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We evaluated the role of regulatory T cells (CD4+ CD25+ Foxp3+ cells, Tregs) in human Mycobacterium tuberculosis infection. Tregs were expanded in response to M. tuberculosis in healthy tuberculin reactors, but not in tuberculin-negative individuals. The M. tuberculosis mannose-capped lipoarabinomannan (ManLAM) resulted in regulatory T cell expansion, whereas the M. tuberculosis 19 kD protein and heat shock protein 65 had no effect. Anti-IL- 10 and anti- TGF-β alone or in combination, did not reduce expansion of Tregs. In contrast, the cyclooxygenase enzyme-2 inhibitor NS398 significantly inhibited expansion of Tregs, indicating that prostaglandin E2 (PGE2) contributes to Treg expansion. Monocytes produced PGE2 upon culturing with heat-killed M. tuberculosis or ManLAM, and T cells from healthy tuberculin reactors enhanced PGE2 production by monocytes. Expanded Tregs produced significant amount of TGF-β and IL-10 and depletion of Tregs from PBMC of these individuals increased the frequency of M. tuberculosis-responsive CD4+ IFN-γ cells. Culturing M. tuberculosis-expanded Tregs with autologous CD8+ cells decreased the frequency of IFN-γ + cells. Freshly isolated PBMC from tuberculosis patients had increased percentages of Tregs, compared to healthy tuberculin reactors. These findings demonstrate that Tregs expand in response to M. tuberculosis through mechanisms that depend on ManLAM and PGE2.
Mycobacterium tuberculosis infects one-third of the world’s population and causes almost 2 million deaths per year . Approximately 90% of infected persons have protective immunity and remain healthy, but 10% of infected individuals develop primary tuberculosis soon after infection or reactivation tuberculosis many years later . The factors responsible for development of active tuberculosis are not well understood. To develop new tools to prevent tuberculosis, it is important to understand the factors that regulate protective immune responses against M. tuberculosis.
T cells play a crucial role in protective immunity against M. tuberculosis and other intracellular pathogens , in part through production of IFN-γ, which is indispensable for resistance to infection [4, 5]. Recently, attention has been focused on regulatory T-cells (Tregs), a subset of CD4+ T-cells that express CD25 and Foxp3 , which can inhibit IFN-γ production by T-cells through production of IL-10 and TGFβ, as well as through mechanisms that depend on cell-to-cell contact [7–9]. Many studies have shown that Tregs play a central role in downregulating the immune response to organ transplants and tumors, and preventing the development of autoimmunity . Tregs have also been shown to dampen the immune response to intracellular pathogens, such as viruses, bacteria and protozoa, primarily in animal models [11,12]. However, limited information is available regarding the capacity of these pathogens to mediate expansion of Tregs in human infection.
To investigate the role of Tregs in human M. tuberculosis infection, we studied the capacity of M. tuberculosis-infected monocytes to expand these lymphocytes, and sought to determine the mechanisms for this expansion. We also compared the frequency of Tregs in PBMC obtained from tuberculosis patients and healthy tuberculin reactors.
To determine whether prior tuberculosis infection affects expansion of Tregs, we first isolated CD4+ cells and autologous monocytes from PBMC of 14 healthy tuberculin reactors and four healthy tuberculin-negativepersons, and cells were cultured in the presence or absence of heat-killed M. tuberculosis. After 5 days, the numbers of CD4+ CD25+ Foxp3+ cells were measured by flow cytometry. Heat-killed M. tuberculosis significantly expanded CD4+ CD25+ Foxp3+ cells in healthytuberculin reactors (1510± 200 vs. 220± 40 cells per 104 CD4+ cells, P< 0.001), in contrast to findings in tuberculin-negativepersons (370 ± 110 vs. 180 ± 40 cells per 104 CD4+ cells, P> 0.1, Fig. 1). For healthy tuberculin reactors, the total number of cells in each well did not change significantly after culture with heat-killed M. tuberculosis or medium alone (3.0 ± 0.3 cells vs. 2.7 ± 0.1 cells per well, with 99% cell viability), indicating that M. tuberculosis elicited an increase in the absolute number of CD4+CD25+FoxP3+ cells Recent studies showed that Tregs express low levels of the IL-7R (CD127) . To confirm that the cells expanded above were Tregs, we measured CD127 expression of Foxp3+ cells. In three healthy tuberculin reactors, 85% of the Foxp3+ cells expressed low levels of CD127 (data not shown).
To determine whether live M. tuberculosis expands Tregs, we cultured CD4+ and autologous monocytesfrom four healthy tuberculin reactors, either with live M. tuberculosis H37Ra or with heat-killed M. tuberculosis. There was no significant difference inexpansion of Tregs in the presence of live or heat-killed M. tuberculosis (1300 ± 430 vs. 1400± 110 cells per 104 CD4+ cells, respectively; P>0.1).
We next wished to identify the mycobacterial components that could contribute to expansion of Tregs. We selected the M. tuberculosis 19-kD lipoprotein as a potential candidate because it is a TLR1/2 agonist and previous studies have demonstrated that TLR2-derived signals increase survival of Tregs . We also selected ManLAM because it induces dendritic cells to produce IL-10 and inhibits production of IL-12 , and this could favor development of Tregs. HSP65 was also evaluated as a control mycobacterial protein. We cultured CD4+ cells and autologous monocytes from PBMC of six healthy tuberculin reactors in medium alone or with the hexameric peptide of the 19-kD M. tuberculosis lipoprotein, ManLAM or HSP65. After 5 days, the numbers of CD4+CD25+Foxp3+ cells were measured by flow cytometry. ManLAM significantly expanded CD4+ CD25+ Foxp3+ cells in healthytuberculin reactors (1040± 120 vs. 220± 40 cells per 104 CD4+ cells, P< 0.001, Fig. 2A). In contrast, the hexameric peptide of the 19-kD lipoprotein and HSP65 had no effect on the expansion of CD4+ CD25+ Foxp3+ cells (Fig. 2A). This suggests that ManLAM mediates expansion of Tregs in M. tuberculosis infection.
ManLAM binds to the mannose receptor of mononuclear phagocytes . To confirm that ManLAM is the major mycobacterial component involved in Treg expansion, we cultured CD4+ cells and autologous monocytes from PBMC of seven healthy tuberculin reactors with ManLAM, inthe presence of anti-CD206 Ab or isotype control Ab (10 μg/ml). Anti-CD206 significantly inhibited ManLAM-mediated expansion of Tregs (280 ± 80 vs 1040 ± 120 cells per 104 CD4+ cells, P < 0.001, Fig. 2B).
We next asked whether induction of heat-killed M. tuberculosis or ManLAM specific Tregs can occur in the absence of natural Tregs (CD4+ CD25+ Foxp3+ cells). We depleted CD4+ CD25+ cells from the PBMC of five healthy tuberculin reactors and cultured CD4+ CD25- (< 1% FoxP3+ cells) with autologous monocytes, and either heat-killed M. tuberculosis or ManLAM, for 5 days. Heat-killed M. tuberculosis or ManLAM converted some of the CD4+ CD25- cells into Foxp3+ cells (1390± 162 vs. 133± 61 and 1172± 154 vs. 133± 61 cells per 104 CD4+ cells, respectively, p < 0.001, Fig. 3).
Next, we asked whether soluble factors produced by M. tuberculosis-activatedmonocytes contribute to expansion of Tregs. Heat-killed M. tuberculosis was added to monocytes from four healthy tuberculin reactors and cultured in Transwells in 12-well plates containing autologous CD4+ cells. Positive controls were CD4+ cells and monocytes cultured in the same wells. Exposure of T-cells to M. tuberculosis-activated monocytes in Transwells increased the number of Tregs from 140 ± 20 to 700 ± 60 cells per 104 CD4+ cells. However, when monocytes and T-cells were cultured in the same well, the number of Tregs increased further to 1390 ± 130 cells per 104 CD4+ cells (p < 0.05, comparing Transwell and direct exposure, Fig. 4). These findings indicate that expansion of Tregs was partially mediated by soluble factors produced by M. tuberculosis-exposed monocytes.
M. tuberculosis-stimulated monocytes produce IL-10 and TGF-β [17, 18], both of which expand Tregs in other experimental systems [19, 20]. However, Abs to IL-10 and TGF-β, alone orin combination, did not reduce expansion of Tregs, when CD4+ cells were cultured with M. tuberculosis–stimulated monocytes (Fig. 5).
M. tuberculosis stimulates human macrophages to produce PGE2 , which can elicit proliferation by Tregs . Because PGE2 is the major end product of the COX-2 enzyme, we added a COX-2 inhibitor, NS398, to CD4+ cells and M. tuberculosis–stimulated monocytes from 7 healthy tuberculin reactors. NS398 markedly reduced the number of Tregs from 1260 ± 160 to 320 ± 40 cells per 104 CD4+ cells, (p < 0.001, Fig. 6). In contrast, HQL79, which inhibits formation of PGD2, had no effect (Fig. 6). These results indicate that PGE2 may be one of the soluble factors produced by M. tuberculosis stimulated monocytes that expand Tregs.
We previously found that M. tuberculosis-activated T-cells produce factors that enhance IL-18 production by monocytes . Therefore, we wished to determine if T-cells increased PGE2 production by monocytes. To do this, CD4+ cells and M. tuberculosis-stimulated monocytes from six healthy tuberculin reactors and five healthy tuberculin-negative donors were cultured for 3 days at a ratio of 9:1, as outlined above. We found that PGE2 levels in the supernatants were only slightly higher in healthy tuberculin reactors (357 ± 128 pg/ml vs 173 ± 29 pg/ml, P>0.1). In this experimental system, the ratio of CD4+ T cells to monocytes is high, and some PGE2 produced by monocytes may be utilized by Tregs for expansion, making it difficult to detect differences in PGE2 production between groups. We therefore cultured monocytes without CD4+ cells, or markedly reduced the ratio of CD4+ cells to monocytes to 1:4, and repeated the experiments in 5 healthy tuberculin reactors and 4 healthy tuberculin-negative persons. Addition of M. tuberculosis increased PGE2 production by monocytes from healthy tuberculin reactors (1445 ± 298 pg/ml vs 346 ± 96 pg/ml, P<0.01) and tuberculin-negative persons (1450 ± 456.4 pg/ml vs 344 ± 62 pg/ml, P= 0.01, Fig. 7A). However, addition of CD4+ cells further increased PGE2 levels in healthy tuberculin reactors 11-fold from 1445 ± 298 pg/ml to 15,617 ± 3337 pg/ml (p < 0.01). In contrast, addition of CD4+ cells had no effect on PGE2 production by monocytes from healthy tuberculin-negative donors (Fig. 7A).
In Figs. 2A and 2B, we found that ManLAM expands Tregs in healthy tuberculin reactors. To determine if ManLAM stimulates PGE2 production by monocytes, we cultured CD4+ cells from 6 healthy tuberculin reactors with their autologous monocytes at a ratio of 1:4, in the presence or absence of ManLAM. After 3 days, PGE2 levels in the culture supernatants of ManLAM-treated cells were increased 8-fold, compared to those of cells cultured in the absence of ManLAM (18,622 ± 233 pg/ml vs 3237± 397 pg/ml, p < 0.01, Fig. 7B).
Tregs are known to produce high concentrations of IL-10 and TGF-β [7–10], whereas activated T-cells that are CD4+CD25+ produce IL-2 and IFN-γ. To determine if M. tuberculosis or ManLAM-expanded CD4+CD25+ cells had a cytokine profile of Tregs or activated T-cells, we evaluated their ability to produce IL-10, TGF-β, IL-2 and IFN-γ. We cultured CD4+ cells and autologous monocytes from PBMC of six healthy tuberculin reactors in medium alone or with M. tuberculosis whole cell lysate or ManLAM. After 3 days, Tregs were isolated as described in the methods section, and the frequency of cells producing TGF-β, IL-10, IFN-γ and IL-2 were determined by ELISPOT. The frequency of IL-10-producing cells in M. tuberculosis-expanded CD4+CD25+ cells was high(94 ± 10 cells per 105 cells), compared with that in M. tuberculosis-expanded CD4+CD25- cells (12.6 ± 7.2 cells per 105 cells, p <0.001, Fig. 8A). Similarly the frequency of TGF-β-producing M. tuberculosis-expanded CD4+CD25+ cells was high(66 ± 7.6 cells per 105 cells), compared with that in M. tuberculosis-expanded CD4+CD25- cells which had no TGF-β-producing cells. In contrast, the frequency of IFN-γ+ and IL-2+ cells in M. tuberculosis-expanded CD4+CD25- cells was extremely high(268 ± 30 and 111 ± 5 cells per 105 cells, respectively), compared with that inexpanded Tregs (2.4 ± 2.4 and 2 ± 1.2 cells per 105 cells, respectively, p <0.001 for both IFN-γ and IL-2, Fig. 8B).
To determine whether Tregs affect the capacity of CD4+ cells to produce IFN-γ in response to M. tuberculosis, we cultured PBMC or Treg-depleted PBMC from 5 healthy tuberculin reactorswith heat-killed M. tuberculosis for 48 h. CD4+ cells were then isolatedfrom PBMC and Treg-depleted PBMC and incubated overnighton an ELISPOT plate to detect IFN-γ-producing cells. Depletionof Tregs increased the frequency of IFN-γ-producing CD4+ cells (286 ± 16.2 per 105 cells vs 120 ±17.5 per 105 cells, p = 0.001, Fig. 9A).
To further determine whether M. tuberculosis-expanded Tregs inhibit functional T-cell responses, we measured their capacity to reduce IFN-γ production by CD8+ cells in response to M. tuberculosis. Tregs expanded by M. tuberculosis-stimulated monocytes decreased the frequency of IFN-γ-producing CD8+ cells by more than 70% (33 ± 8.9 per 105 cells vs 9.4 ±2.3 per 105 cells, p < 0.01, Fig. 9B). In contrast, the M. tuberculosis-expanded CD4+ Treg-depleted fraction (CD4+ CD25- cells), isolated as described in the methods section, had no effect on the frequency of IFN-γ-producing CD8+ cells (180 ± 8.3 per 105 cells vs 183 ± 4.8 per 105 cells).
The above experiments demonstrated that Tregs expand in response to M. tuberculosis-stimulated monocytes in vitro. To determine if these findings correlate with the clinical manifestationsof M. tuberculosis infection in vivo, we measured the numbers of Tregs in tuberculosis patients, who have an ineffective immuneresponse to infection. Flow cytometry showed that the numbers of CD4+CD25+Foxp3+ cells in freshly isolated PBMC were two to three times higher in tuberculosis patients than in healthytuberculin reactors (1939± 284 vs 802 ± 39, cells per 104 CD4+ cells, p = 0.05, Fig. 10).
Recent studies have shown that Tregs can dampen the immune response to intracellular pathogens, such as viruses, bacteria and protozoa, primarily in animal models [11, 12]. However, limited information is available regarding the capacity of these pathogens to expand Tregs in human infection. In this report, we found that, when CD4+ T-cells are exposed to M. tuberculosis-infected monocytes, Tregs expand in healthy tuberculin reactors, but not in tuberculin-negative persons. Tregs were expanded by monocytes treated with ManLAM of M. tuberculosis, and neutralizing antibodies to the mannose receptor, which binds ManLAM, prevented the increase of Tregs. Expansion of Tregs depended on PGE2 production, which was markedly enhanced by culturing monocytes with M. tuberculosis- or ManLAM stimulated T-cells from healthy tuberculin reactors, but not with T-cells from tuberculin-negative donors. Tregs suppressed immune responses, as depletion of Tregs from PBMC increased IFN-γ production by CD4+ cells, and M. tuberculosis-expanded Tregs inhibited IFN-γ production by CD8+ cells. These findings are relevant to the clinical manifestations of infection with M. tuberculosis, because in tuberculosis patients, who have reduced ability to produce IFN-γ in response to M. tuberculosis , the number of Tregs is two to three times higher than in healthy tuberculin reactors.
Recent studies found that Tregs proliferate and accumulate at sites of infection , and prevent efficient clearance of infection in mice infected with M. tuberculosis . In tuberculosis patients, T-cell production of IFN-γ in response to mycobacterial Ags is reduced, compared to findings in healthy tuberculin reactors . Tuberculosis patients had increased numbers of Tregs, and depletion of Tregs enhanced M. tuberculosis-induced IFN-γ production by PBMC, suggesting that Tregs inhibit an effective immune response [27, 28]. Our results confirm and extend these reports by providing additional insight into the mechanisms by which Tregs are induced in mycobacterial disease. We found that monocytes exposed to M. tuberculosis favor expansion of Tregs from CD4+ cells of donors infected with M. tuberculosis, but not from tuberculin-negative donors. In healthy tuberculin reactors, monocytes present mycobacterial antigens to antigen-reactive CD4+ cells, which proliferate and produce IL-2. As antigen-reactive T-cells expand, it is likely that Treg numbers increase in parallel, as Treg expansion is indexed to the number of T-cells producing IL-2 , dampening the immune response and preventing tissue damage. We found that antigen-expanded Tregs were derived from CD4+CD25- cells, rather than from CD4+CD25+FoxP3+ natural Tregs, suggesting that M. tuberculosis expanded Tregs are induced Tregs. Similarly conversion of naive peripheral CD4+CD25- T cells into CD4+CD25+Foxp3+ cells was noted in an autoimmune model system .
It is important to identify the mycobacterial components that favor expansion of Tregs. ManLAM is a major cell wall component of M. tuberculosis that binds to the mannose receptor on antigen-presenting cells. This interaction inhibits dendritic cell maturation and IL-12 production , and therefore has the potential to inhibit T-cell responses. Our study demonstrates that ManLAM induces PGE2 production, which in turn elicits expansion of Tregs. Although ManLAM stimulates dendritic cells to produce IL-10, which can expand Tregs in other experimental systems [19, 20], neutralization of IL-10 did not reduce expansion of Tregs by M. tuberculosis-stimuluated monocytes (Fig. 5). Therefore, our results suggest that PGE2 mediates expansion of Tregs in M. tuberculosis infection through mechanisms that are independent of IL-10, as discussed below. It is intriguing to speculate that M. tuberculosis utilizes ManLAM to activate an anti-inflammatory immunosuppressive program by antigen-presenting cells which can modulate T-cell responses to maintain persistence in the host. Previous studies have demonstrated that expansion and function of Tregs in Candida albicans-infected mice is regulated by TLR2 . In contrast, we found that the hexameric peptide of the 19-kD M. tuberculosis lipoprotein, a TLR1/2 agonist, had no role in Treg expansion, emphasizing that the mechanisms for Treg induction vary in the immune response to different intracellular pathogens. Our results demonstrate that expansion of Tregs in response to M. tuberculosis-stimulated monocytes depends on production of PGE2, an arachidonic acid metabolite that is produced by mononuclear phagocytes. PGE2 contributes to enhanced growth of respiratory syncytial virus in human airway epithelial cells  and to T-cell dysfunction from the murine leukemia retrovirus . PGE2 also mediates enhanced growth of Salmonella in macrophages and inhibits the local Th1 response to Helicobacter pylori [34, 35]. In addition, during the late phases of murine tuberculosis, high PGE2 concentrations downregulate cell-mediated immunity, favoring disease progression . The negative effects of PGE2 have generally been ascribed to dampening of the Th1 response, as PGE2 inhibits IL-12 production by monocytes, and reduces IFN-γ and IL-2 production by Th1 cells [36, 37]. Recent studies showed that PGE2 can also inhibit cell-mediated immunity through enhanced activity of Tregs. PGE2 favors development of human Tregs by inducing FoxP3 promoter activity , and PGE2 acts through E-prostanoid-2 and -4 receptors to increase suppressor activity of Tregs . In addition, certain tumors produce PGE2, which elicits IL-10 secretion by dendritic cells, favoring development of Tregs . Our current findings provide the first evidence that PGE2 contributes to expansion of Tregs in response to an intracellular infection. It will be important to determine if this mechanism contributes to the negative effects of PGE2 on the immune response to viruses and other intracellular bacteria.
Prior reports demonstrated that murine macrophages exposed to M. bovis BCG generate PGE2 in lipid bodies . To determine the cellular source of PGE2 in our experimental system, we obtained purified monocytes and T-cells by positive immunomagnetic selection from cultures of M. tuberculosis-activated monocytes and CD4+ cells. Western blotting revealed that COX-2 was only present in monocytes but not in T-cells (R Vankayalapati, unpublished data). Tregs stimulated with staphylococcal superantigen can also produce PGE2 , but the levels are at least 100-fold lower than those we observed.
Addition of CD4+ cells to M. tuberculosis-activated monocytes increased PGE2 levels in tuberculin reactors, but had no effect on PGE2 production by monocytes from tuberculin-negative donors. Activated T-cells produce IL-17 and IL-32 [42, 43, 44], which are known to increase production of PGE2 by macrophages [45, 46] and PBMC , respectively. Our preliminary findings suggest that IL-17 producing antigen specific T cells are partially responsible for the expansion of Tregs (R Vankayalapati, unpublished data). We speculate that these cytokines may contribute to Treg expansion in our experimental system. PGE2 production and expansion of Tregs may be also mediated in part through cell-to-cell contact between activated T-cells and M. tuberculosis-infected monocytes. Ligation of CD40 induces PGE2 production and favors development of Tregs [47, 48], and CD40:CD40L interactions are observed in the human immune response to M. tuberculosis . ICOS:ICOS ligand interactions also induce expansion of Tregs . Therefore, it is possible that Treg expansion in response to M. tuberculosis is mediated by interactions between CD40L and/or ICOS on CD4+ cells and CD40 and/or ICOS ligand, respectively, on M. tuberculosis-activated monocytes.
In summary, we found that, in healthy tuberculin reactors, ManLAM-stimulated monocytes produce PGE2 and favor development of Tregs. In addition, antigen-activated T-cells interact with M. tuberculosis–stimulated monocytes to increase PGE2 production and expansion of Tregs that inhibit the cell-mediated immune response. Further studies are needed to determine the underlying mechanisms for this phenomenon. An improved understanding of these mechanisms will contribute to the design of more effective antituberculosis vaccines and immunotherapeutic strategies, based on inhibiting Treg development.
Blood was obtained from 24 healthy tuberculin reactors, 5 tuberculin-negative healthy donors and from 9HIV-seronegative patients with culture-proven pulmonary tuberculosis, all of whom had received less than 2 weeks of antituberculosis therapy. Acid-fast stains of sputum were positive in all patients. All studies were approved by the Institutional Review Boards of the University of Texas Health Center at Tyler and the University of Buenos Aires School of Medicine, and informed consent was obtained from all participants.
For flow cytometry, we used FITC anti-CD4, allophycocyanin (APC)-conjugated anti-CD25, PE-conjugated anti-Foxp3, PE-Cy5-conjugated anti-Foxp3 (all from eBioscience, San Diego, CA), FITC anti-CD14, FITC anti-CD8 and PE anti-CD127 (all from BD Biosciences, San Diego, CA). For neutralization of cytokines, we used mAbs to IL-10 (10 μg/ml, BD Biosciences), and TGF-β (10 μg/ml, R & D Systems, Minneapolis, MN). The lipoglycan mannose-capped lipoarabinomannan (ManLAM), HSP65 and M. tuberculosis whole cell lysate were obtained from Dr. J. Belisle, Colorado State University (Fort Collins, CO), heat-killed M. tuberculosis Erdman was providedby Dr. Patrick Brennan, Colorado State University (Fort Collins, CO) and a hexamericpeptide Pam3-Cys-Ser-Ser-Asn-Lys-Ser-OH of the 19 kD M. tuberculosis lipoprotein , was generously donated by Dr. R. Modlin, University of California (Los Angeles, CA).
Surface staining to detect CD4+, CD25+ and CD127+ cells and intracellular staining to detect Foxp3+ cells was performed, using the Cytofix/Cytoperm Pluskit (eBioscience). Controls for each experiment included cellsthat were unstained, cellsto which FITC-, APC conjugated or PE–conjugatedrat IgG had beenadded, and cells thatwere single stained, either for thesurface marker or for Foxp3. We gated on CD4+ lymphocytes, and determined the percentages of CD25+ and Foxp3+ cells. For some experiments, we gated on Foxp3+ cells to detect CD127lo cells using aFACSCalibur (BD Biosciences).
PBMC were isolated by centrifugation over Ficoll-Paque (Amersham Pharmacia Biotech, Piscataway, NJ). Monocytes, CD4+ and CD8+ cells were isolated with magnetic beads conjugated to anti-CD14, anti-CD4 or anti-CD8 (Miltenyi Biotech, Auburn, CA), respectively. Positively selected cells were >95%+, as measured by flow cytometry.
In some experiments, CD4+ cells were culturedin 12-well plates at 2 ×106 cells/well in RPMI 1640 containingpenicillin (Life Technologies, Grand Island, NY) and 10% heat-inactivated humanserum, with or without 2 × 105 autologous monocytes/well. CD4+ cells and monocytes were cultured in the presence or absence of heat-killed M. tuberculosis Erdman (10 μg/ml), ManLAM (10 μg/ml), the hexameric peptide of the 19-kD M. tuberculosis lipoprotein (10 μg/ml) or HSP65 (10 μg/ml) for 5 d at 37°C in a humidified 5% CO2 atmosphere. For some experiments, monocytes were infected with frozen aliquoted stocks of M. tuberculosis H37Ra at a multiplicity of infection of 5:1, as previously described . In some cases, 10 μg/ml of neutralizing Abs to the mannose receptor or 10 μM of the cyclooxygenase enzyme-2 (COX-2) inhibitor NS398 (Cayman Chemicals, Ann Arbor, Michigan) or HQL79 (Cayman Chemicals), which inhibits production of PGD2 (Cayman Chemicals) was added to the cultures. In other experiments, 2 × 105 autologous monocytes exposed to M. tuberculosis, prepared as outlined above, were cultured in Transwell inserts (Costar, Milpitas, CA) in the 12-well plates. The insert contains0.4-μm diameter pores that allow diffusion of solublefactors but not cell to cell contact. CD4+ cells (2 × 106/well) were cultured in a 12-well plate for 5 days.
CD4+ cells and autologous monocytes were cultured in the presence of heat-killed M. tuberculosis Erdman strain (10 μg/ml) for 3 days. Tregs were isolated using the Treg isolation kit (Miltenyi Biotech), which involves positive selection with anti-CD4-conjugated magnetic beads, followed by positive selection with anti-CD25-conjugated magnetic beads. The CD4+CD25-cells were also obtained.
Freshly isolated CD8+ cells from healthy tuberculin reactors were cultured in 12-well plates at 2 ×106 cells/well, in the presence of 2 × 105 autologous monocytes/well. In some wells, 2 × 105 autologous Tregs, isolated from CD4+ cells and monocytes cultured with heat-killed M. tuberculosis, were added. Cells were cultured for 2 days, CD8+ cells were isolated by positive immunomagnetic selection and the frequency of IFN-γ-producing cells was determined by ELISPOT .
Frequency of CD4+CD25+ and CD4+CD25- cells producing IFN-γ, IL-2, TGF-β and IL-10 CD4+CD25+ and CD4+CD25- cells from cultures of CD4+ cells, monocytes and heat-killed M. tuberculosis or M. tuberculosis whole cell lysate, were isolated, using the Miltenyi Biotech Treg isolation kit, as outlined above. Aliquots of CD4+CD25+ and CD4+CD25- cells were placed on ELISPOT plates, and the number of cells that produce latent TGF-β and IL-2 were detected (R & D systems). For detection of IL-10-producing cells, we used the ELISPOT kit from eBioscience. IFNγ-producing cells were detected using anti-human IFN-γγ mAbs 1-DIK and 7-B6–1 as coatingand detection antibodies (Mabtech, Nacka, Sweden), respectively. The wells were developed accordingto the manufacturer’s instructions, and the spots in theair-dried plates were counted with a stereomicroscope.
Monocytes were cultured in the presence or absence of heat-killed M. tuberculosis or ManLAM, and culture supernatants were collected after 3 days and stored at −70°Cuntil PGE2 concentrations were measured by ELISA (Cayman Chemicals). To determine the effect of T cells on PGE2 production by monocytes, CD4+ cells were cultured with M. tuberculosis-stimulated monocytes at two different ratios of 9:1 and 1: 4. In some experiments, CD4+ cells were cultured with ManLAM-stimulated monocytes at a ratio of 1: 4. Culture supernatants were collected after 3 days and stored at −70°Cuntil PGE2 concentrations were measured by ELISA (lower limit of detection: 15 pg/ml).
Results are shown as the mean ± standard error. For data that were normally distributed, comparisons between groups were performed by a paired or unpaired t test, as appropriate. For data that were not normally distributed, the Wilcoxon rank-sum test was used. P values < 0.05 were considered statistically significant.
We thank Dr. John Belisle for provision of ManLAM and HSP65, Dr. Robert Modlin for donating the hexameric peptide of the 19 kD lipoprotein, and Dr. Patrick Brennan for providing heat-killed M. tuberculosis Erdman. This work was supported by grants from the National Institutes of Health (AI054629 and A1063514), the Cain Foundation for Infectious Disease Research, and the Center for Pulmonary and Infectious Disease Control. Peter F. Barnes holds the Margaret E. Byers Cain Chair for Tuberculosis Research.
The authors have no financial conflict of interest.