Spleen mononuclear cells
Peritoneal adherent cells
It has been proposed that pregnancy-specific factors could be responsible for shift the balance of cytokine profiles during maternal immune response from Th1-type reactivity into a "less-damaging" Th2-type reactivity. In the present work, we investigated the in vivo function of human pregnancy-specific glycoprotein (PSG)1a, the major variant of PSG polypeptides released into thecirculation during pregnancy, on the modulation of the innate and adaptive immune response. For this, BALB/c mice were injected with a vaccinia virus-based vector harboring the human PSG1a cDNA (Vac-PSG1a) 4 days before immunization with ovalbumin (OVA) in complete Freund's adjuvant, and the early specific T cell response against OVA was evaluated 8 days post-immunization. We also studied the activation status of spleen and peritoneal monocytes/macrophages (Mo) populations from Vac-PSG1a-treated mice, and explored whether PSG1a-targeted Mo could affect the Th-type commitment by investigating their impact on the differentiation of naive T cells. Our data show that the treatment with Vac-PSG1a is able to induce a state of alternative activation on Mo. Furthermore, the generation of the immune response in the context of these alternatively activated antigen-presenting cells may shift T cell differentiation to Th2-type immunity which is more compatible with a successful pregnancy.
T helper (Th) cells can be divided into Th1 and Th2 cells, according to their cytokine profile upon antigenic stimulation 1, 2. The Th1 subset produces highlevels of IL-2 and IFN-γ causing monocyte/macrophage (Mo) activation, which is important for the protection against intracellular pathogens. The Th2 subset produces IL-4, IL-5 and IL-10 and it is implicated in the inhibition of macrophage activation and the stimulation of antibody production. The selective differentiation of either subset can be significantly influenced by a variety of factors, including cytokine environment, antigen dose, the strength of TCR signaling and costimulatory signals derived from APC 3–5.
Depending on its activation state, APC may be subdivided in (a) pro-inflammatory, "classically activated" APC, which are developed in a type I cytokine environment (INF-γ, IL-12, TNF-α) and are inhibited by type II cytokines (IL-4, IL-10, IL-13), and (b) anti-inflammatory "alternatively activated" APC, which are developed in a type II cytokine environment, secret IL-10 and TGF-β and down-regulate inflammatory processes counteracting NO synthesis through the expression of arginase, which competes with inducible NO synthase for L-arginine as substrate 6. It has been proposed that these subsets of Mo may regulate Th1 and Th2 differentiation, and recently it was clearly demonstrated that the functional direction of the T cell response depends on the type of APC and its previous environmental exposure 7.
Understanding the successful gestation of the histoincompatible fetus in the immunological competent mother's uterus has been a major goal for reproductive immunologists. It has been proposed that during normal pregnancy, even if fetal antigens were presented, the maternal immune response shift the balance of cytokine profiles away from Th1-type reactivity to a Th2-type less-damaging reactivity 8, 9.
Pregnancy-specific glycoproteins (PSG) are a family of highly similar proteins synthesized in large amounts by the placental syncytiotrophoblast 10–12. PSG seems to be essential for successful pregnancy, since low levels of these glycoproteins are associated with spontaneous abortion, intrauterine growth retardation and pre-eclampsia 13, 14. Previously, we demonstrated that mammalian cells infected "in vitro" with a vaccinia virus-based vector harboring the open reading frame of human PSG1a cDNA release high concentrations of an N-glycosylated 72-kDa PSG1a protein (rec-PSG1a) to the culture supernatant, like that secreted by human placenta 15. We used rec-PSG1a to investigate its activity over Mo activation pathways and T cell proliferation.
We demonstrated that rec-PSG1a is able to induce alternative activation of human as well as murine Mo and to modulate the T cell immune response in vivo and in vitro 15. In fact, this glycoprotein is able to suppress the accessory cell-dependent T cell proliferative response, whereas it does not alter the ability of T cells to respond to accessory cell-independent stimuli 15. Furthermore, murine PSG17 and PSG18 mimic the biological effects of human PSG inducing IL-10, IL-6 and TGF-β expression in human Mo 16, and human PSG1, PSG6 and PSG11 induce the secretion of anti-inflammatory cytokines by human and murine Mo 17. These findings prompted us to find out whether PSG1a, when released into circulation, may have a role in shifting the T cell immune response to Th2 pathway through the induction of alternative activation of Mo.
2.1 Vac-PSG1a injection induces in vivo expression of human PSG1a
In vivo expression of rec-PSG1a was analyzed in BALB/c mice treated with a single i.p. injection of Vac-PSG1a at a dose of 107 PFU/mouse. At 24 h after virus administration. Serum rec-PSG1a was immunoprecipitated using polyclonal anti-PSG antibody and then detected by Western blot as a 72-kDa band (Fig. 1A, lane 3). rec-PSG1a from peritoneal fluid was also detected as 72-kDa band (Fig. 1B, lane 2), whereas two fast-migrating bands corresponding to the less glycosylated intracellular form of this protein were observed in whole-cell extracts from peritoneum and lymph nodes (Fig. 1B, lanes 3, 4). In contrast, rec-PSG1a was detected neither in spleen cells from Vac-PSG1a-treated mice (Fig. 1B, lane 5) nor in the same cell populations from control mice (not shown). The rec-PSG1a detected in sera and peritoneal fluids shows the same 72-kDa molecular mass as the one secreted to the culture supernatant of Vac-PSG1a-infected J774 cells (Fig. 1B, lane 1) and the major placental PSG fraction 15, indicating that the protein secreted in vivo correspond to the properly glycosylated form.
2.2 Vac-PSG1a treatment induces Th2-type response by an IL-10-dependent mechanism
With the aim to determine the Th-type response towards OVA generated in Vac-PSG1a-treated mice, the profile of cytokines released by spleen mononuclear cells (SMC) from Vac-PSG1a-treated or control mice cultured with OVA was analyzed. As previously reported for OVA-CFA-immunized BALB/c mice 18, Fig. 2A shows that antigen stimulation of SMC from control mice resulted in a Th1 response characterized by high IFN-γ and IL-2, and low IL-4, IL-5 and IL-10 production. Conversely, OVA-stimulated SMC from Vac-PSG1a-treated mice exhibited high IL-4, IL-5 and IL-10, and low IFN-γ and IL-2 production. When Con A and anti-CD3 were used as stimuli, the results also demonstrated the tendency of the treated mice to secrete polarized Th1 or Th2 cytokines. Similarly, freshly explanted T cells from Vac-PSG1a-treated mice stimulated with PMA and ionomycin showed a reduction in the number of IFN-γ-producing cells as compared to T cells from control mice [21.2%, mean fluorescence intensity (MFI) 555 vs. 11.7%, MFI 438] (Fig. 2B). Interestingly, mitogenic stimuli induced a more significant TGF-β secretion by cells from control compared to Vac-PSG1a-treated mice whereas antigenic stimulus induced the opposite behavior (Fig. 2A). Consistent with these results, Fig. 2C shows that Vac-PSG1a treatment leads to an up-regulation in the expression of GATA-3, the Th2-specific transcription factor, in purified T cells 19.
IL-10 and TGF-β are cytokines commonly associated with the capacity to down-regulate IFN-γ as well as to induce a Th2 differentiation 20–22. In our experimental system, TGF-β seemed not to be involved in T cell suppression and Th2 differentiation because their levels are similar in suppressed and non-suppressed cultures (Fig. 2A). However, IL-10 plays an important role since blocking antibodies against IL-10, but not control rat IgG, reversed the suppression of the proliferative response of SMC from Vac-PSG1a-treated mice (Fig. 3A). In addition, this treatment increased four- and ninefold the IL-2 production and two- and 11-fold the IFN-γ production of SMC from Vac-PSG1a-injected mice (for Con A and anti-CD3 stimulation, respectively; Fig. 3B), and had a weak effect on the proliferation and IFN-γ production of SMC from control mice (Fig. 3A, B). Studying the source of IL-10, we observed that the percentages of CD4+IL-10+ cells and CD11b+IL-10+ cells were increased when SMC from Vac-PSG1a-treated mice were stimulated by OVA in comparison with OVA-stimulated SMC from control mice (Fig. 4). Together, these findings indicate that the IL-10 secreted by both T cells and Mo from Vac-PSG1a-treated mice is involved in suppressing cell proliferation and IFN-γ secretion.
2.3 Vac-PSG1a treatment induces alternative activation of spleen and peritoneal Mo
When we investigated the role of human PSG1a in vivo in the modulation of the arginine metabolism, we observed that the arginase activity of spleen Mo (SpMo) and peritoneal adherent cells (PAC) from Vac-PSG1a-treated mice was increased in comparison with Mo populations from control mice (Fig. 5A). LPS is able to stimulate alternatively, but not classically activated Mo to increase arginine metabolism to produce ornithine and urea 23. When Mo populations from Vac-PSG1a-treated mice were cultured with LPS during 48 h, they increased threefold their production of urea with respect to controls (Fig. 5A), while no differences between the groups were observed for nitrite levels (data not shown).
Moreover, we observed that PAC from Vac-PSG1a-treated mice spontaneously produced higher levels of TGF-β compared to PAC from control mice, and that the stimulation with LPS increased TGF-β secretion (Fig. 5B). Conversely, SpMo from Vac-PSG1a-treated or control mice secreted low levels of TGF-β even after LPS activation. In addition, while unstimulated PAC and SpMo from Vac-PSG1a-treated and control mice did not secrete IL-10, it is highly induced in LPS-activated PAC but only from Vac-PSG1a-treated mice. Similarly, SpMo from Vac-PSG1a-treated mice were able to secrete higher levels of IL-10 than controls after LPS activation. Since in Vac-PSG1a-treated mice not only PAC (which produce rec-PSG1a; Fig. 1B, lane 3) but also SpMo (unable to produce rec-PSG1a; Fig. 1, lane 5) showed signals of alternative activation, our results are consistent with a biological role of the circulating rec-PSG1a in a systemic bias of the Mo metabolism toward an alternatively activated pathway.
2.4 Effects of APC from Vac-PSG1a-treated mice on naive T cell proliferation and cytokine secretion
Because the first signals between T cells and APC could be crucial in determining the subset of Th cells to expand, we investigated the effect of Mo from Vac-PSG1a-treated mice on naive T cell proliferation, cell cycle progression and cytokine production. We observed that PAC from Vac-PSG1a-treated mice profoundly inhibited the mitogen-induced proliferation of naive T cells inducing a strong arrest in G0/G1 phase of the cell cycle (Fig. 6A, B). In addition, Fig. 6C shows that naive T cells stimulated with Con A and co-cultured with PAC from Vac-PSG1a-treated mice secreted significantly lower levels of IL-2 and IFN-γ and slightly increased levels of IL-4 compared to naive T cells stimulated with Con A and co-cultured with PAC from control mice. Besides, only supernatants of those co-cultures with PAC from Vac-PSG1a-treated mice showed detectable levels of IL-5. Neither TGF-β nor IL-10 production showed significant differences between mitogen-stimulated suppressed and non-suppressed cultures. Consistent with this result, we found that blocking antibodies against IL-10 did not have any significant effect on naive T cell proliferation and Th1 cytokine production (data not shown).
Next we investigated whether suppressive soluble factors, such as prostaglandins and NO, are involved in the suppressive effect of PAC from Vac-PSG1a-treated mice. For this, PAC from Vac-PSG1a-treated or control mice were separated from SMC from naive BALB/c mice by a 0.4-μm pore membrane in a transwell culture and then stimulated with Con A. As observed in Fig. 6D and E, the anti-proliferative and cell cycle arrest effects induced by PAC from Vac-PSG1a-injected mice were reversed by transwell separation. Moreover, when SpMo from Vac-PSG1a-treated mice were used as APC in the cultures, they inhibit the Con A-induced proliferation of naive T cells delaying the progression of T cells through the cell cycle and inducing the accumulation of the cells in the S phase of the cell cycle by a mechanism that is IL-10-dependent (not shown). In agreement, transwell culture did not reverse the inhibition of the proliferative response (not shown).
In the present work we demonstrate that treatment with Vac-PSG1a before immunization of BALB/c mice with OVA favors the Th2-type response. In this regard, it is important to remark the major role of PSG1a treatment in the decision process to mount a Th2-type immune response, since both adjuvants, CFA and vaccinia virus, used in our experimental system are strong Th1-type response inductors 18, 24.
In addition to Th2-type cytokines, other compounds such as eicosanoids, glucocorticoids or parasite antigens can modulate arginine metabolism in order to induce alternative activation of Mo 25. In agreement, we have previously demonstrated that rec-PSG1a has a direct effect on human and murine Mo inducing an alternatively activated state 15. Likewise, in this work we demonstrate that Mo populations derived from Vac-PSG1a-treated mice are alternatively activated since they exhibit an enhanced arginase activity and secret TGF-β and/or IL-10 when are stimulated by LPS.
To address the mechanisms by which the Vac-PSG1a treatment influences Th2-type immune response enhancement, we first evaluated the role of the Th1-inhibitory cytokines present in the cultures of SMC from Vac-PSG1a-treated and control mice. Since the levels of TGF-β were similar in suppressed and non-suppressed cultures of SMC, this cytokine did not appear to be involved in the Th-type differentiation and proliferation. Conversely, the levels of IL-10 showed important differences between suppressed and non-suppressed cultures. Furthermore, using blocking antibodies against IL-10 we could reverse the inhibition of the proliferation and Th1-type cytokine synthesis by SMC from Vac-PSG1a-treated mice.
IL-10 was originally termed "cytokine synthesis inhibition factor" due to its ability to inhibit the proliferation of CD4+ T cells very early in the cell cycle, inhibiting IL-2 production and affecting the IL-2 receptor signaling 26. Thus, the inhibitory effects on T cell cycle and cytokine secretion observed in the spleen cells from Vac-PSG1a-treated mice, as well as when SpMo from these mice are co-cultured with naive T cells, are in agreement with the functions described for IL-10. It is important to note that even though T cells from Vac-PSG1a-treated mice showed detectable levels of GATA-3 (which is rapidly induced in T cells upon stimulation under Th2-skewing conditions), blocking antibodies against IL-10 increased the Th1-type cytokine levels reaching similar values to SMC from control mice. These results are in agreement with several in vitro studies that demonstrated that GATA-3 is sufficient, although not very potent, in directing Th2 polarization 19, 27. In addition, our results suggest that when the Th2 phenotype is acquired during pregnancy, it could be not enduring and it could be reversed when the Th2-skewing factors fade away (e.g. when pregnancy is over).
Second, we evaluated the effect of Vac-PSG1a-recruited PAC (the first cell in receipt the danger signal in our experimental model 28) on naive T cell proliferation, cell cycle progression and cytokine production. PAC recruited by Vac-PSG1a injection inhibited the mitogen-induced cell cycle progression of naive T cells by cell-to-cell contact, suggesting a receptor-mediated mechanism. In addition, suppressed T cells produced IL-5, more IL-4 and less IFN γ and IL-2 than naive T cells stimulated in the presence of control PAC. These results suggest that alternatively activated PAC from Vac-PSG1a-treated mice influence naive T cell differentiation not only by inhibiting IFN-γ production allowing a default Th2 pathway, but also by inducing the secretion of Th2 cytokines.
Briefly, after Vac-PSG1a treatment, we have found two different Mo populations; one from spleen (not PSG1a producer and possibly targeted by circulating rec-PSG1a) and another from the peritoneum (rec-PSG1a producer); both having low-level induction of proliferative ability and Th2-biasing activity. SpMo are able to inhibit the antigen-specific and nonspecific T cell proliferation delaying the progression of T cells through the cell cycle and inducing Th2-type cytokine secretion by a mechanism which is IL-10-dependent. On the other hand, PAC do not exert their effects via well-known soluble mediators, but instead, act through direct cellular contact inducing IL-5 secretion. Alternatively activated peritoneal Mo with similar functions have been reported in chronic nematode infections, where they inhibit the T cell cycle progression and appear to play a role in inducing Th2 differentiation 29. Moreover, these Mo also produce IL-10 and TGF-β and apparently play an important role in down-modulating inflammation and immunity 30. In addition, since a source of IL-10 is necessary to induce regulatory T cells 31, the possibility that these Mo populations act to induce regulatory T cells in response to stimulation, is another exciting prospect that we are currently testing.
Pregnancy is characterized by a down-regulation of Th1-type reactivity, and instead a dominant Th2-type reactivity is prevalent. Increased rates of fetal loss are induced by injecting pregnantmice with IL-2, which promotes Th1-type Th cell responses at the expense of Th2-type responses 32. This reinforces the view that a delicate immunological equilibrium is established during gestation that can be subverted by inappropriate activation of Mo or lymphocytes of the innate and adaptive immune systems 33. Sacks et al. 34 have proposed that soluble placental products released directly into the maternal circulation can generate specific pregnancy signals through interaction with the innate immune system. Thus, the innate immunity might be able to distinguish the pregnant from the non-pregnant state producing a signal, so-called "signal P". In the light of this hypothesis, the results reported here suggest that PSG1a may have "signal P" role during pregnancy, inducing a non-inflammatory pathway of activation of the innate immune system and actively contributing to the T cell shift of the maternal cell immunity toward a Th2 phenotype in order to assure successful pregnancy.
4 Materials and methods
4.1 Recombinant vaccinia viruses
Recombinant vaccinia virus vector construction was previously described 15. Viruses isolated from BrdU-resistant cells were tested for their ability to express PSG1a following infection of J774 cells by Western blot analysis of both culture supernatant and protein cell extracts using specific polyclonal antisera (Dako, Denmark).
4.2 Mouse model
Inbred female BALB/c mice, aged 6–8 weeks (CNEA, Argentina) were injected i.p with 107 PFU of Vac-PSG1a (n=6). Age-matched mice were injected with 107 PFU of Vac-wt as controls (n=6). After 4 days, all mice were immunized with 50 μg of OVA (Sigma-Aldrich, St. Louis, MO) emulsified in CFA (Sigma-Aldrich) by i.p. injections. Mice were maintained according to the National Research Council's guide for the care and use of laboratory animals and killed on day 8 post-immunization. In some experiments, age-matched untreated female BALB/c mice were used to obtain naive SMC or T cells.
4.3 Evaluation of the in vivo transgene expression
After 24 h of virus administration, whole-cell extracts of cell suspensions from spleen, peritoneum and lymph nodes were obtained using RIPA buffer. Cell extracts (50 μg) or peritoneal fluid (10 μl) were analyzed by 10% SDS-PAGE and transferred to a nitrocellulose membrane. Serum (500 μl) from vac-PSG-treated mice was incubated with anti-PSG antibody (40 μg) for 1 h at 4 °C. Then, 50 μl of protein A-Sepharose suspension was added and incubated for additional 1 h. The mixture was centrifuged and the pellet resuspended in Laemmli buffer, boiled, analyzed by 10% SDS-PAGE and transferred to a nitrocellulose membrane. PSG protein was revealed by immunoblotting using polyclonal anti-PSG antibodies. Immunocomplexes were visualized by a chemiluminescence reaction (NEN), according to manufacturer's recommendations.
SMC were prepared as previously described 15. For T cell purification, adherent cells were removed from SMC by two rounds of plastic adherence (1 h incubation at 37°C in 10-cm petri dishes), and B cells were depleted by magnetic cell sorting using anti-B220-coated magnetic beads (Dynal, A.S, Oslo, Norway), following the manufacturer's instructions. After this procedure, >95% of CD3+ cells were detected by flow cytometry.
For SpMo purification, SMC were incubated with RPMI 1640 containing 20% FBS at 37°C in 5% CO2 in 10-cm petri dishes (1×107 cells/petri dish). Nonadherent cells were removed after 2-h incubation. Macrophages were detached using an ice bath, washed twice and resuspended in complete medium. This procedure yielded >90% of CD11b+ (Mac-1+) cells, as determined by flow cytometry.
PAC were harvested by thorough washing of the peritoneal cavity with 15 ml RPMI. Then, the cells were washed and incubated with RPMI 1640 containing 20% FBS at 37°C in 5% CO2 in 10-cmpetri dishes to allow the Mo to adhere to the plate. The same procedure was followed to obtain SpMo from SMC. After this procedure, >90% of CD11b+ (Mac-1+) cells were detected by flow cytometry.
4.5 Cultures and proliferation assay
All in vitro cultures were carried out in RPMI complete medium. SMC (1×106/ml) from Vac-PSG1a-treated or control mice were cultured in triplicate in 96-well microcultureplates at 37°C in 5% CO2 with Con A (5 μg/ml), anti-mouse CD3 (2.5 μg/ml), OVA (15 μg/ml) or medium alone during 72 h (mitogens) or 120 h (specific antigen). Culture supernatants were harvested after 72 h of culture for cytokine assays and proliferation was measured by [3H]thymidine incorporation. In some experiments, neutralizing anti-IL-10 (PharMingen) or rat IgG1 (PharMingen) was added to the cultures at the recommended concentration (5 μg/ml).
For SpMo or PAC activation studies, 5×105/ml SpMo or PAC were cultured with LPS from Escherichia coli Serotype 0111:B4 (10 μg/ml, Sigma Aldrich) or medium alone for 48 h before arginase activity determination and supernatant collection to measure NO and cytokine production. For SpMo- or PAC-naive T cells co-cultures, 1×105 SpMo or PAC from Vac-PSG1a-treated or control mice were used to stimulate 1×105 purified naive T cells with Con A or anti-CD3. After 48 h at 37°C, 1 μCi [3H]thymidine was added to each well, andplates were harvested for counting 18 h later.
For analysis of cell contact-dependent mechanisms, 24-well (0.4-μm pore) transwell cell culture chambers (Costar, GB) were used to separate PAC from responder cells. In each well, 1×105 PAC were separated from 5×105 SMC and Con A (5 μg/ml) or anti-mouse CD3 (2.5 μg/ml). Results were identical regardless of whether PAC were cultures in the upper or lower compartments. As control, the same number of cells were cultured together in 24-well plates without a separate membrane. Cells were incubated for 72 h and pulsed with 50 μCi [3H]thymidine per well for the last 18 h of culture. Responder cells were transferred to 96-well plates to be harvested and counted.
4.6 Cytokine assays
Culture supernatants from PAC or SpMo were collected after 48 h in culture for the determination of IL-10, IL-12 and TGF-β. Culture supernatants from SMC or naive T cell cultures were collected after 72 h for the determination of IL-2, IL-4, IL5, IL-10, IFN-γ and TGF-β. The cytokine production was measured using monoclonal antibody pairs purchased from PharMingen following the manufacturer's instructions. Standard curves were generated using known amounts of recombinant mouse cytokines (PharMingen). These ELISA were capable of detecting 0.78 ng/ml INF-γ, 8 pg/ml IL-2, 23.4 pg/ml IL-4, 0.1 ng/ml IL-5, 31.25 pg/ml IL-10 and 100 pg/ml TGF-β.
4.7 Flow cytometry determinations
Intracellular analysis of cytokines was performed using Cytofix/Cytoperm Plus kit (PharMingen) following the manufacturer's instructions. For IFN-γ determination, freshly explanted SMC from Vac-PSG1a-treated or control mice were stimulated in vitro with PMA (30 ng/ml; Sigma-Aldrich) and ionomycin (300 ng/ml; Sigma-Aldrich) at 106 cells/ml for 4 h at 37°C in thepresence of 1 μl GolgiStop (PharMingen). For IL-10 determination, SMC were cultured with OVA (15 μg/ml) at 106 cells/ml for 48 h at 37°C. After 48 h, 1 μl GolgiStop was added and the cells were incubated for another 4 h. Then, the cells were washed with Hanks' balanced salt solution containing 1% BSA and 0.1% NaN3, and stained with PE- or FITC-labeled anti-mouse CD3 or PE-labeled anti-mouse CD11b (Mac-1). After this the cells were washed with staining buffer (PharMingen), and intracellular staining with optimal concentration of PE-labeled anti-IFN-γ, FITC-labeled anti-IL-10 or control PE-/FITC-Ig conjugates (PharMingen) was performed following the manufacturer's instructions. Then the cells were acquired in a Cytoron Absolute cytometer (Ortho Diagnostic System, Raritan, NJ), and analyzed using WinMDI 2.8 (J. Trotter, Scripps Research Institute, La Jolla, CA). The quadrant markers were set based on the staining profile of cells stainedwith the appropriate isotype conjugates.
The DNA content was determined as described by Nicoletti et al. 35. After 48 h of culture with or without the appropriate stimulus, the cells were stained as previously indicated using FITC-labeled anti-mouse CD3 (PharMingen) and the DNA content was evaluated with propidium iodide staining. Ten-thousand events were acquired in a flow cytometer using Cell Cycle program (Ortho Cytoronabsolute) to measure cell cycle phases. Data were analyzed with WinMDI 2.7 and Cylchred 1.0.2 (Cardiff) software.
4.8 Determination of arginase activity
All arginase assays were performed with cell lysates that had been cultured in different conditions, as indicated in the figure legends. Arginase activity was measured according to the alreadydescribed procedure with slight modifications 15, 36.
4.9 Nitric oxide assay
NO was measured as nitrite using the Griess reagent. Culture supernatant was mixed with 100 μl of 1% sulfanilamide, 0.1% N-(1-naphthyl)ethylene-diamine dihydrochloride and 2.5% H3PO4. Optical density, measured at 540 nm in a microplate reader (Bio-Rad, Hercules, CA), was averaged and converted to μM of nitrites using a standard curve of sodium nitrite.
4.10 GATA-3 Western blot analysis
Whole-cell extracts (50 μg) of splenic T cells (1×107) from BALB/c mice treated with Vac-PSG1a or Vac-wt and immunized with OVA-CFA were treated with RIPA buffer, resolved by 12% SDS-PAGE and transferred to nitrocellulose. The immunoblotting was performed with murine monoclonal anti-GATA-3 antibody (1:100; Santa Cruz Biotechnology Inc., Santa Cruz, CA) followed by horseradish peroxide-coupled secondary antibody (Sigma-Aldrich) and visualized using a chemiluminescence substrate (NEN).
4.11 Statistical analysis
Data were analyzed using the Sigma Plot statistical package (Jandel Scientific software). Comparisons between two groups were made using the unpaired t-test. Values were considered statistically significant when p<0.05. All experiments were repeated at least three times with similar results.
We are grateful to Dr. Luis Patrito for his continuous encouragement and support as well as to Dr. Bruno Chatton and Dr. Daniela Slavin for many helpful discussions. We thank the staff of cell culture department of the IGBMC (Illkirch, France) for the technical assistance during recombinant vaccinia virus production. We also thank Eva Acosta Rodriguez for idiomatic corrections. F.L.-D. is a fellow of the Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET) of Argentina. C.C.M., C.L.M., J.L.B. and A.G. are members of the Scientific Career of CONICET. This work was supported by SECyT-UNC, Agencia Córdoba Ciencia and CONICET grants.