CN101126076A - TGF-beta induced adjustment T cell and its forming method and application - Google Patents
TGF-beta induced adjustment T cell and its forming method and application Download PDFInfo
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Abstract
The invention pertains to biomedical technique field, in particular to a TGF-beta induced regulatory T-cell and a synthesis method and the applications thereof. The regulatory T-cell of the invention is formed by combining two cytokines: TGF-Beta and IL-2, and obtained by extrinsically inducing CD4+ cells of autoimmune diseases sufferer, and is recorded as CD4++CD25+Foxp3+ regulatory T-cell, wherein FoxP3 is FoxP3 gene or protein. The regulatory T-cell of the invention can be taken as a medicine preparation that is used for treating the diseases of autoimmune diseases (such as SLE) sufferer.
Description
Technical field
The invention belongs to biomedical neck technical field, be specifically related to the relevant adjusting T cell of a kind of treatment autoimmune disorder (as systemic lupus erythematous, rheumatoid arthritis or the like), and the formation methods and applications that should regulate the T cell.
Background technology
Both at home and abroad to systemic lupus erythematous, the treatment of autoimmune disorders such as rheumatoid arthritis depends on the immunosuppressor of hormones or non-hormone at present.Though the symptom that the treatment of these medicines can the control section patient,, late result is limited.The more important thing is that these medicines of life-time service usually can produce severe complications, the perhaps serious infection of secondary, even the generation of meeting induced tumor.Spending huge medical resource in addition also is a defective.
Middle and later periods nineteen nineties, the immunologist finds to exist in intact animal and the human body a spot of CD4+CD25+ cell (account for greatly normal human CD4+ cell 2 percent).If lack this cell, the animal and human knows from experience the symptom that produces various autoimmune disorders.This group of evidence cell derives from thymus gland, because thymectomized animal lacks the CD4+CD25+ cell, and the development autoimmune disorder.If injection CD4+CD25+ cell can prevent mouse that autoimmune disorder takes place to the mouse of thymusectomy.This group cell is called as " CD4+ of nature regulates the T cell " now.
Lacking the CD4+CD25+ cell of nature and the relation of autoimmune disorder at first is confirmed in the autoimmunity experimental animal model.As BWF1 and SNF1 mouse, idiopathic lupoid acne disease all takes place about 22 weeks of birth, the quantity of (peripheral blood and spleen) CD4+CD25+ cell descends in the present mouse body that studies have shown that morbidity.On the contrary, intravenous injection CD4+CD25+ cell can delay the generation and the development of lupus disease really to this mouse.
Above-mentioned CD4+ regulates the change of properties of T cell and people's SLE takes place and develop that confidential relation is also arranged.Initial several research groups find that at first patient's SLE of active period CD4+CD25+ cell quantity has obvious reduction.Recent findings, Foxp3 gene and albumen can be expressed in CD4+ specifically and regulate in the T cell, and with CD4+ adjusting T cells whose development and function substantial connection are arranged.Like this, identify the quantity of Foxp3+ rather than CD25+ cell, just can reflect accurately that the CD4+ of nature regulates the level of T cell.In fact, the quantity of our recent findings active SLE patients patient's CD4+CD25+Foxp3+ cell descends, and the activity level of this decline and disease is inversely proportional to.The CD4+CD25+ cell inhibiting function that other people have also reported patient SLE recently descends.
Since the CD4+ of nature regulates generation and development that the T cell affects the SLE disease significantly, people attempt to consider that regulating the T cell with CD4+ treats SLE.Because the quantity of CD4+CD25+Foxp3+ positive cell is very limited, the researchist considers the method with amplification, improves the quantity of CD4+CD25+Foxp3+ cell.Though several research groups have proved that the CD4+ of amplification regulates the T cell and still can keep immunoloregulation function and phenotype, and in prevention and the reaction of treatment acute graft versus host, play a part to a certain degree, but the research group in another one Europe finds that the natural CD4+ of amplification repeatedly regulates the T cell and can lose immunosuppressive effect.In addition, the CD4+ of amplification regulates the lymphocyte death that activation-inducing takes place the T cell easily, and this has also limited the therapeutic value of this cell.
Recently, research group (Univ California-San Francisco USA, BLUESTONE professor) LUPUS disease of having used the CD4+CD25+ cell of amplification to treat animal (BWF1) mouse only.In any case this effect is very limited.And the cell that they use is the normal animal CD4+CD25+ cell of amplification rather than the CD4+CD25+ cell in lupus disease source, because the intrinsic defective may take place the CD4+CD25+ cell of lupus and SLE disease.Therefore, whether can increase that these cells are treated SLE and the other autoimmune disorder is still unclear.As seen, this finds patient's SLE adjusting T cell therapy is remained limited.
Summary of the invention
The objective of the invention is to propose a kind of at beta induced adjusting T cell of the TGF_ of autoimmune disorder (as systemic lupus erythematous, rheumatoid arthritis etc.) and forming method thereof and application.
The beta induced adjusting T cell of the TGF_ at autoimmune disorder that the present invention proposes, be to combine by TGF_ β and two cytokines of IL_2, CD4+ cell by external evoked autoimmune disorder patient obtains, specifically be designated as CD4++CD25+Foxp3+ and regulate the T cell, FoxP3 is FoxP3 gene or albumen here.
The formation method that above-mentioned CD4+CD25+Foxp3+ regulates the T cell is as follows:
(1) from autoimmune disorder (as SLE) patient vein, extracts peripheral blood, with FICOLL technology isolated lymphocytes; Use monoclonal antibody (for example anti-monocyte antibody, B cell antibody, CD8+ cell antibody and memory cell antibody etc.) and negative technical point from the NAIVE_CD4+ cell again;
(2) to isolated NAIVE_CD4+ cell, the particulate pearl that wraps up with anti-CD3/CD28 antibody stimulates; Add the TGF_ β of 3-10ng/ml and the IL_2 of 15-30unit/ml then, cultivated 5-6 days;
3, then the CD25 positive cell in the CD4+ cell is separated with MACS particulate pearl.Concrete steps are: remove the cell in conjunction with CD4+ with PE CD25 antibody, the MACS particulate pearl with anti-PE carries out positive separation again, promptly obtains required CD4+CD25+Foxp3+ and regulates the T cell.
The CD4+CD25+Foxp3+ that is obtained by aforesaid method regulates the T cell, can be used as a kind of pharmaceutical preparation, is used for the treatment of autoimmunity (as SLE) disease disease of patient.Concrete grammar is as follows:
Induce the CD4+CD25+Foxp3+ of acquisition to regulate the T injection cell aforesaid method and give the patient, injection volume is 5-10
20Individual above-mentioned adjusting T cell.
In order to obtain better result of treatment, carry out conventional medicine (as using conventional immunosuppressor etc.) can for earlier active period (as SLE) patient and treat a course of treatment, inject above-mentioned CD4+CD25+Foxp3+ then and regulate the T cell.
Because above-mentioned adjusting T cell time to live in animal body is approximately one month, therefore, the present invention can inject once the patient January.
In addition, the present invention also can combine use with above-mentioned adjusting T cell and conventional immunosuppressor and hormone etc.In this process, can reduce the using dosage of hormone and immunosuppressor.
The present invention has following advantage:
L, CD4+CD25+FOXP3+ regulate the T cell and have obvious suppression T proliferation of cells, production of cytokines and cytotoxic activity;
2, CD4+CD25+FOXP3+ regulates the ability that the T cell has obvious suppression B cell generation antibody.
3, CD4+CD25+FOXP3+ adjusting T cell has prevention lupus generation in the tangible body, the ability that the control lupus develops.
3, CD4+CD25+FOXP3+ adjusting T cell has secular provide protection ability in the body.
4, CD4+CD25+FOXP3+ regulates T cell itself does not have obvious toxic and side effects.
5, CD4+CD25+FOXP3+ regulates the using dosage that the T cell can replace immunosuppressor and hormone or reduce immunosuppressor and hormone, is used in combination, and improves autoimmune disorder patients' such as SLE treatment and definite toxic side effect.
Description of drawings
Fig. 1 .TGF-β can induce
The CD4+CD25 negative cells develops into CD4+CD25+Foxp3+ cell diagram.From normal C57BL/6 mouse
The CD4+CD25 negative cells is stimulated 5 days description of drawings by the particulate pearl (by particulate bead concentration of per 10 cells) that anti-CD3/CD28 antibody attaches.IL-2 (20u/ml) or/and TGF-β (2ng/ml) be added in the stimulated cells.The expression level of Foxp3 detects by flow cytometer in the CD25, CD4 and cell.Diagram is the representative of 5 tests that separate.
Fig. 2 .TGF-β can induce
The CD4+CD25 negative cells develops into the CD4+CD25+ cell of expressing the Foxp3 information RNA.From normal C57BL/6 mouse
The CD4+CD25 negative cells was stimulated 5 days by the particulate pearl (by particulate bead concentration of every lO cell) that anti-CD3/CD28 antibody attaches.IL-2 (20u/ml) or/and TGF-β (2ng/ml) be added in the stimulated cells.Cultivate the CD4+ after 5 days, further carried out cell sorting by flow cytometer, isolate the negative hypotype of CD25+ and CD25 then, conventional preparation RNA and reverse transcription become cNDA, by RT-PCR amplification Foxp3 and house-keeping gene HPRT.Diagram is the representative of 5 tests that separate.
Fig. 3 .IL-2 diagram that in the beta induced Foxp3+ cell of TGF-, plays a key role.
The CD4+CD25 negative cells prepares from the mouse of IL-2 gene knockout, stimulates with Fig. 1 similar methods then.The interleukin I L-7 (100ng/ml) that in some culture hole, adds exogenous reorganization, or IL-15 (100ng/ml), or IL-2 (50u/ml).The expression of low cytometric analysis decision Foxp3+.This result has been repeated three times, and has similar result.Annotate: if lack IL-2, the ability that the beta induced Foxp3 of TGF-expresses has disappeared.Can not to induce the expression of Foxp3 be expression in default of CD25 in order get rid of to lack IL-2, and the investigator has also added IL-7, or IL-15, and the two all can induce the expression of CD25, but still can not make the beta induced Foxp3+ of TGF-.Only adding exogenous IL-2, the expression that can recover Foxp3.
Fig. 4 .IL-2 and TGF-β induce and the Foxp3+ that increases in play synergy diagram.From normal C57BL/6 mouse
The CD4+CD25 negative cells is carried out mark by fluorescent dye CFSE, then, stimulates with Fig. 1 similar methods.Different is, in some culture hole, in and the anti-IL-2 antibody (2ug/ml) of IL-2 be added into.For in the decision and the specific effect of IL-2 antibody, the contrast IgG of isomers also is added into.The expression of FoxP3 decides according to the different time (seeing figure) by the dilution of CFSE and the level of intracellular Foxp3 with the amplification situation.The result is 4 independently test representatives;
The beta induced CD4+CD25+ cell of Fig. 5, AIL-2 and TGF-has the obvious in-vitro suppression effect.
The CD4+CD25 feminine gender is stimulated by Fig. 1 similar methods, and then, the CD25+ and the CD25 negative cells that contrast in the beta induced CD4+ cell (Treg) of CD4+ (Tcon) and IL-2 and TGF-are further carried out cell sorting with flow cytometer.These cells are added in the T reacting cells of CFSE mark and the antigen presenting cell in 1: 4 ratio and carry out common cultivation (stimulation of solubility anti-cd 3 antibodies) 3 days.CD4+ proliferation of cells ratio is calculated by the degree of CFSE dilution.The result is 4 test representatives that separate.
The beta induced CD4+CD25+ cell of Fig. 5, BIL-2 and TGF-has the obvious in-vitro suppression effect.
The CD4+CD25 feminine gender is stimulated by Fig. 1 similar methods, and then, the CD25+ and the CD25 negative cells that contrast in the beta induced CD4+ cell (Treg) of CD4+ (Tcon) and IL-2 and TGF-are further carried out cell sorting with flow cytometer.These cells (are seen diagram) according to a certain percentage and are added in the T reacting cells of CFSE mark and the antigen presenting cell and carried out co-cultivation (stimulating with the solubility anti-cd 3 antibodies) 3 days.Total CD4+ hyperplasia situation multiply by the total cellular score of cultivating in the pore by hyperplasia per-cent and decides (B).B is that the average of 4 tests adds standard deviation.
The beta induced BDA/2T cell of Fig. 6, intravenous injection IL-2 and TGF-can prevent the D2T cell induction to the D2B6F1 mouse lupoid acne disease.20x10
6, the fresh T cell of DBA/2 mouse (D2), or the D2 cell adds 20x10
6By the T cell (D2+T of heterologous antigen and IL-2 stimulation
Con), or the D2 cell adds 20x10
6By heterologous antigen, the T cell (D2+T that IL-2 and TGF-β stimulate
TGF β) be injected into respectively in the D2B6F1 mouse, anti-IgG antibody, anti-dsDNA antibody (injection back 1-4 week) and proteinuria (8 week) are detected by ELISA and proteinuria detection method.Annotate: the T cell that IL-2 and TGF-are beta induced, rather than control cells, prevented the ability of the fresh induced t cell lupus of D2 disease significantly.
Fig. 7.The symptom and the survival of lupus disease mice taken place in the beta induced obvious improvement of BDA/2T cell of intravenous injection IL-2 and TGF-.20x10
6, the fresh T cell of DBA/2 mouse (D2), by intravenous injection in the D2B6F1 mouse.After two weeks (mouse falls ill), 5 to 20x10
6By heterologous antigen, the T cell (T that IL-2 and TGF-β stimulate
Reg) be injected into respectively in the D2B6F1 mouse, anti-ds-DNA antibody (Fig. 7 A, injection back 2-12 week) and proteinuria (Fig. 7 B, 4-12 week) are detected by ELISA and proteinuria detection method.The survival of mouse is also monitored up to dead (Fig. 7 C).Accept the D2 cell, but do not accept T
RegThe positive control group of injection cell person, the D2B6F1 mouse of not accepting the D2 cell is the normal control group.Annotate: the T cell that IL-2 and TGF-are beta induced, reduced antibody ds-DNA and the albuminuretic level of the mouse that falls ill significantly, and prolonged the survival of lupus mouse doubly.
The beta induced BDA/2T cell of Fig. 8, intravenous injection IL-2 and TGF-is not induced the lupoid acne disease to the D2B6F1 mouse.20x10
6, the fresh T cell of DBA2 mouse (D2), or by the T cell (D2-T of heterologous antigen and IL-2 stimulation
Med), or by heterologous antigen, the T cell (D2-T that IL-2 and TGF-β stimulate
TGF β) be injected into respectively in the D2B6F1 mouse, anti-IgG antibody, anti-ds-DNA antibody (injection back 1-4 week) and proteinuria (Proteinuria, 8 weeks) are detected by ELISA and proteinuria detection method.Annotate: the T cell that IL-2 and TGF-β handle has lost the ability of inducing the lupus disease, and various indexs are similar to the normal mouse of not accepting injection DBA/2T cell.
Survival is longer in the beta induced DBA/2T cell paste of Fig. 9, IL-2 and TGF-.10x10
6, the fresh T cell of the DBA/2 mouse of CFSE mark (being labeled as Fresh among the figure), or by heterologous antigen, IL-2 stimulates T cell (Tcon), perhaps by heterologous antigen, the T cell (T that IL-2 and TGF-β stimulate
TGFbeta) be injected into respectively in the D2 mouse, the quantity of the CFSE+CD3+T cell in each group mouse spleen is checked in after injection the 1st, 7,14 days.Garden circle expression CFSE+CD3+ positive cell, the intensity of upper right numeral CFSE, the CFSE+CD3+ cell that the lower-left digital watch is total.
Figure 10, can induce the CD4+ cell expressing Foxp3 of children's age and aged BWF1 mouse in conjunction with IL-2 and TGF-β.BWF1 mouse from 8 and 22 ages in week
The CD4+CD25 negative cells is undertaken stimulating in 5 days by the similar technology of Fig. 1.The FoxP3 of low cytometric analysis decision CD4+ cell expresses.Annotate: can induce the CD4+ cell expressing Foxp3 of the aged BWF1 mouse that lupus has taken place in conjunction with IL-2 and TGF-β, though expression level is a little less than young mice.
Figure 11, IL-2 and TGF-β inductive CD4+CD25+ cell from 22 all BWF1 mouse can suppress the generation and the development of lupoid acne disease.1x10
6From the B cell of 22 all B/WF1 mouse purifying or/and 10x10
6The CD4+ cell of purifying be injected into 8 weeks accepted 350 draw in the B/WF1 mouse of radio exposure of dosage.In some group, also accepted 5x10
6The CD4+CD25+ hypotype cell from control cells (Tcon) and IL-2+TGF-beta induced (Treg) of purifying.The concentration of the anti-ds-DNA of serum after 1 month, albuminuretic level determines according to Fig. 5 similar methods after 4 months.Each group comprises 4-6 mouse.
Figure 12, IL-2 and TGF-β can induce active SLE patients patient's CD4+ to develop into CD4+Foxp3+ and regulate the T cell.
The CD4+CD25 negative cells is conventional preparation from normal people and active SLE patients patient, and these cells are stimulated by anti-people's CD3/CD28 antibody.In some group, IL-2 (20u/ml) or/and TGF-β be added in the culturing cell.The expression (A) of Foxp3 in the FACS technology decision cell, value is represented the average and the standard deviation of 10 tests.(B). these cells join in 1: 6 ratio to go to detect in the fresh T cell of CFSE mark and suppress active, and concrete grammar is similar to Fig. 4).
Embodiment
The present invention produces to natural CD4+ with IL-2 zygotic induction peripheral blood with cytokine TGF-β to regulate the similar a group adjusting T cell of cell.The present invention has set up a kind of brand-new CD4+ that induces and has regulated T cell method, replaces the method for the CD4+CD25+ adjusting T cell of amplification nature, is used for the treatment of SLE or other autoimmune disorder.The present invention finds that TGF-β and two cytokines of IL-2 can induce normal animal and human's CD4+ cell to develop into CD4+Foxp3+ and regulate the T cell, and these two cytokines are also had the ability to induce the CD4+ cell of suffering from lupus animal and patient SLE to develop into CD4+Foxp3+ and are regulated the T cell.Therefore, plan of the present invention obtains peripheral blood CD4+ cell from autoimmune disorder patients such as SLE, and behind external process TGF-β and IL-2 inducing culture, separation of C D25+ cell mass obtains CD4+CD25+Foxp3+ and regulates the T cell then.At last, according to the calculating of body surface area, with 10
9The external evoked CD4+CD25+Foxp3+ in the individual left and right sides regulates the T cell to being expelled in the patient body.According to the body internal dynamics and the time to live of the beta induced CD4+CD25+Foxp3+ adjusting T cell of TGF-, the present invention will regulate the T cell to CD4+CD25+Foxp3+ of venous patient injection every month.For assurance obtains best effect, before CD4+CD25+Foxp3+ regulates the T cell therapy, earlier with course of treatment of immunosuppressant treatment.
The particular content branch is described below:
1.TGF-β can induce the CD4+ cytodifferentiation to become CD4+CD25+FOXP3+ and regulate the T cell.
In fact, regulating the T cell is the homogeneity heterology.CD4+ regulates natural CD4+ adjusting T cell and the acquired CD4+ adjusting of the peripheral blood inductive T cell that the T cell comprises the thymus gland source.We find that at first TGF-β has the ability to induce the CD4+CD25-cell of intact animal and normal people's peripheral blood to develop into CD4+ adjusting T cell.These cells have similar phenotypic characteristic to the CD4+ cell of nature, as all expressing CD25, CD122, CTLA-4, GITR etc.Because the CD4+CD25+ that nearest Foxp3 gene of identifying and albumen only are found in nature regulates in the T cell, and conventional activated CD4+CD25+ cell is not expressed Foxp3, like this, Foxp3 just becomes a specificity marker regulating the T cell.
We find that cytokine TGF-β can induce under TCR stimulation situation
The CD4+CD25 negative cells is expressed Foxp3 albumen and mRNA (Zheng Songguo etc., JImmunol.2002 169:4183-4189).(mRNA, the attemperator of protein synthesis Fig. 2) are positioned the CD25+ cell mass for all Foxp3 albumen (Fig. 1) and information RNA.In addition, the beta induced Foxp3 of TGF-also needs the existence of IL-2.If endogenous IL-2 is by after the antibody neutralization, TGF-β can not induce CD4+ cell expressing Foxp3.If with the CD4+ cell of IL-2 knock out mice, the ability that the beta induced Foxp3 of TGF-expresses also disappeared (Fig. 3).More evidence IL-2 and TGF-β are inducing and the CD4+Foxp3+ that increases regulates in the T cell and played a synergy (Fig. 4).
2.TGF-the hyperplasia that beta induced adjusting T cell can suppressor T cell, production of cytokines and lupus morbidity.
TGF-β and IL-2 not only induce the expression of Foxp3, and new inductive CD4+CD25+ cell is transformed into immunosuppressant cell.In the external immune inhibition test of a standard, result of the present invention shows that TGF-β and IL-2 inductive CD4+ cell have very strong immune suppression function.The inductive CD4+ cell hyperplasia of suppressor T cell significantly outside this colony, comprise inhibition (Fig. 5 A) to T hyperplasia rate and total T hyperplasia quantity (Fig. 5 B), in addition, the generation (result does not show) of the cytokine (comprising IFN-γ and IL-2) that they also can suppressor T cell.
Because external immunosuppression can not definitely illustrate similar immune suppression function is arranged also in the body.In order to illustrate this problem, whether the present invention has analyzed further that external TGF-β and IL-2 inductive CD4+ cell have immune suppression function in the body.The present invention has adopted a chronic lupus disease model.The spleen t-cell of 2,000,000,000 DBA/2 mouse of intravenous injection enters in the DBA/2xC57BL/6F1 hybridize mice, after two weeks, the anti-IgG and the antinuclear antibody of typical high titre appears in the F1 mouse, and 6 to 8 weeks began to occur proteinuria and lupus nephritis, and mouse begins death after 16 weeks.The present invention's demonstration, if inject TGF-β and IL-2 inductive adjusting T cell simultaneously at the disease inductive, can prophylactic significantly generation (Fig. 6).In addition; the present invention has also further observed the provide protection to the lupus model of having fallen ill of this group adjusting T cell; studies show that; if 5 to 2,000,000,000 cells of injection in the F1 mouse that has fallen ill; the progress of control disease significantly; particularly can prolong the survival (Fig. 7 A, B and C) of lupus mouse significantly.
3.TGF-beta induced adjusting T cell itself is toxic side effect not.
Spleen cell of injection DBA/2 mouse or T cell can induce the F1 mouse that lupus-like syndrome takes place in the F1 mouse.Whether the present invention has also studied this group cell (the T cell that IL-2 and TGF-are beta induced) to the pathogenic effects of F1.The result shows that if do not pass through the processing of TGF-β, this group still can be induced the lupoid acne disease with the T cell of the DBA/2 mouse that the C57BL/6 non-T cell stimulates.But if the processing of cytokines such as process IL-2 and TGF-β, this group T cell just no longer induces the F1 mouse that lupoid acne disease (Fig. 8) takes place.In addition, we also inject the anti-CD3/CD28 antibody stimulation of process, the beta induced CD4+CD25+ cell of IL-2 and TGF-arrives DBA/2 mouse of the same race, find that these mouse of accepting the CD4+CD25+ cell are without any side effect, important organ, as internal organs such as lung, liver, kidney, the heart and brains, do not find through biopsy how pathologic changes (result does not show).
4.TGF-beta induced adjusting T cell has time to live in the longer body than conventional activated CD4+ cell.
The present invention is by carrying out charcoal oxygen luciferin diacetic acid fat (carboxy fluoresceindiacetate succinimidyl ester to TGF-β and IL-2 inductive T cell, CFSE) mark, and then be expelled in the allogenic animal body, by follow-up analysis, find that they can be survived in vivo at least and reach two weeks above (Fig. 9).In other experimental study,, find that the adjusting T cell of injection can be survived at least one month (result does not show) with not homology (Congenic) sign research of allogenic animal.
5.TGF-can inducing the CD4+ cell of B/WF1 mouse to develop into, β regulates the T cell.
Though TGF-β and IL-2 can induce normal animal and human's CD4+ cell to develop into CD4+ and regulate the T cell, the present invention further studied these two cytokines whether can induce the individuality of suffering from autoimmune disease as: the CD4+ cell of lupus mouse develops into regulates the T cell.Because the B/WF1 mouse can idiopathic generation lupus, from the B/WF1 mouse in 8 weeks (morbidity as yet) and 22 weeks (falling ill), prepare Naive CD4+ cell respectively, we observe, and TGF-β and IL-2 can induce two kinds of mouse
The CD4+ cell develops into the Foxp3+ cell, and the ability that the CD4+ cell of the beta induced 22 all mouse of TGF-develops into the Foxp3+ cell is weaker than 8 all mouse (Figure 10) a little.Further discover, the CD4+ cell of TGF-β and IL-2 inductive 22 all mouse in being expelled to the BWF1 mouse after, can reduce the progress (Figure 11) of the disease of B/WF1 mouse significantly.
6.TGF-can inducing patient's SLE CD4+ cell to develop into, β regulates the T cell.
Develop into and regulate the T cell because ultimate aim of the present invention is the CD4+ cell with TGF-β and these two autoimmune disorder patients such as cytokine induction SLE of IL-2, feed back then and give this patient, hope has no side effect with this or toxic side effect the is less replacement of adjusting T cell and the hormone that is using clinically and the immunosuppressor of non-hormone are treated autoimmune diseases such as SLE.
In this invention research, active SLE patients patient's peripheral blood
After the CD4+CD25-cell is produced, stimulate with anti-CD3/CD28 antibody, test group adds TGF-β and IL-2, and control group only adds IL-2.After 5 to 6 days, the proteic expression of Foxp3 of control group and test group cell is detected, and shows as Fig. 2.TGF-β and IL-2 can induce activated active SLE patients patient's significantly
CD4+ cell expressing Foxp3 albumen, and TGF-β and IL-2 inductive CD4+ cell also can suppress patient's SLE T proliferation of cells.
The concrete operations step is summarized as follows:
1. extract peripheral blood from patient's SLE medium sized vein, with lymphocyte separation medium (Ficoll) technology isolated lymphocytes.Add that with monoclonal antibody (as: anti-monocyte antibody, B cell antibody, CD8+ cell antibody and memory cell antibody etc.) negative triage techniques isolates
The CD4+ cell.
2. to patient SLE
The CD4+ cell, the particulate pearl that wraps up with anti-CD3/CD28 antibody stimulates.In the culture dish of 24 orifice plates, add 2x10
6Individual
The CD4+ cell, 2x10
5The particulate pearl of individual anti-CD3/CD28 antibody parcel and the plain IL-2 of cytokine interleukin (20u/ml) of reorganization be totally one week.In inducing the process of regulating the T cell, we add the TGF-β of 0.5-10ng/ml.According to our pre-experimental result just, we find that the TGF-β of 5ng/ml is optimal condition.In order to get rid of antigen presenting cell to injecting the side effect that is produced in external evoked adjusting T cell and the body subsequently, the present invention is without antigen presenting cell.
3. after cultivating 5 to 6 days, CD25+ cell in the CD4+ cell separates with the small magnetic bead of the German Miltenyi company that meets the GMP standard.Specifically be, collect after the cultured cells that dye with CD25 antibody, 4 ℃, after 20 minutes, CD25 will combine with the CD25+ subgroup in the CD4+ cell, then, the small magnetic bead that adds Miltenyi company is (according to 10ul and 1x10
6The ratio of individual cell adds the amount of magnetic bead) since have on the CD25 antibody phycoerythrin (Phycoerythrin PE), and has the antibody of anti-phycoerythrin on the small magnetic bead of Miltenyi, like this, all CD25+ cells just with magnetic bead formation mixture.Then, by a cell wash-out pillar that magnetic field is arranged, the positive cell that obtains that separates is exactly that CD4+CD25+FOXP3+ regulates the T cell.
4. according to the calculating of body surface area, planned injection 10 of the present invention
9Individual CD4+CD25+FOXP3+ regulates the T cell and gives patient SLE.For guaranteeing maximum efficiency, our plan carries out course of treatment of routine medication at first for the active SLE patients patient, and then, start injection CD4+CD25+ regulates the T cell.
5. because CD4+CD25+FOXP3+ regulates about month of T cell time to live in animal body, plan each month duplicate injection of the present invention once.
6. in regulating T cell therapy process, the present invention also needs to detect closely the concentration of autoantibody and the index of other reflection disease degree and mobility except needs are observed patient's clinical symptom progress.
7. the present invention plans that also CD4+CD25+FOXP3+ is regulated the T cell and is used in combination with immunosuppressor and hormone and treats SLE, reduces the dosage of hormone and immunosuppressor in this process.
Claims (5)
1. one kind at the beta induced adjusting T cell of the TGF_ of autoimmune disorder, it is characterized in that it being to combine by TGF_ β and two cytokines of IL_2, CD4+ cell by external evoked autoimmune disorder patient obtains, specifically be designated as CD4++CD25+Foxp3+ and regulate the T cell, FoxP3 is FoxP3 gene or albumen here.
2. formation method at the beta induced adjusting T cell of the TGF_ of autoimmune disorder is characterized in that concrete steps are as follows:
(1) from autoimmune disorder patient vein, extracts peripheral blood, with lymphocyte separation medium technology isolated lymphocytes; Use monoclonal antibody and negative technical point from the NAIVE_CD4+ cell again;
(2) to isolated NAIVE_CD4+ cell, the particulate pearl that wraps up with anti-CD3/CD28 antibody stimulates; Add the TGF_ β of 3-10ng/ml and the IL_2 of 15-30unit/ml then, cultivated 5-6 days;
(3) then the CD25 positive cell in the CD4+ cell is separated with MACS particulate pearl, promptly obtain required CD4+CD25+Foxp3+ and regulate the T cell.
One kind by the beta induced adjusting T cell of TGF_ as claimed in claim 1 as the application in preparation treatment autoimmune disorder patient's the pharmaceutical preparation.
4. application according to claim 3 is characterized in that the beta induced adjusting T cell ampoule of TGF_ is 5-1020, and injection in every month once.
5. application according to claim 3, it is characterized in that the autoimmune disorder patient is earlier with immunosuppressor or treat a course of treatment, use the beta induced adjusting T cell of TGF_ then, perhaps that TGF_ is beta induced adjusting T cell and immunosuppressor or hormone combine use, and reduce the using dosage of hormone or immunosuppressor mutually.
Priority Applications (1)
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|---|---|---|---|---|
| CN102321580A (en) * | 2011-08-23 | 2012-01-18 | 郑颂国 | Regulatory T cells for treating autoimmune diseases, and preparation method thereof |
| CN103782173A (en) * | 2011-07-01 | 2014-05-07 | 贝克曼考尔特公司 | Regulatory T cells and methods of identifying, obtaining and using to treat immuno-based disorders |
| CN107177547A (en) * | 2017-06-05 | 2017-09-19 | 复旦大学附属中山医院 | It is a kind of to contribute to the external composition for obtaining folliculus regulatory T cells, method and its application |
| CN108004208A (en) * | 2017-12-01 | 2018-05-08 | 南京爱瑞生物科技有限公司 | A kind of method of external evoked T cells with antigenic specificity |
| CN108467852A (en) * | 2010-04-22 | 2018-08-31 | 南加州大学 | Method and composition for expanding and stablizing nature regulatory T cells |
| CN110331131A (en) * | 2019-07-25 | 2019-10-15 | 上海轩锋生物科技有限公司 | A kind of Treg cell abductive approach of optimization |
| CN110478474A (en) * | 2019-08-21 | 2019-11-22 | 启辰生生物科技(珠海)有限公司 | Immunomodulator, vaccine, cell and application |
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| CN108467852A (en) * | 2010-04-22 | 2018-08-31 | 南加州大学 | Method and composition for expanding and stablizing nature regulatory T cells |
| CN103782173A (en) * | 2011-07-01 | 2014-05-07 | 贝克曼考尔特公司 | Regulatory T cells and methods of identifying, obtaining and using to treat immuno-based disorders |
| CN103782173B (en) * | 2011-07-01 | 2018-07-13 | 贝克曼考尔特公司 | Regulatory T-cell and identification obtain and for treating based on immune disorderly method |
| CN102321580A (en) * | 2011-08-23 | 2012-01-18 | 郑颂国 | Regulatory T cells for treating autoimmune diseases, and preparation method thereof |
| CN107177547A (en) * | 2017-06-05 | 2017-09-19 | 复旦大学附属中山医院 | It is a kind of to contribute to the external composition for obtaining folliculus regulatory T cells, method and its application |
| CN107177547B (en) * | 2017-06-05 | 2020-11-24 | 复旦大学附属中山医院 | Composition and method for facilitating in vitro acquisition of follicular regulatory T cells and application of composition and method |
| CN108004208A (en) * | 2017-12-01 | 2018-05-08 | 南京爱瑞生物科技有限公司 | A kind of method of external evoked T cells with antigenic specificity |
| CN110331131A (en) * | 2019-07-25 | 2019-10-15 | 上海轩锋生物科技有限公司 | A kind of Treg cell abductive approach of optimization |
| CN110478474A (en) * | 2019-08-21 | 2019-11-22 | 启辰生生物科技(珠海)有限公司 | Immunomodulator, vaccine, cell and application |
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