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IFNγ in immune-mediated graft failure
AB
C
Figure 6. Successful hematopoietic stem cell transplantation (HSCT) chimerism in interferon (IFN)-γR1-/- mice correlates with low IFNγ activity; circulating CXCL9 levels is a biomarker of in vivo IFNγ activity. Ifngr1-/- mice (expressing the Ly5.2 congenic marker) were intravenously (i.v.) infected with 1,106 CFU of Bacillus Calmette–Guérin (BCG) (strain Pasteur 1173P2). After 14, 20, 28, 35 and 42 days mice were treated i.v. with 100 mg/kg of an isotype control (n=5) or the anti- mIFNγ, XMG1.2 (n=5). At day 21, mice were infused with bone marrow from Ifngr+/+ mice, expressing the Ly5.1 marker, after mild irradiation (550 rads). Chimerism, assessed by determining the surface expression of Ly5.1 and Ly5.2 on lymphocytes, was analyzed by flow cytometry at different time points after HSCT treatment. IFNγ levels were quantified at different time points by ELISA using the Luminex technology. (A) Graph represents the super-imposition of the chimerism (black straight line) and the IFNγ levels (gray dotted line) in the isotype control treated mice. (B) Graph represents the chimerism determined in mice treated with the isotype control (black straight line) or with the XMG1.2 (gray straight line) mAbs. (C) Ifngr1-/- mice were i.v. infected with 1.106 CFU of BCG (strain Pasteur 1173P2). After 14, 20, 28, 35 and 42 days mice were treated i.v. with 100 mg/kg of an isotype control (black straight line; n=5) or the anti-mIFNγ, XMG1.2 (gray straight line; n=5). At day 21, mice were transplanted with bone marrow from Ifngr+/+ mice, expressing the Ly5.1 marker, after mild irradiation (550 rads). At different time points post-BCG infection, circulating CXCL9 levels were quantified by ELISA using the Luminex technology. Ab: antibody.
infusion, while signs and symptoms of GF appear only later (see Table 2). Indeed, the current proposed risk score for GF determined on day +21 after HSCT, based on eight patient and transplant variables, showed good specificity, but low sensitivity.1 The high accuracy of CXCL9 in pre- dicting GF, as indicated by the AUC of 0.905, renders this chemokine an ideal “candidate biomarker”, as stated by the 2014 National Institutes of Health consensus on bio- markers.30,31 CXCL9, also known as monokine induced by γ-interferon (MIG), is a chemokine specifically induced by IFNγ,32 and represent the most sensitive and specific of the soluble factors we analyzed. It binds to the chemokine receptor CXCL3 expressed on naïve T cells, Th1 CD4+ T cells, effector CD8+ T cells, as well as on NK and NKT cells, driving Th1 inflammation. Circulating CXCL9 levels have been shown to reflect the amount of IFNγ produced in organs, such as liver and spleen,25 which are the typical target of inflammation. This strong correlation with IFNγ produced in organs rather than in blood provides an expla- nation why, despite high CXCL9 serum levels, serum lev- els of IFNγ were found to be low or even undetectable in a few of our GF patients. Furthermore, elevated levels of
CXCL9 have been related to graft rejection in solid organ transplantation (such as heart, kidney and lung transplan- tation),33-35 but, to the best of our knowledge, this is the first report demonstrating that the hyperproduction of IFNγ in GF occurring after HSCT results in increased CXCL9 serum levels. Among other cytokines/ chemokines, we also observed increased levels of IL10, an important Th2 cytokine with anti-inflammatory proper- ties, this finding being in agreement with the hyperpro- duction of this molecule recorded in patients with HLH.36
Our results are not only relevant for diagnostic purposes, but also suggest that IFNγ is a potential therapeutic target in GF. Indeed, independently of the mechanism of IFNγ- mediated GF (i.e. direct effect on HSC or HLH-like effect), our results support the investigation of IFNγ neutralization for prevention and/or treatment of GF in patients undergo- ing HSCT. The encouraging efficacy and safety data reported from the ongoing study in primary HLH with emapalumab (NI-0501), an anti-IFNγ monoclonal antibody,17,37 provides additional support for the rationale for using this drug.38 The data we generated in the murine model of GF confirm and extend the role played by IFNγ
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