Cell-cell interaction with APC, not IL-23, is required for naive CD4 cells to acquire pathogenicity during Th17 lineage commitment.

Subpopulations of pathogenic or nonpathogenic Th17 cells were reported to develop when presensitized CD4 cells were activated with their target Ag during polarization by either IL-23 or IL-6 and TGF-ß, respectively. In this study, we generated two Th17 subpopulations by using a system in which naive CD4 cells from TCR transgenic mice specific to hen egg lysozyme (HEL) are polarized with IL-6/TGF-ß and, concurrently, are activated either with HEL presented by APCs, or with anti-CD3/CD28 Abs. Only the former cells were pathogenic, inducing inflammation in eyes expressing HEL. Naive CD4 cells activated by the anti-CD3/CD28 Abs acquired pathogenicity, however, when cocultured with HEL/APC. Importantly, the naive CD4 cells did not acquire pathogenicity when cocultured with APCs stimulated with LPS or when separated from the HEL-presenting cells by a semipermeable membrane. Unlike with presensitized Th17, soluble IL-23 does not participate in pathogenicity acquisition by naive CD4 cells; no pathogenicity was induced by adding IL-23 to cultures activated with anti-CD3/CD28 Abs. Furthermore, Abs against IL-23 or IL-23R did not inhibit acquisition of pathogenicity in cultures of naive CD4 cells activated by HEL/APC. Our data thus show that, unlike presensitized CD4 cells, naive CD4 cells polarized toward Th17 phenotype acquire pathogenicity only by direct interaction with APCs presenting the Ag, with no apparent involvement of soluble IL-23. We suggest that the Th17 lymphocytes derived from naive CD4 cells participate in pathogenic and other immune processes, along with the IL-23-dependent Th17 cells.

The living eye "disarms" uncommitted autoreactive T cells by converting them to Foxp3(+) regulatory cells following local antigen recognition.

Immune privilege is used by the eye, brain, reproductive organs, and gut to preserve structural and functional integrity in the face of inflammation. The eye is arguably the most vulnerable and, therefore, also the most "privileged" of tissues; paradoxically, it remains subject to destructive autoimmunity. It has been proposed, although never proven in vivo, that the eye can induce T regulatory cells (Tregs) locally. Using Foxp3-GFP reporter mice expressing a retina-specific TCR, we now show that uncommitted T cells rapidly convert in the living eye to Foxp3(+) Tregs in a process involving retinal Ag recognition, de novo Foxp3 induction, and proliferation. This takes place within the ocular tissue and is supported by retinoic acid, which is normally present in the eye because of its function in the chemistry of vision. Nonconverted T cells showed evidence of priming but appeared restricted from expressing effector function in the eye. Pre-existing ocular inflammation impeded conversion of uncommitted T cells into Tregs. Importantly, retina-specific T cells primed in vivo before introduction into the eye were resistant to Treg conversion in the ocular environment and, instead, caused severe uveitis. Thus, uncommitted T cells can be disarmed, but immune privilege is unable to protect from uveitogenic T cells that have acquired effector function prior to entering the eye. These findings shed new light on the phenomenon of immune privilege and on its role, as well as its limitations, in actively controlling immune responses in the tissue.

Humoral and cellular immune responses in sheep immunized with a 22 kilodalton exported protein of Mycobacterium avium subspecies paratuberculosis.

An immunogenic 22 kilodalton exported Mycobacterium avium subspecies paratuberculosis (MAP) lipoprotein (P22) was previously identified, and found to belong to the LppX/LprAFG family of mycobacterial lipoproteins. N-terminal polyhistidine-tagged P22 was produced and purified from Escherichia coli. Antibody recognition of P22, and interferon-gamma (IFN-gamma) responses in vitro using blood from a sheep vaccinated with Neoparasec, confirmed its immunogenicity. To evaluate the immunogenicity of P22 in vivo, five sheep were immunized with a single dose containing 0.8 mg recombinant P22 protein in adjuvant. Blood was collected at 4, 13 and 29 weeks post-immunization (p.i.) and tested for anti-P22 antibodies and P22-specific IFN-gamma production. P22-specific antibodies were detected by Western blot analysis in all five Neoparasec-immunized sheep at the three time points. Three out of five P22-immunized sheep produced P22-specific antibodies for up to 13 weeks p.i., and two gave a response at 29 weeks p.i. Recombinant P22 was able to stimulate significant IFN-gamma production in blood of P22-immunized sheep at 13 and 29 weeks p.i. Recombinant P22 also elicited an IFN-gamma response in blood of sheep immunized with Neoparasec.

Macrophage phagocytosis of foot-and-mouth disease virus may create infectious carriers.

Macrophages play critical roles in innate defences against virus infections, particularly pertinent to the rapid immune response required following emergency vaccination against foot-and-mouth disease virus (FMDV). Consequently, macrophage-FMDV interaction was studied in vitro, in the absence of specific antibodies, to mimic the animal early postvaccination. A gradual loss of infectivity and viral antigen was observed over 48 hr, and no evidence of productive virus replication was found. From the pathological viewpoint, an important observation was that the majority of macrophages carried infectious virus for at least 10 hr. Pronase and mild acid treatments showed the virus to be primarily on the cell surface during the first 4 hr. Thereafter, it became internalized (pronase- and pH resistant), but remained infectious for 10-24 hr. The internalization process was dependent on microfilament activity, while the survival of infectious virus related to live virus-dependent inhibition of macrophage protein synthesis. Infectious centre assays demonstrated that this infectious virus - whether on the cell surface or internalized - was actually being released from the cells. This is interesting considering that FMDV is highly pH labile. Together, these characteristics suggest that the virus had been internalized by a process such as macropinocytosis, and fusion with endosomes was delayed or impaired. This mechanism whereby the virus could 'piggyback' on or in the macrophage, becoming internalized but not degraded for at least 10 hr, are important considerations in FMD pathogenesis. Such 'virus-transporting' macrophages would be in a position to carry infectious FMDV to different sites in the body, where it could be released to infect other cells for replication.