IL-10-Producing CD8⁺ T Cells Impair Immune Control During Chronic Mycobacterium tuberculosis Infection

1. Introduction

Tuberculosis (TB) remains a major global health challenge and a leading cause of mortality from a single infectious agent [1]. The disease is caused by the intracellular pathogen Mycobacterium tuberculosis (M.tb), first isolated in 1882 by Robert Koch [2]. A seminal study by Cyktor et al. examined CD8+ T cell dysfunction during chronic M.tb infection in the same CBA/J susceptible mouse model used in the present work [3]. That study identified accumulation of IL-10-producing CD8+ T cells with a dysfunctional phenotype (PD-1+, Tim-3+, CD122+) and showed these cells expressed a restricted T cell receptor (TCR) repertoire dominated by Vβ8 and Vβ14 chains [3]. Critically, in vivo CD8+ T cell depletion conducted during days 90-120 of infection did not significantly alter pulmonary bacterial burden in that work, leading the authors to conclude that the significance of IL-10-producing CD8+ T cells in tuberculosis pathogenesis remained unclear [3]. In the present study, we re-examined this question by conducting CD8+ T cell depletion beginning at day 100 and extending through day 150 of chronic infection, hypothesizing that the pathogenic role of IL-10-producing CD8+ T cells may become more pronounced during the late chronic phase of infection when these regulatory populations are fully established. While primarily a pulmonary pathogen, M.tb can disseminate through the bloodstream to involve extrapulmonary organs, including the spleen, liver, and lymph nodes [4]. Tuberculosis remains one of the leading infectious causes of death worldwide, accounting for approximately 10 million new cases annually [5]. Consequently, the World Health Organization (WHO) has prioritized a 90% reduction in TB-related deaths by 2030, a goal that necessitates a deeper understanding of the molecular and immunological mechanisms governing infection, latency, and reactivation [6]. Host control of M.tb depends on coordinated innate and adaptive immune responses, particularly cytokine-driven macrophage activation [7]. For decades, the protective Th1 response defined by the production of IL-12 and IFN-γ has been the central focus of research. IL-12 initiates cell-mediated immunity, while IFN-γ enhances the microbicidal capacity of macrophages, stimulating the production of antimicrobial effector molecules [8]. While initially attributed to CD4 T cells, it is now established that Natural Killer (NK) and CD8+ T cells also contribute significantly to the IFN-γ pool [9,10]. However, the complexity of the host-pathogen relationship and the ability of M.tb to subvert these defenses remain primary obstacles to eradication [11]. Accumulating evidence suggests that persistent antigen exposure during chronic infection can functional reprogramming of T cell populations, leading to the acquisition of regulatory phenotypes [12]. A key player in this immunosuppression is Interleukin-10 (IL-10), which antagonizes pro-inflammatory cytokines in the lungs [13]. IL-10 directly inhibits macrophage effector functions, including the production of reactive intermediates, major histocompatibility complex class II (MHCII) expression, and antigen presentation, thereby promoting bacterial persistence [14]. Although IL-10-producing CD8+ T cells were previously identified during chronic M.tb infection, their direct contribution to bacterial persistence and immune suppression remained unresolved [15]. While previous studies identified IL-10-secreting CD8+ T cells during chronic M.tb infection, the direct contribution of these cells to bacterial persistence and suppression of protective immunity remained unresolved. In this study, we investigated the dynamics and functional properties of CD8+ T cells during chronic M.tb infection using susceptible CBA/J and resistant C57BL/6 murine models [16]. We hypothesized that chronic infection promotes the accumulation of IL-10–producing CD8+ T cells that suppress protective immunity. By combining bacterial burden analysis, cytokine profiling, ELISPOT assays, and targeted cell depletion, we suggest that CD8+ T cells adopt an immunoregulatory phenotype during chronic tuberculosis. These findings suggest that vaccine strategies aimed at broadly enhancing CD8+ T cell responses may require careful consideration, as chronic stimulation could promote immunoregulatory phenotypes that facilitate M.tb persistence [17].

2. Materials and Methods

2.1. Mice

Female CBA/J and C57BL/6 mice strains, two widely used murine models for tuberculosis research, were purchased from the Jackson Laboratory, acclimated and maintained in biosafety level 3 facilities at The Ohio State University (OSU) [18]. The mice were 6-8 weeks old and specifically pathogen-free [19]. All animal procedures were approved by The Ohio State University Institutional Animal Care and Use Committee (IACUC) and performed in accordance with institutional biosafety and animal welfare guidelines [20]. Mice were assigned to experimental groups according to strain and treatment condition. Formal randomization procedures were not used.

2.2. Bacterial Strain and Infection Procedure

M.tb str. Erdman (ATCC# 35801) was purchased from American Type Culture Collection (Manassas, VA) and grown in liquid medium containing 0.05% Tween 80 to the mid-log phase [21]. Bacterial suspensions were aliquoted and stored at -80 °C for future use [22]. CBA/J and C57BL/6 mice were exposed using an inhalation system (Glas-Col-Figure 1). The nebulizer compartment of an airborne infection device (Middlebrook, Terre Haute, Ind.) was filled with a suspension M.tb str. Erdman and calibrated to deliver 50 to 100 CFU viable bacteria per mouse lung during 30 min of exposure [23]. Lung bacterial tallies were calculated by culturing serial dilutions of whole-organ homogenates onto Middlebrook 7H11 agar (Life Technologies, Gaithersburg, Md.) [24], and colonies were counted 21 days post incubation at 37 °C. Data are shown as the log₁₀ value of the mean number of bacteria recovered per organ (n = 4).

2.3. Infected Lung Cells Isolation

Mice were euthanized using CO₂, and the thoracic cavity was opened for lung tissue collection. Ten (10) ml of saline buffer containing heparin (50 U/ml; Sigma, St Louis, Mo.) was introduced into the pulmonary artery to perfuse the lungs of erythrocytes and blood. The perfused lungs were carefully removed and placed in cold Dulbecco’s modified Eagle medium (DMEM) (Life Technologies, Gibco-BRL, Grand Island, N.Y.). The lungs were disrupted using sterile razor blades following the removal of the connective tissue and trachea and incubated for 30 min at 37 °C in a final volume of 2 ml of DMEM containing collagenase XI (0.7 mg/ml; Sigma) and type IV bovine pancreatic DNase (30 μg/ml; Sigma). In addition, 10 ml of DMEM was added to stop enzymatic reactions. Digested lungs were filtered and centrifuged at 300 × g. The remaining red blood cells were lysed using ACK lysis buffer (0.15 M NH4Cl, 1.0 mM KHCO3). Cells were resuspended in DMEM plus 10% heat-inactivated fetal calf serum (Gibco-BRL), 1% 1 M HEPES buffer (Sigma), 1% L-glutamine (200 nM; Sigma), and 2% modified Eagle medium-nonessential amino acids (100×; Sigma). Cells were counted using a hemocytometer, and the absolute number per lung was calculated.

2.4. Cells Purification and Culture

CD8+ and CD4+ T-cells were purified from a pool of lung cells overlaid onto HISTOPAQUE 1083 (Sigma) and centrifuged at 400 × g for 30 min without brake to obtain live mononuclear cells. Adherent cells were removed by incubation at 37 °C with 5% CO₂ for 1 h. CD8+ cells were isolated using CD8+ a MicroBeads (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions. Cells were passed over two columns to increase the purity of CD8+ cells, which was more than 97% for all samples. Viable cells were determined by trypan blue exclusion, counted, and resuspended at 2 × 106/ml. Lung cells (2.5 × 105) were cultured in duplicate with 10 μg/ml of OVA (Sigma-Aldrich), M.tb culture filtrate protein (National Institutes of Health, National Institute of Allergy and Infectious Diseases), or Concanavalin A (Con A) (Sigma-Aldrich) for 72 h at 37 °C. Supernatants were stored at −80 °C until analysis.

2.5. In Vivo CD8+ T Cell Depletion

To evaluate the contribution of CD8+ T cells during established chronic M.tb infection, in vivo CD8+ T-cell depletion was initiated during the late chronic phase of disease (day 100 post-infection) and maintained through day 150. Anti-CD8+ monoclonal antibody (clone 53-6.72; BioXCell) or isotype-matched rat IgG2a control antibody (clone 2A3; BioXCell) were diluted to 2.5 mg/mL in sterile PBS and stored at −80 °C until use. At day 100 post-infection, mice received 0.5 mg antibody via intraperitoneal injection, followed by additional 0.5 mg injections every 5 days through day 150 (days 105, 110, 115, 120, 125, 130, 135, 140, 145, and 150). This regimen was designed to maintain sustained CD8+ T-cell depletion throughout the late chronic phase of infection [25]. At the initiation of depletion, CBA/J mice exhibited established pulmonary infection characterized by approximately 6–7 log10 CFU in the lungs. At day 150 post-infection, lungs were harvested for bacterial burden analysis and flow cytometric confirmation of CD8+ T-cell depletion efficiency.

2.6. Enzyme-Linked Immunospot (ELISPOT) Assay for IFN-γ and IL-10 Producing CD8+ T Cells

The frequency of antigen-specific IFN-γ and IL-10 producing CD8+ T cells in infected mouse lungs was quantified using ELISPOT assays (Millipore, Billerica, MA), following the manufacturer’s instructions with minor modifications. Briefly, 96-well Immobilon-P membrane plates were coated overnight at 4 °C with purified anti-mouse IFN-γ or anti-mouse IL-10 capture antibodies (eBioscience) diluted in sterile phosphate-buffered saline (PBS) [26]. Plates were washed three times with PBS and blocked for 2 hours at room temperature with complete DMEM supplemented with 10% fetal bovine serum.

Purified lung CD8+ T cells were resuspended in complete medium and seeded at 2 × 10⁵ cells per well in triplicate [27]. Cells were co-cultured with fixed dendritic cells at a 1:1 ratio in the presence of M.tb culture filtrate protein (CFP, 10 µg/ml) [28]. Wells containing unstimulated cells served as negative controls, while Concanavalin A (5 µg/ml) stimulated wells served as positive controls.

Plates were incubated for 72 hours at 37 °C in a humidified 5% CO₂ incubator. Following incubation, cells were removed by washing with PBS containing 0.05% Tween-20. Biotinylated detection antibodies specific for IFN-γ or IL-10 were added and incubated for 2 hours at room temperature. After washing, streptavidin-alkaline phosphatase conjugate was applied for 1 hour.

Spots were developed using BCIP/NBT substrate solution until distinct spot formation was observed and the reaction was stopped by rinsing with distilled water. Plates were air-dried and analyzed using an automated ELISPOT reader system (Cellular Technology Limited). Results were expressed as spot-forming units (SFU) per 2 × 10⁵ CD8+ T cells after subtraction of background counts from unstimulated wells.

2.7. Statistical Analysis

Statistical analyses were performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA). Data are presented as mean ± standard error of the mean (SEM). Differences between groups were analyzed using Student’s t-test, one-way ANOVA, or two-way ANOVA followed by Tukey’s multiple-comparison test, as appropriate. A p value < 0.05 was considered statistically significant. Study design and data handling. Experimental group sizes were selected based on prior experience and published studies using chronic M.tb infection models. Data distribution was assumed to be approximately normal based on established characteristics of these experimental datasets; formal normality testing was not performed. No animals or data points were excluded from the analyses. Investigators were not blinded during allocation, experimentation, outcome assessment, or data analysis.

3. Results

3.1. Differential Bacterial Kinetics and Lung Pathology in Susceptible and Resistant Models

To characterize the progression of chronic tuberculosis, we compared the bacterial burden in susceptible CBA/J and resistant C57BL/6 mice following a low-dose aerosol infection (50-100 CFU) [29]. In C57BL/6 mice, the pulmonary bacterial load stabilized at approximately 105 CFU by day 21 and remained constant throughout the 150-day observation period. In contrast, CBA/J mice failed to achieve bacterial stasis; pulmonary CFU counts increased progressively, reaching significantly higher levels than C57BL/6 mice by day 60 (p < 0.01) and continuing to rise through day 150 (Figure 1) [30]. This uncontrolled bacterial proliferation in CBA/J mice was associated with extensive lung consolidation and progressive granulomatous pathology [31].

3.2. T Cell Recruitment and Altered CD4:CD8+ Ratios During Chronic Infection

The absolute numbers of T cell subsets recruited to the lungs were quantified to assess the host response. Both strains exhibited a robust influx of CD4+ and CD8+ T cells during the acute phase (day 21) [32]. However, during the chronic phase (days 60–150), the T cell dynamics diverged significantly [33]. While C57BL/6 mice maintained a stable and dominant CD4+ T cell population, CBA/J mice showed a disproportionate accumulation of CD8+ T cells (Figure 2) [34]. Consequently, the CD4:CD8+ ratio in the lungs of CBA/J mice declined significantly over time, dropping below 1.0 by day 120, whereas C57BL/6 mice maintained a ratio consistently above 2.0.

3.3. Elevated IL-10 Production in Susceptible CBA/J Lungs

Given the progressive disease in CBA/J mice despite high T cell numbers, we analyzed the cytokine profile of lung homogenates using ELISA to identify potential suppressive factors. While IFN-γ levels were initially comparable, we observed a dramatic and sustained increase in IL-10 concentrations in the lungs of CBA/J mice starting at day 60 (Figure 3). In contrast, C57BL/6 mice exhibited minimal IL-10 production throughout the infection. The onset of significantly elevated IL-10 in CBA/J mice correlated temporally with the loss of bacterial control and the declining CD4:CD8+ ratio [35].

3.4. Accumulation of IL-10-Secreting CD8+ T Cells in Susceptible Mice

To determine the cellular source of IL-10, we performed ELISPOT assays on highly purified (>97%) CD8+ T cells isolated from the lungs during the chronic phase. CD8+ T cells from CBA/J mice exhibited a significantly higher frequency of IL-10-producing spot-forming units (SFU) compared to those from C57BL/6 mice when stimulated with M.tb culture filtrate protein (CFP) (Figure 4). Parallel assays for IFN-γ revealed that while CD8+ T cells in resistant mice remained predominantly IFN-γ producers, a substantial fraction of CD8+ T cells in susceptible mice exhibited an immunoregulatory cytokine profile characterized by elevated IL-10 production [36].

3.5. CD8+ T Cell Depletion Restores Bacterial Control in CBA/J Mice

To evaluate whether these IL-10-producing CD8+ T cells were actively contributing to disease susceptibility, we performed in vivo cell depletion during the chronic phase (starting at day 100) [37]. The depletion of CD8+ T cells in CBA/J mice resulted in a statistically significant reduction in pulmonary bacterial burden by day 150 compared to isotype-treated controls (Figure 5). Furthermore, this reduction in bacterial load was accompanied by a restoration of IFN-γ levels in the lung tissue. These findings support the conclusion that during chronic infection in susceptible hosts, CD8+ T cells adopt a pathogenic, immunoregulatory role that associated with impaired Th1-associated immune responses [38].

4. Discussion

4.1. Comparison with Previous Work and Resolution of Apparent Contradictions

Our findings represent a significant advance over previous investigations of CD8+ T cell function during chronic Mtb infection, particularly with respect to work by Cyktor et al. (2013) that examined the same biological system in the same susceptible CBA/J mouse model [3]. While Cyktor et al. demonstrated that IL-10-producing CD8+ T cells accumulate during chronic infection and display dysfunctional phenotypes (PD-1+, Tim-3+, CD122+), they found that in vivo CD8+ T cell depletion during days 90-120 post-infection did not significantly alter bacterial burden, leading them to conclude that the pathogenic significance of these cells remained uncertain [3]. In contrast, our data support that CD8+ T cell depletion beginning at day 100 and continuing through day 150 post-infection produces a statistically significant reduction in pulmonary CFU and restoration of protective IFN-γ responses [3]. These findings may reflect a temporal dependence in CD8+ T cell-mediated immune suppression during chronic TB [39].

4.2. Temporal Evolution of CD8+ T Cell Pathogenic Function: A Model of Progressive Functional Subversion

We propose that the differing outcomes between our depletion study and that of Cyktor et al. reflect the progressive functional reprogramming of CD8+ T cells as chronic infection advances [3]. Our mechanistic model posits that during the early-to-mid chronic phase (days 60-120), IL-10-producing CD8+ T cells exist in a transitional state characterized by mixed IL-10/IFN-γ secretion or contain a heterogeneous population of cells with variable regulatory capacity [40,41]. During this period, documented by Cyktor et al., the IL-10-suppressive function of CD8+ T cells may be counterbalanced by their residual or concurrent IFN-γ production, explaining why their depletion did not yield a net benefit to bacterial control [3]. Importantly, Cyktor et al. observed that Vβ8+ CD8+ T cells exhibited dual IL-10/IFN-γ secretion profiles, suggesting that individual cells or subpopulations within the expanded CD8+ pool maintained protective capacity even in the presence of robust IL-10 production [3].

In contrast, during the late chronic phase (days 100-150 that we examined), prolonged antigenic exposure and progressive inflammatory stimulation drive further functional commitment of CD8+ T cells toward an IL-10-dominant regulatory phenotype [38]. By this later timepoint, we propose that the CD8+ T cell population has undergone more pronounced immunoregulatory polarization, with IL-10 production becoming the dominant function while protective IFN-γ production is substantially suppressed [42]. This model may explain why late-phase depletion (our work) produces significant bacterial control while early-phase depletion (Cyktor et al.) did not. The extended duration of our depletion regimen (50 days, from day 100-150) versus Cyktor’s protocol (30 days, from day 90-120) also provides a longer window to detect and allow detection of restored protective immunity that emerges after CD8+ removal [43]. This temporal evolution hypothesis is supported by our finding that IFN-γ levels are restored following CD8+ T cell depletion (Figure 5C) [44]. Restoration of protective IFN-γ indicates that CD4+ T cells retain the capacity to produce this critical cytokine but are suppressed by the concurrent presence of IL-10-producing CD8+ T cells [45]. This suppression becomes more profound in the late chronic phase, explaining the benefit of depletion at this timepoint [45]. Early in infection (Cyktor’s timeframe), the suppression by mixed IL-10/IFN-γ CD8+ T cells may be more balanced, such that their removal eliminates both suppressive and protective functions simultaneously, yielding no net benefit [46].

4.3. Methodological Considerations in Comparing Depletion Studies

Several methodological factors likely contribute to the different outcomes between our CD8+ depletion study and that of Cyktor et al., all of which are consistent with the temporal evolution model: (1) Depletion Timing and Duration. Our depletion protocol initiated at day 100 (late chronic phase, established dysfunctional CD8+ population) and continued through day 150. Cyktor et al. depleted from days 90-120 (early-to-mid chronic phase, during initial CD8+ accumulation and transition) [3]. The timing difference is critical: by day 100, bacterial burden in susceptible CBA/J mice has reached plateau levels or is actively progressing with established CD8+ dysregulation; by days 90-120, the CD8+ population is still accumulating and transitioning to a regulatory phenotype [47]. (2) Depletion Intensity and Consistency. Both studies used anti-CD8+ antibody for in vivo depletion, but protocols differed [3]. Cyktor et al. administered 0.5 mg doses at weekly intervals beginning at day 90 through day 120 (approximately 4 injections) [3]. Our study administered 0.5 mg doses at 5-day intervals beginning at day 100 through day 150 (11 injections total), providing more frequent and sustained depletion coverage during the late chronic phase [48]. This more intensive regimen may be required to maintain adequate depletion when CD8+ T cells are maximally activated and proliferating [48]. (3) Endpoint Assessment. Cyktor et al. analyzed bacterial burden at day 125 post-infection (5 days after final antibody) [3]. We analyzed at day 150 (50 days into depletion, at the final injection timepoint) [48]. The longer interval post-final-depletion in their study may have allowed CD8+ T cell repopulation, obscuring the benefit of depletion [48]. Our analysis immediately at the end of sustained depletion may better capture the effect of complete CD8+ suppression [49]. (4) Bacterial Burden Baseline. At the initiation of depletion, bacterial loads differ between studies. In our hands, day 100 CBA/J mice harbor 6-7 log10 CFU; Cyktor et al.’s day 90 mice had lower burden, reflecting differences in inoculum preparation, animal age/sex, or housing conditions that can significantly impact mycobacterial growth kinetics [3]. The higher baseline burden in our depletion cohort may sensitize the system to reveal the suppressive role of CD8+ T cells more clearly. These methodological differences are not contradictions but rather variations that, when interpreted within the temporal evolution framework, become complementary rather than conflicting.

4.4. The Proposed Immunoregulatory Model and Functional Subversion

Based on our findings and synthesis with prior work, we propose an updated model of CD8+ T cell function during chronic TB infection (Figure 6) [50]. We distinguish between two phases: Early-to-Mid Chronic Phase (Days 60-120): CD8+ T Cell Transition [51]. During this period, CD8+ T cells accumulate in the lung and begin acquiring regulatory markers (PD-1, Tim-3, CD122) as documented by Cyktor et al. [3]. Critically, many of these cells maintain dual IL-10/IFN-γ secretion capacity (observed by Cyktor in Vβ8+ populations) [3]. The regulatory function of IL-10-producing CD8+ T cells is present but incompletely predominant [40]. Depletion during this phase removes both suppressive and protective CD8+ populations, yielding no net benefit to bacterial control (Cyktor et al. result) [3].

Late Chronic Phase (Days 120-150): CD8+ T Cell Commitment to Regulation [52]. Prolonged antigenic exposure and chronic inflammation drive progressive functional subversion of CD8+ T cells toward a committed IL-10-dominant regulatory phenotype [53]. IL-10 production may become the predominant function, and the population shifts from mixed cytokine secretion toward predominantly regulatory cells [53]. This complete commitment explains why depletion at this stage produces significant improvement in bacterial control and restoration of protective IFN-γ, the CD8+ population has become primarily suppressive with minimal residual protective capacity [54,55]. This model generates testable predictions regarding CD8+ T cell biology across the infection course: (1) IL-10/IFN-γ co-secreting cells should decrease proportionally from early to late chronic phase, with shift toward an IL-10-dominant phenotype increasing; (2) Exhaustion marker expression (PD-1, Tim-3) should increase with infection duration, reflecting progressive differentiation toward regulatory function; (3) TCR repertoire restriction (as documented by Cyktor et al. for Vβ8 and Vβ14) should remain consistent, indicating the same clonal populations expand but undergo functional evolution rather than clonal replacement [56,57,58].

4.5. CD4:CD8+ Imbalance and Disease Progression

The observed reduction in the CD4:CD8+ ratio in susceptible CBA/J mice during chronic infection (Figure 2C) further supports the pathogenic role of CD8+ T cells [59]. While superficially this appears to reflect impaired CD4+ T cell responses, our data indicates a more nuanced interpretation: the CD4+ T cell compartment retains the capacity to produce IFN-γ (Figure 5C baseline), but this response is actively suppressed by concurrent IL-10-producing CD8+ T cells [53]. CD8+ cell depletion reveals this suppressed CD4+ T cell potential, leading to restored IFN-γ [60]. Thus, the CD4:CD8+ imbalance reflects not primarily CD4+ dysfunction but rather overwhelming suppression of CD4+ responses by an expanded, IL-10-producing CD8+ population [60].

4.6. Implications for Tuberculosis Vaccine Design

Our findings have important implications for TB vaccine development [61]. Current vaccine strategies frequently emphasize induction of broad CD8+ T cell responses, under the assumption that more CD8+ T cells equals better protection [62]. Our work, combined with that of Cyktor et al., demonstrates that this assumption is overly simplistic [3]. The quality, functional commitment, and temporal regulation of CD8+ responses matter as much as their magnitude [39]. These findings suggest that vaccine-induced CD8+ T cell responses may require careful functional regulation during chronic antigen exposure [63]. However, if vaccination fails to rapidly clear infection and instead results in chronic antigenic exposure, the same vaccine-induced CD8+ T cells may progressively reprogram toward an IL-10-dominant suppressive phenotype, eventually contributing to disease rather than protection [64]. Vaccine strategies must therefore either: (1) be designed to clear infection rapidly before CD8+ reprogramming occurs, or (2) be combined with immunomodulatory approaches that prevent IL-10-driven suppression during the chronic phase [63,65].

5. Conclusions

In conclusion, our proposed model highlights the dualistic nature of CD8+ T-cell immunity during TB. In susceptible hosts, prolonged infection drives progressive functional reprogramming of CD8+ T cells from protective effectors toward IL-10-producing regulatory populations that favor bacterial persistence. The temporal dependence of this reprogramming-progressing from mixed IL-10/IFN-γ secretion in early chronic infection toward committed IL-10-dominant regulation in late chronic infection, explains how previous investigations (Cyktor et al., 2013) and the present work can reach apparently contradictory conclusions about CD8+ T cell depletion, when in fact they reveal a dynamic, time-dependent process of immune subversion. Although murine TB models may not fully recapitulate human disease, the identification of this time-dependent functional reprogramming provides a new framework for understanding immune failure during chronic tuberculosis [66]. Future investigations should test specific predictions of the temporal evolution model through multi-timepoint depletion experiments (days 60, 90, 120, 150) combined with single-cell analysis of IL-10/IFN-γ co-expression patterns and TCR clonal identity. Direct blockade of IL-10 receptor signaling during late chronic infection would establish whether the improved bacterial control we observe is IL-10-dependent. Finally, examination of CD8+ T cell function and IL-10 production in clinical TB patients with recurrent or severe disease may reveal whether similar regulatory reprogramming contributes to human TB pathogenesis and susceptibility to reactivation.