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International Immunology Advance Access originally published online on April 19, 2007
International Immunology 2007 19(5):627-633; doi:10.1093/intimm/dxm028
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Requirements for CD4+ T cell levels in acute Mycobacterium bovis strain bacille Calmette Guérin (BCG)-induced granulomas differ for optimal mycobacterial control versus granuloma formation

Laura H. Hogan, Erika Heninger, Rebecca A. Elsner, Heidi A. Vonderheid, Paul Hulseberg, Dominic Co and Matyas Sandor

University of Wisconsin School of Medicine and Public Health, Pathology and Laboratory Medicine, 5468 Medical Science Center, 1300 University Avenue, Madison, WI 53705, USA

Correspondence to: M. Sandor; E-mail: msandor{at}wisc.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Bacille Calmette Guérin (BCG)-induced granulomas contain T cells that express a broad TCR repertoire even at the level of the individual lesion. We have developed a BCG infection model in mice having only one T cell specific for a recombinant BCG epitope expressed in a lipoprotein fusion protein. Here we report that the single T cell model induces well-formed granulomas, but has weaker protection than that conferred by wild-type granulomas. This finding correlates with lower CD4+ T cell recruitment into acute granulomas (3 weeks post infection). Chronic granulomas (6 weeks post infection) contain similar proportions of CD4+ T cells in both models, but in the single T cell model the proportion of leukocyte function-associated antigen-1 low, non-IFN{gamma}-producing CD4+ T cells is lower. In fact, even though it is likely that there are very few, if any, IFN{gamma}+ CD4+ T cells present in the single T cell model, granuloma integrity is not influenced, indicating that high levels of IFN{gamma} are not required for granuloma maintenance. These data underline the importance of early CD4+ T cell recruitment into the granuloma to anti-mycobacterial protection and show that CD4+ T cell levels required for granuloma formation and optimal protection are different. These data also show that T cell repertoire complexity contributes to protection against mycobacteria.

Keywords: inflammation, mycobacterium, T cell receptor


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Granuloma formation in response to chronic mycobacterial infection requires the presence of CD4+ T cells to regulate the formation and ongoing function of protective lesions (1). Granulomas both restrict dissemination of pathogen from the inflammatory site and protect surrounding healthy tissue from immunopathology associated with chronically activated macrophage. At the same time, granulomas maintain a dynamic T cell population reflective of the systemic activated repertoire (1–3) and are able to accumulate recently activated T cells lacking mycobacterial specificity. We have previously shown that in fully immunocompetent mice, the TCR repertoire of T cell populations responding to Mycobacterium bovis strain bacille Calmette Guérin (BCG) infections is heterogeneous even at the level of the individual granuloma lesion (4). However, this diversity does not appear to be essential for granuloma formation, since a single specificity model is able to form granulomas which block dissemination and control bacteria (4). Our single specificity model uses recombinant BCG tagged with a pigeon cytochrome C (PCC) epitope (amino acids 88–104) to infect 5C.C7 Rag2–/– TCR transgenic mice expressing V{alpha}11Vß3 transgene specific for PCC88–104 presentation by I-Ek. Comparison of this model to wild-type strains led us to a significant insight into differential requirements for T cells for various aspects of T cell function.

We made two key observations while comparing the single specificity model to inbred immunocompetent strains. One observation suggested that CD4+ T cell requirements in acute granulomas are different for subsequent optimal bacterial control and for bacterial containment by granulomas. The other observation suggested that post-activated CD4+ T cells producing very little IFN{gamma} are capable of sustaining chronic granulomas. Together, this information has important implications for models of the initiation and ongoing function of granulomatous inflammation.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
In these studies, we used B10.BR (H2k) and C57BL/6 (H2b) mice (Jackson Laboratories, Bar Harbor, ME, USA) and a TCR transgenic strain, 5C.C7 Rag2–/– (Taconic Farms Emerging Models Program, Tarrytown, NY, USA), maintained on a B10.BR background, expressing a, V{alpha}11Vß3 transgene specific for PCC88–104 presentation by I-Ek. Animals were housed in animal facilities at the UW Medical School having AAALAC accreditation and meeting PHS policy. All experiments involving mice were reviewed and approved by the Medical School IACUC in accord with federal guidelines.

Mycobacterial infections
Wild-type BCG (substrain Pasteur, Staten Serum Institute) and rBCG-19 kDa lipoprotein-PCC (4) infections by intra-peritoneal injection were as described previously (4, 5). The rBCG-19 kDa lipoprotein-PCC strain expresses a PCC epitope recognized by 5C.C7 TCR transgene and has been described (4). The dose injected is not lethal and induces a disease that is partially cleared with time. Small pieces of liver were fixed in 10% formalin, prior to being imbedded in paraffin for thin sectioning (8–10 µm). H&E staining was done by the UW Department of Pathology’s Histopathology Service.

Organ load
At 3 and 6 weeks post infection, bacterial organ load was determined by plating serial dilutions of liver homogenates on Middlebrook 7H10 agar plates (Difco, Franklin Lakes, NJ, USA) supplemented with 10% OADC (Difco) and 10 µg ml–1 cycloheximide. Colonies were counted after 3 weeks incubation at 37°C. Data are presented as averaged individual mouse values after log10 transformation. Analysis of variance (ANOVA) was used to determine statistical significance.

Flow cytometry
Isolation of bulk granulomas and splenocytes was described previously (6–8). Splenocytes or granuloma cell suspensions were incubated for 30 min at 4°C with labeled antibodies at saturation, and then washed and analyzed. Unlabeled 2.4G2 anti-Fc receptor antibody (50 µg ml–1) was used to block binding of labeled antibodies to Fc receptors. Cell-surface staining on 10–20 000 events was measured using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA), and data were analyzed using FlowJo (Macintosh version 6.2.1, Tree Star, Ashland, OR, USA) software. Fluorochrome-labeled antibodies were purchased from PharMingen (San Diego, CA, USA) or Sigma (St Louis, MO, USA).

Flow cytometric detection of intracellular IFN{gamma}, tumor necrosis factor and activated caspase
Single-cell suspensions of spleen or granuloma cells were cultured in cRPMI 1640-10% FBS. Cells were activated with 5 µg ml–1 anti-CD3 antibody (145-2C11) for 18 h at 37°C, 5% CO2, followed by an additional 5 h after addition of 1:1000 dilution of Golgistop (PharMingen). Cells were washed once with FACS staining buffer and surface stained for 30 min at 4°C with the indicated antibodies. After washing, cells were permeabilized for 20 min at room temperature in Cytofix/Cytoperm (PharMingen) followed by washing three times with FACS staining buffer + 0.1% saponin, staining with either 4 µg ml–1 anti-IFN{gamma} (PharMingen) or 4 µg ml–1 anti-tumor necrosis factor (TNF) (PharMingen) and unlabeled 2.4G2 at 4°C for 30 min. Cells were then washed three times with FACS staining buffer + 0.1% saponin, fixed and analyzed by four-color flow cytometry. Staining cells for activated caspase 3 was done directly ex vivo using a 1:10 dilution of FITC-conjugated rabbit anti-caspase 3 (cat. no. 559341, PharMingen).

Measurement of secreted cytokines
Samples for cytokine analysis were collected from 1 x 106 liver granuloma cells from a 6-week infection (live by trypan blue exclusion) seeded into 96-well plates in 0.2 ml cRPMI. After 72 h, cell culture supernatants were harvested and stored at –70°C until testing. Cytokine measurements were obtained by a custom multiplex cytokine analysis from Linco Research (St Charles, MO, USA).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
We compared the 5C.C7 Rag–/– single specificity model with inbred models represented by wild-type BCG infection of either B10.BR or C57BL/6 mice. At 3 weeks of infection, wt B10.BR and 5C.C7 liver organ loads are statistically indistinguishable (Fig. 1A). At 6 weeks, liver organ load in the B10.BR mice significantly declines compared with 3 weeks (P ≤ 0.02 versus 3-week B10). Liver organ load in the 5C.C7 model is substantially decreased compared with Rag–/– mice, but is higher than wt B10.BR (P ≤ 0.0005 versus 6-week B10). Thus, quantitative organ load plating shows that the 5C.C7 model is not as effective as the wt model in controlling mycobacterial growth at 6 weeks and shows a clear log difference. Examination of thin liver sections by H&E staining shows well-formed granulomas in both models at 3 and 6 weeks, despite the difference in total organ load at the 6-week time point (Fig. 1B), suggesting a separation of granuloma physical formation from granuloma anti-mycobacterial function. Previously, we reported a much smaller difference between the single T cell model and a wild-type BCG infection model (4) using a semi-quantitative microscopic counting method. Using the more precise method of organ plating revealed an 8- to 10-fold load increase in load not apparent in sections, yet still clearly far more protective than Rag–/–. Whether this discrepancy arises from sampling errors inherent to looking at a finite number of field views, unequal distribution of lesions in the sections, or whether there are staining discrepancies related to dormant bacilli as reported by others cannot be distinguished at this time (9).


Figure 1
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  		Fig. 1 Comparison of organ load in single T cell and wild-type T cell BCG infection models. (A) Serial dilutions of liver homogenates were plated to determine the CFU/liver after BCG infection for 3 or 6 weeks. 5C.C7 represents rBCG-lipo-PCC infection of 5C.C7 Rag2–/–, B10 represents wtBCG infection of B10.BR and Rag–/– is wtBCG infection of B10.BR Rag1–/– mice. Bars represent average values after log10 transformation and error bars represent SEM. Left to right (n = 12, 11, 11, 6 and 3). (B) Pictures illustrate H&E-stained thin liver sections from the indicated groups at 3 and 6 weeks of infection. Microscopy was at x1000 times total magnification under oil. Images show individual well-formed granulomatous lesions.

 
We measured the relative fraction of CD4+ T cells and Mac-1+ cells in the wt and the single specificity models at 3 and 6 weeks of infection (Fig. 2A). At 3 weeks, during the acute phase of the infection, granuloma-infiltrating cells isolated after infection of wild-type mice have a higher proportion of CD4+ T cells compared with the fraction present at 6 weeks (Fig. 2A and B). The 5C.C7 model does not have the same enrichment of CD4+ T cells at the 3-week acute stage (Fig. 2A and B), indicating that either the antigen present is less or other T cells, B cells and/or antibodies absent in the 5C.C7 Rag–/– model may influence recruitment. Mac-1+ cells are a higher proportion of the 5C.C7 Rag–/– cell preparations since they are totally deficient in B cells and CD8+ T cells, and also do not express any non-transgenic CD4+ T cells. The cellular composition of the spleen is not as highly biased toward macrophage because it is not a primary inflammatory site like the granuloma. Statistical analysis of multiple experiments showed that the percentage of CD4+ T cells in lymphocyte-gated granuloma cells was statistically indistinguishable for 6-week 5C.C7 + rBCG-PCC, 6-week wt B10.BR + wild-type BCG and 3-week 5C.C7 by ANOVA (Fig. 2B).


Figure 2
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  		Fig. 2 Comparison of T cell numbers in single specificity and multi-specificity BCG infection models. At 3 and 6 weeks after the indicated infections, splenocytes and granuloma-infiltrating cells were analyzed by flow cytometry. (A) Dot plots show anti-CD4 and anti-Mac-1 antibody surface staining on cells within a large macrophage gate. Numbers represent the fraction of the gated population in boxed areas. (B) Graph shows the average percentage of CD4+ ± SEM in granuloma-infiltrating cells.

 
We also measured leukocyte function-associated antigen-1 (LFA-1) surface expression as a measure of T cell activation (Fig. 3) and intracellular IFN{gamma} (Fig. 4) for granuloma-infiltrating CD4+ T cells by flow cytometry to determine the activation status and effector function of granuloma-infiltrating cells. The data show that in both splenocytes and granuloma-infiltrating cells, LFA-1 expression at 3 weeks is similarly high on cells isolated from both wt B10.BR and 5C.C7 (Fig. 3). At 6 weeks, however, wt B10.BR T cells retain elevated LFA-1 expression levels and 5C.C7 T cells have decreased LFA-1 expression. LFA-1 expression is consistently higher on granuloma-infiltrating cells than on splenocytes as reported in our previous studies (4, 5). Likewise, at both 3 and 6 weeks, granuloma-infiltrating cells from C57BL/6 mice are able to produce IFN{gamma} in a recall response to {alpha}-CD3 measured by intracellular staining (Fig. 4A, left plots). Cells from the 5C.C7 Rag–/– mice produce high levels of IFN{gamma} at 3 weeks (Fig. 4A), but at 6 weeks, IFN{gamma}-positive cells are barely detectable above background (Fig. 4A, bottom right). The post-activated phenotype of the single specificity model at 6 weeks may reflect the absence of a broad T cell population needed to maintain T cell activation. The decrease in activated IFN{gamma}-producing CD4+ T cells does not seem to arise from enhanced rates of apoptosis, since levels of activated caspase 3 staining are comparable between C57BL/6 and 5C.C7 at 6 weeks (Fig. 5).


Figure 3
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  		Fig. 3 Comparison of T cell activation in single specificity and multi-specificity BCG infection models. Histogram plots show LFA-1 expression on CD4-gated splenocytes and granuloma cells from the indicated groups. Numbers represent the LFA-1 high fraction of the gated population and reflect lot-to-lot antibody variation. Staining from a naive B10.BR spleen is included for comparison. The data shown are representative of three to five independent experiments.

 

Figure 4
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  		Fig. 4 Comparison of IFN{gamma} recall in single specificity and multi-specificity BCG infection models. Intracellular staining for IFN{gamma} was performed on splenocytes and granuloma-infiltrating cells from the indicated infections at 3 and 6 weeks. Cells were cultured overnight with 5 µg ml–1 {alpha}-CD3. Golgistop was added for an additional 5 h before intracellular staining. Dot plots show LFA-1 and IFN{gamma}-specific staining on CD4-gated cells. Numbers are the percentage of gated cells in quadrants.

 

Figure 5
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  		Fig. 5 Activated caspase 3 levels at 6 weeks of BCG infection for the two models. Histograms show staining with anti-caspase 3-specific antibodies on CD4+ lymphocyte-gated granuloma-infiltrating cells at 6 weeks of infection. The numbers shown represent the caspase 3+ percentage of the gated population.

 
This observation suggests that while IFN{gamma}-producing T cells are required to eliminate bacteria, non-IFN{gamma} mediated mechanisms or very low levels of IFN{gamma} arising from resting T cells are sufficient for formation of granulomas able to contain the pathogen. One means by which low numbers of T cells can extend their influence is via regulation of macrophage and the induction of regulated macrophage products. One likely candidate for granuloma maintenance is TNF{alpha} (10) produced by activated macrophage. At 6 weeks, granuloma-infiltrating cells from both wt models and the single specificity model are able to make similar levels of TNF during in vitro culture (Table 1). The lack of production of these cytokines to {alpha}-CD3 stimulation suggests that macrophage and other non-T cells are the primary sources (data not shown). Intracellular staining for TNF is also similar between the single specificity and the multi-specificity models (Fig. 6). Both IL-10 and IL-1{alpha} are produced in equivalent amounts by granuloma cells from either model and do not correlate with decreased mycobacterial load (Table 1). However, macrophage senses the different granuloma environment in wild-type versus 5C.C7 Rag–/– mice as they make more IL-6, MIP-1{alpha} and MCP. From this data set, it appears that macrophage production of TNF, IL-10 and IL-1{alpha} levels are similar between the single and multiple T cell models (Table 1) and are sufficient for granuloma formation capable of containing mycobacterial dissemination. It needs to be clarified whether the higher measured levels of IL-6, MIP-1{alpha} and MCP are involved in indirect control of mycobacterial organ load, and the expression levels of these cytokines were not increased by {alpha}-CD3 treatment during culture. Expression of MHC class II on granuloma Mac-1+ cells was independent of the complexity of the T cell repertoire (Fig. 6). This suggests ongoing macrophage activation and maturation in the 5C.C7 model consistent with physical containment of mycobacteria, despite the scarcity of IFN{gamma}-producing CD4+ T cells. Since class II expression on macrophage is dependent upon IFN{gamma}, it is possible that there is a non-T cell source of IFN{gamma} in the granuloma or that the earlier presence of IFN{gamma} is sufficient to keep class II expression high. Overall, macrophage phenotype in the granulomas induced in the two models show many similarities suggesting a multiplicity of macrophage functions, either independent of T cell heterogeneity or requiring a low threshold for activity. Similar data were derived by gene array analysis of Mac-1+ granuloma-infiltrating cells (data not shown). It is likely that elimination of mycobacteria requires more CD4+ T cells and CD4+ T cell-regulated cytokines than do granuloma formation and maintenance.


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  		Table 1 Cytokine production by 106 granuloma cells during 72 h ex vivo culturea

 

Figure 6
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  		Fig. 6 Macrophage phenotype at 6 weeks of infection for the two models. (A) Dot plots show intracellular TNF{alpha} staining and anti-Mac-1 antibody surface staining on granuloma-infiltrating cells within a large macrophage gate. Numbers represent the fraction of the gated population in oval areas. Histograms represent Class II staining on Mac-1+ CD4– cells in a large macrophage gate. Numbers represent percentage gated cells in the indicated region.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The single TCR specificity model represented by rBCG-lipo-PCC infection of 5C.C7 Rag–/– mice provided us with an unexpected opportunity to examine the contribution of CD4+ T cell numbers to optimal control of mycobacterial infection. Importantly, a statistically significant difference in total CD4+ T cell number was evident at 3 weeks during the acute phase of the infection. Differences in granuloma function were not evident until 6 weeks of infection when the host response is chronic. At 6 weeks, granuloma formation in the single specificity model was indistinguishable from that seen in animals with a wild type T cell repertoire. However, bacterial organ load at 6 weeks remained high in the single TCR model and did not decline to a lower steady-state level as observed for B10.BR and C57BL/6 strains. Although fewer in number, the 5C.C7 T cells at 3 weeks expressed roughly equivalent LFA-1 (Fig. 3) and IFN{gamma} recall responses (Fig. 4), suggesting that the primary deficiency responsible for subsequent altered protection at 6 weeks was quantitative rather than qualitative. Acute-stage granulomas are more CD4+ T cell rich than during the chronic stage in various infectious models including mycobacteria (11), Leishmania (12), Histoplasma (13) and Schistosoma (14), indicating the biological significance of CD4+ T cells in early stage granulomas.

The correlation of CD4+ T cells with bacterial control might arise because the multi-specificity model has a heterogeneous TCR repertoire able to respond to diverse BCG epitopes relative to the single antigen recognition available in the single specificity model. The multiple TCR recognition events available in the wild type repertoire result in higher recruitment of CD4+ T cells. In the TCR transgenic model, the amount of PCC antigen is the limiting factor, rather than T cell levels. A variation of this scenario could involve lowered or delayed expression of the PCC transgene during in vivo growth relative to more essential mycobacterial products required for interaction in the host. Alternatively, T cell retention and survival within the granulomatous lesion may be enhanced by interactions of T cell populations having different specificities, leading to higher accumulation in the multi-specificity environment of the immune competent mouse. So-called ‘bystander’ events may play an essential role in regulating the final T cell number. In either model, higher levels of granuloma recruited T cells ultimately lead to better control of organ load. This is consistent with the results of van Pinxteren et al. (15) showing that CD4+ T cells are essential during the acute phase of lung infection, and that anti-CD4 treatment leads to significant increases in bacterial load in the lungs. Nagabhushanam et al. (16) have shown that significant accumulation of antigen-specific T cells in the lung of Mycobacterium tuberculosis-infected mice does not occur until 17 days after infection and corresponds with establishment of bacterial control initiated at ~3 weeks of infection. Significantly, since the single specificity model promotes granuloma formation (Fig. 1B), less CD4+ T cells are needed for granulomatous containment of BCG in the liver than are required for optimal protection. The equivalent caspase 3 staining at 3 weeks (Fig. 5) argues against a model in which cross-talk among T cells of varying specificity regulates the final levels of T cells using the pathway of programmed cell death.

One of the most striking differences in the macrophage phenotype between the two models is the significantly lower production of IL-6 during in vitro culture of granuloma-infiltrating cells from the single specificity model (Table 1). IL-6 production was equivalent to wild type at 3 weeks during the acute response (data not shown). IL-6 is known to be secreted by M. tuberculosis-infected macrophage and can inhibit IFN{gamma}-mediated responses in vitro (16). IL-6 also plays a key role in the differentiation of IL-17-producing regulatory T cells which are thought to suppress Th1 effector functions including IFN{gamma} production (17, 18). Our laboratory is actively investigating the role of IL-6 in chronic immune responses of immunocompetent mice and whether IL-6 is a cause or a result of the T cell insufficiency.

Multiple cell types are known to be present during granulomatous inflammation and a number of them are thought to play an important role in the formation of the structure. Infected and uninfected macrophage are the dominant cell type in granulomas induced by mycobacteria. Some studies have also indicated that B cells play a role in granuloma formation and control of mycobacterial numbers (19, 20). Other studies implicate both neutrophil and granulocyte participation in early events of anti-mycobacterial responses. Polymorphonuclear cells are stimulated by mycobacterium to secrete chemokines (21, 22), and their products may inhibit the growth of mycobacteria (23), suggesting that they may form a significant part of the innate immune response to mycobacterial infection. In addition, others have demonstrated that BCG-derived lipids are potent inducers of neutrophil recruitment (24). At the same time, depletion studies with neutrophil-depleting antibodies indicate that these effector mechanisms are not essential (25) for control of growth in vivo, and excess neutrophil accumulation has been associated with the development of immunopathology (26, 27). In contrast to this conflicting data, CD4+ T cells are the most important regulator of granuloma formation and function, and are the only indispensable cell type for pathogen control (2, 3, 28). Our results from this study suggest that T cell accumulation during the acute response is an important component for eventual control of mycobacteria and these T cells ultimately control anti-mycobacterial responses and macrophage activation during the subsequent chronic phase of the infection.

Scanga et al. (29) reported that CD4+ T cell depletion at the chronic stage of M. tuberculosis infection leads to reactivation, despite the continued presence of IFN{gamma} arising from CD8+ T cells. In our 5C.C7 model, protective function arising from a single CD4+ T cell population can form stable granulomas. Significantly, lower T cell levels during the acute phase has no obvious immediate consequence, but instead leads to a subsequent diminution in activated T cells capable of producing IFN{gamma} and controlling bacterial numbers and alterations in some, but not all, macrophage functions at the chronic stage. However, sufficient T cell and macrophage activity is retained to enable maintenance of granulomas' physical barrier to bacterial dissemination. Previous reports that anergic T cells can maintain granuloma formation are in agreement with our data (30). We are currently investigating the relative contributions of quantitative insufficiency of the lipo-PCC:5C.C7 antigen:TCR interaction relative to the full repertoire of interactions, and the role of antigen-induced anergy in the 5C.C7 model. In conclusion, we report here that quantitative requirements for CD4+ T cells are different for granuloma formation and optimal bacterial control. Furthermore, we present data indicating that granuloma maintenance can be achieved in the absence of IFN{gamma}-producing T cells. It will be interesting to ascertain how many T cell populations are required for full protection and the effect of adding back additional populations of specific and non-specific T cells.


   Acknowledgements
 
This work was supported by the Public Health Service grants R01 AI48087, R01 AI/HL46430 and R21 AI054893 from the National Institutes of Health to M.S.


   Abbreviations
 
ANOVA, analysis of variance
BCG, bacille Calmette Guérin
LFA-1, leukocyte function-associated antigen-1
PCC, pigeon cytochrome C
TNF{alpha}, tumor necrosis factor alpha
CFU, Colony forming units
H&E, Hematoxylin and Eosin

   Notes
 
Transmitting editor: A. Falus

Received 20 December 2006, accepted 22 February 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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