ORLL-NIH001 inhibits expansion of Th17 cells in a dose dependent manner.

<p>(A, B) Naïve CD4⁺ and CD8⁺ T cells were labeled with CFSE, stimulated for 4 days with anti-CD3/anti-CD28 under Th17 polarization condition in medium containing vehicle alone or different doses of ORLL-NIH001 (A). Numbers in the quadrants indicate percent of proliferating or non-proliferating CD4⁺ or CD8+ T cells expressing IL-17. (B) Proliferation of cells in cultures without CFSE was quantified by the Thymidine incorporation assay. Data presented are representative of at least 3 independent experiments.</p

ORLL-NIH001 inhibited proteins that mediate lymphocyte trafficking into retina.

<p>(A, B) LN cells from mice with EAU were re-stimulated with IRBP in medium containing ORLL-NIH001 or commercially available STAT3 inhibitors. Expression or activation of integrins and chemokine receptors were analyzed by FACS. Plots were gated on CD4⁺ T cells and numbers in quadrants indicate percent of CD4⁺ T cells expressing CD11a, CD29, CD44, CD49d, CXCR3, CCR6, α4β7α4β1 or Ly6c. Data presented are representative of 3 independent experiments.</p

ORLL-NIH001 inhibited expansion of mouse uveitogenic Th17 cells.

<p>(A) Draining LN cells from vehicle-treated (control) or drug-treated mice (Protocol 1) were re-stimulated <i>in vitro</i> with IRBP for 3 days and effects of ORLL-NIH001 was assessed by Thymidine incorporation assay. Freshly isolated PBMC (B, C) was isolated from individual mice 4 days post-immunization. The levels of CD3⁺CD4⁺ T cells were quantified using FACS (B) and the number of cytokine-expressing T cells was assessed by intracellular cytokine assay (C). (D, E) Freshly isolated lymph node cells from vehicle or drug-treated mice were re-stimulated ex vivo with IRBP for 3 days and then analyzed by the intracellular cytokine assay. CFSE was added to some cultures (E). Plots were gated on CD3⁺ T cells and numbers in quadrants indicate percent of CD4⁺ T cells expressing IL-17 and/or IFN-γ. Data presented are representative of at least 3 independent experiments.</p

ORLL-NIH001 inhibited the expansion of human Th17 cells.

<p>(A) CD4⁺ T cells isolated from PBMC of healthy human subjects were labeled with CFSE and then stimulated with anti-CD3/CD28 for 4 days in medium containing vehicle alone or different doses of ORLL-NIH001. Numbers in the quadrants indicate percent of proliferating or non-proliferating T cells expressing IL-17 or IFN-γ. (B) Human PBMC were activated with anti-CD3/CD28 abs for 4 days, stimulated with IL-6 in serum-free medium containing vehicle or ORLL-NIH001. The cells were then analyzed for the expression of STAT3, pSTAT1 or β-actin by Western blotting. Activated human (C) or mouse (D–H) CD4 T cells were stimulated with IL-2 or IL-6 and expression of pSTAT1, pSTAT3 and pSTAT5 was detected by Western blotting (C, D, E) or flow cytometry (F, G, H). (E) Western blots (B, C, D) were analyzed using NIH Image Quant program and each band was normalized to corresponding β-actin band and expressed in arbitrary relative protein expression units. Results are representative of 3 independent experiments.</p

STAT3-deficient T cells could not traffic into the retina nor induce EAU.

<p>(A) WT mice or mice with targeted deletion of STAT3 in T cells (CD4-STAT3KO) were immunized with IRBP in CFA and their eyes were enucleated 21 days post-immunization and histological sections through the retina were stained with H&E. Black arrows indicate presence of inflammatory cells in the vitreous (V) and blue arrows depict pathologic foci characterized by the presence of retinal folds and hemorrhage. Red arrow, choroiditis; White arrow, granuloma; green arrow, retinal vasculitis; OpN, optic nerve; V, vitreous; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. (B) Freshly isolated cells from the draining lymph nodes or spleen of WT unimmunized WT control or EAU mice were analyzed by the intracellular cytokine assay. Plots were gated on CD4⁺ T cells and numbers in quadrants indicate percentage of CD4⁺ T cells expressing IL-17 and/or activated STAT3 (pSTAT3). (C) WT or CD4-STAT3KO mice were immunized with IRBP in CFA and T cells present in retina on day-21 post-immunization were detected and quantified by flow cytometry. Numbers in quadrants indicate percentage of CD3⁺ or CD4⁺ T cells or CD4⁺ T cells expressing IL-17 and/or IFN-γ. Data presented are representative of at least 3 independent experiments.</p

ORLL-NIH001 conferred protection from development of severe EAU.

<p>(A) Schematic depiction of immunization protocols used for EAU induction in B10.A mice. Mice treated under Protocol 1 received injections of ORLL-NIH001, starting 1 day before immunization (day −1) and on every other day thereafter until day-17 post-immunization. Protocol 2: mice began receiving the drug on day-5 post-immunization. Mouse eyes were harvested, fixed, embedded in paraffin and stained with H&E. (B, D) Histological analysis showing: inflammatory cells in the vitreous (black arrows), retinal folds (white arrows). OpN, optic nerve; V, vitreous; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. (C, E) EAU scores were calculated for vehicle-treated (control) or ORLL-NIH001-treated mice based on and histology. Similar results were obtained in mice treated under Protocol 1 (B, C) or Protocol 2 (D, E) and data presented are representative of at least 3 independent experiments.</p

IRF8-deficient T cells are more activated during ocular HSV-1 infection.

<p>(A) WT mice were challenged with HSV-1 by corneal scarification and we titrated the gB-tetramer to analyze and characterize the virus-specific CD8⁺ T cell responses in the spleen. (B-D) IRF8KO and WT mice were infected with HSV-1 by corneal scarification and cells isolated from the (B) spleen, or (C) PBMC (C, D) LN on day 8 p.i were analyzed by FACS using the gB-tetramer. The cells were gated on CD3/CD8 and numbers in quadrants indicate percentages of CD8⁺ T cells expressing CD44 and CD8⁺ gB-tetramer⁺ T cells expressing IFN-γ. (E) IFN-γ expression by CD8⁺ T cells was analyzed by real-time qPCR. Data represent at least 3 independent experiments.</p

IRF8 regulates the expansion of memory precursor effector cells (MPECs).

<p>(A) WT or STAT3KO mice were infected with HSV-1 and RNA from sorted CD8⁺ T cells on day 8 p.i were analyzed by qPCR for IRF8 expression. (B-D) WT or CD4-IRF8KO mice were infected with HSV-1 and CD8⁺ T cells were isolated from the spleen on day (B) 8 p.i, day (C) 34 or day 53 p.i and analyzed by FACS. (B) CD8⁺ T cells were first gated on gB-Tetramer+ population and then the percentages of MPEC (KLRG-1^loCD127^hi) and SLEC (KLRG^hiCD127^lo) in gB-Tetramer⁺ population were analyzed. (D) Flow plots represent the total gB-tetramer positive T cell population. The numbers in quadrants indicate percentages of CD8⁺ gB-tetramer-positive T cells expressing KLRG-1 and/or CD127. (E) Genomic DNA extracted from TG were analyzed by qPCR of the HSV-1 genes. (F) WT and CD4-IRF8KO mice were also infected with HSV-1 by the intraperitoneal route (i.p.) and CD8⁺ T cells were isolated from the spleens of infected mice on day 6 p.i, gated on CD8⁺gB-tetramer-positive and numbers in quadrants indicate percentages of T cells expressing KLRG-1 and/or CD127. Data represent at least 3 independent experiments.</p

Adoptive transfer of CD8⁺ deficient T cells reduced viral load and enhanced viral-induced inflammation in HSV-1-infected mice.

<p>(A) CD8⁺ T cells from WT or IRF8 KO mice were MACS sorted and 10 x 10⁶ cells were transferred into CD8αKO mice. After 1 day, mice were ocularly infected with 2 x 10⁵ pfu of HSV-1 via scarification. (B) On day 6 p.i., eye swabs were taken and infectious virus was assessed by plaque assay in duplicates (N = 20). Bars represent the mean pfu ± SEM of virus per cornea. Significance was determined by Mann-Whitney U test. (C-F) Mice were then sacrificed on day 8 p.i. (C) Corneas were harvested, digested with collagenase and inflammatory cells recruited to the cornea were assessed by flow cytometry. Bars represent the mean frequency ± SEM of Ly6C^IntGr-1^hi neutrophils from the CD11b⁺ gate. Flow plots represent the quantity of cells within the cornea. (D & E) Draining lymph nodes were harvested and activated CD8⁺ T cells (D) and virus-specific (gB-positive) CD8⁺ T cells (E) were analyzed. The cells were gated on CD8⁺ population and numbers in the quadrants of the FACS plot represent the frequency of CD44^hi cells while bars on the graphs represent the mean frequency ± SEM of CD44^hi or gB-tetramer specific CD8⁺ T cells. (F) DNA from the TG was harvested (day 8 p.i.) and qPCR was performed with HSV-1-specific primers. Bars in the graph represent the mean DNA level ± SEM of 5 mice.</p

IRF8-deficient T cells exhibit a pre-activated phenotype and more proliferative capacity in response to antigenic stimulation.

<p>(A) IRF8^fl/fl mice were cross-bred with CD4-Cre mice to generate mice with deletion of IRF8 in the CD4 and CD8 compartments (IRF8KO) and the IRF8^fl/fl/Cre alleles were identified by PCR analysis of mouse tail genomic DNA. Presence of the 314bp band indicates <i>Irf8</i>-floxed DNA, while the 214bp band is consistent with size of the endogenous <i>Irf8</i> gene sequence in the wild-type mouse genome. (B, C) Naïve CD8⁺ T cells isolated from the spleen were activated with anti-CD3/CD28 for 3 days and analyzed for IRF8 expression by (B) RT-PCR or (C) western blotting. (D) The TCR-activated WT or IRF8KO CD8⁺ T cells were also analyzed by the thymidine incorporation assay. (E) Naïve T cells isolated from spleen were analyzed by FACS. Numbers in quadrants indicate percentages of T cells expressing the cell surface markers, CD44 or CD62L. Data represent at least 3 independent experiments.</p