CD1d structure and regulation on human thymocytes, peripheral blood T cells,

B cells and monocytes

M. EXLEY,* J. GARCIA,* S. B. WILSON,{ F. SPADA,{1 D. GERDES,* S. M. A. TAHIR,* K. T. PATTON,*

R. S. BLUMBERG", S. PORCELLI,{1 A. CHOTT** & S. P. BALK*{ *Cancer Biology Program, Hematology-Oncology

Division, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA,

{Diabetes Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA, {Lymphocyte Biology Section,

Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital and Harvard Medical School, Boston, MA,

USA, "Gastroenterology Division, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA,

**Department of Clinical Pathology, University of Vienna, Austria

SUMMARY

Human T cells expressing CD161 and an invariant T-cell receptor (TCR) a-chain (Va24invt T cells)

speci®cally recognize CD1d and appear to have immunoregulatory functions. However, the

physiological target cells for this T-cell population, and whether alterations in CD1d expression

contribute to the regulation of Va24invt T-cell responses, remain to be determined. A series of

antibodies were generated to assess CD1d expression, structure and regulation on human lymphoid

and myeloid cells. CD1d was expressed at high levels by human cortical thymocytes and

immunoprecipitation analyses showed it to be a 48 000-MW glycosylated protein. However, after

solubilization, the majority of the thymocyte CD1d protein, but not CD1d expressed by transfected

cells, lost reactivity with monoclonal antibodies (mAbs) against native CD1d, indicating that it was

alternatively processed. Moreover, thymocytes were not recognized by CD1d-reactive Va24invt T-

cell clones. Medullary thymocytes and resting peripheral blood T cells were CD1d±, but low-level

CD1d expression was induced on activated T cells. CD1d was expressed by B cells in peripheral

blood and lymph node mantle zones, but germinal centres were CD1d±. Resting monocytes were

CD1d+ but, in contrast to CD1a, b and c, their surface expression of CD1d was not up-regulated by

granulocyte±macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) activation.

These results demonstrate constitutive CD1d expression by human professional antigen-presenting

cells and that post-translational processing of CD1d may contribute to regulation of the activity of

CD1d-speci®c T cells.

INTRODUCTION

The human CD1 locus encodes a family of proteins that are

structurally related to major histocompatibility complex

(MHC) class I proteins.1±3 CD1a, b and c are most homologous

to each other and are expressed by thymocytes, dendritic cells

and activated monocytes,4±9 while B cells express only CD1c.10

T cells reactive with CD1a, b, or c have been isolated from

peripheral blood, and lipids derived from mycobacteria have

been identi®ed as the antigens recognized by some of these

clones.8,11 CD1d is divergent in sequence from CD1a, b and

c3,12 and its tissue distribution is more widespread, including

cells outside the lymphoid and myeloid lineages.13±15 Possible

CD1d-presented antigens include hydrophobic peptides,

glycosylceramides and glycosylphosphatidylinositol.16±18

Murine CD1d, even in the absence of an exogenous antigen,

is recognized by a population of CD4+ or CD4± CD8± (double

negative, DN), CD161+ (NK1) T cells that use an invariant

Va14-Ja281 T-cell receptor (TCR) a-chain.19±23 A population

of human DN or CD4+ T cells that uses a homologous

invariant Va24-JaQ TCR a-chain has been identi®ed (Va24invt

T cells).24,25 These human T cells also express CD161 (NKR-

P1A), which acts as the major costimulatory molecule, and

speci®cally recognize CD1d.26,27 Functional studies suggest an

Correspondence: Steven P. Balk, Hematology-Oncology Division,

Beth Israel Deaconess Medical Center, HIM Building Room 1047, 330

Brookline Avenue, Boston, MA 02215, USA.

Received 27 October 1999; revised 9 December 1999; accepted

9 December 1999.

Abbreviations: GST, glutathione-S-transferase; b2m, b2-microglo-

bulin; Va24invt T cells, Va24 invariant T cells.

Present address: 1Department of Microbiology and Immunology,

Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx,

NY 1046, USA.

Immunology 2000 100 37±47

# 2000 Blackwell Science Ltd 37

immunoregulatory role for these Va24invt T cells,28±31 but the

precise nature of this role and that of CD1d remain to be

determined. This report describes the development and use of a

new series of CD1d antibodies to assess CD1d expression,

structure and regulation on human lymphoid and myeloid cells.

MATERIALS AND METHODS

Cells and culture

C1R cells, a human leucocyte antigen (HLA)-A and -B

de®cient B-lymphoblastoid cell line, transfected with CD1d

cDNA or a CD1d/CD1a chimeric cDNA, were as described

previously.26 Interleukin (IL)-2 used at 100 U/ml was provided

by the NIH Biological Response Modi®ers Program (Bethesda,

MD). Human thymocytes were obtained from paediatric

patients undergoing cardiac surgery. CD1d-reactive Va24invt

T-cell clones were derived from the peripheral blood of normal

donors, as described previously.26 Functional studies mon-

itored by proliferation and cytokine production were carried

out on resting Va24invt T-cell clones, as described pre-

viously.26,27 Brie¯y, Va24invt T-cell clones (105/well) were

incubated with irradiated stimulator cells in 96-well ¯at-

bottom plates and phorbol 12-myristate 13-acetate (PMA)

(1 ng/ml) was included, as indicated, to maximize costimula-

tion.26 Peripheral blood T-cell activation was carried out using

phytohaemagglutinin (PHA), as described previously.26 Mono-

cyte CD1a, b and c expression was induced by culture in

granulocyte±macrophage colony-stimulating factor (GM-CSF)

and IL-4 (100 units/ml of each),8 plus or minus lipopoly-

saccharide (LPS), for 24 hours.

Antibodies

Monoclonal and polyclonal antibodies were generated against

CD1d-glutathione-S-transferase (GST) fusion proteins and

(a)

C1R CD1a

C1R CD1a

C1R CD1b

C1R CD1c

C1R CD1d

C1R CD1b C1R CD1c

38 M. Exley et al.

# 2000 Blackwell Science Ltd, Immunology, 100, 37±47

against a CD1d-immunoglobulin fusion protein. A pool of

CD1d-GST fusion proteins containing the a1 domain alone,

the a1 and a2 domains, or the a1±a3 domains, were used to

immunize rabbits and mice. Rabbit antiserum was af®nity

puri®ed by passing multiple times through a GST column

followed by absorption to a column of agarose beads (Af®gel,

Bio-Rad, Hercules, CA) covalently conjugated with a mixture

of the three CD1d-GST fusion proteins. Monoclonal anti-

bodies (mAbs) against the pooled CD1d-GST fusion proteins

were generated by fusing hyperimmune BALB/c spleen cells to

murine myeloma (NS-1) cells. One antibody from this screen,

termed D5 (immunoglobulin G2b [IgG2b] isotype), reacted in

immunoblots against the a1 domain of CD1d (S. Balk,

unpublished).

A CD1d-immunoglobulin fusion protein was produced

by ®rst generating a BamHI site at the 3k end of the

CD1d a3 domain, using an oligonucleotide (antisense

CGGGATCCCCCCAGTAGAGGACGATG) and polymer-

ase chain reaction (PCR) ampli®cation. This BamHI site was

then used to fuse the CD1d leader sequence through the a3

domain (ending at the sequence Val±Leu±Tyr±Trp±Gly) to the

Fc portion of murine IgG2a, using an immunoglobulin Fc

expression vector (kindly provided by Dr Terry Strom, Beth

Israel Deaconess Medical Center, Boston, MA).32 The fusion

protein was secreted as a b2-microglobulin (b2m)-associated,

disulphide-linked dimer when expressed in hamster (CHO),

murine (NSO) or human (C1R) cells (S. Balk, unpublished).

CD1d knockout mice (M. Exley et al. submitted) were

immunized with the fusion protein and hybridomas were

subsequently screened by enzyme-linked immunosorbent assay

(ELISA) using the protein. The anti-CD1d mAbs raised against

the CD1d-immunoglobulin fusion protein used in this report

were 27.1, 42.1, and 51.1. Additional antibodies used in this

study included BBM.1, OKT6, 4A76, and M241, which

recognize b2m, CD1a, b and c, respectively.

Flow cytometry

Flow cytometry analyses were carried out using < 1r106 cells

in 50±100 ml of phosphate-buffered saline (PBS) containing

0.05% Na azide and 1% fetal calf serum (FCS). Peripheral

blood mononuclear cells (PBMC) were initially blocked by

incubation in 10% human serum. For indirect immuno¯uor-

escence, the primary antibodies were each used at 10±20 mg/ml

for 20±30 min at 4u. The secondary antibodies were ¯uorescein

(b) Thymocytes

Figure 1. Speci®city of anti-CD1d antibodies and thymocyte CD1d expression by indirect immuno¯uorescence. (a) Polyclonal rabbit anti-CD1d

and mouse monoclonal 27.1, 42.1 and 51.1 anti-CD1d antibodies were tested by indirect immuno¯uorescence against C1R cells stably transfected

with CD1a, b, c or d, as indicated. The top three panels (C1R CD1a, C1R CD1b and C1R CD1c) are the CD1a, b and c transfectants stained with

anti-CD1a (OKT6), anti-CD1b (4A76) and anti-CD1c (M241) monoclonal antibodies (mAbs), respectively. The thick lines represent speci®c

antibodies and the thin lines are control antibodies (normal rabbit or mouse immunoglobulin G [IgG]). (b) Freshly isolated human thymocytes were

stained by indirect immuno¯uorescence with the indicated speci®c (thick lines) or control (thin lines) antibodies.

39CD1d structure and regulation of expression

# 2000 Blackwell Science Ltd, Immunology, 100, 37±47

(a) (d)

(b)

(c)

IgG 27.1 anti-CD1d

IgG

42.1

ant

i-CD

1dIg

G42

.1 a

nti-C

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ant

i-CD

1d

Anti-TCR

Anti-CD19

Anti-CD19

Anti-CD14

Anti-CD14

Donor 1 Donor 2 Donor 3 Donor 4

42.1 anti-CD1d 42.1 anti-CD1d

51.1 anti-CD1d 51.1 anti-CD1d

27.1 anti-CD1d

Figure 2. CD1d expression by peripheral blood lymphocytes and monocytes. Peripheral blood mononuclear cells from two

representative normal donors were analysed by two-colour immuno¯uorescence with the indicated directly ¯uorescein isothiocyanate

(FITC)- or phycoerythrin (PE)-conjugated speci®c and control antibodies. (a) Anti-T-cell receptor ab (TCR ab) versus control or 42.1

anti-CD1d analysis of cells in the resting lymphocyte gate. (b) Anti-CD19 versus control or 42.1 anti-CD1d analysis of cells in the

resting lymphocyte gate. (c) Anti-CD14 versus control or 42.1 anti-CD1d analysis of cells in the monocyte gate. (d) Histogram of

CD1d expression on B cells from two additional donors, examined as described above, gating of the B-cell (CD19+) population.

40 M. Exley et al.

# 2000 Blackwell Science Ltd, Immunology, 100, 37±47

isothiocyanate (FITC)-conjugated anti-mouse or anti-rabbit

F(abk)2 fragments (DAKO, Carpinteria, CA). Direct Ab

conjugates were from Pharmingen or DAKO. The 42.1 anti-

CD1d mAb was conjugated to FITC according to the

manufacturer's protocol (Molecular Probes, Eugene, OR).

Immunoprecipitation and immunoblotting

Cell-surface proteins were radiolabelled with 125I using

lactoperoxidase.33 Alternatively, cells (1±2r107/ml) were sur-

face labelled with 0.5 mg/ml sulphosuccinimidyl-6-(biotina-

mido) hexanoate (NHS-LC-Biotin; Pierce Chemicals,

Rockford, IL) in 25 mM HEPES, pH 8.0, 0.14 M NaCl, for

30 min at 4u. After washing, labelled cells were lysed in

immunoprecipitation buffer (0.15 M NaCl, 50 mM Tris, pH 7.8,

and 0.5% Nonidet P-40 [NP-40]) containing protease inhibi-

tors. For immunoblotting experiments, lysates from unlabelled

cells were prepared similarly.

Immunoprecipitations were performed using antibodies

coupled to protein G (IgG1 mAbs) or protein A±Sepharose

beads (Pierce). To minimize background from eluted IgG,

antibody was covalently coupled to the beads using dimethyl-

pimelimidate. Lysates were cleared by incubation with non-

immune serum bound to protein A± and/or protein G±

Sepharose beads. They were then incubated with speci®c

antibodies coupled to Sepharose beads, washed, eluted in non-

reducing sodium dodecyl sulphate±polyacrylamide gel electro-

phoresis (SDS±PAGE) sample buffer and analysed by SDS±

PAGE under reducing or non-reducing conditions.

After transfer to nitrocellulose, biotinylated proteins were

detected using streptavidin-horseradish peroxidase (HRP) and

enhanced chemiluminescence (ECL) (Amersham, Bucks, UK).

Non-labelled CD1d was detected by immunoblotting using the

af®nity-puri®ed rabbit anti-CD1d antibody, followed by anti-

rabbit immunoglobulin-HRP conjugates and ECL. b2m was

detected by immunoblotting of non-reducing gels with the

BBM.1 mAb. N-linked carbohydrates were removed by

digestion with N-glycanase (Genzyme, Cambridge, MA), as

described previously.33

Immunohistochemistry

Staining with anti-CD1 mAbs was performed on frozen

sections following a 10-min ®xation in acetone. Endogenous

peroxidase was ®rst blocked by incubation in 1% H2O2 in Tris-

buffered saline or in glucose (50 mg/ml) and glucose oxidase

(7 U; Sigma Chemical Co., St Louis, MO). Biotinylated horse

anti-mouse IgG was used as the secondary antibody followed

by Vectostain Elite ABC Reagent (Vector Labs, Burlinghame,

CA). The chromogens were 3-amino-9-ethyl-carbazole or

3,3k-diamino-benzidine.

RESULTS

Speci®city of anti-CD1d antibodies

A panel of human C1R B-cell clones transfected with CD1a, b,

c, or d were used to determine the speci®city of antibodies

generated against CD1d-GST and CD1d-immunoglobulin

fusion proteins. The af®nity-puri®ed rabbit anti-CD1d anti-

body, generated against CD1d-GST fusion proteins, speci®-

cally stained the CD1d transfectant (Fig. 1a, control versus

rabbit anti-CD1d). Similarly, the 27.1 mouse anti-human

(a)

Rabbit anti-CD1d 27.1 anti-CD1d

Fresh monocytes

(b)

Anti-CD1a Anti-CD1b

Anti-CD1c Anti-CD1d

Cultured monocytes

(c)

Anti-CD1a Anti-CD1b

Anti-CD1c Anti-CD1d

Monocytes with LPS

Figure 3. CD1d expression by activated monocytes. (a) Freshly

isolated peripheral blood mononuclear cells in the monocyte gate were

analysed by indirect immuno¯uorescence with rabbit anti-CD1d or

27.1 anti-CD1d antibodies (thick lines) versus control non-immune

rabbit or mouse serum (thin lines). (b) Granulocyte±macrophage

colony-stimulating factor (GM-CSF)- and interleukin-4 (IL-4)-cul-

tured monocytes were analysed by indirect immuno¯uorescence with

anti-CD1a, b, c or d monoclonal antibodies (mAbs) (thick lines) versus

control Abs (thin lines). The CD1d Ab shown was 27.1, but identical

results were obtained with the rabbit anti-CD1d antibody. (c)

Monocytes cultured in GM-CSF, IL-4 and lipopolysaccharide (LPS)

were analysed as described above in (b). FITC, ¯uorescein isothiocya-

nate; FL1-H, FITC channel.

41CD1d structure and regulation of expression

# 2000 Blackwell Science Ltd, Immunology, 100, 37±47

CD1d mAb, raised against a CD1d-IgG fusion protein,

speci®cally recognized CD1d (Fig. 1a, control versus 27.1

anti-CD1d). The 42.1 and 51.1 mouse anti-human CD1d mAbs

similarly reacted strongly with CD1d transfectants, although

they showed some cross-reactivity with CD1b (Fig. 1a). The

42.1 and 51.1 mAbs may also cross-react very weakly with

CD1a and c, as suggested by ¯ow cytometry results (Fig. 1a).

The D5 mouse anti-human CD1d mAb, raised against a CD1d-

GST fusion protein, did not bind to CD1d-transfected C1R

cells or other human CD1d transfectants, as determined by

¯ow cytometry (not shown), but was shown to bind human

CD1d on intact, transfected MDCK cells.34

CD1d expression by thymocytes

Northern blot analyses showed previously that CD1d tran-

scripts were expressed at very low levels by human thymocytes

compared with CD1a.3 Moreover, CD1d was not detected

previously on human thymocytes using a cross-reactive anti-

mouse CD1d mAb.14 Nonetheless, high-level cell-surface

expression of CD1d protein, comparable to that on CD1d

transfectants, was detected on the majority of thymocytes from

multiple donors using each of the anti-CD1d mAbs and the

CD1d-speci®c rabbit polyclonal Ab (Fig. 1b). Thymocyte

CD1d expression was also con®rmed by immunoprecipitations

(see below).

CD1d expression in peripheral blood

Figure 2 shows an analysis of CD1d expression on peripheral

blood cells from two representative normal donors (of < 40

analysed). CD1d expression was undetectable on the vast

majority of resting peripheral blood T cells, as determined by

using the 27.1, 42.1, 51.1 or rabbit anti-CD1d antibodies,

although very small numbers of weakly positive T cells were

sometimes observed (Fig. 2a and data not shown). In contrast,

CD1d staining of essentially all CD19+ B cells was observed

with the 42.1 mAb (Fig. 2B). Analyses of further donors and

the use of multiple antibodies con®rmed CD1d expression on B

cells (Fig. 2d and data not shown). This con®rmed the previous

®nding of CD1d on human B cells using a cross-reactive anti-

mouse CD1d mAb.14 CD1d expression by B cells was not

increased after activation in vitro with pokeweed mitogen, anti-

immunoglobulin M (anti-IgM), or LPS (results not shown).

The group 1 CD1 proteins (CD1a, b and c) are not

expressed on resting monocytes. However, CD1d expression

was clearly detected on freshly isolated CD14+ monocytes

(Figs 2c, 3a). It was shown previously that CD1a, b and c

expression could be strongly induced on monocytes by

stimulation with GM-CSF and IL-4.8,9 To determine whether

CD1d expression was regulated similarly, fresh monocytes

were cultured with GM-CSF and IL-4, plus or minus LPS

(Fig. 3b, 3c, respectively). In both cases there was induction of

CD1a, b and c expression, but surface CD1d expression was

not induced under these short-term culture conditions. These

results demonstrate that CD1d is expressed constitutively by

monocytes and its expression is regulated differently from the

group 1 CD1 proteins (CD1a, b and c).

Although peripheral blood T cells were CD1d±, expression

of CD1d could be induced by T-cell activation in vitro. CD1d

expression was observed within 24 hr of culture (Fig. 4a) and

persisted for at least 5 days (results not shown). Signi®cantly,

the analysis of peripheral blood from healthy donors revealed

some samples with similar low-level CD1d expression by the

majority of T cells (Fig 4b, 27.1 anti-CD1d, week 2). CD1d

expression by these donors did not appear to re¯ect

polymorphisms or other genetic differences in CD1d regula-

tion, as in all cases previous and/or subsequent samples from

the same donors were found to be CD1d± negative (Fig. 4b,

27.1 anti-CD1d, week 1). CD1d expression in each of these

samples correlated with modest levels of CD69 expression,

(a) Unstimulated Stimulated

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Figure 4. CD1d expression by activated T cells. (a) Freshly isolated

peripheral blood mononuclear cells (PBMC) from two donors were

cultured for 24 hr with or without stimulation and then analysed by

indirect immuno¯uorescence with 42.1 anti-CD1d monoclonal anti-

body (mAb) (42.1) (thick lines) or isotype-matched control Ab (thin

lines). Similar results were obtained with the other anti-CD1d mAbs

and the rabbit anti-CD1d Ab (not shown). The very weak staining in

the cells cultured without stimulation was not seen in the freshly

isolated resting T cells (results not shown). (b) Freshly isolated

peripheral blood mononuclear cells isolated 1 week apart from the

same donor were analysed by indirect immuno¯uorescence with the

indicated speci®c mAbs (thick lines) versus control antibodies (thin

lines). FL1-H, FITC channel.

42 M. Exley et al.

# 2000 Blackwell Science Ltd, Immunology, 100, 37±47

indicative of recent activation (Fig. 4b). Therefore, these

studies of CD1d expression in peripheral blood from healthy

donors indicated that CD1d expression by T cells was induced

by activation in vivo.

Immunohistochemical analysis of CD1d expression in thymus

and lymph node

The above ¯ow cytometric analysis showed strong CD1d

expression by most thymocytes. Consistent with this, immu-

nohistochemistry showed relatively uniform CD1d expression

by cortical thymocytes (Fig. 5a). However, there was a striking

down-regulation of CD1d expression by medullary thymo-

cytes, with CD1d+ thymocytes being very rare in the medulla.

CD1a expression was also diminished in the transition from

cortex to medulla, but many CD1a+ cells could nonetheless be

detected in the medulla (Fig. 5b). In lymph node, mantle zone

B cells were strongly CD1d+, while CD1d+ cells in germinal

centres were extremely rare (Fig. 5c). In the interfollicular T-

cell-rich zones, there were scattered, small-to-medium sized

mononuclear cells that were strongly CD1d+. Comparable

results were obtained with each of the anti-CD1d Abs.

Structure of CD1d expressed by human thymocytes

Although ¯ow cytometry with the 27.1, 42.1 and 51.1 mAbs

indicated a relatively high level of CD1d expression by

thymocytes, sequential immunoprecipitations with the

42.1 mAbs from biotinylated thymocyte lysates yielded rela-

tively small amounts of protein (Fig. 6a, lanes 2 and 3).

Comparable low levels of CD1d were immunoprecipitated by

the 27.1 and 51.1 mAbs (results not shown). In contrast,

substantially more CD1d was precipitated from the same

thymocyte lysates by the D5 anti-CD1d mAb (Fig. 6a, lane 4),

with the amount of biotin label being comparable to that

precipitated by the OKT6 anti-CD1a mAb (lane 5, T6). The

proteins precipitated by 42.1 and D5 migrated identically to the

major CD1d protein expressed by transfected C1R cells

(Fig. 6a, lane 6) (< 48 000 MW), and migrated at

37 000 MW after N-glycanase digestion (Fig. 6b), consistent

with thymocyte CD1d being fully glycosylated. These results

indicated that the epitopes recognized by the 27.1, 42.1, and

51.1 mAbs were lost after biotinylation and detergent solubi-

lization on the majority of the thymocyte cell-surface CD1d.

The D5 mAb was generated against a CD1d-GST fusion

protein and recognizes an epitope in the a1 domain (S. Balk,

unpublished). D5 binding to detergent-solubilized CD1d

appears to disrupt the association with b2m, as D5 immuno-

precipitates CD1d from transfected C1R cells without b2m.35

Although D5 immunoprecipitates CD1d, and not CD1a, b or c,

from the respective transfected C1R cells (reference 35 and data

not shown), the speci®city of this mAb was further assessed

directly on thymocyte lysates. To determine whether the

D5 mAb might be cross-reactive with CD1a expressed by

thymocytes (which migrates at the same position on SDS±

PAGE), thymocyte lysates were initially precleared three times

with an anti-CD1a mAb (Fig. 6c, CD1a, lanes 1±3). Immuno-

precipitations were then carried out with D5 on the CD1a

precleared versus fresh thymocyte lysates. The amount of

protein immunoprecipitated by D5 from the precleared lysate

(lane 4) versus fresh lysate (lane 5) was comparable, indicating

that D5 was not cross-reacting with thymocyte CD1a.

Although cross-reactivity of the D5 mAb with CD1b or

CD1c expressed by thymocytes was unlikely, as the apparent

molecular weight of these proteins on SDS±PAGE was lower

(not shown), this was nonetheless further addressed by

determining whether the D5 mAb could immunodeplete

CD1a, b, or c from thymocyte lysates. In these experiments,

CD1a, b and c were detected by immunoblotting with anti-b2m,

as immunoblotting antibodies for each of the group 1 CD1

proteins were not available. D5 was ®rst used to preclear half of

an unlabelled thymocyte lysate, followed by CD1a, b and c

immunoprecipitation and b2m immunoblotting. Figure 6(d)

shows that the amount of CD1a, b and c immunoprecipitated

was similar before (lanes 2±4) and after (lanes 5±7) two

successive preclears with the D5 mAb, which further con®rmed

that the D5 mAb was CD1d speci®c.

To address the possibility that epitopes on CD1d

recognized by the 42.1 and 51.1 mAbs were lost as a result

of biotin labelling, and to further con®rm the speci®city of the

mAbs, unlabelled thymocyte lysates were analysed by immu-

noblotting with the rabbit anti-CD1d Ab. Lysates were cleared

by two immunoprecipitations with the 42.1 mAb (Fig. 7a,

lanes 1 and 2) or 51.1 mAb (lanes 4 and 5), followed by

immunoprecipitation with the D5 mAb (lanes 3 and 6,

respectively). Immunoblotting with the rabbit anti-CD1d Ab

con®rmed that the 42.1 and 51.1 mAbs precipitated only a

small fraction of the total cellular CD1d relative to that

immunoprecipitated by the D5 mAb. In marked contrast to

these results with thymocyte CD1d, most of the CD1d in

lysates from CD1d-transfected C1R cells could be cleared by

immunoprecipitation with the 42.1 mAb (Fig. 7b, sequential

42.1 preclears in lanes 2±4 versus subsequent D5 immuno-

precipitations in lanes 5 and 6). Similar results were obtained

(a) (b) (c)

Figure 5. Immunoperoxidase analysis of CD1 expression in thymus and lymph node. (a) Thymus stained with the 51.1 anti-CD1d

monoclonal antibody (mAb). (b) Thymus stained with the OKT6 anti-CD1a mAb. (c) Lymph node stained with 51.1 anti-CD1d mAb.

Comparable results were obtained with the 27.1 and 42.1 anti-CD1d mAbs (not shown).

43CD1d structure and regulation of expression

# 2000 Blackwell Science Ltd, Immunology, 100, 37±47

with the 27.1 and 51.1 mAbs (results not shown). Taken

together, these results con®rmed biochemically CD1d expres-

sion by human thymocytes and demonstrated a structural

difference, detectable as the loss of one or more epitopes after

solubilization in non-ionic detergent, between a large fraction

of the CD1d protein expressed by thymocytes versus CD1d

expressed by transfected C1R cells.

The cytoplasmic tail of CD1d contains a tyrosine-based

endosomal-targeting motif that appears to be necessary for the

acquisition of speci®c lipid antigens.36 To determine whether

the structural difference between thymocyte and C1R CD1d

could re¯ect a post-translational modi®cation that occurs

during endosomal processing, a mutant CD1d, without this

endosomal targeting motif and containing instead the cyto-

plasmic tail from CD1a (CD1d/a), was examined. Figure 7(b)

shows that the vast majority of the CD1d/a protein could be

immunoprecipitated with the 42.1 mAb, indicating that the

structural difference between thymocyte and C1R CD1d does

not re¯ect a cytoplasmic tail-dependent processing step.

CD1d-speci®c Va24invt T cells do not respond to thymocyte

CD1d

Human Va24invt T cells can be stimulated by CD1d-transfected

cells expressing high levels of CD1d protein, comparable to the

levels on thymocytes, without the addition of an exogenous

antigen.26,27 Therefore, the ability of thymocytes to function as

stimulator cells for Va24invt T-cell clones was assessed. In

contrast to CD1d-transfected C1R and CHO cells, thymocytes

were unable to stimulate Va24invt T-cell clones (Fig. 8). Similar

results were obtained using four distinct Va24invt T-cell clones

(data not shown). Stimulation could not be reconstituted by

inclusion of phorbol ester or light glutaraldehyde ®xation

(Fig. 8), procedures that can circumvent the need for

costimulation in the case of some CD1d+ target cells.26

DISCUSSION

The expression of CD1d in the lymphoid and myeloid lineages

was assessed to identify potential physiological CD1d+ target

cells for CD1d-reactive T cells and to determine whether CD1d

expression was constitutive or regulated. CD1d expression

during T-cell development paralleled CD1a, b and c, which are

expressed at similar levels on immature cortical thymocytes and

down-regulated on mature thymocytes and peripheral blood

T cells.4±6 CD1d expression on peripheral blood B cells

paralleled that of CD1c, which is expressed on a large

proportion of mature B cells.10 However, these similarities in

the regulation of CD1a, b and c expression did not extend to

peripheral blood monocytes, as CD1a, b and c expression by

(a) (b)

(c) (d)

Thymus

42.1 D5 T6 42.1

1 2 3 4 5 6

1 2 3Streptavidin blot

MW (× 103)

66

46

30

MW (× 103)

46

MW (× 103)

14

MW (× 103)

46

30

C1R-d N-glc

Preclear

CD1a CD1d a b c a b c

1 2 3 4 5

Streptavidin blot

1 2 3 4 5 6 7

β2m blot

Figure 6. CD1d immunoprecipitations from thymocytes and speci®city of D5 monoclonal antibody (mAb). (a) Streptavidin blot of

immunoprecipitates from biotinylated thymocyte lysates: lane 1, normal mouse serum; lanes 2 and 3, sequential 42.1 anti-CD1d mAb

precipitations; lane 4, D5 anti-CD1d mAb; lane 5, OKT6 anti-CD1a precipitation; lane 6, 42.1 precipitation from CD1d-transfected

C1R cells. (b) Deglycosylation of CD1d from surface-iodinated thymocyte lysates: lane 1, normal mouse serum immunoprecipitation;

lanes 2 and 3, D5 anti-CD1d mAb immunoprecipitates untreated (lane 2) or treated lane 3 with N-glycanase (N-glc). (c) D5 immuno-

precipitation from CD1a precleared biotinylated thymocyte lysate: lanes 1±4, lysate immunoprecipitated sequentially with OKT6

(anti-CD1a) beads (lanes 1±3) followed by reimmunoprecipitation with D5 (anti-CD1d) beads (lane 4); lane 5, D5 precipitation from

an equal amount of lysate without a CD1a preclear. (d) Immunoprecipitates from unlabelled thymocyte lysates immunoblotted with

BBM.1 anti-b2m mAb: lanes 1±4, fresh thymocyte lysate; lanes 5±7, equivalent amounts of D5 anti-CD1d mAb-depleted thymocyte

lysate. Immunoprecipitations were as follows: lane 1, normal mouse serum; lanes 2 and 5, anti-CD1a (OKT6) mAb; lanes 3 and 6,

anti-CD1b (4A76) mAb; lanes 4 and 7, anti-CD1c (M241) mAb. Samples were analysed on a 15% gel under non-reducing conditions.

44 M. Exley et al.

# 2000 Blackwell Science Ltd, Immunology, 100, 37±47

monocytes is activation dependent.8,9 In contrast, cell-surface

CD1d expression by monocytes was constitutive and not up-

regulated by in vitro activation. Finally, CD1d expression by

mature peripheral blood T cells could be induced in vitro, and

appeared to be induced in vivo, by activation.

The lymph node immunohistochemistry was consistent

with the peripheral blood analysis, showing CD1d expression

primarily in the B-cell-rich mantle zones. Recent reports

showed higher level expression of murine CD1d by splenic

marginal zone B cells.37,38 Human lymph nodes do not have

well-de®ned marginal zones,39 but scattered CD1d+ cells

observed in the interfollicular T-cell zones could re¯ect this B-

cell population (or dendritic cells). Signi®cantly, CD1d+ cells

were not detected in germinal centres, indicating that B-cell

CD1d expression is down-regulated at this stage and that

CD1d may not play a role in the lymphocyte and dendritic cell

interactions that occur in germinal centres.

Immunoprecipitations con®rmed that thymocytes ex-

pressed relatively high levels of CD1d and showed that it

was fully glycosylated. They further showed that thymocyte

CD1d, but not CD1d expressed by transfected cells, lost

epitopes recognized by conformationally sensitive mAbs after

solubilization in non-ionic detergent. The structural differences

between CD1d expressed by thymocytes and CD1d transfec-

tants probably re¯ects post-translational processing at the level

of antigen binding or glycosylation. Correspondingly, CD1d

expressed by thymocytes also appeared to be functionally

distinct from CD1d on transfected cells, as thymocytes

expressing high levels of CD1d protein were unable to stimulate

CD1d-reactive Va24invt T cells. These results indicate that

while CD1d+ thymocytes may mediate the positive selection of

CD1d-reactive T cells,40 they are not the physiological target

cells of mature Va24invt T cells. Further studies are necessary to

determine the relationship between CD1d structure and

function on thymocytes. However, the data suggest that the

role of alternative CD1d processing by thymocytes may be to

promote the positive selection of Va24invt T cells while

preventing negative selection caused by high-af®nity binding

of CD1d by the Va24invt TCR.

Signi®cant human thymocyte CD1d expression was not

detected previously by ¯ow cytometry or immunohistochem-

istry using the cross-reactive rat anti-mouse CD1d mAbs, 1H1

and 3C11,14,15 although these mAbs detected human CD1d on

transfected cells, B cells and intestinal epithelial cells.14,15,33

The 1H1 and 3C11 mAbs similarly failed to immunoprecipitate

CD1d from human thymocytes (data not shown), consistent

with a distinct structure for thymocyte CD1d. We showed

previously, using the 1H1 and 3C11 mAbs, that CD1d was

alternatively processed in human intestinal epithelial cells,

yielding a non-glycosylated and non-b2m-associated protein,33

and these results have recently been con®rmed with additional

antibodies.41 These observations suggest that post-transla-

tional modi®cation may regulate CD1d function in a number

of cell types.

The rate of CD1d turnover in thymocytes appeared to be

extremely slow, based upon the relatively high level of CD1d

protein expression in conjunction with very low message

levels.3 However, immunohistochemistry demonstrated a

marked down-regulation of CD1d protein expression in the

transition from thymic cortex to the medulla. This suggests a

speci®c mechanism to clear CD1d protein from the cell surface

(a)

(b)

Thymus

42.1 D5 51.1 D5

1 2 3 4 5 6

1 2 3 4 5 6 7 8 9 10 11 12

42.1

Anti-CD1d immunoblot

Anti-CD1d immunoblot

D5 42.1 D5

C1R-CD1d C1R-CD1d/a

46 000 MW

46 000 MW

Figure 7. CD1d immunoprecipitations from thymocytes and trans-

fected cells immunoblotted with rabbit anti-CD1d. (a) Immuno-

precipitates from unlabelled thymocytes immunoblotted with

af®nity-puri®ed rabbit anti-CD1d: lanes 1 and 2, primary and

secondary 42.1 monoclonal antibody (mAb) precipitations, respec-

tively; lanes 4 and 5, primary and secondary 51.1 mAb precipitations,

respectively; lanes 3 and 6, D5 anti-CD1d precipitations from 42.1 and

51.1 precleared lysates, respectively. (b) Lysates from C1R cells

expressing wild-type CD1d (lanes 1±6) or a CD1d/a chimeric protein

(lanes 7±12) immunoprecipitated and immunoblotted with af®nity-

puri®ed rabbit anti-CD1d: lanes 1 and 7, normal mouse serum; lanes 2±

4 and 8±10, three sequential immunoprecipitations with 42.1 mAb;

lanes 5, 6 and 11, 12, sequential D5 mAb immunoprecipitations of the

42.1-cleared lysates. All samples were analysed on 12% reducing gels.

Stimulus T cells

No stimulus +

C1R mock +

C1R CD1d +

CHO CD1d (fix) +

Thymocytes (live) +

Thymocytes (fix) +

Thymocytes (live) −

0 20 40 60 80 100 120

% of maximum response

Figure 8. Lack of thymocyte CD1d recognition by Va24invt T cells. A

CD1d-reactive Va24invt clone, DN2.B9, was cultured with the indicated

stimulator cells in the presence of phorbol 12-myristate 13-acetate

(PMA). Proliferation was determined by 3H-thymidine incorporation.

The stimulator cells or DN2.B9 T cells were omitted from the top and

bottom experiments, respectively. The response to the C1R CD1d

transfectants was set at 100%. ®x, light glutaraldehyde ®xation; Mock,

transfected with empty expression vector.

45CD1d structure and regulation of expression

# 2000 Blackwell Science Ltd, Immunology, 100, 37±47

at this developmental stage. This mechanism could involve the

tyrosine-based endosomal targeting signal in the cytoplasmic

tail of CD1d (as well as CD1b and CD1c), but not CD1a.

In contrast to the results presented here in humans,

autoreactive recognition of murine thymocyte CD1d by

homologous murine Va14invt T-cell hybridomas has been

observed.23 This difference could re¯ect less stringent in vitro

requirements for TCR-mediated activation in the murine

hybridomas versus the human Va24invt T-cell clones. Another

signi®cant difference between CD1d in humans and mice is that

murine CD1d is expressed at low levels by both thymocytes and

mature T cells,13,42 and at slightly higher levels by B cells.37,38

We suggest that the expression of group 1 and group 2 CD1

proteins by human thymocytes and their absence in mature

human T cells may be co-ordinately regulated by CD1 locus-

speci®c enhancer and silencer elements, respectively, and that

these elements may have been lost with the deletion of the

group 1 (CD1a, b and c) genes from the murine CD1 locus. If

this is correct, then the regulated expression of CD1d in the

human T-lymphocyte lineage may re¯ect a physiological role in

immunoregulation. Speci®cally, CD1d expression by activated

human T cells may render them as direct in vivo stimulators or

targets of CD1d-reactive T cells. A role for Va24invt T cells in

modulating the function of activated T cells would be

consistent with the loss of Va24invt T cells in a number of

human autoimmune diseases.31,43

ACKNOWLEDGMENTS

We thank Dr T. Strom (Beth Israel Deaconess Medical Center, Boston)

for providing the IgG fusion vector. This work was supported by NIH

R01 grants AI42955 to S. P. B.; AI3319 and DK44319 to S. P. B. and R.

S. B.; DK51362 to R. S. B., AI45051 to S. B. W, AI40135 and an

Arthritis Foundation Investigator Award to S. P.; a Crohns and Colitis

Foundation of America grant to D. G.; and an American Hematology

Society award to K. T. P.

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