in the PbAb cultures (Fig. 2A).

B>Figure 2.

B>Figure 2

Open in a new tab

HEL/APC and PbAb Th17 subpopulations differ in their cytokine production profiles. (A) Expression of RORα and RORγt transcripts by the two Th17 subpopulations, after 4 days of incubation, determined by qPCR. (B) Mean levels ± SEM of IL-17, IL-22 and IL-10 in supernatants collected at different time points of cultures in which the two subpopulations were generated. The data were collected in four independent experiments. (C) Immunostaining for intracellular expression of IL-17, IL-22 and IL-10 of cells from the two Th17 subpopulations. The cells were collected after 4 days in culture and stained with the specific monoclonal antibodies, or isotype controls (“IgG”), as detailed in Shi et al. (10). Please note that HEL/APC Th17, but not PbAb Th17 cells stained for intracellular IL-22 and that different subsets within this subpopulation stained for either IL-17 or IL-22, with just a minor portion of cells co-expressing both cytokines. Also of note is the co-expression of IL-17 and IL-10 by cells of the two Th17 subpopulations. A representative experiment; similar observations were made in another experiment.: p < 0.05.

Next, we analyzed the cytokine production by the two subpopulations, by measuring in the culture supernatants the levels of three Th17 products, IL-17, IL-10 and IL-22, at different time points (Fig. 2B). PbAb cultures, yielding non-pathogenic Th17, produced exceedingly high levels of IL-17, that were notably higher than those produced by HEL/APC cultures, that generate the pathogenic Th17. The two types of cultures produced similar or only moderately different levels of IL-10, but striking differences were seen in the pattern of IL-22 production between the cultures activated by PbAb, or by HEL/APC, with the latter cultures producing IL-22 levels higher by two orders of magnitude. The vigorous production of IL-22 by CD4 cells stimulated by HEL/APC was not affected by addition of Ab against IL-23R, or against 12p40, but was moderately inhibited by the addition of PbAb (Supplemental Fig. 1).

Production of the three cytokines by Th17 cells was also directly demonstrated by flow cytometry. The data of a representative experiment are recorded in Fig. 2C and show that the intracellular staining patterns of the tested cytokines are in accord with our ELISA data. Of interest are the findings that (i) IL-10 was expressed by the same subset of CD4 cells that produced IL-17. This observation is in line with our finding (Fig. 2B), that pathogenic Th17 in our study also secrete IL-10. (ii) In contrast to IL-10, IL-22 production in cultures activated by HEL/APC was localized to a large extent in a subset of CD4 cells that did not co-express IL-17.

The subpopulations of pathogenic and non-pathogenic Th17 also differed in their staining for certain surface antigens. Differences were particularly clear in the expression of CCR5, CCR6 and α4β7 (Fig. 3A). More dramatic differences between the two subpopulations were found in their expression profiles of several chemokines and chemokine receptors (Fig. 3B). Whereas transcripts of CCL20 and CXCR3 were more elevated in PbAb cultures, those of CXCL10, CCL2, CCL22 and CXCL2 were higher in HEL/DC cultures.

Figure 3.

Figure 3

Open in a new tab

The two Th17 subpopulations differ in their expression of surface molecules and certain chemokine and chemokine receptor transcripts. (A) Suspensions of PbAb and HEL/APC Th17 cells, harvested after 4 days in culture, were stained for the indicated surface molecules and examined by flow cytometry. (B) qPCR analysis of Th17 cells collected after 4 days of incubation at the “HEL/DC” or “PbAb” conditions. Transcript expression is presented relative to GAPDH. A representative experiment; similar expression profiles were observed in 4 other independent experiments.

Th17 cells generated by stimulation with HEL/APC or with PbAb were also tested for production of other molecules: cells of the two subpopulations expressed similar levels of CD40L transcripts, but HEL/APC Th17 expressed higher levels of ICOS transcript, whereas moderately higher levels of AHR transcript were expressed by PbAb Th17 cells (Supplemental Fig. 2A). Supernatants of these cultures contained similar levels of GM-CSF and IL-21, but PbAb-stimulated cultures were superior in their production of IL-17F (Supplemental Fig. 2B), in line with their higher levels of IL-17A (Fig. 2B).

Th17 generated by stimulation with either PbAb or HEL/APC proliferated vigorously, but a marked difference was noted in the kinetics of their response, with PbAb-stimulated Th17 cells responding earlier (Supplemental Fig. 3). This difference is in accord with the two mechanisms of stimulation process: the Abs stimulate directly the naïve CD4 cells, whereas stimulation with HEL/APC requires prior processing of the Ag by the APC.

The two Th17 subpopulations differ in their homing to and proliferation in the recipient mouse spleen

As mentioned above, adoptively transferred activated T-cells migrate to the recipient spleen, where they proliferate before invading the target organ (25, 26). To compare between the two Th17 subpopulations for this capacity we adoptively transferred the tested cells into HEL-Tg mice and tracked them in the recipient spleen by the clonotypic Ab, “1G12”. Fig. 4A depicts data of a representative experiment in which 1G12+ cells were identified by flow cytometry in recipient spleens 4 days post cell transfer. The figure shows that Th17 generated by activation with HEL/APC were superior to Th17 generated by PbAb activation in their capacity to accumulate and proliferate in the recipient spleen. Repeated experiments in which recipient spleens were collected on days 2, 4 and 7 post cell transfer are summarized in Fig. 4B, showing the superiority of the HEL/APC Th17 at all tested time points.

Figure 4.

Figure 4

Open in a new tab

HEL/APC Th17 are superior to PbAb Th17 cells in their capacity to migrate to and proliferate in the recipient spleen. Four million Th17 cells of the two subpopulations were adoptively transferred into HEL-Tg mice and spleens of the recipients were collected at the indicated time points. (A) Flow cytometric analysis on day 4, identifying donor cells, positive for the clonotypic antibody, 1G12, among the CD4 population. The proportion of donor cells is higher in the recipients injected with HEL/APC Th17 than in that of the PbAb Th17 recipients. A representative experiment; similar data were obtained in two other experiments. (B) A summary of three experiments, depicting the mean percentage ± SEM of PbAb or HEL/APC Th17 cells (1G12+) among CD4 cells in the recipient spleens, on days 2, 4 or 7 post-cell injection. (C) Proliferation rate of the two subpopulations determined by the CFSE dilution method, at the indicated time points. Th17 cells of the HEL/APC subpopulation proliferated at a faster rate.: p < 0.05. (D) Th17 generated by APC+HEL activation switch to the Th1 phenotype in the recipient spleen more readily than PbAb-generated Th17. Intracellular flow cytometric analysis shows a higher proportion of the former subpopulation expressing IFN-γ on day 4 post-cell injection.

Next, we used the CFSE dilution procedure to compare the two subpopulations of Th17 cells for their proliferation rate in the recipient spleen. As seen in Fig. 4C, the HEL/APC Th17 proliferated considerably more vigorously than the non-pathogenic PbAb cells, a finding in line with their higher proportion among the total spleen CD4 cells (Figs. 4A and 4B).

We have reported previously (10) that Th17 generated by activation with APC+HEL exhibit plasticity and acquire the Th1 phenotype when exposed to other cytokine environment. To compare the two Th17 subpopulations for their plasticity, we collected recipient spleen cells on day 4 post cell injection and analyzed them for intracellular expression of IL-17 and IFN-γ. As shown in Fig. 4D, the proportion of cells expressing IFN-γ was higher among the APC+HEL recipients than among the PbAb recipients.

Naïve CD4 cells activated by plate-bound Abs acquire pathogenicity when co-cultured with DC and HEL

Soluble IL-23 was shown to be critical for acquisition of pathogenicity by CD4 cells pre-immunized against the immunopathogenic Ag (7, 17). No pathogenicity was acquired, however, in our system by naïve CD4 activated in culture by the plate-bound Abs when IL-23 was added to the medium during Th17 polarization, on day 1 (Fig. 5A, upper panel), or even on days 2 and 3 (not shown), when the expression of IL-23R was elevated (Fig. 5B). In contrast, PbAb-activated CD4 cells did acquire pathogenic capacity when DC and HEL were also added to the culture medium; these Th17 cells induced inflammation levels similar to those achieved by Th17 cells of cultures stimulated by DC and HEL (Fig. 5A, upper panel). Importantly, however, no pathogenicity was acquired in cultures in which DC were added alone, or even with LPS, a potent APC stimulant (27-29) (Fig. 5A upper panel). All cultures in these experiments were polarized with IL-6 and TGF-β during the activation process and, as shown in the lower panel of Fig. 5A, Th17 cells generated in all these cultures released high levels of IL-17, despite the differences in their immunopathogenicity, but in agreement with data shown in Fig. 2B. It is of interest, however, that the lowest IL-17 levels were released by the pathogenic cultures activated by only DC and HEL, again in agreement with the data recorded in Fig. 2B, underscoring the IL-17 stimulating capacity of the plate-bound Abs.

Figure 5.

Figure 5

Open in a new tab

Naive CD4 cells activated by PbAb do acquire pathogenicity when DC and HEL are also added to the culture medium. (A) Top frame, severity levels of ocular inflammation induced in recipient mice expressing HEL in their eyes, following adoptive transfer of four million Th17, generated as indicated. The dots represent individual eyes, scored as detailed in Kim et al. (23). Bottom frame, levels of IL-17 secreted by the different Th17 cultures, generated as indicated, following incubation for 4 days. (B) Expression levels of IL-23R transcripts by activated naïve CD4 cells, at different time points. PbAb-stimulated naïve CD4 cells, under Th17 polarizing conditions, were collected at the indicated time points and assayed by qPCR. Freshly isolated naïve CD4 cells were used as control for the assay.

It is also noteworthy that addition of Abs against IL-23 or IL-23 receptor (IL-23R) had no effect on acquisition of pathogenicity by CD4 cells activated by HEL/DC, or by plate-bound Abs and HEL/DC (data not shown).

Cell-cell interaction between naive CD4 and APC is essential for acquisition of pathogenicity by Th17 cells

APC affect T-cells by the release of cytokines and/or by cell-cell interaction (30, 31). To analyze the mechanism whereby APC induce pathogenicity in Th17 in our experimental system, we separated by semi-permeable membranes the HEL presenting cells and the naïve CD4 cells activated with anti-CD3/CD28 Abs (Fig. 6A) and examined the development of pathogenicity by the latter cells. Naïve CD4 cells were also added to the top compartment, along with the APC. Th17 polarizing cytokines (IL-6 and TGF-β) and HEL were added to both compartments. Data collected in repeated experiments are summarized in Fig. 6B, whereas Fig. 6D shows representative eye sections of recipient mice injected with Th17 cells collected from the two compartments. As expected, the CD4 lymphocytes from the top compartment, cultured with HEL/APC, developed pathogenic capacity. In contrast, no pathogenicity was demonstrated by the CD4 cells from the bottom compartment, which were activated by anti-CD3/CD28 Abs. These observations thus provide evidence showing that acquisition of pathogenicity by naïve CD4 cells during polarization toward Th17 phenotype requires cell-cell interaction with APC, rather than soluble cytokines released by APC.

To further examine the observations we made with Th17 subpopulations using the transwell system, we carried out similar experiments in which Th1 cell subpopulations were generated and tested for pathogenicity (Figs. 6C and 6E). Th1 cells were generated by polarization with IL-12 (10, 18). Unlike with Th17, Th1 cells generated by activation with PbAb during polarization did acquire pathogenicity. As shown in Figs. 6C and 6E, Th1 collected from the bottom compartment (activated by PbAb) induced inflammation in recipient eyes, similar to that induce by Th1 collected from the top compartment (activated by HEL/APC). These observations thus underscore the profound difference between Th17 and Th1 populations in their acquisition of pathogenicity; activation of naïve CD4 cells with PbAb during polarization elicits pathogenic capacity in Th1 but not in Th17 cells.

Discussion

As cited above, recent publications revealed the heterogenicity of the Th17 population, with different subpopulations being generated by different procedures (14, 15, 17). Data collected in the present study further extend the information concerning the variety of Th17 subpopulations. These data show that activating naïve CD4 cells with either the Ag presented by APC, or with Abs against CD3/CD28, during polarization with IL-6 and TGF-β, generates two Th17 subpopulations that differ by their immunopathogenic capacity: the former subpopulation is pathogenic, whereas the latter is not. It is of note that the pathogenic and non-pathogenic subpopulations in our study differed from the analogous two Th17 subpopulations described by McGeachy et al. (17) in their cell source, i.e., naïve vs pre-immunized cells, as well as the activation and polarization procedures used for their generation. In addition, the pathogenic Th17 lines of the two studies also differed in their expression of IL-10; pathogenic cells in the study by McGeachy et al. did not produce this cytokine (17), whereas the pathogenic cells in our study did, as shown by both their release and intracellular production of this cytokine (Figs. 2B,C).

The two subpopulations in our study also differed in their production of IL-22, a characteristic product of Th17 cells (32): only cells of the pathogenic Th17 subpopulation produced this cytokine. Interestingly, flow cytometric analysis revealed that the majority of cells that express IL-22 do not co-produce IL-17 (Fig. 2C). More studies are needed to further analyze the biological function(s) of the subset of IL-22 producing Th17 cells and their possible relationship to the “Th22” subpopulation recently identified in humans (33, 34). The capacity to produce IL-22 is not related to pathogenicity of Th17 cells, since treatment of recipients of pathogenic Th17 with anti-IL-22 Ab had no effect on the development of ocular inflammation in these mice (data not shown).

The two subpopulations in our study differed in their profiles of cell surface Ags, chemokines and chemokine receptors and it is conceivable that these differences determined to a large extent their pathogenic capacity. This notion is also supported by the finding that the profiles of chemokines and chemokine receptors of the pathogenic and non-pathogenic Th17 subpopulations generated in our study (Fig. 3B) resembled those of the analogous subpopulations generated by McGeachy et al. (17), despite the remarkable differences in their cell origin, mode of generation and lymphokine profiles.

Our study shows that pathogenic Th17 cells are superior to the non-pathogenic cells in their capacity to migrate into and proliferate in the recipient spleen. The migration was monitored by tracking the injected cells with the clonotypic Ab 1G12 and their proliferation rate was measured by the CFSE dilution method (Fig. 4A, B, C). It is conceivable that these cellular capacities are essential for the pathogenic Th17 cells in their induction of inflammation in the target organ, the eye in the present study. Our study thus provides a new parameter for the capacity of Th populations to migrate into and proliferate in the recipient organs.

In accord with our previous study (10), a portion of Th17 generated by APC+HEL activation acquire the Th1 phenotype in the recipient spleen (Fig. 4D). Lower proportions of Th17 generated by PbAb activation also exhibited phenotype switching, but it seems unlikely this lower activity of the PbAb Th17 cells contributed significantly to their lack of pathogenicity.

Importantly, data collected in the present study show that, unlike pre-sensitized CD4 lymphocytes (17), naïve CD4 cells are not affected by soluble IL-23 during their polarization toward the pathogenic Th17 phenotype. Addition of soluble IL-23 to cultures stimulated with anti-CD3/CD28 Abs had no effect on the generation of non-pathogenic phenotype, whereas addition of APC and HEL to these cultures did yield pathogenic Th17 (Fig. 5A). Furthermore, the generation of pathogenic Th17 from naïve cells could not be blocked in culture by Abs against IL-23 or IL-23R (data not shown).

It is noteworthy that the APC effect of generating pathogenicity in naïve CD4 cells in our experimental system was achieved only when the APC presented the target Ag, HEL; no effect was seen when the APC were added with no addition of HEL, or even when LPS, a potent APC stimulator (27-29), was added to the culture system (Fig. 5A). These data suggested that naïve CD4 cells acquire pathogenicity only by cell-cell interaction with HEL-presenting APC, a notion that was further examined by the transwell method. This method allowed us to test in the same culture system activation of naïve CD4 cells by either cell-cell interaction with APC presenting HEL, or by cytokines released from these APC. Pathogenicity was acquired only by the former mode of activation (Fig. 6B).

In conclusion, data of the present study provide evidence to show that naïve CD4 cells polarized into Th17 phenotype acquire pathogenic capacity only by directly interacting with APC presenting the specific Ag. Our data also show that IL-23, which was found crucial for development of pathogenicity by pre-immunized CD4 cells (4, 7, 12, 17), is not essential for the process in naïve CD4 cells (Fig. 5A). These data thus show, for the first time, that the process which generates Th17 from naïve CD4 is remarkably different from the process that generates Th17 from “mature” pre-immunized Th cells. More investigation is needed to analyze the functions of Th17 originated from naïve cells in the “total” Th17-induced immune response against microbial invasion, or the initiation of pathogenic inflammation. We suggest that the Th17 lymphocytes derived from naive CD4 cells participate in these immune processes, along with, or ahead of, the IL-23-dependent Th17 cells.

Supplementary Material

1

Acknowledgments

We thank Dr. Ronald H. Schwartz for helpful suggestions, Lindsey Nugent for expert assistance, R. Steven Lee for tail DNA analysis, the National Eye Institute Flow Cytometry Core for technical support, and the Histology Core for tissue section preparations.

Footnotes

1

This research was supported by the Intramural Research Program of the National Eye Institute, NIH.

3

Abbreviations used in this paper: Tg, transgenic; HEL, hen egg lysozyme; qPCR, quantitative PCR; IL-23R, IL-23 receptor.

Reference

  • 1.Bettelli E, Oukka M, Kuchroo VK. T(H)-17 cells in the circle of immunity and autoimmunity. Nat Immunol. 2007;8:345–350. doi: 10.1038/ni0407-345. [DOI] [PubMed] [Google Scholar]
  • 2.Weaver CT, Hatton RD, Mangan PR, Harrington LE. IL-17 family cytokines and the expanding diversity of effector T cell lineages. Annu Rev Immunol. 2007;25:821–852. doi: 10.1146/annurev.immunol.25.022106.141557. [DOI] [PubMed] [Google Scholar]
  • 3.Park H, Li Z, Yang XO, Chang SH, Nurieva R, Wang YH, Wang Y, Hood L, Zhu Z, Tian Q, Dong C. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat Immunol. 2005;6:1133–1141. doi: 10.1038/ni1261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Morrison PJ, Ballantyne SJ, Kullberg MC. Interleukin-23 and T helper 17-type responses in intestinal inflammation: from cytokines to T-cell plasticity. Immunology. 2011;133:397–408. doi: 10.1111/j.1365-2567.2011.03454.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Shainheit MG, Lasocki KW, Finger E, Larkin BM, Smith PM, Sharpe AH, Dinarello CA, Rutitzky LI, Stadecker MJ. The pathogenic Th17 cell response to major schistosome egg antigen is sequentially dependent on IL-23 and IL-1β. J Immunol. 2011;187:5328–5335. doi: 10.4049/jimmunol.1101445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.McKenzie BS, Kastelein RA, Cua DJ. Understanding the IL-23-IL-17 immune pathway. Trends Immunol. 2006;27:17–23. doi: 10.1016/j.it.2005.10.003. [DOI] [PubMed] [Google Scholar]
  • 7.McGeachy MJ, Chen Y, Tato CM, Laurence A, Joyce-Shaikh B, Blumenschein WM, McClanahan TK, O’Shea JJ, Cua DJ. The interleukin 23 receptor is essential for the terminal differentiation of interleukin 17-producing effector T helper cells in vivo. Nat Immunol. 2009;10:314–324. doi: 10.1038/ni.1698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Geri G, Terrier B, Rosenzwajg M, Wechsler B, Touzot M, Seilhean D, Tran TA, Bodaghi B, Musset L, Soumelis V, Klatzmann D, Cacoub P, Saadoun D. Critical role of IL-21 in modulating TH17 and regulatory T cells in Behcet disease. J Allergy Clin Immunol. 2011;128:655–664. doi: 10.1016/j.jaci.2011.05.029. [DOI] [PubMed] [Google Scholar]
  • 9.Sutton C, Brereton C, Keogh B, Mills KH, Lavelle EC. A crucial role for interleukin (IL)-1 in the induction of IL-17-producing T cells that mediate autoimmune encephalomyelitis. J Exp Med. 2006;203:1685–1691. doi: 10.1084/jem.20060285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Shi G, Cox CA, Vistica BP, Tan C, Wawrousek EF, Gery I. Phenotype switching by inflammation-inducing polarized Th17 cells, but not by Th1 cells. J Immunol. 2008;181:7205–7213. doi: 10.4049/jimmunol.181.10.7205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Lee YK, Turner H, Maynard CL, Oliver JR, Chen D, Elson CO, Weaver CT. Late developmental plasticity in the T helper 17 lineage. Immunity. 2009;30:92–107. doi: 10.1016/j.immuni.2008.11.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Hirota K, Duarte JH, Veldhoen M, Hornsby E, Li Y, Cua DJ, Ahlfors H, Wilhelm C, Tolaini M, Menzel U, Garefalaki A, Potocnik AJ, Stockinger B. Fate mapping of IL-17-producing T cells in inflammatory responses. Nat Immunol. 2011;12:255–263. doi: 10.1038/ni.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Martin-Orozco N, Chung Y, Chang SH, Wang YH, Dong C. Th17 cells promote pancreatic inflammation but only induce diabetes efficiently in lymphopenic hosts after conversion into Th1 cells. Eur J Immunol. 2009;39:216–224. doi: 10.1002/eji.200838475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Peters A, Lee Y, Kuchroo VK. The many faces of Th17 cells. Curr Opin Immunol. 2011;23:702–706. doi: 10.1016/j.coi.2011.08.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Ghoreschi K, Laurence A, Yang XP, Tato CM, McGeachy MJ, Konkel JE, Ramos HL, Wei L, Davidson TS, Bouladoux N, Grainger JR, Chen Q, Kanno Y, Watford WT, Sun HW, Eberl G, Shevach EM, Belkaid Y, Cua DJ, Chen W, O’Shea JJ. Generation of pathogenic T(H)17 cells in the absence of TGF-β signalling. Nature. 2010;467:967–971. doi: 10.1038/nature09447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Kim JS, Smith-Garvin JE, Koretzky GA, Jordan MS. The requirements for natural Th17 cell development are distinct from those of conventional Th17 cells. J Exp Med. 2011;208:2201–2207. doi: 10.1084/jem.20110680. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • 17.McGeachy MJ, Bak-Jensen KS, Chen Y, Tato CM, Blumenschein W, McClanahan T, Cua DJ. TGF-β and IL-6 drive the production of IL-17 and IL-10 by T cells and restrain T(H)-17 cell-mediated pathology. Nat Immunol. 2007;8:1390–1397. doi: 10.1038/ni1539. [DOI] [PubMed] [Google Scholar]
  • 18.Cox CA, Shi G, Yin H, Vistica BP, Wawrousek EF, Chan CC, Gery I. Both Th1 and Th17 are immunopathogenic but differ in other key biological activities. J Immunol. 2008;180:7414–7422. doi: 10.4049/jimmunol.180.11.7414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Shi G, Ramaswamy M, Vistica BP, Cox CA, Tan C, Wawrousek EF, Siegel RM, Gery I. Unlike Th1, Th17 cells mediate sustained autoimmune inflammation and are highly resistant to restimulation-induced cell death. J Immunol. 2009;183:7547–7556. doi: 10.4049/jimmunol.0900519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Zhang M, Vacchio MS, Vistica BP, Lesage S, Egwuagu CE, Yu CR, Gelderman MP, Kennedy MC, Wawrousek EF, Gery I. T cell tolerance to a neo-self antigen expressed by thymic epithelial cells: the soluble form is more effective than the membrane-bound form. J Immunol. 2003;170:3954–3962. doi: 10.4049/jimmunol.170.8.3954. [DOI] [PubMed] [Google Scholar]
  • 21.Huter EN, Stummvoll GH, DiPaolo RJ, Glass DD, Shevach EM. Pre-differentiated Th1 and Th17 effector T cells in autoimmune gastritis: Ag-specific regulatory T cells are more potent suppressors than polyclonal regulatory T cells. Int Immunopharmacol. 2009;9:540–545. doi: 10.1016/j.intimp.2009.01.022. [DOI] [PubMed] [Google Scholar]
  • 22.Tang J, Zhu W, Silver PB, Su SB, Chan CC, Caspi RR. Autoimmune uveitis elicited with antigen-pulsed dendritic cells has a distinct clinical signature and is driven by unique effector mechanisms: initial encounter with autoantigen defines disease phenotype. J Immunol. 2007;178:5578–5587. doi: 10.4049/jimmunol.178.9.5578. [DOI] [PubMed] [Google Scholar]
  • 23.Kim SJ, Zhang M, Vistica BP, Chan CC, Shen DF, Wawrousek EF, Gery I. Induction of ocular inflammation by T-helper lymphocytes type 2. Invest Ophthalmol Vis Sci. 2002;43:758–765. [PubMed] [Google Scholar]
  • 24.Fujimoto C, Yu CR, Shi G, Vistica BP, Wawrousek EF, Klinman DM, Chan CC, Egwuagu CE, Gery I. Pertussis toxin is superior to TLR ligands in enhancing pathogenic autoimmunity, targeted at a neo-self antigen, by triggering robust expansion of Th1 cells and their cytokine production. J Immunol. 2006;177:6896–6903. doi: 10.4049/jimmunol.177.10.6896. [DOI] [PubMed] [Google Scholar]
  • 25.Chen J, Vistica BP, Takase H, Ham DI, Fariss RN, Wawrousek EF, Chan CC, DeMartino JA, Farber JM, Gery I. A unique pattern of up- and down-regulation of chemokine receptor CXCR3 on inflammation-inducing Th1 cells. Eur J Immunol. 2004;34:2885–2894. doi: 10.1002/eji.200425318. [DOI] [PubMed] [Google Scholar]
  • 26.Flugel A, Berkowicz T, Ritter T, Labeur M, Jenne DE, Li Z, Ellwart JW, Willem M, Lassmann H, Wekerle H. Migratory activity and functional changes of green fluorescent effector cells before and during experimental autoimmune encephalomyelitis. Immunity. 2001;14:547–560. doi: 10.1016/s1074-7613(01)00143-1. [DOI] [PubMed] [Google Scholar]
  • 27.Gery I, Waksman BH. Potentiation of the T-lymphocyte response to mitogens. II. The cellular source of potentiating mediator(s) J Exp Med. 1972;136:143–155. doi: 10.1084/jem.136.1.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Piani A, Hossle JP, Birchler T, Siegrist CA, Heumann D, Davies G, Loeliger S, Seger R, Lauener RP. Expression of MHC class II molecules contributes to lipopolysaccharide responsiveness. Eur J Immunol. 2000;30:3140–3146. doi: 10.1002/1521-4141(200011)30:11<3140::AID-IMMU3140>3.0.CO;2-O. [DOI] [PubMed] [Google Scholar]
  • 29.Ulevitch RJ, Tobias PS. Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Annu Rev Immunol. 1995;13:437–457. doi: 10.1146/annurev.iy.13.040195.002253. [DOI] [PubMed] [Google Scholar]
  • 30.Gutcher I, Becher B. APC-derived cytokines and T cell polarization in autoimmune inflammation. J Clin Invest. 2007;117:1119–1127. doi: 10.1172/JCI31720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Yokosuka T, Saito T. The immunological synapse, TCR microclusters, and T cell activation. Curr Top Microbiol Immunol. 2010;340:81–107. doi: 10.1007/978-3-642-03858-7_5. [DOI] [PubMed] [Google Scholar]
  • 32.Liang SC, Tan XY, Luxenberg DP, Karim R, Dunussi-Joannopoulos K, Collins M, Fouser LA. Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides. J Exp Med. 2006;203:2271–2279. doi: 10.1084/jem.20061308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Eyerich S, Eyerich K, Pennino D, Carbone T, Nasorri F, Pallotta S, Cianfarani F, Odorisio T, Traidl-Hoffmann C, Behrendt H, Durham SR, Schmidt-Weber CB, Cavani A. Th22 cells represent a distinct human T cell subset involved in epidermal immunity and remodeling. J Clin Invest. 2009;119:3573–3585. doi: 10.1172/JCI40202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Wan Q, Kozhaya L, ElHed A, Ramesh R, Carlson TJ, Djuretic IM, Sundrud MS, Unutmaz D. Cytokine signals through PI-3 kinase pathway modulate Th17 cytokine production by CCR6+ human memory T cells. J Exp Med. 2011;208:1875–1887. doi: 10.1084/jem.20102516. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS382475-supplement-1.pdf (108KB, pdf)

RESOURCES