CN109055523A - TNFR2 gene and its application for encoding albumen - Google Patents
TNFR2 gene and its application for encoding albumen Download PDFInfo
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Abstract
The invention discloses the application of TNFR2 gene and its encoded albumen and TNFR2 specific agonist in the drug for preparing and screening treatment autoimmune disease；The present invention discloses a kind of molecular marker on regulatory T cells surface, which is TNFR2.The invention proposes a kind of drugs that TNFR2 agonist can be used as treatment autoimmune disease；Since TNFR2 is expressed in part cell surface with only limiting to, especially expressed in regulatory T cells surface height, TNFR2 participates in differentiation, proliferation and the inhibition function of regulatory T cells, and maintain the existing state of regulatory T cells, thus, the present invention proposes that TNFR2 agonist can treat autoimmune or diseases associated with inflammation, such as multiple sclerosis, rheumatoid arthritis etc., can targeting in TNFR2, the immunoloregulation function for activating TNFR2 signal avoids and blocks serious side effects brought by TNF α signal completely, reduces dosage.
Description
Technical field
The present invention relates to the applications of biomedicine technical field, especially TNFR2 gene and its coding albumen；The present invention is also
It is related to a kind of molecular labeling on biomarker, especially T cell surface；More particularly it relates to TNFR2 agonist,
Its TNFR2 by acting on regulatory T cells surface promotes differentiation, proliferation and function of regulatory T cells etc. to treat certainly
Body immunity or diseases associated with inflammation.
Background technique
It is well known that tumor necrosis factor (Tumor Necrosis Factor, TNF) α is a kind of very important anticusp
Inflammation factor participates in activating and promotes immune response to remove pathogen.And in recent years, more and more report discovery TNF αs are also joined
With immunomodulatory action.Regulatory T cells (Tregs) are maintaining autoimmune tolerance as classical suppressor T lymphocyte subgroup
Stable state and prevention autoimmunity in terms of play a crucial role.If the lazy weight or function of Tregs is impaired, inflammation will lead to
Disease property T cell duration excessive activation, so as to cause the generation of autoimmune disease.
On the other hand, more and more research discovery TNFR2 are expressed in the surface Tregs height, and TNF α can enhance Tregs
Stability and function.And be directed to the inflammatory reaction that TNF α is mediated and the total inhibitor of TNF α occurred, although it is certain from
Certain effect is played in body immunological disease, however, there are some patients not react it, or even long-term use appearance is serious
Side effect.Intracellular section of TNFR1 and TNFR2 does not have function signal access, and the two conduction activates different cellular activities.And
TNFR1 and TNFR2 is unclear to the specifically effect of regulatory T cells.Therefore, TNFR1 and TNFR2 are to inflammatory T cell and tune
The effect of section property T cell need further to study.
Summary of the invention
Based on the above issues, present inventor in the course of the research, studies TNF α/TNFR pairs through a large number of experiments
Tregs differentiation, existing state, proliferation and inhibit function influence process in, it has unexpectedly been found that TNFR2 is in the high table in the surface Tregs
It reaches, TNF α promotes differentiation, proliferation and the inhibition function of Tregs by TNFR2.Based on this discovery, the technical side that inventor proposes
Case includes the following aspects:
In the first aspect, the present invention provides TNFR2 genes to prepare and screen treatment autoimmune or inflammatory
Application in the drug of disease.
In the second aspect, the present invention provides the protein of TNFR2 coded by said gene to prepare and screen treatment itself
Application in the drug of immunity or diseases associated with inflammation.
In the third aspect, the present invention provides TNFR2 specific agonists to prepare and screen treatment autoimmune
Or the application in the drug of diseases associated with inflammation.
Preferably, the autoimmune disease is multiple sclerosis, rheumatoid arthritis, encephalomyelitis or colitis.
In the fourth aspect, the present invention provides a kind of for treating the drug of autoimmune or diseases associated with inflammation, institute
It states drug and contains TNFR2 specific agonist.
According to an embodiment of the invention, the TNFR2 is expressed in the surface Tregs height, activation TNFR2 can promote Tregs
Differentiation, amplification and inhibit function, and TNFR2 lacks the amplification that may cause Tregs and inhibits function impaired, so as to cause from
The generation of body immunological disease.
According to an embodiment of the invention, the invention proposes the differentiation that specificity T NFR2 agonist can mediate iTregs.
TNF α can promote the vitro differentiation of iTreg cell, and as the raising of TNF α concentration induction is that iTreg cell is more.
Be added TNF α time it is more early, induce for iTreg cell it is more.Naive CD4 is not influenced after TNFR1 gene knockout+T is thin
Born of the same parents break up to iTregs, and the differentiation of iTregs is obviously damaged after TNFR2 gene knockout, and stimulate TNF α reactionless.
The invention also provides specificity T NFR2 agonists can mediate the amplification of Tregs.Inventor has found TNFR2 energy
Enough promote the amplification of Tregs, especially when disproportional immune reaction occurs for body, after TNFR2 gene knockout, Tregs's
Amplification ability is obviously damaged, and cannot be expanded to certain amount, thus Tregs cannot effectively play immunosuppression capability, inhibit
The immune response of degree, so as to cause the generation of autoimmunity disease.And specificity T NFR2 agonist can promote Tregs in body
Interior amplification is played the role for the treatment of autoimmune disease.
The present invention, which has also been proposed specificity T NFR2 agonist, can mediate the inhibition function of Tregs.Inventor has found TNF α
The external inhibition function of pretreated Tregs enhances, and the presence of TNF α does not damage the inhibition function of Tregs.But it will
After TNFR2 gene knockout, the inhibition function of Tregs is obviously damaged.From this prompt TNFR2 is when Tregs plays inhibition function
Key effect.And specificity T NFR2 agonist can promote the inhibition function of Tregs, when autoimmunity occurs, expand it and exempt from
Epidemic disease rejection ability plays the role for the treatment of autoimmune disease.
Preferably, the reagent that can make TNFR2 gene overexpression is contained in the drug；Preferably, contain in the drug
It can promote the reagent of the protein active of TNFR2 coded by said gene.As a result, by promoting the expression of TNFR2 gene, and then obtain
More TNFR2 coding proteins are obtained, or promote the protein active of TNFR2 coded by said gene, the differentiation of Tregs can be promoted, expanded
Increase and inhibit function.
Preferably, the autoimmune disease is multiple sclerosis, rheumatoid arthritis, encephalomyelitis or colitis.
At the 5th aspect, the present invention provides a kind of molecular marker on regulatory T cells surface, the molecular markers
Object is TNFR2, and on the regulatory T cells surface of normal person, the expression of TNFR2 is higher than TNFR1；And in autoimmune disease
The regulatory T cells surface of patient, the expression decline of the TNFR2, and the expression of TNFR1 rises.
According to an embodiment of the invention, TNFR1 gene knockout causes it to convert in vivo and in vitro to the direction Th1 and Th17
Ability it is impaired, and the naive CD4 of TNFR2 gene knockout+The ability that T cell is broken up to Tregs is impaired.And TNFR2 gene
The mouse development of knockout is that the symptom of autoimmune disease is heavier compared with WT mouse, and the mouse of TNFR1 gene knockout then can
Fight the generation of autoimmunity disease.The Tregs amplification ability and immunosuppression capability of TNFR1 knock out mice are normal, and
The Tregs amplification ability and immunosuppression capability of TNFR2 gene knockout are obviously damaged.
Preferably, the autoimmune disease is multiple sclerosis or rheumatoid arthritis.
According to an embodiment of the invention, inventor also found the surface Tregs TNFR2 expression after TNFR1 gene knockout
It increases, the surface Tregs TNFR1 expression increases after TNFR2 gene knockout.And inventor also found, in rheumatoid arthrosis
The surface Tregs extracted in scorching and patients with multiple sclerosis peripheral blood, TNFR1 expression increases, and TNFR2 expression
Decline, therefore, inventor further provide TNFR1 and TNFR2 the surface Tregs expression with immune microenvironment
Variation and changes, the two presence overlapping functionally and compensatory；Under noninflammatory environment, TNFR1 and TNFR2 receptor is complete, may
The signal of two receptors maintains homeostasis；Under inflammatory microenvironment, the TNFR1 expression of the surface Tregs energy is increased, this may
So that Tregs is intended to apoptosis, or breaks up to inflammatory cell；And TNFR2 expression declines, stability, proliferative capacity and function
It may can be damaged.Therefore, the specificity T NFR2 agonist that the embodiment of the present invention proposes avoids TNF α completeness inhibitor and leads
The general toxicity of cause and other serious side effects, it is also possible to reduce the expression of the TNFR1 on the surface Tregs indirectly.
In conclusion the invention has the benefit that
Present invention firstly provides a kind of drug that TNFR2 agonist can be used as treatment autoimmune disease, TNF α conducts
A kind of multidirectional function cell factor, receptor TNFR1 wide expression is in systemic cell, main inducing inflammatory reaction；And TNFR2 is only
It is expressed in limitation part cell surface, is especially expressed in regulatory T cells surface height；TNFR2 participates in point of regulatory T cells
Change, be proliferated and inhibit function, and maintains the existing state of regulatory T cells；
TNFR2 agonist proposed by the invention can treat autoimmune or diseases associated with inflammation, such as multiple sclerosis, class
Rheumathritis etc., can targeting in TNFR2, activate the immunoloregulation function of TNFR2 signal, avoid complete blocking
Serious side effects brought by TNF α signal, reduce dosage.
Detailed description of the invention
Fig. 1 is the naive CD4 with Foxp3-GFP+T cell is induced to differentiate into the result figure of iTregs in vitro；Its
Middle a, b and c show that TNF α promotes the differentiation of iTregs in the form of dose-dependent,
D and e shows that TNF α maintains the stability of iTregs,
F shows TNFR1-/-The differentiation of iTregs, TNFR2 are not influenced-/-Keep iTregs differentiation obvious impaired,
G shows that TNF α can promote WT and TNFR1-/-naive CD4+T cell is broken up to the direction iTregs, and right
TNFR2-/-naive CD4+T cell is reactionless；
Fig. 2 is to use TNFR1-/-And TNFR2-/-Mouse extract naive CD4+T cell is in Rag1-/-Enteritis is made on mouse
The result figure of model；
Wherein, a shows the changes of weight of each group Recipient mice after modeling；
B and c shows the colon pathological change of each group Recipient mice after modeling；
D shows the intuitive performance of the spleen, lymphonodi mesenterici and enteron aisle of each group Recipient mice after modeling；
Fig. 3 is the colitis model of Fig. 2 according to an embodiment of the present invention the 21st day and the 28th day after modeling, the spleen of each group
Dirty, lymphonodi mesenterici and enteron aisle lamina propria Foxp3+Cell is in CD4+The result figure of ratio in cell；
Fig. 4 is to WT, TNFR1-/-And TNFR2-/-Mouse carries out the result figure of the modeling of EAE model；
Wherein, a shows the disease symptoms scoring variation of each group mouse after modeling；
After b shows modeling, the pathological change of each group mouse brain and spinal cord；
C shows the CD4 of each group mouse brain after modeling+The ratio of Tregs, Th1 and Th17 cell in T cell；
D shows the CD4 of each group mouse spinal cord after modeling+The ratio of Tregs, Th1 and Th17 cell in T cell.
Fig. 5 is the result figure of the influence expanded in vivo and in vitro of TNFR1 and TNFR2 to Tregs；
Wherein, a~d shows WT, the TNFR1 for not receiving any immune primary stimuli-/-And TNFR2-/-The spleen of mouse and
In lymph node, in CD4+The shared ratio of Tregs difference in T cell；
E~h is shown in WT, the TNFR1 for receiving MOG+PTX stimulation 30 days-/-And TNFR2-/-The spleen of mouse and leaching
In fawning on, in CD4+The shared ratio of Tregs difference in T cell；
I~k shows WT, TNFR1-/-And TNFR2-/-Tregs is extracted in the lymph node of mouse, carries out amplification in vitro
Afterwards, each group Tregs cell surface Ki-67 expression, group of cells sum and Foxp3+Cell absolute number；
Fig. 6 is influence result figure of the TNF α/TNFR to the external function of iTregs；
Wherein, a, b are shown WT mouse naive CD4+Induced t cell is iTregs, is added in Induction Process or not
Add TNF α, compares two groups of iTregs in vitro to CD8+The rejection ability of T cell；
C, d is shown WT mouse naive CD4+Induced t cell is iTregs, then carries out inhibition functional experiment,
TNF α is added or be not added in inhibition system, compares two groups of iTregs in vitro to CD8+The rejection ability of T cell；
E, f is shown WT, TNFR1-/-And TNFR2-/-The naive CD4 of mouse+Induced t cell is iTregs, is compared
Three groups of iTregs is in vitro to CD8+The rejection ability of T cell；
Fig. 7 is WT, TNFR1-/-And TNFR2-/-The result figure that the iTregs in mouse source treats EAE model；
Wherein, a shows the disease symptoms scoring variation of each group mouse；
B shows the pathological change of each group mouse brain and spinal cord；
C, d, e show the CD4 of each group mouse brain+The ratio of Th1 and Th17 cell in T cell；
F, g, h show the CD4 of each group mouse spinal cord+The ratio of Th1 and Th17 cell in T cell；
Fig. 8 is WT, TNFR1-/-And TNFR2-/-The naive CD4 in mouse source+The TNFR1 of T cell and iTregs and
The result figure of the expression of TNFR2；
Fig. 9 is the expression of the TNFR1 and TNFR2 of the Tregs of patients with multiple sclerosis and rheumatoid arthritis patients
Result figure.
Specific embodiment
TNFR2 agonist is put forward for the first time in the application can be used to treat autoimmune or diseases associated with inflammation, effect side
Formula includes: specific recognition TNFR2, activates TNFR2 signal mediated immunity regulatory function.
In autoimmune disease, TNFR2 agonist can selectively activate the immunological regulation function of TNFR2 signal mediation
Can, TNFR2 letter has also been blocked while the inflammatory effect for inhibiting TNFR1 signal to mediate rather than TNF α completeness inhibitor
Number immunoregulation effect, therefore serious side effect can be brought.TNFR1 wide expression in the nearly all cell surface of body,
So that specificity T NFR1 blocking agent also results in corresponding side effect.And cell category expressed by TNFR2 is limited, makes its poison
Property minimize, dosage minimize.
Further, the agonist includes the signal of specific recognition TNFR2.
On the other hand, a kind of surface marker present invention firstly provides TNFR2 as regulatory T cells expresses water
It is flat to be higher than effector T cell.
The present invention also provides the methods using TNFR2 agonist treatment autoimmune disease, comprising steps of
(a) TNFR1 signal inducing inflammatory reaction, TNFR2 signal mediated immunity adjustment effect；And TNFR2 participation is various
The repair and reconstruction of tissue；
(b) based on (a) as a result, TNFR2 agonist specific activation TNFR2 signal, promote point of regulatory T cells
Change, proliferation and function.
Specifically, the immunoregulation effect that TNFR2 agonist specific activation TNFR2 signal mediates, especially promotes
Differentiation, proliferation and the function of regulatory T cells achieve the purpose that inhibit excessive immune reaction, so as to be used to treat itself
Immunity disease.
Present inventor has found that TNFR1 transmitting inflammation cell such as Th1 and Th17 cell divides through a large number of experiments
Change and activates, and TNFR2 mediates differentiation, proliferation and the inhibition function of Tregs cell.And Tregs surface expression is high-caliber
TNFR2 and low-level TNFR1.In the peripheral blood of multiple sclerosis and rheumatoid arthritis patients, the surface Tregs
The decline of TNFR2 expression, and TNFR1 expression increases.In view of the receptor-mediated different signal path of two differences of TNF α,
Therefore and specificity T NFR1 blocking agent or specificity T NFR2 agonist can targeted inhibition immune response, however TNFR1 is wide
General to be expressed in systemic cell, targeting excitement TNFR2 signal can promote differentiation, proliferation and the inhibition function of Tregs more accurately
Can, TNF α complete inhibition agent or TNFR1 blocking agent bring systemic side effects are avoided, while reducing dosage, become
The new method of future therapeutic autoimmunity disease.
Specifically, key played in function in the differentiation of Tregs cell, proliferation and is inhibited to TNFR2 based on inventor
Effect, and the surface Tregs height expresses TNFR2, in autoimmune disorders, under the TNFR2 expression on the surface Tregs
Drop, the invention proposes can treat the new method of autoimmunity disease with specificity T NFR2 agonist.
In addition, the type of the reagent of specific recognition TNFR2 is not particularly limited, as long as being capable of specific recognition cell table
The TNFR2 in face.The reagent for the specific recognition TNFR2 that embodiment according to the present invention can use includes but is not limited to spy
The opposite sex identifies at least one of the antibody of TNFR2 albumen, probe of specific recognition TNFR2mRNA.Specific recognition TNFR2 egg
White antibody can be for the specific binding that TNFR2 antigen-specific sites are designed and prepare, and then pass through antigen-antibody
Reach the TNFR2 of specific activation cell surface.The selectively targeted treatment autoimmune disease that the embodiment of the present invention proposes
Method, i.e. specific activation TNFR2 can reduce dosage in terms for the treatment of autoimmune disease, mitigate total
Tnf inhibitor serious side effects caused by body.
According to a particular embodiment of the invention, hair when inventor is influence of the discussion TNF α for the differentiation of iTregs
Existing TNF α can promote the differentiation, amplification and inhibition function of iTregs.And induction is that Th1 and Th17 is thin after TNFR1 gene knockout
The ability of born of the same parents is impaired, and induction is that the ability of Tregs cell is impaired after TNFR2 gene knockout, TNFR1-/-The surface iTregs
TNFR2 expression increases, TNFR2-/-The TNFR1 expression on the surface iTregs increases.And it is more in autoimmune disease
In the peripheral blood of the hardening of hair property and rheumatoid arthritis patients, inventor has found the TNFR2 expression water on the surface Tregs in an experiment
It is flat to be remarkably decreased, and TNFR1 expression increases.To which inventor obtains specific inhibition TNFR1 or specific activation
TNFR2 has the potential for the treatment of autoimmune disease, and TNFR2 is differentiation, proliferation and the important letter of function for mediating Treg cell
Number conductive protein.
To the differentiation of iTregs, proliferation and the influence of function will be inhibited to carry out in detail TNF α, TNFR1 and TNFR2 below
Description:
According to an embodiment of the invention, naive CD4+T cell can be coated with the magnetic bead of CD3/CD28, IL-2 and
TGF-β is induced in vitro as iTregs.Naive of embodiment of the present invention CD4+T is being induced to differentiate into iTregs, and TNF α ratio is added
The ratio that TNF α induction is not added as iTregs is high, and increases with the raising of TNF α concentration, and TNF α is added more early,
Induction is higher as the ratio of iTregs.Since TNF α plays biological function by its two receptors TNFR1 and TNFR2
's.The embodiment of the present invention has used TNFR1-/-And TNFR2-/-Mouse.Inventor has found TNFR1-/- naive CD4+T is thin
Born of the same parents can successfully be divided into iTregs, and when losing TNFR2 gene, naive CD4+T cell is divided into the ratio of iTregs
It is decreased obviously, total cell number and Foxp3+The absolute number of cell substantially reduces.In addition, rmTNF α (recombination macaque neoplasm necrosis
The factor) WT and TNFR1 can be promoted-/- naive CD4+T cell is broken up to the direction iTregs, and cannot promote TNFR2-/-
naive CD4+T cell is broken up to iTregs.Thus TNFR2 and its downstream signaling pathway also can be in the corresponding TCR thorns of cell
Promote the activation of T cell when swashing as costimulatory molecules.Or during iTregs induction, TNFR2, which expresses Foxp3, to be had
There is synergistic effect, the signal path of this synergistic effect is activated in cytoplasm.
According to an embodiment of the invention, the embodiment of the present invention isolates naive CD4 from WT mouse+T cell, according to mark
Quasi- method induction is at iTregs.Cell is collected after 3 days, removes the coated beads of AntiCD3 McAb/CD28 in culture medium, washs iTregs
Twice to remove cell factor, then the iTregs generated planted into plate again, it is (5 thin that the coated beads of AntiCD3 McAb/CD28 is added
Born of the same parents: 1 bead), it is added or is added without rmTNF α (100ng/ml).After 3 days, the expression of flow cytomery Foxp3.
Inventor has found that when lacking rmTNF α, the Foxp3 expression of iTregs declines rapidly, and rmTNF α stimulation can maintain
The phenotype of iTregs.
According to an embodiment of the invention, the WT naive CD4 that the embodiment of the present invention is extracted+Induced t cell differentiating into T h1
With Th17 cell, in culture medium plus or it is not added rmTNF α.The expression of flow cytomery IFN γ and IL-17A, invention
People has found that the stimulation of TNF α can not enhance WT naive CD4+The vitro differentiation of T cell to Th1 and Th17 cell is horizontal.Hair
Bright people is also to from WT, TNFR1-/-Mouse and TNFR2-/-The naive CD4 of mouse+T cell has carried out Th1 and Th17 differentiation
Induction, the expression of flow cytometry analysis IFN γ and IL-17A.Inventor has found that TNFR1 gene knockout makes IFN γ+
Cell and IL-17A+Cell proportion reduces.And TNFR2-/-CD4+T cell is unaffected.
According to an embodiment of the invention, the embodiment of the present invention is by by naive CD4+T cell intraperitoneal injection is squeezed into
Rag1-/-In Mice Body, colitis model is induced, while naive CD4 can be observed+T cell is in the intracorporal differentiation of Recipient mice
Situation.By WT, TNFR1-/-And TNFR2-/-The naive CD4 of mouse+T cell squeezes into Rag1 respectively-/-In Mice Body, pass through sight
The expression of changes of weight, intestinal wall pathological manifestations and flow cytometry analysis Foxp3, IFN γ and IL-17A is examined to judge
naive CD4+The differentiation situation of T cell.The Rag1 of inventor's discovery WT group-/-Mouse weight gradually mitigates.H&E dyeing display
Colon intestinal wall has serious inflammatory cell infiltration, ulcer, and intestinal wall oedema thickens.TNFR1-/-On group Recipient mice weight can walk unhurriedly
It rises, pathology shows more complete gut wall structure, seldom lymphocytic infiltration.TNFR2-/-Group Recipient mice weight loss is most
It is more, and whole section of intestinal wall Severe edema and the molding excrement of shortage, spleen and mLNs enlargement are most obvious, and inflammatory cell infiltration is most
It is more, structure disturbance.
The 21st day and 28 days execution mouse after modeling.With CD4 in each organ of flow cytomery+In cell
Foxp3+The ratio of T cell.Inventor has found WT and TNFR1 in spleen, lymphonodi mesenterici and enteron aisle lamina propria-/-It is small
The cell differentiation of mouse is Foxp3+The ratio indifference of T cell, and TNFR2-/-The cell differentiation of mouse at Treg cell ratio
Example is minimum.And with the progress of disease, which persistently exists.
According to an embodiment of the invention, choice experiment of embodiment of the present invention Autoimmune Encephalomyelitis (EAE) mouse
Model, selection are all the WT, TNFR1 of female, 8 week old-/-And TNFR2-/-Mouse carries out the induction of EAE model.Observe mouse
Clinical score.Put to death mouse after, observe brain and spinal cord pathological change, flow cytometry analysis spleen, lymph node, brain and
The CD4 of spinal cord+Foxp3 in T cell+、IFNγ+Or IL-17A+The ratio of cell.Inventors have found that TNFR1-/-Mouse energy
Enough to react fully against autoimmune inflammation, without any EAE symptom, and TNFR2 gene knockout is to mention the disease onset time
Before, state of an illness most serious.H&E coloration result shows, TNFR2-/-Inflammatory mononuclear cells infiltration is most tight in the brain and spinal cord of mouse
Weight, and TNFR1-/-The brain and spinal cord of mouse are barely perceivable inflammatory cell.Inventor also found TNFR2-/-Tregs in mouse
In CD4+Ratio in T cell is minimum, and TNFR1-/-Th1 and Th17 cell is in CD4 in mouse+Ratio in T cell is minimum.
According to an embodiment of the invention, whether the embodiment of the present invention can damage Treg for clear TNFR2 gene knockout
Cell amplification ability, we have evaluated WT, TNFR1-/-And TNFR2-/-Mouse Foxp3 before not inducing EAE model+Cell exists
CD4+Underlying scale in T cell.Then three kinds of mouse are given, after being immunized 30 days with MOG+PTX, put to death mouse, fluidic cell
Instrument detection Foxp3 in spleen and lymph node+Cell is in CD4+Ratio in T cell, and be it is immune before compare.I
Also from WT, TNFR1-/-And TNFR2-/-Treg cell is extracted in mouse lymph nodes, it is (thin with the magnetic bead of coating CD3/CD28
Born of the same parents: beads=1:1), rhIL-2 (300U/ml) is cultivated 3 days.It is thin by flow cytomery Ki-67 expression and each group
Born of the same parents' sum and Foxp3+The absolute number of cell.Inventor's discovery TNFR2 in spleen and LN-/-The Tregs ratio of mouse is slightly below
WT and TNFR1-/-Mouse.However, after being immunized 30 days with MOG, in spleen and lymph node, WT and TNFR1-/-In mouse
Tregs increases by 2~4 times, and in TNFR2-/-Tregs cell is in mouse almost without increase.Amplification in vitro experiment is thin by streaming
Born of the same parents' instrument detects Ki-67 expression.We have found that TNFR2-/-The Ki-67 level of Tregs is minimum, and cell state is worst, survival
Cell number it is minimum.
According to an embodiment of the invention, the embodiment of the present invention inhibits the work of function for clear TNF α in vitro to iTregs
With.We observe cell proliferative conditions with CFSE labelling method.After 72 hours are incubated for, baseline control group shows had significant proliferation, and adds
It is similar to when being not added TNF α to enter the baseline group Teffs of TNF α.First by WT naive CD4+Induced t cell is iTregs,
In induction system plus or be not added TNF α, after induction 3 days, washing iTregs twice with remove cell factor and with CFSE- label
Teffs is co-cultured 3 days.The inhibiting effect ratio that inventor's discovery is proliferated Teffs with the pretreated iTreg of rmTNF α without locating in advance
The inhibitory effect of reason is good.In order to which whether the clear TNF α in the cultivating system of Inhibition test influences iTreg to the external of Teffs
Rejection ability, inventors have found that the rejection ability of iTregs does not decline under rmTNF α exposure.In order to further clarify
TNFR1 and TNFR2 is in the effect of the in-vitro suppression capacity of iTregs, we are from WT, TNFR1-/-And TNFR2-/-In mouse spleen
Extract naive CD4+T cell induces as iTregs, has carried out body outer suppressioning experiment under the same terms.TNFR2-/- iTregs
Function is inhibited to be remarkably decreased compared with WT iTregs.
According to an embodiment of the invention, the embodiment of the present invention is in order to which clear TNFR1 and TNFR2 is in the internal inhibition of iTregs
The effect of ability, inventor is by WT, TNFR1-/-And TNFR2-/-The naive CD4 of mouse+Induced t cell is iTregs, observation
Its therapeutic effect to EAE mouse model.Inventor has found that WT iTregs can successfully inhibit the EAE state of an illness, and delay EAE rises
Sick time breaking-out, improves symptom, TNFR1-/-ITregs inhibits function slightly strong compared with WT iTregs, greatly reduces spinal cord and brain
In autoimmune response, the proliferation of Th17 cell and Th1 cell is suppressed, and pathological examination shows seldom monocyte leaching
Profit.However, the knockout of TNFR2 gene has damaged the inhibition function of iTregs, the EAE state of an illness cannot be improved, FACS is in spinal cord and brain
In detect the significant increase of Th17+ cell, pathological section can also see that has a large amount of inflammatory cells to accumulate in brain and spinal cord.
In addition, the expression of the surface Tregs TNFR1 and TNFR2 has the feature that in the embodiment of the present invention
According to an embodiment of the invention, the embodiment of the present invention has detected TNFR1 and TNFR2 in WT, TNFR1-/-、TNFR2-/-
Expression on iTregs.Inventor has found these three naive CD4+T cell express low-level TNFR1 and
TNFR2.After inducing as iTregs, the expression variation of TNFR1 is unobvious, and the expression of TNFR2 increases.And
And in TNFR1-/-On the surface iTregs, TNFR2 expression is higher, and TNFR2-/-The expression of the TNFR1 of iTregs
It increases.It further demonstrates iTregs and expresses high-caliber TNFR2, therefore there is efficient function inhibitio Teffs activity, and lose
TNFR2 expression, TNFR2 are removed-/-ITregs cannot fall off TNFR2, and function is inhibited to be damaged.
According to an embodiment of the invention, the embodiment of the present invention is in order to clear in patients with multiple sclerosis and rheumatoid arthrosis
The CD4 of scorching patient+Whether TNFR1 the and TNFR2 level of Tregs is different compared with Healthy People.Inventor collects Healthy People
With the peripheral blood of MS patient, peripheral blood mononuclear cells is isolated.The table of TNFR1 and TNFR2 on flow cytomery Tregs
It reaches.The Foxp3 of inventor's discovery Healthy People+T cell expresses low-level TNFR1 and high-caliber TNFR2.And multiple
The surface Tregs of sclerosis patients and rheumatoid arthritis patients, the horizontal significant raising of TNFR1, TNFR2 level decrease beyond one
Half.This further demonstrates that TNFR1 is required for causing or promoting reaction before the inflammation in autoimmune disease；And
TNFR2 may be fallen off by Tregs in the form of sTNFR2 to neutralize excessive TNF α, and under the TNFR2 on the surface Tregs level is significant
Drop.But the horizontal low Tregs of TNFR2 can not effectively be proliferated and inhibit Teffs.
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with the drawings and specific embodiments pair
The present invention is described further.The embodiments described below is exemplary, and for explaining only the invention, and should not be understood as pair
Limitation of the invention.Particular technique or condition are not specified in embodiment, according to the literature in the art described technology or
Condition is carried out according to product description.Reagents or instruments used without specified manufacturer is that can be obtained by commercially available
The conventional products obtained.
Embodiment 1naive CD4+External evoked T cell is iTregs
Inventor extracts naive CD4 using the method for autoMACS instrument magnetic bead sorting+T cell.First by the spleen of mouse
Cell carries out T cell enrichment with nylon hair column, and biotin mouse anti-CD8 antibody 6ul, biotin mouse anti-is then added
B220 antibody 8ul, biotin mouse anti-CD11b antibody 1.5ul, biotin mouse anti-CD11c antibody 1.5ul,
Biotin mouse anti-CD49b 1ul is mixed, and 4 DEG C are incubated for 20 minutes.Anti-biotin beads 8ul is added, is mixed,
4 degree are incubated for 15 minutes.AutoMACS instrument carries out the choosing of first step yin, goes unless CD4+Cell.Mouse CD62L-beads is added
8ul, 4 degree are incubated for 15 minutes.AutoMACS instrument carries out the choosing of second step sun.Sun choosing pipe is naive CD4+T cell.Streaming is thin
Born of the same parents' instrument detects naive CD4+T cell purity (> 90%).
Then, inventor is by the WT naive CD4 of extraction+T cell kind is in 96 orifice plates or 48 orifice plates, cultivating system
The middle addition AntiCD3 McAb/coated beads of CD28 (cell: beads=1:5), rhIL-2 (50U/ml) and rhTGF- β (2ng/ml),
It simultaneously will be in the rmTNF α adding hole of various dose (range is from 0 to 100ng/ml).Or same agent is added in different time points
The rmTNF α (100ng/ml) of amount.Same time point collecting cell is expressed by Flow Cytometry Assay Foxp3.Invention human hair
It is higher as the ratio of iTregs than TNF α induction is not added that TNF α is now added, and is increased with the raising of TNF α concentration, and TNF
α is added more early, and induction is higher as the ratio of iTregs, as a result as shown in Fig. 1 a~c.
Inventor is by WT naive CD4+T cell is iTregs by standard law induction.Then plate, cultivating system are planted again
In plus or be not added TNF α.Foxp3 expression is measured by flow cytometer after three days.Inventor has found that TNF α is able to maintain that iTregs
Stability, and the iTregs Foxp3 expression that TNF α is not added is decreased obviously, as a result such as Fig. 1 d, shown in e.
It will be from WT, TNFR1-/-And TNFR2-/-The naive CD4 that mouse is extracted+T cell kind is in 96 orifice plates or 48 orifice plates
In, be added in cultivating system the coated beads of AntiCD3 McAb/CD28 (5 cells corresponding a pearl), rhIL-2 (50U/ml) and
RhTGF- β (2ng/ml), while adding or being not added rmTNF α.Same time point collecting cell passes through Flow Cytometry Assay Foxp3
Expression.Inventor has found TNFR1-/- naive CD4+T cell can successfully be divided into iTregs, and work as and lose TNFR2 gene
When, naive CD4+The ratio that T cell is divided into iTregs is decreased obviously, total cell number and Foxp3+The absolute number of cell is aobvious
It writes and reduces.In addition, rmTNF α cannot promote TNFR2-/- naive CD4+T cell is broken up to iTregs, as a result such as Fig. 1 f, g institute
Show.
Embodiment 2naive CD4+Induced t cell Rag1-/-Mouse is colitis model
Choose the healthy Rag1 with week old-/-Mouse, random point 3 groups.Extract WT, TNFR1-/-Or TNFR2-/-Mouse
naive CD4+T cell, adjustment cell concentration are 3million/ml, are injected intraperitoneally to Rag1-/-Mouse, every mouse
0.6million(200ul).The weight of every mouse is recorded daily.It third week and 4th week after injecting cell, puts to death small
Mouse, the 1mm colon for taking out every mouse are fixed in the formalin of 10% buffering, and paraffin embedding is closed, slice, H&E dyeing.
It is collected from Recipient mice and comes from spleen, the monocyte of mLNs and cLP carry out counting and fluorescent staining.It is true by flow cytometer
Determine Foxp3+, IL-17A+Or IFN γ+Cell is in CD4+Ratio in T cell.Inventor has found TNFR1-/-Group Recipient mice body
Beijing South Maxpower Technology Co. Ltd walks unhurriedly rising, and colitis pathological manifestations are most light, there is more complete gut wall structure, seldom lymphocytic infiltration.TNFR2-/-
Group Recipient mice weight loss most serious is most fast, and whole section of intestinal wall Severe edema and the molding excrement of shortage, spleen and mLNs
Enlargement is most obvious, and inflammatory cell infiltration is most, multiple ulcers, and intestinal wall thickens most obvious, structure organization disorder, Th17 cell proportion
Highest and Tregs cell proportion is minimum, as a result such as Fig. 2 and 3.
Embodiment 3 is with WT, TNFR1-/-And TNFR2-/-Mouse is that study subject induces EAE model
Selection is all Healthy female WT, TNFR1 of 8 week old-/-And TNFR2-/-Mouse is as study subject.It is small for every
Mouse, in incomplete Freund's adjuvant 150ul plus the tubercle bacillus dry powder of 1.2mg inactivation is made the 8mg/ml's containing tuberculosin
Complete Freund's adjuvant 150ul emulsifies it with MOG (2mg/ml) 150ul in tee tube.Take pertussis toxin with no blood
Clear sterile PBS is diluted to 1.25ug/ml.
It is subcutaneously injected in three sites of back of mice, 200ul (250ng/ is injected intraperitoneally in each site 100ul, PTX
Mouse).Every mouse peritoneal injects 250ng pertussis toxin within second day.Repetition in 7th day operates for first day, subcutaneous injection
250ng PTX is injected intraperitoneally in 300ug MOG.Repetition in 9th day operates for second day.
Start within the 5th day after immune for the first time, records clinical score daily, each group calculates average mark daily.EAE is commented
It is divided into 0,1,2,3,4,5 grade.Inventors have found that TNFR1-/-Mouse can be fully against the generation of EAE disease, and TNFR2 gene
Knockout is disease onset time advance, state of an illness most serious, as a result as shown in Figure 4.
4 Tregs amplification in vitro of embodiment
Inventor is from WT, TNFR1-/-And TNFR2-/-In be extracted nTregs, and with the coated beads of AntiCD3 McAb/CD28
(cell: beads=1:1), rhIL-2 (300U/ml) are cultivated 3 days.Ki-67 expression is detected by FACS.We have found that
TNFR2-/-Tregs expression Ki-67 level it is minimum, cell state is worst, and the cell number of survival is minimum, and WT and
TNFR1-/-Tregs can maintain preferable cell state and cell number, express high-caliber Ki-67, as a result as shown in Figure 5.
Embodiment 5iTregs inhibits functional verification in vitro
The T cell 20million being enriched with nylon hair column is marked with 200ul CFSE (10uM).
Antigen deduction cell (antigen presenting cell, APC) in the present embodiment is from T cell enrichment
The cell of nylon Mao Zhuli afterwards, being handled with Co60 gamma-rays 30Gy makes its inactivation.The iTregs of induction is collected, magnetic frame removal is anti-
The coated beads of CD3/CD28 washes the cell factor removed in culture medium twice.By CFSE label T cell (Teffs, 0.3
×106A cells/well), the APC (0.3 × 10 of irradiation6A cells/well), soluble anti-cd 3 antibodies (0.025ug/ml) are seeded in
In flat 96 orifice plate.The iTregs with Teffs different proportion is added in Tregs inhibition group.By using flow cytometer after three days
Detect the proliferative conditions of CD8+ T cell.
Firstly, by WT naive CD4+When induced t cell is iTregs, adds in induction system or TNF α is not added, sends out
The inhibiting effect that the bright current pretreated iTreg of rmTNF α of human hair is proliferated Teffs is better than not pretreated inhibitory effect.
With standard method by WT naive CD4+Induced t cell is iTregs, is added in the cultivating system of Inhibition test
Or TNF α is not added, then carry out Inhibition test.Inventors have found that rmTNF α exposure under, the rejection ability of iTregs not under
Drop.From WT, TNFR1-/-And TNFR2-/-Naive CD4 is extracted in mouse spleen+T cell induces as iTregs, the same terms
Under carried out body outer suppressioning experiment.Inventor has found TNFR2-/-ITregs inhibits function to be remarkably decreased compared with WT iTregs,
As a result as shown in Figure 6.
Inhibit functional verification in 6 iTregs body of embodiment
Selection is all the Healthy female WT mouse of 8 week old as study subject, is randomly divided into 4 groups, every group.Exempt from for the first time
Pass through the WT, TNFR1 of intraperitoneal injection induction to every mouse within the 9th day after epidemic disease-/-Or TNFR2-/-ITregs 2million is thin
Born of the same parents (10million/ml, 200ul).The isometric sterile PBS of blank control group injection.It collects and comes from spleen, MLN, brain, spinal cord
Cell.Foxp3 is determined by FACS+、IL-17A+Or IFN γ+The ratio of T cell.Part brain and spinal cord carry out H&E dyeing.Hair
Bright people has found that WT iTregs can successfully inhibit the EAE state of an illness, and the breaking-out of delay EAE onset time improves symptom, TNFR1-/-
ITregs inhibits function slightly strong compared with WT iTregs, and the knockout of TNFR2 gene has damaged the inhibition function of iTregs, and streaming is thin
Born of the same parents' instrument detects Th17 in spinal cord and brain+The significant increase of cell, pathological section can also see that a large amount of inflammation in brain and spinal cord
Disease cellular accumulation, as a result as shown in Figure 7.
Influence of the 7 TNFR1 or TNFR2 gene knockout of embodiment to TNFR1 or the TNFR2 expression of iTregs
Experimental method: inventor is from WT, TNFR1-/-And TNFR2-/-Naive CD4 is extracted in Mice Body+T cell, and
It is under the same conditions iTregs with standard law induction, then has detected the naive of these three types respectively with flow cytometer
The expression of the TNFR1 and TNFR2 on T cell and the surface iTregs.
Experimental result: result is as shown in figure 8, these three naive CD4+T cell express low-level TNFR1 and
TNFR2.After inducing as iTregs, the expression variation of TNFR1 is unobvious, and the expression of TNFR2 increases.And
And in TNFR1-/-On the surface iTregs, TNFR2 expression is higher, and TNFR2-/-The expression of the TNFR1 of iTregs
It increases, further demonstrates iTregs and express high-caliber TNFR2, therefore there is efficient function inhibitio Teffs activity, and lose
TNFR2 expression, TNFR2 are removed-/-ITregs cannot fall off sTNFR2, and function is inhibited to be damaged.
The variation of the expression of the TNFR1 and TNFR2 of 8 Serum of Patients With Autoimmune Diseases Tregs cell surface of embodiment
For the clear CD4 in patients with multiple sclerosis and rheumatoid arthritis patients+The TNFR1 and TNFR2 of Tregs
Whether level is different compared with Healthy People, and inventor collects the peripheral blood of Healthy People and MS patient, isolates peripheral blood list
Nucleus.The expression of TNFR1 and TNFR2 on flow cytomery Tregs.
Experimental result: result is as shown in figure 9, find the Foxp3 from Healthy People+T cell express low-level TNFR1 and
High-caliber TNFR2；And in the surface Tregs of patients with multiple sclerosis and rheumatoid arthritis patients, the horizontal significant liter of TNFR1
Height, TNFR2 level decrease beyond half；This further demonstrates that TNFR1 is for causing or promoting in autoimmune disease
Reaction is required before inflammation；And TNFR2 may be fallen off in the form of sTNFR2 to neutralize excessive TNF α, and the surface Tregs
TNFR2 level is remarkably decreased.The low Tregs of TNFR2 expression can not effectively be proliferated and inhibit Teffs.
TNFR2 mediates the activation, amplification and inhibition function, TNFR2 of Tregs that can promote tissue repair and reconstruction.Tregs
Surface height expresses TNFR2, and in autoimmune disease, the TNFR1 expression on the surface Tregs is increased, under TNFR2 expression
Drop, the specific agonism TNFR2 and its downstream signal for being are able to maintain that and protect the immunoregulation effect of Tregs, to play
The effect for treating autoimmune disease.And TNFR1 wide expression is in the nearly all cell surface of body, specificity T NFR1 blocking
Agent may also will lead to serious side effect.And TNFR2 then limits to the cell mass for being expressed in derived from hematopoietic precursor cells, small colloid is thin
Born of the same parents etc. limit monoid, so that the specificity T NFR2 agonist is more suitable for treating autoimmune disease, accuracy
It is further increased with specificity, avoids the total inhibitor of TNF α and specificity T NFR1 the blocking agent serious secondary work caused by body
With.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office
It can be combined in any suitable manner in one or more embodiment or examples.In addition, without conflicting with each other, the skill of this field
Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples
It closes and combines.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
Claims (10)
- Application of the 1.TNFR2 gene in the drug for preparing and screening treatment autoimmune or diseases associated with inflammation.
- The protein of 2.TNFR2 coded by said gene is in the drug for preparing and screening treatment autoimmune or diseases associated with inflammation Using.
- Application of the 3.TNFR2 specific agonist in the drug for preparing and screening treatment autoimmune or diseases associated with inflammation.
- 4. described in any item applications according to claim 1~3, which is characterized in that the autoimmune disease is multiple Hardening, rheumatoid arthritis, encephalomyelitis or colitis.
- 5. a kind of for treating the drug of autoimmune or diseases associated with inflammation, which is characterized in that the drug contains TNFR2 spy Specific agonist.
- 6. drug according to claim 5, which is characterized in that TNFR2 gene overexpression can be made by containing in the drug Reagent.
- 7. drug according to claim 5, which is characterized in that TNFR2 coded by said gene can be promoted by containing in the drug Protein active reagent.
- 8. according to the described in any item drugs of claim 5~7, which is characterized in that the autoimmune disease is multiple Hardening, rheumatoid arthritis, encephalomyelitis or colitis.
- 9. a kind of molecular marker on regulatory T cells surface, which is characterized in that the molecular marker is TNFR2, normal The expression on the regulatory T cells surface of people, TNFR2 is higher than TNFR1；And in the regulatory T of autoimmune disease patient Cell surface, the expression decline of the TNFR2, and the expression of TNFR1 rises.
- 10. molecular marker according to claim 9, which is characterized in that the autoimmune disease is multiple hard Change or rheumatoid arthritis.
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| CN105829885A (en) * | 2013-10-17 | 2016-08-03 | 综合医院公司 | Methods of identifying subjects responsive to treatment for an autoimmune disease and compositions for treating the same |
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| CN105829885A (en) * | 2013-10-17 | 2016-08-03 | 综合医院公司 | Methods of identifying subjects responsive to treatment for an autoimmune disease and compositions for treating the same |
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| CN115003811B (en) * | 2020-12-11 | 2023-10-13 | 厦门宝太生物科技股份有限公司 | anti-TNFR 2 antibodies and uses thereof |
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