CN116444646A - Peripheral blood TCR marker sequence of systemic lupus erythematosus, and detection kit and application thereof - Google Patents

Peripheral blood TCR marker sequence of systemic lupus erythematosus, and detection kit and application thereof Download PDF

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CN116444646A
CN116444646A CN202211033659.8A CN202211033659A CN116444646A CN 116444646 A CN116444646 A CN 116444646A CN 202211033659 A CN202211033659 A CN 202211033659A CN 116444646 A CN116444646 A CN 116444646A
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tcr
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张志新
杨鑫
治想林
卓越
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Chengdu Exab Biotechnology Co Ltd
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Abstract

The invention discloses a peripheral blood TCR marker of Systemic Lupus Erythematosus (SLE), a detection kit and application thereof, wherein the marker comprises at least one of proteins shown as sequences SEQ ID NO. 1-100. The invention is based on a high-throughput sequencing method, only a small amount of peripheral blood is needed to be taken, RNA is extracted, an immune spectrum library is established through sample treatment, then a characteristic TCR sequence set in SLE peripheral blood is firstly determined through high-throughput sequencing and TCR data analysis, and then a test result of a sample to be tested is compared with the characteristic TCR sequence set, so that whether SLE exists or not is judged. The invention can simultaneously compare a huge number of SLE characteristic TCR sequences, has higher specificity and sensitivity compared with the single detection of one or more markers, and improves the diagnosis efficiency.

Description

Peripheral blood TCR marker sequence of systemic lupus erythematosus, and detection kit and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a peripheral blood TCR marker of systemic lupus erythematosus, and a detection kit and application thereof.
Background
Systemic lupus erythematosus (Systemic Lupus Erythematosus, SLE) is an autoimmune disease that can cause multiple system damage. The exact cause of this disease is not clear, but many studies have shown that genetic, endocrine, infectious, immune abnormalities, and some environmental factors are all associated with the onset of SLE. Under the interaction of various pathogenesis-related factors, the T lymphocytes are reduced, the regulatory T cell inhibition function is reduced, the B cells are hyperproliferative, a large amount of autoantibodies are generated, and the autoantibodies are combined with corresponding autoantigens in the body to form corresponding immune complexes, and the immune complexes are deposited on the parts of skin, joints, small blood vessels, glomeruli and the like.
In the presence of complement, acute and chronic inflammation and tissue necrosis (such as lupus nephritis) are caused, or antibodies directly act on tissue cell antigens to cause cell destruction (such as binding of specific antigens of erythrocytes, lymphocytes and platelet walls with corresponding autoantibodies to cause hemolytic anemia, lymphopenia and thrombocytopenia respectively), thereby causing multisystemic damage to the body.
1. Clinical symptoms of SLE
SLE may have only 1 organ affected manifestation in early stages, or several organ damaged manifestations may occur in succession. The most common early symptoms are fever, fatigue, weight loss, arthritis (pain) and the like, and the more common early symptoms are skin damage, multiple serositis, nephropathy, central nervous system damage, blood abnormality, digestive tract symptoms and the like. At this point the laboratory test may find autoantibodies (especially antinuclear antibodies, ANA) positive. However, because of the clinical manifestation of SLE, which is variable, the onset of the disease is variable, and therefore early diagnosis and treatment opportunities are easily missed.
1) Heating:
more than 85% of patients can develop fever to varying degrees during the course of the disease, some can continue to develop fever for long periods without other symptoms and obvious laboratory findings, but often with positive ANA.
2) Joint muscle symptoms:
more than 90% of patients have joint pain, often leading symptoms. About half of patients may develop myalgia and muscle weakness during the exacerbation period.
3) Skin damage:
skin lesions can occur in 80% of cases, with rashes being the most common and characteristic of SLE. Typical skin lesions are butterfly-shaped erythema that occur on the face, symmetrically distributed across the bilateral cheeks and bridge of the nose. Skin rashes in the extremities are also very specific and often present with characteristic erythema swelling and telangiectasia in the tips of the digits. 20% -30% of patients are allergic to sunlight. Alopecia and mucosal ulcers are common skin lesions of SLE, and occur in active periods of the disease.
4) Hematology:
almost all patients can undergo hematological changes during the course of the disease, with anemia being the most common. About 10% of patients may develop autoimmune hemolytic anemia, often accompanied by splenomegaly, so as to be misdiagnosed as spleen hyperfunction. SLE patients have high incidence of transfusion reactions and severe reaction, often causing irreversible disease deterioration and even death. Leukopenia is also a common hematological manifestation, with both neutrophil and lymphocyte depletion. About 15% of cases have thrombocytopenia, which can be early manifestations, causing severe bleeding. Also, abnormal platelet aggregation is observed, which often causes skin purpura.
5) Renal lesions:
most commonly. Routine kidney biopsies on SLE patients showed almost all kidney lesions, but only half of cases had clinical symptoms. Lupus nephropathy is mainly nephritis and nephrotic syndrome. The kidney function is normal in early stage, and gradually worsens along with the prolongation of the disease course, and uremia can appear in late stage, which is one of the death reasons of SLE.
6) Symptoms of the cardiovascular system:
heart damage is seen in more than 2/3 of the patients, including pericarditis, myocarditis, endocarditis, etc., with pericarditis being the most common. 2% -8% of patients have coronary lesions, which are often manifested as angina pectoris, myocardial damage, heart failure, etc. Lupus endocarditis often causes heart valve lesions, clinically is very misdiagnosed as rheumatic heart disease, but heart failure symptoms worsen and even die after months or years.
7) The respiratory system:
in 50% -70% of patients, pleura, pulmonary parenchyma and pulmonary blood vessels can all be affected, with pleurisy being the most common. The most common pulmonary dysfunction is a decrease in gas diffusion function, and a decrease in lung capacity. The most serious symptoms of SLE pneumonia are persistent alveolar infiltration, onset of sudden disease, clinical manifestations of severe dyspnea, tachypnea, hypoxia and cyanosis, and the mortality rate of lung lesions is extremely high.
8) The digestive system:
it can occur in more than half of cases, manifesting as abdominal pain, especially as lupus crisis, often misdiagnosed as acute abdomen.
9) Neurological symptoms:
more than about 50% of patients, often afflicting the central nervous system, may develop various forms of neuropathy and psychosis. The symptoms of the mental and nervous system may be the first symptoms, but are more common in the middle or late stages of the disease, which are known as lupus encephalopathy or neuropsychiatric lupus erythematosus.
(1) Epilepsy: the incidence rate of one of common central nervous lesions is 15%, and children patients are higher.
(2) Headache: about 10%, associated with the disease activity.
(3) Central nervous system vascular lesions: about 15% of patients are suffering from hemiplegia and aphasia.
(4) Peripheral neuropathy: manifesting as various sensory and motor disturbances, especially those apparent to the lower extremities.
(5) Hyperactivity disorder: it is manifested as chorea, often as first symptoms, and can repeatedly occur.
(6) Intracranial hypertension or aseptic meningitis: the former is manifested as severe headache, vomiting, and the latter as headache, vomiting, and meningeal irritation.
(7) Mental disorder: common in most cases, the first symptom of SLE, but most occur when the condition is exacerbated. The most common mental symptom is depression. Another common mental disorder is schizophreniform manifestation, possibly associated with cerebrovascular inflammation, and organic lesions of the brain.
10 Five sense organs symptoms: eye symptoms are often manifested, with fundus changes being the dominant.
11 Lymph node): SLE often has a varying degree of lymphadenectasis.
12 Lupus crisis): is a worsening manifestation of SLE. It is manifested by high fever, extreme failure and fatigue of the whole body, severe headache and abdominal pain, and often chest pain. There may also be severe damage to the systems such as myocarditis, heart failure and central nervous system symptoms, manifested as seizures, psychosis and coma, with concomitant local infection or sepsis. Renal failure, for example, can lead to death.
SLE has a variable clinical manifestation and often overlaps other rheumatic diseases, and may also overlap other non-connective tissue disease autoimmune diseases, such as thyroiditis, hyperthyroidism, adrenalitis, pernicious anemia, thrombotic thrombocytopenic purpura, chronic gastritis, etc.
2. Existing methods for diagnosing SLE by inspection:
1) Routine inspection
Blood routine examination can find abnormal blood system, such as anemia, leucocyte count decrease, thrombocytopenia, etc.; erythrocyte sedimentation rate (blood sedimentation) increases during SLE activity and decreases to normal during remission.
Urine analysis can find that the kidneys are affected, manifested as proteinuria, hematuria, cellular and granular forms;
2) Immunological examination
50% of patients are associated with hypoalbuminemia and complement levels are often reduced when the disease is active; 30% of SLE patients are associated with hyperglobulinemia, especially elevated gamma globulin, and elevated serum IgG levels during disease activity.
3) Biochemical examination
Liver function in SLE patients is usually marked by mild to moderate abnormalities, and is usually marked by increased alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) during the course of disease. Part of SLE patients have serious blood lipid metabolic disorder, and have raised inflammatory index and hyperhomocysteinemia.
Abnormal numbers of serum albumin suggest decompensation of kidney function. Serum urea nitrogen and serum creatinine help to judge clinical stage and observe treatment effect when lupus nephritis occurs. Urine microalbumin quantitative detection in kidney function examination is helpful for judging and monitoring kidney damage degree and prognosis.
4) Autoantibody detection
The conventional detection items of SLE related autoantibodies developed clinically at present comprise: anti-Sm antibodies (> 90% specific for SLE, sensitivity 20-30%), anti-dsDNA antibodies (95-100% specific for SLE, sensitivity 30-50%), anti-phospholipid antibodies, and the like.
5) Histopathological examination
Both cutaneous lupus with test positive and "Man Tangliang" glomerular manifestations have higher SLE specificity.
3. Diagnostic criteria for SLE:
diagnosis of SLE relies primarily on clinical manifestations, laboratory examinations, histopathology and imaging examinations.
In SLE classification standards revised in 1997 of American rheumatism Association (ACR), laboratory tests such as hematological abnormality, immunological abnormality and autoantibody positive are clearly listed as diagnosis standards, and the method has important significance for diagnosis, differential diagnosis and judgment of activity and recurrence of SLE:
1) Cheekbone erythema: flattened or higher-up skin permanent erythema throughout the cheekbones often does not involve the nasolabial folds.
2) Discoid erythema: the raised erythema is covered with cuticle and follicular embolism, and the old focus may be skin atrophic scar.
3) Light sensitivity: sun exposure causes skin allergy.
4) Canker sore: the oral cavity or the nasopharynx is painless.
5) Arthritis: non-erosive arthritis, which violates 2 or more surrounding joints, characterized by swelling, pain, or fluid permeability of the joints.
6) Serositis: (1) pleurisy: chest pain, pleural fricatives or pleural effusions; (2) pericarditis: an abnormal electrocardiogram, pericardial fricatives or pericardial effusions.
7) Renal lesions: proteinuria >0.5g/d or++; the cell type may be erythrocyte, hemoglobin, particle type or mixed type.
8) Abnormal nervous system: (1) tic (convulsion): non-pharmaceutical or metabolic disorders such as uremia, ketoacidosis or transient electrolyte disorders.
(2) Psychosis: not the case described above.
9) Hematological abnormalities: hemolytic anemia accompanied by reticulocyte increase and leucopenia<4*10 9 L), at least twice; lymphopenia (.ltoreq.1.5×10) 9 L) at least twice; thrombocytopenia (.ltoreq.100×10) 9 /L), except for drug effects.
10 Immunological abnormalities): LE cell positive; positive for anti-dsDNA antibodies; sm antibody positive; syphilis serum test false positive.
11 Anti-nuclear antibody immunofluorescence anti-nuclear antibody positive or other test titer anomalies equivalent to the method, excluding drug-induced lupus.
The 11 indices include 4 or more of them, and SLE can be diagnosed.
SLE diagnostic criteria set forth by the college of rheumatology of the chinese medical community:
1) Butterfly erythema or discoid erythema.
2) Photo allergy.
3) Canker sore.
4) Non-malformed arthritis or joint pain.
5) Serositis, pleurisy or pericarditis.
6) Nephritis proteinuria or tube urine or hematuria.
7) Nervous system injury tics or psychotic symptoms.
8) Abnormal white blood cells of hemogram<4*10 9 /L or platelets<80*10 9 /L or hemolytic anemia.
9) Lupus cells or anti-dsDNA antibodies are positive.
10 Positive for anti-Sm antibodies.
11 Positive for antinuclear antibodies.
12 Positive lupus band test.
13 Complement is less than normal.
The diagnosis can be confirmed according to above 4, but other collagenous diseases, drug lupus, tuberculosis, chronic active hepatitis and the like should be excluded.
The symptoms of fever and rash are identified by dermatomyositis, adult stell disease (AOSD), systemic vasculitis, infectious diseases, neoplastic diseases and the like; the symptoms mainly including arthritis should be identified with rheumatoid arthritis, acute rheumatic fever and the like; the primary glomerular disease is identified by the principal symptoms of kidney involvement.
On the other hand, clinical manifestations are not obvious, but laboratory examination is sufficient to diagnose SLE, and can be temporarily referred to as subclinical SLE. It is sometimes seen that patients have some symptoms that suggest diagnosis of SLE, but fail to meet the classification criteria for 4 ACRs. Ganczarczyk et al classified this patient as latent lupus, with no significant differences in patient-initiated symptoms and laboratory examinations, but patients with a family history of SLE developed SLE. The latent lupus is considered to be a subtype of SLE at present, the clinical manifestation is slight, the involvement of the kidney and the central nervous system is little, and the prognosis is good.
In summary, SLE is an autoimmune disease with unknown pathogenesis, multiple organ involvement throughout the body, and variable course and symptoms. SLE is easily confused with other diseases in many symptoms early in the course of the disease. At present, a laboratory test diagnosis method with higher specificity exists, but the sensitivity of the existing method is not ideal. Thus, there is a need to find a convenient, inexpensive method of assaying for diagnosis of SLE that is simultaneously highly sensitive and specific.
Disclosure of Invention
The invention aims at: aiming at the defects existing in the prior art, the CDR3 sequence of the TCR in the peripheral blood of SLE patients is used as a brand new marker, and a detection kit and application thereof are provided. The method can accurately and rapidly judge whether the sample to be tested suffers from SLE or not in a noninvasive manner.
The technical scheme adopted by the invention is as follows:
a TCR marker in peripheral blood of Systemic Lupus Erythematosus (SLE), the marker being derived from a TCR variable region CDR3 sequence comprising at least one of the protein sequences set forth in SEQ ID nos. 1-100, as set forth in table 1:
TABLE 1 TCR marker sequences characteristic of SLE
Further, the protein sequence of the marker is a protein with the same function after one or more amino acids are substituted, deleted and/or replaced by the sequence shown in SEQ ID NO. 1-100.
The application of the marker in preparation of a preparation for detecting or treating SLE.
Further, the formulation includes a plasmid, viral vector or nucleic acid fragment containing the T cell receptor of the TCR marker.
A kit for SLE detection comprising an antibody capable of specifically binding to a TCR marker as described above.
A formulation comprising an antibody that specifically binds to a TCR marker as described above; the formulation can be used for diagnosis, prognosis, detection or screening of SLE.
A protein chip for detecting SLE (SLE) comprises a substrate and a specific antibody spotted on the substrate, wherein the specific antibody is an antibody capable of specifically binding with the TCR marker.
The principle of the invention is as follows:
b lymphocytes and T lymphocytes in humans are two important classes of cells in the acquired immune system. B cells recognize antigens via cell surface B Cell Receptors (BCR), which are expressed as antibodies and secreted outside the cell when B cells differentiate into plasma cells. T cells recognize antigens through the cell surface T Cell Receptor (TCR). The diversity of BCR and TCR is the basis for the establishment of the adaptive immune system. BCR diversityTheoretical value is 10 18 Theoretical value of TCR diversity is 10 14 . Among BCR and TCR sequences, epitope 3 (CDR 3) is the most important part determining its antigen specificity, and thus the sequence of CDR3 is considered to represent the properties of BCR, TCR sequences.
In various diseases, the diversity or expression levels of BCR and TCR vary with antigen stimulation. Thus, the occurrence and development of diseases can be tracked by using the result of BCR or TCR high-throughput sequencing. In cells in humans, fragments of senescent proteins are transported to the cell surface and presented to T cells in the immune system via histocompatibility antigen II (MCHII). Antigen fragments presented by normal cells do not elicit an immune response due to the tolerogenic relationship. Once normal cells become cancerous, the mutated gene expresses an abnormal protein, a fragment of which is presented to the cell surface, which causes a targeted immune response in the human immune system. Thus, analysis of BCR or TCR changes can detect tumor development and progression.
According to the markers obtained by the invention, peripheral blood TCR sequences are analyzed by high throughput sequencing to detect whether SLE is present, and the specific steps are as follows:
(1) Obtaining TCR sequences in peripheral blood of SLE patients and control groups of a training set and a verification set through high-throughput sequencing, and analyzing and determining CDR3 amino acid sequences of each TCR; removing a TCR sequence of CDR3 sequence less than 8 amino acid residues and greater than 20 amino acid residues in length; ensuring that the total number of functional TCR sequences per sample is no less than 30000;
(2) Randomly sampling the TCR sequences of each sample without replacement so that the sum of the number of the TCR sequences of each sample is 30000;
(3) Data analysis, determining SLE characteristic TCRCDR3 sequence:
a) Summarizing all CDR3 sequences of a training set control group sample, removing weights, and setting the training set control group sample as a control group TCR sequence set;
b) Summarizing and de-duplicating all CDR3 sequences of a training set SLE patient sample, and removing all CDR3 sequences which appear in a control group TCR sequence set, wherein the obtained CDR3 sequences are defined as a SLE characteristic TCR sequence set;
c) The CDR3 sequences present in the two or more training set SLE patient samples in the SLE-characteristic TCR sequence set were calculated for their SLE-characteristic weights according to the following equation (1). Wherein N is X For training set SLE patient sample number comprising sequence X, C i The number of repeated occurrences (expression levels) of sequence X in the ith training set SLE patient sample containing sequence X. These sequences were ranked from high to low by SLE characteristic weights to screen for representative SLE characteristic TCRCDR3 sequences.
(4) determining the risk of developing SLE in the unknown subject using the SLE characterization index:
a) Immune profiles of the control, SLE patients and cross-validation group samples in the training and validation sets were analyzed for SLE characteristic index according to the following formula (2). Wherein M is S C for the class of CDR3 sequences belonging to the TCR sequence set characteristic of SLE contained in a sample S j The number of repeated occurrences (expression levels) of CDR3 sequences within the sample that are characteristic of SLE for the j-th TCR sequence set.
b) When the SLE characteristic index of a subject of unknown health condition is above or near a certain threshold, the person is at higher risk of suffering SLE; if its SLE characteristic index is below the threshold, it indicates a low risk of suffering from SLE.
The beneficial effects of the invention are as follows:
1. in the invention, an analysis model is established by utilizing TCR high-throughput sequencing data of a control group sample without SLE and SLE patients, and a SLE characteristic TCR sequence is determined. By comparing with the SLE characteristic TCR sequences, whether a sample to be tested has a higher SLE risk or not can be clearly judged;
2. the early SLE can be found by analyzing TCR changes through high-throughput sequencing, and the reaction of T cells in the immune system of a human body to SLE is analyzed according to the characteristic TCR sequence of the SLE, so that the method is a novel detection method;
3. in the invention, as the high-throughput sequencing technology is adopted, and simultaneously, a huge number of characteristic TCR sequences are compared, compared with the single detection of one or more markers, the specificity and the accuracy are higher;
4. the high-throughput sequencing instrument used in the invention has lower cost than that of large-scale imaging equipment and can be outsourced to a third party, and in addition, the labor cost of sampling and processing is lower than that of detecting various markers simultaneously and also lower than that of detecting a large number of cytology, so that the invention greatly reduces the detection cost;
5. the invention only needs to adopt a small amount of peripheral blood, is simple and safe to sample, and is a noninvasive test method;
6. the SLE characteristic TCR sequences described in the present invention are also useful in the immunotherapy of SLE.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a Manhattan scatter plot; it demonstrates TCR sequences comparing control samples to SLE patient samples to determine SLE-characteristic TCR sequences and their expression levels. The abscissa represents the unique TCR CDR3 amino acid sequences in the control TCR set and SLE-characterized TCR set, the control TCR set comprising 2.0 x 10 7 The species unique TCR CDR3 sequences, SLE-characterized TCR sequence set comprises 1.4 x 10 6 A species unique TCR CDR3 sequence; the ordinate represents the logarithmic value of the expression level of a certain TCR sequence in a certain sample/group; SLE-characteristic TCR sequences are shown in isolation magnified scale, as indicated by the arrow; A. by comparing TCR sequences in 1527 training set control samples with TCR sequences in 76 training set SLE patients, a determination was madeSLE-characteristic TCR sequences; B. TCR sequences of an exemplary sample of a control group in a training set; C. TCR sequences of an example sample of SLE patients in a training set; D. TCR sequences of an exemplary sample of a control group in a validation set; E. TCR sequences of an example sample of SLE patients in one validation set. All samples had their own manhattan scatter plot analysis;
FIG. 2 is a Manhattan scatter plot; it shows that the species and expression level of SLE-characteristic TCR sequences in SLE patients in training and validation sets are much higher than their corresponding control samples; the abscissa represents the unique SLE-characteristic TCR CDR3 sequences in each sample, and the ordinate is the logarithmic value of the expression level of each SLE-characteristic TCR sequence in that sample. All samples had their own manhattan scatter plot of the SLE characteristic TCR sequence, where 5 representative manhattan scatter plot displays were taken from each set of samples, respectively;
FIG. 3 is a graph comparing SLE characteristic indices; SLE feature index of SLE patient samples in training set (n=76) is significantly higher than SLE feature index of control group samples (n=1527), SLE feature index of SLE patient samples in validation set (n=25) is significantly higher than SLE feature index of control group samples (n=144) and cross validation group samples (n=36) (: p <0.001, mann-Whitney U-test);
FIG. 4 is a graph of SLE characteristic index for control samples and SLE patient samples in a validation set of ROC curve analysis; auc=1, p <0.001, indicating that the sensitivity and specificity of the assay are 100%, and SLE can be accurately determined;
FIG. 5 is a graph of SLE characteristic indices for cross-validated group samples and SLE patient samples in a ROC curve analysis validation set; auc=0.999, p <0.001, indicating that the sensitivity and specificity of the assay is at or near 100% and SLE can be accurately determined.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the particular embodiments described herein are illustrative only and are not intended to limit the invention, i.e., the embodiments described are merely some, but not all, of the embodiments of the invention. Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without making any inventive effort, are intended to be within the scope of the present invention.
The features and capabilities of the present invention are described in further detail below.
Example 1
By aligning TCR sequences of a large number of control samples with SLE patient samples, a set of SLE-characteristic TCR sequences was determined:
1. samples involved in the analysis:
control data for the training set included 1527 samples (1414 healthy people and 113 non-tumor other mild disease patients); SLE data for the training set included 76 samples of SLE patients diagnosed; control data for the validation set included 144 samples (100 healthy people, 44 other mild disease patients who were not tumor); the validated set of SLE patient data includes 25 confirmed SLE patient samples.
Obtaining TCR gene sequences in peripheral blood of each sample by high-throughput sequencing, and analyzing and determining the amino acid sequence of CDR3 of each TCR; removing a TCR sequence of CDR3 sequence less than 8 amino acid residues and greater than 20 amino acid residues in length; ensuring that the total number of functional TCR sequences per sample is no less than 30000; for uniform alignment analysis, each sample was randomly not put back to extract 30000 TCR sequences.
2. Analyzing TCR sequences of a control sample in the training set and a SLE patient sample to determine a set of TCR sequences characteristic of SLE:
by analyzing and comparing TCR CDR3 amino acid sequence data of 1527 control group samples and 76 SLE patient samples in a training set, a SLE characteristic TCR sequence analysis model is established, and a specific TCR sequence of a SLE patient is determined:
1) Summing all TCR CDR3 amino acid sequences of a control sample in a training set to remove weight, defined as a control TCR sequence set, comprising about2.0×10 7 A unique TCR CDR3 amino acid sequence;
2) Summing all TCR CDR3 amino acid sequences from samples from SLE patients in a training set to remove all sequences present in the control TCR sequence set, the resulting TCR sequence defined as a set of TCR sequences characteristic of SLE, comprising about 1.4X10 6 None of the unique TCRCDR3 sequences was present in 1527 control samples.
3) The analysis results are shown as manhattan scatter plots (fig. 1 and 2). FIG. 1A is a graph showing the TCR CDR3 amino acid sequences unique from the control TCR sequence set and the SLE characteristic TCR sequence set, and the ordinate shows the number of repeated occurrences (expression level) C of a certain CDR3 sequence X in a sample X Is a logarithmic value of (2); FIG. 1B is an example of a Manhattan scatter plot of control samples in a training set, showing that none of the control samples in the training set have SLE feature sequences (all 1527 control samples in the training set have similar Manhattan scatter plots); FIG. 1C is an example of a Manhattan scatter plot of SLE patients in a training set, showing SLE samples in the training set having multiple classes and high expression level SLE signature sequences (all of the 76 SLE patient samples in the training set have similar Manhattan scatter plots).
3. Analyzing the TCR sequences of the validation set samples, validating the SLE characteristic TCR sequence set:
1) Analyzing TCR sequences of a validation set sample according to a SLE characteristic TCR sequence analysis model established by the training set sample analysis: the CDR3 amino acid sequence is identical to any one of the set of TCR sequences of the control group determined in the previous analysis, classified as a TCR sequence of the control group, and the CDR3 amino acid sequence is identical to any one of the set of TCR sequences characteristic of SLE determined in the previous analysis, classified as a TCR sequence characteristic of SLE.
FIG. 1D is an example of a Manhattan scatter plot of a control sample of the validation set, in which only a very small number of SLE characteristic TCR sequences are seen, and in which the expression level is very low (all 144 validation set control samples have similar Manhattan scatter plots); FIG. 1E is an example of a Manhattan scatter plot of a validation set SLE patient, which can be seen to contain a plurality of SLE characteristic TCR sequences of higher expression levels (similar Manhattan scatter plots for all 25 validation set SLE patient samples).
2) All samples had manhattan scatter plots of their own SLE characteristic TCR sequences, and fig. 2 shows manhattan scatter plots of 5 representative samples from the training set control group, the training set SLE patient group, the validation set control group, and the validation set SLE patient, respectively. The results show that in the training set, all control samples have no SLE characteristic TCR sequences, and all SLE patients have SLE characteristic TCR sequences with various types and high expression level; in the validation set, all control samples had very few SLE-characteristic TCR sequences, and all SLE patients had multiple types of SLE-characteristic TCR sequences with higher expression levels.
4. 100 representative SLETCR marker CDR3 amino acid sequences were determined:
the TCR CDR3 sequences present in two or more training set SLE patient samples were pooled for SLE-characteristic TCR sequences, and their SLE-characteristic weights were calculated according to the following formula (1). Wherein N is X For training set SLE patient sample number comprising sequence X, C i The number of repeated occurrences (expression levels) of sequence X in the ith training set SLE patient sample containing sequence X. SLE characteristic weights for these sequences were ranked from high to low, with the top-ranked 100 sequences taken as representative SLE characteristic TCR marker sequences (table 1).
Example 2
The type and expression level of SLE characteristic TCR sequences of SLE patients in the training set and the verification set are obviously higher than those of corresponding control samples, so that SLE patients can be accurately diagnosed according to the type and expression level of SLE characteristic TCR sequences of SLE patients in the training set and the verification set:
1. samples involved in the analysis:
1) Samples from example 1:
control data for the training set included 1527 samples (1414 healthy people and 113 non-tumor other mild disease patients); SLE data for the training set included 76 samples of SLE patients diagnosed; control data for the validation set included 144 samples (100 healthy people, 44 other mild disease patients who were not tumor); the validated set of SLE patient data includes 25 confirmed SLE patient samples.
2) Newly adding a sample:
cross-validation set data for validation set included samples of 37 patients with other immune-related diseases other than SLE (IgA nephropathy, alopecia areata, sjogren's syndrome, rheumatoid arthritis).
TCR sequencing data for all samples were analyzed as in example 1: obtaining TCR gene sequences in peripheral blood of each sample by high-throughput sequencing, and analyzing and determining the amino acid sequence of CDR3 of each TCR; removing a TCR sequence of CDR3 sequence less than 8 amino acid residues and greater than 20 amino acid residues in length; ensuring that the total number of functional TCR sequences per sample is no less than 30000; for uniform alignment analysis, each sample was randomly not put back to extract 30000 TCR sequences.
2. The class and expression level of SLE-characteristic TCR sequences for SLE patients in the training set, validation set were significantly higher than for the corresponding control samples:
to compare the types and expression levels of SLE-characterized TCR sequences between different sets of samples, we defined the SLE-characterized index in each sample according to the following equation (2). Wherein M is S C for the class of CDR3 sequences belonging to the TCR sequence set characteristic of SLE contained in a sample S j The number of repeated occurrences (expression levels) of CDR3 sequences within the sample that are characteristic of SLE for the j-th TCR sequence set.
The analysis results are shown in Table 2 and FIG. 3 below. The SLE characteristic index of 1527 control samples in the training set was 0, while the SLE characteristic index of 76 SLE patient samples in the training set was between 5696 and 19084, so that the SLE characteristic index of SLE patients in the training set was significantly higher than that of the control samples in the training set (p <0.001, mann-Whitney U-test).
SLE characterization index for 144 control samples in the validation set was between 3 and 1054, SLE characterization index for other immune related disease samples for the 37 non-SLE cross-validation set was between 73 and 3263, and SLE characterization index for 25 SLE patient samples in the validation set was between 3222 and 14470. Thus, SLE characterization index for SLE patients in the validation set was significantly higher than SLE characterization index for the control group and cross-validation group samples in the validation set (p <0.001, mann-Whitney U-test).
These results demonstrate that SLE characterization index of SLE patient samples is significantly higher in both training and control groups than in control groups (healthy and non-tumor mild disease patients) and other immune related disease patients that are not SLE.
TABLE 2 SLE characterization index for different sample groups in training and validation sets
And 3, accurately judging SLE by using the SLE characteristic index:
1) ROC curve analysis verifies SLE characterization index for concentrated control group samples and SLE patient samples:
ROC curve (Receiver Operating Characteristic Curve, subject work profile) analysis was performed on SLE profile indices of control samples (including healthy 100 and other mild disease patients 44 who are non-tumor, assigned as negative) and SLE patient samples (25, assigned as positive) in the validation set (fig. 4). The results were AUC (area under AreaUnderCurve, ROC curve) =1, p <0.001, indicating that the use of SLE profile index can clearly distinguish between control and SLE patient samples in the validation set.
According to the hierarchical likelihood ratio calculation (table 3), when the cut off value=2138 is taken, SLE specificity and sensitivity are both judged to be 100%, which indicates that when the cut value is taken, SLE characteristic index can be used to accurately distinguish the control group sample from SLE patient sample in the verification set.
Table 3 data of hierarchical likelihood ratios for the SLE feature index ROC curves for the control group samples and SLE patient samples in the validation set
2) SLE characterization index for ROC curve analysis validation set cross validation group samples and SLE patient samples:
ROC curve analysis was performed on SLE characterization indices of the validation set cross validation group samples (37 other immune related disease patients other than SLE, classified as negative) and SLE patient samples (25, classified as positive) (fig. 5). The results were auc=0.999, p <0.001, indicating that cross-validation group samples in the validation set can be clearly distinguished from SLE patient samples using the SLE profile index.
According to the hierarchical likelihood ratio calculation (table 4), when cut off value=2558 (higher than cut value 2138 determined from the verification set control group vsSLE samples before) is taken, the specificity of SLE is 97.3% and the sensitivity is 100%, which indicates that the cross-verification group sample and SLE patient sample can be well distinguished by using SLE characteristic index; when the cutoff value=1864 (lower than cutoff value 2138 determined from the verification set control vsSLE samples above), it is determined that SLE specificity is reduced to 94.6%, sensitivity is still 100%, indicating that taking the cutoff value, the SLE characteristic index can also be used to better distinguish between the cross-verification set samples and SLE patient samples.
Table 4 data of hierarchical likelihood ratios for SLE characteristic index ROC curves for validation set cross validation group samples and SLE patient samples
In conclusion, the SLE characteristic TCR sequence discovered by the invention has obvious SLE specificity, can be used for accurately judging SLE patients, and has high specificity and sensitivity. These SLE-characterized TCR sequences may also be used in the future to predict the risk of a subject for developing SLE and disease progression, or for biological immunotherapy of SLE.
The detailed description of the present invention is presented in detail, but should not be construed as limiting the scope of protection of the present patent. Various modifications, equivalent substitutions and improvements, etc., which may be made by those skilled in the art without the exercise of inventive faculty, are intended to be included within the scope of the present invention.

Claims (7)

1. A peripheral blood TCR marker for systemic lupus erythematosus, wherein the marker is derived from a TCR variable region CDR3 sequence comprising at least one of the protein sequences set forth in SEQ ID nos. 1-100.
2. The peripheral blood TCR marker of systemic lupus erythematosus according to claim 1, wherein the protein sequence of the marker is a protein with the same function after substitution, deletion and/or substitution of one or more amino acids of the sequences shown in SEQ ID nos. 1-100.
3. Use of a TCR marker as claimed in claim 1 or claim 2 in the manufacture of a formulation for detecting or treating systemic lupus erythematosus.
4. The use according to claim 3, wherein the preparation comprises a T cell receptor comprising the TCR marker, or a plasmid, viral vector or nucleic acid fragment capable of expressing the TCR marker.
5. A kit for systemic lupus erythematosus detection comprising an antibody that specifically binds to the TCR marker of claim 1.
6. A formulation comprising an antibody that specifically binds to the TCR marker of claim 1. The preparation can be used for diagnosing, predicting, detecting or screening the systemic lupus erythematosus.
7. A protein chip for detecting systemic lupus erythematosus, comprising a substrate and a specific antibody spotted on the substrate, wherein the specific antibody is an antibody capable of specifically binding to the TCR marker of claim 1.
CN202211033659.8A 2022-08-26 2022-08-26 Peripheral blood TCR marker sequence of systemic lupus erythematosus, and detection kit and application thereof Pending CN116444646A (en)

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