Detailed description of the invention
Experimental technique is as follows:
1. people's specimen and cell strain
Melanoma, breast carcinoma, breast carcinoma, colon cancer specimen and pairing normal structure come from Saint Louis University's surgery in-patients receiving operations.Normal person's blood leukocytes layer comes (BuffyCoat) certainly in houston, u.s.a Bay area Blood Center.These researchs have obtained the approval of research examination board of school.Ficoll-Paque is separated and obtains peripheral blood lymphocytes (PBMC).The inmature CD4 of people+and CD8+t cell is separated with EasySep enrichment kit (StemCellTechnologies) and obtains.Different types of tumor cell line (melanoma, breast carcinoma, ovarian cancer, carcinoma of prostate, colon cancer and scale cancer) and normal galactophore tissue's derived cell (BN), set up purchased from American. tissue storehouse (ATCC) or this laboratory.Breast carcinoma (BC), colon cancer (CC) cell strain are incubated at keratinocyte culture fluid (containing 25mg/ml Medulla Bovis seu Bubali pituitary extract, 5ng/ml epidermal growth factor, 2mML-glutamine, 10mMHEPES buffer, 2% heat-inactivated fetal bovine serum (FCS) and close streptomycin).Other tumor cell lines are incubated at the RPMI1640 containing 10%FCS.
2. the acquisition of tumor infiltrating lymphocyte
Tumor and normal structure lymphocyte infiltration come from different tumors and normal structure, and method sees reference document [Peng, G., etal.Toll-likereceptor8-mediatedreversalofCD4+regulatoryTcellfunction.Science309,1380-4 (2005) .PengG., etal.Tumor-infiltratinggammadeltaTcellssuppressTanddendr iticcellfunctionviamechanismscontrolledbyuniquetoll-like receptorsignalingpathway.Immunity27,334-8 (2007)].Key step is as follows: with collagenase IV, hyaluronidase and desoxyribose enzymic digestion after tissue chopping; Postdigestive cell, after RPMI1640 washing, is incubated in the RPMI1640 containing the IL-2 of 10% human serum, l-glutamine, 2 mercapto ethanol and 50U/ml.
The synthesis of 3.TLR8 part
(A) the TLR8 part Poly-G3 of synthetic, AG*G*GA.A represents adenine, and G represents guanine.* represent sulfo-phospholipid key to modify.Synthesized by Invitrogen company.
(B) natural TLR8 part ssRNA40/LyoVec, is synthesized by Invivogen company.
(C) negative control part, Poly-T10, T*TTTT*T*T*T*T*T.T represents gland gland pyrimidine.* represent sulfo-phospholipid key to modify.Synthesized by Invitrogen company.
4. aging relevant beta galactosidase (SA-β-Gal) dyeing
In aging T cell, the detection of SA-β-Gal activity is with reference to former document (YeJ., etal.HumanregulatoryTcellsinduceTlymphocytesenescence.Bl ood, 2012,120:2021-2031).Key step is as follows: the inmature CD4 that anti-CD3 activates+or CD8+t cell after 1 day with the ratio of 1:1 and tumor cell or normal structure derived cell Dual culture, is separated and continues cultivation 3 or 5 days.After the cell PBS washing of process, be fixed on 3% formaldehyde, with freshly prepared SA-β-Gal nitrite ion (1mg/mlX-gal, 5mMK3fe [CN]6, 5mMK4fe [CN]6, 2mMMgCl2in PBS, Ph6.0.) 37 DEG C, overnight incubation.Cell after dyeing, after water washs gently, is placed in basis of microscopic observation.
To some experiment, co-culture system contains following TLR part or inhibitor.TLR part comprises: Pam3CSK4 (TLR2 part, 200ng/ml), Poly (I:C) (TLR3 part, 25 μ g/ml), LPS (TLR4 part, 100ng/ml), Flagellin (TLR5 ligand 10 μ g/ml), Loxoribine (TLR7 part, 500 μMs), ssRNA40/LyoVec (TLR8 part 3 μ g/ml) (Invivogen, SanDiego, and oligonucleotide CpG-B (3 μ g/ml), Poly-T3 (3 μ g/ml) and Poly-G3 (3 μ g/ml) (Invitrogen synthesis) CA); CAMP inhibitor comprises: 2 ', 5 '-Didanosine (7ddA, 320 μMs) and two hydrochloride (H89,20 μMs) (Calbiochemistry, SanDiego, CA).MAPK and NF-κ B blocking experiment: tumor cell is through MAPK inhibitor U0126 (10 μMs), SB203580 (10 μMs), with SP600125 (10 μMs) or NF-kB inhibitor APDC (10 μMs) process 3 days, within the 3rd day, add Poly-G3.Continue cultivation 3 days after T cell after process is separated, then detect SA-β-Gal active.
5. flow cytometry analysis
CD4+t cell is after dyeing in the anti-human distinct antibodies cell surface or born of the same parents of PE or FITC labelling, and FACS detects CD4+t cell mark is expressed.Used people's antibody comprises: anti-CD4, anti-CD8, anti-CD28 and anti-phosphorylation ATM, all purchased from BDBiosceinces.All staining cell FACSCalibur flow cytometry analysis, the data obtained is with FLOWJo software analysis (TreeStar).
6. immunoblot experiment
The CD4 that anti-CD3 activates+t cell and MCF7 or PC3 Dual culture 0,1,3 or 5 day.The CD4 of Dual culture+with lysate [50mMTris-HCl (Ph8.0), 1%NP40,250mMNaCl, 50mMNaF, 1mMNa after T cell is separated3vO4, 1mM protein inhibitor (PMSF, AKOLINE, leupeptin) and 1mMDDT] and process.Cell pyrolysis liquid is after protein quantification, and SDS-PAGE is separated, and goes to PDVF film subsequently.Pvdf membrane through primary antibodie and horseradish peroxidase labelling two anti-hatch after chemical luminous substrate process development.In experiment, antibody used comprises: anti-phosphorylation LCK, anti-LCK, anti-phosphorylation-PKA, anti-PKA, anti-phosphorylation CREB, anti-CREB, anti-actin (CellSignalingTechnology, Danvers, MA).In some experiment, add Poly-G3 in culture fluid, the inmature CD4 after process+t cell is used for carrying out immunoblot experiment.
7. functional proliferation experiment
Proliferation experiment is [Peng, G., etal.Toll-likereceptor8-mediatedreversalofCD4 as previously mentioned+regulatoryTcellfunction.Science309,1380-4 (2005) .PengG., etal.Tumor-infiltratinggammadeltaTcellssuppressTanddendr iticcellfunctionviamechanismscontrolledbyuniquetoll-like receptorsignalingpathway.Immunity27,334-8 (2007)].Key step: wrap in 96 well culture plates of quilt at anti-CD3 (2 μ g/ml), 1 × 105from the inmature CD4 of Healthy People fresh separated+the T cell of T cell and tumor or normal tissue cell process in varing proportions (1:10,1:0.5,1:0.2,1:0.1,0:1) Dual culture in containing 2% people AB serum free culture system liquid.Cultivate after 56 hours, with the final concentration in 1 μ Ci/ hole add [3h]-thymus pyrimidine, continues cultivation 16 hours.With scintillation counter measure [3h] the mixing of-thymus pyrimidine.
8.CalceinAM transfer and Cell tracking communication block
MCF7, M628 and PC3 tumor cell is hatched in the PBS containing CalceinAM (5 μMs, Invitrogen, Inc.) 40 minutes (37 DEG C, 5%CO2).The PBS of cell containing 5%FBS washs 2 times, the CD4 activated with the ratio of 1:1 and anti-CD3+t cell Dual culture 1-3 days, adds or does not add 300 μMs of GAP27 (TocrisBioscience) simultaneously in culture fluid.FACS detects the CalceinAM being transferred to T cell.Cell tracking communication blocking experiment, the CD4 that MCF7, M628 or PC3 tumor cell and anti-CD3 activate+t cell Dual culture 1 day, adds in culture fluid or does not add GAP27.Continue cultivation after cell separation after process 3 days, then detect SA-β-Gal and express and cAMP concentration.
The detection of 9.cAMP
Tumor cell or T cell wash three times with pre-cooling PBS, then freeze thawing three times.The concentration (concrete grammar is shown in R & DSystems) of endochylema cAMP is measured with the specific ELISA of cAMP.
The generation of 10.Lentivirus-shRNA and knocking out of tumor cell gene
The design of TLR8, IRAK4, MyD88, ERK1, ERK2, P38a, JNK1, IKKa or random lenti-shRNAs and construction method, and the generation of restructuring lentivirus and shRNA carrying GFP sees above [Peng, G., etal.Toll-likereceptor8-mediatedreversalofCD4+regulatoryTcellfunction.Science309,1380-4 (2005) .PengG., etal.Tumor-infiltratinggammadeltaTcellssuppressTanddendr iticcellfunctionviamechanismscontrolledbyuniquetoll-like receptorsignalingpathway.Immunity27,334-8 (2007)].Tumor cell culture, in 24 well culture plates, adds concentrated lentivirus supernatant (5-10MOI/0.5ml culture fluid) and 8 μ g/ml polybrenes (Sigma), centrifugal 1 hour of room temperature 1000g.After transfection 3-4 days, FACS is separated GFP+and GFP-tumor cell.Further tumor cell (the GFP measuring separation+and GFP-) induce ability that is aging and generation cAMP.TLR8siRNA,5’GGTGGTGCTTCAATTAATA；IRAK4siRNA,5’GCAGCAATGGTTGACATTA；MyD88siRNA,5’GGCACCTGTGTCTGGTCTA。
11. reverse transcription PCRs
Extract total serum IgE with Trizol (Invitrogen), extract cDNA with SuperScriptIIRT test kit, with reference to the description operation of manufacturer.Reverse transcription PCR detects the expression of TLR1 to TLR9mRNA.Each sample mrna expression level is corrected with GAPDH.
12. zooperies
Rag1-/-mice and NOD-scidIL2Rgammanullmice (disappearance T and B cell) is purchased from Jackson laboratory.All zooperies have obtained the approval of SaintLouisUniversity the care of animal committee.Rag1-/-mice and NOD-scidIL2Rgammanullmouse subcutaneous injection is containing Humanmachine tumour 586mel tumor cell (5 × 106) the CD4 of 100 μ lPBS buffer .CD3 antibody (2 μ g/ml) preactivates+t cell (5 × 106/ Mus) or TS CD8+tIL586 cell (5 × 106/ Mus) (can identify specifically and kill 586mel cell) enter control group mice and tumor-bearing mice (tumor size is about 10 × 10mm) through tail vein injection.In parallel laboratory test, respectively at injection CD4+after T cell after 1,4,7 and 10 day, intratumor injection PBS (100 μ l/ Mus), LPS (10,20,50 μ g/100 μ lPBS/ Mus) or Poly-G3 (50 μ g/100 μ lPBS/ Mus).Often organize 5-10 mice.After T cell injects 12 days, collect blood, lymph node (LN), spleen (SP) and tumor; Ficoll separating monocytic cell.The people CD4 injected+t cell is separated with the magnetic bead sorting method (Microbeads, MiltenviBiotec) of antibody bag quilt, recovery, and does phenotype and functional analysis.SA-β-Gal dyes and 3H-thymus pyrimidine mixes test ditto.As for tumor growth and antitumor Immune Experiment, Humanmachine tumour 586mel tumor cell (5 × 106) in 100 μ lPBS subcutaneous injection NOD-scidIL2Rgammanullmice.3rd day, tail vein injection tomour specific CD8+tIL586 cell, give simultaneously or not give Poly-G3 (4 times, 3 days intervals).Tumor size is measured once for every four days, and calculates gross tumor volume.
Experimental result:
1. tumor cell energy inducing T cell changes the aging T cell with inhibit feature into.
In 1.1 tumor infiltrating lymphocytes (TILs), aged cells ratio raises.
Whether is one of mechanism of immune escape for research inducing T cell is aging, we obtain different tumor and normal structure and infiltrate and drench property bar cell, and analyze the ratio of aged cells.First we have detected SA-β-Gal (people's aged cells mark) positive cell ratio.Compared with the lymphocyte of originating with the normal structure of pairing, breast carcinoma, melanoma, tumor of head and neck infiltrating lymphocytes (TILs) comprise a high proportion of SA-β-Gal positive cell (Figure 1A).The loss that CD28 expresses or reduction are another important symbols of aging T cell.Thus we have detected further TILs (be respectively and derive from breast carcinoma, melanoma, tumor of head and neck) or normal galactophore tissue source lymphocyte CD 28 express situation.Result shows, the lymphocyte high expressed CD28 in normal structure source, and in TILs, CD28 significantly declines (Figure 1B).BNT: normal galactophore tissue's source T cell; BTIL, MTIL and HNTIL: be respectively the TILs deriving from breast carcinoma, melanoma, tumor of head and neck.
1.2 tumor cell lines and Naive T cells Dual culture significantly induce Naive T cells aging.
We inquire into tumor cell further, and whether directly inducing T cell is aging.Inmature CD4+t cell is separated from healthy volunteer, after activating with anti-CD 3 antibodies and variety classes tumor cell line (breast carcinoma MCF7, melanoma M586 and M628, colorectal cancer SW480 and CC2, ovarian cancer OC155, carcinoma of prostate DU145 and PC3, scale cancer SSC25 and CAL27) or normal breast cell strain BN2 and BN5 Dual culture.Dual culture, after 1 day, isolates CD4+t cell, and continue cultivation 3 days, then detect aging Relevant phenotype and function.Result shows, inmature CD4+a small amount of SA-β-Gal positive cell is only there is after T cell and normal breast cell strain Dual culture; And with different types of tumor cell line Dual culture after SA-β-Gal positive cell ratio significantly raise (Fig. 2 A).Further analysis CD28 expresses and finds, Naive T cells and different types of tumor cell (instead of normal tissue cell) Dual culture significantly can reduce the expression (Fig. 2 B) of its CD28.
1.3 tumor inducing aged cells have inhibit feature.
We further study the changing function of the aged cells of tumor inducing.[3h]-thymus pyrimidine mix experimental result display, the aging CD4 of variety classes tumor cell induction+t cell is to reactive CD4+t cell has very strong inhibited proliferation (Fig. 3).
2. the endogenous cAMP in tumor source is the critical mediator that inducing T cell is aging.
In 2.1 tumor cells, a large amount of cAMP can pass to the T cell of Dual culture.
Existing research confirms, tumor cell can set up an anaerobic environment, thus causes the rising of tumor locus adenosine and cAMP.Metabolite caused by these anoxias can protect tumor cell from tumour-specific CD4+t cell and CD8+the antineoplastic immune of T cell mediation.CAMP, as one of regulatory T cells (Treg) mechanism playing inhibit feature, can suppress generation and the CD4 of IL-2+t cell propagation [Bopp, T.etal.Cyclicadenosinemonophosphateisakeycomponentofregu latoryTcell-mediatedsuppression.JExpMed204,1303-10 (2007)].Whether these researchs impel us to go to confirm that the cAMP in tumor source is also aging with inducing T cell relevant.Result shows, and cAMP is at tumor cell M628, and the level in PC3 and MCF7 is significantly higher than normal breast cell (Fig. 4 A).With tumor cell M628, PC3 and MCF7 Dual culture after one day, cAMP is at CD4+the level of T cell significantly raises (Fig. 4 B).Existing research display, regulatory T cells transmits cAMP [Bopp by Cell tracking communication to effector T cell, T.etal.Cyclicadenosinemonophosphateisakeycomponentofregu latoryTcell-mediatedsuppression.JExpMed204,1303-10 (2007)].We infer that tumor cell may utilize similar mechanism, transmit cAMP, thus inducing T cell are aging to target T cell.Add cell gap junction inhibitory polypeptide GAP27 in co-culture system after, significantly can reduce cAMP level (Fig. 4 B).And GAP27 significantly can reduce the T cell aging (Fig. 4 C) of tumor cell induction.* p<0.05, * * p<0.01, compares with matched group.
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The T cell of 2.2cAMP inhibitor energy remarkable inhibition tumor cell induction is aging.
Recent research shows, and cAMP is by the activation of PKA-CSK-LCK suppressor T cell.We have detected LCK and cAMP downstream signaling molecule PKA further and cAMP reactive element binds albumen (CREP) level and Expression of phosphorylated level thereof.Result shows, the aging CD4 of PC3 and MCF7 cell induction+lCK, CREB and PKA phosphorylation (Fig. 5 A) in T cell, prompting tumor cell can induce LCK inhibition signal path in aging T.For the effect of further cAMP in tumor inducing T cell is aging, we are at anti-CD3 preactivate CD4+cAMP inhibitor [7ddA (320uM), or H89 (20uM), or both synergy] is added in T cell and tumor cell co-culture system.Found that, the T cell aging (Fig. 5 B) of the remarkable reversing tumor induction of cAMP inhibitor energy.Further, Pharmacological inhibitors 7ddA and H89 pre-treatment of tumor cells significantly can reduce the CD4 of Dual culture+cAMP level (Fig. 5 C) in T cell.* p<0.05, * * p<0.01, compares with matched group.
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The inmature CD4 of the remarkable reversing tumor cell induction of 3.TLR8 part+t cell is aging.
Chronic infection and inflammation are tumorigenic key factors.Multiple clinical before and in zoopery prompting tumor cell different TLR stimulate or promote or the growth [Huang of Tumor suppression, B., etal.TLRsignalingbytumorandimmunecells:adouble-edgedswor d.Oncogene27,218-24 (2008); Paulos, C.M.etal.Toll-likereceptorsintumorimmunotherapy.ClinCanc erRes13,5280-9 (2007); Smits, E.L., etal.TheuseofTLR7andTLR8ligandsfortheenhancementofcancer immunotherapy.Oncologist13,859-75 (2008)].Therefore, we suppose that TLR signal path also can affect the function of the aging and TIL of the T cell of tumor inducing.For this reason, we are by tumor cell line MCF7, PC3 and M628 and anti-CD3 preactivate CD4+t cell Dual culture, and in co-culture system, add different TLR part, then detect CD4+t cell aging.Fig. 6 A shows, and only has the inmature CD4 of the remarkable reversing tumor cell induction of TLR8 part Poly-G3 and ssRNA40+the ability that T cell is aging.For getting rid of TLR part to CD4+there is direct acting possibility in T cell, we have detected TLR part and the CD4 being located away from two healthy volunteers+cell senescence after T cell Dual culture, finds that various TLR part directly can not induce CD4+t cell aging (Fig. 6 B).
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The T cell of 4.TLR8 part reversing tumor induction is aging relates to specificity T LR8 signal path, comprises the signaling molecules such as MyD88, IRAK4, ERK1/2 and P38.
Our nearest experiment confirms the relation of TLR8 signal path and Treg inhibit feature.Poly-G3 and ssRNA40 can reverse naturally-occurring CD4 completely+cD25+treg and tumor source CD4+and CD8+inhibit feature [Peng, G., the etal.Toll-likereceptor8-mediatedreversalofCD4 of gamma delta T reg+regulatoryTcellfunction.Science309,1380-4 (2005); PengG., etal.Tumor-infiltratinggammadeltaTcellssuppressTanddendr iticcellfunctionviamechanismscontrolledbyuniquetoll-like receptorsignalingpathway.Immunity27,334-8 (2007)].Therefore, we want to confirm that Poly-G3 and ssRNA40 reversing tumor cell induction T cell is aging whether also by TLR8 signal path.We utilize viral vector to carry siRNA gene Knockout, the slow virus shRNA of MCF7, PC3 and M628 cell transfecting energy specific knockdown TLR8, MyD88 and IRAK4 or contrast shRNA, then detects Poly-G3 to the impact of the aging ability of its inducing T cell.Result shows, and knocks out tumor cell MCF7, the TLR8 in M628 and PC3, and MyD88, IRAK4 signaling molecule can block Poly-G3 to the inmature CD4 of tumor cell induction+the reverse effect (Fig. 7 A) that T cell is aging.For studying the aging TLR8 signal path downstream specific molecular of modulate tumor inducing T cell further, we block MAPK signal path (JNK, ERK1/2 and P38) in tumor cell with specific inhibitor.As shown in Figure 7 B, with inhibitor U0126 (blocking ERK1/2) pretreatment PC3 and M628 tumor, or significantly can block Poly-G3 to the inmature CD4 of tumor cell induction with U0126 and SB203580 (blocking P38) pretreatment MCF7+the reverse effect that T cell is aging.* p<0.05, * * p<0.01, compares with matched group.
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Inmature CD4+t cell and the MCF7 co-culture of cells of different siRNA transfection, add or do not add Poly-G3
Inmature CD4+t cell and the M628 co-culture of cells of different siRNA transfection, add or do not add Poly-G3
Inmature CD4+t cell and the PC3 co-culture of cells of different siRNA transfection, add or do not add Poly-G3
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Inmature CD4+t cell and MCF7 co-culture of cells, add Poly-G3 and other inhibitor U0126, SB203580, SP600125 or APDC
Inmature CD4+t cell and M628 co-culture of cells, add Poly-G3 and other inhibitor U0126, SB203580, SP600125 or APDC
Inmature CD4+t cell and PC3 co-culture of cells, add Poly-G3 and other inhibitor U0126, SB203580, SP600125 or APDC
5.TLR8 signal is aging by the T cell lowering the cAMP reversing tumor induction in tumor cell.
5.1TLR8 signal path activation energy lowers the cAMP level in tumor cell and in Dual culture T cell.
We further study the impact of TLR8 signal path on the function of the aging T cell of tumor inducing.As Fig. 8 A, [3h]-thymus pyrimidine mixes shown in experiment, and Poly-G3 pretreatment MCF7 can the rejection ability of the remarkable Naive T cells pairing effect T cell of reversing tumor process.Because cAMP is the important medium that tumor inducing T cell is aging, we study further, and whether Poly-G3 reversing tumor cell induction T cell is aging lowers cAMP level in tumor cell due to it.As shown in Figure 8 B, Poly-G3 process can significantly reduce cAMP level in tumor cell.And Poly-G3 pre-treatment of tumor cells also can significantly reduce and cAMP level in the Naive T cells of tumor cell Dual culture (Fig. 8 C).
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5.2 gene knockout MCF7 cell ERK1/2 and p38, or the cAMP level that PC3 cell ERK1/2 signaling molecule can significantly stop Poly-G3 to cause reduces.
We verify whether TLR8 signal downstream passages can participate in regulation and control Poly-G3 reversing tumor intracellular cAMP levels further.We utilize viral vector to carry siRNA gene Knockout.The slow virus shRNA of MCF7 and PC3 cell transfecting energy specific knockdown ERK1, ERK2 or P38 molecule or contrast shRNA, then detects cAMP level in the tumor cell after Poly-G3 process.As shown in Figure 9, knock out ERK1/2 and p38 molecule in MCF7 cell, or ERK1/2 molecule in PC3 cell, significantly can block the cAMP level reduction that Poly-G3 causes.On the contrary, contrast shRNA can not block the cAMP level reduction that Poly-G3 causes.* p<0.01, compares with matched group.
6.TLR8 signal path can reverse the inmature CD4 of human melanoma cell mice Immune inducing in vivo+t cell is aging.
Our experiment in vitro has confirmed that tumor cell energy inducing T cell is aging.Therefore, with animal model, we confirm whether tumor cell induces Naive T cells to be transformed into the aging T cell with inhibit feature in vivo further.We are employment 586mel melanoma cells s inoculation Rag1-/-mice (lacking T and B cell), sets up the mouse model of lotus people 586mel melanoma cell.The inmature CD4 of anti-CD 3 antibodies preactivate+t cell enters the Rag1-/-mice of lotus people 586mel melanoma cell through mouse tail vein injection.After 12 days, collect blood, lymph, spleen and tumor tissues, be separated people CD4+t cell, detects aging and function.As shown in Figure 10 A, at contrast Rag1-/-mice, about 10-15% Naive T cells changes aged cells (SA-β-Gal is positive) into.And at the Rag1 of lotus people 586mel melanoma cell-/-mice, aging T cell significantly increases (>50%), and prompter's tumor cell can induce Naive T cells aging in vivo.We use further [3h]-thymus pyrimidine mix experiment have detected the CD4 reclaimed in Mice Body+t cell pairing effect CD4+the impact of T cell propagation.As shown in Figure 10 B, CD4+t cell proceeds to lotus tumor 586melRag1-/-after mice, there is very strong inhibitory action to the propagation of reaction-ive T cell.We examine whether by regulation and control TLR8 signal path aging to the T cell of preventing mouse interior tumor to induce further.The inmature CD4 of preactivate+t cell injects the Rag1 of lotus people 586mel melanoma cell through caudal vein-/-mice.Inject CD4+after T cell 1,4,7,10 day, intra-tumoral injection TLR8 part Poly-G3.If TLR4 ligand L PS and PBS group are matched group.Inject CD4+within after T cell 12 days, detect the aging and inhibit feature of the T cell reclaimed from organ and tumor tissues.As illustrated in figure 10 c, intra-tumoral injection Poly-G3 can significantly block aging induction and proceed to lotus tumor 586melRag1-/-inmature CD4+t cell is aging.And in LPS and PBS group, we do not observe, and the T cell of tumor inducing is aging to be reversed.Intra-tumoral injection Poly-G3 significantly can reverse and proceed to lotus tumor 586melRag1-/-inmature CD4+the inhibit feature (Figure 10 D) of T cell.
* p<0.05, * * p<0.01, compares with matched group.
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It is aging thus strengthen its antitumor action that 7.TLR8 signal path can block tumor cell induction specificity antineoplastic effector T cell.
The specificity antineoplastic effector T cell that 7.1TLR8 signal path can reverse human melanoma cell Immune inducing in vivo is aging.
Whether we study tumor further can be transformed into aging T cell by tumor specific effector cell in vivo.We inject NOD-scidIL-2Rgamma by under Humanmachine tumour 586mel cell skinnullmice sets up the NOD-scidIL-2Rgamma of lotus melanoma 586melnullmouse model.Human tumor-specific CD8+tIL586 cell injects the NOD-scidIL-2Rgamma of lotus melanoma 586mel through caudal veinnullmice.Within 12 days, reclaim tumour-specific CD8 afterwards+tIL586 cell, detects its aging and inhibit feature.As shown in Figure 11 A, human tumor-specific CD8+tIL586 cell proceeds to the NOD-scidIL-2Rgamma of lotus melanoma 586melnullafter mice, SA-β-Gal positive cell ratio significantly increases.And, reclaim the NOD-scidIL-2Rgamma from lotus melanoma 586nullthe CD8 of mice Different Organs and tumor tissues+tIL586 cell has very strong inhibit feature (Figure 11 B).We study the NOD-scidIL-2Rgamma whether TLR8 signal path can stop lotus melanoma 586mel furthernullaging and the changing function of mouse interior tumor specific C D8+TIL586 cell.Found that, intra-tumoral injection Poly-G3, instead of LPS or PBS, significantly can reduce the NOD-scidIL-2Rgamma of lotus melanoma 586melnullaging CD8 in Mice Body+the ratio (Figure 11 C) of TIL586 cell.In addition, intra-tumoral injection Poly-G3 also can significantly reverse the CD8 proceeding to lotus melanoma 586mel mice+the rejection ability of TIL586 cell.These results of study illustrate that human tumor cells also can become the aged cells with inhibit feature by inducing tumor-specific effector T cell, and TLR8 signal path acts on the change (comprising naivety and effector T cell) that tumor cell can reverse its function and effect.* p<0.05, * * p<0.01, compares with matched group.
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7.2TLR8 signal path is aging and strengthen its antitumor action by Tumor suppression specific T-cells.
We inject NOD-scidIL-2Rgamma by under Humanmachine tumour 586mel cell skinnullmice.After 3 days, vein injects tumour-specific CD8+tIL586mel cell, subsequently at tumor injection injection location TLR8 part Poly-G3.As shown in figure 12, Humanmachine tumour 586mel cell can at NOD-scidIL-2Rgammanullramp in Mice Body; CD8+the growth of the remarkable Tumor suppression of TIL586 cell energy; Tumor locus injection Poly-G3 significantly can strengthen CD8+the antitumor action of TIL586 cell.