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The Journal of Immunology, 2005, 175: 5817-5826.
Copyright © 2005 by The American Association of Immunologists

ES-62, an Immunomodulator Secreted by Filarial Nematodes, Suppresses Clonal Expansion and Modifies Effector Function of Heterologous Antigen-Specific T Cells In Vivo1

Fraser A. Marshall*, Angela M. Grierson*, Paul Garside*, William Harnett{dagger} and Margaret M. Harnett2,*

* Division of Immunology, Infection and Inflammation, University of Glasgow, Glasgow, United Kingdom; and {dagger} Department of Immunology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
ES-62 is a phosphorylcholine-containing glycoprotein secreted by filarial nematodes, which has previously been shown to possess a range of immunomodulatory capabilities. We now show, using a CD4+ transgenic TCR T cell adoptive transfer system, that ES-62 can modulate heterologous Ag (OVA)-specific responses in vivo. Thus, in contrast to the mixed IgG1-IgG2a response observed in control animals, ES-62-treated mice exhibited a Th2-biased IgG Ab response as evidenced by stable enhancement of anti-OVA IgG1 production and a profound inhibition of anti-OVA IgG2a. Consistent with this, Ag-specific IFN-{gamma} produced was suppressed by pre-exposure to ES-62 when T cells were rechallenged ex vivo. However, the response observed was not classical Th2, because although Ag-specific IL-5 production was enhanced by pre-exposure to ES-62, IL-13, and IL-4 were inhibited when T cells were rechallenged ex vivo. Moreover, such T cells produced lower levels of IL-2 and proliferated less upon Ag rechallenge ex vivo. Finally, pre-exposure to ES-62 inhibited the clonal expansion of the transferred Ag-specific CD4+ T cells and altered the functional response of such T cells in vivo, by modulating the kinetics and reducing the extent of their migration into B cell follicles.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Filarial nematodes are arthropod-transmitted parasites of vertebrates including humans. Of the eight species known to infect humans, three, Wuchereria bancrofti, Brugia malayi, and Onchocerca volvulus, represent major causes of morbidity in the tropics (1). Infection of humans with filarial nematodes is long term, with mature worms surviving for >5 years (2), and it is now accepted that such longevity is promoted by parasites modulating the host immune system (3, 4). Thus, filarial nematode infections are characterized by altered T cell effector responses (5), not only in terms of suppression of parasite-specific immune responses (6) but more generally with respect to nonspecific stimuli (7) and bystander Ags, such as routine vaccinations (8, 9). With respect to the nature of the immunomodulation, there is a consensus that it incorporates not only impairment of lymphocyte proliferation but also a bias in production of both cytokines and Abs. With respect to cytokines, there is reduced production of IFN-{gamma} and increased synthesis of IL-10. For Abs, there are imbalances in IgG subclasses: greatly elevated IgG4 (an Ab of little value in eliminating pathogens due to an inability to activate complement or bind with high affinity to phagocytic cells); and decreases in other IgG subclasses. Overall, the picture is of an immune response demonstrating a somewhat suppressed, anti-inflammatory, Th2-like phenotype. It has been speculated that such a phenotype is conducive to both parasite survival and, by limiting pathology resulting from aggressive immune responses, host health.

Many studies have been undertaken to examine the basis for this parasite-driven immunomodulation and it is becoming increasingly clear that excretory-secretory (ES)3 products released by filarial nematodes subvert the host immune system to help maintain infection and parasite survival (10). Thus, we have previously characterized the immunomodulatory activities of one such molecule from the rodent filarial nematode Acanthocheilonema viteae, ES-62 (which has homologues in human filarial nematodes) (11, 12, 13, 14, 15, 16, 17, 18). Specifically, these studies showed that ES-62 inhibits the ability of B and T lymphocytes to respond to ligation of their Ag receptors by rendering cells hyporesponsive to stimulation in vitro (reviewed in Ref.19). It can also bias the immune response toward a Th2 phenotype, thereby preventing the induction of Th1-mediated pathology, which would be deleterious to both host and parasite (15, 16, 17). Consistent with this, we have recently shown that ES-62 possesses immunomodulatory, anti-inflammatory properties in vivo in a model of collagen-induced arthritis (CIA) in DBA/1 mice and that these effects are mediated at least in part through suppression of the collagen-specific Th1 response (20).

Although we were able to demonstrate that ES-62 suppressed collagen-specific proliferation and cytokine production, we were unable to characterize the effects of ES-62 on such Ag-specific cell responses in vivo because of the low frequency of, and the inability to distinguish, Ag-specific lymphocytes in normal mice. Thus, to determine whether ES-62 was mediating its effects by modifying clonal expansion and effector function of Ag-specific T cells, we exploited a model in which T cells bearing a transgenic (tg) TCR specific for the major immunodominant epitope of the model Ag, OVA are transferred into normal BALB/c mice in numbers large enough to trace in vivo with an anti-TCR-specific Ab, but small enough to reflect, and indeed not interfere with, the normal physiological response to Ag (21, 22, 23). Using this model, we now show that exposure to ES-62 in vivo modulates responses to heterologous Ag by inhibition of Ag-specific clonal expansion and follicular migration of the transferred tg T cells in vivo. Moreover, this is associated with a modification of OVA323–339 peptide-dependent T cell effector function (decreased IFN-{gamma}, IL-13, and IL-4; increased IL-5) and Ab (increased IgG1, decreased IgG2a) phenotype.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Mice homozygous for the tg TCR that is specific for chicken OVA (cOVA)323–339 in the context of I-Ad were used as T cell donors. The tg TCR (expressed on 70–80% of the CD4+ T cells from DO.11.10 BALB/c mice and recognizes the major immunodominant epitope of cOVA) was detected by flow cytometry using the clonotypic mAb KJ1.26 (21, 24). Eight-week-old male BALB/c mice (H-2d/d, IgMa) were used as recipients. All animals were specific pathogen-free and were maintained under standard animal house conditions with free access to both water and standard rodent pellets at the University of Glasgow Central Research Facilities in accordance with local and Home Office regulations.

Preparation of ES-62

ES-62 is a major secreted glycoprotein of the rodent filarial nematode A. viteae and homologue of molecules found in filarial nematodes that parasitize humans. The molecule consists of a tetramer of identical 62-kDa monomers that contain phosphorylcholine (PC) attached to N-type glycans (25). ES-62 was purified to homogeneity from spent culture medium of adult A. viteae using endotoxin-free reagents essentially as described previously (11). Purity and identity of each batch was confirmed by a combination of SDS-PAGE and Western blotting. The level of endotoxin in the ES-62 sample was confirmed using an Endosafe kit (Charles River Laboratories). ES-62 is used at a working concentration that has an endotoxin reading of <0.003 U/ml.

Flow cytometry

Analysis of cell surface marker expression was as described previously (23, 26). Briefly, for detection of CD4+DO11.10 tg T cells, the cell suspensions were incubated with PerCP-conjugated anti-CD4 and biotinylated clonotypic anti-TCR Ab KJ1.26 and PE-conjugated streptavidin (all BD Pharmingen) at concentrations previously determined by titration of optimum binding. Immediately before data acquisition, 50 µg/ml propidium iodide (Calbiochem) was added to each sample to enable exclusion of dead cells from the analyses. Cellular fluorescence data was acquired using a BD Biosciences FACSCalibur flow cytometer and analyzed using FlowJo software (Tree Star)

Adoptive transfer of DO.11.10 T cells

Preparation of cell suspensions for adoptive transfer was as described previously (23). Briefly, single-cell suspensions were prepared from peripheral lymph nodes (PLN; axillary, brachial, inguinal, cervical), mesenteric lymph nodes and spleens from DO.11.10 BALB/c mice, and the percentage of KJ1.26+CD4+ DO.11.10 T cells in these preparations was determined by flow cytometric analysis. Cell suspensions containing 2.5 x 106 tg T cells in 200 µl of sterile RPMI 1640 were injected i.v. through the tail vein into nonirradiated, age-matched male BALB/c recipients. Adoptive transfer recipient mice were injected s.c. with 2 µg of ES-62 in PBS three times in total, 2 days before transfer, on the day of transfer, and the following day when mice were also immunized. Such quantities of ES-62 are similar to those used in ameliorating CIA (20) and will give serum concentrations within the range found for PC-containing molecules in filarial nematode infection (e.g., see Refs.18 and 27). In indicated experiments, cells (5 x 107 cells/ml) were labeled with 5 µM CFSE (Molecular Probes) before transfer as described above (23).

Immunization of adoptive transfer recipients

Following adoptive transfer, recipient mice were injected s.c. in the scruff of the neck with 130 µg of OVA-HEL in 100 µl of PBS-50% CFA (Sigma-Aldrich) as described previously (23, 26). For in vivo rechallenge experiments, mice were immunized s.c. in the lower back with 130 µg of OVA-HEL in 100 µl of PBS-50% IFA (Sigma-Aldrich). Conjugated OVA-HEL (0.5 mM) was prepared as described previously (26).

Ex vivo cell cultures for analysis of DNA synthesis and cytokine production

Briefly, on day 10 postimmunization, PLN were removed, and single-cell suspensions were prepared as described previously (26, 28). For analysis of DNA synthesis, cells (2 x 105 cells/well) were cultured in RPMI 1640 supplemented with 2 mM glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, and 10% FCS (Sigma-Aldrich). Cells were stimulated with 10 µg/ml OVA323–339 for 48 h at 37°C in a 5% (v/v) CO2 atmosphere at 95% humidity. Identical results were obtained from cultures in which OVA was provided as source of Ag (results not shown).

DNA synthesis was assessed by pulsing with 0.5 µCi/well [6-3H]thymidine (Amersham Pharmacia Biotech) for the last 4 h of culture. Cells were harvested, and incorporated label was assessed using a Betaplate 96-well harvester system (Amersham Pharmacia Biotech). Results are expressed as mean cpm incorporated ± SEM; n = 3 (of pooled means of triplicate values from three individual mice).

For analysis of cytokines, cells (2 x 106 cells/well in triplicate) were cultured in 24-well plates in RPMI 1640 supplemented with 2 mM glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, and 10% FCS (Sigma-Aldrich). Cells were stimulated with 10 µg/ml OVA323–339 at a final well volume of 1 ml. Cells were cultured for 72 h at 37°C in a 5% (v/v) CO2 atmosphere at 95% humidity. Cell culture supernatant was then removed and frozen at –20°C until analysis. Cytokine ELISA was performed according to the Ab supplier’s recommendations and as described previously (17, 26, 28, 29). IL-2, IL-4, IL-5, IL-10, IFN-{gamma}, and TNF-{alpha} were analyzed using OPTEIA Mouse ELISA kits (BD Pharmingen), and IL-12p40 was analyzed using Ab pairs (BD Pharmingen). The limit of sensitivity was ~20 pg/ml for each cytokine. Cytokine production was also analyzed by using Multiplex Bead Assay (BioSource) according to the manufacturer’s instructions using specific Abs for IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17, IFN-{gamma}, TNF-{alpha}, GM-CSF, and MIP-1{alpha} coated onto the surface of fluorescently encoded microspheres. After incubation with biotinylated detection Ab and streptavidin-RPE, the fluorescence bound to the microspheres was analyzed using a Luminex XMAP system.

Ex vivo analysis of intracellular cytokine production

Briefly, on day 5 postimmunization, PLN were removed, and single-cell suspensions were prepared as described previously (26, 28). For analysis of intracellular cytokines, cells (4 x 106 cells/well) from individual mice (four per group) were cultured in 24-well plates in RPMI 1640 supplemented with 10% FCS, 50 U/ml penicillin, 50 µg/ml streptomycin, and 2 mM glutamine. Cells were restimulated with 1 µg/ml OVA323–339 and 10 µg/ml brefeldin A (Sigma-Aldrich) at a final well volume of 1 ml. Cells were incubated at 37°C in a 5% (v/v) CO2 atmosphere at 95% humidity for 4 h. tg T cells were stained for surface markers as described. Cells were then fixed and permeabilized using the Cytofix/Cytoperm kit (BD Pharmingen) according to the manufacturer’s instructions. After washing, cells were further stained with anti-IFN{gamma}-PE, anti-IL-4-PE, or anti-IL-10-PE or an isotype control of corresponding specificity (all BD Pharmingen) and analyzed using CellQuest software (BD Pharmingen).

Detection of serum Abs from adoptive transfer recipients

Anti-OVA Ig levels in serum were determined as described previously (23, 26) by incubation with 2 µg/ml biotinylated anti-IgG, IgG1, IgG2a, or IgE (BD Pharmingen) Abs for 1 h at 37°C. Plates were then washed and incubated with Extravidin (1/1000; Sigma-Aldrich) for 1 h at 37°C. Plates were washed again, and tetramethylbenzidine Microwell peroxidase substrate (Kirkegaard & Perry Laboratories) was added. All ELISAs were read on a plate reader at 630 nm.

Laser scanning cytometry (LSC)

Sections were stained as described previously (23). Briefly, PLN were frozen in liquid nitrogen in OCT embedding medium (Miles Diagnostic Division) and stored at –70°C. Sections (8 µm) were cut, mounted on microscope slides (Shandon), and stained immediately with biotinylated anti-KJ1.26 and FITC-anti-B220 (BD Pharmingen), for 30 min, washed, and then incubated with streptavidin-HRP for 30 min. After a washing, the cells were treated with biotinylated tyramide (TSA Biotin system; PerkinElmer Life Sciences) for 10 min, washed, and then incubated with streptavidin Alexa Fluor 647 (Molecular Probes) for 30 min. Finally, slides were washed three times and mounted in Vectashield (Vector Laboratories) for analysis by LSC. Tissue maps were generated from these data. Upon these tissue maps, equally sized regions were randomly placed within follicular and paracortical areas. This allowed statistical data on the number and percentage of KJ1.26+ located within B cell follicles to be determined using Wincyte software (Compucyte). Using the relocation feature of the LSC, areas surrounding follicular regions were relocated to and high quality digital images of the fluorescently stained tissue sections were obtained using a Hammamatsu camera and Openlab software (Improvision)

Statistical analysis

Statistical significance was determined by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Exposure to ES-62 in vivo modulates the subsequent Ab response to the model Ag, OVA

Our previous reports demonstrating that ES-62 induced the development of dendritic cells that primed for an anti-inflammatory/Th2 phenotype of T cells in vitro and a predominant Th2-biased ES-62-specific IgG1 immune response in vivo (reviewed in Ref.16) suggested that ES-62 might act to polarize the T cell help provided to B cells (22) in the generation of an Ab response directed against an immunizing Ag. To investigate whether ES-62 induced polarized T cell responses at a functional level in vivo, recipient BALB/c mice that had received 2.5 x 106 OVA-specific TCR tg CD4+ T cells from donor DO.11.10 BALB/c mice were immunized (24 h after transfer) with OVA-HEL in CFA, and serum was removed for up to 20 days to analyze the effect of in vivo exposure to ES-62 on the Ab response to the heterologous model Ag OVA.

To assess whether treatment of mice with ES-62 had significant impact on the phenotype of the OVA-specific IgG-mediated Ab response, we measured levels of the signature Th2 (IgG1) and Th1 (IgG2a) IgG isotypes. Compared with control mice, ES-62-treated mice demonstrated increased production of anti-OVA IgG1; this promotion of IgG1 production is stable up until at least 20 days postimmunization (Fig. 1A and results not shown). In contrast, treatment with ES-62 in vivo was found to inhibit the production of OVA-specific IgG2a (Fig. 1B and results not shown). Analysis of the Ag-specific IgM and total IgG Ab response revealed that this suppression of OVA-specific IgG2a did not reflect defective class switching from IgM to IgG given that ES-62 treatment had negligible effects on IgM production (Fig. 1C) and, indeed, that ES-62-treated mice had higher levels of anti-OVA IgG than control mice (Fig. 1D).



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  		FIGURE 1. Effects of in vivo exposure to ES-62 on the production of OVA-specific Abs. BALB/c recipient control and ES-62-treated mice were injected with 2.5 x 106 CD4+KJ1.26+ T cells. After 24 h, these mice were immunized s.c. with 130 µg of cOVA-HEL in CFA. The production of OVA-specific IgG1 (A) and IgG2a (B) was assessed in mice 10 days after immunization. Five days after immunization, serum was collected, and OVA-specific IgMa was assessed by ELISA (C). The production of total OVA-specific IgG was then assessed in serum collected at 7 days after immunization by ELISA (D). At day 10 postimmunization, mice were challenged with 130 µg of cOVA-HEL in IFA. The production of OVA-specific IgG1 (E) and IgG2a (F) was then assessed in serum collected a further 3 days after in vivo rechallenge with Ag-IFA by ELISA. Data are presented as the means (of pooled means of triplicate values from individual mice) ± SEM of three mice per group. *, p < 0.05 compared with ES-62-treated mice. Data are from a single experiment representative of at least four independent experiments.

 
To determine whether in vivo exposure to ES-62 had any effects on the Ab memory response, in some experiments, recipient BALB/c mice were rechallenged with OVA-HEL in IFA at day 10 post-primary immunization, and serum was removed for up to a further 10 days to examine Ag-specific Ab responses. Consistent with the above data suggesting ES-62 promoted IgG1 production, after in vivo rechallenge with OVA-IFA, ES-62-exposed mice still maintained increased levels of anti-OVA IgG1 relative to control mice (Fig. 1E). Similarly, the inhibition of anti-OVA IgG2a production in ES-62-treated mice was maintained after in vivo rechallenge (Fig. 1F). Collectively, these Ab data suggest that T cells primed in mice treated with ES-62 favor production of IgG1 and are inherently inhibited from promoting IgG2a responses even when subsequently rechallenged with specific Ag.

Exposure to ES-62 in vivo results in modulation of Ag-specific T cell responses after ex vivo Ag rechallenge

Examination of anti-OVA IgG production strongly suggested that exposure to ES-62 in vivo had altered the effector properties of OVA-specific T cells. Therefore, to further characterize the functional phenotype of OVA-specific T cells from mice that were treated with ES-62, OVA-specific proliferation and cytokine production was examined ex vivo. First, T cells from ES-62-treated mice exhibited significantly reduced proliferative capacity following recognition of the immunodominant epitope OVA323–329 ex vivo, compared with control groups (Fig. 2), despite having equivalent numbers of tg T cells in the starting cultures. Second, although ES-62 appeared to promote a Th2-biased Ab response in vivo, there was not a classical Th2 signature to the cytokine profile upon ex vivo rechallenge with OVA323–329 (Fig. 3). As expected, ES-62-treated groups demonstrated decreased production of IL-2, IL-17, and IFN-{gamma}. Moreover, proinflammatory Th1-biasing cytokines that are not classically T cell derived such as IL-12, TNF-{alpha}, IL-6, and MIP-1{alpha} were also inhibited in an Ag-specific manner, suggesting that ES-62 also modulated cognate bidirectional signaling between APCs and T cells. However, although the production of IL-5 was enhanced in ES-62-treated groups, which is consistent with the observed Th2-like Ab response, surprisingly, the production of IL-4 was extremely low and was in fact marginally reduced relative to the control. Moreover, Ag-specific IL-13 production was greatly inhibited compared with control groups. Furthermore, no IL-10 could be detected in either ES-62 or control groups after OVA rechallenge ex vivo (data not shown).



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  		FIGURE 2. Effects of in vivo exposure to ES-62 on Ag-specific CD4+ T cell proliferation after Ag rechallenge ex vivo. BALB/c recipient control and ES-62-treated mice were injected with 2.5 x 106 CD4+KJ1.26+ T cells. After 24 h, these mice were immunized s.c. with 130 µg of cOVA-HEL in CFA. Draining lymph nodes were removed 10 days postimmunization, and cells were stimulated in vitro with 10 µg/ml OVA323–339 peptide. Cells were cultured for 48 h and pulsed with 0.5 µCi/well [6-3H]thymidine for the last 4 h of culture. Incorporated [3H]thymidine was assessed by liquid scintillation counting. Data are presented as the means (of pooled means of triplicate values from three individual mice) from three mice per group ± SEM and are representative of four independent experiments. ***, p < 0.005 compared with relative immunized (Imm) control group. Unimm, Unimmunized.

 


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  		FIGURE 3. Effects of in vivo exposure to ES-62 on Ag-specific CD4+ T cell cytokine production after Ag rechallenge ex vivo. BALB/c recipient control and ES-62-treated mice were injected with 2.5 x 106 CD4+KJ1.26+ T cells. After 24 h, these mice were immunized (Imm) s.c. with 130 µg of cOVA-HEL in CFA. Draining lymph nodes were removed 10 days postimmunization, and cells were stimulated in vitro with 10 µg/ml OVA323–339 peptide for 72 h. Cell culture supernatants were then removed and assessed for production of IL-12 (A), TNF-{alpha} (B), IL-2 (C), IL-6 (D), IL-17 (E), MIP-1{alpha} (F), IFN-{gamma} (G), IL-4 (H), IL-13 (I), and IL-5 (J) by Luminex assay. Data are presented as the means (of pooled means of duplicate values from three individual mice) from three mice per group ± SEM. Data are from a single representative experiment, and the IFN-{gamma}, IL-2, IL-4, IL-5, IL-12, and TNF-{alpha} data are representative of two additional independent experiments using conventional cytokine analysis by ELISA (data not shown). ***, p < 0.005, **, p < 0.01, and *, p < 0.05 compared with the relative immunized group.

 
Pre-exposure to ES-62 inhibits Ag-specific T cell expansion and effector function in vivo

To address how ES-62 mediated the above polarizing effects on the generation and outcome of the OVA-specific immune response in vivo, we exploited the adoptive transfer system used to investigate the effects of exposure to ES-62 on various parameters associated with the priming and development of effector function of Ag-specific T cells in vivo. Thus, recipient BALB/c mice that had received 2.5 x 106 OVA-specific TCR tg CD4+ T cells from donor DO.11.10 BALB/c mice were immunized with OVA-HEL/CFA 24 h after transfer, and draining lymph nodes were removed 3, 5, 7, and 10 days later to examine Ag-specific clonal expansion of the transferred tg TCR T cells by flow cytometry.

In agreement with previous studies (22), peak T cell expansion was observed at 5 days postimmunization in the control group. Mice that had been pretreated with ES-62 also demonstrated peak clonal expansion at day 5; however, the level of expansion was >3-fold lower than the control group (Fig. 4A). After peak expansion at day 5, the percentage of CD4+KJ1.26+ T cells in both control and ES-62-treated groups declined to nonimmunized levels by day 10 postimmunization. As expected, nonimmunized mice from both control and ES-62-treated groups did not demonstrate clonal expansion of the tg OVA-specific T cells, confirming not only that CD4+KJ1.26+ T cells expand and proliferate in an Ag-dependent manner but also that treatment with ES-62 does not induce Ag-independent T cell clonal expansion or depletion (Fig. 4A). The decreased clonal expansion in ES-62-treated mice was also supported by a reduction in the total number of CD4+KJ1.26+ T cells obtained from draining lymph nodes (Fig. 4B). Thus, the decrease in percentage of Ag-specific T cells was not a dilution effect due to increased infiltration of other cells into draining lymph nodes, and, indeed, ES-62-treated mice displayed a reduced lymphocyte cellularity of lymph nodes (results not shown).



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  		FIGURE 4. Effects of ES-62 on Ag-specific CD4+ T cell clonal expansion in vivo. BALB/c recipient control and ES-62-treated mice were injected with 2.5 x 106 CD4+KJ1.26+ T cells. After 24 h, these mice were immunized (Imm) s.c. with 130 µg of cOVA-HEL in CFA. The percentage of CD4+KJ1.26+ T cells in the draining lymph nodes of adoptively transferred recipients was assessed by flow cytometry on days 3, 5, 7, and 10 after immunization. Each time point represents the mean ± SEM for three mice per group (A). At day 5 postimmunization, the total number of CD4+KJ1.26+ T cells (B) contained within draining lymph nodes was also calculated. Data are presented as mean ± SEM from three mice per group and are representative of at least five other independent experiments. ***, p < 0.005 compared with immunized group. At day 5 postimmunization, draining lymph nodes were removed, and the percentage of CD4+KJ1.26+ staining positive for CD69 (C) was assessed by flow cytometry. Data are presented as the mean percent from three mice per group ± SEM and are representative of two independent experiments. BALB/c recipient control and ES-62-treated mice were injected with 2.5 x 106 CD4+KJ1.26+ T cells, which had been labeled with CFSE before transfer. Five days after immunization, draining lymph nodes from unimmunized (D), immunized control (E), and ES-62-treated (F) adoptive transfer recipients were removed, and CFSE fluorescence, a measure of cell division, was assessed in CD4+KJ1.26+ T cells by flow cytometry. Data are shown as histograms of CFSE fluorescence vs percent of CD4+KJ1.26+ T cells and are from one mouse, which is representative of three mice per group, and a total of four independent experiments (for analysis of all four experiments, see Table I).

 
Early T cell activation is accompanied by changes in the expression of certain surface molecules. Specifically, after activation, T cells increase their expression of CD69 and concomitantly decrease the expression of L-selectin (CD62L). Therefore, to examine whether the reduced level of T cell clonal expansion in ES-62-treated mice was due to defective T cell activation, the levels of CD69 and CD62L expressed by transferred Ag-specific T cells were examined. Consistent with activated T cells expressing CD69, KJ1.26+ T cells from both immunized groups expressed similar high levels of CD69. Likewise, activation of KJ1.26+ T cells was accompanied by down-regulation of expression of CD62L, and importantly, there was little difference in the levels of expression between T cells from control or ES-62-treated mice (Fig. 4C and results not shown). Thus, the reduced Ag-specific T cell clonal expansion observed in ES-62-treated mice does not appear to reflect the failure of such T cells to recognize Ag following in vivo exposure to ES-62.

To determine whether the reduction in Ag-specific T cell expansion was due to decreased cell division, cells were labeled with CFSE before adoptive transfer, and the profile of CFSE staining in CD4+KJ1.26+ T cells was analyzed at day 5 postimmunization (Fig. 4, D–F, and Table I). Consistent with the observed lack of clonal expansion observed in nonimmunized mice, the majority of CD4+KJ1.26+ cells in such mice remained nondivided and stained highly for CFSE (Fig. 4D and Table I). In contrast, both immunized groups demonstrated CD4+KJ1.26+ T cell division as evidenced by decreased intensity of CFSE staining, but T cells from ES-62-treated mice demonstrated reduced cell division. In control immunized mice, the majority of CD4+KJ1.26+ T cells had undergone five to six cell divisions (Fig. 4E and Table I), whereas ES-62 treatment not only reduced the percentage of cells dividing the maximal number of times but also increased the percentage of nondividing cells ~2- to 3-fold (Fig. 4F and Table I).


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  		Table I. Effects of in vivo exposure to ES-62 on the levels of cell division of Ag-specific CD4+ T cells in draining lymph nodes after immunization

 
Finally, to demonstrate that the cytokine profile observed in ex vivo cultures of cells pre-exposed to ES-62 in vivo represented modification of the effector function of Ag-specific T cells, intracellular staining of IFN{gamma}, IL-4, and IL-10 production by KJ1.26+ T cells was performed on day 5 lymph node cultures stimulated with OVA323–329 ex vivo (Fig. 5 and results not shown). These studies confirmed that both IFN-{gamma} and IL-4 production was reduced in cells obtained from mice treated with ES-62 (Fig. 5). No difference in IL-10 production could be detected between control and ES-62 groups (results not shown).



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  		FIGURE 5. Effect of in vivo exposure to ES-62 on effector function of Ag-specific cells after ex vivo stimulation with OVA323–339. On day 5 postimmunization, PLN cells were restimulated with 1 µg/ml OVA323–339 and 10 µg/ml brefeldin A for 4 h. tg T cells were stained for surface markers as described in Materials and Methods. Cells were then fixed and permeabilized before being further stained with anti-IFN-{gamma}-PE, anti-IL-4-PE, or the appropriate isotype control and analyzed using CellQuest software. Data are the mean ± SEM of KJ1.26+CD4+ T cells expressing the relevant cytokine, where n = 4 for the individual mice. Imm, Immunized.

 
Effects of ES-62 on the localization of T cells within draining lymph nodes

It has been proposed that to facilitate effective cross-talk and cognate interactions, T and B cells migrate to specific anatomical areas within a lymph node in response to immunization (22). Before immunization, DO.11.10 TCR tg CD4+ T cells are found in the paracortex of the lymph node; however, after antigenic challenge with OVA, these Ag-specific T cells undergo clonal expansion, and a proportion migrate toward the edge of the B cell follicle where they are believed to provide B cell help for Ab production (22). Furthermore, we have recently proposed that to undergo maximal clonal expansion and terminal differentiation and to acquire effector and memory function, CD4+ T cells must migrate through B cell follicles (30). Therefore, to examine the effects of ES-62 on T cell migration and localization within lymph nodes, draining inguinal lymph nodes were removed from control and ES-62-treated mice at 3, 5, and 7 days postimmunization. Tissue sections of these lymph nodes were stained with anti-B220 and KJ.126 Abs to identify B cells and Ag-specific CD4+ T cells, respectively, and were then analyzed by LSC.

Analysis of all sections obtained from control and ES-62-treated mice at 3, 5, and 7 days postimmunization allowed determination of both the absolute number and the percentage of lymph node KJ1.26+ T cells within B cell follicles at each day point to be calculated (Table II) and demonstrated that B cell follicular regions of lymph nodes from ES-62-treated mice contained both lower absolute numbers and lower percentages of total lymph node KJ1.26+ T cells, compared with control mice (Fig. 6, Table II). This indicated that in addition to there being fewer KJ1.26+ T cells in total within the lymph nodes of ES-62-treated mice, as evidenced by both flow cytometry (Fig. 4) and LSC data (Fig. 6), even fewer of these are migrating into B cell follicles and those which do, display slower kinetics of migration (Table II). Thus, ES-62 not only reduces Ag-specific T cell expansion and accumulation in lymph nodes, but it also inhibits the rate and extent of migration of these T cells into B cell follicles.


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  		Table II. Effects of in vivo exposure to ES-62 on the localization of Ag-specific CD4+ T cells within B cell follicles of draining lymph nodes after immunization

 


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  		FIGURE 6. Localization of Ag-specific CD4+ T cells within draining lymph nodes after immunization. BALB/c recipient control mice were injected with 2.5 x 106 CD4+KJ1.26+ T cells. After 24 h, these mice were immunized (Imm) s.c. with 130 µg of cOVA-HEL in CFA. Five days after immunization, draining lymph nodes were removed, and tissue sections were prepared. These were subsequently stained for KJ1.26+ T cells and B220+ B cells, and the number and percentage of KJ1.26+ T cells located within B cell follicle regions were calculated as described in Materials and Methods. A and B, Setting of positive staining KJ1.26+ T cell gate (A) allowing a tissue map of the localization of KJ1.26+ T cells to be generated (B). C and D, Setting of positive staining B220+ B cell gate (C), allowing a tissue map of the localization of B220+ B cells to be generated (D). On this tissue map, equally sized regions were randomly placed within paracortical and follicular areas and were subsequently copied onto the KJ1.24+ T cell tissue map. This allowed unbiased selection of follicular regions containing KJ1.26+ T cells. E KJ1.26+ T cell and B220+ B cell tissue maps were merged together, allowing the statistical information (number of KJ1.26+ T cells and the percentage of KJ1.26+ T cells relative to the whole lymph node) contained within each region to be calculated. F, Using the relocation feature of the LSC, high quality digital images of regions within follicular areas were generated with Openlab software 3.0.9 (Improvision). KJ1.26+ T cells appear as red, and B220+ B cells appear as green, and the appropriate control staining sections are as follows: a tissue section stained with KJ1.26-biotin-streptavidin Alexa Fluor 647 and B220-FITC isotype control (G); staining of a tissue section stained with B220-FITC and KJ1.26 isotype control (H); no Ab stain control (I). Digital images of B cell follicle regions from control (J) and ES-62-treated (K) mice were obtained at days 5 and 7 using the relocation feature of the LSC and Openlab software 3.0.9 (Improvision). KJ1.26+ T cells appear as red, and B220+ B cells appear as green.

 
To further visualize the reduced numbers of KJ1.26+ T cells in the lymph nodes and B cells follicles, and to support the FACS data demonstrating reduced levels of T cell expansion in ES-62-treated mice, representative immunofluorescent images of areas surrounding B cell follicles from control mice and ES-62-treated mice at days 5 and 7 postimmunization are shown (Fig. 6). The images clearly show that compared with control immunized mice, ES-62-treated mice have substantially fewer KJ1.26+ T cells located both in the paracortex and in B cell follicles.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our previous studies have demonstrated that ES-62 possesses immunomodulatory capabilities both in vitro and in vivo. We now show, using a CD4+ tg TCR T cell adoptive transfer system, that ES-62 can modulate heterologous Ag-specific responses in vivo. Thus, in contrast to the mixed IgG1-IgG2a response observed in control animals, ES-62-treated mice exhibited a biased IgG Ab response as evidenced by stable enhancement of anti-OVA IgG1 production and a profound inhibition of anti-OVA IgG2a (Fig. 1). Consistent with this, T cells from ES-62-treated mice produced a reduced IFN-{gamma} and increased IL-5 (but decreased IL-4 and IL-13) cytokine response when rechallenged with Ag ex vivo (Figs. 3 and 5). Exploitation of the adoptive transfer system used to dissect the mechanisms involved revealed that pre-exposure to ES-62 in vivo inhibited the clonal expansion of transferred Ag-specific CD4+ T cells (Fig. 4). This inhibition did not appear to be due to decreased triggering of T cell activation because T cells from ES-62-treated mice had levels of CD69 and CD62L comparable with those from control mice (Fig. 4 and results not shown). Rather, analysis of CFSE staining suggested that this might reflect reduced cell division, given that ES-62 was found not only to suppress the number of cells undergoing maximal rounds of divisions but also to induce the development of a larger population of nondividing Ag-specific T cells (Fig. 4 and Table I). Moreover, in addition to restricting the clonal expansion of the transferred Ag-specific CD4+ T cells, pre-exposure to ES-62 in vivo resulted in a reduced percentage migrating into B cell follicles to participate in B-T cell cooperation (Fig. 6) and these events were associated with a reduced capability of Ag-specific CD4+ T cells to make IFN-{gamma} and IL-4 when rechallenged ex vivo (Fig. 5).

As mentioned above, ES-62 treatment polarized the functional response of OVA-specific T cells toward an IgG1 Ab response in vivo. However, this was not a classical Th2 response as although IL-5 production was enhanced by pre-exposure to ES-62, the Ag-specific production of other Th2 cytokines, IL-13 and IL-4 (although barely detectable), was inhibited in cells derived from such mice. Nevertheless, the phenotype of the response was certainly not proinflammatory or Th1 like, because the production of IL-12, TNF-{alpha}, IL-2, IL-6, IL-17, MIP-1{alpha}, and IFN-{gamma} was also significantly reduced compared with control groups (Fig. 3). Furthermore, because no IL-10 could be detected in either group, suppression of these cytokines did not appear to be due to the induction of IL-10. This lack of IL-10 production was rather surprising given that we had previously demonstrated that the inhibition of ES-62-specific IgG2a production appeared to depend critically on ES-62-induced IL-10 (31). However, it is possible that ES-62, like other filarial products (32, 33), stimulates IL-10 production primarily from B1 cells (34).

IL-4 has been widely reported to be the principal positive regulator of IgG1 production in mice (35), and many studies in human filariasis and animal models of filarial nematode infection have suggested that IL-4 also plays an important role in parasite induced immunomodulation (36, 37). Consistent with this, we have previously shown that whereas naive spleen cells exposed to ES-62 in vitro for 48 h do not produce IL-4 (15), IL-4 production can be detected in animals previously primed and rechallenged in vivo with ES-62 (31). Moreover, using IL-4 knockout mice (31), we have shown that the induction of IgG1 Ab in response to ES-62 is dependent on the production of IL-4. Therefore, it is difficult to interpret our current findings that not only is IL-4 barely detectable after rechallenge ex vivo at day 10, but also its production in response to OVA323–339 is decreased in T cells derived from mice exposed to ES-62 in vivo. Certainly, intracellular staining analysis demonstrating that production of IL-4 by Ag-specific cells is similarly inhibited in day 5 cultures (Fig. 5) suggests that the low levels of IL-4 observed in day 10 ex vivo rechallenge assays do not simply reflect its exhaustion during culture (38). Moreover, this suppression of IL-4 production by Ag-specific T cells, derived from the earlier, clonal expansion stages of the immune response, appears to rule out a transient role for T cell-derived IL-4 in the observed immunomodulation mediated by ES-62. Thus, the IL-4 dependence of IgG1 responses indicated by our IL-4 knockout mouse data (31) referred to above may simply reflect the possibility that the required IL-4 is not produced by Ag-stimulated T cells but, rather, may be derived from alternative sources such as basophils, which have recently been reported to be a major source of IL-4 in human filarial infections (39). Alternatively, it has recently emerged that IL-4-independent mechanisms may be responsible for promoting anti-OVA IgG1 responses. Indeed, IgG1 production has been observed in IL-4 knockout mice (40, 41, 42). Consistent with this, although IL-5 has been widely established to select for IgA rather than IgG1 isotypes, there is also evidence showing that IL-5, which is elevated by ES-62, is important for the promotion of IgG1 Ab (43). Our current data showing that ES-62 elicits some but not all of the traits of a classical Th2 immune response (increased IgG1; decreased IgG2a; elevated IL-5; reduced IL-4, IL-13, and IFN-{gamma}) indicate a nonclassical Th phenotype.

In addition to modifying effector function, exposure to ES-62 inhibited OVA-induced clonal expansion. Consistent with this, ex vivo T cell cultures not only proliferated less upon ex vivo rechallenge (Fig. 2) but also produced corresponding lower levels of IL-2 (Fig. 3) perhaps reflecting the induction of T cell anergy or CD4+CD25+ Tregs. T cells pre-exposed to ES-62 in vivo express increased mRNA levels of Foxp3, GATA-3, c-Maf, and T-bet, following ex vivo restimulation with Ag (results not shown). When taken together with the nonclassical cytokine profiles resulting from exposure to ES-62, these findings are perhaps reminiscent of recent studies reporting nonclassical regulatory T cell phenotypes. For example, Raghavan et al. (44) have recently reported that Ag-specific suppression of murine Helicobacter pylori-reactive immunopathology was mediated by CD4+CD25+ T cells in an IL-5-dependent mechanism. Moreover, heat-killed Listeria monocytogenes, which has been shown to act as a Th1 polarizing adjuvant, induces the development of Th1-like Tr1 cells which express Foxp3 and T-bet and potently suppress the development of airway hyperreactivity. Thus, the simultaneous expression of Foxp3 and T-bet by T cells from ES-62-treated mice could possibly reflect the generation of Th1-like Tr1 cells in a system in which CFA (as opposed to heat-killed L. monocytogenes) is a Th1-polarizing adjuvant (45). However, despite the increase in mRNA levels of these transcription factors following rechallenge ex vivo, no increase in staining of CD25, Foxp3, GATA-3, or T-bet could be detected in Ag-specific cells from ES-62, relative to control mice at sacrifice (results not shown), findings consistent with a recent report that baseline expression levels of T-bet, GATA-3, and Foxp3 were not significantly different between filarial-infected individuals and uninfected but exposed controls (46). These latter results suggest that naturally occurring CD4+CD25+ T regulatory cells and Th1-like Tr1 cells are probably not induced by ES-62 in vivo. Thus, the increases in Foxp3 mRNA levels observed after rechallenge ex vivo may simply reflect recent findings that although Foxp3 expression is primarily restricted to CD4+CD25+ human T cells, it can be induced after activation of either CD4+ or CD8+ T cell clones (47).

Although the relationship between Tr1 and naturally occurring CD4+CD25+ T regulatory cells has not been defined (48), classical Tr1 cells have also been associated with IL-5 production (49). Indeed, closer analysis of the basis of Ag-specific hyporesponsiveness in humans chronically infected with O. volvulus and animal models of filarial nematode infection (50) has revealed that suppression is mediated by IL-10- and/or TGF-{beta}-producing Tr1 cells (8, 51, 52, 53, 54), which mediate suppression of T cell proliferation and IFN-{gamma} production. Although we have not detected elevation of IL-10 or TGF-{beta} release (results not shown) from Ag-specific T cells on rechallenge ex vivo, this does not exclude the possibility that they are produced from other sources (55) during initial T cell priming in vivo leading to differentiation of Tr1 cells (56).

Finally, an explanation for the immunological phenotype underlying filarial nematode infection, in particular the inhibition of T cell proliferation and polarization of antibody/cytokine responses to parasite Ags, has been sought for many years. Previously, we have shown that ES-62 modulates the ability of the immune system to respond to its peptide epitopes (31). Here we show that the parasite product can similarly manipulate the response of the immune system to a heterologous Ag as shown by reduced T cell proliferation, follicular migration, altered cytokine profiles, and the induction of polarized Ab responses. The significance of the reduced follicular migration is as yet not clear given that we have previously shown that both Th1 and Th2 cells can migrate to follicles to support B cell responses (23). Nevertheless, the reduced and slower kinetics of T cell follicular migration observed may, by determining the precise timing of particular B-T cell help interactions, play a role in determining the reduced clonal expansion and induction of the precise Th phenotype of immune response generated. In any case, collectively, these findings raise the possibility that ES-62 can also modulate the immune response to other filarial nematode molecules during infection. It is possible, therefore, that ES-62 may in fact be a significant contributor to the generalized anti-inflammatory-Th2-like response observed during natural infection in humans.


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


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

1 This work was supported by the Medical Research Council and the Wellcome Trust. F.A.M. held a Medical Research Council Doctoral Training Studentship. Back

2 Address correspondence and reprint requests to Dr. Margaret M. Harnett, Division of Immunology, Infection and Inflammation, University of Glasgow, Western Infirmary, Dumbarton Road, Glasgow G11 6NT, United Kingdom. E-mail address: M.Harnett{at}bio.gla.ac.uk Back

3 Abbreviations used in this paper: ES, excretory-secretory; cOVA, chicken OVA; CIA, collagen-induced arthritis; tg, transgenic; PC, phosphorylcholine; PLN, peripheral lymph node; LSC, laser scanning cytometry; CD62L, L-selectin. Back

Received for publication January 20, 2005. Accepted for publication August 16, 2005.


   References
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 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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