CN103487572B - Application of the folliculus helper T cell (Tfh) in autoimmune disease state of an illness early warning - Google Patents

Abstract

" application of the folliculus helper T cell (Tfh) in autoimmune disease state of an illness early warning " of the invention.There is provided a kind of method that detection effect Tfh cell is used to judge autoimmune disease (RA, SLE, SS) active level.The expression rate of effect Tfh cell has stronger concordance, and direct reaction immunocyte imbalance state with mobility scoring (DAS28, SLEDAI, ESSDAI) of traditional autoimmune disease.The index detection method is easy, stable, is conducive to and guiding the condition assessment of Familiar Rheumatism patient treatment.

Description

Role of follicular helper T lymphocytes (Tfh) in early warning of autoimmune diseases application

technical field

The invention relates to the application of follicular helper T cells in the evaluation of disease activity in patients with rheumatoid arthritis, systemic lupus erythematosus and Sjögren's syndrome.

1. Background technology

Among autoimmune diseases, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS) are the three major immune diseases.

Rheumatoid arthritis is a systemic autoimmune disease characterized by multi-joint erosive synovial inflammation, with a prevalence rate of 0.33%. There are about 5 million patients in my country, and most of them are young and middle-aged women. The residual rate reaches 75%. In the second national sampling survey of disabled people in my country in 2006, disabling joint disease ranked second (20.1%) among the 21 causes of physical disability, second only to cerebrovascular disease (20.6%). 79.4% of them were disabled by RA.

Systemic lupus erythematosus is a diffuse connective tissue disease characterized by immune inflammation. Multiple pathogenic autoantibodies in serum and multisystem involvement are two main clinical features of SLE. In the early stage of SLE patients, clinical symptoms such as low-grade fever, facial rash, fatigue, hair loss, and joint pain often appear. In severe cases, neurological symptoms such as convulsions and syncope occur. The prevalence of this disease in my country is about 70/100,000 people, that is, the number of patients is as high as about 900,000, and the 5-year survival rate is only 50-85%. The prognosis is poor and seriously threatens the quality of life of patients. and society has brought a heavy spiritual and economic burden.

Sjögren's syndrome is a systemic autoimmune disease mainly characterized by dry mouth and eyes and involving multiple systems and organs throughout the body. It is mainly manifested as infiltration and destruction of inflammatory cells in exocrine glands such as salivary glands, lacrimal glands, and digestive tract, leading to progressive dry mouth, dry eyes, and visceral involvement. Due to the decrease or stop of saliva and tear secretion, the mouth and eyes are unbearably dry, cracked lips, rampant dental caries or repeated keratitis, conjunctivitis, parotid gland swelling and vasculitis. Severe patients may be accompanied by severe complications such as pulmonary interstitial fibrosis, pulmonary hypertension, renal tubular acidosis, and central nervous system involvement. The incidence rate of this disease in my country is 0.77%, and about 80 million people are affected nationwide. The incidence of malignant tumors such as lymphoma and myeloma in Sjogren's syndrome can be as high as 40 times that of normal people.

At present, there are still the following deficiencies in the diagnosis and treatment of diseases such as RA, SLE and SS at home and abroad: (1) There is a lack of immunological predictive indicators with high sensitivity and specificity. For example, some RA patients have high serum RF but still have little joint damage after 10 years; on the contrary, some RA patients have negative serum RF or low-titer positive serum RF, and have joint deformities and limited mobility within 3 months of the disease course, seriously affecting quality of life of patients. Therefore, the guiding significance of autoantibodies to treatment is limited, which seriously affects the early treatment of RA. It can be seen that finding indicators of target organ involvement or clinical prognosis with high sensitivity and specificity is of great significance for improving the prognosis of RA treatment. (2) In the disease process of RA, SLE and SS, there are no direct immune indicators to evaluate the disease activity to guide the treatment of medical workers. The current evaluation indicators for disease activity are mainly: SLEDAI score for SLE patients, DAS28 for RA patients, and ESSDAI for SS patients. However, there is a lack of direct indicators to evaluate diseases through changes in immune cells , which is crucial for guiding treatment, regulating abnormal immune levels, and evaluating the efficacy of targeted immunotherapy, and its development is imperative.

A large number of studies in recent years have shown that autoantibodies are mainly produced in the germinal center (GC) of peripheral lymphoid organs. CD4+ helper T (Th) cells assist B cells in GC, and immunoglobulin undergoes affinity maturation and subtype switching in GC. The inventors of this study and foreign researchers have re-recognized a group of CD4+ T cells that are located in the germinal center, stably express CXCR5, and have follicular homing ability after combining with CXCR13, called follicular helper T cells (Tfh). Tfh cells can directly assist B cells in GC, select high-affinity B cells to differentiate into memory cells or plasma cells, and maintain humoral immune response. In the study of human diseases, Simpson et al. found that the proportion of cells expressing CD4 ⁺ CXCR5 ⁺ PD-1 ⁺ or CD4 ⁺ CXCR5 ⁺ ICOS ⁺ in the peripheral circulation of SLE patients increased, and the titers of autoantibodies including anti-dsDNA antibodies It is related to end-organ damage of the kidney, but not to the course of the disease, treatment medication and disease activity. In the study of patients with active hepatitis B, it was also found that the proportion of CD4 ⁺ CXCR5 ⁺ T cells in peripheral blood was higher than that of inactive hepatitis B and healthy controls, and the proportion was positively correlated with serum alanine aminotransferase. The percentages of CD4 ⁺ CXCR5 ⁺ PD-1 ⁺ and CD4 ⁺ CXCR5 ⁺ ICOS ⁺ T cells in the peripheral blood of patients with autoimmune thyroid disease also increased, and were positively correlated with the level of anti-thyroid antibodies. The above studies suggest that the proportion of Tfh cells in the peripheral blood of patients with autoimmune diseases is increased, and may be related to the production of autoantibodies.

The inventor's research found that patients with RA, SLE, and SS have high expression of Tfh cells in the active stage of the disease, and this group of cells can be detected by some mature, stable and simple immunological detection methods (such as: flow cytometry). It has a strong correlation with the existing disease activity, and can directly understand the imbalance of immune cells in the body, which has a very high guiding significance for treatment and monitoring efficacy.

2. Summary of the invention

One of the objectives of the present invention is to provide a measurement index capable of effectively evaluating the disease activity of the three main autoimmune diseases of RA, SLE and SS, so as to guide clinical treatment.

Another object of the present invention is to provide a method for detecting effective follicular helper T cells (effective Tfh) in peripheral circulation of patients.

The term "Tfh" used in the present invention refers to lymphocytes with high expression of CD4, CXCR5 and PD-1 on the cell surface.

The inventors found that Tfh cells expressing marker surface molecules such as CD4, CXCR5 and PD-1 could be detected in the peripheral circulation of patients with autoimmune diseases and normal people. Further studies have found that Tfh cells can also be divided into different subgroups, among which effective Tfh cells (effective Tfh) have the characteristics of low expression of CCR7. Our study proved that effector Tfh cells were positively correlated with SLEDAI score in SLE patients, DAS28 in RA patients, and ESSDAI in SS patients. Additionally, in SLE, effector Tfh cells were strongly associated with anti-nucleosome antibodies and anti-dsDNA antibodies, respectively, associated with organ involvement. Therefore, effector Tfh cells can be used as immune cell markers to correctly judge disease activity and directly respond to abnormal immune cell status, which has important guiding significance for clinical treatment.

The purpose of the present invention is to provide a measurement index capable of effectively evaluating the disease activity of the three main autoimmune diseases of RA, SLE and SS, so as to guide clinical treatment. Based on the above studies, the inventors of this study defined lymphocytes with high expression of CD4, CXCR5 and PD-1 and low expression of CCR7 as "effector Tfh" cells, and detected effector Tfh cells in peripheral blood of normal people and patients by flow cytometry , determine the disease activity and the potential of pathogenic autoantibodies through cellular immunology methods, directly reflect the immune abnormal state of patients, guide clinical treatment, and provide objective laboratory basis for evaluating disease conditions.

The cell subgroups invented and verified in this study can be detected by flow cytometry in peripheral blood of patients, and the detection method is well known to those skilled in the art. The detection technology has a stable structure, is simple and feasible, and can detect other subtypes of Tfh cells at the same time.

Description of drawings

Figure 1. Schematic diagram of the principle of flow cytometry.

Figure 2. Flow cytometry results show that the expression of effector Tfh cells is different in normal people, RA, SLE and SS.

Figure 3. Comparison of expression levels of effector Tfh cells in RA, SLE, and SS patients and healthy controls. It can be seen from the figure that the expression levels of effector Tfh cells detected by flow cytometry in the peripheral blood of patients with RA, SLE and SS were significantly higher than those in the normal control group (all P<0.05).

detailed description

Embodiment 1 Specific detection method of effector Tfh cells.

Peripheral blood lymphocytes were separated, the number of cells was about 10 ⁵ -10 ⁶ , labeled with fluorescent antibodies PD-1, CXCR5, CD4 and CCR7, and the CD4 ⁺ CXCR5 ⁺ PD-1 ^high CCR7 ^low cell population was detected by flow cytometry.

This study shows that effector Tfh cells are highly expressed in the peripheral blood of RA, SLE and SS patients, which is significantly higher than that of healthy controls, which is of great significance for the determination of disease populations.

In this study, the expression rate of effector Tfh cells was calculated by flow cytometry, and the detection schematic diagram is shown in Figure 2 of the manual.

Example 2 Determination of Effector Tfh Cells on the Peripheral Blood of RA, SLE, and SS Patients

In this study, 30 cases of rheumatoid arthritis (RA), 26 cases of systemic lupus erythematosus (SLE), 28 cases of Sjogren's syndrome (SS) and 32 cases of normal human peripheral blood lymphocytes were detected to express the effector Tfh cell level. The positive cut-off point was defined as the mean+2SD higher than normal.

This embodiment mainly illustrates the Tfh cells of the present invention in several major autoimmune diseases through the following aspects The meaning of detection:

1. Comparison of the positive expression of effector Tfh cells in RA, SLE, SS and normal people.

In this study, the positive rate of effector Tfh cells in each group of patients and normal people was detected by FACS method, as shown in Table 1. The X ² test was used to compare the data of each group. The results showed that the positive rate of effector Tfh cells in RA, SLE and SS was significantly higher than that of normal people, with p values <0.05.

Table 1 Comparison of the positive rate of effector Tfh cells expressed in different diseases

2. Comparison of expression (%) of effector Tfh cells in different diseases:

The expression levels of effector Tfh cells in RA, SLE and SS patients were significantly higher than those in healthy controls (all P<0.05), as shown in Figure 2 of the specification.

3. The relationship between the expression of effector Tfh cells and the clinical and laboratory indicators of RA patients

As shown in Table 2: In RA patients, there was no statistically significant difference in gender, age, disease duration, etc. between the high-expression and low-expression groups of effector Tfh. The disease activity index DAS28 score and ESR of patients with high expression of effector Tfh cells were higher than those of patients with low expression of effector Tfh (P<0.05). Anti-keratin antibody (AKA), anti-perinuclear factor (APF) and IgM-RF, IgA, IgM and IgG between the two groups

The differences were not statistically significant.

Table 2. Correlation between clinical characteristics and percentages of Tfhem in 38 patients with RA

ESR: erythrocyte sedimentation rate; RF: rheumatoid factor; CRP: C-reactive protein; Anti-CCP: anti-cycliccitrullinated peptide antibody

AKA: antikeratin antibody; APF: antiperinuclear factor

*: Pvalue<0.05

4. The relationship between the expression of effector Tfh cells and the clinical and laboratory indicators of SLE patients

In this study, 25 cases of systemic lupus erythematosus (SLE) and 32 cases of normal human peripheral blood lymphocytes were detected to express the effector Tfh cell level.

As shown in Table 3: In SLE patients, there was no statistically significant difference in gender, age, disease duration, etc. between the effector Tfh high expression group and the low expression group. The SLEDAI score, complement C3, anti-dsDNA, anti-nucleosome antibody and anti-chromatin antibody of patients with high expression of effector Tfh cells were higher than those of patients with low expression of effector Tfh (P<0.05).

Table 3. Correlation between clinical characteristics and percentages of Tfhem in 26 patients with SLE

UrinePRO: 24-hour urine protein; Anti-dsDNA: anti-double-stranded DNA antibody; ANA: antinuclear antibody; Acl: anticardiolipin antibody;

AnuA: anti-nucleosome antibody

*: Pvalue<0.05

5. The relationship between the high expression of effector Tfh cells and the clinical and laboratory indicators of SS patients

In this study, 28 cases of Sjögren's syndrome (SS) and 32 cases of normal people were detected the expression of effector Tfh cells in peripheral blood lymphocytes.

As shown in Table 3: in SS patients, there was no statistically significant difference in gender, age, disease duration, etc. between the high expression and low expression groups of Tfh%. The ESSDAI score, serum IgA level, and anti-SSA antibody positive rate of patients with high expression of effector Tfh cells were higher than those of patients with low expression of effector Tfh (P<0.05).

Table 3. Correlation between clinical characteristics and percentages of Tfhem in26patients with SLE

PIF, pulmonary interstitial fibrosis.

Claims (1)

1. a kind of flow cytometer detection method of the folliculus helper T lymphocyte of non-diagnostic purpose, it is characterised in that by helper T lymphocyte The cd4 cell of the high expression of surface molecular CXCR5 and PD-1 is defined as Tfh cells, separates peripheral blood lymphocyte, and cell number is 10⁵-10⁶Individual, mark fluorescent antibody PD-1, CXCR5, CD4 and CCR7, streaming method detect Patients With Rheumatoid Arthritis and drying CD4 in syndrome virus people's cell⁺CXCR5⁺PD-1^highCCR7^lowCell mass；Effect Tfh cell is calculated by streaming method Expression rate.