Cytek (Commercial Talk) : In-depth analysis of CD34pos hematopoietic stem and precursor cell (HSPC) subpopulations by flow cytometry using a single 20-color panel & 50 Shades of fluorescence to sort rare immune populations

Talk 1 – Thomas Matthes :

In-depth analysis of CD34pos hematopoietic stem and precursor cell (HSPC) subpopulations by flow cytometry using a single 20-color panel

Aleksandra Dufour, Cindy Lanvers, and Thomas Matthes*

Clinical Pathology, Diagnostics Department; and *Hematology Service, Oncology Department, University Hospital Geneva, Switzerland

Objective: Haematopoiesis was the first system for which a tissue-specific stem cell was identified, the haematopoietic stem cell (HSC). It was thought at the beginning that CD34pos cells corresponded to these HSC, but in recent years it became clear that the CD34pos cell population is heterogenous and contains subpopulations of different precursor cells (HSPC) with only <1% corresponding to real HSC, as evidenced by in vivo mouse transplantation assays. Based on multicolor flow cytometry (MFC) with a 20-marker combination in a single tube we performed an in-depth study of the HSPC CD34pos cell compartment in normal bone marrow (BM) samples and in AML patients with the aim to develop an analysis pipeline for application in routine diagnostics. Methods: BM samples were obtained from 7 normal donors, 10 AML patients at diagnosis, 17 follow-up samples after chemotherapy and/or BM transplantation (4 MRD pos; 13 MRD neg), and 13 samples from other diseases. Samples were stained with a 20-color antibody panel and were analyzed on a full spectrum flow cytometer (Northern Lights, Cytek). 1-1.5 x106 events were acquired. Files were analyzed using the Kaluza and Cytobank software (Beckman Coulter). Results: 8 different CD34pos HSPC subpopulations were defined corresponding to HSC and multipotent progenitors, common myeloid progenitors (CMP), lymphoid, myeloid, monocytic, erythroid, megakaryocytic and pDC precursors, respectively. Reference values for these subpopulations in normal BM samples were determined. Analysis of AML samples at diagnosis revealed as yet unsuspected heterogeneity in the leukemic blast populations. Analysis of samples after chemotherapy or BM transplant allowed to analyze reconstitution of the HSPC compartment and the presence or absence of MRD, down to a sensitivity level of 0.1%. Unsupervised analysis of the data files based on dimensionality reduction (t-SNE) and clustering algorithms (FlowSOM) were used to establish a robust analysis pipeline. Conclusion: Quantification of MRD after therapy is increasingly used for risk stratification, prognostication and evaluation of treatment efficiency in hematologic diseases. Using our single 20-color antibody panel and unsupervised clustering methods for the detection of HSPC subpopulations, we established an analysis pipeline for routine follow-up of AML patients which allows us to measure HSPC reconstitution and MRD after treatment. Talk 2 – Cyril Mionnet :

50 Shades of fluorescence to sort rare immune populations

Once B. pertussis, causing agent of the whooping cough, attaches to ciliated epithelial cells on the upper respiratory tract, the lung-resident innate immune cells start to control the initial step of B. pertussis infection. Then, initial recruitment of DCs and macrophages into infected lungs is followed by the recruitment or the expansion of T and B cells and the infiltration of neutrophils and NK cells in order to mediate B. pertussis clearance.

Our objective is to decipher the cellular mechanisms underlying the “cross-talk” between innate immune cells (myeloid populations, NKs/ILCs and granulocytes) and adaptative immune T and B cells that allows an efficient response to eliminate Bordetella pertussis and confers a memory response.
To this aim, we developed a 48 markers panel to follow the behavior of several immune populations in lungs of infected mice. This panel aims to identify immune populations during the time course of infection as well as at steady state. Using this panel on blood, spleen and lymph nodes isolated populations we were able to identify rare populations of T cells appearing only in infected mice. More, using this panel, we sort these populations and clearly isolated among them a population of T progenitors which will give rise to tissue resident memory T cells.