Modulating the T Lymphocyte Immune Response via Secretome Produced miRNA: From Tolerance Induction to the Enhancement of the Anticancer Response

T cells are key mediators of graft tolerance/rejection, development of autoimmunity, and the anticancer response. Consequently, differentially modifying the T cell response is a major therapeutic target. Most immunomodulatory approaches have focused on cytotoxic agents, cytokine modulation, monoclonal antibodies, mitogen activation, adoptive cell therapies (including CAR-T cells). However, these approaches do not persistently reorient the systemic immune response thus necessitating continual therapy. Previous murine studies from our laboratory demonstrated that the adoptive transfer of polymer-grafted (PEGylated) allogeneic leukocytes resulted in the induction of a persistent and systemic tolerogenic state. Further analyses demonstrated that miRNA isolated from the secretome of polymer-modified or control allogeneic responses effectively induced either a tolerogenic (TA1 miRNA) or proinflammatory (IA1 miRNA) response both in vitro and in vivo that was both systemic and persistent. In a murine Type 1 diabetes autoimmune model, the tolerogenic TA1 therapeutic effectively attenuated the disease process via the systemic upregulation of regulatory T cells while simultaneously downregulating T effector cells. In contrast, the proinflammatory IA1 therapeutic enhanced the anticancer efficacy of naïve PBMC by increasing inflammatory T cells and decreasing regulatory T cells. The successful development of this secretome miRNA approach may prove useful treating both autoimmune diseases and cancer

Inhibition of Autoimmune Diabetes in NOD Mice by miRNA Therapy.

Autoimmune destruction of the pancreatic islets in Type 1 diabetes is mediated by both increased proinflammatory (Teff) and decreased regulatory (Treg) T lymphocytes resulting in a significant decrease in the Treg:Teff ratio. The non-obese diabetic (NOD) mouse is an excellent in vivo model for testing potential therapeutics for attenuating the decrease in the Treg:Teff ratio and inhibiting disease pathogenesis. Here we show for the first time that a bioreactor manufactured therapeutic consisting of a complex of miRNA species (denoted as TA1) can effectively reset the NOD immune system from a proinflammatory to a tolerogenic state thus preventing or delaying autoimmune diabetes. Treatment of NOD mice with TA1 resulted in a systemic broad-spectrum upregulation of tolerogenic T cell subsets with a parallel downregulation of Teff subsets yielding a dramatic increase in the Treg:Teff ratio. Moreover, the murine-derived TA1 was highly effective in the inhibition of allorecognition of HLA-disparate human PBMC. TA1 demonstrated dose-responsiveness and exhibited equivalent or better inhibition of allorecognition driven proliferation than etanercept (a soluble TNF receptor). These findings demonstrate that miRNA-based therapeutics can effectively attenuate or arrest autoimmune disease processes and may be of significant utility in a broad range of autoimmune diseases including Type 1 diabetes

IFN-β Facilitates Neuroantigen-Dependent Induction of CD25+ FOXP3+ Regulatory T Cells That Suppress Experimental Autoimmune Encephalomyelitis

This study introduces a flexible format for tolerogenic vaccination that incorporates IFN-β and neuroantigen (NAg) in the Alum adjuvant. Tolerogenic vaccination required all three components, IFN-β, NAg, and Alum, for inhibition of experimental autoimmune encephalomyelitis (EAE) and induction of tolerance. Vaccination with IFN-β + NAg in Alum ameliorated NAg-specific sensitization and inhibited EAE in C57BL/6 mice in pretreatment and therapeutic regimens. Tolerance induction was specific for the tolerogenic vaccine Ag PLP178-191 or myelin oligodendrocyte glycoprotein (MOG)35--55 in proteolipid protein-- and MOG-induced models of EAE, respectively, and was abrogated by pretreatment with a depleting anti-CD25 mAb. IFN-β/Alum--based vaccination exhibited hallmarks of infectious tolerance, because IFN-β + OVA in Alum--specific vaccination inhibited EAE elicited by OVA + MOG in CFA but not EAE elicited by MOG in CFA. IFN-β + NAg in Alum vaccination elicited elevated numbers and percentages of FOXP3+ T cells in blood and secondary lymphoid organs in 2D2 MOG-specific transgenic mice, and repeated boosters facilitated generation of activated CD44high CD25+ regulatory T cell (Treg) populations. IFN-β and MOG35--55 elicited suppressive FOXP3+ Tregs in vitro in the absence of Alum via a mechanism that was neutralized by anti--TGF-β and that resulted in the induction of an effector CD69+ CTLA-4+ IFNAR+ FOXP3+ Treg subset. In vitro IFN-β + MOG--induced Tregs inhibited EAE when transferred into actively challenged recipients. Unlike IFN-β + NAg in Alum vaccines, vaccination with TGF-β + MOG35-55 in Alum did not increase Treg percentages in vivo. Overall, this study indicates that IFN-β + NAg in Alum vaccination elicits NAg-specific, suppressive CD25+ Tregs that inhibit CNS autoimmune disease. Thus, IFN-β has the activity spectrum that drives selective responses of suppressive FOXP3+ Tregs

TA1 results in a decreased <i>in vivo</i> inflammatory response upon allogeneic cells challenge.

<p>In immunologically competent mice (Balb/c), TA1 completely abrogates the inflammatory response to allogeneic (C57Bl/6) splenocytes. Shown are the levels of Treg and Teff (Th17) cell levels in the spleen of naïve (N), allogeneic challenged (C) and TA1 treated (± RNase A treatment) mice 5 days post treatment. TA1 was administered 24 hours prior to administration of the allogeneic splenocytes. As noted, the ‘active’ agent of the TA1 preparation on Treg and Teff levels was fully degraded by RNase treatment (<i>Δc/d</i>, respectively). Dashed lines represents naïve resting levels of Treg or Th17 cells while the solid lines denote the mean Treg and Th17 cells of control mice 5 days post transfusion of unmodified, viable, allogeneic splenocytes. Data shown is the mean ± SD of a minimum of 8 mice per group. <b>*</b> Denotes significantly different (p<0.001) from naïve mice. <b>#</b> Denotes significantly different (p<0.001) from TA1 treated mice. <b>Panel C:</b><i>In vivo</i> effects of saline, naive miRNA or TA1-miRNA on Foxp3⁺, CD25⁺ and CD69⁺ T cells in the spleen and brachial lymph node. For ease of comparison, the cross-hatched regions of the TA1 group represent the mean saline values. Cell populations were determined 5 days post treatment with a minimum of 5 animals per treatment group. (*) Indicates significantly (p<0.001) different from saline group in Panels A-B. In Panel C (#) denotes significantly reduced from sample 1 but still significantly (p<0.01) elevated relative to saline group.</p

Inhibition of Autoimmune Diabetes in NOD Mice by miRNA Therapy.

Autoimmune destruction of the pancreatic islets in Type 1 diabetes is mediated by both increased proinflammatory (Teff) and decreased regulatory (Treg) T lymphocytes resulting in a significant decrease in the Treg:Teff ratio. The non-obese diabetic (NOD) mouse is an excellent in vivo model for testing potential therapeutics for attenuating the decrease in the Treg:Teff ratio and inhibiting disease pathogenesis. Here we show for the first time that a bioreactor manufactured therapeutic consisting of a complex of miRNA species (denoted as TA1) can effectively reset the NOD immune system from a proinflammatory to a tolerogenic state thus preventing or delaying autoimmune diabetes. Treatment of NOD mice with TA1 resulted in a systemic broad-spectrum upregulation of tolerogenic T cell subsets with a parallel downregulation of Teff subsets yielding a dramatic increase in the Treg:Teff ratio. Moreover, the murine-derived TA1 was highly effective in the inhibition of allorecognition of HLA-disparate human PBMC. TA1 demonstrated dose-responsiveness and exhibited equivalent or better inhibition of allorecognition driven proliferation than etanercept (a soluble TNF receptor). These findings demonstrate that miRNA-based therapeutics can effectively attenuate or arrest autoimmune disease processes and may be of significant utility in a broad range of autoimmune diseases including Type 1 diabetes

IFN-ß Facilitates Neuroantigen-Dependent Induction of CD25+ FOXP3+ Regulatory T Cells That Suppress Experimental Autoimmune Encephalomyelitis

This study introduces a flexible format for tolerogenic vaccination that incorporates IFN-ß and neuroantigen (NAg) in the Alum adjuvant. Tolerogenic vaccination required all three components, IFN-ß, NAg, and Alum, for inhibition of experimental autoimmune encephalomyelitis (EAE) and induction of tolerance. Vaccination with IFN-ß + NAg in Alum ameliorated NAg-specific sensitization and inhibited EAE in C57BL/6 mice in pretreatment and therapeutic regimens. Tolerance induction was specific for the tolerogenic vaccine Ag PLP178-191 or myelin oligodendrocyte glycoprotein (MOG)35–55 in proteolipid protein– and MOG-induced models of EAE, respectively, and was abrogated by pretreatment with a depleting anti-CD25 mAb. IFN-ß/Alum–based vaccination exhibited hallmarks of infectious tolerance, because IFN-ß + OVA in Alum–specific vaccination inhibited EAE elicited by OVA + MOG in CFA but not EAE elicited by MOG in CFA. IFN-ß + NAg in Alum vaccination elicited elevated numbers and percentages of FOXP3+ T cells in blood and secondary lymphoid organs in 2D2 MOG-specific transgenic mice, and repeated boosters facilitated generation of activated CD44high CD25+ regulatory T cell (Treg) populations. IFN-ß and MOG35–55 elicited suppressive FOXP3+ Tregs in vitro in the absence of Alum via a mechanism that was neutralized by anti–TGF-ß and that resulted in the induction of an effector CD69+ CTLA-4+ IFNAR+ FOXP3+ Treg subset. In vitro IFN-ß + MOG–induced Tregs inhibited EAE when transferred into actively challenged recipients. Unlike IFN-ß + NAg in Alum vaccines, vaccination with TGF-ß + MOG35-55 in Alum did not increase Treg percentages in vivo. Overall, this study indicates that IFN-ß + NAg in Alum vaccination elicits NAg-specific, suppressive CD25+ Tregs that inhibit CNS autoimmune disease. Thus, IFN-ß has the activity spectrum that drives selective responses of suppressive FOXP3+ Tregs

TA1 administration prevents (p≤0.01) or delays T1D (p≤0.01) progression in NOD mice, alters the ratio of Treg:Teff cells and improves islet histology.

<p><b>Panel A:</b> Mice were treated (<i>i</i>.<i>v</i>.) with either saline or TA1 at 7 weeks of age and blood glucose was measured to a maximum of 30 weeks. Shown are the percentage of animals remaining normoglycemic (<i>left axis</i>). As shown, only 25% of control mice were normoglycemic at week 19 (line <i>a</i>) while 87% of TA1 remained normal. At 30 weeks, 25% of untreated mice and 60% of TA1-treated mice remained normoglycemic. Conversion to T1D correlated in both groups with the log Treg:Teff ratio (<i>left axis</i>). TA1 significantly increased the Treg:Teff ratio in all animals (Diabetic and non-diabetic). The Treg:Teff ratio of normoglycemic saline (● mean 286; range 170–680) and TA1 (○ mean 255; range 140–1040) treated mice (shade box; right) were similar but significantly (p<0.001) higher than diabetic mice. Analysis of the Treg:Teff ratio of diabetic and non-diabetic saline and TA1 mice suggests that an <i>inexact</i> threshold level (box <i>b</i>) in the Treg:Teff ratio may exist for protection against progression to T1D. Also shown are the Treg:Teff ratios for immunocompetent C57Bl/6 and Balb/c mice (Δ, 91.5; ■, 198; respectively) and the asymptomatic 7 week old NOD mice (▲;103). Diabetic tissues were harvested at time of conversion, non-diabetic tissues were harvested at week 30. Diabetic values are the mean ± SD of 12 saline and 6 TA1 treated NOD mice. For diabetic animals (saline and TA1 treated) the mean age of onset ± SD is shown via both text and horizontal bar. Non-diabetic results are the mean ± SD of 4 saline and 9 TA1 treated NOD mice. <b>Panel B:</b> TA1 inhibits pancreatic islet insulitis as demonstrated by the increased number of normal (<i>b3</i>) and peri-insulitis (<i>b2</i>) islets relative to control NOD mice. Untreated NOD mice exhibited virtually no normal islets (either in diabetic or non-diabetic mice) with a heavy preponderance of overt insulitis (<i>b1</i>). The numbers shown at the top of each column represent the number of individual islets graded per condition from a minimum of 5 animals per group.</p

TA1 demonstrates 'drug-like' dosing, efficacy and, consequent to evolutionary conservation, is biologically functional across species.

<p><b>Panels A-B:</b> Treatment of human PBMC with murine-derived TA1 inhibits allorecognition in a human PBMC mixed lymphocyte reaction. The horizontal dashed line represents the mean value for resting cells while the horizontal grey bar represents the mean ± SEM for the control MLR value. <b>Panels C-D:</b> Both etanercept and TA1 exhibit dose dependent inhibition of PBMC proliferation. Relative to etanercept, TA1 shows equivalent (CD4+; Panel C) or better (CD8+; Panel D) inhibition of human PBMC proliferation after 8 and 14 days of culture. (*) denotes significant (p <0.001 or greater) reduction in proliferation relative to the control MLR. (¢) denotes the concentration used for the <i>in vivo</i> murine normal (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145179#pone.0145179.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145179#pone.0145179.g002" target="_blank">2</a>) and NOD (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145179#pone.0145179.g003" target="_blank">3</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145179#pone.0145179.g006" target="_blank">6</a>) studies. Data shown represents the mean ± SEM of 3 experiments.</p

TA1 treatment results in significantly increased Foxp3⁺ (Treg) cells while simultaneously decreasing IL-17A⁺ (Th17) T cells.

<p>Shown are the flow cytometric data for 3 representative animals from the Control Diabetic (Total N = 12) and TA1 Non-Diabetic (Total N = 9) groups presented in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145179#pone.0145179.g005" target="_blank">Fig 5</a></b>. The ratio of Foxp3⁺ to Il-17A⁺ Cells (i.e., the Treg:Teff ratio) is shown in the upper right quadrant. A high Treg:Teff ratio (<i>i</i>.<i>e</i>., > ~200) correlated with maintenance of normoglycemia (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145179#pone.0145179.g003" target="_blank">Fig 3A</a></b>). The gating strategy utilized in shown in the left-most panel. To best reflect the <i>in situ</i> leukocyte subpopulations mediating the autoimmune disease pathology, no exogenous stimulation of the isolated cells was done prior to flow cytometric analysis.</p