Tuberculosis (TB) remains the leading cause of human death from a single infectious agent with a total of 1.4 million deaths in 2019 (1). The outcome of a pulmonary TB infection can either be complete clearance of the pathogen to active tuberculosis (ATB) disease. The percentage of the infected population developing the clinical symptoms of TB remains small with a much higher percentage being able to control the naturally acquired infections (2, 3). This latently infected population largely remains asymptomatic and in some cases even clear the infections (4). Generation of robust T cell responses is critical in the immunity to TB and are responsible for a dynamic balance between the host and pathogen in a latent TB infection (LTBI) (5). While co-morbidities, such as, with HIV is a known factor for the reactivation of LTBI (6), the underlying causes for the susceptibility to the active disease remains unknown. Antigen specific responses to TB infection, including novel features of T cell differentiation have revealed pathways that facilitate the immune control of infection (7). The production of inflammatory cytokines such as gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) are critical in the protection against long-term rampant Mtb growth and loss of these factors leads to heightened Mycobacterium tuberculosis (Mtb) replication and death (8, 9). Indeed, stimulation with Mtb antigens Early Secretory Antigenic Target (ESAT)-6 and Culture Filtrate Protein (CFP)-10 induces IFN-γ and TNF-α production by the CD4⁺ and CD8⁺T cells that may provide tools to study the role of these early responses in protection from a fatal infection.

Characterizing the phenotype and function of these early T cell responses could provide a critical tool to distinguishing latent from active TB disease in future experiments wherein, the macaques would be followed for a longer duration of time (10). The aim of this study is to characterize the early T cell responses in a nonhuman primate (NHP) model of TB. The model recapitulates humans, wherein, the infectious doses differ between individuals. There have been reports of differential impact on functional CD4⁺ and CD8⁺ T cell responses by the disease stage and bacterial burden (11–13). However, there is a paucity of data on the distinguished early adaptive response signatures in a biologically and physiologically relevant animal model. The NHP model of TB serves as an excellent model recapitulating the spectrum of immune responses observed in humans, including the pathology (14, 15). Manipulating the bacteria in a macaque model of TB infection presents a valuable tool to dissect the local immune responses in a TB predominant microenvironment that is not possible in any other animal model (16–18). We hypothesized that measuring the TB-specific T cell responses early in a rhesus macaque model of TB infection could provide a better understanding of the early responses and their potential role in disease progression. Hence, we performed high parameter flow cytometry on stimulated bronchoalveolar lavage (BAL) cells from macaques infected via aerosol, with a low dose and high dose of Mtb, to measure key cytokines in TB infection, IFN-γ, TNF-α and IL-17 produced by CD4⁺ and CD8⁺ T cells in response to ESAT-6/CFP-10 and Mtb Cell Wall Fraction (Mtb CW). This enabled a comprehensive elucidation of the differences in the early responses and provided a potential tool to delineate the disease progression in long-term studies.

Our results clearly outline the differences in the early Mtb-specific T cells responses in a low dose versus higher dose infection in a rhesus macaque model of TB. The macaques exposed to a low-dose controlled Mtb infection were associated with an early IFN-γ and TNF-α response in Mtb-specific CD4+ T cells. A high dose infection caused a significantly higher TNF-α response in the CD8⁺ T cells at 3 weeks post-infection but no noticeable changes in the IFN-γ response this early in infection. TNF-α secreting Mtb-specific CD4⁺ T cells are a promising candidate to differentiate between active and latent TB infections (10, 22). In the study by Harari et al. (22), significant increase in the proportions of Mtb-specific CD4⁺T cells expressing TNF-α was seen in patients with active disease and proposed to be the strongest predictor of diagnosis of active disease. Indeed, commensurate with these findings, we observed a significantly higher TNF-α response in the Mtb-specific CD8⁺ T cells in the group infected with a higher number of bacilli. The difference in our study was that here we compared two different doses of infection of Mtb in a biologically relevant animal model. Though the difference between TNF-α expression by CD4⁺ T cells was not significantly different between low dose and high dose infection groups at week 3, there was a consistent increase in the TNF-α expression from pre-infection to week 3 in the high dose group. Hence, while the low dose elicits an earlier TNF-α response that then remains at similar levels up till 3 weeks post infection, the same response is slower to develop in the higher dose but more robust as the infection progresses. Previous studies have shown the detection of Mtb-specific effector CD4⁺ T cells expressing IFN-γ and/or TNF-α can distinguish between a latent TB and active TB infection (12, 23). A recent study demonstrated that increased amounts of TNF-α in an active TB infection subverted the immune-surveillance by perturbing dendritic cell mediated antigen transportation to the lymph node allowing bacterial reserve (24). Further studies on phenotyping the subsets in our study to distinguish the effector and memory functions could provide a highly discriminatory readout.

IFN-γ producing CD4⁺T cells are the cornerstone of protective immunity in pulmonary Mtb infections (25). In the two doses studied here, the difference in the CD4⁺IFN-γ⁺ response to Mtb antigens, ESAT-6/CFP-10 and Mtb CW, was the highest at 1-week post-infection and diminished by week 3 post-infection. IFN-γ deficient mice studies have demonstrated a lack of survival even in low-dose Mtb infections with progression to active disease (26, 27). This early gamma response in the low dose infection alone could be representative of the protective role of CD4⁺ T lymphocytes in mediating macrophage activation via iNOS expression (27, 28). IFN-γ is known to promote iNOS expression in macrophages that in turn serves to recruit other reactive nitrogen intermediates (RNI) (29). Not only is this early gamma response critical for TB control, it also plays a role in the long-term survival of the host by working synergistically with the early TNF-α responses and thus contributing to the granuloma formation that controls the disease progression (30). Interestingly, we observed a significantly higher CD8⁺IFN-γ⁺ T cells in the high dose group in response to stimulation with Mtb CW at 3 weeks post-infection, but did not see a similar response to ESAT-6/CFP-10 stimulation. While the role of CD4⁺T cells in IFN-γ production in TB is well documented, the role of CD8⁺ T cells in the IFN-γ production in human TB is less well studied. A part of the role of the CD8⁺ T cells has been elucidated in mice experiments, wherein, mice deficient in CD8⁺ T cells were unable to control Mtb infection (31). Additionally, CD8⁺ T cells have been shown to undergo phenotypic and functional changes, comparable to CD4⁺ T cells during pulmonary Mtb infection (32). Mtb-specific CD8⁺ T cells have demonstrated differences in prevalence, frequency, phenotypic and functional profiles in latent versus active TB disease (33). Similar to our findings, a higher Mtb-specific CD8⁺ T cells frequency (60%) was observed in the TB patients compared to 15% in LTBI patients. These CD8⁺ T cell responses were directed against ESAT-6/CFP-10 in vitro stimulation comparable to our study in NHP model. Also, the IFN-γ response in the Mtb-specific CD8⁺ T cells was not very different between active and LTBI cases like our study, in which we did not observe a significant difference in the CD8⁺IFN-γ⁺ T cells in the low dose and high dose when stimulated with ESAT-6/CFP-10.

While Th1 cells plays a distinct role in rendering protection in TB via production of IFN-γ and activating antimicrobial action in macrophages (34), Th17 cells implements neutrophilic inflammation, tissue damage and TB pathology (35). The data on the role of Th17 cells in TB remains controversial with some groups reporting a higher frequency correlating with TB protection in latent patients (36) while others reported lower expression in latent patients and increased frequencies in active or multi-drug resistant patients (37–39). Some are of the verdict that Th17 cells are minimally expressed in TB and do not have a significant role to play in the protection and/or pathology of TB in humans (40, 41). In our study, we observed a significant increase in the IL-17 expressing Mtb-specific CD4⁺ and CD8⁺ T cells in the high dose infection compared to the low dose at 3 weeks post-infection. Mtb infection in humans induces IFN-γ and IL-17 and the main source is the CD4⁺IFN-γ⁺IL-17⁺ T cells (38). Moreover, the antigen-expanded CD4⁺IL-17⁺ T cells correlates with the clinical parameters associated with disease severity. Given these findings, the expansion of Mtb-specific CD4⁺IFN-γ⁺IL-17⁺ T cells has been proposed as a biomarker for prediction of clinical outcome in active TB patients (38). T cells from MDR-TB patients has been shown to express high levels of IL-17 via the strong TLR-2 dependent TGFβ production by antigen-presenting cells (37). Mouse studies mimicking human vaccination post Mtb-exposure verified the presence of increased IL-17 which correlated to lung tissue damage (42). Conversely, protective role of Th17 responses have also been reported in the lung tissue following BCG vaccination (43, 44). However, it is to be noted that it is feasible to observe an increased bacterial burden with a higher initial inoculum that could impact the disease kinetics. While this study aims to identify the very early differences in the adaptive response to Mtb, it will be critical to follow the kinetics over a longer duration in future studies to ascertain the true role of IL-17 in this model. Overall, we have demonstrated a distinct phenotype of Mtb-specific CD4⁺ and CD8⁺T cells following in vitro stimulation with ESAT-6/CFP-10 and Mtb CW early in TB infection in a biologically and physiologically relevant animal model. Further, in depth phenotyping of these subsets into tissue resident memory cells at later time point in future studies would prove instrumental in improving our understanding of these early T cells responses and their correlation to disease progression.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by Texas Biomedical Research Institute IACUC.

RS, DS, JR, and DK designed the study. RS and DS executed the experiments and analyzed the data. RS and DK wrote the manuscript. All authors contributed to the article and approved the submitted version.

This work was primarily supported by NIH grants R01AI111943 and R01AI123047 (to DK and JR), 1 K01 OD031898-01 (to RS) with additional support from NIH grants R01AI111914, R01AI134240, R01AI138587, and U19AI111211 and institutional grants from the Office of the Director, NIH P51OD011133 (to SNPRC), P30 RR00165 and P51OD011132 (to YNPRC), and P30 AI050409 [Emory University Center for AIDS Research (CFAR)].

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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