A novel flow-cytometric based method to assess post-HSCT donor chimerism exploiting RNA hybridization

Abstract

Analysis of donor-recipient chimerism after hematopoietic stem cell transplantation (HSCT) is of pivotal importance for patient's clinical management, especially in the context of mixed chimerism. Patients are routinely monitored for chimerism in sorted subsets of peripheral blood cells. However, measurement of chimerism in sorted immune cell subsets is technically challenging and time consuming. We here propose a novel, flow cytometry-based approach to detect donor cell chimerism in sex-mismatched HSCT. We exploit RNA PrimeFlow™ system, based on RNA hybridization, to detect mRNA from a lysine demethylase encoded by Y chromosome, KDM5D. This approach allows to distinguish male and female derived cells with around 1% sensitivity. The procedure can be coupled with multiparametric immunophenotyping to assess chimerism in specific immune cell subsets without the need for prior FACS-sorting. We apply this method to a cohort of HSCT patients (n = 10) and we show that it is consistent with standard PCR-based method. We also show that different T lymphocyte subsets display variable degrees of donor chimerism, especially in CD8+ T cell compartment where we observe an enrichment for recipient chimerism in central memory T cells. This method can be exploited to advance current knowledge on immune reconstitution focusing on specific subsets avoiding prior FACS-sorting.

Fig. 1. Establishment of a flow-cytometry based method to assess chimerism exploiting RNA hybridization on KDM5D.

a Expression of KDM5D assessed by ddPCR in peripheral blood mononuclear cells (PBMCs) from male and female healthy donors (n = 5 and 3 respectively) confirms the presence of KDM5D mRNA only in male samples. b Scheme with mRNA KDM5D structure showing the regions where the two probes hybridize. c Representative FACS plot showing validation of KDM5D dual probe system on healthy donors. Mixing of samples was performed on white blood cell (WBC) counts with serial dilution of male sample with female cells. n = 4 independent experiments from a total of eight healthy donors. d Percentage of male chimerism from female, male and male:female mixed samples expressed as percentage on total CD45 + RPL13A+ live cells. e Mean fluorescence intensity (MFI) of KDM5D Alexa647+ cells on total CD45+ cells and in main immune cell subsets from male healthy donor (n = 4). f Graph showing technical reproducibility of three independent 1:1 mixes of the same male and female samples and of the same peripheral blood sample of a transplanted patient with mixed chimerism. Coefficient of variation was calculated for the 1:1 mix (0.051) and for the analyzed patient (0.024).

Fig. 2. Validation of flow-cytometry based chimerism on HSCT patients.

a Comparison of total chimerism in HSCT patients (n = 8) performed by STR-PCR (PCR) and RNA Prime flow (FC). Mean ± SEM. b Correlation between the levels of total recipient chimerism detected by PCR and flow cytometry (n = 19 samples. R² = 0.529, p = 0.0004). c Representative FACS plot showing the presence of two distinct populations of cells from donor (in this case male) and recipient (female) origin in the different immune cell subsets and negative control (FMO). d Percentage of recipient chimerism in HSCT patients (n = 8) in the different immune cell subsets. e Kinetics of recipient chimerism detected by STR-PCR and flow cytometry at different timepoints on total peripheral blood samples shows a similar trend. f MFI of KDM5D Alexa647+ cells on total CD45+ cells and in main immune cell subsets in analyzed patients (n = 7) shows similar levels of expression in the different subsets. Mean ± SEM.

Fig. 3. Application of flow-cytometry based chimerism to the study of immune reconstitution.

a Representative FACS plot and gating strategy for T cell analyses (plots on the left) and representative expression of KDM5D on total CD8+ and naïve CD8+ T cells (plots on the right). b Recipient chimerism in CD3+ T cell subsets (n = 7) shows no significant differences among the main subsets. Repeated measures ANOVA with multiple comparison, mean ± SEM. c Recipient chimerism in CD8+ T cell subsets (n = 7) shows a significantly higher recipient chimerism in central memory T cells (CM) compared to terminally differentiated CD45RA+ cells (TEMRA). Repeated measures ANOVA with multiple comparison, mean ± SEM. d Recipient chimerism in CD4+ T cell subsets (n = 7). Repeated measures ANOVA with multiple comparison, mean ± SEM. e Representative FACS plot and gating strategy for B cell analysis and expression of KDM5D on naïve B cells. f Percentage of recipient chimerism in B cell subsets (n = 5). Only subsets with adequate number of events were taken into account for chimerism analysis. Mean ± SEM.