Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0607426 on August 15, 2007

Published online before print August 15, 2007
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(Journal of Leukocyte Biology. 2007;82:1221-1229.)
© 2007 by Society for Leukocyte Biology

XCL1 (lymphotactin) chemokine produced by activated CD8 T cells during the chronic stage of infection with Mycobacterium tuberculosis negatively affects production of IFN-{gamma} by CD4 T cells and participates in granuloma stability

Diane Ordway, David M. Higgins, Joaquin Sanchez-Campillo, John S. Spencer, Marcela Henao-Tamayo, Marisa Harton, Ian M. Orme and Mercedes Gonzalez Juarrero1

Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA

1 Correspondence: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado 80523-1682, USA. E-mail: malba{at}mail.colostate.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD8 T cell immune responses are known not to be essential during the initial stages of infection with Mycobacterium tuberculosis (Mtb), but their presence becomes important as the chronic infection ensues. The basis of this is still not clear. In previous studies, we showed that CD8 T cells have a distinctive positioning in the architecture of the granuloma lesion, with further changes throughout the course of the chronic infection. We have also hypothesized that further movement of lymphocytes once they are within the lung lesions could be associated with the levels of expression of the chemokine XCL1 (lymphotactin). XCL1 is produced mainly by activated CD8 T cells, and its chemotactic activity seems primarily controlling movement of CD4 and CD8 T cells. In this study, using a murine low-dose aerosol infection model coupled with antibody depletion of T cell subsets, we investigated the role of CD8 T cells in the control of the bacterial growth and in the pathogenesis of the disease in mice at early, mid, or late stages of the chronic disease state. Additionally, we also describe for the first time that during Mtb infection, activated CD8 T cells in the lungs produce XCL1 and that this chemokine is capable of controlling IFN-{gamma} production by CD4 T cells.

Key Words: TNF-{alpha} • tuberculosis • immunity • lung • murine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Infection with Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, induces both innate and acquired mechanisms of immunity, but this immunity is not capable of eliminating the bacteria completely, allowing the establishment of a chronic infection. During this chronic infection, the immune system "walls off" the bacterium within a granulomatous lesion in the lungs, which can lie dormant in humans for years. At this stage, the disease is considered latent, but can reactivate at a later time, making the patient highly infectious. At the present time, tuberculosis remains one of the most difficult infectious diseases to control and eradicate, and it is thought that ~2 billion people in the world have latent tuberculosis. It is estimated that at some point in the future, 10% of those cases will reactivate and will result in active tuberculosis [1 ].

To control Mtb growth, CD4 T cell responses are required throughout the infection, whereas only when the chronic infection is established does the role of the CD8 T cell response become more important [2 3 4 5 6 7 ]. We know that the two most important mechanisms attributed to the CD8 T cell compartment, the cytotoxic mechanism mediating direct interaction and killing of specifically targeted cells, and the activation of macrophages via Th1 cytokine production, are functional and effective during the immune response to Mtb infection [4 , 8 ]. However, whereas the cytotoxic mechanisms do not seem to be essential for control of infection [9 10 11 ], the production of Th1 cytokines by CD8 T cells appears to be critical in the later stages of the infection [12 ]. In addition, we also know that the establishment and maintenance of a chronic infection with Mtb not only requires specific mechanisms targeting directly infected cells, it also requires a well-orchestrated movement of lymphocytes needed to form granulomatous lesions in the lungs. In support of this notion, our previous studies [13 ] demonstrated an interesting and unique feature of the CD8+ T cell population within the granulomatous lesion; namely, that during early stages of granuloma formation, CD8+ T cells in the lungs are distributed in an apparent circular formation in the outer layer of the lymphocytic core of the granuloma, while the CD4+ T cells are clearly dispersed throughout the lesion. However, as the chronic infection progresses and tissue destruction and necrosis begin to develop, CD8+ T cells become more interspaced with the CD4+ T cells across the granuloma. These changes observed in the positioning of CD8+ T cells suggested modifications in the chemotactic movement directing the T cell populations as the infection progresses. In this line of thinking, we also found that Mtb-infected mice with low levels of expression of the chemokines XCL1 and MIPβ were unable to direct lymphocyte movement in the lungs toward sites of infection, lost control of the lung bacterial growth, and succumbed to infection in the later stages of the chronic infection [14 ]. XCL1, also known as lymphotactin or ATAC, is the sole member of the C subgroup of chemokines, and more importantly, is produced mainly by activated CD8 T cells (and to a lesser extent by NK and mast cells) [15 ]. The chemotactic activity of XCL1 is restricted to lymphocytes and has no effect on monocytes [16 , 17 ]. XCL1, therefore, represents a chemotactic stimuli primarily aimed for CD4 and CD8 T cells.

Thus, in this study using a low-dose aerosol infection with Mtb in mice, we investigated the importance of the role played by CD8 T cell responses in the control of the bacterial growth and pathogenesis of the disease in the early, middle, and late stages of chronic infection. Additionally, we also describe the first demonstration that activated CD8+ T cells in the lungs produce XCL1 and that high levels of expression of this chemokine in the lungs are associated with a contraction of the CD4+ IFN-{gamma}+ T cell population while not influencing the CD8 response. Thus, this identifies a new role for CD8 T cells both in the control of Th1 CD4 T cells and in maintaining granuloma stability in the latter stages of chronic Mtb infection.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Specific-pathogen-free female C57BL/6 mice, 6 to 8 wk old, were purchased from the Jackson Laboratories (Bar Harbor, ME, USA). Mice were maintained in the biosafety level 3 biohazard facility at Colorado State University and were given sterile water, mouse chow, bedding, and enrichment for the duration of the experiments. The specific pathogen-free nature of the mouse colonies at these facilities is demonstrated by testing sentinel animals. These were shown to be negative for 12 known mouse pathogens. All experimental protocols used in this study were approved by the Animal Care and Use Committee of Colorado State University.

Experimental infections
Mice were infected by low-dose aerosol challenge with Mycobacterium tuberculosis (Mtb) strain Erdman using a Glas-Col (Terre Haute, Inc., Terre Haute, IN) aerosol generator calibrated to deliver 50–100 bacteria into the lungs. To confirm success of bacterial deposition in the lungs, 24 h after challenge, the lungs from 4 mice were used to determine the number of bacteria deposited. Bacterial counts in the lung and spleen (n=5) at each time point of the study were determined as described previously [18 ]. Briefly, bacterial loads in lungs and spleen of infected mice were determined by plating serial dilutions of the organ homogenates on nutrient 7H11 agar and counting colony-forming units after 3 wk incubation at 37°C. We defined the chronic stages of infection as early, middle, or late, and we chose 35, 90, and 150 days post-challenge with Mtb as representative time points for each of the three stages of chronic infection, respectively. Lungs from mice (n=5) in the same groups were harvested for histological and cell population analysis on day 35, 90, and 150 postchallenge. The results shown in this study are representative of three experiments. Specific groups of animals were not used to determine the mortality rate in this study. However, and as expected, reactivation of the bacterial growth correlated with increased morbidity of mice, and when mice became moribund during the study, they were humanely killed.

In vivo depletion of T cells
In this study, we performed in vivo depletion of specific T cell populations using intravenous injections of recognizing specific T cell subsets. Briefly, mice were treated intravenously every 3 days (0.5 mg/dose) for a period of 3.5 weeks prior to the assay time point. The mAbs recognizing CD4 (GK 1.5, ATCC TIB 207, IgG2b) and CD8 (2.43, ATCC TIB 210, IgG2b) molecules were obtained from American Type Culture Collection (Manassas, VA, USA). Control mice received an unspecific rat IgG2 with the same dosing regimen.

Histological analysis
The accessory lung lobe from each mouse was fixed with 10% formalin in PBS. Sections from these tissues were stained using hematoxylin and eosin and examined under an Ix70 Olympus microscope. All pictures were taken with a DP70 Olympus camera.

Lung cell digestion
Single-cell suspensions were prepared as described before [19 ]. Briefly, the lungs were perfused with a solution containing PBS and heparin (50 U/ml; Sigma-Aldrich, St. Louis, MO, USA) through the pulmonary artery and aseptically removed from the pulmonary cavity, placed in media, and dissected. The dissected lung tissue was incubated with complete DMEM (cDMEM media) containing collagenase XI (0.7 mg/ml; Sigma-Aldrich) and type IV bovine pancreatic DNase (30 µg/ml; Sigma-Aldrich) for 30 min at 37°C. The digested lungs were further disrupted by gently pushing the tissue through a cell strainer (BD Biosciences, Lincoln Park, NJ, USA). Red blood cells were lysed with ACK buffer, washed and resuspended in cDMEM. Total cell numbers per lung were determined using a hemocytometer.

Flow cytometric analysis of cell surface markers
Cells suspension from each individual mouse was incubated with mAbs labeled with FITC, PE, Peridinin chlorophyll-a protein (PerCP), or Allophycocyanin (APC) at 4°C for 30 min in the dark. After washing the cells with PBS containing 0.1% sodium azide (Sigma-Aldrich) the cells were incubated with mAbs against CD4 (clone RM4-5, rat IgG2a,k), CD8 (clone 53-6.7, rat IgG2a,k), CD3 (clone 145-2C11, Armenian hamster IgG1,k) molecules and rat IgG2a, IgG2b, IgG1, mouse IgG2a,or hamster IgG were used in this study. All mAbs were purchased from BD PharMingen (San Diego, CA, USA), Serotec Inc. (Raleigh, NC, USA) or eBioscience (San Diego, CA, USA) as direct conjugates to FITC, PE, PerCP, PerCP-Cy5.5 or APC. Data acquisition and analysis for this study were done using a FACscalibur (BD Biosciences, Mountain View, CA, USA) and CellQuest software (BD Biosciences, San Jose, CA, USA), respectively. Analyses were performed with an acquisition of at least T cells 100,000 or 500,000 total events.

Intracytoplasmic cytokine staining
Measurement of intracellular IFN-{gamma} or TNF-{alpha} was conducted by preincubating lung cells with monensin (3 µM), anti-CD3 and anti-CD28 (both at 0.2 µM/106 cells) for 4 h at 37°C, 5% CO2. Thereafter, the cells were first stained for cell surface molecules, as indicated above, and they were washed and permeabilized with Perm Fix/Perm Wash (BD PharMingen). Then, cells were stained for intracellular IFN-{gamma} (XMG1.2, rat IgG1, BD PharMingen) or TNF-{alpha} (MP6-Xt22, Rat IgG1; BD PharMingen) or the corresponding isotype controls for another 30 min. Staining for the chemokine XCL1 was done by intracellular staining method. For this purpose, the cell surface staining procedure was followed by intracellular staining as explained above. XCL1 was recognized using a goat polyclonal antibody specific for the c-terminal portion of mouse XCL1 (M-20, Santa Cruz Biotechnolgy, Santa Cruz, CA, USA) followed by incubation with an anti-goat F(ab)2 antibody conjugate to FITC.

Determination of inflammatory cytokines
A cytometric bead array kit (CBA: BD Biosciences) was used to measure inflammatory cytokines in supernatants obtained from lung cell suspension incubated for 72 h at 37°C with culture filtrate protein (CFP) from Mtb at 2 µg/ml. The CBA mouse inflammatory cytokine assay procedure was performed according to manufacturer instructions. The assay was completed with duplicate samples, and results are representative of two experiments. Values are represented by the mean cytokine (pg/ml) minus the naïve media control. The beads were analyzed on a FACscalibur flow cytometer (BD Biosciences).

Intrapulmonary delivery of XCL1
Mice in late chronic infection (n=3) were anesthesized with ketamine and xylazine, and thereafter, 1 µg of recombinant mouse XCL1 (R&D Systems) in 25 µl sterile PBS was delivered by the intratracheal route. Control mice received 25 µl of sterile PBS. This procedure was repeated every 24 h for three consecutive days. Twenty four hours after the last delivery, the lungs were collected, and the accessory lobule was fixed for histology, while the left lobule was processed as a cell suspension (as indicated above) and used for cell flow cytometry analysis. The other lobules were homogenized in 1 ml of saline and used to determine the concentration of XCL1 using an ELISA kit to detect XCL1 purchased from R&D Systems. Samples were used in triplicate to determine the concentration of XCL1 per lung. Data are represented as the mean ± SD of concentration of XCL1 per group of mice.

Statistical analysis
The results presented in this publication are representative of three experiments. The data are expressed as the mean values (n = 5) from replicated samples from duplicate or triplicate assays. A parametric method, the Student’s t test was used to assess statistical significance between groups of data.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Degree of Mtb growth reactivation after CD4 or CD8 cell depletion in mice during the early, middle, or late stages of chronic infection
First, we determined the degree of increased bacterial growth in the lungs after depletion of CD4 or CD8 cells. For this purpose, mice during the early, middle, or late stages of chronic Mtb infection were treated as indicated above with a regimen of intravenous injections of rat IgG (control mice) or with mAb recognizing the CD4 and CD8 molecules. As shown in Fig. 1 , the bacterial burden in the lungs and spleens of CD4 cell-depleted mice increased by 0.5 to 1.5 log, regardless of the state of the disease. After CD8 cell depletion, only mice treated during the middle or late stages of chronic infection showed such increases in bacterial burden (Fig. 1) . These data confirm previous reports showing that the bacterial growth control in the lung and spleen requires CD4 cells throughout the chronic infection, whereas the requirement for CD8 cells only becomes evident as the chronic infection progresses [3 , 20 , 21 ].


Figure 1
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  		Figure 1. Susceptibility to Mtb growth reactivation after CD4+ or CD8+ cell depletion. Bacterial load in the lungs and spleens of control (solid bars), CD4 (gray bars), or CD8 (white bars) depleted mice infected by a low dose aerosol challenge with Mtb bacilli. Mice at 35, 90, and 150 days postchallenge with Mtb were used as representative time points of early, middle, or late stages of chronic infection respectively. Then, groups of mice in the early, mid- or late chronic infection were treated as indicated in Material and Methods with a regimen of intravenous injections of rat IgG (control mice), or recognizing the CD4 and CD8 molecules (CD4 or CD8c cell-depleted mice, respectively). Results are expressed as the average (n=5) of the bacterial load in each group expressed as Log10 CFU ± SE.

 
Antibody treatment resulted in almost total depletion of CD4+ and CD8+ T cells in the lungs and spleens of mice chronically infected with Mtb
One major concern during the design of this study was whether the antibody treatments were capable of depleting the CD8+ and CD4+ T cells already located in the lung granuloma lesion. For this reason, we included immunohistochemical staining for CD8 and CD4 cells for each group of mice after each treatment. Control mice showed numerous CD4 lymphocytes scattered within the granuloma lesions and CD8 lymphocytes in the periphery of the granuloma. When the CD8 or CD4 cells were depleted in each group of mice, very few CD4 or CD8 lymphocytes could be detected in the granulomas (data not shown).

The overall efficacy of the antibody treatment in the depletion of T cell populations from the lungs and spleens of mice in the early, middle, or late stage of chronic infection was also quantified by flow cytometry. The numbers of total CD4, CD8 T cells in the lungs of control, or depleted mice in the early stage of chronic infection are shown in Fig. 2A . These results demonstrated that treatment with antibodies recognizing CD4 molecules resulted in a 94% depletion of CD4 T cells and a slight increase in the numbers of CD8 T cells when compared with the number of the same cells in the lungs of control mice. On the other hand, treatment with antibodies recognizing the CD8 molecules resulted in 90% depletion of CD8 T cells in the lungs and a slight decrease in the CD4 T cells when compared with the number of the same cells in the lungs of control mice. The efficacy of the treatment for depletion of CD4 and CD8 T cells in the spleen of these mice was also evaluated. The spleens showed 92% and 90% depletion of CD4+ and CD8+ T cells, respectively, when compared with the same populations in spleens of control mice (data not shown). All together, these results suggest that in the lungs of mice after treatment remained ~6% and 10% of CD4 and CD8 T cells, respectively, and that very few of these cells appear to be sequestrated within the granuloma lesions.


Figure 2
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  		Figure 2. Antibody treatment resulted in almost total depletion of CD4+ and CD8+ T cells in the lungs of mice chronically infected with Mtb. (A) Representative flow cytometric dot plots from cell suspension samples of lung cells from a representative mouse in each group. First, cells were gated on FSClow vs. SSC low, followed by a gate in CD3 cells. Thereafter, the plots were gated for CD4+, CD8+ cells in samples from the lungs of control mice, CD4- and CD8-depleted mice. The number within each quadrant represents the percentage of positive cells. (B) Total numbers of CD3/CD4; CD3/CD8 in lung cell suspensions obtained from control (black bars), CD4- (gray bars), and CD8- (white bars) depleted mice in the early, middle, or late stage of infection with Mtb. Results are expressed as the average (n=5) of the total numbers in each group expressed as mean ± SE.

 
Additionally, we compared the effect of depletion of CD4 or CD8 cells during the early chronic infection with the effect of depletion of these cells during the mid- and late chronic infection (Fig. 2B) . When compared with the numbers of T cell populations in the lungs from control mice groups, depletion of CD4 T cells, at any stage of chronic infection, resulted in almost complete removal of CD4 T cells from the lungs, but the number of CD8 T cells were increased. However, depletion of CD8 T cells in the early chronic stage of infection resulted in almost complete removal of CD8 T cells in the lungs, along with also a slightly decreased number of CD4 T cells. If the CD8 T cells were depleted during the mid- or late chronic stages of infection, lack of CD8 T cells resulted in increased numbers of CD4 T cells in these groups of mice. From these results, it was clear that depending on the stage of chronic infection, removal of CD8 T cells have different effect on the influx of other T cells populations. However, removal of CD4 T cells at any stage of chronic infection always resulted in an influx of other T cell populations into the lungs.

We also evaluated the effect of CD4 and CD8 cell depletion in the number of alveolar macrophages and dendritic cells found in the lungs of the same mice. The data obtained showed that the depletion of CD4 and CD8 cells during the chronic stages of infection does not have a drastic effect in the alveolar macrophages or dendritic cell numbers in the lungs. Only when CD4 or CD8 cells were depleted during the late chronic infection was the number of alveolar macrophages dropped by half of the number found in the control mice, with no effect on the number of dendritic cells (data not shown).

Effect of depletion of CD4 or CD8 cells on the histopathology of the granulomatous lesions
Control of bacterial growth in the lungs during chronic infection is dependent on the integrity of the granuloma, so we analyzed the effect of depletion of CD4 or CD8 cells on granuloma lesion development (Fig. 3 ). The lungs of control mice in the early stage of chronic infection showed small granulomas consisting of tight aggregates of lymphocytes with the presence of scattered foamy macrophages. As the chronic infection entered into the mid and late stages, the granulomas became larger with more poorly defined margins. Depletion of CD4 cells at any stage of the chronic infection resulted in changes in the size and organization of the granulomatous lesions. Lungs of depleted mice had increased cell infiltration and thickening of the parenchymal walls. The granulomas had disorganized lymphocytic cores and large numbers of foamy macrophages. The airspace in the lungs of these mice was compromised by the presence of large coalescing granulomas, dense parenchymal walls, and prominent necrosis. In contrast, the effect of CD8 cell depletion in the lungs had a different outcome. Following depletion at an early stage of chronic infection, the granulomas were smaller compared with granulomas in the lungs of isotype control mice. These granulomas had tight aggregates of lymphocytes, a minimal presence of foamy macrophages, and evidence of little parenchymal wall thickening. However, when the CD8 cells were depleted in mice during the middle or late stages of the chronic infection, the changes in the lung histology were similar to that seen in the animals depleted of CD4 cells.


Figure 3
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  		Figure 3. Depletion of CD4 or CD8 cells in mice in early, middle, or late stages of chronic infection affected the histological makeup of the granuloma lesions. The lung sections from mice in the early, middle, or late stage of chronic infection with Mtb [left to right panels, respectively] that were treated with rat IgG (control) or recognizing CD4 or CD8 molecules (CD4- or CD8-depleted mice, respectively) (top to bottom panels, respectively) were stained with hematoxylin and eosin. Total magnification = x4.

 
Effects of depletion of CD4 or CD8 T cells on the overall expression of Th1 cytokines in the lungs
CD4 and CD8 T cells are the main sources of Th1 cytokines during Mtb infection [22 23 24 25 ]. Thus, we determined the ability of cellular suspensions obtained from the lungs of each group of mice to produce Th1 cytokines after stimulation with mycobacterial antigens. In addition, we also quantified the ability of CD4 or CD8 T cells from each group of mice to produce IFN-{gamma} and TNF-{alpha}.

Levels of IFN-{gamma} and TNF-{alpha} in supernatants obtained from cultured lung cells are shown in Fig. 4A . Control mice produced high levels of IFN-{gamma} and TNF-{alpha} in the early stage of chronic infection, but the expression of these cytokines dropped substantially as the infection ensued. Depletion of CD4 T cells in the early, mid- or late chronic stages of the infection resulted in a 10-fold reduction of IFN-{gamma} and TNF-{alpha} secretion. However, depletion of CD8 T cells in mice from early chronic infection resulted in a slight decrease of IFN-{gamma}, whereas if depletion was done in the mid-chronic infection, there was a 2- to 3-fold increase in the expression of IFN-{gamma}. In contrast, the content of TNF-{alpha} in the same cultures was similar to control mice. Finally, when CD4 or CD8 cells were depleted in mice in the late stage of the infection, all groups of mice had lower expression for both cytokines than similar control mice. The results obtained from CD4- and CD8-depleted groups indicated that the major contribution to the Th1 (IFN-{gamma} and TNF-{alpha}) response during the early stage of the chronic infection was due to the CD4 T cell population. However, depletion of CD8 cells in mice during mid-chronic infection resulted in increased expression of IFN-{gamma} but not of TNF-{alpha}.


Figure 4
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  		Figure 4. CD4 or CD8 T cells at any stage of chronic infection affected the overall expression of Th1 cytokines in the lungs. The production of IFN-{gamma} or TNF-{alpha} by either CD4 or CD8 T cells at each stage of chronic infection in control (black bars), CD4 (gray bars), or CD8 (white bars) depleted mice during early, middle, or late chronic infection was demonstrated by CBA assay and intracellular staining. Both sets of results were analyzed using flow cytometric analysis. (A) Graphs represent the contents of IFN-{gamma} and TNF-{alpha} in supernatants obtained from cultures of lung cell suspensions incubated for 72 h at 37°C with culture filtrate protein (CFP) from Mtb at 2 µg/ml and tested by CBA assay. (B) Changes in the percentage of CD4 or CD8 T cells producing IFN-{gamma} or TNF-{alpha} cytokines at each stage of the chronic infection.

 
Finally, the results presented above followed the same trend when we determined the total number of CD4 and CD8 T cells producing Th1 cytokines in each group of mice. Figure 4B shows the specific contribution of CD4 and CD8 cells to the production of IFN-{gamma} or TNF-{alpha}. We observed that in the lungs of control mice, the percentage of CD4 and CD8 T cells producing IFN-{gamma} or TNF-{alpha} dropped by half as the infection progressed. Depletion of CD4 cells in mice at any stage of the chronic infection resulted in a decreased percentage of CD8 T cells producing either IFN-{gamma} or TNF-{alpha} in the lungs. However, while depletion of CD8T cells was higher in the mid-chronic stage of infection compared with control, when CD8 cell depletion was done in the late chronic infection, despite having a higher number of total CD4 T cells (Fig. 2) , the percentage of CD4+ T cells producing IFN-{gamma} in the lungs decreased drastically.

Increased production of XCL1 by activated CD8+ T cells during the chronic infection
XCL1 is produced mainly by activated CD8 T cells and to a lesser extent by NK and mast cells [15 , 26 ], and its activity is associated with inducing lymphocyte movement. Therefore, we investigated whether activated CD8 T cells over the course of the chronic infection were able to produce this chemokine. Figure 5 shows that activated CD8 T cells obtained from the lungs of mice throughout the chronic infection (Fig. 5B) were able to produce XCL1. CD8 T cell activation was demonstrated by high levels of expression of the CD44 marker. CD4 T cells were unable to produce XCL1, and naïve mice did not express XCL1 (data not shown). The number of activated CD8+ T cells producing XCL1 increased over the course of the chronic infection reaching as many as 40% of total lung CD8 T cells during the later stages of the infection. Depletion of CD4+ T cells did not affect the numbers of activated CD8+ T cells producing XCL1.


Figure 5
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  		Figure 5. (A) Increased production of XCL1 by activated CD8+ T cells as the chronic infection progressed. Expression of XCL1 by activated CD8 T cells during the early, mid- or late chronic infection were determined by intracellular staining using specific antibodies recognizing the XCL1 chemokine. Lymphocytes were gated according to their characteristic FSC/SSC and thereafter gated in the CD8 cells. Activated CD8+ T cells were demonstrated by high levels of expression of the CD44 marker. (B) Total number of activated CD8 cells producing XCL1 is defined as CD8 +/CD44 high / XCL1+ in lung cell suspensions from control ({diamondsuit}), CD4 ({blacksquare}), and CD8 ({blacktriangleup}) mice.

 
High levels of pulmonary XCL1 was associated with a decrease in the percentage of CD4T cells producing IFN-{gamma}, but did not affect the percentage of CD8T cells producing IFN-{gamma}
Finally, we wanted to determine the biological effect of XCL1 in the lungs of mice infected with Mtb. For this purpose, we performed intrapulmonary delivery of recombinant murine XCL1 to mice in on day 150 of the infection. After three days of treatment with XCL1, we observed that while high levels of XCL1 in the lung did not affect the total numbers of CD4 or CD8 T cells, the percentages of CD4 cells producing IFN-{gamma} decreased to half, while the percentage of CD8 cells producing IFN-{gamma} were not affected. The major difference observed between XCL1 treated and control mice was in the relative distribution of lymphocytes in the lung lesions. In control mice, the lymphocytes were found in small lymphocyte aggregations (Fig. 6D , upper right photo) and the perivascular aggregations of cells contained large numbers of lymphocytes. In mice treated with XCL1, regions of the peribronchial and perivascular cuffing were increased compared with control mice, and contained numerous lymphocyte and plasma cells (Fig. 6D , bottom right). The lymphocytic cores in the lesions were interspaced with many macrophages. A very interesting finding in these lesions was the presence of apoptotic bodies either free or taken up by macrophages (Fig. 6D bottom left).


Figure 6
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  		Figure 6. (A) High levels of pulmonary XCL1 decreased the percentage of CD4+ producing IFN-{gamma} while maintaining the same percentage of CD8+ producing IFN-{gamma}. Mice in late chronic infection (n=3) were anesthetized, and thereafter 1 µg of recombinant mouse XCL1 (R&D Systems) in 25 µl sterile PBS was delivered by the intratracheal route. Control mice received 25 µl of sterile PBS. This procedure was repeated every 24 h for three consecutive days. Twenty four hours after the last delivery, the lungs of PBS (solid bars) or XCL1 (open bars) treated mice were collected, processed, and assayed for expression of total contents of XCL1 by ELISA (A), total numbers of CD4 and CD8 T cells (B), and percentage of CD4+/IFN-{gamma}+ or CD8+/ IFN-{gamma} (C). (D) Histology of lung sections from mice treated with XCL1 (upper left) or PBS (upper right), showing higher concentrations of lymphocytes and macrophages in the core of the granuloma when compared with PBS-treated mice. Many lymphocytes were found in apoptosis interspaced by macrophages with free or phagocytosed apoptotic bodies (arrows in lower left and right photos), as well as many plasma cells (lower right).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we identified a new mechanism of action for CD8 T cells during acquired immunity to tuberculosis that comes into play during the chronic stage of the infection. CD8 T cells expressing an activated phenotype, but not naïve T cells or activated CD4 T cells, produced the chemokine XCL1. Expression of XCL1 in the lungs resulted in reduced percentage of CD4 T cells producing IFN-{gamma}, while not affecting the percentage of CD8 T cells producing IFN-{gamma}, or the total numbers of CD4 and CD8 T cells. The expression of IFN-{gamma} by CD4 T cells gradually waned during the chronic stage of disease and was associated with production of XCL1 by activated CD8 T cells. It is reasonable to speculate therefore that CD8 T cells influence the CD4 T response during the chronic disease stage by secreting XCL1. Interestingly, when exogenous XCL1 was administered the percentages of IFN-{gamma} producing CD4 T cells were reduced to almost half of those seen in control mice, whereas the total number of CD4 T cells in the lungs was maintained. This supports the previous observation [27 , 28 ] that XCL1 negatively affects the production of IFN-{gamma} by CD4 T cells (Fig. 6B) .

After treatment with exogenous XCL1, regions of perivascular cuffing as well as the lymphocytic core in the granulomatous lesions contained higher numbers of lymphocytes and macrophages than control mice. Many lymphocytes were found in apoptosis and there were as well apoptotic bodies phagocytosed by macrophages (Fig. 6D) . These results are in agreement with other studies indicating that the XCL1 receptor, termed XCR1, is heavily expressed by anergic CD4 T cells and that XCL1 increases apoptosis in lymphocytes [28 ]. Our current hypothesis, therefore, is that during Mtb infection, XCL1 selectively induces apoptosis and anergy in CD4 T cells in the lungs, but future studies will need to be done to confirm this. In support of this hypothesis, the apoptosis of T cells producing IFN-{gamma} in the lungs during chronic mycobacterial infection has already been suggested by the results of another study [29 ]

Our results also indicated that CD4 T cells are the dominant Th1 [IFN-{gamma} and TNF-{alpha}] cell population in the lungs. Thus, during the early stages of chronic infection when production of Th1 cytokines is strongly expressed by CD4 T cells, the removal of CD8 T cells had no effect on control of the bacterial growth. However, as the infection progressed, Th1 cytokines produced by CD4 T cells dropped considerably and hence concomitant removal of CD8 T cells resulted in reactivation of the bacterial growth (Fig. 1) . Despite CD8 T cells being able to compensate for reduced Th1 expression in the CD4 T cell compartment, our results also demonstrated that not only are CD4 T cells required at all stages of the infection but that their presence is also required for expression of Th1 responses by CD8 T cells (Fig. 4) . Interestingly, depletion of CD4 T cells did not affect the number of activated CD8 T cells producing XCL1, and the number of these cells increased significantly during the late chronic infection (Fig. 5B) , suggesting that production of IFN-{gamma} and XCL1 by activated CD8 T cells result from independent mechanisms. Furthermore, we observed that when CD8 T cells were depleted in mice during the midphase of the chronic infection, there was higher expression of IFN-{gamma} than in control mice. This result supported the conclusions reached above that CD8 T cells negatively control the number of CD4 T cells producing IFN-{gamma} (Fig. 6A 6B . However, despite mice in this group having higher expression of IFN-{gamma}, the histological analysis of lung sections and assessment of the bacterial cell load in the lungs demonstrated increased disorganization of the granuloma and reactivation of bacterial growth, indicating that at this stage, CD8 T cells have an important role in the maintenance of the granuloma integrity. Together, these data support the current knowledge that higher levels of expression of IFN-{gamma} responses during acquired immunity to Mtb infection does not serve as the only correlate of protection and indicates that other cytokines and chemokines are also needed to control bacterial growth [30 ].

In this regard, it is known that TNF is essential to the formation and maintenance of the granuloma structure [30 ] and that reduced levels of TNF-{alpha} during Mtb infection results in disorganization of the granulomatous lesions and reactivation of bacterial growth. This was clearly the case in the current study: mice that were depleted of CD4 T cells had a greatly reduced TNF-{alpha} expression and a similar outcome. In contrast, however, in this study, we also found that in mice at the early and middle stages of chronic infection, depletion of CD8 T cells, despite having similar total levels of expression of TNF-{alpha} as the control mice, the cell composition of the granuloma lesion and capacity to control bacterial growth differed greatly. These results demonstrate that TNF-{alpha} is not the only component required for granuloma stability and that CD8 T cells by means of XCL1 production also play an important role in the maintenance of this structure during chronic infection.

These results allow us to hypothesize that XCL1 produced by activated CD8 T cells participates in the control of IFN-{gamma} producing CD4 T cells. Acquired immunity against Mtb infection requires very strong Th1 responses by CD4 T cells but because Th1 responses are not sufficient to eliminate the bacteria completely, the course of infection enters into a chronic stage. In this study, our data suggest that the capacity of CD4 T cell populations to produce IFN-{gamma} appears to be influenced by the chemokine XCL1 produced by activated CD8 T cells. When the capacity of CD4 T cells to express Th1 cytokines is diminished, CD8 T cells are able to compensate for that deficit. Moreover, CD8 T cells are also a very important source of chemokines that are essential to the integrity of the granuloma, including the containment of bacteria in these structures.


   ACKNOWLEDGEMENTS
 
The authors would like to thank the Laboratory Animal Resources staff at Colorado State University for their help in the development of the intratracheal procedures. This work was supported by National Institutes of Health (NIH) grant AI-44072 and NIH, NIAID NOI contract AI-25469.

Received June 23, 2007; revised July 24, 2007; accepted July 25, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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