CN105219711A - The culture system of a kind of CIKs cell and DC-CIKs cell - Google Patents

Abstract

The invention provides the culture system of CIKs cell; PD-1 inhibitor (PD-1IN) is added in CIKs cell culture system; enhance amplification quantity and the function of CIKs cell; and further by setting up DC-CIKs co-culture system; and add the quantity that PD-1IN increases cultivated CIKs or DC-effector cell greatly; decrease cultivated days, and strong (the p & lt of PD-1 inhibitor (PD-1IN) is not comparatively had to the killing activity of tumour cell yet; 0.01).

Description

The culture system of a kind of CIKs cell and DC-CIKs cell

Technical field

The present invention relates to cytokine induced kill cell (cytokine-inducedkillers, CIKs) technical field, particularly, a kind of culture system of CIKs cell and DC-CIKs co-culture of cells system and the application in the cellular immunotherapy Chinese traditional medicine being prepared in tumour thereof is related to.

Background technology

Cancer problem is increasingly severe, and the Prevention and controls of cancer faces huge challenge.IARC of the World Health Organization is thought, following pathogenesis of cancer number every year will with the speed increase of 3% ~ 5%, and estimate that will there be 2,000 ten thousand cancer new cases in the year two thousand twenty whole world, death will reach 1,200 ten thousand.From sickness rate, middle and low income National Cancer sickness rate is far above developed country.Current China cancer is obvious situation occurred frequently, and in city, cancer has accounted for 25% of dead sum, and rural area is 21%, has occurred cancer township and cancer village.Cancer patients is rejuvenation trend.According to " Chinese tumour registration annual report in 2012 " report, the overview of China's tumor incidence and mortality ratio is: every 100,000 people have 286 people to suffer from cancer, has the probability cancer stricken of 22% in life; Every 100,000 people have 181 people to suffer from cancer death, have the probability of 13% to suffer from cancer in life dead.The ratio of men and women's tumor incidence is 1.3:1, and the ratio of tumor mortality rate is 1.65:1.Tumor incidence rises gradually with population ages, and particularly within more than 50 years old, increase and significantly rise with the age, within more than 50 years old, account for more than 80% of all morbidities, annual tumor mortality number is 140-150 ten thousand.

Conventional oncotherapy means, namely perform the operation, radiation and chemotherapy, for progressivity or late tumor still feel simply helpless.Many tumour patients finally die from transfer and the resistance of tumour.The team of Chinese Academy of Sciences Zeng Yixin academician leader finds that chemicotherapy may cause genomic instability, tumour cell transformation becomes tumor stem cell (Cancerstemcells, CSCs), namely produce new tumour " seed ", this may be the important mechanisms recurred after tumor chemoradiotherapy.The object of oncotherapy kills and wounds the residual tumor that can not excise completely of performing the operation, but the intrinsic characteristic of tumour cell and tumor microenvironment limit the curative effect of ripe or just under study for action treatment plan.Tumour cell comes from host, carries the characteristic of host, and therefore various side effect meeting limit treatment window, is difficult to obtain the effective result for the treatment of of tumour.And due to the characteristic of tumour variation, tumour has obvious rebound effect to conventional planning and chemotherapy.Such as, after most of tumour cell destroys by cytotoxic chemotherapy agents, on a small quantity the cell of this drug resistant is still had the ability to become new tumour stove.What is worse, this tumour no longer produces reaction, because tumour cell can produce resistance under the selective pressure of cytotoxic drug to previously effective treatment.Namely to survive under normal operation and the part tumour cell be selected result in the recurrence of tumour and development, transfer.Because tumour cell has a kind of important genetic flexibility, so the resistance to any lethality pressure, can be chosen by evolution in tumor cell colonies, the variation of this similar HIV and the resistance of bacterium.Successfully to treat tumour, the multiple targeting preparation for the different survival mechanisms of tumour cell may be needed.But compare with HIV, tumour cell has larger genome, so available hereditary space is far longer than HIV in tumour cell evolution.It is quite challenging that present proof kills tumour effectively, and the survival mechanisms because Oncogenome can be evolved out in the reaction of the multiple choices pressure to medicine, even if be also like this during conbined usage medicine.Differentiate various survival mechanisms as much as possible in oncology, and find the method suppressing these mechanism.

Usually there are two kinds of solutions: attack tumour cell itself and change over the environment attacked and maintain tumor growth and survival, or make immunity system remove killing tumor cell as tackling infection.Front a kind of strategy is passive with regard to its essence because tumour cell kill and wound by indirectly approach.Such as, anti-angiogene treatment blocks the blood supply of tumour, thus killing tumor cells indirectly.This treatment is not easy induction and produces resistance, because the stroma cell in tumor environment is relatively stable on gene.But due to its passive-type, this treatment is still easily recurred because of the evolution of tumour cell, such as, in anti-angiogene treatment, blood vessel can reproduce again, may because nutrition and low-oxygen environment cause the generation of tumor stem cell.In contrast, a kind of active, more attractive strategy is, in tumour patient, " wake " response to active immunization up, makes it participate in antineoplaston.The Main Function of this strategy is the heterogeneity of evading tumour, and this heterogeneity is the result that tumour cell reacts selective pressure.In this, immunity system is particularly suitable for removing tumour cell remaining on a small quantity, and especially that radiation and chemotherapy is difficult to the stationary phase cells killed or tumor stem cell, and this contributes to extending Sulfurless fixative.In fact, even if this treatment does not cure tumour, and be just be similar to the such sub-clinical state of HIV by tumour cell reversing, tumour can be allowed to tilt to immunity system when the immune monitoring of activation can be escaped, make tumor growth progression slowly or reverse and disappear.

Pay special attention to immune cell therapy tumour both at home and abroad at present.FDA in 2010 ratifies DC cell vaccine treatment prostate cancer and has milestone significance, and the DC vaccine that countries in the world have the various different tumour of 200 multinomial treatment enters clinical I, II phase.In recent years except DC cell vaccine, other immunocytes are also for the cellular immunotherapy of tumour, mainly contain: cytokine induced kill cell (cytokine-inducedkillers, CIKs), tumor-infiltrating lymphocytes (TIL), natural killer cell (Naturalkiller, NK), Capri cell (also claiming the CIK that chain type activates) and gamma delta T cells etc., and the combination of different immunocyte, mainly the effector cell of DC cell-stimulating is (as DC-CIK, and the combination therapy of panimmunity cell DC-CTL), to play the different function of these immunocytes.These immunocytes use clinically, wherein CIK application is comparatively extensive, the effector cell that DC-CIKs or CTL is more simple is good to the result for the treatment of of tumour, mainly due to the interaction between DC and effector cell CIKs and CD8+T, add the specific killing of effector cell to tumour and the activation of effector cell.But the interaction of DC cell and effector cell is very complicated, not only there is the second signal (signal-2) of the first signal (signal-1) that MHC-I-epitope polypeptide mixture and φt cell receptor (TCR) act on and the effect between costimulatory molecules and its acceptor, and the 3rd signal (signal-3) (Fig. 1) that cytokine stimulates.Effector cell activates rear generation co-suppression signal, and to weaken the injury because effector cell's excessive activation produces body, this Inhibitory receptor is called as " immunologic test point (immunecheckpoint) ".Mainly contain PD-1 and co-suppression acceptor, as CTLA-4, B and T lymphocyte Attenuation factor (BandTLymphocyteAttenuator, BTLA or CD272), Tim-3 (TcellImmunoglobulinandMucindomain-3), LAG-3 (LymphocyteActivationGene-3) etc.Wherein PD-1/PD-L1 signal pathway is one of most important approach, a large amount of evidence shows that PD-1 is a co-suppression acceptor, express in the T cell of activation, negative regulator T cell function [DrewM.Pardoll.Theblockadeofimmunecheckpointsincancerimmu notherapy.NATUREREVIEWS|CANCER.2012,12 (253): 252-264.].Such as, PD-1 is relevant with CD8+T cell depletion (Tcellexhausion) at the CD8+T cell high level expression of antigen-activation.Gradually lost effector function by the CD8+T cell consumed, comprise multiplication capacity, expression as the ability of the cytokines such as IL-2, TNF-α and IFN-γ, finally cause apoptosis.The PD-1+CD8+T cell consumed is at the tumor-infiltrating lymphocytes (tumor-infiltratinglymphocytes of tumour, TILs) and chronic viral infection be proven [BadouaC, HansS, MerillonN, etal.PD-1 – ExpressingTumor-InfiltratingTCellsAreaFavorablePrognosti cBiomarkerinHPV-AssociatedHeadandNeckCancer.CancerRes.20 13; 73 (1): 128-138.PaiS.AdaptiveimmuneresistanceinHPV-associatedhea dandnecksquamouscellcarcinoma.OncoImmunology.2013; 2:5, e24065.SznolMandChenL.AntagonistAntibodiestoPD-1andB7-H1 (PD-L1) intheTreatmentofAdvancedHumanCancer.ClinCancerRes.2013; 19 (5): 1021 – 1034.BauzonMandHermistonT.Armedtherapeuticviruses – adisruptivetherapyonthehorizonofcancerimmunotherapy.Fron tiersinImmunology.2014; 5:1-10.Lyford-PikeS, PengS, YoungG, etal.EvidenceforaroleofthePD-1:PD-L1pathwayinimmuneresis tanceofHPV-associatedheadandnecksquamouscellcarcinoma.Ca ncerRes.2013; 73 (6): 1733 – 1741.].But the precise mechanism of the expression inhibiting CD8+T cell function of PD-1 is not understood completely.B7-H1 (PD-L1) is the major ligand that PD-1 mediated immunity suppresses, composition formula is expressed at APCs, can be induced at the cell of lymph sample and non-lymph sample peripheral tissues, Peripheral regulation [SznolMandChenL.AntagonistAntibodiestoPD-1andB7-H1 (PD-L1) intheTreatmentofAdvancedHumanCancer.ClinCancerRes.2013 of the T cell making B7-H1 be applicable to being activated; 19 (5): 1021 – 1034.].It is effective especially that cytokine IFN-γ raises B7-H1, because IFN-gamma reaction original paper is in B7-H1 promoter region.B7-DC (PD-L, the part of another PD-1) expression be substantially confined to marrow sample DCs and scavenger cell, not express in peripheral tissues or multiple cell type widely, therefore, it is made to weaken [SznolMandChenL.AntagonistAntibodiestoPD-1andB7-H1 (PD-L1) intheTreatmentofAdvancedHumanCancer.ClinCancerRes.2013 of less effect in periphery T cell reaction; 19 (5): 1021 – 1034.].Inhibition signal is driven with the PD-1 that anti-CD-3 and anti-CD28 monoclonal antibody stimulate, block the activation [PatsoukisN of the AKT in PI3K activity and its downstream, Li, SariD, etal.PD-1IncreasesPTENPhosphataseActivityWhileDecreasing PTENProteinStabilitybyInhibitingCaseinKinase2.Moleculara ndCellularBiology.2013; 33 (16): 3091 – 3098.].Although the intracellular region of PD-1 is containing the inhibitory motifs (ITIM) on an immunity receptor tyrosine-basis and the conversion motif (ITSM) on an immunity receptor tyrosine-basis, there is inhibition function, find only ITSM by mutation analysis, be not ITIM motif PI3K/AKT activate suppress and T cell increase in be needs.PD-1 recruits SHP-1 and SHP-2, SH-2 structural domain is containing Protein-tyrosine-phosphatase, energy dephosphorylation and inactivation downstream signalling components [HebeisenM, BaitschL, PresottoD, etal.SHP-1phosphataseactivitycounteractsincreasedTcellre ceptoraffinity.JClinInvest.2013; 123 (3): 1044 – 1056.].But the effect of SHP-1/2 is not clear in the suppression of PD-1 mediation, may be there is other and play an important role in T cell suppresses with the interactional signaling molecule of PD-1 intracellular region.The research of integrator gene group method to the adjustment of CD8+T cell expressing PD-1 is utilized to identify a large amount of related signaling molecules.A basic leucine zipper transcription factor in BATF, ATF family, by allowing c-Fos/c-Jun dimer dislocation negative regulator AP-1 transcription factor, may improve consume after PD-1 connects.In addition, Blimp-1 with NFATc1 transcription factor is relevant to the T cell of consume, all regulates the expression of PD-1.According to comparing, Transcription Factor T-bet may suppress PD-1 to express, and promotes and maintains CD8+T cell response.But correlative study does not disclose the true relation between PD-1 expression and the CD8+T signal of consume.

Immunity system usually checks and removes the new cell transformed, and this is the function for monitoring of immunity.Correspondingly, tumour cell may change phenotype in order to immunoreactive attack of escaping.The relation of this " immunoediting " hypothesis general description between the infantile tumour cell and immune response of tumor originates.But a large amount of evidences shows that various different subversiveness process is shown in the follow-up progress of primary tumo(u)r and transfer, survives to keep it.It is the mechanism that tumour maintains uncontrolled growth and transfer that antineoplastic immunity is overturned.Large quantity research represents tumor microenvironment manipulation B7-H1/PD-1 molecular pathways and induction B7-H1 expression and suppresses relevant to tumour immunity and thus allow tumour progression and transfer.B7-H1/PD-1 molecular pathways is the main mechanism of tumor immune escape, and reason is as follows: first, the most important thing is, this approach relates to immune response, particularly at the negative regulation of the peripheral tissues of tumor originates and growth; The second, raise at tumor microenvironment B7-H1, and the neoplasm invasiveness T cell PD-1 activated also raises, therefore, a pernicious suppression loop may be started; 3rd, this approach is interweaved by two-way signaling, and contact is congenital to be regulated with acquired immunity.These factors make PD-1/B7-H1 molecular pathways be in central role in tumour manipulation immune response and its progress, as shown in Figure 1 [DrewM.Pardoll.Theblockadeofimmunecheckpointsincancerimmu notherapy.NATUREREVIEWS|CANCER.2012,12 (253): 252-264.].That is, the B7-H1/PD-1 molecular axis of tumour manipulation causes " good gene " to degenerate.

Immune cell therapy tumour is more and more had an optimistic view of by scientist and clinicist, is also easy to be accepted by patient.But the LAK cell of the eighties in last century and current CIKs, DC-CIKs and DC-CTL etc. achieve some impressive progresses, wherein need to add some cytokines to the cultivation of these cells, as IL-2, the amplification in vitro of immune stimulatory cell.In addition, the cultivation for CIKs needs to add IFN-γ, IL-1 α; Cultivation for DC cell needs IL-4 and GM-CSF, and the maturation of DC needs TNF-α.But adding of these factors may induce DC cell or effector cell to produce Inhibitory receptor, such as, IFN-γ can express PD-1 by inducing T cell, thus weakens T cell and produced strong cellular immunization.Therefore, increase while these immunocytes, induction of the generation of inhibition signal, effector cell is caused to reduce the killing activity of tumour cell, and the cultivation of vitro also causes Cell Telomerase Activity to reduce, telomere shorten and aging, cause the immune cell function fed back to reduce, be difficult to play a role in vivo.Be expected to address these problems [PengW, LiuC, XuC, etal.PD-1BlockadeEnhancesT-cellMigrationtoTumorsbyElevat ingIFN-InducibleChemokines.CancerRes.2012 to the suppression of these immunologic test points; 72:5209-5218.WestE, JinH, RasheedA, etal.PD-L1blockadesynergizeswithIL-2therapyinreinvigorat ingexhaustedTcells.JClinInvest.2013; 123 (6): 2604 – 2615.KearT, JingW, GershanJ, etal.PD-1/PD-L1BlockadeafterTransientLymphodepletiontoTr eatMyeloma.JImmunol.2013; 190 (11): 5620 – 5628.HamidO, RobertC, DaudA, etal.SafetyandTumorResponseswithLambrolizumab (Anti – PD-1) inMelanoma.NEnglJMed2013; 369:134-144.CreelanB.UpdateonImmuneCheckpointInhibitorsi nLungCancer.CancerControl.2014; 21 (1): 80-89].

Summary of the invention

The present invention by adding PD-1 inhibitor (PD-1IN) in the culture system of CIKs cell, enhance amplification quantity and the function of CIKs cell, and further by setting up DC-CIKs co-culture system, and add the quantity that PD-1IN increases cultivated CIKs or DC-effector cell greatly, decrease cultivated days, and PD-1IN strong (p<0.01) is not comparatively had to the killing activity of tumour cell yet.Through the growth of the post-stimulatory effector cell of PD-1IN to the tumour cell of tumor-bearing mice, there is obvious restraining effect.

Technical scheme of the present invention is as follows: a kind of culture system of CIKs cell, adds PD-1IN in CIKs cell culture system.

Described CIKs cell IL-1 α, IFN-γ and anti-CD49d McAb, and IL-2 induction obtains.

The concentration of described PD-1IN is 0.1-10ng/ml.

Application in medicine in the immunocyte consume that above-mentioned CIKs cell culture system causes because of long-term chemotherapy, radiotherapy or advanced tumor in preparation treatment.

A culture system for CIKs cell, adds DCs cell and PD-1IN in the culture system of CIKs cell.

Described CIKs cell IL-1 α, IFN-γ and anti-CD49d McAb, and IL-2 induction obtains.

Described DCs cell IL-4 and GM-CSF stimulates cultivation to obtain.

The ratio of described CIKs cell and described DCs co-culture of cells is 10-50:1.

The concentration of the described PD-1IN added is 0.1-10ng/ml.

The application of culture system in the medicine of preparation treatment tumour of above-mentioned CIKs cell.

Beneficial effect of the present invention is: the culture system of CIKs cell of the present invention, by adding PD-1IN in the culture system of CIKs cell, enhances amplification quantity and the function of CIKs cell; By setting up in the co-culture system of DC-CIKs cell, add the quantity that PD-1IN increases cultivated CIKs or DC-effector cell greatly, decrease cultivated days, and PD-1IN strong (p<0.01) is not comparatively had to the killing activity of tumour cell yet.Through the growth of the post-stimulatory effector cell of PD-1IN to the tumour cell of tumor-bearing mice, there is obvious restraining effect.

Accompanying drawing explanation

Fig. 1 is the schematic diagram of the main signal in DCs cell and initial/Resting T cells interaction process.

Fig. 2 is DCs cell cultures the 4th day, and cell attachment is agglomerating, and part is in half suspended state.

Fig. 3 CIKs cell proliferation phase cloning cluster.

The prematurity of Fig. 4 ADCs cell and ripe phenotype analytical;

Surface molecular CD3, CD4, CD8 and CD56 of Fig. 4 B flow cytometry analysis CIKs cell.

Fig. 5. analyze DC-CIKs cell to the killing activity of Caski by LDH release experiment.

Embodiment

One, the cultivation of dendritic cell (dendriticcells, DCs) cell

1, the cultivation of DCs cell

The DCs (BMDCs) of bone marrow derived carries out inducing according to the experimental technique of ShenL ' s, namely marrow is gone out from its hind leg femur with syringe, medullary cell is collected by a nylon wire, add the erythrocyte cracked liquid splitting erythrocyte of containing ammonium chloride, then cultivate with the RPMI1640 complete culture solution containing 10%FCS, put 37 DEG C, suspension cell is discarded after cultivating 4h in the incubator of 5%CO2, with containing rmGM-CSF (20ng/ml after RPMI1640 many washings, and rmIL-4 (20ng/ml PeproTech), PeproTech) complete culture solution is cultivated, cell is at cultivation 2d and 4d, abandon nutrient solution supernatant, and replace containing mGM-CSF and mIL-4 substratum with fresh, cultivate through 48h, the granulocyte adhered to is not had to be removed.The phenotype of the 4th day DCs is cultivated, as shown in Figure 2 with flow cytometry analysis.Fig. 2 shows that DCs cultivates the 4th day, and cell attachment is agglomerating, and part is in half suspended state.

The cultural method of people DCs cell is as follows: take healthy peripheral blood 80 milliliters, be separated and obtain PBMCs with lymphocyte separation medium, after cell counting, according to 10 ⁶cell/ml is layered on Tissue Culture Flask, and 4 as a child removed suspension cell, and step is below cultivated with mouse DC.

2, DCs Phenotypic examination

Flow cytometer detection is carried out through adenovirus infection with after the fresh medium containing or do not contain LPS carries out stimulation 24h.Cell concn is adjusted to be 2 × 10 ⁵/ ml, adds mouse CD80, CD83, CD86 monoclonal antibody that 1 μ lFITC marks respectively, sets up blank.4 DEG C of lucifuge reaction 30min, after washing, flow cytometer detects.FCM analysis operating process:

1), after DCs cell cultures 7d, centrifugal 800r/min, 7min, abandon supernatant;

2) the PBS washing lotion 1ml containing 2% bovine serum washs 2 times;

3) antibody (CD80, CD83, CD86) the 1 μ l that labels respectively is 5mg/L to final concentration; Negative control group adds FITC and marks sheep anti mouse lgG, and final concentration is 5mg/L;

4) 4 DEG C of lucifuges slightly shake 30min, wash 2 times with 1mlPBS;

5) add 400 μ l1% paraformaldehydes to be fixed;

6) 4 DEG C of dark places are preserved, upper machine analysis.

Two, the cultivation of killer cell (cytokine-inducedkillers, CIKs)

1, the separation and Culture of monocyte (PBMC)

1) C57BL/6 (H-2b) mouse (purchased from Guangdong Province's Experimental Animal Center) is put to death by the method for disconnected neck, with 75% alcohol immersion number minute, asepticly get spleen, mill with the blunt end of syringe, cross 200 order copper mesh, add certain volume PBS with aseptic straw to dilute, anti-cell is agglomerating, improves separating effect;

2) get 50ml centrifuge tube, add Ficoll lymphocyte separation medium, venous blood is slowly being added on lymphocyte separation medium apart from 1cm place on layering liquid interface along centrifugal tube wall, is sure not mixing; Diluted blood is 1.5:1 with the ratio of the post height of lymph parting liquid, and both total posts are high is no more than test tube 2/3;

3) centrifuge tube is put in horizontal centrifuge, the centrifugal 20min of 2000r/min in room temperature.After centrifugal, 4 layers can be divided in pipe: 1. upper strata is blood plasma, most of thrombocyte and blood dilution liquid; 2. lower floor is granulocyte and red corpuscle; 3. middle level is lymphocyte separation medium; 4. the buffy coat at parting liquid and position, blood plasma boundary is mononuclearcell layer;

4) insert buffy coat gently with capillary pipet, along tube wall sucking-off canescence gently mononuclearcell, move into another sterile centrifugation tube;

5) obtained PBMC suspension 1 times of volume PBS is washed 1 time, the centrifugal 5min of 1000r/min, abandons supernatant;

6) add the PBS washing of 2 times of volumes again, suspension cell, manual count plate counts, and the centrifugal 5min of 1000r/min, abandons supernatant, obtains bottom lymphocyte;

7) with content 5% foetal calf serum (FCS) RPMI1640 nutrient solution suspension PBMC, obtaining final concentration is the suspension cell of 5x106/ml; 8) by the cytoactive that the inspection of trypan blue dye liquor is separated: get 2 cell suspensions and add 1 2% trypan blue dye liquor, high power microscopy after 5min, dead cell dyes blueness, viable cell is not painted, count 200 cells, calculate viable cell percentage, general cytoactive should more than 95%.

The cultivation of 2.CIKs cell

By above-mentioned cell with 5x10 ⁶/ ml is added to 75cm ²tissue Culture Flask in, the each 10ul/ bottle of IL-1 α, IFN – γ and against murine CD3 antibody is added in CIKs, IL-210 μ l/ bottle is added after 24h, continue to cultivate, within every 2 days, add with the substratum of front volume equivalent, the 10th day with CD3-PE, CD56-FITC, its surface markers of CD4-FITC, CD8-FITC antibody flow cytomery.

3. Microscopic observation CIKs breeds after being separated.

Basis of microscopic observation CIKs cell proliferation mature condition, as shown in Figure 3, CIKs adds stimulating factor cultivation 24 ~ 48h to start to occur little cloning cluster, and 72h reaches peak, as there is a large amount of suspension cell cloning cluster, need dispel with dropper, and supplements nutrient solution.Observe 2 cell growth status every day, supplement nutrient solution according to cell clone group's growing state and nutrient solution color, this process does not change liquid, a liquid feeding, and along with the amplification of cell, go down to posterity sub-bottle.

Three, flow cyctometry detects DCs, CIKs phenotype

Separation and Culture the 3rd day to the 8th day, low cytometric analysis is used to detect the phenotype of immature and mature DCs cell, CIKs cell.

DCs cell PBS washing is collected once, the centrifugal 10min collecting cell of 1000r/min at above-mentioned different time.Often pipe 10 ⁵individual cell packing 5 measures pipe, adds the monoclonal antibody CD80 (FITC) of anti-mouse respectively, CD83 (FITC), CD86 (PE).Isotype control is mouse IgG, incubated at room 30min.PBS washs 1 time, adds 500 μ lPBS flow cytometer and detects analysis.Utilize software analysis data, as shown in Figure 4 A.

Collect CIKs cell PBS at above-mentioned different time and wash 1 time, the centrifugal 10min collecting cell of 1000r/min, often pipe 10 ⁵individual cell packing 5 measures pipe, adds monoclonal antibody CD3 (FITC)/CD4 (PE), CD3 (FITC)/CD8 (PE), CD3 (the FITC)/CD56 (PE) of anti-mouse respectively.Isotype control is mouse IgG, incubated at room 30min.PBS washs 1 time, adds 500 μ lPBS flow cytometer and detects analysis, utilize software analysis data, as shown in Figure 4 B.

Result shows to cultivate CIKs cell and DCs cell in vitro by one group of cytokine.After DCs cell maturation, its surface marker molecule CD83 and CD80 significantly increases, and CD14 then significantly reduces, but CD40 and CD86 change is not obvious.

Four, the phenotype analytical of CIKs cell when existing containing PD-1IN

The cultivation of CIKs is with description above, and the PD-1IN (0,1ng/ml, 5ng/ml and 10ng/ml) adding different concns for second day cultivated, by the 10th day with flow cytometry analysis its surface markers CD3, CD4, CD8 and CD56.Result shows that the cell proportion of CD3+CD56+ increases along with the increase of PD-1IN concentration.When the concentration added is the PD-1IN of 10ng/ml, CD3+CD56+ ratio reaches 45.8%, far above the ratio (25.2%) of CD3+CD56+ when not having a PD-1IN.And the quantity of CIKs also increases along with the increase of PD-1IN, PD-1IN stimulates the increase of CIKs cell quantity, this means that at the culturist CIKs of CIK be a heterogeneous population, in culturing process, the induction of cytokine may increase the expression of PD-1 in the past, and PD-1IN can block this process, thus be conducive to the amplification of effector cell and the increase of functional cell.

Table 1.10ng/mlPD-1IN is on the impact of CIKs phenotype and quantity

	CIKs(PD-1IN)	CIKs(NO PD-1IN)
			Total cellular score	5.2*10 8*	2.5*10 8
CD3+CD4	9.7％	12.2％
			CD3+CD8+	42.3％*	54.5％
CD3+CD56+	45.8％**	25.2％
			Tregs	13.2％*	20.6％

*p<0.05,**p<0.01

Five, DC-CIKs co-culture system

The cultivation of DCs cell and CIKs cell is according to method above, at the 6th day of DCs cell cultures, be added in DCs cell culture fluid by the concentration of the lysate (1mg/ml) of Caski cell and spend the night, use TNF-α (10 μ g/ml) to stimulate subsequently to spend the night, then mix with 1:50 ratio with the CIKs cell cultivated, and in culture system, add PD-1IN (10 μ g/ml), continue cultivation 6 days, then cell counted and use its phenotype of flow cytometry analysis, the results are shown in Table 2.

Table 2PD-1IN is on the impact of DC-CIKs phenotype and quantity

	DC-CIK(PD-1IN)	DC-CIK(NO PD-1IN)
			Total cellular score	7.4*10 8	3.2*10 8
CD3+CD4	6.8％	10.5％
			CD3+CD8+	35.7％	50.3％
CD3+CD56	56.3％	27.3％
			Tregs	6.1％	18.7％

Six, with DC-CIKs be below the bright PD-1IN of illustration in vivo with the lethal effect of external strengthening to target cell

In order to prove that PD-1IN can strengthening effect cell killing and wounding target cell, we with to cervical cancer cell Caski for research object.

The cultivation of 1.DCs cell and CIKs cell is the same.

The lysate (1mg/ml) adding Caski cell on the 6th day of 2.DCs cell cultures, with the R187 of HBsAg (aa183-191) (FLLTRILTI) in contrast, the concentration adding polypeptide is 5 μ g/ml, after 24 hours, DC cell maturation is stimulated with TNF-α (50ng/ml), then mix with the CIKs cultivating 7 days, ratio is 1:50, after adding the PD-1IN of 5ng/ml in the co-culture system of each ratio mixing after mixing, by the 5th day, phenotype analytical and cell counting are carried out to CIKs, and DC-CIKs cell in varing proportions and target cell Caski cell co-cultivation, blending ratio is 30:1, 50:1 and 100:1, detects the activity of serum lactic dehydrogenase (LDH) with test kit after 24 hours.

In order to detect spontaneous LDH activity, if only have target cell not have effector cell in contrast in culture hole, detect the activity of the serum lactic dehydrogenase (LDH) of maximum release.Add TritonX-100 (1% mass percentage) in culture hole and represent that maximum release is active.The calculation formula of lactate dehydrogenase activity is: (LDHtest – LDHspont)/(LDHtotal – LDHspont)] x100 (Shan, B.E., J.S.Hao, Q.X..Li, andM.Tagawa.AntitumorActivityandImmuneEnhancementofMurin eInterleukin-23ExpressedinMurineColonCarcinomaCellsCellu lar & MolecularImmunology.2006; 3 (1), 49-57).In formula, LDHtest, LDHspont and LDHtotal represent test holes, spontaneous release aperture and maximum release aperture respectively.Three multiple holes are established in experiment, shown in concrete outcome table 3.Table 3 shows that DC-CIKs (PD-1IN) killing activity to target cell Caski is the strongest.

Survival time after table 3.DC-CIK immune mouse

Seven, nude mice experimentation on animals

BALB/C (null) nude mice, age in week is 3-4 week, purchased from Guangdong Province's Experimental Animal Center, feeds in the cage of specific-pathogen free.

After the Caski cell (ATCC) cultivated is washed 3 times with PBS, adjustment cell density is 1*10 ⁷/ ml, every above-mentioned TC-1 cell of mouse hypodermic inoculation 200 μ l, inoculates after 10 days, can touch tumour with hand.PBS, DC-CIKs (NOPD-1IN), DC-CIKs (PD-1IN), CIKs (NOPD-1IN) and CIKs (PD-1IN) groups of cells is divided at random by becoming the mouse of knurl, often organize 10 mouse, intravenous injection total cellular score is 2*10 ⁶, use the same method after one week and inject again once, observe the tumor growth situation of mouse.

Immune above-mentioned each group of mouse once weekly, continuous immunity 2 weeks, namely altogether injects 4 times, every two days length by vernier caliper measurement tumour and wide, calculates the volume size (size of gross tumor volume calculates according to wide 2X long/2) of tumour.When tumor size reaches 2500mm ³time, disconnected neck puts to death mouse.The survival results of mouse as shown in Figure 5.

Result shows: PBS group mouse tumor size in 18-30 days reaches 2500mm3; After with immune cell therapy, the growth of tumour is obviously slowed down, and shows that immunocyte can the growth of Tumor suppression.Compare with CIKs (NOPD-1IN) group, CIKs (PD-1IN) is to the restraining effect of tumour more obviously (p<0.01). compares with DC-CIKs (NOPD-1IN) group, it is completely suppressed that DC-CIKs (PD-1IN) organizes mouse tumor, 2% is wherein had to disappear completely, this demonstrates our guess, meet with the external result of killing and wounding of DC-CIKs (PD-1) to tumour cell.This shows that the interaction in DC-CIK (PD-1IN) Dual culture process can urge the increase of CIK function, and PD-1IN can strengthen the killing activity of DC-CIK to tumour.

Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, its framework form can be flexible and changeable, can subseries product.Just make some simple deduction or replace, all should be considered as belonging to the scope of patent protection that the present invention is determined by submitted to claims.

Claims (10)

1. a culture system for CIKs cell, is characterized in that, adds PD-1IN [MDX-1106 (BMS-936558/ONO-4538, afullyhumanIgG4anti-PD-1mAb)] in CIKs cell culture system.

2. CIKs cell culture system as claimed in claim 1, is characterized in that, described CIKs cell IL-1 α, IFN-γ and anti-CD49d McAb, and IL-2 induction obtains.

3. CIKs cell culture system as claimed in claim 1, it is characterized in that, the concentration of the described PD-1IN added is 0.1-10ng/ml.

4. the application in the CIKs medicine in the immunocyte consume that the CIKs cell culture system as described in any one of claim 1-3 causes because of long-term chemotherapy, radiotherapy or advanced tumor in preparation treatment.

5. a culture system for DC-CIKs cell, is characterized in that, adds DCs cell and PD-1 inhibitor (PD-1IN) in the culture system of CIKs cell.

6. the culture system of CIKs cell as claimed in claim 5, is characterized in that, described CIKs cell IL-1 α, IFN-γ and anti-CD49d McAb, and IL-2 induction obtains.

7. the culture system of CIKs cell as claimed in claim 5, is characterized in that, described DCs cell IL-4 and GM-CSF stimulates cultivation to obtain.

8. the culture system of CIKs cell as claimed in claim 5, it is characterized in that, the ratio of described CIKs cell and described DCs co-culture of cells is 10-50:1.

9. the culture system of the CIKs cell as described in any one of claim 5-8, is characterized in that, the concentration of the described PD-1IN added is 0.1-10ng/ml.

10. the application of culture system in the DC-CIKs cellular therapeutic agent of preparation treatment tumour of the DC-CIKs cell as described in any one of claim 5-9.