An immune marker for tuberculosis and its application

Abstract

This invention relates to the field of biomedical technology, specifically to an immunomarker for tuberculosis and its applications. This invention experimentally demonstrates that the expression level of CD355 (also known as Class I-restricted T cell-associated molecule, or CRTAM) leads to corresponding changes in the bactericidal effect of human peripheral lymphocytes against intracellular Mycobacterium tuberculosis. Animal experiments show that mice lacking the CD355 encoding gene are more susceptible to Mycobacterium tuberculosis infection compared to wild-type mice. This indicates that CD355 is an important target for host-targeted immunotherapy in tuberculosis. This research has promising applications in the development of related products for screening, diagnosis, efficacy evaluation, and prognostic relapse risk assessment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an immune marker for tuberculosis and its application. Background Technology

[0002] Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb) and its complex flora. Currently, TB treatment primarily relies on chemotherapy. However, due to the slow growth of Mycobacterium tuberculosis and the influence of the body's immune microenvironment, the pathogen easily develops drug resistance during treatment. Currently, clinically resistant strains have been identified for all clinically used chemotherapy drugs.

[0003] In recent years, the emergence and prevalence of drug-resistant tuberculosis, especially multidrug-resistant tuberculosis (MDR-TB), have made the prevention and control of tuberculosis extremely challenging. MDR-TB has developed resistance to the most effective first-line clinical drugs, isoniazid and rifampin, making its treatment very difficult, and in some cases, unavailable. Therefore, there is an urgent need to develop new treatment strategies.

[0004] The occurrence, development, and outcome of tuberculosis are closely related to the body's immune status. Adjuvant immunotherapy that enhances the body's immune function plays a crucial role in the treatment of multidrug-resistant tuberculosis.

[0005] Existing technology: The study on the molecular mechanism of the interaction between NK cell receptors and Nectin / Necl family ligands (May 1, 2014, Zhang Yongjun et al.) recorded that: ...the recognition of CRTAM and Necl-2 promotes the killing of tumor cells by NK cells on the one hand, and inhibits the adhesion, aggregation and metastasis of tumor cells to tissues on the other hand... In summary, our study elucidates the molecular basis of the interaction between NK cell receptors such as CRTAM, CD96 and DNAM-1 and their ligands, laying the foundation for the mechanism of NK cell immune recognition and possible subsequent structure-based drug design.

[0006] Chinese patent application CN105247075A discloses a biomarker for diagnosing lung diseases and a method for using it, providing a method for diagnosing interstitial lung disease (ILD). It provides a method for differentiating idiopathic pulmonary fibrosis from other ILDs. It also provides compositions and kits for performing the subject method. Interstitial lung disease (ILD), also known as diffuse parenchymal lung disease (DPLD), represents a variety of conditions that can lead to diffuse remodeling of normal lung tissue, architectural damage, and inflammation that can lead to progressive loss of lung function. In ILD, in addition to the inflammation and fibrosis often seen in the lung parenchyma, the trachea and blood vessels are also significantly affected, and the application mentions the relationship between CRTAM expression levels and interstitial lung disease (ILD).

[0007] However, while the aforementioned literature, whether in existing technology (molecular mechanism study of the interaction between NK cell receptor and Nectin / Necl family ligands (May 1, 2014, Zhang Yongjun et al.)) or patent literature (CN105247075A), touches upon the relationship between CRTAM and immunity, those skilled in the art also know that the two pillars of immunotherapy are: first, enhancing the attack power of immune cells; and second, relieving the inhibitory effect of cancer cells on immunity. Treatment methods include: one is to enhance the attack power of immune cells, and the other is to relieve the inhibitory effect of cancer cells on immunity, allowing immune cells to return to an attacking state against cancer (various immune checkpoint inhibitors). Therefore, it is not self-evident when to enhance the body's immunity to achieve the purpose of treating the disease, and when to suppress the body's immunity to restore the immune cells to an attacking state against cancer.

[0008] In this invention, the inventors discovered for the first time that CD355, or Class I-restricted T cell-associated molecule (CRTAM), is an important target for regulating the host's protective immunity and can serve as an immune marker for tuberculosis, providing a new approach for the treatment of multidrug-resistant tuberculosis. No literature has yet reported on this immune marker for tuberculosis and its applications. Summary of the Invention

[0009] The purpose of this invention is to address the shortcomings of existing technologies by providing an immune marker for tuberculosis and its application.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] In a first aspect, the present invention provides an immune marker for tuberculosis, wherein the immune marker is the expression level of CD355.

[0012] Secondly, the present invention provides the application of the immune markers described above in the preparation of kits for differential diagnosis and / or prognosis of tuberculosis.

[0013] Furthermore, the present invention provides the application of the immunomarkers described above in the preparation of kits for the differential diagnosis and / or prognostic monitoring of multidrug-resistant tuberculosis.

[0014] Preferably, the sole active ingredient in the kit is a reagent for detecting CD355 expression levels.

[0015] Preferably, the indicator used by the kit for monitoring efficacy and prognosis is: a significant increase in CD355 expression in peripheral blood, indicating that the drug is effective and the risk of relapse is low.

[0016] Thirdly, the present invention provides a kit for the differential diagnosis and / or efficacy prognosis monitoring of multidrug-resistant tuberculosis, the kit comprising: a detection kit and an instruction manual, wherein the detection kit includes a substance for detecting CD355 expression, and the instruction manual describes the procedures and indicators for the differential diagnosis of multidrug-resistant tuberculosis and efficacy prognosis monitoring.

[0017] Fourthly, the present invention provides the use of CD355 promoters in the preparation of medicaments for treating tuberculosis.

[0018] Furthermore, the present invention provides the use of CD355 promoters in the preparation of medicaments for treating multidrug-resistant tuberculosis.

[0019] Furthermore, the present invention provides the use of Necl2 promoter in the preparation of medicaments for treating multidrug-resistant tuberculosis.

[0020] Preferably, the promoter is a substance that promotes the expression of CD355 and / or Necl2, and the promoter is selected from small molecule compounds or biological macromolecules.

[0021] The advantages of this invention are:

[0022] This invention is the first to demonstrate that CD355 (also known as Class I-restricted T cell-associated molecule, or CRTAM) is an important protective cytokine in the host's anti-tuberculosis immune response. The CD355 encoding gene exists in both humans and mice, performing similar functions. In human peripheral lymphocytes, increasing CD355 expression enhances the bactericidal effect of lymphocytes against intracellular Mycobacterium tuberculosis, while inhibiting CD355 expression significantly reduces this effect. Furthermore, mice lacking the CD355 encoding gene showed significantly higher lung colony counts after Mycobacterium tuberculosis infection compared to wild-type controls, indicating that these mice are more susceptible to Mycobacterium tuberculosis infection. Therefore, CD355 represents a novel target for host-targeted immunotherapy in the adjuvant treatment of tuberculosis. This invention provides a new approach to the treatment of multidrug-resistant tuberculosis and has strong practical applications. Attached Figure Description

[0023] Appendix Figure 1 This refers to the expression levels of CRTAM and its ligand Necl2. A. Expression levels of CRTAM and Necl2 detected by quantitative PCR in PBMCs from healthy individuals (HC) and tuberculosis patients (TB). B. Expression levels of CRTAM and Necl2 in PBMCs after stimulation with PPD / HMBPP. C. Expression level of Necl2 in THP-1 cells before and after BCG infection stimulation.

[0024] Appendix Figure 2 This study describes the effect of CD355-positive cells on the growth of intracellular Mycobacterium tuberculosis. The colony counts were obtained after co-culturing CD355+CD3+ cells (black columns) and CD355-CD3+ cells (gray columns) of PBMCs from a tuberculosis-resistant population with Mycobacterium tuberculosis-infected hMDM cells for 3 days.

[0025] Appendix Figure 3 This study investigated the effect of altered CD355 expression on the inhibition of intracellular Mycobacterium tuberculosis growth in human PBMCs. A. Human PBMCs were transfected with a lentivirus (Lenti-CD355) overexpressing the CD355 coding gene and an empty vector virus as a control (Lenti-Control). These cells were then co-incubated with macrophages infected with the Mycobacterium tuberculosis standard strain H37Rv. Cell lysates were diluted and plated to count the number of H37Rv colonies (CFU). B. A lentivirus (Sh-CD355) and an empty vector control virus (Sh-NC) were prepared by ligating a shRNA fragment targeting the CD355 coding gene into a vector and transfected into human PBMCs. As described in A, the number of H37Rv colonies (CFU) was then counted.

[0026] Appendix Figure 4 The number of bacterial colonies in the lungs of mice infected with Mycobacterium tuberculosis H37Rv was measured at different time points. The number of bacterial colonies in the lungs of CD355-encoding gene-deleted mice (KO) was significantly higher than that in wild-type control mice (WT) at 1 week (1W, A) and 2 weeks (2W, B) after infection. Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0028] Example 1

[0029] 1 Experimental Methods

[0030] 1.1 Recruitment of the experimental population

[0031] The first group of tuberculosis infection resistant individuals: close contacts were recruited from spouses of sputum-positive pulmonary tuberculosis patients. Spouses who had been in close contact with the patient for more than 2 weeks after the onset of the disease were selected, and their serum specific antibodies against ESAT6 and CFP10 proteins were tested. Individuals who tested positive for antibodies were further screened by TB.SPOT and PPD skin tests. Those who tested negative for both were tested again after 6 months. Individuals who tested negative for both consecutive times were included.

[0032] The second group of tuberculosis patients with positive sputum smears: recruited from tuberculosis patients diagnosed at Shanghai Pulmonary Hospital.

[0033] The third group consists of healthy individuals who have not been infected with tuberculosis: healthy individuals who have not had close contact with tuberculosis patients.

[0034] The recruited personnel must not have any other infectious diseases, chronic obstructive pulmonary disease, or a history of smoking.

[0035] Peripheral blood was drawn from the subjects, and PBMCs were separated from the peripheral blood using the Ficoll centrifugation method according to our established technical system.

[0036] PBMC cells were stimulated with PPD and HMBPP, and their surface was stained with fluorescent antibodies against CD3, CD107a, CRTAM (CD355), and CXCR3. The stained cells were then analyzed or sorted using flow cytometry.

[0037] After sorting the target cell subpopulations, the samples are sent to the company for transcriptome sequencing analysis. Alternatively, following our established technical system, we can extract cellular mRNA and detect the expression levels of specific genes.

[0038] 1.2 Using lentiviral vectors to transfect human PBMC cells and detect the bactericidal effect of PBMCs in vitro. Following our established technical system, we changed the expression level of CD355 in lymphocytes.

[0039] Methods to increase CD355 expression: Construct a lentivirus expressing the CD355 encoding gene and transfect PBMCs to increase intracellular CRTAM expression.

[0040] Methods to reduce CD355 expression: Construct a lentiviral vector to express shRNA targeting CD355, and then transform it into human PBMCs to reduce CD355 expression.

[0041] After altering CD355 expression using the above method, cells were stimulated with PPD and HMBPP. Simultaneously, THP-1 macrophages were infected with Mycobacterium tuberculosis H37Rv at an MOI of 10. Following our established technique, the two cell types were co-cultured for 3 days, cells were lysed, and the diluted cell lysate was plated on 7H10 plates and incubated at 37°C for 4 weeks. Colony counts were then performed.

[0042] 1.3 Mice were challenged with Mycobacterium tuberculosis H37Rv, and the number of bacterial colonies in the lungs was detected by dissection.

[0043] Purchased CD355-encoding gene-deleted mice (KO) and normal wild-type mice. Each mouse was infected with 1 x 10⁵ CFU of Mycobacterium tuberculosis via intranasal instillation. After 1 and 2 weeks of feeding, the mice were dissected, their lungs were isolated, homogenized, diluted, plated, and incubated at 37°C for 4 weeks. The number of colonies was then counted.

[0044] 2 Results

[0045] 2.1 CD355 is highly expressed in lung cells that migrate towards the lungs in individuals resistant to tuberculosis infection.

[0046] To identify an effective immune protective response against Mycobacterium tuberculosis infection, this invention targeted a tuberculosis-resistant population capable of rapidly clearing the infection. The immunological characteristics of cytotoxic effector cells (CXCR3+CD107a+) that rapidly migrate to the lungs in this population were analyzed. Peripheral blood lymphocytes were extracted, and CXCR3+CD107a+ and CXCR3-CD107a- cells were sorted for transcriptome analysis. Transcriptome analysis was also performed on the same cell type derived from tuberculosis patients. The results showed that in the tuberculosis-resistant population, CD355 expression was significantly higher in CXCR3+CD107a+ cells than in CXCR3-CD107a- cells (Table 1). CD355 expression was very low in CXCR3+CD107a+ cells from tuberculosis patients, significantly lower than in the tuberculosis-resistant population (Table 2). These results indicate that CD355 can be highly expressed in cytotoxic cells migrating to the lungs, suggesting that CD355 may play an important role in the body's anti-tuberculosis immune response.

[0047] Table 1. Expression level of CD355 in double-positive cells of tuberculosis infection resistant populations.

[0048]

[0049] Note: (CXCR3+CD107a+, double positive, abbreviated as DP) and (CXCR3-CD107a-, double negative, abbreviated as DN)

[0050] Table 2. Expression levels of CD355 in cells of individuals resistant to tuberculosis infection.

[0051] Gene name Expression levels in DP-R Expression levels in DP-TB log2(change factor) p-value q value CD355 581.537 7.14369 -6.3471 0.00005 0.00322

[0052] Note: (CXCR3+CD107a+, double positive Resister, abbreviated as DP-R, and CXCR3+CD107a+, double positive tuberculosis, abbreviated as DP-TB) in tuberculosis patients.

[0053] 2.2 The expression levels of the genes encoding CD355 and its ligand Necl2 were significantly decreased in tuberculosis patients.

[0054] In the absence of tuberculosis antigen stimulation, the expression levels of the CD355 and its ligand Necl2 encoding genes in PBMCs of healthy individuals were significantly higher than those in tuberculosis patients. Figure 1 A). After stimulation with tuberculosis antigen, the expression levels of CD355 and Necl2 in PBMCs of healthy individuals were significantly higher than those in patients with tuberculosis. Figure 1B). The results showed that the expression levels of both CD355 and its ligand Necl2 were significantly decreased in tuberculosis patients.

[0055] Human macrophages THP-1 were infected with Mycobacterium tuberculosis in vitro, and the expression level of Necl2 was detected by quantitative PCR. The results showed that Necl2 expression was significantly reduced in cells 48 hours after infection. Figure 1 C).

[0056] The above results indicate that Mycobacterium tuberculosis infection inhibits the expression of CD355 and its ligand Necl2.

[0057] 2.3 Tuberculosis antigen-reactive CD355-positive cells can significantly inhibit the growth of intracellular Mycobacterium tuberculosis infection.

[0058] To investigate the anti-tuberculosis effect of CD355-positive cells, the inventors examined the influence of CD355+CD3+ cells on the growth of intracellular Mycobacterium tuberculosis infection. PBMCs from a tuberculosis-resistant population (Resister) were stimulated with PPD / HMBPP and then sorted into CD355+CD3+ cells and CD355-CD3+ cells. CD14+ cells from the same individual were induced into hMDM and infected with Mycobacterium tuberculosis. The two cell types were then co-cultured and plate-seared for bacterial count. The results showed that the bacterial count in the CD355+CD3+ treatment group (black column) was significantly lower than that in the CD355-CD3+ treatment group (gray column). Figure 2 ).

[0059] 2.4 CD355 can regulate the bactericidal function of human lymphocytes.

[0060] To investigate the effect of CD355 on the anti-tuberculosis effect of lymphocytes, the inventors altered the expression level of CD355 and then verified the bactericidal effect of lymphocytes against intracellular Mycobacterium tuberculosis infection. First, human lymphocytes were transfected with a lentiviral vector overexpressing CD355 to increase CD355 expression. Then, PBMCs were co-incubated with H37Rv infected with a standard strain of Mycobacterium tuberculosis for 3 days. After cell lysis, the number of H37Rv colonies (CFU) was counted. The results showed that, compared with the control group (Lenti-Control), the number of colonies in the lentiviral group with CD355 overexpression (Lenti-CD355) treatment group was significantly lower than that in the control group. Figure 3A). Similarly, this invention utilizes a lentiviral vector expressing shRNA targeting the CD355 coding gene to transfect human PBMCs to reduce CD355 expression. The bactericidal effect of PBMC cells on intracellular H37Rv infection was then examined. It was found that the colony count in the CD355-encoding gene-inhibited treatment group (Sh-CD355) was significantly higher than the control. Figure 3 B). The above results indicate that changes in CD355 expression levels will affect the in vitro bactericidal effect of human lymphocytes, meaning that CD355 can regulate the anti-tuberculosis immune function of human lymphocytes.

[0061] 2.5 Mice lacking the CD355 encoding gene are more susceptible to Mycobacterium tuberculosis infection.

[0062] To verify the role of the CD355-encoding gene in the host's resistance to tuberculosis infection, this invention constructed CD355-encoding gene-deficient mice (KO), which were then infected with Mycobacterium tuberculosis, and the number of bacterial colonies in the mice's lungs was detected. The results showed that, compared with wild-type mice (WT), CD355-encoding gene-deficient mice (KO) showed significantly improved resistance to tuberculosis infection one week after infection with Mycobacterium tuberculosis H37Rv. Figure 4 A) and 2 weeks ( Figure 4 The colony counts in B) were all significantly increased. These results indicate that mice lacking the CD355 encoding gene exhibit reduced resistance to Mycobacterium tuberculosis infection, suggesting that CD355 plays a crucial protective role in the host's resistance to Mycobacterium tuberculosis infection.

[0063] 3. Conclusion

[0064] This invention is the first to demonstrate that CD355 (also known as Class I-restricted T cell-associated molecule, or CRTAM) is an important protective cytokine in the host's anti-tuberculosis immune response. The CD355 encoding gene is present in both humans and mice, performing similar functions. In human peripheral lymphocytes, increasing CD355 expression enhances the bactericidal effect of lymphocytes against intracellular Mycobacterium tuberculosis, while inhibiting CD355 expression significantly reduces this effect. Furthermore, mice lacking the CD355 encoding gene showed significantly higher colony counts in their lungs after Mycobacterium tuberculosis infection compared to wild-type control mice, indicating that these mice are more susceptible to Mycobacterium tuberculosis infection. Therefore, CD355 represents a novel target for host-targeted immunotherapy in the adjuvant treatment of tuberculosis.

[0065] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the principle of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. Application of human PBMCs overexpressing CD355 in the preparation of drugs for treating tuberculosis.

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