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IFNγ in immune-mediated graft failure
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Figure 4. Immunohistochemistry characterization of bone marrow (BM) in patients who either did or did not experience graft failure (GF). (A) Comparison of absolute number of CD3+, CD4+, CD8+, CD68+, TIA-1+, perforin+ and granzyme+ cells in BM of GF patients and controls (CTRL). The total number of positive cell for each marker was counted in five fields per sample under 20-fold magnification and reported as Mean±Standard Deviation. (B) Percentages of CD68+ cells with hemophagocytic activity (i.e. showing cellular fragments, erythrocytes and lipid vacuoles in their cytoplasm) in BM of GF patients and CTRL. *P<0.05; **P<0.01; ***P<0.001.
extended our analysis on BM-infiltrating lymphocytes through flow-cytometry in both controls and GF patients. Regarding NK (CD56+/CD3–) and γd T cells (CD3+/CD4–/CD8–) no difference was observed between the two patient groups (data not shown). By contrast, in the αβ T-cell subset, the analysis revealed a significant differ- ence in both CD4 (58.9%±13.4% vs. 7.6%±7.3%, controls vs. GF patients) and CD8 (25.9%±6.1% vs. 66.5%±18.2%, controls vs.GF patients) subsets (P<0.0001 and P=0.0018, respectively) (Figure 5A). We further characterized both CD4+ and CD8+ populations for the expression of memory markers. While no significant difference was detected in the CD4+ subpopulation, the CD8+ subset displayed a sig- nificant enrichment of effector memory T cells (EfM) (CD45RO+/CCR7-) (40.3±24.6% vs. 20.7%±7.3%, GF patients vs. CTRL patients; P=0.034) (Figure 5B and C) and a significant reduction of the naïve subset (CD45RA+/CCR7+) (18.6%±16.6% vs. 28.6%±12.1%, GF patients vs. controls; P=0.014). See Online Supplementary Appendix for further details.
Increasing expression of activation and exhaustion markers on T cells during graft failure
We evaluated the expression of several activation and exhaustion markers on infiltrating cells. As expected, in patients experiencing GF, both CD4+ and CD8+ cells dis- played a significant activation profile, as demonstrated by the overexpression of CD95 (69.2%±23.0% vs. 93.9%±6.9% and 57.9%±27.2% vs. 98.35%±2.0%, con- trols vs. GF patients, respectively; P=0.021 and P=0.002) (Figure 5D) and downregulation of CD127 (recently shown to be associated with prolonged T-cell receptor stimula- tion20) on the proliferating CD8+ cells (69.3%±16.9% vs. 37.9%±18.8%, controls vs. GF patients, respectively; P=0.014) (Figure 5E). The expression of several exhaustion and senescence markers confirmed the status of prolonged activation of T lymphocytes located in the BM of GF patients, such as the upregulation of CD57 (CD57+:
10.2%±10.5% vs. 37.4%±12.4% and 34.7%±17.3% vs. 68.0%±18.8% controls vs. GF patients in CD4 and CD8 respectively; P=0.003 and P=0.011) (Figure 5F). See Online Supplementary Appendix for further details.
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In order to understand if the sole IFNγ-inhibition would be sufficient to prevent GF, we used an established mouse model of GF.13 As previously reported by Rottman et al.,13 the infection of Ifngr1-/- mice with Bacillus Calmette– Guérin (BCG) resulted in a rapid increase of circulating IFNγ levels reaching a concentration of 11,000 pg/mL on day 20 post-infection (Figure 6A). HSCT performed at day 21, i.e. at the peak of IFNγ levels, resulted in poor chimerism as only 5% of the Ifngr1+/+ donor cells engrafted in the BCG-infected Ifngr1-/- recipient mice. After day 21 post-BCG infection, serum IFNγ levels gradually decreased to a steady state level of approximately 100 pg/mL. This decrease in IFNγ serum levels correlated with an increase in chimerism as the Ifngr1-/- recipient mice exhibited 19% HSC engraftment of donor cells at day 84 (Figure 6A). For further assessing the role played by IFNγ in GF, BCG- infected Ifngr1-/- recipient mice were given a neutralizing IFNγ mAb, XMG1.2, pre- and post-HSCT. Neutralization of IFNγ improved engraftment in BCG-infected Ifngr1-/- recipient mice because, at three months after the allograft, 45% of the lymphocytes were of donor origin (i.e. Ly5.1 positive), as compared to 19% in isotype control-treated mice (Figure 6B). In order to assess IFNγ activity and ensure neutralization by XMG1.2, the IFNγ-dependent chemokine CXCL9 was measured. A decrease in CXCL9 serum levels during the XMG1.2 treatment was observed, confirming IFNγ neutralization in contrast to isotype con- trol-treated mice (Figure 6C). Once XMG1.2 treatment was interrupted, at day 42 post-BCG infection, a gradual increase in CXCL9 serum levels was observed, indicating restoration of IFNγ activity.
Interferon-γ drives rejection of donor cells in Ifngr1 mice
haematologica | 2019; 104(11)
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Absolute count
% of positive cells