Adoptive transfer of pre-activated nTreg does not protect animals from IRI.

<p>nTreg were obtained from FoxP3 GFP mouse spleens by flow sorting followed by expansion <i>in vitro</i> for 21 days and further flow-sorting for purity [a–c]. Flow cytometry of spleen and liver confirmed successful transfer [d,e]. There was no difference in injury severity between nTreg supplemented and control animals at 3 hours of reperfusion (n = 4 per group) [f].</p

Adoptive transfer of pre-activated iTreg does not protect animals from IRI.

<p>iTreg were generated <i>in vitro</i> from sorted CD62L high, FoxP3- naïve T cells cultured in the presence of TGFβ and IL-2 for 5 days before flow sorting to maximize purity [a-d]. Successful transfer was confirmed by detection of FoxP3 GFP+cells in spleen [e] and liver [f]. No difference was detected in injury severity between iTreg supplemented and PBS treated control animals at 3 or 24 hours of reperfusion (n = 4–5 per group) [g].</p

<i>In vivo</i> expansion of Treg does not protect animals from IRI.

<p>Mice received IL-2/JES6-A12 complexes or PBS for three consecutive days prior to hepatic ischemia reperfusion insults (n = 8 per group). Animals were culled at 24 hours of reperfusion, and tissues analysed for Treg frequency. There was significant expansion of splenic [a], circulating [b] and hepatic [c] Treg. Injury severity was similar between Treg expanded and control animals, in terms of ALT and histological injury [d,e]. FlowCytomix was used to profile circulating chemo/cytokines. Levels of IL-2 [f], CXCL-10/IP-10 [g] and IL-6 [h] were elevated. Rises in KC/CXCL-1 [i], and GM-CSF [j] did not differ between groups. Other analytes (IL-1α, IL-1β, IL-10, IL-17, IL-17F, IFNγ, MIP-1α) were not detected.</p

Treg antibody depletion does not increase susceptibility to IRI.

<p>Wildtype mice received the CD25 depleting antibody pc61 7 and 2 days or PBS (n = 5–8 per group) prior to ischemia reperfusion injury. Injury severity was no different between Treg intact and Treg depleted animals in terms of ALT [a] or histological injury score [b]. PC61 treatment resulted in significant depletion of CD4+CD25+[c] and CD4+FoxP3+[d] cells. FlowCytomix was used to profile circulating chemo/cytokines. No differences were detected in post-operative rises in CXCL-10/IP-10 [e], KC/CXCL-1 [f], IL-6 [g] and GM-CSF [h] between Treg intact and depleted animals. Other analytes (IL-1α, IL-1β, IL-2, IFNγ, IL-17 and IL-17F, and IL-10) were not detected.</p

Treg mobilization during reperfusion is not enhanced by IPC.

<p>Mice (n = 5–8 per group) were subjected to ischemia reperfusion injury with or without ischemic preconditioning and killed at 3 or 24 hours. Ischemic preconditioning protected the liver from injury both in terms of ALT release [a] and histological injury score [b] measured at 24 hours. CD4+FoxP3+cells were mobilized into the circulation during reperfusion [c]. Hepatic CD4+FoxP3+cells increased over the same time period [d]. Total CD3+lymphocytes were stable throughout reperfusion [e]. FlowCytomix was used to profile circulating chemo/cytokines. Rises were detected in CXCL-10/IP-10 [f], CXCL-1/KC [g], IL-6 [h] and GMCSF [i]. Other analytes (IL-1α, IL-1β, IL-2, IFNγ, IL-17 and IL-17F, and the Treg cytokine IL-10) were not detected.</p

Treg depletion in the FoxP3.LuciDTR mouse does not increase susceptibility to IRI.

<p>FoxP3.LuciDTR mice or wildtype control (n = 5–10 per group) received 25 ng/g DT 24 hours prior to ischemic insult. ALT release [a] and histological injury score [b] did not differ between groups. DT treatment of DTR animals effected almost total depletion of Treg from the circulation [c,d], spleen [e] and liver [f].</p

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<p>Several inflammatory diseases including multiple sclerosis and inflammatory bowel disease have been associated with dysfunctional and/or reduced numbers of Foxp3⁺ regulatory T cells (Treg). While numerous mechanisms of action have been discovered by which Treg can exert their function, disease-specific Treg requirements remain largely unknown. We found that the integrin αv, which can pair with several β subunits including β8, is highly upregulated in Treg at sites of inflammation. Using mice that lacked αv expression or β8 expression specifically in Treg, we demonstrate that there was no deficit in Treg accumulation in the central nervous system during experimental autoimmune encephalomyelitis and no difference in the resolution of disease compared to control mice. In contrast, during a curative T cell transfer model of colitis, Treg lacking all αv integrins were found at reduced proportions and numbers in the inflamed gut. This led to a quantitative impairment in the ability of αv-deficient Treg to reverse disease when Treg numbers in the inflamed colon were below a threshold. Increase of the number of curative Treg injected was able to rescue this phenotype, indicating that αv integrins were not required for the immunosuppressive function of Treg per se. In accordance with this, αv deficiency did not impact on the capacity of Treg to suppress proliferation of naive conventional T cells in vitro as well as in vivo. These observations demonstrate that despite the general upregulation of αv integrins in Treg at sites of inflammation, they are relevant for adequate Treg accumulation only in specific disease settings. The understanding of disease-specific mechanisms of action by Treg has clear implications for Treg-targeted therapies.</p

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<p>Several inflammatory diseases including multiple sclerosis and inflammatory bowel disease have been associated with dysfunctional and/or reduced numbers of Foxp3⁺ regulatory T cells (Treg). While numerous mechanisms of action have been discovered by which Treg can exert their function, disease-specific Treg requirements remain largely unknown. We found that the integrin αv, which can pair with several β subunits including β8, is highly upregulated in Treg at sites of inflammation. Using mice that lacked αv expression or β8 expression specifically in Treg, we demonstrate that there was no deficit in Treg accumulation in the central nervous system during experimental autoimmune encephalomyelitis and no difference in the resolution of disease compared to control mice. In contrast, during a curative T cell transfer model of colitis, Treg lacking all αv integrins were found at reduced proportions and numbers in the inflamed gut. This led to a quantitative impairment in the ability of αv-deficient Treg to reverse disease when Treg numbers in the inflamed colon were below a threshold. Increase of the number of curative Treg injected was able to rescue this phenotype, indicating that αv integrins were not required for the immunosuppressive function of Treg per se. In accordance with this, αv deficiency did not impact on the capacity of Treg to suppress proliferation of naive conventional T cells in vitro as well as in vivo. These observations demonstrate that despite the general upregulation of αv integrins in Treg at sites of inflammation, they are relevant for adequate Treg accumulation only in specific disease settings. The understanding of disease-specific mechanisms of action by Treg has clear implications for Treg-targeted therapies.</p

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<p>Several inflammatory diseases including multiple sclerosis and inflammatory bowel disease have been associated with dysfunctional and/or reduced numbers of Foxp3⁺ regulatory T cells (Treg). While numerous mechanisms of action have been discovered by which Treg can exert their function, disease-specific Treg requirements remain largely unknown. We found that the integrin αv, which can pair with several β subunits including β8, is highly upregulated in Treg at sites of inflammation. Using mice that lacked αv expression or β8 expression specifically in Treg, we demonstrate that there was no deficit in Treg accumulation in the central nervous system during experimental autoimmune encephalomyelitis and no difference in the resolution of disease compared to control mice. In contrast, during a curative T cell transfer model of colitis, Treg lacking all αv integrins were found at reduced proportions and numbers in the inflamed gut. This led to a quantitative impairment in the ability of αv-deficient Treg to reverse disease when Treg numbers in the inflamed colon were below a threshold. Increase of the number of curative Treg injected was able to rescue this phenotype, indicating that αv integrins were not required for the immunosuppressive function of Treg per se. In accordance with this, αv deficiency did not impact on the capacity of Treg to suppress proliferation of naive conventional T cells in vitro as well as in vivo. These observations demonstrate that despite the general upregulation of αv integrins in Treg at sites of inflammation, they are relevant for adequate Treg accumulation only in specific disease settings. The understanding of disease-specific mechanisms of action by Treg has clear implications for Treg-targeted therapies.</p