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IL-13 Production by Regulatory T Cells Protects against Experimental Autoimmune Encephalomyelitis Independently of Autoantigen¹

Javier Ochoa-Repáraz^2,*, Agnieszka Rynda^*, Miguel A. Ascón, Xinghong Yang^*, Irina Kochetkova^*, Carol Riccardi^*, Gayle Callis^*, Theresa Trunkle^* and David W. Pascual^3,*

^* Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717; and Division of Nephrology, Department of Medicine, Johns Hopkins University, Baltimore, MD 21205

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Treatment with an anti-inflammatory Salmonella vaccine expressing enterotoxigenic Escherichia coli colonization factor Ag 1 (CFA/I) proved effective in stimulating protective, potent CD25⁺CD4⁺ regulatory T (T_reg) cells in susceptible mice challenged with experimental autoimmune encephalomyelitis (EAE). Because the Salmonella vector was considerably less protective, we questioned whether altering fimbrial subunit expression to resemble conventional Salmonella expression may impact T_reg cell potency. The Salmonella-CFA/I vaccine was modified to limit fimbrial subunit expression to the intracellular compartment (Salmonella-CFA/I_IC). SJL mice were challenged with proteolipid protein peptide 139–151 to induce EAE and orally treated with one of three Salmonella vaccines 6 days postchallenge. Treatment with Salmonella-CFA/I_IC greatly reduced clinical disease, similarly as Salmonella-CFA/I, by subduing IL-17 and IL-21; however, mechanisms of protection differed as evident by increased IL-13 and IFN- but diminished TGF-β production by T_reg cells from Salmonella-CFA/I_IC-treated mice. Adoptive transfer of T_reg cells from both CFA/I-expressing constructs was equivalent in protecting against EAE, showing minimal disease. Although not as potent in its protection, CD25^–CD4⁺ T cells from Salmonella-CFA/I_IC showed minimal Th2 cells, but vaccination did prime these Th2 cells rendering partial protection against EAE challenge. In vivo IL-13 but not IFN- neutralization compromised protection conferred by adoptive transfer with Salmonella-CFA/I_IC-induced T_reg cells. Thus, the Salmonella-CFA/I_IC vaccine elicits T_reg cells with attributes from both the Salmonella vector and Salmonella-CFA/I vaccines. Importantly, these T_reg cells can be induced to high potency by simply vaccinating against irrelevant Ags, offering a novel approach to treat autoimmune diseases independently of the autoantigen.

	Introduction

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Immunity to Salmonella is typically Th1 cell dependent (1, 2). Consequently, Th1 cell-dominating responses are also elicited when using conventional Salmonella vaccine vectors, which result in elevated IFN--regulated IgG2a Ab production as well as suboptimal mucosal IgA Ab production toward both Salmonella and passenger Ags (1, 2, 3, 4, 5). Previous work performed in our laboratory demonstrated that when mice are orally immunized with a Salmonella vaccine vector expressing the enterotoxigenic Escherichia coli fimbria colonization factor Ag I (CFA/I)⁴ on its cell surface, elevated serum IgG1 and mucosal IgA Ab titers are stimulated (6). This Ab production is supported by a biphasic Th cell response in which Th2 cells are rapidly induced preceding the development of the Th1 cells (6). In addition to stimulating a Th2 cell bias, the Salmonella-CFA/I vaccine failed to elicit the proinflammatory cytokines TNF-, IL-1, IL-1β, and IL-6 following infection of macrophages, unlike the isogenic Salmonella vector strain H647, which readily induced these cytokines (7).

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS) that shares many features with this human neurodegenerative disease (8). EAE is a Th1/Th17 cell-mediated autoimmune disease directed against protein components of CNS myelin, resulting in neurological damage and paralysis. In EAE and MS, encephalitogenic CD4⁺ T cells react against CNS autoantigens and secrete proinflammatory cytokines, such as IFN-, TNF-, IL-2, and IL-17 (9). These proinflammatory components induce the activation of microglia and macrophages, the infiltration of inflammatory cells into the CNS, and eventual axonal damage and demyelination (10, 11, 12). In addition to IL-23, IL-21 might play a role in the amplification of Th17 effector responses (13, 14, 15). A recent report showed that myeloid dendritic cells react to proteolipid protein (PLP), leading to Th17 and not Th1 cell differentiation and thus increasing the expression of TGF-β, IL-6, and IL-23 (16). Recent data have shown that the combination of IL-6 with TGF-β drives the development of pathogenic Th17 cells (12, 17).

Recently, we described that the anti-inflammatory properties of the Salmonella-CFA/I vaccine confer prophylactic (18) and therapeutic (19) protection against EAE in SJL mice. Although not nearly as effective, the isogenic Salmonella vector was able to modestly reduce the EAE clinical scores. Infection with the attenuated Salmonella-CFA/I vaccine elicited FoxP3⁺ CD25⁺CD4⁺ regulatory T (T_reg) cells, producing protective levels of TGF-β (19). Although the Salmonella vector was also able to induce T_reg cells, the costimulation by immune deviation obtained with the Salmonella-CFA/I was not evident and, consequently, protection against EAE was substantially reduced. Neutralization of T_reg cell function in mice immunized with Salmonella-CFA/I provoked a more severe EAE, and adoptive transfer of Salmonella-CFA/I-induced T_reg cells, but not naive T_reg cells, protected against EAE. Adoptive transfer of T_reg cells from mice immunized with Salmonella vector protected against EAE, but to a lesser degree. We concluded that the immunization with attenuated strains of Salmonella expressing irrelevant, non-self Ags induced and expanded T_reg cells that were protective against EAE (19).

In this current work, we queried whether differences in the observed T_reg cell potency were attributed to the stimulation of Th2 cells resulting in immune deviation, because these latter T cells were not elicited by the Salmonella vector. A Salmonella vaccine limiting the CFA/I subunit to its expression in the periplasmic/intracellular compartment (Salmonella-CFA/I_IC) was developed to determine whether the CFA/I fimbrial subunit could then stimulate Th1 rather than Th2 cells and whether this shift in CD4⁺ T cell response would impact the development of potent T_reg cells to treat EAE. In other words, would the Salmonella-CFA/I_IC strain remain anti-inflammatory or would it resemble the proinflammatory, isogenic Salmonella vector? It was found that treatment with Salmonella-CFA/I_IC greatly reduced the EAE clinical scores as effectively as Salmonella-CFA/I and better than the Salmonella vector-treated mice. Both CFA/I vaccines reduced the IFN- produced by CD25^–CD4⁺ T cells but, interestingly, the Salmonella-CFA/I_IC stimulated the generation of IL-13- and IFN--producing T_reg cells that appeared to protect against EAE. In vivo blockade of IL-13 reversed protection conferred by Salmonella-CFA/I_IC-induced T_reg cells, whereas the in vivo blockade of IFN- did not. As evident from this study, oral immunization with Salmonella vaccines enhances multiple subsets of T_reg cells with different regulatory profiles for which their induction is dependent upon the presence and location of heterologous Ags.

	Materials and Methods

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Mice

Female, 6-wk-old SJL mice were obtained from Frederick Cancer Research Facility, National Cancer Institute (Frederick, MD), and The Jackson Laboratory. All mice were maintained at Montana State University Animal Resources Center (Bozeman, MT) under pathogen-free conditions in individual ventilated cages under high efficiency particulate air (HEPA)-filtered barrier conditions and were fed sterile food and water ad libitum. The mice were free of bacterial and viral pathogens as determined by Ab screening and histopathological analysis of major organs and tissues. All animal care and procedures were in accordance with institutional policies (Montana State University) for animal health and well-being.

Salmonella-CFA/I_IC (AP331) construction

The pMA20-asd⁺ recombinant plasmid was constructed by deleting the 2.0-kb SacI DNA fragment from the CFA/I operon (cfaABCE) from pJGX15C-asd⁺ (20). The deleted fragment encodes for an outer membrane usher protein (CfaC), which mediates the ordered assembly of the filamentous heteropolymer and a minor component protein (CfaE) present as a single tip subunit (21). The remaining cfaAB genes are involved in the expression of the major structural subunit (CfaB) and the chaperone (CfaA) promoting subunit folding and transport in periplasmic and intracellular compartments. The ligated plasmid construct was electroporated into the E. coli H681 asd strain. Clones containing the selected plasmid construct were detected and screened by colony immunoblotting, in vivo Ag/Ab slide agglutination, and plasmid miniprep restriction map protocols. The purified plasmids obtained were then electroporated into Salmonella enterica serovar Typhimurium H683 aroA asd strain to obtain the desired balanced lethal Salmonella-CFA/I_IC (AP331) construct. Recombinant strains and the control strain H647 were cultured by using both Luria-Bertani and nutrient agar or broth medium without antibiotics or diaminopimelic acid as previously described (20). Neither H681 nor H683 grows on these media unless the asd⁺ allele is supplied in trans.

Detection of CFA/I fimbrial subunit in Salmonella strains by Western blotting

The absence or presence of the CFA/I fimbrial subunit in S. enterica serovar Typhimurium H647, AP331, and H696 was analyzed by SDS-PAGE and Western blotting. SDS-PAGE gel (15% (w/v) polyacrylamide) was transferred to a 0.2-mm pore size nitrocellulose membrane (Bio-Rad Laboratories). The membrane was incubated in rabbit anti-CFA/I antiserum (produced in house; diluted 1/2,000 in PBS-Tween 20) overnight at 4 °C. After four washes in PBS-Tween 20, a second incubation for 90 min with HRP-conjugated rat anti-rabbit IgG (Southern Biotechnology Associates) diluted 1/1,000 in PBS-Tween 20 was performed. Detection of CFA/I fimbrial subunit, after four new washes in PBS-Tween 20, was achieved upon development with the substrate 4-chloro-1-naphthol chromogen and H₂O₂ (Sigma-Aldrich). The apparent molecular masses were determined by comparing their electrophoretic mobility with that of the following molecular mass markers: (Amersham Biosciences): myosin (220 kDa), phosphorylase b (97 kDa), BSA (66 kDa), OVA (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa), and lysozyme (14.3 kDa).

AP331 subcellular fraction and isolation studies were performed to quantify the level of CfaB expression. AP331 was cultured in Luria-Bertani medium as described above and bacteria were harvested by centrifugation. Subcellular fractionation was performed according to the manufacturer’s directions using the PeriPreps Periplasting kit (Epicentre Biotechnologies) as previously described (22, 23). The different subcellular components were subjected to electrophoresis with 15% SDS-polyacrylamide gel and subsequently transferred to a nitrocellulose membrane for Western blot analysis. A rabbit anti-CFA/I serum was used as primary Ab, and bound Ab was detected using a HRP-conjugated goat anti-rabbit-IgG Ab (Southern Biotechnology Associates) and substrate, as described above.

PLP_139–151 challenge and oral Salmonella vaccination

The encephalitogenic PLP peptide (PLP_139–151; HSLGKWLGHPDKF) was synthesized by Global Peptide Services and purified by HPLC to >90%. For each experiment, female SJL mice (five per group) were challenged s.c. with 200 µg of PLP_139–151 in 200 µl of complete Freund’s adjuvant (18). On days 0 and 2 postchallenge, mice received i.p. 200 ng of Bordetella pertussis toxin (List Biological Laboratories). Six days after PLP_139–151 challenge, mice were given a single oral dose of 5 x 10⁹ CFU of the Salmonella-CFA/I vaccine (6,19; aroA Typhimurium-CFA/I vector vaccine, strain H696, expressing functional CFA/I fimbriae from E. coli), Salmonella-CFA/I_IC (aroA Typhimurium-CFA/I vector vaccine, strain AP331, expressing CFA/I in the periplasmic/intracellular compartment), or its isogenic control strain H647 (Salmonella empty vector) (6, 19). Fimbrial expression was maintained by a plasmid bearing a functional asd gene to complement the lethal chromosomal asd mutation in the parent Salmonella strain. Control groups were treated with PBS. Mice were monitored and scored daily for disease progression (18) as follows: 0, normal; 1, limp tail; 2, hind limb weakness; 3, hind limb paresis; 4, quadriplegia; 5, death.

Mice were also evaluated for the extent of colonization by AP331 and H696 vaccine strains. Mice (five per group) were orally dosed with 5 x 10⁹ CFU of AP331 or H696, and spleens and Peyer’s patches were harvested 1 wk later. Tissues were weighed and homogenized in sterile water for CFU enumeration on MacConkey agar (Difco) and incubated overnight at 37°C (24).

Ab ELISA

CFA/I-specific endpoint titers from the dilution of immune sera or fecal extracts were measured by an ELISA as previously described using purified fimbrial Ag (6) as a coating Ag. Specific reactivity to CFA/I fimbriae was determined using HRP conjugates of goat anti-mouse IgG-, IgG1-, IgG2a-, IgG2b-, and IgA-specific Abs (1 µg/ml; Southern Biotechnology Associates), and ABTS (2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid; Moss) enzyme substrate was used to develop the reaction. The absorbance was measured at 415 nm on a Bio-Tek Instruments ELx808 microtiter plate reader. Endpoint titers were expressed as the reciprocal dilution of the last sample dilution, giving an absorbance of 0.1 OD units above OD₄₁₅ of negative controls after 1 h of incubation.

Histological evaluation of spinal cords

For histological evaluation of tissue pathology, spinal cords were removed 14 days after challenge and fixed with neutral buffered formalin (VWR International), embedded into paraffin, and sectioned at 5 µm. Transverse sections of spinal cords were stained with H&E for pathological changes and inflammatory cell infiltration. Adjacent sections were stained with Luxol fast blue and examined for loss of myelin. Pathological manifestations were scored separately for cell infiltrates and demyelination. Each H&E section was scored from 0 to 4 as follows: 0, normal; 1, cell infiltrate into the meninges; 2, one to four small focal perivascular infiltrates; 3, five or more small focal perivascular infiltrates and/or one or more large infiltrates invading the parenchyma; and 4, extensive cell infiltrates involving 20% or more of the white matter (18, 19). In each Luxol fast blue-stained section, myelin was also scored from 0 to 4: 0, normal; 1, one small focal area of demyelination; 2, two or three small focal areas of demyelination; 3, one to two large areas of demyelination; 4, extensive demyelination involving 20% or more of white matter (18, 19).

Cytokine ELISA

Spleens, mesenteric lymph nodes (MLNs), and head and neck LNs (HNLNs) were aseptically removed 14 days after challenge from PBS-, Salmonella vector-, Salmonella-CFA/I_IC-, and Salmonella-CFA/I-treated groups of mice. Lymphocytes were prepared as previously described (19) and resuspended in complete medium consisting of RPMI 1640 medium supplemented with 1 mM sodium pyruvate, 1 mM nonessential amino acids, penicillin/streptomycin (10 U/ml), and 10% FBS (Atlanta Biologicals). Lymphocytes were cultured in 24-well tissue plates at 5 x 10⁶ cells/ml in complete medium alone or in the presence of OVA (10 µg/ml; Sigma-Aldrich), purified CFA/I fimbriae (10 µg/ml), or PLP_139–151 peptide (30 µg/ml) in a total volume of 1 ml for 60 h at 37° C. The supernatants were collected by centrifugation and stored at –80°C. Capture ELISA was used to quantify, on duplicate sets of samples, the levels of IFN-, IL-4, IL-10, IL-13, IL-17, and TGF-β produced by lymphocytes, as previously described (19). For detection of IL-21 and IL-22, microtiter wells were coated with 2 µg/ml purified goat anti-mouse IL-21 Ab or goat anti-mouse IL-22 Ab, respectively (R&D Systems). After blocking with PBS plus 1% BSA for 2 h at 37°C, washed wells were incubated with cell culture supernatants at 4° C for 24 h. After washing, 0.5 µg/ml biotinylated rat anti-mouse IL-21 mAb or biotinylated goat anti-mouse IL-22 Ab (R&D Systems) was added, respectively, for 90 min at 37° C. Following washing, 1/500 HRP-goat anti-biotin Ab (Vector Laboratories) was added for 1 h at room temperature. After washing, ABTS peroxidase substrate (Moss) was added to develop the reaction.

FACS analysis

Lymphocytes from the HNLNs, MLNs, and spleens were isolated 14 days after challenge, and single cell preparations were prepared as described above (19). To obtain lymphocytes from spinal cords, mice were perfused through the left ventricle with 20 ml of cold PBS, and spinal cords were removed by flushing the vertebral canal with medium and then prepared as previously described (19).

Cells were stained for FACS analysis using conventional methods. To distinguish among neutrophils, monocytes/macrophages, and lymphocytes, staining for CD45 and MHC class II was done as previously described (19). Leukocyte gates were set within the forward and side scatter profiles to exclude resting microglia cells from the spinal cord preparations. To detect neutrophils, cells were stained with SK208 mAb (18, 19), followed by FITC-conjugated donkey anti-rat anti-IgG (Jackson ImmunoResearch Laboratories) and fluorochrome-conjugated anti-CD11b (BD Pharmingen); for macrophages, the fluorochrome-conjugated mAbs CD45 (clone 30-F11), I-A^s (clone 10-3.6), and CD11b (BD Pharmingen) and the PE-conjugated F4/80 mAb (Serotec) were used. T cell subsets were analyzed using fluorochrome-conjugated mAbs (BD Pharmingen) for CD4, CD25, TCRβ, CD8, glucocorticoid-induced TNFR (GITR), CCR6, CTLA-4, and RANKL, and biotinylated TGF-β (R&D Systems). Intracellular staining for FoxP3 was done using fluorochrome labeled-anti-Foxp3 mAb (clone FJK-16s; eBioscience), FITC- or PE-conjugated anti-IFN- Ab, (BD Pharmingen), and biotinylated anti-IL-13 Ab (R&D Systems). Bound fluorescence was analyzed with a FACSCanto flow cytometer (BD Biosciences).

Adoptive transfer studies

Fourteen days after oral immunization with H647, AP331, or H696, total CD4⁺ T cells from spleens, HNLNs, and MLNs were obtained (negative CD4⁺ T cell isolation kit; Dynal Biotech). CD25⁺CD4⁺ and CD25^–CD4⁺ T cells were isolated to >93 and 99%, respectively, by cell sorting (FACSVantage with Turbo-Sort; BD Biosciences) of stained T cells. To test T_reg cell efficacy, 6 x 10⁵ CD25^–CD4⁺ T cells or CD25⁺CD4⁺ T cells were i.v. injected into naive recipients. One day after the adoptive transfer of T cell subsets, mice were challenged with PLP_139–151.

In vivo blockade of IFN- and IL-13

To block IFN-, mice were given i.p. 0.5 mg of anti-IFN- mAb purified from a hybridoma cell line (clone R4 6A2; American Type Culture Collection) on days –1 (before) and 1 and 5 (after) EAE challenge with PLP_139–151. One day before the challenge, AP331- and H647-induced CD25⁺ CD4⁺ T_reg cells were adoptively transferred into mice. Control groups received 0.5 mg of purified rat IgG Ab (AbD Serotec). A separate control group was immunized with PBS 1 day before EAE challenge. All mice were monitored daily for the development of EAE.

To block IL-13, mice were given i.p. 0.5 ml of serum from rabbits immunized with rat IL-13 (25) on day –1 before challenge, and 0.25 ml of serum on days 1 and 5 after EAE challenge with PLP_139–151. The same adoptive transfer protocols were used in this study, and control mice were given equivalent volumes of normal rabbit serum (NRS). All mice were monitored daily for development of EAE.

In vitro T cell assays

To assess cytokine production by T_reg and effector T cells, CD25⁺CD4⁺ T cells and CD25^–CD4⁺ T cells (2 x 10⁵) were stimulated in vitro with anti-CD3 mAb-coated wells (10 µg/ml; BD Pharmingen) plus the soluble anti-CD28 mAb (5.0 µg/ml; BD Pharmingen) or with 30 µg/ml PLP_139–151 for 5 days in complete medium (final volume of 300 µl in a 48-well plate). Capture ELISA was used to quantify triplicate sets of samples to measure cytokines.

Statistical analysis

ANOVA followed by posthoc Tukey test was applied to show differences in clinical scores in treated vs PBS mice and in the T_reg cell kinetic experiments. The Student t test was used to evaluate the differences between variations in cytokine level production, and values of p < 0.05 are indicated.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Oral immunization with Salmonella-CFA/I_IC protects against EAE

Previously, it was shown that oral treatment with a single dose of a S. enterica serovar Typhimurium expressing the CFA/I fimbriae from enterotoxigenic E. coli (Salmonella-CFA/I, strain H696) was able to reduce inflammatory cell infiltration, demyelination, and EAE clinical scores in SJL mice (19). This particular vaccine was exquisite in stimulating elevated levels of T_reg cells in part by immune deviation obtained with CD25^– Th2-type cells. Although T_reg cells were also induced upon vaccination with the Salmonella empty vector, the costimulation of the CD25^– Th2-type cells was not induced, resulting in much less protection (19). Thus, the empty vector was unable to elicit the production of anti-inflammatory cytokines, although it elicited the expansion of T_reg cells, but this was insufficient for protection against EAE when compared with Salmonella-CFA/I. Given these findings, if the CFA/I fimbrial subunit was not expressed on the Salmonella cell’s surface and was made to resemble the Salmonella empty vector, we queried whether the protective efficacy would be compromised, because this construct would be predicted to lack the coinduced anti-inflammatory response by CD25^–CD4⁺ T cells.

To address this question, a S. enterica serovar Typhimurium strain that expresses the CFA/I fimbrial subunit in its periplasmic/intracellular compartment, Salmonella-CFA/I_IC (strain AP331), was constructed (Fig. 1A) by deleting the presumed usher protein and minor subunit (21). To demonstrate expression of the CFA/I fimbrial subunit by AP331, Western blotting was performed using a rabbit polyclonal Ab against CFA/I fimbriae (Fig. 1B) and compared with expression by Salmonella-CFA/I (H696). Similar amounts of the CFA/I fimbrial subunit were observed in whole cell extracts from AP331 and H696 when compared with a purified sample of CFA/I fimbriae, and no expression of the fimbriae was observed by the Salmonella empty vector (H647). The CfaB subunit fractionated mostly with the periplasm and some with the spheroplast, with negligible levels associated with the outer membrane and cell wall (Fig. 1, C and D). Oral vaccination of SJL mice with AP331 elicited serum and mucosal Ab titers against CFA/I fimbriae as demonstrated by ELISA 14 days after vaccination, but they were not as great as those elicited with Salmonella-CFA/I (p < 0.001; Fig. 1, E and F). Differences in these Ab titers did not appear to be attributed to differences in colonization by AP331- or H696-dosed mice. Splenic and Peyer’s patch weights 1 wk after infection were identical, as was splenic colonization; however, the AP331-infected mice showed 50% more reduction (p < 0.001) in colonization of the Peyer’s patches than did the H696-infected mice.

View larger version (43K): [in this window] [in a new window]   FIGURE 1. Expression of CFA/I fimbrial subunit by Salmonella vaccines are equivalent but render different Ab titers. A, The cfaABCE operon for the expression of the CFA/I fimbriae was modified by deleting the cfaCE genes but leaving the fimbrial subunit (CfaB) and its chaperone (CfaA) intact. Restriction sites include XhoI (X), SacI (S), AatII (A), BglII (B), and PvuI (P). B, Equivalent whole cell extracts from Salmonella-CFA/I (H696) and Salmonella-CFA/IIC (AP331), when subjected to SDS-PAGE and Western blot analysis, showed similar amounts of the CFA/I fimbrial subunit being produced. Purified CFA/I fimbriae were used as a positive migration control, and whole cell extracts from Salmonella empty vector (H647) did not react against the polyclonal rabbit anti-CFA/I fimbriae Abs. C and D, Subcellular fractionation of S. enterica serovar Typhimurium strain AP331. AP331 cells were fractionated and enriched for spheroplasts (lane 1), periplasmic membranes (lane 2), outer membranes and cell wall (lane 3), and cell culture supernatant (lane 4) and quantified by densitometric scanning (D). Lane M is the prestained protein m.w. standards. Presented are the results from one of the three experiments. The majority of the CfaB subunit was present in the periplasm (lane 2), some was in the spheroplasts (lane 1), and a minimal amount was associated with the outer membrane (lane 3). None was secreted (lane 4). *, p < 0.001, represents statistical differences between the periplasmic fraction vs each fraction; #, p < 0.001, represents the statistical difference between the spheroplast fraction vs the outer membrane and cell wall fraction. E and F, A single oral immunization with Salmonella-CFA/I or Salmonella-CFA/IIC vaccine increased mucosal (Copro-) and serum IgA (E) and IgG (F) responses to CFA/I fimbriae, but not the Salmonella empty vector (H647). The AP331 strain stimulated less mucosal and serum Abs than the H696 strain. Analysis of IgG subclasses showed significant increases in IgG1, IgG2a, and IgG2b (p < 0.001) in H696-vaccinated mice when compared with AP331-vaccinated mice. Depicted are the means ± SEM from three separate experiments for a total of 15 mice/group. *, p < 0.001 for H696-vaccinated vs AP331- and H696-vaccinated vs H647-vaccinated groups. , p < 0.001 for AP331- vs H647-vaccinated groups.

View larger version (21K): [in this window] [in a new window]   FIGURE 2. Oral Salmonella-CFA/I (H696) and Salmonella-CFA/IIC (AP331) treatments reduced EAE clinical scores in PLP139–151-challenged SJL mice. Mice therapeutically treated 6 days after EAE challenge with H696 or AP331 vaccines showed a significant reduction in their clinical scores, with all mice recovering from EAE. Salmonella vector (strain H647)-treated mice showed some recovery but presented greater clinical scores when compared with PBS-treated mice. Depicted are the combined results from three separate experiments for a total of 15 mice/group; *, p < 0.001 for PBS- vs H647-, H696-, or AP331-treated mice.

Cytokine production was analyzed for sorted CD25^–CD4⁺ T cells from PBS-, H647-, AP331-, and H696-treated mice 14 days post-EAE challenge. Cells were sorted (purity of isolated cells, determined by FACS analysis, >95%) and in vitro costimulated with anti-CD3 and anti-CD28 mAbs (Fig. 3). CD25^–CD4⁺ T cells from PBS-treated mice produced significantly elevated levels of IFN-, IL-17, and IL-21 (p < 0.001) than CD25^–CD4⁺ T cells obtained from H647-, AP331-, or H696-treated mice (Fig. 3A). IFN- and IL-17 production by CD25^–CD4⁺ T cells isolated from H647-treated mice was also significantly enhanced (p < 0.001) when compared with AP331- and H696-treated mice. In contrast, CD25^–CD4⁺ T cells from H696-treated mice produced significantly greater levels (p < 0.001) of the anti-inflammatory cytokines IL-4, IL-10, and TGF-β than those obtained from PBS-, H647-, or AP331-treated mice and greater levels of IL-13 and IL-22 when compared with PBS- and H647-treated mice (p < 0.001). CD25^–CD4⁺ T cells from AP331-treated mice showed significantly higher production (p < 0.001) of IL-4, IL-13, and IL-22 than that obtained from PBS- and H647-treated mice.

View larger version (32K): [in this window] [in a new window]   FIGURE 3. Salmonella-CFA/IIC vaccine therapy elicits IL-13- and IFN--producing Treg and CD25–CD4+ T cells in contrast to Salmonella-CFA/I therapy that stimulates TGF-β-producing Treg and CD25– Th2 cells subsequent to EAE challenge. Cell-sorted CD25–CD4+ T cells (A) and Treg cells (B) from EAE-challenged SJL mice treated with H647, AP331, H696, or PBS were purified on day 14 postchallenge with PLP139–151 and costimulated in vitro with anti-CD3 and anti-CD28 mAbs. Cytokine production was measured from supernatants after 72 h of culture. A, CD25–CD4+ T cells from diseased (PBS-treated) mice produced elevated levels of IFN-, IL-17, and IL-21 with more depressed levels of IL-4, IL-10, IL-13, TGF-β, and IL-22 than any of the Salmonella-treated groups. IFN- and IL-17 production by H647-treated mice was also significantly enhanced when compared with AP331- and H696-treated mice, and AP331 CD25–CD4+ T cells produced more IFN- than H696. CD25–CD4+ T cells from H696-and AP331-treated mice produced more IL-4, IL-13, and IL-22 than PBS- or H647-treated mice; H696 also produced more IL-10 and TGF-β than any of the treatment groups. B, CD25+CD4+ T cells sorted from H647- and AP331-treated mice produced elevated levels of IFN- when compared with cells sorted from PBS-treated or H696-treated mice (p < 0.001). Treg cells from PBS- and H647-treated groups produced minimal levels of the anti-inflammatory cytokines IL-4, IL-10, IL-13, and TGF-β. Treg cells sorted from any of the treatment groups produced no to minimal amounts of IL-17, IL-21, and IL-22. Although both produced equivalent amounts of IL-4, AP331-induced Treg cells showed strikingly enhanced IL-13 while H696-induced Treg cells produced elevated levels of TGF-β. Depicted are the combined results from three separate experiments for a total of 15 mice/group ± SEM. *, p < 0.001 represents differences in cytokine production among PBS-, H647-, AP331-, and H696-treated groups.

Ag-specific cytokine evaluations were also performed on the purified T_reg and CD25^– CD4⁺ T cells. As with the anti-CD3 plus anti-CD28 stimulation (Fig. 3), AP331 vaccine-induced T_reg cells produced elevated IL-13 and IFN- with lesser amounts of TGF-β but similar levels of IL-4 and IL-10 following in vitro restimulation with PLP_139–151 (Fig. 4B). For the CD25^– CD4⁺ T cells, similar trends obtained with PLP_139–151 restimulation as with the anti-CD3 plus anti-CD28 stimulations observed in Fig. 3A were evident (Fig. 4A). Peptide restimulation notably induced increased IL-13 with similar IL-4, IL-10, and TGF-β and depressed IFN- and IL-17 production (Fig. 4A). IL-21 was only produced by CD25^–CD4⁺ T cells from PBS-dosed and -challenged mice.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. Salmonella-CFA/IIC vaccine elicits IL-13- and IFN--producing Treg cells as evidenced by PLP139–151 restimulation. Sorted Treg cells (A) and CD25–CD4+ T cells (B) from SJL mice that were orally dosed with H647, AP331, H696, or PBS on day –14, EAE-challenged on day 0, and purified 14 days after EAE challenge and costimulated in vitro with 30 µg/ml PLP139–151 peptide-cultured cells. Cytokine production was measured from supernatants after 5 days of culture. A, CD25–CD4+ T cells from diseased (PBS-treated) mice produced elevated levels of IFN-, IL-17, and IL-21 with minimal IL-4, IL-10, IL-13, and TGF-β than any of the Salmonella-infected groups. IFN- and IL-17 production by H647-dosed mice was also significantly enhanced when compared with AP331- and H696-dosed mice, and more IFN- was produced by AP331- than H696-dosed mice. CD25–CD4+ T cells from H696-dosed mice produced more IL-4 and IL-10 than PBS- or H647-dosed mice, whereas CD25–CD4+ T cells from AP331-dosed mice produced strikingly more IL-13 than any of the groups; H696-dosed mice produced more TGF-β than any of the treatment groups. B, CD25+CD4+ T cells sorted from H647- and AP331-dosed mice produced elevated levels of IFN- when compared with cells sorted from PBS- or H696-dosed mice (p < 0.001). Treg cells from PBS- and H647-dosed groups produced minimal levels of the anti-inflammatory cytokines IL-4, IL-10, IL-13, and TGF-β. Treg cells sorted from any of the treatment groups produced minimal amounts of IL-17 and IL-21. Both AP331 and H696 produced equivalent IL-4 and IL-10 and AP331-induced Treg cells showed strikingly enhanced IL-13 while H696-induced Treg cells produced elevated levels of TGF-β. Cytokine production after stimulation with PLP139–151 peptide is depicted as the means of 10 mice/group ± SEM corrected for cytokine production by unstimulated cells. *, p 0.001; **, p 0.05 represents differences in cytokine production between PBS-, H647-, and AP331-treated groups vs H696-treated groups.

Oral vaccination with Salmonella-CFA/I_IC induces IL-13- and IFN--producing FoxP3⁺ T_reg cells

To ascertain whether the altered expression of the CFA/I fimbrial subunit in Salmonella would modify the induction of T_reg cells, spleens, MLNs, and HNLNs were harvested from SJL mice 14 days postvaccination with H647, AP331, and H696, as well as from naive SJL mice. The presence of T_reg cells was analyzed by FACS (Fig. 5A). A single oral dose with the Salmonella vaccines induced significant increases in the percentages of CD25⁺CD4⁺ T cells in spleens and LNs when compared with percentages obtained from naive SJL mice (Fig. 5A and Tables III and IV; p < 0.001). Increased FoxP3⁺ CD25⁺ T cells were obtained in mice vaccinated with H696 when compared with the same cells induced in spleens and LNs of H647- and AP331-vaccinated mice (Fig. 5B and Tables III and IV; p < 0.001). In fact, the percentages of induced FoxP3⁺ T_reg cells by Salmonella-CFA/I_IC more resembled those of the Salmonella vector than those of Salmonella-CFA/I.

View larger version (31K): [in this window] [in a new window]   FIGURE 5. Oral vaccination with Salmonella vaccines stimulates the induction of FoxP3+ Treg cells in mice. A and B, Spleens and lymph nodes were harvested from SJL mice 14 days after immunization with Salmonella vaccines, and CD25+CD4+ T cells were analyzed by flow cytometry. Each of the Salmonella vaccines stimulated enhanced expression of Treg cells when compared with naive mice, albeit vaccination with H696 induced the greatest increases in the CD25+CD4+ T cells (A), as well as the percentages of FoxP3+ CD25+CD4+ T cells (B). C, The percentages of FoxP3+ CD25–CD4+ T cells were also enhanced after oral vaccination with each of the Salmonella strains when compared with naive mice. Depicted are the combined results from two separate experiments for a total of 12 samples/group ± SEM *, p < 0.001 for naive vs treated groups, and differences between treated groups.

View larger version (25K): [in this window] [in a new window]   FIGURE 6. Increased expression of IL-13+ and IFN-+ FoxP3+ CD25+CD4+ T cells subsequent to oral vaccination with Salmonella-CFA/IIC (AP331). Spleens and HNLNs from SJL mice immunized with Salmonella vector (H647), Salmonella-CFA/I (H696), and AP331 strains were harvested 14 days postimmunization, and FoxP3+ CD25+CD4+ T cells were analyzed by FACS (A) to determine their cytokine profiles by intracellular staining for IFN-, IL-10, IL-13, or cell surface TGF-β expression (B and C). Splenic and HNLN FoxP3+ Treg cells obtained from AP331-dosed mice shared features of both H647- and H696-immunized mice. Resembling H647-dosed mice, an elevated percentage of IFN-+ Treg cells and a reduced level of TGF-β+ Treg cells were obtained; resembling H696-dosed mice, an elevated percentage of IL-13+ Treg cells was observed. The percentage of IL-10+ Treg cells was the same for each immunized group and tissue evaluated. Insets in B represent the isotype control for intracellular cytokine staining. Depicted are the results from 6–9 mice/group; *, p < 0.001; **, p = 0.006; and ***, p = 0.040 represent differences in cytokine-positive FoxP3+ Treg cells from H647-vaccinated mice vs AP331- or H696-vaccinated mice; , p < 0.001, represents differences in cytokine-positive FoxP3+ Treg cells from AP331-vaccinated mice vs H696-vaccinated mice.

Similar evaluations were performed with CD25^–CD4⁺ T cells. Clearly, a subset of these cells from mice immunized with H696 showed enhanced expression of FoxP3 when compared with naive, H647, and AP331 CD25^–CD4⁺ T cells in spleens and LNs (p < 0.001; Fig. 5C and Tables III and IV, respectively). Interestingly, receptor activator of NF-B ligand (RANKL) was found to be associated mostly with LN T_reg cells and CD25^–CD4⁺ T cells and not with splenic CD4⁺ T cells (Tables III and IV). Past studies have shown these RANKL⁺ T cells to be mostly associated with pathogenic CD4⁺ T cells (26, 27).

Adoptive transfer of CFA/I fimbriae-induced T_reg cells protects against EAE

Previous studies have shown that T_reg cells obtained from EAE-induced mice were able to protect when adoptively transferred into susceptible naive mice and subsequently challenged with myelin Ags to induce EAE (28, 29, 30). In contrast to innate T_reg cells, it was previously shown that Salmonella-CFA/I-induced T_reg cells were able to potently protect against EAE (19). To understand differences in the mechanism of protection conferred by Salmonella-CFA/I_IC by Salmonella-CFA/I immunization, cell-sorting experiments were performed 14 days postimmunization with H647, AP331, or H696 Salmonella strains to obtain T_reg cell and CD25^–CD4⁺ T cell subsets to evaluate their cytokine profile after in vitro stimulation with anti-CD3 and anti-CD28 mAbs. T_reg cells sorted from Salmonella-CFA/I_IC-immunized mice produced significantly (p < 0.001) greater levels of IFN- when compared with H647- or H696-induced or naive T_reg cells (Fig. 7B). AP331-induced T_reg cells also produced significantly greater IL-10 and IL-13 when compared with naive, H647-, and H696-induced T_reg cells (p < 0.001). In contrast, H696-induced T_reg cells produced significantly more TGF-β and IL-4, which is consistent with what was previously reported (19). Minimal to no IL-17, IL-21, or IL-22 was observed with any of the vaccinated or naive groups.

View larger version (31K): [in this window] [in a new window]   FIGURE 7. Oral immunization of SJL mice with Salmonella-CFA/IIC induces Treg cells to produce elevated levels of IFN- and IL-13 but does not induce immune deviation. A and B, CD25–CD4+ T cells (A) and CD25+CD4+ Treg cells (B) from SJL mice vaccinated with H647, AP331, or H696 and from naive mice were costimulated in vitro with anti-CD3 and anti-CD28 mAbs, and cytokine production was measured from culture supernatants after 72 h. A, CD25–CD4+ T cells from AP331- vaccinated mice failed to show any significant difference in cytokine production from naive mice. H647 CD25–CD4+ T cells showed enhanced IFN- and H696 CD25–CD4+ T cells showed elevated production of IL-4, IL-10, and IL-13. B, Treg cells elicited by AP331 vaccination produced enhanced levels of IFN-, IL-10, and IL-13 when compared with Treg cells from H647- or H696- vaccinated or naive mice. In contrast, H696-induced Treg cells produced elevated levels of IL-4 and TGF-β. Depicted are the combined results from two separate experiments for a total of 12 samples/group ± SEM. *, p < 0.001 represents differences in cytokine production between Salmonella-vaccinated groups and naive mice.

To determine the potency of T_reg cells induced by Salmonella-CFA/I_IC when compared with Salmonella-CFA/I, adoptive transfer experiments were performed. SJL mice were orally immunized with a single dose of H647, AP331, or H696, and 14 days after immunization CD25^–CD4⁺ T cells and T_reg cells were sorted from spleens and HNLNs (purity of isolated cells was determined by FACS analysis: >90% for CD25⁺CD4⁺ T_reg cells and 99% for CD25^– CD4⁺ T cells; Fig. 8A). T_reg or CD25^–CD4⁺ T cells (6 x 10⁵) were i.v. injected into naive recipient mice, and 1 day later the mice were challenged with PLP_135–151. Adoptive transfer of mice with CD25^– CD4⁺ T cells from H647-dosed mice failed to confer protection against EAE challenge, and no significant differences were observed when compared with the PBS-treated control mice that developed the expected EAE disease (Fig. 8B). Recipient mice given CD25^–CD4⁺ T cells from AP331 donor mice showed a significant delay in the EAE clinical onset when compared with H696-induced CD25^–CD4⁺ T cells (p < 0.001); however, 15 days postchallenge no significant differences were observed between both groups. Thus, recipient mice given CD25^–CD4⁺ T cells obtained from mice dosed with either CFA/I vaccine were equally protected. In a similar fashion, donor T_reg cells obtained from H696 or AP331 mice and adoptively transferred into challenged recipient mice were able to confer nearly complete protection (Fig. 8B), albeit via different mechanisms. In both recipient groups, a significant reduction in the EAE clinical scores was observed in challenged mice when compared with the challenged PBS-treated mice. No statistical differences were observed in the minimal to no clinical scores of mice treated with AP331 and H696 T_reg cells.

View larger version (36K): [in this window] [in a new window]   FIGURE 8. Adoptive transfer of Treg cells and, to a lesser extent, CD25–CD4+ T cells from mice orally immunized with Salmonella-CFA/IIC (AP331) conferred protection against EAE. A, CD25–CD4+ T cells and CD25+CD4+ T cells were sorted from spleens and HNLN of SJL mice 14 days postimmunization with AP331, Salmonella-CFA/I (H696), or Salmonella vector (H647). Purity of isolated cells was determined by FACS analysis (>90% for Treg cells and 99% for CD25–CD4+ T cells). B, Treg or CD25–CD4+ T cells (6 x 105) from each Salmonella vaccination group were i.v. injected into naive recipient mice and, 1 day later, mice were challenged with PLP135–151. Adoptive transfer of Treg cells from AP331- and H696-dosed mice conferred nearly complete protection against EAE when compared with the PBS-treated mice, whereas H647-Treg cells were only partially protective. Adoptive transfer of CD25–CD4+ T cells from AP331- and H696-dosed mice were partially protective, although the AP331-induced CD25–CD4+ T cells showed a significant delay in the EAE clinical outcome when compared with H696-induced CD25–CD4+ T cells (p < 0.001); by 15 days postchallenge, however, no significant differences were observed between these groups. H647-induced CD25–CD4+ T cells did not confer any protection against EAE. Depicted results are from a total of five mice per group. *, p < 0.001, for adoptively transferred CD25+ CD4+ T cells vs PBS-dosed mice; , p < 0.001 for adoptively transferred CD25–CD4+ T cells vs PBS-dosed mice.

Evaluation of cytokines produced by T_reg cells subsequent to oral immunization with Salmonella-CFA/I_IC suggested that IL-13 and IFN- (Figs. 6 and 7) may be important for the observed protective responses obtained in the adoptive transfer studies (Fig. 8). To determine the relative importance of IL-13 and IFN- in the protection conferred by AP331-induced T_reg cells, in vivo cytokine neutralization studies were performed at the time of adoptively transferred T_reg cells to assess their impact upon protection against EAE (Figs. 9 and 10). As before, 6 x 10⁵ T_reg cells sorted from PBS-, H647-, and AP331-immunized SJL mice were adoptively transferred (i.v.) into naive SJL recipient mice and, 1 day after, EAE was induced. For IL-13 neutralization, 0.5 ml of rabbit anti-rat IL-13 serum (25) was administered 1 day before EAE induction and two additional doses of 0.25 ml were each given on days 1 and 5 post-EAE challenge (Fig. 9). In vivo neutralization of IL-13 resulted in the loss of protection conferred by adoptively transferred T_reg cells obtained from AP331. Treatment with anti-IL-13 or NRS upon EAE-challenged mice given T_reg cells obtained from H647-immunized mice had no significant effect upon the disease course. Likewise, treatment of PBS-group with anti-IL-13 or NRS did not impact EAE clinical scores. These results demonstrate that IL-13 is essential for the protection conferred by T_reg cells induced upon immunization with the AP331 vaccine.

View larger version (19K): [in this window] [in a new window]   FIGURE 9. IL-13 produced from mice adoptively transferred with Treg cells from Salmonella-CFA/IIC (AP331)-immunized mice protects against EAE. Cell-sorted 6 x 105 Treg cells from Salmonella vector (H647)- and AP331-immunized SJL mice were adoptively transferred (i.v.) into naive SJL recipient mice and, 1 day later, EAE was induced with PLP139–151. To neutralize IL-13, rabbit antiserum (0.5 ml) was given 1 day before EAE induction and two additional doses (0.25 ml each) were given on days 1 and 5 post-EAE challenge (thick arrows). Equivalent volumes of NRS were given to control groups. In vivo neutralization of the IL-13 of recipient mice given AP331-induced Treg cells reversed the protection conferred by these T cells, and their clinical scores resembled mice adoptively transferred with H647-induced Treg cells. Anti-IL-13 treatment had no impact upon EAE in PBS-treated or mice adoptively transferred with Treg cells from H647-immunized mice. NRS treatments had no negative impact upon the various treated groups. Depicted results are from a total of five mice per group. *, p < 0.001, for anti-IL-13 vs NRS treatment with the adoptively transferred Treg cell groups; , p < 0.001 for PBS vs anti-IL-13 and NRS treatment with the adoptively transferred Treg cell groups.

View larger version (20K): [in this window] [in a new window]   FIGURE 10. In vivo neutralization of IFN- fails to augment clinical disease in mice adoptively transferred with CD25+CD4+ T (Treg) cells from Salmonella-CFA/IIC (AP331)-vaccinated mice. Cell-sorted 6 x 105 Treg cells obtained from Salmonella vector (H647)- or AP331-vaccinated SJL mice were adoptively transferred (i.v.) into naive SJL recipient mice and, 1 day later, EAE was induced with PLP139–151. To neutralize IFN-, 0.5 mg of anti-mouse IFN- mAb was given 1 day before EAE induction and two additional doses of 0.25 mg were given on days 1 and 5 post-EAE challenge (thick arrows). Equivalent amounts of normal rat IgG Ab were given to control groups. In vivo neutralization of the IFN- of recipient mice given AP331-induced Treg cells only delayed EAE onset and did not cause a loss in the ability to confer protection when compared with similarly treated mice given rat IgG, suggesting that IFN- is not required for protection by the AP331 vaccine. In contrast, in vivo neutralization of the IFN- of recipient mice given H647-induced Treg cells significantly reduced their clinical scores when compared with similarly treated mice given rat IgG, suggesting that IFN- contributes to EAE when using the Salmonella vector vaccine. In vivo IFN- neutralization showed a slight delay in PBS-treated mice, but similar EAE clinical outcomes as those of rat IgG-treated mice were observed. Depicted results are from a total of five mice per group. *, p < 0.001 for anti-IFN- treatment vs IgG treatment within the same adoptively transferred Treg cell groups; , p < 0.001 for PBS vs anti-IFN-- or rat IgG-treated Treg cell groups.

	Discussion

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In EAE, encephalitogenic T cells secrete inflammatory cytokines, inducing the activation of macrophages and microglial cells and the infiltration of inflammatory cells from peripheral lymphoid tissues (10, 11). Recent studies have reported the potential for developing therapeutic T_reg cells independently of autoantigen for treating EAE (18, 19) by down-regulating inflammatory mediators and inhibiting autoimmune aggressive reactions. Alternatively, significant efforts have focused on treating autoreactive T cells by vaccinating with altered peptide ligands (30, 31), isolating T_reg cells from diseased animals (29), or stimulating T_reg cells in vitro (30). Because it is often unclear to which myelin epitope(s) MS patients may be reacting, a strategy not relying upon known epitopes may be advantageous. Along these lines, we demonstrated that by using a live attenuated S. enterica serovar Typhimurium strain expressing on its cells’ surface, the enterotoxigenic E. coli CFA/I fimbriae was able to protect SJL mice against EAE by preventing encephalitogenic T cells from entering the CNS (18). Likewise, oral treatment with the vaccine ceased subsequent demyelination by the stimulation of FoxP3⁺ CD25⁺CD4⁺ T cells (19).

In an attempt to disrupt the therapeutic potential of Salmonella-CFA/I vaccine, this current work queried whether altering the expression of the CFA/I fimbrial subunit within the Salmonella cell’s periplasmic/cytoplasmic compartments would dampen protective immunity to EAE. Instead, these studies revealed that the stimulation of different T_reg cell subsets became evident by differences in cytokine profiles and cell surface expression of various regulatory-associated molecules. Nonetheless, the protective efficacy against EAE was conserved, but via different mechanisms. One advantage of this new construct, Salmonella-CFA/I_IC (strain AP331), is that the host response against the fimbrial subunit was dampened (lessened Ab titers) but still retained protective qualities. It is not as evident that the presence of a CD25^– Th2 cell subset was responsible for immune deviation as was found with the intact Salmonella-CFA/I vaccine. In this latter construct, there is a dominance of IL-4-, IL-10-, IL-13-, and TGF-β-producing Th2 cells by CD25^–CD4⁺ T cells and, although not as protective as their induced T_reg cells, these Th2 cells clearly contribute to protection against EAE (this study and Ref. 19). However, the AP331-induced CD25^–CD4⁺ T cells do seem to prime for immune deviation, as evident by the 73-fold increased production of IL-13 following challenge. Likewise, immunization with Salmonella-CFA/I_IC enhanced the production of IL-13 by T_reg cells by 5.6-fold subsequent to EAE challenge. Despite these differences in capacity to produce IL-13, it was quite evident that nearly all of the protective activity is with the AP331-induced T_reg cells, because adoptive transfer of these T_reg cells, not their corresponding CD25^–CD4⁺ T cells, was capable of imparting nearly full protection against EAE. Given this finding, in vivo neutralization of IL-13 upon adoptive transfer of AP331-induced T_reg cells reversed its ability to protect against clinical EAE, and the manifestation of EAE in these mice was similar to that induced in mice adoptively transferred with Salmonella vector-induced T_reg cells. Although a portion of the Salmonella-CFA/I_IC-induced T_reg cells produced IFN-, in vivo neutralization of IFN- upon adoptive transfer of AP331-induced T_reg cells had no effect upon the development of EAE. Thus, the protection conferred by Salmonella-CFA/I_IC is mostly served by IL-13-producing T_reg cells.

To aid in discerning how the two CFA/I-expressing Salmonella vaccines differed, studies were performed to assess potential phenotypic differences between the induced T_reg cells. The Salmonella-CFA/I (strain H696) vaccine clearly elicited >90% of the CD25⁺CD4⁺ T cells that were FoxP3⁺, whereas CD25⁺CD4⁺ T cells from Salmonella-CFA/I_IC varied from 40–70%; yet, both vaccines induced similar splenic levels of FoxP3⁺ CD25^–CD4⁺ T cells. CTLA-4 has also been shown to be expressed by T_reg cells (32, 33, 34). In some cases, activated T_reg cells express higher levels of CTLA-4, down-regulating the T cell proliferative responses induced by dendritic cells by modifying their B7 costimulatory pathways (35). Our results showed that CTLA-4 was more prevalent on Salmonella-CFA/I-induced T_reg cells than in T_reg cells from naive mice or those induced by the Salmonella empty vector or Salmonella-CFA/I_IC. Other regulatory-associated molecules were also evaluated, including GITR (29, 36) and CCR6 (37, 38). GITR expression was only modestly enhanced on LN T_reg cells from Salmonella vector- or AP331-immunized mice; however, CCR6 expression was elevated for splenic T_reg cells and again for T_reg cells from H647- and AP331-immunized mice. RANKL expression was examined as a possible means to help identify pathogenic CD4⁺ T cells as others have shown for periodontal disease (26) and diabetes (27). Instead, higher percentages of LN RANKL⁺ T_reg cells were found among T cells from H696-vaccinated mice, as well as among LN CD25^–CD4⁺ T cells. Thus, while T_reg cells from Salmonella-CFA/I-vaccinated mice showed distinctive expression of FoxP3 and CTLA-4, a distinctive phenotype for the T_reg cells from Salmonella-CFA/I_IC was not as evident.

The relevance of IL-13 upon protection against EAE was quite evident from its in vivo neutralization, showing loss of protection by the Salmonella-CFA/I_IC-induced T_reg cells. Clearly, the restimulation assays of CD4⁺ T cell subsets, as well as FACS analysis of FoxP3⁺ T_reg cells, showed dominance of IL-13. Only a few studies have evaluated the relevance of IL-13 to EAE. Using recombinant TCRs with a covalently bound encephalitogenic peptide, it was shown to be partially protective against EAE, which was, in part, attributed to enhanced IL-13 production as well as to other anti-inflammatory cytokines (39). The observed induction of IL-13 may be IL-25-dependent, because a recent study has shown that IL-4 and IL-13 are depressed in IL-25-deficient mice challenged with EAE (40). Moreover, it was shown that the protection conferred by IL-25 was lost in IL-13-deficient mice (40). These collective findings, along with our observations, showed that IL-13 represents alternative therapeutics for treating autoimmune diseases. Of interest, IL-22 was induced upon oral vaccination with either of the CFA/I fimbrial subunit-expressing Salmonella vaccines, less was detected in naive mice or in mice orally vaccinated with the Salmonella empty vector, and no IL-22 was detected after EAE challenge. This association with an anti-inflammatory response has been previously reported, showing that IL-22-deficient mice are susceptible to ConA-induced hepatocyte inflammation (41). However, in our study only minimal IL-22 levels were detected. IL-22 has been recently shown to be produced during myelin oligodendrocyte glycoprotein-induced EAE, but development of EAE in IL-22-deficient mice was not affected (42); thus, its presence may have minimal impact upon EAE.

Another feature of the Salmonella-CFA/I_IC vaccine was its ability to stimulate IFN-⁺ T_reg cells, and thus, we questioned the significance of this finding. In vivo neutralization of IFN- did not affect the potency of the adoptively transferred T_reg cells from the Salmonella-CFA/I_IC-vaccinated mice. The potency of these IFN-⁺ T_reg cells to treat EAE might not have been affected because of the presence of other T_reg cells producing IL-10 and IL-13, and these T_reg cells may limit the action of the IFN-⁺ T_reg cells. Such observations contrasted with what was obtained when a similar treatment paradigm was applied to mice adoptively transferred with T_reg cells from Salmonella vector-immunized mice. Instead of being minimally therapeutic, protection was now conferred by Salmonella vector-induced T_reg cells as noted by the reduced clinical scores subsequent to PLP_139–151 challenge. Thus, the in vivo neutralization of IFN- significantly augmented the protection by the Salmonella vector-induced T_reg cells. These variable results obtained after IFN- blockade might be related to our experimental approach, because in vivo neutralization of IFN- not only might neutralize potential production of IFN- by T_reg cells or affect their activation or potency, but also reduce the biological activities of the cytokine during the entire course of the disease. The role of IFN- in the development or even in the resolution of EAE has remained in a quagmire until the recent discovery of IL-17, subsequently identified as the primary inflammatory mediator responsible for the development of EAE (43, 44, 45). Neutralization of IFN- via mAbs (46, 47), IFN--deficient mice (48, 49), or IFN- receptor-deficient mice (50) has resulted in enhanced EAE, suggesting that IFN- is important for protection by possibly dampening IL-17 generation as recently suggested (11, 51, 52). In contrast, delayed neutralization of IFN- after and not upon initial EAE challenge with anti-IFN- mAbs protected EAE-susceptible animals (53, 54). Examination of what may be occurring upon IFN- neutralization during adoptive transfer with AP331-induced T_reg cells revealed a notable absence of an effect, suggesting that the FoxP3⁺ IFN-⁺ T_reg cells are not pathogenic and that the produced IFN- has a minimal impact in the presence of the coproduced regulatory cytokines. In contrast, because the Salmonella vector produces minimal amounts of regulatory cytokines, its production of IFN- may be more pathogenic in this vaccination scheme, and, upon neutralization of IFN- during adoptive transfer with H647-induced T_reg cells, enhanced protection is obtained.

It was originally hypothesized that altering the expression of the CfaB fimbrial subunit could impact the types of Th cells induced, as previously observed in altering FanC expression for K99 fimbriae by Salmonella (23). Although the induced Th cells were altered, as described here with the AP331 strain, protection against EAE was still maintained. Both AP331 and H696 strains produced similar levels of CfaB subunit despite differences in location of expression, e.g., AP331 that localized CfaB to cytoplasm/periplasm as determined in this study and H696 that localized CfaB to the cell surface as determined by electron microscopy (6). Thus, the differences in Th cell responses must be attributed to differences in the mode of Ag recognition and how APCs are recognizing/displaying the CfaB subunit. Future studies will address this observation in greater detail, because these studies could reveal potential mechanisms of action by simply altering the compartment in which passenger Ags are expressed within Salmonella vaccine vectors.

The described work suggests that regulatory T cells, in particular, CD25⁺CD4⁺ T cells, have varied patterns of cytokine production displaying the conventional pattern of regulatory cytokines, as well as displaying IFN-. Not surprisingly, this latter finding may be expected when adapting live attenuated Salmonella vaccines. Again, in the absence of autoantigen, the varied Salmonella vaccines are protective against EAE, and the mechanism of protection is also varied. The data show that the mode of CFA/I fimbrial subunit expression impacts the type of T_reg cells induced. When the fimbrial subunit is on the cell surface, >90% of CD25⁺CD4⁺ T cells are FoxP3⁺ and produce a number of regulatory cytokines, including IL-4, IL-10, IL-13, and TGF-β. In addition, an impact via immune deviation is also induced by the stimulation of CD25^– Th2 cells. Upon internalization of the fimbrial subunit, there is a reduction in the percentage of FoxP3⁺ T_reg cells, as well as a notable bias for IL-13. The stimulation of CD25^– Th2 cells is less noticeable, but the data suggest that there is a priming effect for the generation of IL-13 that is greatly augmented during EAE challenge. Despite these differences in mechanisms, the Salmonella-CFA/I_IC vaccine is equally protective against EAE, as is the Salmonella-CFA/I vaccine. Added to this mix, the Salmonella vector is also protective against EAE when IFN- is neutralized. Thus, these findings further expand the potential repertoire of T_reg cell subtypes that can be induced by Salmonella vaccines and may be beneficial for a variety of other autoimmune diseases. Moreover, these studies suggest that vaccines can be designed to elicit the desired T_reg cell subtype.

	Acknowledgments

 
We thank Dr. Mark A. Jutila, Veterinary Molecular Biology, Montana State University, Bozeman, MT, for kindly providing the SK208 mAb, Larrisa Jackiw for the cell-sorting experiments, and Nancy Kommers for assistance in preparing this manuscript.

	Disclosures

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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 This work was supported by Public Health Service Grant AI-41123 and, in part, by Montana Agricultural Station and U.S. Department of Agriculture Formula Funds. The Veterninary Molecular Biology flow cytometry facility was, in part, supported by National Institutes of Health/National Center for Research Resources, Centers of Biomedical Excellence Grant P20 RR-020185 and by an equipment grant from the M. J. Murdock Charitable Trust.

² Current address: Department of Microbiology and Immunology, Dartmouth Medical School, HB 7937, Dartmouth-Hitchcock Medical Center, Rubin Building, Room 710, 1 Medical Center Drive, Lebanon, NH 03756.

³ Address correspondence and reprint requests to Dr. David W. Pascual, Veterinary Molecular Biology, P. O. Box 173610, Montana State University, Bozeman, MT 59717. E-mail address: dpascual{at}montana.edu

⁴ Abbreviations used in this paper: CFA/I, colonization factor antigen I; CFA/I_IC, CFA/I expressed in the periplasmic/intracellular compartment; EAE, experimental autoimmune encephalomyelitis; GITR, glucocorticoid-induced TNFR; HNLN, head and neck lymph node; LN, lymph node; MLN, mesenteric lymph node; MS, multiple sclerosis; NRS, normal rabbit serum; PLP, protein proteolipid; RANKL, receptor activator of NF-B ligand; T_reg, regulatory T cell.

Received for publication January 22, 2008. Accepted for publication May 12, 2008.

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