Lymphocyte profiles in the peripheral blood of TB patients
Most researchers have studied changes of lymphocyte subsets by means of proportion method in peripheral blood of TB patients, and the results were inconsistent. In some studies, the percentages of CD4+ T cells were decreased, while the percentages of CD8+ T cells were unchanged (15). Some studies showed that CD4+ T and NK cells were reduced while CD8+ T and CD19+ B cells were increased, especially in advanced or disseminated TB (11). Morais-Papini et al. reported that the absolute numbers of NK cells, NKT cells, CD4+ T cells and CD19+ B cells in the TB group decreased significantly when compared to the controls, the percentage of CD19+ B cells and NKT cells also reduced, but the percentage of CD4+ T cells increased (16). Guglielmetti et al. observed reduction in the absolute numbers of CD4+ T cells but no difference in the percentage of these cells (9). A study from Mexico reported that the percentages and absolute numbers of B cells were significantly lower in pulmonary TB patients than in healthy donors, the percentages and absolute numbers of T cells were similar in TB patients and healthy donors, and no significant differences in percentages of CD4+ or CD8+ T cells between TB patients and healthy donors (17). We have noted that high percentage of lymphocyte subsets does not mean a high absolute count. A false high proportion may be due to the reduction of other lymphocyte subsets, and the proportions can be completely kept in the normal rage when all the lymphocyte subset counts decreased or increased simultaneously. In our study, the absolute counts of T lymphocytes, CD4+, CD8+, NK, NKT, and B lymphocytes based on large clinical data were analyzed, and we found that the counts of each subsets hardly exceed the reference range, and 75.3% of TB patients had one or more of the six subsets below the reference range (14). These results showed that TB patients displayed an altered lymphocyte profile in the peripheral blood. Therefore, we still need absolute counts of these subsets based on large data to draw a definite conclusion.
Lymphocyte subsets and clinical features
Tollerud believed that the count fluctuation of CD4+, CD8+ T cells would not exceed 10% from 20 to 70 age years old in healthy people (18). But in our patients studied, the absolute counts of six cell subsets were at a high level in the 26–44 age group and then decreased with the increase of age after 45 years old, and the absolute counts of CD4+ T cell subsets was particularly affected by age. We speculated that the pathogenesis for young TB patients (< 26 years old) may be related to insufficiency of immune cells; for middle aged patients (26–44 years old), the pathogenesis may be increased infection opportunities; for old patients (> 60 years old), their immune function had decreased to a low level, which were manifested by the lowest absolute counts of lymphocyte subsets. Therefore, the old age was a key factor affecting the immune status of TB patients. The fifth national TB epidemiological sampling survey in China found that the proportion of elderly TB patients (> 60 years old) was as high as 48.8% (19). Other study also manifested that T and B lymphocyte number in elderly TB patients were significantly decreased, and their immune function was lower than that in young and middle-aged patients, which directly affected the treatment effect and cure time of elderly patients (20). Therefore, during the treatment course of senile TB patents, in addition to using chemicals to kill M. tb, the clinicians should pay more attention to the immune regulation of patients, such as giving the immunoregulator to improve cellular immune function, which may be helpful to control disease. Gender factor also had a certain impact on the immunity of patients, the lymphocyte subset counts especially NKT cells in the males was higher than female, which was not completely consistent with that the male was susceptible to TB genetically and the TB incidence in the male was significantly higher than that in the female (19). This may be due to that the reduction of lymphocyte counts are not the only determinant leading to TB.
Although the average absolute counts of each subset were not significantly affected by the etiological factor, the percentage of TB patient with CD8+ T lymphocyte counts lower than the reference range was obviously higher in etiological positive patients (40.8%) than that in negative patients (25.4%), which may be due to that CD8+ T cells play an important part in protective immunity against M. tb and can limit pathogen growth by lysis of M. tb-infected cells (21). Therefore, we speculated that the patients confirmed with positive etiological detection results may have insufficient CD8+ T cell immune function, which brings challenges to the control on M. tb spread.
The IGRAs have been widely used in clinical auxiliary diagnosis of TB. However, IGRAs have certain false-negative rates in TB patients (22). In our study, the average count in each subset in PTB patients were higher in IGRA-positive group than in IGRA-negative group, especially T lymphocyte and CD4+ T lymphocyte. In IGRA-negative group, the T and NKT lymphocyte counts below the reference range were in 54.2% and 50.0% patients respectively. Therefore, in clinic, if there is a suspicious TB patient with negative IGRA result, it is necessary to distinguish whether it is true-negative, not infected with M. tb, or false-negative due to insufficient lymphocyte count or functional deficiency. It has been reported that lymphocyte subsets were strongly associated with immune response in both QFT-Plus and T-SPOT, and the patients with CD4+ T cell ≥ 650/ L and CD8+ T cell ≥ 400/ L had significantly higher positivity rates in both QFT-Plus and T-SPOT, which was a good evidence of our view (23).
Cellular immunity has been considered to play a key role in anti-TB immunity, however the roles of humoral immunity are unclear in regulating the immune response against M. tb (8), but some studies showed that B cells also played a role in anti-TB immunity by antibody interacting with cellular immunity (24). In our study the absolute counts of B lymphocyte did not show significant difference between the IgG-positive and IgG-negative group, but the absolute counts of T and CD8+ T lymphocyte decreased significantly in the IgG-positive group. We cannot fully explain this result, but previous studies (25) have shown that B lymphocytes induced by M. tb antigens can differentiate into efficient and short-lived plasma cells and secrete specific antibodies to play an anti-TB role; some of them can differentiate into long-lived memory B cells, when they encounter pathogens again, they can differentiate into new plasma cells and memory B cells quickly, and produce a large number of antibodies. Therefore, when the immune response shifted from Th1 to Th2 in TB patients, B cells differentiated into plasma cells to secrete antibodies, which leaded to the reduction of B cell in peripheral blood.
Owing to the negative charge of sialic acid on the surface of erythrocytes membrane, erythrocytes can repel each other and keep the distance of about 25 nm between each other, and they can disperse, suspend and sink slowly ex vivo. The increase of fibrinogen and immunoglobulin in the plasma leads to a marked increase of erythrocyte sedimentation rate (ESR) in TB patients. We found that the absolute counts of T cells, CD4+ T cells, CD8+ T cells and B cells were lower in patients with elevated ESR, while NK and NKT cells were almost unaffected. Because the ESR of TB patients can reflect the severity of the infectious disease to some extent (26, 27), we speculated that the counts of T, CD4+ T, CD8+ T, and B lymphocyte can better reflect the severity of the disease.
X-ray or CT lesion grading for pulmonary involvement was adopted for disease assessment. In our study, the absolute counts of T, CD4+ T, CD8+ T, and B lymphocyte decreased significantly with the increase of the numbers of pulmonary lobes involved; and the absolute counts of T and CD8+ T cell had a greater impact on whether there were cavities or not. It had been reported that the numbers of T, CD4+ (16, 28), CD8 +, and B lymphocyte (16) in patients with unilateral pulmonary lobe lesions were higher than those in patients with bilateral pulmonary lobe lesions, which was consistent with our results. Thus, insufficiency of lymphocyte count has a great impact on the progression and severity of TB and the lymphocyte subset detection is important for TB patients with extensive lesion.
NK cells are not only the first barrier of anti-TB immunity in human, but also play an important regulatory role in the anti-TB immune responses (29). In our study, the NK cell absolute counts below the reference range were in 49.2% extra-PTB patients, which was higher than the PTB patients. We speculated that the insufficient number of NK cells may be associated with the extrapulmonary dissemination of TB.
Lymphocyte subsets and complications
Among 22 PTB patients with six subset counts all lower than the reference ranges, most of them had diabetes mellitus, hypoproteinemia, anemia and liver injury. Therefore, we further studied the lymphocyte subsets changes influenced by diabetes, anemia and low serum albumin.
Diabetes mellitus and active TB interact with each other through blood glucose level, immunity and other factors, forming a reciprocal vicious circle (30, 31). On one hand, the metabolic disorder and immune injury in patients with diabetes mellitus promote the incidence and development of TB (30); on the other hand, TB also aggravate metabolic disorder of diabetic patients (30), nearly 13% of pulmonary TB patients complicated with diabetes (32), which poses a serious threat to the lives of patients (33). We found that the TB patients complicated with diabetes mellitus had lower T, CD4+, CD8+, and B lymphocytes counts, and other study also showed that coincident diabetes altered the cellular subset distribution of T cells, B cells, dendritic cells and monocytes in active TB (34). We also found that the NKT cells were higher in TB patients complicated with diabetes. NKT is a unique subset of T lymphocytes, having both T cell receptors and NK cell receptors on their cell surfaces. NKT cells are functionally distinct from conventional CD4+ and CD8+ T cells, responding rapidly to lipid rather than peptide antigens, and secreting large amounts of Th1 and Th2 cytokines (35). It was reported that NKT cells significantly increased in TB patients complicated with type 2 diabetes mellitus (36), which was consistent with our results. The reason may be due to the high bacillary burden existed in these patients (37).
In this study, the T, CD4+, CD8+, NK, and B cells in TB patients with anemia and low serum albumin were significantly decreased. The albumin was synthesized and secreted to extracellular by liver cells instead of being reserved in liver. Normal albumin level can represent the normal liver function and reflect the nutrition and health status of the host to a certain extent.
Based on all the data above, we think that it is necessary to evaluate the immune status of TB patients by lymphocyte subsets detection. Especially for those complicated with diabetes mellitus, hypoproteinemia, anemia, and liver injury, the lymphocyte subsets detection can help clinicians make comprehensive judgments and formulate treatment plans suitable for individuals. If necessary, immune intervention can be provided to these patients to promote the recovery of immune function.