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Comparative Study
. 2009 May 1;182(9):5268-75.
doi: 10.4049/jimmunol.0800681.

Autoantigen Immunization at Different Sites Reveals a Role for Anti-Inflammatory Effects of IFN-gamma in Regulating Susceptibility to Experimental Autoimmune Encephalomyelitis

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Free PMC article
Comparative Study

Autoantigen Immunization at Different Sites Reveals a Role for Anti-Inflammatory Effects of IFN-gamma in Regulating Susceptibility to Experimental Autoimmune Encephalomyelitis

Silvia Pastor et al. J Immunol. .
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Abstract

Experimental autoimmune encephalomyelitis is induced in B10.PL (H-2(u)) mice by immunization with the immunodominant N-terminal epitope of myelin basic protein, Ac1-9. In the present study, we show that the site of immunization impacts disease incidence and severity. This effect is more marked in female mice than in males. Although immunization in the flanks is effective in eliciting disease, delivery of Ag in the footpad and tailbase results in poor induction. Analyses of the immune responses in female mice following different immunization regimens indicates that resistance to disease is accompanied by higher levels of IFN-gamma and CD11b(+)Gr-1(int) myeloid cells. Such myeloid cells are known to have a suppressive function, and consistent with this knowledge, blockade of IFN-gamma results in increased disease activity and decreased levels of splenic CD11b(+)Gr-1(int) cells. Conversely, injection of adjuvants (CFA or Pam(3)CSK(4)) in the footpad decreases experimental autoimmune encephalomyelitis incidence and severity. Our study indicates that the site of immunization can impact the magnitude of the ensuing inflammatory response, and that at a certain threshold a protective, regulatory circuit can be elicited.

Conflict of interest statement

Disclosures

The authors have no financial conflict of interest.

Figures

FIGURE 1
FIGURE 1
Induction of EAE in male and female B10.PL following immunization with Ac1-9 peptide at different sites. Male and female mice (five mice per group) were immunized in the footpad/tailbase or in the flanks with 200 µg of the N-terminal-acetylated epitope of MBP, Ac1-9, emulsified in CFA supplemented with 4 mg/ml Mycobacterium. A total of 200 ng of pertussis toxin was i.p. administered on days 0 and 2 after immunization. The clinical score of EAE was determined daily as described in Materials and Methods. Disease incidence (number of mice showing disease symptoms/total number of mice) for each group was as follows: females, 2/5 mice (footpad/tailbase-immunized) and 4/5 mice (flank-immunized); males, 4/5 mice (footpad/tailbase-immunized) and 4/5 mice (flank-immunized). p < 0.01, significant differences determined by Mann-Whitney U test were observed between female B10.PL mice immunized in the footpad/tailbase vs the flanks from days 17–23. No significant differences were found in male mice following immunization at different sites. Data are representative of at least four independent experiments.
FIGURE 2
FIGURE 2
Analyses of immune responses in female mice following immunization in the footpad and tailbase or flanks. Female B10.PL mice were immunized in the flanks or footpad/tailbase as described for Fig. 1. A, Percentage of Ag-specific tetramer-positive CD4+ T cells (CD4+tet+) determined using PE-labeled MBP1-9[4Y]:I-Au tetramers and PerCP-labeled anti-CD4 Abs in pooled (inguinal, popliteal, and cervical) LNs, spleens, and percentage of total CD4+ T cells or macrophages in CNS samples, from mice at days 6, 8, 10, 13, and 15 after immunization. Samples were pooled from two mice per time point and treatment. In vivo proliferation of CD4+ T cells was evaluated by i.p. injection of BrdU before harvesting LNs and spleens. Percentage of CD4+BrdU+ cells was determined by flow cytometry. B, Percentage of CD4+IFN-γ+-, CD4+TNF-α+-, and CD4+IL-17+-producing T cells from mice following 6, 8, 10, 13, and 15 days immunization. Splenocytes were incubated with 10 µg/ml Ac1-9 for 4 h in the presence of Golgi Plug and cytokine production assessed by intracellular cytokine staining. Data in B show results from analyses of spleen cells pooled from two mice per time point and treatment. EAE scores of mice on day of sacrifice were 0, except for flank-immunized mice on day 10 (scores of 0 and 3), day 13 (scores of 0 and 1), and day 15 (scores of 0 and 2). Data are representative of at least two independent experiments. p < 0.05, p < 0.02, or p < 0.01 for significant differences determined by Mann-Whitney U test as indicated.
FIGURE 3
FIGURE 3
EAE incidence and severity, absolute numbers and functional responses of Ag-specific T cells in male and female mice following footpad/tailbase immunization. Male or female B10.PL or Vβ172.10 mice were immunized with 200 µg of Ac1-9 in the footpad and tailbase, and B10.PL mice (but not Vβ172.10 mice) were treated with pertussis toxin as described for Fig. 1. A, Disease score in male (M) and female (F) Vβ172.10 mice. Disease incidence was 5/5 mice for males, and 2/5 for females. p < 0.05 for significant differences determined by Mann-Whitney U test observed between male and female Vβ172.10 mice from days 12–35. B, Analyses of a number of Ag-specific tetramer-positive CD4+ T cells (CD4+tet+) using PE-labeled MBP1-9[4Y]:I-Au multimers/tetramers (5), FITC-labeled anti-Vα2, and PerCP-labeled anti-CD4 Abs following immunization of mice. Percentage of CD4+tet+ T cells (Vα2+ or Vα2, as indicated in respective quadrants) in spleens and in pooled inguinal and popliteal LNs in male and female mice on day 10 (B10.PL mice) and day 4 (Vβ172.10 mice). Data in B show results from analyses of spleens or pooled inguinal and popliteal LNs from three mice per group. C, Number of cells per mouse in spleens, LNs (inguinal and popliteal), and CNS following immunization. Data shown are derived from pooled organs from three mice per group. Mice were sacrificed on days 11 and 18 postim-munization for B10.PL mice and on days 4 and 11 postimmunization for Vβ172.10 mice. Analyses of the number of Ag-specific CD4+ T cells were conducted using PE-labeled MBP1-9[4Y]:I-Au tetramers (5, 9), FITC-labeled anti-Vα2, PerCP-labeled anti-CD4, and allophycocyanin-labeled anti-CD45 Abs. Histogram plots represent the number of CD4+tet+ T cells or macrophages, except for CNS in B10.PL mice where the number of total CD4+ T cells are shown due to the reduced number of CD4+tet+ T cells. *, p < 0.05 for significant differences determined by Mann-Whitneys U test between male and female mice. D, Proliferation and IFN-γ secretion of splenocytes isolated from male (M) and female (F) mice at day 11 postimmunization with Ac1-9 (MBP) following in vitro restimulation with Ac1-9. Cells were pooled from n = 2 to 3 mice per group. Error bars indicate SEs. Differences between male and female mice were not significant as determined by Kruskal-Wallis test. Data are representative of at least two independent experiments, with disease experiments (in A) being conducted three times.
FIGURE 4
FIGURE 4
Induction of myeloid cells following immunization of female B10.PL mice at different sites. A, Female mice were injected in the footpad with PBS or CFA and CD11b+Gr-1high and CD11b+Gr-1int myeloid cell populations analyzed by FSC/SSC and Gr1/CD11b parameters in pooled spleens 10 days later. Percentage of myeloid subsets in total splenocytes are shown. B, Gr-1high and Gr-1int myeloid cell populations in spleens were analyzed as in A for female mice immunized in footpad/tailbase or flanks (as described for Fig. 1) at different days postimmunization. C, Absolute numbers of Ag-specific tetramer-positive CD4+ T cells (CD4+tet+), CD11b+Gr-1high, and CD11b+Gr-1int myeloid cells for the same animals that were used to generate data shown in B. Data in A and C show results from analyses of organs pooled from two mice per group. For B and C, EAE scores of mice on day of sacrifice were 0, except for flank-immunized mice on day 10 (scores of 0 and 3), day 13 (scores of 0 and 1), and day 15 (scores of 0 and 2). p < 0.05 or p < 0.01 for significant differences determined by Mann-Whitney U test between flank- and footpad/tailbase-immunized mice. Data are representative of at least two independent experiments.
FIGURE 5
FIGURE 5
Impact of IFN-γ blockade and adjuvants on EAE induction in female mice. A, Female B10.PL mice (five mice per group) were immunized in the flanks (control group), or in the footpad/tailbase with 200 µg of Ac1-9 emulsified in CFA. Pertussis toxin was delivered i.p. on days 0 and 2 postimmunization (200 ng). Mice immunized in the footpad/tailbase were treated with 100 µg of anti-IFN-γ Ab, or isotype-matched control Ab, on days 6 and 9 postimmunization. Both flank-immunized mice and mice immunized in the footpad/tailbase followed by treatment with anti-IFN-γ Ab showed significantly higher EAE scores relative to footpad/tailbase-immunized mice treated with isotype control. p < 0.05 by Mann-Whitney U test for days 16–30. No significant difference between flank-immunized mice and footpad/tailbase-immunized mice treated with anti-IFN-γ Ab was found. B, Analysis of the number of CD11b+Gr1int cells in spleens of female B10.PL mice following treatment of mice as in A. Data shown represent mean cell number for individual spleens isolated from a total of n = 4–7 mice for each time point shown, and are compiled from three different experiments. Mice treated with anti-IFN-γ Ab show a significantly lower number of CD11b+Gr1int cells in spleens relative to isotype control treated mice on days 13 and 15. p = 0.012 by Mann-Whitney U test. C, Female B10.PL mice (five mice per group) were injected in the footpad with PBS, CFA, Pam3CSK4, or IFA, immediately following immunization with Ac1-9 in the flanks. Mice treated in the footpad with CFA showed lower clinical scores relative to animals treated with PBS or IFA. p < 0.05 for comparison with PBS for days 14–21 or p < 0.05 for comparison with IFA for days 17–19 determined by Mann-Whitney U test. No significant difference was found when CFA and Pam3CSK4 treatments were compared. Data shown are representative of at least two independent experiments.

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