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

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

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

Blockade of TGF-β via (A) neutralizing antibody, (B) TGF-β soluble receptor, C) TGF-βreceptor kinase at the time of the intracameral injection of OVA on the development of DTH suppression.

<p>Intracameral injection of OVA included either vehicle only or isotype antibody control (AC positive control) or various TGF-β blocking treatments. The DTH measurements were performed 7 days after these groups were immunized with OVA. Each experiment, 5 mice/group, was conducted 3X.</p

Intracameral (AC) injection results in infiltration of, F4/80⁺, CD11b^hi Gr1^hi monocytes and this process is greatly reduced in CCR2 or CCL2 null animals.

<p>Sixteen hours post intracameral injection of OVA irides were recovered from 10 eyes. A single cell suspension of the pooled irides was prepared and the cells were stained for specific monocyte markers as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043182#s2" target="_blank">material and methods</a> and compared the same with naive group or with cells from irides of either CCR2 null or CCL2 null animals treated identically. (A) Initial gating on the scatter plot is done as shown. A comparison between naïve iris monocytes and iris- gated monocytes recovered from mice receiving an intracameral injection of antigen (AC) stained with anti-CD11b, F4/80 and anti- Gr1. Only the group receiving an intracameral injection contains cells that are CD11b^hi, F480⁺and Gr1^hi. CD11b^hi but F4/80⁻ cells are also observed in mice receiving an intracameral injection of antigen. The figure is representative of 8 experiments (P<0.001). (B) The composite histogram shows that CD11b^hi population in AC groups but not in naïve group. The CD11b^hi F4/80⁺ Gr1^hi cells are increased in AC wild type group but only marginally increased in CCL2^−/− group after AC injection. (C) The bar diagrams shows the F/480⁺ cell population present in the wild type and CCL−/− mice receiving an intracameral injection as cells/10⁶ acquired. The figures are representative of 2 experiments. (D) AC injection results in neutrophil infiltration. Cells are gated on a population of higher SSC and intermediate FSC which in AC injection group show a Ly6G^hi peak. This population is absent in naive iris. Ly6G cells are F4/80 negative and Ly6C intermediate. (E) Comparison of 2 populations of cells in the AC injected iris group, based on their scatter properties. Ly6G hi cells are abundant in the R1 region but also are present in R2 region. These cells are also CD11b^hi.</p

AC- PBMCs from Anti-TGF-β-treated animals do not transfer DTH suppression to OVA however intravenous injection of AC- PBMCs to those animals receiving anti-TGF-β+ OVA intracameral injections rescues the suppression of DTH.

<p>(A) Anti-TGF-β treatment at the time of intracameral injection of antigen blocks the ability of AC-PBMCs to induce the suppression of DTH. Twenty-four hr after intracameral injection of OVA and anti-TGF-β, PBMCs were recovered and 1×10⁶ recovered cells were injected IV into the naïve mice. These recipient mice were subsequently immunized with OVA/CFA. Seven days after immunizing, footpads of the mice were challenged subcutaneously and the relative increase in footpad swelling was compared between the immunized control mice and naïve mice. Each group in the final DTH measurement had 5 mice. The experiment was repeated 3X, (B) Animals receiving intracameral injections of anti-TGF-β +OVA were injected IV at 24 hours with AC-PBMCs recovered from animals receiving intracameral injections of OVA only. The recipient mice sere then immunized with OVA/CFA. Seven days after immunizing DTH measurements were done as described above. The experiment was repeated twice.</p

F4/80 and CD11b⁺ cells infiltrating the iris after the intracameral injection of antigen also express Ly6C, 7/4 (Ly 6B) and CD45.

<p>Preparations of naïve iris cells and cells recovered after an intracameral injection of antigen were stained with anti- F4/80 and (A) Ly6C and 7/4 (B) show a characteristic CD45 peak C) Ly6G<b>^lo</b> or negative, CD115, CD49b⁺, CD62L<b>^lo</b>. The figures are representative of 2 experiments. All the isotype controls in the histograms are shown as shaded.</p

Regulation of the increase in TNF-α and CCL2 in the anterior chamber after an intracameral injection.

<p>Aqueous humor was collected at at 3 and 6 hour intervals following the intracameral injection of OVA. CCL2 and TNF-α levels in the aqueous humor were measured by ELISA. (A) Neutralization of TNF-α prevents the early rise of CCL2. Mice received an intracameral injection of OVA +/– anti-TNF-α antibody or isotype control. Neutralization of TNF-α via anti-TNF-α antibody at the time of AC injection significantly (P<0.05) reduced the early (3 hr) rise of (CCL2) MCP-1 but not at 6 hours. The data were pooled from 2 independent experiments and a minimum of 6 replicates were used in each group for each experiment. (B) Neutralization of TGF-β via a neutralizing antibody or TGF-β receptor kinase inhibitor (SB-431542) results in a significant reduction(P<0.05) in the rise of TNF-α induced by an intracameral injection of OVA. The data were pooled from 2 independent experiments. A minimum of 6 replicates were used for each experiment. (C) Neutralization of TGF-β via anti-TGFβ neutralizing antibody, Soluble TGF-β receptor or by TGF-β receptor kinase inhibitor (SB-431542) does not influence the CCL2 levels in the aqueous humor.(D) Neutralization of TGF-β by intracameral injection of anti-TGF-β with OVA does not block the infiltration of monocytes into the anterior chamber. Irides were removed 16 hr after intracameral injection and cell suspensions stained for Gr1 and CÎ11b. Experiments were repeated 3X.</p

CCL2, CCL7 and TNF-α levels in the aqueous humor after intracameral injection.

<p>Aqueous humor was collected at different time intervals following the intracameral injection of OVA. CCL2, CCL7 and TNF-α levels in aqueous humor were detected by ELISA and are expressed as pg/ml. The data were pooled from 4 independent experiments for CCL2, 2 independent experiments for CCL7 and TNF-α and 2 independent experiments for MCP-3. A minimum of 6 replicates were used for each experiment.</p