Tuberculosis (TB) is a chronic respiratory infectious disease caused by Mycobacterium tuberculosis (Mtb). According to the latest World Health Organization (WHO) report, there were approximately 10.6 million new cases and 1.3 million deaths worldwide in 2022 (WHO, 2023). Worldwide, it is estimated that one-fourth of the population is infected with Mtb, and 5–10% of those infected are at risk of further progression to active TB (ATB) (Paton et al., 2019). Mtb can infect any organ system in the body with pulmonary TB (PTB) being its most common presentation, and the rest as extrapulmonary TB (EPTB). Among them, tuberculous meningitis (TBM) represents the most severe form of TB, with high morbidity and mortality, and with more than 100,000 new cases reported by the WHO every year (Poh et al., 2021; Li et al., 2022).

TB is a typical bacterial infectious disease and also an immune-related disease, which is affected by a variety of factors, such as bacterial load and host immunity, especially local anti-TB immunity of the lung. After Mtb invasion, the initial acute immune response against Mtb is initiated by the innate immune system of the body to eliminate the pathogen. Innate immune cells include macrophages, natural killer (NK) cells, dendritic cells, etc., which represent important components of the innate immune defense mechanisms. They mainly play the role of phagocytosis and antigen presentation, clearing Mtb in macrophages and producing cytotoxic effects to destroy lysed target cells (Garand et al., 2018; Choreño-Parra et al., 2020, 2021). Subsequently, the adaptive immune responses are activated, in which the cellular immune responses mediated by T cells (including CD4⁺ and CD8⁺ T cells) and their production of Th1 cytokines (such as IFN-γ and IL-2) play a major anti-TB role, and the humoral immune responses mediated by B cells and antibodies exert an adjuvant effect (Mi et al., 2021; Wu Xueqiong, 2022). Therefore, the activation of the body’s immune system and inflammatory responses play a critical role in the immune pathogenesis of diseases. Studies have found that these changes in immune responses were closely related to the occurrence and development of this disease (An et al., 2022). However, most current research has only been conducted in ATB patients, latent TB infection (LTBI), or tuberculous pleurisy, and few studies have reported the clinical immunological characteristics of TBM patients.

Presently, the pathogenesis of TBM is still insufficiently understood. Researchers in the last century have found that Mtb infected the central nervous system long before TBM patients showed symptoms, but the associated clinical symptoms did not manifest immediately in the brain (Rich, 1933). NK cells are a unique group of granular lymphocytes of the innate immune system with innate cytotoxicity and immune regulatory ability, playing a crucial role in the early stages of infection. NK cells can present signals to infected dendritic cells and macrophages to assist the body in clearing Mtb (Esin et al., 2013; Choreño Parra et al., 2017). NK cells can also exert classical cytotoxic effects by secreting perforin and granzymes to clear mycobacterium and Mtb-infected host cells (Brill et al., 2001; Lu et al., 2014). Additionally, NK cells can secrete cytokines to regulate the immune response, such as IFN-γ, TNF-α, and IL-22 (Feng et al., 2006; Zhang et al., 2006). However, little work has been reported on the phenotypic changes of NK cells in TBM patients. Only several studies found that the NK cells in the peripheral blood of TBM patients were lower than those in healthy controls, and CD56^brightCD16⁻ NK cells were significantly lower than those in LTBI population and PTB patients, while neutrophils and classical monocytes were significantly higher, but the number of TBM patients enrolled was limited (van Laarhoven et al., 2019; Choreño-Parra et al., 2021; Shridhar et al., 2022). There were no studies on the mechanisms involved in the immune response after Mtb infection in TBM patients. Therefore, the study of the number of NK cells and the adaptive immune status of TBM patients can provide new insights and ideas to elucidate the pathogenesis of TBM.

Currently, flow cytometry (FCM) is commonly used to analyze peripheral blood lymphocyte subsets in TB patients to assess immune status, immune function, and immune balance. In this study, we analyzed the changes in the percentages and absolute counts of CD3⁺, CD4⁺, CD8⁺, NK, NKT, and B lymphocytes in peripheral blood of TBM patients, non-severe TB (NSTB) patients, LTBI persons, and normal controls (NC) through FCM. In addition, the relationship between the number of immune cells in TBM patients and the disease severity, bacterial load in the body, cytokines, and antibody levels, as well as the changes in NK cells in the TBM population were emphatically analyzed, providing a reference basis for further understanding of the immune mechanism of TBM and clinical evaluation of the immune status of TBM patients.

When Mtb invades the body, the host’s innate immune response is rapidly activated to play an early defense role and then initiates a specific cellular immune response leading to the formation of tuberculous granuloma. Cellular immunity is the main immune protection mechanism for TB, and TB will be developed when the human immune function becomes unbalanced or abnormal. TBM occurs if Mtb succeeds in crossing the blood–brain barrier through blood circulation or other means when the infection is not effectively controlled. The pathogenesis of TBM is still unclear, and it is known that T lymphocyte-mediated chronic inflammation is an important link in the body’s resistance to Mtb. The reduction of T lymphocytes and loss of their function are common pathological changes in the occurrence and development of TBM. NK cells, as important innate immune cells, can exert cytotoxicity in the initial stage of Mtb infection, kill extracellular Mtb, trigger the effector mechanisms of the macrophage and dendritic cell, and limit the intracellular growth of Mtb. In addition, NK cells can produce cytokines (such as IFN-γ, IL-12, etc.) and regulate the activation and function of Mtb-specific T cells (Esin and Batoni, 2015), thereby participating in the immune defense process of LTBI and active PTB (Choreño-Parra et al., 2021). However, there is currently limited research on NK cells in TBM, and their role in TBM is not yet fully understood.

Previous studies have shown that TB patients, including TBM and NSTB, were significantly immunocompromised, and their absolute counts of lymphocyte subsets were significantly lower than those of the NC and LTBI populations (Al Majid and Abba, 2008; Mi et al., 2021; Wu Xueqiong, 2022), which is consistent with the findings of our study. If CD3⁺ T, CD4⁺ T, CD8⁺ T, and NK cells are significantly reduced in PTB patients, it indicates that the body is prone to expand the range of lesions, appear cavities, and even spread outside the lungs to further develop into patients with EPTB including TBM (Guglielmetti et al., 2013). The study by Choreno Parra et al. demonstrated that NK cells were only activated during the active phase of Mtb infection and entered tissues (Choreño-Parra et al., 2021). Our study also found that compared with NSTB patients, the decrease of NK cells in TBM patients was particularly noticeable, indicating that the local innate immune responses of the lungs in TBM patients may be significantly attenuated, which causes the spread of Mtb in the lungs, thereby leading to the occurrence of TBM (Choreño-Parra et al., 2021). It is also possible that peripheral blood NK cells migrate to the central nervous system and produce pro-inflammatory cytokines, causing clinically common brain injury or other symptoms. Van Laarhoven et al. also found that the αβT, γδT, and NK cells in the blood of TBM patients were significantly reduced compared with PTB and healthy control groups, while the increased leucocyte activation and a predominance of αβT and NK cells in CSF were associated with better survival (van Laarhoven et al., 2019). Also, TBM patients have shown increased myeloid cell responses with diverse immune functions (van Laarhoven et al., 2019). In addition, flow cytometry confirmed the presence of αβT, γδT, B, NK, NKT, and MAIT cells in the CSF of TBM patients, with NK cells being the most abundant cell type besides αβT cells (Dieli et al., 1999; Simmons et al., 2005; van Laarhoven et al., 2019). The above literature studies have indirectly supported our findings that NK cells in TBM patients may be transferred from peripheral blood to CSF, but the number and functional changes of the transfers are still unknown. Therefore, we will simultaneously study the lymphocyte subsets in both CSF and peripheral blood of TBM patients to analyze the distribution, quantity, and function of lymphocyte subsets, especially NK cells in different clinical specimens of these patients, in order to discover more effective blood and CSF biomarkers and provide more reliable indices for future host-directed treatment of TBM.

At present, there are two methods for analyzing lymphocyte subsets in clinical practice. Among them, the relative counting method is to analyze the percentage of each subset of cells in the total number of lymphocytes, so the results are easily affected by changes in various cell subsets. The absolute counting method can understand the independent changes of various cell subsets. The change in the absolute count of one lymphocyte subset does not affect the changes in the absolute count of other cell subsets, but may affect the changes in the relative count of lymphocyte subsets (Morais-Papini et al., 2017). In this study, the percentage of NK cells in the TBM and NSTB groups significantly decreased, leading to significant increases in the percentages of CD3⁺ and CD4⁺ T cells, but the absolute counts of these three types of cells were significantly reduced. Guglielmetti et al. also found that the absolute count of all lymphocyte subsets in patients with severe TB decreased, but the percentage of all lymphocyte subsets may not change significantly, still in the normal range (Guglielmetti et al., 2013). Therefore, it is recommended to simultaneously detect and analyze the relative and absolute counts of lymphocyte subsets in TB patients, to objectively and accurately reflect the immune cell status of TB patients. Our findings provided further evidence that TBM was associated with a decrease in peripheral blood lymphocytes, which was also closely related to disease severity (Guglielmetti et al., 2013). In particular, the decreases of NK cells and B cells were closely associated with the degree of disease spread and bacterial load in TBM patients, which is consistent with the results of previous studies (Davoudi et al., 2008; Yang Ming et al., 2017), suggesting that severe TBM patients may have immune cell exhaustion and more severe cellular immunosuppression in the body.

In the process of adaptive immune against TB, effector CD4⁺ and CD8⁺ T cells activated play a major role in anti-TB. As is well known, IFN-γ in peripheral blood is mainly produced by activated CD4⁺ effector T cells and a small amount of CD8⁺ effector T cells. IFN-γ, as a macrophage activating factor (MAF), induces the release of hydrogen peroxide (H₂O₂) and mediates antibacterial effects. In addition, IFN-γ, also as an immune modulator, mainly limits neutrophil recruitment and tissue inflammation-induced damage by inhibiting CD4⁺ T cell-mediated IL-17 generation (Saghazadeh and Rezaei, 2022). IFN-γ plays an important role in immune-mediated inhibition on Mtb infection. Our study found that the absolute counts of lymphocyte subsets in IGRA (+) TBM patients were higher than those in the IGRA (−) group, except for B cells (Yu Shan et al., 2021). The absolute counts of peripheral blood lymphocytes in IGRA (+) patients were mostly within the normal range, that is, the positive rate of IGRA in the patients with the absolute count of peripheral blood lymphocytes within the normal range was significantly higher than that in other patients (Xueqiong, 2020). These results demonstrated that the elevation of IFN-γ may be the key to host defense against TBM. Our study showed that the negative rate of IGRA in TBM patients was as high as 41.25% (33/80), significantly higher than that of PTB, and there was a trend of positive correlation between negative IGRA and the severity of TBM, which is consistent with the results of previous studies (Di and Li, 2018; Kim et al., 2018). It has been found that IGRA-negative TB patients had higher mortality, suggesting that IGRA-negative results may be a risk factor for the development of TBM, which may be caused by the reduction of CD4⁺ T cells and CD8⁺ T cells (Ngai et al., 2007; Nguyen et al., 2018; Yamasue et al., 2020). Multiple meta-analyses have shown that advanced age, low peripheral blood lymphocyte count, and decreased NKT cells may be common risk factors for false-negative IGRA (Nguyen et al., 2018; Yamasue et al., 2020), suggesting that cellular immune function is impaired in TBM patients (Wu Xueqiong, 2022). In addition, the absolute number of NKT cells in IGRA (+) TBM patients was significantly higher than that in the IGRA (−) group, which may be due to the proliferation of NKT cells to recognize glucolipid antigens after Mtb infection. It has been found that NKT cells were significantly higher in “rapid responders” (Kulpraneet et al., 2007; Pandey et al., 2019). Subsequently, activated NKT produces large amounts of IFN-γ and promotes the increase of other T lymphocyte subsets to combat Mtb invasion. Therefore, double detection of T lymphocyte subset absolute count and IGRAs in clinical practice may assist in identifying immunocompromised TB patients for timely immune intervention (Xu et al., 2018).

The humoral immune response of the body is mainly the specific antibody response generated by the differentiation of mature B lymphocytes induced by Mtb antigen into plasma cells, which plays a protective role in anti-TB immunity in collaboration with cellular immunity (Encinales et al., 2010; Achkar et al., 2015; Jacobs et al., 2016; Lu et al., 2016). Here we provide the first comparison of the changes in lymphocyte subsets in the peripheral blood of TBM patients with negative and positive antibodies. Our results showed that the absolute counts of all lymphocyte subsets in TBM patients in the Ab (−) group were higher than those in the Ab (+) group, suggesting that Th1-type protective immune response was dominant in TBM patients in the Ab (−) group, while T and B lymphocytes in the peripheral blood of TBM patients in the Ab (+) group were reduced, especially CD4⁺ T cells and B lymphocytes were significantly reduced, which may be caused by the migration of B cells to peripheral lymphoid organs, possibly indicating a decrease in the number of “protective” B cell subsets and an increase in potential pathological B cell subsets (Rijnink et al., 2021). After being stimulated by antigens, mature B cells migrated to peripheral lymphoid organs and became activated B cells, which then differentiate into plasma cells and increase the synthesis and secretion of antibodies, leading to the patient’s immune imbalance, with Th1-type immunity shifting toward Th2-type immunity, resulting in a weakened Th1-type immune response and an enhanced Th2-type immune response. This may be the reason why Ab (+) TBM patients have a positive correlation with disease severity. B cells not only produce antibodies, but also an antigen-presenting cell (APC) that produces pro-inflammatory and anti-inflammatory cytokines, such as IL-1β, IL-10, IL-17, IL-21, and TNF-α (du Plessis et al., 2016), which play an important role in the production and regulation of anti-TB immunity. In addition, we studied for the first time the changes in the number of peripheral blood immune cells in TBM patients at different BMRC stages. It was found that there was no significant difference in the lymphocyte absolute count between BMRC-stage I and stage II TBM patients, and the decrease was particularly obvious in BMRC-stage III TBM patients, and the proportion of the cases with NK cells and B cells below the normal reference value was significantly higher than that in BMRC-stage I and stage II patients. It is suggested that the decrease of NK cells may be one of the factors leading to the increase in the severity of clinical TBM patients, and the body’s immune response will also decrease, resulting in the decline of its resistance ability to Mtb, and the number of people with immune abnormalities will also increase (Holub et al., 2015; Thao et al., 2018). Thwaites et al. have found that among the HIV-negative TBM patients, the mortality was 20% in BMRC-stage I patients, 30% in BMRC-stage II patients, and 55% in BMRC-stage III patients (Tunkel, 2005). Therefore, in the early diagnosis and clinical treatment monitoring process of TBM patients, when the absolute count of various lymphocyte subsets in patients plummeted, especially NK cell changes, it is necessary to timely judge the disease progression to adjust the clinical treatment regimen, reduce mortality, prevent disease deterioration, and improve prognosis.

The main limitations of this study are as follows: (1) Due to the low incidence of TBM, the sample size of this study is not large enough, especially for severe TBM patients with BMRC-stage III; (2) This study focused on the number and function of immune cells in peripheral blood, without simultaneously studying immune cells in CSF. Therefore, we will further study the distribution and functional changes of immune cells, especially NK cells, in CSF of TBM patients in future studies.

In conclusion, the absolute counts of lymphocyte subsets, especially NK cells, in the peripheral blood of TBM patients were significantly reduced. Moreover, the reduction of these immune cells was closely related to the disease severity and had a certain correlation with cellular and humoral immune responses. Therefore, this study understood the clinical immunological characteristics of TBM patients, which helps to better understand the immune mechanism of TBM caused by Mtb infection. It can provide reliable indicators for evaluating the immune status of TBM patients in clinical practice and provide the experimental basis for future host-directed therapy of TBM patients.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.

The studies involving humans were approved by the Ethics Committee of the 8th Medical Center of the PLA General Hospital (approval number: 309201904081530). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

JM: Data curation, Formal Analysis, Writing – original draft. YiL: Data curation, Validation, Writing – original draft. YX: Data curation, Validation, Writing – original draft. WS: Supervision, Writing – original draft. YaL: Supervision, Writing – original draft. HA: Writing – review & editing. XW: Conceptualization, Methodology, Project administration, Supervision, Writing – review & editing. JL: Conceptualization, Writing – review & editing.