Production and characterization of anti-porcine CXCL10 monoclonal antibodies.

Research on C-X-C motif chemokine ligand 10 (CXCL10) has been widely reported for humans and select animal species, yet immune reagents are limited for pig chemokines. Our goal is to provide veterinary immunologists and the biomedical community with new commercial immune reagents and standardized assays. Recombinant porcine CXCL10 (rPoCXCL10) protein was produced by yeast expression and used to generate a panel of α CXCL10 monoclonal antibodies (mAbs). All mAbs were assessed for cross-inhibition and reactivity to orthologous yeast expressed CXCL10 proteins. Characterization of a panel of nine α PoCXCL10 mAbs identified six distinct antigenic determinants. A sensitive quantitative sandwich ELISA was developed with anti-PoCXCL10-1.6 and -1.9 mAb; reactivity was verified with both rPoCXCL10 and native PoCXCL10, detected in supernatants of peripheral blood mononuclear cells stimulated with rPoIFNγ or PMA/Ionomycin. Immunostaining of in vitro rPoIFNγ stimulated pig spleen and blood cells verified CXCL10 + cells as CD3-CD4-CD172+, with occasional CD3-CD4 + CD172 + subsets. Comparison studies determined that α PoCXCL10-1.4 mAb was the ideal mAb clone for intracellular staining, whereas with α PoCXCL10-1.1 and -1.2 mAbs were best for immunohistochemistry analyses. These techniques and tools will be useful for evaluating swine immune development, responses to infectious diseases and vaccines, as well as for improving utility of pigs as an important biomedical model.

Development and Characterization of New Monoclonal Antibodies Against Porcine Interleukin-17A and Interferon-Gamma.

Current research efforts require a broad range of immune reagents, but those available for pigs are limited. The goal of this study was to generate priority immune reagents for pigs and pipeline them for marketing. Our efforts were aimed at the expression of soluble swine cytokines and the production of panels of monoclonal antibodies (mAbs) to these proteins. Swine interleukin-17A (IL-17A) and Interferon-gamma (IFNγ) recombinant proteins were produced using yeast expression and used for monoclonal antibody (mAb) production resulting in panels of mAbs. We screened each mAb for cross-species reactivity with orthologs of IL-17A or IFNγ and checked each mAb for inhibition by other related mAbs, to assign mAb antigenic determinants. For porcine IL-17A, the characterization of a panel of 10 mAbs identified eight different antigenic determinants; interestingly, most of the mAbs cross-reacted with the dolphin recombinant ortholog. Likewise, the characterization of a panel of nine anti-PoIFNγ mAbs identified four different determinants; most of the mAbs cross-reacted with dolphin, bovine, and caprine recombinant orthologs. There was a unique reaction of one anti-PoIFNγ mAb that cross-reacted with the zebrafish recombinant ortholog. The αIL-17A mAbs were used to develop a quantitative sandwich ELISA detecting the yeast expressed protein as well as native IL-17A in stimulated peripheral blood mononuclear cell (PBMC) supernatants. Our analyses showed that phorbol myristate acetate/ionomycin stimulation of PBMC induced significant expression of IL-17A by CD3+ T cells as detected by several of our mAbs. These new mAbs expand opportunities for immunology research in swine.

Feeding lactobacilli impacts lupus progression in (NZBxNZW)F1 lupus-prone mice by enhancing immunoregulation.

Although the relationship between autoimmunity and microorganisms is complex, there is evidence that microorganisms can prevent the development of various autoimmune diseases. Lactobacilli are beneficial gut bacteria that play an important role in immune system development. The goals of this study were to assess the ability of three different strains of lactobacilli (L. casei B255, L. reuteri DSM 17509 and L. plantarum LP299v) to control lupus development/progression in (NZBxNZW)F1 (BWF1) lupus-prone mice before and after disease onset, and identify the mechanisms mediating protection. BWF1 mice fed with individual L. casei or L. reuteri before disease onset exhibited delayed lupus onset and increased survival, while feeding L. plantarum had little impact. In vitro treatment of BWF1 dendritic cells with individual lactobacilli strains upregulated IL-10 production to various extents, with L. casei being the most effective. The protection mediated by L. casei was associated with upregulation of B7-1 and B7-2 by antigen presenting cells, two costimulatory molecules important for regulatory T cell (Treg) induction. Moreover, feeding L. casei lead to increased percentages of CD4+Foxp3+ Tregs and IL10-producing T cells in the lymphoid organs of treated mice. More importantly, mice fed L. casei after disease onset remained stable for several months, i.e. exhibited delayed anti-nucleic acid production and kidney disease progression, and increased survival. Therefore, feeding lactobacilli appears to delay lupus progression possibly via mechanisms involving Treg induction and IL-10 production. Altogether, these data support the notion that ingestion of lactobacilli, with immunoregulatory properties, may be a viable strategy for controlling disease development and progression in patients with lupus, i.e. extending remission length and reducing flare frequency.

Combination Therapy Using IL-2/IL-2 Monoclonal Antibody Complexes, Rapamycin, and Islet Autoantigen Peptides Increases Regulatory T Cell Frequency and Protects against Spontaneous and Induced Type 1 Diabetes in Nonobese Diabetic Mice.

Regulatory T cells (Treg) play a crucial role in the maintenance of self-tolerance. In this study, we sought to expand Ag-specific Tregs in vivo and investigate whether the expanded Tregs can prevent or delay the development of type 1 diabetes (T1D) in the NOD mouse model. NOD mice were treated with a combination of IL-2/anti-IL-2 Ab complex, islet Ag peptide, and rapamycin. After the combined treatment, CD4(+)CD25(+)Foxp3(+) Tregs were significantly expanded in vivo, they expressed classical Treg markers, exerted enhanced suppressive functions in vitro, and protected against spontaneous development of T1D in NOD mice. Moreover, treated mice were almost completely protected from the adoptively transferred, aggressive form of T1D caused by in vitro-activated cytotoxic islet Ag-specific CD8 T cells. Protection from T1D was transferrable by Tregs and could be attributed to reduced islet infiltration of immune cells as well as the skewing of the immune response toward a Th2 cytokine profile. This new method of peripheral immune regulation could potentially contribute to development of novel immunotherapeutic strategies to prevent the development of T1D or to promote tolerance to islet transplants without using immunosuppressive drugs for long terms.

Evaluation of immunosuppressive function of regulatory T cells using a novel in vitro cytotoxicity assay.

Naturally occurring regulatory T cells (Tregs) play a pivotal role in the maintenance of self-tolerance due to their intrinsic immunosuppressive activity. Currently, a number of human clinical trials are being conducted to investigate the roles of Tregs in treating various immune-mediated disorders. Traditionally, the suppressive activity of Tregs is measured using either a thymidine incorporation assay, which is a radioactive assay; or CFSE based flow cytometry assay, which requires a relatively large number of cells. Consequently, there is an increasing need to develop novel alternative bioassays that can characterize various aspects of the immunosuppressive function of Tregs in vitro. In this study, using murine clonal CD8(+) T cells specific for an islet antigen as responder T cells, we first established a novel, sensitive and quantitative in vitro luminescence based cell viability assay to measure cytotoxicity. Then we used this assay to measure if Tregs could inhibit the cytotoxicity of CD8 effector T cells. This assay does not involve the use of radioisotopes and only needs relatively low number of Tregs. Since normally Tregs only constitute 5-10% of peripheral CD4(+) T cells, this advantage is noteworthy compared with other methods. With the assay we developed, we demonstrated that regulatory T cells (Tregs) could inhibit the antigen-specific killing of an adherent target cell monolayer by the CD8(+) cytotoxic T cells. We observed more inhibition when Tregs and CD8 killer T cells were incubated during the in vitro activation (stimulation) stage of the cytotoxic T lymphocytes (CTL) than when they were added later at the start of the effector phase. Interestingly, Tregs from B6 mice demonstrated higher suppression of CD8(+) T cell killing than Tregs from NOD mice. Moreover, IL-2/anti-IL-2 mAb complexes induced expansion of Tregs in vivo, as well as enhancing the Treg's suppressive activity per cell. Therefore, this novel non-radioactive, luminescence based cytotoxicity assay mediated by clonal islet antigen-specific CD8 T cells can be used to measure, characterize, and quantitate the immunosuppressive activity of natural Tregs, representing a useful approach to characterize the functions of Tregs in the setting of autoimmune diseases and to elucidate the mechanisms for Treg cell-mediated immunoregulation.

NOD dendritic cells stimulated with Lactobacilli preferentially produce IL-10 versus IL-12 and decrease diabetes incidence.

Dendritic cells (DCs) from NOD mice produced high levels of IL-12 that induce IFNγ-producing T cells involved in diabetes development. We propose to utilize the microorganism ability to induce tolerogenic DCs to abrogate the proinflammatory process and prevent diabetes development. NOD DCs were stimulated with Lactobacilli (nonpathogenic bacteria targeting TLR2) or lipoteichoic acid (LTA) from Staphylococcus aureus (TLR2 agonist). LTA-treated DCs produced much more IL-12 than IL-10 and accelerated diabetes development when transferred into NOD mice. In contrast, stimulation of NOD DCs with L. casei favored the production of IL-10 over IL-12, and their transfer decreased disease incidence which anti-IL-10R antibodies restored. These data indicated that L. casei can induce NOD DCs to develop a more tolerogenic phenotype via production of the anti-inflammatory cytokine, IL-10. Evaluation of the relative production of IL-10 and IL-12 by DCs may be a very useful means of identifying agents that have therapeutic potential.

Discrete TCR repertoires and CDR3 features distinguish effector and Foxp3+ regulatory T lymphocytes in myelin oligodendrocyte glycoprotein-induced experimental allergic encephalomyelitis.

Regulatory T lymphocytes (Tregs) expressing the Foxp3 transcription factor are critical modulators of autoimmunity. Foxp3(+) Tregs may develop in the thymus as a population distinct from conventional Foxp3(-) αß T cells (Tconvs). Alternatively, plasticity in Foxp3 expression may allow for the interconversion of mature Tregs and Tconvs. We examined >160,000 TCR sequences from Foxp3(+) or Foxp3(-) populations in the spleens or CNS of wild-type mice with experimental allergic encephalomyelitis to determine their relatedness and identify distinguishing TCR features. Our results indicate that the CNS-infiltrating Tregs and Tconvs arise predominantly from distinct sources. The repertoires of CNS Treg or Tconv TCRs showed limited overlap with heterologous populations in both the CNS and the spleen, indicating that they are largely unrelated. Indeed, Treg and Tconv TCRs in the CNS were significantly less related than those populations in the spleen. In contrast, CNS Treg and Tconv repertoires strongly intersected those of the homologous cell type in the spleen. High-frequency sequences more likely to be disease associated showed similar results, and some public TCRs demonstrated Treg- or Tconv-specific motifs. Different charge characteristics and amino acid use preferences were identified in the CDR3ß of Tregs and Tconvs infiltrating the CNS, further indicating that their repertoires are qualitatively distinct. Therefore, discrete populations of Tregs and Tconvs that do not substantially interconvert respond during experimental allergic encephalomyelitis. Differences in sequence and physical characteristics distinguish Treg and Tconv TCRs and imply dissimilar Ag recognition properties.

APC activation restores functional CD4(+)CD25(+) regulatory T cells in NOD mice that can prevent diabetes development.

BACKGROUND: Defects in APC and regulatory cells are associated with diabetes development in NOD mice. We have shown previously that NOD APC are not effective at stimulating CD4(+)CD25(+) regulatory cell function in vitro. We hypothesize that failure of NOD APC to properly activate CD4(+)CD25(+) regulatory cells in vivo could compromise their ability to control pathogenic cells, and activation of NOD APC could restore this defect, thereby preventing disease. METHODOLOGY/PRINCIPAL FINDINGS: To test these hypotheses, we used the well-documented ability of complete Freund's adjuvant (CFA), an APC activator, to prevent disease in NOD mice. Phenotype and function of CD4(+)CD25(+) regulatory cells from untreated and CFA-treated NOD mice were determined by FACS, and in vitro and in vivo assays. APC from these mice were also evaluated for their ability to activate regulatory cells in vitro. We have found that sick NOD CD4(+)CD25(+) cells expressed Foxp3 at the same percentages, but decreased levels per cell, compared to young NOD or non-NOD controls. Treatment with CFA increased Foxp3 expression in NOD cells, and also increased the percentages of CD4(+)CD25(+)Foxp3(+) cells infiltrating the pancreas compared to untreated NOD mice. Moreover, CD4(+)CD25(+) cells from pancreatic LN of CFA-treated, but not untreated, NOD mice transferred protection from diabetes. Finally, APC isolated from CFA-treated mice increased Foxp3 and granzyme B expression as well as regulatory function by NOD CD4(+)CD25(+) cells in vitro compared to APC from untreated NOD mice. CONCLUSIONS/SIGNIFICANCE: These data suggest that regulatory T cell function and ability to control pathogenic cells can be enhanced in NOD mice by activating NOD APC.

Deficiency in NOD antigen-presenting cell function may be responsible for suboptimal CD4+CD25+ T-cell-mediated regulation and type 1 diabetes development in NOD mice.

Various defects in antigen-presenting cells (APCs) and T-cells, including regulatory cells, have been associated with type 1 diabetes development in NOD mice. CD4(+)CD25(+) regulatory cells play a crucial role in controlling various autoimmune diseases, and a deficiency in their number or function could be involved in disease development. The current study shows that NOD mice had fewer CD4(+)CD25(+) regulatory cells, which expressed normal levels of glucocorticoid-induced tumor necrosis factor receptor and cytotoxic T-lymphocyte-associated antigen-4. We have also found that NOD CD4(+)CD25(+) cells regulate poorly in vitro after stimulation with anti-CD3 and NOD APCs in comparison with B6 CD4(+)CD25(+) cells stimulated with B6 APCs. Surprisingly, stimulation of NOD CD4(+)CD25(+) cells with B6 APCs restored regulation, whereas with the reciprocal combination, NOD APCs failed to activate B6 CD4(+)CD25(+) cells properly. Interestingly, APCs from disease-free (>30 weeks of age), but not diabetic, NOD mice were able to activate CD4(+)CD25(+) regulatory function in vitro and apparently in vivo because only spleens of disease-free NOD mice contained potent CD4(+)CD25(+) regulatory cells that prevented disease development when transferred into young NOD recipients. These data suggest that the failure of NOD APCs to activate CD4(+)CD25(+) regulatory cells may play an important role in controlling type 1 diabetes development in NOD mice.