CN116115744A - Application of antigen peptide preparation in preparation of oral mucosa immunity enhancing in vivo tissue resident memory T cell kit - Google Patents
Application of antigen peptide preparation in preparation of oral mucosa immunity enhancing in vivo tissue resident memory T cell kit Download PDFInfo
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- CN116115744A CN116115744A CN202310077957.5A CN202310077957A CN116115744A CN 116115744 A CN116115744 A CN 116115744A CN 202310077957 A CN202310077957 A CN 202310077957A CN 116115744 A CN116115744 A CN 116115744A
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- oral
- resident memory
- preparation
- tissue resident
- oral mucosa
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Abstract
The invention discloses an application of an antigen peptide preparation in preparing an oral mucosa immunity enhancing in vivo tissue resident memory T cell kit, and relates to the field of biological medicine. Provides the application of the antigen peptide preparation in preparing an oral mucosa immunity enhancing in vivo tissue resident memory T cell kit, wherein the antigen peptide preparation comprises polypeptide OVA and complete Freund's adjuvant. According to the application, through the sublingual submucosal immune antigen peptide, the proportion and the number of the tissue resident memory T cells Trm in a body can be improved in a short time, the Trm can rapidly participate in a defense reaction after pathogen invasion again, and the generation of inflammatory cytokines is promoted, so that local immune monitoring and rapid protection on reinfection are provided, the oral mucosa barrier is enhanced, and further, the occurrence and the development of oral squamous cell carcinoma are effectively prevented and treated.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to application of an antigen peptide preparation in preparation of an oral mucosa immunity enhancing in-vivo tissue resident memory T cell kit.
Background
The memory recognition of the antigen by the immune system is the basis for long-term immune protection and vaccination of the body, and in recent years, in addition to the previously known central memory T cells and effector memory T cells, a tissue resident memory T cell (tissue resident memory T cells, trm) has been found to be involved in memory recognition of the antigen. Trm specifically expresses CD69 and CD103, and unlike memory T cells in the circulatory system, trm is mainly present in peripheral barriers such as skin, respiratory tract, intestinal tract, etc. The Trm of mice can be maintained for several months in the peripheral barrier, while the Trm where human tissues reside locally can be maintained for several years, which has important protective effects against bacterial infection, parasitic infection, viral infection, tumorigenesis and the like. Trm can rapidly participate in defensive responses following re-invasion by pathogens, including promoting inflammatory cytokine production, thereby providing local immune surveillance and rapid protection against re-infection.
The oral mucosal barrier is one of the most important peripheral barriers in humans and is exposed to a variety of immunogenic stimuli over a long period of time, including various commensal microbiota, food and airborne antigens. Under these antigenic stimuli, trm at the mucosal site of the oral cavity is constantly forming and alternating. The content and state of Trm in the oral mucosal barrier determine the function of mucosal immune monitoring, and very low levels of Trm may lead to loss of immune monitoring and thus the occurrence of inflammatory or neoplastic lesions in the oral mucosa. Epithelial-derived oral squamous cell carcinoma (oral squamous cell carcinoma, OSCC) is the most common malignant disease of oral mucosa, severely affects survival and appearance of patients, and can effectively control occurrence and development of OSCC by enhancing Trm proportion in local tissues through submucosal immunity and enhancing local immune surveillance.
The prior art reports a method for generating tissue resident memory-like T cells in vitro comprising: (a) obtaining an initial population of T cells; (b) Culturing the starting population of T cells under hypoxic conditions or in the presence of a hypoxia inducer to produce early effector cells; (c) The early effector cells are further cultured in the presence of transforming growth factor β1 (TGF- β1) to produce Trm-like T cells. However, exogenous Trm-like cells generated in vitro may have host specificity, have a certain immune rejection with patients, and at present, domestic and foreign researches have not reported about methods for enhancing oral mucosa barrier Trm in vivo, nor reported about the potential of the methods for controlling OSCC after application.
Disclosure of Invention
The invention provides application of an antigen peptide preparation in preparing an oral mucosa immunity enhancing in-vivo tissue resident memory T cell kit, and enhances oral mucosa barrier Trm expression by using an oral mucosa immunity antigen peptide mode so as to realize in-vivo enhancement of the oral mucosa barrier Trm, reduce immune rejection and provide a solution for prevention and treatment of oral malignant tumor.
In order to solve the technical problems, one of the purposes of the invention is to provide an application of an antigen peptide preparation in preparing an oral mucosa immunity enhancing in vivo tissue resident memory T cell kit, wherein the antigen peptide preparation comprises a polypeptide OVA and a complete Freund adjuvant.
Preferably, the polypeptide OVA is a polypeptide OVA 257-264 。
As a preferable scheme, the oral mucosa immunity enhancing in vivo tissue resident memory T cell kit also comprises pharmaceutically acceptable auxiliary materials.
As a preferred scheme, the mass ratio of the complete Freund's adjuvant to the polypeptide OVA is 1:1 to form an emulsified formulation.
Preferably, when the subject is a mouse, the immune dose is 1. Mu.g/g.
In order to solve the technical problems, the second object of the invention is to provide a T cell kit for enhancing the in vivo tissue resident memory of oral mucosa immunity.
In order to solve the technical problems, the invention provides an application of the oral mucosa immunity-enhancing in-vivo tissue resident memory T cell kit in preparing medicines for treating or preventing oral squamous cell carcinoma.
Preferably, the oral squamous cell carcinoma is an epithelial-derived oral squamous cell carcinoma.
In order to solve the technical problems, the fourth object of the invention is to provide a medicine for treating or preventing oral squamous cell carcinoma.
Compared with the prior art, the embodiment of the invention has the following beneficial effects:
1. according to the research of the embodiment of the application, the emulsified preparation is prepared by mixing the polypeptide OVA with the complete Freund adjuvant, and the proportion and the number of resident memory T cells Trm in a body can be improved in a short time by immunizing the emulsified preparation under tongue mucosa at least 1 time, and CD8+ tissue resident memory T cells are obviously increased after 3-6 weeks of sublingual immunization of mice.
2. After the resident memory T cells of local tissues are enhanced by the sublingual submucosal immune antigen peptide, trm can rapidly participate in defense reaction after pathogen re-invasion, including promotion of inflammatory cytokine production, thereby providing local immune monitoring and rapid protection against re-infection, enhancing the oral mucosa barrier and effectively preventing and curing occurrence and development of oral cancer.
Drawings
Fig. 1: the tongue tissue morphology of the Immune group (Immune group) and the Control group (Control group) of the mice immunized under the tongue mucosa for 3 weeks and 6 weeks in the embodiment of the invention is changed;
fig. 2: a ratio flow type representative graph (A) and a ratio change statistical analysis graph (B) of CD69+CD103+CD4+Trm cells in tongue tissues at 3 weeks and 6 weeks of a polypeptide immune group and a control group after mouse sublingual submucosal immunization in the embodiment of the invention;
fig. 3: a ratio flow type representative graph (A) and a ratio change statistical analysis graph (B) of CD69+CD103+CD8+Trm cells in tongue tissues of a mouse after sublingual submucosal immunization of the embodiment of the invention and a control at 3 weeks and 6 weeks;
fig. 4: the design scheme of the animal model for inducing the oral cavity cancer of the mice by 4NQO after sublingual submucosal immunization in the second embodiment of the invention;
fig. 5: representative general figures of oral lesions in mice of sublingual submucosa immune carcinoma induction group (4nqo+immune) and control carcinoma induction group (4nqo+control) in example two of the present invention;
fig. 6: is a representative graph of pathological changes of oral lesions at different stages of 4NQO induced simple Hyperplasia (Hyperplasia), abnormal Hyperplasia (Dysplsia) and invasive Carcinoma (Carcinoma);
fig. 7: the number and statistical analysis of mice with different lesions in the sublingual submucosa immune cancer induction group and the control cancer induction group in the second embodiment of the invention;
fig. 8: the flow type representative graph (A) of the secretion ratio of IFN gamma and GZMB, perforin of CD44+CD8+T cells in the oral lesion tongue tissue of mice in the sublingual mucosa immunity cancer induction group and the control cancer induction group in the embodiment II of the invention and the statistical analysis graph (B-D) of the secretion ratio difference of IFN gamma and GZMB, perforin are shown.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It will be appreciated that the medicament according to embodiments of the present invention may also be formulated into a variety of suitable clinical dosage forms by adding various pharmaceutically acceptable excipients, including but not limited to the following: tablets, capsules, oral liquids, oral granules or oral powders, and the like. The pharmaceutically acceptable auxiliary materials include, but are not limited to, diluents, wetting agents, binders, flash-disintegrating agents, lubricants, color, flavor and fragrance modifiers, solvents, solubilizers, co-solvents, emulsifiers, antioxidants, metal complexing agents, preservatives, pH modifiers and the like. Further, diluents, including, for example, starches, sucrose, celluloses, inorganic salts, and the like; wetting agents including, for example, water, ethanol, and the like; binders including, for example, starch slurry, dextrin, sugar, cellulose derivatives, gelatin, povidone, polyethylene glycol, and the like; disintegrants including, for example, starch, sodium carboxymethyl starch, low-substituted hydroxypropylcellulose, sodium dicarboxymethyl cellulose, surfactants, etc.; lubricants, including, for example, talc, calcium stearate, magnesium lauryl sulfate, polyethylene glycol, and the like; color, flavor, and taste flavoring agents, including, for example, coloring agents, sweeteners, fragrances, mucilages, and the like; solvents including, for example, water, glycerol, ethanol, and the like; solubilizing agents including, for example, tweens, sellers, sulphates, sulphonates and the like; cosolvents including organic acids (e.g., citric acid) and salts thereof, inorganic salts, polyethylene glycol, and the like; emulsifying agents including span, glycerol fatty acid ester, acacia, gelatin, agar, sodium alginate, etc.; antioxidants including, for example, sulfites, ascorbic acid, gallic acid and salts thereof; metal complexing agents including, for example, disodium edetate, polycarboxylic acid compounds, and the like; preservatives including, for example, parabens, quaternary ammonium compounds, chlorhexidine acetate, and the like; pH adjusting agents include, for example, hydrochloric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphates, citrates, and the like.
Example 1
Constructing a mouse sublingual submucosa polypeptide immune animal model, respectively obtaining materials 3 weeks and 6 weeks after mouse sublingual submucosa immunization, and detecting relevant cell phenotype changes in mouse lingual lymph nodes, wherein the method comprises the following steps of:
(1) Polypeptide immunization preparation: OVA of 257-264 Mixing with Complete Freund's Adjuvant (CFA) at a mass ratio of 1:1, repeatedly injecting and mixing with syringes at two sides of the three-way tube to obtain emulsified polypeptide immune preparation as polypeptide immune group, and immunizing with corresponding amount of CFA in control group.
(2) Experimental animals: c57BL/6WT mice of 6-8 weeks old were selected and kept in SPF environment.
(3) The immunization method comprises the following steps: after anesthetizing a mouse by using sodium pentobarbital with the concentration of 30mg/Kg, pulling out the tongue of the mouse, injecting an emulsified immune preparation into the tongue abdomen by using an insulin needle, gradually injecting 25 mu l of the immune preparation after 1cm of backward injection, finishing immunization once, carrying out submucosal immunization for 1 time, periodically observing the eating condition and physiological state of the mouse, detecting the change of the number of resident memory T cells in the oral mucosa of the mice of a polypeptide immunization group and a control group after 3 weeks of immunization, and detecting the change of the number of resident memory T cells in the oral mucosa of the mice of the polypeptide immunization group and the control group after 6 weeks of immunization.
(4) Digestion of oral mucosal tissue:
A. partially dissected lingual tissue was fixed in 4% paraformaldehyde, the remaining lingual tissue and other mucosal tissue were transferred to RPMI-1640 medium (10% FBS, 10mg/ml collagenase IV, 5mg/ml collagenase I, 1mg/ml DNase I) with 200ul digestive enzymes added, and the tissue was minced in centrifuge tubes to about 1-2mm 3 Incubating for 60 minutes at a constant temperature of 37 ℃ and 150 rpm;
B. transferring the digested tissue suspension to a 70 mu m cell filter screen, fully grinding by a grinding rod to obtain a cell suspension, centrifuging at 1500rpm and 4 ℃ for 5 minutes to obtain a cell precipitate, adding 1ml of prepared 1X erythrocyte lysate, fully blowing and uniformly mixing, performing room-temperature lysis for 2 minutes, adding 5 times of PBS (phosphate buffer solution) of 2% FBS (FBS) to dilute the cell suspension, centrifuging at 1500rpm and 4 ℃ for 5 minutes to obtain a lysed cell precipitate, and adding 1ml of PBS to resuspend the precipitate to obtain a single cell suspension for later use.
(5) Superficial dye flow staining of oral mucosal tissue:
A. transferring the single cell suspension prepared in the step (4) into a flow tube, centrifuging at 2000rpm and 4 ℃ for 3 minutes, adding 100 μl of dead living dye (1:200) prepared with PBS, incubating at room temperature for 15 minutes, washing cells with 3ml of PBS containing 2% FBS, stopping dying the dead living cells, centrifuging at 2000rpm and 4 ℃ for 3 minutes;
B. 100 μl of flow-through surface antibody suspension (CD 3, CD4, CD8, CD69, CD 103) in PBS with 2% FBS was added to the cell pellet; 3ml PBS containing 2% FBS was used to wash cells to terminate staining, 200. Mu.l-500. Mu.l PBS was added for flow-on-machine analysis.
As shown in FIG. 1, the tongue tissue of mice after submucosal immunization is infiltrated by immune cells in the tissue of the polypeptide immunized group, partial myofiber areas are replaced by infiltrated immune cells, and the tongue mucosa tissue morphology of mice of the control group is not obviously changed.
Further, the change of sublingual submucosa immunity to Trm cell phenotype is detected by flow cytometry, and the result is shown in figure 2, and after the sublingual submucosa immunity is found for 3 weeks, the proportion of CD69+CD103+CD4+Trm cells in the tongue tissue of the polypeptide immune group is obviously increased compared with the control group; after 6 weeks of immunization, the proportion of cd69+cd103+cd4+trm cells in the polypeptide immunized group was significantly increased compared to the control group; also, as shown in fig. 3, cd69+cd103+cd8+trm cells in tongue tissue were not significantly different from the control group after 3 weeks of immunization, but at 6 weeks, the proportion of cd69+cd103+cd8+trm cells in the polypeptide immunized group was significantly higher than that in the control group.
The above experimental results show that, in this embodiment, after the sublingual mucosa immune polypeptide treatment is performed on the mice, the content of the tissue resident memory T cells Trm in the lingual mucosa of the mice can be increased within 3-6 weeks, so as to enhance local immune monitoring.
Example two
After confirming the quantity and proportion of the Trm cells in the tongue tissue of the sublingual submucosa polypeptide immunity, the embodiment constructs a 4NQO cancer induction mouse model after the sublingual submucosa immunity so as to further explore the influence of the sublingual submucosa immunity on the occurrence and development of the oral cancer of the mouse:
(1) Experimental animals: 6-8 week old C57BL/6WT mice were selected and equally divided into two groups: sublingual submucosal polypeptide immunized groups and control immunized groups were raised in an SPF environment.
(2) The experimental steps are as follows: adopting the method of sublingual submucosa immunization in the embodiment, respectively carrying out polypeptide preparation immunization and control CFA immunization under the lingual mucosa of the mice, changing the drinking water of the mice in the experimental group and the control group into the drinking water containing 100 mg/L4-nitroquinoline-1-oxide (4 NQO) after 6 weeks of immunization, and replacing the drinking water with normal drinking water after 16 weeks of feeding; after normal drinking water is fed for 4 weeks, oral mucosa of the mice is extracted, and the change of immune cells under the mucosa is detected.
(3) Digestion of oral mucosal tissue:
A. partially dissected lingual tissue was fixed in 4% paraformaldehyde, the remaining lingual tissue and other mucosal tissue were transferred to RPMI-1640 medium (10% FBS, 10mg/ml collagenase IV, 5mg/ml collagenase I, 1mg/ml DNase I) with 200ul digestive enzymes added, and the tissue was minced in centrifuge tubes to about 1-2mm 3 Incubating for 60 minutes at a constant temperature of 37 ℃ and 150 rpm;
B. transferring the digested tissue suspension to a 70 mu m cell filter screen, fully grinding by a grinding rod to obtain a cell suspension, centrifuging at 1500rpm and 4 ℃ for 5 minutes to obtain a cell precipitate, adding 1ml of prepared 1X erythrocyte lysate, fully blowing and uniformly mixing, performing room-temperature lysis for 2 minutes, adding 5 times of PBS (phosphate buffer solution) of 2% FBS (FBS) to dilute the cell suspension, centrifuging at 1500rpm and 4 ℃ for 5 minutes to obtain a lysed cell precipitate, and adding 1ml of PBS to resuspend the precipitate to obtain a single cell suspension for later use.
(4) Flow staining of immune cell intracellular factors of oral mucosa tissue:
A. applying the cell sediment in the step (3), adding 200 mu l of 1X Cell Stimulation Cocktail prepared from complete culture medium after re-suspending, fully blowing and evenly mixing, re-suspending cells, and carrying out 5% CO at 37 DEG C 2 Stimulation was performed for 5 hours in a cell incubator, washed with 3ml PBS, terminated, followed by staining for 15 minutes at room temperature with 100. Mu.l of PBS-formulated dead-living dye (1:200);
B. next, 20 μl of 2% fbs-containing PBS-formulated surface marker suspension (CD 3, CD4, CD8, CD 44), incubation at 4 ℃ for 30 min, 3ml of 2% fbs-containing PBS for stop staining, centrifugation at 2000rpm at 4 ℃ for 3 min, cell pellet addition to 200 μl of IC Fixation working solution for cell Fixation, 4 ℃ for 30 min, 1x membrane-disrupting reagent for stop Fixation, intracellular factor flow antibody suspension (granzyme b, ifny, performin) formulated with membrane-disrupting stop solution, overnight incubation staining at 4 ℃ and washing of stained cells with PBS, centrifugation followed by appropriate PBS re-suspension, flow assay of cell function.
As shown in fig. 5-6, 4 NQO-induced oral cancer (carpinoma) was generally seen with exogenous tumor on the tongue, locally palpable infiltrates, histomorphology manifested by epithelial layer keratinized bead formation, incomplete basal lamina, disturbed basal lamina, and visible cancer cell infiltration foci in muscle tissue; dysplastic lesions (Dysplsia) are generally seen as localized white plaque-like changes in tongue tissue, pathologically manifested by thickening of the epithelial layer, disturbance of basal lamina, changes in cell polarity, and deep staining of the nucleus; the simple Hyperplasia lesions (hyperstasia) are close to normal tissue, but the epithelium is thickened, the pathology is represented by Hyperplasia of the epithelium, but the basal layer is closely arranged, and the cell morphology is not changed. After immunization with sublingual polypeptides, as shown in fig. 7, the incidence of oral cancer in mice is 20%, the incidence of abnormal hyperplasia lesions is 40%, and the incidence of simple hyperplasia is 40%; the incidence rate of oral cancer of mice in the control group is 60%, the incidence rate of abnormal hyperplasia lesions is 30%, and the incidence rate of simple hyperplasia is 10%. It can be seen that 4 NQO-induced oral carcinogenesis in mice is inhibited after immunization with sublingual submucosal polypeptides.
Meanwhile, the effect function of CD8+ T cells residing in tongue tissues is detected, and the result is shown in figure 8, compared with the control group, the capability of the CD44+CD8+ T cells of the polypeptide immune group to secrete anti-tumor factors IFNgamma and GZMB, perforin is obviously enhanced.
The foregoing embodiments have been provided for the purpose of illustrating the general principles of the present invention, and are not to be construed as limiting the scope of the invention. It should be noted that any modifications, equivalent substitutions, improvements, etc. made by those skilled in the art without departing from the spirit and principles of the present invention are intended to be included in the scope of the present invention.
Claims (9)
1. The application of the antigen peptide preparation in preparing an oral mucosa immunity enhancing in vivo tissue resident memory T cell kit is characterized in that the antigen peptide preparation comprises polypeptide OVA and complete Freund's adjuvant.
2. The use of the antigenic peptide preparation of claim 1 for the preparation of a kit for oral mucosal immunity enhancing in vivo tissue resident memory T cells, wherein said polypeptide OVA is a polypeptide OVA 257-264 。
3. The use of the antigenic peptide preparation of claim 1 for the preparation of a kit for oral mucosa immunopotentiating in vivo tissue resident memory T cells, wherein said kit for oral mucosa immunopotentiating in vivo tissue resident memory T cells further comprises pharmaceutically acceptable excipients.
4. The application of the antigen peptide preparation in preparing a kit for oral mucosa immunity enhancement in vivo tissue resident memory T cells as claimed in claim 1, wherein the mass ratio of the complete freund's adjuvant to the polypeptide OVA is 1:1 to form an emulsified formulation.
5. The use of the antigenic peptide preparation of claim 1 for the preparation of a kit for oral mucosal immunopotentiation of tissue resident memory T cells in vivo, wherein the immunization dose is 1 μg/g when the immunized subject is a mouse.
6. A kit for enhancing in vivo tissue resident memory T-cells based on oral mucosa immunity as claimed in any one of claims 1-5.
7. Use of the oral mucosa immune-enhancing in vivo tissue resident memory T cell kit of claim 6 in the preparation of a medicament for the treatment or prevention of oral squamous cell carcinoma.
8. The use of a T cell kit for oral mucosa immunopotentiating in vivo tissue resident memory as claimed in claim 7, in the manufacture of a medicament for the treatment or prophylaxis of oral squamous cell carcinoma, wherein said oral squamous cell carcinoma is an epithelial-derived oral squamous cell carcinoma.
9. A therapeutic or prophylactic agent based on oral squamous cell carcinoma as claimed in claim 7 or 8.
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| US20180325952A1 (en) * | 2015-11-06 | 2018-11-15 | Regents Of The University Of Minnesota | Activation of resident memory t cells for cancer immunotherapy |
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