CN107365748A - The memory immune cell of PMNC induction and application - Google Patents

The memory immune cell of PMNC induction and application Download PDF

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CN107365748A
CN107365748A CN201710712565.6A CN201710712565A CN107365748A CN 107365748 A CN107365748 A CN 107365748A CN 201710712565 A CN201710712565 A CN 201710712565A CN 107365748 A CN107365748 A CN 107365748A
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路春光
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Abstract

The present invention relates to immunocyte targeted therapy technical field, more particularly to a kind of human peripheral blood mononuclear cell is induced to differentiate into memory immune cell, the TCM cells of the PMNC induction of freezing provide the foundation applied to clinical cancer therapy, tumor patient T cell multiplication capacity difference and Immune anergy can be solved the problems, such as 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 PMNC induction and application
Technical field
The present invention relates to immunocyte targeted therapy technical field, more particularly to a kind of human peripheral blood mononuclear cell induction point Turn to memory immune cell（TCM）, further relate to memory immune cell（TCM）Application.
Background technology
Its antineoplastic immune is recovered or strengthened to adoptive cell therapy technology to break tumor patient maincenter and peripheral immune tolerance Function provides a kind of effective method.But many problems demands at present also be present and solve, including：1) abundant number how is obtained The tumor-reactive T cells of amount are to determine the most critical factor of adoptive cell therapy effect；2) immunocyte fed back at present Especially cloning CD8+T cells are difficult to long-term surviving in vivo, the strong influence clinical efficacy of adoptive cell therapy.
Memory t cell in vivo again meet with same antigen when can prompt activation play immunological effect, while have from My updating ability, using the teaching of the invention it is possible to provide more long-term immunoprotection, may be provided 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.
The content of the invention
In order to solve the problem of immunocyte is difficult to long-term surviving in vivo in adoptive cell therapy technology, lacks targeting； This application provides a kind of PMNC from cryopreservation（PBMC）In isolate and purify memory t cell（TCM）, The high abductive approach of TCM induced activation rates, then by tomour specific tcr gene transfection into autologous TCM, there is immune cell therapy Long-term effect and targeting.
What the present invention was obtained through the following steps：
The present invention preserves human peripheral blood mononuclearcell using cryopreservation technology, in advance the immunocyte of storage health, when When patient needs treatment in the future, recover and isolate and purify TCM, and it is killer T cell to induce, tomour specific tcr gene transfects Autologous TCM, makes immune cell therapy have targeting.Solve tumor patient memory t cell multiplication capacity difference and Immune anergy The problem of.
A kind of human peripheral blood mononuclear cell cryopreservation isolates and purifies with memory t cell after recovery, induced activation With tomour specific tcr gene transfection into autologous TCM method, comprise the following steps：
Healthy People PBMC cryopreservation resuscitations, TCM is therefrom isolated and purified, after studying Activated in Vitro, the effect T in CIK cell and TCM sources Cell（TE）The difference of cell differentiation phenotype.And CIK and TCM is modified with specific for tumour antigen tcr gene, specify tcr gene Modify CIK and TCM antineoplastic immune function.
1.CIK is induced：PBMC is according to cell density (0.4-1.2) × 106/ mL is resuspended with X-VIVO15, adds 300- 2000u/mL IFN-γs and 50-100u/mL ATP Fiber differentiation CIK cells, second day complement factor 50-200u/mL IL-1 α, 500-2000u/mL IL-2,500-1000 u/mL IL-7,50-200ng/mL CD3 monoclonal antibodies, 50-200ng/mL CD28； Hereafter cell growth state is observed daily, and the X-VIVO15 culture mediums containing 500-2000u/mL IL-2 were added every 1-2 days.
2.TCM sorting, stimulation and gene transfects
(1) magnetic bead negativity screening CD8+T cells, secondary positive sorting TCM.
(2) collaboration stimulates antibody/cell factor primary stimulus（The 1st day after culture）.With 300-2000u/mL IFN-γs, Anti-human 50-200ng/mL CD3 monoclonal antibodies, 50-200ng/mL CD28 monoclonal antibodies and 500-2000u/mL recombined humans IL-2 and 50-200u/mL IL-1 α complete primary stimulus.Every 2 days half amounts change liquid, add 500-2000u/mL recombinant human il-2s, 500-1000 u/mL IL-7 and 500-1000 u/mL recombined humans IL-15.
（3）Recombinate TCR Adenovirus Transfections（The 3rd day after culture）CIK cell and TCM cells.
（4）Carry out tumour antigen and stimulate again within 5 days after culture.
（5）Trypan blue staining obtains growth curve after CIK cell and the activation of TCM stimulated in vitro（To in vitro culture to 35 days）.
（6）Cell quantity peak period after the detection activation of flow cytometry fluorescence in situ hybridization（14th day）CIK cell with TCM sources TE telomere relative lengths.
3.CIK and TCM sources TE cell differentiation phenotypes：Flow cytometry CIK cell is with TCM after stimulating and activating Mark CIK and TCM such as CD45RO, CD62L, CD44, CCR7, CD28 sources TE cell differentiations phenotype note developed by molecule and dynamic change （The 33rd day after in vitro culture）.
6.TCR genes transfection CIK and TCM source TE antineoplastic immune function：
（1）External source tcr gene expression efficiency after Flow cytometry Adenovirus Transfection（The 3-14 days after transfection）；
（2）CTL activity：Tumour antigen is stimulated one day after again, and target cell is marked with calcein（HepG-2, SMMC-7721, MCF-7）
Detection is respectively derived from CIK cell, TCM, tcr gene transfection CIK cell, the tcr gene transfection TCM different effects of each group TE Target compares the percent specific lysis of target cell.
（3）Antibody blocking experiments：Anti-human HLA-A2 antibody is incubated altogether with HLA-A2 positive target cells HePG-2 and SMMC-7721 After educating, calcein method for releasing detects the CTL activity influence that antibody blocking transfects TCM source TE on tcr gene.
（4）Cell cracking nucleoprotein secretion：Tumour antigen stimulates one day after again, and each group TE cytosiies are in target cell 4 hours after HEPG2, SMMC7721.The perforin and granzyme point newly synthesized in intracellular cytokine dyeing method detection T cell Secrete.
（5）Cytokine secretion：Tumour antigen stimulates one day after again, each group TE cytosiies in target cell HEPG2, After SMMC77-721, MCF24 hour, IFN-r in ELISA method detection cell culture supernatant, IL-2 contents.
7. statistical analysis
Experimental data is represented with mean scholar standard deviation.Statistical analysis is carried out using SPSS16.0 statistical softwares.Each index is advanced Row normality and homogeneity test of variance.The horizontal data of two factor two compares the variance analysis using 2x2 Factorial Designs（Difference group is thin Born of the same parents' anti-tumor activity compares, and antibody suppresses experiment, the cytokine secretion of each group cell.Using the variance analysis of duplicate measurements Complete the cell quantity analysis of each group cell different time place.Different groups of cell telomere lengths are entered using one-way analysis of variance Row analysis.Work as P<When 0.05, being considered as difference has statistical significance.
Preferred steps（2）TCM sortings, stimulation and tcr gene transfection：The 1st day after separation, with 1000IU/ml IFN-γs, Anti-human 130ng/ml CD3 monoclonal antibodies, anti-human 110ng/ml CD28 monoclonal antibodies and recombined human 1000IU/ml IL-2, 120u/mL IL-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 humans IL-15.
PBMC activity is saved using Cryopreservation Technology, this experiment is confirmed using the PMNC energy freezed Enough directional separation and purification TCM, after external evoked activation, the effector T cell in TCM sources（TE）Its amplification ability, cell purity and TCM cell of the cytotoxic activity with the direct inductive formation of fresh cells does not have obvious difference.In Process of in vitro, with CIK cell is compared, and the TE cells from TCM cells have higher proliferation times；Tcr gene transfection TCM cell deriveds TE's CTL activity has antigentic specificity and MHC restricted.
This experimental result provides for the TCM cells of the PMNC induction of freezing applied to clinical cancer therapy Basis, according to the therapeutic scheme of tumor patient recovery cell at any time the existing anti-tumor immunotherapy method can be made cleverer Work is changeable, solves the problems, such as tumor patient T cell multiplication 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, stomach cancer, leaching Bar knurl etc..
Beneficial effects of the present invention：
1）The present invention provides base for the TCM cells of the PMNC induction of freezing applied to clinical cancer therapy Plinth, the poor and immune nothing of tumor patient T cell multiplication capacity can be solved according to the therapeutic scheme of tumor patient recovery cell at any time 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.
Embodiment
The present invention is further described with reference to specific embodiment：
Embodiment 1
First, the separation of PBMC cells is with freezing：
The separation of a.PBMC cells：Peripheral blood is not more than 35 mL with electronic suction assisting device according to every pipe, and average mark to 50 mL centrifuges Guan Zhong, 931 g, 8 min are centrifuged, draw upper plasma into 50 mL centrifuge tubes, take 1.5 mL in EP pipes, mark, -80 DEG C Preserve.Haemocyte is diluted with NA so that haemocyte：NA is 1:1, corresponding A liquid upper strata is slowly uniformly added to after mixing, is formed Complete interface.596 g, centrifuge 20 min.It can be seen that obvious layering.First layer supernatant is abandoned in suction, carefully gently by the second layer White cellular layer is drawn in the different mL centrifuge tubes of cleaning 50 respectively.Mixed respectively to each Guan Zhongjia NA, dilution, be settled to 40 ML/ is managed.931 g, centrifuge 8 min.Supernatant is abandoned in suction, adds NA that cell is resuspended.Freeze before processing and leave and take cell suspension counting and vigor Measure.
B.PBMC cell freezings preserve：Cell is resuspended in autogamy freezen protective liquid after purification, gently mixes rearmounted -20 DEG C Less than 5-15 min to 4 DEG C of refrigerator cooling balance, while cryopreservation tube is put 4 DEG C of refrigerators and balances 15 min.It is distributed into 2-10mL In cryopreservation tube, tube wall carries out mark.Be placed on sample freezing storing box, be transferred to program temperature reduction box instrument, most at last cell be stored in- In 196 DEG C of liquid nitrogen, falling temperature gradient is as follows：
（a）4 DEG C of waits, programmed cooling instrument is put into product；
（b）0 DEG C is down to 5 DEG C/min, keeps 5min；
（c）- 10 DEG C are down to 2 DEG C/min, keeps 5min；
（d）- 45 DEG C are down to 1 DEG C/min, keeps 35min；
（e）- 90 DEG C are down to 5 DEG C/min, keeps 5min；
（f）Terminate.
2nd, recovered after PBMC freezings：The recovery cell after freezen protective 1,2,4,8,12 and 24 months：Take out cryopreservation tube, Rapidly in 37 DEG C of water-baths of input, and constantly gently shake, ensure liquid in pipe 80% dissolving in 1 min, cell is drawn onto In a certain amount of DMEM cleaning solutions, and wash cryopreservation tube 1 time, move into 50 mL centrifuge tubes, 233 g centrifuge 5 min.Abandon Clearly, appropriate culture medium is added, is gently mixed, 500 microlitres is left and taken and does counting survey cytoactive.
3rd, cell count and trypan exclusion stain survey cytoactive
Automatic Blood Cell Analyzer detects cell sample, reads the WBC numerical value shown on screen.According to formula cell number T= WBC*V calculates TCS.Prepare two 1.5 mL Ep pipes, 95 μ l DMEM solution are added in the 1st Ep pipe, at the 2nd The trypan blue solutions of 30 μ l 0.4% are added in Ep pipes.After cell sample fully mixes, draw 5 μ l samples and add 95 μ l DMEM In solution, cell suspension is made with suction pipe piping and druming mixing.Take 30 μ l cell suspensions to add in 30 μ l trypan blue solutions to mix After even, the suspension of 10 microlitres of cells and dye liquor is taken, four quadrant viable counts in Microscopic observation tally（C）Be dyed to indigo plant The dead cell number of color（N）, cytoactive is calculated using following equation：Living cell rate（%）= [100-10N/C]×100%
4th, 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%.
Embodiment 2
On the basis of embodiment 1, sorting purifying TCM cells, and external evoked and transfection is carried out, it is as follows
1.T cell subsets magnetic bead sortings：
（1）Cell count.
（2）Prepare single cell suspension（With the nylon net filter in 30 apertures）.
（3）Add buffer solution for cleaning cell, centrifugation, 1500rpm, 5min（4-8℃）
（4）Add the buffer solution of 80ul/107 cell.
(5) the antibody coating magnetic bead of 20ul/107 cell is added.
（6）4-8 DEG C is incubated 10 minutes.
（7）Add the buffer solution for cleaning cell of 1ml/107 cell and centrifuge, 1500rpm, 5min（4-8℃）.
（8）Cell is resuspended in the buffer solution for adding 500ul/107 cell.
（9）Splitter is got out, and MS posts are cleaned with buffer solution 500ul.
(10) cell suspension is carefully added to sorting post basal surface.
(11) 500ul wash buffer pillar is used,（New liquid is added during each liquid noresidue）Altogether three times
(12) pillar is removed into magnetic field into a suitable container, slightly firmly rinsed with 1ml buffer solutions.
(13) cell is collected, is screened for CD8+ negativity, collection step（11）The cell suspension of outflow；
For CD62L, CD44 positive-selecting, collection step（12）The cell suspension of outflow.
2. TCM stimulation transfects with gene
（1）The PBMC that separation obtains is resuspended with X-VIVO15, with anti-human 50-200ng/mL CD3 monoclonal antibodies, 50-200ng/ The anti-human CD28 monoclonal antibodies of mL and 500-2000u/mL recombinant human il-2s complete primary stimulus.Every 2 days half amounts change liquid, add 500-2000u/mL recombinant human il-2s, 500-1000 u/mL IL-7 and 500-1000 u/mL recombined humans IL-15.
（2）Recombinate TCR Adenovirus Transfections（The 3rd day after culture）CIK cell and TCM cells.It is by PBMC adjustment concentration 1x106In/mlX-VIVO15 culture mediums.Recombinant virus particle is added in different MOI values ratios, is incubated in 37 DEG C of cell culture incubators Liquid is changed after 12H, continues to cultivate.
（3）Carry out tumour antigen and stimulate again within 5 days after culture.
（4）Trypan blue staining obtains growth curve after CIK cell and the activation of TCM stimulated in vitro（To in vitro culture to 35 My god）.
Embodiment 3
On the basis of embodiment 2 is formulated a, induce the selection of formula
The 1st day after separation, with 1000IU/ml IFN-γs, anti-human 130ng/ml CD3 monoclonal antibodies, anti-human 110ng/ml CD28 monoclonal antibodies and recombined human 1000IU/ml IL-2,120u/mL IL-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 humans IL-15.
The PBMC that separation obtains is resuspended with X-VIVO15, is resisted with anti-human 130ng/mL CD3 monoclonal antibodies, 110ng/mL People CD28 monoclonal antibodies and 1000u/mL recombinant human il-2s complete primary stimulus.Every 2 days half amounts change liquid, add 1000u/mL weights Group human IL-2 and 800 u/mL recombined humans 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 groups are control group：1000IU/ml IL-2+ 130ng/mL CD3+110ng/mL CD28+ 800 u/mL IL-15；
B groups：1000IU/ml IFN-γ+1000IU/ml IL-2 +130ng/mL CD3+110ng/mL CD28+800 u/mL IL-15；
C groups：1000IU/ml IFN-γ+120u/mL IL-1α+1000IU/ml IL-2+130ng/mL CD3+110ng/mL CD28+ 800 u/mL IL-15；
D groups：1000IU/ml IFN-γ+120u/mL IL-1α+800 u/mL IL-7+1000IU/ml IL-2+800 u/mL 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/mL IL-2 and 800 u/mL The X-VIVO15 culture mediums of human IL-15.In culture the 3rd, 6,9,12,14d, cell count is carried out using blood counting instrument；Training 14d is supported, group and control group TCM cell CD45RO+CD62L+ positive expressions are frozen using Flow cytometry.
Embodiment 4
1. flow cytometry fluorescence in situ hybridization detects telomere relative length
CD8+T cells and TCM telomere lengths are detected with PNA Kit/FITC kits.It is negative with the Epstein-Barr virus of known telomere length Cell calculates telomere relative length to T lymphocytic leukemia cells system CCRF-CEM as a control group.To exclude telomere between sample Length variation, telomere length index is calculated with non-activated PBMC.
Telomere length index calculates according to the following formula：
Telomere length index=different subgroups T cell telomere relative length/unactivated PBMC telomere relative lengths
2. recombinate adenovirus hominis Ad5F35-TRVA-TRBV transfection CIK and TCM cells
Restructuring adenovirus hominis Ad5F35-TRVA-TRBV transfects CIK cell and TCM respectively with the ratio of MOI=100.
3.CFSE labels targets cell detection CTL activities.
Antigen stimulates one day after again, marks HEPG2, SMMC-7721, MCF-7 cell with CFSE, it is thin that detection derives from CIK Born of the same parents, TCM, tcr gene transfection CIK, the TE effector cell of tcr gene transfection TCM cells compare the spy of various cells in different effects Opposite sex cracking percentage.
Percent specific lysis=experimental group fluorescent value-spontaneous release group fluorescent value）/（Maximum release group fluorescent value-from Send out release group fluorescent value）x100%
4. Flow cytometry T cell film surface markers are expressed.
In each detection time point, with the anti-human CD3 of fluorescence labeling, CD8, CD62L, CD44, CD45RO, CD28, CCR7
Each group TE cells are dyed etc. monoclonal antibody.Flow cytometry positive cell ratio.
5. the flow cytometer detection of intracellular cytokine：
Antigen stimulates one day after again, and detection each group TE cytosiies are 4 small after target cell HEPG2, SMMC-7721
When perforin and granzyme positive cell ratio.Newly synthesized into the cell with the detection of intracellular cytokine dyeing method
Perforin and granzyme B secretion.
The key instrument and reagent used in the present invention is as follows：
Serum free medium (X-VIVO15), Pague plus separating liquids, recombinanthumanifn-γ, recombinant human il-2, recombined human IL- 15th, the anti-human CD3 monoclonal antibodies of IL-7, mouse and the anti-human CD28 monoclonal antibodies 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.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not limited by embodiment System, it is other it is any without departing from the present invention Spirit Essences with made under principle change, modification, combine, replacement, simplification should be Equivalence replacement mode, is included within protection scope of the present invention.

Claims (10)

  1. A kind of 1. memory immune cell of PMNC induction, it is characterised in that by fresh separated or pass through The PMNC recovered after freezing sub-elects memory immune cell by screening and the secondary positive, after induction i.e. .
  2. 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- adds 300-2000u/mL IFN-γs, anti-human 50-200ng/mL CD3 Clonal antibody, 50-200ng/mL CD28 monoclonal antibodies and 500-2000u/mL recombinant human il-2s and 50-200u/mL IL-1 α Complete primary stimulus；
    （2）Every 2 days half amounts change liquid, add 500-2000u/mL recombinant human il-2s, 500-1000 u/mL IL-7 and 500-1000 U/mL recombined human IL-15, incubation time are 12-20 days.
  3. 3. memory immune cell according to claim 2, it is characterised in that turned after induction using restructuring TCR adenovirus Dye.
  4. 4. memory immune cell according to claim 2, it is characterised in that step（1）It is middle to use memory immune cell X-VIVO15 is resuspended.
  5. 5. memory immune cell according to claim 2, it is characterised in that step（1）The concentration of middle resuspension is (0.4- 1.2)×106/mL。
  6. 6. memory immune cell according to claim 2, it is characterised in that step（1）Middle PMNC weight After outstanding, it is mono- to add 500-1000u/mL IFN-γs, anti-human 90-200ng/mL CD3 monoclonal antibodies, 90-200ng/mL CD28 Clonal antibody and 500-1000u/mL recombinant human il-2s and 90-200u/mL IL-1 α complete primary stimulus.
  7. 7. memory immune cell according to claim 2, it is characterised in that step（2）In add 500-1000u/mL weight Group human IL-2,500-1000 u/mL IL-7 and 500-1000 u/mL recombined humans IL-15.
  8. 8. memory immune cell according to claim 2, it is characterised in that
    （1）PMNC be resuspended after, with 1000IU/ml IFN-γs, anti-human 130ng/ml CD3 monoclonal antibodies, Anti-human 110ng/ml CD28 monoclonal antibodies and recombined human 1000IU/ml IL-2,120u/mL IL-1 α complete primary stimulus；
    （2）Every 2 days half amounts change liquid, add 1000IU/ml recombinant human il-2s, 800 u/mL IL-7 and 800 u/mL recombined humans IL-15。
  9. A kind of 9. application of the memory immune cell any one of claim 1-8 in clinical cancer therapy medicine.
  10. 10. application according to claim 9, it is characterised in that the tumour includes but is not limited to kidney, maligna element Knurl, lung cancer, liver cancer, colorectal cancer, stomach cancer or lymthoma.
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