División Inmunogenética, Hospital de Clínicas, Facultad de Medicina, Universidad de Buenos Aires, Argentina
Correspondence: María del Carmen Salamone, Ph.D., División Inmunogenética, Hospital de Clínicas José de San Martín, Av Córdoba 2351, 3° Piso, (1120) Buenos Aires, Argentina. E-mail: marysasinec{at}is.com.ar
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Key Words: activated PBMC • phytohemagglutinin • NK • CD1d isotope
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1, 2, 3,
transmembrane, and cytoplasmic-3' UT) [2
,
5
]. This relative simple structure acquires complexity by
different, alternative splicing mechanisms [7
]. In human tissues CD1a–c and CD1-d, like molecules appear to differ in their tissue distribution and possibly in their function. CD1a–c are primarily thymic antigens, which are expressed on immature cortical thymocytes. Within the periphery, CD1a–c are expressed in a restricted fashion on certain antigen-presenting cells such as B lymphocytes, Langerhans cells, and activated monocytes [8 9 10 11 12 13 14 ]. In addition, we have also demonstrated the cytoplasmic expression of the classic CD1 molecules on activated, peripheral T lymphocytes [15 , 16 ]. Recent investigations had shed light on the function of CD1 antigens, suggesting that they may constitute a third pathway of antigen presentation. Evidence now exists for two types of specific T cell recognition mediated by CD1 proteins: a direct T cell reactivity to CD1 proteins and a CD1-dependent presentation of nonproteic, microbial antigens to specific T cells [13 , 17 18 19 20 21 ]. Elimination of CD1d1 gene by homologous recombination in knockout mice revealed a phenotype with normal numbers of CD4+ and CD8+ T cells but a marked reduction in the number of natural killer (NK) T cells [22 23 24 25 26 ]. Human CD1d has been shown to exhibit a prominent expression in human intestinal epithelial cells as well as in epithelial cells of a wide variety of tissue, hepatocytes, thymocytes, and some other cell types [27 28 29 ]. In the present study we investigated the presence of the CD1d antigen on activated peripheral blood mononuclear cells (PBMC) by flow cytometry, reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot analysis, and immuperoxidase staining. We demonstrated that CD1d isotype is expressed on activated, normal, mature T lymphocytes with a behavior similar to classic CD1 antigens. Furthermore, we explored whether mitogenic and allogenic activation could modulate the expression of this glycoprotein in mature T cells, in an attempt to further delineate its potential functions in the context of the effector mechanisms of the adaptive immune response.
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Cell cultures
PBMC were obtained from normal donors with confirmed negative
serology for hepatitis B virus, human immunodeficiency virus, hepatitis
C virus, and Chagas’ disease. PBMC were purified from buffy coat,
through Ficoll-Hypaque gradient sedimentation, and washed twice with
phosphate-buffered saline (PBS). Cells (1x106 cells/ml)
were suspended in Iscove’s modified Dulbecco’s medium (IMDM)
supplemented with 10% fetal calf serum (FCS), gentamicine (100
µg/ml), and fungizone (20 µg/ml) and used directly.
For mitogenic stimulation, PBMC were cultured in complete medium with 1 µg/ml phytohemagglutinin (PHA; Murex Diagnostics, UK) for different time periods at 37°C. In another set of experiments, PBMC were depleted of adherent cells by incubation on plastic Petri dishes. After 2 h of culture at 37°C, nonadherent cells were removed and used as a T cell-enriched subpopulation.
In addition, PBMC purified from peripheral blood of unrelated donors
were used as a source of effector and stimulator cells in mixed
lymphocyte reaction (MLR). Responder cells were cultured at 37°C in
96-well plates at 1 x 105/200 µl cells/well with
mitomycin C-treated PBMC as stimulators (1x105/200 µl
cells). Cells were collected after 0, 3, 4, and 5 days of culture.
After washing, dead cells were excluded by viability gradient. Then,
viable PBMC fractions were incubated with an anti-CD3 mAb (UCHT1)
followed by incubation with anti-mouse Ig-coated magnetic beads.
Positive-selected cells were used as a source of purified T cells. The
purity of these fractions checked by fluorescein-activated cell sorter
(FACS) analysis was 
96%. As control of allogenic stimulation, cells
cultured for 5 days were pulsed with 3H-thymidine for
18–24 h during the last day of culture. Proliferation was measured by
3H-thymidine incorporation in a liquid scintillation
counter.
Cell lines
CD1-transfected cells lines 10B3 (transfected with CD1A gene),
ER1 (transfected with CD1B gene), NR7 (transfected with CD1C gene),
CIRD, ER3 (transfected with CD1D gene), EL4 (mouse thymoma), or
CIR/mock (human lymphoblastoid cell line) were used as negative
controls and maintained at 37°C in RPMI 1640 medium supplemented with
10% FCS, 50 µg/ml gentamicin. All cell lines were kindly provided by
Dr. Cesar Milstein (MRC) except CIRD and CIR/mock, which were kindly
supplied by Dr. Steve Porcelli (Albert Einstein College of
Medicine, Bronx, NY). The pre-T, MOLT-4 cell line was grown in IMDM and
supplemented similarly.
FACS analysis
Cells (106/ml) were fixed in 2% formaldehyde in PBS
for 15 min, washed, and permeabilized with methanol for 30 min on ice.
Cells were washed again with PBS and incubated with a solution of
glycine/PBS (0.1 mg/ml) for 15 min at room temperature. After washing,
indirect immunofluorescence assay was performed by incubating
permeabilized or nonpermeabilized cells for 30 min at room temperature,
with appropriated amounts of NOR3.2 mAb. IgG1 isotype control antibody
was included to establish background fluorescence. After washing, a
fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse (GaM)
F(ab')2 Ig (GAM/FITC, Dako Corp., Carpinteria, CA) was
added for 30 min on ice. Cells were then washed twice with RPMI
1640/FCS, and the viability of nonpermeabilized cells was evaluated by
exclusion with propide iodide. In experiments where permeabilized cells
were compared with nonpermeabilized cells, dead cells were excluded by
viability gradient. Immunofluorescence was analyzed by flow cytometry
in a FACStar plus® (Becton Dickinson, San Jose, CA).
Reverse transcription
Total poly (A+) RNA was prepared from cell lines or activated
PBMC by using guanidinium thiocyanate and oligo (dt)-cellulose
microcolumns (Pharmacia P-L Biochemicals, Uppsala, Sweden), according
to the manufacturer’s recommended protocol. RT was carried out in a 50
µl reaction mixture containing 1x RT buffer, 10 mM dithiothreitol
(DTT), 0.25 mM of each of the four deoxyribonucleotide triphosphates,
20 pmol poly-dT, and 80 units of RNasin inhibitor (Promega, Madison,
WI). After 3 min at 65°C, 2 units of RT were added, and the reaction
was incubated for 60 min at 42°C.
cDNA amplification
After RT, cDNAs were amplified by PCR. The reaction was
performed in a 50 µl reaction mixture containing 1 µl cDNA, 10 pmol
of each 5' primers (specific for the 3 domain) and 3' primer
(specific for the 3' UT region), 10x PCR buffer, 0.5 mM dNTPs, and 2
units of TaqPol (Promega). The reaction was incubated for 35 cycles of
denaturation, 30 sec at 92°C; annealing, 1 min at 55°C; and
extension, 3 min at 72°C, followed by a final extension for 10 min at
72°C. Reaction was also performed without cDNA as a control for PCR
contamination. The oligonucleotide sequence used for CD1d has been
demonstrated previously [7
]: (CD1D) (5') AGT TTA CGT AAT
GAA TTC GGG CAC TCA GCC AGG GGA CAT CCT GCC CAA (3') and (5') GTC AAT
ATC TAT GGA TCC GAT ACA AGT TTG CAC ACC TTT GCA CTT CTG (3'); 6 µl of
the amplified products was loaded in a 2% agarose microgel with 1x
TBE (Tris borate/EDTA electrophoresis buffer) [89 mM
Tris/borate/2.5 mM ethylenediaminetetraacetate (EDTA)]. Bands were
visualized by ethidium bromide staining under UV light.
Western blot analysis
Cells (PBMC and the nonadherent T cell fraction) cultured at
1 x 106 cells/ml were collected in PBS and
centrifuged at 1000 g for 10 min. The MOLT-4, pre-T cell
line was used as controls of positive reaction. In another set of
experiments, the T cell fraction was purified from resting and
activated PBMC by Dynals beads® as described above. The cell pellets
were washed twice with PBS, resuspended in 1 ml ice-cold lysis buffer
containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40
(NP-40), 10 mM EDTA, and a protease inhibitor cocktail [0.2 mM
phenylmethylsulfonyl fluoride (PMSF), 0.1% aprotinin, 0.7 µg/ml
leupeptin, 1 µg/ml pepstatin A, 10 mM iodoacetamide, and 1 mM sodium
vanadate], and left on ice for 30 min. The solution was then
centrifuged at 4°C for 10 min at 10,000 g, and the
resultant cell lysate was mixed 1:1 with 2x sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer.
Briefly, the suspension was then boiled for 5 min, cooled on ice, and
resolved on a 10% polyacrylamide slab gel. After electrophoresis, the
separated proteins were electroblotted onto nitrocellulose membranes
(Bio-Rad, Richmond, CA) and probed with a 1:100 dilution of the NOR3.2
mAb. Blots were then incubated with a 1:2000 dilution of a horseradish
peroxidase-conjugated anti-mouse IgG (Sigma Chemical Co., St. Louis,
MO). The reaction was finally developed using 4-chloro-1-naphthol
(Sigma). Rainbow protein molecular weight (MW) markers were from
Bio-Rad.
Data analysis
Protein bands were analyzed with a Fotodyne Image Analyzer®
(Fotodyne, Inc., Hartland, WI). Results were expressed as optical
densities by means of the Image Quant software.
Immunoperoxidase staining
Multitest slides (ICN Biomedicals Inc., Horsham, PA) were
treated with 30 µl/well of a 20% solution of poly-l-lysine (Sigma).
After overnight incubation at 4°C, glass slides were washed for 10
min in distilled water and air-dried. PBMC (5x105) were
added to each well of coated glass slides and incubated for 30 min at
4°C. Cells were fixed and permeabilized as described above.
Endogenous peroxidase was inhibited by preincubation with 0.1%
phenylhydrazine hydrochloride in PBS at 37°C for 30 min. The first Ab
was incubated overnight at 4°C followed by 2 h at 37°C. Slides
were washed twice in PBS for 15 min. A GaM Ab (Sigma) was then
incubated for 60 min at 37°C. After rinsing in PBS, slides were
incubated with a mouse monoclonal PAP (peroxidase
anti-peroxidase) complex (Sigma) for 60 min at 37°C. Peroxidase
activity was detected using 3'-diaminobenzidine tetrahydrochloride
(DAB) as developing agent. The reaction was stopped by dilution in PBS,
and slides were mounted for microscopic observation using buffered
glycerin.
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FACS staining revealed a clear increase in the mean fluorescence intensity (MFI) in permeabilized MOLT-4 as well as in ER3- and CIRD-transfected cell lines (EL4 and CIR/mock cells transfected with a CD1D cDNA construct, respectively; Fig. 1 ). The labeling was specific, because NOR3.2 mAb failed to recognize CD1A-(10B3)-, CD1B-(ER1)-, and CD1C-(NR7)-transfected cell lines and EL4 or CIR/mock, which were used as negative controls (Fig. 1) . After the experimental conditions were established on cell lines, expression of CD1d antigen was analyzed on resting and activated PBMC. Stimulation with PHA for 48–72 h increased CD1d reactivity on PBMC, as clearly shown by increased MFI when compared with the staining of nonstimulated PBMC population (Fig. 2 ). After two days of mitogenic stimulation, the expression of CD25 and anti-HLA-DR antigens (used as controls of activation) was >55% and 50%, respectively (not shown).
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Figure 1. Detection of CD1d expression by flow cytometry. Cells (106
cells/ml) were fixed for 15' in 2% formaldehyde in PBS, washed and
permeabilized with methanol for 30' at 4°C, incubated with NOR3.2 mAb
or with an irrelevant isotype mouse control Ab, and stained with a
FITC-conjugated GaM F(ab')2 Ig. Staining
pattern of MOLT-4, ER3, CIRD, 10B3, ER1, NR7, and EL4 cell lines on
nonpermeabilized (A) and permeabilized cells (B). The figure shows a
clear increase on the MFI on MOLT-4, ER3, and CIRD cell lines after
formaldehyde and methanol treatment. Histograms represent the analysis
of 10,000 cells and show the relative cell number (y-axis)
plotted against the relative intensity of green fluorescence
(x-axis). FACS analysis of the NOR3.2 mAb reactivity is
shown by shaded histograms, and the irrelevant mAb is presented in
outlined histograms.
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Figure 2. Reactivity of NOR3.2 mAb in nonpermeabilized (A) and permeabilized (B)
resting and PHA-activated PBMC, which were cultured in the presence of
1 µg/ml PHA for 0, 48, and 72 h. Fixation and permeabilization
were performed under the same experimental conditions described. Flow
cytometry analysis of four donors revealed a clear increase in CD1d
protein expression between 48 to 72 h after activation. Histograms
represent the analysis of 10,000 cells and show the relative cell
number (y-axis) plotted against the relative intensity of
green fluorescence (x-axis). FACS analysis of the NOR3.2 mAb
reactivity is shown by shaded histograms, and the irrelevant mAb is
presented in outlined histograms. Results are representative of three
independent experiments.
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3
and 3' untranslated region of the CD1D gene. A single band representing
the transcript encoding the membrane isoform [7
] was
observed in ER3 (CD1d-transfected; Fig. 3
, lane b) and the MOLT-4, pre-T cell line (not shown). Consistent
with the cytofluorometric study, the same single band observed in
positive controls was present in all three donors analyzed after PBMC
were subjected to PHA stimulation for 72 h (Fig. 3
, lane c). No
band was detected on resting PBMC in three of the individuals analyzed
(Fig. 3
, lane d). However, a weak band was detected in a fourth donor
(result not shown).
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Figure 3. Analysis of CD1D mRNA expression. cDNA was amplified by PCR for 35
cycles, and the products were electrophoresed in a 2% polyacrylamide
gel. A single band representing the membrane isoform was amplified in
ER3, CD1D-transfected cell lines (lane b) and 72-h, PHA-activated PBMC
(lane c). Resting PBMC did not show any band in three different
individuals (lane d). Size markers, 100 bp DNA ladder (lane a).
Results are representative of three independent experiments.
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Figure 4. Western blot analysis of CD1d expression. PBMC (A) were cultured in the
presence of PHA for 0 (lane a), 24 (lane b), 48 (lane c), and 72 (lane
d) h. The MOLT-4, pre-T cell line was used as a positive control (lane
e). Cells were collected, washed, and homogenized in the presence of
protease inhibitors. Equal amounts of protein (30 µg) were subjected
to SDS-PAGE on a 10% polyacrylamide slab gel and immunoblotted with
the NOR3.2 CD1d mAb (1 µg/ml). (B) Densitometric profile representing
the amount of CD1d protein expressed by resting and activated PBMC and
by the MOLT-4, pre-T cell line used as control. Bands corresponding to
the gels described in A were analyzed using a Fotodyne Image
Analyzer® (Fotodyne, Inc.), and the results were expressed as optical
densities by means of the Image Quant software. Values on the
x-axis represent arbitrary densitometric units (A.U.). (C
and D) Expression of CD1d protein product on the T cell-enriched
population. PBMC were depleted of adherent cells and used as a T
cell-enriched subpopulation (see Materials and Methods). Cells were
cultured and analyzed under the same experimental conditions described
in A and B. (C) Enriched T cell fraction stimulated with PHA for 0
(lane a), 24 (lane b), 48 (lane c), and 72 (lane d) h. (D)
Densitometric profile. Values on the x-axis represent A.U.
Results are representative of three independent experiments.
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Figure 5. Western blot analysis of CD1d expression on purified T cells activated
by alloantigens. PBMC purified from peripheral blood of unrelated
donors were used as the source of effector and stimulator cells in MLR.
Responder cells were cultured at 37°C in 96-well plates at 1 x
105/200 µl cells/well with mitomycin C-treated PBMC as
stimulators (1x105/200 µl cells). Cells were collected
after 0 (lane a), 3 (lane b), 4 (lane c), and 5 (lane d) days of
culture. After washing, dead cells were excluded by viability gradient.
Then, viable PBMC fractions were incubated with an anti-CD3 mAb (UCHT1)
followed by incubation with anti-mouse Ig-coated magnetic beads.
Positively selected cells were used as a source of purified T cells.
The purity of this fraction at all times tested was 96%. The MOLT-4,
pre-T cell line was used as positive control (lane e). Cells were
collected, washed, and homogenized in the presence of protease
inhibitors. Equal amounts of protein (30 µg) were subjected to
SDS-PAGE on a 10% polyacrylamide slab gel and immunoblotted with the
NOR3.2 CD1d mAb (1 µg/ml). (B) Densitometric profile representing the
amount of CD1d protein expressed by resting and activated T cells and
by the MOLT-4, pre-T cell line used as control. Protein bands were
analyzed using a Fotodyne Image Analyzer® (Fotodyne, Inc.), and the
results were expressed as optical densities by means of the Image Quant
software. Values on the x-axis represent A.U. Results are
representative of two independent experiments.
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Figure 6. Immunocytochemical detection of CD1d using the NOR3.2 mAb. PBMC were
stimulated with PHA for 0 or 72 h. Cells were collected, fixed,
and permeabilized under the same experimental conditions described for
flow cytometry. (A) ER3 CD1D-transfected cell lines. (B) MOLT-4, pre-T
cell line. (C) Resting and activated PBMC. The figure shows the
immunoperoxidase staining pattern compatible with the presence of CD1d
protein in the cytoplasm and associated to the cell membrane. PBMC also
showed a perinuclear staining pattern. Black arrows indicate membrane
and cytoplasmic labeling. Red arrows show the perinuclear staining
pattern. Results are representative of three independent
experiments._art>
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Previously, we have shown the cytoplamic expression of CD1a–c antigens on mature T lymphocytes after PHA activation [15 , 16 ]. The human CD1d molecule appears to differ from classic CD1a, b, and c proteins in many aspects, such as tissue distribution, expression, and functions. However, our study demonstrates that the CD1d isotype behaves similarly as classic CD1 antigens in mature T cells. Peripheral blood lymphocytes cultured in the presence of PHA have been shown to strongly re-express CD1d, as demonstrated by flow cytometry, RT-PCR of the corresponding transcript, and Western blot analysis of the protein product. The densitometric profile revealed a peak of maximal expression of CD1d protein 48 h after PHA activation. The level of CD1d protein product expressed by the blast cells at 48 h was greater than that observed in 72 h-activated T lymphocytes. Moreover, it was also greater than immunoreactivity raised by the MOLT-4 pre-T cell line. In an attempt to gain insight into the biologic relevance of our results, the kinetic expression of CD1d after allogenic stimulation was also explored. Western blot analysis clearly showed the expression of CD1d antigen on alloantigen-activated T cells. The densitometric profile revealed that CD1d expression increased significantly at day 3 following stimulation. After four days of culture, the amount of CD1d protein started to decrease in a time-dependent manner. In both mitogenically and allogenically activated T cells, CD1d expression showed a kinetic that peaked earlier than the time when maximal proliferation is usually observed.
It should be highlighted that a weak immunoreactivity was found by Western blot in resting PBMC, although the transcript of CD1D was generally undetecd by RT-PCR. Similar discrepancies have been documented for other CD1d molecules [28 ]. This observation suggests that a potential posttranscriptional mechanism could be involved in the stabilization of the protein product. Effects attributed to a posttranscriptional regulation have been suggested previously for other members of CD1 family. An exclusion was observed between the classic CD1 (CD1A, C) genes when two or more members of this family were cotransfected into COS cells [32 ]. Herein, we conclude that CD1d antigen is induced in a mature T cell after activation, as has been shown previously for other CD1 antigens [15 , 16 ]. We do not rule out the possibility that the CD1d isotype could also be regulated by other members of the CD1 family.
In the present study, we used the NOR3.2 mAb, an antibody that recognized CD1d antigen under denaturing conditions [30 ], and developed experimental strategies for its use in immunocytochemistry and flow cytometry studies. We demonstrated the specific staining of NOR3.2 on CD1d-transfected cells and studied the expression of the CD1d antigen after T cell activation. Immunocytochemical studies of the subcellular distribution of this protein revealed that CD1d was localized at the cytoplasmic compartment and at the plasma membrane. The perinuclear staining observed in activated PBMC resembles that observed in permeabilized CD1b-transfected cells under denaturing conditions, where the accumulation of CD1b heavy chains in the endoplasmic reticulum has been demonstrated [33 ]. Although CD1d has been detected at the cytoplasm and plasma membrane, the strong cytoplasmic staining pattern suggests that it behaves mainly as an intracellular protein in activated T cell blasts. Human CD1b, CD1c, and CD1d molecules contain a YXXZ motif (tyrosine-amino acid-amino acid-hydrophobic amino acid) in the cytoplasmic tail that is known to direct proteins to subcellular compartments by AP-1 and AP-2 interactions [34 35 36 37 ]. Probably, the CD1d cytoplasmic tail motif is responsible for the intracellular localization and regulates its cell surface expression in mature, activated T lymphocytes. The cytoplasmic tail motif, the subcellular localization, and the regulated expression of the CD1d molecule are biochemical features that remind other T cell regulatory markers, such as CD152 (CTLA-4). Surface expression of CTLA-4 peaks at 48 h following cell activation, returning to background levels at 96 h. This antigen expresses a YVKM motif in its cytoplasmic tail involved in the intracellular localization and biological function [38 39 40 ]. The role of the CD1d antigen on activated, mature T cells still remains to be elucidated, but these similarities suggest a potential role as a regulatory or costimulatory molecule involved in peripheral tolerance.
Other potential functions related to the expression of CD1d on T cells could be associated to the control or activation of NKT cells. It has been suggested that at least two mechanisms exist, by which NKT cells exert their effector functions: a CD1d-restricted and a non-CD1d-restricted mechanism [22 , 41 42 43 ]. In mice, the production of IL-4 by NKT cells correlates with their ability to prevent the onset of autoimmunity [44 , 45 ]. In humans, autoimmune disease like multiple sclerosis and insulin-dependent diabetes mellitus is associated with the loss of NKT cells or IL-4-producing NKT cells [46 , 47 ]. According to these observations and our results, it could be speculated that expression of CD1d in conventional, activated T cells could be an alternative mechanism to achieve homeostasis within the immune system and prevent the development of autoimmune disease after a normal response to microbial antigens, by maintaining the pool of the NKT cell population.
It has been also demonstrated that activation of mouse NKT cells, may induce innate immunity indirectly via a rapid and selective induction of NK cell proliferation and cytotoxicity [48 ]. Flow cytometry studies demonstrated NK cell proliferation in the mixed lymphocyte reaction [49 ]. Our results show clearly that CD1d levels are strongly up-regulated in response to alloantigens. Therefore, the CD1d molecule could also be involved in the proliferation of additional cells subsets (i.e., NK cells), triggering an indirect tissue damage, for instance in transplant rejection. Because NKT cells constitute a heterogeneous cell population that may be activated by different antigens [42 , 50 51 52 53 54 ], it also could be speculated that expression of CD1d on activated T cells could have different implications, according to the class of antigen that has been presented and the type of NKT cell subpopulation that has been activated.
Taken together, the results presented in this study indicate that, upon activation, mature T lymphocytes can strongly re-express CD1d antigen. Although, the role of the CD1d antigen on activated, mature T cells remains to be elucidated, these observations give light to a new ontogeny for CD1d isotype in the T cell lineage. It is expressed initially at low levels in thymus [37 ], its expression is down-modulated on mature, circulating, resting T cells, being strongly re-expressed after activation.
Received April 14, 2000; revised August 28, 2000; accepted September 27, 2000.
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ß+ T cells Nature 372,691-694[Medline]24+ CD4+ NKT cells activated by -glycosylceramide-pulsed monocyte-derived dendritic cells J. Immunol. 164,4458-4464-galactosylceramide by natural killer T cells is highly conserved through mammalian evolution J. Exp. Med. 188,1521-1528/ß T cell receptor (TCR)+CD4-CD8- (NKT) thymocytes prevent insulin-dependent diabetes mellitus in nonobese diabetic (NOD)/Lt mice by the influence of interleukin (IL)-4 and/or IL-10 J. Exp. Med. 187,1047-105614-J281 transgenic nonobese diabetic mice against diabetis J. Exp. Med. 188,1831-183924JQ antigen receptor in patients with systemic sclerosis J. Exp. Med. 182,1163-116824JQ T cells in type 1 diabetes Nature 391,177-181[Medline]14 NKT cells by glycosylceramides Science 278,1626-1629-galactosylceramide specifically stimulates V14+ NK T lymphocytes J. Immunol. 161,3271-3281This article has been cited by other articles:
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