CN109082410A - The memory immune cell of peripheral blood mononuclear cells induction and application - Google Patents

The memory immune cell of peripheral blood mononuclear cells induction and application Download PDF

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CN109082410A
CN109082410A CN201810919520.0A CN201810919520A CN109082410A CN 109082410 A CN109082410 A CN 109082410A CN 201810919520 A CN201810919520 A CN 201810919520A CN 109082410 A CN109082410 A CN 109082410A
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路春光
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Abstract

The present invention relates to immunocyte targeted therapy technical fields, in particular to a kind of human peripheral blood mononuclear cell is induced to differentiate into memory immune cell, the TCM cell of the peripheral blood mononuclear cells induction of freezing provides the foundation applied to clinical cancer therapy, can solve the problems, such as tumor patient T cell proliferative capacity difference and Immune anergy according to the therapeutic scheme of tumor patient recovery cell at any time；The immunocyte fed back at present especially cloning effector T cell is difficult to long-term surviving in vivo, the strong influence clinical efficacy of adoptive cell therapy.Memory t cell has long-acting Memorability, transfects TCM by tcr gene, it is cell targeted to assign TCM.

Description

The memory immune cell of peripheral blood mononuclear cells induction and application
Technical field
The present invention relates to immunocyte targeted therapy technical field, in particular to a kind of human peripheral blood mononuclear cell lures It leads and is divided into memory immune cell (TCM), further relate to the application of memory immune cell (TCM).
Background technique
Adoptive cell therapy technology is antitumor to break tumor patient maincenter and peripheral immune tolerance, restoring or enhancing its Immune function provides a kind of effective method.However there is also the solutions of many problems demands at present, comprising: 1) how to be filled The tumor-reactive T cells of dosis refracta are to determine the most critical factor of adoptive cell therapy effect；2) what is fed back at present is immune Cell especially cloning CD8+T cell is difficult to long-term surviving in vivo, the strong influence clinic of adoptive cell therapy Curative effect.
Memory t cell in vivo again meet with same antigen when can prompt activation play immunological effect, while have from My updating ability, is capable of providing more long-term immunoprotection, may provide for adoptive cell therapy more particularly suitable immune Effector cell.It is expected to establish the antineoplastic immune of longer-term after feeding back in vivo.Separation memory t cell simultaneously passes through tcr gene Transfection is expected to provide the tumor-reactive T cells with stronger survival ability and effector function.
Summary of the invention
It is difficult to long-term surviving in vivo in order to solve immunocyte in adoptive cell therapy technology, lacks asking for targeting Topic；Memory t cell is isolated and purified in the peripheral blood mononuclear cells (PBMC) that this application provides a kind of from cryopreservation (TCM), the high abductive approach of TCM induced activation rate controls immunocyte then by tomour specific tcr gene transfection into autologous TCM Treating has long-term effect and targeting.
What the present invention was obtained through the following steps:
The present invention saves human peripheral blood mononuclearcell using cryopreservation technology, in advance the immunocyte of storage health, when It when patient needs to treat in the future, recovers and isolates and purifies TCM, and inducing is killer T cell, the transfection of tomour specific tcr gene Self TCM makes immune cell therapy have targeting.Solve tumor patient memory t cell proliferative capacity difference and Immune anergy The problem of.
After a kind of human peripheral blood mononuclear cell cryopreservation and recovery memory t cell isolate and purify, induced activation With the method for tomour specific tcr gene transfection into autologous TCM, comprising the following steps:
Healthy People PBMC cryopreservation resuscitation therefrom isolates and purifies TCM, after studying Activated in Vitro, the effect T in CIK cell and the source TCM The difference of cell (TE) cell differentiation phenotype.And CIK and TCM is modified with specific for tumour antigen tcr gene, specify tcr gene Modify the antineoplastic immune function of CIK and TCM.
1.CIK induction: PBMC is according to cell density (0.4-1.2) × 106/ mL is resuspended with X-VIVO15, adds 300- 2000u/mLIFN- γ and 50-100u/mL ATP Fiber differentiation CIK cell, second day complement factor 50-200u/mLIL-1 α, 500-2000u/mLIL-2,500-1000 u/mL IL-7,50-200ng/mLCD3 monoclonal antibody, 50-200ng/mLCD28；Hereafter every Its observation cell growth state, every 1-2 days X-VIVO15 culture mediums of the addition containing 500-2000u/mLIL-2.
Sorting, stimulation and the gene of 2.TCM transfects
(1) magnetic bead negativity screens CD8+T cell, secondary positive sorting TCM.
(2) collaboration stimulation antibody/cell factor primary stimulus (after culture the 1st day).With 300-2000u/mLIFN- γ, resist People 50-200ng/mL CD3 monoclonal antibody, 50-200ng/mL CD28 monoclonal antibody and 500-2000u/mL recombined human IL- 2 and 50-200u/mLIL-1 α completes primary stimulus.Every 2 days half amounts change liquid, add 500-2000u/mL recombinant human il-2,500- 1000 u/mL IL-7 and 500-1000 u/mL recombined human IL-15.
(3) TCR Adenovirus Transfection (after culture the 3rd day) CIK cell and TCM cell are recombinated.
(4) 5 days progress tumour antigens stimulate again after cultivating.
(5) trypan blue staining obtains growth curve after CIK cell and the activation of TCM stimulated in vitro (in vitro culture to 35 days).
3.CIK and the source TCM TE cell differentiation phenotype: Flow cytometry CIK cell and TCM are after stimulation activation CD45RO, CD62L, CD44 etc. mark the source CIK and TCM TE cell differentiation phenotype and remember developed by molecule.
The antineoplastic immune function of the 6.TCR gene transfection source CIK and TCM TE:
CTL activity: tumour antigen stimulates one day after again, is respectively derived from CIK cell, TCM, tcr gene with mtt assay detection detection CIK cell is transfected, each group TE difference effect target that tcr gene transfects TCM compares target cell (HepG-2SMMC-7721, MCF-7) Percent specific lysis.
7. statistical analysis
Experimental data is indicated with mean scholar standard deviation.It is for statistical analysis using SPSS16.0 statistical software.Each index is advanced Row normality and homogeneity test of variance.Two factors, two horizontal data compares (different to organize carefully using the variance analysis of 2x2 Factorial Design Born of the same parents' anti-tumor activity compares, and antibody inhibits test, the cytokine secretion of group of cells.Using the variance analysis of duplicate measurements Complete the analysis of group of cells different time place cell quantity.Using one-way analysis of variance to different groups of cell telomere lengths into Row analysis.As P < 0.05, it is considered difference with statistical significance.
Preferred steps (2) TCM sorting, stimulation and tcr gene transfection: the 1st day after separation, with 1000IU/ml IFN-γ, Anti-human 130ng/ml CD3 monoclonal antibody, anti-human 110ng/ml CD28 monoclonal antibody and recombined human 1000IU/ml IL-2, 120u/mLIL-1 α completes primary stimulus.Every 2 days half amounts change liquid, add recombinant human il-2 (final concentration of 1000IU/ml), 800 U/mL IL-7 and 800 u/mL recombined human IL-15.
The activity of PBMC is saved using Cryopreservation Technology, this experiment is confirmed using the peripheral blood mononuclear cells energy freezed Enough directional separation and purification TCM, after external evoked activation, effector T cell (TE) in the source TCM its amplification ability, cell purity and TCM cell of the cytotoxic activity with the direct inductive formation of fresh cells does not have apparent difference.In Process of in vitro, with CIK cell is compared, and the TE cell from TCM cell has higher proliferation times；Tcr gene transfects TCM cell origin TE's CTL activity has antigentic specificity and MHC restricted.
This experimental result provides for the TCM cell of the peripheral blood mononuclear cells induction of freezing applied to clinical cancer therapy Basis can keep the existing anti-tumor immunotherapy method cleverer according to the therapeutic scheme of tumor patient recovery cell at any time Work is changeable, solves the problems, such as tumor patient T cell proliferative capacity difference and Immune anergy.
Disease of the present invention includes but is not limited to kidney, malignant mela noma, lung cancer, liver cancer, colorectal cancer, gastric cancer, leaching Bar tumor and disease of viral infection include but unlimited virus hepatitis, tuberculosis and AIDS etc..
Beneficial effects of the present invention:
1) present invention provides base applied to clinical cancer therapy for the TCM cell of the peripheral blood mononuclear cells induction of freezing Plinth can solve tumor patient T cell proliferative capacity difference and immune nothing according to the therapeutic scheme of tumor patient recovery cell at any time It can problem；
2) immunocyte fed back at present especially cloning effector T cell is difficult to long-term surviving, strong influence in vivo The clinical efficacy of adoptive cell therapy.Memory t cell has long-acting Memorability, transfects TCM by tcr gene, assigns TCM It is cell targeted.
Detailed description of the invention
Fig. 1 is the TCM cellular morphology of fresh (left figure in Fig. 1) with the mononuclearcell induction for freezing rear (right figure in Fig. 1) Feature,
Fig. 2 is TCM ratio before and after magnetic bead sorting,
Fig. 3 is to supply 1 to freeze growth curve after a group CIK cell, TCM and fresh group of TCM Activated in Vitro culture,
Fig. 4 is to supply 2 to freeze growth curve after a group CIK cell, TCM and fresh group of TCM Activated in Vitro culture,
Fig. 5 is to supply 3 to freeze growth curve after a group CIK cell, TCM and fresh group of TCM Activated in Vitro culture,
Fig. 6 is the growth curve of cell under different condition of culture,
Fig. 7 is killing activity n=3 that different effect cell compares HePG2 cell in different effect targets,
Fig. 8 is killing activity n=3 that different effect cell compares MCF-7 cell in different effect targets,
Fig. 9 is killing activity n=3 that different effect cell compares HePG2 cell in different effect targets,
Figure 10 is killing activity n=3 that different effect cell compares MCF-7 cell in different effect targets,
Figure 11 is Flow cytometry TCM cell membrane surface marker molecule CD3+CD8+CD62L+CD45RO+ expression,
Figure 12 is the expression of Flow cytometry TCM cell membrane surface marker molecule CD62L+CD44+.
Specific embodiment
Invention is further explained combined with specific embodiments below:
Embodiment 1
One, the separation of PBMC cell with freeze:
The separation of a.PBMC cell: peripheral blood is not more than 35mL, average mark to 50mL centrifuge tube according to every pipe with electronic suction assisting device In, 931 g are centrifuged 8min, and upper plasma is drawn into 50mL centrifuge tube, takes 1.5mL in EP pipe, is marked, -80 DEG C of preservations. Haemocyte is diluted with NA, so that haemocyte: NA 1:1 uniformly slowly adds to corresponding A liquid upper layer after mixing, is formed complete Interface.596 g are centrifuged 20min.It can be seen that obvious layering.It inhales and abandons first layer supernatant, it is carefully gently that the white of the second layer is thin Born of the same parents' layer is drawn to respectively in different clean 50mL centrifuge tubes.Respectively to each Guan Zhongjia NA, dilution is mixed, and is settled to 40mL/ pipe. 931 g are centrifuged 8min.It inhales and abandons supernatant, add NA that cell is resuspended.Cell suspension counting and vitality test are left and taken before freezing processing.
B.PBMC cell freezing saves: cell is resuspended in autogamy freezen protective liquid after purification, mixes gently -20 DEG C of postposition Cryopreservation tube hereinafter, put 4 DEG C of refrigerator balance 15min simultaneously by cooling 5-15min to 4 DEG C of the balance of refrigerator.It is distributed into 2-10mL jelly It deposits in pipe, tube wall carries out mark.It is placed on sample freezing storing box, program temperature reduction box instrument is transferred to, cell is finally stored in -196 In DEG C liquid nitrogen, falling temperature gradient is as follows:
(a) 4 DEG C of waitings, until product is put into programmed cooling instrument；
(b) it is down to 0 DEG C with 5 DEG C/min, keeps 5min；
(c) -10 DEG C are down to 2 DEG C/min, keep 5min；
(d) -45 DEG C are down to 1 DEG C/min, keep 35min；
(e) -90 DEG C are down to 5 DEG C/min, keep 5min；
(f) terminate.
Two, recover after PBMC freezing: the recovery cell after freezen protective 1,2,4,8,12 and 24 months: taking out cryopreservation tube, It rapidly in 37 DEG C of water-baths of investment, and constantly gently shakes, guarantees liquid in pipe 80% dissolution in 1min, cell is drawn onto one It in quantitative DMEM cleaning solution, and washs cryopreservation tube 1 time, moves into 50mL centrifuge tube, 233 g are centrifuged 5min.Supernatant is abandoned, Appropriate culture medium is added, mixes gently, leaves and takes 500ml and does counting survey cell activity.
Three, cell count and trypan exclusion stain survey cell activity
Automatic Blood Cell Analyzer detects cell sample, reads the WBC numerical value shown on screen.According to formula cell number T= WBC*V calculates total number of cells.Prepare two 1.5mLEp pipes, 95 μ l DMEM solution is added in the 1st Ep pipe, in the 2nd Ep 30 μ l, 0.4% trypan blue solution is added in pipe.After cell sample mixes well, draws 5 μ l samples and 95 μ l DMEM solution are added In, it is mixed with suction pipe piping and druming and cell suspension is made.It takes 30 μ l cell suspensions to be added in 30 μ l trypan blue solutions after mixing, takes The suspension of 10ml cell and dye liquor four quadrant viable counts (C) and is dyed to the dead thin of blue in microscopic observation tally Born of the same parents' number (N) calculates cell activity: living cell rate (%)=[100-10N/C] × 100% using following equation
Four, the PBMC rate of recovery
The PBMC rate of recovery is calculated, using formula: PBMC cell recoveries=(cell number before cell number/separation after separation) × 100%.
Observe under inverted microscope: TCM cell is in suspension growth, and uniform in size, refractivity is consistent, the part after 4d is cultivated Cell significantly increases, it is seen that cell colony, karyon density are reinforced, and volume increases, and after birth is smooth, has no protrusion, it is fresh with freeze The TCM cell of mononuclearcell induction afterwards on form (being shown in left figure and right figure in Fig. 1) without significant difference respectively.
Embodiment 2
On that basis of example 1, sorting purifying TCM cell, and external evoked and transfection is carried out, it is as follows
1.T cell subsets magnetic bead sorting:
(1) cell count.
(2) prepare single cell suspension (with the nylon net filter in 30 apertures).
(3) buffer solution for cleaning cell, centrifugation, 1500rpm, 5min(4-8 DEG C is added)
(4) buffer of 80ul/107 cell is added.
(5) antibody that 20ul/107 cell is added is coated with magnetic bead.
(6) it is incubated for 10 minutes for 4-8 DEG C.
(7) the buffer solution for cleaning cell of 1ml/107 cell is added and is centrifuged, 1500rpm, 5min(4-8 DEG C).
(8) cell is resuspended in the buffer that 500ul/107 cell is added.
(9) splitter is got out, and cleans MS column with buffer 500ul.
(10) cell suspension is carefully added to sorting column bottom surface.
(11) pillar is rinsed with the buffer of 500ul, (new liquid is added when each liquid noresidue) is total three times
(12) pillar is removed into magnetic field into a suitable container, is slightly firmly rinsed with 1ml buffer.
(13) cell is collected, CD8+ negativity is screened, the cell suspension of collection step (11) outflow；
For CD62L, CD45RO positive-selecting, the cell suspension of collection step (12) outflow.
The TCM rate of recovery is calculated, using formula: TCM cell recoveries=(PBMC cell before cell number/separation after TCM sorting Number * TCM%) × 100%.
The yield of TCM is 19 ± 2%.See Fig. 2.
2. the stimulation of TCM and gene transfect
(1) PBMC that separation obtains is resuspended with X-VIVO15, with anti-human 50-200ng/mL CD3 monoclonal antibody, 50-200ng/ The anti-human CD28 monoclonal antibody of mL and 500-2000u/mL recombinant human il-2 complete primary stimulus.Every 2 days half amounts change liquid, add 500-2000u/mL recombinant human il-2,500-1000 u/mL IL-7 and 500-1000 u/mL recombined human IL-15.
(2) TCR Adenovirus Transfection (after culture the 3rd day) CIK cell and TCM cell are recombinated.It is by PBMC adjustment concentration 1x106In/mlX-VIVO15 culture medium.Recombinant virus particle is added in different MOI value ratios, is incubated in 37 DEG C of cell incubators Liquid is changed after 12H, continues to cultivate.
(3) 5 days progress tumour antigens stimulate again after cultivating.
(4) growth curve is (in vitro culture to 35 after trypan blue staining obtains CIK cell and the activation of TCM stimulated in vitro It).
As shown in Fig. 3,4,5: after experience monoclonal antibody/cell with tumour antigen stimulation because activating.The CIK of in vitro culture is thin All there is rapid amplifying in born of the same parents and TCM quantity, and reach peak simultaneously with the 14th day or so of in vitro culture.Hereafter cell quantity It is on a declining curve, but the cell quantity of TCM is more stable, and decrease speed is slower compared with CIK cell.CIK group and TCM group cell Between quantity variance have statistical significance (F=426.56, P < 0.001), fresh group and freeze group TCM iuntercellular quantity variance without Statistical significance (F=1.75, P=0.21).
(5) hundred grades that the preparation of TCM composition should all be required in GMP in above-mentioned cell cultivation process and in following embodiment Toilet or station carry out, the quality safety of strict guarantee cell and its preparation.
Embodiment 3
On the basis of embodiment 2 is formulated a, the selection of induction formula is carried out
The 1st day after separation, with 1000IU/ml IFN-γ, anti-human 130ng/ml CD3 monoclonal antibody, anti-human 110ng/ml CD28 monoclonal antibody and recombined human 1000IU/ml IL-2,120u/mLIL-1 α complete primary stimulus.Every 2 days half amounts change liquid, Add recombinant human il-2 (final concentration of 1000IU/ml), 800 u/mL IL-7 and 800 u/mL recombined human IL-15.
The PBMC that separation obtains is resuspended with X-VIVO15, anti-with anti-human 130ng/mL CD3 monoclonal antibody, 110ng/mL People CD28 monoclonal antibody and 1000u/mL recombinant human il-2 complete primary stimulus.Every 2 days half amounts change liquid, add 1000u/mL weight Group human IL-2 and 800 u/mL recombined human IL-15.Using addition IFN-γ, IL-1 α, again on traditional TCM cell culture basis Group human cell factor IL-7.
Experiment is divided into four groups,
A group is control group: 800 u/mL IL-15 of 1000IU/ml IL-2+130ng/mL CD3+110ng/mL CD28+；
B group: 1000IU/ml IFN-γ+1000IU/ml IL-2+130ng/mLCD3+110ng/mL CD28+800 u/mL IL-15；
C group: 1000IU/ml IFN-γ+120u/mL IL-1 α+1000IU/ml IL-2+130ng/mLCD3+110ng/mL CD28+ 800 u/mL IL-15；
D group :+800 u/mL IL-7+1000IU/ml IL-2+800 u/mL of 1000IU/ml IFN-γ+120u/mL IL-1 α CD3+110ng/mL CD28+800 u/mL IL-15。
Hereafter cell growth state is observed daily, is recombinated every addition in 1-2 days containing 1000u/mLIL-2 and 800 u/mL The X-VIVO15 culture medium of human IL-15 carries out cell count using Countstar calculating instrument.Difference group iuntercellular quantity variance With statistical significance (F=189.12, P < 0.001).See Fig. 6.
Note: cell culture 6d rise, D group TCM group cell number is more compared with A, B, C group TCM cell number, difference have conspicuousness (P< 0.05)
Embodiment 4
1. mtt assay detects the killing activity that different effect cell compares different tumour cells in different effect targets
1) it is directed to the killing activity of different target cells: respectively by CIK group, antigenic stimulus CIK group, TCR transfection CIK group and through stimulating TCR transfects CIK group, acts on HePG2 and MCF-7 tumor cell line with different effect target ratio MCF-7.
Tumour cell HePG2 and MCF-7 concentration is adjusted to 1x105, is inoculated into 96 orifice plates, the hole 100ul/.
Target is imitated than being respectively 5:1,10:1,20:1, if (it is thin that target is only added in control group (CIK is only added), target cell control group Born of the same parents), maximum cracking group (1%Tritonx-100 is added), each group sets 5 multiple holes.Each group effector cell tumor-killing is detected after 4 hours Activity.
Fig. 7 the results show that tcr gene transfection group can promote CIK cell identification killing HePG2 cell, kill by the tumour of CIK Wound activity with effect target than rise and improve, difference group iuntercellular percent specific lysis difference it is statistically significant (F= 603.1, P < 0.001)
Antigenic stimulus after being transfected by tcr gene, further improves anti-tumor activity.
Each group effector cell is directed to the killing activity of MCF-7 cell
Tcr gene transfection is invalid to the breast cancer cell MCF-7 for promoting CIK cell identification AFP negative to express, AFP antigenic stimulus It is invalid for the killing activity of MCF-7 to CIK cell is improved, difference group iuntercellular percent specific lysis no statistical difference Meaning (F=1.531, P=0.213), but CIK cell anti-tumor activity is improved with effect boots than rising, and difference has statistics meaning Adopted (F=28.31, P < 0.001).See Fig. 8.
2) CTL activity of the tumour-specific tcr gene transfection source the TCM TE to HePG2 and MCF-7 tumor cell line
Influence for the transfection of identification tcr gene to TCM identification specificity, sets HePG2 and MCF-7 as target cell, has rated base Because transfecting TCM, untransfected TCM, gene transfects CIK cell, and untransfected CIK cell generates after tumour antigen stimulates activation in advance TE cell in the lytic capacity of different effect target ratios (5:1,10:1,20:1), every group is done three repetitions.
As shown in Figures 9 and 10, tcr gene transfection TCM effectively facilitates T cell cracking AFP+ target cell HePG2.Difference group is thin Intercellular percent specific lysis difference is statistically significant (F=108.1, P < 0.001)
Tcr gene transfection is on AFP- breast cancer cell MCF-7 cracking without influence, difference group iuntercellular percent specific lysis difference
Not statistically significant (F=2.17, P=0.06) prompts the effect of tcr gene transfecting T cells to have good antigen-specific Property.
Embodiment 5
Recombinate TCR Retroviral Transfer (after culture the 3rd day) CIK cell and TCM cell.
According to 7.1 sequence of liver cancer specific gene TCRa12-2 and V β, design primer, by pcr clone target gene, Recombined human retrovirus PLXPXSN-TCRa12-2-IRES-V β 7.1 is constructed, with HEK293 cell Packing Intact virus Grain, -80 DEG C of refrigerators save virion, transfect CIK cell and TCM respectively with the ratio of MOI=100.
1) retrovirus saves liquid (- 80 DEG C) and melts in 37 DEG C of water-baths.
2) virus liquid is added to six orifice plates after coating, every hole 1ml.
3) it places 4 hours for 32 DEG C.
4) virus is sucked out and saves liquid.
5) cell suspension to be transfected is added in every hole.
4. Flow cytometry T cell film surface marker molecule is expressed.
In culture the 13rd day, with the anti-human CD3 of fluorescent marker, CD8, CD62L, the dyeing of the monoclonal antibodies such as CD44, CD45RO Each group TE cell, flow cytometry positive cell ratio.See Figure 11,12.
Key instrument and reagent used in the present invention are as follows:
Serum free medium (X-VIVO15), Pague plus separating liquid, recombinanthumanifn-γ, recombinant human il-2, recombined human IL- 15, the anti-human CD3 monoclonal antibody of IL-7, mouse and the anti-human CD28 monoclonal antibody of mouse.II grade of Biohazard Safety Equipment (Termoscitific), low temperature Desktop Desktop centrifuge (Beckman Coulter), ultra low temperature freezer and carbon dioxide culture Case (Termoscitific) and stream type cell analyzer.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by the limit of embodiment System, other any changes made without departing from the spirit and principles of the present invention, modification, combination, substitution, simplification should be Equivalence replacement mode, is included within the scope of the present invention.

Claims (10)

1. a kind of memory immune cell of peripheral blood mononuclear cells induction, it is characterised in that by fresh separated or pass through The peripheral blood mononuclear cells recovered after freezing sub-elects memory immune cell by screening and the secondary positive, after induction i.e. ?.
2. memory immune cell according to claim 1, it is characterised in that induction differentiation operation is as follows:
(1) it is resuspended after the separation of memory immune cell, it is mono- that 300-2000u/mLIFN- γ, anti-human 50-200ng/mL CD3 is added Clonal antibody, 50-200ng/mL CD28 monoclonal antibody and 500-2000u/mL recombinant human il-2 and 50-200u/mLIL-1 α Complete primary stimulus；
(2) every 2 days half amounts change liquid, add 500-2000u/mL recombinant human il-2,500-1000 u/mL IL-7 and 500-1000 U/mL recombined human IL-15, incubation time are 12-20 days.
3. memory immune cell according to claim 2, it is characterised in that use recombination TCR retrovirus after induction Transfection.
4. memory immune cell according to claim 2, it is characterised in that step uses memory immune cell in (1) X-VIVO15 is resuspended.
5. memory immune cell according to claim 2, it is characterised in that the concentration being resuspended in step (1) is (0.4- 1.2)×106/mL。
6. memory immune cell according to claim 2, it is characterised in that peripheral blood TCM cell is resuspended in step (1) Afterwards, 500-1000u/mLIFN- γ, anti-human 90-200ng/mL CD3 monoclonal antibody, 90-200ng/mL CD28 Dan Ke is added Grand antibody and 500-1000u/mL recombinant human il-2 and 90-200u/mLIL-1 α complete primary stimulus.
7. memory immune cell according to claim 2, it is characterised in that add 500-1000u/mL weight in step (2) Group human IL-2,500-1000 u/mL IL-7 and 500-1000 u/mL recombined human IL-15.
8. memory immune cell according to claim 2, it is characterised in that
(1) peripheral blood mononuclear cells be resuspended after, with 1000IU/ml IFN-γ, anti-human 130ng/ml CD3 monoclonal antibody, Anti-human 110ng/ml CD28 monoclonal antibody and recombined human 1000IU/ml IL-2,120u/mLIL-1 α complete primary stimulus；
(2) every 2 days half amounts change liquid, add 1000IU/ml recombinant human il-2,800 u/mL IL-7 and 800 u/mLIL-15.
9. a kind of memory immune cell of any of claims 1-8 is in clinical cancer therapy and viral infection disease Application in medicine.
10. application according to claim 9, it is characterised in that the tumour includes but is not limited to kidney, maligna element Tumor, lung cancer, liver cancer, colorectal cancer, gastric cancer or lymthoma and disease of viral infection include but unlimited virus hepatitis, tuberculosis and AIDS etc..
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