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A method for culturing and expanding CD8 T cells

A cell and nuclear cell technology, applied in the field of biomedicine, can solve the problems of unacceptable and difficult patients

Active Publication Date: 2022-05-27
SHANGHAI ICELL BIOTECH +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Obviously, such a large amount of blood collection is clinically difficult, and it is not easy for patients to accept it.

Method used

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  • A method for culturing and expanding CD8 T cells
  • A method for culturing and expanding CD8 T cells
  • A method for culturing and expanding CD8 T cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1 The production process flow of antigen-specific CD8 T cells of the present invention ( figure 2 )

[0067] 1. MLPC-plus activates and expands antigen (CMV-pp65) specific CD8 T cells

[0068] 1) Collect 20ml of peripheral blood, heparin anticoagulation

[0069] 2) PBMCs were collected by Ficoll gradient centrifugation and washed 3 times with PBS

[0070] 3) Resting overnight in 10% FCS-RPMI

[0071] 4) After adding 20% ​​K3EC, centrifuge and discard the supernatant, adjust PBMC to 1x 10 with 3% SG-AIM-V 7 / ml

[0072] 5) Add 1 μg / ml CMV-pp65 antigenic peptide (Miltenyi, Miltenyi, Cat. No. 130-093-435), incubate at 37°C for 2hr

[0073] 6) Supplement 3% SG-AIM-V to 4ml, add 4ml 2x CM, culture and passage in 6-well plate

[0074] 7) Collect cells after 7 days of culture, adjust cells to 1x 10 7 / ml

[0075] 8) Add 1 μg / ml CMV-pp65 antigenic peptide and incubate at 37°C for 2hr

[0076] 9) Fix K3EC cells with paraformaldehyde

[0077] 10) 3% SG-AIM-V...

Embodiment 2

[0086] Example 2 Construction of K562-aAPC (K3EC)

[0087] 1. Basis for the selection of costimulatory molecules

[0088] The main purpose of constructing K3EC is to provide a costimulatory signal for MLPC to activate antigen-specific CD8 T cells. It is currently known that the costimulatory signals required to activate such T cells can be provided by multiple receptors, of which CD28 is the most functional costimulatory receptor. CD28 belongs to the Ig superfamily (IgSF), and its cytoplasmic tail contains three motifs: YMNM, TPRRP and PYAP. When CD28 binds to B7 ligand (CD80 / 86), YMNM activates AP1 (activating protein 1), NF-kB (κB nuclear factor) and NFAT (activating T cell nuclear factor) through PI3K-AKT pathway and Grb2-Vav-Sos pathway ) resulted in Bcl-xL anti-apoptotic protein and IL2 cytokine expression. The PYAP motif can also activate AP1, NF-kB and NFAT transcription factors through the Grb2-Vav-Sos pathway, while the TPRRP motif mainly activates the tyrosine kin...

Embodiment 3

[0107] Example 3 Polyclonal T cell proliferation assay

[0108] aAPC usually needs to be inactivated (ie, the ability to proliferate) before being used for T cell co-culture. This inactivation method includes irradiation, mitomycin treatment, and paraformaldehyde fixation. In order to evaluate whether K3EC still has costimulatory activity after fixation, CD3 antibody was used to provide polyclonal TCR signal, and 4% paraformaldehyde-fixed K3EC provided costimulatory signal. The proliferation activity of T cells was detected by the CFSE dilution method, that is, PBMCs were labeled with CFSE (20 μM). At the same time, a CD3 antibody control group was set up in the plate, and 1 μg / ml CD3 antibody was added to each group. Detection of CD3 after 2 and 5 days of culture + Cells and CD3 + CFSE Low proliferating cells. CD3 + Proliferating cells % = (CD3 + CFSE Low cells / CD3 + cells) x 100%

[0109] Experimental results

[0110] After 2 days of culture, it showed that T ce...

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PUM

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Abstract

The present invention provides a method for expanding and culturing CD8 T cells, that is, in the presence of the following substances, inducing, expanding and culturing CD8 T cells: K3EC cells; the K3EC cells express CD2 ligand CD58 and NKG2D ligand MICA / B K562 engineered cells with CD137 ligand CD137L are used to provide the second signal of co-stimulatory receptors to activate CD8 T cells; peptide overlapping libraries of target antigens are used to provide TCR first signals to activate CD8 T cells. This method realizes the rapid and efficient expansion of antigen (CMV-pp65)-specific CD8 T cells; the CD8 T cells obtained through the expansion have high clinical application value in the prevention and treatment of CMV infection and the treatment of CMV-related cancers.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and particularly relates to a method for culturing and expanding T cells. Background technique [0002] CD8 T cells, also known as cytotoxic T lymphocytes (CTL), are an important line of defense for the adaptive immune system to construct antigen-specific immune protection mechanisms. CD8 T cells specifically recognize antigens through T cell receptors (TCRs), which are expressed in the form of immunogenic peptide epitopes embedded in MHC-I molecules (pMHCI for short) on the cell surface. [0003] Using modern cell engineering techniques to enrich and expand antigen-specific CD8 T cells from bodily fluids (such as blood) or tissues (such as tumor lesions) on a large scale, and then infuse them into patients in need of treatment, this The treatment option is called adoptive T cell therapy or Tcell therapy. Clinical data reveal that this T cell therapy can control refractory viral infections...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0783C12N5/10
CPCC12N5/0638C07K14/70535C07K14/70503C12N2501/515C12N2501/2302C12N2501/2307C12N2501/2315C12N2502/30
Inventor 董强刚张艳周瑾王春慧赵雅宁严小敏张光辉戴果鲜邵小燕
Owner SHANGHAI ICELL BIOTECH
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