Pertussis toxin reduces the cellular infiltrate in the DTH response to IRBP peptide_161–180.

<p>C57BL10.RIII mice were immunized with IRBP peptide _161–180, CFA +/− PTX. Seven days P.I. a footpad was challenged with intradermal IRBP peptide_161–180. Twenty four hr after challenge footpads were harvested, sectioned and stained with H&E. Section is 10X and represents footpad of one of three mice.</p

EAE is reduced by a challenge to the footpad with MOG_35–55 peptide.

<p>EAE was assessed fourteen days after C57BL/6 mice were immunized with MOG peptide_{35<b>–</b>55}, CFA and PTX or immunized and a footpad challenged with intradermal MOG peptide_{35<b>–</b>55} or IRBP peptide_{161<b>–</b>180} 7 days P.I. Following immunization, animals were kept under observation to score the disease. The study was done in a blinded fashion. The EAE scale was as follows: 0  =  normal, 1  =  limp tail, 2  =  paraparesis with a clumsy gait, 3  =  hind limb paralysis, 4  =  quadriplegia, 5  =  death. Data represents the mean score +/− S.E.M. of five mice/point. The experiment was done twice. DTH: delayed type hypersensitivity, chl: challenge</p

PTX potentiates the production of Th1 cytokines by splenocytes.

<p>C57BL10.RIII mice were immunized with IRBP peptide_161–180, CFA +/− PTX. Splenocytes were obtained from immunized mice on day 7 or 14 days P.I. Splenocytes were cultured at 37°C. in flat bottom 12 well plates at a concentration of 1×10⁷ cells/well and stimulated with 100 µg IRBP peptide or medium only in a volume of 0.5 ml. Supernatants from 3 cultures were harvested 24 h post in-vitro culture and stored at −20°C. Supernatants were assayed by ELISA for TNF-α or IFN-γ ˜.</p

The expansion of AC-SPL suppressor cells that suppress the expression of DTH is strain and antigen dose -dependent.

<p>Seven days after mice were immunized with TNP-BSA, a footpad was challenged with epicutaneous PCl. Footpad thickness was measured before and 24 hr after challenge. Five thousand AC-SPL cells were injected into a footpad of mice immunized with TNP-BSA immediately after the footpad was challenged with epicutaneous PCl. Footpad thickness was measured before and 24 hr after challenge and swelling computed. (A): Effect of immunizing dose of antigen on DTH-induced swelling in BALB/c (A) C57BL/6 mice (C), Generation of suppressive AC-SPL cells is antigen dose dependent (B): BALB/c, D: C57BL/6. Data represents the mean swelling +/− S.E.M. of 12 mice/group , 3 experiments. * p<0.05,NS: not significant.</p

The RsW50 of regulatory spleen cells is decreased by enriching for CD8⁺ regulatory spleen cells.

<p>The footpads of mice immunized with TNP-BSA and CFA 9 days previously received id CD8⁺ cells or unseparated AC-SPL cells prepared as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022496#pone-0022496-g001" target="_blank">Fig. 1</a> immediately after the footpad was challenged with epicutaneous PCl. Swelling was measured 24 hr later. The data is pooled from two separate experiments with six mice /group. NAÏVE: non-immunized mice, IMM: immunized mice that did not receive regulatory spleen cells, CD8⁺: immunized mice that received CD8⁺ regulatory spleen cells. p<0.01. Straight lines were generated by curve fit software.</p

Immunization reduces the RSw50 of AC-SPL cells.

<p>(A) BALB/c mice received an injection of TNP-BSA into the anterior chamber. Seven days after receiving an intracameral injection of TNP-BSA, some mice were immunized with TNP-BSA/CFA. Spleen cells were recovered from the immunized, AC-injected mice (AC-IMM) and mice that received an injection of antigen into the AC only (AC-ONLY) one week after the immunization of AC-injected mice or one week after intracameral injection only. Twenty-five thousand AC-SPL cells were injected into the footpads of TNP-BSA-immunized mice immediately after the footpads were challenged with epicutaneous PCl. (B) Immunization-induced increase in suppression is antigen-specific. Seven days after mice received an intracameral injection of TNP-BSA, the mice were immunized with TNP (AC-TNP), TNP-IMM or OVA (AC-TNP-OVA-IMM). Seven days after immunization, spleens were recovered from the mice and recovered spleen cells injected into the footpad of TNP-BSA-immunized mice immediately after the footpad was challenge with epicutaneous PCl. Swelling was measured 24 hr later. The data represents the mean RsW50 +/− the standard error of the mean for three experiments, 9 mice/group. *p<0.02.</p

PTX reduces DTH to MOG_{35–<b>55</b>}<b> peptide but not to OVA.</b>

<p>(A) C57BL/6 mice were immunized with MOG _35–55 peptide or OVA, CFA +/− PTX. Seven days P.I. footpads of the mice were challenged with intradermal MOG peptide_35–55 or OVA respectively. Swelling was measured 24 and 48 hr later. Data represents the mean swelling (µm) +/−S.E.M. of five mice/group. The experiment was done twice. (B) C57BL10.RIII mice were immunized with OVA_265–280 peptide, CFA +/− pertussis toxin. Seven days P.I, footpads were challenged with 50 µg OVA_35–55 peptide and swelling measured 24 hr post-challenge.Data represents the mean +/−S.E.M. of 4 mice. NS: not significant.</p