CN103520198A - Method for preventing tumor-induced T cell aging and reversing immunosuppression capability of tumor-induced T cell, and use of tumor-induced T cell in antitumor immunological therapy - Google Patents

Abstract

The invention provides a method for preventing tumor-induced T cell aging and reversing immunosuppression capability of the tumor-induced T cell. A ligand of a TOLL like receptor 8 is provided to a cell; a TOLL like receptor 8 (TLR8) signal channel in a tumor cell is activated; the tumor-induced T cell aging is blocked up, so as to improve the antitumor immunity. The ligand of the TOLL like receptor 8 is characterized by being oligonucleotides and comprising adenine, guanine, a main chain connecting bond for connecting the guanine and nuclease resistance residue of adjacent nucleic acid bases (preferably a chemical bond is phosphorothioate); a nucleic acid synthesis sequence is 5'-AGG...GA-3'; G represents guanine; A represents adenine; G and G are modified by a dithio phosphate ester bond; the number of G may be 3 to10 in difference; an acid synthesis sequence of a common ligand Poly-G3 is 5'-AGGGA-3'. By injecting TLR8 ligand Poly-G3 into the tumor, the inhibitory effect of a CD8<+>T cell can be significantly enhanced.

Description

Stop tumor cell induction T cell senescence and reverse the method for its immunosuppressant ability and the purposes in anti-tumor immunotherapy

Technical field

Activating TOLL sample receptor 8(TLR8) signal is as novel and effectively immunotherapy of tumors medicine and/or tumor vaccine adjuvant.

Background technology

Increasing evidence shows effective, functional CD4 ⁺and CD8 ⁺t cell is brought into play pivotal role in the immune surveillance of tumor and antineoplastic immune.Thereby how to regulate and control immunocyte and make it identify efficiently and remove the New Policy that tumor cell has become treatment aggressive and metastatic tumo(u)r.Current many immunotherapy methods as cytokine, tumor live vaccine and adoptive immunotherapy etc., show certain effect in preclinical phase test, but the overall clinical effectiveness of these immunization therapies not good enough [Rosen berg, S.A., Yang, J.C.& Rsetifo, N.P.Cancer immunotherapy:moving beyond current vaccine.Nat.Med10,909-15 (2004)].Studies have shown that at present inhibition microenvironments that tumor forms by various strategy are major obstacles of transferring immunologic function in body and obtaining effective antitumor immunity curative effect.Recruitment and amplification inhibition tumor infiltrating lymphocyte are one of immunosuppressant main mechanisms of tumor inducing.Tumor cell can increase and recruit dissimilar tumor infiltrating immunosuppressant cell; comprise regulatory T cells (Treg); cause tolerance dendritic cell (DC); macrophage and bone marrow transplantation cell (the MSCs) [Roncarolo in tumor source; M.G.; et al.Interleukin-10-secreting type1regulatory T cells in rodents and humans.Immunological reviews212,28-50 (2006); Peng G., et al.Tumor-infiltrating gammadelta T cells suppress T and dendritic cell function via mechanisms controlled by unique toll-like receptor signaling pathway.Immunity27,334-8 (2007)].In addition, tumor cell can be secreted inhibitive factor (IL-10, TGF-β and IDO), and expression inhibiting molecule (FasL and PD-L1) directly suppresses amplification the inducing T cell apoptosis of tumour-specific T cell.Therefore how immunosuppressant microenvironment, how to understand better tumor inducing is set up and the molecular mechanism that maintains can provide theoretical foundation for developing new tumor vaccine and Immunotherapy Strategy.Strengthen exploitation reverse immunosuppressant treatment New Policy is to carry out the main challenge that successful clinical immunization therapy faces simultaneously.

Cell senescence is the limited multiplication capacity that goes down to posterity occurring in human fibroblasts incubation for describing at first.Recent research prompting is aging also occurs in people's immunocyte, thereby causes the immune dysfunction relevant to normal aging.Research is found, aging CD8 ⁺the increasing of T cell is present in the patient of chronic viral infection and some tumour patient as pulmonary carcinoma, breast carcinoma and tumor of head and neck etc.Aging T cell often has significant phenotype to change, as the rise of permanent loss, cell cycle arrest and Cell cycle-related genes p53, p21 and the p16 of costimulatory molecule CD28 expression.The more important thing is that aging T cell generating function changes, comprise kill capability disappearance, there is potential negative regulation function [Vallejo, V.N.CD28extinction in human T cells:altered functions and the program of T-cell senescence.Immunol Rev205,158-69 (2005)].Therefore, understand better the effector function how molecular mechanism that in tumor microenvironment, aging T cell produces and research to recover aging tumour-specific immunocyte antineoplastic immune and treatment are had to crucial meaning.

TLR activator has become an active research field as the medicine of vaccine adjuvant or anti-curing oncoma and infectious disease.[Wang,R.F.,et?al.Toll-like?receptors?and?immune?regulation:implications?for?cancer?therapy.Oncogene，27,181-9(2008)]。Multiple TLR part, comprises that imiquimod (TLR7) and CpG (TLR9) part have remarkable effect to the treatment of tumor.These TLR parts are the apoptosis of tumor cells of the corresponding TLR of abduction delivering directly, or strengthens tumor-infiltrated inherent immunity and tumour-specific T cell function.On the contrary, many some TLR signal paths that studies confirm that, as LPS and Loxoribine (Loxoribine) can promote generation and the development of tumor.It is complicated in the regulation and control of tumor and immunocyte that the result of these contradictions further illustrates TLR signal path, and because of different tumors and different TLR part different.Therefore the regulation and control of, understanding better TLR signal path in tumor cell and tumor infiltrating immunocyte can not only further contribute to the molecular mechanism of identifying tumour immunity pathology also will provide New Policy for developing effective tumor therapeuticing method.TLRs also plays an important role to the function of regulation and control Treg.We studies confirm that, the part of people TLR8 comprises that the Poly-G oligonucleotide of synthetic and native ligand (ssRNA40) can directly reverse naturally-occurring CD4 ⁺cD25 ⁺the CD4 in Treg cell and tumor source ⁺, CD8 ⁺and the inhibit feature of gamma delta T reg cell [Peng, G., et al.Toll-like receptor8-mediated reversal of CD4 ⁺regulatory T cell function.Science309,1380-4 (2005) .Peng G., et al.Tumor-infiltrating gammadelta T cells suppress T and dendritic cell function via mechanisms controlled by unique toll-like receptor signaling pathway.Immunity27,334-8 (2007)].

Summary of the invention

Goal of the invention: the effector function that recovers aging tumour-specific immunocyte.

Technical scheme: our nearest research discloses human tumor cells can induce inmature and tumor effect T cell senescence.The endogenous cyclisation AMP (cAMP) in tumor source is the main medium that inducing T cell is aging.Our research further confirms, the ability of the remarkable reversing tumor cell induction of TLR8 activator Poly-G3 and ssRNA40 T cell senescence.Poly-G3 can Effective Regulation tumor cell TLR8 signal path and block the effect of tumor source cAMP.We find by mouse tumor model and immunization therapy model, by Poly-G3, activate tumor cell TLR8 signal path and can effectively prevent the also caused Naive T cells of reversing tumor and tumour-specific T cell senescence, and can reverse its inhibit feature, thereby significantly strengthen anti-tumor immunotherapy effect.Our research clearly illustrates the depression effect of people TLR8 signal path energy reversing tumor microenvironment mediation and can be switched to effect microenvironment.Our research is for utilizing TLR8 part to provide strong foundation as novel with effective immunotherapy of tumors medicine and/or tumor vaccine adjuvant.

The invention provides a kind of method that stops tumor cell induction T cell senescence and reverse its inhibition ability, the part of TOLL sample receptor 8 is provided to cell, activate TLR8 signal path in tumor cell, blocking-up tumor cell induction T cell senescence.TLR8 part is by activating ERK1/2 and the P38 in TLR8 signal path downstream in tumor cell, and blocking-up tumor source endogenous cyclisation AMP is the generation of cAMP.The part of TOLL sample receptor 8, is a kind of oligonucleotide, and between the nuclease resistance residue that comprises adenine, guanine and connection guanine and adjacent nucleic acid base, main chain is connected chemical bond and forms, and its nucleic acid composition sequence is: 5 '-AGG ... GA-3 '; G represents guanine, and A represents adenine, between G and G, for chemical bond, modifies, and chemical bond is thiophosphate.The number of G can 3-10 not etc., preferably the sour composition sequence of part Poly-G3 is 5 '-AGGGA-3 '.

Tumor cell comprises mouse tumor cell and human tumor cells.

Human tumor cells comprises breast carcinoma, melanoma, carcinoma of prostate, ovarian cancer, colorectal cancer, pulmonary carcinoma, hepatocarcinoma and tumor of head and neck and other solid tumors.

The part of TOLL sample receptor 8 is for immunotherapy of tumors medicine or tumor vaccine adjuvant; And the mode of available injection is in tumor, subcutaneous, or systemic vein injection.

Beneficial effect:

1, found a tumor cell induction immunosuppressant new mechanism, be about to naivety/effector T cell and be converted into the aging T cell with inhibit feature.

2, the endogenous cAMP in confirmation tumor cell source is relevant with the induction of T cell senescence.

3, further confirm to activate that TLR8 signal path in tumor cell can be blocked tumor cell induction Naive T cells and tumour-specific effector T cell is aging and reverse its inhibit feature, thereby improve antineoplastic immune.

4, further illustrated the concrete adjustment signal path of TLR8 at tumor cell.The immunosuppressant strategy that these researchs are blocked tumor inducing for research and development provides new approaches and means.

4, TLR8 part is blocked the generation of tumor source cAMP by activating TLR8 signal path (comprising MyD88, IRAK4, ERK1/2 and P38) in tumor cell, and then blocks tumor cell induction T cell senescence.

5, we utilize melanoma 586mel lotus tumor Rag1 ^-/-mouse model proves, swollen intratumor injection TLR8 part Poly-G3 can significantly block the inmature CD4 that proceeds to tumor-bearing mice ⁺t cell senescence and reverse its inhibit feature.

6, we utilize melanoma 586mel lotus tumor NOD-scid IL-2Rgamma ^nullmouse model proves, swollen intratumor injection TLR8 part Poly-G3 can significantly block the tumour-specific effector T cell CD8 that proceeds to tumor-bearing mice ⁺the aging induction of TIL586 also reverses its inhibition ability.

7, we find by mouse tumor immunization therapy model, and swollen intratumor injection TLR8 part Poly-G3 can significantly strengthen CD8 ⁺the tumor-inhibiting action of TIL586 cell.

Accompanying drawing explanation

Fig. 1 has shown that in tumor infiltrating lymphocyte (TILs), aged cells ratio raises;

Fig. 2 has shown aged cells ratio liter in tumor infiltrating lymphocyte (TILs);

Fig. 3 has shown that the aged cells of tumor inducing has very strong inhibit feature;

Fig. 4 has shown that the endogenous cAMP in tumor source is the crucial medium that inducing T cell is aging;

Fig. 5 has shown the significantly T cell senescence of inhibition tumor cell induction of cAMP inhibitor;

Fig. 6 has shown the inmature CD4 of the remarkable reversing tumor cell induction of TLR8 part ⁺t cell senescence;

Fig. 7 has shown the related TLR8 signal path of T cell senescence of TLR8 part reversing tumor induction, comprises MyD88, IRAK4, ERK1/2 and P38;

Fig. 8 has shown that TLR8 signal is by lowering the T cell senescence of the cAMP reversing tumor induction in tumor cell;

Fig. 9 shown and knocked out ERK1/2 or p38 signaling molecule in MCF7 cell, or knocks out ERK1/2 signaling molecule in PC3 cell and can significantly block cAMP level in the tumor cell that Poly-G3 causes and reduce;

Figure 10 has shown that TLR8 signal path can reverse the inmature CD4 of induction in human melanoma cell Mice Body ⁺t cell senescence;

Figure 11 has shown and activates TLR8 signal in tumor to block the tumour-specific effector T cell of tumor inducing aging;

Figure 12 has shown that swollen intratumor injection TLR8 part Poly-G3 can significantly strengthen NOD-scid IL-2Rgamma ^nullthe T cell antineoplastic immune of mice.

The specific embodiment

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 (Buffy Coat) certainly in houston, u.s.a Bay area Blood Center.These researchs have obtained the approval of research examination board of school.The separated peripheral blood lymphocytes (PBMC) that obtains of Ficoll-Paque.The inmature CD4 of people ⁺and CD8 ⁺t cell obtains with EasySep enrichment test kit (StemCell Technologies) is separated.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), purchased from setting up in U.S.'s tissue bank (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 hypophysis extract, 5ng/ml epidermal growth factor, 2mM L-glutamine, 10mM HEPES 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, method see reference document [Peng, G., et al.Toll-like receptor8-mediated reversal of CD4 ⁺regulatory T cell function.Science309,1380-4 (2005) .Peng G., et al.Tumor-infiltrating gammadelta T cells suppress T and dendritic cell function via mechanisms controlled by unique toll-like receptor signaling pathway.Immunity27,334-8 (2007)].Key step is as follows: after tissue chopping with collagenase IV, hyaluronidase and desoxyribose enzymic digestion; Postdigestive cell, after RPMI1640 washing, is incubated in the RPMI1640 of the IL-2 that contains 10% human serum, l-glutamine, 2 mercapto ethanol and 50U/ml.

Synthesizing of 3.TLR8 part

(A) the TLR8 part Poly-G3 of synthetic, AG*G*GA.A represents adenine, G represents guanine.* represent the modification of sulfo-phospholipid key.By Invitrogen company, synthesized.

(B) natural TLR8 part ssRNA40/LyoVec ，You Invivogen company is synthetic.

(C) negative control part, Poly-T10, T*TTTT*T*T*T*T*T.T represents gland gland pyrimidine.* represent the modification of sulfo-phospholipid key.By Invitrogen company, synthesized.

4. aging relevant beta galactosidase (SA-β-Gal) dyeing

The former document of the detection of SA-β-Gal activity reference in aging T cell (Ye J., et al.Human regulatory T cells induce T lymphocyte senescence.Blood, 2012,120:2021-2031).Key step is as follows: the inmature CD4 that anti-CD3 activates ⁺or CD8 ⁺t cell is cultivated after 1 day altogether with the ratio of 1:1 and tumor cell or normal structure derived cell, separated and continue to cultivate 3 or 5 days.After the cell PBS washing of processing, be fixed on 3% formaldehyde, with freshly prepared SA-β-Gal nitrite ion (1mg/ml X-gal, 5mM K ₃fe[CN] ₆, 5mM K ₄fe[CN] ₆, 2mM MgCl ₂in PBS, Ph6.0.) 37 ℃, overnight incubation.Cell after dyeing, after water washs gently, is placed in micro-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 μ M), ssRNA40/LyoVec (TLR8 part 3 μ g/ml) (Invivogen, San Diego, CA) and oligonucleotide CpG-B (3 μ g/ml), Poly-T3 (3 μ g/ml) and Poly-G3 (3 μ g/ml) (Invitrogen is synthetic); CAMP inhibitor comprises: 2 ', 5 '-Didanosine (7ddA, 320 μ M) and two hydrochloride (H89,20 μ M) (Calbiochemistry, San Diego, CA).MAPK and NF-κ B blocking experiment: tumor cell is through MAPK inhibitor U0126 (10 μ M), SB203580 (10 μ M), and SP600125 (10 μ M) or NF-kB inhibitor APDC(10 μ M) process 3 days, within the 3rd day, add Poly-G3.After T cell separation after processing, continue to cultivate 3 days, then detect SA-β-Gal active.

5. flow cytometry analysis

CD4 ⁺t cell is after dyeing in the anti-human distinct antibodies cell surface of PE or FITC labelling or born of the same parents, and FACS detects CD4 ⁺t cell sign is expressed.People's antibody used comprises: anti-CD4, anti-CD8, anti-CD28 and anti-phosphorylation ATM, and all purchased from BD Biosceinces.All staining cells FACSCalibur flow cytometry analysis, FLOWJo software analysis (Tree Star) for the data obtained.

6. immunoblot experiment

The CD4 that anti-CD3 activates ⁺t cell and MCF7 or PC3 cultivate 0,1,3 or 5 day altogether.The CD4 cultivating altogether ⁺after T cell separation with lysate [50mM Tris-HCl (Ph8.0), 1%NP40,250mM NaCl, 50mM NaF, 1mM Na ₃vO ₄, 1mM protein inhibitor (PMSF, AKOLINE, leupeptin) and 1mM DDT] process.Cell pyrolysis liquid is after protein quantification, and SDS-PAGE is separated, goes to subsequently PDVF film.Pvdf membrane chemical luminous substrate after two anti-hatching of primary antibodie and horseradish peroxidase labelling is processed and is developed.In experiment, antibody used comprises: anti-phosphorylation LCK, anti-LCK, anti-phosphorylation-PKA, anti-PKA, anti-phosphorylation CREB, anti-CREB, anti-actin (Cell Signaling Technology, Danvers, MA).In some experiment, in culture fluid, add Poly-G3, the inmature CD4 after processing ⁺t cell is used for carrying out immunoblot experiment.

7. functional proliferation experiment

Proliferation experiment is [Peng, G., et al.Toll-like receptor8-mediated reversal of CD4 as previously mentioned ⁺regulatory T cell function.Science309,1380-4 (2005) .Peng G., et al.Tumor-infiltrating gammadelta T cells suppress T and dendritic cell function via mechanisms controlled by unique toll-like receptor signaling pathway.Immunity27,334-8 (2007)].Key step: at anti-CD3(2 μ g/ml) in 96 coated well culture plates, 1 * 10 ⁵inmature CD4 from Healthy People fresh separated ⁺the T cell that T cell and tumor or normal tissue cell are processed in varing proportions (1:10,1:0.5,1:0.2,1:0.1,0:1) is incubated at altogether 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 to cultivate 16 hours.With liquid flashing counting device measure [ ³h] the mixing of-thymus pyrimidine.

8.Calcein AM shifts and the blocking-up of iuntercellular connecting communication

MCF7, M628 and PC3 tumor cell hatch in containing in the PBS of Calcein AM (5 μ M, Invitrogen, Inc.) 40 minutes (37 ℃, 5%CO2).PBS washing containing 5%FBS 2 times for cell, with the ratio of 1:1 and the CD4 of anti-CD3 activation ⁺t co-culture of cells 1-3 days, adds or does not add 300 μ M GAP27(Tocris Bioscience simultaneously in culture fluid).FACS detects the Calcein AM that is transferred to T cell.Iuntercellular connecting communication blocking experiment, the CD4 that MCF7, M628 or PC3 tumor cell and anti-CD3 activate ⁺t co-culture of cells 1 day, adds or does not add GAP27 in culture fluid.After cell separation after processing, continue to cultivate 3 days, then detect SA-β-Gal and express and cAMP concentration.

The detection of 9.cAMP

Tumor cell or T cell are with pre-cooling PBS washing three times, and then freeze thawing is three times.(concrete grammar is shown in R&amp with the specific ELISA of cAMP, to measure the concentration of endochylema cAMP; D Systems).

Knocking out of the generation of 10.Lentivirus-shRNA and tumor cell gene

Design and the construction method of TLR8, IRAK4, MyD88, ERK1, ERK2, P38a, JNK1, IKKa or random lenti-shRNAs, and carry the restructuring lentivirus of GFP and the generation of shRNA sees above [Peng, G., et al.Toll-like receptor8-mediated reversal of CD4 ⁺regulatory T cell function.Science309,1380-4 (2005) .Peng G., et al.Tumor-infiltrating gammadelta T cells suppress T and dendritic cell function via mechanisms controlled by unique toll-like receptor signaling pathway.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, the separated GFP of FACS ⁺and GFP ^-tumor cell.Further measure separated tumor cell (GFP ⁺and GFP ^-) induce aging and produce the ability of cAMP.TLR8siRNA,5’GGTGGTGCTTCAATTAATA;IRAK4siRNA,5’GCAGCAATGGTTGACATTA;MyD88siRNA,5’GGCACCTGTGTCTGGTCTA。

11. reverse transcription PCRs

With Trizol (Invitrogen), extract total RNA, with SuperScript II RT test kit, extract cDNA, 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-scid IL2Rgamma ^nullmice (disappearance T and B cell) is purchased from Jackson laboratory.All zooperies have obtained the approval of Saint Louis University the care of animal committee.Rag1 ^-/-mice and NOD-scid IL2Rgamma ^nullmouse subcutaneous injection is containing Humanmachine tumour 586mel tumor cell (5 * 10 ⁶) the CD4 of 100 μ l PBS buffer .CD3 antibody (2 μ g/ml) preactivates ⁺t cell (5 * 10 ⁶/ Mus) or TS CD8 ⁺tIL586 cell (5 * 10 ⁶/ Mus) (586mel cell be can identify specifically and kill) and control group mice and tumor-bearing mice (tumor size is about 10 * 10mm) entered through tail vein injection.In parallel laboratory test, respectively at injecting 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 l PBS/ Mus) or Poly-G3 (50 μ g/100 μ l PBS/ Mus).Every group of 5-10 mice.The injection of T cell, after 12 days, is collected blood, lymph node (LN), spleen (SP) and tumor; Ficoll separating monocytic cell.The people CD4 injecting ⁺t cell is separated with the coated magnetic bead sorting method of antibody (Microbeads, Miltenvi Biotec), recovery, and does phenotype and functional analysis.It is the same that SA-β-Gal dyeing and 3H-thymus pyrimidine mix test.As for tumor growth and antitumor Immune Experiment, Humanmachine tumour 586mel tumor cell (5 * 10 ⁶) in the subcutaneous injection of 100 μ l PBS NOD-scid IL2Rgamma ^nullmice.The 3rd day, tail vein injection tomour specific CD8 ⁺tIL586 cell, gives simultaneously or does 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 for research inducing T cell is aging, be one of mechanism of immune escape, we and normal structure infiltrates pouring property bar cell, and analyze the ratio of aged cells if obtaining different tumors.First we detected SA-β-Gal(people aged cells sign) positive cell ratio.Compare with the lymphocyte that the normal structure of pairing is originated, breast carcinoma, melanoma, tumor of head and neck infiltrating lymphocytes (TILs) comprise a high proportion of SA-β-Gal positive cell (Figure 1A).Another important symbol that the loss that CD28 expresses or reduction are aging T cell.Thereby we further detected TILs(be respectively derive from breast carcinoma, melanoma, tumor of head and neck) or the lymphocyte CD 28 expression situations in normal galactophore tissue source.Result shows, the lymphocyte high expressed CD28 in normal structure source, and CD28 significantly decline (Figure 1B) in TILs.BNT: normal galactophore tissue's source T cell; BTIL, MTIL and HNTIL: be respectively the TILs that derives from breast carcinoma, melanoma, tumor of head and neck.

It is aging that 1.2 tumor cell lines and Naive T cells are cultivated remarkable induction Naive T cells altogether.

We further inquire into tumor cell, and whether directly inducing T cell is aging.Inmature CD4 ⁺t cell separation is 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 cultivate altogether.Cultivate altogether after 1 day, isolate CD4 ⁺t cell, and continue to cultivate 3 days, aging relevant phenotype and function then detected.Result shows, inmature CD4 ⁺after cultivating altogether, only there is a small amount of SA-β-Gal positive cell in T cell and normal breast cell strain; And cultivate altogether rear SA-β-Gal positive cell ratio significantly raise (Fig. 2 A) with different types of tumor cell line.Further analyze CD28 and express discovery, Naive T cells and different types of tumor cell (rather than normal tissue cell) are total to the expression (Fig. 2 B) that cultivation can significantly reduce its CD28.

1.3 tumor inducing aged cells have inhibit feature.

We have further studied the changing function of the aged cells of tumor inducing.[ ³h]-thymus pyrimidine mixes experimental result and shows, 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 crucial medium that inducing T cell is aging.

In 2.1 tumor cells, a large amount of cAMP can pass to the T cell of common cultivation.

Existing studies confirm that, tumor cell can be set up an anaerobic environment, thereby causes the rising of tumor locus adenosine and cAMP.Metabolite due to these anoxias can protect tumor cell to avoid tumour-specific CD4<sup TranNum="191">+</sup>t cell and CD8<sup TranNum="192">+</sup>the antineoplastic immune that T is cell-mediated.CAMP brings into play one of mechanism of inhibit feature as regulatory T cells (Treg), can suppress generation and the CD4 of IL-2<sup TranNum="193">+</sup>t cell proliferation [Bopp, T.et al.Cyclic adenosine monophosphate is a key component of regulatory T cell-mediated suppression.J Exp Med204,1303-10 (2007)].Whether these researchs impel us to go to confirm that the cAMP in tumor source is also aging relevant with inducing T cell.Result demonstration, 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 cultivate after one day altogether, and cAMP is at CD4<sup TranNum="194">+</sup>the level of T cell significantly raise (Fig. 4 B).Existing studies show that, regulatory T cells transmits cAMP[Bopp by iuntercellular connecting communication to effector T cell, T.et al.Cyclic adenosine monophosphate is a key component of regulatory T cell-mediated suppression.J Exp Med204,1303-10 (2007)].We infer that tumor cell may utilize similar mechanism, transmit cAMP, thereby inducing T cell are aging to target T cell.In co-culture system, add after cell gap junction inhibitory polypeptide GAP27, can significantly reduce cAMP level (Fig. 4 B).And GAP27 can significantly reduce the T cell senescence (Fig. 4 C) of tumor cell induction.* p < 0.05, * * p < 0.01, with matched group comparison.

(B）

(C)

2.2cAMP inhibitor is the T cell senescence of inhibition tumor cell induction significantly.

Recent research demonstration, cAMP is by the activation of PKA-CSK-LCK suppressor T cell.We have further detected LCK and cAMP downstream signaling molecule PKA and cAMP reactive element binding albumen (CREP) level and Expression of phosphorylated level thereof.Result shows, PC3 and the aging CD4 of MCF7 cell induction<sup TranNum="205">+</sup>lCK in T cell, CREB and PKA phosphorylation (Fig. 5 A), prompting tumor cell can be induced LCK inhibition signal path in aging T.For the further effect of cAMP in tumor inducing T cell senescence, we are at anti-CD3 preactivate CD4<sup TranNum="206">+</sup>in T cell and tumor cell co-culture system, add cAMP inhibitor [7ddA (320uM), or H89 (20uM), or both synergy].Found that, cAMP inhibitor is the T cell senescence (Fig. 5 B) of reversing tumor induction significantly.And medicine inhibitor 7ddA and H89 pretreatment tumor cell can significantly reduce the CD4 of common cultivation<sup TranNum="207">+</sup>cAMP level in T cell (Fig. 5 C).* p < 0.05, * * p < 0.01, with matched group comparison.

(B)

(C)

The inmature CD4 of the remarkable reversing tumor cell induction of 3.TLR8 part ⁺t cell senescence.

Chronic infection and inflammation are tumorigenic key factors.A plurality of clinical before and in zoopery prompting tumor cell different TLR stimulate or promote or suppress the growth [Huang of tumor; B.; et al.TLR signaling by tumor and immune cells:a double-edged sword.Oncogene27,218-24 (2008); Paulos, C.M.et al.Toll-like receptors in tumor immunotherapy.Clin Cancer Res13,5280-9 (2007); Smits, E.L., et al.The use of TLR7and TLR8ligands for the enhancement of cancer immunotherapy.Oncologist13,859-75 (2008)].Therefore, we suppose that TLR signal path also can affect the T cell senescence of tumor inducing and the function of TIL.For this reason, we are by tumor cell line MCF7, PC3 and M628 and anti-CD3 preactivate CD4 ⁺t co-culture of cells, and in co-culture system, add different TLR parts, then detect CD4 ⁺t cell aging.Fig. 6 A shows to only have the inmature CD4 of the remarkable reversing tumor cell induction of TLR8 part Poly-G3 and ssRNA40 ⁺the ability of T cell senescence.For getting rid of TLR part to CD4 ⁺there is direct acting possibility in T cell, we have detected TLR part and the CD4 that is located away from two healthy volunteers ⁺cell senescence after T co-culture of cells, finds that various TLR parts can not directly induce CD4 ⁺t cell senescence (Fig. 6 B).

(A)

The T cell senescence of 4.TLR8 part reversing tumor induction relates to specificity T LR8 signal path, comprises the signaling molecules such as MyD88, IRAK4, ERK1/2 and P38.

Our nearest experiment has confirmed the relation of TLR8 signal path and Treg inhibit feature.Poly-G3 and ssRNA40 can reverse naturally-occurring CD4 completely<sup TranNum="232">+</sup>cD25<sup TranNum="233">+</sup>treg and tumor source CD4<sup TranNum="234">+</sup>and CD8<sup TranNum="235">+</sup>the inhibit feature of gamma delta T reg [Peng, G., et al.Toll-like receptor8-mediated reversal of CD4<sup TranNum="236">+</sup>regulatory T cell function.Science309,1380-4 (2005); Peng G., et al.Tumor-infiltrating gammadelta T cells suppress T and dendritic cell function via mechanisms controlled by unique toll-like receptor signaling pathway.Immunity27,334-8 (2007)].Therefore, we want to confirm that whether Poly-G3 and ssRNA40 reversing tumor cell induction T cell senescence are also by TLR8 signal path.We utilize viral vector to carry siRNA gene Knockout, MCF7, PC3 and M628 cell transfecting energy specificity knock out slow virus shRNA or the contrast shRNA of TLR8, MyD88 and IRAK4, then detect the impact of Poly-G3 on the aging ability of its inducing T cell.Result shows, knocks out tumor cell MCF7, the TLR8 in M628 and PC3, and MyD88, IRAK4 signaling molecule can be blocked Poly-G3 to the inmature CD4 of tumor cell induction<sup TranNum="237">+</sup>the reverse effect of T cell senescence (Fig. 7 A).For further studying the aging TLR8 signal path downstream specific molecular of modulate tumor inducing T cell, 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-up ERK1/2) pretreatment PC3 and M628 tumor, or with U0126 and SB203580(blocking-up P38) pretreatment MCF7 can significantly block Poly-G3 to the inmature CD4 of tumor cell induction<sup TranNum="238">+</sup>the reverse effect of T cell senescence.* p < 0.05, * * p < 0.01, with matched group comparison.

(A)

Inmature CD4 ⁺the MCF7 co-culture of cells of T cell and different siRNA transfections, adds or does not add Poly-G3

Inmature CD4 ⁺the M628 co-culture of cells of T cell and different siRNA transfections, adds or does not add Poly-G3

Inmature CD4 ⁺the PC3 co-culture of cells of T cell and different siRNA transfections, adds or does 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 by lowering the T cell senescence of the cAMP reversing tumor induction in tumor cell.

5.1TLR8 signal path activation energy is lowered in tumor cell and is cultivated altogether the intracellular cAMP level of T.

We have further studied 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, the inhibition ability of the Poly-G3 pretreatment MCF7 Naive T cells pairing effect T cell that significantly reversing tumor is processed.Because cAMP is the important medium of tumor inducing T cell senescence, whether we further study Poly-G3 reversing tumor cell induction T cell senescence because it lowers cAMP level in tumor cell.As shown in Figure 8 B, Poly-G3 processes and can significantly reduce cAMP level in tumor cell.And Poly-G3 pretreatment tumor cell also can significantly reduce cAMP level in the Naive T cells of cultivating altogether with tumor cell (Fig. 8 C).

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5.2 gene knockout MCF7 cell ERK1/2 and p38, or PC3 cell ERK1/2 signaling molecule can significantly stop the cAMP level that Poly-G3 causes to reduce.

We further verify whether TLR8 signal downstream passages can participate in cAMP level in Poly-G3 reversing tumor cell.We utilize viral vector to carry siRNA gene Knockout.MCF7 and PC3 cell transfecting energy specificity knock out slow virus shRNA or the contrast shRNA of ERK1, ERK2 or P38 molecule, then detect cAMP level in the tumor cell after Poly-G3 processes.As shown in Figure 9, knock out ERK1/2 and p38 molecule in MCF7 cell, or ERK1/2 molecule in PC3 cell, can significantly 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, with matched group comparison.

6.TLR8 signal path can reverse the inmature CD4 of induction in human melanoma cell Mice Body ⁺t cell senescence.

Our experiment in vitro has confirmed that tumor cell energy inducing T cell is aging.Therefore, we further confirm with animal model whether tumor cell induces Naive T cells to be transformed into have the aging T cell of inhibit feature in vivo.We are employment 586mel melanoma cell subcutaneous vaccination Rag1 ^-/-mice (lack 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, separated people CD4 ⁺t cell, detects aging and function.As shown in Figure 10 A, at contrast Rag1 ^-/-mice, approximately 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%), prompter's tumor cell can induce Naive T cells aging in vivo.We further use [ ³h]-thymus pyrimidine mixes experiment and detected the CD4 from reclaiming in Mice Body ⁺t cell pairing effect CD4 ⁺the impact of T cell proliferation.As shown in Figure 10 B, CD4 ⁺t cell proceeds to lotus tumor 586mel Rag1 ^-/-after mice, the propagation of reaction-ive T cell is had to very strong inhibitory action.Whether we further research can prevent by regulation and control TLR8 signal path the T cell senescence of mouse interior tumor induction.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, swollen intratumor 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 from the aging and inhibit feature of the T cell of organ and tumor tissues recovery.As shown in Figure 10 C, swollen intratumor injection Poly-G3 can significantly block aging induction and reverse proceeds to lotus tumor 586mel Rag1 ^-/-inmature CD4 ⁺t cell senescence.And in LPS and PBS group, the T cell senescence that we do not observe tumor inducing is reversed.Swollen intratumor injection Poly-G3 can significantly reverse and proceed to lotus tumor 586mel Rag1 ^-/-inmature CD4 ⁺the inhibit feature of T cell (Figure 10 D).

* p < 0.05, * * p < 0.01, with matched group comparison.

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Thereby 7.TLR8 signal path can be blocked aging its antitumor action that strengthens of tumor cell induction specificity antineoplastic effector T cell.

The specificity antineoplastic effector T cell that 7.1TLR8 signal path can reverse induction in human melanoma cell body is aging.

Whether we further study tumor can be transformed into tumor specific effector cell aging T cell in vivo.We will inject NOD-scid IL-2Rgamma under Humanmachine tumour 586mel cell skin<sup TranNum="323">null</sup>mice is set up the NOD-scid IL-2Rgamma of lotus melanoma 586mel<sup TranNum="324">null</sup>mouse model.Human tumor-specific CD8<sup TranNum="325">+</sup>tIL586 cell injects the NOD-scid IL-2Rgamma of lotus melanoma 586mel through caudal vein<sup TranNum="326">null</sup>mice.After 12 days, reclaim tumour-specific CD8<sup TranNum="327">+</sup>tIL586 cell, detects its aging and inhibit feature.As shown in Figure 11 A, human tumor-specific CD8<sup TranNum="328">+</sup>tIL586 cell proceeds to the NOD-scid IL-2Rgamma of lotus melanoma 586mel<sup TranNum="329">null</sup>after mice, SA-β-Gal positive cell ratio significantly increases.And, reclaim from the NOD-scid of lotus melanoma 586 IL-2Rgamma<sup TranNum="330">null</sup>the CD8 of mice Different Organs and tumor tissues<sup TranNum="331">+</sup>tIL586 cell has very strong inhibit feature (Figure 11 B).We further study the NOD-scid IL-2Rgamma whether TLR8 signal path can stop lotus melanoma 586mel<sup TranNum="332">null</sup>aging and the changing function of mouse interior tumor specific C D8+TIL586 cell.Found that swollen intratumor injection Poly-G3, rather than LPS or PBS can significantly reduce the NOD-scid IL-2Rgamma of lotus melanoma 586mel<sup TranNum="333">null</sup>aging CD8 in Mice Body<sup TranNum="334">+</sup>the ratio of TIL586 cell (Figure 11 C).In addition, swollen intratumor injection Poly-G3 also can significantly reverse the CD8 that proceeds to lotus melanoma 586mel mice<sup TranNum="335">+</sup>the inhibition ability of TIL586 cell.These result of study explanation 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, with matched group comparison.

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7.2TLR8 signal path strengthens its antitumor action by suppressing tumour-specific T cell senescence.

We will inject NOD-scid IL-2Rgamma under Humanmachine tumour 586mel cell skin ^nullmice.After 3 days, vein injects tumour-specific CD8 ⁺tIL586mel cell, subsequently at tumor injection position injection TLR8 part Poly-G3.As shown in figure 12, Humanmachine tumour 586mel cell can be at NOD-scid IL-2Rgamma ^nullramp in Mice Body; CD8 ⁺tIL586 cell can significantly suppress the growth of tumor; Tumor locus injection Poly-G3 can significantly strengthen the antitumor action of CD8+TIL586 cell.

Claims (9)

1. stop tumor cell induction T cell senescence and reverse the method for its immunosuppressant ability, it is characterized in that, the part of TOLL sample receptor 8 is provided to cell, activate TLR8 signal path in tumor cell, blocking-up tumor cell induction T cell senescence.

2. prevention tumor cell induction T cell senescence according to claim 1 reverse the method that it suppresses ability, it is characterized in that, the part of TOLL sample receptor 8, it is a kind of oligonucleotide, between the nuclease resistance residue that comprises adenine, guanine and connection guanine and adjacent nucleic acid base, main chain is connected chemical bond and forms, and its nucleic acid composition sequence is: 5 '-AGG ... GA-3 '; G represents guanine, and A represents adenine, is that chemical bond modifies between G and G, the number of G can 3-10 not etc.

3. prevention tumor cell induction T cell senescence according to claim 2 reverse the method that it suppresses ability, is characterized in that, the part of TOLL sample receptor 8, and the sour composition sequence of part Poly-G3 is 5 '-AGGGA-3 '.

4. according to the prevention tumor cell induction T cell senescence described in claim 2 or 3 and reverse the method that it suppresses ability, it is characterized in that, the part of TOLL sample receptor 8, chemical bond is thiophosphate.

5. prevention tumor cell induction T cell senescence according to claim 1 and 2 reverse the method that it suppresses ability, it is characterized in that, TLR8 part is by activating TLR8 signal path in tumor cell, be ERK1/2 and the P38 in downstream, blocking-up tumor source endogenous cyclisation AMP is the generation of cAMP.

6. prevention tumor cell induction T cell senescence according to claim 1 and 2 reverse the method that it suppresses ability, is characterized in that, tumor cell comprises mouse tumor cell and human tumor cells.

7. prevention tumor cell induction T cell senescence according to claim 6 reverse the method that it suppresses ability, it is characterized in that, human tumor cells comprises breast carcinoma, melanoma, carcinoma of prostate, ovarian cancer, colorectal cancer, pulmonary carcinoma, hepatocarcinoma and tumor of head and neck and other solid tumors.

8. for people's reversing tumor immunosuppressant vaccine, it is characterized in that, the part of the TOLL sample receptor 8 described in claim 2 or 3 or 4 is used for to immunotherapy of tumors medicine or tumor vaccine adjuvant.

The part injection system of 9.TOLL sample receptor 8, is characterized in that, in the part injection tumor of the TOLL sample receptor 8 in claim 2 or 3 or 4, subcutaneous, or systemic vein injection.

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