citations

                    Induced expression of CCL19 promotes the anti-tumor ability of CAR-T cells by increasing their infiltration ability
                

Authors:

Jian-Fei Hu , Zu-Wei Wang , Cheng-Yu Liao , Zhi-Wen Chen , Feng-Ping Kang , Cai-Feng Lin , Tian-Sheng Lin , Long Huang , Yi-Feng Tian , Shi Chen

In:

Source: Frontiers in Immunology
Publication Date: (2022)
Issue: 13: 958960

Research Area:

Immunotherapy / Hematology

Gene Expression

Cells used in publication:

T cell, human stim.: Species: human; Tissue Origin: blood

Platform:

4D-Nucleofector® X-Unit

Experiment

Human T-cells were activated by the Dynabeads™ Human T-Activator CD3/CD28 (cat. no. 11161D; Thermo Fisher
Scientific, Inc.) and cultured for 24 h. Subsequently, the lentivirus was added with a multiplicity of infection of 2. The
cells were cultured with X-VIVO-15 (Lonza Group, Ltd.) with 100 IU/ml interleukin (IL)-2 (cat. no. 200-02; PeproTech China). The medium was changed every 2 days and the cells were cultured for 10 days after activation.
The T cells were activated and transduced with lentivirus as previously described. In total, 2×106 CAR-T cells were electroporated, 50pM CAS9 protein (Novoprotein, E365) and 300 pM single guide (sg) RNA were incubated together for 10min at 25°C to obtain Cas9/ sgRNA ribonucleoprotein. CAR-T cells were resuspended in 20 mlP3 Buffer(Lonza Group, Ltd.), combined with RNP and electroporated using a 4D-Nucleofector™(Lonza Group, Ltd.) with pulse code
E0115. Next, CAR-T cells were cultured with T cell medium as previously described for 4-5 days. The sgRNA targeting sequence in the CCR7 gene was 5'-CGCAACTTTGAGCGCAACA-3’ (CRISPRD HSPD0000007879).

Abstract

Background: Chimeric antigen receptor-engineered T cell (CAR-T) therapy has shown promising potential for anti-cancer treatment. However, for pancreatic ductal adenocarcinoma (PDAC), the lack of infiltrative ability of these CAR-T cells leads to sub-optimal treatment outcome.

Methods: Chemokine (C-C motif) ligand 19 (CCL19), the expression of which is regulated by the nuclear factor of activated T cell pathway, was transfected into targeting mesothelin CAR-T cells (mesoCAR-N19) using NFAT regulating element. It was expressed in activated CAR-T cells by OKT3 or mesothelin+ tumor cells but not in inactive cells. The migratory ability of these CAR-T cells was then measured. Subsequently, functional identification of these CAR-T cells was performed in vivo. In addition, the tumor lytic activity and proliferation of the CAR-T cells were measured in vitro. The degree of CAR-T cell infiltration and distribution into the PDAC tumors was examined using the immunohistochemical staining of hCD3 and the detection of CAR gene copy number by quantitative PCR. Finally, the functional assessment of chemokine (C-C motif) receptor 7 knock-out was performed in the CAR-T cells.

Results: Through in vitro Transwell assays, it was demonstrated that mesoCAR-N19 can be specifically expressed in CAR-T cells activated by tumor cells compared with conventional mesothelin CAR-T (mesoCAR) cells. We also observed that upregulating the expression of CCL19 can increase the recruitment of additional T cells. In vivo studies subsequently revealed that this highly specific recruitment of T cell infiltration is associated with enhanced tumor-suppressive activities downstream.

Conclusion: Induced expression of CCL19 can promote the anti-tumor ability of CAR-T cells by increasing their infiltrative ability. This study potentially uncovered novel method of activating CAR-T cells to enhance their infiltrative capacities, which offers a novel direction for PDAC treatment.

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