A Peptide from the Beta-strand Region of CD2 Protein that Inhibits Cell Adhesion and Suppresses Arthritis in a Mouse Model

This is the peer reviewed version of the following article: Satyanarayanajois, S. D., Büyüktimkin, B., Gokhale, A., Ronald, S., Siahaan, T. J. and Latendresse, J. R. (2010), A Peptide from the Beta-strand Region of CD2 Protein that Inhibits Cell Adhesion and Suppresses Arthritis in a Mouse Model. Chemical Biology & Drug Design, 76: 234–244. doi:10.1111/j.1747-0285.2010.01001.x, which has been published in final form at http://doi.org/10.1111/j.1747-0285.2010.01001.x. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.Cell adhesion molecules play a central role at every step of the immune response. The function of leukocytes can be regulated by modulating adhesion interactions between cell adhesion molecules to develop therapeutic agents against autoimmune diseases. Among the different cell adhesion molecules that participate in the immunological response, CD2 and its ligand CD58 (LFA-3) are two of the best-characterized adhesion molecules mediating the immune response. To modulate the cell adhesion interaction, peptides were designed from the discontinuous epitopes of the β-strand region of CD2 protein. The two strands were linked by a peptide bond. β-Strands in the peptides were nucleated by inserting a β-sheet-inducing Pro-Gly sequence with key amino acid sequences from CD2 protein that binds to CD58. Using a fluorescence assay, peptides that exhibited potential inhibitory activity in cell adhesion were evaluated for their ability to bind to CD58 protein. A model for peptide binding to CD58 protein was proposed based on docking studies. Administration of one of the peptides, P3 in collagen-induced arthritis (CIA) in the mouse model, indicated that peptide P3 was able to suppress rheumatoid arthritis in mice

Type I interferon signaling attenuates regulatory T cell function in viral infection and in the tumor microenvironment

<div><p>Regulatory T cells (Tregs) play a cardinal role in the immune system by suppressing detrimental autoimmune responses, but their role in acute, chronic infectious diseases and tumor microenvironment remains unclear. We recently demonstrated that IFN-α/β receptor (IFNAR) signaling promotes Treg function in autoimmunity. Here we dissected the functional role of IFNAR-signaling in Tregs using Treg-specific IFNAR deficient (IFNAR^fl/flxFoxp3^YFP-Cre) mice in acute LCMV Armstrong, chronic Clone-13 viral infection, and in tumor models. In both viral infection and tumor models, IFNAR^fl/flxFoxp3^YFP-Cre mice Tregs expressed enhanced Treg associated activation antigens. LCMV-specific CD8⁺ T cells and tumor infiltrating lymphocytes from IFNAR^fl/flxFoxp3^YFP-Cre mice produced less antiviral and antitumor IFN-γ and TNF-α. In chronic viral model, the numbers of antiviral effector and memory CD8⁺ T cells were decreased in IFNAR^fl/flxFoxp3^YFP-Cre mice and the effector CD4⁺ and CD8⁺ T cells exhibited a phenotype compatible with enhanced exhaustion. IFNAR^fl/flxFoxp3^YFP-Cre mice cleared Armstrong infection normally, but had higher viral titers in sera, kidneys and lungs during chronic infection, and higher tumor burden than the WT controls. The enhanced activated phenotype was evident through transcriptome analysis of IFNAR^fl/flxFoxp3^YFP-Cre mice Tregs during infection demonstrated differential expression of a unique gene signature characterized by elevated levels of genes involved in suppression and decreased levels of genes mediating apoptosis. Thus, IFN signaling in Tregs is beneficial to host resulting in a more effective antiviral response and augmented antitumor immunity.</p></div

Transcriptome analysis of Foxp3⁺ Tregs from LCMV infected Treg-specific IFNAR-deficient mice.

<p>(<b>A</b>) PCA was performed on day 5 LCMV Armstrong infected Foxp3^YFP-Cre and IFNAR^fl/fl x Foxp3^YFP-Cre mice sorted CD4⁺YFP⁺ Treg cells RNA-seq samples (4 samples in each group). (<b>B</b>) Scatter plot showing the comparison of global gene expression profiles of Tregs between Armstrong infected Foxp3^YFP-Cre and IFNAR^fl/fl x Foxp3^YFP-Cre mice. Total of 586 genes were significantly differentially expressed and colored, 249 genes (red) were down regulated, and 337 genes (blue) upregulated in IFNAR^fl/fl x Foxp3^YFP-Cre mice (fold change 1.5 and above, adjusted <i>P</i> < 0.05). (<b>C</b>) GSEA for non-IFN related genes, showing enrichment plot for natural Treg vs. T conv DN gene set (36 out 42 genes were enriched in core, Enrichment score: 0.566, <i>P</i> < 0.01, FDR: 0.0) with positive enrichment in IFNAR^fl/fl x Foxp3^YFP-Cre mice Tregs relative to Foxp3^YFP-Cre mice Tregs. (<b>D</b>) Heat map showing the significant differential expression of 32 Treg-signature genes (fold change 1.5 and above, adjusted <i>P</i> < 0.05), as previously reported [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006985#ppat.1006985.ref049" target="_blank">49</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006985#ppat.1006985.ref050" target="_blank">50</a>], differentially expressed genes were normalized by z-score. (<b>E</b>) PCA was performed on day 25 LCMV Cl-13 infected Foxp3^YFP-Cre and IFNAR^fl/fl x Foxp3^YFP-Cre mice sorted CD4⁺YFP⁺ Treg cells RNA-seq samples (5 samples in each group). (<b>F</b>) Scatter plot showing the comparison of global gene expression profiles of Tregs between Cl-13 infected Foxp3^YFP-Cre and IFNAR^fl/fl x Foxp3^YFP-Cre mice. Total of 36 genes were significantly differentially expressed and colored, 23 genes (red) were down regulated, and 13 genes (blue) upregulated in IFNAR^fl/fl x Foxp3^YFP-Cre mice (fold change 1.5 and above, adjusted <i>P</i> < 0.05). (<b>G</b>) Heat map showing the significant differential expression of 22 non-IFN related genes (fold change 1.5 and above, adjusted <i>P</i> < 0.05, normalized by z-score). (<b>H</b>) Heat map showing the significant differential expression of 14 genes from LCMV Armstrong infected Foxp3^YFP-Cre mice and IFNAR^fl/fl x Foxp3^YFP-Cre mice Tregs, which were in consistent with transcriptome from LCMV Cl-13 infected mice Tregs (fold change 1.5 and above, adjusted <i>P</i> < 0.05, normalized by z-score). Armstrong infection transcriptome data obtained from an experiment involving four mice per group (<b>A-D</b> and <b>H</b>), and transcriptome data from Cl-13 infection, involved an experiment with five mice per group (<b>E-G</b>).</p

IFNAR signaling in Tregs modulates the generation of antiviral effector T cells in acute LCMV infection.

<p>(<b>A</b>) LCMV titers were determined from Armstrong virus infected IFNAR^fl/fl, IFNAR^fl/fl x Foxp3^YFP-Cre and IFNAR^-/- mice sera on indicated days. (<b>B-D</b>) Splenocytes harvested from day 14 virus infected IFNAR^fl/fl and IFNAR^fl/fl x Foxp3^YFP-Cre mice were stained for GP33, NP396 and GP276 tetramers and analyzed for Tet⁺ T cells within CD8⁺CD44⁺ cells. (<b>E-G</b>) Spleen cells stimulated with GP33, NP396, GP276 and Golgi Stop for 5 hours at 37 ^oC. Frequencies and absolute numbers of IFN-γ⁺ and TNF-α⁺ cytokine producing cells within CD8⁺CD44⁺GP33 Tet⁺, CD8⁺CD44⁺NP396 Tet⁺ and CD8⁺CD44⁺GP276 Tet⁺ T cells from day 14 acute LCMV infected mice are shown. (<b>H</b> and <b>I</b>) Spleen cells were analyzed (as in <b>B</b>-<b>D</b>) for GP66 Tet⁺ T cells, and IFN-γ⁺ and TNF-α⁺ producing cells were assessed among gated CD4⁺Foxp3^-CD44⁺GP66 Tet⁺ T cells from day 14 acute LCMV infected mice. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001, and **** <i>P</i> < 0.0001 (unpaired two-tailed Student’s <i>t</i>-test). Data are shown from two to three experiments involving three to nine mice per group (<b>A</b>), and representative of two independent experiments (<b>B-I)</b> with four to five mice per group (Mean±SEM).</p

Absence of IFNAR signaling in Tregs results in enhanced virus-specific T cell exhaustion.

<p>(<b>A</b> and <b>B</b>) Kinetics of PD1 expression was shown on gated CD8⁺ and CD4⁺Foxp3^- T cells during chronic LCMV infection. (<b>C</b> and <b>D</b>) Percentage and total numbers of PD1⁺ EOMES⁺ cells among gated CD8⁺ and CD4⁺Foxp3^- T cells were plotted from days 25, and 46 Cl-13 infected mice. (<b>E</b> and <b>F</b>) PD1⁺CD39⁺ cells frequencies and numbers were estimated within CD8⁺ T and CD4⁺Foxp3^- T cells from day 46 Cl-13 infected mice. (<b>G</b> and <b>H</b>) PD1 expression was evaluated within CD8⁺CD44⁺GP33 and CD8⁺CD44⁺GP276 Tet⁺ T cells from Cl-13 infected mice on indicated days. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, and *** <i>P</i> < 0.001 (unpaired two-tailed Student’s <i>t</i>-test). Data represent from two to five experiments (<b>A</b> and <b>B</b>), representative of two experiments (<b>C-H</b>), on indicated days with three to four mice per group in each experiment (Mean±SEM).</p

Absence of IFNAR signaling in Tregs results in decreased virus-specific CD8⁺ T cells and cytokine production.

<p>(<b>A</b> and <b>B</b>) Spleen cells of chronic LCMV infected (days 25 and 46) IFNAR^fl/fl and IFNAR^fl/fl x Foxp3^YFP-Cre mice were analyzed for GP33 Tet⁺ and NP396 Tet⁺ T cells within CD8⁺CD44⁺ T cells. (<b>C</b> and <b>D</b>) Spleen cells from (days 25 and 46) Cl-13 infected mice were stimulated with GP33, NP396, and Golgi stop for 5 hours at 37 ^oC. Frequencies and absolute numbers of IFN-γ⁺ and TNF-α⁺ cytokine producing cells within CD8⁺CD44⁺GP33 Tet⁺ and CD8⁺CD44⁺NP396 Tet⁺ T cells are shown. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, and *** <i>P</i> < 0.001 (unpaired two-tailed Student’s <i>t</i>-test). Data shown (<b>A-D</b>) from a representative and two experiments involving three to eight mice per group (Mean±SEM).</p

Absence of IFNAR signaling potentiates viral persistence in chronic LCMV infection.

<p>(<b>A</b>) LCMV titers were determined from Cl-13 infected IFNAR^fl/fl, IFNAR^fl/fl x Foxp3^YFP-Cre, and IFNAR^-/- mice sera on indicated days. (<b>B</b>) Lung and kidney tissues from day 46 Cl-13 infected IFNAR^fl/fl and IFNAR^fl/fl x Foxp3^YFP-Cre mice were assessed for detection of LCMV. (<b>C</b>) IFNAR^fl/fl and IFNAR^fl/fl x Foxp3^YFP-Cre mice spleen cells on day 25 and day 46 Cl-13 infection were analyzed for Foxp3⁺ Treg among CD4⁺ T cells. (<b>D</b>) CD44⁺ and CD62L⁺ cells were determined within gated Foxp3⁺ T cells on indicated days during chronic LCMV infection. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001, and **** <i>P</i> < 0.0001 (unpaired two-tailed Student’s <i>t</i>-test). Data are shown from three to four experiments (<b>A</b>) involving three to fourteen mice, representative of two independent experiments (<b>B</b>) from four mice per group, and two to three experiments (<b>C</b> and <b>D</b>) on indicated days involving six to thirteen mice per group (Mean±SEM).</p