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

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

The systemic immunomodulatory effects of TA1 therapy are further evidenced by the tolerogenic skewing of the immune cell subpopulations in the spleen and brachial lymph node.

<p>Similar to the pancreatic lymph node, the spleen and brachial lymph node show increased tolerogenic cell populations and decreased proinflammatory cells following TA1 administration at 7 weeks of age. 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. Non-diabetic results are the mean ± SD of 4 saline and 9 TA1 treated NOD mice. 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

TA1 therapy dramatically alters the expression of multiple pro-inflammatory (A) and tolerogenic/anergic (B) T cell subsets in the pancreatic lymph node.

<p>In addition TA1 favored pro-tolergenic changes in DC (CD11c+) cells (B). 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. Non-diabetic results are the mean ± SD of 4 saline and 9 TA1 treated NOD mice. Representative flow cytometic data are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145179#pone.0145179.s001" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145179#pone.0145179.s002" target="_blank">S2</a> Figs 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

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

Therapeutic Cells via Functional Modification: Influence of Molecular Properties of Polymer Grafts on In Vivo Circulation, Clearance, Immunogenicity, and Antigen Protection

Modulation of cell surface properties via functional modification is of great interest in cell-based therapies, drug delivery, and in transfusion. We study the in vivo circulation, immuogenicity, and mechanism of clearance of hyperbranched polyglycerol (HPG)-modified red blood cells (RBCs) as a function of molecular properties of HPGs. The circulation half-life of modified cells can be modulated by controlling the polymer graft concentration on RBCs; low graft concentrations (0.25 and 0.5 mM) showed normal circulation as that of control RBCs. Molecular weight of HPG did not affect the circulation of modified RBCs. HPG grafting on RBCs reduced CD47 self-protein accessibility in a graft concentration-dependent fashion. HPG-grafted RBCs are not immunogenic, as is evident from their similar circulation profile upon repeated administration in mice and monitoring over 100 days. Histological examination of the spleen, liver, and kidneys of the mice injected with modified RBCs revealed distinct differences, such as elevated iron deposits and an increase in the number of CD45 expressing cells at high graft concentration of HPGs (1.5 mM); no changes were seen at low graft concentration. The absence of iron deposits in the white pulp region of the spleen and its presence in the red pulp region indicates that the clearance of functional RBCs occurs in the venous sinuses mechanical filtering system, similar to the clearance of unmodified senescent RBCs. HPG modification at grafting concentrations that yield long circulation in mice produced camouflage of a large number of minor blood group antigens on human RBCs, demonstrating its utility in chronic transfusion. The normal circulation, nonimmunogenic nature, and the potential to modulate the circulation time of modified cells without toxicity make this HPG-based cell surface modification approach attractive for drug delivery and other cell-based therapies