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,
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Departments of
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Pathology and
Cell Biology and
Center for Blood Research, Harvard Medical School, Boston, MA 02115; and
Faculty of Science, Charles University, Prague, Czech Republic
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A mouse strain in which all I-Ab molecules are replaced by I-Ab fused at the C terminus of the 
-chain with green fluorescent protein has been described recently (4). Such animals are phenotypically normal with respect to their immune system. We generated dendritic cells (DCs) by culture of bone marrow cells as described elsewhere (4, 5). class II-enhanced green fluorescent protein (eGFP) molecules in bone marrow-derived DCs are located in late endocytic compartments concentrated around the microtubule organizing center in vesicles as well as in short tubules. Upon contact with an Ag-specific T cell, class II-eGFP molecules are rapidly recruited to the DC/T cell contact zone by polarization of the tubulovesicular endosomes, but only when cognate Ag is available (4). Fusion of class II-eGFP-positive endosomes with the plasma membrane was demonstrated in live DCs (6), consistent with the possibility that such polarized endosomes may transport specific peptide-loaded class II MHC molecules to the cell surface. An APC thus responds to the encounter with an Ag-specific T cell in a manner that may help sustain the chain of events leading to full activation of the T cell.
When a TCR on an Ag-specific T cell is ligated by the correct peptide-class II MHC complex, T cells respond by the formation of specialized structures, referred to as supramolecular activation clusters (SMACs) or immunological synapses (7, 8). The serial engagement of several T cell receptors is apparently required to surpass a threshold required for activation of the responding T cell (9, 10, 11, 12). Molecules such as TCR, CD2, CD3, CD28, and protein kinase C-
, lck, and fyn cocluster at and near the T cell surface (7, 8, 13, 14, 15, 16). A central SMAC is surrounded by the peripheral SMAC in which the integrin LFA-1 and the cytoskeletal protein talin are concentrated (8). The involvement of T cell surface molecules TCR, CD2, CD28, and LFA-1 in the induction of polarized endosomal tubulation in the DCs must therefore be explored.
We here describe cell surface requirements of DCs and T cells for T cell-polarized class II -positive tubular endosomes in DCs. In addition to the Ag-specific interaction between class II MHC and TCR, requirements for endosomal tubulation include the mobility of proteins in the lipid bilayer of the plasma membrane, the ligation of cell adhesion molecules, and induced protein expression on the surface of activated T cells. An unexpected result is the observation that a T cell can distinguish between peptide-loaded class II MHC complexes depending on their mode of peptide acquisition. Whereas T cell ligation of endosome-derived peptide/class II MHC allowed endosomal tubulation, DCs loaded with exogenously added peptide induced no morphological transformation of endosomal compartments upon interaction with Ag-specific T cells. We propose that the microenvironment of class II MHC molecules that acquired their antigenic cargo in endosomes may be important for polarized tubulation of class II -positive compartments. Activation of naive T cells could benefit from such spatial and temporal conservation of peptide-loaded class II MHC molecules on the surface of the APC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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I-Ab-eGFP knock-in mice (4) and transgenic mice that express an OVA-specific TCR (OTII) (17) have been described. Mice were housed in a barrier facility and studies were performed according to institutional guidelines for animal use and care.
Cell preparation and culture
DCs were generated as described previously (4, 5). Briefly, bone marrow cells from class II-eGFP mice were flushed from the femurs and tibia and resuspended in DMEM (Life Technologies, Grand Island, NY) supplemented with 10% FCS, 10 ng/ml GM-CSF (PeproTech, Rocky Hill, NJ) and 1 ng/ml IL-4 (Boehringer Mannheim, Indianapolis, IN). Cells were cultured on 25-mm circular coverslips in six-well plates (5 x 106 cells/well), with changes of medium every second day; nonadherent cells were removed by gentle washing. Naive T cells were obtained either directly from lymph nodes of OTII mice in which CD4 T cells represent 25% of all of the cells or purified from spleen and/or lymph node using a T cell purification kit (Miltenyi Biotec, Auburn, CA), resulting in >95% pure T cell preparation. OTII cells were stained with nuclear dye Hoechst 33258 (Molecular Probes, Eugene, OR).
Flow cytometry
mAbs used for cell typing as well as for T cell activation and blocking experiments were the following: anti-CD69-PE (Armenian hamster clone H1.2F3), anti-CD4-allophycocyanin (rat clone L3T4), anti-TCR-biotin (hamster clone H57-597), anti-TCR-V5.1/5.2-FITC (mouse clone MR9-4), anti-CD71-biotin (rat clone C2), anti-CD2-biotin (rat clone RM2-5), anti-LFA-1-biotin (rat clone 2D7), anti-CD40 ligand (CD40L)-biotin (Armenian hamster clone MR1), and anti-CD28-biotin (Syrian hamster clone 37.51) and were obtained from BD Biosciences (Mountain View, CA). Biotin-conjugated Abs were combined with PE-conjugated streptavidin for flow cytometry. Cells were analyzed on a BD Biosciences FACScan instrument.
Fixation of T cells
In some experiments, OTII cells were fixed using mild conditions: 2 x 106 cells/ml were incubated at room temperature (RT), 30 s in 0.05% glutaraldehyde, then 0.2 M glycine was added for 90 s before stopping the fixation reaction by addition of 10 vol of cold PBS. Robust fixation of OTII cells was performed by incubating cells in 3% paraformaldehyde (2 x 106 cells/ml, 10 min, RT). For both fixation conditions, cells were washed twice in PBS and once in medium before addition to DC cultures. To arrest processing of Ag and loading of peptide on class II MHC molecules in live DCs, cells were incubated at 2 x 106 cells/ml in 0.05% glutaraldehyde (30 s)/0.2 M glycine (90 s). Before the addition of T cells, DCs were washed twice in PBS and once in medium.
Cycloheximide (CHX) treatment and inhibition of cell surface molecule ligation using blocking mAbs
To block T cell surface molecules LFA-1 and CD2, T cells were incubated on ice 1 h with 10 µg/ml of the relevant mAb before washing and adding to the DCs. To block newly expressed CD40L and CD28, the relevant mAb was also included in the DC/T cocultures (also 10 µg/ml). For CHX treatment, OTII cells were incubated at 37°C in the presence of 25 µg/ml CHX for 1 h and washed three times before addition to DCs. For experiments with activated T cells, OTII cells were activated overnight with PMA (1.7 µg/ml) and ionomycin (114 µg/ml) or with plate-bound anti-TCR Abs. Plates were coated by incubation with anti-TCR Abs (5 µg/ml in 0.1 M NaHCO3/Na2CO3, pH 9.5, overnight (ON)).
Peptide
The OVA-derived OTII epitope ISQAVHAAHAEINAGR (OVA323–339, ISQ in short) (18) was synthesized on an advanced ChemTech 40 channel peptide synthesizer using standard F-moc chemistry. Its molecular mass was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
Ag presentation and real-time imaging of class II-eGFP in DCs
At day 5 of culture, OVA (40 µM; Sigma-Aldrich, St. Louis, MO) or ISQ peptide (20 µM) was added to DCs. OVA uptake and processing was allowed for 4 h at 37°C, after which DCs were washed and 1 x 106 OTII T cells were added. Plates were centrifuged (1200 rpm, 1 min at RT) to induce rapid contact of T cells with DCs. When using ISQ peptide as Ag, binding of peptide to surface-exposed empty class II MHC was allowed for 1 h at RT. DCs were then washed three times with medium. Naive OTII T cells were obtained from lymph nodes of OTII-transgenic mice. T cells (1 x 106) were labeled with the nuclear dye Hoechst 33258 (Molecular Probes), washed, and added to each coverslip containing DC. Conjugates of DCs and T cells were analyzed for the induction of class II-eGFP-positive tubular compartments between 1 and 2 h of T cell addition using an inverted Zeiss 200M microscope equipped with a x63 objective (1.4 aperture PanApo) supplied with a 37°C open perfusion temperature-controlled chamber (20/20 Technology, Wilmington, NC). We used Slidebook (Intelligent Imaging Innovation, Denver, CO) for image acquisition and data processing.
At least 100 DCs were analyzed for the induction of tubulation and were scored independently by two individuals.
Activation of T cells by culture with Ag-presenting DCs
DCs were allowed to endocytose different concentrations of crystalline OVA for 2 h and were washed. Lymph node cells from naive OTII mice were added (1 x 106) and were incubated for 6 h (in duplicate experiments). Activation of OTII T cells was gauged by the increase in cell surface expression of the early activation marker CD69 (by flow cytometry). For the time course of OVA presentation by DCs, DCs were allowed to endocytose 40 µM OVA for 20 min to 6 h at 37°C, fixed by addition of 0.05% glutaraldehyde (30 s, RT) and 0.2 M glycine (90 s, RT), and were washed three times. OTII T cells (1 x 106) were then added to DCs and activation of T cells was assessed as above after 24 h.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To visualize the intracellular distribution of class II MHC molecules, we made use of I-Ab-eGFP knock-in mice. We generated DCs from bone marrow by culturing them with GM-CSF and IL4 as described previously (4). DCs are generally cultured in plastic tissue culture dishes, but for epifluorescence microscopy, bone marrow precursors were grown on glass coverslips contained in regular plastic dishes. The culture of DCs on a modified surface may affect DC differentiation from bone marrow precursors or produce a change in activation status of DCs once fully developed. Therefore, we examined DCs that were cultured in plastic tissue culture dishes, on glass coverslips, or on poly-L-lysine-coated coverslips for DC yield and activation status, as judged by direct visualization of class II-eGFP and staining for CD11c and CD86 (our unpublished observations). We did not observe a significant difference in the development of CD11c/class II double-positive cells grown on different surfaces. After 5 days of culture, a third of the cells expressed class II-eGFP, at least 85% of which expressed the DC marker CD11c and were therefore considered DCs. The majority of DCs, whether grown on plastic or glass, exhibited low levels of CD86 (our unpublished observations) and we consider DCs from these cultures early/intermediate-type DCs (19).
Ag loading requirement for tubulation of class II-containing endosomal compartments in DCs
Before addressing the signals required for the induction of T cell-polarized endosomal tubulation, we examined the time required of effective Ag loading and presentation. DCs were allowed to endocytose OVA (40 µM, 37°C) for periods of 20 min to 6 h and were then fixed to arrest all further processing of Ag. Naive T cells from transgenic mice that express an I-Ab-restricted TCR specific for OVA (OTII) were purified from lymph nodes and added to the fixed DCs. Activation of T cells was gauged by the increase in CD69 expression after 24 h of further incubation.
Two hours of OVA uptake suffices for display of peptide-loaded class II MHC molecules to activate 30% of OTII cells (CD69high), with a further increase to 50% after 4 h and 80% after 6 h (Fig. 1a). In all subsequent Ag presentation experiments, DCs were allowed to endocytose OVA for 4 h before confrontation with T cells.
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Because of the number of different parameters examined here, we present the data as the percentage of DCs that express tubulating endosomes, calculated based on observation of at least 100 DCs. With 40 µM OVA, 40–60% of all of the DCs examined had class II -positive tubular endosomes. The percentage of DCs that express tubular endosomes was normalized by setting the maximum response as 100%. We analyzed DCs after engagement of Ag-specific T cells by analysis of images rather than movies, and therefore this study focuses on the conditions required for tubulation rather than dynamic endosomal transport. For representative images of DCs that express tubular endosomes, we refer to Boes et al. (4).
We observed that T cells evoke class II-eGFP tubules in DCs in an Ag concentration-dependent manner. As little as 0.4 µM OVA triggers formation of tubules in some DCs, the maximal response being observed at 40 µM OVA (Fig. 1b). We have no estimate of the success of conversion of intact OVA into the relevant peptide-MHC complex.
Is there a correlation between the concentration of OVA required for endosomal tubulation in DCs and the minimal dose necessary to activate naive T cells? The activation status of OTII T cells was analyzed after 6 h of culture with OVA-loaded DCs. The Ag dose that induces T cell-directed endosomal tubules in DCs correlates well with the Ag dose required to activate naive T cells, as judged from up-regulation of the early activation marker CD69 (Fig. 1c). Little up-regulation of CD69 was observed when OTII T cells were cocultured with DCs loaded with OVA at a concentration of 0.4 µM, but a significant number of activated T cells was present after coculture with DCs that had endocytosed 40 µM OVA. Subsequent experiments were thus performed using 40 µM OVA endocytosed by DCs for 4 h.
Mild fixation compromises the ability of T cells to induce tubular endosomes in DCs
Cytoskeleton-driven clustering of T cell membrane proteins at the T cell/APC interface plays an important role in T cell activation (7, 8, 13). Are there analogous requirements to induce polarized endosomal rearrangement in DCs upon contact with Ag-specific T cells? To interfere with rearrangements of surface proteins, OTII T cells were fixed using 0.05% glutaraldehyde or using a stronger fixative (3% paraformaldehyde). To assess the effect of these fixations on membrane protein clustering, naive purified OTII T cells were labeled with fluorescein-coupled anti-TCR Ab and PE-coupled anti-CD4 Ab (20). In untreated naive T cells, a 30-min incubation at 37°C induces complete colocalization of TCR and CD4 (Fig. 2a, top panels). When cells were fixed using 0.05% glutaraldehyde, some clusters of TCR and CD4 developed, but no full cap was produced (Fig. 2a, middle panels). Strong fixation using 3% paraformaldehyde completely blocked the ability of TCR and CD4 to cluster in the membrane (Fig. 2a, bottom panels). Of note, the conditions of fixation of OTII T cells were verified by examining up-regulation of CD69, when fixed T cells were exposed to DCs loaded with OVA peptide. Expression of CD69 was not induced in T cells that were fixed using either cross-linking reagent (data not shown).
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Fixation of OTII T cells using paraformaldehyde blocked 90% of polarized endosomal tubulation in DCs (Fig. 2b). Thus, the maintenance of normal membrane dynamics appears to be essential to induce tubulation. T cell fixation using glutaraldehyde resulted in 40% reduction in tubulation. Glutaraldehyde fixation, but not paraformaldehyde fixation, allowed for some clustering of surface glycoproteins TCR and CD4 (see Fig. 2a), which therefore appears sufficient for the induction of tubulation in some DCs. Thus, the mobility of proteins in the T cell membrane is essential for their ability to induce tubulation.
Adhesion requirements between DCs and Ag-specific T cells to induce tubular endosomes
To examine whether adhesion molecule-mediated binding is important for induction of endosomal tubulation, we blocked these interactions using mAbs to adhesion molecules LFA-1 and CD2. Both LFA-1 and CD2 are present at the interface between APC and T cells during Ag-specific contact (21). First, the blocking assay was optimized using mAb to the TCR as a positive control and mAb to transferrin receptor (TfR, or CD71) as a negative control Ab unlikely to affect tubulation. Inhibition of interaction of TCR using anti-TCR mAb blocked 90% of tubulation. Thus, in this system, endosomal tubulation is dependent on the interaction with Ag-specific TCR, as demonstrated previously (4). Ab-mediated blocking of TfR interaction did not affect tubulation significantly compared with untreated DC/T cell conjugates (Fig. 3a).
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Activated T cells induce more endosomal tubulation in DCs
Alterations in surface expression of proteins on the T cell plasma membrane during activation may enhance their ability to induce extended endosomes in interacting DCs. Do activated Ag-specific T cells stimulate more endosomal tubulation in DCs compared with their naive counterparts? Purified OTII cells were preactivated with PMA/ionomycin or with plate-bound anti-TCR Abs (ON) and were then added to OVA-loaded DCs. Both methods induce strong activation of T cells as verified by increased CD69 expression (data not shown). Induction of tubulation by activated OTII T cells remained strictly dependent on the presence of Ag, as was the case with naive Ag-specific T cells. Both methods of activation increased the ability of OTII T cells to induce polarized class II-eGFP-positive tubules in DCs (Fig. 4a). OTII T cells that were activated using PMA/ionomycin increased DCs that express tubulating endosomes by 28%, whereas anti-TCR mAb used as activating agents increased the number of DCs that express extended endosomes by 68% compared with naive OTII T cells.
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A candidate ligand induced upon T cell activation is CD40L. We therefore assayed the possible involvement of CD40L in inducing the rearrangement of endosomal compartments in DCs through ligation of CD40 on the DC. First, is CD40L induced at the cell surface during T cell activation? Naive OTII T cells were purified from lymph nodes and were activated using plate-bound anti-TCR Abs. Activation-induced cell surface expression of CD40L on the OTII plasma membrane, with and without pretreatment with CHX to block de novo synthesis of proteins, was measured over a 6-h culture period. Expression of CD40L on OTII T cells increased as soon as 2 h after activation (Fig. 4b). The increased ability of activated T cells to induce endosomal tubulation in DCs therefore correlates with induced expression of CD40L on the T cells.
We then examined whether the induction of CD40L on activated T cells stimulates tubulation of endosomal compartments in interacting DCs. Blocking mAb to CD40L (10 µg/ml) was added simultaneously with naive OTII lymph node cells to Ag-loaded DC cultures (40 µM OVA for 4 h), and mAb were kept in the cultures throughout the experiment. DCs were then monitored for the formation of class II-positive tubules between 3 and 4 h after T cell addition by epifluorescence microscopy.
Although inhibition of CD28 ligation using anti-CD28 mAb did not significantly reduce tubulation, treatment with mAb to CD40L resulted in a 60% reduction of endosomal tubulation compared with untreated T cells (Fig. 4c). Pretreatment of T cells with CHX also resulted in inhibition of tubulation comparable to anti-CD40L mAb treatment. We conclude that the CD40-CD40L interaction is involved in the induction of tubular endosomes during T cell activation, although other molecules must play a role as well and on a whole the process is likely to be complex. The involvement of CD40-CD40L interaction in endosomal extensions is probably limited to interaction with newly activated T cells, but is irrelevant for initiation of the response.
DC membrane requirements to induce tubular endosomes in DCs
Most surface class II MHC segregate constitutively into cholesterol- and glycosphingolipid-enriched plasma membrane microdomains (22), and reorganization of proteins into microdomains at the interface of a T cell and an APC plays an important role in T cell activation (21, 22, 23). In APC, class II -positive microdomains at the plasma membrane may be a manifestation of the transient preservation of the membrane composition of transport vesicles that recently inserted in the plasma membrane. Do T cells normally encounter peptide-loaded class II MHC in a spatial configuration that is enriched for Ag-loaded class II molecules and costimulatory molecules? The presence of surface-displayed empty class II MHC or self-peptide-loaded class II MHC is probably not enhanced in tetraspan-expressing microdomains (23). How would T cells respond to peptide-loaded class II MHC, when the cognate epitope was administered in the form of a synthetic peptide from the outside?
DCs were treated with synthetic peptide or with intact OVA Ag before or after fixation using glutaraldehyde. DCs were then washed and OTII lymph node cells were cocultured with the DCs for 24 h (37°C). Both routes of peptide acquisition result in equally effective stimulation of naive T cells, as judged from the ensuing increase in expression of the activation marker CD69 by unfixed DCs (Fig. 5a). Loading of synthetic peptide onto class II MHC does not require endosomal processing and should therefore be largely unaffected by prior fixation of DCs, whereas endosomal processing requires enzymatic activity of live DCs, abrogated by prior fixation of DCs (24, 25). Indeed, up-regulation of CD69 by OTII T cells was severely compromised by fixation of the DCs before addition of intact OVA Ag, but not when DCs loaded with synthetic peptide were used after fixation. Class II MHC molecules that capture synthetic peptide from the outside can activate naive T cells even when the DCs are fixed.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We describe here signals exchanged between DCs and T cells that are likely involved in polarized endosomal tubulation. Because of the large number of conditions tested, we relied on still images of DCs that were cocultured with Ag-specific T cells rather than time-lapse movies. In the current study, we therefore refrain from statements regarding the transport of class II MHC to the cell surface and focus on the requirements to generate polarized endosomal tubulation toward the Ag-specific T cell.
Endosomal tubulation that occurs in DCs after engagement of Ag-specific T cells relies on the rearrangement of membrane proteins on the T cell surface. A partial reduction of the mobility of membrane proteins, as effectuated by glutaraldehyde fixation of T cells, correlates with diminished tubulation, whereas the harsher paraformaldehyde fixation results in a complete block in T cell-polarized tubulation. Therefore, we conclude that rearrangement of proteins on the T cell surface is required for Ag-specific T cells to induce tubular endosomes in DCs.
The initial interaction between APC and T cells is probably mediated by low-affinity interactions, thus allowing the serial scanning for Ag on the surface of different APC (28). Such initial contacts between APC and T cells are probably mediated by adhesion molecules with long extracellular domains (28). The adhesion molecules LFA-1 and CD2 are involved in both the stabilization of DC-T cell interaction as well as the initiation of intracellular signaling (29, 30). We show here that the induction of tubular endosomes in DCs relies on the interaction of DCs with T cells, mediated at least in part by adhesion molecules LFA-1 and CD2 as inferred from Ab inhibition experiments (Fig. 3), although we were unable to achieve complete inhibition.
The activation of T cells induces cell surface expression of molecules that may influence their ability to induce rearrangement of endosomal compartments in DCs (31). OTII T cells were activated by treatment with either PMA/ionomycin or plate-bound anti-TCR mAb. Compared with naive OTII T cells, activated OTII T cells induced more endosomal tubulation in DCs. We show that in OTII T cells, cell surface expression of CD40L emerges after 2 h of coculture with DCs that were allowed to endocytose intact OVA. A reduction of tubulation was found in DCs that were cocultured with anti-CD40L mAb-treated OTII T cells, whereas anti-CD28 mAb only had a minimal effect. Thus, T cell surface expression of CD40L does stimulate tubulation of class II-eGFP-positive compartments in DCs.
In this regard, it may be of interest to point out that class II MHC-positive compartments in B cells are also subject to rapid remodeling, in response to ligation of the B cell receptor (32). We consider it likely that the ability of class II-positive endosomal compartments to undergo such structural rearrangements may be a more general property of APC and may have a beneficial effect on the proper display of class II MHC-peptide complexes on the surface of the APC.
APC can regulate the distribution of their surface class II MHC within the plane of the membrane to microdomains before contact with CD4 T cells (33, 34). Specialization is achieved through the microdomains that contain members of the tetraspanin family, such as CD9, CD81, and CD82. These tetraspanins form two-dimensional networks in certain areas of the plasma membrane (35, 36). Approximately 10% of human surface class II MHC molecules are found in such tetraspanin-positive domains, and in human cells can be differentiated phenotypically from other surface class II MHC using a mAb, FN1, that probably recognizes clusters of at least two class II MHC molecules (23, 33, 34, 37).
In the endosomal pathway, where protein-lipid sorting occurs, structural and functional microdomains are already formed (38). Moreover, the segregation of specific peptide-loaded class II MHC molecules into microdomains was demonstrated in endosomes as well as on the plasma membrane (23). Class II MHC recently loaded with antigenic peptide may be enriched in microdomains, whereas empty class II MHC or pre-existing peptide-loaded class II MHC may be present in a more even distribution in the lipid membrane (23).
The activation of naive or resting T cells generally requires coengagement of costimulatory molecules between the APC and T cells. Upon arrival of endosomal vesicles at the cell surface, costimulatory molecules such as CD86 remain clustered with class II MHC (39). Our results suggest that the clustered presence of peptide-loaded class II MHC and costimulatory molecules may be essential for T cell-polarized tubulation, a behavior that may be important early on in the DC-T cell interaction (21, 23). In contrast, T cell activation after 24 h of coculture with DCs (as judged by up-regulation of CD69) occurs irrespectively of the initial gathered presence of CD86 with specific peptide-loaded class II MHC.
Loading the DC surface with exogenously provided peptide necessarily results in a local density of class II MHC complexes and of the auxiliary molecules required for tubulation that is distinct from that found at new insertion sites (see model, Fig. 6). This would explain the differences in the tubulation response to T cells observed for intact protein- vs peptide-loaded DCs (Fig. 5). DCs display large numbers of peptide-receptive class II MHC molecules at their surface, generated presumably through loss of bound peptide. If the formation of polarized tubules is essential to activate naive T cells in the lymph node, then inadvertent activation of T cells by DCs with passively acquired peptides would be less likely. This aspect of T cell activation may help avoid unwanted T cell responses and contribute to establish peripheral tolerance. Seen from that perspective, it is not only the maturation or activation status of a DC that helps determine its immunogenic or tolerogenic properties: the local distribution of surface proteins is an additional factor to be taken into account.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hidde Ploegh, Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115. E-mail address: ploegh{at}hms.harvard.edu
3 Abbreviations used in this paper: ER, endoplasmic reticulum; DC, dendritic cell; eGFP, enhanced green fluorescent protein; SMAC, supramolecular activation cluster; CHX, cycloheximide; CD40L, CD40 ligand; RT, room temperature; ON, overnight; TfR, transferrin receptor.
Received for publication June 20, 2003. Accepted for publication September 17, 2003.
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References |
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- and -chain genes under the control of heterologous regulatory elements. Immunol. Cell Biol. 76:34.[Medline]
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