Characterization of CD4 and CD8 T cells producing IFN-γ in human latent and active tuberculosis

Tuberculosis 90 (2010) 346e353

Contents lists available at ScienceDirect

Tuberculosis journal homepage: http://intl.elsevierhealth.com/journals/tube

IMMUNOLOGICAL ASPECTS

Characterization of CD4 and CD8 T cells producing IFN-g in human latent and active tuberculosis Cesar M. Rueda a, d, Nancy D. Marín a, e, Luis F. García a, b, f, *, Mauricio Rojas a, b, c, f a

Grupo de Inmunología Celular e Inmunogenética (GICIG), Instituto de Investigaciones Médicas, Universidad de Antioquia, Medellín, Colombia Centro Colombiano de Investigación en Tuberculosis (CCITB), Medellín, Colombia c Unidad de Citometría de Flujo, Sede de Investigación Universitaria, Universidad de Antioquia, Medellín, Colombia b

a r t i c l e i n f o

s u m m a r y

Article history: Received 26 January 2010 Received in revised form 27 August 2010 Accepted 7 September 2010

Patients with pulmonary tuberculosis (PTB) frequently have reduced IFN-g production in response to mycobacterial antigens, compared to individuals with latent Mycobacterium tuberculosis infection (LTBi). However, it is not clear whether this reduced responsiveness is restricted to a particular T cell subset. Herein, PBMCs from 26 PTB patients, 30 household contacts (HHCs) of PTB, and 30 tuberculin positive (TSTþ) healthy subjects not recently exposed to PTB, were stained with CFSE and stimulated non-specific (PPD) for 120 h, and specific (CFP-10/ESAT-6) and latency (HSpX) mycobacterial antigens for 144 h and the percentage of CD4þ and CD8þIFN-gþ T cells responding determined by flow cytometry, in addition to their memory phenotype by the CD45RO and CD27 expression. PTB had decreased frequency of both CD4þ and CD8þ precursor cells, as well as decreased number of CD4þIFN-gþ cells in response to all antigens, whereas CD8þIFN-gþ cells were decreased in response to PPD and ESAT-6, but not to CFP-10 and HSpX. HHCs exhibited the highest precursor frequencies and IFN-g responses, irrespective of the antigen employed. The CD4þ/CD8þ cell ratios showed that in response to PPD CD4þ precursor and IFN-g-producer cells are more frequent than their CD8þ counterparts, and that PTB have a decreased CD4þIFN-gþ/CD8þIFN-gþ ratio in response to PPD, CFP-10, and ESAT-6. CD4þIFN-gþ and CD8þIFN-gþ cells exhibited a central memory phenotype (CD45ROþCD27þ), irrespective of the group of subjects and the antigen used for stimulation. In conclusion, PTB patients had a decreased percentage of CD4þ and CD8þ precursor cells and CD4þIFN-gþ. HHCs exhibited the highest frequency of CD4þ and CD8þ precursors and CD4þIFN-gþ-producing cells. Ó 2010 Published by Elsevier Ltd.

Keywords: Tuberculosis CD4 CD8 IFN-g RD1 antigens Latency

1. Introduction Tuberculosis (TB) remains as one of the major health problems worldwide.1 The failure of the epidemiological surveillance programs, the increase of the human immunodeficiency virus (HIV)-TB coinfection, and the emergence of multidrug resistant strains have contributed to its persistence and increased prevalence in many countries.2 Most infected individuals develop an immune response able to contain the microorganism, although the bacteria are not completely eliminated, leading to a latent infection (LTBi).3 Latency is * Corresponding author. Grupo de Inmunología Celular e Inmunogenética, Sede de Investigación Universitaria, Carrera 53 #61-30 Lab 410, Medellín, Colombia. Tel.: þ57 4 219 6446; fax: þ57 2 219 6455. E-mail address: [email protected] (L.F. García). d CMR was supported by the “Programa de Sostenibilidad”, Vicerrectoría de Investigaciones, Universidad de Antioquia, Medellín, Colombia. e NDM is recipient of a predoctoral scholarship from Colciencias, Bogotá, Colombia. f LFG and MR share senior authorship. 1472-9792/$ e see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.tube.2010.09.003

considered a dynamic event in which an active immune response keeps the persistent mycobacteria under control.4,5 Reactivation of the mycobacteria may occur in 5e10% of LTBi individuals during their life time, leading to active TB. Two to four weeks after the infection, specific CD4þ and CD8þ T cell responses are evident. Among other cytokines, these cells produce IFN-g and Tumor Necrosis Factor (TNF)-a6 that enhances the effector mechanisms of innate immunity, and IL-2 which is critical for the clonal expansion and differentiation of specific T cells.7 The adaptive immune response against Mycobacterium tuberculosis, encompasses the expansion of CD4þ and CD8þ effector cells and the generation of long-lived memory T cells.8e10 CD4þ T cells are considered the main source of IFN-g, which is a critical cytokine for anti-TB response in both humans and mice that plays an important role in the anti-mycobacterial protective immune response.11e13 Mice deficient in CD4þ T cells are highly susceptible to M. tuberculosis infection although their IFN-g production is only transiently diminished.14 Compelling evidence for the role of CD4þ cells in humans is provided by HIVþ individuals who are highly

C.M. Rueda et al. / Tuberculosis 90 (2010) 346e353

susceptible to TB reactivation and re-infection.15,16 CD8þ T cells are also an important source of IFN-g during in vivo M. tuberculosis infection17e19 and in vitro in response to mycobacterial antigens.20e23 However, the CD4/CD8 ratio of IFN-g production under different experimental or clinical conditions is not fully elucidated. Decreased lymphocyte proliferation and production of IFN-g, IL2 by peripheral blood mononuclear cells in TB patients, compared to healthy controls, have been reported in cultures stimulated with different mycobacterial preparations.24e27 The diminished IFN-g production correlates with the severity of the disease and the development of extra pulmonary TB.28 On the other hand, it has been proposed that increased levels of IFN-g production in individuals recently exposed to an active TB case are indicative of actively replicating mycobacteria and is thus a biomarker of high risk susceptibility.29 This hypothesis is supported by our findings in a cohort of household contacts of patients with active TB, in which incident cases were more frequent among high-IFN-g producers.30 However, the type of T cells involved in these contrasting conditions, as well as the antigen-specific T cells in LTBi individuals have not been completely identified. Measurement of T cell proliferation and IFN-g production have been traditionally carried out following Purified Protein Derivative (PPD) stimulation, which do not differentiate among M. tuberculosis infection, BCG vaccination and exposure to environmental mycobacteria. However, the discovery of the region of differentiation 1 (RD-1), present in the genome of M. tuberculosis and few environmental mycobacteria, but absent in most non-tuberculous mycobacteria strains and Mycobacterium bovis BCG31e34 led to the development of more specific diagnostic assays.35 The RD-1 genes encoded the 6 kDa early secretory antigenic target of 6 (ESAT-6) and the 10 kDa secreted protein from culture filtrated (CFP-10).36 These proteins are highly immunogenic and induce T cell proliferation and production of IFN-g.37 Thus ESAT-6 and CFP-10 have been used to identify individuals infected with M. tuberculosis, independently of active or latent infection, from those BCG vaccinated or exposed to environmental mycobacteria.35,38 Another protein used to identify latent infection is Rv2031c, also known as a-crystalline, HSpX or 16 kDa antigen, which is a cytosolic heat shock protein that is expressed as a result of an induced state of metabolic persistence by the intracellular environmental stress conditions of macrophages.39,40 HSpX is recognized by T cells from household contacts of smear-positive PTB patients and patients with pulmonary TB and its induction of IFN-g production correlates with M. tuberculosis infection.41,42 Since the events underlying TB reactivation are still not completely understood, the comparison of the anti-mycobacterial immune response in patients with PTB and LTBi could help to elucidate these events. This may also lead to the development of early diagnostic procedures for individuals at risk of TB reactivation and the development of new strategies for TB control. In this study we quantified the percentage of CD4þ and CD8þ T cell precursors producing IFN-g in response to PPD, CPF-10, ESAT-6 and HSpX antigens in long-term PBMC cultures of pulmonary tuberculosis patients (PTB), healthy household contacts (HHC) highly exposed to M. tuberculosis that had not developed active disease, and healthy tuberculin positive individuals (TSTþ) with no demonstrated recent exposure to TB patients. We also determined the memory phenotype of CD4 and CD8 IFN-g-producing cells in these individuals. Our results show that in TB patients cell cultures stimulated with PPD and RD1 antigens, there are reduced frequencies of precursor CD4þIFN-gþ and CD8þIFN-gþ cells, compared with HHCs and TSTþ healthy individuals. In HHC proliferation and production of IFN-g was mediated by both T cell subpopulations, but mainly by CD4þ T cells. We also show that HSpX poorly stimulated IFN-g production in all of the groups studied. Finally, under our culture

347

conditions CD4 and CD8 cells producing IFN-g exhibited a central memory phenotype. 2. Materials and methods 2.1. Reagents and media RPMI-1640 was obtained from Gibco-BRL (Grand Island, NY, USA), penicillinestreptomycin and the Limulus amebocyte Lysate (LAL) kit from Cambrex-BioWhittaker (Walkersville, MD, USA), Dulbeco’s PBS, Histopaque, bovine serum albumin (BSA) and Brefeldin A (BFA) from SigmaeAldrich (St Louis, MO, USA). Bicinchoninic acid (BCAÔ) was purchased from PIERCE (Rockford, IL, USA). Carboxyfluorescein succinimidyl ester (CFSE) was purchased from Invitrogen (Eugene, OR, USA), monoclonal anti-CD4-PE-Cy5 (clone RPA-T4), anti-CD8-PE-Cy5 (clone HIT8a), anti-IFN-g-PE (clone B27), anti-CD4-PerCPCy5 (clone OKT-4), anti-CD8-Pacific blue (clone OKT-8), anti-CD27-FITC (clone M-T271), anti-CD45RO-APC (Clone UCHL-1), anti-IFN-g PECy7 (clone 4S.B3) and the isotype controls IgG1 g-PE Cy5, IgG1 g-PE were obtained from BD-Pharmigen (San Diego, CA, USA). 2.2. Mycobacterial antigens Culture filtrate proteins (CFP), rHSpX, rCFP-10 and rESAT-6 were obtained from Colorado State University, Fort Collins CO (TB Vaccine Testing and Research Materials Contract NIH, NIAID N01-AI40091). Protein concentration was confirmed by BCAÔ and their integrity was tested by polyacrylamide gel electrophoresis. PPD used for skin test (TST, RT23) and for tissue culture (RT50) were purchased from Staten Serum Institute (Copenhagen, Denmark). All media, reagents and antigens were endotoxin-free (<0.15 EU/ml) as assessed by the LAL test. The optimal concentrations of antigens were established in preliminary titration experiments (data not shown). 2.3. Patients and controls The groups of individuals participating in this study were: 2.3.1. Pulmonary tuberculosis patients (PTB) Twenty-six patients with pulmonary TB (PTB), whose diagnosis was confirmed by ZiehleNeelsen staining of sputum smears, positive bacterial culture, biopsy or bronchoalveolar lavage (BAL) direct examination and culture, were recruited through the Secretaría de Salud de Medellín and the TB Control Program of the Servicio Seccional de Salud de Antioquia, Colombia, as part of a large cohort study previously published.30 All TB patients were HIV negative. The diagnostic tests for TB and HIV were performed in the laboratories associated with the health centers in which the patients received primary care. Blood samples were obtained before or within the first 2 weeks of anti-TB treatment. 2.3.2. Household contacts of patients with active PTB (HHC) Thirty HHC who regularly slept or stayed most of the day (about 10 h daily, 4 days a week) at the residence of the TB index case during the symptomatic period. The HHC were a random sample of a group of 18 families from an ongoing cohort study of 2060 HHCs.30 In addition, all HHC were positive for IFN-g production in response to CFP-10 (cutoff value: 22 pg/ml) as determined by ELISA in 144 h culture supernatants30 and had TST indurations 10 mm. HHC were all healthy, without clinical signs or symptoms of active TB at the time of the study and met the other inclusion criteria.

348

C.M. Rueda et al. / Tuberculosis 90 (2010) 346e353

2.3.3. Tuberculin skin test positive (TSTþ) individuals Thirty healthy laboratory and service personnel with TST 10 mm and no history of previous TB or recent exposure to TB patients were studied as a control group. All subjects participating in this study read and signed a written informed consent form previously approved by the Ethics Committee of the Facultad de Medicina of the Universidad de Antioquia. Exclusion criteria were: HIVþ diabetes, cancer, autoimmune diseases, immunosuppressive treatment, previous TB.

washed and fixed with 2% paraformaldehyde, permeabilized and intracellularly stained with anti-IFN-g PECy7 for 1 h at 4  C light protected. Thereafter, cells were washed and 1  105 cells were acquired in a FACS Canto II Flow Cytometer (BD-Pharmingen, San Diego, CA, USA). For the analysis, cells were selected within the CD4 or CD8 gate, and the phenotype (CD27 and CD45RO) of IFNg-producing cells was determined. The phenotypes were defined as follows: Central memory cells, CD27þCD45ROþ; effector memory cells, CD27CD45ROþ; effectors cells, CD27CD45RO; and naïve or resting cells, CD27þCD45RO.9,44e48

2.4. Tuberculin skin test 2.7. Statistical analysis TST was performed after the blood sample was obtained by intradermal injection on the volar face of the arm of 0.1 ml of PPD RT23 (2 UT). Indurations were measured 48e72 h later and interpreted as positive if the diameter was 10 mm. 2.5. Cell cultures and detection of precursor cells and intracellular IFN-g by flow cytometry PBMCs were obtained from 20 ml of heparinized venous blood samples by centrifugation on Histopaque for 30 min at 900g. Cells were washed with DPBS and cell viability, as determined by trypan blue exclusion, was >95% in all experiments. CFSE staining was done as described by Lyons43 with some modifications. Briefly, 5  106 PBMCs were resuspended in 10 ml DPBS and treated with 1 mm CFSE for 15 min at 37  C. Cells were washed twice with DPBS and resuspended in 5 ml of RPMI-1640 supplemented with 10% heat-inactivated autologous serum, 100 U/ml of penicillin and 100 mg/ml of streptomycin for 30 min at 37  C and incubated for an additional 45 min. One hundred thousand CFSE-labeled PBMC/well were plated in triplicate on 96 well U-bottom plates (Costar-Corning, Lowell, MA) in RPMI-1640 medium supplemented with 100 IU/ml penicillin, 100 mg/ml streptomycin and 10% heat-inactivated autologous serum in the presence or absence of 10 mg/ml of either PPD or HSpX and 5 mg/ml of CFP-10 or ESAT-6. Non-stimulated cultures were used as controls. Cultures were incubated at 37  C, 5% CO2 for 120 h for PPD and 144 h for the other antigens. In preliminary experiments, CFSE-labeled PBMCs from HHCs were stimulated with PHA for 96 h to verify that the system worked properly, and the optimal time-point for each antigen was determined (data not shown). Four hours before the end of the culture, 4 mg/ml of BFA were added to each well. At the end of incubation, cells were harvested, the triplicates pooled, washed with DPBS, and then separately stained with anti-CD4-PE-Cy5, anti-CD8-PE-Cy5 or the respective isotype controls for 30 min, fixed with 2% paraformaldehyde (PFA) for 30 min, permeabilized with a buffer containing 0.5% Tween 20 and 0.2% bovine serum albumin (BSA), and intracellularly stained with anti-IFN-g-PE or IgG1 g-PE as isotype control. Cells (1  105) were acquired in a three-color Coulter Epics XL flow cytometer (Beckman Coulter, Hialeah, FL, USA) and data analysis was performed by gating CD4þ and CD8þ cells versus SSC. Thereafter the number of generations and precursors cells was calculated using the Flow-Jo 7.6 software (Tree Star, Inc. Ashland, OR). 2.6. Identification of IFN-g-producing CD4þ and CD8þ memory phenotype In selected experiments, the cultured cells were collected, washed with PBS and incubated in blocking buffer (2% PHS, 0.1% sodium azide in PBS) for 15 min/4  C. Anti-CD4-PerCPCy5 plus antiCD8-Pacific blue, anti-CD27-FITC and anti-CD45RO-APC (BD-Pharmingen), or their respective isotype controls, were added for 30 min at room temperature light protected. Then, cells were

The frequency of IFN-g-producing cells was calculated by subtracting non-stimulated control values from the stimulated ones. CD4þ/CD8þ ratios were calculated by dividing each individual CD4þ cell percentage by each individual CD8þ cell percentage. Medians were compared using the KruskaleWallis test and Dunn’s post-test. The frequency of females and males, the ranges of age and the percentages of vaccinated individuals among the groups were compared by the Chi-square test (c2). Statistical analyses were done using GraphPad Prism 5 (San Diego, CA, USA). Differences were considered significant when the p  0.05. 3. Results 3.1. Demographic and clinical characteristics of subjects studied Table 1 shows the demographic and clinical characteristics of the subjects studied. There were not significant differences among the groups regarding age or gender. There were no differences in the age of TSTþ individuals, HHCs, and PTB patients. BCG scar was found in 87% TSTþ individuals, 83% HHCs and 72% PTB (ns). In TSTþ individuals and HHCs, the TST induration diameter was 10 mm in all of them as defined in the inclusion criteria. Among PTB patients, the indurations were variable; 9 had 0 mm, 3 had 1e9 mm, 8 had 10e19 mm and 6 had 20 mm of induration. In this group, 44% had a family history of tuberculosis. The parameters of tuberculin and TB family history in the PTB group were not compared with other groups because they were part of the inclusion criteria for TSTþ and HHC. The group of PTB included 25 patients with pulmonary TB and 1 with simultaneous pulmonary and lymph node tuberculosis. TB was diagnosed in 80% of the patients by smear-positive sputum and 20% were diagnosed based on clinical evidence and confirmed by chest X rays (4%), smear-positive BAL (4%), biopsy (4%) and culture (8%). 3.2. PTB patients have decreased frequency of CD4þ and CD8þ precursor T cells responding to mycobacterial antigens To determine the frequency of CD4þ and CD8þ precursor cells and CD4þIFN-gþ and CD8þIFN-gþ in the different groups, cultured Table 1 Clinical and demographic characteristics of Tuberculin Skin Test Positive (TSTþ) controls, Household Contacts (HHCs) of TB patients, and patients with tuberculosis (TB).

Age(years) X  SD % Female/Male BCG scar (%) TST Diameter (mm) X  SD (Range) Family history of TB (%)

TST þ n ¼ 30

HHC n ¼ 30

PTB n ¼ 26

34  11

30  20

30  18

47/53 87 16  5 (10e25)

50/50 83 17  4 (10e23)

52/48 72 11  8 (0e26)

0

100

44

There were not significant differences among groups (c2 p > 0.05).

C.M. Rueda et al. / Tuberculosis 90 (2010) 346e353

349

CFSE-labeled PBMCs, stimulated or not with mycobacterial antigens, were stained with anti-CD4 and anti-CD8 followed by intracellular staining with anti-IFN-g and the percentage of CD4þ and CD8þ T cell precursors and IFN-g-producing cells were determined by flow cytometry. Figure 1 shows a representative experiment in a TSTþ subject in which cells were gated according to expression of CD4þ (R1) and CD8þ (R10 ); within these gates, IFN-gþ cells were defined in a second gate (R3 and R30 ), then the higher peaks from the histograms were used to fit the distribution of each generation (Figure 1B). It was estimated that in response to PPD stimulation both CD4þ and CD8þ cells underwent about 8 cycles of cell division and IFN-g was produced mainly by proliferating CD4þ cells. This gating strategy allowed us to define the percentages of total IFN-gþ cells. IFN-gþ precursor cells were calculated with the histograms of CD4þIFN-gþ and CD8þIFN-gþ cells and total precursors (proliferating ones) within the CD4þ and CD8þ cell gates. PTB patients had a lower percentage of total CD4þ precursor cells in response to PPD, CFP-10 ESAT-6 and HSpX compared with HHCs (Figure 2AeD; p < 0.001 for all antigens). Comparing PTB with TSTþ, this percentage was lower in response to PPD and HSpX (Figure 2A and B; p < 0.01). The response of HHCs was higher than that of TSTþ

Figure 2. Proliferation of CD4þ cells in PBMC cultures from TSTþ individuals (n ¼ 30), HHCs (m ¼ 30) and PTB patients (n ¼ 26) stimulated with (A) PPD (10 mg/ml), (B) HSpX (10 mg/ml), (C) CFP-10 (5 mg/ml) and (D) ESAT-6 (5 mg/ml). Groups were compared by the KruskaleWallis test and Dunn’s multiple comparison post-test. Significant differences are indicated by *p < 0.05, **p < 0.01, ***p < 0001.

individuals in response to PPD (Figure 4; p < 0.01), CFP-10 (Figure 2C; p < 0.001) and ESAT-6 (Figure 2D; p < 0.001), but not in response to HSpX (Figure 2B). It should be noted that in general the percentage of total CD4þ precursor cells was higher in cultures stimulated with PPD, followed by CFP-10, ESAT-6 and HSpX responding cells. As shown in Figure 3, PTB patients also had a lower percentage of total CD8þ precursor cells compared with HHCs in response to

Figure 1. A e Strategy to determine the precursors of proliferating and IFNg-producing CD4þ and CD8þ T cells. PBMCs were stained with CFSE and stimulated with PPD (10 mg/ml). After 120 h culture cells were stained with anti-CD4-PE-Cy5 and anti-CD8-PE-Cy5. Cells were fixed, permeabilized, and stained with anti-IFN-g-PE as described in Materials and methods. The analysis was performed with the Flow-Jo software gating lymphocytes on FSC and SSC dot plots. Then, FL-1-H\CFSE versus FL-3H\CD4-PE-Cy5 and FL-3-H\CD8-PE-Cy5 contour plots were generated. The percentage of total precursors (proliferating cells) was calculated in the regions gated on CD4þ (R1eR2) or CD8þ (R10 eR20 ) cells. IFN-gþ precursor cells were analyzed on CFSElow CD4þ (R3eR4) or CD8þ (R30 eR40 ) cells. B) Histogram depicting the fitted distribution to calculate the number of generations and precursors. The figure shows a representative experiment in a TSTþ individual. The inset shows the CFSE fluorescence and IFNg in a negative control harvested at 120 h after PPD stimulation.

Figure 3. Proliferation of CD8þ cells in PBMC cultures from TSTþ individuals (n ¼ 30), HHCs (m ¼ 30) and PTB patients (n ¼ 26) stimulated with (A) PPD (10 mg/ml), (B) HSpX (10 mg/ml), (C) CFP-10 (5 mg/ml) and (D) ESAT-6 (5 mg/ml). Groups were compared by the KruskaleWallis test and Dunn’s multiple comparison post-test. Significant differences are indicated by *p < 0.05, **p < 0.01, ***p < 0001.

350

C.M. Rueda et al. / Tuberculosis 90 (2010) 346e353

PPD, HSpX, CFP-10 and ESAT-6 (Figure 3B; p < 0.001 for all antigens), and to PPD (Figure 3A; p < 0.001) and HSpX (Figure 3B, p < 0.05) compared with TSTþ individuals. HHCs had higher percentage of precursor CD8þ cells than TSTþ individuals in response to PPD and HSpX (Figure 3A and B; p < 0.05), and to CFP10 and ESAT-6 (Figure 3C and D; p < 0.001). To further establish CD4þ and CD8þ participation in the response to the antigens used among the groups studied, the CD4þ/CD8þ ratio of total precursor cells was calculated for each antigen. These ratios were similar among the groups irrespective of the antigen (Table 2, left columns). However, the CD4þ/CD8þ precursor ratios were higher in response to PPD than to the other antigens, suggesting that the proliferative response elicited by PPD is predominantly given by total CD4þ precursor cells, while in response to HSpX, CFP-10 and ESAT-6, CD8þ cells proliferate at a larger proportion, albeit to a lesser extent than CD4þ cells. 3.3. PTB patients have decreased frequency of CD4þIFN-gþ and CD8þIFN-gþ T cells responding to mycobacterial antigens A comparison of the percentage of IFN-g-producing cells showed that PTB patients had lower frequencies of CD4þIFN-gþ precursors than HHCs in response to PPD, CFP-10, ESAT-6 (Figure 4A, C, and D; p < 0.001) and HSpX (Figure 4B; p < 0.05). These frequencies were also lower compared to TSTþ individuals stimulated with PPD (Figure 4A; p < 0.001), but there were no differences between TSTþ and PTB patients in the percentage of CD4þIFN-gþ cells in response to HSpX, CFP-10 and ESAT-6 (Figure 4BeD). The response of HHCs was higher than TSTþ in response to CFP-10 (p < 0.01) and ESAT-6 (p < 0.001) (Figure 4C and D), but not in response to PPD and HSpX (Figure 4A and B). Similar to the frequency of precursor cells, the percentage of CD4þIFN-gþ cells was generally higher in response to PPD, followed by CFP-10, ESAT-6 and the lower percentage was found in response to HSpX. The frequency of CD8þIFN-gþ cells was lower in PTB patients compared to HHCs in response to PPD (p < 0.05) (Figure 5A), and ESAT-6 (Figure 5D; p < 0.001), and to TSTþ individuals in response to PPD (Figure 5A; p < 0001). In response to HSpX and CFP-10, the percentage of CD8þIFN-gþ was not different among the three groups studied (Figure 5B and C). Regarding CD4þIFN-gþ/CD8þIFN-gþ ratios (Table 2, right columns), in response to PPD IFN-gþ cells were mainly CD4þ T cells in all groups, but the CD4/CD8 ratio was higher in HHCs than in PTB patients (p < 0.01), suggesting a greater participation of CD8þ T cells in the production of IFN-g in PTB. For the other antigens the CD4þIFN-gþ/ CD8þIFN-gþ ratio was closer to one, except CFP-10 by HHCs in whom the production of IFN-g was mainly due to CD4þ cells. Taken together, these findings suggest that CD4þ cells are the main IFN-g producer in response to PPD stimulation in all groups studied, but it is produced in almost equal proportions by CD4þ and CD8þ cells by TSTþ and PTB in response to HspX, CFP-10 and ESAT-6, whereas in HHCs higher

Figure 4. Percentage of CD4þIFN-gþ T cells in PBMC cultures from TSTþ individuals (n ¼ 30), HHCs (m ¼ 30) and PTB patients (n ¼ 26) stimulated with (A) PPD (10 mg/ml), (B) HSpX (10 mg/ml), (C) CFP-10 (5 mg/ml) and (D) ESAT-6 (5 mg/ml). Groups were compared by the KruskaleWallis test and Dunn’s multiple comparison post-test. Significant differences are indicated by *p < 0.05, **p < 0.01, ***p < 0001.

percentage of CD4þIFN-gþ T cells were found in response to PPD, CFP10 and ESAT-6, and CD8þIFN-gþ T cells in response to HspX. 3.4. CD4þ and CD8þ T cells producing IFN-g in TSTþ, HHCs and PTB have a central memory phenotype The forehead results were obtained in long-term PBMC cultures (120 h and 144 h), thus it was expected that the cells proliferating and producing IFN-g under these experimental conditions correspond to the central memory T cells.49,50 To assess this assumption, cultured cells stimulated with PPD, CFP-10 or ESAT-6 for 120 h were stained with anti-CD4 and anti-CD8 plus anti-CD45RO and anti-CD27 followed by intracellular staining with anti-IFN-g. Figure 6 shows a representative experiment with cells from a PTB patient stimulated with CFP-10 in which virtually all CD4þ and CD8þ IFN-g-producing cells were CD45ROþCD27þ, confirming the T central memory phenotype. This phenotype was the same for CD4þ and CD8þ cells from TSTþ, HHCs and PTB irrespective of the antigen used for stimulation (data not shown). 4. Discussion The results presented herein show that both CD4þ and CD8þ precursor T cells proliferate and produce IFN-g, but their proportion is different among the groups studied depending upon the antigen

Table 2 CD4þIFN-gþ/CD8þIFN-gþ cell ratios and CD4þCFSElow/CD8þCFSElow cell ratios in PBMC cultures stimulated with mycobacterial antigens. Antigen

CD4þ/CD8þ Precursors ratio* TSTþ

HHC

PPD HSpX CFP-10 ESAT-6

4.68 3.42 2.99 2.13

5.71 2.54 2.63 1.63

(3.04e8.27) (1.76e6.31) (1.74e5.12) (1.68e3.25)

CD4þIFN-gþ/CD8þIFN-gþ Ratioy

(3.02e9.26) (1.36e4.30) (1.62e4.26) (0.96e2.16)

PTB

TSTþ

HHC

4.98 (3.31e8.42) 3.04 (1.12e4.86) 1.71(0.81e3.86) 1.56 (1.02e2.37)

2.30 1,00 1.31 1.00

3.95 0.81 1.95 1.44

(1.15e5.13) (0,49e2,87) (0.37e2.96) (0.37e3.33)

PTB (2.86e6.87) (0.14e6.27) (1.10e5.24) (0.70e3.10)

1.57 1.00 1.00 0.83

(0.54e3.38){ (0.33e1.70) (0.27e2.21)** (0.20e3.25)

þ þ * The CD4 /CD8 precursor ratios (left columns) were based on individual values from Figures 2 and 3 as described in Materials and methods. The values represent the median (interquartile range) of the ratios. y The CD4þIFN-gþ/CD8þIFN-gþ ratios (right columns) were established from the individual values shown in Figures 4 and 5 as described in Materials and methods. The values represent the median (interquartile range) of the ratios. { HHC versus PTB p < 0.01. ** HHC versus PTB p < 0.05.

C.M. Rueda et al. / Tuberculosis 90 (2010) 346e353

Figure 5. Percentage of CD8þ IFN-gþ T cells in PBMC cultures from TSTþ individuals (n ¼ 30), HHCs (m ¼ 30) and PTB patients (n ¼ 26) stimulated with (A) PPD (10 mg/ml), (B) HSpX (10 mg/ml), (C) CFP-10 (5 mg/ml) and (D) ESAT-6 (5 mg/ml). Groups were compared by the KruskaleWallis test and Dunn’s multiple comparison post-test. Significant differences are indicated by *p < 0.05, **p < 0.01, ***p < 0001.

used. The percentage of CD4þ precursor cells and CD4þ producing IFN-g in response to the antigens used was higher in HHCs, intermediate in TSTþ subjects and lower in PTB patients. The percentage of CD8þ precursor cells was also higher in HHCs than in TSTþ subjects and PTB patients, but the difference between the last two groups was observed only for cultures stimulated with PPD and HSpX. CD8þ IFN-g-producing cells were significantly decreased in PTB, compared with HHCs, only for cultures stimulated with PPD and ESAT-6. To further analyze CD4þ and CD8þ cell participation in the response to the antigens used among the groups studied, the CD4/ CD8 precursor ratios were calculated. This analysis clearly showed that there was more CD4þ than CD8þ precursor cells responding to

351

all the stimuli used in the three groups studied. The highest ratios were found in response to PPD where CD4þ cells proliferated 4.5- to 6-fold more than CD8þ cells, while for the other antigens the ratios were smaller. A different situation was found for the CD4þIFN-gþ/ CD8þIFN-gþ cell ratios. In cultures stimulated with PPD CD4þ cells were the main IFN-g producers, whereas in cultures stimulated with HSpX, CFP-10 and ESAT-6 the ratios were close to 1 or even below. These findings suggest that stimulation with a mixed antigenic preparation, like PPD, favors the production of IFN-g by CD4þ precursor cells, whereas stimulation with purified antigens tends to stimulate IFN-g production by both CD4þ and CD8þ cells in variable proportions. The results also show that PTB patients have a decreased frequency of both CD4þ and CD8þ precursors compared with HHCs and TSTþ individuals, but without differences in the CD4þ/CD8þ ratio among the three groups. However, the production of IFN-g by CD4þ and CD8þ had a different profile in the different groups depending upon the stimulus employed. In cultures stimulated with PPD, this ratio was smaller in PTB patients than in HHCs, but CD4þ cells were still the main IFN-g producers. However; in CFP-10, ESAT-6 and HSpX stimulated cultures, PTB patients seem to have a more profound decrease in the number of CD4þIFN-gþ cells, compared to TSTþ and HHCs, resulting in a higher proportion of CD8þIFN-gþ cells. These results are in agreement with previous reports in which both CD4þ and CD8þ cells are markedly reduced in TB patients.51 The highest frequencies of precursor and CD4þIFN-gþ and CD8þIFN-gþ cells were detected in individuals with latent TB, whether HHC or TST, in response to PPD, suggesting that the protective response against M. tuberculosis requires cells able to proliferate in addition to produce IFN-g in response to mycobacterial antigens. It is important to remember that we did 120e144 h cultures in which, contrary to the short (24 h) cultures, stimulated cells have the possibility to proliferate and differentiate into IFN-g producer cells.49,50,52 In TB patients the decreased frequency of CD4þ T cells and CD8þ precursors of IFN-g-producing cells in response to mycobacterial antigens could be related to the concomitant decrease in the production of IL-224,53 the increase in production of regulatory cytokines like the transforming growth factor beta (TGF-b), and IL-10 produced by regulatory T cells (Tregs),54e56 associated with the generation of a permissive

Figure 6. Phenotype of IFN-g-producing CD4þ and CD8þ T cells. PBMCs from a PTB patient were stimulated with CFP-10 for 144 h, thereafter the cells were stained with antiCD4-PerCPCy5.5 plus anti-CD8-Pacific Blue, anti-CD27-FITC and anti-CD45RO-APC, followed by fixation, permeabilization and intracellular staining with anti-IFN-gPECy7. The memory phenotype (CD27 versus CD45RO) was defined within the gate of CD4þIFN-gþ and CD8þIFN-gþ cells. Upper boxes show CD4þ cells; lower boxes show CD8þ cells. Figure shows a representative experiment in a PTB patient stimulated with CFP-10. The same results were obtained in 7 PTB patients and 7 HHCs (not shown).

352

C.M. Rueda et al. / Tuberculosis 90 (2010) 346e353

environment for M. tuberculosis replication and the diminished number of functional T cells by either decreased clonal expansion of T cells due to anergy57 or activation-induced cell death.58,59 It would be important to analyze the intracellular presence of other cytokines, like IL-2 and TNF-a to determine the multifunctional nature of the responding cells in the different groups, since it is well accepted that IFN-g alone is not sufficient to provide protection against M. tuberculosis.8,46,47,60 Also, it would be important to determine whether CD8þ cells producing IFN-g are different from those able to kill infected macrophages, as recently shown in mice.61 Different studies have shown that PBMC from TB patients, compared with TSTþ healthy controls, have decreased lymphocyte proliferation and IFN-g production in response to different mycobacterial preparations.24,62 However, in these reports the T cell subpopulations producing IFN-g were not identified. Our data are consistent with some reports in which the decreased proliferative response and the production of IFN-g in TB patients affect both T cell subpopulations.63 However, it would be important to determine whether these changes are also occurring with the T cells present at the affected pulmonary level, as recently reported.47,64 The differences in the percentage of CD4þ and CD8þ precursors and IFN-g-producing cells among the groups studied may be related to the level of exposure to M. tuberculosis. The higher frequency of these cells in HHCs is likely a consequence of the exposure to high amounts of mycobacteria during their cohabitation with the smear-positive index cases. In the TSTþ group without known recent exposure, intermediate percentages of IFN-gproducing cells in response to CFP-10 and ESAT-6 were found. Interestingly, these differences were not observed in PPD stimulated cultures where HHCs and TSTþ individuals had comparable percentages of IFN-g-producing cells. It is possible that in the TSTþ group the response to PPD reflects the memory response to other mycobacterial stimuli, such as BCG vaccination or exposure to environmental mycobacteria. Other authors have reported that in long-term cultures re-stimulation with specific antigens improves detection of IFNg-producing cells65; however, preliminary experiments in our system showed that re-stimulation with PPD, but not with the other antigens, induced lymphocyte death, thus we selected single time points for each antigen detecting the maximal peak of cells producing this cytokine. One important difference between our culture conditions and the ones used by other investigators18,20,21,23 is the use of recombinant proteins rather than peptides. However, the finding that in the response to HSpX, ESAT-6 and CFP-10 the CD4/CD8 ratios are close to one in the three groups of subjects studied suggest that CD8þ cells were indeed recognizing these molecules at the same extent that CD4þ cells. The CD45ROþCD27þ phenotype found in our long-term cultures for both CD4 and CD8 IFN-g producers support that indeed these cells correspond to central memory T cells.9,46,47 It has been recently reported that PBMC cultures from TSTþ individuals stimulated RD-1 antigens for 9 days, IFN-g-producing CD4þ T cells exhibited the phenotype CD45RACD27þ, which is associated with central memory, while in patients with moderate TB predominated the CD45RACD27 phenotype of effector memory T cells.9 Also, in short-term cultures it has been reported that the predominant cell responding to PPD, CFP-10 and ESAT-6 in individuals with a history of previous TB has the CD45ROþCD27 of T effector memory cells.48 The response to HSpX was potentially very interesting since it is a cytosolic protein, under the control of the DosR regulon, which requires determined conditions to be expressed, such as the presence of nitric oxide, low oxygen tension, cultures in stationary phase and during the course of infection of activated macrophages.66 Again, TB patients had a decreased frequency of CD4þ and CD8þ precursor cells compared to HHCs. There were no differences

between HHCs and TSTþ subjects for CD4þ precursors, suggesting that during latent infection there is indeed a response to HSpX,67 albeit not as strong as the response to RD1 antigens. Taken together, our results clearly demonstrate differences in the frequency of CD4þ and CD8þ precursor T cells and antigenspecific CD4þIFN-gþ and CD8þIFN-gþ T cells among patients with active TB and healthy LTBi individuals, depending on the degree of exposure to mycobacteria. In individuals with latent TB infection the cytokine production is mainly mediated by CD4þ T cells in response to PPD, CFP-10 and ESAT-6, while in active disease decreased percentages in both subpopulations are detected. These observations will allow a better understanding of the immunopathogenesis of TB and the opportunity to develop new strategies for diagnosis. Acknowledgements The authors thank the Tuberculosis Control Programs of the Servicio Seccional de Salud de Antioquia and the Secretaria de Salud de Medellín for allowing us to have access to the patients and their clinical records. The authors recognize the valuable help of Sara C. París and Blanca L. Ortiz, and Luis F. Barrera for their critical reading of the manuscript. We also acknowledge the patients, their household contacts and the volunteers that kindly participated in this study. Funding: This study was supported by Colciencias (Bogotá, Colombia), grants 11150416335, 111540820488 and 34261817270, and Programa de Sostenibiloidad (Vicerrectoría de Investigaciones, Universidad de Antioquia, Medellín, Colombia). Competing interests:

The authors have no conflicts of interest.

Ethical approval: This study was approved by the Ethics Committee of the Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia. References 1. World Health Organization. Global tuberculosis control: epidemilogy, strategy, financing: WHO report 2009. Geneva, Switzerland: World Health Organization; 2009. 2. Nunn P, Williams B, Floyd K, Dye C, Elzinga G, Raviglione M. Tuberculosis control in the era of HIV. Nat Rev Immunol 2005;5:819e26. 3. Andersen P. Vaccine strategies against latent tuberculosis infection. Trends Microbiol 2007;15:7e13. 4. Salgame P. Host innate and Th1 responses and the bacterial factors that control Mycobacterium tuberculosis infection. Curr Opin Immunol 2005;17:374e80. 5. Lin PL, Flynn JL. Understanding latent tuberculosis: a moving target. J Immunol 2010;185:15e22. 6. Flynn JL. Lessons from experimental Mycobacterium tuberculosis infections. Microbes Infect 2006;8:1179e88. 7. Wang J, Wicker LS, Santamaria P. IL-2 and its high-affinity receptor: genetic control of immunoregulation and autoimmunity. Sem Immunol 2009;21: 363e71. 8. Mueller H, Detjen AK, Schuck SD, Gutschmidt A, Wahn U, Magdorf K, et al. Mycobacterium tuberculosis-specific CD4þ, IFNgþ, and TNFaþ multifunctional memory T cells coexpress GM-CSF. Cytokine 2008;43:143e8. 9. Goletti D, Butera O, Bizzoni F, Casetti R, Girardi E, Poccia F. Region of difference 1 antigen-specific CD4þ memory T cells correlate with a favorable outcome of tuberculosis. J Infect Dis 2006;194:984e92. 10. Kamath A, Woodworth JSM, Behar SM. Antigen-specific CD8þ T Cells and the development of central memory during Mycobacterium tuberculosis infection. J Immunol 2006;177:6361e9. 11. Cooper AM, Dalton DK, Stewart TA, Griffin JP, Russell DG, Orme IM. Disseminated tuberculosis in interferon gamma gene-disrupted mice. J Exp Med 1993;178:2243e7. 12. Flynn JL, Chan J, Triebold KJ, Dalton DK, Stewart TA, Bloom BR. An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection. J Exp Med 1993;178:2249e54. 13. Vanham G, Toossi Z, Hirsch CS, Wallis RS, Schwander SK, Rich EA, et al. Examining a paradox in the pathogenesis of human pulmonary tuberculosis: immune activation and suppression/anergy. Tuber Lung Dis 1997;78:145e58.

C.M. Rueda et al. / Tuberculosis 90 (2010) 346e353 14. Caruso AM, Serbina N, Klein E, Tribold K, Bloom BR, Flynn J. Mice deficient in CD4 T cells have only transiently diminished levels of IFN-g, yet succumb to tuberculosis. J Immunol 1999;162:5407e16. 15. Girardi E, Goletti D, Antonucci G, Ippolito G. Tuberculosis and HIV: a deadly interaction. J Biol Regul Homeost Agents 2001;15:218e23. 16. Chen ZW. Immunology of AIDS virus and mycobacterial co-infection. Curr HIV Res 2004;2:351e5. 17. Flynn JL, Goldstein MM, Triebold KJ, Koller B, Bloom BR. Major histocompatibility complex class I-restricted T cells are required for resistance to Mycobacterium tuberculosis infection. Proc Nat Acad Sci USA 1992;89:12013e7. 18. Grotzke JE, Lewinsohn DM. Role of CD8þ T lymphocytes in control of Mycobacterium tuberculosis infection. Microbes Infect 2005;7:776e88. 19. Sud D, Bigbee C, Flynn JL, Kirschner DE. Contribution of CD8þ T cells to control of Mycobacterium tuberculosis infection. J Immunol 2006;176:4296e314. 20. Lalvani A, Brookes R, Wilkinson RJ, Malin AS, Pathan AA, Andersen P, et al. Human cytolytic and interferon gamma-secreting CD8þ T lymphocytes specific for Mycobacterium tuberculosis. Proc Nat Acad Sci USA 1998;95:270e5. 21. Smith SM, Klein MR, Malin AS, Sillah J, Huygen K, Andersen P, et al. Human CD8þ T cells specific for Mycobacterium tuberculosis Secreted antigens in tuberculosis patients and healthy BCG-vaccinated controls in The Gambia. Infect Immun 2000;68:7144e8. 22. Serbina NV, Lazarevic V, Flynn JL. CD4þ T cells are required for the development of cytotoxic CD8þ T cells during Mycobacterium tuberculosis infection. J Immunol 2001;167:6991e7000. 23. Shams H, Klucar P, Weis SE, Lalvani A, Moonan PK, Safi H, et al. Characterization of a Mycobacterium tuberculosis peptide that is recognized by human CD4þ and CD8þ T cells in the context of multiple HLA alleles. J Immunol 2004;173:1966e77. 24. Sánchez FO, Rodríguez JI, Agudelo G, García LF. Immune responsiveness and lymphokine production in patients with tuberculosis and healthy controls. Infect Immun 1994;62:5673e8. 25. Hussain R, Kaleen A, Shahid F, Dojki M, Jamil B, Mehmood H, et al. Cytokine profiles using whole-blood assays and discriminate between tuberculosis patients and healthy endemic controls in a BCG-vaccinated population. J Immun Meth 2002;264:98e108. 26. Palazzo R, Spensieri F, Massari M, Fedele G, Frasca L, Carrara S, et al. Use of whole-blood samples in in-house bulk and single-cell antigen-specific gamma interferon assays for surveillance of Mycobacterium tuberculosis infections. Clin Vaccine Immunol 2008;15:327e37. 27. Hinks TSC, Dosanjh DPS, Innes JA, Pasvol G, Hackforth S, Varia H, et al. Frequencies of region of difference 1 antigen-specific but not purified protein derivative-specific gamma interferon-secreting T cells correlate with the presence of tuberculosis disease but do not distinguish recent from remote latent infections. Infect Immun 2009;77:5486e95. 28. Sodhi A, Gong J, Silva C, Qian D, Barnes PF. Clinical correlates of interferon g production in patients with tuberculosis. Clin Infect Dis 1997;25:617e20. 29. Andersen P, Doherty TM, Pai M, Weldingh K. The prognosis of latent tuberculosis: can disease be predicted? Trends Mol Med 2007;13:175e82. 30. del Corral H, París SC, Marín ND, Marín DM, López L, Henao HM, et al. IFNg response to Mycobacterium tuberculosis, risk of infection and disease in household contacts of tuberculosis patients in Colombia. PLoS One 2009;4:e8257. 31. Cole ST, Brosch R, Parkhill J, Garnier T, Churcher D, Harris D, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998;393:537e44. 32. Garnier T, Eiglmeier K, Camus JC, Medina N, Mansoorn H, Pryor M, et al. The complete genome sequence of Mycobacterium bovis. Proc Nat Acad Sci USA 2003;100:7877e82. 33. Mattow J, Jungblut PR, Schaible UE, Mollenkopf H-J, Lamer S, Zimny-Arndt U, et al. Identification of proteins from Mycobacterium tuberculosis missing in attenuated Mycobacterium bovis BCG strain. Electrophoresis 2001;22:2936e46. 34. van Pinxteren LAH, Ravn P, Agger EM, Pollock J, Andersen P. Diagnosis of tuberculosis based on the two specific antigens ESAT-6 and CFP10. Clin Diagn Lab Immunol 2000;7:155e60. 35. Pai M, Dheda K, Cunningham J, Scano F, O’Brien R. T-cell assays for the diagnosis of latent tuberculosis infection: moving the research agenda forward. Lancet Infec Dis 2007;7:428e38. 36. Cockle PJ, Gordon SV, Lalvani A, Buddle BM, Hewinson RG, Vordermeier HM. Identification of Novel Mycobacterium tuberculosis antigens with potential as diagnostic reagents or subunit vaccine candidates by comparative genomics. Infect Immun 2002;70:6996e7003. 37. Al-Attiyah R, Mustafa AS. Characterization of human cellular immune responses to novel Mycobacterium tuberculosis antigens encoded by genomic regions absent in Mycobacterium bovis BCG. Infect Immun 2008;76:4190e8. 38. Pai M, Riley LW, Colford J. Interferon-g assays in the immunodiagnosis of tuberculosis: a systematic review. Lancet Infect Dis 2004;4:761e76. 39. Chan J, Flynn J. The immunological aspects of latency in tuberculosis. Clin Immunol 2004;110:2e12. 40. Hu Y, Movahedzadeh F, Stoker NG, Coates ARM. Deletion of the Mycobacterium tuberculosis a-crystallin-like hspX gene causes increased bacterial growth in vivo. Infect Immun 2006;74:861e8. 41. Wilkinson RJ, Wilkinson KA, De Smet KA, Haslov K, Paslov G, Singh M, et al. Human T- and B-cell reactivity to the 16kDa alpha-crystallin protein of Mycobacterium tuberculosis. Scand J Immunol 1998;48:403e9.

353

42. Geluk A, Lin MY, van Meijgaarden KE, Leyten EM, Franken KL, Ottenhoff TH, et al. T-cell recognition of the HspX protein of Mycobacterium tuberculosis correlates with latent M. tuberculosis infection but not with M. bovis BCG vaccination. Infect Immun 2007;75:2914e21. 43. Lyons AB. Analysing cell division in vivo and in v itro using flow cytometric measurement of CFSE dye dilution. J Immun Meth 2000;243:147e54. 44. Sallusto F, Geginat J, Lanzavecchia A. Central memory and effector memory T cell subsets: function, generation, and maintenance. Rev Immunol 2004;22:745e63. 45. Schiott A, Lindstedt M, Johansson-Lindbom B, Roggen E, Borrebaeck CA. CD27CD4þ memory T cells define a differentiated memory population at both functional and transcription levels. Immunol 2004;113:363e70. 46. Scriba TJ, Kalsdorf B, Abrahams DA, Isaacs F, Hofmeister J, Black G, et al. Distinct, specific IL-17- and IL-22-producing CD4þ T cell subsets contribute to the human anti-mycobacterial immune response. J Immunol 2008;180:1962e70. 47. Kalsdorf B, Scriba TJ, Wood K, Day CL, Dheda K, Dawson R, et al. HIV-1 infection impairs the bronchoalveolar T-cell response to mycobacteria. Am J Respir Crit Care Med 2009;180:1262e70. 48. Tapaninen P, Korhonen A, Pusa L, Seppala I, Tuuminen T. Effector memory T-cells dominate immune responses in tuberculosis treatment: antigen or bacteria persistence? Int J Tuberc Lung Dis 2010;14:347e55. 49. Hanekom WA, Dockrell HM, Ottenhoff THM, Dpherty TM, Fletcher H, McShane H, et al. Immunological outcomes of new tuberculosis vaccine trials: WHO panel recommendations. PLoS Med 2008;5:e145. 50. Leyten EMS, Arend SM, Prins C, Cobelens FGJ, Ottenhoff THM, van Dissel JT. Discrepancy between Mycobacterium tuberculosis-specific gamma interferon release assays using short and prolonged in vitro incubation. Clin Vaccine Immunol 2007;14:880e5. 51. Shams H, Wizel B, Weis SE, Samten B, Barnes PF. Contribution of CD8þ T cells to gamma interferon production in human tuberculosis. Infect Immun 2001;69:3497e501. 52. Cehovin A, Cliff JM, Hill PC, Brookes RH, Dockrell HM. Extended culture enhances sensitivity of a gamma interferon assay for latent Mycobacterium tuberculosis infection. Clin Vaccine Immunol 2007;14:796e8. 53. Millington KA, Innes JA, Hackforth S, Hinks TSC, Deeks JJ, Dosanjh DPS, et al. Dynamic relationship between IFN-g and IL-2 profile of Mycobacterium tuberculosis-specific T cells and antigen load. J Immunol 2007;178:5217e26. 54. Guyot-Revol V, Innes JA, Hackforth S, Hinks T, Lalvani A. Regulatory T cells are expanded in blood and disease sites in patients with tuberculosis. Am J Resp Crit Care Med 2006;173:803e10. 55. Burl S, Hill PC, Jeffries DJ, Holand MJ, Fox A, Lugos MD, et al. FOXP3 gene expression in a tuberculosis case contact study. Clin Exp Immunol 2007;149:117e22. 56. Roberts T, Beyers N, Aguirre A, Walzl G. Immunosuppression during active tuberculosis is characterized by decreased interferon-g production and CD25 expression with elevated Frokhead Box P3, transforming growth factor-b, and interleukin-4 mRNA levels. J Infect Dis 2007;195:870e8. 57. Zea AH, Culotta KS, Ali J, Mason C, Park H-J, Zabaleta J, et al. Decreased expression of CD3z and nuclear transcription factor kappa B in patients with pulmonary tuberculosis: potential mechanisms and reversibility with treatment. J Infect Dis 2006;194:1385e93. 58. Hirsch CS, Toossi Z, Vanham G, Johnson JL, Peters P, Okwera A, et al. Apoptosis and T cell hyporesponsiveness in pulmonary tuberculosis. J Infect Dis 1999;179:945e53. 59. Hirsch CS, Toossi Z, Johnson JL, Luzze H, Ntambi L, Peters P, et al. Augmentation of apoptosis and interferon-gamma production at sites of active Mycobacterium tuberculosis infection in human tuberculosis. J Infect Dis 2001;183:779e88. 60. Seder RA, Darrah PA, Roederer M. T-cell quality in memory and protection: implications for vaccine design. Nat Rev Immunol 2008;8:247e58. 61. Einarsdottir T, Lockhart E, Flynn JL. Cytotoxicity and secretion of gamma interferon are carried out by distinct CD8 T cells during Mycobacterium tuberculosis infection. Infect Immun 2009;77:4621e30. 62. Bhattacharyya S, Singla R, Dey AB, Prasad HK. Dichotomy of cytokine profiles in patients and high-risk healthy subjects exposed to tuberculosis. Infect Immun 1999;67:5597e603. 63. Martins MV, Lima MC, Duppre NC, Matos HJ, Spencer JS, Brennan PJ, et al. The level of PPD-specific IFN-g-producing CD4þ T cells in the blood predicts the in vivo response to PPD. Tuberculosis 2007;87:202e11. 64. Jafari C, Ernst M, Strassburg A, Greinert U, Kalsdorf B, Kirsten D, et al. Local immunodiagnosis of pulmonary tuberculosis by enzyme-linked immunospot. Eur Resp J 2008;31:261e5. 65. Black GF, Thiel BA, Ota MO, Parida SK, Adegbola R, Boom WH, et al. Immunogenicity of novel DosR regulon-encoded candidate antigens of Mycobacterium tuberculosis in three high-burden populations in Africa. Clin Vaccine Immunol 2009;16:1203e12. 66. Sherman DR, Voskuil M, Schnappinger D, Liao R, Harrell MI, Schoolnik GK. Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding alpha-crystallin. Proc Nat Acad Sci 2001;98:7534e9. 67. Rogerson BJ, Jung YJ, LaCourse R, Ryan L, Enright N, North RJ. Expression levels of Mycobacterium tuberculosis antigen-encoding genes versus production levels of antigen-specific T cells during stationary level lung infection in mice. Immunol 2006;118:195e201.