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(Journal of Leukocyte Biology. 2001;69:207-214.)
© 2001 by Society for Leukocyte Biology

Activation-induced expression of CD1d antigen on mature T cells

María del C. Salamone, Gabriel A. Rabinovich, Ana K. Mendiguren, Gabriela V. Salamone and Leonardo Fainboim

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, we investigated the expression of human CD1d antigen on activated mature T cells. Expression of this glycoprotein was found to be highly regulated and dependent on PHA stimulation. Flow cytometry studies using the NOR3.2 antibody, which recognized CD1d under denaturing conditions, showed a clear increase in its expression after PHA stimulation. Expression of this molecule after PHA activation was confirmed by analysis of its corresponding transcript by RT-PCR. A single band representing mRNA for CD1d membrane isoform was observed in activated PBMC as well as in ER3 CD1D-transfected and MOLT-4, pre-T cell lines, which were used as controls. Western blot analysis revealed an activation-dependent increase in CD1d protein expression when PBMC and enriched T cells were activated for different time periods. Activation-dependent expression of CD1d antigen was also confirmed in allogenic-activated T cells, suggesting that this event could have biological significance. Finally, immunocytochemical studies showed the presence of this protein at the plasma membrane accompanied by a cytoplasmic and perinuclear distribution. Results presented herein provide the first experimental evidence showing that CD1d antigen is present on circulating, activated T lymphocytes, suggesting that its expression is dependent on the activation state of the cells. Elucidation of the molecular mechanisms implicated in the activation-dependent expression of this nonclassical antigen will provide new insights into the understanding of antigen presentation and immune regulation.

Key Words: activated PBMC • phytohemagglutinin • NK • CD1d isotope


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD1 molecules constitute a family of nonclassical, major histocompatibility complex (MHC) molecules mapped to a cluster on chromosome 1, which can be classified on the basis of sequence homology into two different classes. The first class consists of human CD1A, B, C, and the homologous rabbit CD1B ("classic CD1 antigens"); the second includes the human CD1D gene and its homologues in mouse, rat, and rabbit ("CD1D-like"). The human CD1E gene is equally related to both groups [1 2 3 4 5 6 ]. At the level of gene organization, CD1 consists of multiple exons with functional domains of the polypeptide encoded by separate exons (5' UT-leader, {alpha}1, {alpha}2, {alpha}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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies
CD1d antigen was identified by the NOR3.2 [immunoglobulin G1 (IgG1)] monoclonal antibody (mAb), kindly provided by Dr. Cesar Milstein (MRC, Cambridge, UK). Mouse nonrelevant mAb IgG1 isotype was used as a negative control. The activation state of the cells was evidenced by their reactivity with anti-CD25 (TAC) mAb and anti-HLA-DR mAb.

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 {alpha}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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of CD1d molecule was analyzed by immunochemical and immunocytochemical methods using the NOR3.2 mAb. This mAb, obtained by immunization with the fusion protein expressed in bacteria, was unable to stain the pre-T cell lines NH17 and MOLT-4 by indirect immunofluorescence, probably because of its failure to recognize the antigen under native conditions [30 ]. To study the expression of CD1d antigen under denaturing conditions, a protocol for flow cytometry assay was established using 2% formaldehyde and methanol at 4°C.

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.

 
To confirm expression of CD1d and to rule out the possibility of cross-reaction with an unrelated CD1 molecule, we analyzed the expression of the mRNA encoded by the CD1D gene by RT-PCR and the MW of the identified protein by Western blot analysis. For this purpose, mRNA from ER3, MOLT-4 cell lines, and activated PBMC were retrotranscribed, and the cDNAs were then analyzed by PCR using specific primers for {alpha}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.

 
Western blot analysis confirmed the presence of a single, 49-kD protein band corresponding to CD1d antigen on activated PBMC. Expression of CD1d antigen increased in a time-dependent manner when cells were exposed to PHA for periods of 24, 48, and 72 h (Fig. 4 A , lanes b, c, and d, respectively). Kinetic studies indicated that CD1d expression increased three- to fourfold after 24 h of PHA stimulation, compared with resting PBMC (Fig. 4A , lane b vs. lane a). As clearly shown by the densitometric profile, the highest CD1d reactivity was detected after 48 h of PHA activation, revealing a seven- to eightfold increase compared with nonstimulated PBMC (lane c vs. lane a). Additionally, a fivefold increase was observed after 72 h of PHA stimulation compared with resting cells (lane d vs. lane a). It is interesting that 48 h of PHA stimulation triggered an increase in CD1d protein levels greater than that found constitutively in the pre-T cell line MOLT-4, which was used as control (rate 1.6:1; Fig. 4A and 4B ). Furthermore, when a T cell-enriched population was obtained by plastic adherence and further stimulated with PHA for different time periods, Western blot analysis showed absence of CD1d immunoreaction in the resting T cell-enriched fraction (Fig. 4C , lane a). However, the 49-kd protein band increased clearly when cells were exposed to PHA for 24, 48, and 72 h (Fig. 4C , lanes b, c, and d, respectively).



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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.

 
Expression of CD1d after allogenic stimulation was also studied by Western blot analysis, in an attempt to explore whether this upregulation could also occur in a biological system. The maximal increase in CD1d antigen was observed on positively selected T cells after 3 days of allogenic stimulation (Fig. 5 A , lane b). The densitometric profile is shown in Fig. 5B . The detected protein began to decrease by day 4 after allogenic stimulation (ratio 4:1; day 3 vs. day 4), as shown in Fig. 5A , lane c. Expression was downregulated significantly by day 5 following allogenic stimulation (Fig. 5 A , lane d). Expression of CD1d in the MOLT-4 cell line is shown as control of immunoreaction (lane e).



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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.

 
To investigate the subcellular localization of CD1d, we analyzed the staining pattern of this antigen by immunocytochemistry under the same fixation and permeabilization conditions described for flow cytometry. The staining of ER3 and MOLT-4 confirmed that, under these experimental conditions, the presence of CD1d was detected at the level of the cytoplasmic compartment and at the plasma membrane (Fig. 6A and B ). Nontransfected EL4 cells served as negative controls showing no reactivity by immunocytochemistry (unpublished results). Finally, immunoperoxidase staining of activated PBMC showed a specific labeling of CD1d antigen compatible with a specific cytosolic and membrane localization for this protein. Although most of the CD1d protein was localized intracellularly in activated T cells, membrane expression was clearly evident, suggesting that activation-induced expression of this protein could have biological relevance. The cytoplasmic staining pattern was also accompanied by a specific perinuclear labeling, compatible with the presence of CD1d antigen in the endoplasmic reticulum.



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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>

 
Taken together, these results demonstrate that total and surface expression of CD1d antigen is highly regulated in response to the activation state of mature circulating T cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results presented in this study provide the first experimental evidence showing that CD1d is expressed differentially in mature PBMC and T cells, according to the activation state of the cells. Although CD1 has been implicated in the presentation of nonpeptide, mycobacterial antigens [13 , 17 18 19 20 21 ], tissue distribution and additional biological function of these antigens remain to be elucidated. The CD1 family has been associated with activation events previously. In this regard, it has been described that stimulation of classic CD1 molecules in human pre-T cell lines induced a rapid mobilization of intracellular [Ca2+] and an increase in interleukin (IL)-2 synthesis [31 ].

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.


   ACKNOWLEDGEMENTS
 
This work was supported in part by grants from University of Buenos Aires (JM21) and Conicet (PEI N° 0185/98). M. d. C. S. and L. F. are members of the scientific career of Conicet. G. A. R. is a recipient of a post-doctoral fellowship from Conicet. G. V. S. is a fellow from Conicet. We thank N. Rubinstein for kind assistance.

Received April 14, 2000; revised August 28, 2000; accepted September 27, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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