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Keywords:

  • Infectious diseases;
  • Memory cells;
  • Tuberculosis;
  • T cells;
  • Vaccination

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Definition of protective immunity induced by effective vaccines is important for the design of new pathogen control strategies. Inactivation of the PhoP response-regulator in Mycobacterium tuberculosis results in a highly attenuated strain that demonstrates impressive protective efficacy in pre-clinical models of tuberculosis. In this report we demonstrate that the protection afforded by the M. tuberculosis phoP mutant strain is associated with the long-term maintenance of CD4+ T-cell memory. Immunization of mice with SO2 resulted in enhanced expansion of M. tuberculosis-specific CD4+ T cells compared with vaccination with the BCG vaccine, with an increased frequency of these cells persisting at extended time-points after vaccination. Strikingly, vaccination with SO2 resulted in sustained generation of CD4+ T cells displaying a central memory phenotype, a property not shared by BCG. Further, SO2 vaccination markedly improved the generation of polyfunctional cytokine-secreting CD4+ T cells compared with BCG vaccination. The improved generation of functionally competent memory T cells by SO2 correlated with augmented recall responses in SO2-vaccinated animals after challenge with virulent M. tuberculosis. This study defines a mechanism for the protective effect of the SO2 vaccine and suggests that deletion of defined virulence networks may provide vaccine strains with potent immuno-stimulatory properties.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Tuberculosis (TB) remains a leading cause of death worldwide, with an estimated 1.8 million deaths per year 1. The current vaccine, Mycobacterium bovis Bacille Calmette-Guérin (BCG), has failed to control the spread of TB and a more effective vaccine is required urgently. One of the strategies proposed in rational TB vaccine development is to identify the transcriptional networks contributing to virulence in Mycobacterium tuberculosis and remove specific genes from the pathogen, thereby creating an attenuated vaccine candidate that retains a diverse repertoire of immunodominant epitopes 2. With strict protocols to ensure removal of virulence, this strategy is gaining acceptance from key regulatory bodies as a feasible and safe approach to develop much-improved vaccines against TB 3.

Protective immunity against intracellular pathogens such as M. tuberculosis is critically dependent on the functional quality of specific T cells that have been induced following vaccination. These Ag-specific memory cells are defined by their ability to migrate to peripheral tissue and respond rapidly to secondary infection by releasing effector cytokines such as IFN-γ and TNF. These cells also have the capacity to respond to the cytokine interleukin (IL)-7 and IL-15, which facilitates the persistence of these clones within the overall Ag-specific T-cell repertoire 4. The generation of such polyfunctional T cells appears to be an important correlate of vaccine-induced protection against M. tuberculosis 5, 6. Both CD4+ and CD8+ T cells contribute to protective immunity against M. tuberculosis; however, CD4+ T cells appear to play a predominant role (reviewed in 7). The recent evidence suggests that the chronic nature of M. tuberculosis impacts on the functional capacity of pathogen-specific CD4+ T cells suggesting that modified, attenuated versions of M. tuberculosis that induce sustained generation of memory CD4+ T-cell responses may prove beneficial in TB control 8–10.

Two-component systems (TCSs) are a feature of many bacterial species, enabling the detection and response to environmental stimuli. TCSs comprise a membrane-associated sensor kinase and a nuclear transcription factor responsible for gene activation or repression in response to signals detected by its cognate sensor protein. TCSs have been reported to play an important role in virulence regulation in numerous bacterial pathogens, such as Salmonella typhimurium and Yersinia pestis 11. In M. tuberculosis, the phoP gene corresponds to Rv0757 and encodes the transcription factor of the PhoP/PhoR TCS, one of the 11 TCSs in M. tuberculosis 12, 13. Disruption of the PhoP/PhoR TCS has been shown to have a profound effect on M. tuberculosis gene expression and virulence. Removal of PhoP attenuates M. tuberculosis growth in murine macrophages and infected mice 14, 15. PhoP/PhoR regulates genes required for the synthesis of complex mycobacterial lipids implicated in M. tuberculosis virulence, including sulfolipids, diacyltrehaloses and polyacyltrehaloses 16, 17 and the PhoP/PhoR TCS controls secretion of the early secreted antigen, 6 kDa (ESAT-6), an important virulence factor and antigenic component of M. tuberculosis 18. A phoP mutant of M. tuberculosis MT103 (SO2) confers protective immunity in murine models of M. tuberculosis 15, 19 and protection against both pulmonary and disseminated M. tuberculosis challenge in the guinea pig 20, 21. The recent studies in non-human primates showed that SO2 was more protective than BCG against pulmonary M. tuberculosis challenge, as assessed by lung bacterial counts and chest X-ray scores 22. Consequently, this vaccine is now being prepared for testing in humans 23.

Although the microbiological effects of the PhoP attenuation in M. tuberculosis and the protective capacity of the SO2 vaccine candidate are well established, the mechanisms by which the phoP-inactivated strain modulates host immunity are poorly defined. In this study, we investigated the influence of phoP-expression on the development of T-cell memory responses after vaccination with the SO2 vaccine. Characterization of the temporal and spatial kinetics of Ag-specific CD4+ T-cell immunity against SO2 revealed that inactivation of the PhoP–PhoR TCS results in the long-term maintenance of functionally competent Ag-specific T-cell immunity.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

SO2 expands Ag-specific CD4equation image T-cell responses after vaccination

The phoP-mutant of M. tuberculosis is a highly protective vaccine when assessed in a number of pre-clinical models of tuberculosis 15, 19–21. We confirmed this finding in a low-dose aerosol C57BL/6 mouse model of M. tuberculosis infection. Mice vaccinated with SO2 significantly controlled M. tuberculosis load in the lung and spleen after aerosol challenge compared with unvaccinated animals (Fig. 1A and B). SO2-immunized hosts showed a consistent reduction in M. tuberculosis burden in the lung and spleen compared with BCG-immunized mice, with differences most apparent in the spleen (Fig. 1A and B). In order to determine the basis for the protective potential of SO2-induced immunity, we examined the capacity of SO2 vaccination to induce the proliferation and maintenance of M. tuberculosis Ag85B-reactive CD4+ T cells 24 that had been adoptively transferred into congenic hosts. Compared with BCG vaccination, SO2 delivery led to a distinct increase in the total number of blood-borne, Ag85B-reactive p25 T cells (Fig. 2). This increase was most evident at days 14 and 21, where the SO2 strain demonstrated a significant four to five-fold increase in p25 T-cell number compared with BCG-vaccinated animals (Fig. 2D). This phenomenon was also seen at sites important for the initiation of mycobacterial immunity; when CFSE-labelled p25 T cells were transferred to WT recipients and then vaccinated with SO2, marked expansion of the p25 T cells was apparent at both 7 and 28 days post-vaccination in the draining lymph nodes (DLNs) and spleens of recipient mice (Fig. 3). At day 7, this expansion was significantly increased compared with the BCG-vaccinated groups in the DLNs, spleen and lung, both in terms of the extent of CFSE dilution (Fig. 3A) and the number of activated p25 CD4+ T cells (Fig. 3B–D). At day 28, the number of activated p25 T cells in SO2-primed mice was still markedly elevated compared with BCG-vaccinated mice at all sites (Fig. 3E–G). These data indicate that vaccination with the SO2 strain results in the activation and proliferation of Ag-specific T cells that exceed those seen with the BCG vaccine.

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Figure 1. Protective efficacy of the PhoP mutant against M. tuberculosis. Mice (five per group) were vaccinated s.c. with either 5×105 CFU of M. bovis BCG or phoP-inactivated M. tuberculosis (SO2) or left unvaccinated (unv). At 12 weeks post-vaccination, mice were challenged by aerosol with 100 CFU M. tuberculosis H37Rv. Twenty-eight days post-infection, the mice were sacrificed and the number of viable bacterial CFU determined in tissue homogenates of the (A) lung and (B) spleen. Data are presented as the mean log10 transformed bacterial CFU+SEM and are representative of two independent experiments. The significance of differences between groups was determined by ANOVA (*p<0.05, **p<0.01) as shown.

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Figure 2. Vaccination with SO2 results in marked expansion of antigen-specific CD4+ T-cell responses in the blood. 5×105 p25 splenocytes (CD45.1+) were i.v. transferred on day 0 into C57BL/6 mice (CD45.2+, four per group per experiment), which were either left unvaccinated (unv) or were vaccinated sc with 5×105 CFU of BCG or SO2 on the next day. At days 1, 7, 14, 21, 28 and 70, mice were bled and the number of p25 CD4+ T cells enumerated by multi-parameter flow cytometry. p25 CD4+ T cells were also analysed for CD62L and CD44 expression. (A–C) Representative flow cytometry plots for (A) unvaccinated, (B) BCG-vaccinated and (C) SOS-vaccinated mice at day 7 are presented. (D) The total number of blood-borne p25 CD4+ CD44hiCD62Llo T cells at the indicated timepoints are shown. Data are the mean±SEM and are representative of one of the two independent experiments. The significance of differences between SO2 and BCG groups were determined by ANOVA (*p<0.05).

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Figure 3. Activation and expansion of antigen-specific CD4+ T cells in multiple sites after SO2 immunization. Mice (CD45.2+, four per group per experiment) received CFSE-labeled p25 CD4+ T cells and were immunized with BCG and SO2, as described in Fig. 2. (A) Representative flow cytometry plots showing p25-specific CD4+ T-cell populations in the DLNs of unvaccinated (unv), BCG- or SO2-vaccinated mice as characterized by CD62L and CFSE or CD44 staining. (B–G) Total numbers of CD44hiCD62Llo p25 CD4+ T cells in the DLNs, spleen and lung at (B–D) day 7 and (E–G) day 28 after vaccination. Data are the mean+SEM and are representative of one of the two independent experiments.

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Persistence of memory T cells after vaccination with the SO2 vaccine

The data in Fig. 2 indicate that vaccination with the SO2 strain results in more pronounced expansion of CD4+ T cells compared with BCG; however, T-cell numbers contracted to similar levels by 70 days post-vaccination. When the distribution of these persisting, circulating T cells at day 70 was examined in more detail, a clear distinction in the persistence of memory T-cell subsets was observed in mice that received SO2. In the blood, most CD4+ p25 T cells in SO2- or BCG-vaccinated animals displayed a central memory T (TCM)-cell phenotype (CD44hiCD62LhiCD127hi) (Fig. 4A and data not shown). TCM cells were markedly elevated in the blood of SO2-vaccianted mice compared with animals that had received BCG (Fig. 4B). In the spleen a similar pattern was observed, with the number of TCM cells elevated in SO2-vaccinated animals compared with other groups (Fig. 4C). Only small numbers of p25 T cells were detected in the lung at day 70, and no difference was observed between groups (Fig. 4D).

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Figure 4. Persistence of memory T cells after delivery of the SO2 vaccine. Mice (CD45.2+, four per group per experiment) received p25 CD4+ T cells and were immunized with BCG and SO2, as described in Fig. 2. (A) Representative flow cytometry plots showing p25-specific CD4+ T-cell populations in the blood of unvaccinated (unv), BCG- or SO2-vaccinated mice as characterized by CD62L and CD44 staining at day 70 post-vaccination. (B–D) Total numbers of CD44hiCD62LhiCD127hi p25 CD4+ T cells in the (B) blood, (C) spleen and (D) lung at day 70 post-vaccination. Data are the mean+SEM and are representative of one of the two independent experiments. The significance of differences between groups was determined by ANOVA (**p<0.01) as shown. NS, non significant.

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We also examined the potential of Ag-reactive T cells at early (day 7) and late (day 70) stages of vaccination to secrete pro-inflammatory cytokines. At day 7 in the spleen, vaccination with SO2 led to a striking increase in the proportion (Fig. 5A) and number (Fig. 5C) of IFN-γ-TNF+ p25 CD4+ T cells compared with that seen following BCG vaccination. BCG vaccination did lead to the generation of IFN-γ+TNF+ p25 T cells; however, these cells were only detected in relatively low numbers compared with the effects of SO2 vaccination (Fig. 5C). At day 70 post-vaccination, the SO2-vaccinated cohort maintained a significant increase in the number of splenic p25-specific IFN-γ+TNF+ p25 T cells as compared with the BCG-primed cohort, with numbers in the BCG-vaccinated group near baseline levels (Fig. 5B and D). These results indicate that both the attenuated BCG and SO2 organisms are capable of inducing functional CD4+ T cells; however, the magnitude of the SO2 vaccine-induced response is significantly greater than BCG at later stages post-vaccination.

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Figure 5. Improved cytokine secretion after vaccination SO2. Mice (CD45.2+, four per group per experiment) received p25 CD4+ T cells and were immunized with BCG and SO2, as described in Fig. 2. At days 7 and 70, p25 CD4+ T cells from the DLNs, lung and spleen were re-stimulated overnight with p25 peptide and examined by intracellular IFN-γ and TNF-α staining. Representative data from the spleen at (A) day 7 and (B) day 70 are shown. (C, D) The total numbers of IFN-γ+TNF-α+ cells at (C) day 7 and (D) day 70. Data are the mean+SEM and are representative of one of the two independent experiments. The significance of differences between groups was determined by ANOVA (*p<0.05, **p<0.01) as shown. NS, non-significant.

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SO2 vaccination results in enhanced Ag-specific secondary T-cell responses to M. tuberculosis challenge

We hypothesized that the improved T-cell memory after SO2 immunization would lead to a more robust secondary antigen-specific immune response, compared with that afforded by BCG vaccination, following aerosol challenge with the M. tuberculosis pathogen. To test this, mice were first immunized sc with BCG or SO2 and after 70 days infected via aerosol challenge with H37Rv (Fig. 6A). At 14 days post-secondary challenge, the number of donor effector p25 T cells in the spleen (CD4+CD44hiCD62Llo) were significantly elevated in the SO2-vaccinated group (Fig. 6B). By contrast, the total number of splenic p25 T cells in the BCG-vaccinated groups was not significantly greater than non-vaccinated mice, indicating delayed recall of vaccine-induced memory cells to secondary infection after BCG delivery (Fig. 6B). In the lung CD4+ p25 T cells were present at lower numbers compared with the spleen, but both BCG- and SO2-vaccinated groups resulted in p25 T-cell proliferation after secondary infection compared with non-vaccinated mice (Fig. 6C). SO2 resulted in significantly increased numbers of p25-specific T cells compared with BCG-immunized animals in the lung (Fig. 6C).

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Figure 6. Enhanced antigen-specific secondary T-cell responses to M. tuberculosis challenge in SO2-vaccianted mice. (A) Schematic of the experimental procedure. Mice (CD45.2+, four per group per experiment) received p25 CD4+ T cells and were immunized with BCG and SO2, as described in Fig. 2. At day 70, mice received an aerosol infection of M. tuberculosis H37Rv and 14 days post-infection, the cellular phenotype of responding p25 cells in the spleen and lung was determined. (B–D) Total numbers of (B, C) p25 CD4+CD44hiCD62Llo cells and (D, E) p25 CD4+ IFN+TNF+ cells in the spleen and the lung are shown. Data are the mean+SEM and are representative of one of the two independent experiments. The significance of differences between groups was determined by ANOVA (*p<0.05, **p<0.01) as shown. NS, non-significant.

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The generation of polyfunctional T cells (p25 CD4+IFN-γ+TNF+) mirrored the response seen when examining total activated p25 T-cell numbers, with the SO2 strain clearly the most capable of generating polyfunctional T cells after secondary challenge with M. tuberculosis (Fig. 6D and E). BCG led to the detectable levels of p25 CD4+IFN-γ+TNF+ T cells in the spleen and lung; however, the extent of secondary expansion was clearly lower compared with the SO2-vaccinated cohort (Fig. 6D and E). Together, the data indicate that the phoP mutant confers a strong secondary CD4+ T-cell response to Ag85B upon M. tuberculosis challenge, and this response is of increased magnitude compared with that induced by the BCG vaccine.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

This study demonstrates that PhoP-inactivated M. tuberculosis results in significant expansion, differentiation and maintenance of Ag-specific CD4+ T cells after vaccination of mice. By use of an adoptive transfer system employing T-cell receptor transgenic CD4+ T cells specific for Ag85B, Ag-specific CD4+ T cells generated in response to the BCG vaccine were found to be significantly lower compared with the phoP-inactivated SO2 strain. Vaccination with SO2 resulted in improved expansion of specific CD4+ T cells early after vaccination and delayed the contraction phase of reactive T cells compared with BCG (Fig. 2). While it has previously been shown that the level of Ag appears to dictate the degree of T-cell expansion after mycobacterial infection 25, this does not appear to provide the basis for heightened SO2 immunity, as both SO2 and BCG do not differ significantly in the expression of Ag85B, as assessed by RT-PCR (Pinto et al., unpublished). These data position the SO2 vaccine candidate as a potential vaccine for human use with improved immune-stimulating properties compared with the BCG vaccine. Additionally, the work implies that specific factors associated with phoP expression may contribute to the relative inhibition of Ag-specific responses during WT M. tuberculosis infection. This is a hypothesis that we are actively investigating.

The development of T-cell memory to chronic pathogens is associated with the generation of TCM cells that form the Ag-experienced pool capable of proliferating upon secondary contact with Ag 26. In this report, we demonstrate that at extended time-points post-vaccination, mice vaccinated with the phoP-inactivated strain had increased numbers of TCM cells than their BCG-primed counterparts. This had clear functional consequences, as T cells from SO2-vaccinated mice were more capable of differentiation into functional effector cells upon challenge with M. tuberculosis (Fig. 6). This maintenance of TCM cells following SO2-vaccination contrasts with the apparent exhaustion of TCM cells in other systems, such as CD8+ T-cell immunity to viral infections 27 and in the lungs of mice chronically infected with pathogenic mycobacteria 28, 29. We considered that such an effect might be due to the attenuated phenotype of the SO2 strain, which reduces Ag persistence and thus limits Ag-driven exhaustion of Ag-specific T cells. Interestingly, the BCG vaccine was a relatively poor-inducer of TCM cells at day 70 post-vaccination. One possible explanation for the poor-functional quality of BCG-induced CD4+ T cells may relate to the cytokine pattern expressed by the cells at chronic stages of infection. At day 70, the number of IFN-γ+TNF+ p25-specifc CD4+ T cells after BCG delivery was significantly lower compared with SO2-vaccinated mice (Fig. 5). This absence of multifunctional T cells at this late stage may be indicative of a poor control of infection, given that multifunctional T cells expressing a combination of at least IFN-γ and TNF are associated with protection 30. Other promising vaccination strategies, such as the BCG-prime, Modified Vaccinia Virus Ankara Strain expressing M. tuberculosis Antigen 85A (MVA-85A) boost regimen, also results in the expansion of long-lived multifunctional T cells expressing IFN-γ, TNF and IL-2 31.

The inability of the BCG vaccine to expand p25 CD4+ T-cell numbers to high levels post-vaccination (Figs. 2 and 3) and the low levels of TCM cells and cytokine secretion generated at extended time points (Figs. 4 and 5) reinforces the hypothesis that BCG may be a poor inducer of long-lived memory T-cell responses 28, 32. It has been assumed that this was due to the high level of attenuation of the vaccine and the resulting clearance of Ag, which is required to maintain protective CD4+ T cells. CD4+ T cells appear more dependent on continued Ag exposure for their survival compared with CD8+ T cells 33. In our model, however, BCG persisted to the same extent as the SO2 strain, with higher levels of SO2 present at early timepoints in the spleen (Supporting Information Fig. 1). As Ag dose appears to be crucial for the early priming of mycobacterial T cells 25, it is possible that higher levels of SO2 may contribute to a difference in responses. Nevertheless, as BCG immunization resulted in a lower frequency of TCM cells and reduced efficacy, as compared with the SO2 strain, it is possible that this low level of TCM development may manifest as reduced protection at extended time points post-vaccination. This correlates with the perceived lack of BCG-induced protection in adults compared with children vaccinated at birth 34.

In conclusion, this report provides a mechanism for the increased immunity afforded by the phoP-inactivated mutant vaccine candidate, and suggests that virulence-associated transcriptional networks regulated by the PhoP/PhoR TCS may play an important role in the generation, persistence and recall function of T-cell immunity to immunodominant Ags of M. tuberculosis. Indeed, in preliminary investigations we have observed that generation of T-cell memory is enhanced in SO2-vaccinated animals compared with those infected with virulent M. tuberculosis, suggesting expression of PhoP by M. tuberculosis may serve to inhibit protective immune responses (Nambiar et al., unpublished). This study also provides important information on the immune correlates associated with protective immunity to infection with M. tuberculosis. Safety and improved protection against M. tuberculosis for SO2 has been demonstrated in guinea pigs and non-human primates 15, 21, 22 and an unmarked M. tuberculosis mutant lacking both phoP and fadD26 (MTBVAC) is moving forward to the good manufacturing practice (GMP) production phase and is projected to enter clinical trials in the near future (Martin et. al., personal communication). It is hoped that extended studies with MTBVAC in humans, strengthened by the immunological analysis of the mechanism of action of the phoP mutant at the cellular level, will pave the way for the introduction of a superior vaccine to protect against tuberculosis.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Bacterial strains and growth conditions

Mycobacterial strains were grown in Middlebrook 7H9 broth with 10% albumin-dextrose-catalase enrichment (BD Diagnostics, Sparks, USA). The PhoP mutant SO2 is derived from the M. tuberculosis MT103 clinical isolate 14. When required, the antibiotics kanamycin (25 μg/mL) was added to liquid and/or solid medium for recombinant mycobacterial cultures. Mycobacteria were enumerated on Middlebrook 7H11 agar supplemented with 10% oleic acid-albumin-dextrose-catalase enrichment (BD Diagnostics).

Animals

C57BL/6 mice were obtained from ARC (Perth, Australia) and p25 CD4+ TCR transgenic mice (specific for residues 240–254 of M. tuberculosis Antigen 85B (Ag85B)) 24 were obtained from Prof. K. Takatsu (University of Tokyo, Japan) and Prof. J. Ernst (New York University School of Medicine, NY, USA). This study was carried out in strict accordance with the recommendations in the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (National Health and Medical Research Council of Australia). Animal experiments were performed with the approval of the University of Sydney Animal Care and Ethics Committee (approval number K75/5-2009/3/4964).

Assessment of protective efficacy

For assessment of vaccine efficacy assays, C57BL/6 mice (five per group) were first immunized sc with 5×105 CFU BCG Pasteur or SO2, and rested for 12 weeks. Mice were then challenged with aerosol M. tuberculosis H37Rv using a Middlebrook airborne infection apparatus (Glas-Col, Terre Haute, IN, USA) as previously described with an infective dose of approximately 100 viable bacilli per lung 35.

Immunogenicity studies

Lymph nodes from p25 mice were prepared and labeled with CFSE (Molecular Probes-Invitrogen, USA) as described 35. C57BL/6 mice (CD45.2) received iv 5×105 CFSE-labeled p25 lymph node cells (CD45.1) and the next day were immunized sc with 5×105 CFU of BCG or SO2 (3–5 mice per group). At selected time-points organs were harvested, single-cell suspensions prepared and stained with anti-mouse mAb fluorochrome conjugates using pre-titrated concentrations: CD3, CD4, CD8α, CD44, CD62L, CD45.2, CD45.1 (BD Biosciences, San Jose, USA). For some experiments mice were challenged with low-dose aerosol M. tuberculosis and immunogenicity examined at 14 days post-vaccination. For intracellular cytokine staining, cells were prepared as above and stimulated overnight in the presence of the p25 peptide FQDAYNAAGGHNAVF (10 μg/mL) and Brefeldin A (10 μg/mL), then permeabilized and stained for intracellular IFN-γ and TNF using Cytofix/Cytoperm™ and Perm/Wash™ reagents (BD Biosciences). For tracking of peripheral blood-borne p25 T cells, approximately 200 μL of blood per mouse was collected, erythrocytes lysed in RBC lysis buffer (BioLegend, San Diego, USA) and cells washed in PBS containing 2% fetal bovine serum prior to immunostaining.

Flow cytometry acquisition and analysis

Samples were acquired on an LSR-II (Becton Dickinson, USA) analysed using FlowJo™ analysis software (Tree Star, Macintosh Version 8.8.4). The gating strategy involved single-cell selection using Forward Scatter-Height versus Forward Scatter-Area and Side Scatter-Height versus Side Scatter-Area exclusion of doublets, then selection of relevant populations according to surface or intracellular markers (Supporting Information Fig. 2). The absolute number of cell subsets within an organ was calculated by multiplying the population event count by the quotient of the total organ cell number (as pre-determined by haemocytometer counts) and the total number of acquired events per sample.

Statistical analysis

The significance of the differences for linear and log-transformed assays was evaluated by one-way ANOVA with pair-wise comparison of multi-grouped data sets achieved using the Bonferroni post-hoc test.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

The authors are grateful to Professor J. Ernst (New York University School of Medicine, NY) for assistance with breeding and acquisition of the p25 transgenic mice. This work was supported by NHMRC Project Grant 570768 (J. A. T., W. J. B.), the Rebecca L Cooper Medical Research Foundation (J. A. T., W. J. B.), the Spanish Ministerio de Ciencia e Innovacion (BIO2008-01561) and the European NEWTBVAC FP7 241745 Project (C.M.) J. K. N. is a recipient of an Australian Postgraduate Award and J. A. T. is a recipient of a Biomedical Career Development Award from the National Health and Medical Research Council of Australia (NHMRC). They thank Lisa Leotta for technical support and Dr Ben Roediger for helpful discussion.

Conflict of interest: The authors declare no commercial or financial conflict of interest.

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  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

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