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* Division of Rheumatology, Immunology and Allergy,
Immunology Research Division, Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115;
Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461; and
Center for Health Sciences, Division of Infectious Diseases, Department of Medicine, University of California Los Angeles School of Medicine, Los Angeles, CA 90095
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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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The human CD1 gene family is a nonpolymorphic gene cluster
located on chromosome 1, unlinked to the MHC locus on chromosome 6
(1). The CD1 gene family consists of five isoforms
designated CD1A, -B, -C,
-D, and -E. These are divided into two groups
based on protein sequence homology, with group 1 consisting of the
CD1a, -b, and -c isoforms and the group 2 consisting of only the CD1d
isoform (2, 3). Unlike the MHC, which presents peptides to
T cells, CD1 has evolved the unique capacity to present lipids and
glycolipids derived from microbes to Ag-specific T cells. These T cells
have potentially important effector functions for control of
infections, including release of bactericidal factors, secretion of
IFN-
, and cytolytic functions (4, 5). Interestingly,
all of the Mycobacterium tuberculosis lipid reactive T cells
are restricted to one of the group 1 CD1 isoforms while no
CD1d-restricted T cells specific for bacterial lipids have been
described. These data suggest an important role for the group 1 CD1
proteins in the immune response to infection.
In humans, CD1 proteins are expressed on DC in both lymphoid and
nonlymphoid tissues including lymph nodes, Langerhans cells in the
epidermis, and dermal DC (3). CD1 is also up-regulated
following cytokine-mediated maturation of peripheral blood monocytes
into immature DC. These APC are capable of stimulating CD1-restricted T
cells specific for bacterial lipid Ags in vitro. Examples of
CD1-restricted human T cell lines have been found for all of the major
T cell phenotypes including
CD4+/TCR
+,
CD8+/TCR+,
CD4-CD8-/TCR+,
and TCR
+ (4, 6, 7, 8, 9). In
addition, patients recently infected with M. tuberculosis
have significant CD1-restricted T cell proliferative responses to
a lipid Ag compared with uninfected controls (10).
However, the in vivo significance of these T cell responses remains
unknown.
Previously, we have described an extended group 1 CD1 gene family in guinea pigs. The guinea pig CD1 (gpCD1) gene family has expanded to include four homologs of CD1b (gpCD1B1, -B2, -B3, and -B4) and three of CD1c (gpCD1C1, -C2, and -C3) and a single CD1E gene (gpCD1E) (11, 12). In addition, we have recently demonstrated that immunization of guinea pigs with lipid Ags is capable of eliciting CD1-restricted T cell responses (13). It is not possible to study group 1 CD1-restricted T cells in mice and rats as these species have only CD1d (group2) homologs (14). Therefore, we have been developing the guinea pig as a small animal model to assess the functional role of group 1 CD1 Ag presentation in vivo. In this study, we describe the expression of group 1 CD1 isoforms in tissue DC and on purified DC isolated from guinea pig spleen. Purification of DC from spleen was facilitated by treatment of guinea pigs with a murine fms-like tyrosine kinase 3 (Flt-3) ligand mouse IgG2a Fc fusion protein (Flt-3LFc). In addition, we show that two different gpCD1b isoforms traffic to distinct endosomal compartments within DC. These findings have important implications for the potential role of the different CD1 isoforms in Ag presentation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hartley strain guinea pigs were obtained from Elm Hill Breeders (Chelmsford, MA). Strain 2 guinea pigs were bred in-house. Guinea pigs were housed under specific pathogen-free conditions at the Dana-Farber Cancer Institute animal facility. BALB/c mice were purchased from Taconic Farms (Germantown, NY). Mice were housed and maintained in the animal facility of Brigham and Women’s Hospital (Boston, MA) under virus Ab-free conditions. All animals were treated and maintained in accordance with institutional standards.
Construction and purification of murine Flt-3 ligand (Flt-3L) IgGFc
The cDNA coding sequence for the secreted form of murine Flt-3L was amplified by PCR from a murine T cell cDNA using standard techniques (15). The 5' primer (5'-aaaagaattccatgaggggtccccggcaga-3') and 3' primer (5'-cggggtaccgtggatccttctgcctgggccgaggctctgggag-3') were used to generate a product containing the nucleotides encoding the secreted domains of the murine Flt-3L. A plasmid construct containing the nonlytic Fc coding sequence of the murine IgG2a was kindly provided by X. Xiao Zheng (Beth Israel Deaconess Medical Center, Boston, MA) (16). The IgG2a Fc coding sequence was previously engineered to eliminate the binding of the Fc region to cell surface FcR (16). A second mutation in the Fc domain also abrogates complement binding to the fusion protein (16). A DNA fragment containing the coding sequences for the nonlytic Fc coding region was isolated using restriction enzyme digestion with BamHI and XbaI. The amplified Flt-3L PCR product was then digested with restriction enzymes EcoRI and BamHI to generate compatible cohesive ends ligated to the coding sequence of the murine nonlytic Fc and inserted into pCI-neo (Promega, Madison, WI) to create a construct with the murine Flt-3L coding sequences at the 5' and the nonlytic murine IgG2a Fc domain coding sequences at the 3' end. This construct was transfected into CHO cells using Superfect reagent (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Cells were subjected to selection with G418 (Invitrogen, Carlsbad, CA) and cloned by limiting dilution. A clone secreting high levels of the fusion protein was detected by an ELISA screening assay. Briefly, plates were coated with anti-mouse IgG, A, and M (Zymed Laboratories, San Francisco, CA) in PBS followed by blocking with 1% BSA in PBS. After incubating the supernatants in the wells for 1 h, the fusion protein was detected with a biotinylated anti-mouse Flt-3L Ab (R&D Systems, Minneapolis, MN), followed by incubations with alkaline phosphatase-conjugated streptavidin and paranitrophenyl phosphate. A single clone was expanded to 10 L in a stirred vessel and the fusion protein was purified using protein A Sepharose affinity chromatography. The endotoxin content of the purified Flt-3LFc protein was <1 EU/ml as assessed by Pyrotell assay (Associates of Cape Cod, Falmouth, MA). Biochemical analysis of the fusion protein was conducted by PAGE. Purified Flt-3LFc protein was electrophoresed on a 12% acrylamide gel and stained with Coomassie brilliant blue or analyzed by Western blotting (17). For Western blotting, the fusion protein was detected using an anti-Flt-3L anti-serum (R&D Systems). The bands were visualized using HRP-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and Renaissance chemiluminescence reagent (NEN, Boston, MA).
Preparation and analysis of mouse DC
BALB/c mice were given daily i.p. injections with either PBS
alone or 10 µg of Flt-3Fc fusion protein in PBS for 9 days. On day
10, mice were sacrificed and their spleens were removed. After RBC
lysis, spleen cells were directly stained with Abs. To determine the
expression of various cell surface markers, 1 x
106 cells were suspended in anti-FcR Ab 2.4G2
(American Type Culture Collection (ATCC), Rockville, MD) supernatant to
block nonspecific Ab binding. Abs to various cell surface markers were
added at the appropriate dilution and the cells were incubated for 20
min on ice in the dark. Cells were washed twice with FACS buffer (PBS
with 2% FCS) and then resuspended in 2% paraformaldehyde for
analysis. Flow cytometry was performed using a FACSCalibur cytometer
(BD Biosciences, San Jose, CA) with CellQuest 3.0 software for
acquisition and analysis. The following Abs were used for flow
cytometry analysis: control hamster IgG (A19-4), control rat IgG2b
(A95-1), CD11c (HL3), and CD11b (M1/70). All of these Abs were
directly conjugated with FITC or PE and were obtained from BD
PharMingen (San Diego, CA).
Preparation of guinea pig DC
Guinea pigs were injected i.p. daily with 0.1 mg purified Flt-3LFc protein in 3 ml PBS for 9 days. No adverse reactions to the treatment were observed. Animals were euthanized on day 10 by sodium pentobarbital overdose. Spleen and other tissues were removed under sterile conditions. DC were obtained from the spleens using published techniques (18). Briefly, spleens were injected with 20 ml of DMEM media (Invitrogen) containing 100 U/ml collagenase type IV (Worthington Biochemical, Lakewood, NJ) and incubated at 37°C for 15 min. The spleens were then transferred to 400 U/ml collagenase and mechanically disrupted, followed by repeated pipetting until a single-cell suspension was generated. The resulting suspension was centrifuged and the pellet resuspended in ACK for 2 min to lyse RBC and then washed with RPMI. The resulting splenocyte pellet was resuspended in 20 ml BSA solution (1.080 g/ml) and 5 ml added to 15-ml conical tubes and overlaid with RPMI. Gradients were centrifuged 3000 rpm in a Sorvall RT-7 centrifuge for 30 min at 4°C. The floating cells were then removed from the top of the gradients and washed with RPMI. Cells were then allowed to adhere to plastic petri dishes for 90 min followed by removal of nonadherent cells. Adherent cells were then incubated for 18 h in RPMI medium. Enriched DC were then harvested and used for analysis.
Guinea pig-specific mAbs and FACS
The mAbs used for guinea pigs in these studies include BCD1b3.1 (specific for human CD1b and cross-reactive with the gpCD1b2, -b4, and -c3 isoforms) that has been described previously (11, 12, 19); P3 was used as the nonspecific mouse IgG1 isotype control Ab (20); 1D12.1 is specific for gpCD1b1 (11); 6B5 is a guinea pig pan-CD1 mAb (11); MSgp9 (Serotec, Raleigh, NC) is a pan B cell Ab specific for gpCD1b3 (11, 21). IVA12 (ATCC) is a pan-human MHC class II specific Ab that cross-reacts with guinea pig MHC class II.
FACS analysis was performed as previously described (22). Briefly, primary Abs were added to single-cell suspensions at saturating concentrations for 1 h, washed with staining buffer (PBS containing 2% FCS and 0.01% azide), and then incubated for 1 h with 30 µg/ml FITC-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories). After staining, cells were washed and analyzed with a FACSort flow cytometer (BD Biosciences). The forward and side scatter profiles of the analyzed cells were used to gate on the monocyte subpopulation. Dead cells were excluded using propidium iodide (Sigma-Aldrich, St. Louis, MO).
Histology and immunohistochemistry
Samples of all tissues were fixed in 10% buffered formalin for at least 24 h. Following this, fixed tissues were embedded in paraffin, sectioned at 5 µm, and stained with H&E using standard techniques.
Unfixed tissue samples used for immunohistochemistry were mounted in OCT compound (Tissue-Tek, Torrance, CA), frozen in liquid nitrogen and stored at -80°C. Single-cell suspensions of enriched DC were cytospun onto glass slides. Cytospun cells and frozen tissue sections (5 µm) were fixed in acetone for 10 min, air dried, and stained with a primary Ab as indicated and detected by an indirect immunoperoxidase method using avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, CA) and 3-amino-9-ethylcarbazole (Sigma-Aldrich) as the chromogen. Some cytospun DC preparations were stained with Diff-Quik according to the manufacturer’s instructions (Fischer Scientific, Pittsburgh, PA).
Immunofluorescence
Double labeling was performed as described previously (23). Briefly, isolated guinea pig DC were adhered to glass coverslips by cytospin followed by staining with mAb to either gpCD1b1 or gpCD1b3 isoforms and MHC class II. The CD1 Abs were detected with Texas Red-conjugated donkey F(ab')2 Ab to mouse IgG (Jackson ImmunoResearch Laboratories). The IVA12 Ab (anti-MHC class II) was directly conjugated with Alexa-488 (Molecular Probes, Eugene, OR). For Ag-pulsed cells, guinea pig DCs were incubated for 4 h with purified M. tuberculosis lipoarabinomannan (LAM) (20 µg/ml; kindly supplied by J. Belisle, Colorado State University, Fort Collins, CO). At the end of the culture, the LAM-pulsed cells were washed and adhered on glass slides by cytospin. The cells were then fixed and permeabilized, and double-labeled with mouse mAbs to the specified gpCD1 isoforms and a rabbit polyclonal Ab specific for mycobacterial LAM. The CD1 Abs were detected as described above. The rabbit anti-LAM was detected with FITC-conjugated donkey (Fab'2) Ab to rabbit IgG (Jackson ImmunoResearch Laboratories). The labeled cells were examined using a Leica TCS-NT confocal laser scanning microscope fitted with krypton and argon lasers as described previously (23).
Scanning electron microscopy
Purified guinea pig DC were resuspended in RPMI medium and placed onto poly-L-lysine-treated glass coverslips for 30 min at 37°C. Cells were then fixed with 5% glutaraldehyde/2.5% paraformaldehyde/0.06% picric acid in 0.1 M cacodylate buffer (pH 7.4) for 1 h at room temperature. Fixed cells were then treated with 1% osmium tetroxide for 1 h at room temperature. Following dehydration cells were critical point dried, mounted, and sputter-coated with palladium. Specimens were examined using a JEOL JSM-5600LV scanning electron microscope (JEOL, Peabody, MA).
Phagocytosis assay
The M. tuberculosis H37Rv strain expressing green
fluorescent protein (GFP) was a generous gift from Dr. J. Ernst
(24). Bacteria were cultured in Middlebrook 7H9 medium
supplemented with albumin/dextrose/catalase to midlog growth. Bacteria
were then washed once in tissue RPMI medium containing 10% FCS,
filtered through a 5 µm filter, counted, and added to wells
containing gpDC growing on glass coverslips at an multiplicity of
infection of 
5:1. Cells were then incubated at 37°C/5%
CO2 for 4 h. Cells were then fixed with 2%
paraformaldehyde and stained with Alexa-594 (Molecular Probes)
conjugated 6B5 mAb. Stained cells were then visualized using a Nikon
Eclipse 800 microscope (Nikon, Melville, NY).
Mixed lymphocyte reaction
Strain 2 guinea pigs were treated with Flt-3LFc or PBS as a vehicle control and DC were isolated as described above. A single-cell suspension of whole strain 2 guinea pig splenocytes was also generated. Nylon wool nonadherent T cells were isolated from the spleen of a Hartley strain guinea pig and used as responder cells for the assay. A single-cell suspension from whole spleen of the same Hartley guinea pig was used to generate autologous stimulators. The purified DC and autologous stimulators were gamma-irradiated with a dose of 5000 rad. Stimulators were plated in 96-well plates starting at 2 x 105/well and titered out 2-fold. The Hartley responder T cells were plated at 2 x 105/well. Cells were incubated for 5 days at 37°C/5% CO2, pulsed with [3H]-thymidine (1 µCi/well), harvested, and counted.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously described the expression of group 1 CD1
proteins on cells in lymphoid and nonlymphoid tissues of guinea pigs
(11, 12). Of particular interest is the expression of CD1
on DC in lymphoid tissues since these are critical cells for presenting
Ags to T cells. Tissue sections of lymph nodes from naive guinea pigs
were stained with Ab 6B5 (pan gpCD1) and detected by an
immunoperoxidase technique. Fig. 1
A shows a large proportion of
B cells in the secondary lymphoid follicles that are positive for CD1
as we have described previously (11, 12). In addition, a
small but distinct population of CD1-positive cells was detected in the
paracortical regions of the nodes which are predominantly the T cell
areas (Fig. 1A, arrows). Higher magnification revealed that
these cells exhibited a dendritic morphology with interdigitating
processes that project into the surrounding tissue (Fig. 1, B and C). The location and pattern of staining in
lymph nodes is consistent with a guinea pig DC population and is
similar to that seen in other species. Because of the potential
importance of DC in priming CD1-restricted T cells, we characterized
this cell population in greater detail.
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DC represent a rare subpopulation of bone marrow-derived cells
within lymphoid organs, including the spleen and lymph nodes. Typical
yields from mouse spleen are <1 million cells. This makes isolation of
pure DC in sufficient numbers for in vitro analysis technically
difficult. To overcome this problem, we generated a recombinant Flt-3L
fusion protein which has been shown previously to significantly
increase the number of DC in vivo (25). The murine Flt-3L
cDNA coding for the secreted form of the protein was inserted upstream
of a modified murine IgG2a Fc domain coding sequence. Supernatant from
a cell line transfected with the plasmid was used to purify Flt-3LFc
protein using protein A affinity chromatography. A sample of purified
protein was analyzed by PAGE and Western blot to confirm the molecular
mass and identity of the purified material. The molecular mass
of the purified protein is 66 kDa which is consistent with an intact
glycosylated fusion protein (Fig. 2A, lane 1). A
Western blot using antiserum to Flt-3L was performed to confirm the
immunoreactivity of the Flt-3L portion of the fusion construct. A major
immunoreactive species was visualized at the same molecular mass as
that of the Coomassie stained gel (Fig. 2A, lane
2). We then confirmed the bioactivity of the purified Flt-3LFc
protein in mice since the murine Flt-3L coding sequences were used in
the construct. Whole splenocyte suspensions from Flt-3LFc
protein-treated mice were subjected to FACS analysis (Fig. 2B). A clear increase in the percentage of
CD11c+ cells was observed (2.2–17.8%) following
Flt-3LFc treatment. These data indicate that the recombinant Flt-3LFc
protein had in vivo bioactivity and that it increased the splenic DC
population consistent with previously described activity.
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We next tested the murine Flt-3LFc fusion protein in guinea pigs
in an attempt to quantitatively increase the DC population in lymphoid
tissues, thereby facilitating their isolation and further analysis in
vitro. Because Flt-3L is highly conserved and functionally cross-reacts
among several mammal species, we reasoned that murine Flt-3L would also
be bioactive in the guinea pig (26). Guinea pigs were
treated with Flt-3LFc as described in Materials and Methods.
Gross examination of the spleen (Fig. 3A) and lymph nodes (Fig. 3B) during necropsy revealed significant increases in size
and mass of the Flt-3LFc-treated guinea pig tissues when compared with
vehicle-injected control animals. Histological examination of the
inguinal lymph nodes taken from vehicle- and Flt-3LFc-treated animals
showed extensive infiltration of nonlymphocytic mononuclear cells
throughout the cortex of the Flt-3LFc-treated animals only (Fig. 3, D and F). In contrast, PBS-treated animals had
normal appearing nodes with lymphocytes predominating (Fig. 3, C and E). Histological examination of the spleens
also demonstrated extensive infiltration of nonlymphocytic mononuclear
cells (data not shown). In addition, both the lung and liver had
perivascular infiltrates (data not shown).
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5-fold greater than
animals treated with PBS (vehicle). Typical cell yields from
Flt-3LFc-treated guinea pigs were 5–6 x
106 cells compared with <1 million for a
negative control (PBS-injected) animal. Phase contrast microscopy of
the cultured nonadherent cell population showed numerous cells with
thin processes and folds emanating from the cell surface, consistent
with a DC phenotype (data not shown). This enriched guinea pig splenic
DC preparation was characterized further. Scanning electron microscopy
was used to visualize the cellular ultrastructure of isolated guinea
pig DC (Fig. 4, A and
B). Numerous cells with prominent dendritic processes and
cell surface folds were present in these preparations. A cytospin
preparation of the DC stained with Diff-Quik revealed many cells with
long cytoplasmic extensions emanating from the cell body consistent
with the dendritic morphology observed in situ (Figs. 1
C and
4, A–C). The length of the projections relative to the size
of the cell body is consistent with DCs as opposed to macrophages (D.
Friend, unpublished observations). In addition, the nucleus of
DC often have indentations and an irregular morphology which is also
evident here (Fig. 4, C and D). Cytospin
preparations were stained with Abs specific for gpCD1 and visualized by
immunoperoxidase technique. Numerous cells exhibiting dendritic
morphology were stained with the 6B5 Ab demonstrating that CD1 is
highly expressed on this population of splenic DC elicited by Flt-3LFc
fusion protein (Fig. 4D). Using this technique, we estimated
the purity of the cell preparations from Flt-3L-treated guinea pigs to
be 70% DC while the vehicle-treated animals were 30% DC or
less. The remaining contaminating cells were likely to be T cells based
on FACS (data not shown).
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G). The CD1 protein is also expressed on
purified DC (Fig. 4H). Among these cells, there appears to
be two subpopulations with a medium or high expression of MHC class II
and CD1 possibly reflecting the maturation state of the cells. These
data correlate with the CD1 Ab staining of the cytospins and are
consistent with the known expression of MHC and CD1 Ag-presenting
molecules on DC from both human and mouse (27).
A functional hallmark of immature DCs is that they are highly
phagocytic, which facilitates their role as APCs. We tested the
phagocytic capacity of purified guinea pig DC in vitro using
GFP-expressing M. tuberculosis bacteria. Purified DC were
pulsed with viable bacteria, fixed, and then stained with anti-CD1
Ab. Microscopic examination revealed numerous bacteria associated with
CD1-positive cells confirming the phagocytic capacity of the purified
DC (Fig. 4E).
Another distinguishing feature of DC is their capacity to stimulate an
MLR. We used DC isolated from Flt-3LFc- or vehicle-treated strain 2
guinea pigs (inbred) as stimulatory target cells. Purified T cells from
a Hartley strain (outbred) guinea pig were used as responders (Fig. 4I). These data show a more potent activating potential of
the Flt-3LFc-derived DC compared with cells isolated from PBS-treated
animals or whole splenocytes. Although equal cell numbers were used in
the assay, the reduced yield of DC from the vehicle-treated control
animal results in a lower percentage of DC (30%) in the cell
preparation. This may account for the reduced proliferation by
allogeneic responders. Thus, the murine Flt-3LFc fusion protein induces
the expansion of a splenic cell population that have both the
morphological and functional characteristics consistent with DC
expansion.
CD1 isoforms traffic to different endosomal compartments in guinea pig DC
Previous studies of human CD1 proteins have demonstrated differential trafficking within the endosomal system of the CD1a, -b, and -c isoforms (23, 28, 29). This has been postulated to facilitate lipid Ag sampling from different subcellular compartments of the endocytic system. Inspection of the C-terminal residues of the gpCD1 proteins revealed that all of the group 1 CD1 isoforms possess putative tyrosine-based endosomal sorting motifs with the exception of the gpCD1b3 isoform (12). Based on this observation, we predicted that the gpCD1b3 protein would traffic in a pattern distinct from the other gpCD1 isoforms, possibly similar to human CD1a, the only human CD1 isoform that lacks the cytoplasmic tyrosine-based motif and which localizes mainly in early endosomes and on the cell surface. This observation was confirmed using a gpCD1b3 transfectant cell line (11). In contrast, we predicted that the remaining gpCD1 isoforms would traffic to the lysosomal compartment, similar to human CD1b and CD1c isoforms that contain a similar tyrosine-based sorting motif (30). Therefore, we characterized the differential trafficking of the CD1 isoforms expressed in guinea pig DC to examine this phenomenon in freshly isolated cells that naturally express these proteins.
The trafficking of distinct gpCD1b isoforms was compared in DC to
characterize the differential trafficking of two closely related CD1
isoforms. We first identified two mAbs, 1D12.4 and MSgp9, that
recognize the gpCD1b1 and gpCD1b3 isoforms, respectively, and do not
cross-react with other gpCD1 isoforms (11). Isolated and
permeabilized splenic DC from Flt-3LFc-treated animals were stained
with Abs to either gpCD1b1 or gpCD1b3 (Fig. 5, A and D) to
localize the compartmental expression of the isoforms. Cells were
colabeled with Ab to MHC class II which is expressed both on the cell
surface and in late endosomes/lysosomes (Fig. 5, B and
E). Double labeling of the DC for gpCD1b1 and MHC class II
revealed colocalization of CD1 and MHC class II (Fig. 5C,
arrow). These data are consistent with gpCD1b1 localization in the late
endosome or lysosome compartment. This same labeling procedure was also
conducted using the MSgp9 Ab specific for gpCD1b3 that lacks the
tyrosine-based sorting motif (Fig. 5, D–F). Most of the
staining for gpCD1b3 is on the cell surface with a small but distinct
punctate staining present inside the cell (Fig. 5D). As
predicted, the gpCD1b3 protein does not colocalize with the MHC class
II inside the cell but rather remains primarily on the cell surface
(Fig. 5F). Analysis of the DC preparations by FACS gave
similar results with the gpCD1b1 isoform staining weakly on the cell
surface (Fig. 5N) while gpCD1b3 isoform showed greater
surface staining (Fig. 5O). These data indicate that the
gpCD1b1 and gpCD1b3 isoforms have distinct patterns of expression in DC
that may allow sampling of different endosomal compartments, and mirror
the differential localization of human CD1b and CD1a isoforms,
respectively.
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I, arrows). These data support the
presence of CD1 in lysosomes together with a mycobacterial glycolipid
Ag known to be presented by human CD1b (31). Conversely,
when we stained for gpCD1b3 and LAM, there was little or no
colocalization of the molecules (Fig. 5L). Therefore, the
gpCD1b3 isoform might not have access to LAM molecules that are taken
up by DC. These data demonstrate that individual isoforms, even within
the same CD1b group, have distinct endosomal trafficking patterns that
could facilitate broad surveillance of the intracellular environment
for microbial Ags. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We used the in vivo administration of Flt-3LFc to increase the number
of DC in guinea pigs. In these studies, we demonstrate that a murine
Flt-3LFc reagent increases the number of DC in guinea pigs and
thus facilitates the isolation of these cells from guinea pig tissues.
Characterization of guinea pig DC demonstrates that these cells have a
phenotype similar to DC from other mammalian species. Unfortunately,
available Abs against cell surface proteins that are markers for mouse
and human DC, such as CD11c and CD83, fail to cross-react with guinea
pig cells (data not shown). However, the cell surface protein CD1 is
highly expressed on DC in lymphoid (Fig. 1) and nonlymphoid tissues in
guinea pigs (11, 12). Therefore, we tracked the guinea pig
DC through the isolation procedure by a combination of morphological
criteria (dendritic morphology) and cell surface markers (CD1 and MHC
class II). Consistent with the phenotype of human and mouse DC, guinea
pig DC express both CD1 and MHC class II. Morphologically, isolated
guinea pig DC exhibit fine cytoplasmic extensions (dendritic processes)
when cultured ex vivo. Functionally, the DC support MLR responses and
are capable of phagocytosis, also consistent with the DC phenotype.
Thus, using both functional and morphological criteria, we have
successfully isolated DC from guinea pigs that are comparable to those
described from other mammalian species.
Unlike humans that have single CD1b and CD1c isoforms, guinea pigs have
an expanded set of the individual CD1b and CD1c isoforms. For example,
guinea pigs have four members of the CD1b isoform group compared with
the single CD1b isoform of humans. The individual gpCD1b isoforms have
80% homology between the individual CD1b isoforms
(12). Interestingly, we noted previously that no homolog
of the human CD1A gene was found among the gpCD1 genes
identified thus far (12). However, examination of the
predicted protein sequence revealed the absence of a tyrosine-based
sorting motif on the C terminus of the gpCD1b3 protein while the other
isoforms contained this motif (12). This is a hallmark of
the mammalian CD1a protein which is the only group 1 CD1 isoform to
lack a tyrosine motif in its cytoplasmic tail. The consensus
tyrosine-based endosomal sorting motif used by CD1 proteins is YXXZ
(where Y = tyrosine; X = any amino acid; Z = bulky
hydrophobic). Such motifs have been shown previously to direct
intracellular traffic through the interaction of a family of adapter
protein complexes (32, 33). In contrast, the human CD1a
isoform lacks this sorting motif and remains primarily
surface-localized and traffics through early recycling endosomes
(23). We previously proposed that gpCD1b3 might traffic in
a manner similar to CD1a and thus fulfill the functional role of
sampling the recycling endosomal compartment for lipid Ags
(12). This observation was supported by immunofluorescence
microscopy using a cell line transfected with gpCD1 genes
(11). Confocal microscopy of the tissue-derived DC
described in this study also showed surface staining of the gpCD1b3
isoform (Fig. 5). In contrast, we found that in tissue-derived DC the
gpCD1b1 isoform colocalized with MHC class II, possibly in late
endosomes. Using LAM as a model CD1 lipid Ag, we demonstrated that
traffic of this Ag to late endosomes prevented colocalization of the Ag
with the gpCD1b3 isoform. These data indicate that the gpCD1b1 and
gpCD1b3 isoforms localize to distinct cellular compartments which may,
in turn, result in the presentation of different subsets of lipid
molecules to CD1-restricted T cells.
If indeed the guinea pig genome lacks a true homolog of CD1A, then it suggests that there may be strong evolutionary pressure in most mammalian species for a compensatory mechanism to sample lipid Ags from the recycling endosomal compartment. Sequence analysis clearly indicates that the gpCD1b3 isoform is a member of the CD1b isoform group although the trafficking pattern resembles that of the human CD1a isoform (11, 12). Thus, the guinea pig may have adapted one of the four gpCD1b isoforms to serve the same functional role as CD1a. Fine mapping of the YXXZ targeting motif has shown that substitution of the tyrosine (Y) residue is sufficient to prevent trafficking of the human CD1b protein to late endosomes (34). The gpCD1b1, -b2, and -b4 isoforms all use the TAT codon (Tyr) while the gpCD1b3 isoform has a TGT codon (Cys) at the same position. Thus, a single base change (A to G) at the second position of the tyrosine codon of an ancestral gpCD1B gene would result in a nonsynonymous mutation. This change could account for the predominantly cell surface location of the gpCD1b3 isoform. The capacity to broadly survey the endosomal system may be vital for effective immune responses to intracellular microbial infections that can occupy various subcellular compartments. Therefore, this may be an example of functionally convergent evolution in which a non-CD1a isoform has mutated to serve the functional role of sampling Ags from the early endosomal compartment.
The guinea pig provides an alternative small animal model that has advantages over the mouse for certain infectious diseases. Vaccine testing for a range of human pathogens has been conducted using the guinea pig animal model. These include models of tuberculosis, anthrax, Ebola virus, and chlamydia where the mouse is either resistant to infection or exhibits a different pathology (35, 36, 37, 38). The characterization of DC in the guinea pig provides a unique opportunity to gain insight into the immunological capacity of this animal model.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Christopher C. Dascher, Division of Rheumatology, Immunology and Allergy, Department of Pathology, Brigham & Women’s Hospital, Harvard Medical School, Smith 514, 1 Jimmy Fund Way, Boston, MA 02115. E-mail address: cdascher{at}rics.bwh.harvard.edu
3 Abbreviations used in this paper: DC, dendritic cell; Flt-3, fms-like tyrosine kinase 3; Flt-3L, Flt-3 ligand; Flt-3LFc, Flt-3L mouse IgG2a Fc fusion protein; gpCD1, guinea pig CD1; LAM, lipoarabinomannan; GFP, green fluorescent protein.
Received for publication July 25, 2002. Accepted for publication October 18, 2002.
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T cell pool. J. Immunol. 162:366./ cells. Eur. J. Immunol. 20:703.[Medline]
/ repertoire. J. Exp. Med. 171:1597.This article has been cited by other articles:
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S. K. Dougan, A. Salas, P. Rava, A. Agyemang, A. Kaser, J. Morrison, A. Khurana, M. Kronenberg, C. Johnson, M. Exley, et al. Microsomal triglyceride transfer protein lipidation and control of CD1d on antigen-presenting cells J. Exp. Med., August 15, 2005; 202(4): 529 - 539. [Abstract] [Full Text] [PDF]
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are DC from a Flt-3LFc-treated strain 2 guinea pig;
are DC from PBS-treated strain 2 guinea pig; 
are strain 2
guinea pig whole splenocytes; 
are autologous splenocytes from the
responder Hartley strain guinea pig. The x-axis of the
graph reflects the total number of cells added to the well from each
preparation. The actual DC in each well is dependent upon the purity of
the preparation. The Flt-3L-treated guinea pig splenic preparation was
