Detecting 24 Kinds of Cytokines Via Flow Cytometry Aimplex Kit Is an Effective Way to Monitor CRS after CAR-T Cells Infusion
Authors: Wang Dongchu , Li Xiang , Qiu Liyuan , Fu Shizhe , Cui Lingyun , Wang Hui , Lu Peihua 
Published: November 2020

Introduction: Cytokine release syndrome (CRS) is a serious side effect of chimeric antigen receptor-T cell (CAR-T) therapy. Effective cytokine monitoring can provide support for early prevention and treatment. As more and more promising markers have been identified to be associated with CRS responses, traditional ELISA method is inapplicable because it's sample-consuming, low flexibility, and difficult to detect multiple cytokines simultaneously. Aimplex kit, a new high-throughput technique based on flow cytometry(FCM) and micro beads has been testified a effective way to monitor multiple cytokines in peripheral blood(PB) samples. If a new suitable cytokines panel is established to monitor the variation curve of multiple cytokines in patients' PBs post CAR-T therapy, study the relationship between clinical symptoms and the lab results, even build a cytokines database of CAR-T therapy, it would offer a promising support to prevent and handle CRS. On the other hand, by studying the relationship between cytokines and activated cells, we might investigate mechanisms of CRS and explore more related markers.
Methods: A two tubes panel was designed to detect 24 cytokines, including IFN-γ/IL-1β/IL-2/IL-4/IL-5/IL-6/IL-8/IL-10/IL-12p70/IL-17A/IL-17F/IL-22/TNF-α/TNF-β, and sCD25/GM-CSF/IL-15/MCP-1/GranzymeB/Reg3A/ST2/TNFRSF1A/Elafin/MIP-1 alpha. 50 PB samples from complete response(CR) patients without CAR-T therapy were detested as normal controls to establish the normal values of 24 cytokines. 81 patients who infused CAR-T cell in Hebei Yanda Ludaopei Hospital from January to June 2020 were selected. Serum were collected on 0d, 4d, 7d, 11d, 15d, 20d and 30d after infusion, and 24 cytokines were detected by FCM Aimplex. Of which 31 patients were selected for methodological comparison between FCM and ELISA by detecting four routine cytokines, IL-6, IFN-γ, TNF-α and sCD25. 66 Patients were divided into 2 groups according to clinical manifestations, 60 patients without or mild CRS were classified as low grade CRS group, 6 patients with grade 2 or 3 CRS were classified as high grade CRS group.
Results: The comparative test showed that a good relationship between the results of ELISA and Aimplex kit, showing similar time-cytokines variation curve of PBs after CAR-T treatment. The concentrations of IL-2, IL-10, IL-12p70, IL-17A, IL-17F, MCP-1, ST-2s, IL-5, IL-6 and IFN-γ were significantly higher in high CRS grade group. By comparing the proportion of CAR-T+, CD8+ and CD4+ T cells in CD3+ cells and the concentration of cytokines in PB specimens, there were obvious positive correlations between percentages of CAR-T+, CD8+ T cells and most cytokines, and negative correlation between proportion of CD4+ T cells and most cytokines.
Conclusions: Detecting 24 kinds of cytokines by Aimplex kit is a promising method, which is time and specimens saving, and can cost-effectively reflect the cytokine situation in PBs from patients post-CAR-T treatment. Increasing the sample size and establishing a cytokines database will be helpful for monitoring, early prevention and control of side effects after CAR-T treatment.

Disclosures
No relevant conflicts of interest to declare.