Method for quantitatively analyzing immune response strength promoted by adjuvant and application of method in adjuvant screening

Abstract

The invention provides a method for quantitatively analyzing immune response strength promoted by an adjuvant and application of the method in adjuvant screening, and relates to the technical field of vaccine adjuvants. According to the method for quantitatively analyzing the immune response strength promoted by the adjuvant, the immune response level of the to-be-detected adjuvant can be predicted only by taking the control adjuvant as a reference substance and taking the number of KikGR red fluorescent cells 6-120 hours after immunostimulation of the combined antigen as an index. The method has the characteristics of noninvasive and continuous tracing, simplifies the flow and period of adjuvant effect evaluation, improves the screening efficiency, reduces the research and development period, and provides an effective technical means for rapid screening of adjuvants.

Description

Technical Field

[0001] This invention relates to the field of vaccine adjuvant technology, and in particular to a method for quantitatively analyzing the strength of an adjuvant in promoting an immune response and its application in adjuvant screening. Background Technology

[0002] Vaccine adjuvants play a crucial role in enhancing adaptive immune responses to diseases such as SARS-CoV-2, hepatitis B, and influenza. Their primary function is to strengthen humoral immune responses against antigens. Traditional adjuvants include aluminum salts (Alum) and squalene emulsions (such as MF59). Recent research has focused on developing Toll-like receptor (TLR) agonist adjuvants to enhance T-cell responses and cytokine production, such as the TLR9 agonist CpG1018 and the TLR7 / 8 agonist R848. Meanwhile, TLR3 agonists such as polynucleotide polymers (poly I:C) and TLR4 agonists such as monophosphatidic acid A (MPLA) have also shown potential in enhancing immune responses. Furthermore, STING agonists, as a novel class of adjuvants, significantly enhance antiviral and antitumor immune responses by activating the interferon gene pathway. These advances in TLR and STING agonist research point the way to developing more effective vaccine adjuvants.

[0003] Despite this, traditional methods for evaluating the immunogenicity of adjuvants in vaccine development suffer from significant time delays, typically requiring 30 to 60 days to complete the assessment, making the development cycle excessively long. To shorten this cycle, there is an urgent need to develop new methods for rapid evaluation and screening of adjuvants. Many adjuvant-assisted vaccines primarily promote antigen presentation by activating innate immune cells in the early stages before injection site and lymph node (LN) drainage. Conventional dendritic cells (cDCs) are important APCs that guide effector T cells and induce adaptive immune responses. cDCs can be divided into two distinct subsets based on their location: LN-resident cDCs (Res cDCs) and migrating cDCs (Mig cDCs). Res cDCs continuously migrate from the bloodstream into the LN, receiving antigens through lymphatic drainage or transfer to other cells. Conversely, Mig cDCs reside in parenchymal tissues and carry exogenous antigens to the dLN, not only stimulating primary T cells but also transferring antigens to Res cDCs. Elimination of Mig cDCs can impair T-cell-dependent immune responses following skin immunization. From a technical perspective, migratory dendritic cells (Mig cDCs) play a crucial role in the early stages of immune responses, particularly in anti-infective and anti-tumor immune responses, where they are key cell subsets for activating CD4+ and CD8+ T cells. Numerous studies have shown that Mig cDCs effectively present antigens to T cells via antigen presentation, activating CD4+ and CD8+ T cells using major histocompatibility complexes MHCII and MHCII peptide antigen complexes. Based on this finding, this invention proposes changes in the absolute number of Mig cDCs in draining lymph nodes (dLNs) as an early indicator for predicting adjuvanted vaccine-triggered T-cell responses against specific viruses or tumors.

[0004] Based on this, this study proposes a reasonable method for screening and evaluating adjuvants and their combinations based on early monitoring of Mig cDC recruitment. This method will show great potential and value in practical applications. However, current research still has two unresolved issues: First, there is currently a lack of a method for efficiently and clearly tracing Mig cDCs. Under normal physiological conditions, the distinction between Mig cDCs and Res cDCs is usually based on the difference in their surface MHC-II and CD11c expression levels. However, under inflammatory conditions, this marker-based distinction becomes blurred because the expression differences of these two cell subsets on MHC-II and CD11c narrow, making them difficult to clearly distinguish. Second, there is a lack of in-depth understanding of the quantitative relationship between the number of adjuvant-promoted Mig cDC recruitment and the intensity of CD4+T and CD8+T cell responses. To address these two issues, this invention develops a precise tracing and quantification technique for Mig cDCs, and simultaneously defines the quantitative relationship between the number of Mig cDCs and the intensity of the immune response by enhancing and reducing the number of Mig cDCs, constructing a rapid adjuvant evaluation system with the number of Mig cDCs as the key indicator. This will not only provide a scientific basis for the early screening of adjuvants, but will also promote the progress of vaccine development and improve its efficiency and accuracy. Summary of the Invention

[0005] The first objective of this invention is to provide a method for quantitatively analyzing the number of Mig cDCs recruited by adjuvants, in order to solve the above-mentioned problems.

[0006] The second objective of this invention is to provide a method for predicting the intensity of adjuvant-induced immune response using the number of Mig cDCs as a key indicator, and to enable its application in high-throughput adjuvant screening.

[0007] To achieve the above objectives, the following technical solution is proposed:

[0008] In a first aspect, the present invention provides a method for quantitatively analyzing the intensity of adjuvant-induced immune responses, comprising the following steps:

[0009] First, the skin of KikGR transgenic mice was irradiated with ultraviolet light, causing the KikGR protein in the irradiated skin to change from green fluorescence to red fluorescence, thus enabling the tracing of skin-derived dendritic cells. Then, the control adjuvant combined with antigen and the test adjuvant combined with antigen were subcutaneously injected into the red fluorescent skin areas of the KikGR mice. Finally, the draining lymph nodes of the mice were isolated 6-120 hours after immunization. The number of KikGR red fluorescent cells recruited by the control adjuvant combined with antigen in the draining lymph nodes was used as a control indicator, and the number of KikGR red fluorescent cells recruited by the test adjuvant was compared with it to predict the strength of the immune response to the test adjuvant.

[0010] The control adjuvant is an adjuvant that enhances the strength of the immune response.

[0011] As a further technical solution, the draining lymph nodes of mice were isolated 24-48 hours after immunization.

[0012] As a further technical solution, the immune response includes one or more of humoral immune response and cellular immune response;

[0013] Preferably, the humoral immune response includes one or more of the following: promoting CD4+ T cell activation, proliferation, and long-term memory; promoting CD4+ Tfh cell differentiation and assisting B cell function; promoting B cell activation, high-frequency mutation, class conversion, and long-term memory; enhancing binding antibody and neutralizing antibody levels; and promoting protective effects against infection.

[0014] Preferably, the cellular immune response includes one or more of the following: promoting CD8+ T cell activation, proliferation, killing function, long-term memory, promoting CD4+ T cell differentiation into Th1 cells and assisting CD8+ T cell function, promoting protective effects against infection, and promoting anti-tumor effects.

[0015] As a further technical solution, the power of the ultraviolet light irradiation is 100-400mW / cm². 2 The irradiation time is 1-10 minutes;

[0016] The wavelength of the ultraviolet light is 405-436nm.

[0017] As a further technical solution, the skin includes back skin and / or abdominal skin.

[0018] As a further technical solution, the area of ​​skin irradiated with ultraviolet light in the KikGR transgenic phototransformed mice is 2-6 cm². 2 .

[0019] As a further technical solution, the control adjuvant includes one or more of aluminum adjuvant, squalene emulsion, TLR agonist adjuvant, STING agonist adjuvant, and cytokine adjuvant;

[0020] Preferably, the squalene emulsion includes MF59; the TLR agonist adjuvant includes one or more of CpG, R848, MPLA, and polyI:C; the STING agonist adjuvant includes cGAMP; and the cytokine adjuvant includes GM-CSF.

[0021] As a further technical solution, the control adjuvant is a combination of CpG and R848 adjuvants.

[0022] As a further technical solution, the antigen includes one or more of the following: pattern antigen (ovalbumin), tumor antigen, and pathogenic microbial antigen.

[0023] As a further technical solution, the draining lymph nodes include inguinal lymph nodes and / or superficial cervical lymph nodes.

[0024] As a further technical solution, the number of KikGR red fluorescent cells recruited in the draining lymph nodes was counted by flow cytometry.

[0025] Secondly, the present invention provides the application of the above-mentioned method for quantitatively analyzing the strength of adjuvant-induced immune response in adjuvant screening.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] In the field of vaccine development, traditional methods for evaluating the immunogenicity of adjuvants suffer from significant time delays, typically requiring 30 to 60 days to complete the assessment, making the development cycle excessively long. To shorten this cycle, there is an urgent need to develop a new method for rapidly evaluating and screening adjuvants. This invention only requires a control adjuvant (e.g., aluminum adjuvant, MF59 adjuvant, or a combination of CpG and R848 adjuvant) as a reference, combined with the number of KikGR red fluorescent cells 6-120 hours after antigen immunization stimulation as an indicator, to predict the immune response level of the test adjuvant. This significantly reduces the development cycle for adjuvant evaluation and enables rapid screening of superior adjuvants. Attached Figure Description

[0028] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0029] Figure 1 A. Use phototransformation signals from KikGR mice to label skin-derived Mig cDCs; B. Shave the fur of KikGR mice and expose the skin to 200 mW / cm². 2 Photoconversion was performed by irradiating the skin with 436nm ultraviolet light for 4 minutes, causing the skin to change from KikGR green fluorescence (507 / 517nm) to KikGR red fluorescence (583 / 593nm). The intensity, location, and size of the KikGR red fluorescence were observed using a portable fluorescence detector (GFPfinder-2101) under 488nm laser and 520nm filter conditions; B. At 200mW / cm²... 2After 24 hours of irradiation under 436 nm ultraviolet light, skin samples that had undergone photoconversion and those that had not were collected. Using a fluorescence confocal microscope (Lecai), the green fluorescence of KikGR was observed at 488 / 520 nm, and the red fluorescence at 530 nm / 588 nm. C. Different intensities of ultraviolet light (100, 200, and 400 mW / cm²) were used. 2 A) Photoconversion was performed on shaved skin from KikGR mice, and then KikGR red fluorescence was measured at 0, 24, 48, and 72 hours post-photoconversion using small animal in vivo techniques. D) The intensity of the KikGR red fluorescence signal was analyzed, with unconverted skin serving as a negative control, at 100, 200, and 400 mW / cm². 2 E. Detection of intensity light 3 hours after conversion; F. Number of KikGR red cells in draining lymph nodes; G. Total number of lymph node cells.

[0030] Figure 2 Dynamic changes in the number of KikGR erythrocytes and Mig cDCs activated by adjuvant combined with OVA; A. Phototransformation of shaved skin from KikGR mice, exposing the skin to 400 mW / cm². 2 Irradiation with 436nm ultraviolet light for 4 minutes, followed by subcutaneous injection of CR108, MF59, or Alum adjuvant plus OVA or OVA alone at the phototransformed skin sites 3 hours after phototransformation, with PBS as a negative control. 24 hours after injection, the proportion of KikGR red blood cells and MHCII in KikGR red blood cells in the draining lymph nodes were measured using FACS. hi CD11c med B. Using the same batch of mice, the concentration of MHCII in the entire lymph node was measured by anti-MHCII and anti-CD11c antibodies at 0, 12, 24, and 48 hours after injection. hi CD11c med C. Analyze the precise number of Mig cDCs; D. Analyze the dynamic number of kikGR red blood cells in the entire lymph node at 0, 12, 24 and 48 hours after injection using FACS; E. Analyze the correlation between the number of kikGR red blood cells and the number of Mig cDCs in the draining lymph nodes.

[0031] Figure 3Adjuvant combined with OVA induces anti-tumor immune response; A. Schematic diagram of experimental design: Female C57BL / 6 mice were subcutaneously inoculated with E.G7-OVA cells (5*10^5 cells / mouse) on their right back until the tumor length reached 5mm. Starting from days 1, 4, and 7, CR108, MF59, Alum plus OVA, or OVA alone were administered near the tumor. PBS was used as a negative control. Mean growth curve, animal survival rate, tumor weight, and CD45+CD3+C tumor infiltration were measured. B. D4+ T cells and CD45+CD3+CD8+ T cells; C. Mean growth curves showing the mean ± standard error of independent experiments (n=8); D. Survival rate of mice was determined on day 17 (n=4); E. Tumor weight of each group was determined on day 8; F. The number of tumor-infiltrating CD45+CD3+CD8+ T cells was determined on day 8 using FACS (n=4); G. The number of tumor-infiltrating CD45+CD3+CD4+ T cells was determined on day 8 using FACS (n=4);

[0032] Figure 4A. Schematic diagram of the experimental design: On day 0, CFSE-labeled OTI CD45.1+CD3+CD8+ T cells were injected intravenously into CD45.2C57BL / 6 mice at a dose of 1 x 10^6 cells. Subsequently, on day 1, mice were subcutaneously immunized with CR108, MF59, Alum plus OVA, or OVA alone, with PBS as a negative control. Three days later, the percentage of CD45.1+CD3+CD8+TCRVα2+ T cells was analyzed in draining lymph nodes, non-draining lymph nodes, and spleen, and the T cell proliferation index was analyzed by flow cytometry using the CFSE mitotic peak. B. Visualization of CFSE+CD45.1+TCRVα2+CD8+ T cells in draining lymph nodes. Histogram overlay; C and D. Analysis of T cell proliferation index and percentage of CD45.1+CD3+CD8+TCRVα2+ T cells in each CFSE mitotic peak in draining lymph nodes; E. Display of histogram overlay of CFSE+CD45.1+TCRVα2+CD8+ T cells in draining lymph nodes, non-draining lymph nodes, and spleen; F, G, and H. Measurement of percentage of CD45.1+TCRVα2+CD8+ T cells in draining lymph nodes, non-draining lymph nodes, and spleen; I. C57BL / 6 mice were subcutaneously inoculated with CR108+OVA, MF59+OVA, Alum+OVA, OVA alone, or PBS on days 0 and 14. On day 21 after the first immunization, target cells treated with OVA257-264 peptide were stained with high or low concentrations of CFSE and then intravenously injected into immunized mice. Spleens were collected 20 hours after injection, and spleen cells were analyzed by flow cytometry to determine the proportion of OVA-specific cell killing (n = 5-6, mean ± standard deviation).

[0033] Figure 5 Adjuvant combined with OVA activates antigen cross-presentation in KikGR erythrocytes; A. Shave the fur of KikGR mice and expose the skin to 400mW / cm². 2 After 4 minutes of irradiation under 436nm ultraviolet light, and 3 hours later, CR108, MF59, Alum plus OVA, or OVA alone were injected into the phototransformed skin sites. PBS was used as a negative control. The results showed MHCII... med CD11c hi Res cDC and MHCII hi CD11c med The upper part of the Mig cDC cell population was analyzed by flow cytometry for MHCII in the draining lymph nodes 48 hours after drug administration. med CD11c hi Res cDC (bottom left) or MHCIIhi CD11c med A. Expression level of the MHC-IOVA257-264(SIINFEKL) complex on Mig cDCs (lower right); B and C. Analysis of the number of MHCI SIINFEKL+Res cDCs and MHCI SIINFEKL+Mig cDCs; D. Determination of the level of the MHC-IOVA257-264(SIINFEKL) complex on KikGR erythrocytes by FACS at 48 hours post-injection; E. Measurement of the number of MHCI SIINFEKL+KikGR erythrocytes; F. Analysis of the correlation between the number of MHCI SIINFEKL+KikGR erythrocytes and the number of MHCI SIINFEKL+Mig cDCs.

[0034] Figure 6 Adjuvant combined with OVA-activated KikGR erythrocytes directly induced CD8+ T cell activation; A. Schematic diagram of experimental design: The fur of KikGR mice was shaved off, exposing the skin to 400mW / cm². 2 Irradiation with 436nm ultraviolet light for 4 minutes, followed by injection of CR108, MF59, or Alum plus OVA into the phototransformed skin sites 3 hours later. 48 hours later, KikGR erythrocytes and MHCII were isolated from the lymph nodes by sorting. hi CD11c med Mig cDC and MHCII med CD11c hi Res cDC, untreated OTI CD8+ T cells were isolated from OTI mice, and 5*10^3 KikGR erythrocytes and MHCII were respectively added to the cells. hi CD11c med Mig cDC or MHCII med CD11c hi Res cDCs were co-cultured with 5*10^4 OTI CD8+ T cells for three days, and CFSE was measured by FACS. lo A. The number of OTI CD8+T (CD45.1+CD3+CD8+TCRVα2+) T cells; B. Detection of CFSE after co-culture with KikGR erythrocytes. lo OTI CD8+ T cell count; C. Detection and MHCII hi CD11c med After co-culturing Mig cDC erythrocytes, CFSE lo OTI CD8+ T cell count; D. Detection and MHCII med CD11c hi After co-culturing with Res cDC, CFSElo Number of OTICD8+ T cells;

[0035] Figure 7 Blocking CCR7 impairs CD8+ T cell activation. The experimental procedure involved shaving the fur of KikGR mice and exposing the skin to 400 mW / cm². 2 After irradiation with 436nm ultraviolet light for 4 minutes, 3 hours later, each mouse was subcutaneously injected with 10ug of anti-CCR7 IgG2a blocking antibody at the phototransformed skin site, with 10ug of IgG2a antibody injected as an isotype control. 6 hours later, CR108 or MF59 plus OVA was subcutaneously injected into the α-CCR7 injection site; A, B, and C. 48 hours after injection, KikGR erythrocytes and MHC II in the lymph nodes were measured by FACS. hi CD11c med Mig cDC and MHCII med CD11c hi Res cDC count; D. Schematic diagram of experimental design: 1*10^6 CFSE-labeled OTI CD8+ T cells were transferred to each mouse. Subsequently, mice were subcutaneously injected with 10ug of anti-CCR7 IgG2a blocking antibody, with 10ug of IgG2a antibody injected as an isotype control. One day later, CR108 or MF59 plus OVA were injected into the antibody injection site. PBS was used as a negative control. Three days after immunization, CFSE in lymph nodes, non-lymph nodes, and spleen were analyzed by FACS. lo CD45.1+CD3+CD8+TCRVα2+ T cells. E. Showing CFSE in lymph nodes, non-lymph nodes, and spleen cells. lo Histogram overlay of CD45.1+TCRVα2+CD8+ T cells; F, G, and H. Measurement of CFSE in lymph nodes, non-lymph nodes, and 2*10^6 spleen cells. lo The number of CD45.1+TCRVα2+CD8+ T cells;

[0036] Figure 8 Female C57BL / 6 mice were subcutaneously inoculated with E.G7-OVA cells (5*10^5 cells / mouse) on their right back until the tumor length reached 5 mm. Starting from day 1, 7 and 13, 10 μg of anti-CCR7IgG2a blocking antibody was subcutaneously injected near the tumor. 10 μg of IgG2a antibody was injected as an isotype control. 6 hours later, CR108 plus OVA was subcutaneously injected into the α-CCR7 injection site, and the growth curve was continuously measured (n=4-5). Detailed Implementation

[0037] The embodiments and examples of the present invention will be described in detail below with reference to the implementation methods and examples. However, those skilled in the art will understand that the following implementation methods and examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] In a first aspect, the present invention provides a method for quantitatively analyzing the intensity of adjuvant-induced immune response, comprising the following steps:

[0039] First, the skin of KikGR transgenic mice was irradiated with ultraviolet light, causing the KikGR protein in the irradiated skin to change from green fluorescence to red fluorescence, thus tracing skin-derived dendritic cells. Then, based on demonstrating a causal and quantitative relationship between the number of Mig cDCs recruited by the adjuvant and the intensity of the induced immune response, the control adjuvant combined with antigen and the test adjuvant combined with antigen were subcutaneously injected into the red fluorescent skin areas of KikGR mice. Finally, 24-48 hours post-immunization, the draining lymph nodes of the mice were isolated. The number of KikGR red fluorescent cells recruited by the control adjuvant combined with antigen in the draining lymph nodes was used as a control indicator, and the number of KikGR red fluorescent cells recruited by the test adjuvant was compared to this indicator to predict the intensity of the immune response to the test adjuvant.

[0040] The control adjuvant is an adjuvant that enhances the strength of the immune response.

[0041] Preferably, the draining lymph nodes of mice are isolated 24-48 hours after immunization.

[0042] In this invention, the immune response includes one or more of humoral immune response and cellular immune response;

[0043] The humoral immune response includes one or more of the following: promoting CD4+ T cell activation, proliferation, and long-term memory; promoting CD4+ Tfh cell differentiation and assisting B cell function; promoting B cell activation, high-frequency mutation, class conversion, and long-term memory; enhancing binding antibody and neutralizing antibody levels; and promoting protective effects against infection.

[0044] The cellular immune response includes one or more of the following: promoting CD8+ T cell activation, proliferation, killing function, long-term memory, promoting CD4+ T cell differentiation into Th1 cells and assisting CD8+ T cell function, promoting protective effects against infection, and promoting anti-tumor effects.

[0045] KikGR mice are a genetically engineered mouse strain whose cells throughout the body carry the photoconvertible fluorescent reporter gene Kikume Green-Red (KikGR). When a designated region of a KikGR mouse is irradiated with ultraviolet light, the KikGR protein in that region irreversibly changes from green fluorescence to red fluorescence. Following adjuvant stimulation, the accumulation of Mig cDCs in the dLN typically lasts 1-2 days. However, within this timeframe, the sustained expression of the KikGR green gene leads to a corresponding weakening of the KikGR red signal. To overcome this limitation and maintain a clear distinction between KikGR green and KikGR red cells, this invention optimizes the photoconversion conditions based on existing techniques to improve the quantitative accuracy of Mig cDC quantification in KikGR mice.

[0046] The inventors have discovered that, in some optional embodiments, the power of the ultraviolet light irradiation can be, for example, but is not limited to, 100 mW / cm². 2 200mW / cm 2 300mW / cm 2 Or 400mW / cm 2 The preferred value is 400mW / cm 2 The irradiation time can be, for example, but not limited to, 1 min, 3 min, 6 min or 10 min; the wavelength of the ultraviolet light is 405-436 nm.

[0047] When ultraviolet light is at 400mW / cm 2 After continuous irradiation for 4 minutes, the KikGR red signal can be effectively maintained for more than 3 days without decay.

[0048] In some alternative implementations, the skin includes, but is not limited to, back skin or abdominal skin.

[0049] In a preferred embodiment, the skin is the back skin adjacent to the inguinal lymph nodes.

[0050] In some alternative embodiments, the area of ​​skin of the KikGR transgenic phototransformed mouse irradiated with ultraviolet light is 2-6 cm². 2 .

[0051] In some alternative embodiments, the control adjuvant includes one or more of aluminum adjuvants, squalene emulsions, TLR agonist adjuvants, STING agonist adjuvants, and cytokine adjuvants;

[0052] Preferably, the squalene emulsion includes MF59; the TLR agonist adjuvant includes one or more of CpG, R848, MPLA, and polyI:C; the STING agonist adjuvant includes cGAMP; and the cytokine adjuvant includes GM-CSF.

[0053] In some alternative implementations, the antigen includes, but is not limited to, one or more of the following: pattern antigen (ovalbumin), tumor antigen, and pathogenic microbial antigen.

[0054] In some alternative implementations, the draining lymph nodes include inguinal lymph nodes and / or superficial cervical lymph nodes.

[0055] In some alternative implementations, the number (absolute number) of KikGR red fluorescent cells recruited in the draining lymph nodes is counted by flow cytometry.

[0056] Secondly, the present invention provides the application of the above-mentioned method for quantitatively analyzing the strength of adjuvant-induced immune response in adjuvant screening.

[0057] The method for quantitatively analyzing the immune effect of adjuvants provided by this invention can quickly achieve high-throughput screening of excellent adjuvants based on the strength of the immune response promoted by the adjuvant to be tested, providing new tools and ideas for vaccine development and greatly shortening the time for evaluating the immune effect of vaccines.

[0058] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0059] The main experiments of this invention are as follows: First, to establish the quantitative relationship between the number of Mig cDCs and the initiation of CD8+ T cells in vivo. This invention uses CFSE-labeled OTI CD8+ T cells for adoptive transfer in vivo. By measuring the number of OTI cell proliferations, the quantitative relationship between the number of Mig cDCs recruited by different adjuvants and the level of CD8+ T cell initiation is determined. Furthermore, this invention uses a functional blocking antibody against CCR7 to block the number of Mig cDCs in draining lymph nodes, analyzing the inhibitory effect of reduced Mig cDC numbers on the level of CD8+ T cell initiation, indicating a direct quantitative relationship between the number of Mig cDCs recruited and the number of CD8+ T cells initiated. Second, to establish the quantitative relationship between the number of Mig cDCs and their anti-tumor effect in vivo. This invention establishes an E.G7 subcutaneous tumor model, and in the tumor model, the quantitative relationship between the number of adjuvant-promoted Mig cDCs and their anti-tumor effect is determined. Furthermore, this invention uses a functional blocking antibody against CCR7 to block the number of Mig cDCs in draining lymph nodes, and analyzes the inhibitory effect of reduced Mig cDC numbers on anti-tumor efficacy, demonstrating a quantitative relationship between the number of Mig cDCs recruited and the anti-tumor efficacy.

[0060] The specific experimental procedures are as follows:

[0061] Example 1: Labeling of skin-derived migratory dendritic cells using KikGR mice

[0062] This embodiment uses the phototransformation signature of KikGR mice to label skin-derived migratory dendritic cells (MigcDCs). KikGR mice are genetically modified to express the KikGR protein, an engineered fluorescent protein that can transform from KikGR green fluorescence (KikGR-green) to KikGR red fluorescence (KikGR-red) under ultraviolet light. Through phototransformation, skin-derived cells are made to carry KikGR red fluorescence, which is used to track the dynamic changes of this cell group in draining lymph nodes (dLNs). Optimal phototransformation conditions have been screened in this embodiment.

[0063] 1. Method

[0064] 1.1 Light Conversion and Live Animal Imaging

[0065] STOCK Tg(CAG-KikGR)33Hadj / J mice (KikGR) were purchased from Jackson Laboratories. Female KikGR mice were anesthetized, and then the abdominal skin was exposed to ultraviolet light from a 436nm curing device at 100, 200, or 400 mW / cm². 2Irradiation with high power for 4 minutes. On days 1, 2, and 3 after light conversion, KikGR green fluorescence was collected using an in vivo imaging system (IVIS Spectrum CT, PerkinElmer, Waltham, USA), acquired under EX / EM filters at 465nm / 520nm, and KikGR red fluorescence was detected under EX / EM filters at 535nm / 600nm. The intensity of KikGR red fluorescence was quantified, and the size and location of the fluorescence on the skin were observed.

[0066] 1.2 Isolation and Flow Cytometry of Draining Lymph Nodes

[0067] Inguinal lymph nodes were isolated from immunized mice, homogenized by grinding and filtering through a 40 μm sieve to prepare a single-cell suspension. Individual cells from the lymph nodes were analyzed using a viability marker (Fixable Viability Difference Fluorescent Tablet). TM 780 (eBioscience) and the following antibodies (Biolegend and eBioscience) were stained for 15 minutes. To analyze the absolute number of KikGR red blood cells, 16 μL of Precision CountBeads was added to a 400 μL cell loading system. TM (Biolegend), KikGR red signal was detected via the PE channel. Flow cytometry was performed using LSR Fretessa (BD Biosciences), and data were analyzed using FlowJo software (BD Biosciences). The absolute number of cells was calculated using the following formula 1.

[0068]

[0069] 2. Conclusion

[0070] 2.1 Skin cells were labeled using the light conversion characteristics of KikGR mice.

[0071] KikGR mouse skin with fur removed was exposed to ultraviolet light at a wavelength of 436 nm and an intensity of 200 mW / cm². 2 The process lasted for 4 minutes to achieve light conversion. As expected, confocal microscopy confirmed that 24 hours after light conversion, the KikGR-green fluorescence in the exposed skin completely transformed into KikGR red fluorescence. Figure 1 In the A group, no KikGR green fluorescent residue was detected compared to skin not exposed to ultraviolet light. Figure 1 (B in the text). This demonstrates that in KikGR mice, irradiation of the skin with 436nm ultraviolet light can effectively label skin cells and completely distinguish unirradiated cells.

[0072] 2.2 Optimization of light conversion conditions

[0073] Following adjuvant stimulation, the accumulation of Mig cDCs in the dLN typically lasts for 1–2 days. However, within this timeframe, the KikGR red signal is attenuated due to the continued expression of the KikGR green gene. To overcome this limitation and maintain a clear distinction between KikGR green and KikGR red cells, the light intensity of the skin photoconversion near the dLN was increased from 100 mW / cm². 2 Increased to 400mW / cm 2 The light was applied continuously for 4 minutes. The results showed that increasing the light intensity effectively maintained the KikGR red signal for more than 3 days without decay. Figure 1 (C and D in the text).

[0074] 2.3 Evaluation of inflammatory response caused by phototransformation.

[0075] Increase the light intensity to 400 mW / cm 2 Lower light intensities did not induce additional KikGR erythrocytes into the dLN, and did not cause dLN swelling compared to unirradiated skin dLN. Figure 1 (E and F in the text). These findings indicate that 400 mW / cm 2 The light intensity can be safely maintained for 3 days to observe the dynamic changes of Mig cDC migration to dLN.

[0076] Example 2: Under adjuvant-induced inflammatory conditions, the change in the number of KikGR erythrocytes was consistent with that of Mig cDC.

[0077] Mig cDC migration to dLNs is a key factor in initiating an immune response. This study used KikGR red fluorescence to track the dynamic migration of these cells. To evaluate the different recruitment characteristics exhibited by Mig cDCs activated by different adjuvants, this study first used 436 nm ultraviolet light at 400 mV / cm². 2 KikGR mice underwent photoconversion by irradiating their skin with a specific intensity for 4 minutes. Subsequently, ovalbumin (OVA) was subcutaneously inoculated at the photoconversion site. OVA was combined with different adjuvants and adjuvant combinations, including the TLR9 and TLR7 / 8 agonists CpG and R848 (CR108), aluminum salts (Alum), and MF59. An equal volume of PBS was injected as a blank control. dLNs were isolated at 1, 12, 24, and 48 hours post-immunization and analyzed using specific antibodies and flow cytometry, along with MHC-II and CD11c markers, to identify Mig cDC and Res cDC populations.

[0078] 1. Method

[0079] 1.1 Vaccine Immunization

[0080] To study dendritic cell migration, female KikGR mice underwent photoconversion on their dorsal skin, followed by subcutaneous injection of different treatments at the central site of the KikGR red fluorescent region. Inguinal lymph nodes (iLNs) were collected from mice at 1, 12, 24, and 48 hours post-immunization to determine the percentage and exact number of Mig cDCs and Res cDCs. Immunotherapy formulations included: PBS, 10 μg OVA (Sigma) alone, 10 μg OVA plus 100 μg Alum (Invivogen), 10 μg OVA plus 50 μl MF59 (MF59 solution prepared by Advaccine Biopharmaceuticals Co., Ltd.), and 10 μg OVA plus 20 μg CR108 adjuvant and 20 μg R848 adjuvant (4 mg / ml CpG1018 and 1 mg / ml R848 stock solution prepared by Advaccine Biopharmaceuticals Co., Ltd.). Immunization volumes were brought to 100 μl with PBS.

[0081] 1.2 Isolation and Flow Cytometry of Draining Lymph Nodes

[0082] Inguinal lymph nodes were isolated from immunized mice, homogenized by grinding and filtering through a 40 μm sieve to prepare a single-cell suspension. Individual cells from the lymph nodes were analyzed using a viability marker (Fixable Viability Difference Fluorescent Tablet). TM 780 (eBioscience) and the following antibodies (Biolegend and eBioscience) were stained for 15 minutes. For Mig cDC analysis, Res cDC was stained with the following antibodies: anti-CD11c-BV605 or APC (N418), anti-IA / IE-PerCP / cy5.5 or BV421 (M5 / 114.15.2). For KikGR red cells analysis, staining was not required; KikGR red signal was detected via the PE channel. To determine the absolute number of these cell subpopulations, 16 μL of Precision Count Beads was added to a 400 μL cell loading system. TM (Biolegend) Flow cytometry was performed using LSL Fortessa (BD Biosciences), and the data were analyzed using FlowJo software (BD Biosciences). The absolute number of cells was calculated using Equation 1.

[0083] 2. Conclusion

[0084] 2.1 Most of the KikGR red blood cells after adjuvant stimulation were Mig cDC subsets.

[0085] Analysis showed that approximately 70-80% of KikGR erythrocytes were CD11c at 24 hours after adjuvant activation. med MHCI Ihi Mig cDC population. However, when only OVA and PBS were administered as a control group, the proportion of the Mig cDC population in KikGR erythrocytes within the dLN was very small ( Figure 2 (A) This indicates that the proportion of Mig cDC from the skin to dLN can be increased by adding adjuvants.

[0086] 2.2 Under adjuvant-induced inflammatory conditions, the number of KikGR erythrocytes changed in a manner consistent with that of Mig cDC.

[0087] Analysis showed that within the dLN, CD11c immune activation occurs when combined with CR108 and OVA (CR108+OVA). med MHCII hi The number of Mig cDCs increased significantly. This increase began 12 hours after immunization, peaked at 24-48 hours, at which point an estimated 30,000 CR108+OVA-activated Mig cDCs flowed into dLNs. Conversely, MF59 combined with OVA (MF59+OVA) activated Mig cDCs peaked at 24 hours, with an estimated influx of 15,000 cells, and rapidly decreased within 48 hours. The trend in the number of MF59+OVA-activated Mig cDCs was similar to that of aluminum adjuvant combined with OVA (Alum+OVA), but the number of Mig cDCs stimulating Alum+OVA was much smaller. Figure 2 (B in the text) After inoculation with different adjuvants in combination with OVA or with OVA alone, a highly significant correlation was observed between the number of Mig cDCs and KikGR erythrocytes. Figure 2 The dynamic changes in KikGR erythrocyte migration are very similar to the trend of Mig cDC (C). Figure 2 (D in the text). Therefore, KikGR mice are a valuable tool for analyzing the dynamic changes in Mig cDC numbers after vaccine and adjuvant immunization. KikGR mice offer a significant advantage in tracking Mig cDC models, as they can clearly identify cells from the skin injection site. These results collectively suggest that changes in KikGR erythrocyte counts under adjuvant-induced inflammatory conditions can reflect changes in the Mig cDC population.

[0088] Example 3: Correlation between efficient recruitment of KikGR erythrocytes and anti-tumor effects

[0089] Given the well-established role of Mig cDCs in initiating cellular immune responses, this study aimed to explore whether the number of KikGR erythrocytes recruited into dLNs is related to the antitumor efficacy of the vaccine. An E.G7 subcutaneous tumor model was established in C57BL / 6 mice by subcutaneous injection of 1 x 10^6 OVA-specific E.G7 tumor cells. Tumor volume exceeded 5 mm. 3 Subsequently, treatment was administered on days 1, 4, and 7 with CR108+OVA, Alum+OVA, MF59+OVA, or OVA alone.

[0090] 1. Method

[0091] 1.1 Preparation of tumor cells and construction of subcutaneous tumor models

[0092] E.G7-OVA cells were donated by Dr. Minghui Zhang (Tsinghua University). E.G7-OVA cells were cultured in a 37°C, 5% CO2 incubator using RPMI 1640 (Meilun Biotechnology, MA0215) medium containing 10% heat-inactivated fetal bovine serum (HI)-FBS (BI, 04-001-1ACS) and 1% penicillin-streptomycin (BI, 03-031-1B). Cells were passaged using 0.25% trypsin-EDTA (Gibco, 25200056). To construct a subcutaneous tumor model, female C57BL / 6 mice were selected as experimental subjects. Each mouse underwent a subcutaneous injection of E.G7-OVA cell suspension into one side of its back. Specifically, each mouse was injected with 5 × 10^6 E.G7-OVA cells suspended in 100 μL of PBS buffer, ensuring uniform mixing before injection. To accurately monitor tumor growth, calipers were used as the measuring tool, and the length and width of the tumor were measured every two days. The tumor volume was calculated using the standard ellipsoid formula, i.e., (length × width^2) / 2, which can accurately reflect changes in tumor size.

[0093] 1.2 Immunotherapy

[0094] To treat an E.G7-OVA tumor model, female C57BL / 6 mice were subcutaneously immunized three times every three days with different immunotherapy formulations after the E.G7-OVA tumor length exceeded 5 mm. The immunotherapy formulations included: PBS, 10 μg OVA alone (Sigma), 10 μg OVA plus 100 μg Alum (Invivogen), 10 μg OVA plus 50 μl MF59 (MF59 solution prepared by Advaccine Biopharmaceuticals Co., Ltd.), and 10 μg OVA plus 20 μg CR108 adjuvant and 20 μg R848 adjuvant (4 mg / ml CpG1018 and 1 mg / ml R848 stock solution prepared by Advaccine Biopharmaceuticals Co., Ltd.). The immunization volume was brought to 100 μl with PBS.

[0095] 1.3 T cell count of tumor infiltrating cells

[0096] Tumors in mice were excised and digested at 37°C for 45 minutes in a digestion buffer containing 1000 U / ml DNase I and 2 mg / ml collagen IV. The digested tumor tissue was then pulverized using the plunger end of a syringe to prepare a single-cell suspension, which was filtered through a 40 μm cell sieve. The tumor cell suspension was stained with anti-CD45-AF700, anti-CD3-BV421, and anti-CD8-PerCP / cy5.5 in the dark for 15 minutes. After staining, 16 μL of Precision Count Beads was added to a 400 μL cell loading system. TM (Biolegend) Flow cytometry was performed using LSL Fortessa (BD Biosciences), and the data were analyzed using FlowJo software (BD Biosciences). The absolute number of cells was calculated using Equation 1.

[0097] 2. Conclusion

[0098] 2.1 Evaluation of the efficacy of immunotherapy for tumors.

[0099] OVA+CR108 treatment significantly reduced tumor size after the second immunization. Conversely, mice treated with Alum+OVA, MF59+OVA, or OVA alone continued to show a similar tumor growth trend to the PBS control group, failing to effectively control tumor growth. Figure 3 (B in the text). The survival rate of the tumor model treated with OVA+CR108 reached 100%, while the tumor models treated with other adjuvants in combination with OVA all reached ethical death before day 14. Figure 3 (C in the text). Further analysis revealed that, compared with other adjuvants combined with OVA treatment, mice treated with OVA+CR108 showed a significant reduction in tumor weight after the third immunization. Figure 3 (D in the text). In summary, these results suggest a strong association between the degree of KikGR red cell accumulation in dLNs and adjuvant-enhanced antitumor efficacy of vaccines.

[0100] 2.2 Evaluation of antitumor immune response.

[0101] To assess the level of anti-tumor cellular immune response, the number of tumor-infiltrating CD8+ and CD4+ T cells was measured after the third immunization. Compared with CR108+ OVA treatment, MF59+ OVA or Alum+ OVA treatment did not show a corresponding increase in CD8+ T cell infiltration. Figure 3 In the figure, E represents the group on the horizontal axis. Figure 3 The D in the text is the same. Interestingly, after treatment with CR108+OVA or MF59+OVA, the number of infiltrating CD4+ T cells was significantly reduced compared to Alum+OVA and OVA alone. Figure 3 In the figure, F represents the group on the horizontal axis. Figure 3 (The D in the text is the same). These results suggest that using CR108 as an adjuvant for tumor vaccines may be more effective than MF59 or Alum in activating CD8+ T cells.

[0102] Example 4: Correlation between efficient recruitment of KikGR red blood cells and in vivo CD8+ T cell activation and cytotoxic function

[0103] To explore the correlation between the efficient recruitment of KikGR erythrocytes and the in vivo activation of CD8+ T cells, this study employed in vivo adoptive transfer of CFSE-labeled OTI CD8+ T cells (which specifically recognize the OVA257-264 sequence) and injected them into... C57BL / 6 mice (1 x 10^6 cells per mouse). Then, 24 hours later, these mice were inoculated with CR108+OVA, MF59+OVA, Alum+OVA, OVA alone, or PBS (control). 72 hours after vaccination, dLNs were isolated and the proliferation of OVA-specific OTI CD8+ T cells was detected by flow cytometry.

[0104] To explore the correlation between the efficient recruitment of KikGR red blood cells and the in vivo activation and killing function of CD8+ T cells, this study used in vivo killing experiments for verification.

[0105] 1. Method

[0106] For the in vivo CD8+ T cell initiation experiment, the following procedure was followed: Spleens were isolated from OTI mice and crushed using the plunger end of a syringe. The spleen cell suspension was filtered through a 40 μm cell sieve. CD8+ T cells were purified using a CD8 T cell isolation kit (Biolegend) according to the manufacturer's protocol. The purified CD8+ T cell suspension was then stained with 5 μM CellTrace CFSE (Biolegend) PBS solution in the dark for 20 minutes. At the end of staining, the cells were washed twice with PBS and resuspended at a concentration of 1 x 10^6 cells / 100 μL PBS. 100 μL of the cell suspension was injected intravenously into each mouse. Twenty-four hours later (day 0), mice were subcutaneously injected with different formulations including: PBS, 10 μg OVA (Sigma) alone, 10 μg OVA plus 100 μg Alum (Invivogen), 10 μg OVA plus 50 μl MF59 (MF59 solution prepared by Advaccine Biopharmaceutical Co., Ltd.), and 10 μg OVA plus 20 μg CR108 adjuvant and 20 μg R848 adjuvant (4 mg / ml CpG1018 and 1 mg / ml R848 stock solution prepared by Advaccine Biopharmaceutical Co., Ltd.). The immunization volume was brought to 100 μl with PBS. On day 3, draining lymph nodes (dLNs) were collected and the lymph node cell suspensions were stained with anti-CD3-eFluro450, anti-CD8-PerCP / cy5.5, anti-CD45.1-PE, and anti-TCRVα-APC antibodies. CFSE+ OTI cells were detected in a flow cytometry-gated CD3+CD8+CD45.1+TCRVα2+ cell sequence. Results were presented as CD3+CD8+CD45.1+TCRVα2+ CFSE+ OTI cells. o The absolute number of cells (i.e., cells that have undergone at least one division cycle) or CD3+CD8+CD45.1+TCR Vα2+CFSE lo Percentage expression of each mitotic peak in the cell.

[0107] For experiments on cytotoxic function, the following methods were used: Naive C57BL / 6 mice were subcutaneously immunized on days 0 and 14, with immunizing agents including CR108, MF59, Alum with OVA, OVA alone, and PBS. On day 21 after the first immunization, the mice were sacrificed and spleen cells were collected. The collected spleen cells were divided into two equal samples. One sample was treated with 10 μg / 1x10^7 cells / mL of OVA257-264 peptide at 37°C for 1 hour. These cells were then stained with 5 μM CFSE (CFSE^hi). The other sample was treated with intact culture medium and stained with 0.5 μM CFSE (CFSE^lo). The CFSE^hi and CFSE^lo stained cells were mixed 1:1 to form a cell suspension with a concentration of 2x10^7 cells / mL.

[0108] The mixed cell suspension was injected intravenously into control or immunized mice. Twenty hours post-injection, spleens were isolated from the mice. Spleen cells were collected and analyzed by flow cytometry to determine the percentages of CFSE^hi and CFSE^lo cells. The percentage of OVA257-264-specific lysis was calculated using the following formula: Specific lysis (%) = [(mean control group CFSE^hi% - immunized group CFSE^hi%) / mean control group CFSE^hi%] × 100%.

[0109] 2. Conclusion

[0110] 2.1 The more adjuvants promote the recruitment of Mig cDC, the higher the level of CD8+T response activation.

[0111] Analysis showed that, compared with mice inoculated with MF59+OVA, Alum+OVA, or OVA alone, mice inoculated with CR108+OVA had significantly higher levels of CFSE proliferation in their dLN. lo The percentage of OTI CD8+ T cells was the highest. Figure 4 (B in the text). Furthermore, the proliferation induced by MF59+OVA was similar to that induced by Alum+OVA, and higher than that induced by OVA alone (…). Figure 4 (C in the text). Further analysis showed that mice inoculated with CR108+OVA had a higher proportion of CFSE. lo CD8+ T cells underwent 6 or 7 divisions, while MF59+OVA, Alum+OVA, or OVA alone resulted in fewer cell divisions. Figure 4 (D in the text). These results generally indicate that efficient recruitment of KikGR erythrocytes is closely associated with CD8+ T cell activation.

[0112] 2.2 The key site for the initiation of adjuvant-stimulated CD8+ T cell responses is the dLN.

[0113] In contrast to dLN, a lower percentage of OTI CD8+ T cells was observed in the spleen and non-draining lymph nodes (non-dLN), and the proportion of proliferating cells was low regardless of the type of vaccine administered (CR108+OVA, MF59+OVA, Alum+OVA, or OVA alone). Figure 4 In EH, G, and H, the groups on the horizontal axis are the same as those in F. Therefore, the results showing proliferation mainly in the dLN, but not in the spleen and non-dLN, reflect that Mig cDCs in the dLN are key cells promoting the initiation of CD8+ T cells.

[0114] 2.3 The more Mig cDCs recruited by the adjuvant, the stronger its ability to promote CD8+T killing.

[0115] In vivo CD8+ T cell cytotoxicity assay results showed ( Figure 4 In the study (I), after two immunizations, CR108+OVA significantly increased the killing rate of CD8+ T cells against target cells treated with OVA257-264 peptide, compared to MF59+OVA or Alum+OVA. This finding indicates that CR108+OVA can elicit a CD8+ T cell response to a specific antigen and possesses the ability to kill tumor cells. This cytotoxic ability is closely related to the effective recruitment of KikGR erythrocytes.

[0116] Example 5: KikGR red blood cells have the function of cross-presenting antigens.

[0117] Building upon previous observations of the correlation between effective aggregation of KikGR erythrocytes and their anti-tumor effects and CD8+ T cell activation, this study aimed to explore the possibility of KikGR erythrocytes engaging in antigen cross-presentation. An antibody recognizing the MHC I molecule-binding CD8+ epitope peptide OVA257-264 (SIINFEKL) was used to clarify the antigen cross-presentation function of these cells.

[0118] 1 Method

[0119] Inguinal lymph nodes were isolated from immunized mice, homogenized by grinding and filtering through a 40 μm sieve to prepare a single-cell suspension. Individual cells from the lymph nodes were analyzed using a viability marker (Fixable Viability Difference Fluorescent Tablet). TM780 (eBioscience) and the following antibodies (Biolegend and eBioscience) were stained for 15 minutes. To analyze the cross-presentation of the OVA-SIINFEKL peptide on MHC I by cDCs, single-cell suspensions were stained with the following antibodies: anti-CD11c-BV605 (N418), anti-IA / IE-PerCP / cy5.5 (M5 / 114.15.2), and anti-H-2Kb OVA257-264-APC (eBio25-D1.16). To analyze CD8+ OTI T cells, the following antibodies were used: anti-CD45.1-PE (A20), anti-CD3e-BV510 (145-2C11), anti-CD8α-BV650 (53-6.7), and anti-TCRVa2-APC (B20.1). After staining, add 16 μL of Precision Count Beads to a 400 μL cell loading system. TM (Biolegend) Flow cytometry was performed using LSL Fortessa (BD Biosciences), and the data were analyzed using FlowJo software (BD Biosciences). The absolute number of cells was calculated using Equation 1.

[0120] 2. Conclusion

[0121] KikGR red blood cells have antigen cross-presentation capabilities.

[0122] The results showed that approximately 3-15% of KikGR erythrocytes expressed MHC I-OVA257-264 (SIINFEKL) on their surface. Regardless of the adjuvant used to enhance the immune response, KikGR erythrocytes and MigcDCs consistently expressed a greater amount of MHC I-OVA257-264 (SIINFEKL) on their surface compared to Res cDCs stimulated with the same adjuvant. Figure 5 AC in Figure 5 The group of the horizontal axis in B is related to Figure 5 The C in the text is the same. It is noteworthy that, compared to MF59-activated KikGR erythrocytes, CR108-activated KikGR erythrocytes showed a significantly higher absolute number of cells expressing MHC I-OVA257-264 (SIINFEKL) on their surface. Figure 5 (D and E in the text). The absolute number of Mig cDC and KikGR erythrocytes expressing MHC I-OVA257-264 (SIINFEKL) showed similar fluctuations in adjuvant-stimulated levels. Figure 5 (F in the text). These findings suggest a role for skin-derived KikGR erythrocytes in antigen cross-presentation.

[0123] Example 6: KikGR red blood cells directly induce CD8+ T cell initiation

[0124] To further investigate whether adjuvant-activated KikGR erythrocytes directly initiate CD8+ T cell initiation, this study subcutaneously immunized KikGR mice at photoconversion sites with CR108+OVA, MF59+OVA, or Alum+OVA. Forty-eight hours post-vaccination, dLNs were harvested to prepare single-cell suspensions, and KikGR erythrocytes, along with Mig cDCs and Res cDCs isolated using CD11c and MHC-II markers, respectively, were separated by FACS. OTI CD8+ T cells were then isolated using a CD8+ T cell isolation kit and labeled with CFSE fluorescein. These three distinct cell subpopulations were co-cultured at 5 x 10^3 cells per well with purified CFSE+OTI CD8+ T cells at 5 x 10^4 cells per well.

[0125] 1. Method

[0126] Mig cDC, Res cDC and KikGR red blood cell sorting and in vitro co-culture with T cells

[0127] Secretory draining lymph nodes were extracted from immunized KikGR mice and homogenized through a 40 μm sieve to prepare single-cell suspensions. The cell suspensions were stained with anti-CD11c-BV605 and anti-IA / IE-PerCP / cy5.5 for 15 minutes. Cell sorting was performed by flow cytometry using a BDFACSAria II (BD Biosciences). OTI CD8+ T cells were isolated using the same method as in in vivo CD8+ T cell proliferation assays and stained with CFSE. Sorted Mig cDCs and T cells were resuspended in RPMI 1640 medium containing 10% FBS, 1% penicillin, and 1% streptomycin, and seeded in 96-well plates. Cells were co-cultured at 37°C and 5% CO2 for 3 days, and then stained with anti-CD3-eFluro450, anti-CD8-PerCP / cy5.5, anti-CD45.1-PE, and anti-TCRVα-APC. CFSE+ OTI cells were detected in a flow cytometry gating system for the CD3+CD8+CD45.1+TCRVα2+ cytometry pattern. Results were presented as CD3+CD8+CD45.1+TCRVα2+ CFSE cells. lo The absolute number of cells (i.e., cells that have undergone at least one division cycle) or CD3+CD8+CD45.1+TCR Vα2+CFSE loPercentage expression of cell division peaks. After staining, 16 μL of Precision Count Beads were added to a 400 μL cell loading system. TM (Biolegend) Flow cytometry was performed using LSL Fortessa (BD Biosciences), and the data were analyzed using FlowJo software (BD Biosciences). The absolute number of cells was calculated using Equation 1.

[0128] 3. Conclusion

[0129] Adjuvant-activated KikGR erythrocytes directly induced the proliferation of OTI CD8+ T cells.

[0130] Notably, KikGR erythrocytes activated by CR108+OVA showed the highest CFSE. lo The absolute number of OTI CD8+ T cells was the highest, followed by MF59+ OVA, and then Alum+ OVA. Figure 6 (B) Alloadjuvant-activated KikGR erythrocyte-induced CFSE lo The absolute number of OTI CD8+ T cells was similar to that induced by Mig cDCs, and significantly higher than that induced by RescDCs. Figure 6 (C in the text). Furthermore, no significant differences were observed between Res cDCs isolated from dLNs activated by different adjuvants (C in the text). Figure 6 (D in the original text). These results indicate that the selected adjuvant primarily affects the cross-presentation capacity of KikGR erythrocytes, rather than the cross-presentation capacity of Res cDCs. Overall, these results directly demonstrate the effect of adjuvant strength on the cross-presentation capacity of KikGR erythrocytes and highlight the different behaviors of Mig cDCs and Res cDCs in the early stages of the immune response.

[0131] Example 7: Limiting the recruitment of KikGR red blood cells affects the initial activation of CD8+ T cells.

[0132] Compared to Res cDCs, Mig cDCs significantly overexpress the chemokine receptor CCR7. During adjuvant-induced inflammation, Mig cDCs with high CCR7 expression can respond to CCL19 / CCL21 chemokines and migrate to dLNs. To investigate the effect of reduced Mig cDC numbers on the initial activation of CD8+ T cells, this study used a functional blocking antibody against CCR7 to reduce the number of Mig cDCs migrating from the skin to dLNs due to adjuvant activation via subcutaneous injection, thereby assessing the proliferation level and anti-tumor immune response of OTI CD8+ T cells. First, this study examined the changes in the number of Mig cDCs in dLNs after blocking with the functional blocking antibody against CCR7.

[0133] 1. Method

[0134] To analyze the inhibitory effect of CCR7 blockade on the recruitment of Mig cDCs, on day 0, rat IgG2A isotype controls (R&D systems) and CCR7 function blocking antibody were subcutaneously injected at the photoconversion sites of KikGR mice at a dose of 10 μg / 100 μl. Three hours later, the blocking sites were immunized with PBS, 10 μg OVA plus 50 μl MF59, and 10 μg OVA plus CR108 (20 μg each of CpG1018 and R848). Forty-eight hours later, single-cell suspensions of draining lymph nodes were prepared and stained with anti-CD11c-BV605 and anti-IA / IE-PerCP / cy5.5 for 15 minutes. Flow cytometry analysis of Mig cDCs, Res cDCs, and KikGR erythrocytes was performed using an LSR Tortessa (BD Biosciences).

[0135] To analyze the inhibitory effect of reduced Mig cDC recruitment on the initial initiation of CD8+ T cells, CD8+ T cells from OTI mice were sorted using a CD8+ T cell isolation kit (BioLegend) and CFSE staining, following previously described methods. Stained T cells were intravenously injected into C57BL / 6 mice at a concentration of 1 x 10^6 cells / 100 μl on day -2, and subcutaneously injected on day -1 with 10 μg / 100 μl of CCR7 function blocking antibody (R&D systems) and rat IgG2A isotype control (R&D systems). These C57 mice were divided into several groups and subsequently immunized on day 0 with PBS, 10 μg OVA plus 50 μl MF59, and 10 μg OVA plus CR108 (20 μg each of CpG1018 and R848), respectively. On day 4 of immunization, single-cell suspensions were collected from draining lymph nodes and spleen and analyzed by flow cytometry using anti-CD3-eFluor450, anti-CD8-PercP / cy5.5, anti-CD45.1-PE, and anti-TCRVα-APC staining.

[0136] To analyze the inhibitory effect of reduced Mig cDC recruitment on the antitumor activity of the vaccine, female C57BL / 6 mice were subcutaneously inoculated with E.G7-OVA cells (5*10^5 cells / mouse) on their right back until the tumor reached 5 mm in length. Starting on days 1, 7, and 13, 10 μg of anti-CCR7 IgG2a blocking antibody was subcutaneously injected near the tumor, with 10 μg of IgG2a antibody injected as an isotype control. Six hours later, CR108 plus OVA was subcutaneously injected at the α-CCR7 injection site, and growth curves were continuously measured.

[0137] 2. Conclusion

[0138] 2.1 KikGR erythrocytes are recruited to draining lymph nodes in a CCR7-CCL19 / CCL21-dependent manner.

[0139] The results showed that, regardless of whether MF59+OVA or CR108+OVA was administered, the number of Mig cDCs in dLN was significantly lower than that in the isotype control. Figure 7 In contrast, the absolute number of Res cDCs was not affected by the functional blocking antibody against CCR7 (A). Figure 7 In B, the group on the horizontal axis of the graph is... Figure 7 (The A in the text is the same). More importantly, the number of red blood cells in KikGR was significantly reduced, consistent with the trend of decreasing number of Mig cDCs ( Figure 7 In the figure, C represents the group on the horizontal axis. Figure 7The A in the data is the same, indicating that the recruitment of KikGR erythrocytes and Mig cDCs are significantly correlated with CCR7-CCL19 / CCL21 signaling.

[0140] 2.2 KikGR erythrocytes play a key role in the initial activation of CD8+ T cells.

[0141] Based on the above results, this study further investigated the inhibitory effect of the reduction in KikGR erythrocyte count on the initial activation of CD8+ T cells after blocking with a functional blocking antibody against CCR7. The specific experimental method was as follows: 1 x 10^6 CFSE-labeled OTI CD8+ T cells were adoptedively transferred to each C57BL / 6 mouse via tail vein. 24 hours later, 10 μg of anti-CCR7 blocking antibody was subcutaneously injected near the skin drainage lymph node area; the control group received 10 μg of the same type antibody. Then, 3 hours later, CR108+OVA, MF59+OVA, or PBS were inoculated at the same antibody injection site. The proliferation of OTI CD8+ T cells was assessed 3 days after immunization. Figure 7 (D in the text). The results showed that whether immunized with CR108+OVA or MF59+OVA, blocking the migration of Mig cDCs to dLN led to the proliferation of OTI CD8+ T cells (CFSE). lo The number of cells was significantly reduced. Figure 7 (E and F in the text). This result clarifies that the reduced recruitment of Mig cDCs in the dLN inhibits the initial activation and proliferation of CD8+ T cells, indicating a direct causal relationship between the recruitment of Mig cDCs in the dLN and the initial activation of CD8+ T cells. Consistent with previous observations, CFSE was observed in the spleen and non-draining lymph nodes, regardless of CCR7 blockade. lo The number of OTI CD8+ T cells is very low. Figure 7 In the figure, G and H represent the groups on the horizontal axis. Figure 7 (The F in the text is the same). This spatial distribution characteristic of OTI CD8+ T cells further illustrates that Mig cDCs in draining lymph nodes play a key role in the initial activation of CD8+ T cells.

[0142] 2.3 KikGR erythrocytes play a key role in anti-tumor immune responses.

[0143] Based on the above results, this study further investigated the inhibitory effect of the reduction in KikGR red blood cell count after blocking with a functional blocking antibody against CCR7 on anti-tumor immunity. The results are as follows: Figure 8 The results showed that CCR7 blockade significantly suppressed the antitumor effect compared to the isotype control. This indicates that the number of Mig cDCs in draining lymph nodes plays a crucial role in antitumor immunity.

[0144] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for quantitatively analyzing the strength of an immune response promoted by an adjuvant, characterized by, The method comprises the following steps: First, the skin of a KikGR transgenic phototransformed mouse is irradiated with ultraviolet light, so that the KikGR protein of the irradiated skin is converted from green fluorescence to red fluorescence, thereby realizing tracing of skin-derived dendritic cells; then, a control adjuvant combined antigen and a test adjuvant combined antigen are respectively injected subcutaneously into the red fluorescent skin region of the KikGR mouse; finally, the draining lymph nodes of the mouse are isolated after 6-120 hours of immunization, and the number of KikGR red fluorescent cells recruited by the control adjuvant combined antigen is taken as a control index, and the number of KikGR red fluorescent cells recruited by the test adjuvant is compared with the control index, so as to predict the strength of the immune response of the test adjuvant; The control adjuvant is an adjuvant capable of enhancing the strength of the immune response. Preferably, the draining lymph nodes of the mouse are isolated after 24-48 hours of immunization.

2. The method of claim 1, wherein, The immune response comprises one or more of a humoral immune response and a cellular immune response. Preferably, the humoral immune response comprises one or more of promoting CD4+ T cell activation, proliferation, long-term memory, CD4+ Tfh cell differentiation and helper function to B cells, promoting B cell activation, high-frequency mutation, class switching, long-term memory, enhancing the level of binding antibodies and neutralizing antibodies, and promoting the protective effect against infection. Preferably, the cellular immune response comprises one or more of promoting CD8+ T cell activation, proliferation, killing function, long-term memory, promoting CD4+ T cell differentiation to Th1 and helper function to CD8+ T cells, and promoting the protective effect against infection and the effect against tumor.

3. The method of claim 1, wherein, The power of the ultraviolet light irradiation is 100-400 mW / cm 2 , the irradiation time is 1-10 min; and the wavelength of the ultraviolet light is 405-436 nm.

4. The method of claim 1, wherein, The skin comprises back skin and / or abdominal skin.

5. The method of claim 1, wherein, The area of the skin of the KikGR transgenic phototransformed mouse irradiated with the ultraviolet light is 2-6 cm 2 .

6. The method of claim 1, wherein, The control adjuvant comprises one or more of an aluminum adjuvant, a squalene emulsion, a TLR agonist adjuvant, a STING agonist adjuvant, and a cytokine adjuvant. Preferably, the squalene emulsion comprises MF59; the TLR agonist adjuvant comprises one or more of CpG, R848, MPLA, and poly I:C; the STING agonist adjuvant comprises cGAMP; and the cytokine adjuvant comprises GM-CSF.

7. The method of claim 1, wherein, The antigen comprises one or more of a model antigen, a tumor antigen, and a pathogenic microorganism antigen.

8. The method of claim 1, wherein, The draining lymph nodes comprise inguinal lymph nodes and / or superficial cervical lymph nodes.

9. The method of claim 1, wherein, The number of KikGR red fluorescent cells recruited in the draining lymph nodes is counted by flow cytometry.

10. Use of the method for quantitatively analyzing the strength of an immune response promoted by an adjuvant according to any one of claims 1-9 in adjuvant screening.

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