Method for in-vitro amplification of CD8+T cell and cell subset of CD8+T cell

A cell subset and cell technology, applied in the field of cell biology, can solve the problems of lack of rapid expansion of PD1+CD8+ T cells, difficulty in expansion, and difficulty in meeting anti-tumor immunity and anti-infection immunity, and achieves improved expansion. Efficiency, improve the effect of functional repair

Active Publication Date: 2017-06-13
SHANGHAI INNOVATIONAL CHANGAN BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the specific functional cell subsets necessary for anti-tumor immunity, such as PD-1+CD8+T cells, TEM CD8+T cells, etc., are difficult to expand under conventional culture conditions, and so far there is no rapid expansion of PD1 in vitro. +CD8+ T cell approach
It has been reported in the literature that the in vitro expansion efficiency of PD1+CD8+T cells sorted from cancer patients is limited, and the ability of the expanded cells to secrete IFN-γ is generally less than 20%, which is difficult to meet the requirements of anti-tumor immunity and anti-infection immunity. need

Method used

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  • Method for in-vitro amplification of CD8+T cell and cell subset of CD8+T cell
  • Method for in-vitro amplification of CD8+T cell and cell subset of CD8+T cell
  • Method for in-vitro amplification of CD8+T cell and cell subset of CD8+T cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Isolation of Peripheral Blood Mononuclear Cells

[0034] 1. Take 20ml of heparin-anticoagulated human peripheral blood in a centrifuge tube, dilute the peripheral blood with normal saline 1:1, and mix well;

[0035] 2. Take another new 50mL centrifuge tube, add 15ml of lymphocyte separation solution (ficoll), and then according to the volume ratio of ficoll: blood dilution solution 1:2, slowly add the mixed blood dilution solution along the tube wall to the upper layer of lymphocyte separation solution , so that the two form a clear layer, centrifuge at 3000rpm for 30min;

[0036] 3. After centrifugation, absorb the mononuclear cell layer into a new 50ml centrifuge tube, add 30ml X-VIVO-15 medium to wash once, centrifuge at 800g for 5min, and discard the supernatant.

[0037] 4. Add 20ml of X-VIVO-15 medium, blow and aspirate to mix, centrifuge at 200g for 10 minutes at room temperature, and discard the supernatant. Add 10ml X-VIVO-15 medium to resuspend an...

Embodiment 2

[0038] Example 2: Toll-like receptor agonists effectively stimulate the proliferation of CD8+ T cells.

[0039] 1. According to Example 1, healthy human peripheral blood PBMCs were isolated.

[0040] 2. Use EasySep™ Negative Selection Human CD8+ T cell Enrichment kit (Stemcell: 19053) to sort CD8+ T cells.

[0041] 3. Use Cell Proliferation Dye eFluor 670 (ebioscience: 65-0840-90) to stain the sorted CD8 cells.

[0042] 4. Place the cells in a 96-well U-bottom plate, 1×10 per well 5 cell.

[0043] 5. Add the following stimulators and Toll-like receptor agonists (invivogen: tlr-kit1hw, humanTLR1-9 Agonist KIT) in groups

[0044] (1) Blank;

[0045] (2) Anti-human CD3 antibody;

[0046] (3) Anti-human CD3 antibody + anti-human CD28 antibody;

[0047] (4) Anti-human CD3 antibody + TLR1 / 2 agonist-Pam3CSK4 (0.1-1μg / ml);

[0048] (5) Anti-human CD3 antibody + TLR2 agonist-HKLM (10 8 cells / ml);

[0049] (6) Anti-human CD3 antibody + TLR3 agonist-Poly(I:C) (10ng-10ug / ml);

...

Embodiment 3

[0060] Example 3: Toll-like receptor agonists effectively stimulate the proliferation of CD8+ T cell subsets.

[0061] Toll-like receptor agonists can effectively stimulate CD8+ T cell subset central memory T cells (T CM :Centralmemory T cell and effector memory T cells (T EM :effector memory T cell) proliferation.

[0062] 1. Isolation of healthy human peripheral blood PBMC

[0063] 2. Use EasySep™ Negative Selection Human CD8+ T cell Enrichment kit (stemcell: 19053) to sort CD8+ T cells.

[0064] 3. Use Cell Proliferation Dye eFluor 670 (ebioscience: 65-0840-90) to stain the sorted CD8 cells.

[0065] 4. Place the cells in a 96-well U-bottom plate, 1×10 per well 5 cells.

[0066] 5. Add the following stimulators and Toll-like receptor agonists in groups (invivogen: tlr-kit1hw, humanTLR1-9 Agonist KIT.)

[0067] (1) Anti-human CD3 antibody;

[0068] (2) Anti-human CD3 antibody + TLR1 / 2 agonist-Pam3CSK4 (0.1-1μg / ml);

[0069] (3) Anti-human CD3 antibody + TLR6 / 2 agonis...

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Abstract

The invention establishes a method for in-vitro amplification of a CD8+T cell and a cell subset of the CD8+T cell. By adding a TLR1 / 2 agonist, a TLR2 / 6 agonist and a TLR5 agonist into an in-vitro culture system for conventionally culturing and amplifying the CD8+T cell or conjunctively utilizing the agonists, the amplification efficiency of the CD8+T cell can be remarkably improved; besides, by utilizing a TLC agonist, the functional CD8+T cell subsets which are difficult to amplify under a conventional culture condition are amplified, such as PD-1+CD8+T cells and TEM CD8+T cells; and the CD8+T cells and the functional cell subsets can be rapidly and largely propagated under the common and continuous stimulation of a TLRs agonist, recombinant cell factors IL-2, IL-7 and IL-15, an anti-human CD3 antibody and anti-human CD28 antibody coated magnetic beads.

Description

technical field [0001] The invention belongs to the field of cell biology, and in particular relates to a method for inducing and expanding CD8+ T cells and functional cell subgroups in vitro. Background technique [0002] CD8+ T cells are important effector cells involved in anti-tumor and anti-infection immunity. Activated CD8+ T cells can rapidly release high concentrations of cytokines such as IFN-γ and IL-2, and have a wide range of immune regulation effects. In acute infection, CD8+ T cells are stimulated by antigens, activate and proliferate in large quantities, differentiate into effector CD8+ T cells (CTL), and kill infected cells by secreting perforin and granzyme B, or by The Fas-FasL pathway induces apoptosis in infected cells. When the pathogen is eliminated, a small number of effector CD8+ T cells differentiate into memory CD8+ T cells. When the same antigen reappears, the memory CD8+ T cells in the body will rapidly proliferate and differentiate into effector...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0636C12N2501/51C12N2501/515C12N2501/2307C12N2501/2315C12N2501/2302C12N2501/999C12N2501/998C12N2501/056C12N2501/052C12N5/0638C12N2509/10
Inventor 徐建青张晓燕邱趁丽
Owner SHANGHAI INNOVATIONAL CHANGAN BIOLOGICAL TECH CO LTD
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