Institute for Animal Health, Compton, Newbury, Berkshire, United Kingdom
Correspondence: Dr. J. C. Hope, Institute for Animal Health, Compton, Newbury, Berkshire, RG20 7NN, United Kingdom. E-mail: Jayne.Hope{at}BBSRC.ac.uk
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in response to BCG-infected DC. CD3- CD8+
NK-like and CD3+ CD8+ T cells were evident
within the responding CD8+ population. The response was not
MHC-restricted. The NK-like CD3- cells were the major
population producing IFN-. The presence of mycobacteria-reactive,
IFN--secreting CD8+ NK cells in neonatal calves may have
important consequences for the induction of a Th1-biased immune
response.
Key Words: mycobacteria • IFN- • NKT cells • antigen-presenting cells
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(IFN-) and the subsequent activation or
lysis of infected antigen-presenting cells (APC) are crucial to the
induction of antimycobacterial immunity. The importance of IFN- in
antimycobacterial immune responses was demonstrated in IFN- gene
knockout mice, which do not survive challenge with Mycobacterium
tuberculosis or the avirulent vaccine strain Mycobacterium
bovis bacillus Calmette-Guerin (BCG) [2
,
3
]. Humans genetically defective in production of IFN-
or the IFN- receptor also show increased susceptibility to
mycobacterial infection [4
], suggesting a central role
of this cytokine in host defense. In BCG-vaccinated and M.
bovis-infected cattle, the secretion of IFN- by
CD4+ and CD8+ T cells has been demonstrated and
is an important element of the response to mycobacterial challenge
[5
, 6
].
For many intracellular pathogens such as Listeria monocytogenes,
Toxoplasma gondii, and mycobacterial species [7
,
8
], the rapid production of IFN- by natural killer
(NK) cells is an important element in the early host-resistance
mechanism [9
], and this may be important in skewing the
immune response toward a Th1 bias. The activation of NK cells for
IFN- secretion is largely dependent on interleukin (IL)-12 and IL-18
[10
, 11
]. Dendritic cells (DC) are a major
source of these cytokines [12
, 13
] and are
the only APC capable of inducing responses in naïve T
lymphocytes. Because of this, DC are likely to be central to the
induction of antimycobacterial immune responses. Secretion of IL-12 and
IL-18 by DC may be important early in the activation of NK cells, as
well as T lymphocytes, following mycobacterial infection and form an
essential part of the "bridge" linking innate and adaptive
immunity. DC are also shown to express NK receptors, which are
important in recognition events [14
].
In addition to NK cells, NKT cells, which share many properties with NK
cells, also have features common to the T-cell lineage, such as
expression of T-cell receptors and CD4 or CD8 [15
,
16
]. These cells are able to produce large quantities of
IFN-, which requires the presence of IL-12 [17
], and
may also be important in innate responses to pathogens
[15
]. It has been shown that NKT cells may respond
preferentially to lipid antigens presented by CD1 molecules
[18
, 19
], a pathway that has been proposed
to be important in the presentation of mycobacterial antigens
[20
]. In addition, it was demonstrated that NKT cells
recognizing the lipid 
-GalCer required the presence of DC for
activation and secretion of IFN- [21
]. It seems
likely therefore that NK and NKT cells will be activated as a
consequence of the interaction of DC and mycobacteria.
Innate immunity is particularly important in the neonate, where adaptive immune responses are not yet established. It has been suggested that vaccination of neonates may be more efficient than vaccination of adults, especially in the case of BCG. The variable efficacy of BCG vaccination shown for man [22 ] and cattle [23 ] has been linked to previous exposure to environmental mycobacteria, which may induce an inappropriately biased immune response [24 , 25 ]. In studies conducted in the Gambia [26 , 27 ], vaccination with BCG at birth was shown to induce a Th1-biased immune response, whereas vaccination later in life, presumably after environmental exposure to mycobacterial species, was also associated with nonprotective Th2-type cytokine secretion [26 ]. Innate immune mechanisms present in the neonate, such as the rapid activation of NK cells following antigenic exposure, may serve to boost the Th1 bias and enhance protection. The role of innate mechanisms and the effects of environmental mycobacteria are important considerations in designing vaccination strategies for the prevention of tuberculosis.
We have investigated the response of neonatal calves to mycobacterial
antigens using DC as APC. We show here that in calves as young as 1 day
old, there is a population of CD8+ T cells that respond to
mycobacterial antigens. The CD8+ cells respond to
BCG-infected DC by proliferating and by production of IFN-.
CD3- (NK-like) and CD3+ CD8+ cells
comprised the proliferating population that responded to DC infected
with BCG, and the response was found to be non-major histocompatibility
complex (MHC)-restricted. The CD3- CD8+ cells
were the major population producing IFN-.
The presence of mycobacteria-reactive, NK-like CD8+ cells in neonatal calves may have important consequences for the induction of strong Th1-biased immunity upon infection with virulent mycobacteria or upon vaccination.
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Experimental animals
British Holstein-Friesian calves (Bos taurus) were
derived by hysterotomy into a gnotobiotic isolator [28
].
At approximately 8 weeks of age, calves were transferred from the
isolator to a specific pathogen-free unit. Blood samples were also
taken from conventionally reared calves at 1 day, 1 week, and 3 weeks
of age. Some of the cattle used were from a family of MHC-defined
animals, derived and held at the Institute for Animal Health
(Berkshire, UK) [29
]. APC were derived from MHC
homozygous A31/A31 cattle. Neonatal CD8+ cells were
isolated from calves that were the progeny of an A31/A31 bull and
therefore expressed the MHC haplotype A31. In some experiments, DC were
derived from a second animal with the MHC haplotype A18/A18. In
experiments where non-MHC-defined animals were used, autologous DC and
T cells were derived from the same animal.
A group of five gnotobiotic calves (aged 3 weeks) were immunized subcutaneously with 106 cfu BCG Pasteur as previously described [5 ], and the immune response was monitored.
Generation of bovine blood-derived DC
Bovine monocytes were cultured with granulocyte-macrophage
colony-stimulating factor (GM-CSF) and IL-4 to derive DC by a slight
modification of the method described previously [5
].
Peripheral blood mononuclear cells (PBMC) were derived as described
[5
]. In some cases, PBMC were stored in liquid nitrogen
in 10% dimethyl sulfoxide (DMSO)-fetal calf serum (FCS); these were
washed extensively prior to use. Fresh or frozen, PBMC were incubated
with anti-human, CD14-labeled, super-paramagnetic particles
(Miltenyi-Biotech, Bergisch Gladbach, Germany), and labeled cells were
isolated from a Midimacs column (Miltenyi-Biotech) according to the
manufacturer’s instructions. The purity of the cells was evaluated by
flow cytometry and shown in each case to be >98%. Cell viability was
>95%. Cells were adjusted to 8 x 105/ml in
RPMI-1640 medium containing Glutamax-1 (Life Technologies, Paisley,
UK), 10% heat-inactivated FCS, 5 x 10-5
M 2-mercaptoethanol (ME), 50 µg/ml gentamycin [tissue culture medium
(TCM)], 200 U/ml COS cell-derived bovine recombinant (r)IL-4
[30
], and 0.2 U/ml bovine rGM-CSF (units based on
induction of one-half maximal proliferation in bone marrow precursor
cells); 3 ml of this suspension was added per well of six-well plates.
After 3 days of culture, DC were harvested, washed, and resuspended in
TCM without gentamicin. At this time, the cells had acquired morphology
and surface phenotype similar to the cattle monocyte-derived DC
described previously [31
]. DC were cultured for an
additional 2 h with 100 cfu/cell of BCG or in TCM alone (control
DC) and then washed extensively; viable cells were counted. Under these
conditions, >80% of DC were infected with BCG as determined by
Ziehl-Nielsen staining (unpublished results). In all assay systems,
similar results were obtained with BCG Pasteur and BCG Copenhagen.
Purification of lymphocytes
CD4+ and CD8+ cells were isolated from
PBMC following staining with monoclonal antibody (mAb) CC8 or CC63
{mouse immunoglobulin G (IgG)2a; specific for bovine CD4 and CD8,
respectively; [32
]} and anti-mouse IgG2a
super-paramagnetic particles (Miltenyi-Biotech). The purity of the
cells, as evaluated by flow cytometry, was >97%.
Proliferation assays and measurement of IFN-
Purified CD4+ and CD8+ lymphocytes
(105/well) were incubated in triplicate with
104-irradiated DC (20 Gy from a 137Cs source)
in a total volume of 200 µl TCM. Cultures were incubated for 5 days
at 37°C, and 37 mBq [3H]-thymidine (3H-TdR;
DuPont, Stevenage, UK) was added for the final 18 h of culture.
Proliferation was assessed by ß-scintillation counting of
incorporated 3H-TdR. Results are expressed as counts per
minute (cpm) ± SD.
Supernatants were removed from parallel cultures of DC and
CD4+ or CD8+ lymphocyte on day 4 and assessed
for IFN- by enzyme-linked immunosorbent assay (ELISA) as previously
described [33
]. Results are expressed as pg per ml.
Flow cytometric analyses of responding T lymphocytes
Expression of CD3 or the 
T-cell receptor (TCR) by
CD8+ T cells was examined by staining cells with mAb to
CD8 (CC63; IgG2a) and CD3
(MM1A; IgG1; [34
]) or
the TCR (GB21a; IgG2b; [35
]). mAb MM1A recognizes
CD3 [36
] and may not detect CD3
expressed on NK
cells. However, for clarity herein, those cells that did not show
positive staining with mAb MM1A are referred to as CD3-.
Bound antibody was detected with fluorescein isothiocyanate (FITC) or
phycoerythrin (PE)-labeled anti-mouse, isotype-specific reagents
(Southern Biotechnology Associates, Birmingham, AL). The phenotype of
the CD3- CD8+ cells was examined further by
three-color immunofluorescent staining using biotinylated mAb to CD45RB
(CC76; IgG1; [37
]), CD45RO (IL-A116; IgG3;
[38
]), and CD11b (CC94; IgG1; [32
]). In
these experiments, CD8 expression was detected with mAb CC63 directly
conjugated to allo-phycocyanin (CC63-APC), and biotinylated antibody
binding was detected with streptavidin-PE (Southern Biotechnology
Associates). Expression of CD8 was determined by labeling with mAb CC63
(anti-CD8) and CC58, which recognize CD8ß but not CD8
[39
]. Those cells that were stained positive with CC58
and CC63 were considered to be CD8ß, whereas those that are
recognized by CC63 but not CC58 are CD8-positive. In addition,
mAb CC84 was used. The antigen recognized by this mAb has been shown
previously to be expressed by bovine peripheral blood monocytes
[40
] and a population of circulating CD2+
CD3- cells, which may be NK cells (unpublished results).
Immunofluorescent staining was analyzed using Win-MDI software.
Detection of intracytoplasmic IFN- expression
Purified CD8+ cells (105/well) were
incubated with BCG-infected or control DC for 3 days at 37°C.
Brefeldin-A (10 µg/ml), with or without phorbol 12-myristate
13-acetate (PMA; 50 ng/ml) and ionomycin (1 µg/ml), was added for the
final 4 h of culture. Expression of CD3 was detected following
staining with mAb MM1A, and bound mAb was detected with goat anti-mouse
IgG1-PE. The cells were then fixed with 1% paraformaldehyde and
permeabilized (permeabilization solution; Becton Dickinson, Oxford,
UK), and IFN- expression was detected with mAb 6H5 (antibovine
IFN-; IgG2a; [41
]). Bound anti-IFN- antibody was
detected with goat anti-mouse IgG2a-FITC (Southern Biotechnology
Associates). Immunofluorescent staining was analyzed using Win-MDI
software.
Detection of IL-12 and IL-18 mRNA in DC
On day 3 of DC culture, 10 cfu/cell of BCG or TCM was
added, and the DC were cultured for another 24 h. In some
cultures, 1 ng/ml IFN- was added for 24 h prior to the addition
of BCG. Conventional polymerase chain reaction (PCR) was performed as
described previously [33
]. Briefly, mRNA was extracted
from cells following lysis using the Dynabeads mRNA DIRECT kit. The
mRNA was reverse-transcribed using oligo-dT primer and avian
myeloblastosis virus reverse transcriptase (RT). Aliquots of
cDNA were used in PCR for amplification of ß actin, IL-12 p40, and
IL-18. The primer sequences for p40 were: forward,
5'-GCAGTACACCTGTCACAAAG-3', and reverse, 5'-CTACCACGACCTCAATAAGC-3'.
These primers were generated based on published sequences (EMBL
accession numbers U14416 and U11815). IL-18 primers were: forward,
5'-ACTTTGGCAAACTTGAACTTAAG-3', and reverse,
5'-CTAGTTCTGGTTTTGAACAGTGAACAT-3', and were generated on sequence
information obtained from Dr. Declan McKeever (ILRI, Kenya). The
predicted PCR product sizes were IL-12 p40, 325 bp, and IL-18, 470 bp,
and these were confirmed by comparison with 100 bp ladder (Invitrogen,
Paisley, UK). Primers for ß actin were as described previously
[33
].
Statistical analysis
Statistical analyses were performed using a paired
Student’s t-test. P values of <0.05 were
considered significant.
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 View larger version (7K): [in a new window] |
Figure 1. CD8+ cells isolated from gnotobiotic calves proliferate in
response to BCG-infected DC. CD4+ and CD8+
cells were purified from gnotobiotic calves at 1 day (n=3;
a), 1 week (n=3; b), or 3 weeks of age (n=14; c).
Blood-derived DC were incubated with M. bovis BCG (100
cfu/cell) or cultured with TCM (control) for 2 h, then washed,
irradiated, and 104 cultured with 105
CD4+ (open bars) or CD8+ cells (solid bars) for
5 days. 3H-TdR was added for the final 18 h of
culture. Results are expressed as mean cpm ± SD of
triplicate wells. *, P > 0.05 compared with control
DC. **, P > 0.01 compared with control DC.
|
 View larger version (7K): [in a new window] |
Figure 2. CD8+ cells from 1-day-old, conventionally reared calves
proliferate in response to BCG-infected DC. CD4+ and
CD8+ cells were purified from conventionally reared calves
at 1 day (n=3; a), 1 week (n=3; b), or 3 weeks of
age (n=12; c). Blood-derived DC were incubated with M.
bovis BCG (100 cfu/cell) or cultured with TCM (control) for 2 h, then washed, irradiated, and 104 cultured with
105 CD4+ (open bars) or CD8+ cells
(solid bars) for 5 days. 3H-TdR was added for the final
18 h of culture. Results are expressed as mean cpm ±
SD of triplicate wells. *, P > 0.05 compared
with control DC.
|
in response to
BCG-infected DC by ELISA. CD8+ cells isolated from
gnotobiotic calves at 1 week (5/5 calves; P=0.04; Fig. 3 b
) and 3 weeks of age (9/12 calves; P=0.008; Fig. 3c
)
secreted IFN- when cultured with BCG-infected DC. However, although
we detected IFN- secretion in response to BCG-infected DC in
CD8+ cells isolated from gnotobiotic and conventionally
reared calves at 1 day of age, this was not elevated significantly
compared with the response to control DC (Fig. 3a
; unpublished
results). No IFN- secretion was detected from CD4+ T
cells (unpublished results).
 View larger version (6K): [in a new window] |
Figure 3. Secretion of IFN- by mycobacteria-reactive CD8+ cells
isolated from gnotobiotic calves. CD8+ cells were purified
from gnotobiotic calves at 1 day (n=5; a), 1 week
(n=5; b), or 3 weeks of age (n=14; c).
Blood-derived DC were incubated with M. bovis BCG (100
cfu/cell) or cultured with TCM (control) for 2 h, then washed,
irradiated, and 104 cultured with 105
CD8+ cells for 4 days. Supernatants were assessed for
IFN- by ELISA. Results are expressed as pg/ml. *, P > 0.05 compared with control DC. **, P > 0.01
compared with control DC.
|
(Fig. 4d)
. However, by week 3 post-vaccination (when
the animals are aged 6 weeks), these CD8+ responses could
not be detected. CD4+ T-cell responses were detected from
week 6 onward (Fig. 4a
and 4b)
. BCG-specific CD8+
cell-proliferative responses were more transient, and significant
proliferative responses were detected only at weeks 9 and 12
post-vaccination (P<0.05; Fig. 4c
). IFN- secretion by
CD8+ cells post-BCG vaccination was not significantly
different whether BCG-infected DC or control DC were used (Fig. 4d)
.
 View larger version (21K): [in a new window] |
Figure 4. Proliferation and IFN- secretion by CD4+ and
CD8+ lymphocytes derived from BCG-vaccinated, neonatal
calves. Gnotobiotic calves were vaccinated with BCG. At the times
indicated, CD4+ (a, b) and CD8+ (c, d) cells
were purified and cultured with BCG-infected (black bars) or control DC
(gray bars). On day 5, 3H-TdR was added for the final
18 h of culture. Results are expressed as the mean cpm ±
SD. IFN- secretion was assessed in supernatants from
parallel cultures derived at 4 days and is expressed as pg/ml. For
proliferation and IFN-, the mean ± SD for a group
of five animals is shown. *, P > 0.05 compared with
control DC. **, P > 0.01 compared with control DC.
|
TCR+ cells or CD8+ CD3+ cells was
determined by flow cytometry on day 5 (Fig. 5
). No increase in the percentage of T cells was observed
following stimulation with BCG-infected DC (unpublished results),
despite an evident increase in the total number of responding cells
present within the total live lymphocyte gate (Region 1, Fig. 5a
) or in
the large proliferating cell gate (Region 2, Fig. 5a
). In contrast, in
4/4 calves assessed, there was a significant increase in the number of
CD3- CD8+ cells present within the large
proliferating cell gate (Fig. 5b
, representative example at week 1).
Further analysis of the CD3- CD8+ cells
present within the large cell gate (Fig. 6a
and b) demonstrated that the majority of these cells
expresses the CD8ß heterodimer recognized by mAb CC58 (Fig. 6c)
,
and a proportion expresses the CD8 homodimer
(CC58-). There was no expression of the TCR by
these cells (Fig. 6d)
, and the majority expressed moderate levels of
CD45RB (Fig. 6e)
together with low-to-moderate expression of CD45RO
(Fig. 6f)
. Approximately 40–50% of the CD3-
CD8+ cells also expressed CD11b (Fig. 6g)
. The vast
majority of these cells (>90%) also expressed the uncharacterized
antigen recognized by mAb CC84 (Fig. 6h)
. In contrast, the
CD3+ CD8+ cells did not express CD11b or the
CC84 antigen but did express high levels of CD45RO, and the majority
was also TCR-positive (unpublished results), indicating that
these were T lymphocytes.
 View larger version (43K): [in a new window] |
Figure 5. Phenotypic analysis of CD8+ cells from gnotobiotic calves.
CD8+ cells purified from a gnotobiotic calf were cultured
for 5 days with BCG-infected (a, b) or control DC (c, d). CD3 and CD8
expression by cells within the large proliferating cell gate (R2) was
assessed by two-color flow cytometry. The percentage of
CD3- CD8+ cells within R2 is indicated. One
representative experiment of four is shown.
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 View larger version (32K): [in a new window] |
Figure 6. Surface phenotype of CD3- CD8+ cells.
CD8+ cells purified from a gnotobiotic calf were cultured
for 5 days with BCG-infected DC. CD3- CD8+
cells were identified within the large cell gate (R1). Expression of
CD8ß (c), TCR (d), CD45RB (e), CD45RO (f), CD11b (g), and
the uncharacterized Ag recognized by mAb CC84 (h) by the gated
CD3- CD8+ cells (R2) was assessed by
three-color flow cytometry. Open histograms indicate isotype-control
staining. One representative animal is shown.
|
in response to
BCG-infected DC and CD3 was assessed in
fixed and permeabilized cells by flow cytometry (Fig. 7
). The large, proliferating cells were assessed as above. In
cultures without PMA and ionomycin stimulation, there were few IFN-
cells detected (unpublished results). However, upon addition of PMA and
ionomycin, IFN--expressing cells were detected readily. After
stimulation with BCG-infected DC, greater than 80% of the
IFN--expressing cells were within the CD3- population
(Fig. 7b) . The percentage of IFN--positive CD3- cells
increased significantly in response to BCG (lower right quadrant, Fig. 7b
) compared with the response to control DC (lower right quadrant,
Fig. 7d ). In contrast, although the number of CD3+ cells
expressing IFN- was increased following stimulation with
BCG-infected (upper right quadrant, Fig. 7b
) compared with control DC
(upper right quadrant, Fig. 7d
), this was much less significant than
the increase noted for CD3- cells. Staining with
isotype-matched control antibodies is indicated in Figure 7a
(BCG-stimulated CD8+) and Figure 7c
(control DC-stimulated
cells).
 View larger version (35K): [in a new window] |
Figure 7. Intracytoplasmic expression of IFN- by CD8+ cells
following stimulation by BCG-infected DC. CD8+ cells
purified from a gnotobiotic calf were cultured for 3 days with
BCG-infected (a, b) or control DC (c, d). Brefeldin-A, PMA, and
ionomycin were added for the final 4 h of culture. Coexpression of
intracellular IFN- with surface CD3 (b, c) by cells within the large
cell gate (R1) was assessed by flow cytometry. Quadrants were set
according to isotype-matched control antibody staining (a, c).
|
 View larger version (10K): [in a new window] |
Figure 8. The CD8+ cell-proliferative response to BCG-infected DC is
not MHC-restricted. CD8+ cells were purified from
gnotobiotic calves at 3 weeks of age. DC were prepared from MHC-matched
(a) or -mismatched (b) animals and incubated with M. bovis
BCG (100 cfu/cell) or cultured with TCM (control) for 2 h. The DC
were washed, irradiated, and 104 cultured with
105 CD8+ cells for 5 days. 3H-TdR
was added for the final 18 h of culture. Results are expressed as
mean cpm ± SD of triplicate wells. One representative
experiment of three is illustrated.
|
alone (Fig. 9a
, lane 4) induced low
expression of p40 IL-12 mRNA, and there was enhancement of p40 IL-12
mRNA upon stimulation with IFN- and BCG (Fig. 9a
, lane 6). IL-18
transcripts were detected in DC independent of BCG stimulation (Fig. 9b
, lane 3), indicating that bovine DC constitutively express IL-18
mRNA. However, IL-18 mRNA expression appeared to be increased following
incubation with IFN- alone (lane 4), BCG alone (lane 5), or BCG plus
IFN- (lane 6). Levels of ß-actin were similar under all conditions
(unpublished results).
 View larger version (88K): [in a new window] |
Figure 9. IL-12 and IL-18 mRNA expression by DC. DC were cultured for 3 days. BCG
(10 cfu/cell) was added to the DC for an additional 24 h. Where
indicated, 1 ng/ml IFN- was added for 24 h prior to the
addition of BCG. mRNA was isolated, and IL-12 p40 (a) and IL-18 (b)
expression was determined by RT-PCR. The sizes of the PCR products are
indicated by comparison with a 100-base pair ladder. Lane 1, 100 base
pair ladder; lane 2, negative control; lane 3, DC alone; lane 4,
DC + IFN-; lane 5, DC + BCG; lane 6, DC + BCG +
IFN-.
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is a central component of the response. Although
antigen-specific T cells can produce large quantities of IFN-
upon activation, the initial response to pathogen and polarization
of the subsequent T-cell response are likely to depend on rapid
cytokine secretion by cells of the innate immune system, notably NK and
NKT cells. The secretion of large amounts of IFN- by these cells is
dependent on the production of IL-12 and IL-18 by DC. Thus, the
interaction of DC and NK or NKT cells is required to skew the immune
response toward a Th1 phenotype, and this may be particularly important
following infection with intracellular bacteria such as salmonella and
mycobacterial species [7
].
We have demonstrated that in immunologically naïve, neonatal
animals, there is a population of mycobacteria-reactive
CD8+ cells, which respond to mycobacteria-infected DC by
proliferating and secreting IFN- A large proportion of these
CD8+ cells are CD3-, indicating that these are
likely to be NK cells rather than NKT or classical T cells, both of
which express CD3. The mAb used in this study (MM1A) detects CD3 but
not CD3, which has been shown to be expressed on human and murine NK
cells [36
, 42
]. However, the lack of
staining of these cells with mAb MM1A strongly suggests the lack of
expression of the TCR/CD3 complex by these NK cells. The absence of
defined markers for NK cells in cattle makes it difficult to be more
precise as to the nature of these cells. However, the lack of
expression of CD3 combined with secretion of IFN- and their
importance in innate responses to mycobacteria as demonstrated in
immunologically naïve animals suggest that the cells described
here are NK-like. A proportion of the mycobacteria-reactive
CD3- CD8+ cells expressed CD8 and CD11b,
both of which are shown to be expressed on NK cells
[43
44
45
]. However, unlike NK cells in other species, the
majority of the NK-like cells described here expressed CD8ß
heterodimers rather than CD8 homodimers. The reason for this
heterogeneity of CD8 expression by bovine NK cells is not known.
However, it is likely that there are other subsets of NK cells present
within cattle and that the phenotype of these subsets may be diverse.
The diversity of NK cell phenotype may relate to tissue distribution
and may also change with age or immunological status of the animal. For
example, putative NK cells that have been identified in older cattle
were shown not to express CD8 (unpublished results). In addition, cells
with NK activity isolated from adult cattle were shown to express only
CD8 [46
]. Thus, it is likely that examination of
other subsets of NK cells in cattle will identify NK populations that
express CD8 exclusively, as has been described for human and
porcine NK cell populations [43
44
45
]. The differential
expression of CD8 or CD8ß may have implications for the
interaction of these cells with MHC class I-expressing cells
[47
], but the significance of this in the bovine system
is not known.
The NK cells did not express the TCR, and their proliferation
was not restricted by MHC class I, suggesting that these cells are
TCR-negative, NK-like cells. The majority of the NK-like cells
expressed the uncharacterized antigen recognized by mAb CC84. Within
the periphery, the majority of cells expressing the CC84 antigen are
CD14-positive monocytes. However, in addition, there is a small
population of CC84 Ag-positive cells that are CD14-negative but that
coexpress CD2, CD11b, and CD45RB and are phenotypically similar to the
NK cells described herein (ref. [48
]; unpublished
results). These NK-like cells also respond strongly to IL-15, an NK
growth factor (unpublished results). Therefore, it seems likely that
this antibody may be useful in the identification of bovine NK cells.
The CD3- CD8+ cells were the major producers
of IFN- following culture of the CD8 cells with BCG-infected DC,
indicating that these might be important in biasing the immune response
toward the Th1 phenotype. Taken together, the data suggest that
BCG-infected DC stimulate the activation of NK cells that are present
in neonatal calves.
In addition to the CD3- CD8+ NK-like cells,
there is also proliferation of CD3+ CD8+ T
cells following culture with BCG-infected DC. These CD3+
CD8+ cells were shown to be CD11b- and
CC84-antigen-negative, CD45RO-positive T lymphocytes, the majority of
which expressed the TCR. In mouse spleen-cell cultures
stimulated with Burkholderia pseudomallei or L.
monocytogenes, it has been demonstrated that there was significant
bystander activation of CD3+ CD8+ T cells, as
well as specific stimulation of NK cells [9
]. This
secondary activation of CD8+ T cells served to potentiate
the overall response to antigen. An important role was suggested for
IL-12 and IL-18 in this response, and IL-12 and IL-18 have been
suggested previously as important mediators of innate responses.
Infection of bovine DC with BCG has been shown here to induce increased
expression of IL-12, which is likely to be important in the stimulation
of bovine NK activity. The expression of IL-12 by DC following
infection with BCG was augmented by IFN-, suggesting a positive
feedback-loop mechanism whereby IL-12 potentiates IFN- secretion,
which in turn up-regulates IL-12 expression. The constitutive
expression of IL-18 by bovine DC may also contribute to the induction
of IFN- secretion by bovine NK cells. Infection of DC with BCG also
increased the expression of IL-18 mRNA, suggesting this cytokine may be
involved in the responses induced following BCG infection of DC. The
interaction among IL-12, IL-18, and IFN- is likely to be central to
the initiation of Th1-biased, immune responses.
The cytolytic capacity of NK cells is a distinguishing feature of this
cell population. However, we (unpublished results) and others
[49
, 50
] have not been able to successfully
demonstrate cytotoxicity of bovine NK cells. This has been attributed
to the lack of suitable target cell lines that are sensitive to bovine
NK cell-mediated lysis. One of the few studies of bovine NK cytolytic
activity used bovine Herpes virus-infected target cells
[51
], but there have been no studies of NK-mediated
lysis shown against "classical" NK targets such as YAC-1 cells.
Current studies are aimed to develop assay systems to measure NK
cytolysis. Recently, Jacobs and colleagues [52
]
described subsets of human NK cells that are characterized
phenotypically as CD56bright or CD56dim. These
subsets varied reciprocally with respect to IFN- production and
cytolytic capacity. Thus, the CD56bright cells that
secreted high levels of IFN- were poorly cytolytic and were
suggested to regulate immune responses by cytokine secretion rather
than by cytolysis [52
]. The bovine NK cells
described herein may represent an equivalent subset of NK cells that
secrete high IFN- with little cytolytic capacity and as such, may be
important regulators of Th1-biased immune responses.
The mycobacteria-reactive CD8+ cells were identified in
gnotobiotic calves up to at least 3 weeks of age. However, in calves
reared conventionally, and thus exposed to environmental microbial
stimuli from birth, these cells were only identified within the first
few days of birth. This may reflect movement of these cells from the
periphery to other sites such as the gut or total removal of these
NK-like cells. It seems possible that the exposure of these animals to
environmental microbial stimuli may affect the response or availability
of the CD8+ mycobacteria-reactive, NK-like cells. In
support of this hypothesis, within 3 weeks following BCG vaccination of
gnotobiotic calves, mycobacteria-reactive CD8+ cells were
not present in the periphery. However, BCG-reactive, memory
CD8+ T cells were observed in calves 9 and 12 weeks
post-BCG vaccination. Vaccination of neonates with BCG was shown to
induce vigorous antigen-specific CD4+ T-cell responses,
which were similar in magnitude to those observed in older,
conventionally reared animals [5
]. These data suggest
that vaccination of neonatal animals with BCG induces effective immune
responses. Vaccination with BCG later in life has previously been shown
to provide variable levels of protection against virulent challenge in
cattle and humans [22
, 23
]. It has been
suggested that exposure to environmental mycobacteria and subsequent
induction of inappropriately biased immune responses are the major
factors affecting BCG efficacy [24
, 25
].
Studies of infants in the Gambia showed that vaccination at birth was
associated with Th1 bias and secretion of IFN-, whereas vaccination
after a few months was less likely to induce IFN- and was associated
with more IL-4 production [26
]. In this study,
nonvaccinated infants acquired responsiveness to purified protein
derivative within 2 months of birth, and this was associated
with significant IL-4 secretion, suggesting Th2 bias
[26
]. If exposure to environmental bacteria alters the
innate response to mycobacteria such that there is reduced stimulation
of NK cells and IFN- secretion, then this may not allow maximal Th1
stimulation and result in altered Th bias upon mycobacterial challenge.
This may have important consequences for the induction of protection by
antimycobacterial vaccines and should be a consideration when designing
control and vaccination strategies. The data described herein for
M. bovis BCG in cattle may be relevant for studies of the
immune response in human newborns. Cattle are immunologically competent
from one-third to halfway through gestation and are born with a
competent but immature immune system [53
]. In this
respect, neonatal calves are similar to newborn humans and are unlike
mice, suggesting that results obtained in neonatal cattle may be more
relevant to human studies than those performed in mice.
Innate IFN- production by peripheral blood cells derived from young,
immunologically naïve cattle has also been demonstrated
recently in response to antigens from Mycobacterium avium subsp.
Paratuberculosis [54
]. Although the cell population
responsible for the IFN- secretion was not determined definitively,
the majority of the IFN--secreting cells was suggested to be NK
cells, and a proportion of these were CD8+. Thus, secretion
of innate IFN- by NK cells may be an important feature of the early
immune response to mycobacteria in cattle.
In summary, we describe a population of CD8+ cells in
neonatal calves that respond to mycobacterial antigens. The presence of
these mycobacteria-reactive, NK-like CD8+ IFN--secreting
cells in neonatal calves may have important consequences for the
induction of strong Th1-biased immunity following vaccination or upon
virulent mycobacterial challenge.
Received August 21, 2001; revised October 11, 2001; accepted October 15, 2001.
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interferon by natural killer cells from Toxoplasma gondii-infected SCID mice: regulation by interleukin-10, interleukin-12, and tumour necrosis factor Infect. Immun. 62,2818-2824 producing CD4+ natural killer T cells by Mycobacterium bovis bacillus Calmette Guerin Eur. J. Immunol. 29,650-659[Medline] production by CD4+ T lymphocytes Eur. J. Immunol. 31,1531-1535[Medline] production Vet. Immunol. Immunopathol. 68,193-207[Medline] TcR Vet. Immunol. Immunopathol. 52,275-283[Medline] production in cattle in response to MPP14, a secreted protein from Mycobacterium avium subsp. Paratuberculosis Scand. J. Immunol. 54,306-313[Medline]This article has been cited by other articles:
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