Dominant expansion of CD4+, CD8+ T and NK cells expressing Th1/Tc1/Type 1 cytokines in culture-positive lymph node tuberculosis.

Lymph node culture-positive tuberculosis (LNTB+) is associated with increased mycobacterial antigen-induced pro-inflammatory cytokine production compared to LN culture-negative tuberculosis (LNTB-). However, the frequencies of CD4+, CD8+ T cells and NK cells expressing Th1/Tc1/Type 1 (IFNγ, TNFα, IL-2), Th17/Tc17/Type 17 (IL-17A, IL-17F, IL-22) cytokines and cytotoxic (perforin [PFN], granzyme [GZE] B, CD107a) markers in LNTB+ and LNTB- individuals are not known. Thus, we have studied the unstimulated (UNS) and mycobacterial antigen-induced frequencies of CD4+, CD8+ T and NK cells expressing Th1, Th17 cytokines and cytotoxic markers using flow cytometry. The frequencies of CD4+, CD8+ T and NK cells expressing cytokines and cytotoxic markers were not significantly different between LNTB+ and LNTB- individuals in UNS condition. In contrast, upon Mtb antigen stimulation, LNTB+ individuals are associated with significantly increased frequencies of CD4+ T cells (PPD [IFNγ, TNFα], ESAT-6 PP [IFNγ, TNFα], CFP-10 PP [IFNγ, TNFα, IL-2]), CD8+ T cells (PPD [IFNγ], ESAT-6 PP [IFNγ], CFP-10 PP [TNFα]) and NK cells (PPD [IFNγ, TNFα], ESAT-6 PP [IFNγ, TNFα], CFP-10 PP [TNFα]) expressing Th1/Tc1/Type 1, but not Th17/Tc17/Type 17 cytokines and cytotoxic markers compared to LNTB- individuals. LNTB+ individuals did not show any significant alterations in the frequencies of CD4+, CD8+ T cells and NK cells expressing cytokines and cytotoxic markers compared to LNTB- individuals upon HIV Gag PP and P/I antigen stimulation. Increased frequencies of CD4+, CD8+ T and NK cells expressing Th1/Tc1/Type 1 cytokines among the LNTB+ group indicates that the presence of mycobacteria plays a dominant role in the activation of key correlates of immune protection or induces higher immunopathology.

Introduction

Tuberculosis (TB) is a major infectious disease with 10 million active cases and 1.3 million deaths reported worldwide [1]. Infection with Mycobacterium tuberculosis (Mtb) is associated with various states of disease progression such as latent TB (LTB), pulmonary TB (PTB) and extra-pulmonary TB (EPTB) [2]. While the incidence of overall TB cases is falling in developing nations, the incidence of EPTB is not decreasing significantly with higher mortality and morbidity rate observed globally. EPTB represents between 15–20% of all forms of TB cases [3]. Among them, lymph node tuberculosis (LNTB) or tuberculous lymphadenitis is the typical manifestation responsible for 20% of cases with the peripheral involvement and cervical region being most usually affected [3-6]. Excisional biopsy or fine-needle aspiration cytology (FNAC) is preferred for pathologic examination; however, still, culture (either solid or liquid) or nucleic acid amplification test (NAAT) based confirmation of the Mycobacterium bacilli remains the gold standard for diagnosis [7]. Therefore, diagnosis of LNTB is very difficult due to the occurrence of various infectious and non-infectious diseases which are often associated with a similar clinical state. Moreover, the immune responses between the culture positive and negative LNTB phenotype remain unclear.

We have previously shown lymph node TB culture-positive (LNTB+) individuals are associated with elevated Mtb antigen-induced levels of type 1, type 17, pro-inflammatory cytokines and reduced TGFβ levels upon comparison with lymph node TB culture-negative (LNTB−) individuals [8]. Similarly, LNTB patients displayed significantly reduced CD4+, CD8+ T cells and natural killer (NK) cells expressing cytotoxic markers compared to peripheral blood [9]. We showed that LNTB individuals were associated with increased (TNFα, IL-17A) and decreased (IL-1α, IL-1β, IL-18) pro-inflammatory cytokines in lymph nodes (LN) compared to whole blood [10]. However, understanding the correlates of immune protection at the site of infection or affected LNs has not been studied. Hence, it is important to examine the relationship of diverse cytokines/cytotoxic markers and their role in immune activation at sites of Mtb infection to improve our understanding of the differences in pathogenesis between LNTB+ and LNTB-. Therefore, our present study is the first to examine the cytokine and cytotoxic marker expressing CD4+, CD8+ T cells and NK cell frequencies in LNTB+ and LNTB- individuals. We demonstrate that LNTB+ individuals are predominantly associated with heightened Mtb antigen-induced CD4+, CD8+ T cells and NK cell frequencies expressing Th1/Tc1/type 1 cytokines compared to LNTB- individuals. Overall, our results indicate that the existence of mycobacteria plays a dominant role in the activation of key correlates of immune protection or induces higher immunopathology.

Materials and methods

Study groups

We recruited lymph node TB culture-positive (hereafter LNTB+, n = 18) and lymph node TB culture-negative (hereafter LNTB-, n = 10) patients and performed the experiments. The demographics and hematological data of the study population were reported previously [8] and the same set of lymph node (LN) samples was used for this study. LNTB+ group was characterized based on the excision biopsy exhibiting positive for Mycobacterium tuberculosis (Mtb) on liquid cultures and LNTB- (culture grades 0 [no or <19 colonies]) group was characterized based on the negative results for Mtb detection in liquid cultures. LNTB- individuals were diagnosed on the basis of histopathology. Both the study groups were negative for HIV infection and were not administered any steroids. The study was approved by the ethics committee of the National Institute of Research in Tuberculosis (NIRTIEC2010007). We have obtained written informed approval from both the study groups.

Lymph node isolation, culture and antigen stimulation

The LN cells from LNTB+ and LNTB- individuals were isolated, cultured and either unstimulated, stimulated with Mtb (PPD [Staten’s Serum Institute], ESAT-6, CFP10 [BEI resources, 10 μg/mL]), non-Mtb (HIV Gag PP, (AIDS Reagent Program, Division of AIDS, NIAID, NIH), 10 μg/mL) and positive control (phorbol myristate acetate/Ionomycin [P/I], (Calbiochem, San Diego, CA), 12.5 and 125 ng/mL) antigens [8, 9]. Before culturing, the LNs were transported in the Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco) after biopsy and washed two times in RPMI 1640 medium. Further, the LNs were chopped into smaller pieces, treated with deoxyribonuclease (DNase, Sigma-Aldrich) and liberase (collagenase I and II, 0.1 mg/mL, Sigma-Aldrich) and incubated at 37°C for 20–30 minutes. The cells were filtered using an 80–100 μm filter (Becton Dickinson, BD), washed, centrifuged (2,600 rpm for 10 minutes) and mixed using complete RPMI (RPMI, fetal calf serum [FCS], HEPES buffer, antibiotic cocktail [gentamycin, penicillin, streptomycin]). The cells were counted using trypan blue (Bio-WhittakerTM, Walkersville, MD) and evenly dispersed (2 million cells in 2mL/well) in culture plates (12-well, Costar), stimulated with the above-mentioned condition. To that, Brefeldin A solution (10 μg/mL, BD) was added after 2 hours and incubated for 18 hours at 37°C. Once incubation was over, LN cells were washed with 1X PBS and fixed using cyto fix/perm (BD, 20 minutes in dark conditions). Finally, the cells were washed with 1X permeabilization buffer (Invitrogen) and stored at -80°C in phosphate buffer saline (PBS, Lonza)/ dimethyl sulphoxide (DMSO, Sigma-Aldrich) until further use.

Extracellular and intracellular staining of lymph nodes

The LN cells were thawed at 37°C, washed with 1X PBS and spun at 2,600 rpm for 10 minutes. The supernatants were discarded and the LN cells were mixed with 1X permeabilization buffer. Further, the cells were stained first only with surface antibodies and incubated for 30–60 minutes, washed with 1X permeabilization bufferTM [BD Biosciences] and followed by staining with intracellular antibodies (cytokines and cytotoxic markers) and incubated overnight at 4°C. Once the incubation was over, the cells were washed with permeabilization buffer and spun at 2,600 rpm for 10 minutes. The supernatants were discarded and LN cells were mixed with 1X PBS and acquisition was performed. The surface markers used were CD3, CD4, and CD8 (all purchased from BD Biosciences) and CD56 (ebioscienceTM). The intracellular markers used were Th1 [IFNγ (BD Biosciences), TNFα (BD Biosciences), IL-2 (ebiosciences)], Th17 [IL-17A (Miltenyi Biotech), IL-17F and IL-22 (R&D systems)] cytokines and cytotoxic markers [perforin (BD Pharmingen), granzyme B (Invitrogen- ebioscienceTM), CD107a (BD Biosciences)]. The volume added and the other details of the antibodies were given in S1 Table. Flow cytometry (eight-colour, FACSCanto II, Diva software v.6) was performed for the sample acquisition (Becton Dickinson). Lymphocyte gating was determined by using forward vs side scatter and a total of 100,00 million cellular events were obtained. FlowJoTM (version 10) software was used to analyze the data and CD4+, CD8+ T cells and CD56+ NK cell gating strategies for cytokines and cytotoxic markers were determined with the help of FMO. We represented our data as CD4+, CD8+ and NK cell frequencies expressing cytokine(s) and cytotoxic markers. The baseline (following media stimulation) values of each cytokine and cytotoxic markers were used to determine the frequency; whereas, the net frequencies were represented for Mtb, non-Mtb and PMA/I antigen stimulation.

Analysis

The significant (P) values were calculated using the statistical software GraphPad PRISM (version 9.3) software (Graph-Pad Software, Inc., San Diego, CA). The geometric means (GM) were calculated by central tendency and the difference between the two groups was calculated using the nonparametric Mann-Whitney U test.

Results

LNTB+ individuals are associated with increased frequencies of CD4+ Th1 cytokines

We show the CD4+ and CD8+ T cells and NK cell gating strategy in S1A Fig and FMO population of CD4, CD8 and CD56 markers in S1B Fig. The representative plots of cytokine and cytotoxic markers (CD4+/CD8+ T cells and NK cells) of the study population are shown in S2 and S3 Figs. We measured the frequencies of CD4+ T cells expressing Th1 (IFNγ, TNFα, IL-2), Th17 (IL-17A, IL-17F, IL-22) cytokines and cytotoxic (PFN, GZE B, CD107a) markers between LNTB+ and LNTB- individuals (Fig 1). We show upon unstimulated (UNS) condition (Fig 1A), the frequencies of CD4+ T cells expressing Th1 and Th17 cytokines and cytotoxic markers were not significantly different between LNTB+ and LNTB- individuals. The frequencies of CD4+ T cells expressing Th1 (IFNγ, TNFα and/or IL-2) cytokines but not Th17 cytokines and cytotoxic markers were significantly increased between LNTB+ and LNTB- individuals upon stimulation with Mtb (PPD, ESAT-6 PP, CFP-10 PP) antigens (Fig 1B–1D). As shown in Fig 1E, the frequencies of CD4+ T cells expressing Th1 and Th17 cytokines and cytotoxic markers were not significantly different between LNTB+ and LNTB- individuals upon stimulation with HIV Gag PP antigen. Finally, upon P/I antigen stimulation, CD4+ T cells expressing Th1 and Th17 cytokines as well as cytotoxic marker frequencies are not significantly different between the study groups (Fig 1F). Hence, LNTB+ individuals are associated with increased frequencies of mycobacterial antigen-induced CD4+ T cells expressing Th1 cytokines.

Fig 1

LNTB+ individuals are associated with enhanced antigen-induced frequencies of CD4+ T cells expressing Th1 (IFNγ, TNFα) cytokines.

Lymph node cells from Lymph node (LN) culture-positive tuberculosis (LNTB+, shown in blue colored round circle, n = 18) and LN culture-negative tuberculosis (LNTB-, shown in red-coloured round circle, n = 10) individuals were either unstimulated (UNS) or stimulated with mycobacterial (PPD, ESAT-6 PP, CFP-10 PP), HIV Gag PP and positive (P/I) control antigens for 18 hours. LN cells were harvested, stained and examined using multicolour flow cytometry. (A) UNS (B-D) Mtb antigen (E) HIV Gag PP and (F) positive control (P/I) antigen cultured CD4+ T cell expressing frequencies of Th1 (IFNγ, TNFα, IL-2), Th17 (IL-17A, IL-17F, IL-22) cytokines and cytotoxic (PFN, GZE B, CD107a) markers were illustrated. The geometric mean was shown using a bar and the Mann-Whitney U test was implemented to calculate the p values and the statistically significant value is represented as p<0.05. For each individual, we have given the values as net frequencies and they were obtained by deducting the unstimulated frequencies from the antigen-stimulated frequencies.

LNTB+ individuals associated with increased frequencies of CD8+ Tc1 cytokines

We analyzed CD8+ T cells expressing Tc1 (IFNγ, TNFα, IL-2), Tc17 (IL-17A, IL-17F, IL-22) cytokines and cytotoxic (PFN, GZE B, CD107a) marker frequencies between LNTB+ and LNTB- individuals (Fig 2). We show the unstimulated frequencies of CD8+ T cells expressing Tc1 and Tc17 cytokines and cytotoxic markers were not significantly different between LNTB+ and LNTB- individuals (Fig 2A). We show the frequencies of CD8+ T cells expressing Tc1 (IFNγ in PPD and ESAT-6 PP, TNFα in CFP-10 PP) cytokines but not Tc17 cytokines and cytotoxic markers were significantly increased between LNTB+ and LNTB- individuals upon Mtb antigen-induced stimulation (Fig 2B–2D). In contrast, CD8+ T cells expressing Tc1 and Tc17 (except IL-17F) cytokines and cytotoxic marker frequencies were not significantly different between LNTB+ and LNTB- individuals upon stimulation with HIV Gag PP antigen (Fig 2E). Finally, upon P/I antigen stimulation, CD8+ T cells expressing Tc1 and Tc17 cytokines and cytotoxic marker frequencies are not significantly different between the study groups (Fig 2F). Hence, LNTB+ individuals are associated with increased frequencies of mycobacterial antigen-induced CD8+ T cells expressing Tc1 cytokines.

Fig 2

LNTB+ individuals are associated with enhanced antigen-induced frequencies of CD8+ T cells expressing Tc1 (IFNγ, TNFα) cytokines.

LN cells from LNTB+ (n = 18) and LNTB- (n = 10) individuals were either unstimulated (UNS) or stimulated with mycobacterial, HIV Gag PP and positive (P/I) control antigens for 18 hours. LN cells were harvested, stained and analyzed using multicolour flow cytometry. (A) UNS (B-D) Mtb antigen (E) HIV Gag PP and (F) P/I antigen-stimulated CD8+ T cell expressing frequencies of Tc1 (IFNγ, TNFα, IL-2), Tc17 (IL-17A, IL-17F, IL-22) cytokines and cytotoxic (PFN, GZE B, CD107a) markers were illustrated. The geometric mean was shown using a bar and the Mann-Whitney U test was performed to calculate the p values and p<0.05 is denoted as statistically significant. For each individual, we have given the values as net frequencies and they were obtained by deducting the unstimulated frequencies from the antigen-stimulated frequencies.

LNTB+ individuals are associated with increased frequencies of NK cells expressing Type 1 cytokines

We analyzed the frequencies of NK cells expressing Type 1 (IFNγ, TNFα, IL-2), type 17 (IL-17A, IL-17F, IL-22) cytokines and cytotoxic (PFN, GZE B, CD107a) markers between LNTB+ and LNTB- individuals (Fig 3). We show the unstimulated frequencies of NK cells expressing Type 1 and Type 17 cytokines and cytotoxic markers were not significantly different between LNTB+ and LNTB- individuals (Fig 3A). As we have shown in Fig 3B–3D, the frequencies of NK cells expressing Type 1 (IFNγ and TNFα in PPD and ESAT-6 PP, IFNγ in CFP-10 PP) cytokines but not Type 17 cytokines and cytotoxic markers were significantly increased in LNTB+ compared to LNTB- individuals upon Mtb antigen-induced stimulation. In contrast, NK cells expressing Type 1 and Type 17 cytokines and cytotoxic (except CD107a) marker frequencies were not significantly different between LNTB+ and LNTB- individuals upon stimulation with HIV Gag PP antigen (Fig 3E). Finally, upon P/I antigen stimulation, NK cells expressing Type 1 and Type 17 cytokines and cytotoxic frequencies are not significantly different between the study groups (Fig 3F). Hence, LNTB+ individuals are associated with increased Mtb antigen-induced NK cells expressing Type 1 cytokine frequencies.

Fig 3

LNTB+ individuals are associated with enhanced antigen-induced frequencies of NK cells expressing type 1 (IFNγ, TNFα) cytokines.

LN cells from LNTB+ (n = 18) and LNTB- (n = 10) individuals were either unstimulated (UNS) or stimulated with mycobacterial (PPD, ESAT-6 PP, CFP-10 PP), HIV Gag PP and positive (P/I) control antigens for 18 hours. LN cells were harvested, stained and analyzed using multicolour flow cytometry. (A) UNS (B-D) Mtb antigen (E) HIV Gag PP and (F) P/I antigen-stimulated CD4+ T cell expressing frequencies of type 1 (IFNγ, TNFα, IL-2), type 17 (IL-17A, IL-17F, IL-22) cytokines and cytotoxic (PFN, GZE B, CD107a) markers were illustrated. Geometric mean was shown using a bar and Mann-Whitney U test was performed to calculate the p values and p<0.05 is denoted as statistically significant. For each individual, we have given the values as net frequencies and they were obtained by deducting the unstimulated frequencies from the antigen-stimulated frequencies.

Discussion

Investigating the immune-mediated cytokines and cytotoxic markers produced at the infection site or affected LNs is crucial and provides knowledge on whether they can mediate immune protection or induce pathology. Previous studies have shown that LNs are the collective place of Mtb infection in both cattle and non-human primates [11, 12]. We and others have shown the immunological responses associated with LNs of Mtb infected patients and compared them with biopsied cervical LNTB, healthy individuals, pulmonary TB and other diseases [8, 13–15]. However, the knowledge of pathogen-specific tissue-related immune alterations induced by bacteriological burden is scarce. Also, there are no reports have shown the Mtb antigen-induced frequencies of CD4+, CD8+ T cells and NK cells expressing Th1/Tc1/type 1, Th17/Tc1/type 17 cytokines and cytotoxic markers among culture-positive (LNTB+) and culture-negative (LNTB-) LNTB disease. Thus, we have studied the same in our present study and shown that LNTB+ individuals are associated with enhanced Th1/Tc1/type 1 (IFNγ, TNFα) cytokines (not the other cytokines and cytotoxic markers) compared to LNTB- individuals.

CD4+ T cells produce both Th1 and Th17 cytokines to control the disease [16]. Th1 immunity, especially both IFNγ and TNFα plays an instrumental role in the progress of defensive immune responses in the fight against TB disease [17, 18]. Th17 cells function by the induction of IL-17A and IL-17F cytokines which are instrumental in facilitating cellular immunity to both extra and intracellular microbes, including Mtb [19, 20]. CD4+ T cells induce cytotoxic mechanisms by secreting cytotoxic (granzyme B and perforin) granules and might kill the target cells in an antigen-specific fashion through direct interaction and able to recognize some explicit marker proteins or transcription factors [21]. Our data exhibit significantly elevated frequencies of Th1 (IFNγ and TNFα) cytokines in LNTB+ individuals compared to LNTB- individuals. The frequencies are mycobacterial antigen-specific; since there are no significant differences were observed either in the unstimulated condition or in the HIV antigen stimulation. Our earlier data have also shown similar findings on type 1 cytokines in LNTB+ than LNTB− group upon Mtb antigen stimulation [8]. Our data also suggests that there are no significant differences were observed in the IL-2 or Th17 cytokines and cytotoxic markers in both unstimulated and antigen-stimulated conditions. Overall, we show the crucial role of Th1 expressing cytokines in the disease pathogenesis of LNTB. The higher frequencies of Th1 cytokines could enhance disease severity or might reflect the increased antigen or bacterial load in the LNs of LNTB [8, 10].

Similar to CD4+ Th1 cells, CD8+ Tc1 cells express Tc1 (IFNγ, TNFα, IL-2) cytokines which are essential during Mtb infection [22]. Along with the above cytokines CD8+ T cells might induce Tc17 cytokines, especially IL-17 which have been shown in TB disease [23]. Notably, CD8+ T cells can induce cytolytic ability through granule (perforin, granzymes, granulysin) production to kill Mtb directly or Fas-Fas ligand interaction to stimulate apoptosis [22]. We previously described that the LNTB+ group is known to associate with increased Mtb antigen-stimulated type 17 (IL-17F, IL-22) cytokines than LNTB− group [8]. We show that the frequencies of CD8+ T cell expressing Tc1 (IFNγ, TNFα) cytokines were significantly elevated in LNTB+ individuals compared to LNTB- individuals. Again, our results illustrate higher frequencies of CD8+ T cell expressing Tc1 cytokines potentially indicating greater Mtb antigen load and bacterial burden in LNTB infection [24].

NK cell-mediated responses are crucial during the initial phase of immune protection against intracellular microbes. They also play an important function in linking innate and adaptive immune responses and could produce type 1 and type 17 cytokines [25, 26]. Our study has shown that NK cells expressing type 1 cytokine frequencies were significantly enhanced in LNTB+ individuals compared to LNTB- individuals. The changes observed in the frequencies of type 1 cytokines are pathogen-specific. We previously revealed that cytotoxic markers were significantly decreased in LNs compared to peripheral blood in LNTB disease [9]. Reduced frequencies of type 1 cytokines might be associated with poor clearance of mycobacteria and impaired activation of innate immunity therefore associated with enhanced disease pathogenesis in LNTB+ individuals compared to LNTB- individuals [8, 27].

Overall, we show that culture-positive LNs have increased CD4/CD8/NK cells expressing Th1/Tc1/type 1 cytokine responses that might occur due to increased bacteriological burden in the culture-positive affected LNs. However, our study has certain limitations by not examining the LNs of the normal healthy individuals or the other chronic disease patients which are of future interest. The other future interest is to examine the Th2 cytokine frequencies between the LNTB+ and LNTB- population. Also, the other limitation of our study is being cross-sectional, not a longitudinal study, and showing the data with a smaller sample size.

Lymph node tuberculosis (LNTB) gating strategy for CD4+, CD8+ T cells and NK cells.

(A) Lymphocytes are gated on LN sample and further gated on single cells. From single cells CD3+ T cells and NK (CD3-CD56+) cell population were gated. CD3+ T cells were further gated for CD4+ and CD8+ T cells. (B) FMO population of CD4+, CD8+ T cells and NK cells.

(PDF)

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LNTB representative plots of CD4+/CD8+ T cells.

(A) Th1 (IFNγ, TNFα, IL-2) cytokines, (B) Th17 (IL-17A, IL-17F, IL-22) cytokines, (C) cytotoxic (PFN, GZE B, CD107a) markers upon UNS, Mtb (PPD) antigen stimulation and positive antigen (P/I) control stimulation.

(PDF)

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LNTB representative plots of NK cells.

(A) Th1 (IFNγ, TNFα, IL-2) cytokines, (B) Th17 (IL-17A, IL-17F, IL-22) cytokines, (C) cytotoxic (PFN, GZE B, CD107a) markers upon UNS, Mtb (PPD, CFP-10 PP) antigen stimulation and positive antigen (P/I) control stimulation.

(PDF)

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Antibodies panel used in the study.

(DOC)

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3 Feb 2022

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Reviewer #1: In general, the manuscript needs significant review before submission. Authors should proof read and correct all grammar before any submission. There is also lack of consistency in the abbreviations used throughout the manuscript. Abbreviations used to indicate LNTB+ and – should be consistent. Methods section lacks details of protocols and reagents used. All the figures need to follow consistent formatting. Unfortunately, the manuscript is not up to standard of publishing yet.

Reviewer #2: Comments

The introduction section should state clearly what is the hypothesis and justification of the study. It is not clear why the comparisons made by the authors between LN+ and LN- extra pulmonary tuberculosis contribute to a better understanding of anti-TB immunity. This is relevant since one of the caveats of the study is the lack of analysis of specimens from healthy individuals.

Lines 102-107. Please add the rationale for this conclusion.

Lines 103-104. I won’t call these cells antigen specific cells just because they expressed cytokines after stimulation with Mtb components. To do so, the authors should have carried out tetramer staining.

Methods are short. Despite authors refer to a previous investigation, they should have at least described the origin and disease characteristics of the individuals who donated lymph node tissues. Also, did you stain lymph node samples for Mtb to be completely sure that LN- do not have bacilli?

Although the limitation is acknowledged, the authors should try to analyze healthy control samples.

Results:

Supplementary figure 1. The gate on NK cells is missing CD56 high NK cells. Also, it is curious that you show a big and discrete population of CD3+CD56+ cells. Can you add a plot showing the FMO controls for this analysis?

Supplementary figures 2 and 3 are not necessary but the authors should definitively illustrate the differences they claimed by adding the correspondent plots to figures 1-3

Did you analyze the frequency of T cells expressing both Th1 and Th2 cytokines?

Discussion should clarify how these data are relevant for the study of extra pulmonary tuberculosis and what is the gap in knowledge covered by this investigation. What’s different from previous analyses of lymph nodes with TB? What does it mean to find increased cytokine and cytotoxic functions of T and NK cells only in samples with viable Mtb but not Mtb- samples?

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Reviewer #2: Yes: Dr. Joaquin Zúñiga

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15 Feb 2022

Dear Reviewers,

Thanks for your valuable comments for our manuscript. We have given the responses to all the comments raised by both the reviewers.

Comments Response Line numbers revised

Reviewer 1

In general, the manuscript needs significant review before submission. Authors should proof read and correct all grammar before any submission. There is also lack of consistency in the abbreviations used throughout the manuscript. Abbreviations used to indicate LNTB+ and – should be consistent. Methods section lacks details of protocols and reagents used. All the figures need to follow consistent formatting. Unfortunately, the manuscript is not up to standard of publishing.

As suggested by the reviewer now we have done grammar corrections throughout the manuscript and uniform abbreviations are incorporated and figures are formatted. Multiple places

Abstract:

Abstract should not be in split into sections. Please combine background, methods, results and conclusions together and submit as one paragraph. As suggested by the reviewer we have now combined the abstract section and given as a single paragraph. Lines 27-47

Line 30: It should be (LNTB-) We apologize and now we have corrected LNTB- in the line number 30. Now it is in Line 29.

Introduction:

Introduce what LN stands for e.g. lymph node (LN) before using abbreviation. If you’re using LN for lymph node throughout manuscript, please use this abbreviation. As suggested by the reviewer we have already included the expansion of lymph node tuberculosis (LNTB) in the introduction. line number 70.

Briefly explain the significance of your research and why it’s important to distinguish between LNTB+ and – As suggested by the reviewer now have given our study significance in the introduction as “Hence, it is important to examine the relationship of diverse cytokines/cytotoxic markers and their role in immune activation at sites of Mtb infection to improve our understanding of the differences in pathogenesis between LNTB+ and LNTB-“. Line 91, 151-154

Line 95-97, TBL LNs are you referring to LNTB+? Avoid using TBL and LNTB+ interchangeably. Stick to one term As suggested by the reviewer now we have removed TBL and used LNTB here and thereafter throughout the manuscript. Now it is in line 87 and other places

Line 98- provide e.g. of increased cytokines and decreased cytokines. Otherwise your statement is too vague. As suggested by the reviewer now we have revised the sentence as “We showed that LNTB individuals were associated with increased (TNFa, IL-17A) and decreased (IL-1a, IL-1b, IL-18) pro-inflammatory cytokines than whole blood”. Now it is in line 90

Methods:

Lymph node isolation, culture and antigen stimulation.

How were the lymph nodes extracted? Were cells purified before use? If so what component? Or were they used directly?

As suggested by the reviewer now we have included the extraction procedure. The cells were filtered using 80-100 μm filter (Becton Dickinson, BD). It is now included in the materials and methods section.

Where the lymph nodes used for culture immediately after removal or were they stored and used later? Please indicate these steps No, the cells were stored in -80o C in 1X phosphate buffer saline (PBS)/ dimethyl sulphoxide (DMSO). Then the cells were thawed at 37o C and washed with PBS before staining. It is now included in the materials and methods section.

Authors need to mention concentrations of each stimulation used. As suggested by the reviewer now we have included the concentrations It is now included in the materials and methods section.

Please detail all exact protocols used. As suggested by the reviewer now we have given the complete protocol. It is now included in the materials and methods section.

Make heading of this section “Extra cellular and intra cellular staining of lymph nodes”.

As suggested by the reviewer now we have changed the title as “Extra cellular and intra cellular staining of lymph nodes”. It is now included in the materials and methods

Please provide list of antibodies used in the form of a supplementary table, with cat#, manufacturer, volume of antibody used in final volume.

We have provided the list of antibodies, catalogue number, manufacturer details and antibody volume in Supplementary table Supplementary table 1

Provide detail protocols, i.e. step by step on the antibody staining procedure.

As suggested by the reviewer now we have given the detailed staining procedure. It is now included in the materials and methods

Mention the manufacturer names of the permeabilization buffer and media used.

As suggested by the reviewer now we have given the manufacturer names of the permeabilization buffer (Invitrogen) and RPMI media (Gibco). It is now included in the materials and methods

Provide recipes of cell culture media and any and all buffers with their respective manufacturer names.

As suggested by the reviewer now we have included the manufacturer names of cell culture media and buffers It is now included in the materials and methods

What is the FlowJo version number?

As suggested by the reviewer now we have included the FlowJo version number It is now included in the materials and methods

Results:

Figure 1: Use different colours to indicate two cohorts (black/red). Axis labels are too small and difficult to read. Please increase text size. Use consistent labeling to indicate LNTB+/- not cult+ and cult – on figures. This is applicable to entire manuscript where authors are not consistent with using the abbreviations.

The same comments apply to figure 2 and 3.

As suggested by the reviewer now we have revised the figures and also changed the x-axis and throughout the manuscript from cul+/- to LNTB+/-.

Figures 1, 2, 3

Supplementary figures 1, 2, 3: Use same format for gating strategy. If using pseudo colour, stick to pseudo colour if not stick to contour plots for all three. Increase font size of the labels and axis. Clearly mark in each plot the population of cells you’re referring to and use consistent formatting. Use the same formatting for every figure. Keep it consistent.

As suggested by the reviewer, now we have given the supplementary figures (1, 2, 3) in pseudo colour and increased the font size of the labels.

Supplementary Figures

Figure 2 and 3: Show one representative plot from an unstimulated for negative and one positive control for each cytokine to give readers an idea of where the gates were set. You do not need to show plots for each stim condition.

As suggested by the reviewer, now we have revised the representative plots and now included one negative (UNS), one antigen stimulation (PPD) and one positive control stimulation (P/I) plots and shown in the supplementary figure 1A.

Supplementary Figures 2, 3

Discussion:

Line 303-305: How did you arrive to this conclusion? Where are the ref papers to back up your claim?

As suggested by the reviewer now we have included the reference number in the line number (now the line number is 403-404).

Discussion section

Line 314-316: Ref?

As suggested by the reviewer now we have included the reference number in the line number (now the line number is 413-415).

Discussion section

Line 324-326: ref? As suggested by the reviewer now we have included the reference number in the line number (now the line number is 418, 437-439).

Discussion section

Lines 102-107. Please add the rationale for this conclusion.

As suggested by the reviewer, now we have included the rationale as: However, understanding the correlates of immune protection at the site of infection or affected LNs has not been studied. Hence, it is important to examine the relationship of diverse cytokines/cytotoxic markers and their role in immune activation at sites of Mtb infection to improve our understanding of the differences in pathogenesis between LNTB+ and LNTB-. Introduction section

Reviewer 2

The introduction section should state clearly what is the hypothesis and justification of the study. It is not clear why the comparisons made by the authors between LN+ and LN- extra pulmonary tuberculosis contribute to a better understanding of anti-TB immunity. This is relevant since one of the caveats of the study is the lack of analysis of specimens from healthy individuals

Comments Response Line numbers revised

Lines 102-107. Please add the rationale for this conclusion.

As suggested by the reviewer, now we have included the rationale as “Hence, it is important to examine the relationship of diverse cytokines/cytotoxic markers and their role in immune activation at sites of Mtb infection to improve our understanding of the differences in pathogenesis between LNTB+ and LNTB”. Introduction section

Lines 103-104. I won’t call these cells antigen specific cells just because they expressed cytokines after stimulation with Mtb components. To do so, the authors should have carried out tetramer staining. As suggested by the reviewer, now we have changed the antigen specific to antigen induced in the introduction section.

Introduction section and multiple places in the manuscript

Methods are short. Despite authors refer to a previous investigation, they should have at least described the origin and disease characteristics of the individuals who donated lymph node tissues. Also, did you stain lymph node samples for Mtb to be completely sure that LN- do not have bacilli? As suggested by the reviewer now we have given the detailed staining procedure. Yes, we did the staining for both the groups (LNTB+, LNTB-). LNTB+ group were characterized based on the excision biopsy exhibiting positive for Mycobacterium tuberculosis (Mtb) on liquid cultures and LNTB- (culture grades 0 [no or <19 colonies]) group were characterized based on the negative results for Mtb detection in liquid cultures. LNTB- individuals were diagnosed on the basis of histopathology. Materials and methods section

Although the limitation is acknowledged, the authors should try to analyze healthy control samples.

Yes, we agree with the reviewer point, but there is no feasibility or ethical committee approval to obtain the lymph nodes from healthy individuals. Thus, we have included as a limitation. Discussion section

Results:

Supplementary figure 1. The gate on NK cells is missing CD56 high NK cells. Also, it is curious that you show a big and discrete population of CD3+CD56+ cells. Can you add a plot showing the FMO controls for this analysis?

As suggested by the reviewer, now we have gated the CD56 high population and included in the supplementary figure 1A and we have included the FMO of CD4, CD8 and CD56 population in the supplementary figure 1B.

Supplementary figure A and 1B

Supplementary figures 2 and 3 are not necessary but the authors should definitively illustrate the differences they claimed by adding the correspondent plots to figures 1-3

The other reviewer suggested to include one from negative, Mtb antigen and positive antigen stimulation. Thus, now we included UNS, PPD and P/I plot alone in the Supplementary figures 2 and 3.

Supplementary figures

Did you analyze the frequency of T cells expressing both Th1 and Th2 cytokines?

No, we have measured the frequencies of Th1 cytokines alone and we will try to measure the Th2 cytokines in our future studies. It is now included in the discussion section as “The other future interest is to examine the Th2 cytokine frequencies between the LNTB+ and LNTB- population”. Discussion section

Discussion should clarify how these data are relevant for the study of extra pulmonary tuberculosis and what is the gap in knowledge covered by this investigation. What’s different from previous analyses of lymph nodes with TB?

We now given the relevance of the study as “However, the study of pathogen-specific tissue related immune alterations induced by bacteriological burden is scarce”. It is now included in the discussion section. Discussion section

What does it mean to find increased cytokine and cytotoxic functions of T and NK cells only in samples with viable Mtb but not Mtb- samples?

It is not the Mtb- samples, we compared the cytokines and cytotoxic markers between lymph node (LNs) and whole blood of same individuals infected with Mtb and shown there was significant alterations were observed between the LNs and whole blood [8, 9, in the introduction]. Introduction references 8, 9.

Submitted filename: Reviewer Comments Plos One Final.doc

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8 Mar 2022

10 Mar 2022

Reply to Reviewer,

Reviewer #1: Overall there is a definite improvement in the new draft. However, authors will benefit from another round of proof reading before the final submission.

Response: Thank you for the valuable comments on our manuscript.

211 T cells expressing Tc1 and Tc17 (expect IL-17F) cytokines and cytotoxic marker frequencies: change expect to except

Response: We apologize for the spelling error and now we changed expect to except.

213 Finally, upon P/I antigen stimulation, CD4+ 213 T cells 214 expressing Tc1 and Tc17 cytokines and cytotoxic marker frequencies are not significantly different between the study groups (Figure 2F).

This is CD8 and not CD4.

Response: We apologize for the typographical mistake and now we changed CD4 to CD8.

Figure 1-3 would benefit from a legend to state which stimulation condition is demonstrated in each row of graphs. This will help reader easily navigate and identify plots.

Response: As suggested by the reviewer now we included the legends for the stimulation in figures 1-3.

Discussion

As suggested by the reviewer now we have included the reference number in the line number (now the line number is 413-415). Discussion section Line 324-326: ref?

Response: We have included the reference (reference number 24) in line number 324-326.

24. Kumar NP, Sridhar R, Hanna LE, Banurekha VV, Jawahar MS, Nutman TB, Babu S. Altered CD8(+) T cell frequency and function in tuberculous lymphadenitis. Tuberculosis (Edinb). 2014; 94(5):482-93. doi: 10.1016/j.tube.2014.06.007.

As suggested by the reviewer now we have included the reference number in the line number (now the line number is 418, 437-439). Discussion section

Line 413-415, 418, 437-439 does not indicate to the correct lines in this version of the draft. Please indicate the correct line numbers for reviewers in the future.

Response: We apologize and now we have included the correct line numbers (line numbers 292-294 and 302-304 in the clean version of the manuscript) along with the references.

24. Kumar NP, Sridhar R, Hanna LE, Banurekha VV, Jawahar MS, Nutman TB, Babu S. Altered CD8(+) T cell frequency and function in tuberculous lymphadenitis. Tuberculosis (Edinb). 2014; 94(5):482-93. doi: 10.1016/j.tube.2014.06.007.

8. Kathamuthu GR, Moideen K, Sridhar R, Baskaran D, Babu S. Enhanced mycobacterial antigen-induced pro-inflammatory cytokine production in lymph node tuberculosis. Am J Trop Med Hyg. 2019; 100(6):1401-1406. doi:10.4269/ajtmh.18-0834

27. Kathamuthu GR, Kumar NP, Moideen K, Sridhar R, Baskaran D, Babu S. Diminished type 1 and type 17 cytokine expressing - Natural killer cell frequencies in tuberculous lymphadenitis. Tuberculosis (Edinb). 2019; 118:101856. doi: 10.1016/j.tube.2019.101856.

Submitted filename: Reply to Review Comments Revision II.doc

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16 May 2022

Dominant expansion of CD4+, CD8+ T and NK cells expressing Th1/Tc1/Type 1 cytokines in culture-positive lymph node tuberculosis

PONE-D-21-36622R2

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18 May 2022

PONE-D-21-36622R2

Dominant expansion of CD4+, CD8+ T and NK cells expressing Th1/Tc1/Type 1 cytokines in culture-positive lymph node tuberculosis

Dear Dr. Kathamuthu:

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