Expression of Notch Pathway Components in Primary Samples of Allogeneic Hematopoietic Cell Transplant Patients with Chronic Graft Versus Host Disease

Introduction: Chronic graft versus host disease (crGvHD) is a major cause of morbidity and mortality after allogeneic hematopoietic cell transplant (allo-HCT). The exact pathogenesis of crGvHD remains unclear, as the process comprises a complex immune reaction with both T and B cells leading to chronic inflammation and collagen deposit. The role of Notch pathway in regulating different cells of immune system is well-recognized and has been implied in the pathogenesis of acute graft versus host disease in different mouse allo-HCT models. Recently, Radojcic et al (Blood 2018), using a mouse model of sclerodermatous cGVHD, showed that the blocking of Dll1/Dll4-mediated Notch signals provided maximum protection if used early after transplant in a preventative fashion. Finally, human data suggest cooperation of Notch with B-cell receptor signaling in crGVHD.

Objective: We sought to analyze the correlation of the components of Notch pathway in the tolerance to GvHD of patients (pts) who received allo-HCT.

Patients and Methods: This was a cross-sectional study. The samples were collected from 18 pts, classified according to 2014 NIH consensus criteria, and 13 health controls, between 2010 to 2016, at our Institution, after written informed consent was obtained. We divided pts into three groups: tolerant (pts that have never developed crGvHD or who had withdrawn from all immunosuppression therapy (IST) for at least 6 months and without signs and symptoms of active crGvHD at the time of blood collection), crGvHD with IST, and crGvHD without IST. PBMC were isolated by Ficoll-Paque method. We used mirVAna^TMmiRNA isolation kit (Ambion/Life Technology, CA) for RNA extraction, according to manufacturer instruction. We performed quantitative PCR for the following genes; NOTCH1, NOTCH2, JAG1, JAG2, DLL1 and DLL4. ANOVA followed by Bonferroni´s post hoc test was used for statistical analysis.

Results: Pts characteristics are shown in Table 1. Pts were distributed across the groups as follows: 8 (44.5%) in tolerant, 6 (33%) were in crGvHD with immunossupressive therapy (IST), and 4 (22%) were in crGvHD without IST group. In the health-control group, 7 were male (54%) and median age was 35 years (range 19-50). Interestingly, when all compared with the control group, tolerant group presented a significant lower expression of NOTCH1 (-7,6 fold-change; p=0.02), NOTCH2 (-10,6 fold-change, p=0.0006), DLL1 (-5,89 fold-change; p=0.03) and JAG2( -4,11 fold-change; p=0.04, respectively). Chronic GvHD without IST and crGvHD with IST also presented a lower expression of NOTCH2 (-13,13 fold-change; p=0.02; -11,81 fold-change; p=0.03, respectively).

Conclusion: To our knowledge, this study is the first to evaluate the gene expression of components of Notch pathway in human samples of crGvHD. Our preliminary results showed that, surprisingly, the expression of key components of Notch signaling is downmodulated in PBMC of pts who underwent allo-HCT, tolerant to crGVHD. Our next step is to confirm those findings in a larger cohort and to analyze Notch pathway in subsets of lymphocytes of patients undergoing allo-HCT. Until then, whether Notch signaling is critical for the human allogeneic lymphocytes during GVHD remains unclear.

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