Abdi, Camillo Ricordi, Mohamed H. Sayegh, Antonello Pileggi and ...

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15 DOI: 10.1161/CIRCULATIONAHA.112.123653 JNK/SAPK1, p70/S6 or STAT5 in CD4 + T cells was unaffected (Figures 7B, 7C and 7D, respectively). We then confirmed by western blot that oATP dose-dependently inhibits STAT3 phosphorylation (Figure 7F). To demonstrate that the effect of oATP is STAT3- phosphorylation-inhibition-dependent, we tested the effect of Colivelin treatment, a peptide that promotes STAT3 phosphorylation 36, 37 in an add-back experiment. We first confirmed that Colivelin (Col) was able to restore the activation-induced phosphorylation of STAT3 in a dose- dependent manner in oATP-treated CD4 + T cells by western blotting (Figure 7G). Moreover, in an ELISPOT assay, in which naïve CD4 + T cells were stimulated with 0.5 g/ml anti-CD3-Ig and anti-CD28-Ig, treatment with Colivelin was shown to significantly revert oATP-mediated suppression uppression of IFN--producing IFN--producing cells (Figure 7H). The effect of oATP and Colivelin Colive veli li lin on on STA STAT3 TA TAT3 T3 phosphorylation and T cell function was also evaluated following cardiac transplantation. BA BALB BALB/c LB LB/c /c hea hearts ea eart rts we were re transplanted into C57BL/6 6 mi mmice, ce, and STAT3 phos phosphorylation osph phorylation was assessed by western blot in CD4 + by western blot t in CD4 D4 T cells isolated from splenocytes of mice 7 days after transplantation. + T cel el ells ls iso sola lated from splen en enocyt yt ytes es of mice 7 da days aft fter er tra rans ns n pl p an anta tati tion on on. oA oATP oATP-treated TP TP-t -tre tre reat at ated ed mice mic ice e displayed di disp spla la laye y d reduced re r du duce ced levels le l ve vels ls of off phosphorylated pho ho hosp sp spho ho h ry ryla la late ted d ST STAT STAT3 AT AT3 co comp compared mp mpar ar ared ed to un untr untreated trea ea eate ted d mice, mice, and para paralleling rall ll llel el elin in ing g th the e re resu results su sult l s ob oobtained ta tained ed in vit vitro, it i ro ro, the th the e use us use e of Col Colivelin ol oliv iv i el elin in was as abl able ble to ree reestablish ee eestablish STAT3 phosphorylation in CD4 + T cells of oATP-treated mice (Figures 7I and 7J). From a functional point of view, the use of Colivelin greatly abrogated the effect of oATP on allograft survival (Figure 7K). These data demonstrate that P2X7R signaling is crucial for T cell activation and Th1/Th17 generation and that this effect is STAT3-dependent. In vivo short-term P2X7R targeting prevents coronary vasculopathy in a model of chronic heart cardiac transplant rejection In order to evaluate the importance of P2X7R signaling and targeting in a clinically relevant setting, we tested the effect of oATP treatment in a model of cardiac transplant chronic rejection Downloaded from http://circ.ahajournals.org/ by guest on December 19, 2012
16 DOI: 10.1161/CIRCULATIONAHA.112.123653 (bm12 donors to C57BL/6 recipients). In this model, C57BL/6 mice do not acutely reject bm12 cardiac allografts, but transplanted hearts develop transplant-associated coronary vasculopathy. Cardiac allograft pathology was assessed 40 days after transplantation of bm12 hearts into C57BL/6 mice. Advanced coronary vasculopathy and severe lymphocyte and macrophage interstitial and vascular infiltration was observed in untreated mice (Figures 8A1-8A4) as compared to oATP-treated mice (Figures 8B1-8B4), which displayed only mild cellular infiltration as well as absence of coronaropathy (Figures 8C and 8D, respectively). oATP-treated mice also showed a reduced number of IFN--producing cells (Figure 8E) and an increased number of IL-4-producing cells (Figure 8F) in an ELISPOT assay when splenocytes were challenged with donor antigens. Discussion Di Disc sc scus us ussi sion ion The e introduction in i troduction on of no nove novel vel im immu immunosuppressive muno nosu su supp ppressive drug drugs ug ugs has as led ed to o signif significant ific icant t im impr improvement prov ovem emen ent in sho shorthort- termerm rm car cardiac ar ardi di diac ac tra transplant ranspl pl plan ant su surv survival r iv ival al rat rates at ates es but ut has s been be been en unable una nabl bl ble to sig significantly ig ignifi fi fica ca c nt ntly ly imp improve mp mpro ro rove ve allograft all llog ogra ra raft ft survival in the long-term 1, 2 urvival in th the e lo long ng ng-t -t -ter er erm Furthermore, current immunosuppressive regimens are associated 1, 2 Fur ur u th ther er e mo m re re, , cu curr rren en ent t im immu mu m no nosu su supp pp ppres es e sive ve reg eg egim im imen ens s ar are e as associated with multiple complications including cancer, opportunistic infections, diabetes, kidney failure, and hypertension 2-4 . In order to improve transplantation outcomes, it is therefore critical to continue development of novel strategies in order to lessen the need for life-long immunosuppression and to achieve stable graft acceptance. We thus studied the role of the ionotropic purinergic P2X receptors (a family of receptors with largely unknown function in the alloimmune response) in cardiac transplant rejection and tolerance. Aside from signal 1 (TCR engagement) and signal 2 (costimulatory molecule interaction), soluble signals have also been recognized as major players in T cell activation; we Downloaded from http://circ.ahajournals.org/ by guest on December 19, 2012
Page 1 and 2: Long-Term Heart Transplant Survival
Page 3 and 4: Abstract: 2 DOI: 10.1161/CIRCULATIO
Page 5 and 6: 4 DOI: 10.1161/CIRCULATIONAHA.112.1
Page 7 and 8: infiltrate, 3: heavy cell infiltrat
Page 9 and 10: 8 DOI: 10.1161/CIRCULATIONAHA.112.1
Page 11 and 12: 10 DOI: 10.1161/CIRCULATIONAHA.112.
Page 15: 14 DOI: 10.1161/CIRCULATIONAHA.112.
Page 23 and 24: activation at the immune synapse. B
Page 25 and 26: Table 1. Patient characteristics (D
Page 31 and 32: H1 H2 J1 J J2 Figure 1 A B C D E F
Page 33 and 34: Figure 3 Untreated D7 oATP-treated
Page 35 and 36: Figure 5 A P2X1R P2 P2X1 X1R P2X4R
Page 37 and 38: Figure 7 MFI A 2000 500 500 250 0 p
Page 39 and 40: IMMUNOLOGICAL METHODS Adoptive tran
Page 41 and 42: CIRCULATIONAHA/2012/123653-R2 expre
Page 43: P2X7R GAPDH T-bet ROR-γ A B C GAPD

Abdi, Camillo Ricordi, Mohamed H. Sayegh, Antonello Pileggi and ...

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