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Toxins 2010, 2(8), 2007-2027; doi:10.3390/toxins2082007
Abstract: LT-R192G, a mutant of the thermolabile enterotoxin of E. coli, is a potent adjuvant of immunization. Immune responses are generally analyzed at the end of protocols including at least 2 administrations, but rarely after a prime. To investigate this point, we compared B and T cell responses in mice after one and two intrarectal immunizations with 2/6 rotavirus-like particles (2/6-VLP) and LT-R192G. After a boost, we found, an unexpected lower B cell expansion measured by flow cytometry, despite a secondary antibody response. We then analyzed CD4+CD25+Foxp3+ regulatory T cells (Tregs) and CD4+CD25+Foxp3− helper T cells after in vitro (re)stimulation of mesenteric lymph node cells with the antigen (2/6-VLP), the adjuvant (LT-R192G) or both. 2/6-VLP did not activate CD4+CD25+Foxp3− nor Foxp3+ T cells from non-immunized and 2/6-VLP immunized mice, whereas they did activate both subsets from mice immunized with 2/6-VLP in the presence of adjuvant. LT-R192G dramatically decreased CD4+CD25+Foxp3+ T cells from non-immunized and 2/6-VLP immunized mice but not from mice immunized with 2/6-VLP and adjuvant. Moreover, in this case, LT-R192G increased Foxp3 expression on CD4+CD25+Foxp3+ cells, suggesting specific Treg activation during the recall. Finally, when both 2/6-VLP and LT-R192G were used for restimulation, LT-R192G clearly suppressed both 2/6-VLP-specific CD4+CD25+Foxp3− and Foxp3+ T cells. All together, these results suggest that LT-R192G exerts different effects on CD4+CD25+Foxp3+ T cells, depending on a first or a second contact. The unexpected immunomodulation observed during the recall should be considered in designing vaccination protocols.
Because systemic immunization is not optimal to induce local immune effectors and requires multiple injections, mucosal immunization is an important goal of vaccine development to protect against pathogens that use mucosa as portals of entry. However, several factors such as antigen degradation in the digestive tract, compartmentalization of the responses at the mucosa of antigen delivery as well as mucosal tolerance make mucosal vaccination with non replicating antigens complex. To generate strong mucosal immune responses, mucosal adjuvants have been proposed. The cholera toxin, CT, and the thermolabile enterotoxin of Escherichia coli, LT, which are well known diarrheagenic toxins produced by V. cholerae and enterotoxinogen E. coli, respectively, are potent mucosal adjuvants for abrogating mucosal tolerance and inducing strong B and T cell immune responses against coadministered antigens. To overcome the enterotoxicity and use them as adjuvant in humans, non toxic mutants of the A subunit have been developed, among them, the protease site mutant of LT, LT-R192G. This mutant retains adjuvant properties in experimental models [1,2,3,4] and has been tested in clinical trials [5,6,7]. However, although many studies have reported about the effects of the whole toxins or their mutants on different innate or adaptive immune cells that could explain the adjuvant effect (reviewed in [1,2,3,4,8,9]), the precise mechanism of action of these adjuvants has not been completely elucidated. Of note, the comparison of their different effects on immune responses after a prime and a boost, using the same route and the same immunogen, has not been documented. This comparison may bring relevant cognitive information and, in addition, it may be useful to optimize protocols of immunization.
To better understand early events induced after mucosal priming with a non-replicating antigen, we previously traced rotavirus (RV)-specific B cells by flow cytometry (FCM), after a single intranasal (IN) or intrarectal (IR) administration of RV virus-like particles (2/6-VLP) in mice, in the presence of LT-R192G [10,11]. 2/6-VLP coadministered intranasally or intrarectally with LT-R192G, in protocols including at least 2 immunizations, have been shown to induce strong T and B cell responses as well as protection against experimental infection [12,13]. With both routes, we have shown, after a prime, high expansion of specific B cells in different lymphoid tissues, which depend on the route of immunization. A substantial proportion of these cells expressed CD5 and was considered B-1a cells. Unexpectedly, we found that a second IN immunization in the same conditions did not increase the frequency of specific B cells on day 7 following the second immunization, whereas a secondary systemic and mucosal antibody response was observed . As LT-R192G is a potent mucosal adjuvant, this result was therefore difficult to explain. We hypothesized that the massive B cell expansion observed when the adjuvant was used for immunization was probably regulated during the second contact. Such modulation may be important to avoid potential deleterious autoreactivity of CD5+ expressing B-1a cells, as well as T cell-mediated inflammation. Regulatory T lymphocytes (Tregs) are important in suppressing the activation, proliferation and differentiation of T and B cells, and thus control immune responses [14,15]. Classically, Tregs are divided into two major subtypes: natural Tregs (nTregs) and peripherally inducible Tregs (iTregs). CD4+ nTregs develop in the thymus and express CD25 and the forkhead box P3 transcription factor, Foxp3 . CD4+ iTregs include different subtypes, among which is a subpopulation that shares the same phenotype, CD25+Foxp3+, with nTregs . In mice, Foxp3 does not make it possible to distinguish between nTregs and iTregs but is considered a useful marker to distinguish between CD4+ T cells with a presumed regulatory-suppressive function and other CD4+ T cells .
In this work, to investigate more in details the effects of LT-R192G after a prime and a boost, we first compared the primary and secondary specific B cell response induced by IR immunization with 2/6-VLP in the presence or in the absence of LT-R192G. Then, we analyzed specific CD25+CD4+ T cells, both Foxp3+ and Foxp3−, from different lymphoid tissues, in in vitro response to 2/6-VLP, LT-R192G or both. Quantitative analysis reflecting activation and/or proliferative responsiveness was performed using cell frequency, CD25 and/or Foxp3 mean fluorescence intensity of both subsets, as well as IL-2 production .
2. Materials and Methods
Pathogen-free, adult, female BALB/c mice (6–8 weeks of age) were obtained from Iffa-Credo (L’Arbresle, France) or from our in-house facilities. Study protocols were approved by the local institutional animal care committee. No mouse had evidence of previous RV infection, as determined by serum antibody titres.
2.2. VLP Preparation and Adjuvant
Two different VLP, containing RV VP2 and VP6 (2/6-VLP) used for immunization or GFP-Δ92VP2 and VP6 used for flow cytometry, were produced in the baculovirus system as described previously . Briefly, Sf9 insect cells were coinfected with two recombinant baculoviruses expressing the protein VP6 of the bovine RF strain and an authentic or a modified GFP-VP2 at a multiplicity of infection higher than 5PFU/cell. VLP were collected 5–7 days post infection and purified by density gradient centrifugation in CsCl.
LT-R192G, a LT mutant, the thermolabile enterotoxin of Escherichia coli, was used as the adjuvant. LT-R192G is a mutant of LT containing a single amino acid substitution that alters the site of proteolytic cleavage within the region joining A1 and A2. This mutation is associated with the reduced ability to induce an accumulation of cAMP in cultured cells as well as reduced enterotoxicity in experimental animals and humans when compared to native LT .
2.3. Immunization and Sample Collection
The mice were intrarectally immunized on day 0 with either NaCl, 10 µg LT-R192G alone, 10 µg 2/6-VLP alone or mixed with 10 µg LT-R192G. Prior to immunization, the mice were anesthetized by intraperitoneal administration of a mixture of ketamine (80 mg/kg) and xylazine (16 mg/kg). An additional group of mice was given two doses of 10 µg 2/6-VLP with 10 µg LT-R192G, or 2/6-VLP or LT-R192G alone on day 0 and on day 14. The mice were killed at different time points post-immunization (2, 4, 7 or 14 days) and the different lymphoid tissues, rectal follicles (RF), lumbar lymph nodes (LLN), mesenteric lymph nodes (MLN), Peyer’s patches (PP) and spleen were removed. Serum and faecal samples were collected and stored at −20 °C.
2.4. Measurement of Rotavirus-Specific Antibodies in Serum and Fecal Samples
Antibody titres in serum and faecal samples were determined by ELISA. Microtitre plates were coated overnight at 4 °C with 100 ng of 2/6-VLP in 100 µL of 0.1 M carbonate/bicarbonate buffer, pH 9.6. The wells were blocked with PBS containing 5% non-fat dry milk for 45 min at 37 °C. Faecal samples were made 10% (wt/vol) by suspension in PBS, pH 7.4. Serial twofold dilutions in PBS-5% non-fat dry milk of serum (starting at 1/100) or faecal extracts (starting at 1/40) were added to the wells and incubated for 40 min at 37 °C. After three washes with PBS-0.05% Tween 20, the plates were incubated for 30 min at room temperature with 1/5000 dilution of biotin-labelled goat anti-mouse IgA, IgG or IgM (Cell Lab, Beckman Coulter). The plates were washed, and 1/4000 of peroxidase-labeled avidin (Cell Lab, Beckman Coulter) was added for 30 min at room temperature. The colour reaction was developed with TMB-Peroxidase Substrate Kit (AbCys.S.A), stopped with 100 µL of H2SO4 0.4N and A450 was determined. A sample was considered positive if the optical density of the sample well was >0.1 plus the mean of the OD values of the negative control wells. Titres were defined by the inverse of the highest serum dilution giving a positive signal. Negative serum and fecal samples (titre < 100 and <40, respectively) were arbitrarily assigned titres of 50 and 20 (half of 100 and 40), respectively, for statistical calculations .
2.5. Preparation of Cells from RF, LLN, MLN, PP and Spleen
Single-cell suspensions were prepared by mechanical dissociation, filtered on 40-µm-pore nylon meshes and washed with incomplete medium (RPMI-1640 supplemented with 0.3% glucose, 100 U penicillin per mL, and 100 µg streptomycin per mL). The cells were counted, labelled and analyzed immediately by FCM or resuspended (4 × 106/mL) in complete medium (incomplete medium plus 10% heat-inactived FCS, 2 mM L-glutamine, 1 mM sodium pyruvate) for in vitro restimulation.
2.6. In Vitro Restimulation
Cells from either immunized or non-immunized mice (4 × 105 cells/well) were cultured in 96-well plates in the presence of different doses of 2/6-VLP, LT-R192G, 2/6-VLP and LT-R192G or RPMI medium only. The T-cell mitogen concanavalin A (5 µg/mL) was used as the positive control. The cells were incubated at 37 °C with 5% CO2 and harvested on day 2 and day 4 after restimulation for flow cytometry analysis, and the culture supernatants were frozen at −40 °C until IL-2 assay.
2.7. FCM Assays
2.7.1. Rotavirus Specific B Cell Quantification
To detect RV-specific B cells, we used an FCM assay as previously described . Cells from different lymphoid tissues were washed once with PBS 1% BSA 0.1% sodium azide buffer. Pellets containing 2 × 106 cells were incubated with a mixture of PE Cy-chrome-labelled anti-B220 (Clone RA3-6B2, Pharmingen, San Diego, CA, USA), biotinylated anti-IgD (Clone AM9.1, Pharmingen), PE-labelled anti-CCR9 (Clone 248918, R&D Systems, Minneapolis, MN, USA) or anti-CD5 (Clone 53-7.3, Pharmingen) or anti-α4β7 (clone DATK32, Pharmingen) and GFP-2/6-VLP (to detect RV-specific B lymphocytes) for 30 min in the dark at 4 °C. The cells were washed and then labelled with streptavidine-RED613 (Gibco-BRL, Scotland, UK). After incubation for 30 min in the dark at 4 °C, the cells were washed, resuspended in buffer containing PBS 1% BSA 0.1% sodium azide, and analysed on a flow cytometer (LSR II, Becton Dickinson, San Jose, CA, USA). Approximately 3 × 105 cells were acquired. Analysis was done as described previously . Small lymphocytes were distinguished from large lymphocytes by their light-scatter profile. Three types of B cell subsets were analysed: a large B220int IgD− subset representing extrafollicular B cells, a large B220high IgD− subset representing germinal center B cells, and a small B220high IgD− subset consisting of memory and germinal center B cells . To delineate RV-specific cell populations and to control for the specificity of the staining, the cells were stained, omitting GFP-VLP, and the quadrant position was fitted eventually, after comparison with identical B cell subsets from non-immunized mice. Absolute numbers of RV-specific B cells were obtained by the quantification of RV-specific B cells among total cells that expressed B220 (B220high plus B220low). In order to compare samples, the number of RV-specific B cells was referred to 105 total B220+ cells .
2.7.2. Analysis of CD4+CD25+Foxp3+ T Cells and CD4+CD25+Foxp3− T Cells
CD4+CD25+Foxp3+ T cells and CD4+CD25+Foxp3− T cells were quantified using the Mouse Regulatory T cell Staining kit #2 (w/APC Foxp3 FJK-16s, FITC CD4, PE CD25; eBioscience, San Diego, USA). Briefly, pellets containing 4x105 cells in 100 µL PBS 1% BSA 0.1% sodium azide, were incubated with a mixture of FITC-labelled anti-CD4 and PE-labelled anti-CD25 for 30 min in the dark at 4 °C. The cells were washed and resuspended in 1 mL of freshly prepared fixation/permeabilization working solution and then incubated at 4 °C for 30 min in the dark. The cells were washed with permeabilization buffer and then labelled with APC-labelled anti-Foxp3 for 30 min in the dark. After incubation, the cells were washed, resuspended in buffer containing PBS 1% BSA 0.1% sodium azide, and analysed by flow cytometry. Approximately 5 × 104 cells were acquired. Lymphocytes were first identified by their light-scatter profile, and T cell subsets were then identified by CD4 expression. CD25+Foxp3+ and CD25+Foxp3− T cells were identified by binding of CD25 and Foxp3 within CD4+ T cells. The mean fluorescence intensity (MFI) for PE and APC were analyzed to detect variations in CD25 and Foxp3 expression, respectively.
2.8. IL-2 Assay
The IL-2 level in culture supernatants was determined by ELISA, using the AbC601MST Mouse IL-2 Module Set kit (AbCys.S.A). Briefly, microtitre plates were coated with 100 µL of anti-IL-2 monoclonal antibody and incubated overnight at 4 °C. The wells were blocked with PBS containing 0.5% BSA and 0.05% Tween 20 at 4 °C overnight. After two washes with PBS containing 0.05% Tween 20, 50 µL of supernatants were added and incubated with the biotinylated anti-IL-2 monoclonal antibody for 2h. The plates were washed, peroxidase-labeled streptavidine was added and the plates were incubated for 1h. The colour reaction was developed with TMB-Peroxidase Substrate Kit (AbCys.S.A), stopped with 100 µL of H2SO4 0.4 N and A450 was determined.
For B cell analysis, results were expressed as means with SEM. Statistical analysis was performed using SigmaStat software. Pairwise comparisons between non-immunized and immunized mice were made using the Mann-Whitney non-parametric U-test. P-values less than 0.05 were considered statistically significant.
For T cell analysis, comparison of cell frequency, CD25 and Foxp3 MFI between stimulated and non stimulated wells was done by using the Wilcoxon paired non-parametric signed-rank test. P-values less than 0.05 were considered statistically significant. In addition, comparisons between non-immunized and immunized mice were made using the Mann-Whitney unpaired non-parametric U-test.
3.1. Primary and Secondary Specific B Cell Responses Induced by IR Immunization with 2/6-VLP with or without LT-R192G
3.1.1. The Secondary Response Induced by IR Immunization with 2/6-VLP and LT-R192G Showed a Serum and Fecal Antibody Response but a Lower RV-Specific B Cell Expansion
Mice were immunized once or twice with 2/6-VLP or NaCl in the presence of adjuvant, and the RV-specific B cell response analyzed as described previously . On day 7 after the second dose, a secondary antibody response was observed in serum (IgA 3.7 vs. 1.7 log and IgG 5 vs. 1.7 log, for the secondary and primary response, respectively) and in feces (IgA 3.4 vs. 1.4 log)(Figure 1), as already shown with the IN route of immunization .
A RV-specific B cell response, measured by flow cytometry using GFP-2/6-VLP, was found on day 7 in RF, LLN and MLN, but not in PP and the spleen, as previously reported after a single immunization (Figure 2A) . However, the secondary B cell response was significantly lower than the primary response within the large B220high B cells (1.5–2% vs. 7–11% depending on the lymphoid tissue) and the small lymphocytes (0.4–1% vs. 1.5–5%)(Figure 2A). This result was confirmed when the B cell frequency was expressed as absolute numbers of RV-specific B cells/105 total B220+ cells (Figure 2B). For the large B220int lymphocyte subset, no major difference was observed between the primary and the secondary response, but in this case, no massive expansion was observed during the primary response (Figure 2B). As the lower frequency observed could be the consequence of an earlier expansion during the secondary response, we further analyzed the B cell response on day 2 and 4 after a second dose and on day 4 after a single dose (Figure 2C). The results clearly confirmed the absence of a massive B cell expansion during the secondary response, despite a trend towards a higher response on day 4 for the B220int subset, which is consistent with the kinetics of a secondary antibody response.
Of note, α4β7, CCR9 and CD5 expression by RV-specific B cells was not different in the primary and secondary responses (<15%, 30–50% and 30–75%, respectively, data not shown).
All together, these results show that, after two immunizations with 2/6-VLP and LT-R192G, the massive expansion observed during the primary response was no longer observed during the secondary response despite a secondary antibody response.
3.1.2. The Secondary Response Induced by IR Immunization with 2/6-VLP in the Absence of Adjuvant Showed a Similar RV-Specific B Cell Expansion to that in the Primary Response and a Secondary Serum IgG Antibody Response
When mice were immunized with 2/6-VLP in the absence of adjuvant, a significant IgG antibody response was observed on day 7 after two immunizations (Figure 1). Concerning the RV-specific B cell response, we have previously shown a great variability among mice after one immunization with 2/6-VLP alone. Furthermore, when no adjuvant was used, we only observed a response in LLN. After two immunizations, a very similar response was observed in terms of heterogeneity and intensity (6 vs. 5.2% in B220high large B cells, 1.5 vs. 1% in B220int cells and 1.8 vs. 1.8% in small lymphocytes, on day 7 following the first and second immunization in LLN, data not shown). In addition, as after one immunization, no significant response was observed in MLN after two immunizations in the absence of adjuvant.
3.2. Primary and Secondary in Vitro T Cell Responses to 2/6-VLP, LT-R192G or Both, from Non-Immunized Mice and Mice Immunized with 2/6-VLP with or without LT-R192G: Analysis of CD4+CD25+Foxp3− and CD4+CD25+Foxp3+ T Cells
Non-immunized mice and mice immunized with 2/6-VLP, with or without LT-R192G, were sacrificed on day 14 and cells from different lymphoid tissues (4 × 105 cells/well) were cultured in the presence of antigen (5 µg/mL), adjuvant (5 µg/mL) or both for 4 days. CD4+CD25+Foxp3− and CD4+CD25+Foxp3+ T cells were analyzed by flow cytometry and IL-2 was quantified in culture supernatants. Results were similar for all the organs or tissues analyzed (i.e., LLN, MLN, PP and the spleen), but as they contain a high number of cells allowing multiple wells for in vitro culture, we focussed on MLN and performed statistical analysis only for MLN. RF were not analyzed because the number of cells harvested was not sufficient for in vitro restimulation.