CD8^+ T cells sustain vaccination-induced immunity against dissemination of contained tuberculosis in immunosuppressed hosts

Domain: biology
Published: 2026-03-24
Editorial outcome: minor_revision

Abstract
About two billion people are latently infected with Mycobacterium tuberculosis (Mtb), which can reside in multiple organs, including the lymphatics. The risk of latent Mtb infection (LTBI) reactivation increases with immunosuppression, such as HIV coinfection, yet the immunological correlates that maintain LTBI remain largely elusive. Using a mouse model of contained lymphatic Mtb infection we dissect the drivers of containment versus reactivation. We show that immunosuppression-induced dissemination of lymphatic Mtb and ensuing progressive disease can be prevented by vaccination with BCG or recombinant BCG even in the absence of CD4+ T cells. Multi-parameter imaging, spatial transcriptomics and network analysis reveal that anti-CD4-mediated immunosuppression triggers distinct repositioning of non-CD4 immune cells at the edge of TB lesions in cervical lymph nodes. Although B cell numbers increase, they prove dispensable for Mtb containment during CD4+ T cell loss. Using immune cell-deficient mice, cell depletion and adoptive transfers, we reveal that CD8+ T cells mediate vaccination-induced prevention of Mtb dissemination in the absence of CD4+ T cells, informing LTBI management in immunocompromised individuals.

Introduction
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains the leading infectious cause of death by a single pathogen globally, with about 1.3 – 1.5 million deaths annually. In addition, it is estimated that about two billion people are latently infected with Mtb without showing signs of active disease 1. Latent Mtb infection (LTBI) is often contained within granulomatous lesions that can occur in multiple organs. However, immunosuppressed individuals often fail to control LTBI, and the risk of LTBI reactivation increases with human immunodeficiency virus (HIV) infection, type 2 diabetes, malnutrition, autoimmune diseases, and following organ transplantation 2,3. While reinfection has been associated as the main driver of TB disease among individuals co-infected with HIV in high-TB burden countries 4, reactivation of LTBI appears to be a significant cause of disease in low burden countries 5. A hallmark of HIV infection and several other immunosuppressive conditions is the impaired function and reduction or loss of CD4+ T cells. These immune cells are crucial for TB control, maintenance of the structural integrity of granulomas, and a primary target for vaccine-induced protection against TB 6,7. However, due to the discrepancy between immunological parameters obtained in animal models and humans, the specific role of CD4+ T cells in LTBI immunity is still debated, especially in immunocompromised individuals 8.

Recognising that LTBI shares common features with lymphatic diseases 9, we previously developed a mouse model to study LTBI reactivation following the loss of CD4+ T cells 10. This lymphocentric murine TB model, which involves ear dermis infection, reflects several immunological aspects of human LTBI. In this model, Mtb spreads to the lungs only in mice depleted of CD4+ T cells after intradermal (i.d.) infection, while immunocompetent mice contain Mtb within the draining lymph nodes (LNs), mimicking aspects of LTBI. Historical descriptions of human TB and observations in non-human hosts suggest that TB has lymphatic characteristics, and that pulmonary pathology primarily aids disease transmission 9. This lymphocentric view of TB is supported by findings that LNs, bone marrow, spleen, thymus, tonsils and adenoids are persistently infected with Mtb in humans and non-human primates and can serve as a source of LTBI reactivation 11-15. In the absence of immunosuppression, this model mimics many of the immunological and cellular processes that are central to asymptomatic Mtb infection in humans 16. While the infected cell types, the innate and adaptive immune responses, lymph node dissemination and Mtb replication dynamics are similar, the tissue microenvironment differs, and lung-specific pathology is minimal. For those reasons, the ear dermis infection model is increasingly recognized as a suitable tool to investigate immune responses associated with contained extrapulmonary Mtb infection 17,18.

Importantly, we have previously shown that Bacillus Calmette-Guérin (BCG) vaccination can prevent the dissemination of contained murine Mtb infection with anti-CD4 monoclonal antibodies (mAb), indicating that vaccine-induced CD4+ T cells are not essential to prevent the spread of lymphatic murine LTBI 19. This finding raised the fundamental question of which immune cells control Mtb containment within the lymphatics after BCG vaccination in the absence of CD4+ T cells. Given the importance of managing and reducing LTBI to achieve the WHO's End TB Strategy 20, the contained murine lymphatic Mtb infection offers an opportunity to study LTBI reactivation from a lymphocentric perspective, and provides a valuable platform to explore the immunological requirements for LTBI containment versus dissemination in the context of vaccination. While macrophages and monocytes express CD4 in humans, they do not in mice 21. This allows the study of CD4+ T cell depletion in the presence of other immune cells, such as macrophages/monocytes. Mtb uses alveolar macrophages to disseminate, survive, and replicate; these cells are also critical to initiate the formation of granulomas 22,23. Moreover, monocytes are altered in individuals with LTBI, so they may play a role in the reactivation of LTBI 24. In this context, this mouse model selectively mimics the impaired function or loss of CD4+ T cells in human LTBI.

Until recently, the dissection of the complex interactions between Mtb and the host immune system, which lead to the establishment of LTBI, predominantly relied on histopathological studies and cellular analyses of disrupted tissues or the extraction of DNA and RNA from blood. With the emergence of spatial tissue profiling in conjunction with multiplex imaging and advances in analytical methodologies, it is now possible to map and analyse the organisation and activity of thousands, or even millions, of cells, genes, and molecules in the tissue 25. This approach has been used to map TB granulomas in the lungs, revealing the tissue architecture and cellular interactions in TB. It shows that Mtb infected human lung tissue comprises at least four types of non-necrotising structures in addition to necrotising granulomas 26. Furthermore, spatial transcriptomics has provided unprecedented insights into the pathogenesis of Alzheimer’s disease, Multiple Sclerosis, skin diseases and various types of cancers in different organs 27-30.

In this work, we employ a multi-pronged approach using several experimental strategies to dissect the immunological requirements of lymphatic LTBI containment. The first approach utilises histopathological and multi-parameter spatial transcriptomics techniques to delineate the LN architecture following lymphatic Mtb infection with and without prior vaccination with BCG or the recombinant BCG (rBCG) strain BCG::ESAT-6-PE25SS (abbreviated as PE25 in Figures) and assesses extrapulmonary organs for TB dissemination. In parallel, we perform Mtb infection and adoptive cell transfer in various mouse strains lacking distinct immune cell subsets and monitor their ability to resist Mtb spread. Our results give detailed high-dimensional insight into the cellular compositions during Mtb dissemination and BCG-mediated containment of Mtb in the lymphatics (Supplementary Figure 1). Understanding the immune correlates of protective versus permissive host response has significant implications for designing preventative and therapeutic strategies to combat LTBI and its reactivation.

Results
BCG and BCG::ESAT-6-PE25SS vaccination prevent Mtb dissemination in a murine lymphatic LTBI model

Recombinant BCG strains have attracted considerable attention as potential substitutes for BCG 31,32. We and others have developed candidate TB vaccines by integrating immunogenic proteins from the Region of Difference 1 (RD1) locus of Mtb into BCG. The first-generation rBCG strains containing RD1 from Mtb were considered inappropriate to progress to human trials due to prolonged persistence and increased virulence in immunocompromised hosts 33. However, next-generation rBCG candidates, such as Mtb::ESX1Mmar and BCG::ESAT-6-PE25SS 34,35 aim to maintain the enhanced immunogenicity while preventing the detrimental aspects of Mtb-derived RD1. To enhance the likelihood of potential translation to clinical trials, it is important to evaluate rBCG strains in different pre-clinical models of TB.

To determine if BCG::ESAT-6-PE25SS can prevent the breakdown of contained lymphatic Mtb infection and the spread of Mtb to non-lymphatic organs, six- to eight-week-old C57BL/6 mice were grouped and treated as described in the Materials and Methods section and illustrated in Fig. 1a. Mice did not show clinical signs of active TB (Fig. 1b), and gained weight throughout the experiment (Fig. S2a-c).

Mice that only received Mtb infection contained the bacteria in the lymphatics (cervical lymph nodes (cLNs) and spleen) with negligible Mtb dissemination to the lungs (Fig. 1c-e). As previously shown 10,19, anti-CD4 mAb treatment led to a progressive increase in bacterial burden in LNs, spleens and lungs, with the most pronounced level of dissemination seen at 120 days after Mtb infection (Fig. 1c-e). While vaccination with BCG and BCG::ESAT-6-PE25SS did not reduce the level of Mtb bacilli in the lymphatic organs relative to anti-CD4 mAb-treated mice, it significantly reduced and, in some cases, BCG even prevented the spread of bacteria to the lungs (Fig. 1c-e). Apart from 2 animals that harboured a single colony of BCG in the lung or a single colony of BCG::ESAT-6-PE25SS in the spleen, all Mtb-uninfected control animals showed no detectable vaccine persistence in any of the organs at the 120-day time-point (Fig. 1f). Strikingly, when cervical LNs and lungs were assessed for histopathological changes by measuring the percent of tissue affected by Mtb (affected area, positive signal, magenta) in contrast with the unaffected area (normal in disease, negative signal, green) implementing an automated pixel function in QuPath (Fig. 1g, j), it became apparent that BCG and BCG::ESAT-6-PE25SS significantly reduced measurable pathology from day 30 onwards in the cLNs (Fig. 1h), and led to a significant reduction of lung pathology at 120 days after infection (Fig. 1k). Representative images show the area affected at 120 days after Mtb infection in cLNs and lungs (Fig. 1i, l). Collectively, those results indicate that vaccination with BCG or BCG::ESAT-6-PE25SS significantly reduces the spread of Mtb from the lymphatic system to the lung and prevents the induction of lung pathology in this model of Mtb containment breakdown. Hence, for subsequent analyses we will refer to three distinct scenarios: i) natural containment (Mtb infection only); dissemination (Mtb infection plus anti-CD4 mAb treatment); and vaccine-mediated containment (vaccination with BCG or BCG::ESAT-6-PE25SS followed by Mtb infection and anti-CD4 mAb treatment) (illustrated in Fig. 1k).

Mtb dissemination occurs across multiple lymphatic and non-lymphatic organs

Given that TB can occur in multiple organs 36, we investigated whether reactivation of contained Mtb infection in our model impacts additional lymphatic and non-lymphatic organs and whether vaccination can prevent this. To this end, histopathological analyses and automated scoring were conducted on mLNs, femurs, livers, brains, and salivary glands. Like in the cLNs (Fig. 1h), anti-CD4 mAb treatment led to significantly enhanced histopathology in mLNs from 60 days after Mtb infection. Vaccination with BCG and BCG::ESAT-6-PE25SS markedly and significantly reduced the level of mLN damage (Fig. 2a; Fig S3a). Histopathological changes in liver, femurs and bone marrow were also seen at different time points across anti-CD4 mAb and vaccination groups (Fig. 2b, c; Fig. S3b, c). No pathological changes were observed in either the brain or the salivary glands in any of the treatment groups at 120 days after infection (Fig. S3d). In line with these histopathological results, we also found significantly increased tissue area occupied by Mtb in the cLNs, the lungs, the mLNs, the livers, and the bone marrow of anti-CD4 mAb-treated animals using Ziehl-Neelsen staining (Fig. 2d). Both BCG strains significantly reduced this area in cLNs, lungs and mLNs to the level of Mtb-infected mice that did not receive anti-CD4 mAb treatment. Furthermore, BCG also significantly reduced the area occupied by Mtb in livers and bone marrows compared with Mtb-infected only mice, while the reduction seen with BCG::ESAT-6-PE25SS reached statistical significance in the liver. Collectively and consistent with data shown in Figure 1, these results show that anti-CD4 mAb-mediated dissemination of LTBI occurs across multiple lymphatic organs, the lung and the liver, but not the brain or the salivary glands, and that vaccination can prevent this spread.

Containment of lymphatic LTBI is associated with changes in the composition and positioning of immune cells in the LNs

To determine the immunological mechanisms that may be associated with natural containment (Mtb-only) vs anti-CD4 mAb treatment-mediated dissemination vs vaccination-mediated containment (BCG or BCG::ESAT-6-PE25SS with anti-CD4 mAb) we first performed multiplex immunohistochemistry (IHC) in the entire cLNs and left lung lobes using eight fluorescent antibodies against CD3, CD4, CD8, B220, CD11b, CD11c, CD161c, Mtb and DAPI for the nuclei (Fig. 3a-d). Enumeration of single positive cell types across the whole cLNs confirmed an increase in Mtb-infected cells in the anti-CD4 mAb-treated group relative to the Mtb-only group and groups that received anti-CD4 mAb treatment but had been vaccinated prior with BCG or BCG::ESAT-6-PE25SS (Fig. 3e; Fig. S4a). In line with our previously published flow cytometry-based results 10,19, Multiplex IHC also confirmed the efficient depletion of CD4+ cells in all groups that had received anti- CD4 mAb (Fig. 3e). Similar observations occurred when single positive cells of each marker were enumerated across the full lung lobes (Fig. S4b, c). Interestingly, anti-CD4 mAb treatment with or without prior vaccination led to a significant increase in B cells in the cLNs and a trend towards increased CD8+ T cells in the lungs. Collectively, these analyses showed that there are numerical changes of distinct immune cell subsets in cLNs and lung that are either driven by Mtb infection, by anti-CD4 mAb treatment and/or by vaccination.

We subsequently extended the image analysis to determine cells positive for Mtb plus an additional cellular marker used in the multiplex IHC panel (Fig. 3f; Fig. S4d ). In both cLNs and lungs, Mtb-infected cells, including non-classical phagocytic and non-phagocytic subsets 37-39, were significantly increased in the anti-CD4 mAb-treated dissemination group across all the double-positive populations except Mtb+CD8+ in cLNs and Mtb+B220+ in the lungs (Fig. 3f; Fig. S4d). In almost all double positive cell populations across cLNs and lungs, the vaccinated groups showed a similar profile to the Mtb-only group. The only combination where all groups showed near-identical cellular abundance were Mtb+CD8+ cells in the cLN. Collectively, these results correlated with the increased Mtb burden observed in the anti-CD4 mAb-treated group with CFU plating (Fig. 1c, e), H&E (Fig. 1h, k) and ZN histopathology (Fig. 2d). The findings suggest that control of lymphatic LTBI through vaccination may be linked to changes in migration, abundance or functionality of B cells or CD8+ T cells.

To measure if Mtb containment is also associated with an altered immune cell localisation around Mtb-infected cells, we performed proximity measurements. To this end, the distance of over 13 million cells expressing one of eight different markers in relation to Mtb-infected cells was analysed across cLNs and lungs (Fig. 3g, h; Fig. S4e, f). In cLNs, a noticeable shift of all single-positive cells towards Mtb-infected cells in the anti-CD4 mAb-treated dissemination group was observed (Fig. 3g). As a general pattern in the cLNs, the Mtb-only group (blue) and the BCG-vaccinated group (black) showed similar cellular distances from Mtb-infected cells across most cell types, except CD8+ and CD11c+ cells (Fig. 3h). Likewise, the anti-CD4 mAb-treated group (red) clustered together with the BCG::ESAT-6-PE25SS-vaccinated group (purple) across the same cell types (Fig. 3h). CD11c+ cells were the closest cell type to Mtb-infected cells in all anti-CD4 mAb-treated groups (Fig. 3g). Even though CD8+ cell proportions did not increase in the LNs following anti-CD4 mAb treatment (Fig. 3e) they showed a consistent pattern of a similarly short distance from Mtb-infected cells in all anti-CD4 mAb treated groups (Fig. 3g, h). Despite increasing in relative abundance (Fig. 3e), B220+ cells showed the largest distance to Mtb-infected cells in all groups (Fig. 3g, h). In the lungs, like the cLNs, there was a noticeable and highly significant shift towards Mtb-infected cells in all single-positive cells within the anti-CD4 mAb dissemination group (red) (Fig. S4e, f).

When taking into account abundance (Fig. 3e, f; Fig. S4c, d) and distance measurement (Fig. 3g, h; Fig. S4e, f), it appears that natural- and BCG-mediated containment of lymphatic LTBI may be linked to an influx and a repositioning of immune cells in the infected cLNs and the lungs. In particular, B220+ B cells increased in the cLNs, and maintained the largest distance to Mtb-infected cells; CD8+ T cells showed a reduced distance to Mtb; and CD161c+ NK cells tightly clustered around Mtb-infected cells in lung tissue.

Spatial transcriptomics reveals distinct pathways of LTBI containment and dissemination at the edge of LN lesions

To assess whether anti-CD4 mAb-mediated Mtb dissemination and vaccine-mediated containment are associated with distinct transcriptomics signatures within defined regions of the cLNs, we performed NanoString GeoMx-based spatial transcriptomics across 30 regions of interest (ROIs) that were selected within normal tissue (in both naïve and disease samples) taken either from the lesion or the edge of a lesion of the cLNs across all groups (Fig. 4a, b). A UMAP analysis revealed some clustering of ROIs based on both tissue location and treatment group (Fig. 4c). We performed differentially expressed genes (DEGs) analysis observing significant differences between ROIs derived from all tissue locations (Fig. 4d). Despite many observed differences, comparing all 30 ROIs independently of tissue location identified no significant DEGs between Mtb infection and anti-CD4 mAb-mediated dissemination relative to naïve mice (Fig. S5a). Significant differences were however observed in three comparisons: separated naïve tissue vs natural containment (Mtb only) (Fig. S5b), natural containment vs anti-CD4 mAb-mediated dissemination (Fig. S5c) and anti-CD4 mAb-mediated dissemination vs vaccine-mediated containment (Fig. S5d, e). Pathway analysis of DE genes using Ingenuity Pathway Analysis (IPA) revealed that many immune system-related pathways were enriched with a predicted activation (Fig. S5f, g). Overall, the anti-CD4 mAb dissemination group vs Mtb-only group showed the largest difference followed by both vaccination groups, illustrating key differences between dissemination and containment of LTBI on a transcriptional level. MHC-I-mediated antigen presentation and B cell receptor signalling were identified in the most highly significant pathways differentiating LTBI containment from anti-CD4 mAb-mediated dissemination (Figs. S5f, g). Strikingly, this aligned with our observations that B cells and CD8 cell numbers and distribution respectively were altered following anti-CD4-mediated dissemination of LTBI (Fig. 3),

When natural containment (Mtb only), dissemination (anti-CD4) and vaccination-mediated containment (BCG/PE25) were compared across ROIs derived from normal tissue, few DEGs were up- or downregulated (Fig. 4e). In contrast, we observed many significant DEGs between natural containment, anti-CD4 mAb-mediated dissemination and vaccine-mediated containment within the lesion and at the edge of a lesion (Fig. 4f, g). Notably, most significant DEGs (FDR <0.05 light blue; <0.01 dark blue) were identified at the edge of a lesion rather than within the lesion, particularly when comparing natural with vaccination-mediated containment (Fig. 4f, g). Interestingly, many immune-cell related pathways, including neutrophil degranulation and T and B cell-associated pathways were all strongly enriched when comparing the edge of the lesion in natural containment vs vaccination-mediated containment (Fig. 4g, h). Likewise, MHC-I-mediated antigen presentation and neutrophil degranulation differentiated the edge of the lesion in anti-CD4 mAb dissemination from vaccine-mediated containment. While neutrophil degranulation is likely a response to bacterial burden 40 and neutrophil swarming 41, T and B cell signatures support the findings from the multiplex IHC analysis (Fig. 3). The expression of cytotoxic genes was not statistically different between treatment groups and tissue types, possibly due to the lack of single cell resolution with NanoString GeoMx (Fig. 4i).

Using NanoStringNCTools to assign cell types (Fig. 4j; Fig. S6a-f), we observed significant changes in the proportions of B cells (cd19), CD4 cells (cd4), dendritic cells (Itgax), and NK cells (Klrblc), again in accordance with our key findings from the multiplex IHC analysis (Fig. 3). These changes largely distinguished natural LTBI containment from anti-CD4 mAb treatment (Fig. 4j, i; Fig. S6a-f). Consistently, cell changes were most pronounced at the edge of the lesion (Fig. S6b, c). Most notably, the overall increase in B cells following anti-CD4 mAb treatment (previously observed by multiplex IHC) was localised to the edge of the lesion and further associated with a marked decline of Cd19 within the lesion (Fig. 4j).

Differences in cytokine (Fig. S6g-i) and chemokine (Fig. S6j-l) gene expression were also observed within the lesion and their edges. While in some cases increased cytokine or chemokine expression was only seen in the anti-CD4 mAb dissemination group (lesion / edge of lesion: Il1a, Il1b, Il18 and lesion only Ifnγ, Tnf, Cxcl10, Cxcl13), likely reflecting the increased inflammation associated with higher bacterial burden, some cytokines/chemokines increased with anti-CD4 mAb treatment more broadly (lesion: Ccl2, Ccl4, Cxcl9; edge of lesion: Tnf, Ccl2, Ccl5, Ccl19, Cxcl9, Cxcl10, Cxcl13) or were increased with containment (lesion: Ccl5, Ccl21a; edge of lesion: Ifnb1, Ccl22).

Collectively, these results support significant repositioning of immune cells in cLNs in response to vaccination and depletion of CD4+ T cells and revealed that transcriptomic changes associated with dissemination and containment of lymphatic LTBI are predominantly localised at the edge of a lymphatic lesion.

Lethal dissemination of Mtb occurs in the combined absence of lymphocytes and is independent of innate responses

Cumulatively, our analyses thus far inferred that lymphatic LTBI containment may be associated with an expansion and/or repositioning of immune cells, particularly B cells, CD8+ T cells and NK cells at the edge of the cLN lesions. To experimentally address the role of these cells for lymphatic LTBI containment, we induced lymphatic Mtb infection in a variety of genetically modified mouse strains with and without vaccination. First, we infected wild-type C57BL/6 mice, B6.muMT-/- mice that lack mature B cells 42, B6.Rag1-/- mice that lack mature B and T cells 43 and B6.Rag2-/-Il2rg-/- mice that lack mature B, T and NK cells 44 i.d. with Mtb. Two groups of C57BL/6 mice received anti-CD4 mAb treatment either alone or in addition to Mtb infection and naïve mice were also included as controls (Fig. 5a). While all C57BL/6 mice and all B6.muMT-/- mice survived Mtb infection for 120 days (Fig. 5b, c), 100 % of infected Rag1-/- mice succumbed to the infection with a median survival of 71 days (Fig. 5d). Severely immunocompromised B6.Rag2-/-Il2rg-/- mice only survived for an average of 56 days (Fig. 5e). These results indicated that the absence of CD4+ T cells or B cells alone does not lead to death, whereas the combined absence of B and T cells does. The results also implied that NK cells may play a minor contribution to LTBI containment in the absence of B and T cells. As expected, and in line with results reported in Figure 1, anti-CD4 mAb-treated C57BL/6, B6.Rag1-/- and B6.Rag2-/-Il2rg-/- mice also showed high levels of CFU in the cLNs, lung and spleen (Fig. 5h) as well as significantly elevated histopathological damage in cLNs (Fig. 5i, j) and lungs (Fig. 5k, l). However, when we enumerated the Mtb burden in surviving C57BL/6 and B6.muMT-/- mice, we were intrigued to observe that B6.muMT-/- mice showed almost no signs of bacterial spread nor a productive infection in the lymphatics (Fig. 5f-l). RNA sequencing of fixed cLNs also revealed that the transcriptomic response of B6.muMT-/- mice to lymphatic Mtb infection differed from C57BL/6 as well as from immunocompromised B6.Rag1-/- and B6.Rag2-/-Il2rg-/- mice (Fig. 5m). Furthermore, pathway analysis of RNAseq data revealed that severe infection in immunocompromised B6.Rag1-/- and B6.Rag2-/-Il2rg-/- mice and anti-CD4 mAb-treated C57BL/6 mice is associated with a particularly strong neutrophil degranulation signature relative to B6.muMT-/- mice. Instead, Mtb-infected B6.mu MT-/- mice were associated with class I MHC-mediated antigen processing and presentation (Fig. S7a, b).

We reasoned that if BCG vaccination-mediated containment of LTBI was driven by innate immune mechanisms, then vaccination of B6.Rag1-/- or B6.Rag2-/-Il2rg-/- mice before Mtb infection should extend their survival (Fig. 5o). However, vaccination with BCG or BCG::ESAT-6-PE25SS did not extend the survival of B6.Rag1-/- mice (Fig. 5r) and only marginally extended the survival of B6.Rag2-/-Il2rg-/- mice (Fig. 5s). Also, vaccination did not change the survival of B6.muMT-/- or C57BL/6 mice (Fig. 5p, q). Collectively, these results indicated that prevention of severe disease and death after Mtb dissemination may either be driven by B and T cell interactions or by a compensatory effect of CD8+ T cells. The results also demonstrated that vaccine-mediated LTBI containment is likely driven by adaptive immune responses with only a minor contribution of innate immunity in the absence of adaptive immune cells (e.g., T/B cell-dependent innate training).

CD8+ T cells mediate LTBI containment following anti-CD4 mAb treatment and B cells are dispensable

Next, we wanted to further quantify the relative contributions of B cells and different T cell subsets in the containment of LTBI. For this, we first established an adoptive transfer system that would allow us to dissect the contributions of cell subsets on mouse survival (Fig. 6a). We confirmed that adoptive transfer of LN- or spleen-derived cells from wild type C57BL/6 mice into B6.Rag2-/-Il2rg-/- mice can reverse the 100% mortality observed after Mtb infection into 100% survival (Fig. 6b). This provided a tractable system to transfer LN cells that were depleted of either T cells, B cells or both (Fig. 6c). Almost all mice that received total LN cells, regardless of whether cells came from naïve or vaccinated donors, survived for 120 days (Fig. 6d). In line with the results seen in B6.muMT-/- mice, the survival data clearly showed that the absence of B cells from transferred cells can be compensated for, with 100% of mice that received cells from B cell depleted vaccinated animals surviving for 120 days and approximately 70% of mice that received cells from B cell depleted naïve mice surviving for 120 days (Fig. 6e). In contrast, mice that received either CD3+ (Fig. 6f) or CD3+ and CD19+ cell-depleted LN cells (Fig. 6g) succumbed to the dissemination of Mtb infection between 40 and 80 days, which was indistinguishable from B6.Rag2-/- Il2rg-/- mice that had received no cells. These results mirrored the survival data seen in B6.Rag1-/- mice (Fig. 6d) but pointed towards a critical role for T cells rather than B cells in mediating LTBI containment.

Given that vaccination prevented dissemination and pathology despite the depletion of CD4+ cells (Fig. 1), the most likely explanation for the results obtained above, was that containment of lymphatic Mtb is mediated by CD8+ T cells. To definitively assess the relative contributions of CD4+ T cells, CD8+ T cells and B cells to the prevention of lethal Mtb dissemination, we performed additional experiments in which C57BL/6 wild-type mice and B cell-deficient B6.muMT-/- mice were vaccinated or left unvaccinated and were subsequently treated with anti-CD4 mAb, anti-CD8 mAb or both (Fig. 6h). Both vaccine strains induced TB10.4:H-2Kb+ mycobacteria-specific CD8+ T cells, including in the cLNs (Fig. 6i). Strikingly, in both genetic backgrounds only mice treated with both anti-CD4 and anti-CD8 mAbs succumbed to the infection (Fig. 6j), with similar kinetics to B6.Rag1-/- mice (Fig. 6d) and B6.Rag2-/-Il2rg-/- mice that had received T cell-depleted LN cells (Fig. 6e). While the overall dependency on CD8+ T cells for survival in the absence of CD4+ T cells was identical between unvaccinated and vaccinated mice, as expected, vaccinated anti-CD4/CD8 mAb-treated mice showed a survival increase of approximately 20-30 days compared to unvaccinated mice (Fig. 6j). In line with the survival data, the CFU results show that CD8+ T cell depletion by itself does not lead to dissemination of latent Mtb infection to the lung (Fig. 6k, l). However, combined CD4/CD8 T cell depletion leads to dissemination of contained Mtb infection. Following vaccination, two anti-CD4/CD8 mAb-treated C57BL/6 mice survived until day 120. While these two mice had reached an ethical endpoint, organs could be collected prior to euthanasia. As expected, very high Mtb CFU were recovered from cLNs, spleen and lungs of those two mice. Taken together, our results clearly demonstrate that LTBI containment is driven by T cells and that B cells are dispensable for this process (Fig. 6j, k). While the absence of either CD4+ or CD8+ T cells can be compensated for, regardless of whether B cells are present or not (C57BL/6 vs B6.muMT-/-), the combined absence of both CD4+ and CD8+ T cells was always lethal. Our results also show that NK cells alone cannot compensate for the loss of T cells (Fig. 6m).

Collectively, our data provide a comprehensive assessment of the cellular requirements for the lymphatic containment of otherwise fatal Mtb dissemination and provide strong evidence that CD8+ T cell-mediated immunity should be targeted to avoid LTBI reactivation when CD4+ T cell immunity is compromised, such as in the context of HIV co-infection.

Methods
Bacterial cultures

The BCG SSI (abbreviated to BCG; ATCC no. 35733) and BCG::ESAT-6-PE25SS 41, (abbreviated to PE25 in Figures) and Mtb H37Rv strain were cultured in Middlebrook 7H9 broth enriched with 10% Albumin Dextrose Catalase (ADC) (Cat. No. 271310 and 212352, respectively, Becton, Dickinson and Company (BD), 0.5% glycerol (Cat. No. AJA242, Ajax Finechem), and 0.2% Tween 80 (vol/vol) (Cat. No. 822187, Sigma-Aldrich). BCG::ESAT-6-PE25SS and Mtb H37Rv were grown in the presence of 50 µl/ml kanamycin and 10 µl/ml ampicillin, respectively. The cultures were shaken at 80-90 rpm at 37°C until they reached the log phase with an OD of 0.6-0.8. The bacterial cultures were then harvested, washed, aliquoted in 10% glycerol and kept at -80°C until use.

Mice

Six- to eight-week-old female C57BL/6 (C57BL/6JOzarc; JAX stock #000664) mice were obtained from the Animal Resources Centre (ARC) Perth, WA, Australia. Male and female C57BL/6, B6.muMT-/- (JAX stock #002288) 42, B6.Rag1-/- (B6.129S7-Rag1tm1Mom/Arc; JAX stock #002216) 43 and B6.Rag2-/-Il2rg-/- (JAX stock #014593) 44 mice were bred and maintained in specific pathogen-free conditions at the James Cook University Bioresource Facility of the Australian Institute of Tropical Health and Medicine (AITHM) in Townsville, QLD, Australia. Experimental and control animals were co-housed, and before performing experiments, the animals were acclimatised to the facility for at least seven days. The mouse rooms were maintained with a 12-hour light/dark cycle, a temperature of 21 ± 2°C, and a humidity of 55% ± 10%. The mice were housed in ventilated cages with bedding consisting of irradiated fine corn cobs, enriched with a cardboard roll, Bed R Nest, cotton, and some pieces of wood for them to chew. Water was treated by reverse osmosis and sterilised by UV light. The irradiated mouse diet was obtained from Specialty Feeds (WA, Australia). Both food and water were provided ad libitum. All experimental procedures were approved by the Animal Welfare and Ethics Committee of James Cook University (approval number A2794). Each mouse was monitored for clinical signs of disease and/or weighed throughout the study.

Vaccinations and infections

Mice were immunised via subcutaneous (s.c.) injection in the base of the tail with 100µl of phosphate buffer saline (PBS) (Cat. No. 10010023, Gibco) containing 1×106 CFU of BCG or BCG::ESAT-6-PE25SS. Sixty days after vaccination, mice were immunosuppressed with an intraperitoneal (i.p.) injection of anti-CD4 (aCD4) mAb (clone GK1.5; 200µg/mouse; Walter and Eliza Hall Institute of Medical Research (WEHI) antibody facility) or anti-CD8 (aCD8) mAb (clone 2.43; 200µg/mouse; WEHI antibody facility) (Table S1) and infected intradermally (i.d.) in the ear with 1×103 CFU of Mtb. aCD4 and aCD8 antibody injections continued weekly for the duration of the experiment. Naïve, vaccination-only, immunosuppressed-only, and infection-only groups were included as controls. The experimental setup is shown in the Figures. Samples were collected at 30 , 60 , and 120 days after Mtb infection.

Disease indicator score

The clinical signs of disease for immunisations, immunosuppression and infections in C57BL/6, B6.muMT-/-, B6.Rag1-/- and B6.Rag2-/-Il2rg-/- mice were scored from 0 to 3 according to approved animal ethics project A2794 as follows: a) Appearance: 0 = Shiny fur, clear eyes, 1 = Fur appears untended with or without a lesion in the skin, 2 = Scruffy fur, discharge from eyes and nose, eyes sunken and anus dirty or sore, 3 = Very scruffy fur, blood from orifices, abnormal body posture; b) Behaviour/movement: 0 = Normal contact with other mice, active movement, 1 = Little contact with other mice, 2 = Little contact with other mice, avoid movement, 3 = Complete isolation, noises, self-harm, no-movement, lying on side; c) Reaction to stimuli: 0 = adequate, 1 = Slightly delayed, 2 = Significantly delayed or weak, 3 = No reaction; d) Breathing: 0 = Normal; 1 = increased breathing, 2 = increased breathing with sporadic increase in abdominal breathing, 3 = constant abdominal breathing; and e) Weight loss: 0 = none, 1 = Less than 10%, 2 = 10% to 14%, 3 = ≥15%. Mice were euthanised by cervical dislocation by trained and experienced staff when they reached a score of 3 on any criterion.

Sample collection

Mice were euthanised by cervical dislocation and spleens, cLNs and right lung lobes were collected for Mtb CFU enumeration. Mesenteric LNs (mLNs), lungs, brains, salivary glands, livers, femurs, cLNs, and left lung lobes were fixed in 4% paraformaldehyde for 24 hours. After being fixed, the samples were transferred into 70% ethanol until they were processed for histopathological studies.

CFU enumeration

Spleens, cLNs and lungs were homogenised in Nasco Whirl-Pak bags or gentleMACS tubes containing 1 ml of PBS enriched with 0.05% Tween 80. Tenfold dilutions (N, -1, -2) were plated onto Middlebrook 7H11 (Cat. No. 212203, BD) agar plates supplemented with 0.02% glycerol, 0.05% Tween 80, 10% oleic albumin dextrose catalase (OADC) (Cat. No. 212240, BD), 25 µl/ml ampicillin and 10 µl/ml cycloheximide (Cat. No. A9518 and C7698, respectively, Merk). Agar plates were sealed, wrapped in aluminium foil, and incubated aerobically at 37°C, and CFU were counted after four weeks.

Histopathological studies

Tissue samples were embedded in paraffin and sectioned into 4-micrometre-thick slices. Slices were transferred onto microscope slides, dewaxed, and stained using the following methods:

a) Hematoxylin and Eosin (H&E)

mLNs, brains, salivary glands, livers, femurs, cLNs, and left lung lobes were stained with H&E. Briefly, dewaxed tissues were hydrated, stained with hematoxylin, differentiated, blued, and then stained with Eosin. Subsequently, the tissues were dehydrated, cleared, and cover-slipped before analysis. Scoring of H&E slides was performed blinded.

b) Ziehl-Neelsen (ZN)

mLNs, brains, salivary glands, livers, femurs, cLNs, and left lung lobes were stained with ZN. In brief, dewaxed tissues were hydrated, stained with Carbol Fuchsin, Ziehl-Neelsen, and differentiated in acid alcohol. Afterwards, tissues were counterstained with methylene blue, dehydrated in ethyl alcohol, cleared, and cover-slipped before analysis.

c) 9-colour Opal Immunohistochemistry (IHC)

cLNs and left lung lobes were stained with a 9-colour Opal IHC Multiplex panel (Akoya) by the Advanced Histotechnology Facility of the WEHI, Melbourne, Victoria, Australia. The 9-colour opal IHC panel included the following antibodies: Mtb (Cat. No. ab905, clone pAb, Abcam), CD3 (Cat. No. A045229-2, clone pAb, Dako), CD4 (Cat. No. 14-9766-82, clone 4SM95, Invitrogen), CD8a (Cat. No. HS361-008, clone Rb321E9, Synaptic systems), B220 (antibody from WEHI), CD161c/NK1.1 (Cat. No. 24395S, clone E6Y9G, Cell Signalling Technology), CD11c (Cat. No. 97585, clone D1V9Y, Cell Signalling Technology), CD11b (Cat. No. ab133357, clone EPR1344, Abcam), and Spectral DAPI (Cat. No. FP1490, Akoya Bioscience).

d) Whole Transcriptome Analysis (WTA)

cLNs were stained using the NanoString GeoMx Mouse Whole Transcriptome Assay (version 3.0.0.182) by NanoString Technologies, Inc. (Seattle, Washington, USA). Thirty regions of interest (ROIs) were selected based on areas with different histopathological changes seen in H&E. ROIs were derived from 8 mice per experimental group and from 2 naïve controls, and included three areas: I) Normal in Naïve = no pathological changes in mice that did not receive treatment, and Normal in disease = no pathological changes were seen in tissues from Mtb-only infected mice, immunosuppressed and Mtb-infected, and vaccinated-immunosuppressed and Mtb-infected; II) Lesion = tissue resembling granuloma; and III) Edge of the Lesion = border of the tissue resembling granuloma.

Digital image analysis

Tissues stained with H&E and ZN were scanned at 20× and 40× using the PhenoImager HT (Akoya) and the Aperio LV1 (Leica Biosystems), respectively, while the Multiplex IHC slides were scanned using the PhenoImager HT (Akoya) at 20×. QuPath versions 3.1-5.1 were used to analyse H&E, ZN and Multiplex IHC slides. Each LN and lung lobe were selected for the three different staining techniques. The areas affected by Mtb were identified, and their respective percentages were calculated for H&E. In the ZN staining, Mtb were identified, and the proportion of the area occupied by the bacteria was calculated. In the multiplex IHC, cell nuclei were identified using DAPI before cellular segmentation. Subsequently, two methodologies were applied. Briefly, each cell was determined by a threshold and classified according to the marker expressed by a threshold, and subsequently, multiple markers were used to train a machine-learning classification of the cell types for each cell. Once each cell was classified according to its phenotype, cells were enumerated, and distances among cells were analysed 70. Cellular deconvolution and counts for estimating the abundance of cell types within each ROI were performed using the GeoMx DSP Analysis Suite. The analysis utilised the DSPPlugSpatialDecon and the scripting file SpatialDecon_plugin.R alongside the cell profile metrics ImmuneAtlas_cellFamily_ImmGen.

Cell isolation and adoptive transfer

Spleen and LNs were collected aseptically from naïve and vaccinated C57BL/6 donor mice. The tissues were dissociated using a sterile three cc syringe plunger and passed through a 70μm cell strainer. Red cells were lysed using red cell lysis buffer.

To deplete CD3+ and/or CD19+ cells prior to adoptive cell transfer, LNs were disrupted as described above. The cell separation was carried out using the CD3e MicroBead kit and CD19 MicroBeads (Miltenyi Biotec). Briefly, single-cell suspensions were labelled with CD3- or CD19-conjugated immunomagnetic beads and then depleted using a MACS system (Miltenyi Biotec). Further enrichment was achieved by passing the negative fraction through a second MACS column. Flow cytometry analysis showed that approximately 90-98% of CD3+ (CD3-FITC; clone 17A2; Cat. No. 555274; BD Biosciences) and/or CD19+ (CD19-PE-Cy7; clone IDL3; Cat. No. 552854; BD Biosciences) cells were effectively removed.

For adoptive transfer experiments 71, B6.Rag2-/-Il2rg-/- recipient mice received 5.7 ×105 to 7 ×106 cells via intravenous (i.v.) injection into the tail vein. The recipient mice were infected with Mtb i.d. 1-7 days after cell transfer.

Flow cytometry

Identification of mycobacteria-specific CD8+ T cells was performed by using antibodies against CD3-A700 (clone 500 A2; Cat. No. 557984), CD4-PerCP-Cy5.5 (clone RM 4-5; Cat. No. 55094), CD8-BV510 (clone 53-6.7; Cat. No. 563068 ) and CD44-BV421 (clone IM7; Cat. No. 563970); all purchased from BD Biosciences. Monomers of H-2Kb Mtb TB10.4 4-11 IMYNYPAM were provided by the NIH Tetramer Core Facility, USA. Monomers were tetramerized with Streptavidin-APC (Agilent Technologies) in house, according to NIH Tetramer Core Facility instructions. Single cells were incubated with Fixable Viability stain 780 (BD Biosciences) for 10 minutes at room temperature and washed with FACS buffer solution. Samples were then incubated with the H-2Kb Mtb TB10.4 tetramer (1:50 dilution) for 1 hour at room temperature and washed with FACS buffer solution. Cells were then incubated for 30 minutes with surface antibodies (1:200 dilution) on ice and washed with FACS buffer solution. Samples were resuspended in 150 µl of blank calibration particles 6.0 - 6.4 µm (BD Biosciences) diluted in FACS buffer solution (1:74) for subsequent enumeration. Samples were analysed using a FACSCanto-II or FortessaX20 analyser (BD Biosciences) (Table S1).

RNA sequencing from fixed cervical lymph nodes

RNA extraction, library preparation, sequencing and initial data processing were performed by Lexogen NGS Services, Lexogen GmbH, Austria. In brief, RNA from cLNs was isolated using PureLink FFPE RNA Isolation Kit (ThermoFisher). Samples were characterized by UV-Vis spectrophotometry (Nanodrop2000c, Thermo Fisher) and RNA integrity was assessed on a Fragment Analyser System using the DNF-471 RNA Kit (15 nt) (Agilent). To remove genomic DNA contaminants, the samples were treated with DNase I. The libraries were constructed using Lexogen’s QuantSeq 3' mRNA-Seq Library Prep Kit FWD. Prepared libraries were quality controlled on a Fragment Analyser System using the DNF-474 and the HS-DNA kit (1-6000 bp) (Agilent). cDNA libraries were sequenced with an Illumina NextSeq 2000 platform using 100 nt single end read length, with 5 million reads on average per sample. Sequencing quality control of the raw reads was assessed using FastqQC software, and adapter sequences were removed with cutadapt 72. Alignment to the Mus musculus reference genome (GRCm38, Ensembl release 102) and read counting were performed using STAR 73 and featureCounts 74. The DESeq2 R package 75 was used to perform differential gene expression analysis.

Oligonucleotides for library synthesis including the first strand oligo(dT) primer, second strand synthesis random primers, and library amplification primers are propitiatory to Lexogen. Illumina sequencing primers are available on page 27 of the Lexogen QuantSeq user guide - https://www.lexogen.com/wp-content/uploads/2023/07/191UG444V0111_QuantSeq_FWD_UDI_V2_Kits_2023-07-12.pdf.

Data analysis & statistics

Data analysis was performed using different software: GraphPad Prism versions 9.3.1 to 10.6.1; R and RStudio version 4.3.1. R packages included NanoStringNCTools, GeomxTools, GeoMxWorkflows and GeoMx-NGS gene panel version Mm_R_NGS_WTA_v1.0. For image analysis and representative images, QuPath versions v0.3.1 to v0.5.0, Image J version 1.53 and GIMP versions 2.10.30 to 2.10.36 were used. The significance of DEGs in the volcano plots was determined using a false discovery cut-off rate (FDR) < 0.05. The Venn diagrams were plotted using significant DEGs for each comparison with the VennDiagram package in R. The pathway analyses were done using the QIAGEN Ingenuity pathway analysis (IPA) version 24.0.2. Flow cytometry data was collected using BD FACSDiva software and analysed using FlowJo version 10.8.1 Results are presented as data means ± SEM. Statistical comparisons were performed using Mann-Whitney test, One-way ANOVA with Kruskal-Wallis test with Dunn’s multiple comparison test, or Long-rank (Mantel-Cox) test 2-way ANOVA with Tukey’s multiple comparison test and Fisher’s exact test. Data were considered significant when *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In the manuscript “CD8+ T cells sustain vaccination-induced immunity against reactivation of latent 2 tuberculosis in\nimmunosuppressed hosts” Miranda-Hernandez et al. investigate how latent infection with Mycobacterium tuberculosis (Mtb)\ncan be controlled in an immune compromised hosts. Utilizing a mouse model of latent lymphatic Mtb infection to dissect the\nimmunological mechanisms underlying Mtb containment versus reactivation the authors demonstrate that\nimmunosuppression-mediated reactivation of Mtb and the subsequent induction of progressive disease relies on the\ninduction of CD8+ T cells, rather than CD4+ T cells. While monocytes and macrophages in humans express CD4+, the sole\nexpression of CD4 on T cells in this mouse models allows the selective study the impaired function or loss of CD4+ T cells\nin human LTBI. THeir results give detailed high-dimensional insight into the cellular compositions during Mtb reactivation\nand BCG-mediated containment of Mtb in the lymphatics.\nOverall, this is a well-designed study that dissects for the first time the contribution of the individual immune subsets to the\ndissemination and control of Mtb infection in mice. This mouse model relies on anti-CD4 blockage as immune compromised\nconditions. The Mtb reactivation experiments and the data showing that reactivation occurs across multiple organs is elegant\nand supports the importance of utilizing a mouse model.\nMinor Concerns:\n• The authors do not perform the concurrent experiment of depleting the CD8 T cells. This would demonstrate the role of\nCD4 T cells alone, which seems to be the antigen specific subsets in humans.\n• What antigen specific T cell response do the BCG vaccines induce?\n• It is unclear how the authors define antigen specific T cells? How can the authors be sure that the effect is antigen specific?\nCan more details be provided?", "reviewer_2": "This manuscript describes a large series of experiments conducted in a mouse model of latent lymphatic Mtb infection that\naim to dissect the immunological mechanisms capable of preventing Mtb reactivation and subsequent progressive disease.\nThe authors first demonstrated that immunosuppression-mediated reactivation of lymphatic LTBI can be prevented by\nvaccination with BCG or recombinant BCG, even in the absence of CD4+ T cells. They then performed antibody-mediated\ncell depletion combined with multi-parameter imaging and spatial transcriptomics to show a redistribution of immune cells (B\ncells and CD8+ T cells) at the edge of TB lesions. They further demonstrated through adoptive transfer experiments that\nprevention of reactivation was mediated by CD8+ T cells.\nThis reviewer is not an expert on the mouse model of Mtb infection and, as such, is not able to fully evaluate the novelty of\nthese findings.\nOverall, the experiments are well conducted, the results support the conclusions made, and limitations—such as the fact that\nthe infection in the animals does not follow the natural route, and that the analysis of immune cells at the edge of TB lesions\nwas not performed using single-cell analysis—are discussed.\nI do not have any relevant comments except to congratulate the authrs for a technically well executed work.", "reviewer_3": "In this manuscript, Miranda-Hernandez and colleagues leverage the ear model of Mycobacterium tuberculosis infection to\nassess the requirement of CD4+ T cells and the impact of vaccination on control in this mouse model that seeks to\nrecapitulate human-like immune control. This manuscript brings together a large number of animal studies that represent an\nimmense amount of work. However, because it is so sprawling, it can be difficult to track what are the major, well-supported\nconclusions and what are provocative observations, and in places the authors use short-hand that can be misleading. I\nwould suggest aggressive editing for readability.\nSpecific concerns are as follows:\n(1) The manuscript uses an Mtb ear infection model which allows Mtb to establish but remain controlled in the mouse lymph\nnodes –at least in the absence of immunocompromise. The authors have previously posited this as a model for latent\ninfection in people –and here use the term LTBI throughout. While I agree that this model is very interesting because it\nreflects immune control at the level of the lymph node, I think there is disagreement in the field as to what sort of human\ndisease is represented. Duffy et al., JID 2022, posit the model reflects asymptomatic TB infection – not overt latency. I find it\ninappropriately speculative to term the different mouse groups “LTBI” and “Reactivation” in the Results\n(2) There are many relatively minor edits that would make the paper easier to read.\na. The Figures can be hard to read which does a disservice to the volume of data. In many of the CFU plots, it is difficult to\ntell which statistical comparisons were done and not significant vs not done. The images (esp in Fig 5J,L) are tiny and fonts are\noften very small.\nb. To improve clarity, the authors should consider cutting observations that are not part of core narrative (Some examples\ninclude time point by time point weight comparisons, differences in liver and femurs between groups at single time points,\nMtb reduction in liver but not bone marrow in BCG-Esat6-PE25SS, enumeration of transcriptional changes etc…).\nc. The CD4 depletion is a key aspect of the study. Would it be helpful to put data that quantitatively addresses the efficacy\nand durability of the depletion in Figure 1 rather than only addressing it as an aside in Figure 3E?\n(3) The weight curves are not intuitive and could use some contextualization. Mice in both\nvaccine groups –even in the absence of aCD4--gain less weight than naïve mice (in 1C), which seems surprising. Also hard to\nunderstand why this is not true for mice receiving BCG:Esat6-PE25+Mtb or Mtb in 1D? Would it be helpful to explicitly\naddress BCG persistence as a driver of phenotypes and CFU (assume not a big problem from 1I but this would be very\nconfusing to a non-expert as written).\n(4) The spatial data is both very interesting and hard to follow. Could the authors please clarify text interpreting the imaging\nat several points. The authors seem to be interpreting compositional changes in B cell and CD8 cell representation after\nCD4 depletion (3E,F) as a change in absolute number (line 420-422)? I did not understand presentation of co-occurrence\ndata – which seems to put Mtb in nonphagocytic cells (3G,H)? I am sure that describing distance features is hard, but it is\nalso difficult to follow this section (438-463).\n(5) The identification of the lesion edge as the zone of CD4 effect is particularly interesting. Clearer images and legible\nvolcano plots of DEGs (4E is truly impossible to read) would be helpful here.\n(6) Given interest in CD8 population, did spatial transcriptomic panel include any cytotoxicity genes that might provide\ninsight into CD8 cell functionality?\n(7) The studies of immunodeficient mice + adoptive transfers in Figures 5 and 6 are the mechanistic heart of this work –\nwould be helpful to cut non-core parts of earlier Figures to get to this sooner + be able to display legibly.\n(8) Is there evidence of moonlighting as has been described in NHP (that CD8 cells acquire partial set of CD4 functions in\nthe setting of CD4 depletion?)\n(9) Although the authors describe rBCG as no improvement over BCG, at many points the data suggest that BCG—Esat6-\nPE25SS is actually less biologically active than BCG. Do the authors have a hypothesis as to why this might be the case?"}, "author_response": "NCOMMS-25-44227: Miranda-Hernandez et al.\nPoint-by-point reply to reviewers’ comments:\nThis document outlines how we have addressed the comments and concerns raised by the\nreviewers. We have copied the original reviewer’s comment (in italics) and our response to\nthem follows immediately below in blue. Line numbers refer to the revised manuscript with\ntrack changes.\nReviewer #1 (Remarks to the Author):\nIn the manuscript “CD8+ T cells sustain vaccination-induced immunity against reactivation\nof latent 2 tuberculosis in immunosuppressed hosts” Miranda-Hernandez et al. investigate how\nlatent infection with Mycobacterium tuberculosis (Mtb) can be controlled in an immune\ncompromised hosts. Utilizing a mouse model of latent lymphatic Mtb infection to dissect the\nimmunological mechanisms underlying Mtb containment versus reactivation the authors\ndemonstrate that immunosuppression-mediated reactivation of Mtb and the subsequent\ninduction of progressive disease relies on the induction of CD8+ T cells, rather than CD4+ T\ncells. While monocytes and macrophages in humans express CD4+, the sole expression of CD4\non T cells in this mouse models allows the selective study the impaired function or loss of CD4+\nT cells in human LTBI. THeir results give detailed high-dimensional insight into the cellular\ncompositions during Mtb reactivation and BCG-mediated containment of Mtb in the\nlymphatics.\nOverall, this is a well-designed study that dissects for the first time the contribution of the\nindividual immune subsets to the dissemination and control of Mtb infection in mice. This\nmouse model relies on anti-CD4 blockage as immune compromised conditions. The Mtb\nreactivation experiments and the data showing that reactivation occurs across multiple organs\nis elegant and supports the importance of utilizing a mouse model.\nWe thank the reviewer for the very positive feedback on our work. We have addressed their\ntwo minor concerns in the revised manuscript.\nMinor Concerns:\n• The authors do not perform the concurrent experiment of depleting the CD8 T cells. This\nwould demonstrate the role of CD4 T cells alone, which seems to be the antigen specific subsets\nin humans.\nThank you for this comment. CD8 T cell depletion in wild-type C57BL/6 mice and B cell\ndeficient B6.muMT-/- mice was previously shown in Figure 6H (now Figure 7J). In both mouse\nstrains CD8 T cell depletion by itself did not lead to mortality. In contrast, all mice with\ncombined CD4/CD8 T cell depletion succumbed due to the reactivation of latent Mtb infection.\nTo further substantiate the role of CD8 T cell depletion we have now also included the CFU\ndata from those experiments. In line with the survival data, the CFU results show that CD8 T\ncell depletion by itself does not lead to dissemination of latent Mtb infection to the lung. Only\ncombined CD4/CD8 T cell depletion leads to lethal reactivation of contained infection. Due to\nthe fact all the CD4/CD8 T cell depleted mice succumbed prior to the experimental endpoint\nat day 120, no CFU data are available for this group (indicated with a mortality symbol in\nFigure 7K).\nHowever, while the manuscript was under review we have also performed additional antibody-\nmediated depletion experiments in vaccinated C57BL/6 and B6.muMT-/- mice. While the\noverall dependency on CD8 T cells for survival in the absence of CD4 T cells was identical, as\nexpected, vaccinated CD4/CD8 mAb-treated mice showed a survival increase of\napproximately 20-30 days compared to unvaccinated mice (see Figure R1-1 K, L below). In\nthis situation, two CD4/CD8 mAb-treated C57BL/6 mice survived until day 120. While these\n2 mice were moribund, organs could just be collected prior to euthanasia. As expected, very\nhigh Mtb CFU were recovered from all organs of those 2 mice, indicating that combined\nCD4/CD8 T cell depletion leads to a dramatic dissemination of Mtb that cannot be contained\nby the host and is fatal. These results further substantiate that CD8 T cells sustain immunity\nagainst reactivation of latent tuberculosis in the absence of CD4 T cells, the key message of\nthis study. The results have now also been included in Figure 7 of the revised manuscript (lines\n1836-2043).\nFigure R1-1: Extraction form revised Figure 6 (now Figure 7) showing survival and CFU\nresults following CD4, CD8 or CD4/CD8 T cell depletion in vaccinated or unvaccinated\nC57BL/6 and B6.muMT-/- mice.\n• What antigen specific T cell response do the BCG vaccines induce? It is unclear how the\nauthors define antigen specific T cells? How can the authors be sure that the effect is antigen\nspecific? Can more details be provided?\nWhile we did not comment on antigen-specificity in our manuscript, we will clarify the\nreviewer’s question below.\nUsing MHC tetramers, we and many other groups have previously shown that mycobacterial\nantigen-specific T cells are being induced following BCG vaccination (Perdomo, Zedler et al.\n2016). We have also previously shown that BCG::ESAT-6-PE25SS induces specific T cell\nresponses to ESAT-6 (Heijmenberg, Husain et al. 2021). Given that BCG contains >4,000\nproteins, induction of a Th1 response, presence of polyfunctional T cells, expressing IFNγ,\nTNF and IL2; or a cytotoxic T cell response are sometimes used as surrogates of T cell\nactivation in responses to BCG vaccination (Moliva, Turner et al. 2017). Given that our\nanalyses relied on NanoString GeoMx spatial transcriptomics that is based on regions of\ninterest (ROIs) within the tissue rather than single cells, specific cytokine responses at a single\nT cell level cannot be extracted from this data set. Single cell resolved spatial transcriptomics\ntechnology, such as NanoString CosMx, was not yet available while this study was conducted\nto define antigen-specific T cells in the lungs of vaccinated/infected mice. We had discussed\nthis as a direction for future investigations.\nFurthermore, given that a similar antimicrobial response was obtained for BCG- and\nBCG::ESAT6-PE25SS- vaccinated hosts against Mtb challenge in this study, it can only be the\nredundant antigens present in both BCG and BCG::ESAT6-PE25SS, rather than the unique\nantigens of BCG::ESAT6-PE25SS, that must have contributed to the T cell response. However,\nmore detailed studies are warranted to confirm this notion.\nNevertheless, to address the reviewer’s comments and to further substantiate that both BCG\nvaccine strains induce mycobacteria-specific CD8 T cell responses, we have now performed\nadditional tetramer-based T cell analyses to show that both vaccine strains induced TB10.4:H-\n2Kb-specific T cells in the spleen, the cervical and the inguinal lymph nodes (see Figure R1-\n2).\nThis new data has now been included in the revised Figure 7I, and we have discussed the role\nof shared and unique antigens in the BCG strains in the manuscript (lines 2296-2311).\nFigure R1-2: Representative FACS plots and enumerations of TB10.4:H-2Kb-specific CD8 T\ncells in spleen, cervical lymph nodes (cLN) and inguinal lymph nodes (iLN) following\nvaccination with BCG or BCG::ESAT-6-PE25SS. Now included as revised Figure 7I.\nReviewer #2 (Remarks to the Author):\nThis manuscript describes a large series of experiments conducted in a mouse model of latent\nlymphatic Mtb infection that aim to dissect the immunological mechanisms capable of\npreventing Mtb reactivation and subsequent progressive disease.\nThe authors first demonstrated that immunosuppression-mediated reactivation of lymphatic\nLTBI can be prevented by vaccination with BCG or recombinant BCG, even in the absence of\nCD4+ T cells. They then performed antibody-mediated cell depletion combined with multi-\nparameter imaging and spatial transcriptomics to show a redistribution of immune cells (B\ncells and CD8+ T cells) at the edge of TB lesions. They further demonstrated through adoptive\ntransfer experiments that prevention of reactivation was mediated by CD8+ T cells.\nThis reviewer is not an expert on the mouse model of Mtb infection and, as such, is not able to\nfully evaluate the novelty of these findings.\nOverall, the experiments are well conducted, the results support the conclusions made, and\nlimitations—such as the fact that the infection in the animals does not follow the natural route,\nand that the analysis of immune cells at the edge of TB lesions was not performed using single-\ncell analysis—are discussed.\nI do not have any relevant comments except to congratulate the authors for a technically well\nexecuted work.\nWe thank the reviewer for their praise and enthusiasm on our work.\nReviewer #3 (Remarks to the Author):\nIn this manuscript, Miranda-Hernandez and colleagues leverage the ear model of\nMycobacterium tuberculosis infection to assess the requirement of CD4+ T cells and the impact\nof vaccination on control in this mouse model that seeks to recapitulate human-like immune\ncontrol. This manuscript brings together a large number of animal studies that represent an\nimmense amount of work. However, because it is so sprawling, it can be difficult to track what\nare the major, well-supported conclusions and what are provocative observations, and in\nplaces the authors use short-hand that can be misleading. I would suggest aggressive editing\nfor readability.\nWe thank the reviewer for their positive feedback. We have addressed their comments to further\nstreamline the manuscript.\nSpecific concerns are as follows:\n(1) The manuscript uses an Mtb ear infection model which allows Mtb to establish but remain\ncontrolled in the mouse lymph nodes –at least in the absence of immunocompromise. The\nauthors have previously posited this as a model for latent infection in people –and here use the\nterm LTBI throughout. While I agree that this model is very interesting because it reflects\nimmune control at the level of the lymph node, I think there is disagreement in the field as to\nwhat sort of human disease is represented. Duffy et al., JID 2022, posit the model reflects\nasymptomatic TB infection – not overt latency. I find it inappropriately speculative to term the\ndifferent mouse groups “LTBI” and “Reactivation” in the Results.\nThank you for this comment. We have now incorporated the Duffy et al. paper into the\nintroduction (lines 229-235) to better reflect the different TB disease manifestations (latency,\ncontainment, asymptomatic disease) that have been associated with the contained ear-dermis\ninfection model. As appropriate, we have replaced many of the ‘LTBI’ occurrences in the text\nwith ‘contained’ or ‘asymptomatic’ Mtb infection and ‘Reactivation’ with ‘dissemination’\nthroughout the manuscript. We have also changed the group designation shown in Figure 1L\n(now Figure 2K) from ‘Reactivation’ to ‘Dissemination’.\nIt is also important to note that one of the reasons for our extensive analysis of multiple different\norgans, including femurs, salivary glands, brains and livers, was to further characterise to what\nextent our lymphocentric infection model represents different human TB disease states. As\nReviewer 1 put it: “The Mtb reactivation experiments and the data showing that reactivation\noccurs across multiple organs is elegant and supports the importance of utilizing a mouse\nmodel.”\nWe strongly believe that the comprehensive analysis of containment vs. dissemination in the\near dermis infection model performed here, will stimulate vigorous discussion and further\nuptake of this model by the field.\n(2) There are many relatively minor edits that would make the paper easier to read.\na. The Figures can be hard to read which does a disservice to the volume of data. In many of\nthe CFU plots, it is difficult to tell which statistical comparisons were done and not significant\nvs not done.\nThank you. To address this, we have now changed the way statistical comparisons are shown,\nby including downward facing lines. In most instances, statistical comparisons were done\nrelative to the anti-CD4 mAb-treated group. We have now specified in the Figure legends which\nstatistical comparisons were made and that only significant differences are shown.\nThe images (esp in Fig 5J,L) are tiny and fonts are often very small.\nThank you. We have carefully gone through each Figure and have now increased the size and\nfont size in many of the Figure panels (e.g., new Fig 1; Fig 2G, J, I, L; Fig 3A-C; Fig5C, E, F,\nG, H, I; Fig 6F, G, H, I J, L, K, M , N, O; Fig 7H, J; Fig S2A, B, C, D; Fig S4A, B, C, D, E) to\naddress this concern. Please also see our response to your question below, regarding the\nchanges to Figures and Supplementary Figures.\nb. To improve clarity, the authors should consider cutting observations that are not part of core\nnarrative (Some examples include time point by time point weight comparisons, differences in\nliver and femurs between groups at single time points, Mtb reduction in liver but not bone\nmarrow in BCG-Esat6-PE25SS, enumeration of transcriptional changes etc…).\nThank you. Given that reviewer 1 and 2 commented and praised the comprehensive nature of\nour analyses, including the investigations in multiple organs, in our revision we have attempted\nto strike a compromise between removing observations that are not part of the core narrative\nand moving them to Supplementary Figures. As requested by the reviewer, we have now\nremoved the weight comparisons from Figure 1 (now Figure 2). Days 30 and 60 results of the\nhistological analyses of liver, femur and mesenteric lymph nodes have now been removed, and\nthe corresponding manuscript text has been shortened. Likewise, histological analyses of\nsalivary glands and brains have been removed from Figure 2 (now Figure 3). In addition, we\nhave removed all of the IHC lung data from Figure 3 (now Figure 4) and shortened the text\naccordingly. Likewise, most of the enumerations of transcriptional changes previously shown\nin Figure 4H-J (except CD19+ cells) have also been moved to Supp Fig 5D-F and the p-value-\nbased pathway analysis shown in Fig. 4F has been removed.\nBelow is a summary of all changes across the Figures:\n- Graphical Abstract: Changed into new Figure1\n- Figure 1: Changed to new Figure 2; weight comparisons moved to Fig S1\n- Figure 2: Changed to new Figure 3; moved day 30 and day 60 results for mLN, liver,\nand femurs as well as brain and salivary gland data to Fig S2\n- Figure 3: changed to new Figure 4; IHC data from lung moved to Fig S3\n- Figure 4: changed to Figure 5; moved transcriptomic counts to Fig. S5; simplified\ncanonical pathway analysis\n- Figure 5: changed to Figure 6; increased font and image sizes\n- Figure 6: changed to Figure 7; now includes additional data on antigen-specificity and\nCFU results\n- Supplementary Figure 1: new\n- Supplementary Figure 2: new\n- Supplementary Figure 3: previously Fig S1; now contains IHC data from lungs\n- Supplementary Figure 4: previously Fig S2; increased image and font sizes\n- Supplementary Figure 5: previously Fig S3; includes transcriptomics-based cell counts\n- Supplementary Figure 6; previously Fig S4; minor changes to mouse strain names\n- Supplementary Figure 7; new gating strategy for FACS data shown in Figure 7\nc. The CD4 depletion is a key aspect of the study. Would it be helpful to put data that\nquantitatively addresses the efficacy and durability of the depletion in Figure 1 rather than\nonly addressing it as an aside in Figure 3E?\nThank you for this comment. The efficacy and durability of the CD4 T cell depletion measured\nby flow cytometry was shown in our previous manuscripts (Kupz et. al., Plos One, 2016;\nSathkumara et al. Front Immunol. 2019). Given that the depletion of CD4 T cells quantified by\nimmunohistochemistry (IHC) in Figure 3E (now Figure 4E) is part of the larger Opal IHC\nanalysis, we have decided not to move this quantification to Figure 1. Instead, we have included\nan additional explanation into the manuscript (line 744) to refer to our previous studies.\n(3) The weight curves are not intuitive and could use some contextualization. Mice in both\nvaccine groups –even in the absence of aCD4--gain less weight than naïve mice (in 1C), which\nseems surprising. Also hard to understand why this is not true for mice receiving BCG:Esat6-\nPE25+Mtb or Mtb in 1D? Would it be helpful to explicitly address BCG persistence as a driver\nof phenotypes and CFU (assume not a big problem from 1I but this would be very confusing to\na non-expert as written).\nThank you. As the reviewer had mentioned in their comment above that it will be beneficial to\ncut observations that are not part of core narrative, we have decided to move the weight curves\nto Supplementary Information as they don’t add value to the key message. In line with this, we\nhave also significantly shortened the text describing the weight results.As the reviewer had\nrightly identified, there is no difference in BCG persistence as shown in Figure 1I (now Fig\n2F). We have also update the text to better reflect that Fig 2F refers to BCG persistence rather\nthan Mtb burden (lines 338-344).\n(4) The spatial data is both very interesting and hard to follow. Could the authors please clarify\ntext interpreting the imaging at several points. The authors seem to be interpreting\ncompositional changes in B cell and CD8 cell representation after CD4 depletion (3E,F) as a\nchange in absolute number (line 420-422)? I did not understand presentation of co-occurrence\ndata – which seems to put Mtb in nonphagocytic cells (3G,H)? I am sure that describing\ndistance features is hard, but it is also difficult to follow this section (438-463).\nThank you very much for this comment. We have now carefully reviewed the section describing\nthe spatial IHC data and moved the lung IHC results to Supplementary Figure 3 to simplify\nthis section.\nThe reviewer rightly identified that Figures 3E, F (now Fig 4E; Fig S3C) indeed show\npercentage of cells per organ. We have corrected this in the text.\nWe have also rectified instances in which data shown in Figure 3J was accidentally called out\nas Figure 3I (now Figure 4G, H).\nWe have also included additional references to show that co-localization of Mtb with non-\nclassical phagocytic cells and nonphagocytic cells has indeed been observed previously (line\n754).\n(5) The identification of the lesion edge as the zone of CD4 effect is particularly interesting.\nClearer images and legible volcano plots of DEGs (4E is truly impossible to read) would be\nhelpful here.\nThank you. We have now increased the size of all volcano plots and DEGs in Figure 4 (now\nFigure 5) and Supplementary Figure 4.\n(6) Given interest in CD8 population, did spatial transcriptomic panel include any cytotoxicity\ngenes that might provide insight into CD8 cell functionality?\nThank you. Yes, the spatial transcriptomics panel included cytotoxicity genes, and we have\nnow included a heatmap showing expression of cytotoxic genes as part of the spatial\ntranscriptomics analyses shown in Figure 5I. The corresponding manuscript text (lines 1033-\n1035) has been adjusted accordingly.\n(7) The studies of immunodeficient mice + adoptive transfers in Figures 5 and 6 are the\nmechanistic heart of this work – would be helpful to cut non-core parts of earlier Figures to\nget to this sooner + be able to display legibly.\nThank you. Please see our comments above on how we have cut some results from Figures 1-\n4 (now Fig 2-5), keeping in mind the comments raised by reviewers 1 & 2. We have also\nincreased the size of histological images and font sizes in Figures 5 and 6 (now Figs 6 and 7)\nto improve legibility.\n(8) Is there evidence of moonlighting? It has been described in NHP (that CD8 cells acquire\npartial set of CD4 functions in the setting of CD4 depletion?)\nThank you. As mentioned above, NanoString GeoMx does not allow spatial resolution to the\nsingle cell level. As such, it is not possible to look at moonlighting in the spatial data. However,\nwe have now included this as an additional point into the discussion (lines 2319-2320).\n(9) Although the authors describe rBCG as no improvement over BCG, at many points the data\nsuggest that BCG—Esat6-PE25SS is actually less biologically active than BCG. Do the authors\nhave a hypothesis as to why this might be the case?\nThank you. We have now expanded the Discussion to further reflect on the differences in BCG\nand BCG::ESAT6-PE25SS (lines 2297-2307). “It is known that the secretion of ESAT-6 leads\nto additional immune activation and the perforation of the phagosomal membrane. Whether\nthis leads to specific immune- or stromal cell alterations in the cLNs with implications for Mtb\ncontainment remains to be investigated. Similarly, although we experimentally confirmed the\nexpression of functional Mtb ESAT-6 by our rBCG strain, the amount of ESAT-6 required, and\nits cytoplasmic sustainability in APCs to induce a persistent immune activation in vaccinated\nanimals were not explored. Thus, further detailed analysis of optimal ESAT-6 levels and the\nkinetics of immune activation at different ESAT-6 levels in APCs are warranted to optimize the\nimmunogenicity and/or protective efficacy of our rBCG. However, given that most rBCGs only\ndiffer from BCG in a very small number of antigens, our study implies that the mechanisms of\nvaccination-mediated containment are largely driven by factors that are shared between BCG\nand rBCGs. Future studies should investigate which components of BCG drive containment\nand if containment can also be induced by subunit TB vaccine candidates.”.\nReferences:\nHeijmenberg, I., A. Husain, H. D. Sathkumara, V. Muruganandah, J. Seifert, S. Miranda-\nHernandez, R. S. Kashyap, M. A. Field, G. Krishnamoorthy and A. Kupz (2021). \"ESX-5-targeted\nexport of ESAT-6 in BCG combines enhanced immunogenicity & efficacy against murine\ntuberculosis with low virulence and reduced persistence.\" Vaccine.\nMoliva, J. I., J. Turner and J. B. Torrelles (2017). \"Immune Responses to Bacillus Calmette-Guérin\nVaccination: Why Do They Fail to Protect against Mycobacterium tuberculosis?\" Front Immunol\n8: 407.\nPerdomo, C., U. Zedler, A. A. Kühl, L. Lozza, P. Saikali, L. E. Sander, A. Vogelzang, S. H. E.\nKaufmann and A. Kupz (2016). \"Mucosal BCG Vaccination Induces Protective Lung-Resident\nMemory T Cell Populations against Tuberculosis.\" mBio 7(6)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have comprehensively answered all concerns.", "reviewer_2": "No further comments.", "reviewer_3": "This is a thoughtful revision that addresses my concerns. I am again impressed with the scope of work in the manuscript and\nbelieve it is an important contribution to the field."}, "author_response": ""}]

Decision letter