Pharmaceutical composition for regulating and controlling peripheral residence capability of T cells and application of pharmaceutical composition

Abstract

The invention provides a pharmaceutical composition for regulating and controlling the peripheral residence capacity of T cells and application of the pharmaceutical composition. The pharmaceutical composition comprises a beta-AR antagonist and a vaccine, or a beta-AR antagonist and an immunotherapeutic drug; or a beta-AR agonist and an anti-inflammatory agent; or CXCL16 or an analogue thereof and a vaccine; or CXCL16 or an analogue thereof and an immunotherapeutic drug; or a substance and an anti-inflammatory preparation for reducing the production of CXCL16 or inhibiting CXCL16. By analyzing a bidirectional regulation mechanism of sympathetic nerves for expression of epithelial cells CXCL16, a neural signal mediated T cell residence dynamic balance system is established for the first time, technical breakthrough is achieved in the two directions of enhancing immune response and inhibiting excessive inflammation, and clinical application universality and transformation potential are achieved.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedicine, and more particularly to a pharmaceutical composition for regulating the peripheral residence ability of T cells and applications thereof. Background Art

[0002] Since memory T cells were classified into circulating memory T cells (TCIRCs) and non-circulating types in 1991, scientific research has long focused on the systemic immune functions of TCIRCs. A 2009 study discovered that some CD8+ memory T cells can permanently reside in peripheral tissues such as the skin. This population of memory T cells, which do not enter the circulation and colonize peripheral tissues, was formally named tissue-resident memory T cells (TRMs). TRMs are widely distributed in organs such as the skin, mucosal barriers, lungs, reproductive tract, and gastrointestinal tract. Due to their unique peripheral tissue residence, they have become key effector cells in maintaining immune surveillance. When TRM cells recognize invading pathogens, they rapidly release pro-inflammatory factors such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), as well as chemokines such as XCL1, activating surrounding epithelial tissues and promoting epithelial cells to produce antibacterial and antiviral proteins to enhance innate immunity. Simultaneously, they recruit immune effector cells such as natural killer (NK) cells, B cells, and circulating memory CD8+ T cells to the site of infection to collaboratively eliminate pathogens. Furthermore, TRM cells themselves can directly lyse infected target cells by secreting cytotoxic molecules such as perforin and granzymes, thereby forming a multi-layered immune clearance network.

[0003] TRM differentiation mechanism and functional regulation

[0004] TRMs are currently primarily believed to originate from circulating effector T cells. Their development follows an orderly three-stage regulatory process: Initially, circulating effector T cells, mediated by chemokine receptors such as CXCR3 and CCR10, migrate to sites of infection or inflammation and undergo clonal expansion. Once in peripheral tissues, these cells initiate a differentiation program under the influence of microenvironmental signals such as TGF-β, IL-7, IL-15, and retinoic acid, activating key transcription factors such as Hobit and Blimp1. These transcription factors inhibit the KLF2 / TCF1-S1PR1 migration pathway while upregulating the expression of adhesion molecules such as CD103 and CD69, leading to the formation of a characteristic tissue-resident transcriptional profile. These adhesion molecules have become hallmarks of the TRM phenotype. In addition, the loss of expression of homing receptors such as CCR7 and CD62L works together with the upregulation of adhesion molecules to block the cell's migration pathway, thereby ensuring its stable residence in the tissue; ultimately, the TGF-β and other signals continuously provided by the local microenvironment not only drive the terminal differentiation of TRM, but also maintain its long-term survival through epigenetic regulatory mechanisms, completing the transformation from circulating effector T cells to tissue-resident memory cells.

[0005] Breakthroughs in tumor immunotherapy and vaccine development

[0006] Due to the characteristics of TRM that they reside stably in peripheral organs for a long time and rapidly participate in immune responses, their application in the field of tumor immunotherapy and vaccine development has received widespread attention: clinical evidence shows that TRM cells are significantly enriched in a variety of human tumors of epithelial origin (such as melanoma, breast cancer, etc.) and play a core anti-tumor role. It is worth noting that the infiltration density of TRM in tumor tissue is significantly positively correlated with the patient's prognosis. This characteristic not only allows it to directly participate in the anti-tumor immune response, but also serves as a dynamic monitoring indicator for evaluating the efficacy of cancer vaccines. The latest studies have confirmed that TRM plays a key role in tumor immune surveillance, especially in inhibiting early melanoma. Its anti-tumor mechanism not only involves direct cytotoxic effects, but also can induce tumor cells to enter a growth arrest state through the TNFα signaling pathway. In addition, cases of immunotherapy resistance are closely related to abnormal TRM function, and immune checkpoint blockade therapy (ICB) can activate the in situ proliferation ability of TRM, thereby enhancing the anti-tumor response and providing a new treatment strategy for overcoming drug resistance;

[0007] In the field of vaccine development, strategies to induce TRMs are driving revolutionary breakthroughs. Studies have shown that by optimizing vaccination methods, tissue-specific TRM populations can be precisely induced, achieving long-lasting immune protection. Several studies have provided compelling evidence for this: 1) Intravenous BCG vaccination can generate a high proportion of antigen-specific TRMs in the lungs of primates, significantly enhancing defense against Mycobacterium tuberculosis; 2) Transcutaneous scarification activates Langerhans cells in the skin, simultaneously forming a TRM defense network in the skin and lungs; 3) Intranasal adenovirus vaccination can stably generate TRM populations in the respiratory tract; 4) Subcutaneous vaccination combined with local chemokine application can target the migration of activated T cells to the genital mucosa, effectively preventing HSV-2 infection; 5) Hepatocyte-targeted adenovirus vaccines combine systemic immunity with local antigen presentation to generate large numbers of CD8+ TRMs in the liver, significantly enhancing malaria prevention (already in clinical trials). These achievements provide key technical pathways for the development of TRM-targeted vaccines.

[0008] Given the unique advantages of TRM in infectious disease prevention and tumor immunotherapy, targeted induction of TRM formation and improvement of TRM function have become key development directions in tumor treatment and novel vaccine design. However, the protective efficacy of TRM cells is highly dependent on their number. Currently, in tumor treatment and vaccine development, the problem of insufficient TRM numbers leading to protection failure needs to be urgently addressed:

[0009] 1. Complexity and tissue-specific differences in microenvironmental signal regulation

[0010] The developmental program of TRM and its homeostasis maintenance are strictly regulated by the dynamic regulatory network of cytokines / chemokines in the local microenvironment, but the requirements for regulatory signals in different anatomical sites show significant heterogeneity. For example, skin TRM relies on TGF-β to maintain long-term survival, while liver TRM has a shortened half-life due to the lack of TGF-β signaling, making it difficult to form a stable protective population. Although current vaccine strategies based on mucosal or systemic delivery can effectively induce the generation of TRM, due to the significantly different response efficiency of different parenchymal organs to immune induction signals, the induction efficacy in specific tissue microenvironments is limited, and ultimately it is impossible to break through the population density threshold required for effective immune protection.

[0011] 2. Insufficient precise regulation of differentiation pathways

[0012] TRMs primarily originate from circulating effector T cells. Their differentiation process is regulated by transcription factors (e.g., Hobit and Blimp1), requiring simultaneous inhibition of migration-related pathways (e.g., S1PR1) and upregulation of adhesion molecules (e.g., CD103 and CD69). However, the TRM differentiation program is highly sensitive to local microenvironmental perturbations, and the induction thresholds and molecular regulatory networks at different anatomical sites have yet to be systematically elucidated. Existing evidence suggests that intervention strategies targeting a single signaling pathway (e.g., TGF-β or IL-15) face significant regulatory bottlenecks due to the lack of multidimensional signaling synergy, making it difficult to achieve large-scale reconstruction of functional TRM populations.

[0013] 3. Insufficient targeting of vaccine delivery systems

[0014] Although current vaccines (such as mRNA vaccines or adenoviral vectors) can induce TRM through tissue-specific delivery (such as intranasal and liver targeting), the efficiency of antigen presentation and local microenvironment regulation remain suboptimal. For example, although transcutaneous immunization strategies can initiate effector T cell responses, they rely on exogenous chemokine gradients to guide the migration of effector T cells across barriers to target organs. Their migration efficiency is limited by the spatiotemporal expression patterns of chemokine receptors and the physical limitations of tissue barriers, making it difficult to form an effective density of TRM populations at specific barrier sites (such as the genital mucosa). Moreover, when engineered chemokines are used as adjuvants, they have inherent defects such as poor molecular conformational stability and complex large-scale preparation processes, which seriously restrict the clinical translational application potential of this strategy.

[0015] T cells in autoimmune diseases

[0016] The massive accumulation and retention of T cells in peripheral tissues is the core pathogenesis of various autoimmune diseases, such as psoriasis, systemic lupus erythematosus (SLE), and alopecia areata. In psoriasis, Th17 cells drive excessive keratinocyte proliferation and skin inflammatory responses by secreting proinflammatory cytokines such as IL-17 and IL-22. Systemic lupus erythematosus is characterized by abnormal activation of autoreactive T cells. These cells promote B cells to produce antinuclear antibodies and attack multiple organ systems throughout the body. T cells in SLE patients undergo significant metabolic reprogramming in peripheral tissues, manifested by enhanced glycolysis and mitochondrial oxidative phosphorylation, further exacerbating inflammation. Alopecia areata is closely related to CD8+ T cells infiltrating hair follicles and attacking hair follicle stem cells, leading to local immune imbalance and hair loss. Treatment options for these diseases include immunosuppressants such as glucocorticoids, methotrexate, and cyclosporine, as well as biologics such as TNF-α inhibitors (e.g., etanercept), IL-17 / IL-23 inhibitors (e.g., secukinumab), and JAK inhibitors, which work by inhibiting T cell activation or blocking inflammatory cytokines. However, current treatments still face multiple challenges. Response rates to traditional drugs and biologics are insufficient, with 30%-40% of patients developing drug resistance or relapse. Furthermore, long-term use of these drugs can lead to abnormal T cell metabolic reprogramming, leading to secondary infections, increased risk of tumors, and antibody failure. CAR-T therapy is limited by its multi-million-dollar treatment costs and complex manufacturing processes, and long-term B cell depletion increases the probability of severe infection. New metabolic-modulating drugs (e.g., OXPHOS inhibitors) present challenges in targeted screening due to T cell heterogeneity, with an average development cycle of over 10 years. More critically, T cell subsets and metabolic profiles vary significantly between patients, making existing standardized treatment regimens incapable of meeting the individualized needs of most patients, a major obstacle to precision medicine.

[0017] Peripheral tissue-resident T cells act as a double-edged sword, acting as a crucial link between immune defense and pathological damage. Their precise regulation has become crucial for overcoming current immunotherapy bottlenecks. In the fields of anti-tumor and anti-infection, TRMs (Tregs) directly determine vaccine protection and immune checkpoint therapy response rates by rapidly activating multidimensional immune responses in situ. In autoimmune diseases, aberrantly resident pathogenic T cells drive tissue-specific inflammatory damage. However, existing regulatory approaches are limited by core issues such as tissue microenvironment heterogeneity, insufficient coordination of differentiation signals, and low targeting efficiency of delivery systems, making it difficult to effectively manipulate peripheral T cells. Therefore, there is an urgent need to develop a low-cost, broadly applicable novel strategy to regulate TRM differentiation, survival, and function. This would not only overcome the protective density threshold of tumor vaccines but also provide a therapeutic pathway for selectively eliminating pathogenic TRMs in autoimmune diseases. Summary of the Invention

[0018] A method and pharmaceutical composition for bidirectionally regulating T cell peripheral resident capacity based on the sympathetic nerve signaling-β-AR-CXCL16 pathway are provided. This method combines commonly used clinical drugs (β-AR antagonists, agonists, and other β-AR inhibitory methods) with existing vaccines, immunotherapy regimens, or anti-inflammatory agents to overcome the complexity and limitations of traditional peripheral T cell regulation technologies and achieve the following goals:

[0019] 1. Reduce treatment costs: No need to rely on expensive chemokines or complex delivery systems, directly utilize approved β-AR antagonists and agonists to synergistically regulate TRM cells;

[0020] 2. Simplified operation process: Compatible with a variety of vaccines (such as mRNA vaccines, adenoviral vectors), immunotherapies (such as PD-1 inhibitors) and anti-inflammatory preparations, without the need for additional tissue targeting design.

[0021] The present disclosure provides a pharmaceutical composition for bidirectionally regulating the peripheral residence ability of T cells based on the sympathetic nerve signal-β-AR-CXCL16 pathway, comprising a β-AR antagonist and a vaccine; or a β-AR antagonist and an immunotherapy drug; or a β-AR agonist and an anti-inflammatory preparation; or CXCL16 or its analogs and a vaccine; or CXCL16 or its analogs and an immunotherapy drug; or a substance for reducing the production of CXCL16 or inhibiting CXCL16 and an anti-inflammatory preparation.

[0022] In some embodiments, the β-AR antagonist includes at least one of propranolol, nadolol, sotalol, timolol, carteolol, penbutolol, oxprenolol, pindolol, butoxamine, Zenidolol, labenolol, and different salt forms of the above drugs (such as hydrochloride, maleate, etc.).

[0023] In some embodiments, the β-AR agonist includes at least one of norepinephrine, isoproterenol, clenbuterol, terbutaline, salmeterol, salbutamol, vilanterol, olodaterol, formoterol and different salt forms of the above drugs (such as hydrochloride, maleate, etc.).

[0024] In some embodiments, the CXCL16 or its analogs include at least one of CXCL16 protein, CXCL16 splice variants, post-translationally modified forms of CXCL16, short peptides designed based on the CXCL16 receptor binding domain, and small molecule drugs targeting the CXCL16 / CXCR6 signaling pathway.

[0025] In some embodiments, the substance for reducing the production of CXCL16 or inhibiting CXCL16 includes at least one of a CXCL16 antibody, a small molecule inhibitor targeting CXCL16, an agent targeting the CXCL16 gene to inhibit and / or knock out the CXCL16 gene, an agent targeting RNA transcribed from the CXCL16 gene to inhibit the translation of CXCL16, an agent that inhibits the expression of CXCL16 at the cellular level, a nanobody, or a protein binder.

[0026] In some embodiments, the vaccine comprises at least one of an inactivated vaccine, a live attenuated vaccine, a recombinant protein vaccine, a virus-like particle vaccine, a DNA vaccine, an mRNA vaccine, and an adenovirus vector vaccine.

[0027] In some embodiments, the immunotherapy drug includes at least one of an immune checkpoint inhibitor, an adoptive cell therapy preparation, an immunomodulator, and an active immune stimulant. In some embodiments, immune checkpoint inhibitors include all drugs targeting immune checkpoints, such as PD-1 / PD-L1 inhibitors, CTLA-4 inhibitors, and LAG-3 inhibitors. In some embodiments, adoptive cell therapy preparations include CAR-T, TCR-T, TIL (tumor infiltrating lymphocytes), NK cell therapy preparations, etc. In some embodiments, immunomodulators include cytokines (IL-2, IFN-α), chemokines, Toll-like receptor agonists, etc. In some embodiments, active immune stimulants include cancer vaccines, oncolytic viruses, and other drugs that can activate the host immune system.

[0028] In some embodiments, the anti-inflammatory drug comprises at least one of a non-steroidal anti-inflammatory drug, a steroidal anti-inflammatory drug, an inflammatory mediator antagonist, and an immunomodulatory cytokine.

[0029] The present disclosure also provides a use of the above-mentioned pharmaceutical composition, which is used to upregulate or downregulate the peripheral residence ability of T cells based on the sympathetic nerve signal-β-AR-CXCL16 pathway.

[0030] In some embodiments, the pharmaceutical composition includes a β-AR antagonist and a vaccine; or a β-AR antagonist and an immunotherapy drug; or CXCL16 or its analogues and a vaccine; or CXCL16 or its analogues and an immunotherapy drug; and the pharmaceutical composition is used to upregulate the peripheral residence ability of T cells.

[0031] In some embodiments, the pharmaceutical composition includes a β-AR agonist and an anti-inflammatory preparation; or a substance for reducing the production of CXCL16 or inhibiting CXCL16 and an anti-inflammatory preparation; the pharmaceutical composition is used to downregulate the peripheral residence ability of T cells for adjuvant treatment of autoimmune diseases.

[0032] In some embodiments, methods for enhancing TRM generation include: blocking the β-AR signaling pathway through a β-AR antagonist; upregulating the expression of the chemokine CXCL16 in the target tissue to promote the migration of circulating CD8+ T cells to the target tissue; and driving CD8+ T cells to differentiate into antigen-specific tissue-resident memory T cells and promote their long-term residence through the synergistic effect of TGF-β and adhesion molecule E-cad in the local microenvironment.

[0033] In some embodiments, local injection of CXCL16 can directly induce the formation of TRMs.

[0034] In some embodiments, the method is used to rapidly clear peripheral tissue T cells, including: activating the β-AR signaling pathway through a β-AR agonist; inhibiting the expression of chemokine CXCL16, TGF-β and adhesion molecule E-cad in target tissues, reducing the residence capacity of peripheral T cells, causing them to leave the periphery, and reducing the level of local inflammation.

[0035] In some embodiments, knocking out the Cxcl16 gene or injecting CXCL16 antibodies can induce downregulation or even elimination of peripheral resident T cell levels.

[0036] In some embodiments, the target tissue comprises skin, respiratory mucosa, reproductive tract mucosa, or tumor tissue.

[0037] The present invention significantly improves the generation efficiency and long-term residence ability of antigen-specific TRM by inhibiting sympathetic nerve activity and targeting the β-AR pathway; activating the sympathetic nerve-β-AR pathway significantly reduces the level of peripheral T cells and reduces the degree of inflammation in the target area. Experiments have confirmed that after blocking norepinephrine (NE)-mediated β-AR activation, the expression of epithelial cell CXCL16 chemokine is significantly upregulated, driving the directional migration of circulating CD8+T cells to the skin, respiratory mucosa and tumor tissue. In this process, TGF-β and E-cad in the local microenvironment form a synergistic regulatory network, which promotes the differentiation of migrating T cells into functional TRM populations. Conversely, treatment with β-AR agonists can lead to a significant decrease in the expression levels of epithelial cell CXCL16, TGF-β and E-cad, thereby weakening the TRM differentiation ability. Further functional verification showed that CXCL16 plays a core role in TRM regulation: exogenous supplementation of CXCL16 can significantly increase the density of mucosal TRM, while the use of CXCL16 antibodies or gene knockout of Cxcl16 can greatly reduce or even completely eliminate the epithelial TRM cell population. In terms of application verification, data from the OVA infection model showed that the number of antigen-specific TRM in the β-AR signal inhibition group was significantly higher than that in the control group by 5-10 times, and the protective immune response lasted for more than 60 days. In the low-dose mRNA vaccine (10 7In the nasal immunization model of 10 PFU, the combination of β2-AR specific antagonist Zenidolol or broad-spectrum antagonist propranolol can increase the density of CD8+ TRM in the nasal mucosa to that of high-dose vaccine (10 8 In contrast, local injection of the β-AR agonist isoproterenol can rapidly reduce the number of skin TRMs. This approach achieves efficient regulation of peripheral T cells at a very low cost (the price of related inhibitors and agonists is only 1 / 50 of that of biologics) by regulating the sympathetic nerve-β-AR-CXCL16 axis, breaking through the limitations of traditional technologies that rely on high-dose antigens or tissue-targeted vectors, and providing an efficient and universal solution for the development of tumor immunotherapy, mucosal vaccines, and the treatment of autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Sympathetic nerve-EpiSCs interaction is shown to regulate skin CD8 + T RM Schematic diagram of cell recruitment (SN: sympathetic nerve, epithelial cell: epithelial cell).

[0039] Figure 2a The mouse operation schematic diagram and immunofluorescence staining image of Example 1 are shown, wherein the dot-shaped cells are CD8+ T cells.

[0040] Figure 2b The statistical data of CD8+ T cells in the skin of the left and right sides of Example 1 are shown.

[0041] Figure 3a The mouse operation schematic diagram and immunofluorescence staining image of Example 2 are shown, wherein the dot-shaped cells are CD8+ T cells.

[0042] Figure 3b The statistical data of CD8+ T cells in the skin of the left and right sides of Example 2 are shown.

[0043] Figure 4a The statistical analysis results of the relevant genes after Nadolol treatment in Example 3 are shown.

[0044] Figure 4b The statistical analysis results of the relevant genes after NE or ISO treatment in Example 3 are shown.

[0045] Figure 5a A schematic diagram of the experimental operation of Example 4 is shown.

[0046] Figure 5b The immunofluorescence staining image of Example 4 is shown, wherein the cells indicated by arrows are CD8+ T cells.

[0047] Figure 6a The immunofluorescence staining image of Example 5 is shown, in which the dot-shaped cells are CD8+ T cells.

[0048] Figure 6b The statistical data of CD8+ T cells in the skin of the left and right sides of Example 5 are shown.

[0049] Figure 7a Flow cytometry images of Example 6 are shown, showing a significant increase in OT-1 cells in Gi-DREADD mice.

[0050] Figure 7b Graphs showing flow cytometry statistical analysis of Example 6 are shown.

[0051] Figure 8 The statistical analysis results of Example 7 are shown. DETAILED DESCRIPTION

[0052] The following embodiments may enable those skilled in the art to more fully understand the present disclosure, but are not intended to limit the present disclosure in any way.

[0053] Unless otherwise stated, the materials and reagents used in the following examples can be obtained from commercial sources.

[0054] In response to the two key issues of protective immune response defects caused by insufficient number of tissue-resident memory T cells in tumor treatment and vaccine development, and the difficulty in effectively intervening in excessive pathological T cell infiltration in autoimmune diseases, this paper discloses a new mechanism for regulating TRM based on the newly discovered sympathetic nerve-β-AR-CXCL16 signaling axis. Based on the newly discovered sympathetic nerve-β-AR-CXCL16 signaling axis regulation mechanism, a bidirectional regulation method targeting this pathway has been developed. Through commonly used clinical drugs (β-blockers / agonists) or regulating CXCL16 signals, the positioning and function of T cells in specific tissues can be precisely regulated: the directional recruitment and long-term residence of TRM cells are enhanced at tumor or vaccination sites, and the clearance of abnormal T cells is promoted in autoimmune lesions. Figure 1 As shown in the results, inhibition of β-AR can relieve the transcriptional inhibition of NE on epithelial cell CXCL16, drive circulating T cells to migrate to the epidermis, and at the same time, the synergistic effect of TGF-β and E-cad in the local microenvironment promotes T cell differentiation into a stably resident TRM population; on the contrary, activation of β-AR reduces the expression levels of CXCL16, TGF-β and E-cad, weakening the ability to form TRM.

[0055] Molecular pathways of sympathetic nerve signaling regulating TRM differentiation

[0056] Enhanced release of chemokine CXCL16: Blocking β-AR signaling can relieve the inhibitory effect of norepinephrine (NE) on epithelial cells, significantly upregulate the expression of CXCL16 in epithelial cells, and recruit circulating CD8+ T cells to migrate to epithelial tissues such as the skin and mucous membranes.

[0057] Microenvironment remodeling: After NE signaling is blocked, epithelial cells simultaneously overexpress TGF-β and the adhesion molecule E-cadherin (E-cad), forming a local microenvironment that is conducive to TRM differentiation. TGF-β drives T cell differentiation, activates the transcription factor Hobit / Blimp1, and inhibits the expression of migration-related genes (such as S1PR1 and KLF2). E-cad enhances the adhesion of T cells to epithelial cells by binding to CD103 (αE integrin), promoting the long-term residence of TRM.

[0058] Reverse regulation by β-AR agonists: After activating β-AR using β-AR agonists, epithelial cell CXCL16, TGF-β and E-cad are significantly reduced, thereby inhibiting the level of peripheral T cells through multiple pathways.

[0059] Multiple synergistic effects of β-AR antagonists / agonists

[0060] By using β-AR antagonists or agonists (such as propranolol, nadolol, or isoproterenol), the following synergistic effects are achieved:

[0061] Multi-factor synergistic control: The NE-β-AR signaling axis can simultaneously regulate the expression of CXCL16, TGF-β, and E-cad, effectively controlling the recruitment, retention, and expansion of peripheral T cells;

[0062] Directed control of antigen-specific TRMs: Under vaccine or tumor antigen stimulation, T cells recruited to target tissues efficiently differentiate into antigen-specific TRMs in the local microenvironment, with a formation efficiency 3-5 times higher than traditional methods. Negative regulation can rapidly reduce the number of pathological T cells and lower the level of inflammation.

[0063] Cross-tissue applicability: This mechanism is effective in a variety of epithelial barriers, including the skin, respiratory mucosa, reproductive tract, and tumor tissue, breaking through the limitations of traditional methods that rely on tissue-specific delivery.

[0064] Example 1:

[0065] Homeostatic model: Topical application of nadolol to the skin can significantly increase the number of TRMs at steady state:

[0066] In the present embodiment, 100 μL of saline / 2.5 mM nadolol was injected into the left and right sides of the dorsal skin of mice. Three days later, the injected skin was sampled and whole-tissue immunofluorescence staining was performed. The CD8 antibody was used to observe and count the number of CD8+ T cells; the K14 antibody was used to distinguish the epidermis from the dermis; and 4',6-diamidino-2-phenylindole (DAPI) was used to label the cell nuclei. Figure 2a Schematic diagram of mouse operation and immunofluorescence staining image, in which the dot-shaped cells are CD8+T cells. Figure 2b The statistical data of CD8+ T cells in the skin of the left and right sides show that Nadolol treatment significantly increased their density. This experiment shows that β-AR antagonists can significantly increase the level of peripheral resident T cells (P<0.001).

[0067] Example 2:

[0068] Steady-state model: Topical application of isoproterenol (ISO) to the skin can significantly reduce the number of TRMs at steady-state:

[0069] In the present embodiment, 100 μL of saline / 2.5 mM isoproterenol (ISO) was injected into the left and right sides of the mouse back skin. Three days later, the injected skin was sampled and whole-tissue immunofluorescence staining was performed. CD8 antibody was used to observe and count the number of CD8+ T cells; K14 antibody was used to distinguish the epidermis from the dermis; and DAPI was used to mark the cell nucleus. Figure 3a Schematic diagram of mouse operation and immunofluorescence staining images, in which the dot-shaped cells are CD8+ T cells. Figure 3b The statistical data of CD8+ T cells in the skin of the left and right sides show that ISO treatment significantly reduced their density. This experiment shows that β-AR agonists can significantly reduce the level of peripheral resident T cells (P<0.001).

[0070] Example 3:

[0071] In vitro T cell retention gene detection: T cell retention genes were significantly upregulated after treatment of human epithelial cells cultured in vitro with Nadolol, while they were significantly downregulated after treatment with NE or ISO:

[0072] In this example, human epithelial cells cultured in vitro were seeded into 12-well plates. After reaching a cell density of 60%, cells were treated with normal saline (CTRL), Nadolol, norepinephrine (NE), or ISO, with three independent replicates per group. After 12 hours of treatment, cells were harvested and mRNA was extracted for qPCR analysis of genes associated with T cell retention. Figure 4a Statistical analysis results of related genes after Nadolol treatment; Figure 4b Results after nephrase or isothiocyanate (ISO) treatment are shown. The figure shows a rapid and significant increase in T cell retention-related genes, particularly CXCL16 and TGFβ, after Nadolol treatment. Following nephrase and isothiocyanate treatment, CXCL16, TGFβ, and E-cad (CDH1) were significantly downregulated. This experiment demonstrates that β-AR antagonists and agonists can effectively regulate T cell retention-related genes, particularly CXCL16, TGFβ, and E-cad.

[0073] Example 4:

[0074] In vivo CXCL16 validation experiment: Intradermal injection of CXCL16 in mice can rapidly increase the level of T cells:

[0075] In the present embodiment, 100 μL of 300 ng of fetal bovine serum (BSA) / CXCL16 protein was injected into the left and right sides of the mouse back skin. Three days later, the injected skin was sampled and frozen sections of the skin tissue were performed and immunofluorescence staining was performed. CD8 antibody was used to observe and count the number of CD8+ T cells; DAPI was used to mark the cell nucleus. Figure 5a Schematic diagram of experimental operation; Figure 5b This is an immunofluorescence staining image, where the arrows point to CD8+ T cells. This experiment demonstrates that CXCL16 can rapidly and significantly increase the level of peripherally resident T cells.

[0076] Example 5:

[0077] In vivo CXCL16 gene knockout validation experiment: Specific knockout of the Cxcl16 gene in mouse epithelial cells can rapidly induce the loss of resident T cells:

[0078] In the examples, the Cxcl16 gene was specifically knocked out in the epithelial cells of the skin of transgenic mice (K14-Cxcl16 cko). Three weeks later, samples were collected from the left and right injected skin and whole-tissue immunofluorescence staining was performed. The CD8 antibody was used to observe and count the number of CD8+ T cells; the K14 antibody was used to distinguish the epidermis from the dermis; and DAPI was used to label the cell nuclei. Figure 6a This is an immunofluorescence staining image, in which the dot-shaped cells are CD8+T cells; Figure 6b Statistical data of CD8+T cells in the skin of the left and right sides showed that knockout of the Cxcl16 gene could significantly reduce the level of peripheral resident T cells (P<0.001).

[0079] Example 6:

[0080] Ovalbumin (OVA) infection model: Ovalbumin is a model antigen commonly used to study immune responses (e.g., allergic reactions and T cell activation). The present disclosure discloses that during ovalbumin infection, combined with the use of drugs to inhibit sympathetic nerve activity, a 5-10-fold increase in OVA-specific TRMs can be achieved, with this effect persisting for up to two months.

[0081] In this example, wild-type mice (CTRL) and sympathetic inhibitory mice (Gi-DREADD) were first transplanted with 106 OVA-specific CD8+ T cells (OT-1) via tail vein injection. One day after transplantation, the mice were inoculated with 100 μL of a mixture of 0.1 mg OVA and an immune adjuvant (CFA) on their backs. On days 5-7, azidopine (CNO, 5 μg per mouse) was injected intraperitoneally to suppress sympathetic nerve activity. 50 days after OVA inoculation, flow cytometric analysis of the skin of the OVA-infected mice was performed. Figure 7a This is a flow cytometry image showing a significant increase in OT-1 cells in Gi-DREADD mice; Figure 7b This is a flow cytometry statistical analysis chart, which shows that sympathetic nerve inhibition can significantly increase the number of resident OT-1 cells in the skin and maintain it for 2 months.

[0082] Example 7:

[0083] Nasal mucosal application model of β-AR antagonist combined with mRNA vaccine: Low-concentration mRNA vaccine (10^7, NC) alone could not induce significant CD8+ TRM cell formation in the nasal mucosa, while high-concentration mRNA vaccine (10 8 PFU, PC) as a positive control can induce a more obvious CD8+ T cell response. 7 PFU) combined with β-AR antagonist - Zenidolol hydrochloride (β2-AR specific antagonist, Zeni) / Propranolol hydrochloride (Panβ-AR antagonist, ProH) can increase CD8+T cells to a high concentration of mRNA vaccine (10 8 PFU) levels of treatment.

[0084] In this embodiment, influenza virus mRNA vaccine (10 8 PFU), influenza virus mRNA vaccine (10 7 PFU), influenza virus mRNA vaccine (10 7 PFU) + 2.5mM Zeni and influenza virus mRNA vaccine (10 7The mice were treated with a single intranasal drip of 2.5 mM ProH (20 μL per mouse) containing 100 μM β-actin (PFU) of β-actin (PFU) and 2.5 mM ProH (all treatments were performed using normal saline as the solvent). Two weeks later, the nasal cavities of the mice were sampled, decalcified, and frozen sections were prepared. The sections were analyzed using immunofluorescence staining, and the number of CD8+ T cells in the nasal mucosa was measured throughout the sections. Figure 8 For statistical analysis, the results showed that when the low concentration of mRNA vaccine (10 7 PFU) combined with β-AR antagonist-Zeni / ProH can increase CD8+T cells to a high concentration of mRNA vaccine (10 8 The results showed that the mice did not show abnormal symptoms when treated with β-AR antagonists at a low PFU level. This experiment shows that the combination of β-AR antagonists and vaccines can significantly increase the potency of low-concentration vaccines.

[0085] The present disclosure reveals the regulatory effect of the sympathetic nerve-β-AR-CXCL16 signaling axis on TRM formation: blocking the β-AR signal can relieve the inhibition of NE on epithelial cells, significantly upregulate the chemokine CXCL16, and recruit circulating CD8+ T cells to migrate to target tissues such as the skin and mucosa; reshape the local microenvironment: synchronously induce epithelial cells to highly express TGF-β (drive TRM differentiation) and adhesion molecule E-cadherin (promote T cell retention), enhance the formation ability of TRM, and promote the long-term retention of TRM; on the contrary, activating β-AR reduces the expression levels of CXCL16, TGF-β and E-cad, weakening the formation ability of TRM.

[0086] The present disclosure also reveals the synergistic effect of β-AR antagonists / agonists. The present disclosure utilizes beta-blockers / agonists (such as propranolol, nadolol, isoproterenol) that have been widely used in clinical practice to bidirectionally control the expression of CXCL16, TGF-β and E-cad by regulating the NE signaling pathway, thereby affecting T cell recruitment, retention and expansion; under the stimulation of vaccines or tumor antigens, the efficiency of antigen-specific TRM generation is increased by 3-5 times, and the clearance of abnormal T cells in autoimmune lesions is promoted, breaking through the bottleneck of traditional methods. The scheme disclosed in the present disclosure shows cross-tissue universality: it is applicable to a variety of epithelial barriers such as skin, respiratory mucosa, reproductive tract and tumor tissue, without the need for complex tissue targeting design. In addition, the technical solution disclosed in the present invention has low cost, high compatibility and rapid transformation potential: β-AR antagonists / agonists are approved generic drugs, and the cost is only 1 / 10-1 / 50 of biological agents (such as cytokines and antibodies); no exogenous chemokines or gene editing technology are required, and multi-tissue TRM amplification can be achieved through oral or local administration; it is fully compatible with existing vaccines (mRNA, adenovirus vectors), immune checkpoint inhibitors (PD-1 / PD-L1 inhibitors) and adoptive cell therapy; β-AR antagonists / agonists have accumulated decades of cardiovascular disease medication data, and side effects are controllable (such as slowing heart rate); and existing drugs can be directly reused, skipping the new drug development cycle, and clinical trials of combination therapy can be immediately carried out.

[0087] The present invention effectively controls the migration efficiency and long-term residence ability of peripheral T cells by bidirectionally regulating sympathetic nerve activity and targeting the β-adrenergic receptor signaling pathway. Experiments have confirmed that after blocking the activation of β-AR by norepinephrine, the expression level of the epithelial cell chemokine CXCL16 increases, driving the directional migration of circulating CD8+ T cells to the skin, respiratory mucosa and tumor tissue. At the same time, the synergistic effect of TGF-β and the adhesion molecule E-cadherin in the local microenvironment promotes the efficient differentiation of migrating T cells into TRM. In addition, treatment with β-AR agonists can lead to a significant decrease in the expression levels of CXCL16, TGF-β and E-cad in epithelial cells, thereby weakening the TRM differentiation ability. Further functional verification shows that CXCL16 plays a core role in TRM regulation: exogenous supplementation of CXCL16 can significantly increase the density of mucosal TRM, while the use of CXCL16 antibodies or genetic knockout of Cxcl16 can greatly reduce or even completely eliminate the epithelial TRM cell population. In terms of application verification, data from the OVA infection model showed that the number of antigen-specific TRMs in the β-AR signal inhibition group was significantly higher than that in the control group by 5-10 times, and the protective immune response lasted for more than 60 days. 7In the nasal immunization model of 10 PFU, the combination of β2-AR specific antagonist Zenidolol or broad-spectrum antagonist propranolol can increase the density of CD8+ TRM in the nasal mucosa to that of high-dose vaccine (10 8 In contrast, local injection of the β-AR agonist isoproterenol can rapidly reduce the number of skin TRMs. This approach achieves efficient regulation of peripheral T cells at a very low cost (the price of related inhibitors and agonists is only 1 / 50 of that of biologics) by regulating the sympathetic nerve-β-AR-CXCL16 axis, breaking through the limitations of traditional technologies that rely on high-dose antigens or tissue-targeted vectors, and providing an efficient and universal solution for the development of tumor immunotherapy, mucosal vaccines, and the treatment of autoimmune diseases.

[0088] The present disclosure specifically involves two types of application schemes: the enhanced scheme combines β-AR antagonists with vaccines or immunotherapy drugs to antagonize the sympathetic nerve-β-AR signaling pathway, induce epithelial cells to upregulate CXCL16 expression, and thereby enhance the ability of T cells to migrate to peripheral tissues such as the skin and mucosa. At the same time, high expression of CXCL16 by epithelial cells in conjunction with TGF-β / E-cad signals can significantly enhance the residence characteristics of T cells in target tissues, thereby efficiently forming and amplifying tissue-resident memory T cells. This scheme breaks through the limitations of traditional technologies that rely on high-dose antigens or tissue-targeted vectors, achieves efficient amplification of TRM at a low cost, and provides a universal solution for tumor immunotherapy and mucosal vaccine development; the inhibitory scheme targets T cell-mediated autoimmune diseases, combines β-AR agonists with anti-inflammatory drugs, and inhibits the expression of the CXCL16 / TGF-β / E-cad signaling pathway, reducing the chemotactic recruitment and residence ability of pathological T cells to target organs, reducing the level of inflammation, and achieving adjuvant treatment for autoimmune diseases.

[0089] Those skilled in the art should understand that the above embodiments are merely exemplary embodiments and that various changes, substitutions, and alterations may be made without departing from the spirit and scope of the present disclosure.

Claims

1. A pharmaceutical composition, characterized in that Comprising a β-AR antagonist and a vaccine; or a β-AR antagonist and an immunotherapy drug; or a β-AR agonist and an anti-inflammatory preparation; or CXCL16 or its analogues and a vaccine; or CXCL16 or its analogues and an immunotherapy drug; or a substance for reducing the production of CXCL16 or inhibiting CXCL16 and an anti-inflammatory preparation.

2. The pharmaceutical composition according to claim 1, characterized in that The β-AR antagonist includes at least one of propranolol, nadolol, sotalol, timolol, carteolol, penbutolol, oxprenolol, pindolol, butoxamine, Zenidolol, labenolol and salts of the above drugs.

3. The pharmaceutical composition according to claim 1, characterized in that The β-AR agonist includes at least one of norepinephrine, isoproterenol, clenbuterol, terbutaline, salmeterol, salbutamol, vilanterol, olodaterol, formoterol and salts of the above drugs.

4. The pharmaceutical composition according to claim 1, characterized in that The CXCL16 or its analogs include at least one of CXCL16 protein, CXCL16 splice variants, CXCL16 post-translationally modified forms, short peptides designed based on the CXCL16 receptor binding domain, and small molecule drugs targeting the CXCL16 / CXCR6 signaling pathway.

5. The pharmaceutical composition according to claim 1, characterized in that The substance for reducing the production of CXCL16 or inhibiting CXCL16 includes at least one of a CXCL16 antibody, a small molecule inhibitor targeting CXCL16, an agent targeting the CXCL16 gene to inhibit and / or knock out the CXCL16 gene, an agent targeting RNA transcribed from the CXCL16 gene to inhibit the translation of CXCL16, an agent that inhibits the expression of CXCL16 at the cellular level, a nanobody or a protein binder.

6. The pharmaceutical composition according to claim 1, characterized in that The vaccine includes at least one of an inactivated vaccine, a live attenuated vaccine, a recombinant protein vaccine, a virus-like particle vaccine, a DNA vaccine, an mRNA vaccine, and an adenovirus vector vaccine.

7. The pharmaceutical composition according to claim 1, characterized in that The immunotherapy drug includes at least one of an immune checkpoint inhibitor, an adoptive cell therapy preparation, an immunomodulator, and an active immune stimulant.

8. The pharmaceutical composition according to claim 1, characterized in that The anti-inflammatory drug includes at least one of non-steroidal anti-inflammatory drugs, steroidal anti-inflammatory drugs, inflammatory mediator antagonists, and immunoregulatory cytokines.

9. A use of a pharmaceutical composition, characterized in that: The pharmaceutical composition is a pharmaceutical composition according to any one of claims 1 to 8, and is used to upregulate or downregulate the peripheral residence ability of T cells based on the sympathetic nerve signal-β-AR-CXCL16 pathway.

10. The use according to claim 9, characterized in that The pharmaceutical composition includes a β-AR antagonist and a vaccine; or a β-AR antagonist and an immunotherapy drug; or CXCL16 or its analogues and a vaccine; or CXCL16 or its analogues and an immunotherapy drug; and the pharmaceutical composition is used to upregulate the peripheral residence ability of T cells.

11. The use according to claim 9, characterized in that The pharmaceutical composition includes a β-AR agonist and an anti-inflammatory preparation; or a substance for reducing the generation of CXCL16 or inhibiting CXCL16 and an anti-inflammatory preparation; the pharmaceutical composition is used to downregulate the peripheral residence ability of T cells for auxiliary treatment of autoimmune diseases.

Images