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The influence of BCG vaccination on the cellular immune response in immune guinea pigs
infected with Mycobacterium tuberculosis.
Diane Ordway†, Marcela Henao-Tamayo, Crystal Shanley, Erin E. Smith, Gopinath Palanisamy,
Baolin Wang, Randall J. Basaraba, and Ian M. Orme.
Running Title: Effect of BCG vaccination on M. tuberculosis infection.
Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology,
Colorado State University, Fort Collins, Colorado, 80523.
†Correspondence: Dr. Diane Ordway, Department of Microbiology, Immunology and Pathology,
Colorado State University, Fort Collins, Colorado, 80523-1682. Phone 970-491-7469, Fax 970-
491-5129. E-mail address: [email protected]
Keywords: Mycobacterium tuberculosis, Guinea pig, T cells, B cells, Heterophils, Vaccine
1 This work was supported by NIH grants AI-054697 and AI070456. The authors do not have
commercial or other associations that might pose a conflict of interest.
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Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.00019-08 CVI Accepts, published online ahead of print on 28 May 2008
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Abstract
Mycobacterium bovis Bacillus Calmette-Guérin (BCG) currently remains the only
licensed vaccine for the prevention of tuberculosis. In this study we used a newly described flow
cytometric technique to monitor changes in cell populations accumulating in the lungs and
lymph nodes of naïve and vaccinated guinea pigs challenged by low dose aerosol infection with
virulent Mycobacterium tuberculosis. As anticipated, vaccinated guinea pigs controlled the
growth of the challenge infection more efficiently than controls. This early phase of bacterial
control in immune animals was associated with increased accumulation of CD4 and CD8 T cells,
including cells expressing the activation marker CD45, as well as macrophages expressing Class
II MHC molecules. As the infection continued the numbers of T cells in the lungs of vaccinated
animals waned whereas the numbers of these cells expressing CD45 increased. Whereas BCG
vaccination reduced the influx of heterophils (neutrophils) into the lungs, an early B cell influx
was observed in these vaccinated animals. Overall, vaccine protection was associated with
reduced pathology and lung damage in the vaccinated animals. These data provide the first direct
evidence that BCG vaccination accelerates the influx of protective T cell and macrophage
populations into the infected lungs, diminishes the accumulation of non-protective cell
populations, and reduces the severity of lung pathology.
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Introduction
Well over two million people now die each year from tuberculosis (5-7, 9, 12, 23). At
present the only available vaccine against tuberculosis, M. bovis Bacillus Calmette-Guérin
(BCG), has proven unreliable in being able to protect against pulmonary tuberculosis in adults
(2, 4, 32). To date, the specific activity of BCG has thought to lie in its ability to generate a state
of immunological memory in the host and thus accelerate the emergence of a TH1 protective
response upon infection (8, 18, 32). However, recent studies seem to imply that the ability of
BCG to induce stable memory T cell populations may be lacking (19). Hence, further research
into the actual immunological activity of BCG is warranted to guide rational vaccine design.
The two small animal models used most often for preclinical TB vaccine screening are
the low-dose aerosol mouse and guinea pig models (1, 25, 26, 31, 33). The majority of this work
has been carried out in various mouse models due to their low cost and the wealth of
immunological reagents (32). Low-dose aerosol infection of the guinea pig with M. tuberculosis
produces a well-characterized disease that shares important morphologic and clinical features
with human tuberculosis (25, 26). The ability to be able to precisely characterize the protective
immune response induced by BCG during M. tuberculosis infection in the guinea pig would
greatly improve the usefulness of this animal model for the testing and evaluation of urgently
needed new vaccines.
Effective resistance to Mycobacterium tuberculosis infection is mediated by both innate
and adaptive mechanisms of immunity (8). After pulmonary infection with M. tuberculosis,
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alveolar macrophages and dendritic cells phagocytose the bacilli, and it is thought that dendritic
cells carry both intact bacteria and their antigens to draining lymph nodes where recognition by
T cells generate cell mediated immunity (10, 24, 29). Macrophages present antigen via both
class-I and class-II MHC molecules, resulting in effector CD4 and CD8 T cells which secrete
cytokines including gamma interferon (IFN-i) giving rise to macrophage activation and
intracellular killing of the organism (8, 34). The development of a pulmonary granuloma is
orchestrated both by chemokines and cytokines resulting in a continuous recruitment of
lymphocytes, granulocytes, macrophages, dendritic cells and monocytes to the local site of the
infection (37-38). Control of the infection relies on this granulomatous response, which is an
organized cellular network acting to restrain mycobacterial growth and limit dissemination.
The progression of tuberculosis in guinea pigs can be divided into acute, subacute and
chronic stages of infection based on the pattern of bacterial growth and dissemination, as well as
patterns of pulmonary and extra-pulmonary pathology (37, 38). The acute phase consists of a two
week period of rapid bacterial growth and dissemination of the infection to draining lymph
nodes. The subacute phase runs from approximately week two to week four and is characterized
by the emergence of a stationary phase of bacterial replication. The primary granuloma develops
during the acute and subacute stages of infection and has a characteristic central core of necrosis
which overtime may become mineralized. Finally, while the actual growth of the infection is
relatively slow, the chronic stage in guinea pigs is characterized by the development of necrosis
and secondary lesion progression, killing the animal in about 100-150 days.
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In this study we used a new flow cytometric approach (30), combined with
immunohistochemistry, in order to characterize how BCG vaccination alters the immune
responses toward M. tuberculosis infection.. These guinea pigs were able to more rapidly contain
and control M.tuberculosis growth at the peak of acquired immunity compared to unvaccinated
control guinea pigs. The lung granulomas present in these BCG vaccinated guinea pigs were
smaller than in control animals, less parenchymal inflammation, and slower progression of lung
pathology. There was an earlier influx of CD4+ and CD8+ T cells expressing the activation
marker CD45+ in the immune animals. In addition, BCG vaccinated animals showed an early
presence of increased numbers of macrophages upregulating MHC class II molecules. During
chronic infection, BCG vaccination reduced the influx of heterophils (neutrophils) into the lungs
which has been associated with tissue damage. These data provide the first direct evidence that
BCG vaccination induces both qualitative and quantitative immunological differences which can
be related to bacterial growth and lung pathology.
Materials and Methods
Guinea pigs
Female outbred Hartley guinea pigs (~500 g in weight) were purchased from the Charles
River Laboratories (North Wilmington, MA, USA) and held under barrier conditions in a
Biosafety Level III animal laboratory. The specific pathogen-free nature of the guinea pig
colonies was demonstrated by testing sentinel animals. All experimental protocols were
approved by the Animal Care and Usage Committee of Colorado State University.
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Experimental infections in guinea pigs
Guinea pigs were challenged using a Madison chamber aerosol generation device, by
delivering Mycobacterium tuberculosis H37Rv at a low-dose aerosol of 20 bacilli. Animals were
then assayed for lung, mesenteric lymph node, spleen bacterial loads, histology and cell
homogenates for flow cytometric analysis on days 5, 20, 30, 60 and 90 of the infection. Bacterial
counts in the organs of guinea pigs (n = 4) at each time point of the study were determined by
plating serial dilutions of homogenates of lungs on nutrient 7H11 agar and counting colony-
forming units after 3 weeks incubation at 37oC.
BCG vaccination
Groups of 4 animals were vaccinated with BCG Pasteur injected by the intradermal route
at a dose of 103 viable bacilli and then rested for 1 month prior to aerosol challenge. No adverse
reactions at the injection site were noted after injection of BCG.
Histological analysis in guinea pigs
The lung lobes and lymph nodes from each guinea pig were fixed with 4%
paraformaldehyde in phosphate buffered saline (PBS). Sections from these tissues were stained
using haematoxylin and eosin and the Ziehl-Neelsen stain for acid-fast bacilli. In guinea pigs the
concurrent progression of lung and lymph node lesions was evaluated using a histological
grading system (38). The method for grading ganulomatous lesions was based on inflammatory
cell numbers and their infiltrative distribution pattern in the organs assayed. Briefly, scoring of
the pathology of lung sections to provide a cumulative score is done as follows: Primary lesion scoring,
0 – no primary lesion present, 1 – sparse to few foci of primary lesions present, 2 – multiple foci of
primary lesions present, 3 – multiple foci of primarylesions present with the majority coalescing.
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Primary lesions with necrosis, 0 – Primary lesion with the majority showing no necrosis, mineralization
or fibrosis, 1 – primary lesions present with < 30% necrosis, 2 – primary lesions present with 35-65%
necrosis, 3 – primary lesions present with 70-100% necrosis. Primary lesions with mineralization, 0 –
Primary lesion with the majority showing no necrosis, mineralization or fibrosis, 1 – primary lesions
present with < 30% minerlization, 2 – primary lesions present with 35-65% minerlization, 3 – primary
lesions present with 70-100% minerlization. Primary lesions with fibrosis, 0 – Primary lesion with the
majority showing no necrosis, mineralization or fibrosis, 1 – primary lesions present with < 30% fibrosis,
2 – primary lesions present with 35-65% fibrosis, 3 – primary lesions present with 70-100% fibrosis.
Secondary lesion scoring, 0 – no secondary lesions present, 1 – sparse to few foci of secondary lesions
present, 2 – multiple foci of secondary lesions present, 3 – multiple foci of secondary lesions present with
the majority coalescing. A veterinary pathologist completed the blind scoring of lung and lymph node
lesions which was based on randomly selected sections in a representative experiment from four infected
control guinea pigs and four BCG vaccinated immune guinea pigs at indicated times after infection.
Immunohistochemistry.
Once removed from the thoracic cavity, the cranial lobe of the lungs were embedded in
OCT, frozen in liquid nitrogen then stored at -80°C. Serial sections, 8- to 10-µm thick, from each
lung were cut on a cryostat (Leica; CM 1850) using the Instrumedics tape transfer system, fixed
in cold acetone, and air dried. The sections were washed, and nonspecific antibody binding was
blocked with 3% BSA-PBS solution. Thereafter, the sections were incubated overnight at 4°C
with one of the following purified primary antibodies listed in the Flow cytometric analysis of
cell surface markers section below (Serotec Inc, Raleigh, NC). All sections were washed three
times in PBS and incubated with the secondary detection antibody F(ab’)2 rabbit anti-mouse
conjugated to HRP (Serotec Inc, Raleigh, NC). Finally, the reaction was developed using
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aminoethylcarbazole (BioGenex, San Ramon, CA) as substrate. The sections were
counterstained with Meyer’s hematoxylin and thereafter mounted with crystal/mount (BioGenex,
San Ramon, CA). Our experiments utilized different lung lobes of the guinea pig for
immunohistochemistry and flow cytometry because we have shown the lesions are evenly
distributed throughout the guinea pigs infected lung by evaluating the histopathology and
magnetic resonance imaging of pulmonary lesions in guinea pigs (20).
Organ cell digestion
To prepare single cell suspensions the lungs and lymph nodes were perfused with 20.0 ml
of a solution containing PBS and heparin (50 U/ml; Sigma-Aldrich, St. Louis, MO) through the
pulmonary artery and the caudal lobe aseptically removed from the pulmonary cavity, weighted
and 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 ug/ml; Sigma-Aldrich) for 30 minutes at 37°C. The digested lungs
were further disrupted by gently pushing the tissue twice through a cell strainer (BD Biosciences,
Lincoln Park, NJ). Red blood cells were lysed with ACK buffer, washed and resuspended in
cDMEM. Total cell numbers per lung and lymph node were determined by using a
haemocytometer and then calculating the cell number per 1.0 gram of tissue.
Flow cytometric analysis of cell surface markers
Single cell suspensions from the lungs and portions of the whole spleens and lymph
nodes were prepared as recently described (30). Thereafter cell suspensions from each
individual guinea pig were incubated first with antibodies to CD4 (clone FITC CT7) (36), CD8
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(clone FITC CT6) (36), pan T cell (clone APC CT5) (35, 36), CD45 (clone RPE IH-1) (13),
neutrophils and eosinophils (clone RPE MIL4) (14), B cell (clone FITC MsGP9) (16),
macrophage (clone FITC MR-1) (21) and class II (clone RPE Cl.13.I) (36, 39) antibodies at 4°C
for 30 minutes in the dark and after washing the cells with PBS containing 0.1% sodium azide
(Sigma-Aldrich). In addition, membrane permeabilisation using Leucoperm (Serotec Inc,
Raleigh, NC) was completed according to the instructions prior to staining with macrophages
(21) and MHC Class II (27) antibodies. Data acquisition and analysis were done using a
FACscalibur (BD Biosciences, Mountain View, CA) and CellQuest software (BD Biosciences,
San Jose, CA). Compensation of the spectral overlap for each fluorochrome was done using CD4
or MIL4 or CD3 antigens from cells gated in the FSClow
versus SSClow
; FSCmid/high
versus
SSCmid/high
; SSClow
versus MIL4+; SSC
high versus MIL4
neg and SSC
high versus MIL4
+ region
respectively. Analyses were performed with an acquisition of at least T cells 100,000 total
events.
Statistical analysis in guinea pigs
Data is representative of two experiments and presented using the mean values from
individual guinea pigs within each group (n=4) and ± standard error of the mean. A parametric
method, the Student’s t-test, was used to assess statistical significance between groups of data.
Results
BCG vaccination slows the growth of the challenge infection over the early stages of the disease
process.
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Guinea pigs were exposed to approximately 20 bacilli of M. tuberculosis which was
based on a reproducible day one animal necropsy and plating of whole organ homogenates on
agar plates. These guinea pigs were evaluated for bacterial loads in the lung and lymph nodes at
indicated time points (Fig.1). Control guinea pigs showed an increase of approximately 5.5
logs10 in lungs over the first 20 days of infection (Fig.1A), followed by a chronic phase of
disease. A similar rise in numbers to those seen in the lungs was observed in the draining lymph
nodes [mediastinal lymph node cluster] (Fig.1B).
As expected, BCG vaccination reduced the growth of M. tuberculosis in the lungs and
lymph nodes 20 days after infection. The mean lung log10 cfu of M. tuberculosis from immune
animals (Fig.1A) was significantly reduced compared to control animals, as was the mean log10
cfu recovered from the lymph nodes (Fig. 1B).
Fig.1C and D shows mean lesion pathology scores for the lungs and lymph nodes in
control and vaccinated animals. In controls, the severity of the pathology in both tissues (Fig.1C,
1D) worsened progressively over time, with a considerable surge after day 30, as we previously
also showed due to progression of secondary lesions (30). In contrast, this increased at a much
slower rate in vaccinated animals, particularly in the lungs.
Differences in the granulomatous response between control and vaccinated guinea pigs.
Consistent with earlier observations (30), on day 5 of the infection the lung lesions
consisted of small aggregations of resident cells close to major airways and blood vessels (Fig.
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2A, panel A). By day 30, lesions in the lungs and lymph nodes consisted of foci of mixed
inflammation with areas of central necrosis composed of nuclear and cytoplasmic debris (Fig.
2A, panel B). In contrast, the vaccinated guinea pigs showed smaller granulomatous lesions
consisting of minimal foamy macrophages with lymphocytes positioned in tight aggregates
which were devoid of central necrosis (Fig. 2A, panel D and Fig. 2B, panel D). During the
chronic phase of the infection lung lesions in control guinea pigs continued to progress to form
foci of coalescing inflammation that effaced large areas of pulmonary (Fig.2A, panel C) and
lymph node (Fig.2B, panel C) tissue by day 90 of the infection. In these animals debris within
the necrotic centers of the lung lesions had become mineralized (Fig.2A, panel C) and was
surrounded by epithelioid macrophages, whereas the vaccinated animals showed granulomatous
lung lesions with minimal central necrosis (Fig. 2A, panel F) and minimal lymph node tissue
necrosis (Fig. 2B, panel F).
Kinetics of the T cell response in control and vaccinated animals.
Using a recently described flow cytometric technique (24), we evaluated the influx of
numbers of T cells into the lungs and lymph nodes of vaccinated and control guinea pigs over the
course of the infection. This technique utilizes SSClow
versus MIL4neg
gating to clearly delineate
lymphocytes as well as other cell populations, and was applied to this study. As shown in Figure
3, between days 5 and 20 of the infection we observed a substantial and more rapid rise in
numbers of CD4+ and CD8
+ T cells (Figs.3A, B) in the lungs and lymph nodes of vaccinated
guinea pigs compared to controls. This early increased in T cell numbers observed in the
vaccinated animals was coincident with an observed decline in bacterial numbers in the
vaccinated group. However, in control animals by day 30, CD4+ and CD8
+ T cell numbers
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peaked, reflective of the progression of secondary lesions in this group. This increase of CD4+
and CD8+ T cell numbers in the lungs of control animals was not sustained however, and
declined after 30 days. Despite this, numbers of these cells still remained higher in the
vaccinated animals compared to those in controls during the chronic stage of the disease.
The up-regulation of CD45 (leukocyte common antigen) molecules on T cells indicates
signal transduction of antigen receptor signalling during immune responses and hence is an
indication of T cell activation (13, 36). We took advantage of an available monoclonal antibody
to evaluate the percentage of cells expressing CD45 on CD4+ and CD8
+ T cells over the course
of the infection in control and vaccinated guinea pigs. This technique utilizes SSC versus FSC
gating on the viable lymphocyte population demonstrated in Fig. 3E and the isotype control (Fig.
3F).
As shown in Fig. 3C and 3D, CD45 was up-regulated on both CD4+ and CD8
+ cells much
more rapidly in the vaccinated animals, and these levels were sustained through day 90 of the
experiment. Small rises in CD45 expression were observed on CD4+ and CD8
+ T cells in control
guinea pigs over the first month in both the lungs (Fig. 3C) and lymph nodes (Fig. 3D) but this
was not sustained. As shown in Fig. 3G the flow cytometric dot plots from BCG vaccinated
guinea pigs showed early expression of CD45 on CD4 and CD8 T cells compared to the control
animals. It is evident that there is more T cell CD45 expression utilizing this gating technique
and may include some yet unidentified fine populations of cells such as NK or けh T cells which
co-express CD4+ and CD45+ or dendritic cells co-expressing CD8+. Flow cytometry in the
guinea pig is a newly developed technique and with the development of more commercially
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available flow cytometric reagents identification of these fine populations which co-express
multiple markers will be possible.
Increased numbers of macrophages in the lungs of vaccinated animals after challenge infection.
We used the monoclonal antibody MR-1 to track macrophages in the lungs and draining
lymph nodes (21). This technique utilizes SSC versus FSC gating on the viable granulocyte
population demonstrated in Fig. 4C and the isotype control (Fig. 4D). As shown in Fig. 4A and
B, there was a substantial increase of macrophages in the lungs and lymph nodes of vaccinated
guinea pigs. As shown in Fig. 4E the flow cytometric dot plots from BCG vaccinated guinea pigs
showed increased expression of MHC Class II on MR-1+ macrophages compared to the control
animals. Interesting, only small numbers of macrophages in the lungs or lymph nodes of control
animals stained positive for Class-II (39). In vaccinated animals the presence of macrophages in
the lungs was much earlier and more prominent, with most cells expressing Class-II. A similar
pattern was seen in the lymph nodes, but here only about 25% were positive for Class-II.
Kinetics of influx of B lymphocytes and heterophils.
We previously showed that both B cells and heterophils enter the lung lesions,
accumulating in numbers after a decline in CD4 cells seen in naïve infected animals (30). To
determine if these cells could still accumulate in vaccinated animals we analyzed tissues by flow
cytometry using the SSClow
versus MIL4neg
gating to clearly delineate the MIL4+ and B cell
populations. As shown in Fig. 5A and B, we unexpectedly observed a rapid increase in the
number of B cells in the lungs over the first 30 days of infection in the vaccinated guinea pigs,
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which then flattened. In controls, B cell numbers remained low through day 30 and then
increased progressively, consistent with our early studies (30). As shown in Fig. 5C the flow
cytometric dot plots from BCG vaccinated guinea pigs showed more B cells compared to the
control animals during subacute infection. An increase in heterophil [MIL4+] numbers was seen
in controls; this was diminished by vaccination. A similar kinetics was seen in the draining
lymph node cluster (Fig. 5B), although here the influx of heterophils in control animals was
earlier, consistent with the rapid necrosis seen in these tissues in control animals. As shown in
Fig. 5 D the flow cytometric dot plots from BCG vaccinated guinea pigs showed less heterophils
compared to the control animals during chronic disease.
Positioning of T cells revealed by immunohistochemical staining.
Having established cell numbers in the lungs and lymph nodes, in a final series of studies
we used immunohistochemical staining of lung sections to determine the actual organ
distribution of these cell types (Fig. 6).
On day 30 of the infection aggregations of CD4 cells were observed in large rims
surrounding the developing central necrotic core of the primary lesions in the lungs (Fig 6A, 6B).
These aggregates were less prominent in vaccinated animals (Fig 6C, 6D) in primary lesions
(secondary lesions (data not shown) were far less prominent in these animals indicating that they
were prevented by the vaccination process, as suggested by our previous pathologic evaluation of
histology and magnetic resonance imaging data (20). In control animals, CD8 cells were also
detected, but more scattered and more towards the periphery of the lesions (Fig 6E, 6F). A
similar distribution was seen in vaccinated animals, with less CD8+ cells again forming a diffuse
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rim on the edge of the lesions (Fig 6G, 6H). Macrophages were widely distributed in clusters
(consistent with “sheets” of epithelioid macrophages found in the granuloma), but far more
prominent in the vaccinated animals (Fig. 6K, 6L) consistent with their more rapid influx
indicated by the flow cytometric data. Again consistent with that analysis, B cells were scattered
across lesions in control animals, mostly on the periphery of lesions (Fig 6M, 6N), but were
more obvious in vaccinated animals as loose clusters of cells (Fig 6O, 6P). As we reported before
(24), heterophils were almost exclusively associated with the areas of central necrosis (Fig 6Q-T)
and increased in control animals.
Discussion
The results of this study show the protective properties of BCG vaccination at the peak of
acquired immunity results in the generation of immunity in the lung leading to a reduction in
bacterial growth by about 1 log (15). In addition, the protective properties of BCG are associated
with reduced lesion scores indicative of reduced number and size of granulomas combined with
less T cell infiltration and parenchymal inflammation which is directly associated with bacterial
containment. The immune guinea pigs also showed a delay in the secondary granuloma
progression, in the lungs. These results are supported by other murine and human studies
showing reduced granuloma size is associated with immune responses able to control bacterial
growth (29).
One central finding in this study is that immune animals show a substantial increase in
CD4 T cell numbers during the acute and subacute phases of infection, thereafter levelling off as
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bacterial control is established [many of which expressed the activation marker CD45] compared
to the control animals. We observed a two-fold increase in CD4 cells in the lungs by day 5 of the
challenge infection in the vaccinated guinea pigs, an observation reminiscent of our earlier
studies in the mouse model (19). In addition, an early increased CD8 T cell response was also
seen in the vaccinated guinea pigs, although this occurred more slowly compared to the CD4
response. These flow cytometric observations were confirmed by immunohistochemical staining
of lung tissues showing as BCG vaccinated animals establish bacterial control, less CD4+ and
CD8+ T cells are present in immune animals compared to controls. Murine studies (18, 19) have
shown that reduced numbers of bacteria in BCG vaccinated mice infected with M. tuberculosis is
associated with a pre-existing pulmonary Th1 response characterized by fewer numbers of CD4+
T cells producing IFN-け. Immune guinea pigs have been shown to produce protective cytokines
such as (IFN-け and TNF-g) during the subacte phase of infection (25, 26, 27). However, in
contrast to murine models in the guinea pig this rapid increase in CD4 T cell numbers is
followed by CD4+T cell decline and resurgence of disease.
Staining with an antibody to CD45 provided an insight into numbers of activated T cell
numbers, potentially indicating antigen specificity (8). Increased numbers of these cells remained
sustained in vaccinated animals both within the lungs and, interestingly, in the lymph nodes
despite the increasing pathological damage in these latter tissues. Thus one of the protective
properties of BCG vaccination may be the ability of T cells to upregulate and express CD45.
Although we lack the reagents to be as yet more definitive, these observations do allow us to
hypothesize that BCG is causing a more rapid focusing of effector T cells into the infected
tissues, and that while the total numbers of CD4 and CD8 cells clearly drop, the numbers of
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CD45+ activated cells appear to be sustained. A similar situation is suggested by observations in
the mouse model (19).
We still do not fully understand why the CD4+ and CD8+ T cell numbers decline during
chronic disease. However, we know that the drop in CD4 and CD8+ T cell numbers appears to
represent a true effect and not caused by the tissues processing technique nor a dilution by other
cell types entering the granulomas. Our previous studies (38) have confirmed this as the
lymphocyte “mantle”, an early characteristic of the guinea pig granuloma, showed reduction in
the density of immunohistochemical staining after day 30. This was further confirmed in
subsequent flow cytometric and immunohistochemical studies (30) that CD4 cells took up a
position surrounding the developing core of necrosis, but by day 60 this layer was smaller and
staining for CD4 more diffuse. We know that in the draining lymph nodes the early T cell
response was much more rapid, but then dropped considerably during chronic infection as
necrosis developed in this organ. Also the draining lymph nodes are the first extra-pulmonary
lymphoid tissues to encounter the bacilli and the first site of rapidly progressive destructive
pathology (37, 38). The dendritic cells in the draining lymph nodes are responsible for antigen
presentation to naïve T cells and T cell priming (10, 8, 24). We hypothesize that in chronic
infection the rapid involvement and destruction of the draining lymph nodes, may cause dendritic
cells to become unable to function as antigen presenting cells, eliminating T cell priming causing
these cell to be depleted in the lungs during the chronic infection. Presently, a commercially
available antibody does not exist for dendritic cells in the guinea pig model and thus we are
currently using cross-reactive dendritic cell markers to further investigate this hypothesis.
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Another important finding is the early increase in numbers of macrophages present in the
lungs in the vaccinated guinea pigs. We hypothesize that macrophage influx into the lungs is due
to production of recruiting chemokines and appropriate integrin expression on local blood vessel
walls which may be a very early event in these animals. Moreover, most of these cells stained
positive for expression of Class II MHC molecules. In control animals, in contrast, the response
was much smaller and slower. Moreover, very few of these cells expressed Class II even by day
90. A faster influx of macrophages in vaccinated guinea pigs was also observed in the lymph
nodes, but in stark contrast in this organ macrophages in both vaccinated and control groups
poorly expressed Class II. Thus, these results together support the hypothesis that a protective
property of BCG vaccination in guinea pigs is that macrophages have a better ability to activate
and to function as antigen presenting cells by upregulation of MHC class II molecules. It is
known that M. tuberculosis infects macrophages and that these cells are capable of eliminating
the bacteria very efficiently after activation by IFN-i (24, 27, 29). Until we can directly measure
IFN-i secretion by T cells we cannot explain this, but it suggests that the T cells capable of
entering the lymph nodes are either not making IFN-i [we can only measure expression as yet]
or are not getting close enough to these macrophages to activate them. The second option seems
more likely, especially given the difficulty T cells probably encounter in moving through the
lymph node tissues due to the rapid development of necrosis.
BCG vaccinated guinea pigs showed reduced numbers of heterophil influx during chronic
infection compared to controls. We further confirmed this by immunohistochemical staining of
heterophils which were localized to foci of central necrosis in the primary lesions and were
reduced in BCG vaccinated animals compared to controls. Previous studies (30) have shown that
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large numbers of heterophils are present within airway lumens associated with secondary lesions
during the chronic stage of the disease. One of the protective properties of BCG vaccination is
that the secondary granuloma formation is delayed and this could be the reason for a reduction in
heterophils. We have previously suggested (30) that the presence of heterophils in the foci of the
primary lesion may be directly related to lesion necrosis and may reflect the continual
degranulation of heterophils in these lesions. In addition, heterophils are directly related with
Th2 immunity, and the cell phenotype responsible for the immunopathogenesis of asthma and
allergic rhinitis (30, 39). Therefore, one protective property of BCG vaccination in the guinea pig
model may be to limit heterophil accumulation in primary lesions and airway lumens associated
with secondary lesions resulting in reduced tissues necrosis.
Unexpectedly, we observed a rapid influx of B cells early into the lungs of the vaccinated
guinea pigs, which then ceased. We speculate that the observed B cell influx may be simply due
to integrin expression on blood vessels as B cells produce pro-inflammatory cytokines, express
chemokine receptors such as CXCR5 and CCR7 that may promote their migration to
inflammatory sites (30). However, in the mouse model at least there is no evidence these cells
aid or interfere with protective immunity (8), although they are found in large sheets in the
granulomas in murine tuberculosis (8).
This new information on the protective properties of BCG vaccination in the guinea pig
model, we propose, could act as a novel template with which to compare the efficacy of new
vaccine candidates in this important animal model. Potential surrogate markers for protection in
the guinea pig model could be an early increased CD45 expression on CD4 and CD8 T cells and
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early MHC class II expression on macrophages as a measure of vaccine efficacy. One advantage
of this would be to shorten experiments by focusing on the subacute phase of infection rather
than waiting for the chronic disease on days 60 and 90. Our study showed another protective
property of BCG vaccination in this model is reduced heterophil influx during chronic infection.
The presence of reduced heterophils could be utilized for a potential surrogate marker for
protection in post M. tuberculosis exposure vaccine testing.
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Figure Legends
Figure 1. Bacterial growth in the lungs and draining lymph nodes in control and
vaccinated guinea pigs infected with M. tuberculosis.
Bacterial growth in organs from control (n=4) (circles)and vaccinated (n=4) (squares) guinea
pigs receiving a low dose aerosol of M. tuberculosis was assayed in the lung (A) and lymph
nodes (B). Groups of control and immune M. tuberculosis infected guinea pigs organs were
assayed for bacterial loads on day 5, 20, 30, 60 and 90 post challenge. Results are expressed as
the mean Log10 bacilli [colony forming units] (CFU) (± SEM, n=4). The degree of
histopathology (C, D) was determined using a lesion scoring system (36) that showed the
significant extent of lung and lymph node disease in the controls compared to the vaccinated
animals. (Student t-test, *pø0.05).
Figure 2. Changes in lung pathology in control and BCG vaccinated guinea pigs.
Representative photomicrographs from sections of formalin-fixed and paraffin embedded lung
and lymph node tissues are shown, collected on days 5, 30 and 90 after M. tuberculosis
challenge. On day 5, perivascular and peribronchiolar accumulations of resident epithelioid
macrophages, granulocytes and lymphocytes were found located in the parenchyma, as well as
close to major airways and blood vessels, in control and vaccinated guinea pigs. By day 30,
lesions in both sets of panels consisted of foci of mixed inflammation with areas of central
necrosis (arrows) composed of nuclear and cytoplasmic debris, whereas this necrosis was not
observed in either organs in vaccinated animals. By day 90, lesions in control animals had
progressed to form multifocal to coalescing inflammation that effaced large areas of tissue
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compared to the minimal tissue damage seen in the vaccinated guinea pigs. In controls
substantial dystrophic mineralization was taking place (arrows) whereas in vaccinated animals
there was minimal necrosis and abundant functional tissue remaining (arrow). Size bar: 20 µm.
Figure 3. Numbers of T cell influx into the lungs and lymph nodes of control and
vaccinated animals. Lung and lymph node cells obtained from control H37Rv infected guinea
pigs (n=4) and BCG-vaccinated (n=4) guinea pigs were assayed by flow cytometry, and numbers
of cells per 1.0 gram of tissue were calculated from these assays. Panels A and B show the total
numbers of MIL4neg
CD4+ cells (circles) and MIL4neg
CD8+ cells (squares), respectively, in the
lungs of control (open symbols) and BCG-vaccinated guinea pigs (closed symbols). Panels C
and D show the totalcells/1.0 gram of tissue of CD4+
CD45+ (circles) and CD8
+ CD45
+ (squares)
T cells in the lungs (C) and lymph nodes (D) of control (open symbols) and BCG-vaccinated
guinea pigs (closed symbols). Panel E shows a representative flow cytometric dot plot from an
M. tuberculosis infected guinea pig demonstating the lymphocytic gating technique utilizes SSC
versus FSC gating. Panel F shows the isotype control. Panel G shows represenative flow
cytometric dot plots from control and BCG vaccinated animals infected 20 days with M.
tuberculosis showing an increase in percentages of CD4+CD45+ and CD8+CD45+ T cells in the
BCG vaccinated animals. Results are expressed as the mean cells/1.0 gram of tissue of each
analyzed cell population (± SEM, n=4). (Student t-test, *pø0.05) compared to H37Rv control
guinea pigs.
Figure 4. Kinetics of influx of macrophages into infected tissues and their expression of
Class-II MHC molecules.
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Lung and lymph node cells obtained from control (n=4) and vaccinated (n=4) guinea pigs were
assayed by flow cytometry for numbers of cells expressing MR-1 on macrophages (circles), with
further analysis of the numbers of these cells that were expressing Class II molecules (squares)
on days 5, 20, 30, 60 and 90 of the infection. Data is shown for the lungs (A) and draining lymph
node cluster (B). Panel C shows a represenative flow cytometric dot plot from an M. tuberculosis
infected guinea pig demonstating the granulocytic gating technique utilizes SSC versus FSC
gating. Panel D shows the isotype control. Panel E shows representative flow cytometric dot
plots from control and BCG vaccinated animals infected 20 days with M. tuberculosis showing
an increase in percentages of macrophage class II in the BCG vaccinated animals. Results are
expressed as the mean number of macrophages (MR-1) and class II molecule expression on cells
(106) /1.0 gram lung tissue (± SEM, n=4). (Student T-test, *pø0.05) compared to control guinea
pigs.
Figure 5. Numbers of B cell and MIL4+ heterophil (granulocyte) influx into the lungs and
lymph nodes.
Lung and lymph node cells obtained from control (n=4) and vaccinated (n=4) guinea pigs were
assayed by flow cytometry for B cells on days 5, 20, 30, 60 and 90 of the infection. Panels A and
B show the total numbers of MIL4neg
B cells (circles) and MIL4+ granulocytes (squares) in the
lungs and lymph nodes of control (open symbols) and vaccinated guinea pigs (closed symbols).
Panel C shows representative flow cytometric dot plots from control and BCG vaccinated
animals infected 30 days with M. tuberculosis showing an increase in percentages of B cells in
the BCG vaccinated animals. Panel D shows representative flow cytometric dot plots from
control and BCG vaccinated animals infected 60 days with M. tuberculosis showing an increase
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in percentages of MIL4+ cells in the control animals. Results are expressed as the numbers of
MIL4neg
B cells and MIL4+ granulocytes (± SEM, n=4). (Student t-test, *pø0.05) compared to
control guinea pigs.
Figure 6. Immunohistochemical staining of cell populations in the lungs of control and
vaccinated animals.
Representative photomicrographs from lung sections after 30 days of M. tuberculosis infection
in the Control H37Rv infected and the BCG-vaccinated groups showing staining patterns for
immunohistochemical analysis of the tissue locations of CD4+ cells (A-D), CD8
+ cells(E-H),
macrophages (I-L), B cells (M-P) and heterophils (Q-T) I control (left panels) and BCG
vaccinated animals (right panels). C denotes necrotic core of the primary granuloma. Arrow
depicts the cellular phenotype. Size bars are 100 µm for each left set of panels, and 10 µm for the
high power images on the right.
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