CN112331344A

CN112331344A - Immune state evaluation method and application

Info

Publication number: CN112331344A
Application number: CN202011262746.1A
Authority: CN
Inventors: 林莉娅; 王谢
Original assignee: Shenzhen Fanyin Medical Co Ltd
Current assignee: Shenzhen Fanyin Medical Co Ltd
Priority date: 2020-11-12
Filing date: 2020-11-12
Publication date: 2021-02-05
Anticipated expiration: 2040-11-12
Also published as: CN113707316B; CN113707316A; CN112331344B

Abstract

The invention relates to an immune state evaluation method and application, and belongs to the technical field of immune evaluation. The method comprises the following steps: immune repertoire analysis: extracting a target sample genome to be evaluated, and constructing an immune repertoire library; performing high-throughput sequencing, analyzing the clone diversity of TCR and/or BCR, and obtaining an immune repertoire index value; and (3) immune function analysis: taking a target blood sample to be evaluated, dividing the target blood sample into a control sample and a stimulation sample, adding a stimulus into the stimulation sample to enable immune cells in the stimulation sample to generate an immune response reaction, detecting the expression difference of immune components before and after stimulation, and analyzing to obtain an immune function score; and (3) data analysis: and synthesizing the immune repertoire analysis result and the immune function analysis result to obtain an immune age, and obtaining the immune state of the target to be evaluated according to at least one of the immune age, the immune function score and an immune repertoire index value. The method combines a high-throughput immune repertoire sequencing technology and an immune function detection method, and comprehensively evaluates the immune state from different layers.

Description

Immune state evaluation method and application

Technical Field

The invention relates to the technical field of immune evaluation, in particular to an immune state evaluation method and application.

Background

Human beings are everywhere and are not surrounded by microorganisms, and microorganisms are everywhere. When a certain infectious disease is epidemic, a part of people is infected and sometimes spread, and a tendency of epidemic is formed. However, not all people are infected, and some people do not develop diseases even if infected, reflecting that this part of the human body forms the ability to resist foreign substances, which is called immune status.

The immune system is the material basis of the body for taking charge of immune function, and comprises three parts of immune organs, immune cells and immune molecules. Its main functions are defense, monitoring and elimination, and are divided into two categories, non-specific immunity and specific immunity. The immune system has two sides, the normal working immune system can keep the healthy operation of all aspects of functions of the organism, and when the immune system is damaged or out of control, the immune state of the organism is reduced, and people are easy to suffer from diseases. Therefore, the immune balance is important, and how to accurately and scientifically evaluate the immune state is an important technology.

The traditional immunological detection method for evaluating the immune state comprises the following steps: five items of immunity, namely detection of immune function. And detecting the content of the immunoglobulin and the complement in the blood. ② blood routine, a cell counting method is used for analyzing the quantity of white blood cells in peripheral blood, and the increase of the quantity of white blood cells indicates that inflammation reaction exists in vivo. Checking the positive rate of antibody by enzyme-linked immune method or immunofluorescence method, the positive rate of different antibody is related to the occurrence of various autoimmune diseases. Fourthly, infection immunity detection, namely detection of antibodies related to specific bacteria and viruses. Fifthly, the immune state is indirectly evaluated through factors influencing the immune function, such as diet, living habits, mental health and the like. At present, most of detection institutions of partial hospitals and third parties can know the number and proportion of various immune cell populations through flow cytometry sorting. However, the conventional immunological detection methods have certain limitations, and cannot comprehensively evaluate the immune state.

In recent years, methods for studying immune and health diseases at a molecular level by using an immune repertoire high-throughput sequencing technology are also widely used. The Immune Repertoire (IR) is defined as the sum of the T lymphocyte surface receptor (TCR) and the B lymphocyte surface receptor (BCR) of the adaptive Immune system of the body at any particular time. During the T/B cell maturation process, VDJ gene rearrangement occurs, V, D and J gene are randomly selected and connected in series during the rearrangement process to form a variable region of TCR/BCR, and base random insertion and deletion can occur at a junction part, so that almost every newly generated TCR/BCR gene of T/B cells is different from each other to form a huge TCR/BCR immune repertoire, and the organism is endowed with the capability of recognizing various antigens. And the encoding gene of the TCR/BCR is analyzed through high-throughput sequencing, so that the immune repertoire can be decoded and used for analyzing the diversity and specificity of the immune system.

With age, lymphocyte diversity decreases, resulting in decreased immune function. Excessive internal and external environmental stresses and diseases can lead to excessive and premature consumption of the immune system, resulting in significantly less immune diversity than a peer.

Therefore, a method for precisely evaluating the immune status is needed, which can precisely find a way to maintain the normal operation of the immune system based on fully understanding the immune status, so as to better protect against the occurrence of diseases.

Disclosure of Invention

In view of the above, there is a need for an immune status assessment method for non-diagnostic therapeutic purposes, which comprehensively assesses the ability of an individual to resist disease, starting from the combination of immune response function and antigen recognition ability detection.

An immune status assessment method for non-diagnostic therapeutic purposes, comprising:

immune repertoire analysis: taking a biological sample of a target to be evaluated, extracting genomic DNA or RNA from the biological sample, and constructing an immune repertoire library; performing high-throughput sequencing, analyzing the clone diversity of TCR and/or BCR, and obtaining an immune repertoire index value;

and (3) immune function analysis: taking a blood sample of a target to be evaluated, dividing the blood sample into a control sample and a stimulation sample, taking the stimulation sample, adding a stimulant into the stimulation sample to enable immune cells in the stimulation sample to generate immune response reaction, then respectively detecting the expression difference of immune components in the control sample and the stimulation sample before and after stimulation, and analyzing to obtain an immune function score;

and (3) data analysis: and synthesizing the immune repertoire analysis result and the immune function analysis result to obtain the immune age of the target to be evaluated, and obtaining the immune state of the target to be evaluated according to at least one of the immune age, the immune function score and the immune repertoire index value.

In the previous investigation, the complex function of the immune system needs to maintain balance of various immune approaches and modes to achieve a better immune state, and the conventional evaluation methods cannot comprehensively and comprehensively evaluate the immune system. Although there are also methods for evaluating immune function by detecting a stress immune response, immune function is only a part of the intensity of immune state; there are also partial evaluations by the magnitude of the vaccine immune response, the influence of immune cells and immune molecules. However, there is no protocol for comprehensive assessment of immune status.

Therefore, the present inventors have conducted extensive studies on the functional mode of the immune system and have proposed that the ability of individuals to resist diseases can be obtained by comprehensively evaluating the systemic immune state from the cellular level and the molecular level by combining the antigen recognition ability and the immune response function, respectively.

The immune state evaluation method is combined with a high-throughput immune repertoire sequencing technology and an immune function detection method to evaluate the immune state from different layers. On one hand, an immune repertoire library is constructed for sequencing, and the clone diversity of TCR or BCR is analyzed; on the other hand, the immune cell is stimulated to generate immune response by stimulating the sample, and the immune factor change before and after stimulation is compared, so that the immune level of the organism is comprehensively evaluated.

It will be appreciated that the types of biological samples used for the immunohistochemical library analysis described above may be: blood or its components (including whole blood, PBMC or lymphocytes, etc.), tissues, etc. need only be capable of extracting the genome therefrom for analysis.

The sample for immune function analysis needs to adopt a blood sample or a blood sample composition (comprising whole blood, PBMC or lymphocytes and the like) because immune cells in the sample are required to generate immune response, and has high practical value in terms of convenient sampling and detection analysis and evaluation by peripheral blood.

In one embodiment, in the data analysis step, the immune status of the target to be evaluated is preferably analyzed with an immune age and/or an immune function score.

In one embodiment, in the step of analyzing immune function, the immune component is mRNA of each immune cell obtained by immune cell typing, RNA in the control sample and the stimulated sample is extracted respectively, an RNA-seq library is constructed, and high-throughput sequencing is performed to obtain the expression difference of mRNA before and after stimulation of each type of immune cell in the control sample and the stimulated sample.

The mRNA used for counting the expression difference of the immune components is selected according to the influence on the immune function, for example, the mRNA can be selected according to the types of different mRNAs and corresponding different types of immune cells by referring to the annotation of Nextseq software.

It can be understood that the immune component can also be protein, that is, the immune protein is taken as a detection target, and the purpose of evaluating the immune function of the body can be realized by directly analyzing the difference of the expression amount of the immune protein before and after stimulation through proteomics.

In one embodiment, in the step of analyzing immune function, each immune component is defined as an immune molecule, and the normalized value of each immune molecule is calculated according to the following formula:

normalized value x-min/(max-min)

In the formula: x is the expression value of the immune molecule of the target to be evaluated;

the minimum value is the minimum value of the expression quantity of the immune molecules in a population sample;

the maximum value is the maximum value of the expression quantity of the immune molecules in a population sample;

and calculating the immune function score related to the stimulation according to the stimulation type and the following formula:

in the formula: n is the number of immune molecules counted under the stimulation type, g is the normalized value of the immune molecules i after normalization, and alpha is the weight.

Because the expression quantities of different immune molecules have differences and cannot be compared at the same level, in the analysis, the expression quantities of the immune molecules are normalized firstly, so that the different immune molecules are compared at the same order of magnitude, and the evaluation value is more comprehensive and accurate.

Furthermore, each immune molecule can be further converted into an immune function score through the formula, so that the immune state can be evaluated more intuitively. Wherein, the stimulation type is bacteria, virus or the type generating mucosal immune stimulation source, and the like, and the importance degree of different immune molecules in the comprehensive immune function can be reflected by adjusting the weight alpha, so that the overall immune state can be more accurately evaluated.

It is understood that, due to the individual differences of the organisms, in order to reduce the influence of the individual differences on the evaluation result, the population samples (such as not less than 50 cases) are tested to obtain the universal range of the immune molecules in the population, so that the maximum and minimum values in the population samples are corrected, thereby further providing the evaluation accuracy.

In one embodiment, α is obtained by: the correlation coefficient or linear fitting value of the ratio of the normalization value obtained before and after stimulation of certain immune molecules in the healthy population sample and the real age is used as the weight of the index.

It is understood that the healthy population is determined according to general medical evaluation criteria and can be adjusted according to specific situations.

In one embodiment, the stimulation type refers to: simulating at least one of a bacteria-related stimulus, a virus-related stimulus, a T cell activation stimulus.

In one embodiment, the immune molecule comprises: at least one of IL1B, IL6, CXCL8, CCL19, CCL20, CD14, CD36, IL12B, MAPK3, RPS6KA1, CCL24, TLR1, MAPKAPK3, TLR6, ZBTB7B, IL23A, TLR A, SIGIRR, LOXL A, PELI A, IL A, AHR, CD A, DUSP A, NCKAP A, IFNB A, IL12A, IRF A, MEF 2A, MAP2K A, RELA, STAT5A, TLR A, IL 17A, TIRAP, CXCL A, IRF A, XCL A, RIPK A, TWSG A, NLRP A, FOS, if3672, ifs, if3672, SOCS A, vscs A, VSIR A, and vsml A.

It can be understood that the immune molecules which can reflect the most influential immune function can be extracted according to the influence degree of immune components on the immune function, and the inventor finds that the immune function can be estimated by the immune molecules after experimental screening and repeated verification, so that the immune level of an organism can be accurately and comprehensively reflected.

In one embodiment, in the step of analyzing immune function, the stimulus is a representative substance selected from a group consisting of a stimulus simulating at least one of a viral stimulus, a bacterial stimulus and a T cell activation stimulus.

It will be appreciated that the above mentioned stimuli can be selected from substances disclosed in the conventional art which are capable of representing different types of immune stimulation.

In one embodiment, the immune function analysis step, wherein the stimulus that mimics viral stimulation comprises: TLR7/8, 3 pRNA; stimuli that mimic bacterial stimulation include: TLR 4; stimuli that mimic T cell activation stimulation include: at least one of Anti-CD3, Anti-CD28, Phytohemagglutinin (PHA), concanavalin A (ConA), pokeweed mitogen (PWM), Staphylococcus aureus enterotoxins A-E (SEA-E), Extoxin (EXT), Mycoplasma Arthritides Mitogen (MAM), Yersinia enterocolitica membrane proteins, and protein products of mouse retroviruses. The stimulators are matched, so that most of immune stimulation sources in a real environment can be simulated well and comprehensively, and the immune response function of an organism after stimulation can be evaluated accurately and objectively.

In one embodiment, in the step of analyzing the immune repertoire, the data analysis step calculates the immune age according to the following method:

in the formula: v1 is an index value of the immune repertoire, m is the number of indexes, W1 is the corresponding weight of each index of the immune repertoire, V2 is the score of immune function, n is the number of indexes, W2 is the corresponding weight of each index of the immune function, and beta is the proportion of the immune repertoire;

the immune repertoire indexes include: at least one of VJ gene usage diversity, immune cell type and immune cell homogeneity;

the VJ gene usage diversity was calculated by the following method: solving the aroma index of all V-J pairings and frequencies thereof in TCR and/or BCR obtained by analyzing the immune repertoire, namely obtaining the use diversity value of the VJ gene;

the immune diversity was calculated by the following method: calculating the aroma index of all TCR and/or BCR clones and frequencies obtained by analyzing the immune repertoire, wherein the aroma index is the immune diversity value;

the immune cell species were calculated by the following method: taking the number of all TCR and/or BCR clone varieties obtained by immune repertoire analysis as the value of immune cell varieties;

the immune cell homogeneity is calculated by the following method: all TCR and/or BCR clones obtained by immune repertoire analysis and frequency are subjected to a Cheney index calculation, and the Cheney index is an immune cell homogeneity value.

The fragrance concentration index, the King's index and the like are calculated according to a conventional method.

The immune age is an index for integrating immune repertoire and immune function, and is obtained by performing integrated immune state evaluation on individuals, wherein the immune age is lower than or equal to actual age, which indicates that the immune system is normal, and conversely, indicates that the immune state is abnormal.

In one embodiment, in the data analysis step,

w1 was obtained by the following method: taking the correlation coefficient or linear fitting value of the real age and the index as the weight of the index;

w2 was obtained by the following method: taking the correlation coefficient or linear fitting value of the real age and the index as the weight of the index;

beta means the proportion of immune repertoire that affects the immune system.

Through the adjustment of the method, the immune age and the immune state can be more accurately evaluated. The combination of the immune repertoire and the immune function is more accurate than unilateral evaluation, and the two are equally important and reflect different aspects of an immune system, for example, the immune repertoire reflects the antigen recognition capability and the immune function reflects the cellular immune response capability.

In one embodiment, the immune status assessment method for non-diagnostic therapeutic purposes further comprises the step of establishing a reference database, wherein the reference database is established by the following steps: and selecting a whole blood sample of the healthy population, performing immune repertoire analysis and immune function analysis according to the method, and performing data analysis to obtain immune repertoire reference data and immune function reference data of the healthy population, namely obtaining a reference database.

The reference database is established based on immune group database data and immune function reference data of healthy people in all ages, so that an immune potential evaluation model based on the immune group database can be established, the immune potential evaluation model is compared with data of healthy people, the immune state is accurately evaluated, the sub-health state is prompted, and the individual disease risk can be further evaluated.

The invention also discloses application of the immune state assessment method in preparing a reagent and equipment for immune state assessment.

It will be appreciated that the apparatus described above may comprise an integrated detector or a combination of apparatus providing each functional module separately.

The invention also discloses an immune state evaluation system, which comprises:

the immune repertoire analysis module is used for analyzing and obtaining an immune repertoire index value, and the immune repertoire index value is obtained by the following method: taking a biological sample of a target to be evaluated, extracting genomic DNA or RNA from the biological sample, and constructing an immune repertoire library; performing high-throughput sequencing, analyzing the clone diversity of TCR and/or BCR, and obtaining an immune repertoire index value;

the immune function analysis module is used for analyzing and obtaining an immune function score, and the immune function score is obtained by the following method: taking a blood sample of a target to be evaluated, dividing the blood sample into a control sample and a stimulation sample, taking the stimulation sample, adding a stimulant into the stimulation sample to enable immune cells in the stimulation sample to generate immune response reaction, then respectively detecting the expression difference of immune components in the control sample and the stimulation sample before and after stimulation, and analyzing to obtain an immune function score;

a data analysis module: and the immune state of the target to be evaluated is obtained according to at least one of the immune age, the immune function score and the immune repertoire index value.

In one embodiment, the immune status evaluation system further comprises a reference database module, wherein the reference database module is used for storing healthy population immune group library reference data, and the healthy population immune group library reference data is obtained by the following method: and (3) selecting a whole blood sample of the healthy population, and performing immune repertoire analysis and immune function analysis according to the method to obtain immune repertoire reference data of the healthy population.

In one embodiment, in the immune function analysis module, the immune component is mRNA of each immune cell obtained by immune cell typing, RNA in the control sample and the stimulated sample is extracted respectively, an RNA-seq library is constructed, and high-throughput sequencing is performed to obtain expression difference of mRNA before and after stimulation of each type of immune cells in the control sample and the stimulated sample.

In one embodiment, the immune function analysis module defines each immune component as an immune molecule, and calculates the normalized value of each immune molecule according to the following formula:

normalized value x-min/(max-min)

In one embodiment, the immune function analysis module comprises a stimulator that simulates stimulation by a virus: TLR7/8, 3 pRNA; stimuli that mimic bacterial stimulation include: TLR 4; stimuli that mimic T cell activation stimulation include: at least one of Anti-CD3, Anti-CD28, Phytohemagglutinin (PHA), concanavalin A (ConA), pokeweed mitogen (PWM), Staphylococcus aureus enterotoxins A-E (SEA-E), Extoxin (EXT), Mycoplasma Arthritides Mitogen (MAM), Yersinia enterocolitica membrane proteins, and protein products of mouse retroviruses.

In one embodiment, in the immune repertoire analysis module, the data analysis step calculates the immune age according to the following method:

beta means the proportion of immune repertoire that affects the immune system.

Compared with the prior art, the invention has the following beneficial effects:

the immune state evaluation method for non-diagnostic treatment purposes combines a high-throughput immune repertoire sequencing technology and an immune function detection method to evaluate the immune state from different layers. On one hand, an immune repertoire library is constructed for sequencing, and the clone diversity of TCR or BCR is analyzed; on the other hand, the immune cell is stimulated to generate immune response by stimulating the sample, and the immune factor change before and after stimulation is compared, so that the immune level of the organism is comprehensively evaluated.

The method can comprehensively evaluate systemic immune state by combining immune response function and antigen recognition ability from cell level and molecular level, respectively, and obtain individual disease resistance ability, and has the advantages of comprehensive and accurate evaluation.

Drawings

FIG. 1 is a schematic flow chart of the immune status assessment method in example 1;

FIG. 2 is a circular histogram of the evaluation of the anti-bacterial immune function of the volunteers in example 2;

FIG. 3 is a circular histogram of the evaluation of the antiviral immune function of the volunteers in example 2;

FIG. 4 is a circular histogram of the evaluation of the T cell activation function of the volunteers in example 2;

FIG. 5 is a scatter plot of the results of the volunteer immune function assessment in example 2;

FIG. 6 is a circular histogram of the evaluation of the anti-bacterial immune function of the volunteers in example 3;

FIG. 7 is a circular histogram of the evaluation of the antiviral immune function of the volunteers in example 3;

FIG. 8 is a circular histogram of the evaluation of the T cell activation function of the volunteers in example 3;

FIG. 9 is a scattergram of the results of the evaluation of immune function of volunteers in example 3;

FIG. 10 is a schematic diagram showing the immunological age assessment of the general population at different ages in example 5.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The starting reagents used in the following examples were all commercially available unless otherwise specified.

Example 1

An immune state assessment method is realized by combining a high-throughput immune repertoire sequencing technology and an immune function detection technology, the flow of the immune state assessment method is shown in figure 1, and the immune state assessment method comprises the following steps:

first, immune repertoire analysis.

Taking a whole blood sample of a target to be evaluated, drawing 5ml of peripheral blood for later use by vein, taking 1ml of whole blood as an immune repertoire analysis sample, extracting genome (DNA or RNA) in the sample, and constructing the immune repertoire library; high-throughput sequencing is performed, clonal diversity of TCR and/or BCR is analyzed, and immune cell typing is performed, for example, IMonitor data analysis software can be used to obtain data such as lymphocyte variety number and the like.

And secondly, analyzing immune function.

Taking a whole blood sample of a target to be evaluated, dividing the whole blood sample into a control sample 1ml and a stimulation sample 3ml, taking the stimulation sample, adding a stimulant shown in the following table, performing simulated stimulation to enable immune cells in the stimulation sample to generate immune response reaction, then culturing at 37 ℃ for 6h, then collecting cells before and after stimulation, performing RNA extraction, constructing an RNA-seq library, performing high-throughput sequencing, performing expression profile analysis related to immune response after sequencing, and thus detecting and obtaining the expression difference of immune components in the control sample and the stimulation sample before and after stimulation respectively, and analyzing to obtain an immune function score.

TABLE 1 simulated stimulation protocol

In this example, the transcriptional group data was analyzed to classify immune cells, and the immune components were mrnas of each immune cell obtained by classifying immune cells, and qualitative and quantitative analysis of mRNA expression differences before and after stimulation of each type of immune cells was performed to determine the signal pathways affecting immune functions.

Specifically, each immune component is defined as an immune molecule, and the normalized value of each immune molecule is calculated according to the following formula:

normalized value x-min/(max-min)

In the formula: x is the expression quantity of the immune molecules of the target to be evaluated;

and calculating the immune function score related to the stimulation according to the expression difference of immune molecules under the experimental conditions of the stimulation types such as bacteria-related stimulation, virus-related stimulation and T cell activation stimulation according to the following formula:

in the formula: n is the number of immune molecules counted under the stimulation type, g is the normalized value of the immune molecules i after normalization, and alpha is weight;

in this example, the following immune molecules were selected for evaluation: IL1B, IL6, CXCL8, CCL19, CCL20, CD14, CD36, IL12B, MAPK3, RPS6KA1, CCL24, TLR1, MAPKAPK3, TLR6, ZBTB7B, IL23A, TLR A, SIGIRR, LOXL A, PELI A, IL A, AHR, CD A, DUSP A, NCKAP A, IFNB A, IL12A, IRF A, MEF 2A, MAP2K A, RELA, STAT5A, TLR A, IL A, TIRAP 17A, CXCL A, IRF A, XCL A, RIPK A, NLRP A, FOS, IF3672, SOCS A, VSIR A, JAML A, TWSP A.

α is obtained by the following method: the correlation coefficient or linear fitting value of the ratio of the normalization value obtained before and after stimulation of certain immune molecules in the healthy population sample and the real age is used as the weight of the index.

Namely, according to different stimulation types, an antibacterial immune function score, an antiviral immune function score and a T cell activation function evaluation value are obtained.

And thirdly, analyzing data.

And synthesizing the immune repertoire analysis result and the immune function analysis result to obtain the immune age of the target to be evaluated, and obtaining the immune state of the target to be evaluated according to the immune age and/or the immune function score to obtain the evaluation results of each index of the antiviral function, the antibacterial function and the immune defense line (T cell activation stimulation) function.

In this example, the immunization age was calculated as follows:

The V2 includes the antibacterial immune function score, the antiviral immune function score, the T cell activation function evaluation value, and the like calculated above.

The above weight W1 is obtained by: taking the correlation coefficient or linear fitting value of the real age and the index as the weight of the index; the weight W2 is obtained by: taking the correlation coefficient or linear fitting value of the real age and the index as the weight of the index; beta means the proportion of immune repertoire that affects the immune system, and in this example, it is considered that immune repertoire analysis and immune function analysis are equally important, and alpha is set to 0.5.

The immune age comprehensive immune repertoire and the immune function index are obtained by comprehensively evaluating the immune state of an individual, wherein the immune age is lower than or equal to the actual age, which indicates that the immune system state is normal, and conversely, indicates that the immune state is abnormal.

Example 2

An application example of the immune state assessment method.

1. Sample collection

An informed consent was signed with one volunteer, 5ml venous blood was drawn with EADT tube on the premise of fasting without any discomfort and disease in one month, and the tube was inverted up and down several times to prevent clotting.

2. Sample processing

1ml of whole blood was taken into a 2ml EP tube, one portion was used for DNA extraction to construct an immune repertoire (in this example, an immune repertoire library was constructed using DNA), and the other portion was used for RNA extraction to construct a transcriptome library (control sample), and the amount of mRNA expression before stimulation was analyzed. The remaining 3ml of whole blood (stimulated sample) is preferentially processed and needs to maintain cellular viability.

It should be noted that in order to maintain the cell activity and to perform the immune response, the test should be performed immediately after the blood drawing, and if the test cannot be performed immediately, the cell activity can be maintained by freezing with liquid nitrogen and revived with time.

3. Whole blood simulated stimulation

Protocols that mimic the stimulation of the immune system to produce non-specific and specific immune responses are shown in the table below. A stimulation mixture is prepared by using stimulators TLR7 (TLR), TLR4 and Anti-CD3/CD28, then is added into an EDTA tube in which a whole blood sample is stored for stimulation culture, and is cultured for 6 hours at 37 ℃.

TABLE 2 specific simulated stimulation protocol

4. RNA extraction

Whole Blood before and after stimulation was subjected to RNA extraction using HiPure Blood RNA Mini Kit (R4161-01, magenta), and the procedures were carried out according to the instructions of the magenta-compatible extraction Kit. Used for immune function analysis.

5. DNA extraction

1ml of whole Blood collected in a 2ml EP tube was extracted with HiPure Blood DNA Midi Kit I (D3112-02, magenta) according to the instructions of the magenta extraction Kit. For performing immune repertoire analysis.

6. Library construction

6.1 transcriptome library construction

1000ng of RNA before and after stimulation were taken, and a transcriptome Library was constructed according to the standard protocol of TruSeq Stranded mRNA Library Prep Kit (RS-122-2101, Illumina).

6.2 construction of immune repertoire library

1.2 μ g of DNA was initiated to create a library of TCR β chains (TRBs) and the TRB VJ regions were captured and enriched by multiplex PCR, as described in the prior art (Systematic Comparative Evaluation of Methods for Investigating the TCR β region, DOI:10.1371/journal. pole.0152464).

7. Sequencing computer

The 3 libraries constructed above were placed in a NextSeq^TMThe 550Sequencing System is hosted. Sequencing data of the transcriptome library before and after stimulation are 6G/piece, and the reading length is 2x 75 bp; the sequencing data of the immune repertoire library is 2G/one, and the read length is 2x 100 bp.

8. Data analysis

The transcriptome library was aligned using RNA-Seq Alignment software and expression abundance analysis using RNAEX expression software. Both of these software were used in BaseSpace Sequence Hub (Illumina genomics computing platform).

The sequencing data of the immune Repertoire was processed using IMonitor software, and the Analysis procedure was as described in the prior art (IMonitor: A Robust Pipeline for TCR and BCR repeat Analysis, doi: 10.1534/genetics.115.176735).

9. Immune status assessment result visualization

The sequencing data is processed through an analysis process, and the result data is visualized by ggplot2 in the R language.

9.1 immune repertoire analysis

And (3) processing sequencing data of the immune repertoire by IMonitor software, analyzing the clone diversity of TCR and/or BCR, and parting immune cells to obtain the variety and the number of the lymphocytes.

9.2 analysis of immune function

The data comparison of the transcriptome library is carried out by RNA-Seq Alignment software, expression abundance analysis is carried out by RNAEX express software, and immune function score is obtained by analysis, which is concretely as follows:

defining each immune component as an immune molecule, and calculating the normalized value of each immune molecule according to the following formula:

normalized value x-min/(max-min)

in this example, the following immune molecules were selected for evaluation: IL1B, IL6, CXCL8, CCL19, CCL20, CD14, CD36, IL12B, MAPK3, RPS6KA1, CCL24, TLR1, MAPKAPK3, TLR6, ZBTB7B, IL23A, TLR A, SIGIRR, LOXL A, PELI A, IL A, AHR, CD A, DUSP A, NCKAP A, IFNB A, IL12A, IRF A, MEF 2A, MAP2K A, RELA, STAT5A, TLR A, IL A, TIRAP 17A, CXCL A, IRF A, XCL A, RIPK A, NLRP A, FOS, IF3672, SOCS A, VSIR A, JAML A and VSSP A.

The immune response of the immune molecules against different types of stimuli is shown in FIGS. 2-4, and the circular histograms of FIGS. 2, 3 and 4 show the extent of antibacterial, antiviral and mucosal immunity (T cell activation immunity), respectively. The height of the cylinder represents the ratio of the normalized expression before stimulation to the normalized expression after stimulation.

And calculating the immune function score of the volunteer under different stimulation types according to the immune function score formula.

Wherein α is obtained by the following method: the correlation coefficient or linear fitting value of the ratio of the normalization value obtained before and after stimulation of certain immune molecules in the healthy population sample and the real age is used as the weight of the index.

The results of the immune function assessment of the volunteers of this example are shown in FIG. 5, wherein the immune function scores of the volunteers for antibacterial, antiviral and mucosal immunity (i.e., T cell activation immunity) are 92, 88 and 80, respectively.

It is understood that the degree of the standard antibacterial, antiviral and immunopotentiation (T cell activation stimulation) in the normal state of the population can be evaluated by accumulating the data of the healthy population, and then the antibacterial, antiviral and immunopotentiation (T cell activation stimulation) level of the test subject can be evaluated by detecting the test subject.

9.3 comprehensive evaluation.

The immunization age was calculated as follows:

the immune repertoire indexes include: VJ gene usage diversity, immune cell type and immune cell homogeneity;

The above-mentioned important indexes of the present example are shown in the following table by analyzing the data of the immune repertoire.

TABLE 3 results of immune repertoire indices

And the evaluation result is obtained by establishing a model according to the immune group database data of the healthy population and comparing the model with the threshold range of the reference data.

And calculating the index values of the immune function and the immune repertoire to obtain the immune age of the target to be evaluated, and comprehensively evaluating the immune state by the immune age, wherein the immunological age of the volunteer is 24 years, the actual age is 23 years, and the immunological age is slightly higher than the actual age. The evaluation conclusion is that: the volunteer has weak antiviral ability and low antigen recognition ability, and should be well prevented from epidemic virus infection.

In fact, the volunteer reported low immunity and was susceptible to cold, but healed within a week with seasonal episodes of rhinitis and pharyngitis, consistent with the immune status obtained by the assessment method of this example.

Example 3

An application example of the immune status evaluation method was evaluated according to the method of example 2.

The immune response of the volunteer immune molecules of this example against different types of stimuli is shown in FIGS. 6-8, and the circular histograms of FIGS. 6, 7, 8 show the extent of antibacterial, antiviral and mucosal immunity (T cell activation immunity), respectively. The height of the cylinder represents the ratio of the normalized expression before stimulation to the normalized expression after stimulation.

The result of reanalysis of the volunteer immune function assessment of this example is shown in FIG. 9. Wherein the immune function scores of the volunteer for antibacterial, antiviral and mucosal immunity (i.e. T cell activation immunity) are 93, 92 and 93 respectively.

TABLE 4 results of immune repertoire indices

According to analysis and calculation, the immunological age of the volunteer is 25 years, the actual age is 25 years, and the immunological age is consistent with the actual age. The evaluation conclusion is that: the volunteers had normal immune function.

In fact, the volunteer described no discomfort in the near future, the diet worked more normally, and no cold for 2 years. By detecting the whole body, the method can better cope with the invasion of the external antigens, and suggests to keep good habits, which are consistent with the immune state obtained by the evaluation method of the embodiment.

Example 4

An immune status assessment system comprising: the system comprises an immune repertoire analysis module, an immune function analysis module and a data analysis module.

The immunohistochemical library analysis module is used to analyze the clonal diversity of the TCR and/or BCR, perform immunocytotyping, obtain the number of lymphocyte species, and perform the method steps of example 2.

The immune function analysis module is used for analyzing the obtained immune function score and executing the method steps of the embodiment 2.

And the data analysis module synthesizes the immune repertoire analysis result and the immune function analysis result to obtain the immune age of the target to be evaluated, and analyzes and obtains the immune state of the target to be evaluated according to the immune age and/or the immune function score.

Example 5

By using the specific method of example 2, 100 samples of volunteers were analyzed to obtain immunological age values of the samples, and then divided into groups according to age groups, to obtain immunological age evaluation results of general population of different age groups as shown in fig. 10.

As can be seen from the figure, according to the estimation situation of the immunological age of common individuals in different age groups, the immunological age of most healthy people is close to the actual age, which shows that the estimation method can objectively embody the body immunity level.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. An immune status assessment method for non-diagnostic therapeutic purposes, comprising:

2. The method of claim 1, wherein the immune component in the immune function analysis step is mRNA of each immune cell obtained by immune cell typing, RNA in the control sample and the stimulated sample is extracted respectively, an RNA-seq library is constructed, and high-throughput sequencing is performed to obtain the expression difference of mRNA before and after stimulation of each type of immune cell in the control sample and the stimulated sample.

3. The method for assessing immune status for non-diagnostic therapeutic purposes according to claim 1, wherein in the immune function analysis step, each immune component is defined as an immune molecule, and the normalized value of each immune molecule is calculated according to the following formula:

normalized value x-min/(max-min)

4. The method for immune status assessment for non-diagnostic therapeutic purposes according to claim 3, characterized in that α is obtained by: the correlation coefficient or linear fitting value of the ratio of the normalization value obtained before and after stimulation of certain immune molecules in the healthy population sample and the real age is used as the weight of the index.

5. The method for immune status assessment for non-diagnostic therapeutic purposes according to claim 3, wherein said stimulation type refers to: simulating at least one of a bacteria-related stimulus, a virus-related stimulus, a T cell activation stimulus.

6. The method for immune status assessment for non-diagnostic therapeutic purposes according to claim 4, characterized in that said immune molecule comprises: at least one of IL1B, IL6, CXCL8, CCL19, CCL20, CD14, CD36, IL12B, MAPK3, RPS6KA1, CCL24, TLR1, MAPKAPK3, TLR6, ZBTB7B, IL23A, TLR A, SIGIRR, LOXL A, PELI A, IL A, AHR, CD A, DUSP A, NCKAP A, IFNB A, IL12A, IRF A, MEF 2A, MAP2K A, RELA, STAT5A, TLR A, IL 17A, TIRAP, CXCL A, IRF A, XCL A, RIPK A, TWSG A, NLRP A, FOS, if3672, ifs, if3672, SOCS A, vscs A, VSIR A, and vsml A.

7. The method for immune status assessment for non-diagnostic therapeutic purposes according to claim 1, wherein in said step of immuno-functional analysis said stimuli are selected from representative substances mimicking at least one of the stimuli viral, bacterial and T-cell activation.

8. The method of claim 7, wherein the step of analyzing immune function comprises the step of simulating a stimulus that is stimulated by a virus, wherein the stimulus that is stimulated by a virus comprises: TLR7/8, 3 pRNA; stimuli that mimic bacterial stimulation include: TLR 4; stimuli that mimic T cell activation stimulation include: at least one of Anti-CD3, Anti-CD28, phytohemagglutinin, concanavalin A, pokeweed mitogen, staphylococcal enterotoxins A-E, exfoliative toxins, mycoplasma arthritides mitogen, Yersinia enterocolitica membrane proteins, and mouse retroviral protein products.

9. The method for assessing the immunological status of a subject used for non-diagnostic treatment according to claim 1, wherein in the data analysis step, the immunological age is calculated according to the following method:

10. The method for immune status assessment for non-diagnostic therapeutic purposes according to claim 9, wherein in said data analysis step,

beta means the proportion of immune repertoire that affects the immune system.

11. The method for immune status assessment for non-diagnostic therapeutic purposes according to any of claims 1-10, further comprising a reference database establishment step of: and (3) selecting a whole blood sample of the healthy population, performing immune repertoire analysis and immune function analysis according to the method, and performing data analysis to obtain an immune repertoire of the healthy population and immune function reference data, namely a reference database.

12. Use of the immune status assessment method of any one of claims 1-11 in the preparation of reagents and devices for performing immune status assessment.

13. An immune status assessment system, comprising:

14. The immune status assessment system according to claim 13, further comprising a reference database module for storing healthy population immune repertoire reference data obtained by: and (3) selecting a whole blood sample of the healthy population, and performing immune repertoire analysis and immune function analysis according to the method to obtain an immune repertoire of the healthy population and immune function reference data.