Tuberculosis 95 (2015) 40e47

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IMMUNOLOGICAL ASPECTS

Human B cells produce chemokine CXCL10 in the presence of Mycobacterium tuberculosis specific T cells Soren T. Hoff a, *, 1, Ahmed M. Salman b, 1, Morten Ruhwald a, Pernille Ravn c, Inger Brock d, Nabila Elsheikh e, Peter Andersen a, Else Marie Agger a a

Statens Serum Institut, Department of Infectious Disease Immunology, Copenhagen, Denmark Ain Shams University, Faculty of Science, Department of Biochemistry, Cairo, Egypt Copenhagen University Hospital Hillerød, Department of Infectious Diseases, Denmark d Copenhagen University Hospital Hillerød, Department of Clinical Microbiology, Denmark e Al Azhar University, Molecular Immunology Unit, Faculty of Medicine, Cairo, Egypt b c

a r t i c l e i n f o

s u m m a r y

Article history: Received 7 July 2014 Received in revised form 14 October 2014 Accepted 14 October 2014

Background: The role of B cells in human host response to Mycobacterium tuberculosis (Mtb) infection is still controversial, but recent evidence suggest that B cell follicle like structures within the lung may influence host responses through regulation of the local cytokine environment. A candidate for such regulation could be the chemokine CXCL10. CXCL10 is mainly produced by human monocytes, but a few reports have also found CXCL10 production by human B cells. The objective of this study was to investigate CXCL10 production by human B cells in response to in vitro stimulation with Mtb antigens. Methodology/principal findings: We analyzed human blood samples from 30 volunteer donors using multiparameter flow cytometry, and identified a subgroup of B cells producing CXCL10 in response to in vitro stimulation with antigens. T cells did not produce CXCL10, but CXCL10 production by B cells appeared to be mediated via IFN-g and dependent on contact with antigen-specific T cells recognizing the antigen. Conclusion: Human B cells are able to produce CXCL10 in an IFN-g and T cell contact-dependent manner. The present findings suggest a possible mechanism through which B cells in part may influence granuloma formation in human tuberculosis (TB) and participate in infection control. © 2014 Elsevier Ltd. All rights reserved.

Keywords: B cells CXCL10 Peptides Mycobacterium tuberculosis Granuloma

1. Introduction Tuberculosis (TB) remains a major global health problem. Most cases of Mycobacterium tuberculosis (Mtb) infections are localized to the lungs, where the infection is controlled by the host immune system and remains latent [1]. Disregulation of immune control, e.g. by HIV infection, increases the risk of progression from latent infection to active TB disease, but the mechanisms involved in containment and progression remain incompletely understood. In particular, the role of B cells in human TB is still controversial.

* Corresponding author. Department of Infectious Disease Immunology, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark. Tel.: þ45 3268 3496; fax: þ45 3268 3035. E-mail addresses: [email protected] (S.T. Hoff), [email protected] (A.M. Salman), [email protected] (M. Ruhwald), [email protected] (P. Ravn), Inger. [email protected] (I. Brock), [email protected] (N. Elsheikh), [email protected] (P. Andersen), [email protected] (E.M. Agger). 1 STH and AMS contributed equally to this work. http://dx.doi.org/10.1016/j.tube.2014.10.005 1472-9792/© 2014 Elsevier Ltd. All rights reserved.

Recent evidence suggests that B cells may have a more pronounced role than usually perceived [2e4]. For a long time, the boundary between the center of the tuberculous granuloma and the surrounding T cells has been regarded as the main focus for hostepathogen interactions. However, a range of papers have shown the presence of B cell aggregates situated further away from the necrotic center and often clustered around small blood or lymphatic vessels. These aggregates include Mtb-containing macrophages mixed with large numbers of B cells and surrounded by CD4þ and CD8þ T cells [5e7]. When staining for a marker of proliferation (the Ki-67 antigen), Ulrichs et al. found that the main focus for proliferation was within the B cell aggregates e not the inner border of the tuberculous granuloma [6]. This suggests that these follicle-like B cell structures are an active site for hostepathogen interaction. Only few studies have used B cell knock-out (BKO) mice to investigate the mechanism by which B cells may influence the immune response to Mtb and with conflicting conclusions. Bosio et al. infected BKO mice with a clinical isolate of Mtb and found the granuloma formation and pathology in

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the lungs of the BKO mice to be less severe compared to wild type mice. The change could be reversed by transfers of B cells before the infection but, importantly, not with transfer of serum antibodies from infected wild-type mice [8]. This less severe pathology would suggest that B cell infiltration to the lungs may have a damaging influence on control of the infection, perhaps by amplifying the host inflammatory response. In direct contrast to this, a study by Maglione et al. found that BKO mice aerosol-infected with Mtb (Erdman strain, 100 CFU) had exacerbated pathology in the lungs and elevated recruitment of neutrophils compared to the wild-type mice. The exacerbated pathology could be reversed by adoptive transfer of B cells. In this case, B cells seem to play a role as moderators of the inflammatory host response and in the optimal containment of the bacillus [9]. Although the conclusions from the two studies are conflicting, it is apparent that B cells do have a distinct influence on the host response with regard to pathology and granuloma formation, since in both cases this outcome was noticeably changed in mice lacking B cells. Furthermore, since transfer of B cells reversed the observed changes and transfer of serum antibodies failed to do so, B cell interaction with other immune competent cells or B cell production of cytokines or chemokines appears to be crucial. In relation to Mtb infection and control, the chemokine CXCL10 (also known as interferon-gamma inducible protein 10, or IP-10) has been of clinical interest for some time. Elevated levels of CXCL10 are found in blood, urine and pleural effusions from patients with active TB [10e15], in HIV/TB co-infected patients developing immune reconstitution inflammatory syndrome [16], in delayed type hypersensitivity-reactions to Purified Protein Derivative (PPD) [17], and in Mtb-infected granulomas in humans as well as in macaques [18,19]. Recently the measurement of antigen-specific CXCL10 release has been proposed as a sensitive marker for immune-based diagnosis of Mtb infection [20,21]. CXCL10 is primarily known to be a strong chemo-attractant for activated Th1 T cells and Natural Killer cells, but also regulatory T cells and other cells expressing the chemokine receptor CXCR3. A number of studies have reported on the expression of CXCL10 in vivo and in vitro, and CXCL10 has been shown to be expressed in particular by human monocytes/macrophages [17,21,22] as well as by keratinocytes [23], neutrophils [24] and eosinophils [25]. Three studies have shown production of CXCL10 by human B cells [26e28]. In one case by transcriptomics after CD40L/IL-21 activation, and in two cases CXCL10 production was observed in response to co-culture of human B cells with class B (and to a lesser degree class C) CpG oligodeoxynucleotides (ODN), indicating a Toll-like receptor 9-mediated effect on B cells [26,27]. By contrast, another study specifically did not find CXCL10 production in human B cells upon CpG ODN stimulation (class A, B or C) [29]. The reason for this apparent discrepancy is unknown. In the present study we investigated CXCL10 production by human B cells in response to in vitro stimulation with Mtb antigens. Using multiparameter flow cytometry, we report that CXCL10 was expressed in a subpopulation of human B cells upon Mtb antigenic exposure, and that this CXCL10 production was dependent on antigen recognition by antigen-specific T cells. 2. Materials and methods 2.1. Study population The study population consisted of 30 volunteer donors from Copenhagen, Denmark. Nine donors had a known history of Mtb infection (MTB group) either as latent Mtb infected (N ¼ 5) or prior diagnosis of TB disease (N ¼ 4). Sixteen donors were healthy volunteers who had received a rin (BCG) vaccination at least 8 Vaccine Bacillus CalmetteeGue

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weeks prior to blood draw (BCG group). Five donors were healthy volunteers with no known exposure to Mtb and no history of BCG vaccination. Written informed consent was obtained, and the study was  for Københavns og Fredapproved by Den Videnskabsetiske Komite eriksbergs Kommuner (approvals KF 01-369/98 and KF 01-300471). 2.2. Cell preparation and purification of B and T cell subsets All donors provided between 30 and 80 ml whole blood from which peripheral blood mononuclear cells (PBMCs) were isolated and frozen in liquid nitrogen for batched use later. Purification of B cell subset alone or T and B cell subsets together was done by positive selection for CD19 and/or CD3 with the EasySep™ purification kit (StemCell technologies). Procedure was done according to the manufacturer's instructions (www.stemcell.com). Control experiments showed >95% purity for both purification procedures. After purification cell were incubated, stained and analyzed as described below. 2.3. Multiparameter flow cytometry Cells were thawed and rested overnight, then incubated at 37  C with 5% CO2 in Serum free AIM V medium supplemented with 1% Penicillin/Streptomycin (Invitrogen/Gibco) for 20 h followed by 5 h incubation in the presence of BFA (Brefeldin A solution, BD fastimmune). Cells were incubated either with culture medium alone or with one of the following stimulatory antigens: Tuberculin PPD RT-50 (Statens Serum Institut) 5 mg/ml (PPD), TB10.4 peptide mix consisting of overlapping 18-mer peptides used at 2 mg/ml per peptide (JPT peptides, Berlin, Germany), single 18-mers individual TB10.4 peptides at 5 mg/ml, Pokeweed (PWMe) at final dilution of 1:100,000 from stock in Phosphate buffered saline pH 7.4 (PBS) þ 1% Fetal bovine serum. TB10.4 (Rv0288) is a mycobacterial protein expressed both by Mtb and BCG [30]. After incubation cells were washed and Fc receptors were blocked with Human Fc Receptors Binding Inhibitor (eBiosciences) for 20 min on ice. Subsequently, cells were stained for extra-cellular markers for 20 min at room temperature (RT). Extracellular staining included cell viability dye GrViD (Invitrogen), APC-eFluor780-conjugated CD19 (eBioscience) and PerCp-Cy5.5-conjugated CD14 (BD Pharmingen) and depending on the specific experiment APC-conjugated CD27 (BD Pharmingen), APC-AlexaFluor750 conjugated CD3 (eBiosciences), APC-conjugated CXCR3 (BD Pharmingen) or PerCp-Vy5.5-conjugated CD16 (BD Pharmingen). Cells were washed twice with wash buffer, then permeabilized with Cytofix/Cytoperm solution (BD Pharmingen) for 30e60 min at RT, washed twice in Perm/Wash buffer (BD Pharmingen). Intra-cellular staining was done for 20 min at RT with PEconjugated or in certain experiments biotin-conjugated CXCL10 (BD Pharmingen) or APC-conjugated IFN-g (eBioscience). Finally cells were washed twice with Perm/Wash buffer, fixed, and analyzed with a FACS Canto flow cytometer. The procedure was done as earlier described in detail by Lamoreaux et al. [31]. 2.4. Measurement of IFN-g by ELISA PBMCs were thawed, rested overnight and subsequently incubated for five days at 37  C with 5% CO2 with TB10.4 single 18-mers peptides or with culture medium alone. Subsequently IFN-g was measured by in-house ELISA in the supernatants as described earlier [32] 2.5. Measurement of CXCL10 by ELISA CXCL10 was determined in cell culture supernatants by an inhouse sandwich ELISA using an in-house murine capture mAb

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(Clone IM2, provided by M. Ruhwald) and biotinylated murine detection mAb (Becton Dickinson). The assay quantification range was 30e2000 pg/ml, with a lower limit of detection of 5 pg/ml. Samples were measured in duplicates at 1:3 and 1:7 dilution to accommodate expected concentrations within the range of the standard curve. The measurement that best fit the range of the assay is presented. The assay has full linearity with dilution (data not shown).

incubated for 60 min with increasing concentrations of a blocking antibody against IFNGR1 (CD119; BD Pharmingen; 0.1e50 mg/mL) or against the IFN-a receptor chain 2 (MMHAR-2; Calbiochem; 0.01e25 mg/mL) prior to incubation with the highest interferon concentration used in the first condition. Third, cells were preincubated as just described, but instead of IFN-g or IFN-a, cells were stimulated for 20 h with PPD (5 mg/mL). 2.8. Statistics

2.6. Transwell experiments B cells were purified and placed in a transwell insert (Transwell 96-wells Corning culture plate). PBMCs from the same donor were place beneath the microporous membrane (1 mm pore size). Transwells prevent contact between the cells in the upper and the lower compartments, but allows any soluble secretions or cytokines to pass freely between the two compartments. Both compartments were incubated with PWM as described above. After incubation cells from each compartment were transferred separately to V shaped cell culture plates, stained and analyzed as described above.

Data analysis was done using the software FlowJo (Treestar version 8.8.6) and GraphPad Prism version 5 (GraphPad). Statistical analyses were done using GraphPad Prism software, version 5 (GraphPad). Significance levels were calculated using KruskaleWallis nonparametric test with Dunn's post test (Figure 1) or one-way analysis of variances with Dunnet post test corrections (Figures 2e4). 3. Results 3.1. B cells from sensitized individuals produce CXCL10 in response to in vitro stimulation with PPD

2.7. IFN-g and IFN-a experiments In two parallel experiments PBMCs from two donors were tested under 3 different conditions. First, cells were incubated for 20 h with one of six increasing concentrations of human IFN-g (Thermo Scientific; ranging from 625 to 10,000 pg/mL) or IFN-a (Kordia/Tebu-bio; 25e400 IU/mL). Second, cells were pre-

We compared the frequencies of B cells producing CXCL10 after in vitro stimulation of PBMCs between 9 donors with a known history of Mtb infection (MTB), 13 BCG-vaccinated donors (BCG) and 5 control donors (CON). Cells were stimulated with PWMe, PPD or with cell culture medium alone and stained for intra-cellular CXCL10. Gating for single cells, live cells, CD19 and for intra-

Figure 1. B cells from sensitized individuals produce CXCL10 in response to in vitro stimulation with PPD. PBMCs from 9 donors with a known Mtb infection (MTB), 13 BCGvaccinated donors (BCG), or from 5 BCG-unvaccinated control donors (CON) were incubated with PPD. Subsequently, the cells were stained for cell viability (GrViD), CD14 and CD19, and intracellular CXCL10 or PE-isotype control (PE-ISO). (A) Gating for one representative donor is shown. Cells were gated for single cells, then live cells, CD19 and finally CXCL10-positive cells after incubation with culture media only (MEDIA), PPD or Pokeweed mitogen (PWMe). PE-isotype stained cells were incubated with PPD. (B) Scatter plot showing the frequencies of CXCL10 positive B cells after PPD incubation with background frequencies (MEDIA) subtracted. Black line indicates median for each group. *p < 0.05, KruskaleWallis test.

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cellular CXCL10, we found evident CXCL10 production in CD19þ B cells (Figure 1A). We also found a marked CXCL10 production in CD14þ monocytes with median MFI values on average 2 and a half times greater than B cell-derived CXCL10, but none in the T cell (CD3þ) or Natural Killer cell (CD16þ) subpopulations (data not shown). There were no significant difference in the frequencies of B cells producing CXCL10 in MTB group (median 2.04%; range 0.16e7.19%) compared to BCG group (median 1.38%; range 0.08e6.71%) but both groups had significantly higher than responses than the control group (median 0.0%; range 0.0e0.50%). Additionally, cell culture supernatants had significantly higher CXCL10 concentrations compared to controls (data not shown). 3.2. The B cell CXCL10 response is induced independent of CXCR3 on the B cell surface Since B cells themselves may express the CXCL10-receptor CXCR3 on their surface, we subsequently investigated whether the observed CXCL10 response could be due to extracellular binding of free CXCL10 to B cells expressing CXCR3. PPD-stimulated PBMCs were stained with PE-conjugated antibodies to CXCL10, with PEconjugated isotype control antibodies or with blocking antibodies of the same clone as the PE-conjugated CXCL10 antibodies in various combinations (Figure 2). Staining for intra-cellular CXCL10 gave a marked B cell CXCL10 response as seen before. Staining with PE-conjugated isotype control antibodies gave no response as did staining for extra-cellular CXCL10 only. Addition of competing antibodies to the intracellular CXCL10 antibodies gave a significantly reduced response. Furthermore, addition of the blocking antibodies during the extra-cellular staining step gave no significant change. This confirms that the observed B cell CXCL10 responses are not due to extracellular binding of free CXCL10 to the B cell surface.

Figure 3. Interferon gamma (IFN-g) but not type I Interferon (IFN-a) induces CXCL10 production in the B-cell subset. In two parallel experiments (A and B) PBMCs from two donors, selected for known responses, were incubated under 3 different conditions. With increasing concentrations of human IFN-g (A) or IFN-a (B) Cells were preincubated with increasing concentrations of anti-human IFN-g-receptor antibodies or anti-human IFN-a-receptor blocking antibodies prior to IFN-g or IFN-a incubation. The pre-incubation step was repeated but instead of interferon cells were incubated with PPD. Data points indicate mean ± SD of two technical replicates, *p < 0.05 by Dunnett's Multiple Comparison Test.

3.3. Interferon gamma (IFN-g) but not type I interferon (IFN-a) induces CXCL10 production by the B cell subset

Figure 2. The B cell CXCL10 response is not due to extra cellular binding of CXCL10. Human PBMCs were stimulated with PPD and subsequently divided into 5 groups each with a different staining scheme (AeE): (A) Intracellular staining with PE-conjugated anti-CXCL10 antibodies only. (B) Extracellular staining with PE-conjugated antiCXCL10 antibodies only. (C) Intracellular staining with PE-conjugated anti-CXCL10 antibodies mixed with blocking biotin-conjugated anti-CXCL10 antibodies at an excess concentration. (D) Extracellular staining with blocking biotin-conjugated anti-CXCL10 antibodies at an excess concentration, followed by intracellular staining with PEconjugated anti-CXCL10 antibodies. (E) Intracellular staining with PE-conjugated isotype control antibodies. The bars indicate mean frequency of CXCL10 positive B cells. Error bars indicate standard deviation of technical replicates. Mean frequencies in B, C, D and E were compared to A. *p < 0.05, ***p < 0.001, NS, Not Significant. Data from one representative experiment out of two is shown.

In a range of cell types, CXCL10 is known to be induced by IFN-g. We wanted to investigate if this is also the case with B cells (Figure 3A and B). PBMCs from two selected donors were tested under 3 different conditions: In experiment A, a significant dose-related increase in the frequencies of CXCL10 positive B cells was seen for both donors in response to incubation with increasing levels of added IFN-g. The response to IFN-g could furthermore be blocked by performing a pre-incubation step with IFN-g receptor-blocking antibodies. The effect of stimulation with PPD could be blocked by IFN-g receptor-blocking antibodies in a manner very similar to blocking of soluble IFN-g (Figure 3A). In contrast, IFN-a did not induce CXCL10 in B cells and blocking the IFN-a receptor did not significantly block a PPDinduced response (Figure 3B). 3.4. T cell recognition of Mtb antigens induces B cells to produce CXCL10 An obvious source of IFN-g is T cells. We thus wanted to investigate whether the B cell CXCL10 response was dependent on recognition of the antigens by the T cells. We selected four

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Figure 4. T cell recognition of M. tuberculosis antigens induces B cells to produce CXCL10. (A) PBMCs from four selected donors were tested for recognition of T cell epitopes in the Mtb protein TB10.4. The PBMCs were stimulated with 9 single 18-mer peptides (p1 to p9) spanning the entire TB10.4 protein with 8 amino acid overlaps, or with culture medium alone (Media). Cells were stimulated for five days, thereafter supernatants were tested for IFN-g by ELISA. Bars indicate mean ± SD IFN-g in pg/ml. Measured from 3 technical replicates. (B) PBMCs from the same 4 donors were tested by flow cytometry for B-cell CXCL10 production in response to in vitro stimulation with the same TB10.4 overlapping peptides (p1ep9) or with culture medium alone (Media). Bars indicate the mean ± SD of 3 technical replicates. Peptide responses were compared to the un-stimulated control (Media) by one-way analysis of variance using Dunnets post test. *p < 0.05, **p < 0.01, NS, Not Significant.

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donors. Via a pre-screening, three of these had a known detectable B cell CXCL10 response to a pool of overlapping peptides to the Mtb protein TB10.4, and one with no response to TB10.4 (data not shown). We tested for T cell epitope recognition, by measuring IFN-g responses separately to nine single 18-mer TB10.4 peptides spanning the entire protein. Testing was done by stimulating PBMCs for five days with the peptides and subsequently measuring the IFN-g concentration in the supernatants by IFN-g ELISA (Figure 4A). In parallel, PBMC samples from the same four donors were tested for B cell CXCL10 responses to the same nine TB10.4 peptides (Figure 4B). Comparing the two experiments, we saw a clear association between B cell CXCL10 responses and T cell IFN-g responses. In two cases a peptide gave a significant IFN-g response without an accompanying B cell CXCL10 response, but all peptides which induced a B cell CXCL10 response were also recognized by T cells. Thus only T cell epitopes were able to induce B cell CXCL10 production, suggesting that T cell recognition of antigens induces B cells to produce CXCL10. 3.5. B cell CXCL10 production is dependent on T cell contact To further investigate the role of T cells in the B cell CXCL10 production, we looked at whether purified B cells were capable of producing CXCL10 in response to stimulation without contact to T cells. In two different experimental set-ups, purified cells from selected donors were incubated with either PPD or a mix of nine TB10.4 single peptides (Figure 5A), or with soluble IFN-g or Pokeweed (PWMe; Figure 5B). In both set-ups, purified B cells alone did not to respond to any stimulation, whereas the addition of T cells was sufficient to elicit B cell CXCL10 responses. All unstimulated controls had very low or no responses indicating that potential non-specific T cell activation due to the purification procedure did not take place or at least did not influence the B cell CXCL10 response (data not shown). As the staining panel included a live/dead cell marker we were able to exclude that low responses were due to large numbers of dead cells (data not shown). In a third experimental setup (Figure 5C), we incubated purified Bcells in one transwell compartment and PBMCs from the same donor in the other compartment and incubated both in the presence of PWMe. We found that purified B cells that were separated from contact with the T cell subset in the PBMC compartment, but exposed to any soluble signals originating from the PBMC compartment, did not respond. As a control, B cells

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within the PBMC populations did produce CXCL10. Taken together this indicates that B cells need contact with T cells to initiate the CXCL10 response. 4. Discussion The evidence of follicle-like B cell aggregate in the lungs of TB patients [6] as well as in Mtb infected mice [3] has led to investigations of how B cell might modulate immune responses to Mtb and through which cytokines. Khader et al. recently reported that IL-23 and to a lesser degree IL-17 and IL-22 are critical for the formation of these B cell aggregates [33]. A study by Zhang et al. supported this and furthermore reported an association between the formation of the B cell aggregates and containment of Mtb. In their study, the B cell follicle formation was also associated with increased IL-17 and IL-22 expression in the human lung tissue from TB patients. Moreover they showed that B cells regulate antigen-specific IL-17 and IL-22 production ex vivo [34]. It appears that B cells play at least a partial role in regulating the local immune response to Mtb and that the mechanism for this may be through regulation of the local cytokine chemokine environment at the site of host/pathogen interaction during Mtb infection. An obvious candidate for such regulation would be CXCL10 [20]. In this study, we investigated CXCL10 production in human B cells in response to in vitro stimulation with Mtb antigens. We show that CXCL10 was produced in a small population of B cells in a manner dependent on recognition of the antigen by antigenspecific T cells. Our results suggest that B cell CXCL10 induction is at least partly mediated via IFN-g and requires contact with Mtbspecific T cells. This is interesting in the light of work by Fuller et al. who used in situ hybridization in macaques infected with Mtb (Erdman strain) to measure expression of mRNAs encoding the CXCR3 ligands CXCL9, CXCL10 or CXC11. They found high expression profiles of all three, localized to both solid and caseous granulomas in macaques, and the expression of the chemokines was higher in macaques with advanced pulmonary disease than in animals with minimal or moderate disease. Expression of mRNAs encoding CXCR3 were strongly co-localized with the expression of its three ligands, suggesting that recruitment of CXCR3þ cells is involved in granuloma formation [19]. This finding is supported by a study by Seiler et al. who found that granuloma formation was strongly reduced in terms of numbers, size and density in CXCR3 knock-out mice after aerosol

Figure 5. B cell CXCL10 production is dependent on T cell contact. In three experimental set-ups, B cells alone or T and B cells together were purified from selected donors. (A) Cells were subsequently stimulated with PPD or TB10.4 peptide mix (A) or with Pokeweed or IFN-g (B). In the third set-up purified B cells were separated from total PBMCs from the same donor by a microporous membrane in a transwell system (C). Cells in both compartments were incubated with pokeweed. Data points represent frequencies of CXCL10 positive B cells for individual donors. Black lines represent medians.

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infection with Mtb (H37Rv strain) [35]. In contrast, a study by Chakravarty et al. found no structural differences of the granuloma in CXCR3 knock-out mice compared to wild type, and surprisingly, CD4þ cell counts were higher in the CXCR3-deficient mice and the bacterial burden lower in the mice lacking CXCR3 than in the wild type mice [36]. Taken together, this points towards the hypothesis that the function of the chemokines interacting with CXCR3 is more complicated than merely cell recruitment [37]. As an example, Bromley et al. found that interaction between CXCR3 and CXCL10 adversely affected T cell priming [38]. Such effects might attenuate, or regulate, the host response to Mtb, explaining the findings in the CXCR3 knock out experiments. However, in the present study, B cells appear to require cell contact with T cells as CXCL10 responses in purified B cells could not be initiated e not by antigenic stimulation, not by incubation with soluble gamma interferon, and not when separated from T cells by a microporous membrane. Only when mixed with T cells, could B cell CXCL10 responses be induced. The exact mechanism of the required T- and B cell contact are difficult to elucidate from the results of this study, but in-vivo responses would probably need to be initiated by antigen exposure. In this regard, T cell recognition of the antigens seem sufficient, since specific recognition of the exact same TB10.4 single 18-mer peptides by the B cell receptor and the T cell receptor is highly unlikely. The simplest explanation is that the observed B cell CXCL10 responses seen in this study are independent of the B cell specificity and that no B cell epitopes were present. One possible mechanism could be that peptides are presented on the B cell MHC class II molecules as well as on all other antigen presenting cells (APCs). Subsequently, T cells specific for the peptides interact with the B cell MHC and coreceptors, and generates a signal transduction in the B cell which in turn induces the CXCL10 production. This raises an interesting question. As receptor-mediated antigen uptake and subsequent presentation by antigen-specific B cells is known to be many times more efficient than non-specific antigen uptake and presentation by other APCs [39], the presence of antigenspecific memory B cells at the site of host/pathogen interaction in the lung tissue may act as an amplifying mechanism. By detecting even very small concentrations of antigen and via production of chemokines such as CXCL10, the antigen-specific B cells could initiate very fast and early immune responses and granuloma formation. This may well be of importance even if the magnitude of CXCL10 signaling from the granuloma will later be dominated by macrophages. Further studies are needed to elucidate this hypothesis. The main limitations to this study are the small dataset and that no other CXCR3 agonists were investigated. Also, this study did not investigate different B cell subsets. Pilot data not included here have shown the CXCL10 positive B cells to express a CD20 high, CD27 intermediate to high and CXCR3 high phenotype, and ongoing work will investigate this further. In conclusion, assuming that the B cell CXCL10 response to Mtb antigens demonstrated here also takes place in vivo during Mtb infection; the present findings suggests a possible mechanism through which B cells may influence granuloma formation, and thus control of the infection in human TB.

Acknowledgments We thank Joshua Woodworth for proofreading. We also thank Martine G. Aabye for advice and assistance with CXCL10 ELISA assay.

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Human B cells produce chemokine CXCL10 in the presence of Mycobacterium tuberculosis specific T cells.

The role of B cells in human host response to Mycobacterium tuberculosis (Mtb) infection is still controversial, but recent evidence suggest that B ce...
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