Patterns of T and B cell responses to Mycobacterium tuberculosis membrane-associated antigens and their relationship with disease activity in rheumatoid arthritis patients with latent tuberculosis infection.

This study was aimed at exploring whether latent tuberculosis infection (LTBI) contributes to the pathogenesis of immune-mediated inflammatory diseases in a TB endemic setting. We screened 198 rheumatoid arthritis (RA) patients with tuberculin skin test (TST) and studied 61 (median DAS28-ESR = 6.3) who were positive. Whole blood T cell proliferative responses to Mycobacterium tuberculosis (Mtb) membrane (MtM) antigens, including the latency-induced protein alpha crystallin (Acr), were determined by flow cytometry using Ki67 expression as the marker for nuclear proliferation. Serum antibody levels were determined by ELISA. Follow-up investigations (at 3-6, 9-12 and 15-18 months after baseline) were performed in 41 patients who were classified empirically as 'high' (HR-T/HR-B) or 'low' (LR-T/LR-B) responders based on their dynamic T cell or antibody responses. Significant correlations were seen between baseline T cell responses to MtM and Acr, and between IgG, IgA and IgM antibody responses to MtM. However, no correlation was seen between T and B cell responses. At all time points during the follow-up, T cell responses to both antigens (except for MtM at one point) were significantly higher in HR-T (n = 25) than LR-T (n = 16) patients. Levels of IgA and IgM (but not IgG) antibodies to MtM were also significantly higher in HR-B (n = 13) than LR-B (n = 28) at all time points. Importantly, HR-T patients exhibited significantly higher baseline and follow-up DAS28 scores than LR-T. Ten (of 61) patients had a history of TB and developed RA 6 years (median) after contracting TB. Three new TB cases (1 from TST-positive and 2 from TST-negative groups) emerged during the follow-up. Our results suggest that persistently elevated T cell responses to Mtb antigens may contribute to disease activity in RA.

Introduction

Rheumatoid arthritis (RA) is an immune-mediated inflammatory disease (IMID) caused by interplay of genetic and environmental factors that dysregulate the immune system [1]. Treatment with disease modifying anti-rheumatic drugs (DMARDs) acting as tumour necrosis factor-alpha (TNF) blockers makes IMID patients susceptible to TB [2] due mainly to reactivation of latent TB infection (LTBI), defined as ‘a state of persistent immune responsiveness to Mycobacterium tuberculosis (Mtb) antigens without clinically manifested TB’ [3]. In some population-based studies [4, 5] history of TB was found associated with RA suggesting that Mtb could also play a role in pathogenesis of RA which, like TB, may begin in the airways and lungs [6]. In early RA disease, germinal centre-like structures are seen in the lungs wherein activated B cells produce antibodies to citrullinated protein antigens (ACPAs). Seropositive RA patients harbour citrullinated proteins in their bronchial tissue and this abnormality was not associated with smoking [7, 8]. Pulmonary TB patients also show seropositivity for ACPA and rheumatoid factor (RF) [9] suggesting that Mtb could trigger the production of these antibodies which are otherwise considered as hallmarks of RA.

Several studies have indicated that immune-inflammatory responses to Mtb, as well as non-tuberculous mycobacteria (NTM), can drive the pathogenesis of IMIDs and signaling via toll-like receptors (TLRs) plays an important role in this process [10, 11]. Cell membrane-associated glycolipids and lipoproteins of Mtb serve as microbe-associated molecular patterns (MAMPs) recognized by the pattern-recognition receptors (PRRs, including TLRs) of innate immune cells [11]. The pro-inflammatory cytokines produced by these cells, besides orchestrating innate immunity against the invading microbe, can trigger pathogenesis of RA and other IMIDs [6]. Antigenic constituents of Mtb membrane (MtM) are also potent inducers of adaptive immunity which, in turn, may contribute to the pool of pro-inflammatory cytokines [10, 12]. Mtb ‘immunoproteome’ is rich in membrane-associated and secreted proteins [13] and certain MtM-associated proteins identified by us [14] and others [15] were found capable of inducing strong T cell responses in persons with LTBI. In addition, MtM antigens are also strong inducers of B cell responses leading to production of high titres of antibodies [16-18]. The MtM-associated stress protein alpha crystallin (Acr, Rv2031c) sustains Mtb during its latent phase [19]. Its overproduction by the dormant bacilli and occurrence within ‘Mtb complex’ make Acr an important latency-associated antigen [20]. As shown by us [17] and others [21], it induces strong T- and B cell responses in persons with LTBI. Acr was also shown to induce T cell responses in RA patients harbouring LTBI [22].

Immune responses to Mtb are dynamic in nature. T cell responses vary according to the bacterial load and decline with treatment [23, 24]. Specificity and isotypes of antibodies produced by B cells also vary according to the state of infection. While persons with LTBI preferentially produce antibodies to MtM-associated antigens, those with active TB do so against ‘secreted’ antigen of Mtb [13, 25]. In a longitudinal study [26], TB patients showed higher IgA levels against Acr than subjects with LTBI. In another study [27], consistently elevated levels of IgA against Mtb antigens were considered a risk factor for progression to active TB disease. However, despite the apparent similarities in their dynamic trends, T and B cell responses to Mtb may not go hand in hand. A cohort of household contacts from TB endemic area was shown to harbour antibodies to Acr and other MtM-associated antigens while remaining negative for T cell responses determined by tuberculin skin test (TST) or interferon gamma release assay (IGRA) [28].

India is home to over a quarter of TB patients diagnosed worldwide [29] and majority of apparently healthy Indians harbour LTBI [30, 31]. Further, the dynamic nature of host’s immunity is believed to determine whether LTBI will progress to active TB disease or regress to a ‘self-limiting’ state [32]. Against this backdrop, and the indications that Mtb could serve as an environmental trigger for IMIDs, we undertook this study aimed at exploring whether the RA patients harbouring LTBI exhibit distinct T and B cell response patterns against MtM-associated antigens and whether such patterns have any relationship with their disease activity.

Materials and methods

Ethics statement

The study protocol was approved by Institutional Ethics Committee of SGPGIMS (IEC code: 2016-149-IMP-EXP). All participants provided a written informed consent.

Study group

One hundred ninety eight RA patients attending the Rheumatology clinic at our hospital were examined for fulfilment of the following inclusion criteria: Adults with active disease [33], taking ≤10 mg prednisolone/day, no sign or symptom of TB or any other co-morbidity, and a positive TST. Sixty one (30.8%) patients met these criteria and were recruited for the study.

Tuberculin skin test

TST was performed as per recommendations of IUATLD and WHO [34]. Accordingly, 5 tuberculin units (TU) of PPD (Arkray Healthcare, Surat, India) were injected intradermally on the left forearm. After 48–72 hours, mean diameter of induration was measured using a calliper. TST was considered as positive if the induration was 10 mm or more.

Blood samples

At the baseline and follow-up intervals, 1 ml venous blood was collected in heparin tubes (for T cell assays) and 3 ml was collected in plain tubes (to get sera for antibody assays).

Mtb antigens

Mtb cell-membrane (MtM) was isolated by using a previously described protocol [17]. Briefly, 3–4 week old culture of Mtb (strain H37Ra, ATCC25177) was harvested and sonicated. Cell lysate was differentially centrifuged to obtain membrane as sediment and cytosol as supernatant. Protein was estimated by the modified Lowry’s method [17] and aliquots were stored at -80°C. Identity of Mtb was ascertained by immune-chromatographic detection of MPT64 [17] using a kit (‘SD Bioline TB Ag MPT64 Rapid’, Abbott, USA). Recombinant Acr protein (LRP-0019C) was purchased from Lionex GmbH, Germany.

T cell proliferation assay

A previously described protocol [17] was used with some modifications. In brief, blood samples (diluted 1:10) were dispensed (1 ml/well) in 24-well culture plates and incubated with test antigens (MtM or Acr, 5μg/ml) or controls (culture medium as negative and PHA as positive control) for 5 days in a CO2 incubator. Harvested cells were stained with fluorescent anti-CD3 antibody and RBCs were lysed. Leukocytes were fixed with 2% paraformaldehyde and permeabilised with 0.2% Triton-X100. Washed cells were stained with fluorescent anti-Ki67 antibody. Cells were finally washed and suspended in PBS. Data on 105 cells in lymphocyte gate was acquired on a flow cytometer (BD FACS Canto-II) and analysed with FlowJo software (Tree Star). Percent responder cells (CD3+Ki67+) for antigens or mitogen (PHA) were determined by subtracting corresponding values for culture medium. Gating strategy is shown in S1 Fig in S1 File and representative flow plots are shown in S2 Fig in S1 File.

ELISA

A previously described protocol [17] was used. In brief, ELISA plates were coated with antigens (10 μg/ml MtM or 1 μg/ml Acr) or coating-buffer and blocked with 2% skimmed milk powder in tris-buffered saline containing 0·05% Tween 20 (TBS-T). Diluted test sera (1:500 for MtM or 1:100 for Acr) were dispensed in antigen and buffer-coated wells and incubated. Washed plates were re-incubated with peroxidase-conjugated antibodies to human IgG, IgA or IgM. Plates were finally washed and incubated with the substrate (o-phenylene diamine). Reaction was stopped with 7% H2SO4 and optical densities (ODs) were read at 492 nm. For each serum, mean OD with buffer was subtracted from mean OD with antigen and expressed as ΔOD.

Classification of patients as ‘high’ or ‘low’ responders for Mtb antigens

Forty one patients (out of 61 recruited) returned for at least one of the 3 follow-up investigations scheduled at 3–6, 9–12 and 15–18 months after baseline. They were classified as ‘high’ or ‘low’ responders for T cell or antibody responses based on the following empirical criteria: For T cell responses, patients showing >2 fold increase (from baseline) in response to either or both antigens (MtM/Acr), at one or more time points, were classified as high responders (HR-T). Conversely, low responders (LR-T) were those who showed ≤2 fold increase in response to both the antigens at all time points. For antibody responses, patients showing >2 fold increase in the level of any antibody isotype (IgG, IgA or IgM), at one or more time points, were classified as high responders (HR-B). Low responders (LR-B) were those who showed ≤2 fold increase in all antibody isotypes at all time points.

Statistical analysis

Non-parametric statistical methods were applied. Correlation between two variables was assessed by Spearman’s rho. Difference between two proportions was assessed by Fisher’s exact test. Differences between paired datasets were assessed by Wilcoxon test and those between unpaired datasets were assessed by Mann-Whitney test. P values <0.05 were considered as significant. All calculations were performed using Graphpad Prism software.

Results

Baseline characteristics of study subjects

Most (78.7%) of the 61 TST-positive RA patients enrolled for the study were females and about half of them were on low-dose (≤10 mg/day) prednisolone (Table 1).

Table 1

Baseline characteristics of the study subjects.

S. No.	Variables	Values
1.	Number of patients (females)	61 (48)
2.	Median age in years (inter-quartile range or IQR)	41 (31–50)
3.	Number with RF/ACPA	49/46
4.	Median disease duration in years (IQR)	4 (1.5–6)
5.	Median DAS28-ESR (IQR)	6.3 (5.6–7.2)
6.	Number using ≤10 mg prednisolone/day	34
7.	Median TST induration in mm (IQR)	13 (12–18)
8.	Number with BCG vaccination scar	34
9.	Number with history of cured TB	10

TST responses were not affected by BCG

Out of 198 patients who were screened for inclusion in this study, BCG scar was present in 55.7% (34/61) of TST-positive and 45.3% (62/137) of TST-negative patients. This difference was not statistically significant (P = 0.2179) suggesting that BCG vaccination at birth (as practised in India) did not affect TST results in the adults.

MtM induced higher T and B cell responses than Acr

Baseline T cell proliferative responses to PHA (median, IQR; 72, 40 to 86), MtM (0.78, 0.45 to 1.8) and Acr (0.33, 0.12 to 0.72) in 58 study subjects (samples from 3 had technical issue) are shown in Fig 1A. The responses to MtM were significantly higher than to Acr. Even so, a strong correlation (r = 0.69) was seen between the two responses (Fig 1B) suggesting that MtM could provide a more sensitive alternative to Acr for monitoring the T cell responses to Mtb.

Fig 1

Baseline T cell proliferative responses of the study subjects (n = 58).

Panel A shows percent responder T cells (CD3+Ki67+) against the mitogen (PHA) and Mtb antigens (MtM and Acr). Though the responses to Acr were significantly lower than to MtM, a significant correlation was seen between the two (Panel B). P (Mann-Whitney) and r (Spearman’s) values are shown in the panels.

Baseline antibody responses to MtM and Acr are shown in Fig 2A. Anti-MtM IgG levels (median ΔOD, IQR; 0.28, 0.14 to 0.5) were significantly higher than IgA (0.11, 0.08 to 0.2) and IgM (0.2, 0.1 to 0.33) and IgM levels were also significantly higher than IgA. Despite these differences, significant correlations were seen between all 3 isotypes (Fig 2B–2D) indicating that they could broadly be targeting the same set of antigens. Acr did not elicit any appreciable antibody response (0, -0.01 to 0.03 for IgG; 0, -0.02 to 0.02 for IgA and -0.01, -0.04 to -0.54 for IgM) which suggested that MtM could work as a more sensitive alternative to Acr for monitoring of anti-Mtb antibody responses also. In view of these results, further analyses of antibody responses were based on anti-MtM antibodies alone.

Fig 2

Baseline B cell responses of the study subjects (n = 58).

Levels (ΔOD = mean OD of antigen-coated minus mean OD of buffer-coated wells) of anti-MtM IgG antibodies were highest followed by IgM and IgA (Panel A). Despite the significant differences, levels of all 3 antibody isotypes correlated with each other (Panels B-D). Acr did not produce any appreciable antibody response. P (Mann-Whitney) and r (Spearman’s) values are shown in the panels.

We did not observe any correlation between baseline T cell and antibody responses against MtM (S3 Fig in S1 File) suggesting that the antigenic determinants eliciting these responses could, at least partly, be non-overlapping.

Patients showed distinct immune response profiles against Mtb antigens According to the applied criteria (see Materials and Methods), 25 patients (out of 41 who were followed-up) were categorised as ‘high’ responders (HR-T) and the remaining 16 as ‘low’ responders (LR-T) for T cell responses to the Mtb antigens (MtM and Acr). Likewise, 13 patients were high responders (HR-B) and 28 were low responders (LR-B) for B cell (antibody) responses to MtM. Consistent with the observed lack of correlation between baseline T and B cell responses, no association (P = 0.5134) was seen between HR-T and HR-B, or LR-T and LR-B patient categories (S1 Table in S1 File).

Sequential T cell responses (log2 fold-change from baseline responses) of HR-T and LR-T patients against MtM and Acr are displayed in Fig 3A. Both antigens showed comparable response profiles, consistent with the observed correlation between two responses at the baseline. At all 3 time points of follow-up, responses to both the antigens (except for MtM at 15–18 months) in HR-T were significantly higher than LR-T patients. Interestingly, a progressively rising trend was seen in the responses, particularly of LR-T patients who showed a significant rise in response to Acr between 3–6 and 15–18 months.

Fig 3

Sequential T and B cell responses of the study subjects (n = 41).

Dotted lines denote two-fold (log2 = 1) higher response from the baseline (0). Significant differences between high (HR-T, n = 25) and low (LR-T, n = 16) T cell responders were seen for Acr at all 3 and for MtM at 2 follow-up intervals. Responses in LR-T were mostly below baseline but showed a rising trend which attained statistical significance in case of Acr (Panel A). Significant differences were also seen between high (HR-B, n = 13) and low (LR-B, n = 28) B cell responders, for anti-MtM IgA and IgM (but not IgG) antibodies at all time points (Panel B). P values for observed differences (Mann-Whitney test for inter-group and Wilcoxon test for intra-group comparisons) are shown on top of the selected columns.

Sequential antibody responses to MtM (Fig 3B) were distinctly different from the corresponding T cell responses. A significantly higher (P = 0.0143) proportion of patients fell under the LR-B (68%) compared with LR-T (39%) category, suggesting that RA patients with LTBI were able to mount relatively weak antibody responses. Moreover, unlike T cell responses, antibody levels (log2 fold-change from the baseline) of HR-B as well as LR-B patients remained almost stable throughout the follow-up period. Interestingly, though statistically significant differences were seen in IgA and IgM levels of HR-B and LR-B patients at all time points, IgG levels did not differ at any point.

Altogether, these results suggest that the RA patients with LTBI may fall into distinct categories with respect to their longitudinal immune responses against Mtb antigens. They also underscore the role played by immunoglobulin isotypes in shaping the overall antibody response.

Prescribed treatment did not affect the T or B cell response profiles

During the follow-up, patients were treated with prednisolone (up to 10 mg/day) along with methotrexate (up to 25 mg/week). In addition, some patients also received hydroxychloroquine (up to 400 mg/day). The proportions of HR and LR patients (for T as well as B cell responses) receiving these drugs were comparable (S4 Fig in S1 File), suggesting that the administered treatment did not have any remarkable effect on the observed immune response profiles.

Patients with persistently high T cell responses to Mtb antigens also showed high disease activity

DAS28-ESR scores of HR-T and LR-T patients at the baseline and at two follow-up intervals are shown in Fig 4A. As expected, disease activity in all patients declined with treatment. However, at all time points, disease activity scores in HR-T remained significantly higher than LR-T patients. In addition, the duration of disease was significantly shorter in HR-T patients (Fig 4B). These results suggested that RA patients with persistently high T cell response to Mtb antigens could also exhibit high disease activity which could be attained shortly after disease onset.

Fig 4

Association of RA disease activity with T cell responses to Mtb antigens.

Disease activity scores (DAS28-ESR) of patients who were high responders for T cells (HR-T) were significantly higher than those of low responders (LR-T) at the baseline (BL) and also at 2 follow-up intervals (F1 and F2, Panel A). Additionally, disease duration (years) was significantly shorter in HR-T than LR-T patients (Panel B). DAS28-ESR scores of high and low responders for B cells (HR-B and LR-B) at the baseline as well as follow-up intervals did not show any significant difference (Panel C). Disease duration also showed no difference (Panel D). P values (Mann-Whitney) are shown on top of the selected columns.

Unlike T cell responses, antibody responses did not show any association with disease activity or duration (Fig 4C, 4D) suggesting that antibodies to Mtb antigens may have a relatively lesser relevance for the pathogenesis of RA.

Association between TB and RA

Out of 61 patients recruited for the study, 10 (16.4%) had a history of cured TB, of whom 7 had suffered from pulmonary TB. The median time taken for the onset of clinically active RA after contracting TB was 6 years (Table 2).

Table 2

Association between TB and RA.

Patient No.a	Age/Sex	BCG	TST (mm)	TB Type	Time since TB cure (Years)	Time-lag between TB cure and RA onset (Years)
1	46/F	1b	12	Lymphnodal	22	7
2	60/F	0	10	Pulmonary	15	13
3	25/F	1	19	Pulmonary	10	6
4	36/F	1	18	Endometrial	6	2
5	58/M	0	20	Pulmonary	24	15
6	49/F	1	16	Pulmonary	Not known	Not known
7	22/F	0	12	Pleural	6	4
8	49/F	1	18	Pulmonary	15	4
9	37/F	0	19	Pulmonary	14	2
10	32/F	1	15	Pulmonary	14	9
				Median (IQR)	14 (8–19)	6 (3–11)

aNine patients (except No. 4) were positive for RF. Seven patients (except Nos.1, 2 and 4) were positive for ACPA. b1 = BCG scar-positive, 0 = scar-negative.

All 198 patients (82% females) who were screened for inclusion in the study were monitored for development of TB over the study period. One new TB case emerged in the TST-positive (n = 61) and 2 new cases emerged in TST-negative (n = 137) patient groups. Thus the proportions of incident cases in both groups were comparable.

Discussion

In absence of a ‘gold standard’ method for detection of LTBI, we chose TST for several reasons. As comparisons of TST and IGRA in the same population have not provided evidence that one test is superior to the other, WHO has recommended that both are equivalent options, with TST being the method of choice in resource-constrained settings [3]. The choice of 10 mm reaction as cut-off for positivity was aimed at detecting those patients who may have a greater propensity to develop TB. In a large TST trial from south India [35], reactions >10 mm showed a higher association with TB development than those <10 mm. A TBNET study [36] has also shown that TST cut-off of 10 mm is most appropriate for the diagnosis of LTBI in RA patients since loss in sensitivity by increasing the cut-off from 5 to 10 mm is marginal while there is substantial gain in specificity. The rate of TST positivity in our patients (30.8%) was comparable to that (30%) in another study on RA patients from India, despite the authors choosing to use a double strength (10 TU) of PPD [37].

BCG vaccination at birth (as recommended by WHO and practised in India) did not affect the TST responses, which is consistent with another study on RA patients [38] and our earlier study on health care workers (HCWs) [31]. Apprehensions that TST could be confounded by BCG have not found support in a meta-analysis of 24 studies comprising 240,203 subjects [39]. The study concluded that BCG given in infancy had minimal effect on TST, particularly after 10 years of vaccination. The same meta-analysis also included 18 studies comprising 1,169,105 subjects to conclude that false positive TST due to NTM infection was minimal (up to 2.3%) and reached the upper limit only in areas of high NTM and low TB prevalence (e.g., USA and Europe). This and other similar observations have prompted WHO to recommend that BCG vaccination should not be a determining factor in selecting a test (TST or IGRA) [3]. Nearly half (55.7%) of our study subjects were on low-dose (≤10 mg) prednisolone suggesting that their treatment status did not affect TST responses. Several prior studies have also shown that corticosteroids (up to 15 mg/day) or DMARDs do not affect TST responses [38, 40, 41].

Baseline T and B cell responses to MtM were significantly higher than to Acr which was expected since MtM comprises, besides Acr, several putative antigens of Mtb [14, 16]. We had also shown that MtM induces comparable and apparently mycobacterium-specific CD4+ and CD8+ T cell responses [14, 31]. Despite the difference in intensities, a strong correlation was seen between T cell responses to MtM and Acr, similar to that reported by us in HCWs [17]. The serum IgG levels against MtM were significantly higher than IgA and IgM. Even so, levels of all 3 antibody isotypes correlated with each other indicating that they could broadly be targeting the same set of antigens. Contrary to responses induced by it in HCWs and TB patients [17, 26], Acr did not induce any remarkable antibody response in RA patients owing, most probably, to their dysregulated immune system [1]. Overall, these results indicated that MtM could serve as a more sensitive alternative to Acr for monitoring the T as well as B cell responses to Mtb [17]. Lack of correlation between T and B cell responses was another notable observation suggesting that they could be directed at non-overlapping antigenic determinants. This discordance was also flagged in a recent study [28] wherein a group of household contacts who were negative for TST and IGRA had shown antibodies to MtM-associated antigens. In related studies [42, 43], RA patients had also shown antibodies to mycobacterial proteins PknG, PtpA and MAP_4027. Though the authors had sourced these proteins from Mycobacterium avium subsp. paratuberculosis (MAP), they are also present in Mtb.

The patients classified as ‘high’ T cell responders (HR-T) showed significantly higher responses to MtM and Acr than ‘low’ responders (LR-T) at all 3 time points (except for MtM at one point) of the follow-up. Since T cell responses to Mtb decline with reduction in bacterial load [23, 24], HR-T patients could be considered as having a higher load of infection. A progressive rise in T cell responses, as seen by us, could further indicate an ongoing or persistent infection [32]. Sequential antibody (IgA and IgM, though not IgG) responses to MtM were also significantly higher in HR-B than LR-B patients at all time points. Antibodies to Mtb may perpetuate LTBI by restricting the spread of infection [16, 18]. The respiratory mucosa contains an abundance of IgA and a lower level of IgM, both of which can protect mucosal epithelium from invasion by Mtb. In RA patients also, a prominent IgA response precedes the joint inflammation [6]. Unlike the rising trends seen in T cell responses, antibody responses in either category of patients remained almost stable throughout the follow-up, suggesting that they may have reached a plateau even before baseline investigations. This finding resonates with the fact that RA-associated antibodies can be present in blood several years prior to disease onset [1, 6]. Compared with HR-T, significantly lesser proportion of patients fell in the HR-B category, suggesting that their B cell responses may be relatively subdued. No associations were seen between HR-T and HR-B or LR-T and LR-B categories of patients which reflected the lack of correlation between T and B cell responses at the baseline. We did also not observe any effect of treatment on the T or B cell responses which is consistent with the reports showing that corticosteroids and DMARDs, at the doses administered by us, did not affect TST responses [38, 40, 41].

An important observation of this study was significantly higher disease activity scores (DAS28) in HR-T than LR-T patients at baseline as well as during the follow-up. RA patients characteristically express HLA-DR4 [44] which points to a critical role of T cell responses in disease development. Analysis of T cell responses in RA has mostly remained focused on autoantigens identified by autoantibodies and candidate peptides for the selected autoantigens (e.g., citrullinated proteins) are defined by their binding to HLA-DR alleles associated with seropositive RA [45]. However, a major limitation of this approach is its inability to look for other antigens which could cause T cell activation particularly when one third of RA patients are seronegative [6]. A major study has shown that T cell responses to Mtb antigens are also executed through HLA-DR4 and suggested that this could drive the pathogenesis of RA [46]. In addition, cloned T cells from synovial fluid of RA patients have shown reactivity to Mtb antigens [47]. The antibody responses against Mtb, on the other hand, did not show any association with disease activity. In an earlier study also, antibodies to the Mtb antigens PknG and PtpA did not show such an association [42] suggesting that the anti-Mtb antibodies may not play a significant role in RA pathogenesis.

The fact that 16% of our enrolled patients had a history of TB also points to Mtb as a possible ‘environmental’ factor for RA. A recent study from Taiwan has found evidence of past TB, detected by chest x-ray, in a similar proportion of IMID patients, mostly of RA [48]. In another large Taiwanese cohort, 2.38% RA patients presented a history of TB whereas TB prevalence in the control population was 1.36% [5]. Our patients developed RA 6 years (median) after contracting TB whereas in Taiwanese cohort [5] the median time to develop RA was 3.61 years. These differences could be reconciled with the differences in cohort sizes or demography.

We followed all 198 patients who were screened for selection of study subjects for the development of TB. Three new TB cases (1 in TST-positive and 2 in TST-negative patients) emerged during the study period. As lifetime risk of conversion of LTBI to active TB is 5–10% [3], this period could be considered as grossly inadequate. However, another plausible reason for lower than expected TB incidence in our patients could the fact that most (82%) of them were females. In a large trial in south India [35], risk of culture positive TB in males (5%) was 3 fold higher than females (1.6%) over a follow-up period of 15 years. Comparable incidences of TB in TST-positive and TST-negative groups raise concerns about sensitivity of the methods in use for the detection of LTBI. In an earlier study, also from India, comparable proportions of TB cases emerged from TB contacts who were either positive or negative for TST or IGRA [49]. Sensitivity of these assays is also being debated on a wider scale [3, 48] and the need for more sensitive assays for a reliable detection of LTBI cannot be overemphasised [28, 31].

Mycobacteria can drive IMIDs in many ways. While the persistently high T cell responses to Mtb antigens seen by us in a subset of RA patients with high disease activity signify an adaptive immune response, part of it could also have resulted from the ‘bystander’ activation of T cells [12]. Activation of PRRs on innate immune cells by the pathogens can lead to bystander activation of autoreactive TH1 or TH17 cells through production of T cell promoting cytokines [50]. Moreover, T cells also express TLRs and corresponding agonists (e.g., mycobacterial antigens) can induce the proliferation of effector as well as regulatory T cells [51]. Molecular mimicry between mycobacterial and host antigens is another important pathogenic mechanism. An Mtb-specific arthritogenic T cell clone was found to react with cartilage proteoglycan [52]. Likewise, the mycobacterial 65 kDa heat-shock protein has shown cross-reactivity with synovial tissue [53]. RA patients harbour antibodies to a peptide epitope of MAP_4027 (this antigen is also present in Mtb) which cross-reacts with interferon regulatory factor 5 (IRF5). This structural mimicry could contribute to the pathogenesis of IMIDs by dysregulating the signalling by IRF5 which is a central regulator of inflammatory responses [43, 54]. The regions of low TB prevalence (e.g., USA, Canada and Europe) show a high prevalence of MAP which could substitute for Mtb as an environmental trigger for IMIDs [55].

A major limitation of this study was less than desirable compliance of patients to the follow-up schedules. Ours is a tertiary care referral hospital where patients come from distant places. Typically, they are given medicines for 3 months and asked to return for next round of consultations. However, patients also have the option of a telephonic consultation. To partially offset this limitation, and also of a small sample size, we chose to apply a stringent cut-off (>2 fold increase over baseline) for classification of a patient as high responder.

To conclude, our study suggests that RA patients with LTBI may fall into distinct categories showing a persistently high or persistently low T cell response to Mtb antigens; and those showing a high response may also exhibit higher disease activity. The precise mechanisms by which preclinical RA is transformed into a clinically classifiable disease are not yet known [1, 6] and the possibility that Mtb may play a role in this transition is very much alive [56]. This possibility needs to be considered and explored, particularly in patients who are residents of a highly TB endemic area such as India. Since RA develops gradually through preclinical stages, it can be targeted much before the joint inflammation or damage sets in. There are instances when the use of anti-TB drugs has improved the outcome in RA patients [57].

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2 Jul 2021

PONE-D-21-17868

Patterns of T and B cell responses to Mycobacterium tuberculosis membrane-associated antigens and their relationship with disease activity in rheumatoid arthritis patients with latent tuberculosis infection

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Additional Editor Comments (if provided):

I've personally reviewed the manuscript and I need that you respond to the following point:

The TST response may be positive due to other mycobacterial infection, do the authors have taken in consideration to perform IFN gamma assay in order to be more specific detection MTB latent Infection?

As the 1 reviewer suggested, do other environmental or opportunistic mycobacteria (causing chronic infections) trigger RA as well?

Thank you

Leonardo A. Sechi

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript, Shashi Kant Kumar, et al investigated the immune response to Mtb membrane antigens in RA patients with LTBI. The study is interesting and as well the results also.

However, the following are some concerns:

Different microbial agents are involved as triggers factors in RA, and TLR signalling is extremely important in host defence. In order to complete the immunopathology of RA and to better understand the molecular pathways triggered by Tb infection, the authors are invited to explain in more detail this signalling. In addition, recent in vivo studies identified the importance of Interferon Regulatory Factor 5 (IRF5) in the pathogenesis of RA as a new link between the pathogenic activation of RNA-sensing Toll-like receptors and proinflammatory cytokine production in inflamed joints of arthritic mice. Other than that, in human studies has been found a strong immune response against IRF5 suggesting IRF5 as a target of immune response in RA.

Some articles below are suggested that will certainly give more strength to the work enriching the content.

- Almuttaqi H et al. Advances and challenges in targeting IRF5, a key regulator of inflammation. FEBS J. 2019 May;286(9):1624-1637. doi: 10.1111/febs.14654. Epub 2018 Sep 21.

- Bo M et al. Antibody response to homologous epitopes of Epstein-Barr virus, Mycobacterium avium subsp. paratuberculosis (MAP) and IRF5 in patients with different connective tissue diseases and in mouse model of antigen-induced arthritis. J Transl Autoimmun. 2020 Mar 17;3:100048. doi: 10.1016/j.jtauto.2020.100048. eCollection 2020.

- Duffau P et al. Promotion of Inflammatory Arthritis by Interferon Regulatory Factor 5 in a Mouse Model. Arthritis Rheumatol. 2015 Dec;67(12):3146-57. doi: 10.1002/art.39321.

- Bo M et al. Interferon regulatory factor 5 is a potential target of autoimmune response triggered by Epstein-barr virus and Mycobacterium avium subsp. paratuberculosis in rheumatoid arthritis: investigating a mechanism of molecular mimicry. Clin Exp Rheumatol. 2018 May-Jun;36(3):376-381. Epub 2018 Jan 15.

Also, the authors have mentioned the role played by others Mycobacteria in RA, and appears interesting the role played by Mycobacterium avium subsp. paratuberculosis as a possible trigger factor recently discovered in the aetiology of RA. It would better give more details writing a short and concise paragraph on the role of Mycobacterial infections in RA.

Finally, a recent review on the role of Mycobacterial infections in RA is suggested below in order to complete your manuscript.

- Bo M et al. Role of Infections in the Pathogenesis of Rheumatoid Arthritis: Focus on Mycobacteria. Microorganisms. 2020 Sep 23;8(10):1459. doi: 10.3390/microorganisms8101459.

Best wishes

**********

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Reviewer #1: No

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Submitted filename: Review final. Comments..docx

Click here for additional data file.

12 Jul 2021

Response to Academic Editor

1. The TST response may be positive due to other mycobacterial infection, do the authors have taken in consideration to perform IFN gamma assay in order to be more specific detection MTB latent Infection?

Response: Going by the available literature (Ref 31, 38, 39 of the manuscript) and WHO recommendations (Ref 3 of the manuscript), TST results are least likely to be affected by BCG vaccination (if given in infancy) or NTM infection. We have further elaborated this point in the revised Discussion (paragraph 2).

2. As the 1 reviewer suggested, do other environmental or opportunistic mycobacteria (causing chronic infections) trigger RA as well?

Response: Yes, they do. We have made necessary amendments in the Introduction (para 2) and Discussion (para 3, 5 and 8).

Response to Reviewer-1

1. Different microbial agents are involved as triggers factors in RA, and TLR signalling is extremely important in host defence. In order to complete the immunopathology of RA and to better understand the molecular pathways triggered by Tb infection, the authors are invited to explain in more detail this signalling. In addition, recent in vivo studies identified the importance of Interferon Regulatory Factor 5 (IRF5) in the pathogenesis of RA as a new link between the pathogenic activation of RNA-sensing Toll-like receptors and proinflammatory cytokine production in inflamed joints of arthritic mice. Other than that, in human studies has been found a strong immune response against IRF5 suggesting IRF5 as a target of immune response in RA.

Response: Thanks for providing this insight. We have made necessary amendments in the Introduction (para 2) and inserted a new paragraph (para 8) in the Discussion which hopefully addresses these aspects.

2. Some articles below are suggested that will certainly give more strength to the work enriching the content.

(i) Almuttaqi H et al. Advances and challenges in targeting IRF5, a key regulator of inflammation. FEBS J. 2019 May;286(9):1624-1637. doi: 10.1111/febs.14654. Epub 2018 Sep 21.

(ii) Bo M et al. Antibody response to homologous epitopes of Epstein-Barr virus, Mycobacterium avium subsp. paratuberculosis (MAP) and IRF5 in patients with different connective tissue diseases and in mouse model of antigen-induced arthritis. J Transl Autoimmun. 2020 Mar 17;3:100048. doi: 10.1016/j.jtauto.2020.100048. eCollection 2020.

(iii) Duffau P et al. Promotion of Inflammatory Arthritis by Interferon Regulatory Factor 5 in a Mouse Model. Arthritis Rheumatol. 2015 Dec;67(12):3146-57. doi: 10.1002/art.39321.

(iv) Bo M et al. Interferon regulatory factor 5 is a potential target of autoimmune response triggered by Epstein-barr virus and Mycobacterium avium subsp. paratuberculosis in rheumatoid arthritis: investigating a mechanism of molecular mimicry. Clin Exp Rheumatol. 2018 May-Jun;36(3):376-381. Epub 2018 Jan 15.

Response: We have incorporated references (ii) and (iv) in the revised Introduction (para 2) and Discussion (para 3, 5 and 8). However, we did not include (i) and (iii) since we felt that ref (ii), (iv) and another one suggested by you (see below, point 4) cover the necessary information. All along, we were wary of exceeding our brief since our present work does not address these aspects.

3. Also, the authors have mentioned the role played by others Mycobacteria in RA, and appears interesting the role played by Mycobacterium avium subsp. paratuberculosis as a possible trigger factor recently discovered in the aetiology of RA. It would better give more details writing a short and concise paragraph on the role of Mycobacterial infections in RA.

Response: Thanks for the suggestion. We have added a new paragraph 8 in the revised Discussion.

4. Finally, a recent review on the role of Mycobacterial infections in RA is suggested below in order to complete your manuscript.

Bo M et al. Role of Infections in the Pathogenesis of Rheumatoid Arthritis: Focus on Mycobacteria. Microorganisms. 2020 Sep 23;8(10):1459. doi: 10.3390/microorganisms8101459.

Response: We have now incorporated this reference (Introduction para 1).

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

21 Jul 2021

Patterns of T and B cell responses to Mycobacterium tuberculosis membrane-associated antigens and their relationship with disease activity in rheumatoid arthritis patients with latent tuberculosis infection

PONE-D-21-17868R1

Dear Dr. Sudhir Sinha

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Leonardo A. Sechi, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have adequately addressed comments raised by reviewers and I feel that this manuscript is now acceptable for publication.

**********

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

23 Jul 2021

PONE-D-21-17868R1

Patterns of T and B cell responses to

Dear Dr. Sinha:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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