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P. Merli et al.
previously demonstrated by Rottman et al.13 Moreover, we also show that the sole neutralization of IFNγ, without the administration of anti-IL12 (employed in the experiments reported by Rottman et al.),13 is able to improve engraft- ment. The observation that decreased CXCL9 production correlates with improved HSCT chimerism provides fur- ther support to a therapeutic intervention aimed at neutral- izing IFNγ-pathway signaling. Finally, the data obtained in the three patients treated on a CU basis indicate that the use of an anti-IFNγ monoclonal antibody is safe also in a very fragile population, namely infants with a previous GF undergoing a second HSCT.
Four out of the seven patients we studied who under- went BM aspirate and biopsy showed evidence of hemo- phagocytosis. Indeed, it has been shown that an increased number of hemophagocytic macrophages in the BM obtained 14±7 days after HSCT is associated with higher risk of death due to GF.39 Moreover, in a cohort of adult patients receiving cord blood transplantation, GF was strictly related to the occurrence of HLH manifestations.23 Recently, in a retrospective study on peri-engraftment BM samples from 32 adult patients, Kawashima et al. pro- posed two histological measures, namely macrophage ratio and CD8+ ratio (defined as the ratio between the macrophage or CD8+ lymphocyte number on the total nucleated cell number), as predictors of GF at day +14.15
Despite some preliminary studies characterizing host T cell expansion in patients with GF,15,16 no information is available regarding the phenotype of these cells. Our data indicate an active role of T lymphocytes in mediating GF. As previously reported,15 in these patients, the mononu- clear infiltrate is mainly constituted by cytotoxic CD8+ lymphocytes with a predominant effector memory phe- notype. This population was demonstrated to be activat- ed, proliferating and cytotoxic, expressing specific mole- cules, such as Granzyme B, Perforin and TIA-1, involved in target-killing, as well as various activation and prolifer- ation markers. Interestingly, we observed that CD8+ lym- phocyte expansion is predominantly polyclonal, suggest- ing that the immune response is directed towards several antigens and not against few immunodominant epitopes. However, a significant enrichment of certain β clones was found. The cytopathic effect was clearly demonstrated by apoptotic cells surrounding proliferating T cells, which are long-term activated, as demonstrated by the expression of several exhaustion markers.40,41 Furthermore, the remain- ing γ/d and CD4+ T-cell populations are similarly express- ing exhaustion markers, underlying an over-stimulated environment. Notably, a particular behavior was observed
in the NKT-cell population with a significant reduction of CD8+ NKT, probably due to their activation and a signifi- cant increase of CD4+ NKT. The role of these cells is yet to be fully elucidated, although they were shown to be able to prevent pancreatic islet transplant rejection, but also to sustain CD8+ T-cell expansion.42,43 Given these data, a treatment able to interrupt the overproduction of mole- cules responsible for inflammation,33 such as an anti-IFNγ, could be beneficial in this setting.
Fifty percent of tested patients had anti-HLA antibodies: all those with positivity >5,000 MFI received a desensiti- zation therapy in order to lower the antibody title with the aim of reducing the risk of GF. Although we cannot exclude a role of anti-HLA antibodies in causing GF in our patients, all five positive patients showed increased values of IFNγ and/or related cytokines after HSCT. Thus, we can hypothesize that there may be a common final pathway and/or combined action (like that reported in solid organ transplantation)44 between humoral and cellular mecha- nisms sustaining GF.
Limitations of this study are the lack of a validation cohort and the relatively small number of patients includ- ed in the study. Another important limitation is that most patients experiencing GF that we report were transplanted from a PMFD after a TCD procedure (both being well- known risk factors for GF);2,3 thus, our results should be further validated in other transplant settings, especially when post-transplant pharmacological graft-versus-host disease prophylaxis is used. Indeed, the use of calcineurin inhibitors or other immunosuppressive agents can modify IFNγ (and related cytokines) secretion kinetics.45
Overall, our data suggest that immune-mediated GF may share clinical and laboratory characteristics with HLH. Besides providing evidence for further investigating the use of markers to allow a non-invasive, prompt iden- tification of patients at high risk of developing this severe complication of HSCT, the increased serum levels of IFNγ and CXCL9 found in GF patients provide a rationale for investigating a targeted therapy (i.e. anti-IFNγ therapy) in this complication. We are currently designing a clinical trial on the use of emapalumab for prevention and/or treatment of GF in patients at high risk of developing this complication.
Funding
This work was supported by “Ricerca corrente” (Ministero della Salute) (PM), Investigator Grant 2015 Id. 17200 by Associazione Italiana per la Ricerca sul Cancro (AIRC) (FL) and by Novimmune SA, Switzerland.
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