Detection method and application of cytomegalovirus specific T cells

Abstract

The invention relates to the technical field of biology, in particular to a detection method and application of cytomegalovirus specific T cells. According to the detection method and application of the cytomegalovirus specific T cells, the method has the advantages of being stable in experimental result, high in specificity and sensitivity, easy to operate and the like, and CMV-CMI individualized monitoring of transplanted patients can be rapidly achieved. The method is helpful for accurately identifying CMV reactivation high-risk patients after hematopoietic stem cell transplantation. CMV reactivation layering prevention after transplantation is realized under the guidance of CMV-CMI, so that low-risk patients are prevented from being over-treated, and the sanitary economic cost is reduced; it is guaranteed that high-risk patients receive sufficient and sufficient treatment courses of treatment, an individualized CMV infection prevention strategy is formed, CMV reactivation after transplantation is prevented to the maximum extent, the non-relapse death rate after transplantation is reduced, and long-term survival of transplanted patients is improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for detecting cytomegalovirus-specific T cells and an application thereof. Background Art

[0002] Cytomegalovirus (CMV) is a double-stranded DNA virus belonging to the subfamily of herpesviruses in the order Herpesvirales. About 80-90% of adults have been infected with CMV. When CMV infects healthy people, body reactions such as fever and fatigue may occur, but no obvious clinical symptoms will be caused, and the infection will eventually remain latent in the human body. In the field of allogeneic hematopoietic stem cell transplantation (allo-HSCT), in order to prevent the occurrence of immune rejection and improve the success rate of transplantation, immunosuppressants are used clinically to suppress the function of the immune system. At this time, the latently infected CMV virus in the recipient's body or in the exogenous transplant is reactivated and replicated in large quantities, and the CMV DNA load in the patient's serum increases significantly. This is called CMVDNAemia. Further, the CMV virus may invade the target organ and develop into CMV disease, causing damage to different organs of the patient, including the lungs, gastrointestinal tract, brain, spinal cord or eyes, which ultimately affects the survival of the transplant and even the patient.

[0003] In the absence of effective prevention, approximately 60%-95% of allogeneic hematopoietic stem cell transplant patients experience a CMV reactivation within 100 days after transplantation, of which 20%-30% may progress to CMV disease with high mortality and disability rates. In August 2022, the CMV virus terminase complex inhibitor letermovir was approved by the National Medical Products Administration for the prevention of CMV reactivation in adult recipients after transplantation, with a limit of 100 days after transplantation. Although letermovir significantly reduced the incidence of early CMV reactivation after transplantation, the risk of late CMV reactivation after discontinuation of the drug was significantly increased. How to further optimize the prevention strategy for CMV reactivation after transplantation is an urgent clinical issue to be addressed.

[0004] At present, the occurrence of viremia is clinically judged by quantitative detection of CMV virus copy number through fluorescent quantitative PCR technology, which dynamically reflects the in vivo replication of the virus. In patients after transplantation, timely and effective immune reconstruction can reduce the risk of infection. The latest European leukemia anti-infection guidelines point out that CMV specific T cell immunity (CMV-CMI) monitoring plays an important role in the era of letermovir, but there is currently no unified CMV-CMI monitoring method. Many research teams at home and abroad have found that CMV-specific T cells that secrete interferon γ (IFN-γ) play an important role in regulating CMV reactivation. Therefore, detecting CMV-specific T cells that secrete IFN-γ is a reliable experimental means to evaluate the quality of CMV-CMI reconstruction.

[0005] Currently, the CMI detection methods used in international organ transplantation or hematopoietic stem cell transplantation are divided into two categories: the QuantiFERON-CMV test, which mainly detects the release dose of IFN-γ; the CMV-enzyme-linked immunosorbent spot (ELISPOT) test, which mainly detects the quantification of IFN-γ releasing cells based on the enzyme-linked immunosorbent assay. However, the QuantiFERON-CMV test is only used to detect CMV-specific CD8 + T cells, CD4 cannot be detected + The experimental results show the secretion of IFN-γ, not the absolute count of CMV-specific T cells. The experimental results can only indirectly reflect the CMV-specific CD8 with immune function. + The results of T cell function are not intuitive enough. However, the CMV-ELISpot assay can simultaneously detect CMV-specific CD8 + and CD4 + T cells are currently recognized as the gold standard for detection. Based on the CMV-ELISpot assay, some research centers have developed an ELISpot assay combined with flow cytometry cytokine staining technology to detect different cytokines secreted by CMV-specific T cells to reflect T cell effector capacity, but this technology is limited by the cumbersome experimental steps, long time consumption, and no specific threshold, and cannot be widely used in clinical practice; some research centers have also developed an ELISpot assay combined with IFN-γ capture microsphere technology to reflect T cell effector capacity by comparing ratios, but the experimental results are comparative values, and the absolute count of CMV-specific T cells cannot be obtained.

[0006] In summary, providing a highly sensitive and specific method for detecting CMV-specific T cells is the key to solving the problem of late-onset cytomegalovirus infection in patients undergoing hematopoietic stem cell transplantation. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a method and application for detecting cytomegalovirus-specific T cells. The method has the characteristics of stable experimental results, high specificity, high sensitivity, and simple operation, and can quickly realize individualized monitoring of CMV-CMI in transplant patients. This method helps to accurately identify patients at high risk of CMV reactivation after hematopoietic stem cell transplantation. Under the guidance of CMV-CMI, stratified prevention of CMV reactivation after transplantation is achieved, thereby avoiding excessive treatment of low-risk patients and reducing health economic costs; ensuring that high-risk patients receive sufficient and adequate treatment, forming an individualized CMV infection prevention strategy, preventing CMV reactivation after transplantation to the greatest extent, reducing non-relapse mortality after transplantation, and improving the long-term survival of transplant patients.

[0008] To this end, the present invention provides the following technical solutions: In a first aspect, the present invention provides a method for detecting cytomegalovirus-specific T cells in an optional embodiment, comprising the following steps: S1: After centrifugation of the mononuclear cell liquid layer containing the blood sample to be tested, add the pre-warmed RPMI1640 culture medium, mix and collect, count the cells and calculate the cell viability. After the cell viability is qualified, adjust the cell concentration to 2.5×10 6 Finally, the cells were plated in a 96-well plate pre-coated with IFN-γ antibody, with 2.5×10 cells per well. 5 , adding CMV virus-specific mixed antigen peptides or plant lectins for incubation; S2: After lysing the cells in the well plate, wash them, add biotin-labeled IFN-γ antibody, incubate, wash them twice after incubation, add enzyme-labeled avidin, incubate them twice, wash them three times after the second incubation, then add the color development substrate solution, incubate and develop color, and finally count the spots.

[0009] Preferably, in step S1, the method for preparing the mononuclear cell liquid layer containing the blood sample to be tested comprises the following steps: adding PBS buffer to the blood sample to be tested and mixing evenly, then adding it to the upper layer of Ficoll lymphocyte separation fluid, and centrifuging and precipitating to obtain the mononuclear cell liquid layer containing the blood sample to be tested.

[0010] Preferably, in step S1, the qualified condition of the cell viability is 70% or more. The cell concentration is adjusted and the cultured cells are incubated by adding serum-free medium for adjustment and incubation; and / or, the CMV virus-specific mixed polypeptide antigens are IE-1 and pp65. In step S1, the incubation time is 16-20 hours; and / or, the incubation environment is a carbon dioxide concentration of 5% and a temperature of 36-38°C.

[0011] Preferably, in step S2, the incubation time is 1 hour; and / or the secondary incubation time is 1 hour.

[0012] Preferably, the method further comprises: S3: judging the result of spot counting to determine whether the cytomegalovirus-specific T cell immunity is reconstituted at a high level or at a low level. The standard for the high level of cytomegalovirus-specific T cell immunity reconstitution is: T (IE1) ≥ 50 or T (pp65) ≥ 100, and the standard for the low level of cytomegalovirus-specific T cell immunity reconstitution is: T (IE1) < 50 and T (pp65) < 100.

[0013] In a second aspect, the present invention provides, in an optional embodiment, an application of the above-mentioned method for detecting cytomegalovirus-specific T cells in achieving individualized monitoring of CMV-CMI in transplant patients.

[0014] Compared with the prior art, the present invention has one of the following beneficial effects: 1. This technology uses two CMV-specific antigen peptides (IE-1 and pp65) to stimulate cells, so that T cells that can react with CMV-specific peptides can be effectively and comprehensively detected, avoiding the problem of one-sided detection results when using a single peptide, and can also avoid non-specific T cell detection, thereby improving detection specificity and sensitivity.

[0015] 2. The CMV-ELISPOT detection technology provided by the present invention is based on the classic enzyme-linked immunosorbent spot technology, and has the characteristics of stable experimental results, high specificity, high sensitivity, etc., and can quickly complete the individualized monitoring of CMV-CMI in transplant patients.

[0016] 3. The CMV-CMI detection-guided cytomegalovirus infection prevention provided by the present invention is the first clinical application technology for CMV infection management after hematopoietic stem cell transplantation in China and abroad. Preliminary retrospective studies have shown that it can achieve the purpose of stratified prevention of CMV after transplantation, reduce the problem of delayed CMV reactivation that needs to be solved urgently, and improve the long-term survival of transplant patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a flow chart of the method for detecting cytomegalovirus-specific T cells in Example 1 of the present invention; Figure 2 This is a schematic diagram of the ELISpot assay in Example 1 of the present invention; Figure 3 This is a schematic diagram of the spot counting results using an ELISPOT automatic plate reader in Example 1 of the present invention; Figure 4This is a schematic diagram of the threshold for determining whether cytomegalovirus-specific T cell immunity is reconstituted at a high level or at a low level in Example 1 of the present invention; Figure 5 The figure is a schematic diagram of the results of the CMV-CMI reconstruction ratios of patients with different transplant types 100 days after transplantation in Example 2 of the present invention, wherein MRD is a sibling fully matched donor, HID is a haplotype donor, and MUD is an unrelated fully matched donor; Figure 6 6A is a schematic diagram of the cumulative incidence of delayed CMV reactivation after discontinuation of letermovir in Example 2 of the present invention, wherein 6A is the cumulative incidence of delayed CMV reactivation in the experimental group (CMV-CMI guided) and the control group (Fixed duration), Figure 6 B is the cumulative incidence of late CMV reactivation in the control low-risk group (Fixed duration: Low-risk), control high-risk group (Fixed duration: High-risk), trial low-risk group (CMV-CMI guided: Low-risk), and trial high-risk group (CMV-CMI guided: High-risk) Figure 7 This is a schematic diagram of the overall survival rate comparison results in Example 2 of the present invention, wherein: Figure 7 A is the overall survival rate of the experimental group (CMV-CMIguided) and the control group (Fixed duration), Figure 7 B is the overall survival rate of the control low-risk (Fixed duration: Low-risk), control high-risk (Fixed duration: High-risk), trial low-risk (CMV-CMI guided: Low-risk), and trial high-risk groups (CMV-CMI guided: High-risk). DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0020] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0021] Example 1 See also Figure 1The method for detecting cytomegalovirus-specific T cells provided by the present invention comprises the following steps: 1. Isolation and stimulation of peripheral blood mononuclear cells Peripheral blood was collected from patients after transplantation, and peripheral blood mononuclear cells (PBMCs) were separated by Ficoll density gradient centrifugation. The liquid layer containing PBMCs was transferred to a sterile centrifuge tube, and after centrifugation and precipitation, the pre-warmed RPMI 1640 culture medium was added and mixed and collected. Subsequently, the cells were mixed in serum-free medium for cell counting and cell viability confirmation to ensure that the cell viability was not less than 70%, and the cell concentration was adjusted to 2.5×10 6 Cells / mL were plated at this density in a 96-well plate (pre-coated with IFN-γ antibody). A negative control well, an antigen well, and a positive control well were set for each assay sample. No stimulant was added to the negative control well, CMV virus-specific mixed polypeptide antigens (IE-1 and pp65) were added to the antigen well, and plant lectin was added to the positive control well. The microplate was placed in a CO2 cell culture incubator for 16-20 hours.

[0022] 2. ELISpot assay ELISA process Figure 2 As shown. First, lyse the cells in the microplate, wash, add the 1:100 diluted biotin-labeled IFN-γ antibody to each experimental well, and incubate at 37°C for 1 hour. After washing, add the 1:100 diluted enzyme-labeled avidin to each experimental well, and incubate at 37°C for 1 hour. Wash and dry. Then, add the color substrate solution to each experimental well and incubate for color development. Finally, choose the end time of color development according to the spot generation, and use the ELISPOT automatic plate reader to count the spots. See Figure 3 .

[0023] 3. CMV-CMI result judgment If the number of spots in the negative control well is less than 10, the experiment can be considered qualified for quality control; the number of spots in the positive control well should be greater than 20 or spread throughout the reaction well. T(IE1) is the difference between the number of spots in the IE1 antigen well and the negative control, and T(pp65) is the difference between the number of spots in the IE1 antigen well and the negative control. When T(IE1) ≥50 or T(pp65) ≥100, it can be judged that CMV-specific T cell immunity is highly reconstituted, otherwise CMV-specific T cell immunity is low-level reconstitution, see Figure 4 .

[0024] Example 2 Different transplantation types have different CMV-CMI reconstruction characteristics A total of 182 patients undergoing hematopoietic stem cell transplantation were analyzed and divided into an experimental group (n=104, letrozole was discontinued when CMI-CMI reconstruction met the target) and a control group (n=78, CMV-CMI was not monitored and letrozole was uniformly discontinued at d100). First, the characteristics of CMV-CMI reconstruction in different transplant types were analyzed. The results showed that the median time for sibling fully transplanted recipients to complete CMI reconstruction after transplantation was 86 (range: 63-88) days, while that for unrelated donor-type transplants was 115 (range: 65-180) days, and that for haplotype-type transplants was 120 (range: 88-180) days. Therefore, at 100 days after transplantation, most sibling fully transplanted patients (70%, [7 / 10]) could complete CMV-CMI reconstruction, while only 28.6% (4 / 14, p=0.043) of unrelated donor-type transplanted patients and 28.8% (23 / 80, p=0.038, see Figure 5 The above results suggest that most patients with sibling homozygous transplants can stop taking letrozole after 100 days, and prevention should be extended for patients with unrelated donors or haplotype transplants according to the status of CMV-CMI reconstruction.

[0025] CMV-CMI testing guides the reduction of delayed CMV reactivation after transplantation The cumulative incidence of CMV reactivation after cessation of letermovir prophylaxis in the experimental group was significantly lower than that in the control group (9.7% vs. 24.8%, p=0.019, see Figure 6 A). According to the risk factors of CMV reactivation after transplantation (donor CMV IgG <72 AU / mL, R-DRI high risk or very high risk, combined with acute graft-versus-host disease), all patients included in the analysis were divided into high-risk group (control high-risk group, n=16; experimental high-risk group, n=20) and low-risk group (control low-risk group, n=58, experimental high-risk group, n=88). The results showed that the cumulative incidence of CMV reactivation after the cessation of letermovir prophylaxis in the high-risk-experimental group was significantly lower than that in the high-risk-control group (12.5% ​​vs. 46.3%, p=0.048, see Figure 6 B).

[0026] CMV-CMI detection improves long-term survival in transplant patients From the perspective of long-term survival of patients, CMV-CMI can improve the overall survival rate of the experimental group (89.1% vs. 73.8%, p=0.042, see Figure 7 A). In addition, the overall survival rate of patients in the high-risk-experimental group was significantly higher than that in the high-risk-control group (85.7% vs. 71.6%, p=0.19, see Figure 7 B).

[0027] Although the principles of the present invention are described in detail above in conjunction with the preferred embodiments of the present invention, those skilled in the art should understand that the above embodiments are merely explanations of the exemplary implementations of the present invention, and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute limitations on the scope of the present invention, and any obvious changes such as equivalent transformations, simple replacements, etc. based on the technical solution of the present invention, without departing from the spirit and scope of the present invention, fall within the protection scope of the present invention.

Claims

1. A method for detecting cytomegalovirus-specific T cells, characterized in that: The following steps are involved: S1: After centrifugation of the mononuclear cell liquid layer containing the blood sample to be tested, add pre-warmed RPMI 1640 culture medium, mix and collect, count the cells and calculate the cell viability. After the cell viability is qualified, adjust the cell concentration to 2.5×10 6 The cells were plated in a 96-well plate pre-coated with IFN-γ antibody, with 2.5×10 cells per well. 5 , adding CMV virus-specific mixed antigen peptides or plant lectins for stimulation and incubation; S2: After the cells in the well plate are lysed, they are washed, biotin-labeled IFN-γ antibody is added, incubated, washed twice after incubation, enzyme-labeled avidin is added, incubated twice, washed three times after the second incubation, and then the color substrate solution is added, incubated for color development, and finally the spots are counted; In step S1, adjusting the cell concentration and incubating the cultured cells is to add serum-free medium for adjustment and incubation; The CMV virus-specific mixed antigen polypeptides are IE-1 and pp65; The object of the detection method is hematopoietic stem cells.

2. The method for detecting cytomegalovirus-specific T cells according to claim 1, characterized in that: In step S1, the method for preparing the mononuclear cell liquid layer containing the blood sample to be tested comprises the following steps: PBS buffer is added to the blood sample to be tested and mixed evenly, and then added to the upper layer of Ficoll lymphocyte separation liquid, and density gradient centrifugation precipitation is performed to obtain the mononuclear cell liquid layer containing the blood sample to be tested.

3. The method for detecting cytomegalovirus-specific T cells according to claim 1, characterized in that: In step S1, the qualified condition of the cell viability is 70% or more.

4. The method for detecting cytomegalovirus-specific T cells according to claim 1, characterized in that: In step S1, the incubation time is 16-20 hours; and / or, The incubation environment is a carbon dioxide concentration of 5% and a temperature of 36-38°C.

5. The method for detecting cytomegalovirus-specific T cells according to claim 1, characterized in that: In step S2, the incubation time is 1 hour; and / or, The secondary incubation time is 1 hour.

6. The method for detecting cytomegalovirus-specific T cells according to claim 1, characterized in that: The method further comprises: S3: Determine the result of spot counting to determine whether cytomegalovirus-specific T cell immunity is reconstituted at a high level or at a low level.

7. The method for detecting cytomegalovirus-specific T cells according to claim 6, characterized in that: The standard for high-level reconstruction of cytomegalovirus-specific T cell immunity is: T (IE1) ≥ 50 or T (pp65) ≥ 100, The standard for low-level reconstruction of cytomegalovirus-specific T cell immunity is: T (IE1) <50 and T (pp65) <100.

8. Use of the method for detecting cytomegalovirus-specific T cells according to any one of claims 1 to 7 in achieving individualized monitoring of CMV-CMI in transplant patients.

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