Despite the availability of specific chemotherapy since the 1950s has contributed to drastically reducing the number of cases (1), the WHO Global Tuberculosis Report of 2021 still defines tuberculosis (TB) as one of the top causes of death from a single infectious agent worldwide (ranking above HIV/AIDS) (2). The main reasons for the “re-emergence” of TB are the increased onset of socio-economic disparities related to migratory flow from developing countries and the increase in immunosuppressive diseases or, anyhow, diseases requiring immunosuppressive treatments (3).

Host defense mechanisms play a key role in determining the clinical course and the eventual different manifestations of such infection (4). After the inhalation of tubercle bacilli, several scenarios may follow. Inhaled bacilli may be trapped in the upper respiratory airways and expelled by the mucociliary clearance or may reach the alveoli being engulfed by alveolar macrophages in phagosomes and destroyed. Sometimes, a part of bacilli manages to escape from this killing mechanism, replicates within the phagocytic cells, and leads to their destruction. The consequent production of inflammatory cytokines and chemokines results in the in-site recruitment of additional macrophages and neutrophils, which, once again, try to engulf mycobacteria without destroying them. About 2–3 weeks after infection, a T-cell response (both CD4+ and CD8+) leads to the granuloma formation (5). The tuberculous granuloma is an organized structure, formed by specialized macrophages harboring and surrounding the mycobacteria at the center and a rim of lymphocytes, that acts as a barrier to preventing the spread of infection. Over time, the granuloma usually undergoes a productive reaction, in which prevails the formation of a special granulation tissue tending to transform into fibrotic tissue and to heal by scarring (6, 7).

Immunosuppressive conditions, involving particular cell-mediated immunity or a tumor necrosis factor alfa (TNF-α) deficiency, can limit the formation of a well-organized granuloma. This results in the persistence of an exudative reaction (i.e., liquid material in the interstitial space with a tendency to develop caseous necrosis), favoring, therefore, the spread of infection and the development of active disease (7, 8). A subsequent state of immune suppression can also provoke the re-activation of the disease starting from bacillary elements that eventually remained “walled-up alive” inside a granuloma, being, therefore, capable of resuming their virulence (6). Malnutrition, with the consequent immune deficits that derive from it, as well as poor hygienic-sanitary and socio-economic conditions, represent particularly relevant risk factors for re-infection, especially in the population of immigrants (8).

Based on the evolution of the pulmonary lesions, it is radiographically possible to distinguish between mainly “productive” and mainly “exudative” TB forms (9, 10). Productive TB is characterized by more or less extensive fibrous and retracted areas with circumscribed and calcified caseous centers (nodules) or frank cavity lesions with strongly thickened walls (caves). These lesions typically affect the apical and posterior segments of the upper lobes or the upper segments of the lower lobes (10). Exudative radiological manifestations are lobar or segmental consolidations (tuberculous pneumonia) and pleural effusions (9) frequently involving atypical locations, such as the lower segments of the lower lobes, the middle lobe, the lingula, and the anterior segment of the upper lobes. These forms may also be multifocal (11).

The aim of this study was to evaluate the immunological status of patients with active pulmonary TB compared to an age and sex-matched group of hospitalized patients with other bacterial community-acquired pneumonia (CAP). Additionally, we tried to assess the possible association between an eventual alteration in the circulating lymphocyte subsets and the development of different radiological patterns of active TB (i.e., productive forms with typical location or exudative forms with atypical locations) and whether a different geographic provenience may influence the variability in the immunological status and in the radiological patterns of presentation of TB.

In our study, patients with TB showed global lymphocytopenia with a significantly lower count and percentage of CD4+T-cells in the peripheral blood than patients affected by other bacterial CAP. This finding accords with previous studies that suggested a decreased CD4+ T-lymphocyte count in the peripheral blood of patients with TB (12–20). Moreover, no significant differences were observed in the percentages of CD19+ B-lymphocytes and CD16+56+ NK cells between the TB and CAP groups, while the percentage of CD8+ T-lymphocytes was even increased. This data could be interpreted as indicating that the global reduction in the absolute count of total lymphocytes observed in our patients with TB is more pronounced for CD4+ T-lymphocytes.

It is generally accepted that CD4+ T-lymphocytes are an essential component of protective immunity against tuberculosis (21, 22). As a confirmation, patients with AIDS, which is characterized by a specific blood reduction of these cells, have a higher incidence and severity of tuberculosis (17, 23). Following infection, M. tuberculosis is internalized by macrophages and dendritic cells and trapped in a vacuole called a phagosome. Here, mycobacterial polypeptides are digested and the resulting antigenic peptide fragments are loaded onto major histocompatibility complex (MHC) Class II molecules through which they are presented to CD4+ T-cells. The main function of CD4+ T-cells in immunity against TB is to differentiate into T-helper type 1 (Th-1) effector cells and produce interferon-gamma (IFN-γ), which directly activates macrophages for controlling infection, and TNF-α, which induces apoptosis of macrophages infected with mycobacterium and guides the granuloma formation (4, 24). Peripheral CD4+ T-cell reduction in patients with TB has already been interpreted as a consequence of an augmented pooling of these cells at the site of infection. This hypothesis has been sustained by molecular and clinical studies (19, 25–27). Other authors have suggested an important shift toward the pulmonary local compartment to explain the transient lymphocytopenia observed during the acute phase of other bacterial CAP (28). Anyhow, we must not forget that TB itself may determine a further depletion in the CD4+ T-cells compartment. M. tuberculosis and its constituents are known to stimulate monocytes in producing an array of cytokines among which the transforming growth factor beta (TGF-β) is characterized by suppressive action on CD4+ T-lymphocytes (29, 30). Further evidence come from the finding of lower levels of CD4+ T-lymphocytes in HIV/TB subjects compared to subjects with only HIV (31). In addition, the decreased CD4⁺ T-cell counts in patients with TB have been shown to return to normal levels after the correct anti-TB chemiotherapy (12, 32, 33).

The role of CD8⁺ T-lymphocytes in the control of M. tuberculosis infection is still controversial. CD8+ T-lymphocytes are MHC Class I-restricted cells typically involved in cytotoxicity during adaptive immune responses. Like CD4+ T-lymphocytes, CD8+ cells are also capable of producing interleukin 2 (IL-2), IFN-γ, and TNF-α (34). MHC Class I molecules gain their peptide ligands primarily from the cytoplasm. Despite M. tuberculosis being usually found within the phagosomes of infected macrophages, either the bacteria, and the bacterial products can gain access to the cytosol (35). As a result, CD8+ T-cells are able to recognize infected cells and eliminate them through different mechanisms, such as the Fas–Fas ligand interaction, the TNF-mediated killing, and the release of perforin, granzyme, and granulysin by granule exocytosis. As a confirmation, CD8+ T-cells from patients with TB have been shown to exhibit greater cytotoxic activity than healthy donors (36). In addition, a subset of unconventional CD8+ T-cells has been identified that can recognize M. tuberculosis-specific antigens presented by a variety of other non-classical MHC I-like molecules, namely, MHC Ib and CD1a-d (37). Lipoarabinomannan (LAM) seems to be the most potent mycobacterial lipid antigen in activating a specific type of polycytotoxic CD1b-restricted CD8+ T-cells (38, 39). These LAM-responsive unconventional CD8+ T-cells co-express perforin, granzyme B, and granulysin and may have a key role in infection control (38). Available data on CD8+ T-lymphocytes count in patients with TB are contrasting. In some studies, the CD8⁺ T-cells count was significantly decreased (15, 16, 40, 41), while in other studies it was unmodified (13, 15) or increased (14, 32) compared to healthy controls. In our study, the finding of a significantly decreased CD4+/CD8+ ratio in patients with TB compared to patients with CAP might be explained by assuming both a peripheral depletion of the CD4+ T-cells compartment and an increase in the peripheral CD8+ T-cells population. This is evident from the CD4+ and CD8+ percentages in patients with TB compared to the CAP group.

Also, CD16+56+ NK cells and CD19+ B-lymphocytes may be found in the constitution of the TB granuloma (6). NK cells are capable of secreting IL-2, IFN-γ, and TNF-α without requiring any priming, thus representing bridge effectors between the innate and adaptive immune systems (42). During the early phase of M. tuberculosis infection, NK cells may provide an early source of IFN-γ that seems to be crucial for establishing the optimal proinflammatory cytokine milieu in shaping the T-cell response toward a Th-1-protective phenotype. NK cells can also exert cytotoxic activity against infected macrophages that are not well-equipped to control the intracellular growth of mycobacteria, with their consequent release into the extra-cellular space. This may allow NK cells to interact directly with whole bacteria or bacteria-derived products through the recognition of mycobacterial ligands by activating NK cell receptors (43, 44). B-lymphocytes can act as antigen-presenting cells that engulf antigens or whole mycobacterial bacilli and present them to T-cells in the granuloma. As a result, B-cells contribute to the induction of CD4+ T-cells responses to TB. In addition, B-cell antibodies may elicit effector functions on extra-cellular mycobacteria such as opsonization, which facilitates bacterial phagocytic uptake, antibody-dependant cellular cytotoxicity, and complement activation, which can further enhance opsonization and bacterial lysis, but also phagocytosis through complement receptors (45). Very few data are available in the literature about CD16+56+ NK cells and CD19+ B-lymphocytes count in patients with TB (46). In our study, active TB was associated with significant positive odds ratios in finding an absolute count of CD4+ T-lymphocytes, CD19+ B-cells, and CD16+56+ NK cells below normal ranges compared to other bacterial CAP. Only the odds assessing an actual circulating depletion for the CD8+ T-cells compartment did not reach a statistical significance. As no statistically significant difference was detected in the percentage distribution of CD19+ B-lymphocyte and CD16+56+ NK cells between the TB and the CAP group, we can speculate that the achievement of statistical significance only in the absolute count of these lymphocyte subsets is probably affected by a numeric reduction in peripheral blood subsequent to their pooling in the infected lung parenchyma.

Patients with TB affected by exudative forms with atypical locations showed a greater lymphocytopenia with a lower absolute count in all the lymphocyte subsets compared to patients with TB with productive forms and typical locations. In exudative TB forms with atypical locations, the reduction in either the absolute count or percentage reached a statistical significance only for the CD19+ B-lymphocytes. These data suggest that the coordinated response of the humoral and cellular adaptive immune system against M. tuberculosis may have a key role in the development of those encapsulating and fibrotic productive reactions giving rise to nodular or cavitary lesions that typically involve the apical and posterior segments of the upper lobes and in the upper segments of the lower lobes. Alternatively, an impaired humoral immunity may lead to a greater prevalence of exudative processes characterized by liquid and corpuscular exudation and modest connective reaction that frequently involve the lower segments of the lower lobes, the middle lobe, the lingula, and the anterior segment of the upper lobes or have a multifocal involvement (Figure 1). In this regard, studies on mice suggested that CD19+ B-lymphocytes may regulate the level of granulomatous reaction, cytokine production, and T-cell response (47). Other studies are needed to establish the role of reduced B-cell counts in patients with TB.

The current TB epidemiological situation in Italy is characterized by a low incidence in the native population, with a concentration of cases in risk groups, such as immigrants. In this study, the majority of TB cases affecting foreign-born people were detected in black Sub-Saharan Africans. These subjects showed a statistical reduction in the total count and percentage of CD4+ T-lymphocytes compared to white Italian and other European patients with TB, despite they were younger compared to the latter. In the absence of further predisposing factors for immunosuppression (i.e., enrolled subjects were HIV-negative and free from immunosuppressive diseases, as exclusion criteria), TB cases in black Sub-Saharan Africans might represent forms of newly acquired infection or reactivation, favored either by a state of malnutrition or by poor hygienic-sanitary and socio-economic conditions (i.e., urban overcrowding and poverty). Anyhow, this suggestion does not fully explain the higher prevalence of atypical/exudative forms in this TB population. This difference in immune responses might be mostly genetic/inherited from ancestors. According to the US Centers for Disease Control and Prevention (CDC), the TB case rate is eight times higher for Black or African American persons than for non-Hispanic White persons (48). A possible explanation is that white people of European descent are characterized by a more effective immune response to TB infection compared to black people of Sub-Saharan African descent. It is known that European emigrants brought Koch's bacillus to America. The same must be said of other countries which in the past, before the arrival of the Europeans, were totally free from the disease, while today are noted as areas of endemic TB, such as Sub-Saharan Africa. As a result, the less advantageous exudative response detected in Sub-Saharan Africans might be the result of a less effective natural selection in an environment in which there were fewer mycobacteria compared to Europe. Further genetic and molecular studies are required to confirm this suggestion.

Several limitations should be noticed in our study. First, the number of patients with TB enrolled was small. Indeed, our dedicated ward for respiratory diseases is located in a South Europe country with low TB incidence [in 2020, the incidence of tuberculosis in Italy was 6.6 cases per 100,000 people (49)], which makes it more difficult to collect a large number of patients. However, we were able to collect a number of patients that is comparable with other similar studies on the topic (13, 16, 17, 32, 41). Second, the analysis did not include a control group of healthy subjects for comparison. That is why this study was the result of observations made on a population of hospitalized patients requiring medical care. Anyhow, we tried to overcome this limitation by comparing the absolute count in the different lymphocyte subsets assessed in both the TB and CAP to the lower limits of the respective standard reference ranges in terms of the odds ratio. Third, the relatively small sample size in this study did not allow to pre-establish an ideal sample size for the subgroup analyses targeted for different radiological patterns (typical/productive or atypical/exudative forms) and ethnicities (black Sub-Saharan Africans or East European). Furthermore, “Africans” and “Europeans” subjects included in this study are not clearly entirely representative of the highly heterogeneous groups of people who originated in these two different continents. Despite these limitations, we believe that our suggestions could represent interesting starting points for further studies evaluating the potential interaction of active tuberculosis with the host's immune system.

The reduction in the absolute count of lymphocyte subsets in our patients with TB may suggest increased compartmentalization at the site of infection compared to other bacterial CAP. Such depletion of circulating lymphocytes was more pronounced for the CD4+ T-cells subset, as it was evident from the CD4+ T-cells absolute count and percentage observed in patients with TB compared to the CAP group. On the contrary, the percentage of CD8+ T-lymphocytes was increased, influencing the statistically significant reduction of the CD4+/CD8+ ratio. Interestingly, exudative forms with atypical locations of pulmonary TB were associated with a significantly lower absolute count and percentage of CD19+ B-lymphocytes. This association may explain an abnormal granulomatous response resulting in suboptimal bacterial control. Finally, it should be noted that our black Sub-Saharan African patients, although younger, exhibited greater peripheral impairment in the CD4+ T-lymphocytes compartment and a greater prevalence of exudative and atypical forms of TB compared to white European patients with TB. The nutritional status of our patients has not been objectively evaluated in our study. Similarly, a molecular and genetic study assessing a different host's immune response has not been performed. In this regard, future studies are required to evaluate the potential interactions between M. tuberculosis and eventual underlying constitutive and environmental factors in depressing the immune system. Despite our suggestions needs confirms, examining lymphocyte subsets in patients with TB may be of great value in evaluating the immune function of these patients.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Local Ethics Committee of the Policlinico Universitario Riuniti of Foggia (Institutional Review Board Approval N° 17/CE/2014). The patients/participants provided their written informed consent to participate in this study.

MF was the guarantor of the content of the manuscript and including the data and analysis. GS, DL, EG, IC, and MF contributed to the conception and design of the study. DL, EG, IC, CQ, PS, PT, ES, and DP contributed to the acquisition, the analysis, and the interpretation of data. All the authors contributed to drafting the work, revising it critically, read, approved the final version of the paper, and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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