CD1 expression is not affected by human peptide transporter deficiency

CD1 Expression Is Not Affected by Human Peptide Transporter Deficiency Daniel Hanau, Dominique Fricker, Thomas Bieber, Marie-Emmanuelle Esposito-Farese, Huguette Bausinger, Jean-Pierre Cazenave, Lionel Donato, Marie-Marthe Tongio, and Henri de la Salle

ABSTRACT: Conventional major histocompatibility complex class I molecules are highly polymorphic and present peptides to cytotoxic T cells. These peptides derive from the proteolytic degradation of endogenous proteins in the cytosol and are translocated into the endoplasmic reticulum by a peptide transporter consisting of two transporter associated with antigen processing (TAP) molecules. Absence of this transporter leads to the synthesis of unstable peptide free class I molecules that are weakly expressed on the cell surface. Mouse nonconventional class I molecules (class Ib) may also present TAPdependent peptides. In humans, CD1 antigens are nonconventional class I molecules. Recently, we characterized

a human HLA class I deficiency resulting from a homozygous TAP deficiency. We show here that CD la and -c are normally expressed on epidermal Langerhans cells of the TAP-deficient patients, as are CDla, -b, and -c on dendritic cells differentiated in vitro from monocytes. Moreover, the CDla antigens present on the surface of the dendritic cells are functional, since they internalize by receptor-mediated endocytosis gold-labeled F(ab') 2 fragments of an anti-CDla mAb. This suggests either that CD1 molecules are empty molecules, that they are more stable than empty conventional class I proteins, or that CD1 molecules present TAP-independent peptides. Human Immunology 41, 61-68 (1994)

ABBREVIATIONS 132m ~2-microglobulin ER endoplasmic reticulum GM-CSF granulocyte-macrophage colony-stimulating factor HLA human leukocyte antigen

IL-4 mAb MHC TAP

interleukin 4 monoclonal antibody major histocompatibility complex transporter-associated with antigen processing

INTRODUCTION Conventional major histocompatibility complex (MHC) class I molecules (reviewed by Germain and Margulies [1]) are cell surface glycoproteins associated noncovalently with ~2-microglobulin (~2m). These highly polymorphic molecules are expressed ubiquitously and

From the Hlstocompatibillty Laboratory (D.H., D.F., T.B., M.E.E.-F., H.B., M.-M.T., H.d.l.S.) and INSERM U311 (J.-P.C.), Regional Center for Blood Transfusion; and the Pediatric Service (L.D.), H@itaux Universitaires de Strasbourg, HSpital de Hautepierre, Strasbourg, France; and the Laboratory for Immunodermatology (T.B.), Department of Dermatology, University of Munich Medical School, Munich, Germany. Address reprint requests to Dr. Daniel Hanau, Laboratoire d'Htstocompatibilit~, Centre R~gional de Transfusion Sanguine de Strasbourg, I0 rue Spielmann, 67085 Strasbourg Cedex, France. Human immunology 41, 61-68 (1994) © American Society for Histocompatibiliry and Immunogenetics, 1994

function as carrier proteins for the transport of endogenously derived self- and non-self-peptides and for their presentation to CD8 ÷ T cells. These peptides derive from the proteolytic degradation of endogenous proteins in the cytosol and are translocated into the endoplasmic reticulum (ER) by a peptide transporter consisting of two homologous polypeptides, the transporter associated with antigen processing (TAP) molecules, TAP1 and TAP2. In the ER, binding of 8- to 10-amino-acid M H C allele-specific peptides into the groove formed by the ot 1 and ot2 domains of the class I heavy chain stabilizes these molecules and releases the class I - ~ 2 m heterodimers from the chaperone molecules. The class I - ~ 2 m - p e p t i d e complexes then move to the cell surface. In the absence 61 0198-8859/94/$7 00

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of peptides, some "empty" class I-[32m heterodimers reach the cell surface, but these unstable molecules rapidly unfold or dissociate if the cells are not incubated with appropriate peptides or at reduced temperature. Nonconventional MHC class I molecules (reviewed by Stroynowski and Lindahl [2])--distributed in mice over the H-2Q, T, and M regions and in humans over the HLA-E, F, and G regions--are structurally similar to conventional MHC class I molecules. In particular, their amino acid sequence suggests that, like the conventional class I molecules, they should fold into structures capable of forming a peptide-binding groove. Unlike the conventional class I molecules, however, the nonconventional proteins are relatively nonpolymorphic and are expressed in lower amounts, often in a tissue-restricted manner. Recent studies have shown that nonconventional class I proteins, like conventional MHC class I molecules, bind peptides. At least for mouse H-2M3, Qa-lb, and Qa-2 molecules, these peptides result from intracellular processing and are delivered by TAPdependent transport. Although not encoded by the MHC, CD1 antigens are also structurally similar to conventional MHC class I molecules [3]. In particular, their tx 1 and o~2 domains may form a peptide-binding groove. Moreover, like the nonconventional class I proteins, CD1 molecules are nonpolymorphic and are expressed in a tissue-specific manner involving about 85% of thymocytes, normal circulating and neoplastic B cells, intestinal epithelial cells, epidermal Langerhans cells, and dermal dendritic cells. Hence, if CD 1 antigens present the same general characteristics as the nonconventional class I molecules, do they share the same function, i.e., are the CD1 molecules peptide carriers? In a first step to approach this question, we demonstrate in the present study that CD1 expression is TAP independent. PATIENTS AND METHODS

Monoclonal antibodies. The monoclonal antibodies (mAbs) IOT6a (IgG1), IOT6b (IgG2a), and IOT6c (IgG1) directed against CDIa, CDlb, and CDlc molecules, respectively, were obtained from Immunotech (Marseille, France). W6/32, a mAb (IgG2a) directed against HLA class I, was purchased from Dako (Trappes, France), while the anti-CD14 mAb (IgG2b) was obtained from Becton Dickinson (Mountain View, CA, USA). FITC-conjugated, affinity-isolated F(ab') 2 fraction of a sheep-anti-mouse immunoglobulin (Ig) antibody (Silenus, Hawthorn, Victoria, Australia) was used for immunofluorescence labeling procedures. Rabbitanti-mouse antibody (Dakopatts, Hamburg, Germany) and a l k a l i n e - p h o s p h a t a s e m o u s e - a n t i - a l k a l i n e phosphatase complexes (Dakopatts) were employed for

D. Hanau et al.

immunolabeling and mouse IgG1, IgG2a, and IgG2b (Sigma, St. Louis, MO, USA) for isotype controls. Preparation of F(ab') 2 fragments of the anti-CDla mAb and labeling of these fragments with colloidal gold particles of 12-nm diameter were performed as already reported

[4]. Patients. Family E originates from Marocco and includes the parents who are first cousins and five children. One of the children, a 15-year-old girl, suffers from chronic bacterial sinobronchial infections but has not apparently been subject to frequent viral diseases. HLA serotyping revealed that while HLA class II alloantigens were expressed on her peripheral blood mononuclear cells, HLA class I molecules were not detectable. Genetic and molecular analysis demonstrated that the deficiency was due to a homozygous substitution in the TAP2 gene that introduces a stop codon in the first third of the coding sequence [5].

In situ immunolabeling on cryosections. The 6-1~m cryosections prepared from a skin biopsy specimen from the affected child were air dried, fixed for 10 minutes in acetone, and then processed for immunohistochemistry by using either IOT6a, IOT6b, IOT6c, or W6/32 mAbs or the corresponding isotype controls and the alkalinephosphatase-anti-alkaline-phosphatase technique as described previously [6]. Cryosections prepared from skin biopsy specimens from normal individuals served as controis.

Monocyte preparation. Monocytes from the affected child and from normal individuals were isolated from leukocyte concentrates by plastic adherence and detached by incubation at 37°C in phosphate-buffered saline (PBS) containing 0.53 mM EDTA. More than 85% of the adherent cells were CD14 + but negative for CDla, CDlb, and CDlc as determined by immunofluorescence analysis. To induce CD1 expression, monocytes were cultured in RPMI 1640 (Gibco BRL, Paisley, UK) with 10% heat-inactivated fetal calf serum (Gibco BRL) in the presence of 50 ng/ml recombinant human granulocytemacrophage colony-stimulating factor (GM-CSF) (PeproTech, Rocky Hill, NJ, USA) and 200 U/ml recombinant human interleukin 4 (IL-4) (PeproTech) [7]. Cell culture was carried out in a fully humidified 5% CO 2 atmosphere at 37°C.

Immunofluorescence analysis. Cells (5 × 105/100 I~1 medium) were first incubated for 15 minutes on ice and then centrifuged at 4°C and the pellet incubated for 30 minutes at 4°C with 50 I~l normal human AB serum to block nonspecific Fc-receptor-mediated binding of mAbs. Following a second centrifugation at 4°C, the cells were resuspended in cold PBS and indirect immu-

TAP Independent CD1 Expression

nofluorescence was performed using either anti-CD14, anti-CD1 (a, b, and c), or W6/32 mAbs or the corresponding isotype controls in a first step (4°C, 30 minutes) and FITC-conjugated sheep-anti-mouse Ig antibody in a second step (4°C, 30 minutes). Following the labeling procedure, 10,000 cells per sample were analyzed on a FACScan apparatus (Becton-Dickinson).

Direct immunogold-labeling procedure. Monocyte-derived dendritic cells obtained after 7 days of incubation in the presence of GM-CSF and IL-4 were cooled to 15°C for 15 minutes. Gold-conjugated F(ab')2 fragments of the antiC D l a mAb were added for 1 hour at a final dilution of 1%, after which the cells were warmed from 15°C to 37°C, left at 37°C for 8 minutes, and fixed for electron microscopy.

Preparation of cell samples for transmission electron microscopy. The cell suspension (500 gtl) was maintained at 37°C and fixed by adding an equal volume of fixative solution, previously warmed to 37°C and composed of 1.5% glutaraldehyde in 0.1 M Na cacodylate buffer containing 1% sucrose, p H 7.3. After 10 minutes, the mixture was centrifuged, the supernatant discarded, and the cell pellet resuspended and further fixed for 1 hour at 37°C with the same fixative solution. Following a second centrifugation, the samples were incubated for 1 hour at room temperature with 1% tannic acid in 0.05 M Na cacodylate buffer, pH 7.0. The cells were then postfixed for 1 hour at 4°C with 1% osmium tetroxide in 0.1 M Na cacodylate buffer, p H 7.3, washed once more in 0. ! M Na cacodylate buffer for 10 minutes at room temperature, and dehydrated with successively increasing concentrations of ethanol (50%, 70%, 80%, 95%, and 100%). Finally, the samples were incubated overnight in Epon-absolute alcohol (1/1, vol/vol) and embedded in Epon. Ultrathin sections, stained with lead citrate, were examined under a Siemens Elmiscope 102 electron microscope (60 kV). RESULTS

Expression of CD1 antigens in the absence of HLA class I molecules. The expression of HLA class I molecules and CD 1 antigens was studied in a skin biopsy sample taken from the affected child. Whereas no staining was visible in the whole skin section with the anti-HLA class I mAb W 6 / 3 2 (Fig. 1A), epidermal Langerhans cells were strongly labeled using the anti-CDla mAb (Fig. 1B) and more weakly using the a n t i - C D l c mAb (data not shown), which also stained some dendritic dermal cells. No staining was observed using the anti-CDlb mAb or isotype controls. In control experiments using skin biopsy samples from healthy individuals, the W6/32 anti-

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HLA class I mAb labeled the epidermal cells (Fig. 1A') and the anti-CD1 mAbs stained the epidermal Langerhans cells (Fig. 1B').

CDla, -b, and -c antigens can be induced on TAP2-deficient monocytes. Porcelli et al. [7] have reported that the expression of CDla, -b, and -c can be induced on peripheral blood monocytes by a combination of the cytokines GM-CSF and IL-4. We therefore isolated monocytes from the TAP2-deficient patient, incubated them for 60 hours in the presence of GM-CSF and IL-4, and analyzed the cells by indirect immunofluorescence. Whereas no CD1 antigens were expressed by freshly isolated monocytes, after 60 hours of incubation in the presence of GM-CSF and IL-4, high levels of CD la, -b, and -c were observed on these cells (Fig. 2A), while the expression of CD14 was strongly decreased (data not shown). In control experiments with monocytes from normal individuals, we usually observed a lower percentage of CD1positive cells, especially in the case of CDla, which was expressed on no more than 50% of the cells (Fig. 2B).

CD Ia antigens at the surface of TAP2-deficient dendritic cells maintain their ability to internalize by receptor-mediated endocytosis. In 1993, Dellabona et al. [8] reported that monocytes incubated for 6-8 days in the presence of GM-CSF and IL-4 still expressed high levels of CD1 antigens and became large and nonadherent, with a typical veiled appearance. Hence we maintained the monocytes of the TAP2-deficient patient for 7 days in culture. At this time, we indeed observed morphologic changes, i.e., cells with a dendritic morphology either emanating from cell clusters or isolated, although some of these dendritic cells presented only small processes projecting from the cell bodies (Fig. 3). Since we have shown in previous work that C D l a antigens internalize by receptor-mediated endocytosis when epidermal Langerhans cells are incubated in the presence of an anti-CDla mAb [4], we incubated the C D l a + dendritic cells of the TAP2-deficient child with gold-labeled F(ab') 2 fragments of the anti-CD la mAb IOT6a. As shown in Fig. 4, the C D l a antigens expressed at the surface of the TAP2-deficient dendritic cells maintained their capacity to internalize by receptor-mediated endocytosis, i.e., by coated pits, coated vesicles, and endosomes. Thus the "functionality" of the CD la antigens was still present in the C D l a + cells despite the absence of peptide transporter. DISCUSSION In the present study carried out on a TAP2-deficient patient, we demonstrated that, despite the absence of expression of HLA class I antigens, (a) C D I a and -c

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FIGURE 1 Immunocytochemical staining of the skin with anti-class-I, and -CDla mAbs. Using the anti-HLA class I mAb W6/32, no staining of the whole skin section of the TAP2-deficient patient was observed (A) while epidermal Langerhans cells are strongly labeled using the anti-CDla mAb (B). On a skin biopsy sample taken from a healthy individual, staining is observed with W6/32 (A') and antiCDla (B') mAbs. antigens were normally expressed on the surface of dendritic epidermal and dermal cells and (b) CD la, -b, and -c antigens could be induced on the surface of monocytes. It therefore appears that CD1 expression is not affected by peptide-transporter deficiency. Similarly, CD lb antigens may be expressed by a mutant human T2 cell line that lacks a large M H C class II region, including the T A P and proteasome genes [7]. The mean fluorescence intensity of CD 1-positive cells was higher in the cytokine-treated monocytes of the TAP2-deficient patient than in monocytes of normal individuals treated in the same way. This may be a consequence of the instability of the HLA class I - ~ 2 m com-

D. Hanau et al.

plexes in the TAP2-deficient cells, which could provide a greater number of [32m free molecules to stabilize the CD1 heavy chains. Indeed, C D l a molecules can be stabilized by the addition of exogeneous ~2m [9] (unpublished data). Alternatively, since the percentage of CD 1positive cells was higher when the monocytes of the TAP2-deficient patient were used, these monocytes may respond more efficiently to GM-CSF and IL-4. The latter explanation seems the most likely, since after 7 days of incubation the expression of C D l a on the T A P 2 deficient and on normal monocyte-derived dendritic cells was similar (data not shown). Two explanations may be proposed to account for our observations. Firstly, CD1 molecules may be peptide filled, these peptides being delivered to the lumen of the ER by a TAP-independent pathway [10]. Such a mechanism was observed in the mutant T2 cells where it explained the presence of a few HLA-A2 molecules at the cell surface. Indeed, the HLA-A2 molecules in these cells could be filled by peptides corresponding to fragments of signal sequences [11, 12] that mediate the translocation

TAP Independent CD1 Expression

65

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FIGURE 3A and B Morphologic aspects of monocytes incubated for 7 days in the presence of GM-CSF and IL-4. Cells with a dendritic morphology are seen either emanating from cell clusters (arrowheads) or isolated, some of the dendritic cells presenting only small processes projecting from the cell bodies (arrows).

Dendritic

FIGURE 2 Flow-cytometric analysis of the surface expression ofCDla, -b, and -c on monocytes. (A) Whereas no CDla antigens are expressed by freshly isolated peptide transporterdeficient monocytes (D = 0), high levels of CD la, -b, and -c are observed after 60 hours of incubation in the presence of GM-CSF and IL-4. (B) CD1 molecules can be induced on monocytes isolated from a healthy individual, but a lower percentage of CD 1-positive cells is observed and the intensity of fluorescence appears to be lower. of proteins into the ER. These HLA-A2 molecules were also capable of presenting some viral peptides encoded by minigenes introduced into the cytosol of the T2 cells by stable transfection, whether these peptides were preceded [13] or not [14] by an ER signal sequence. A pathway of this type also seems to exist in the mouse TAP2-deficient

RMA-S cells, which can present viral epitopes after exposure to increased viral doses when large amounts of viral peptides are available in the cytosol [15, 16]. Alternatively, CD 1 molecules may be empty molecules. In this case, they would be more stable than empty conventional class I molecules, since classic M H C class I molecules are very poorly expressed at the cell surface in the absence of peptide transporter. Furthermore, CD1 proteins would be more stable than the nonconventional HLA-E molecules, as these molecules are only slightly expressed at the surface of a transfected mouse myeloma [17] in the absence of appropriate peptide ligands. This hypothesis would explain why [32m dissociates easily from immunopurified C D l a at a room (or higher) temperature [18]. It would also explain the dissociation of

66

FIGURE 4 Transmission electron micrograph of monocytederived dendritic cells incubated successively for 1 hour at 15°C and for 8 minutes at 37°C with gold-labeled F(ab')2 fragments of an anti-CD la mAb. Four coated pits (arrowheads) are visible on A and two coated vesicles (arrowheads) on B. Gold-labeled endosornes (E) and observed on B and C and gold-labeled lysosomes (L) on D. Scale bar, 1 I~m.

D. Hanau et al.

~2 m from the C D l a heavy chain at the surface of Langerhans cells [9, 19] or the M O L T - 4 T cell line [9, 20], where extracellular 132m easily exchanges w i t h 132m bound to CD la [9, 2 I] and where extracellular 132m may modulate the stability or expression of CD la epitopes at the surface of the MOLT-4 cell line [9] (unpublished data).

TAP Independent CD1 Expression

Porcelli et al. [7] reported that C D l b antigens induced on monocytes by the cytokines GM-CSF and IL-4 presented exogenously supplied Mycobacterium tuberculosis antigens to CD4 8 - o~]3 T cells. This CDlb-restricted presentation required, as for the M H C class-II-restricted responses, both antigen uptake and processing. How may we reconcile these different observations and hypotheses? The CD la and -b molecules might reach the cell surface while still empty and hence unstable. However, in the course of this progression--or in the course of reinternalization by receptor-mediated endocytosis--the CD1 molecules might enter the endocytic pathway to yield a functional and stable complex. Such an "association" between M H C class I molecules and processed antigens has recently been reported in murine macrophages [22].

ACKNOWLEDGMENTS

We thank J.-C. Garaud for providing us with gold-labeled mAb, P. Baisser and R. Bury for expert technical assistance, R. Dujol for photography, and J. Mulvihill for reviewing the English of the manuscript. This work was supported by INSERM (CRE 930606) by the Deutsche Forschungsgemeinschaft (SFB 217/D4) and the CentreRdgional de Transfusion Sanguine de Strasbourg. M.-E.E.-F. is grateful for a fellowship from the French Minist&e de la Rechercheet de la Technologie.

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20. Schmitt DA, Hanau D, Cazenave J-P, Bieber T: Are C D l a antigens on Langerhans cells empty class I-like molecules that come out in the cold? J Invest Dermatol 97:160, 1991. 21. Bernabeu C, van de Rijn M, Lerch PG, Terhorst CP: [3z-Microglobulin from serum associates with MHC class

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