Antigen Load Governs the Differential Priming of CD8 T Cells in Response to the Bacille Calmette Guérin Vaccine or Mycobacterium tuberculosis Infection This information is current as of September 25, 2021. Anthony A. Ryan, Jonathan K. Nambiar, Teresa M. Wozniak, Ben Roediger, Elena Shklovskaya, Warwick J. Britton, Barbara Fazekas de St. Groth and James A. Triccas J Immunol 2009; 182:7172-7177; ; doi: 10.4049/jimmunol.0801694 Downloaded from http://www.jimmunol.org/content/182/11/7172

References This article cites 40 articles, 23 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/182/11/7172.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on September 25, 2021 • Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Antigen Load Governs the Differential Priming of CD8 T Cells in Response to the Bacille Calmette Gue´rin Vaccine or Mycobacterium tuberculosis Infection1

Anthony A. Ryan,*‡ Jonathan K. Nambiar,*‡ Teresa M. Wozniak,* Ben Roediger,* Elena Shklovskaya,* Warwick J. Britton,*† Barbara Fazekas de St. Groth,* and James A. Triccas2*‡

One reason proposed for the failure of Mycobacterium bovis bacille Calmette Gue´rin (BCG) vaccination to adequately control the spread of tuberculosis is a limited ability of the vaccine to induce effective CD8 T cell responses. However, the relative capacity of the BCG vaccine and virulent Mycobacterium tuberculosis to induce activation of CD8 T cells, and the factors that govern the initial priming of these cells after mycobacterial infection, are poorly characterized. Using a TCR transgenic CD8 T cell transfer Downloaded from model, we demonstrate significant activation of Ag-specific CD8 T cells by BCG, but responses were delayed and of reduced magnitude compared with those following infection with M. tuberculosis. The degree of CD8 T cell activation was critically dependent on the level of antigenic stimulation, as modifying the infectious dose to achieve comparable numbers of BCG or M. tuberculosis in draining lymph nodes led to the same pattern of CD8 T cell responses to both strains. Factors specific to M. tuberculosis infection did not influence the priming of CD8 T cells, as codelivery of M. tuberculosis with BCG did not alter the /magnitude of BCG-induced T cell activation. Following transfer to RAG-1؊/؊ recipients, BCG and M. tuberculosis-induced CD8 http://www.jimmunol.org T cells conferred equivalent levels of protection against M. tuberculosis infection. These findings demonstrate that BCG is able to prime functional CD8 T cells, and suggest that effective delivery of Ag to sites of T cell activation by vaccines may be a key requirement for optimal CD8 T cell responses to control mycobacterial infection. The Journal of Immunology, 2009, 182: 7172–7177.

ational design of vaccines requires an understanding of M. tuberculosis (7). Therefore an understanding of the factors that the host determinants necessary for resistance to patho- facilitate the activation of CD8 T cells is important for the devel- R gens. An effective vaccine against Mycobacterium tuber- opment of effective strategies to control mycobacterial infections. culosis should mimic the natural immune response to infection, M. tuberculosis is a facultative intracellular pathogen that re- by guest on September 25, 2021 generating a large repertoire of both CD4 and CD8 T cells re- sides within phagosomes of infected cells, thereby promoting Ag sponding to protective mycobacterial Ags. Although CD4 T cells entry into the MHC-class II presentation pathway. M. tuberculosis are known to play a central role in protection against M. tubercu- infection of both humans and mice, however, leads to the gener- losis (1), there is mounting evidence that CD8 T cells are also ation of pathogen-specific CD8 T cell responses (8). Cross-pre- important in antimycobacterial immunity. Mice deficient in CD8 T sentation of mycobacterial Ags by dendritic cells (DCs)3 may play cells succumb rapidly to infection with M. tuberculosis (2), and a role, as evidenced by the demonstration that transfer of apoptotic CD8 T cells are both expanded during M. tuberculosis infection vesicles from infected macrophages to bystander DCs can stimu- and recruited to sites of bacterial burden (2, 3). Vaccines that elicit late CD8 T cell responses (9, 10). Early reports demonstrated that CD8 T cells, such as viruses encoding mycobacterial Ags, can M. tuberculosis could directly deliver Ag to the class I processing induce high levels of protective immunity (4, 5), and subunit vac- pathway, while M. bovis bacille Calmette Gue´rin (BCG) was less cines inducing CD8 T cells can protect mice deficient in CD4 T able to activate CD8 T cells in these studies (11). This suggested cells from M. tuberculosis (6). The protective effect of CD8 T cells egress into the cytoplasm from the endosomal compartment may is most apparent late in tuberculosis infection in mice, suggesting be a property of M. tuberculosis infection that promotes CD8 T this subset may be important in the control of latent infection with cell activation. Indeed, a recent report suggests that virulent M. tuberculosis and Mycobacterium leprae can translocate into the cytosol of infected DCs, possibly allowing Ags to be directly ‡Microbial Pathogenesis and Immunity Group, Discipline of Infectious Diseases, presented on MHC class I molecules, a property that was not *Centenary Institute of Cancer Medicine and Cell Biology, and †Discipline of Med- icine, University of , shared by BCG (12). Differences in CD8 T cell activation by different mycobacterial strains may also be influenced by other Received for publication May 27, 2008. Accepted for publication March 23, 2009. factors associated with infection. M. tuberculosis infection elic- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance its a substantial inflammatory response, characterized by the with 18 U.S.C. Section 1734 solely to indicate this fact. release of numerous inflammatory cytokines and chemokines 1 This work was supported by the National Health and Medical Research Council of (13). Proinflammatory cytokines play an important role in Australia. J.A.T. is supported by an NHMRC Career Development Award, and B.F. de S.G. is supported by an NHMRC Principal Research Fellowship. A.A.R., B.R., and J.K.N. are supported by Australian Postgraduate Awards and T.M.W. is the recipient of the Faculty of Medicine Postgraduate Scholarship. 3 Abbreviations used in this paper: DC, dendritic cell; BCG, M. bovis bacille Calmette 2 Address correspondence and reprint requests to Dr. James A. Triccas, Discipline of Gue´rin; DLN, draining lymph node; RD1, region of deletion 1. Infectious Diseases and Immunology, University of Sydney, Sydney, Australia. E-mail address: [email protected] Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0801694 The Journal of Immunology 7173 shaping the proliferation and maintenance of CD8 T cells in software (TreeStar). The CFSE profile of dividing cells was analyzed as models of viral and bacterial infection (reviewed in Ref. 14). It previously described (19, 20). is yet to be determined if inflammatory processes or mecha- Detection of cytokine-producing cells nisms specific to M. tuberculosis infection contribute to the initiation of CD8 T cell response to mycobacteria. Single cell suspensions were prepared, resuspended at a concentration of 3 ϫ 106 cells/ml, and either left unstimulated or stimulated overnight with To delineate the factors that influence early priming of CD8 T the OVA peptide SIINFEKL (10 ␮g/ml) and brefeldin A (10 ␮g/ml). Cell cells upon encounter of mycobacteria by the host immune re- suspensions were washed with FACS buffer (2% FCS, PBS) and surface sponse, we have compared the ability of the BCG vaccine or vir- stained with CD8, CD45.1, or CD45.2 fluorochrome conjugates (BD ulent M. tuberculosis expressing a recombinant Ag to induce the Pharmingen). Following the surface staining, cells were washed with activation of Ag-specific CD8 T cells after infection of mice. We FACS buffer and permeabilized with Cytofix/Cytoperm buffer (BD Pharm- ingen). Cells were then washed in FACS buffer and intracellular cytokines found that BCG was able to induce specific activation of CD8 T were detected with anti-IFN-␥ FITC and anti-TNF-PE conjugates (BD cells, but the response was delayed and of a lesser magnitude when Pharmingen). compared with infection with M. tuberculosis. This reduced acti- Ϫ Ϫ T cell transfer to RAG-1 / mice and survival studies vation of T cells by BCG was associated with reduced bacterial load in the draining lymph nodes (DLN) at the time of T cell C57BL/6 mice (n ϭ 5) were infected s.c. with 1 ϫ 107 CFU BCG or 5 ϫ 4 priming. Ag load was the key determinant of CD8 T activation 10 CFU M. tuberculosis. Four weeks postinfection, the DLN were har- vested and CD8 T cells were purified by positive selection using MACS after mycobacterial infection, as delivery of equivalent numbers of separation (Miltenyi Biotec). A total of 106 CD8 T cells were injected i.v. BCG and M. tuberculosis to the DLN resulted in equivalent CD8 into RAG-1Ϫ/Ϫ mice. Purified CD8 T cells from naive mice were included

T cell responses. Further, Ag-independent processes associated as controls. One day posttransfer, mice were exposed to M. tuberculosis Downloaded from with M. tuberculosis infection did not alter the priming of CD8 T H37Rv (ATCC 27294) using a Middlebrook airborne infection apparatus ϳ cells in response to BCG. BCG-induced CD8 T cells were fully (Glas-Col) with an infective dose of 100 viable bacilli per lung (21). Infected mice were monitored daily and culled if they displayed signs of ill functional, as infection with either BCG or M. tuberculosis in- health, including reduced activity, ruffling of fur, and weight loss exceed- duced CD8 T cells with an equivalent protective capacity ing 15% of the weight loss of age-matched controls. against virulent M. tuberculosis infection. These results dem- Statistical analysis onstrate that the BCG vaccine can prime a functional CD8 T http://www.jimmunol.org/ cell response, and that the level of Ag present during the initial Statistical analysis of the log10 transformed data was conducted using encounter with naive T cells dictates the magnitude of CD8 T ANOVA. Fisher’s protected least significant difference ANOVA post hoc test was used for pair-wise comparison of multigrouped data sets. For cell activation in response to mycobacterial infection. cumulative survival experiments, survival was calculated using a Kaplan- Meier nonparametric survival plot, and significance was assessed by the Materials and Methods Mantel-Cox log rank test. Differences with p Ͻ 0.05 were considered to be Bacterial strains and growth conditions significant. Mycobacterial strains were grown in Middlebrook 7H9 broth with 10% Results albumin-dextrose-catalase enrichment (Difco). When required, the antibi- Differential priming of CD8 T cells in the draining lymph nodes by guest on September 25, 2021 otics kanamycin (25 ␮g/ml) and/or Hygromycin B (50 ␮g/ml) were added to liquid and/or solid medium for recombinant mycobacterial cultures. My- following infection with BCG or M. tuberculosis cobacteria were enumerated on Middlebrook 7H11 agar supplemented with To examine in detail the priming of CD8 T cell after mycobacterial 10% oleic acid-albumin-dextrose-catalase enrichment (Difco). infection, we developed recombinant strains of M. tuberculosis or Animals BCG that secrete a truncated form of the c-terminal region of the Ϫ Ϫ OVA protein (named M.tb:OVA or BCG:OVA, respectively). Male B6.SJL/PtprCa (CD45.1), female C57BL/6 and RAG-1 / mice aged 6–8 wk were purchased from the Animal Research Centre. OT-I trans- Western blotting of cell lysates and supernatants indicated that genic mice (CD45.2) were a gift of Professor W. Heath (Walter and Elisa similar amounts of Ag were expressed by the two strains (data not Hall Institute, Melbourne, Australia) and were bred in the Centenary In- shown). Mice were adoptively transferred with CFSE-labeled OT-I stitute animal facility. Mice were maintained in specific pathogen-free con- T cells, which recognize a class I epitope of OVA (22), and sub- ditions in the Centenary Institute animal facility under ethical approval from the Sydney University Animal Ethics committee. sequently infected with equivalent doses of BCG:OVA or M.tb: OVA. At day 3 postinfection, M.tb:OVA infection had induced Construction of recombinant mycobacterial strains OT-I CD8 T cells (defined as CD45.2ϩCD45.1ϪCD8ϩ cells) to Construction of BCG secreting residues 230–359 of the OVA protein undergo rapid cell division and differentiation in the DLN, as as- (BCG:OVA) has been previously described (15). To construct M. tuber- sessed by CFSE levels and CD62L staining (Fig. 1A). In contrast, culosis secreting the same OVA fragment, plasmid pJEX87 (15) was trans- little proliferation of OT-I CD8 T cells was observed in naive mice formed into M. tuberculosis Mt103 strain (16). Expression of OVA 230–359 or those infected with BCG:OVA. In M.tb:OVA-infected mice, was confirmed by Western blotting, using the 9E10 mAb specific for the ϳ c-myc epitope tag fused to the C- terminal end of the OVA fragment (17). 9-fold more T cells were recruited into cell division on day 3 compared with BCG:OVA (Fig. 1B), and this was reflected in the CFSE labeling, adoptive transfer, and infection of mice larger proportion of cells of a CFSEint or CFSElow phenotype after Single cell suspensions of lymph nodes from male donor OT-I were pre- infection with M.tb:OVA (Fig. 1C). In addition, the total number pared by collagenase and DNase treatment, and cells labeled with 5 ␮M of OVA-specific CD8 T cells was significantly greater in the DLNs 5-(and-6)-CFSE as previously described (18). Five ϫ 105 CFSE-labeled of M.tb:OVA-infected animals at day 3 postinfection (Fig. 1D). By a OT-I LN cells were injected i.v. into B6.SJL/PtprC host mice. One day day 7 postinfection, however, the majority of OT-I CD8 T cells posttransfer of CFSE-labeled cells, mice were infected via s.c. injection low with varying doses of recombinant mycobacteria. On days 3 and 7 postin- expressed a CFSE differentiated phenotype after BCG:OVA in- fection, the activation of transferred T cells in the DLN (popliteal, inguinal, fection (Fig. 1A), although the total number of OT-I CD8 T cells para-aortic), spleen, and perfused lungs was analyzed by flow cytometry. was 10-fold lower than in the M.tb:OVA group (Fig. 1F) and more Single cell suspensions of organs were prepared, incubated with anti- cells remained CFSEhigh after BCG:OVA (Fig. 1C). M.tb:OVA Fc␥RIII/II (clone 2.4G2) and stained with Abs for the following markers using appropriate fluorchromes and concentrations (BD Pharmingen): CD8 also resulted in a greater proportion and absolute number of OTI or CD4, CD44, CD62L, CD45.1, CD45.2. Samples were acquired using a CD8 T cells expressing the effector cytokines IFN-␥ and TNF in LSR-II flow cytometer (BD Biosciences) and data analyzed using FlowJo the DLN at day 7 postinfection (Fig. 1, D and E). Evaluation of the 7174 INDUCTION OF CD8 T CELLS BY MYCOBACTERIAL INFECTION Downloaded from http://www.jimmunol.org/

FIGURE 1. Activation of OT-I CD8 T cells in the draining lymph nodes following mycobacterial infection. B6.SJL/PtprCa mice were injected i.v. by guest on September 25, 2021 with 5 ϫ 105 CFSE-labeled OT-I transgenic LN cells and 1 day later 6 infected s.c. with 10 CFU of BCG:OVA or M.tb:OVA. At 3 or 7 days FIGURE 2. Distribution and differentiation state of activated OT-I CD8 postinfection, the CFSE and CD44 profile of transferred OT-I CD8 T cells T cells induced by BCG or M. tuberculosis. OT-I T cell transfer and in- in the DLNs was determined (A). The division states are represented as fection with BCG:OVA or M.tb:OVA was performed as described in Fig. high int low Ͼ CFSE (hi), CFSE (divisions 1–5, int) or CSFE (divisions 6, 1, and the CFSE profile of OT-I CD8 T cells was determined in the spleen low). The percentage of OT-I CD8 T cells (defined as CD45.2؉CD45.1Ϫ ϩ and lung at day 7 (A). The total numbers of OT-I CD8 T cells in the spleen CD8 cells) recruited into division on day 3 was determined (B), together (B) and lung (C) is also shown. The CD44 and CD62L profiles of OT-I with the proportion of OT-I CD8 T cells in division states based on CFSE CD8 T cells was determined as depicted in D (example shows DLN at day ␥ levels (C). The IFN- /TNF cytokine profiles (D) and absolute number (E) 7) and proportion of OT-I T cells displaying a CD44highCD62Llow pheno- ␥ϩ ϩ of IFN- TNF OT-I CD8 T cells in the DLN at day 7 was also deter- type is shown for each organ (E). Data are the means Ϯ SEM for three or mined. The total number of OT-I CD8 T cells in the DLN (F) or bacterial four mice per group and are representative of two independent experi- Ϯ load (G) at days 3 and 7 is also shown. Data are the means SEM for three ments. The significances of differences between the groups were deter- .(p Ͻ 0.05; NS, not significant ,ء) to four mice per group and are representative of two independent experi- mined by ANOVA ments. The significances of differences between the groups were deter- .(p Ͻ 0.05; NS, not significant ,ء) mined by ANOVA

T cells was restricted to the DLN at this timepoint (data not number of bacteria present in the DLN after infection indicated shown). At day 7, infection with either BCG or M. tuberculosis led there was more M.tb:OVA than BCG:OVA present at both day 3 to migration of activated OT-I CD8 T cells from the DLN to the and 7 postinfection, and this difference was greatest at day 7 (Fig. spleen and the lung (Fig. 2A). Predominately CFSElow cells were 1G). Taken together, these results indicate that BCG is able to present at these sites, consistent with the circulation of these ac- induce the priming of CD8 T cells, although the response invoked tivated cells following initial T cell priming in the DLN. Greater by M. tuberculosis was more rapid and of a greater magnitude. numbers of OVA-specific CD8 T cells had circulated to the spleen and lungs after M.tb:OVA infection when compared with infection Circulation of activated CD8 T cells following BCG or with BCG:OVA (Fig. 2, B and C), most likely due to the increased M. tuberculosis infection numbers of activated OT-I CD8 cells generated in the DLN by We next compared the distribution and phenotype of activated M.tb:OVA (Fig. 1F). OT-I CD8 T cells following infection with BCG or M. tuberculo- Analysis of cell surface marker expression on OT-I CD8 T cells sis. No proliferation or recirculation of activated OT-I CD8 T cells in DLNs at day 7 postM.tb:OVA infection revealed the majority of was observed at day 3 in either the spleen or lungs of mice infected activated T cells displayed a CD44highCD62Llow phenotype, while with the recombinant strains, suggesting the activation of the CD8 the proportion of cells of this phenotype were significantly reduced The Journal of Immunology 7175

FIGURE 4. Coinfection of mice with BCG:OVA and M. tuberculosis does not influence the activation of OT-I CD8 T cells. B6.SJL/PtprCa mice were injected i.v. with 5 ϫ 105 CFSE-labeled OT-I transgenic LN cells and one day later vaccinated s.c. with 106 BCG:OVA, 106 M.tb:OVA or coin- jected with 106 BCG:OVA and 106 M. tuberculosis. At day 7 postinfection the CFSE profile of transferred OT-I CD8 T cells was determined (A), together with the total number of OT-I CD8 T cells (B). Data are the Ϯ FIGURE 3. Ag load dictates the differential priming of OT-I CD8 T means SEM for four mice per group and are representative of two cells after infection with BCG or M. tuberculosis. B6.SJL/PtprCa mice independent experiments. The significances of differences between the .(p Ͻ 0.05; NS, not significant ,ء) ϫ 5 groups were determined by ANOVA were injected i.v. with 5 10 CFSE-labeled OT-I transgenic LN cells and Downloaded from 1 day later infected s.c. with 5 ϫ 104 M.tb:OVA or 107 BCG:OVA and the bacterial load in the DLN at day 7 determined (A). From these mice the CFSE/CD62L profiles (B) and total number (C) of OT-I CD8 T cells was OT-I cells were detected after infection with the modified doses of determined. The IFN-␥/TNF cytokine profiles (D) and absolute number (E) M.tb:OVA and BCG:OVA. ϩ ϩ of IFN-␥ TNF OT-I CD8 T cells in the DLN at day 7 is also shown. Data To determine whether factors relating specifically to M. tuber- are the means Ϯ SEM for four mice and are representative of two inde- culosis infection may influence the priming of CD8 T cell re- pendent experiments. The significances of differences between the groups sponses, we codelivered wild-type M. tuberculosis (not expressing http://www.jimmunol.org/ Ͻ ء were determined by ANOVA ( , p 0.05; NS, not significant). OVA) and BCG:OVA and assessed the impact on the induction of CD8 T cell responses directed against BCG. Codelivery of M. tuberculosis with BCG:OVA did not enhance the priming of spe- D E after infection with BCG:OVA (Fig. 2, and ). More pro- cific CD8 T cells, as an equivalent response was observed in mice nounced down-regulation of CD62L on activated OT-I CD8 T infected with BCG:OVA alone (Fig. 4A). Infection with an equiv- M. tuberculosis cells after infection is consistent with an increased alent dose of M.tb:OVA led to greater proliferation of OT-I CD8 differentiation of the T cells after encounter with the pathogen. cells (Fig. 4B) and a markedly greater number of these cells in the Similarly, in the spleen and lungs of mice, OVA-specific CD8 T DLN (Fig. 4C). Together, these data suggest that the differential cells activated in response to M.tb:OVA displayed a significantly by guest on September 25, 2021 high low Ag load in the DLN after infection with M. tuberculosis or BCG, greater proportion of CD44 CD62L cells than those present rather than factors specific to M. tuberculosis infection, is the key E after BCG:OVA infection (Fig. 2 ). These results indicate that determinant accounting for the differential priming of CD8 T cell M. tuberculosis infection with either BCG or results in CD8 T cell responses in our model. circulation to peripheral sites after activation in the DLN, and this effect is greater after encounter with M. tuberculosis. CD8 T cells from BCG- or M. tuberculosis-infected mice confer comparable survival against aerosol M. tuberculosis challenge The magnitude of CD8 T cell responses is governed by Ag load in the DLN at the time of T cell priming To determine whether the equivalent priming of OT-I CD8 T cells observed following high dose BCG or low dose M. tuberculosis We considered two possible causes for the differential priming of CD8 T cell responses by BCG and M. tuberculosis. First, increased Ag load at sites of T cell activation after M. tuberculosis infection may account for the differences observed. Alternatively, factors specific to M. tuberculosis may influence the response, such as mechanisms to facilitate entry to the MHC-I Ag-processing path- way (12), or the induction of inflammatory responses that are as- sociated with M. tuberculosis infection (13). We first addressed the issue of Ag dose, as results from Fig. 1G suggested an association between CD8 T cell activation and bacterial load. We delivered differing doses of BCG:OVA and M.tb:OVA to arrive at equiva- lent numbers of bacteria present in the DLN at day 7 postinfection. FIGURE 5. Protection afforded by CD8 T cells activated by M. tuber- We determined that infection with 1 ϫ 107 CFU of BCG:OVA and culosis or BCG. C57BL/6 mice (five per group) were infected s.c. with 1 ϫ 5 ϫ 104 M.tb:OVA led to ϳ1 ϫ 103 CFU of each strain in the 107 CFU BCG or 5 ϫ 104 CFU M. tuberculosis. Four weeks postinfection, 6 DLN at day 7 (Fig. 3A). After infection of mice, which had pre- the DLN were harvested and CD8 T cells were purified and 10 CD8 T Ϫ/Ϫ viously received CFSE-labeled OT-I CD8 T cells, with these mod- cells were injected i.v. into RAG-1 recipient mice. Transfer of purified CD8 T cells from naive mice and mice receiving no T cells were included ified doses of M.tb:OVA or BCG:OVA we observed that the pro- as controls. One day posttransfer, mice were infected with aerosol M. tu- liferation of OT-I CD8 T cells in the DLN was identical between berculosis and were monitored for survival. Data are representative of two the two groups, both in terms of the division profile for each strain independent experiments. Survival was calculated using a Kaplan-Meier (Fig. 3B) and the total number of OT-I CD8 cells that were re- nonparametric survival plot, and significance was assessed by the Mantel- p Ͻ 0.05 considered to be ,ء covered from the DLN (Fig. 3C). In addition, a comparable pro- Cox log rank test with differences of ϩ ϩ portion (Fig. 3D) and number (Fig. 3E) of IFN-␥ TNF CD8 significant. 7176 INDUCTION OF CD8 T CELLS BY MYCOBACTERIAL INFECTION infection translated into comparable abilities of activated CD8 T MHC class I occurs by a proteosome and TAP-dependant process. cells to protect against virulent M. tuberculosis challenge. CD8 T In the vacuolar model, peptides generated in the phagosome are cells from M. tuberculosis- or BCG-infected mice were purified loaded on to MHC class I molecules and processing does not re- and adoptively transferred into T cell-deficient RAG-1Ϫ/Ϫ recipi- quire TAP or trafficking through the endoplasmic reticulum. Ags ents. Mice were subsequently infected via the aerosol route with such as OVA can be presented to CD8 T cells by the two pathways M. tuberculosis and survival of immunocompromised mice was (31, 32), and both cytosolic and alternative models of Ag presen- Ϫ Ϫ monitored. RAG-1 / mice that received CD8 T cells from naive tation have been shown to contribute to the recognition of myco- donors or had received no T cells survived to ϳ50 days after M. bacterial Ags (33–35). It is possible that both these pathways con- tuberculosis challenge (Fig. 5). Transfer of CD8 T cells from mice tribute to CD8 T cell activation after BCG or M. tuberculosis Ϫ Ϫ immunized with BCG or M. tuberculosis resulted in RAG-1 / - infection, and the greater Ag load available for MHC class I load- deficient mice surviving significantly longer than control mice, ing after infection with M. tuberculosis results in improved T cell with mean survival times of 125 and 130 days respectively (Fig. priming. However, BCG lacks genes contained in the region of 5). No statistically significant difference was observed between deletion 1 (RD1), which is associated with cytosolic entry by M. mice that had received BCG- or M. tuberculosis-activated CD8 T tuberculosis (12), and it is tempting to speculate that the absence cells. These results suggest that BCG is competent at generating of RD1 directs BCG-derived Ags to the vacuolar pathway of Ag protective CD8 T cell responses, and delivery of equivalent doses presentation. This is particularly relevant as the vacuolar pathway of BCG or M. tuberculosis to the DLN induced CD8 T cells with appears less efficient than the phagosome-to-cytosol pathway in an equivalent functional capacity to protect against infection. the generation of immune responses in vivo (30). It should be noted that the absence of RD1-encoded proteins does not appear to Downloaded from Discussion affect the capacity of DCs to present secreted M. tuberculosis Ags The BCG vaccine has been unable to adequately control the spread to human CD8 T cells via the cytosolic pathway (35), however of tuberculosis, however the immunological basis for the limited detailed comparison of the precise Ag processing and presentation protective efficacy of the vaccine is unknown. In this report, we mechanisms used to activate either BCG- or M. tuberculosis-re- have addressed the long-held view that BCG is a poor inducer of active CD8 T cells has yet to be performed. CD8 T cells, which may limit the protective capacity of the vac- Our results demonstrate that the presence of M. tuberculosis http://www.jimmunol.org/ cine. We provide two lines of evidence to suggest this may not be itself did not appear to influence the rate of priming of BCG:OVA- the case. First, we demonstrate that BCG can induce activation of reactive CD8 T cells (Fig. 4), despite the fact that M. tuberculosis Ag-specific CD8 T cells, with extensive proliferation of transferred induces significant inflammatory response after infection. This was OT-I CD8 T cells by 7 days postinfection (Fig. 1). This supports confirmed by an increased cellular infiltrate in the DLNs of M. recent data in humans showing generation of a robust CD8 T cell tuberculosis/BCG:OVA coinfected mice at day 7 postinfection as response in BCG-vaccinated newborns (23), and the activation of compared with those infected with BCG:OVA (data not shown). CD8 T cells in animal models of BCG infection (24, 25). Second, Inflammatory cytokines, such type-I IFNs or IL-12, play an im- we show that BCG-induced CD8 T cells can protect against M.

portant role in determining the extent of CD8 T cell expansion by guest on September 25, 2021 tuberculosis infection, and these cells are as protective as T cells during viral infection (36, 37). The magnitude of CD8 T cell ex- derived from M. tuberculosis infected animals (Fig. 5). This ob- pansion is programmed early after Ag encounter (28), and the level servation indicates that BCG and M. tuberculosis express a similar of inflammatory cytokines induced very early after M. tuberculosis repertoire of protective CD8 T cell epitopes. Further, these data suggest that the absence in BCG of M. tuberculosis-specific pro- coinfection with BCG may not be sufficient to influence T cell teins recognized by CD8 T cells may not be a major contributing priming. How inflammatory processes influence other stages of the factor to the variable efficacy of BCG in humans, despite that fact CD8 T cell response after mycobacterial infection, such as the that some M. tuberculosis-specific Ags can induce protective CD8 extent of T cell contraction and the generation of protective mem- T cell responses in animal models (26). ory T cell responses, is currently under investigation in our The initial priming of OT-I CD8 T cells after BCG infection was laboratory. delayed and of a reduced magnitude when compare with infection Protection against M. tuberculosis relies on the ability of Ag- with M. tuberculosis (Fig. 1), and this effect correlated with re- specific T cells to respond in the lung upon secondary exposure to duced Ag load in the DLN (Fig. 3). This may explain the previous Ag (38), and as such analysis of the distribution and differentiation observation of reduced generation of CD8 T cells recognizing the of activated T cells following vaccination may reveal important TB10.4 Ag of M. tuberculosis upon infection of mice with myco- correlates of the protective response. After infection of mice with bacterial strains of differing virulence (25). These data, together either BCG or M. tuberculosis activated T cells circulated rapidly with a recent study by Russell et al. (27), demonstrate that the to other sites such as the spleen and lung, however a greater num- well-documented dependence on Ag load for shaping the rate of ber of cells were detected after infection with M. tuberculosis (Fig. CD8 T cell activation during viral and acute bacterial infections 2). Indeed, at the timepoint analyzed, the number of OT-I CD8 in (28) also extends to chronic bacterial infection. There are a number the lung after BCG:OVA was not significantly greater than that of possible mechanisms that may account for the differences in seen in noninfected mice (Fig. 2C). This may reflect one limitation CD8 T cell priming observed in our study. Cross-presentation of of BCG vaccination, however it remains to be determined how mycobacterial Ags via the transfer of apoptotic vesicles from in- these low numbers of Ag-experienced cells respond to secondary fected macrophages to bystander DCs may be important (10). stimulation with Ag. Ag-specific CD8 effector cells accumulate in Macrophage apoptosis has been shown to be more pronounced the lung more rapidly than naive cells in response to pulmonary after infection with M. tuberculosis compared with BCG (9), and influenza challenge (39), and we are currently investigating this model would predict that Ag loads transferred to DCs would whether differences in the capacity of candidates tuberculosis vac- be greater after M. tuberculosis infection. Additionally, exogenous cines to invoke expansion and migration of effector T cells to the Ag can be cross-presented to CD8 T cells by at least two distinct lung correlates with protective efficacy against M. tuberculosis. intracellular pathways (29, 30). In the cytosolic model, Ag is trans- We also observed that unlike infection with M. tuberculosis, in- ferred from the phagosome to the cytoplasm and presentation via fection with BCG did not result in dissemination of the vaccine The Journal of Immunology 7177 from the site of infection to the lung (data not shown). The reten- protective efficacy of a Mycobacterium tuberculosis auxotroph vaccine. Infect. tion of residual Ag dictates the activation state and migratory pat- Immun. 67: 2867–2873. 17. Spratt, J. M., A. A. Ryan, W. J. Britton, and J. A. Triccas. 2005. Epitope-tagging tern of Ag-specific CD8 T cells after pulmonary influenza infec- vectors for the expression and detection of recombinant proteins in mycobacteria. tion (40), and it would be of interest to determine whether a Plasmid 53: 269–273. 18. Fulcher, D. A., A. B. Lyons, S. L. Korn, M. C. Cook, C. Koleda, C. Parish, requirement of effective antituberculosis vaccines is sustained de- B. Fazekas de St Groth, and A. Basten. 1996. The fate of self-reactive B cells livery of Ag at sites of M. tuberculosis infection, such as the lung. depends primarily on the degree of antigen receptor engagement and availability In conclusion, we demonstrate that immunization with the BCG of T cell help. J. Exp. Med. 183: 2313–2328. 19. Fazekas de St Groth, B., A. L. Smith, W. P. Koh, L. Girgis, M. C. Cook, and vaccine results in the activation of functional CD8 T cell re- P. Bertolino. 1999. Carboxyfluorescein diacetate succinimidyl ester and the vir- sponses, and the reduced levels of CD8 T cell activation after gin lymphocyte: a marriage made in heaven. Immunol. Cell Biol. 77: 530–538. 20. Shklovskaya, E., and B. Fazekas de St Groth. 2006. Severely impaired clonal infection with BCG compared with infection with M. tuberculosis ϩ deletion of CD4 T cells in low-dose irradiated mice: role of T cell antigen is due to inefficient delivery of Ag to the DLNs. These data suggest receptor and IL-7 receptor signals. J. Immunol. 177: 8320–8330. that rationale design of new vaccines that aim to improve upon 21. Triccas, J. A., E. Shklovskaya, J. Spratt, A. A. Ryan, U. Palendira, B. Fazekas de St Groth, and W. J. Britton. 2007. Effects of DNA- and Myco- BCG would ideally enhance the delivery of protective Ags to sites bacterium bovis BCG-based delivery of the Flt3 ligand on protective immunity to of Ag presentation, to maximize CD8 T cell responses. Mycobacterium tuberculosis. Infect. Immun. 75: 5368–5375. 22. Hogquist, K. A., S. C. Jameson, W. R. Heath, J. L. Howard, M. J. Bevan, and F. R. Carbone. 1994. T cell receptor antagonist peptides induce positive selection. Acknowledgments Cell 76: 17–27. We thank Prof. W. Heath (Walter and Elisa Hall Institute, Melbourne, 23. Murray, R. A., N. Mansoor, R. Harbacheuski, J. Soler, V. Davids, A. Soares, Australia) for providing the OT-I transgenic mice. A. Hawkridge, G. D. Hussey, H. Maecker, G. Kaplan, and W. A. Hanekom. 2006. Bacillus Calmette Guerin vaccination of human newborns induces a specific, functional CD8ϩ T cell response. J. Immunol. 177: 5647–5651. Downloaded from Disclosures 24. Dudani, R., Y. Chapdelaine, H. Faassen Hv, D. K. Smith, H. Shen, L. Krishnan, The authors have no financial conflict of interest. and S. Sad. 2002. Multiple mechanisms compensate to enhance tumor-protective CD8ϩ T cell response in the long-term despite poor CD8ϩ T cell priming ini- tially: comparison between an acute versus a chronic intracellular bacterium ex- References pressing a model antigen. J. Immunol. 168: 5737–5745. 1. Mogues, T., M. E. Goodrich, L. Ryan, R. LaCourse, and R. J. North. 2001. The 25. Billeskov, R., C. Vingsbo-Lundberg, P. Andersen, and J. Dietrich. 2007. Induc- relative importance of T cell subsets in immunity and immunopathology of air- tion of CD8 T cells against a novel epitope in TB10.4: correlation with myco-

borne Mycobacterium tuberculosis infection in mice. J. Exp. Med. 193: 271–280. bacterial virulence and the presence of a functional region of difference-1. J. Im- http://www.jimmunol.org/ 2. Flynn, J. L., M. M. Goldstein, K. J. Triebold, B. Koller, and B. R. Bloom. 1992. munol. 179: 3973–3981. Major histocompatibility complex class I-restricted T cells are required for re- 26. Wu, Y., J. S. Woodworth, D. S. Shin, S. Morris, and S. M. Behar. 2008. Vaccine- ϩ sistance to Mycobacterium tuberculosis infection. Proc. Nat. Acad. Sci. USA 89: elicited CFP10-specific CD8 T cells are sufficient to mediate protection against 12013–12017. Mycobacterium tuberculosis infection. Infect. Immun. 76: 2249–2255. 3. Feng, C. G., A. G. Bean, H. Hooi, H. Briscoe, and W. J. Britton. 1999. Increase 27. Russell, M. S., M. Iskandar, O. L. Mykytczuk, J. H. Nash, L. Krishnan, and in ␥ interferon-secreting CD8ϩ, as well as CD4ϩ, T cells in lungs following S. Sad. 2007. A reduced antigen load in vivo, rather than weak inflammation, aerosol infection with Mycobacterium tuberculosis. Infect. Immun. 67: causes a substantial delay in CD8ϩ T cell priming against Mycobacterium bovis 3242–3247. (bacillus Calmette-Guerin). J. Immunol. 179: 211–220. 4. McShane, H., A. A. Pathan, C. R. Sander, S. M. Keating, S. C. Gilbert, 28. Jabbari, A., and J. T. Harty. 2006. The generation and modulation of antigen- K. Huygen, H. A. Fletcher, and A. V. Hill. 2004. Recombinant modified vaccinia specific memory CD8 T cell responses. J. Leukocyte Biol. 80: 16–23. virus Ankara expressing antigen 85A boosts BCG-primed and naturally acquired 29. Rock, K. L., and L. Shen. 2005. Cross-presentation: underlying mechanisms and anti-mycobacterial immunity in humans. Nat. Med. 10: 1240–1244. role in immune surveillance. Immunol. Rev. 207: 166–183. by guest on September 25, 2021 5. Wang, J., L. Thorson, R. W. Stokes, M. Santosuosso, K. Huygen, A. Zganiacz, 30. Burgdorf, S., and C. Kurts. 2008. Endocytosis mechanisms and the cell biology M. Hitt, and Z. Xing. 2004. Single mucosal, but not parenteral, immunization of antigen presentation. Curr. Opin. Immunol. 20: 89–95. with recombinant adenoviral-based vaccine provides potent protection from pul- 31. Shen, L., L. J. Sigal, M. Boes, and K. L. Rock. 2004. Important role of cathepsin monary tuberculosis. J. Immunol. 173: 6357–6365. S in generating peptides for TAP-independent MHC class I crosspresentation in 6. Derrick, S. C., C. Repique, P. Snoy, A. L. Yang, and S. Morris. 2004. Immuni- vivo. Immunity 21: 155–165. zation with a DNA vaccine cocktail protects mice lacking CD4 cells against an 32. Wick, M. J., and J. D. Pfeifer. 1996. Major histocompatibility complex class I aerogenic infection with Mycobacterium tuberculosis. Infect. Immun. 72: presentation of ovalbumin peptide 257–264 from exogenous sources: protein con- 1685–1692. text influences the degree of TAP-independent presentation. Eur. J. Immunol. 26: 7. van Pinxteren, L. A., J. P. Cassidy, B. H. Smedegaard, E. M. Agger, and 2790–2799. P. Andersen. 2000. Control of latent Mycobacterium tuberculosis infection is 33. Canaday, D. H., C. Ziebold, E. H. Noss, K. A. Chervenak, C. V. Harding, and ϩ ϩ dependent on CD8 T cells. Eur. J. Immunol. 30: 3689–3698. W. H. Boom. 1999. Activation of human CD8 ␣␤TCR cells by Mycobac- 8. Woodworth, J. S., and S. M. Behar. 2006. Mycobacterium tuberculosis-specific terium tuberculosis via an alternate class I MHC antigen-processing pathway. CD8ϩ T cells and their role in immunity. Crit. Rev. Immunol. 26: 317–352. J. Immunol. 162: 372–379. 9. Schaible, U. E., F. Winau, P. A. Sieling, K. Fischer, H. L. Collins, K. Hagens, 34. Lewinsohn, D. M., M. R. Alderson, A. L. Briden, S. R. Riddell, S. G. Reed, and ϩ R. L. Modlin, V. Brinkmann, and S. H. Kaufmann. 2003. Apoptosis facilitates K. H. Grabstein. 1998. Characterization of human CD8 T cells reactive with antigen presentation to T lymphocytes through MHC-I and CD1 in tuberculosis. Mycobacterium tuberculosis-infected antigen-presenting cells. J. Exp. Med. 187: Nat. Med. 9: 1039–1046. 1633–1640. 10. Winau, F., S. Weber, S. Sad, J. de Diego, S. L. Hoops, B. Breiden, K. Sandhoff, 35. Lewinsohn, D. M., J. E. Grotzke, A. S. Heinzel, L. Zhu, P. J. Ovendale, V. Brinkmann, S. H. Kaufmann, and U. E. Schaible. 2006. Apoptotic vesicles M. Johnson, and M. R. Alderson. 2006. Secreted proteins from Mycobacterium crossprime CD8 T cells and protect against tuberculosis. Immunity 24: 105–117. tuberculosis gain access to the cytosolic MHC class-I antigen-processing path- 11. Mazzaccaro, R. J., M. Gedde, E. R. Jensen, H. M. van Santen, H. L. Ploegh, way. J. Immunol. 177: 437–442. K. L. Rock, and B. R. Bloom. 1996. Major histocompatibility class I presentation 36. Curtsinger, J. M., C. M. Johnson, and M. F. Mescher. 2003. CD8 T cell clonal of soluble antigen facilitated by Mycobacterium tuberculosis infection. Proc. Nat. expansion and development of effector function require prolonged exposure to Acad. Sci. USA 93: 11786–11791. antigen, costimulation, and signal 3 cytokine. J. Immunol. 171: 5165–5171. 12. van der Wel, N., D. Hava, D. Houben, D. Fluitsma, M. van Zon, J. Pierson, 37. Kolumam, G. A., S. Thomas, L. J. Thompson, J. Sprent, and K. Murali-Krishna. M. Brenner, and P. J. Peters. 2007. M. tuberculosis and M. leprae translocate 2005. Type I interferons act directly on CD8 T cells to allow clonal expansion from the phagolysosome to the cytosol in myeloid cells. Cell 129: 1287–1298. and memory formation in response to viral infection. J. Exp. Med. 202: 637–650. 13. Saunders, B. M., and W. J. Britton. 2007. Life and death in the granuloma: 38. Feng, C. G., W. J. Britton, U. Palendira, N. L. Groat, H. Briscoe, and A. G. Bean. immunopathology of tuberculosis. Immunol. Cell Biol. 85: 103–111. 2000. Up-regulation of VCAM-1 and differential expansion of ␤ integrin-ex- 14. Haring, J. S., V. P. Badovinac, and J. T. Harty. 2006. Inflaming the CD8ϩ T cell pressing T lymphocytes are associated with immunity to pulmonary Mycobacte- response. Immunity 25: 19–29. rium tuberculosis infection. J. Immunol. 164: 4853–4860. 15. Ryan, A. A., T. M. Wozniak, E. Shklovskaya, M. A. O’Donnell, 39. Cerwenka, A., T. M. Morgan, and R. W. Dutton. 1999. Naive, effector, and B. Fazekas de St Groth, W. J. Britton, and J. A. Triccas. 2007. Improved pro- memory CD8 T cells in protection against pulmonary influenza virus infection: tection against disseminated tuberculosis by Mycobacterium bovis BCG secreting homing properties rather than initial frequencies are crucial. J. Immunol. 163: murine GM-CSF is associated with expansion and activation of antigen present- 5535–5543. ing cells. J. Immunol. 179: 8418–8424. 40. Zammit, D. J., D. L. Turner, K. D. Klonowski, L. Lefrancois, and L. S. Cauley. 16. Jackson, M., S. W. Phalen, M. Lagranderie, D. Ensergueix, P. Chavarot, 2006. Residual antigen presentation after influenza virus infection affects CD8 T G. Marchal, D. N. McMurray, B. Gicquel, and C. Guilhot. 1999. Persistence and cell activation and migration. Immunity 24: 439–449.