tuberculosis in aged γδ t cell gene disrupted mice

Tuberculosis in aged gd T cell gene disrupted mice

J. Turnera,*, A.A. Frankb, J.V. Brooksa, P.M. Mariettaa, B. Vesoskya,I.M. Ormea

aDepartment of Microbiology, Mycobacteria Research Laboratories, Colorado State University,

Fort Collins, CO 80523, USAbDepartment of Pathology, Colorado State University, Fort Collins, CO 80523, USA

Received 17 July 2000; received in revised form 7 September 2000; accepted 8 September 2000

Abstract

In young mice exposed to aerosol infection with Mycobacterium tuberculosis removal of the gd T

cell population by targeted gene disruption does not affect the expression of host resistance, but does

in¯uence the integrity of the early granulomatous response. The current study demonstrates that in

aged gd T cell gene disrupted mice similar immunopathologic changes ensued in both gene knockout

and wild type control mice. Changes in cell surface marker expression, evident in other gene knock-

out models, was not observed in the aged gd T cell knockout mice. These data imply that gd T cell

functions previously observed in young mice become much less important as the animal ages.

q 2001 Elsevier Science Inc. All rights reserved.

Keywords: Murine; gd T cell; Aging; Infectious disease; M. tuberculosis; Lung

1. Introduction

The role of gd T cell receptor-bearing T lymphocytes in immunity to intracellular

bacterial pathogens remains elusive. It was initially proposed that gd T cells acted as an

innate ®rst line of defense (Janis et al., 1989; Inoue et al., 1991), re¯ecting their distribu-

tion in the skin and mucosal surfaces (Bonneville et al., 1988; Kuziel et al., 1987). In terms

of immunity to Mycobacterium tuberculosis infections, an early report showed that gd T

cells quickly accumulated at the site of inoculation with dead mycobacteria delivered in an

oil adjuvant vehicle (Grif®n et al., 1991). This followed, however, by the demonstration

that the gd T cell in¯ux following live mycobacterial infection was more modest and

followed the course of the infection (Grif®n et al., 1991).

J. Turner et al. / Experimental Gerontology 36 (2001) 245±254 245

Experimental Gerontology 36 (2001) 245±254www.elsevier.nl/locate/expgero

0531-5565/01/$ - see front matter q 2001 Elsevier Science Inc. All rights reserved.

PII: S0531-5565(00)00199-6

* Corresponding author. Tel.: 11-970-491-6587; fax: 11-970-491-1815.

E-mail address: [email protected] (J. Turner).

It is known that gd T cells can recognize mycobacterial antigens (O'Brien et al., 1989;

Tsukaguchi et al., 1995; Kabelitz et al., 1990), and can secrete gamma interferon [IFN-g]

(Garcia et al., 1997), the key cytokine in immunity to tuberculosis (Cooper et al., 1993;

Flynn et al., 1993). However, a more recent study (D'Souza et al., 1997) using mice in

which the gene encoding the constant region of the gamma chain had been disrupted by

targeted gene technology [ªgd knockout miceº] revealed that the absence of this T cell

population did not appreciably alter the capacity of the animal to express protective

immunity to an aerosol infection with M.tuberculosis. What was apparent, however,

was that the integrity of the early granulomatous response was altered, taking a pyogenic

form in which the usual mononuclear cell in¯ux was mixed with polymorphonuclear cells,

predominantly neutrophils. Accordingly, we have proposed (Orme and Cooper, 1999) that

the primary role of gd T cells during pulmonary tuberculosis is to control, probably

through secretion of appropriate chemokines, the orderly formation of the granuloma

structure.

It is well established (Weksler, 1995; Miller, 1995) that when mice age they undergo

dysfunctional changes in terms of their immune response, which gives rise to an increased

susceptibility to infections, tumors, and other diseases. In immunity to tuberculosis, old

mice undergo an early period of transient resistance to pulmonary disease (Cooper et al.,

1995) which remains poorly understood, followed later by progressive disease and even-

tual death. Changes in protective immunity are more subtle than dramatic, and include a

slower expression of certain cell surface markers (Ernst et al., 1993; Orme and Roberts,

1998), and a poor constitutive IL-12 response in lung tissue (Cooper et al., 1995).

Because gd T cells seem to contribute to appropriate granuloma formation, we hypothe-

sized whether this cell population would play a similar role as the animal ages. To address

this, aging wild type control mice and age-matched gd T cell knockout (KO) mice were

infected by aerosol with M.tuberculosis and the course of the infection followed against

time. It was found, in similarity to young mice, that the lack of gd T cells did not in¯uence

the early course of the infection. Where they differed, however, was in terms of the

observation that a pyogenic in¯ammation was not seen in the old mice, which instead

showed a progression in lung pathology similar to aged controls. These data therefore

imply that the anti-in¯ammatory role proposed for gd T cells in young mice appears to be

much less important in mice as they age.

2. Materials and methods

2.1. Mice

Speci®c pathogen free female 6±8 week old C57BL/6, and T-cell receptor Cg mutant

mice (C57BL/6j-Tcrd) were purchased from Jackson Laboratories (Bar Harbor, Maine).

Mice used for aging studies were kept in house for approximately 16±24 months and

maintained with sterile water, bedding, and chow. Infected mice were kept in ABL-3

biohazard facilities throughout the experiments. The speci®c pathogen free nature of

the mouse colonies was demonstrated by testing sentinel animals. These were shown to

be negative for 12 known mouse pathogens.

J. Turner et al. / Experimental Gerontology 36 (2001) 245±254246

2.2. Bacterial infections

M.tuberculosis Erdman was originally obtained from the Trudeau Mycobacteria Collec-

tion, Saranac Lake, N.Y. Bacteria were grown in Proskauer±Beck liquid medium contain-

ing 0.05% Tween 80 to mid log phase and frozen in aliquots at 2708C until needed. Mice

were infected by the aerosol route with a low dose of bacteria. Brie¯y, the nebulizer

compartment of an airborne infection apparatus (Middlebrook, Terre Haute, IN.) was

®lled with a suspension of bacteria resulting in the uptake of 50±100 viable bacteria

per lung during a 30 min exposure. The numbers of viable bacteria in the lung, and spleen

were determined at various timepoints by plating serial dilutions of whole organ homo-

genates onto Middlebrook 7H11 agar (Life Technologies, Gaithersburg, MD) and count-

ing bacterial colonies after 21 d of incubation at 378C. The data are expressed as log10

value of the mean number of bacteria recovered per organ �n � 4 animals).

2.3. Histological analysis

The lower right lung lobe was obtained from each mouse and in¯ated with, and stored

in, 10% formal saline. Tissues were prepared routinely for light microscopy and sectioned

with lobe orientation designed to allow for maximum surface area of each lobe to be seen.

Tissue was stained with hematoxylin and eosin to determine granuloma formation. Tissue

sections were examined by a veterinary pathologist without prior knowledge of grouping,

and data is shown as one photograph representative of the individual groups.

2.4. Flow cytometry

A single cell suspension was prepared from spleens as described previously (Orme et

al., 1993). Pooled spleen cells from each individual group were incubated with speci®c

antibody (FITC, PE, or biotin labeled at 25 mg/ml) for 30 min at 48C and in the dark. After

two washes in D-RPMI lacking biotin and phenol red (Irvine Scienti®c, Santa Ana, CA)

cells were then incubated with Red670 labeled streptavidin (Gibco-BRL, Grand Island,

NY) for a further 30 min. Cells were washed three times prior to analysis on a Becton

Dickinson FACSCalibur. Lymphocytes were gated by forward and side scatter, and CD41

and CD81 T cells characterized by the presence of speci®c ¯uorescent labeled antibody.

Cell surface markers analyzed were ¯uorescein labeled CD4 or CD8, phycoerythrin

labeled CD44, and biotin labeled CD45RB, CD62L, CD11a, and CD54. Appropriate

isotype control antibodies were included in each analysis. All antibodies were purchased

from Pharmingen (San Diego, CA). Data was analyzed using CellQuest (Becton

Dickinson, San Diego, CA).

2.5. Isolation of cells from infected lungs

Mice were euthanised and the pulmonary cavity opened. The lung was cleared of blood

by perfusing through the pulmonary artery with 10 ml of saline containing 50 units/ml of

heparin (Sigma, St Louis, MO). Lungs were removed from the pulmonary cavity and

placed in cold RPMI (Life Technologies, Gibco-BRL, Grand Island, NY). After removing

the connective tissue and trachea, the lungs were disrupted using sterile razor blades and



Fig. 1. Growth curve of M. tuberculosis in old wild type and gd T cell de®cient mice. Wild type (open circles), and

mice de®cient in gd T cells (closed circles), were infected with M. tuberculosis via the aerosol route at 16±24

months in age. The growth curve of M. tuberculosis in the lung (A) and spleen (B) was similar for both wild type

and gd T cell de®cient aging mice. Data are expressed as the mean value from four individual mice, plus SEM.

Representative graph from two independent experiments.

incubated for 45 min at 378C in a ®nal volume of 2 ml RPMI containing collagenase XI

(0.7 mg/ml, Sigma) and type IV bovine pancreatic DNAse (30 g/ml, Sigma). 10 ml of

RPMI was added to stop the action of the enzyme. Digested lungs were then gently

dispersed through a nylon screen, and centrifuged at 300 g. Remaining red blood cells

were lysed using Gey's solution. Cells were resuspended in de®cient RPMI (Irvine

Scienti®c, Santa Ana, CA). Lung cells were stained with anti-CD3e and anti-gd TCR

and analyzed on a FACSCalibur as described previously.

3. Results

3.1. Early resistance against M. tuberculosis in old mice is not modulated in the absence of

gd T cells

Aging gd T cell gene KO mice, and age-matched wild type control animals were

infected with a low dose aerosol inoculum of M.tuberculosis and the ability of these

animals to initially control the infection was assessed. It was observed (Fig. 1A) that

aged gd T cell gene KO mice were no more susceptible to M.tuberculosis infection

than wild type control mice over the ®rst 70 days in the lung. In similarity to observations

in young mice, dissemination to the spleen was moderately delayed in the gd T cell gene

disrupted mice (Fig. 1B). Data is presented from four mice per group at each individual

timepoint, and is representative of two independent experiments.

3.2. Cellular in¯ux into the lung is not impaired in gd T cell de®cient mice

We have observed elsewhere (Cooper et al., 1995) that, if anything, old mice exhibit an

initially faster cellular in¯ux into the infected lung tissues compared to young mice and

this is associated with an early, transient, increased resistance. The experiments described

above did not support a direct protective role for gd T cells in old mice but it remained


Fig. 2. Cellular in¯ux in the lung of infected old gd T cell de®cient mice was not altered. Wild type mice (A) and

gd T cell de®cient mice (B) were both shown to have lesions consisting of macrophages and lymphocytes after

45 d of infection with M. tuberculosis.


Table 1

gd T cell populations within the lungs of infected wild type mice. Lung cells were isolated from old or young wild

type mice which had been aerosol infected with M. tuberculosis. Cells were stained with anti-CD3e and anti-gd

TCR. Percent gd T cells within the lung were calculated using Cellquest software and expressed as the mean

value from four mice plus SEM. Statistical analysis was calculated using the Student t test

Time post infection Mean value (n� 4) SEM P value

Day 14 young 4.20 2.0 0.377

Day 14 old 7.00 2.5

Day 21 young 1.68 0.3 0.050

Day 21 old 3.30 1.1

Fig. 3. Chemokine expression in the lungs of infected old gd T cell de®cient mice. Lung lobes were collected

from individual mice at intervals throughout the infection. Total RNA was isolated, transcribed, and probed using

primers speci®c for HPRT, TNF, MIP-1a , and MCP-1. Three individual old wild type mice (WT), and three

individual gd T cell de®cient mice (gd-KO) are presented at either 21 (A) or 45 (B) d post infection.


Fig. 4. Homing molecules expression was unaltered in gd T cell ef®cient old mice. Splenocytes from infected old

mice were stained with ¯uorescent labeled antibody against several T cell surface antigens. Lymphocytes were

gated by forward and side scatter, and CD41 (panel A) and CD81 (panel B) T cells characterized by the presence

of speci®c ¯uorescent labeled antibody. Wild type mice is the shaded histogram, gd T cell de®cient mice the open

histogram, solid line.

possible that they could be in¯uencing the granulomatous response, as observed in young

mice (D'Souza et al., 1997).

It was found however, that no discernable differences were apparent in lung pathology

between aged controls and gd T cell KO mice over this early stage of the disease, and that,

furthermore, no evidence was found for any extensive pyogenic response known to occur

at this time in young mice (D'Souza et al., 1997). Instead, as shown here by representative

sections (Fig. 2) a similar pattern of a mixed lymphocyte and macrophage cellular in¯ux

was observed in both sets of mice. During the ®rst 21 days of infection with M. tubercu-

losis, old wild type mice showed a modest increased in®ltration of gd T cells into the

infected lung in comparison to young mice (Table 1) although these differences did not

alter the course of infection.

3.3. gd T cells in¯uence early chemokine production in the infected lung

gd T cells have previously been shown to in¯uence the cellular in¯ux into the infected

lung, and therefore the expression of several chemokines were measured in the lungs of

wild type and gd KO mice. Lung tissue was collected throughout the course of the

experiment, RNA isolated, and probed for the presence of TNF, MCP-1, MIP-1a and

RANTES. As Fig. 3A demonstrates, in the absence of gd T cells, old mice which have

been infected with M. tuberculosis expressed more TNF and MIP-1a within the lung

tissues than do the young mice after 21 d of infection. MCP-1 was scarcely detectable

within the lungs of either group after 21 d. Despite these early differences in chemokine

expression, at 45 days post infection (Fig. 3B) the detection of chemokine message was

equal within the lungs of infected young and old mice. RANTES was hardly detectable at

either 21 or 45 days post-infection (data not shown).

3.4. gd T cells do not in¯uence the expression of homing molecules on lymphocytes

We have recently observed elsewhere (Turner et al., unpublished observations) that the

expression of activation and/or homing cell surface molecules on T cells in old mice can

sometimes differ if a gene KO mouse model is used. For example, we have documented

substantial differences in marker expression by CD41 T cells in aged CD81 T cell KO

mice that do not occur in aged wild type controls. To determine if this was the case in gd T

cell KO mice spleen cells from infected mice were analyzed for several cell markers. As

shown in Fig. 4 no differences were observed.

4. Discussion

The results of this study show that the absence of a functional gd T cell population in

aged mice did not compromise their ability to express resistance to a pulmonary tubercu-

losis infection. These mice were also capable of mounting an adequate and necessary

lymphocytic granulomatous response. Finally, no compromise of the ab T cell population,

at least in terms of cell marker expression, was observed in the gd T cell KO mice. These

data thus support the hypothesis that gd T cells play a minimal role in aged mice infected

with tuberculosis.


In fact, very little is known about the gd T cell in old individuals. There is some

suggestion that the gd T cell does appear to undergo a transition from a polyclonal limited

T cell receptor repertoire to a form of oligoclonal T cell receptor expression in the elderly

(Giachino et al., 1994). This shift may be in response to repeated exposure to conserved

antigens, but the possibility that the reduction in the gd T cell repertoire may have

consequences in terms of the ability to control intracellular bacterial infections is not

supported by the ®ndings of the study presented here.

In fact, the only major difference seen in this study was the complete absence of any

breakdown in the integrity of the granuloma in the aged gene KO mice. In a previous study

(D'Souza et al., 1997), we consistently observed a pyogenic form of granulomatous

response in young gene KO mice infected with tuberculosis by the respiratory route, in

which the cellular in¯ux comprised of a mixture of lymphocytes and macrophages, but

also a similar number of neutrophils. This was most prominent in these young animals

around 30±40 days of the infection, but was not seen in the current study over this time

frame or at any other time.

Based upon the observations in young mice, we have hypothesized elsewhere (D'Souza

et al., 1997) that the role of gd T cells in pulmonary tuberculosis is not protective, as others

have suggested (Ladel et al., 1995), but instead involves the correct orderly formation of

the granuloma. These cells appear to be important in the production of chemokines such as

MCP-1, although whether this is a direct secretion by the gd T cells or from local lung

tissue cells stimulated by TNF secretion by gd T cells remains unresolved (Orme and

Cooper, 1999). Either way, the presence of gd T cells in young mice seems to contribute to

the regulation of cell traf®cking. We did observe some minimal early differences in the

production of both MCP-1 and TNF in the lung tissues of gd-KO mice however, contrary

to expectations, this production was enhanced in the gd KO mice. It does however,

con®rm the hypothesis that gd T cells are involved in cellular recruitment but as has

been demonstrated here, this does not appear to be of great consequence on the course

of M. tuberculosis in the lungs of old mice.

The lack of need for the gd T cells in the old mice may re¯ect compensatory mechan-

isms by other T cells. Early resistance in old mice to pulmonary tuberculosis seems to be

associated with an enhanced capacity to recruit lymphocytes into the lungs, and recent

studies in our laboratory (Turner et al., submitted for publication) seem to suggest that the

predominant population in such animals express the CD81 CD44hi phenotype. It is well

known that such cells are capable of producing TH1-type cytokines including IFN-g and

thus their early presence may obviate the need for the presence of gd T cells. If so, then this

mechanism differs signi®cantly from known mechanisms operative in young mice in

which CD41 T cells are of primary importance whereas the role of CD81 T cells seems

to occur much later when the infection is in a chronic state (D'Souza et al., submitted for

publication). Such issues thus continue to support the contention that the regulation of

immunity in the aged lung is still poorly understood.

Acknowledgements

This work was supported by NIH grant AG-06946.


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tuberculosis in aged γδ t cell gene disrupted mice

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