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* Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital, and Harvard Medical School, Boston, MA 02115; and
Antigenics Inc., Woburn, MA 01801
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-microglobulin on the surface of dendritic
cells and other APCs. The CD1 proteins show limited but significant
homology to MHC class I and II Ag-presenting molecules that present
peptide Ags to T cells (2). This homology of structure
provided a clue to the possible function of CD1 proteins in the
presentation of foreign Ags to T cells. However, CD1 proteins are
distinct from MHC-encoded molecules by the marked abundance of
hydrophobic amino acids in the membrane distal domains. This finding
suggested that CD1 proteins might have a different type of Ag binding
site than the peptide binding groove found in MHC molecules
(2). This prediction has been confirmed by the crystal
structure of a mouse CD1 protein, which shows a deep internal cavity
formed by the 
1 and 2 domains which is lined almost entirely by
hydrophobic amino acid side chains (3)
The unique structure of the Ag binding groove of CD1 suggested that CD1
binds and presents something other than peptides. Direct evidence now
confirms this hypothesis, as several studies have shown that the human
group 1 CD1 proteins (i.e., the group composed of CD1a, CD1b, and CD1c)
mediate specific T cell recognition of mycobacterial lipid and
glycolipid Ags (4). Ags identified to date include mycolic
acid, glucose monomycolate, lipoarabinomannan, and other
phosphoglycolipids (5, 6, 7, 8). T cells recognizing
mycobacterial lipid Ags presented by CD1 can release IFN-
and show
cytolytic activity upon contact with lipid Ag-pulsed target cells and
mycobacteria-infected monocytes. Furthermore,
CD8+ CD1-restricted T cells have also been
recently shown to mediate a direct microbicidal effect on intracellular
microorganisms by granulysin-mediated killing (9). These
activities suggest a proinflammatory role for these T cells in type 1
cellular immune responses that are critical for the elimination of
intracellular pathogens such as Mycobacterium tuberculosis
(MTb)5 and
Mycobacterium leprae (10).
Whereas previous work establishes the function of CD1 in Ag recognition by T cells, the critical question of the role of this system in host defense against infection in vivo has not been examined. Evaluation of the in vivo role of the CD1 system in the protective immune response to pathogens such as mycobacteria will require the development of a suitable animal model in which such responses are generated and can be studied. In the case of MTb infection, such an animal model would ideally be one that accurately replicates the important immunopathological and clinical features of human tuberculosis and also must have a CD1 system comparable to that which is present in humans. Mice and rats are probably not suitable animal models in this case because they express only CD1d-like molecules and lack obvious homologs of the human group1 CD1 proteins (i.e., CD1a, b, and c) which are all known to present microbial lipid Ags (4). In contrast, our previous studies suggested that the guinea pig may prove to be an excellent animal model for studying group1 CD1-dependent immune responses in MTb infection (11).
We have already reported that the guinea pig genome contains an extended family of CD1 genes, with at least eight distinct CD1 homologs (11). Based on analyses of predicted amino acid sequences, it was possible to classify each guinea pig CD1 gene as a homolog of one specific human CD1 isoform. This revealed that the guinea pig has genes that encode multiple homologs of human CD1b (i.e., guinea pig CD1B1, B2, B3, and B4) and CD1c (CD1C1, C2, and C3), in addition to at least one gene encoding a CD1e-like protein. In the current study, we demonstrate the existence of CD1-restricted nonpeptide lipid Ag-reactive T cells in guinea pigs. These CD1-restricted T cell responses were shown to be induced in vivo following immunization with MTb lipid Ags and led to the generation of cytotoxic T cell responses restricted by several different members of the guinea pig group 1 CD1 family. These findings provide further justification for the use of the guinea pig as a relevant small animal model for study of the in vivo role of CD1-mediated immune responses to lipid Ags in host defense against MTb and other microbial pathogens.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Outbred female Hartley guinea pigs were purchased from Charles River Breeding Laboratories (Wilmington, MA). Inbred strain 2 guinea pigs were obtained from Tenovus Research Laboratory (Southampton, U.K.) and bred in our animal facility at the Dana-Farber Cancer Institute. Animals were housed under specific pathogen-free conditions and all animal experiments were performed according to the Guidelines on Animal Welfare approved by the committee on animal use at Harvard Medical School and the Brigham and Women’s Hospital.
Monoclonal Abs
CT5 (anti-pan guinea pig T cell), CT6 (anti-guinea pig CD8), and CT7 (anti-guinea pig CD4) were obtained from Serotec (Raleigh, NC). CD1F2/6B5 (mouse IgG1), reactive with all of the currently known isoforms of guinea pig group 1 CD1, was produced in our laboratories from a hybridoma cell line generated from splenocytes of a mouse hyperimmunized with guinea pig CD1 proteins (12). A hybridoma line producing mAb IVA12 (anti-human MHC class II monomorphic determinant and cross-reactive to guinea pig MHC class II) was obtained from the American Type Culture Collection (Manassas, VA).
Purification of lipid Ags from MTb
Lipid Ags were purified as previously described
(13). Briefly, total sonicates (10 mg dry weight
bacteria/ml) of MTb (strain H37Ra) in PBS were extracted with four
volumes of chloroform/methanol (2:1, v/v) according to the method of
Folch et al. (14). After phase separation, the organic
phase was collected, dried by rotary evaporation, and resuspended in
the original starting volume in chloroform for storage. For further
subfractionation of MTb total lipids, silica column chromatography was
used (5). After evaporation of the solvent from the MTb
total lipid extract, the lipids were resuspended in hexane and passed
over open silicic acid columns to which all major lipid classes bind.
The columns were then eluted with solvents of increasing polarity
(chloroform followed by acetone and finally methanol) to elute the
major lipid fractions in order of increasing polarity (neutral lipids
followed by glycolipids and phospholipids in sequential fashion). After
elution from silicic acid columns, all lipid fractions were dried under
a nitrogen stream and then redissolved in chloroform for storage.
Mycolic acids were released from dried MTb cell walls by saponification
and purified using C18 reverse-phase HPLC as
previously described (5). The composition of lipid
preparations was assessed using analytical TLC with development of TLC
plates by charring in sulfuric acid to reveal all lipid species.
Further characterization of lipid preparations was conducted using
special stains of TLC plates that revealed the presence of carbohydrate
(orcinol), amino groups (ninhydrin), and phosphorous (molybdenum blue).
In addition, to assess the possibility of protein contaminants, amino
acid composition using standard methods of acid hydrolysis followed by
reverse-phase HPLC was conducted by the Harvard Microchemistry Facility
(Cambridge, MA). Based on this analysis, the maximum possible levels of
protein contamination in lipid Ag fractions used in this study were as
follows: MTb total lipid, 4.05%; neutral lipid fraction, 0.06%;
glycolipid fraction, 0.19%; phospholipid fraction, 6.41%; and mycolic
acid, 0.33%. These values most likely represent overestimates of
protein content since the majority of amino acids detected (
70%)
were alanine and glutamate/glutamine, probably originating from
mycobacterial peptidoglycan rather than proteins (15).
Consistent with this, silver staining of SDS-PAGE gels (sensitivity of
<10 ng for a single protein band) revealed no visible band in a
100-µg sample of the MTb total lipid Ag preparation, indicating
protein contamination of <0.01% for any single protein constituent
(data not shown). Each of the lipid fractions was dried under a stream
of nitrogen and then resuspended by sonication into complete medium
(RPMI 1640; Life Technologies, Grand Island, NY) with 10% FCS (HyClone
Laboratories, Logan, UT) and additional supplements as previously
described for in vitro use in T cell recognition assays
(16).
Liposome preparation for immunization of guinea pigs with MTb lipid Ags
Liposomes containing MTb lipid Ags with or without adjuvants were prepared using the dehydration/rehydration method (17, 18). Briefly, 50 mg of 1,2-distearoyl-sn-glycero-3-phosphocholine (Avanti, Alabaster, AL) and 25 mg of cholesterol (Avanti) were dissolved in 5 ml of chloroform and added to a clean round-bottom vacuum flask. For those preparations that contained monophosphoryl lipid A (MPL) as adjuvant, 1 ml of chloroform containing 1 mg of MPL from Salmonella minnesota R595 (Ribi, Hamilton, MT) was added (19). Approximately 2 mg of each of the MTb lipid Ag fractions (glycolipids, neutral lipids, phospholipids, and mycolic acids as indicated) dissolved in chloroform were added, and the entire lipid mixture was dried to a thin film using a Rotovap flash-evaporator (Buchler Instruments, Fort Lee, NJ). Five milliliters of distilled water was then added to the flask, and the lipid was suspended into the solution as liposomes by sonicating in a water bath sonicator (Fisher Scientific, Pittsburgh, PA). For those preparations that contained QS-21 as adjuvant, 150 µg of QS-21 (Antigenics, Woburn, MA) was added during the rehydration step. In some experiments, liposomes containing OVA were made by adding soluble OVA (900 µg) during the rehydration step. Liposome suspensions were lyophilized for storage at -20°C and immediately before use were resuspended by adding an appropriate volume of PBS and passing three times through a 25-gauge needle.
Immunization of guinea pigs with lipid Ags
Specific pathogen-free outbred female Hartley strain guinea pigs
(250–300 g) or inbred strain 2 guinea pigs (250–300 g) were immunized
s.c. with purified lipid Ags of MTb in liposomes with or without
adjuvant (QS-21 or MPL). For each immunization, animals received
liposomes containing a mixture of some or all of the MTb lipid Ags
(glycolipids, neutral lipids, phospholipids, and mycolic acids) as
indicated. In each case, the amount of immunogen used per animal
contained 500–600 µg of each lipid component, and the dose of QS-21
or MPL applied as adjuvant was 50 or 300 µg per immunization,
respectively (19, 20). In some experiments, guinea pigs
were immunized with bone marrow-derived dendritic cells (BM-DCs) pulsed
with MTb total lipid Ags. BM-DCs were obtained from bone marrow
cultures supplemented with human GM-CSF (300 U/ml; PeproTech, Rocky
Hill, NJ) as described elsewhere (12). BM-DCs were pulsed
overnight with MTb total lipid extract (30 µg/ml) and, after washing
three times with PBS, Ag-pulsed BM-DCs (2 x
107 cells total per immunization per animal) were
injected s.c. into two or three sites on the abdomen. For
CD1-restricted CTL induction (see Figs. 3
and 4), guinea pigs were
immunized one, two, or three times, as specified, with a mixture of
liposomes reconstituted with MTb lipid Ags (600 µg each of
glycolipids, neutral lipids, and phospholipids) along with QS-21 (50
µg) and MPL (300 µg). Animals were sacrificed at the indicated time
points, and primary ex vivo CTL responses were analyzed as described
below.
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T cells were enriched from the spleens of immunized guinea pigs by passage through nylon wool fiber columns. T cells (5–10 x 104/well) were cultured in complete medium as described in triplicate in 96-well flat-bottom microtiter plates with or without irradiated (5000 rad) CD1+ BM-DCs or autologous nylon wool-adherent spleen cells (1–5 x 104/well) in the presence or absence of Ag (16). To assess CD1 restriction, purified mAb CD1F2/6B5 (reactive with guinea pig CD1b1, b2, b3, b4, c1, c2, and c3) or isotype-matched nonbinding control mAb (P3, mouse IgG1) was added at a final concentration of 20 µg/ml. Cultures were incubated for 3–4 days at 37°C in a 5% CO2 incubator, pulsed with [3H]thymidine (1 µCi/well, 6.7 Ci/mmol; NEN Life Science Products, Boston, MA), and incubated for an additional 5–6 h. The plates were harvested on a Tomtec 96-well plate harvester (Wallac, Turku, Finland) and thymidine incorporation was measured with a Betaplate liquid scintillation counter (Wallac).
Cytotoxic assays
Guinea pig cell lines transfected with expression vector constructs encoding the guinea pig CD1b1, b2, b3, b4, c2, or c3 proteins (12) or mock-transfected cells (i.e., stably transfected with empty vector) were labeled with 51Cr for 2 h. The target cells were cultured overnight at 37°C in a 5% CO2 incubator in complete medium with or without MTb total lipid extract (10 or 20 µg/ml), and then harvested and extensively washed. The labeled target cells were plated in quadruplicate in 96-well V-bottom microtiter plates with effector T cells at a range of different E:T ratios in a total volume of 150 µl of complete medium and incubated at 37oC for 4 h. Samples of supernatant (25 µl) were harvested and counted in a gamma counter. Total release of 51Cr was determined by addition of 1% Triton X-100 to wells containing target cells, and spontaneous release was determined from wells in which Ag-pulsed or Ag-unpulsed target cells were cultured without effector T cells. The data were calculated as percent specific lysis according to the following formula: (100 x (cpm release from experimental sample - cpm spontaneous release)/(cpm maximum release - cpm spontaneous release)).
For the source of splenic effector T cells for primary CTL responses,
guinea pigs (strain 2 or Hartley as specified) were immunized with MTb
lipid Ag mixture (600 µg each of glycolipids, neutral lipids, and
phospholipids) reconstituted in liposomes along with 50 µg of QS-21
and 300 µg of MPL as adjuvants. Strain 2 guinea pigs immunized once
were sacrificed at 2 wk after the primary immunization (S2-005, Fig. 3
A) or sacrificed at 4 wk after the primary immunization
(S2-007/8, Fig. 3C). In some experiments, strain 2 guinea
pigs were immunized twice (primary and boosted at week 4) and
sacrificed 12 wk after the second immunization (S2-003, Fig. 3C); Hartley strain guinea pigs were boosted twice after the
primary immunization at 4 and 8 wk and sacrificed 24 wk after the last
immunization (H0408, Fig. 3C). To prepare effector T cells,
spleen T cells from MTb lipid Ag-immunized guinea pigs were enriched by
nylon wool columns and then cultured for 6 days in complete medium
containing a mixture of MTb total lipid (10 µg/ml) and glycolipid
fraction (30 µg/ml) in the presence of mitomycin C (Sigma-Aldrich,
St. Louis, MO)-treated splenic adherent cells as APCs and 0.5 nM human
rIL-2. In preliminary experiments, we found that in vitro stimulation
with MTb glycolipid Ag in combination with MTb total lipid Ags induced
better CTL responses than stimulation with glycolipid Ag alone,
possibly due to enhancement of bystander help from
CD4+ T cells activated by the broader antigenic
challenge or some other adjuvant activity of the total lipid
preparation. At the end of the 6-day culture period, viable T cells
were purified by Ficoll-Hypaque density centrifugation (specific
gravity, 1.107) and used as effector T cells in CTL assays with MTb
lipid Ag-pulsed CD1-expressing target cells.
Generation of CD1-restricted T cell lines
An inbred strain 2 guinea pig was s.c. immunized twice with MTb
lipid Ags (mixture of 600 µg each of glycolipids, neutral lipids, and
phospholipids) in liposomes along with QS-21 (50 µg) and MPL (300
µg) at 4-wk intervals. The animal was further boosted with 2 x
107 syngeneic BM-DCs that had been pulsed
overnight with MTb total lipid extract (30 µg/ml). The spleen was
harvested 2 wk after the last immunization and splenic T cells were
first enriched using a nylon wool column (Polysciences, Warrington,
PA). CD4+ T cells were then depleted by staining
with anti-CD4 mAb (CT7) followed by magnetic separation using goat
anti-mouse Ig-coupled Dynabeads (Dynal Biotech, Lake Success, NY)
according to the manufacturer’s instructions. The resulting
CD4-depleted T cells were cultured with MTb total lipid extract (20
µg/ml) in the presence of irradiated (5000 rad) syngeneic BM-DCs in 1
ml of complete medium (3 x 106 T cells with
3 x 105 BM-DCs in 1 ml of medium/well of a
24-well plate). Four days after the start of culture, half of the
medium was replaced with fresh complete medium containing 0.25 nM human
rIL-2 (PeproTech). Subsequently, cultures were fed every 3–4 days by
replacing half of the medium with fresh complete medium containing 0.25
nM IL-2. Live cells were collected after 2 wk of expansion and
restimulated using BM-DCs plus MTb total lipid extract. Cultures were
carried through four similar cycles of Ag stimulation (in some cases
preceded by immunomagnetic depletion of residual
CD4+ T cells) followed by expansion in medium
containing IL-2 to generate line S2-1031, which showed CD1-restricted
reactivity to MTb lipids.
Statistical analyses
Data were analyzed by unpaired t test using GraphPad Prism (GraphPad Software, San Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our initial experiments focused on the induction of specific T
cell proliferative responses following the immunization of guinea pigs
with suitable preparations of MTb lipid Ags. To produce uniform
suspensions of the lipid Ags in a form that was likely to be
immunogenic, we incorporated a mixture of three MTb lipid Ag fractions
(glycolipids, neutral lipids, and mycolic acids) into liposomes. To
provide a suitable adjuvant, we incorporated either MPL or QS-21 into
the immunogen. Four weeks after immunization with the
liposome-entrapped MTb lipid Ag mixture with or without adjuvant,
guinea pigs were sacrificed and T cells were purified from their
spleens by passage over nylon wool columns. The resulting T
cell-enriched populations were >95% T cells based on FACS analysis
using mAb CT5 and were 70% CD4+ (mAb CT7) and
20% CD8+ (mAb CT6); data not shown). These cells
were tested for proliferative responses to the MTb lipid Ag fractions
using irradiated nylon wool column-adherent spleen cells as APCs (60%
positive by FACS for CD1 expression with mAb CD1F2/6B5; data not
shown). As shown in Fig. 1A,
nylon wool column-passed splenic T cells from guinea pigs immunized
twice with liposome-entrapped MTb lipid Ags without adjuvant showed
very weak proliferative responses to total MTb lipid Ags and no
responses to the glycolipid, neutral lipid, or mycolic acid fractions.
In contrast, immunization of guinea pigs with liposomes containing MTb
lipids plus either QS-21 (Fig. 1B) or MPL (Fig. 1, C and D) as an adjuvant induced significant
proliferative responses by spleen T cells to the MTb total lipid and
glycolipid preparations. Splenic T cells purified from naive
unimmunized guinea pigs showed only minimal, if any, responses to
mycobacterial lipid Ags in vitro (Fig. 1D and additional
data not shown). We could not raise significant T cell responses to
neutral lipids or mycolic acids from these guinea pigs using these
immunization protocols, suggesting either that these lipid Ag fractions
are less immunogenic or that the frequency of T cells specific to them
is very low.
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As an alternative method for inducing T cell responses against MTb
lipid Ags in vivo, we immunized guinea pigs with lipid Ag-pulsed DCs
without the addition of chemical adjuvants. DCs expressing CD1
molecules were generated from guinea pig bone marrow cells using in
vitro cultivation with human rGM-CSF (12). Fig. 2A shows a representative FACS
profile of guinea pig BM-DCs at day 10 after culture of bone marrow
cells. When gated on the high forward scatter population, nearly 90%
of the BM in these cultures expressed high levels of MHC class II and
CD1 proteins on their surfaces. Given the known properties of DCs in
the stimulation of primary T cell responses in vivo in humans and mice,
we tested the ability of guinea pig BM-DCs to prime lipid Ag-specific
responses following MTb lipid Ag-loading ex vivo and injection back
into animals (21).
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, B and C).
As shown in Fig. 2B, an outbred Hartley guinea pig that was
immunized with BM-DCs pulsed with MTb lipid Ag showed strong
proliferative responses to MTb glycolipid and phospholipid fractions.
The high level of background proliferation observed in this case
(medium alone) was most likely due to the simultaneous sensitization of
alloreactive T cells, since Hartley guinea pigs are not fully inbred.
To rectify this potential problem, we conducted analogous experiments
using inbred strain 2 guinea pigs as the BM-DC donor and as the
recipient immunization with BM-DCs pulsed with MTb lipid Ag. As shown
in Fig. 2C, in vivo immunization of an inbred strain 2
guinea pig with MTb lipid Ag-pulsed BM-DCs of strain 2 origin also
induced significant proliferative responses of splenic T cells to total
MTb-extractable lipids in vitro, as well as a strong response to the
phospholipid fraction and a weak but significant response to the
glycolipid fraction. In vivo sensitization of strain 2 guinea pigs
using MTb lipid Ag-pulsed BM-DCs was repeated three times, showing
consistent levels of T cell proliferation to total MTb lipid Ags and
phospholipids (stimulation index of 3.2–5.5 compared with medium
alone) and a lesser level of proliferation to glycolipids (stimulation
index, 1.7–2.6), with minimal or no responses to neutral lipids. In
similar experiments conducted in parallel, we also observed that guinea
pigs immunized with splenic DCs pulsed with the total MTb lipid Ag
extract showed proliferative responses to MTb lipid Ags upon in vitro
challenge (data not shown). These results indicated that BM-DCs and
splenic DCs expressing CD1 could present mycobacterial lipid Ags to T
cells in vivo, thus leading to lipid Ag-specific recall responses that
could be detected by T cell proliferation ex vivo. Ex vivo CTL responses from guinea pigs immunized with mycobacterial lipid Ags
In humans, the majority of group 1 CD1-restricted T cell lines
reactive with mycobacterial lipid Ags that have been studied to date
exhibit cytotoxic activity against lipid Ag-pulsed DCs or
CD1-expressing transfectant cells along with proliferative responses
and IFN- production. Therefore, we next examined whether the MTb
lipid Ag-reactive T cells arising after immunization of guinea pigs
showed cytotoxic activity against MTb lipid Ag-pulsed target cells.
Based on the results described in Fig. 1, we immunized naive strain 2
or Hartley guinea pigs with MTb lipid Ags (mixture of glycolipid,
neutral lipid, and phospholipid fractions) entrapped in liposomes with
the combination of QS-21 and MPL as adjuvants. Effector T cells were
prepared from immunized guinea pigs by in vitro stimulation of splenic
T cells with MTb total lipid extract (10 µg/ml) and
glycolipid-enriched fraction (30 µg/ml) for 6 days. We first tested
CTL responses against lipid Ag-pulsed syngeneic strain 2 BM-DCs, which
we have shown to express high levels of multiple different guinea pig
CD1 proteins (12). As shown in Fig. 3A, the effector T cells
derived from the MTb lipid Ag-immunized guinea pigs lysed the target
BM-DCs pulsed with MTb glycolipids or total lipids significantly above
the background level of target cells that were not pulsed with MTb
lipids. Similar to the proliferative responses observed after MTb lipid
Ag immunization using the liposome vehicle with QS-21 or MPL as
adjuvant (Fig. 1), effector T cells in cytolytic assays showed the
strongest reactivity to the MTb total lipid Ag preparation and the
glycolipid-enriched fraction, but also showed a trend toward weak
recognition of BM-DCs pulsed with the neutral lipid-enriched
fraction.
Next, we examined these cytotoxic T cell responses for their dependence
on the expression of particular CD1 molecules by the target cells. As
described elsewhere, we generated a new mAb (CD1F2/6B5) against guinea
pig CD1 proteins which recognized all seven of the currently identified
isoforms of guinea pig group 1 CD1 proteins (12). As shown
in Fig. 3B, the cytolytic activity of effector T cells
obtained from lipid Ag-immunized guinea pigs against BM-DCs pulsed with
glycolipids or total lipids were significantly inhibited by
anti-CD1 mAb, but not by isotype-matched control mAb (P3, mouse
IgG1). These results strongly suggested that mycobacterial lipid
Ag-reactive cytotoxic T cells elicited in guinea pigs by immunization
with MTb lipid Ags were at least partially CD1 restricted.
To address which of the multiple different group 1 CD1 proteins of the
guinea pig were involved in CTL responses evoked by immunization with
MTb lipid Ags, we used a panel of guinea pig cell lines stably
transfected with one of six different forms of guinea pig CD1 as target
cells in CTL assays. Subclones of the guinea pig cell line 104C1 each
transfected with a construct encoding one of six different guinea
pig CD1 proteins (i.e., guinea pig CD1b1, b2, b3, b4, c2, or c3)
or with empty vector alone (mock transfectant cells) (12)
were pulsed with the MTb glycolipid fraction and used as target cells
for CTL assay (note that guinea pig CD1c1 was not included in this
analysis because we were unable to achieve stable high levels of
expression of this isoform by transfecting 104C1 cells). The MTb
glycolipid fraction was used for this analysis because this fraction
was consistently active in preliminary CTL experiments (Fig. 3A and additional data not shown) and represented a more
purified form of lipid Ag without significant protein or peptide
contamination. As shown in Fig. 3C, the CTL responses of
either inbred strain 2 or Hartley guinea pigs immunized with MTb lipid
Ags in liposomes plus MPL and QS-21 showed consistent recognition of
MTb glycolipid-pulsed transfectants expressing either guinea pig CD1b1
or guinea pig CD1c2. Note that one animal (Hartley guinea pig H0408)
was immunized with lipid Ag three times and sacrificed 6 mo after the
final immunization to investigate the long-term memory responses of
lipid Ag-reactive CD1-restricted CTLs in this system. The appearance of
similar levels of CTL activity in this animal that was rested for an
extended period of time compared with the animals that had been more
recently boosted strongly suggested that a long-term CD1-restricted
memory CTL response can be induced following MTb lipid Ag
immunization.
A more detailed analysis of the effects of key parameters of the CTL
reaction (glycolipid Ag concentration used for pulsing targets, and E:T
ratio) was conducted for CTL recognition of targets expressing guinea
pig CD1b1 and guinea pig CD1c2 (Fig. 4).
As shown in Fig. 4, guinea pig CD1b1 and c2 but not mock-transfected
104C1 were recognized by effector T cells from strain 2 guinea pigs
immunized twice with lipid Ags in liposomes with QS-21 plus MPL, and
the level of lysis varied directly with the MTb glycolipid Ag
concentration and the E:T ratio. Furthermore, to exclude the
possibility of random variations between different 104C1 transfectant
clones accounting for the observed CTL lysis, we analyzed the CTL
responses using multiple independently derived subclones of
104C1-expressing guinea pig CD1b1 (two clones: clone 16 and clone 18)
or guinea pig CD1c2 (three clones: clone 5, clone 11, and clone 12).
All of these subclones behaved similarly as target cells in these
assays, yielding specific lysis values that were significantly greater
than the background level obtained with the mock transfectant cells
(data not shown). Taken together, these results indicated that
immunization with MTb lipid Ags elicited MTb glycolipid-specific CTL
responses in vivo that were restricted by at least two different group
1 guinea pig CD1 proteins.
Phenotype of MTb lipid Ag-specific T cells elicited in immunized guinea pigs
To gain further insight into the types of CD1-restricted T cells
that arise following MTb lipid Ag immunization in vivo, we examined the
phenotype of the lipid-reactive T cells with respect to expression of
CD4 and CD8 coreceptor molecules. Since the T cells showed CTL
activity, we reasoned that at least a fraction of them would belong to
the CD8 single-positive fraction or possibly to the
CD4-CD8- double-negative
(DN) fraction which has also been associated with CTL effector activity
(13, 22). Thus, to confirm that MTb lipid Ag-reactive T
cells reside in the CD4-negative fraction (i.e., among CD8
single-positive or DN T cells), we performed the following experiments.
Hartley guinea pigs were s.c. immunized with OVA and a mixture of MTb
lipid Ags (glycolipids, neutral lipids, and phospholipids) incorporated
into liposomes along with the QS-21 and MPL. OVA was added
concomitantly as a control, representing a foreign protein Ag that
should elicit peptide-specific CD4+ T cell
responses that could be used to monitor the effectiveness of the
immunization. At 4 wk after immunization, T cells were enriched from
the spleens of sacrificed animals by nylon wool column (purity >90%)
and then further fractionated by immunomagnetic depletion of
CD4+ T cells (residual CD4+
T cell contamination <5%). The proliferative responses to MTb lipid
Ags and OVA of the T cell-enriched fraction and the CD4-depleted T
cell-enriched fraction were then examined in the presence of irradiated
autologous BM-DCs as APC. As shown in Fig. 5A, splenic T cells isolated
from an immunized guinea pig 4 wk after MTb lipid Ag priming showed
significant dose-dependent proliferation to MTb total lipid Ags and to
OVA. Depletion of CD4+ T cells led to a
marked reduction in the OVA-specific T cell proliferation, but had
little effect or even slightly increased the proliferative responses to
the MTb total lipid Ag (Fig. 5B). This suggested that the T
cell fraction responding to MTb lipid Ags was mainly or exclusively
composed of CD4-CD8-
and/or CD8+ T cells. This finding was confirmed
by preliminary experiments designed to determine optimal conditions for
establishing CD1-restricted MTb lipid Ag-specific T cell lines using
CD4-depleted splenic T cells from inbred strain 2 animals similarly
immunized with MTb lipid Ags. As shown in Fig. 5C,
CD4-depleted T cell fractions from immunized strain 2 guinea pigs again
responded to MTb whole lipid extract and to the glycolipid-enriched Ag
fraction. These results provided further support for the conclusion
that CD4-negative T cells were involved in the proliferative responses
to these Ags.
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The ability to detect proliferative and cytotoxic T cell responses
to MTb lipid Ags prompted us to establish CD1-restricted MTb lipid
Ag-specific T cell lines to facilitate more detailed in vitro analyses.
Since the CD4 depletion studies indicated that CD1-restricted T cells
primed by MTb lipid immunization of guinea pigs may be predominantly
CD4 negative, we used CD4-depleted splenic T cells from strain 2 guinea
pigs immunized with liposome-entrapped purified MTb lipid Ags along
with QS-21 and MPL and boosted with lipid Ag-pulsed BM-DCs to initiate
cultured T cell lines. These T cells were cultivated in the presence of
CD1+ BM-DCs from inbred strain 2 guinea pigs and
pulsed with an MTb whole lipid extract. To prevent possible overgrowth
of any residual CD4+ T cells during the culture,
the CD4 depletion was repeated three times during the course of
culture. After two stimulations using syngeneic
CD1+ BM-DCs, the third and fourth stimulations
were conducted with CD1+ BM-DCs from allogeneic
outbred Hartley guinea pigs to favor presentation of lipid Ags by
nonpolymorphic Ag-presenting molecules such as CD1. After the
sixth restimulation, one T cell line was obtained (designated as
S2-1031) that had expanded sufficiently to permit detailed analysis.
FACS analysis revealed that line S2-1031 was composed predominantly of
CD4-CD8- and
CD8+ T cells, with only 5.2%
CD4+ cells (Fig. 6A).
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B).
Isotype-matched control mAb (mAb P3, mouse IgG1) had no effect on these
proliferative responses (data not shown).
To further dissect the CD1 restriction of this MTb lipid Ag-reactive T
cell line, CTL responses were determined using transfectants of cell
line 104C1 that selectively expressed each of the cloned guinea pig CD1
isoforms. T cell line S2-1031 lysed MTb lipid Ag-pulsed 104C1 cells
stably transfected with guinea pig CD1b1, -b2, and -c3, but not mock
transfectants in an Ag dose-dependent manner, as shown in Fig. 7. Consistent with the findings obtained
in the proliferation assays with this T cell line, we could not clearly
detect significant CTL responses (i.e., >10% specific lysis) to
more highly purified lipid Ag fractions (purified mycolates or the MTb
glycolipid, neutral lipid, and phospholipid-enriched fraction;
data not shown). Thus, although the precise lipid Ag specificity
of line S2-1031 cannot yet be determined, the results provided clear
evidence for the restriction of its MTb-specific CTL activity by at
least three members of the guinea pig group 1 CD1 family.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Evidence supporting the hypothesis that MTb lipid Ag-specific CD1-restricted T cells play a role in host defense against microbial pathogens comes from the recent finding that human CD1c-restricted T cells specific for a mycobacterial isoprenoid glycolipid Ag are seen in individuals previously infected with MTb, but not in naive donors (8). CD1-restricted T cells specific for mycobacterial Ags have also been derived from the active skin lesions of leprosy patients, and effective cell-mediated immunity is correlated with expression of group 1 CD1 molecules by the DCs in leprosy skin lesions (23, 24). Furthermore, these CD1-restricted mycobacterial lipid Ag-specific T cells have been found to be biased toward secretion of Th1 type cytokines and can kill CD1+ target cells pulsed with lipid Ags while concurrently producing the bactericidal protein granulysin (25). Together, these data provide strong circumstantial evidence that CD1-restricted T cells specific for foreign lipid Ags may play a role in host defense against bacterial infections.
In humans, CD1-restricted responses were initially found among
CD4-CD8- DN T cells, although
subsequent reports also confirmed the existence of CD1-restricted T
cells that were CD4 (23) or CD8 single positive
(13). In our analysis of MTb lipid-immunized guinea pigs,
we found that CD1-restricted lipid Ag-specific T cells were prominent
in the CD4-negative fraction of splenic T cells, consistent with CD1
restriction being mainly a feature of CD8+ or DN
T cells in this experimental setting. These CD4-negative CD1-restricted
T cells were associated with both proliferative and cytotoxic
activities, similar to what has been described for human CD1-restricted
T cells reactive with mycobacterial lipid Ags. In the case of human MTb
lipid-reactive T cells, Ag recognition has typically been associated
with production of IFN-, a key cytokine in the immune response to
pathogenic mycobacteria. We are not yet able to test whether this is a
property of the CD1-restricted T cells that develop after MTb lipid
immunization in the guinea pig because of the current lack of reagents
for the specific detection of IFN- or other cytokines in this
species.
Although our current study established the priming of Ag-specific T cell responses following immunization with suitably adjuvanted and reconstituted mycobacterial lipid preparations, it is also of interest to determine whether such responses develop during actual infections with mycobacterial pathogens. Our previous studies on PBL from human donors with previous mycobacterial infection strongly support the view that CD1-restricted T cells responses to specific lipid Ags do develop as a result of natural infections with MTb (8). In the guinea pig model, preliminary experiments to determine whether such T cell responses are primed in animals infected with MTb strain H37Ra or bacillus Calmette-Guérin have demonstrated lymphocyte proliferative responses to mycobacterial lipid Ags similar to those elicited upon immunization with MTb lipids (K. Hiromatsu, unpublished data). However, we have thus far failed to detect CD1-restricted CTL activity in lymphocytes isolated from guinea pigs infected with MTb strain H37Ra. This raises the possibility that active mycobacterial infection may be associated with suppression of certain aspects of the immune response that can be elicited by isolated lipid vaccination, possibly reflecting a significant immune evasion strategy of the pathogen, and suggests that vaccination by live attenuated mycobacteria could actually be less effective than vaccination with purified subunit formulations in some cases. This is a potentially important issue and its resolution will require extensive further studies using the guinea pig model and other animal models of MTb infection.
The conservation of multiple homologs of human CD1b and CD1c in the guinea pig strongly suggests that the group 1 CD1 molecules may have evolved specialized functions early in the course of mammalian evolution (11). The guinea pig model may also provide additional insights into the selective pressures that may have led to the diversification of CD1 into its multiple distinct isoforms. From studies of the subcellular localization and Ag-presenting properties of human CD1a, b, and c molecules, it has been postulated that these separate CD1 isoforms may each be specialized to efficiently survey different intracellular compartments for lipid Ags (26, 27, 28). If this is correct, then it would appear that the different isoforms of group 1 CD1 are not functionally redundant, but are specialized to complement each other in providing detection of foreign or abnormal self-lipids. Interestingly, in our studies of the subcellular localization of the various guinea pig CD1 isoforms, we have found that guinea pig CD1 proteins show at least three distinct patterns of intracellular localization (12). Given our current findings on the ability of several guinea pig CD1 proteins to present MTb lipid Ags to T cells, it will be of interest to determine in future studies whether the different guinea pig CD1 molecules involved in these responses acquire their lipid Ags in distinct subcellular compartments of APCs.
Considerable work remains to be done to identify the specific MTb lipids that are recognized by guinea pig CD1-restricted T cells. As shown in the current study, MTb lipid Ag-immunized guinea pigs frequently gave positive T cell responses to the MTb glycolipid-enriched fraction after immunization with a broad mixture of mycobacterial lipid Ags, whereas highly purified mycolic acids and a neutral lipid-enriched fraction appeared not to be well recognized in most cases. Analysis of amino acid content of the various lipid preparations that we have used does indicate a possibility of low levels of residual protein contamination in some of these, since up to 6.4% amino acid content on a weight basis was detected in the total lipid extract and phospholipid-enriched fractions. However, analysis by SDS-PAGE and ultrasensitive silver staining failed to reveal any detectable protein contaminants in our lipid Ag preparations, and the profile of amino acids present suggest that most of these are derived from peptidoglycan rather than from proteins. Thus, although we cannot completely rule out at this point the possibility that trace amounts of highly immunogenic protein Ags in our lipid preparations could contribute to some of the T cell responses we have observed, this appears to be unlikely. It is also noteworthy that in several cases we demonstrated guinea pig CD1-restricted recognition of the MTb glycolipid-enriched fraction, which represents material eluted with acetone from a silicic acid column and contains only trace amounts of detectable amino acids. Finally, the restriction of these responses by guinea pig CD1 proteins provides further evidence in favor of lipid rather than protein Ag targets, since work on human and murine CD1 has strongly linked these proteins to the binding and presentation of lipids rather than proteins or peptides.
Our current data provide further support for the possibility of using the guinea pig as an animal model in which to test the hypothesis that augmentation of group 1 CD1-restricted mycobacterial lipid Ag-specific T cell responses can be used as a component of an effective vaccination strategy against MTb infection (29). This appears to us to be an appealing strategy, given that CD1 genes are largely nonpolymorphic and that the CD1 target ligands (i.e, complex lipids and glycolipids) are essential components of bacterial cells that may be less variable in their structures than the more highly mutable peptide Ags that are targets of MHC-restricted T cells. Currently, Mycobacterium bovis bacillus Calmette-Guérin is the only available vaccine against tuberculosis, and its efficacy had been disputed despite extensive use in humans and testing in the field. CD1-mediated presentation of lipid Ags may provide a completely new vaccination strategy that could offer advantages to new protein subunit vaccines or live attenuated mycobacterial vaccines that are currently under development. Thus, the guinea pig model may provide valuable opportunities for testing the efficacy of lipid Ag vaccination in a small animal model that includes the potential for a prominent group 1 CD1-restricted T cell response.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461.
3 Current address: Astra-Zeneca Pharmaceuticals, 1800 Concord Pike, Wilmington, DE 19850.
4 Address correspondence and reprint requests to Dr. Steven A. Porcelli at the current address: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Forchheimer Building, Room 416, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail address: porcelli{at}aecom.yu.edu
5 Abbreviations used in this paper: MTb, Mycobacterium tuberculosis; MPL, monophosphoryl lipid A; BM-DC, bone marrow-derived dendritic cell; DN, double negative.
Received for publication February 26, 2002. Accepted for publication April 26, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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+ T cells. Nature 372:691.[Medline]
T cell pool. J. Immunol. 162:366./
T cells. Eur. J. Immunol. 21:2999.[Medline]
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) or 25 µg/ml (
)) or with medium only (•).
