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The Journal of Immunology, 2006, 177: 8456-8465.
Copyright © 2006 by The American Association of Immunologists, Inc.

Interactions between T Cells Responding to Concurrent Mycobacterial and Influenza Infections1

Dominic O. Co*,{dagger}, Laura H. Hogan*, Jozsef Karman*, Erika Heninger*, Shoua Vang*, Krisna Wells{ddagger}, Yoshihiro Kawaoka{ddagger} and Matyas Sandor2,*,{dagger}

* Department of Pathology and Laboratory Medicine, School of Medicine and Public Health, {dagger} Program in Cellular and Molecular Biology, and {ddagger} Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD4+ T cells are central in mediating granuloma formation and limiting growth and dissemination of mycobacterial infections. To determine whether T cells responding to influenza infection can interact with T cells responding to Mycobacterium bovis bacille Calmette-Guérin (BCG) infection and disrupt granuloma formation, we infected mice containing two monoclonal T cell populations specific for the model Ags pigeon cytochrome c (PCC) and hen egg lysozyme (HEL). These mice were chronically infected with PCC epitope-tagged BCG (PCC-BCG) and acutely infected with HEL epitope-tagged influenza virus (HEL-flu). In these mice, PCC-BCG infection is much more abundant in the liver than the lung, whereas HEL-flu infection is localized to the lung. We observe that both T cells have access to both inflammatory sites, but that PCC-specific T cells dominate the PCC-BCG inflammatory site in the liver, whereas HEL-specific T cells dominate the HEL-flu inflammatory site in the lung. Influenza infection, in the absence of an influenza-specific T cell response, is able to increase the activation state and IFN-{gamma} secretion of PCC-BCG-specific T cells in the granuloma. Activation of HEL-specific T cells allows them to secrete IFN-{gamma} and contribute to protection in the granuloma. Ultimately, infection with influenza has little effect on bacterial load, and bacteria do not disseminate. In summary, these data illustrate complex interactions between T cell responses to infectious agents that can affect effector responses to pathogens.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Various CD4+ T cells are critical in the long-term control of mycobacterial infections by orchestrating the formation of granulomas. The host-pathogen interface of the granuloma is where mycobacteria live within infected macrophage and where the host immune response controls bacterial growth and dissemination. CD4+ T cell-deficient mice are unable to form granulomas and eventually succumb to infection (1, 2, 3). Adoptive transfer of CD4+ T cells to CD4+ T cell-deficient mice is sufficient to restore granuloma formation and protection (4). The repertoire of granuloma-infiltrating CD4+ T cells is broad, even at the level of individual granulomas (5). However, infection of monoclonal pigeon cytochrome c (PCC)3-specific CD4+ TCR transgenic (Tg) mice (5CC7 RAG–/–) with PCC epitope-tagged recombinant Mycobacterium bovis bacille Calmette-Guérin (BCG), or PCC-BCG, demonstrates that a monoclonal T cell population is sufficient to mediate protective granuloma formation (5).

Although CD8+ T cells are the primary T cells controlling influenza infection, CD4+ T cells also contribute to protection and the establishment of T cell memory (6). Transfer of CD4+ T cell clones can reduce viral titers, and CD4+ T cells can in some circumstances exhibit direct cytolytic activity (7). CD4+ T cells are also important in providing B cell help for the production of protective Ab against influenza infection (8).

To examine how CD4+ T cell responses to BCG and influenza interact, 5CC7 RAG–/– mice expressing a monoclonal T cell population specific for PCC were infected with PCC-BCG. These mice were then adoptively transferred with spleen cells from hen egg lysozyme (HEL)-specific CD4+ 3A9 RAG–/– TCR Tg mice and infected with recombinant influenza virus expressing HEL (HEL-flu). This provided a system in which PCC and HEL Ags are localized in anatomically distinct sites.

We tested how BCG-specific and influenza-specific CD4+ T cells distribute between the two inflammatory sites and how these two T cells would interfere with each other. In addition, we examined how these interactions affect granuloma formation, dissemination, and control of BCG. We observe that activated T cells have access to both the HEL-flu inflammatory site in the lung as well as the PCC-BCG inflammatory site in the liver. However, T cells specific for local Ag dominate each site. In addition, we observe that activation of HEL-flu-specific T cells down-regulates the activation phenotype of recombinant BCG-targeted T cells in the granuloma, but that activated HEL-flu-specific T cells may compensate for this effect. Finally, we find that infection with influenza does moderately increase bacterial load, but bacterial load remains within the range of adequate control. These data demonstrate that T cells responding to an acute respiratory infection can modulate host responses to an ongoing BCG infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

B10.BR RAG1–/– mice were a gift of D. Sant’Angelo (Memorial Sloan Kettering Cancer Center, New York, NY). 5CC7 RAG2–/– mice (9) specific for PCC residues 88–104 in the context of I-Ek were purchased from Taconic Farms Emerging Models Program and bred onto the B10.BR RAG1–/– background. 3A9 mice (10) specific for HEL residues 46–61 in the context of I-Ak were purchased from The Jackson Laboratory and bred onto the B10.BR RAG1–/– background. Mice were housed at the University of Wisconsin Animal Care Unit (Madison, WI) according to the guidelines of the Institutional Animal Care and Use Committee.

Infections

Creation of kanamycin-resistant PCC-BCG from BCG Pasteur strain (Staten Serum Institut, Copenhagen, Denmark) was previously described (5). Growth and preparation of frozen stocks for infection were as described (5). For infection, 7 x 106 CFU of BCG were i.p. injected. WSN-HEL (HEL-flu) recombinant influenza virus was generated by reverse genetics from the A/WSN/33 parent strain wild-type influenza (wt flu) and produced as previously described (11). For intranasal infection, mice were lightly anesthetized with isoflurane and 2.5 x 105 PFU of either HEL-flu or wt flu was pipetted into the nose of the sleeping mouse.

Cell isolation and flow cytometry

For bronchoalveolar lavage (BAL), the trachea was exposed and cannulated with a 20-gauge catheter. Cells were isolated by washing the lungs three times with 1 ml of PBS, and the cell pellet was washed once with HBSS. Isolation of splenocytes and granuloma-infiltrating cells was performed as previously described (5) to produce single cell suspensions. A total of 106 cells were incubated for 30 min on ice with saturating concentrations of labeled Abs and 40 µg/ml unlabeled 2.4G2 mAb to block binding to Fc receptors and washed three times. For intracellular staining, cells were first surface stained as outlined, then fixed for 20 min in Cytoperm/Cytofix (BD Pharmingen). Cells were then washed in staining buffer (PBS plus 1% BSA) containing 0.1% saponin and stained with saturating concentrations of intracellular Abs for 30 min on ice. Cells were washed in staining buffer containing 0.1% saponin. Fixed samples were analyzed on a BD Biosciences FACSCalibur. Flow cytometric data was analyzed using FlowJo software version 4.6.1 (Tree Star). For assessment of intracellular IFN-{gamma}, cells were cultured with complete RPMI 1640 plus 10% FBS or complete RPMI 1640 plus 10% FBS with 5 µg/ml anti-CD3 (145-2C11) in the presence of GolgiStop (BD Pharmingen) for 6 h at 37°C in 5% CO2. Cells were harvested and washed twice with staining buffer and stained as outlined above for intracellular IFN-{gamma}. Fluorochrome-labeled Abs against CD4 (RM5-4), LFA-1 (7D4), TCR Vbeta3 (KJ25), IFN-{gamma} (XMG1.2), and activated caspase-3 (C92-605) were purchased from BD Pharmingen. Abs against TCR Vbeta3 (KJ25), I-Ak (10-2.16), Mac-1 (M1/70), and Vbeta8 (F23.1) were produced from hybridomas and labeled by standard methods with biotin, FITC, or Cy5.

Immunofluorescence

All incubations were performed at room temperature unless otherwise stated. Thick cryosections (5 µm) were cut from OCT embedded tissues and fixed for 30 min in 4% paraformaldehyde in PBS. Sections were then washed three times with PBS and outlined with a Pap pen. Sections were blocked for 30 min with 1% BSA and 40 µg/ml 2.4G2 Ab and stained for 30 min with Alexa Fluor 568-labeled KJ25 (anti-Vbeta3) and FITC- labeled F23.1 (anti-Vbeta8) in the presence of 1% BSA and 40 µg/ml 2.4G2. Unbound Ab was washed away by three washes in PBS and sections were coverslipped with Gel/Mount (Biomeda). Confocal images were acquired on a Bio-Rad MRC-1024 maintained by the W. M. Keck Laboratory for Biological Imaging (University of Wisconsin, Madison, WI).

TUNEL Staining

The 5-µm thick cryosections were cut from O.C.T. embedded liver tissue samples and fixed for 30 min in 4% paraformaldehyde in PBS, then washed three times with PBS and outlined with a Pap pen. Sections were blocked with 40 µg/ml 2.4G2 Ab in 1% BSA for 30 min and then stained for 30 min using Alexa Fluor 568-labeled KJ25 (anti-Vbeta3) and Alexa Fluor 647-labeled F23.1 (anti-Vbeta8). FITC-labeled TUNEL staining was performed following the manufacturer’s protocol (Roche Diagnostics). Confocal images were acquired on a Bio-Rad MRC-1024 maintained by the W. M. Keck Laboratory for Biological Imaging (University of Wisconsin, Madison, WI).

Histopathology

Tissue was fixed in 10% neutral-buffered formalin and processed for paraffin embedding by standard methods. Thick sections (5 µm) were stained with H&E for tissue morphology and by the Ziehl-Neelsen method to identify acid-fast bacilli (AFB). To quantitate granuloma lesion size, digital images of H&E stained sections were acquired at x400 total magnification, and the granuloma area was determined by outlining each lesion in the Scion Image program version 1.62c (NIH Image software). Quantitation of granuloma burden was performed by counting the number of liver granulomas per field at x100 total magnification. To quantitate bacterial load, the number of AFB per lesion were counted at x1000 total magnification on Ziehl-Neelsen-stained slides.

Organ load

Bacterial colony formation was determined by plating serial dilutions of liver homogenates on Middlebrook 7H10 agar plates (Difco) supplemented with 10% oleic acid-dextrose-catalase, 50 µg/ml kanamycin, and 10 µg/ml cycloheximide. Colonies were counted after 3 wk of incubation at 37°C. Data are presented as individual mouse values after a log 10 transformation.

RT-PCR

Homogenized liver and lung tissue was lysed in TRIzol (Invitrogen Life Technologies), and total RNA isolated according to manufacturer’s instructions. cDNA was prepared using MMLV reverse transcriptase (Invitrogen Life Technologies) and PCR performed using beta2-microglobulin primers (sense) 5'-TGACCGGCTTGTATGCTATC-3' and (antisense) 5'-CAGTGTGAGCCAGGATATAG-3' and influenza matrix protein primers (1) 5'-GGACTGCAGCGTAGACGCTT-3' and (2) 5'-CATCCTGTTGTATATGAGGCCCAT-3' (12) as previously described.

Databases for cross-reactivity

HEL and PCC sequences were searched against the mycobacterial sequence (<www.sanger.ac.uk/Projects/M_bovis/>) and influenza sequence (<www.flu.lanl.gov>) databases using the BLAST (Basic Local Alignment Search Tool; National Center for Biotechnology Information).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Influenza and BCG infections create two anatomically distinct inflammatory sites

The 5CC7 RAG–/– animals infected i.p. with PCC-BCG for 3 wk have a high level of bacteria in liver granulomas, and a large portion of splenic, and granuloma-infiltrating T cells are activated as measured by LFA-1 surface expression. At 5 wk, bacterial load is low and liver granulomas induced by T cells prevent dissemination of bacteria to the lung (Fig. 1B). At that stage, most T cells in both spleen and granulomas express low levels of LFA-1, but also low levels of the naive T cell marker CD45RB, suggesting an Ag-experienced but resting phenotype (data not shown). This model is an example of chronically controlled mycobacterial infection. As a model of concurrent acute infection, we chose intranasal infection with murine-adapted influenza A/WSN/33 to test whether granulomatous control of BCG will be affected. WSN replicates in the lungs, but not the liver of intranasally infected mice (13). After intranasal infection, viral titers on liver tissue demonstrate that no infectious virus can be found in liver, at a time when virions are abundant in lungs (Fig. 1A1). No viral RNA is detectable in liver tissues from the same mice (Fig. 1A2). In contrast, i.p. infection with BCG results in an infection that is largely localized to the liver, and spares the lung as judged by microscopic examination of Ziehl-Neelsen-stained liver and lung sections (Fig. 1B) and by >100-fold fewer CFU by quantitative microbiology (see Fig. 8C). Because in RAG–/– animals there is a strong dissemination of BCG to the lung after initial infection, 5CC7 T cells are clearly responsible for the protective granuloma formation observed as reported previously (5). These data establish that the two infectious agents, BCG and influenza, are restricted to two different anatomical sites in this model.


Figure 1
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  		FIGURE 1. BCG and influenza are localized to anatomically distinct inflammatory sites. A1, Liver and lung tissue homogenates from mice infected intranasally with WSN were titered on monolayers of MDCK cells. A2, RT-PCR was performed on RNA isolated from the same liver and lung tissue shown in A1 using primers specific for murine beta2-microglobulin (beta2-m) and influenza matrix protein gene (M gene). B, The 5-µm thick sections from paraffin-embedded liver (left) and lung (right) tissue of PCC-BCG-infected 5CC7 RAG–/– mice coinfected with HEL-flu were stained by the Ziehl-Neelsen method for AFB. Arrowheads, Individual AFB. Original magnification was x1000.

 

Figure 8
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  		FIGURE 8. Granuloma formation and control of bacterial growth and dissemination are not affected significantly by influenza infection. A, Top row is representative micrograph of H&E stained thin liver sections at total original magnification of x400. Arrowheads, Individual AFB. Quantitation of granuloma burden (bottom left) and granuloma size (bottom right) is also presented. B, Top row is representative micrograph of Ziehl-Neelsen-stained thin liver sections at total original magnification of x1000. Quantitation of AFB load (bottom) is presented. Error bars represent SEM. C, Liver and lung homogenates from infected animals were plated on 7H10 agar plates at different dilutions, and colonies were counted after 3 wk of incubation at 37°C. The experimental groups contained three to five mice each.

 
3A9 T cells do not cross-react with PCC-BCG

To test how CD4+ 5CC7 T cells responding to two different infectious agents interact with each other, 3A9 T cells were adoptively transferred into 5CC7 RAG–/– mice at a point when control of BCG has been established. We have three lines of evidence that 3A9 T cells do not cross-react with PCC-BCG. First, a BLAST search of the Mycobacterium tuberculosis genome for sequences encoding amino acid sequences similar to the HEL 46–61 epitope revealed no matches. Second, 3A9 T cells cocultured with PCC-BCG do not up-regulate activation markers and do not expand, whereas 5CC7 T cells activated both responses (data not shown and (5). In addition, 3A9 T cells adoptively transferred into PCC-BCG infected 5CC7 RAG–/– mice do not up-regulate the activation marker LFA-1 (data not shown), demonstrating that PCC-BCG does not activate 3A9 T cells in vivo. Also, 3A9 T cells do not expand in PCC-BCG-infected 5CC7 RAG–/– mice infected with wt flu, suggesting that they do not cross-react with influenza Ags in vivo (Fig. 2B, upper left histogram). With regard to cross-reactivity of 5CC7 T cells for influenza Ags, a BLAST search performed on the Influenza Sequence Database did not identify any significant matches. Also, although nearly all splenic 3A9 T cells up-regulate LFA-1 in response to HEL-flu infection (Fig. 2B, lower left histogram), very little increase in activation is noted on 5CC7 T cells when mice are infected with either wt flu or HEL-flu (Fig. 2A, middle and lower left histograms), indicating that 5CC7 T cells do not cross-react with wt flu or HEL-flu in vivo.


Figure 2
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  		FIGURE 2. LFA-1 expression of 5CC7 and 3A9 T cells in spleen, BAL, and liver granulomas in PCC-BCG coinfected with influenza 5CC7 RAG–/– mice. Cell preparations from mice in Fig. 3 were stained for LFA-1 and analyzed by flow cytometry. A, Histograms represent LFA-1 levels on 5CC7 T cells (CD4+ Vbeta3+ lymphocytes). B, Histograms represent LFA-1 levels on 3A9 T cells (CD4+ Vbeta8+ lymphocytes). Number shown for each histogram represents the percentage of Tg T cell population expressing high levels of LFA-1. Data are representative of three experiments each having three mice per group.

 
In vivo expansion of 3A9 T cells in response to HEL-flu infection

PCC-BCG-infected 5CC7 RAG–/– mice, which control PCC-BCG infection, were adoptively transferred with 3A9 T cells, generating a mouse with two monoclonal CD4+ T cell populations. In contrast to adoptive transfer of TCR Tg T cells into RAG–/– mice, there is little if any homeostatic expansion of 3A9 T cells in 5CC7 RAG–/– mice (Fig. 3, top). Infection with HEL-flu results in a rapid systemic expansion of 3A9 T cells in these mice (Fig. 3, bottom). At day 6, whereas 3A9 T cells only constitute ~4% of splenic CD4+ T cells in of PCC-BCG plus HEL-flu-infected 5CC7 RAG–/– mice, >80% have an activated phenotype by LFA-1 expression (Fig. 2B). Because only ~10% of splenic 5CC7 T cells in PCC-BCG-infected mice are LFA-1high, the number of activated (LFA-1high) 3A9 and 5CC7 T cells is comparable. As this pattern closely repeats in various experiments, all additional experiments were performed at day 6 postinfection with influenza.


Figure 3
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  		FIGURE 3. 3A9 T cell expansion in response to HEL-flu infection. PCC-BCG-infected 5CC7 RAG–/– mice were adoptively transferred with 3A9 RAG–/– spleen cells and infected intranasally with wt flu or HEL-flu. Spleen cells were harvested at days 5, 6, and 7 and stained with Abs against CD4 and Vbeta8. Histograms represent Vbeta8 expression on CD4+ cells in the lymphocyte gate. Each number represents the percentage of CD4+ T cells positive for Vbeta8. Data are representative of three experiments.

 
Coinfection with influenza and BCG induces inflammation in the liver and lungs of 5CC7 RAG–/– mice

PCC-BCG infection of 5CC7 RAG–/– mice induces granulomatous inflammation in the liver, and the extent and character of the inflammation, is similar to that found in mice superinfected with wt flu or HEL-flu (Fig. 4 left panels compare with Fig. 8A). The lungs of PCC-BCG-infected 5CC7 RAG–/– mice have little if any inflammation and in the sections that we examined, no granulomas were found. However, influenza infection induced strong inflammation that is even more severe in HEL-flu-infected animals reflected in the increased inflammatory infiltration and less air space visible in the sections (Fig. 4, right panels). As previous data showed that influenza infection is present in the lungs and not the liver of infected mice, and that BCG infection is confined to the liver, these data show that both agents induce inflammation that is mostly restricted to the respective organ.


Figure 4
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  		FIGURE 4. Inflammation in the lungs and liver in BCG coinfected with influenza 5CC7 RAG–/– mice. Sections from paraffin-embedded liver and lungs of 5CC7 RAG–/– mice infected with PCC-BCG alone or intranasally with either wt flu or HEL-flu were stained with H&E. Total magnification is x100.

 
T cells have access to both BCG and influenza inflammatory sites but dominate where their Ag is present

Having established two separate inflammatory sites, one containing HEL in the lung and the other PCC in the liver, we tested how the two monoclonal T cell populations specific for these different peptides would distribute between these two inflammatory sites in the context of these two infectious agents. To address this question, spleen cells, granuloma-infiltrating cells, and BAL cells were isolated and stained to determine the relative proportions of the two Tg T cell populations in the doubly infected, 3A9 adoptively transferred 5CC7 RAG–/– mice. Fig. 5A shows that HEL-specific T cells undergo a 10-fold expansion in response to infection with HEL-flu vs wt flu (5.7 ± 2.3% of CD4+ T cells are HEL-specific in HEL-flu-infected mice vs 0.5 ± 0.25 in wt flu-infected mice; mean ± SD of n = 3 experiments). HEL-specific T cells can be found both in the granuloma and in the lungs of HEL-flu-infected mice. However, HEL-specific T cells significantly outnumber PCC-specific cells in the BAL of HEL-flu-infected mice (69.0 ± 14.0% of CD4+ T cells are HEL-specific). Conversely, PCC-specific cells outnumber HEL-specific cells in the granuloma by a lower ratio (15.3 ± 12.1% of CD4+ T cells are HEL-specific).


Figure 5
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  		FIGURE 5. T cells have access to influenza and BCG inflammatory sites, but accumulate preferentially where their cognate Ag is present. 5CC7 RAG mice were infected i.p. with PCC-BCG. At 5 wk postinfection, mice were adoptively transferred with 3A9 RAG spleen cells and infected intranasally with HEL-flu or with wt flu as a control. Tissues were harvested 6 days after infection with influenza. A, Flow cytometric analysis of PCC-specific (Vbeta3+) vs HEL-specific (Vbeta8+) CD4+ lymphocytes in spleen, granuloma, and BAL is presented. Numbers represent percentage of total CD4+ lymphocytes. B, Confocal microscopy on frozen sections stained with Alexa Fluor 568-labeled KJ25 (anti-Vbeta3, red) and Alexa Fluor 488-labeled F23.1 (anti-Vbeta8, green) is shown. Total original magnification is x600. C, The absolute number of 3A9 and 5CC7 T cells recovered from lungs by BAL is presented. Data are representative of three experiments. D, The absolute number of 3A9 and 5CC7 T cells recovered from granuloma-infiltrating leukocytes is presented. Data are representative of three experiments each having three or more mice per group.

 
Confocal microscopy on frozen sections of liver and lung tissue was also used to determine the distribution of Tg T cells. Frozen liver and lung sections were stained with Abs against TCR Vbeta3 to identify PCC-specific T cells and against TCR Vbeta8 to identify HEL-specific T cells. As shown in Fig. 5B, HEL-specific cells predominate in lungs of HEL-flu-infected mice, whereas PCC-specific cells predominate in the liver granulomas. In addition, these data indicate that HEL-specific T cells are mostly localized in granulomatous lesions and are rarely found in the liver parenchyma. These data confirmed previous work that observed activated T cells have access to inflammatory sites independent of specificity. However, the difference in the ratio of 5CC7 T cells to 3A9 T cells between the BCG inflammatory site and the influenza inflammatory site suggests that other factors besides T cell activation state play a role in determining what T cells accumulate at an inflammatory site.

In the BAL, reproducibility of the cell yield allowed us to determine the absolute number of 5CC7 and 3A9 T cells infiltrating the lungs (Fig. 5C). There is clearly a 10-fold increase in the number of 3A9 T cells infiltrating the lungs of mice infected with HEL-flu as compared with wt flu-infected mice. In contrast, the absolute number of 5CC7 T cells in the lungs was similar in all groups. Thus, 5CC7 T cells have access to the lung, but influenza-induced inflammation did not increase their recruitment to that site. In contrast, recovery of granuloma-infiltrating cells is much less reproducible, restricting the interpretation of absolute numbers. Despite the fact that these numbers are questionable on technical grounds, these data clearly support experiments (Fig. 5, A and B), indicating that there are more 5CC7 T cells in the granulomas of HEL-flu-infected mice than there are 3A9 T cells.

In summary, activated T cells have access to both the influenza and BCG inflammatory sites. However, T cells dominate the site where their specific Ag is expressed.

Influenza infection increases expression of LFA-1 on 5CC7 T cells in granulomas

Having determined the distribution of 5CC7 and 3A9 T cells during concurrent infection, we examined their activation phenotype at the different inflammatory sites. 3A9 T cells in the spleen of wt flu-infected mice expressed low levels of LFA-1, whereas nearly all 3A9 T cells in the spleen of HEL-flu-infected mice were LFA-1high (Fig. 2B, left column). Similarly, most of the granuloma- and lung-infiltrating 3A9 T cells in HEL-flu-infected mice were LFA-1high (Fig. 2B, bottom row, middle and right panels), demonstrating that 3A9 T cells in HEL-flu-infected mice are acutely activated at all sites. Interestingly, although few 3A9 T cells infiltrate granulomas and BAL of wt flu-infected mice (Fig. 5A, left column), most 3A9 T cells also expressed high levels of LFA-1 (Fig. 2B, top row), suggesting selection for activated T cells. However, the significance of this finding is restricted because these represent very few cells (see Fig. 5).

As mentioned earlier, at this stage in BCG infection, the number of activated 5CC7 T cells in spleen and granuloma is decreased relative to earlier time points (Fig. 2A, upper row). The higher level of LFA-1 expression on 5CC7 T cells in the BAL probably reflects the preference of higher activated T cells for nonspecific recruitment to the lung. Interestingly, infection with wt flu more than doubles the number of LFA-1high 5CC7 T cells in both the spleen and the granuloma, probably by an Ag-independent mechanism. The mechanism by which LFA-1 is up-regulated on 5CC7 T cells in these mice is not clear, but this response correlates with higher levels of CD25 and CD69 expression as well as with an increased ability to secrete IFN-{gamma} as assayed by intracellular cytokine staining (5%, data not shown, vs 10.8%, see Fig. 6). Interestingly, in the presence of another activated T cell, this influenza-induced activation is much smaller. In the presence of a large number of acutely activated 3A9 T cells, the proportion of LFA-1high 5CC7 T cells is decreased in the granulomas and lungs of HEL-flu-infected mice relative to wt flu-infected mice. Again, this decrease in LFA-1, CD69, and CD25 is also reflected in the proportion of 5CC7 T cells secreting IFN-{gamma} in the granuloma (see Fig. 7A). These data suggest that recruitment of acutely activated nonspecific T cells into the granuloma can interfere with the activation and function of local Ag-specific T cells in both the granuloma and the lungs. These data suggest that the 5CC7 T cell compartment senses the activation state of the 3A9 T cells.


Figure 6
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  		FIGURE 6. Influenza-specific 3A9 T cells are preferentially eliminated from the granuloma by apoptosis. Frozen sections of liver (A) and lung (B) tissue from PCC-BCG-infected mice coinfected with wt flu or HEL-flu were stained for 5CC7 T cells, 3A9 T cells, and TUNEL as a marker of apoptosis. Bars, 50 µm. Arrow points to a TUNEL positive.

 

Figure 7
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  		FIGURE 7. Influenza-specific 3A9 T cells secrete IFN-{gamma} and increase macrophage activation in BCG-induced liver granulomas. A, Spleen and granuloma cells were cultured for 6 h with anti-CD3 and stained for intracellular IFN-{gamma}. Dot plots represent Vbeta3 surface staining and intracellular IFN-{gamma} staining on CD4+ gated lymphocytes. Number in each corner represents the percentage of gated cells in the indicated quadrant. Vbeta3-negative events shown in top left of each quadrant represent IFN-{gamma} production by 3A9 T cells as confirmed by Vbeta8 staining (data not shown). B, Spleen and granuloma cells were stained with Mac-1 and anti-I-Ak Ab and analyzed by flow cytometry. The number in each quadrant represents the mean fluorescence intensity on Mac-1+ cells. Data are representative of three experiments each having three mice per group.

 
Activation of 3A9 T cells by HEL-flu infection results in similar low levels of activated caspase-3 in granuloma-infiltrating 3A9 and 5CC7 T cells

One possible mechanism for the differential accumulation of 5CC7 vs 3A9 T cells in the granuloma is that local Ag may favor survival of local Ag-specific T cells. To address this issue, the level of activated caspase-3 was determined as a marker for apoptosis. We observe that similar proportions (~10%) of both 3A9 (local Ag-nonspecific) and 5CC7 (local Ag-specific) T cells in HEL-flu-infected mice express high levels of activated caspase-3 in the granuloma at that time point.

These findings are confirmed by combined TUNEL and TCR staining (Fig. 6). Note that the differential distribution of 3A9 and 5CC7 T cells in the liver and lung shown in Fig. 5 is confirmed. HEL-Flu in the presence of 3A9 T cells results in more cell death in the lungs and livers of infected mice that correlates with the heavier inflammation (Fig. 4). However, very few T cells colocalize with TUNEL staining, consistent with caspase staining described. Interestingly, although most 3A9 T cells are not apoptotic, there are more apoptotic 3A9 T cells than 5CC7 probably due to acute activation. Still, both caspase and TUNEL data suggest that differential cell death is not the major mechanism of differential distribution of 3A9 and 5CC7 T cells at the inflammatory site.

HEL-flu-activated 3A9 T cells contribute IFN-{gamma} and increase macrophage activation in BCG-induced granulomas

One of the major effector functions of CD4+ T cells in the control of mycobacterial infection is the secretion of IFN-{gamma} and subsequent macrophage activation. To test the effect of HEL-flu infection on the effector function of PCC-BCG-specific CD4+ T cells, staining for intracellular IFN-{gamma} was performed (Fig. 7A). It is clear that in HEL-flu-infected mice, a fraction of both 5CC7 (Vbeta3+) and 3A9 (Vbeta3–) T cells in the spleen can produce IFN-{gamma}. In the granuloma, infection with HEL-flu decreases the fraction of granuloma-infiltrating 5CC7 T cells secreting IFN-{gamma} relative to granuloma-infiltrating 5CC7 T cells from mice infected with wt flu. This finding is consistent with decreased LFA-1 expression observed on granuloma-infiltrating 5CC7 T cells of HEL-flu superinfected mice relative to wt flu superinfected mice in Fig. 2A and might reflect a competition of these two T cells in the granuloma. Although HEL-flu infection decreased the proportion of granuloma-infiltrating 5CC7 (Vbeta3+) T cells secreting IFN-{gamma}, the proportion of 3A9 (Vbeta3–) T cells secreting IFN-{gamma} increased. As a result, the total proportion of CD4+ T cells secreting IFN-{gamma} in granulomas of HEL-flu-infected mice is similar to or greater than the proportion in granulomas of wt flu-infected mice. These data suggest that although HEL-flu infection partially suppresses IFN-{gamma} secretion by 5CC7 T cells in the granuloma, HEL-flu activated 3A9 T cells can compensate for this and potentially contribute to protection through the secretion of IFN-{gamma}. In addition, these data suggest that in mice coinfected with HEL-flu, 3A9 T cells are the main source of IFN-{gamma} in the granuloma, despite the fact that 3A9 T cells constitute a smaller proportion of granuloma-infiltrating T cells.

Expression of MHC class II is dependent on secretion of IFN-{gamma}, and up-regulation of MHC class II is suggestive of IFN-{gamma} production in situ. As shown in Fig. 7B, HEL-flu infection did not appear to up-regulate MHC class II on splenic macrophages relative to wt flu infection. However, infection with HEL-flu moderately increased surface expression levels of MHC class II on granuloma infiltrating macrophages (Fig. 7B). Taken together, these data suggest that although IFN-{gamma} secretion by granuloma-infiltrating 5CC7 T cells in HEL-flu-infected mice is decreased, activated 3A9 T cells are able to provide enough IFN-{gamma} to up-regulate MHC class II expression on macrophages in granulomas. These finding suggest that T cells induced by different infectious agents can collaborate in activation of macrophage to express anti-mycobacterial functions.

Effect of HEL-flu infection on control of bacteria and granuloma formation

Continuous activity of CD4+ T cells is necessary for maintaining granuloma function and preventing reactivation, expansion, and dissemination of bacteria (14). We tested whether infection with influenza and the induced CD4+ T cell response would affect the effectiveness of granulomatous immune responses. It has been reported that in wt mice, infection with influenza promoted dissemination of mycobacterium from liver to lung (15) and suppression of delayed-type hypersensitivity responses (16). Fig. 8A shows representative micrographs of granulomas from 5CC7 RAG–/– mice infected with PCC-BCG alone or PCC-BCG and either wt flu or HEL-flu. No significant differences were noted in granuloma morphology, and measurements of granuloma area revealed no statistically significant differences in the average granuloma size between the different groups (Fig. 8A, bottom right), indicating that the 3A9 T cells do not increase granuloma size. However, measurement of the number of granulomas per field (granuloma burden) demonstrated a statistically significant (p = 0.002) increase in the granuloma burden of wt flu-infected mice relative to mice infected with BCG alone (Fig. 8A, bottom left), as well as a statistically significant (p = 0.001) increase in the granuloma burden of HEL-flu-infected mice relative to wt flu-infected mice or mice infected with BCG alone (p = 2.19 x 10–8). This demonstration captured our interest as these data suggest that in the virus-infected animals granulomas leak bacteria that may induce new lesions. However, the biological significance of this observation is not clear because this 2-fold difference in granuloma burden is still within the range of sampling error, even though these data are derived from two sections of seven or more animals per group. Nonetheless, these data prompted us to examine bacterial dissemination carefully as shown in Fig. 8C. Together these data suggest that granuloma size is regulated because increased granuloma cell numbers result in an increase in the number of granulomas rather than an increase in granuloma size.

To determine whether infection with HEL-flu had any effect on bacterial control, the number of AFB per lesion was counted on thin liver sections stained by the Ziehl-Neelsen method and by quantitative bacterial cultures of liver and lung homogenates (Fig. 8, B and C, respectively). Mice infected with wt flu or HEL-flu demonstrated a slight increase (p = 0.0526, 0.0342, respectively) in the number of AFB per lesion relative to mice infected with PCC-BCG alone (Fig. 8B, bottom). There was no statistically significant difference in the number of AFB per lesion between wt flu-infected and HEL-flu-infected mice. However, quantitative microbiology demonstrates a slight decrease in the mycobacterial load which is not statistically significant. Our interpretation of these data is that influenza infection has little or no effect on bacterial control. Given the observed slight increase in granuloma load, we examined dissemination of mycobacteria from granuloma and did not observe any difference. AFB were rarely observed outside of lesions or in the lungs of PCC-BCG and influenza doubly infected mice, suggesting that influenza infected 5CC7 RAG–/– mice were able to control dissemination similarly to 5CC7 RAG–/– mice infected with PCC-BCG alone. Accordingly, lung CFUs remained at the lower limit of detection using this method for all groups.

Taken together, these data suggest that in this system at the observed time point, protection against mycobacteria and pathology are similar between PCC-BCG-infected mice with or without influenza coinfection.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The granuloma represents an important host-pathogen interface at which the host restricts mycobacterial growth and dissemination and at which mycobacteria persist (17). CD4+ T cells are essential to mediate granuloma formation and prevent bacterial growth and dissemination (18, 19). Granulomatous lesions in general and mycobacterium-induced granulomas in particular contain a heterogenous T cell repertoire even at the level of individual granulomas (5, 20, 21). Many of the recruited T cells are BCG-specific as judged by reactivity to mycobacterial purified protein derivative, but many are not BCG-specific. It is not known whether these nonspecific T cells help or harm protection against mycobacterial infection. In the present work, we use a system consisting of two monoclonal T cell populations specific for two different and anatomically separated infections, influenza in the lung, and BCG in the liver, to ask three questions. First, how will the two T cells distribute between the two inflammatory sites when both T cells are specific for a local Ag in one inflammatory site but not in the other? Second, will the T cell response to influenza alter the T cell response to BCG? In other words, can two concurrent T cell responses induced at different anatomical sites interfere with each other? Finally, will influenza infection diminish protective granuloma formation and allow for reactivation of latent BCG infection? Although the dual monoclonal T cell system has inherent limitations, it allows easy identification of BCG-specific and BCG-nonspecific T cells and may reveal properties of T cells interactions that might not be apparent in the context of the normal repertoire.

The major findings are as follows: T cells activated by HEL-flu infection have access to both influenza- and BCG-induced inflammatory sites, but T cells specific for local Ag dominate each site. This differential distribution does not appear to result from differential cell death. Next, although most PCC-BCG-specific 5CC7 T cells have a resting phenotype in chronic infection, infection with wt flu increases the proportion of LFA-1high and IFN-{gamma} secreting 5CC7 T cells in the granuloma. Activation of 3A9 T cells by recombinant influenza slightly decreases the proportion of LFA-1high and IFN-{gamma}-secreting 5CC7 cells. Finally, despite infection with influenza, bacterial numbers and granuloma structure remain in the range of adequate control. These data demonstrate that secondary infections can modulate the function of recombinant BCG-targeted T cells and that activated virus-specific CD4+ T cells can home into granulomatous lesions.

Access of T cells to inflammatory sites

Activated T cells have access to inflammatory sites regardless of specificity for local Ag (22, 23, 24) and our results confirm this finding. This underlines the importance of understanding the interaction between specific and nonspecific T cells. Remarkably, although the number of activated PCC-BCG-specific and HEL-flu-specific T cells was comparable in the systemic compartment, the ratio of the two T cells differed between the liver and lung inflammatory sites with a preferential accumulation of local Ag-specific T cells. This dominance of local Ag-specific T cells can result from differential entry of T cells into inflammatory sites or differential survival, proliferation, and retention at these sites.

Differential entry of T cells to inflammatory sites is mediated by an array of chemokines and adhesion molecules. Lung homing T cells express a unique pattern of adhesion molecules and chemokine receptors (25), but liver homing chemokines and adhesion molecules have not yet been identified. In addition, T cells primed by dendritic cells in Peyer’s patches and mesenteric lymph nodes can imprint T cells to home to the intestine by expression of {alpha}4beta7 integrin (26, 27). Thus, activation of HEL-specific T cells in mediastinal lymph nodes may bias their migration back to the lung, whereas priming of PCC-specific T cells in liver draining lymph nodes may bias their return to liver granulomas. Alternatively, the influenza-induced inflammatory site may express a different set of chemokines than the BCG-induced granuloma. Several systematic studies have examined chemokines produced by purified protein derivative bead-induced granulomas (28, 29), in vivo and in vitro M. tuberculosis infection (30, 31, 32, 33), and influenza infection (34) and those studies reveal some differences. In summary, it is possible that differential expression of T cell molecules required for trafficking, differential chemokine gradients or differential sites of priming may bias T cell accumulation in inflammatory sites in an Ag-independent manner.

At the same time, the presence of Ag probably plays a central role in accumulation by its known capacity to enhance survival, proliferation, and retention of local Ag-specific T cells. Preferential accumulation of OVA-specific T cells at an OVA-containing inflammatory site was not associated with proliferation at the site as measured by short-term BrdU labeling (35). Instead, it was suggested that preferential accumulation is due to selective retention at the site mediated by CD62E. Our data also demonstrate an increase in local Ag-specific T cells in both the influenza and BCG inflammatory sites. In the same vein, cycling cells are rarely observed in granuloma-infiltrating cells by analysis of forward scatter scans, propidium iodide staining, or staining for proliferating cell nuclear Ag, which is a marker of cells in cycle (data not shown). The macrophage dominant nature of granulomas makes detection of apoptosis difficult because apoptotic bodies are rapidly phagocytosed. Nevertheless, in this study, we show that few 5CC7 or 3A9 T cells are activated caspase-3- or TUNEL-positive. A somewhat higher proportion of granuloma-infiltrating 3A9 T cells in HEL-flu-infected mice are TUNEL-positive, but these represent a small fraction of the total granuloma-infiltrating 3A9 T cells. This finding suggests that enhanced survival is not a likely mechanism for preferential accumulation of 5CC7 T cells in the granuloma. This simplified system gives us a unique model to analyze the relative involvement of Ag-dependent and Ag-independent mechanisms in accumulation of T cells in inflammatory sites.

Interactions between immune responses to concurrent infection

Concurrent infections are likely to occur in chronic infections such as tuberculosis. However, little is known about how immune responses to different pathogens interact. We report that infection with influenza in the absence of a T cell response appears to increase LFA-1 expression of granuloma-infiltrating and BAL 5CC7 T cells. This effect seems to correlate with an increase in the LFA-1 expression of systemic (splenic) 5CC7 T cells. This "bystander activation" could occur through several mechanisms. Less bacterial control would increase PCC Ag in granulomas and enhance T cell activation. However, this option was not observed. Alternatively, influenza might possess or induce expression of epitopes that are cross-reactive with 5CC7 T cells. This possibility also seems unlikely given that no sequences matching the PCC epitope were found using a BLAST search. In addition, the activation observed in the spleen of 5CC7 mice in response to wt flu infection is quite minimal when compared with activation of 3A9 T cells in response to HEL-flu infection (compare Fig. 2A, left middle histogram with Fig. 2B, left bottom histogram). Thus, putative cross-reactivity is likely to be weak. A third possibility is that influenza-induced cytokines may promote "true" bystander activation of influenza-nonspecific cells, which has been suggested previously (36). Influenza infections are known to induce type I IFN, TNF, and IL-6 among other cytokines (37), which can induce production of IFN-{gamma} by human peripheral blood T cells in the absence of Ag (38). Type I IFN induces IL-15 secretion and bystander activation of CD8+ as well as CD4+ T cells (39, 40). In addition, a combination of IL-2, IL-6, and TNF has been shown to induce T cell activation in vitro (41). Recent development of MHC tetramer technology has demonstrated that >40% of CD8+ T cells in primary influenza are specific for influenza, suggesting that bystander activation is minimal in the context of a normal T cell repertoire (42). It is clear that the effects of influenza infection on the host immune response are complex.

We also report that the arrival of HEL-flu-activated 3A9 T cells to both the influenza inflammatory site and the BCG-induced granuloma decreases the influenza-mediated activation of PCC-BCG-specific T cells in granuloma and lung, and decreased LFA-1 expression correlates with decreased secretion of IFN-{gamma}. This decreased activation may reflect competition between the two different T cell populations for resources. T cells have been shown to compete with each other for peptide-MHC complexes at the surface of APC (43, 44, 45, 46). However, this competition is greater between T cells specific for the same peptide-MHC complex than for T cells specific for different peptide-MHC complexes (47), and 3A9 and 5CC7 T cells are restricted by different class II Ags. Some have also suggested that T cells may compete for cytokines such as IL-2 (48). Recent work has also demonstrated that recently activated T cells can inhibit the activation of naive T cells in vitro (49). Further work will be needed to determine the mechanism by which this response occurs, but it is clear that ongoing T cell responses interfere with each other, and that nonspecific activated T cells can alter specific T cell responses in the granuloma.

In previous work, using a similar two T cell network, we observed that recombinant BCG-targeted T cells were activated early during infection, but this activation was down-regulated during chronic infection. In this system, we found that activated nonspecific T cells competed with activated recombinant BCG-targeted T cells during acute infection. In the present work, infection with influenza causes bystander activation of recombinant BCG-targeted T cells at the chronic stage of BCG infection. Activation of HEL-specific T cells by HEL-flu infection competes with activated recombinant BCG-targeted T cells similar to acute infection in our previous work. Taken together, these results raise the possibility that activated nonspecific T cells compete with activated recombinant BCG-targeted T cells.

Effect of secondary infection on control of BCG infection

Our results suggest complex effects of secondary infection on the T cell response to BCG infection. Influenza infection is known to cause immunosuppression (50) and in particular to suppress tuberculin delayed-type hypersensitivity (16). Influenza infection has also been shown to interfere with immunity to Listeria, another intracellular pathogen of macrophages (51). Infection with influenza has been reported to allow dissemination of i.p. infected M. tuberculosis from liver granulomas to the lung (15). In the present work, we observed little effect on bacterial control or pathology. Granuloma formation appeared normal in these mice and no bacteria could be found that had disseminated outside of lesions or to the lungs of these mice. It should be noted that a prior work followed M. tuberculosis infection for 3 wk following influenza infection, whereas our study followed the mice for 1 wk. Effects on dissemination might be more apparent at later time points. Our results are consistent with the lack of any strong association reported in the clinical literature between influenza infection and reactivation of tuberculosis.

Interestingly, we report that 3A9 T cells present in granulomas of mice infected with HEL-flu are able to produce IFN-{gamma} and up-regulate expression of class II on macrophage. This finding suggests that activated T cells may participate in qualitatively similar immune responses and cooperate with each other in providing protection against infectious agents. Infection of Mycobacterium avium-infected mice with the Th2-inducing pathogen Schistosoma mansoni was able to skew the normally Th1 type M. avium granulomas to Th2 behavior (52). However, infection with the Th2 biasing helminth Nippostrongylus brasiliensis did not interfere with control of pulmonary BCG infection despite reduced Th1 cytokine secretion by T cells in the lungs of these mice (53). This is surprising given that IFN-{gamma} and other Th1 cytokines are known to be critical in maintaining control of mycobacterial infections. It would be interesting to see whether we would observe similar results in this two T cell system, coinfecting with BCG and a Th2-inducing infectious agent.

In summary, we show that T cells responding to two separate infectious agents in anatomically distinct sites have access to both inflammatory sites, but dominate where their Ag is present. In addition, nonspecific T cells can interfere with local Ag-specific T cells in the granuloma. Finally, nonspecific T cells can contribute to protection. A two T cell system can focus attention on basic principles that may facilitate understanding of T cell interactions in multiple infections.


   Acknowledgments
 
We thank Stefan Hamm for assistance in production of WSN and WSN-HEL virus stocks, Khen Macvilay for assistance in granuloma preparations, and Shin Il Kim for technical assistance. We also thank M. Suresh for critical reading of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants R01 AI48087, R01 AI/HL46430, and R21 AI054893 (to M.S.). Back

2 Address correspondence and reprint requests to Dr. Matyas Sandor, Department of Pathology and Laboratory Medicine, Medical Sciences Center, Room 5468, University of Wisconsin, 1300 University Avenue, Madison, WI 53706. E-mail address: msandor{at}wisc.edu Back

3 Abbreviations used in this paper: PCC, pigeon cytochrome c; BCG, bacille Calmette-Guérin; HEL, hen egg lysozyme; BAL, bronchoalveolar lavage; AFB, acid-fast bacilli; Tg, transgenic; wt, wild type. Back

Received for publication June 22, 2006. Accepted for publication October 5, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Saunders, B. M., A. A. Frank, I. M. Orme, A. M. Cooper. 2002. CD4 is required for the development of a protective granulomatous response to pulmonary tuberculosis. Cell. Immunol. 216: 65-72. [Medline]
  2. Ladel, C. H., J. Hess, S. Daugelat, P. Mombaerts, S. Tonegawa, S. H. Kaufmann. 1995. Contribution of {alpha}/beta and {gamma}/{delta} T lymphocytes to immunity against Mycobacterium bovis bacillus Calmette Guérin: studies with T cell receptor-deficient mutant mice. Eur. J. Immunol. 25: 838-846. [Medline]
  3. Ladel, C. H., I. E. Flesch, J. Arnoldi, S. H. Kaufmann. 1994. Studies with MHC-deficient knock-out mice reveal impact of both MHC I- and MHC II-dependent T cell responses on Listeria monocytogenes infection. J. Immunol. 153: 3116-3122. [Abstract]
  4. Feng, C. G., W. J. Britton. 2000. CD4+ and CD8+ T cells mediate adoptive immunity to aerosol infection of Mycobacterium bovis bacillus Calmette-Guérin. J. Infect. Dis. 181: 1846-1849. [Medline]
  5. Hogan, L. H., K. MacVilay, B. Barger, D. Co, I. Malkovska, G. Fennelly, M. Sandor. 2001. Mycobacterium bovis strain bacillus Calmette-Guérin-induced liver granulomas contain a diverse TCR repertoire, but a monoclonal T cell population is sufficient for protective granuloma formation. J. Immunol. 166: 6367-6375. [Abstract/Free Full Text]
  6. Belz, G. T., D. Wodarz, G. Diaz, M. A. Nowak, P. C. Doherty. 2002. Compromised influenza virus-specific CD8+-T-cell memory in CD4+-T-cell-deficient mice. J. Virol. 76: 12388-12393. [Abstract/Free Full Text]
  7. Doherty, P. C., D. J. Topham, R. A. Tripp, R. D. Cardin, J. W. Brooks, P. G. Stevenson. 1997. Effector CD4+ and CD8+ T-cell mechanisms in the control of respiratory virus infections. Immunol. Rev. 159: 105-117. [Medline]
  8. Topham, D. J., P. C. Doherty. 1998. Clearance of an influenza A virus by CD4+ T cells is inefficient in the absence of B cells. J. Virol. 72: 882-885. [Abstract/Free Full Text]
  9. Seder, R. A., W. E. Paul, M. M. Davis, B. F. de St. Groth. 1992. The presence of interleukin-4 during in vitro priming determines the lymphokine-producing potential of CD4+ T cells from T cell receptor transgenic mice. J. Exp. Med. 176: 1091-1098. [Abstract/Free Full Text]
  10. Ho, W. Y., M. P. Cooke, C. C. Goodnow, M. M. Davis. 1994. Resting and anergic B cells are defective in CD28-dependent costimulation of naive CD4+ T cells. J. Exp. Med. 179: 1539-1549. [Abstract/Free Full Text]
  11. Walker, W. S., M. R. Castrucci, M. Y. Sangster, R. T. Carson, Y. Kawaoka. 1997. HEL-Flu: an influenza virus containing the hen egg lysozyme epitope recognized by CD4+ T cells from mice transgenic for an {alpha}beta TCR. J. Immunol. 159: 2563-2566. [Abstract]
  12. van Elden, L. J., M. Nijhuis, P. Schipper, R. Schuurman, A. M. van Loon. 2001. Simultaneous detection of influenza viruses A and B using real-time quantitative PCR. J. Clin. Microbiol. 39: 196-200. [Abstract/Free Full Text]
  13. García-Sastre, A., R. K. Durbin, H. Zheng, P. Palese, R. Gertner, D. E. Levy, J. E. Durbin. 1998. The role of interferon in influenza virus tissue tropism. J. Virol. 72: 8550-8558. [Abstract/Free Full Text]
  14. Scanga, C. A., V. P. Mohan, K. Yu, H. Joseph, K. Tanaka, J. Chan, J. L. Flynn. 2000. Depletion of CD4+ T cells causes reactivation of murine persistent tuberculosis despite continued expression of interferon {gamma} and nitric oxide synthase 2. J. Exp. Med. 192: 347-358. [Abstract/Free Full Text]
  15. Volkert, M., C. Pierce, F. L. J. Horsfall, R. J. Dubos. 1947. The enhancing effect of concurrent infection with pneumotropic viruses on pulmonary tuberculosis in mice. J. Exp. Med. 86: 202-213.
  16. Massanari, R. M.. 1979. Suppression of tuberculin hypersensitivity during influenza infection in mice. Infect. Immun. 24: 501-507. [Abstract/Free Full Text]
  17. Cosma, C. L., D. R. Sherman, L. Ramakrishnan. 2003. The secret lives of the pathogenic mycobacteria. Annu. Rev. Microbiol. 57: 641-676. [Medline]
  18. Flynn, J. L., J. Chan. 2001. Immunology of tuberculosis. Annu. Rev. Immunol. 19: 93-129. [Medline]
  19. Sandor, M., J. V. Weinstock, T. A. Wynn. 2003. Granulomas in schistosome and mycobacterial infections: a model of local immune responses. Trends Immunol. 24: 44-52. [Medline]
  20. Hogan, L. H., M. Wang, M. Suresh, D. O. Co, J. V. Weinstock, M. Sandor. 2002. CD4+ TCR repertoire heterogeneity in Schistosoma mansoni-induced granulomas. J. Immunol. 169: 6386-6393. [Abstract/Free Full Text]
  21. Mempel, M., P. Musette, B. Flageul, C. Schnopp, R. Remling, G. Gachelin, P. Kourilsky, J. Ring, D. Abeck. 2002. T-cell receptor repertoire and cytokine pattern in granuloma annulare: defining a particular type of cutaneous granulomatous inflammation. J. Invest. Dermatol. 118: 957-966. [Medline]
  22. Topham, D. J., M. R. Castrucci, F. S. Wingo, G. T. Belz, P. C. Doherty. 2001. The role of antigen in the localization of naive, acutely activated, and memory CD8+ T cells to the lung during influenza pneumonia. J. Immunol. 167: 6983-6990. [Abstract/Free Full Text]
  23. Stephens, R., D. A. Randolph, G. Huang, M. J. Holtzman, D. D. Chaplin. 2002. Antigen-nonspecific recruitment of Th2 cells to the lung as a mechanism for viral infection-induced allergic asthma. J. Immunol. 169: 5458-5467. [Abstract/Free Full Text]
  24. Ely, K. H., L. S. Cauley, A. D. Roberts, J. W. Brennan, T. Cookenham, D. L. Woodland. 2003. Nonspecific recruitment of memory CD8+ T cells to the lung airways during respiratory virus infections. J. Immunol. 170: 1423-1429. [Abstract/Free Full Text]
  25. Campbell, J. J., C. E. Brightling, F. A. Symon, S. Qin, K. E. Murphy, M. Hodge, D. P. Andrew, L. Wu, E. C. Butcher, A. J. Wardlaw. 2001. Expression of chemokine receptors by lung T cells from normal and asthmatic subjects. J. Immunol. 166: 2842-2848. [Abstract/Free Full Text]
  26. Mora, J. R., M. R. Bono, N. Manjunath, W. Weninger, L. L. Cavanagh, M. Rosemblatt, U. H. Von Andrian. 2003. Selective imprinting of gut-homing T cells by Peyer’s patch dendritic cells. Nature 424: 88-93. [Medline]
  27. Stagg, A. J., M. A. Kamm, S. C. Knight. 2002. Intestinal dendritic cells increase T cell expression of {alpha}4beta7 integrin. Eur. J. Immunol. 32: 1445-1454. [Medline]
  28. Chiu, B. C., C. M. Freeman, V. R. Stolberg, E. Komuniecki, P. M. Lincoln, S. L. Kunkel, S. W. Chensue. 2003. Cytokine-chemokine networks in experimental mycobacterial and schistosomal pulmonary granuloma formation. Am. J. Respir. Cell Mol. Biol. 29: 106-116. [Abstract/Free Full Text]
  29. Chiu, B. C., X. Z. Shang, V. R. Stolberg, E. Komuniecki, S. W. Chensue. 2002. Population analysis of CD4+ T cell chemokine receptor transcript expression during in vivo type-1 (mycobacterial) and type-2 (schistosomal) immune responses. J. Leukocyte Biol. 72: 363-372. [Abstract/Free Full Text]
  30. Rhoades, E. R., A. M. Cooper, I. M. Orme. 1995. Chemokine response in mice infected with Mycobacterium tuberculosis. Infect. Immun. 63: 3871-3877. [Abstract]
  31. Sadek, M. I., E. Sada, Z. Toossi, S. K. Schwander, E. A. Rich. 1998. Chemokines induced by infection of mononuclear phagocytes with mycobacteria and present in lung alveoli during active pulmonary tuberculosis. Am. J. Respir. Cell Mol. Biol. 19: 513-521. [Abstract/Free Full Text]
  32. Saukkonen, J. J., B. Bazydlo, M. Thomas, R. M. Strieter, J. Keane, H. Kornfeld. 2002. Beta-chemokines are induced by Mycobacterium tuberculosis and inhibit its growth. Infect. Immun. 70: 1684-1693. [Abstract/Free Full Text]
  33. Algood, H. M., J. Chan, J. L. Flynn. 2003. Chemokines and tuberculosis. Cytokine Growth Factor Rev. 14: 467-477. [Medline]
  34. Wareing, M. D., A. B. Lyon, B. Lu, C. Gerard, S. R. Sarawar. 2004. Chemokine expression during the development and resolution of a pulmonary leukocyte response to influenza A virus infection in mice. J. Leukocyte Biol. 76: 886-895. [Abstract/Free Full Text]
  35. Reinhardt, R. L., D. C. Bullard, C. T. Weaver, M. K. Jenkins. 2003. Preferential accumulation of antigen-specific effector CD4 T cells at an antigen injection site involves CD62E-dependent migration but not local proliferation. J. Exp. Med. 197: 751-762. [Abstract/Free Full Text]
  36. Tripp, R. A., S. Hou, A. McMickle, J. Houston, P. C. Doherty. 1995. Recruitment and proliferation of CD8+ T cells in respiratory virus infections. J. Immunol. 154: 6013-6021. [Abstract]
  37. Van Reeth, K.. 2000. Cytokines in the pathogenesis of influenza. Vet. Microbiol. 74: 109-116. [Medline]
  38. Sareneva, T., S. Matikainen, M. Kurimoto, I. Julkunen. 1998. Influenza A virus-induced IFN-{alpha}/beta and IL-18 synergistically enhance IFN-{gamma} gene expression in human T cells. J. Immunol. 160: 6032-6038. [Abstract/Free Full Text]
  39. Tough, D. F., S. Sun, J. Sprent. 1997. T cell stimulation in vivo by lipopolysaccharide (LPS). J. Exp. Med. 185: 2089-2094. [Abstract/Free Full Text]
  40. Sun, S., X. Zhang, D. F. Tough, J. Sprent. 1998. Type I interferon-mediated stimulation of T cells by CpG DNA. J. Exp. Med. 188: 2335-2342. [Abstract/Free Full Text]
  41. Unutmaz, D., P. Pileri, S. Abrignani. 1994. Antigen-independent activation of naive and memory resting T cells by a cytokine combination. J. Exp. Med. 180: 1159-1164. [Abstract/Free Full Text]
  42. Belz, G. T., W. Xie, P. C. Doherty. 2001. Diversity of epitope and cytokine profiles for primary and secondary influenza A virus-specific CD8+ T cell responses. J. Immunol. 166: 4627-4633. [Abstract/Free Full Text]
  43. Kedl, R. M., W. A. Rees, D. A. Hildeman, B. Schaefer, T. Mitchell, J. Kappler, P. Marrack. 2000. T cells compete for access to antigen-bearing antigen-presenting cells. J. Exp. Med. 192: 1105-1114. [Abstract/Free Full Text]
  44. Min, B., G. Foucras, M. Meier-Schellersheim, W. E. Paul. 2004. Spontaneous proliferation, a response of naive CD4 T cells determined by the diversity of the memory cell repertoire. Proc. Natl. Acad. Sci. USA 101: 3874-3879. [Abstract/Free Full Text]
  45. Ge, Q., A. Bai, B. Jones, H. N. Eisen, J. Chen. 2004. Competition for self-peptide-MHC complexes and cytokines between naive and memory CD8+ T cells expressing the same or different T cell receptors. Proc. Natl. Acad. Sci. USA 101: 3041-3046. [Abstract/Free Full Text]
  46. Hayball, J. D., B. W. Robinson, R. A. Lake. 2004. CD4+ T cells cross-compete for MHC class II-restricted peptide antigen complexes on the surface of antigen presenting cells. Immunol. Cell Biol. 82: 103-111. [Medline]
  47. Kedl, R. M., B. C. Schaefer, J. W. Kappler, P. Marrack. 2002. T cells down-modulate peptide-MHC complexes on APCs in vivo. Nat. Immunol. 3: 27-32. [Medline]
  48. Khetan, S., K. B. Sainis, S. Rath, R. Kamat. 1993. Murine CD8+ T suppressors against mycobacterial 65-kDa antigen compete for IL-2 and show lack of major histocompatibility complex-imposed restriction specificity in antigen recognition. Eur. J. Immunol. 23: 2440-2447. [Medline]
  49. Duthoit, C. T., P. Nguyen, T. L. Geiger. 2004. Antigen nonspecific suppression of T cell responses by activated stimulation-refractory CD4+ T cells. J. Immunol. 172: 2238-2246. [Abstract/Free Full Text]
  50. Kavetsky, R. E., Z. D. Savtsova, V. I. Struk, L. V. Yakimenko, Y. A. Umansky. 1977. Effect of influenza virus on the immune responsiveness of animals. Acta Virol. 21: 109-113. [Medline]
  51. Gardner, I. D.. 1981. Suppression of antibacterial immunity by infection with influenza virus. J. Infect. Dis. 144: 225-231. [Medline]
  52. Sacco, R., M. Hagen, M. Sandor, J. V. Weinstock, R. G. Lynch. 2002. Established TH1 granulomatous responses induced by active Mycobacterium avium infection switch to TH2 following challenge with Schistosoma mansoni. Clin. Immunol. 104: 274-281. [Medline]
  53. Erb, K. J., C. Trujillo, M. Fugate, H. Moll. 2002. Infection with the helminth Nippostrongylus brasiliensis does not interfere with efficient elimination of Mycobacterium bovis BCG from the lungs of mice. Clin. Diagn. Lab. Immunol. 9: 727-730. [Medline]



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