Human CD1d Functions as a Transplantation

Antigen and a Restriction Element in Mice

Bin Wang, Taehoon Chun, Ingrid C. Rulifson, Mark Exley,
This information is current as Steven P. Balk and Chyung-Ru Wang
of January 31, 2013. J Immunol 2001; 166:3829-3836; ;
http://www.jimmunol.org/content/166/6/3829

Downloaded from http://jimmunol.org/ at Albert Einstein College of Medicine on January 31, 2013
References This article cites 55 articles, 42 of which you can access for free at:
http://www.jimmunol.org/content/166/6/3829.full#ref-list-1
Subscriptions Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscriptions
Permissions Submit copyright permission requests at:
http://www.aai.org/ji/copyright.html
Email Alerts Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/cgi/alerts/etoc

The Journal of Immunology is published twice each month by

The American Association of Immunologists, Inc.,
9650 Rockville Pike, Bethesda, MD 20814-3994.
Copyright © 2001 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Human CD1d Functions as a Transplantation Antigen and a
Restriction Element in Mice1

Bin Wang,* Taehoon Chun,* Ingrid C. Rulifson,† Mark Exley,‡ Steven P. Balk,‡ and
Chyung-Ru Wang2*
To study the potential functions of human CD1d (hCD1d), we developed transgenic (Tg) mice that ectopically express hCD1d
under the control of H-2Kb promoter. High levels of hCD1d expression were detected in all Tg tissues tested. Skin grafts from the
Kb/hCD1d Tg mice were rapidly rejected by MHC-matched non-Tg recipient mice, suggesting that hCD1d can act as transplan-
tation Ags. Furthermore, we were able to elicit hCD1d-restricted CD8ⴙ CTLs from mice immunized with Kb/hCD1d Tg spleno-

Downloaded from http://jimmunol.org/ at Albert Einstein College of Medicine on January 31, 2013
cytes. These CTLs express TCR rearrangements that are distinct from invariant TCR of NK T cells, and secrete significant
amounts of IFN-␥ upon Ag stimulation. Analysis with various hCD1d-expressing targets and use of Ag presentation inhibitors
indicated the recognition of hCD1d by CTLs did not involve species or tissue-specific ligands nor require the processing pathways
of endosomes or proteasomes. Additionally, the reactivity of hCD1d-specific CTLs was not affected by acid stripping followed by
brefeldin A treatment, suggesting that CTLs may recognize a ligand/hCD1d complex that is resistant to acid denaturation, or
empty hCD1d molecules. Our results show that hCD1d can function as an alloantigen for CD8ⴙ CTLs. The hCD1d Tg mice
provide a versatile model for the study of hCD1d-restricted cytolytic responses to microbial Ags. The Journal of Immunology,
2001, 166: 3829 –3836.

C D1 molecules are MHC-unlinked class Ib molecules that
have been conserved throughout mammalian evolution
(1). Although the overall structure of CD1 resembles that
of MHC class Ia molecules, the sequence homology between CD1
and MHC class Ia is only between 25% and 30% (2). Unlike class
Ia molecules, CD1 is relatively nonpolymorphic, and its expres-
sion is TAP independent (3, 4). CD1 proteins can be divided into
two distinct groups: group 1 CD1, including human CD1 (hCD1)3
a, b, and c, and group 2 CD1, consisting of hCD1d and two mouse
CD1 molecules, mCD1.1 and mCD1.2 (1, 5). They differ in their
sequence, tissue distribution, and possibly function. Group 1 CD1
are expressed by cortical thymocytes (6), a subset of B cells (7),
and APCs, such as dendritic cells, and activated monocytes (8).
The tissue distribution of group 2 CD1 molecules is more wide-
spread. hCD1d is expressed on most thymocytes, activated T cells,
peripheral B cells, monocytes (9), and intestinal epithelial cells
(IEC) (10, 11). The expression of hCD1d on IEC is predominately
localized to the apical and lateral regions of small and large intes-
tinal epithelia (12), placing it in a critical location for interaction
*Gwen Knapp Center for Lupus and Immunology Research, Committee on Immu-
nology and Department of Pathology, and †Ben May Institute for Cancer Research,
University of Chicago, Chicago, IL 60637; and ‡Cancer Biology Program, Hematol-
ogy/Oncology Division, Beth Israel Deaconess Medical Center and Harvard Medical
School, Boston, MA 02215
Received for publication October 11, 2000. Accepted for publication January 4, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants AI43407 (to
C.R.W.) and AI42955 (to S.P.B.).
2
Address correspondence and reprint requests to Dr. Chyung-Ru Wang, University of
Chicago, Gwen Knapp Center for Lupus and Immunology Research, Room 412, 924
East 57th Street, Chicago, IL 60637-5420. E-mail address: cwang@midway.
uchicago.edu
3
Abbreviations used in this paper: hCD1, human CD1; ␣-GalCer, ␣-galactosylcer-
amide; ␤2m, ␤2-microglobulin; IEC, intestinal epithelial cell(s); m, mouse; Tg,
transgenic.
with intraepithelial lymphocytes. mCD1 is expressed on cells of
multiple hemopoietic lineages, including B and T cells, macro-
phages, and dendritic cells (13–15). However, the expression of
CD1 on mouse IEC (mIEC) remains controversial, as anti-CD1
mAbs differ in detection of CD1 expression on mIEC (13–16).
Human group 1 CD1 can present lipid and glycolipid Ags de-
rived from mycobacterial cell wall to different subsets of T cells,
including CD4⫹ T cells (17), CD8⫹ T cells (18), and CD4⫺CD8⫺
T cells (19, 20). Group 2 CD1 can also bind lipid Ags, such as
glycosylceramide and phospholipids (21, 22). Additionally,
mCD1.1 has been shown to bind hydrophobic peptides as well
(23). Both hCD1d and mCD1.1 can be recognized by a unique
subset of T cells, the NK T cells, which express a restricted range
of TCRs bearing a single invariant V␣-chain (V␣14J␣281 in mice
and V␣24J␣Q in humans) paired with limited sets of V␤-chain
(24 –26). NK T cells can recognize CD1d in the absence of exog-
enous Ags, but their reactivity can be enhanced by the addition of
synthetic lipid Ags, such as ␣-galactosylceramide (␣-GalCer) (3)
(21, 27). Upon activation, NK T cells promptly produce large
amounts of cytokines, in particular IL-4. Several studies suggest
that NK T cells may have important functions in regulating im-
mune responses (28 –31). In addition to NK T cells, T cells ex-
pressing diverse TCR ␣- and ␤-chains have also been found to
recognize mCD1. These include some CD4⫹ T cells from class
II-deficient mice (32, 33), some CD8⫹ CTLs from mice immu-
nized with plasmid DNA containing chicken OVA (34), and from
mice immunized with a mCD1 transfectant coated with CD1-bind-
ing peptide (23, 35). However, no hCD1d-restricted CD4⫹ or
CD8⫹ CTLs have been isolated to date.
Expression of two biochemically distinct forms of hCD1d, ␤2-
microglobulin (␤2m)-associated and non-␤2m-associated hCD1d,
has been demonstrated (9, 12, 36). The ␤2m-associated hCD1d is
a mature 48-kDa glycoprotein, which is expressed on the surface
of thymocytes and B cells. The non-␤2m-associated hCD1d is a
37-kDa nonglycosylated isoform, which is predominately ex-
pressed on the IEC. The functional role of hCD1d on IEC has been

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00

3830
implicated by studies showing that anti-hCD1d mAb inhibit IEC-
induced proliferation of CD8⫹ T cells (37). Recently, it was also
shown that cross-linking hCD1d on the surface of human IEC with
the anti-CD1d Ab specifically induced epithelial IL-10 expression,
which may serve to dampen the epithelial proinflammatory signals
(38).
Little is known about the Ag presentation by hCD1d other than
its ability to present ␣-GalCer to CD4⫺CD8⫺ or CD4⫹ NK T
cells. To further the study of hCD1d functional properties, we have
derived transgenic (Tg) mice expressing hCD1d molecules. We
report in this work that hCD1d behaves as a transplantation Ag in
mice, as observed in rapid rejection of skin grafts from hCD1d Tg
mice. Furthermore, hCD1d Tg spleen cells are effective in induc-
ing CD8⫹ hCD1d-specific CTLs in normal mice. The hCD1d-
specific T cells are capable of killing both hCD1d-positive mouse
and human cells, suggesting CTLs recognize hCD1d as an intact
molecule. Our data demonstrate that, similar to hCD1a, b, and c,
hCD1d could function as a restriction element for CTLs.
Materials and Methods
Generation of Kb/hCD1d Tg mice
Genomic clone containing full-length hCD1D was isolated from human
genomic library (Stratagene, La Jolla, CA). The chimeric gene in which
hCD1D gene was driven by the H2-Kb promoter was constructed as shown
in Fig. 1. Chimeric DNA fragment was injected into the pronuclei of fer-
tilized eggs of (C57BL/6 (B6) ⫻ CBA)F1 mice to produce Tg founder
mice. Tg-positive mice were identified by PCR using primers specific for
hCD1D exon 2 (5⬘-CGAGGGCCCCACGCCGGGCGATA-3⬘) and exon 3
(5⬘-CAGAGAGCGGACGGTGTCC-3⬘). Two lines of Tg mice, line 1 and
line 3, were established by crossing Tg founder mice with B6 mice. Line
1 was chosen for further backcrossing with B6 mice because it showed
higher surface expression of hCD1d than line 3 (data not shown). The Tg
mice used in this study have been backcrossed four to seven generations
onto B6 background.
Expression of hCD1d mRNA in Tg mice
RNA was extracted from various tissues of Tg mice with TRIzol reagent
(Life Technologies, Grand Island, NY). cDNA was prepared using random
hexamer primers, and amplified by PCR using primers specific for hCD1D
exon 2 and exon 3. The amount of template cDNA used in each reaction
was normalized to the amount of hypoxanthine phosphoribosyltransferase
mRNA amplified with primers 5⬘-GTTGGATACAGGCCAGACTTT
GTTG-3⬘ and 5⬘-GAGGGTAGGCTGGCCTATAGGCT-3⬘.
Cell lines and CD1 transfectants
RMA-S and L929 cells were transfected with Kb/hCD1D chimeric gene by
electroporation, followed by G418 selection and FACS analysis to generate
lines stably expressing hCD1d. The derivation of RMA-S and L929 trans-
fectants expressing mCD1.1 or mCD1.2, and C1R transfectants expressing
CD1a, b, c, d, or CD1d/a chimeric protein have been described previously
(26, 39). Human cell lines, U937, THP-1, K-562, Jurkat, MOLT-3,
MOLT-4, Raji, and JY, were kindly provided by Gijs van Seventer (Uni-
versity of Chicago, Chicago, IL). All transfectants and human cell lines
were maintained and cultured in RPMI 1640 medium (Life Technologies),
supplemented with 10% heat-inactivated FCS (Sigma, St. Louis, MO), 2
mM L-glutamine, 100 U/ml penicillin and streptomycin, and 50 ␮M 2-ME
(RPMI 10). Human intestine epithelial cell lines (HT29, T84, and CACO2)
were kindly provided by Eugene Chang (University of Chicago). Con A-
and LPS-induced blasts were prepared by incubating spleen cell suspension
(5 ⫻ 106) with Con A (2.5 ␮g/ml; Sigma) or LPS (5 ␮g/ml; Sigma) for
72 h in RPMI 10. Bone marrow-derived macrophages were obtained by
culturing bone marrow cells (2 ⫻ 105 cells/ml) for 6 days in RPMI 10
supplemented with 30% L929 cell supernatant.
Abs, cell preparations, and FACS analysis
The following Abs were purchased from PharMingen (San Diego, CA):
anti- CD11a (2D7), anti-CD102 (3C4), anti-CD54 (3E2), FITC anti-
H2-Kb (AF6-88.5), FITC anti-CD8␣ (53-6.7), PE anti-CD4 (RM4-5),
PE anti-NK1.1 (PK136), FITC anti-H2-IAb (M5114), biotinylated goat
anti-mouse IgG2b, and biotinylated goat anti-mouse IgG1. The hCD1d-
specific Abs, 51.1 (mIgG2b) and 42.1 (mIgG1), have been described
previously (9). PK136, anti-mouse NK1.1; YTS.169.69, anti-mouse
HUMAN CD1d Tg MICE
CD8; GK1.5, anti-mouse CD4; 16-1-11N, anti-H2-Kk; Y3, anti-H2-Kb;
B22, anti-H2-Db; BB7.2, anti-HLA-A2 (mIgG2b); and 4D12, anti-
HLA-B5 (mIgG1) were obtained from the American Type Culture Col-
lection (ATCC, Manassas, VA).
Single cell suspensions from thymus, spleen, lymph nodes, and Peyer’s
patches were prepared using standard procedure and stained in immuno-
fluorescence buffer (HBSS containing 2% FBS and 0.1% NaN3) using
combinations of fluorescent-conjugated Abs for 30 min at 4°C. The IEC
were prepared and purified through the discontinuous 25/40/70% Percoll
gradient centrifugation, as described by Yamamoto et al. (40). Cells that
layered between the 40 and 25% interface were collected as IEC. For
isolating hepatic epithelial cells, mouse livers were perfused for 10 min
with perfusion I medium (in mmol/L: NaCl, 120; KCI, 5; KH2PO4, 0.4;
Na2HPO4, 0.2; NaHCO3, 25; EGTA, 0.5; D-glucose, 5.5; pH 7.4), then for
10 min with perfusion II medium (in mmol/L: NaCl, 120; KCI, 5; KH2PO4,
0.2; NaHCO3, 25; MgSO4, 0.4; MgCl2, 0.5; CaCl2, 3; D-glucose, 5.5; pH
7.4) containing 0.05% collagenase, 0.5 ml insulin (5 mg/ml), and 0.8 U
trypsin inhibitor per unit tryptic activity in the collagenase (41). Then hepa-
tocytes were minced, and separated on a Percoll density gradient described
Downloaded from http://jimmunol.org/ at Albert Einstein College of Medicine on January 31, 2013
as above. Thymic stromal cell suspensions were prepared by digesting fetal
thymi in 0.1% trypsin, 0.5 mM EDTA for 40 min at 37°C. Digestion was
stopped by addition of immunofluorescence (IF) buffer. After mechanical
disruption of the lobe, cells were harvested and washed two times with IF
buffer before cell surface staining experiments. Cells (106) were stained
with anti-hCD1d, followed by biotin-conjugated goat anti-mouse IgG2b
and a third incubation with streptavidin-conjugated PE and FITC anti I-Ab.
The stained cells were analyzed by flow cytometry using a FACSCalibur
(Becton Dickinson, Mountain View, CA) with the CellQuest software.
Skin grafting
Female hCD1d Tg⫹ or Tg⫺ mice (6 – 8 wk old) were used as donors.
Full-thickness sections of skin (⬃1 ⫻ 1 cm in size) were harvested from
the tail of donors and grafted onto the dorsal side of female C57BL/6.
Bandages were removed on day 7 post transplant, and grafts were moni-
tored for 55 days for evidence of rejection. Rejection was defined as com-
plete necrosis of the skin grafts.
Generation of anti-hCD1d CTLs
B6 ⫻ CBA/F1 mice were primed with 107 irradiated hCD1d⫹ splenocytes
(in B6 background) through i.p. injection and footpad injection. After 10
days, lymphocyte suspensions were prepared from draining lymph nodes
and spleen, and then cultured with irradiated hCD1d⫹ splenocytes (2–5 ⫻
106 cells/ml) in RPMI 10 medium. One week later, cultures were restim-
ulated with hCD1d⫹ splenocytes and maintained in supplemented Mischell
Dutton medium (SMDM) with IL-2 supplement (20 U/ml). IL-2 for re-
stimulations was partially purified from the supernatant of EL4.IL2 cells
(ATCC). After that, the CTLs were restimulated weekly with the irradiated
L929/hCD1d transfectants. CTL clones were established by limiting dilu-
tion method in the presence of rIL-2 (10 U/ml; PharMingen) and 2.5 ⫻ 104
irradiated L929/hCD1d transfectants. The established clones were main-
tained by weekly stimulation with irradiated L929/hCD1d transfectants.
The CTL activity was tested by 51Cr release assay, as described below.
CTL assay
One million target cells were labeled with 50 ␮Ci [51Cr]sodium chromate
(Amersham, Arlington Heights, IL) for 45 min at 37°C. Target cells (1 ⫻
104 cells) were incubated with effector T cells in round-bottom microtiter
wells. After 4 h at 37°C, 100 ␮l of supernatant from each well was col-
lected and assayed for 51Cr release. The percentage of specific 51Cr release
was calculated by the following equation: (experimental release ⫺ spon-
taneous release)/(maximal release ⫺ spontaneous release) ⫻ 100. For Ab-
blocking studies, the CTL activity of clones against various targets was
tested in the presence of 25% of supernatant or 30 ␮g/ml of purified mAbs.
Inhibitor treatment of target cells
Target cells were incubated with lactacystin (40 ␮M; Calbiochem, San
Diego, CA), chloroquine (20 ␮M; Sigma), or brefeldin A (1 ␮g/ml, Sigma)
for 18 h before CTL assay (42, 43). For the acid-stripping experiment,
target cells (RMA-S/hCD1d, RMA, and P388) were washed with HBSS,
and incubated for 90 s with acid-stripping medium (0.3 M glycine-HCl and
1% BSA in water, pH 2.4) at cell densities of 2 ⫻ 107 cells/ml (43). Culture
medium (100⫻ vol) was added to neutralize pH. Cells were washed three
times and suspended in RPMI 10 at 106/ml. Completeness of acid stripping
was assessed by flow cytometric analysis on RMA and P388 cells using
anti-Kb and anti-Kd Ab, respectively.
The Journal of Immunology 3831

Cytokine assay
CTLs (105 cells/well) were cultured with the same number of irradiated
stimulators in round-bottom 96-well plate in a total volume of 200 ␮l/well.
After 48 h, the supernatants were harvested for cytokine assay. A sandwich
ELISA was used to determine the concentration of IFN-␥, IL-4, and IL-2.
Abs specific for cytokines and recombinant mouse cytokines were obtained
from PharMingen and used according to the manufacturer’s directions.

Cloning and sequencing of TCR genes

Total RNA was isolated from CTL clones using TRIzol reagent (Life Tech-
nologies). First strand cDNA synthesis and PCR of dC-tailed cDNA were
conducted with 5⬘ rapid amplification of cDNA end (RACE) system (Life
Technologies, Rockville, MD), according to the manufacturer’s protocol.
In brief, first strand cDNA synthesis was done using Superscript II reverse
transcription and C␣-specific primer (5⬘-CAGGAGGATTCGGAGTC
CCA-3⬘) or C␤-specific primer (5⬘-CCAGAAGGTAGCAGAGACCC-3⬘).
The synthesized cDNA was then isolated with GlassMax DNA Isolated

Downloaded from http://jimmunol.org/ at Albert Einstein College of Medicine on January 31, 2013
Spin Cartridge, tailed with TdT, and amplified by PCR. Oligonucleotide
primers used were as follows: abridged anchor primer (5⬘-ACTAG
TACGGGIIGGGIIGGGIIG-3⬘) and C␣-inner primer (5⬘-CTGTCCT
GAGACCGAGGATC-3⬘) for TCR ␣ gene amplification; and abridged an-
chor primer and C␤-inner primer (5⬘-CCTGGGT GGAGTCACA
TTTCTC-3⬘) for TCR ␤ gene amplification.
The PCR products were cloned into pGEM-T vector (Promega, Madi-
son, WI). Positive clones were screened by nested PCR with abridged
anchor primer pairing with the primer specific for either C␣ (5⬘-ACTGG
TACACAGCAGGGTCTG-3⬘) or C␤ (5⬘-CCTGGGTGGAGTCACATT
TCTC-3⬘). Nucleotide sequences were determined by PCR sequencing
method using Taq DNA polymerase, BigDye Terminator Cycle Sequenc-
ing Kit (PE Applied Biosystems, Foster City, CA).

Results
Expression of hCD1d in Kb/hCD1D Tg mice
The expression of hCD1D in the different tissues of Tg mice was
examined by RT-PCR. hCD1D message was detected from all
tissues tested from Tg animals, including thymus, spleen, lymph
nodes, liver, kidney, intestine, and skin (Figs. 1 and 2A). Cell sur-
face expression of hCD1d in the Tg mice was determined by im-
munofluorescence staining with mAbs specific to hCD1d
(mAb51.1) (Fig. 2B). High levels of surface expression can be
detected on the majority of lymphocytes isolated from spleen and
lymph nodes of the Tg animals, suggesting the hCD1d epitope
recognized by mAb51.1 was not affected by the species origin of
the associated ␤2m. A bimodal staining pattern was observed from
Tg thymocytes with high levels of hCD1d expression on mature
thymocytes (CD3high) and low levels of hCD1d expression on im-
mature thymocytes (CD3low) (data not shown). Surface expression
of hCD1d can also be detected on epithelial cells from the thymus,
intestine, and liver (Fig. 2B). The expression pattern of hCD1d in
the Tg mice is similar to that of H2-Kb molecules.
hCD1d is a strong transplantation Ag in mice
To determine whether hCD1d can serve as a transplantation Ag,
B6 mice were grafted with tail skin from hCD1d Tg⫹ and Tg⫺
FIGURE 1. Construction of Kb/hCD1d chimeric gene. The H2-Kb/
hCD1D chimeric gene constitutes the 5⬘-flanking region of H2-Kb and
exons 1– 6 and 3⬘-flanking region of hCD1D. Exons (closed boxed) encode
leader (L), extracellular (␣1, ␣2, and ␣3), transmembrane (T), cytoplasmic
(CTY), and 3⬘ untranslated regions (3⬘UT) are shown. H, HindIII; N, NcoI;
P, PvuII; S, SmaI; X, XbaI; B, BamHI; E, EcoRI.
FIGURE 2. hCD1d expression in Kb/hCD1d Tg mice. A, RT-PCR anal-
ysis of hCD1D expression in thymus (T), spleen (Sp), liver (L), kidney (K),
intestine (I), and skin (S) of Tg mice. Hypoxanthine phosphoribosyltrans-
ferase mRNA expression was used as an internal control. B, Flow cyto-
metric analysis of cell surface expression of hCD1d and H2-Kb in Tg mice.
Specific fluorescence profiles (closed histogram) obtained with either anti-
hCD1d (51.1) or anti-H2-Kb (AF6-88.5) were overlayed onto background
profiles (open histogram) obtained with isotype control Abs. Similar results
were obtained with the other anti-hCD1d mAb, 42.1 (data not shown).
littermate control mice. The donor mice used in skin graft exper-
iments were backcrossed seven generations onto B6 background.
All skin grafts (n ⫽ 10) from hCD1d⫹ mice were rapidly rejected
with a mean survival time of 14 days. Five of six skin grafts from
hCD1d⫺ mice showed no sign of rejection and survived for at least
55 days. One of the hCD1d⫺ skin grafts was rejected at day 28,
which might be due to the remaining minor histocompatibility Ag
disparities. These results reveal that hCD1d molecules are recog-
nized as functional transplantation Ags in mice (Fig. 3).
Generation of CTLs against hCD1d
To investigate whether hCD1d is capable of stimulating hCD1d-
restricted CTL response, we immunized B6 ⫻ CBA/F1 (H-2b/k)
mice with hCD1d Tg⫹ splenocytes. The lymphocytes isolated
from the primed mice were stimulated with irradiated Tg⫹ spleno-
cytes in vitro. After 2 wk of culture with Tg⫹ splenocytes (H-2b
background), the CTLs were restimulated with H-2-mismatched
hCD1d-transfected L929 cells (H-2k background), to eliminate
H-2b-restricted CTLs. RMA-S/hCD1d (H-2b) and L929/hCD1d
(H-2k) transfectants were used to screen hCD1d-specific CTLs.
Two CTL lines, BN1 and BN4, which lysed both transfectants but
not untransfected parental cells, were established from two indi-
vidual mice. Lysis of H-2-mismatched hCD1d-positive cells indi-
cated that these CTL lines recognized hCD1d as an intact molecule
and not as an hCD1d-derived peptide presented by a mouse MHC
3832 HUMAN CD1d Tg MICE

FIGURE 3. Rejection of skin grafts from Kb/hCD1d Tg mice by B6

recipients. Skins from tails of transgene-positive mice or transgene-nega-
tive littermates were grafted onto the flank of a recipient mouse. The grafts

were covered with bandages. Seven days later, grafts were observed and
scored daily for 55 days. Rejection was defined as complete necrosis of the
skin graft. All hCD1d Tg⫹ grafts (n ⫽ 10) were rapidly rejected with a
mean survival time of 14 days, while Tg⫺ skin grafts (n ⫽ 6) had pro-
longed survival time (p ⬍ 0.002).
molecule (Fig. 4). Inhibition of target cell lysis by H2-Kb, Db, or
hCD1d-specific mAb showed that only the Ab to hCD1d exerted a
complete inhibitory effect, which further supports the notion that
BN1 and BN4 CTLs recognized hCD1d as a restriction molecule
(Fig. 4).
Characteristics of anti-hCD1d CTLs
FACS analysis of BN1 and BN4 showed that they are CD8⫹CD4⫺
and negative for NK cell surface markers (data not shown), sug-
gesting that they are distinct from the NK T cell subset. Consistent
with this finding, both lines did not express invariant V␣14J␣281
transcript (data not shown), the canonical TCR rearrangement
found in most of the mCD1-restricted NK T cells. Thus, we used
5⬘-rapid amplification of cDNA end (RACE) protocol followed by
DNA sequencing to determine the TCR usage of CTL clones de-
rived from line BN1 and BN4. All four clones from line BN1
expressed V␣5J␣41 and V␤8.3D␤2J␤2.4, and all eight clones de-
rived from line BN4 expressed V␣17J␣25 and V␤2D␤1J␤1.6.
These CTL clones secreted significant amounts of IFN-␥ upon Ag
stimulation, but not IL-2 or IL-4 (Table I). CTL clones derived
from these two CTL lines were used for further study.
Specificity of anti-hCD1d CTLs
Unlike some mouse and human NK T cells that can recognize CD1
molecules from both species (44), BN1 and BN4 CTLs did not
recognize mCD1.1- and mCD1.2-transfected RMA-S cells (Fig.
5). These CTLs lysed various cell types derived from hCD1d⫹ Tg
mice, including Con A blasts, LPS blasts, kidney fibroblasts, and
Downloaded from http://jimmunol.org/ at Albert Einstein College of Medicine on January 31, 2013
FIGURE 4. Recognition of hCD1d by CTL lines is not H2 restricted.
Untransfected and hCD1d-transfected RMA-S or L929 cells were labeled
with 51Cr, and used as targets in a standard cytotoxicity assay. The CTLs
were incubated with target cells in the presence of an anti-hCD1d mAb
(51.1) or anti-H2-Kb/Db mAbs (Y3 and B22) or medium alone. The E:T
ratio was 3:1. The results are representative of two experiments.
bone marrow-derived macrophages, suggesting that the recogni-
tion of hCD1d by these CTLs did not involve tissue-specific li-
gands. To test whether these CTLs can recognize hCD1d ex-
pressed on human cells, we examined the reactivity of these CTLs
with a variety of human cell lines. Fig. 6A shows that BN1 and
BN4 readily lysed several human cell lines, including MOLT-3,
MOLT-4, Jurkat, THP-1, and JY. An effective lysis of hCD1d⫹
human cells by mouse anti-hCD1d-specific CTLs suggested the
recognition did not involve a species-specific ligand. The degree of
lysis by CTLs to different cell lines largely correlated with the
levels of hCD1d surface expression on the cell lines, as detected by
anti-hCD1d Ab, 51.1 (Fig. 6B). It is worth noting that both CTLs
showed similar reactive patterns toward various target cells, with
the exception that BN4 consistently had low reactivity to MOLT-4
and bone marrow-derived macrophages from Tg mice.
Reactivity of hCD1d-restricted CTLs to various CD1-transfected
C1R cells
To determine whether anti-hCD1d CTLs recognize other hCD1
molecules, human CD1A-, B-, C-, or D-transfected C1R cells were
used as targets. Both clones showed preferential reactivity toward
hCD1d transfectant (Fig. 7). Clone BN1 showed some cross-reac-
tivity to CD1c and CD1a transfectants, while BN4 did not react
with CD1a, b, c transfectants, even at the high E:T ratio (data not
shown).

Table I. Characteristics of anti-hCD1d CTLs

Phenotypea TCR Usageb Cytokine Productionc

CD4 CD8 NK1.1 V␣ J␣ V␤ D␤ J␤ IFN-␥ IL-2 IL-4

BN1 ⫺ ⫹ ⫺ AV5S2 41 BV8S3 2 2.4 40.5 – –

BN4 ⫺ ⫹ ⫺ AV17S1 25 BV2S1 1 1.6 16.1 – –
a
Anti-hCD1d CTL clones were stained with anti-CD4, anti-CD8, or anti-NK1.1 and analyzed by flow cytometry.
b
The TCR usages of hCD1d-reactive T cell clones were determined by RT-PCR with 5⬘-RACE system as described in Materials and Methods. The gene segments used to
encode the TCRs were defined by comparing the sequences to the NCBI Genebank database using the BLAST algorithm. Nomenclature for V␣ and V␤ family numbers is based
on the recommendation (57). These sequence data are available from GeneBank under accession number AF296659-62.
c
CTL clones were stimulated with the splenocytes from Kb/hCD1d Tg mice or B6 mice. The supernatants were harvested at 48 h post stimulation. The IFN-␥, IL-2, and IL-4
cytokine levels were determined by ELISA. Results are shown as U/ml of cytokine from 105 of CTLs. CTLs cultured with B6 splenocytes did not secrete detectable amounts
of IFN-␥ (⬍1 U/ml). –, Cytokine levels were below detection limit (⬍0.05 and ⬍1 U/ml for IL-2 and IL-4, respectively).
The Journal of Immunology 3833

FIGURE 5. Target specificity of two hCD1d-restricted CTL clones,

BN1 and BN4. The hCD1d-restricted CTLs were incubated with 51Cr-

Downloaded from http://jimmunol.org/ at Albert Einstein College of Medicine on January 31, 2013
labeled target cells for 4 h at an E:T ratio of 3:1. Target cells were RMA-S
cells; hCD1d-, mCD1.1-, or mCD1.2-transfected RMA-S cells; and LPS
blasts, Con A blasts, fibroblasts, or macrophages from hCD1d-Tg mice.
The percent specific lysis of cells isolated from Tg-negative littermate was
⬍5%.

Similar to CD1b and CD1c, the cytoplasmic tails of hCD1d

contain a putative tyrosine base-sorting motif, Y-X-X-Z (in which
X is any amino acid, and Z is a hydrophobic amino acid) (45).
Several studies demonstrated that this motif is required for CD1b,
c, and d internalization and access to endosomal compartments
(46, 47). Therefore, we examined the reactivity of CD1d-specific
CTLs to C1R line expressing the chimeric hCD1d molecules
(hCD1d/a), which lacks the endosomal targeting sequence. Fig. 7
FIGURE 6. hCD1d-restricted CTLs recognize hCD1d-positive human
shows that the hCD1d/a transfectants could be killed as efficiently cell lines. A, BN1 and BN4 CTLs were used to coculture with 51Cr-labeled
as the hCD1d transfectants, suggesting that the recognition of these C1R, C1R/hCD1d transfectants, MOLT-3, MOLT-4, Jurkat, U937, THP-1,
CTLs did not involve endosomally derived ligands. K562, JY, or Raji human cell lines for 4 h at an E:T ratio of 3:1. The
To gain insights into the cellular processes required to generate cytotoxic response of BN1 and BN4 against the targets was measured in
epitopes recognized by these anti-hCD1d CTLs, inhibitors known triplicate in a 51Cr release assay. B, Flow cytometric analysis of cell surface
to interfere with discrete stages of Ag processing were used, spe- expression of hCD1d on various human cell lines. Cells were stained by
cifically for proteasomal degradation (lactacystin) and endosomal indirect immunofluorescence with the mAb 51.1.
acidification (chloroquine) requirements. As shown in Fig. 8A, nei-
ther lactacystin nor chloroquine affected the recognition of hCD1d- ment for accessory molecules by BN1 and BN4 was studied by Ab
specific CTLs, suggesting that epitopes recognized by anti-hCD1d blocking of the cytotoxic response against RMA-S/hCD1d and
CTLs do not involve proteasomally derived peptides and endoso- L929/hCD1d transfectants (Fig. 9). The result demonstrated that
mally processed Ags. Acid treatment of MHC class I⫹ target cells anti-CD11a (LFA-1) completely blocks the CTL killing of RMA-
has been shown to denature class I/peptide complexes, resulting in S/hCD1d transfectants, but has no effect on L929/hCD1d transfec-
the loss of recognition by CTLs (43, 48). However, treatment of tants. Anti-CD54 (ICAM-1) and anti-CD102 (ICAM-2) also par-
RMA-S/hCD1d transfectants with glycine/HCl (pH 2.4) followed tially blocked the lysis of RMA-S/hCD1d transfectants by both
by brefeldin A (to minimize new hCD1d expression) did not sig-
nificantly diminish the killing by hCD1d-specific CTLs, suggest-
ing that these CTLs recognized either a ligand/hCD1d complex
that is resistant to acid denaturation, or empty hCD1d molecules.
However, we cannot eliminate the possibility that the number of
remaining epitopes for CTLs in acid-stripped target cells may still
be above the threshold levels of CTL recognition due to very high
levels of epitope expression by transfectants. To verify the efficacy
of various treatments, parallel experiments have been performed
using CTLs specific to H2-M3, a mouse class Ib molecule. Fig. 8B
showed that lactacystin, brefeldin A, and acid treatment plus
brefeldin A effectively blocked the allorecognition of M3-specific
T cells.

Effect of various Abs on CTL response
Accessory molecules, such as ICAM/LFA-1, are known to play
important roles in hCD1d-restricted NK T cell-mediated cytotox-
icity (49), and therefore we examined the role of these molecules
in cytotoxicity mediated by hCD1d-restricted CTLs. The require-
FIGURE 7. Cytotoxic activity of CD1d-restricted CTLs against various
CD1-transfected C1R cells. BN1 and BN4 CTLs were incubated with 51Cr-
labeled C1R transfectants expressing the CD1a, CD1b, CD1c, CD1d, or
CD1d/a chimeric protein. Percent specific lysis of each transfectant is
shown for an E:T ratio of 3:1. The results shown are representative of one
of two independent experiments.
3834
FIGURE 8. Effect of metabolic inhibitors and
acid stripping on the recognition of CD1d-restricted
CTLs and H2-M3-restricted CTLs. A, RMA-S/
CD1d transfectants were treated with lactacystin,
chloroquine, brefeldin A, or acid, as described in
Materials and Methods, and used as targets for a
CTL assay with clone BN4. B, P388 cells were
treated with the indicated inhibitors and used as tar-
gets for alloreactive anti-M3 CTLs. The E:T ratios
are shown in the figure. Results were comparable in
two experiments.
CTLs. The differential effect of anti-LFA-1 and anti-ICAMs Abs
on hCD1d-transfected RMA-S (LFA-1⫹, ICAMs⫹) and L929 cells
(LFA-1⫹, ICAMs⫺) correlated with the expression levels of
LFA-1 and ICAMs on these two target cell lines (data not shown).
This result indicates that an LFA-1/ICAM interaction is critical for
the cytotoxicity of BN1 and BN4 for hCD1d-expressing T cell
line, but not fibroblast cell lines, and the requirement for accessory
molecules by BN1 and BN4 to exert CTL activity is similar. Al-
though these CTL clones express CD8 on their cell surface, anti-
CD8 Ab did not block the CTL response, suggesting the affinity of
the TCRs and hCD1d is sufficient enough to trigger the CTL
response.
Discussion
Unlike hCD1a, b, and c, the surface expression of hCD1d on pe-
ripheral blood monocytes is not up-regulated upon activation with
inflammatory cytokines (9). This property may explain why no
hCD1d-restricted CTLs have been derived to date using in vitro
activated human monocytes as APCs. To overcome this limitation
and to examine the functional potential of hCD1d, we have gen-
erated hCD1d Tg mice that expressed high levels of hCD1d on
many cell types. We have shown that, similar to other HLA class
I molecules that have been expressed in Tg mice (50), hCD1d
mediates a transplantation rejection response and elicits CTLs that
FIGURE 9. Effect of various Abs on CTL response. CTL clones were
incubated with 51Cr-labeled RMA-S/hCD1d to L929/hCD1d transfectants
for 4 h in the presence of mAbs against CD8␣, CD11a (LFA-1), CD54
(ICAM-1), and CD102 (ICAM-2), or medium alone.
HUMAN CD1d Tg MICE
Downloaded from http://jimmunol.org/ at Albert Einstein College of Medicine on January 31, 2013
recognize hCD1d as alloantigen. The hCD1d-specific CTLs rec-
ognized their target Ag regardless of whether they were expressed
on H-2-mismatched mouse cells or on human cells; the results
suggested these CTLs recognized hCD1d molecules as restriction
elements, which was confirmed by anti-hCD1d mAb blocking.
Most of the allo-specific CTLs to MHC class I molecules rec-
ognize epitopes that are dependent on both MHC molecules and
specifically bound peptides (51–54). In contrast, our data showed
that the recognition of hCD1d by anti-hCD1d CTLs may not in-
volve a specific ligand, as hCD1d-specific CTLs can react with
various hCD1d-expressing cells of either mouse or human origin.
Thus, the hCD1d-restricted CTLs may recognize empty hCD1d
molecules or, alternatively, the target could be a complex of
hCD1d and a broadly distributed, conserved cellular Ag. If both
BN1 and BN4 CTLs recognized empty hCD1d, one would expect
that the reactivity of CTLs to different targets would entirely de-
pend on the surface expression levels of hCD1d on the target cells;
furthermore, the reactivity patterns of both CTLs against varied
hCD1d-expressing cells should be the same. Although this was the
case to a certain extent, differential reactivity between BN1 and
BN4 to some hCD1d⫹ target cells was detected. For example, BN4
had lower reactivity to hCD1d⫹ bone marrow-derived macro-
phages and MOLT-4 cell line than BN1 at all ranges of E/T ratio
tested (Figs. 5 and 6). Our Ab-blocking experiment indicated the
requirement for accessory molecules is similar between BN1 and
BN4 (Fig. 9). Therefore, it is most likely that differential activity
between BN1 and BN4 may depend on the relative abundance of
the conserved cellular ligand(s) for hCD1d on different cells. Be-
cause the recognition of hCD1d-restricted CTLs is resistant to acid
denaturation and independent of TAP and proteasomal activity, it
is possible that anti-hCD1d-specific CTLs may recognize nonpep-
tide Ags, probably cellular lipid Ags, in the context of hCD1d.
This notion is supported by the recent finding that some mCD1-
restricted T cells recognized cellular phospholipids in a CD1-de-
pendent manner (55).
The response of hCD1d-specific CTLs to hCD1d-expressing tar-
get cells was not enhanced by addition of ␣-GalCer or glycosyl-
ation variants of ceramides (data not shown). These results indi-
cated that hCD1d-specific CTLs have different ligand specificity
from CD1d-restricted NK T cells. This is consistent with the find-
ing that the hCD1d-specific CTLs did not express characteristic
invariant TCR found in CD1-restricted NK T cells. Our analysis of
TCR sequences of hCD1d-specific CTLs showed that all CTL
The Journal of Immunology
clones derived from each individual mouse have identical DNA
sequences. The homogeneity of CTL population from each indi-
vidual mouse may be due to the limited TCR repertoire against
hCD1d molecules and/or preferential clonal expansion during in
vitro restimulation.
Although the expression of hCD1d on human IEC lines, HT29,
T84, and CACO2, has been demonstrated (56), we found that
hCD1d-specific CTLs fail to recognize the target Ag expressed on
these IEC lines (data not shown). It has been shown that the ma-
jority of hCD1d on IEC is nonglycosylated and non-␤2m associ-
ated (36). Our data suggested that hCD1d-specific CTLs could
distinguish the different conformational state of hCD1d molecules.
Tg mice expressing HLA class I and class II molecules have
been used to provide a suitable animal model for the study of the
functions of HLA molecules. The ability of hCD1d to serve as a
restriction element in allorecognition for CD8⫹ CTLs suggested
that similar to group 1 CD1, hCD1d might present unique micro-
bial Ags to CTLs. We are attempting to challenge the hCD1d Tg
mice with various bacterial pathogens and examine whether the
hCD1d might play a role as a restriction element for the microbial
Ags in vivo. Crossing hCD1d Tg mice onto CD1-deficient back-
ground will further provide an animal model to study the func-
tional role of hCD1d in T cell development and immune response
against infectious disease.
Acknowledgments
We thank Dr. Yasuhiko Koezuka (Kirin Brewery, Gunma, Japan) for pro-
viding ␣-GalCer; Dr. Kistern Fischer Lindahl (University of Texas South-
western Medical Center, Dallas, TX) for providing anti-M3 CTLs; Hanh
Nguyen for critical reading of the manuscript; and Miriam Fay for tech-
nical assistance.
References
1. Porcelli, S. A. 1995. The CD1 family: a third lineage of antigen-presenting mol-
ecules. Adv. Immunol. 59:1.
2. Calabi, F., and C. Milstein. 1986. A novel family of human major histocompat-
ibility complex-related genes not mapping to chromosome 6. Nature 323:540.
3. Brutkiewicz, R. R., J. R. Bennink, J. W. Yewdell, and A. Bendelac. 1995. TAP-
independent, ␤2-microglobulin-dependent surface expression of functional
mouse CD1.1. J. Exp. Med. 182:1913.
4. Holcombe, H. R., A. R. Castano, H. Cheroutre, M. Teitell, J. K. Maher,
P. A. Peterson, and M. Kronenberg. 1995. Nonclassical behavior of the thymus
leukemia antigen: peptide transporter-independent expression of a nonclassical
class I molecule. J. Exp. Med. 181:1433.
5. Jullien, D., L. Brossay, P. A. Sieling, R. L. Modlin, and M. Kronenberg. 1996.
CD1: clues on a new antigen-presenting pathway. Res. Immunol. 147:321.
6. Cattoretti, G., E. Berti, A. Mancuso, L. D’Amato, R. Schiro, D. Soglio, and
D. Delia. 1987. An MHC class I related family of antigens with widespread
distribution on resting and avtivated cells, In Leukocyte Typing III: White Cell
Differentiation Antigens. A. J. McMichael, ed. Oxford University Press, Oxford,
p. 89.
7. Small, T. N., R. W. Knowles, C. Keever, N. A. Kernan, N. Collins, J. O’Reilly,
B. DuPont, and N. Flomenberg. 1987. M241 (CD1) expression on B lympho-
cytes. J. Immunol. 138:2864.
8. Kasinrerk, W., T. Baumruker, O. Majdic, W. Knapp, and H. Stockinger. 1993.
CD1 molecule expression on human monocytes induced by granulocyte-mac-
rophage colony-stimulating factor. J. Immunol. 150:579.
9. Exley, M., J. Garcia, S. B. Wilson, F. Spada, D. Gerdes, S. M. Tahir, K. T. Patton,
R. S. Blumberg, S. Porcelli, A. Chott, and S. P. Balk. 2000. CD1d structure and
regulation on human thymocytes, peripheral blood T cells, B cells and mono-
cytes. Immunology 100:37.
10. Blumberg, R. S., C. Terhorst, P. Bleicher, F. V. McDermott, C. H. Allan,
S. B. Landau, J. S. Trier, and S. P. Balk. 1991. Expression of a nonpolymorphic
MHC class I-like molecule, CD1D, by human intestinal epithelial cells. J. Im-
munol. 147:2518.
11. Canchis, P. W., A. K. Bhan, S. B. Landau, L. Yang, S. P. Balk, and
R. S. Blumberg. 1993. Tissue distribution of the non-polymorphic major histo-
compatibility complex class I-like molecule, CD1d. Immunology 80:561.
12. Somnay-Wadgaonkar, K., A. Nusrat, H. S. Kim, W. P. Canchis, S. P. Balk,
S. P. Colgan, and R. S. Blumberg. 1999. Immunolocalization of CD1d in human
intestinal epithelial cells and identification of a ␤2-microglobulin-associated
form. Int. Immunol. 11:383.
13. Brossay, L., D. Jullien, S. Cardell, B. C. Sydora, N. Burdin, R. L. Modlin, and
M. Kronenberg. 1997. Mouse CD1 is mainly expressed on hemopoietic-derived
cells. J. Immunol. 159:1216.
3835
14. Mandal, M., X. R. Chen, M. L. Alegre, N. M. Chiu, Y. H. Chen, A. R. Castano,
and C.-R. Wang. 1998. Tissue distribution, regulation and intracellular localiza-
tion of murine CD1 molecules. Mol. Immunol. 35:525.
15. Roark, J. H., S. H. Park, J. Jayawardena, U. Kavita, M. Shannon, and
A. Bendelac. 1998. CD1.1 expression by mouse antigen-presenting cells and
marginal zone B cells. J. Immunol. 160:3121.
16. Bleicher, P. A., S. P. Balk, S. J. Hagen, R. S. Blumberg, T. J., Flotte, and
C. Terhorst. 1990. Expression of murine CD1 on gastrointestinal epithelium.
Science 250:679.
17. Sieling, P. A., M. T. Ochoa, D. Jullien, D. S. Leslie, S. Sabet, J. P. Rosat,
A. E. Burdick, T. H. Rea, M. B. Brenner, S. A. Porcelli, and R. L. Modlin. 2000.
Evidence for human CD4⫹ T cells in the CD1-restricted repertoire: derivation of
mycobacteria-reactive T cells from leprosy lesions. J. Immunol. 164:4790.
18. Rosat, J. P., E. P. Grant, E. M. Beckman, C. C. Dascher, P. A. Sieling,
D. Frederique, R. L. Modlin, S. A. Porcelli, S. T. Furlong, and M. B. Brenner.
1999. CD1-restricted microbial lipid antigen-specific recognition found in the
CD8⫹ ␣␤ T cell pool. J. Immunol. 162:366.
19. Beckman, E. M., S. A. Porcelli, C. T. Morita, S. M. Behar, S. T. Furlong, and
M. B. Brenner. 1994. Recognition of a lipid antigen by CD1-restricted ␣␤⫹ T
cells. Nature 372:691.
Downloaded from http://jimmunol.org/ at Albert Einstein College of Medicine on January 31, 2013
20. Sieling, P. A., D. Chatterjee, S. A. Porcelli, T. I. Prigozy, R. J. Mazzaccaro,
T. Soriano, B. R. Bloom, M. B. Brenner, M. Kronenberg, P. J. Brennan, and
R. L. Modlin. 1995. CD1-restricted T cell recognition of microbial lipoglycan
antigens. Science 269:227.
21. Kawano, T., J. Cui, Y. Koezuka, I. Toura, Y. Kaneko, K. Motoki, H. Ueno,
R. Nakagawa, H. Sato, E. Kondo, H. Koseki, and M. Taniguchi. 1997. CD1d-
restricted and TCR-mediated activation of V␣14 NKT cells by glycosylceram-
ides. Science 278:1626.
22. Joyce, S., A. S. Woods, J. W. Yewdell, J. R. Bennink, A. D. De Silva,
A. Boesteanu, S. P. Balk, R. J. Cotter, and R. R. Brutkiewicz. 1998. Natural
ligand of mouse CD1d1: cellular glycosylphosphatidylinositol. Science
279:1541.
23. Castaño, A. R., S. Tangri, J. E. W. Miller, H. R. Holcombe, M. R. Jackson,
W. D. Huse, M. Kronenberg, and P. A. Peterson. 1995. Peptide binding and
presentation by mouse CD1. Science 269:223.
24. Arase, H., N. Arase, K. Ogasawara, R. A. Good, and K. Onoe. 1992. An NK1.1⫹
CD4⫹CD8⫺ single-positive thymocyte subpopulation that expresses a highly
skewed T cell antigen receptor family. Proc. Natl. Acad. Sci. USA 89:6506.
25. Lantz, O., and A. Bendelac. 1994. An invariant T cell receptor ␣ chain is used by
a unique subset of major histocompatibility complex class I-specific CD4⫹ and
CD4⫺CD8⫺ T cells in mice. J. Exp. Med. 180:1097.
26. Exley, M., J. Garcia, S. P. Balk, and S. Porcelli. 1997. Requirements for CD1d
recognition by human invariant V␣24⫹ CD4⫺CD8⫺ T cells. J. Exp. Med. 186:
109.
27. Spada, F. M., Y. Koezuka, and S. A. Porcelli. 1998. CD1d-restricted recognition
of synthetic glycolipid antigens by human natural killer T cells. J. Exp. Med.
188:1529.
28. Denkers, E. Y., T. Scharton-Kersten, S. Barbieri, P. Caspar, and A. Sher. 1996.
A role for CD4⫹NK1.1⫹ T lymphocytes as major histocompatibility complex
class II independent helper cells in the generation of CD8⫹ effector function
against intracellular infection. J. Exp. Med. 184:131.
29. Cui, J., N. Watanabe, T. Kawano, M. Yamashita, T. Kamata, C. Shimizu,
M. Kimura, E. Shimizu, J. Koike, H. Koseki, et al. 1999. Inhibition of T helper
cell type 2 cell differentiation and immunoglobulin E response by ligand-acti-
vated Va14 natural killer T cells. J. Exp. Med. 190:783.
30. Singh, N., S. Hong, D. C. Scherer, I. Serizawa, N. Burdin, M. Kronenberg,
Y. Koezuka, and L. Van Kaer. 1999. Cutting edge: activation of NK T cells by
CD1d and ␣-galactosylceramide directs conventional T cells to the acquisition of
a Th2 phenotype. J. Immunol. 163:2373.
31. Burdin, N., L. Brossay, and M. Kronenberg. 1999. Immunization with ␣-galac-
tosylceramide polarizes CD1-reactive NK T cells towards Th2 cytokine synthe-
sis. Eur. J. Immunol. 29:2014.
32. Cardell, S., S. Tangri, S. Chan, M. Kronenberg, C. Benoist, and D. Mathis. 1995.
CD1-restricted CD4⫹ T cells in major histocompatibility complex class II-defi-
cient mice. J. Exp. Med. 182:993.
33. Wang, B., T. Chun, and C.-R. Wang. 2000. Comparative contribution of CD1 on
the development of CD4⫹ and CD8⫹ T cell compartments. J. Immunol. 164:739.
34. Lee, D. J., A. Abeyratne, D. A. Carson, and M. Corr. 1998. Induction of an
antigen-specific, CD1-restricted cytotoxic T lymphocyte response in vivo. J. Exp.
Med. 187:433.
35. Behar, S. M., T. A. Podrebarac, C. J. Roy, C.-R. Wang, and M. B. Brenner. 1999.
Diverse TCRs recognize murine CD1. J. Immunol. 162:161.
36. Balk, S. P., S. Burke, J. E. Polischuk, M. E. Frantz, L. Yang, S. Porcelli,
S. P. Colgan, and R. S. Blumberg. 1994. ␤2-microglobulin-independent MHC
class Ib molecule expressed by human intestinal epithelium. Science 265:259.
37. Panja, A., R. S. Blumberg, S. P. Balk, and L. Mayer. 1993. CD1d is involved in
T cell-intestinal epithelial cell interactions. J. Exp. Med. 178:1115.
38. Colgan, S. P., R. M. Hershberg, G. T. Furuta, and R. S. Blumberg. 1999. Ligation
of intestinal epithelial CD1d induces bioactive IL-10: critical role of the cyto-
plasmic tail in autocrine signaling. Proc. Natl. Acad. Sci. USA 96:13938.
39. Chen, Y. H., B. Wang, T. Chun, L. Zhao, S. Cardell, S. M. Behar, M. B. Brenner,
and C.-R. Wang. 1999. Expression of CD1d2 on thymocytes is not sufficient for
the development of NK T cells in CD1d1-deficient mice. J. Immunol. 162:4560.
40. Yamamoto, M., K. Fujihashi, K. Kawabata, J. R. McGhee, and H. Kiyono. 1998.
A mucosal intranet: intestinal epithelial cells down-regulate intraepithelial, but
not peripheral, T lymphocytes. J. Immunol. 160:2188.
3836
41. Corasanti, J. G., N. D. Smith, E. R. Gordon, and J. L. Boyer. 1989. Protein kinase
C agonists inhibit bile secretion independently of effects on the microcirculation
in the isolated perfused rat liver. Hepatology 10:8.
42. Lewinsohn, D. M., M. R. Alderson, A. L. Briden, S. R. Riddell, S. G. Reed, and
K. H. Grabstein. 1998. Characterization of human CD8⫹ T cells reactive with
Mycobacterium tuberculosis-infected antigen-presenting cells. J. Exp. Med. 187:
1633.
43. Shi, Y., K. D. Smith, and C. T. Lutz. 1998. TAP-independent MHC class I
peptide antigen presentation to alloreactive CTL is enhanced by target cell incu-
bation at subphysiologic temperatures. J. Immunol. 160:4305.
44. Brossay, L., M. Chioda, N. Burdin, Y. Koezuka, G. Casorati, P. Dellabona, and
M. Kronenberg. 1998. CD1d-mediated recognition of an ␣-galactosylceramide
by natural killer T cells is highly conserved through mammalian evolution.
J. Exp. Med. 188:1521.
45. Ohno, H., S. Ono, N. Hirayama, S. Shimada, and T. Saito. 1994. Preferential
usage of the Fc receptor ␥ chain in the T cell antigen receptor complex by ␥/␦ T
cells localized in epithelia. J. Exp. Med. 179:365.
46. Sugita, M., R. M. Jackman, E. van Donselaar, S. M. Bewhar, R. A. Rogers,
P. J. Peters, M. B. Brenner, and S. A. Porcelli. 1996. Cytoplasmic tail-dependent
localization of CD1b antigen-presenting molecules to MIICs. Science 273:349.
47. Rodionov, D. G., T. W. Nordeng, K. Pedersen, S. P. Balk, and O. Bakke. 1999.
A critical tyrosine residue in the cytoplasmic tail is important for CD1d inter-
nalization but not for its basolateral sorting in MDCK cells. J. Immunol. 162:
1488.
48. Storkus, W. J., H. J. D. Zeh, R. D. Salter, and M. T. Lotze. 1993. Identification
of T-cell epitopes: rapid isolation of class I-presented peptides from viable cells
by mild acid elution. J. Immunother. 14:94.
HUMAN CD1d Tg MICE
49. Takahashi, T., M. Nieda, Y. Koezuka, A. Nicol, S. A. Porcelli, Y. Ishikawa,
K. Tadokoro, H. Hirai, and T. Juji. 2000. Analysis of human Va24⫹ CD4⫹ NKT
cells activated by ␣-glycosylceramide-pulsed monocyte-derived dendritic cells.
J. Immunol. 164:4458.
50. Faulkner, L., L. K. Borysiewicz, and S. Man. 1998. The use of human leukocyte
antigen class I Tg mice to investigate human immune function. J. Immunol.
Methods 221:1.
51. Heath, W. R., M. E. Hurd, F. R. Carbone, and L. A. Sherman. 1989. Peptide-
dependent recognition of H-2Kb by alloreactive cytotoxic T lymphocytes. Nature
341:749.
52. Heath, W. R., K. P. Kane, M. F. Mescher, and L. A. Sherman. 1991. Alloreactive
T cells discriminate among a diverse set of endogenous peptides. Proc. Natl.
Acad. Sci. USA 88:5101.
53. Wang, W., S. Man, P. H. Gulden, D. F. Hunt, and V. H. Engelhard. 1998. Class
I-restricted alloreactive cytotoxic T lymphocytes recognize a complex array of
specific MHC-associated peptides. J. Immunol. 160:1091.
54. Chattopadhyay, S., M. Theobald, J. Biggs, and L. A. Sherman. 1994. Conforma-
tional differences in major histocompatibility complex-peptide complexes can
result in alloreactivity. J. Exp. Med. 179:213.
55. Gumperz, J. E., C. Roy, A. Makowska, D. Lum, M. Sugita, T. Podrebarac,
Y. Koezuka, S. A. Porcelli, S. Cardell, M. B. Brenner, and S. M. Behar. 2000.
Downloaded from http://jimmunol.org/ at Albert Einstein College of Medicine on January 31, 2013
Murine CD1d-restricted T cell recognition of cellular lipids. Immunity 12:211.
56. Colgan, S. P., V. M. Morales, J. L. Madara, J. E. Polischuk, S. P. Balk, and
R. S. Blumberg. 1996. IFN-␥ modulates CD1d surface expression on intestinal
epithelia. Am. J. Physiol. 271:C276.
57. Arden, B., S. P. Clark, D. Kabelitz, and T. W. Mak. 1995. Mouse T-cell receptor
variable gene segment families. Immunogenetics 42:501.