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MacrophagesExpression upon Differentiation ofCytoplasmic Tail Allows Regulated A Novel Trafficking Signal within the HLA-C

CollinsPennelope K. Blakely, Anna Q. Yaffee and Kathleen L. Malinda R. Schaefer, Maya Williams, Deanna A. Kulpa,

http://www.jimmunol.org/content/180/12/7804doi: 10.4049/jimmunol.180.12.7804

2008; 180:7804-7817; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2008 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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A Novel Trafficking Signal within the HLA-C CytoplasmicTail Allows Regulated Expression upon Differentiationof Macrophages1

Malinda R. Schaefer,* Maya Williams,† Deanna A. Kulpa,‡ Pennelope K. Blakely,‡

Anna Q. Yaffee,¶ and Kathleen L. Collins2*†‡§

MHC class I molecules (MHC-I) present peptides to CTLs. In addition, HLA-C allotypes are recognized by killer cell Ig-likereceptors (KIR) found on NK cells and effector CTLs. Compared with other classical MHC-I allotypes, HLA-C has low cell surfaceexpression and an altered intracellular trafficking pattern. We present evidence that this results from effects of both the extra-cellular domain and the cytoplasmic tail. Notably, we demonstrate that the cytoplasmic tail contains a dihydrophobic (LI) inter-nalization and lysosomal targeting signal that is partially attenuated by an aspartic acid residue (DXSLI). In addition, we provideevidence that this signal is specifically inhibited by hypophosphorylation of the adjacent serine residue upon macrophage differ-entiation and that this allows high HLA-C expression in this cell type. We propose that tightly regulated HLA-C surface expressionfacilitates immune surveillance and allows HLA-C to serve a specialized role in macrophages. The Journal of Immunology, 2008,180: 7804–7817.

M ajor histocompatibility complex class I (MHC-I)3 mol-ecules are necessary for presentation of Ags to naiveCD8� CTLs through engagement with the TCR and

CD8. There is evidence that professional APCs are required forthis step, as they have unique costimulatory molecules needed forthis process (1). APCs are thought to activate naive CD8� T cellsby internalizing exogenous Ags and presenting them in associationwith MHC-I in a process known as cross-presentation (2). ActivatedCTLs then mature into effector cells, which have the capacity to killcells bearing foreign epitopes through the release of perforin and gran-zymes and via the activation of apoptotic pathways (3).

MHC-I can also be recognized by MHC-I-specific inhibitoryreceptors, such as killer cell Ig-like receptors (KIR) (4). Thesereceptors are classically thought to be expressed on NK cells tofacilitate the eradication of virally infected or tumorigenic cellsthat have down-modulated MHC-I. More recently, evidence hasbeen accumulating that these receptors are also up-regulated withthe acquisition of cytotoxic function in CD8� T cells (5). In thiscase they may serve as an important negative feedback mechanism

that aids in the prevention of autologous damage by raising thethreshold for cell lysis (5).

There are three major subtypes of classical MHC-I moleculesthat serve these roles. HLA-A, B, and C all have the capacity topresent viral Ags to CTLs. Additionally, almost all HLA-C mol-ecules are recognized by KIRs. Perhaps because of the crucial roleof HLA-C as an inhibitory molecule capable of sending a domi-nant-negative signal (6), HLA-C is normally expressed at low lev-els at the cell surface. HLA-C H chain mRNA is unstable (7), theHLA-C H chain protein is not stably expressed at the cell surface,and it does not associate efficiently with the MHC-I L chain (�2-microglobulin) (8–10). Additionally, HLA-C presents a more re-stricted repertoire of peptides causing it to be retained in the en-doplasmic reticulum (ER) in complex with the TAP, which isresponsible for transporting peptides into the ER for MHC-I load-ing. The retained HLA-C is then eventually degraded in the ER(8). The addition of HLA-C-specific peptides has been shown torelease HLA-C from TAP in vitro (8) and to increase the cellsurface expression of HLA-C (11).

We examined the expression of HLA-Cw*0401 relative toHLA-A*0201 in a variety of cell types, including T cell lines,primary T cells, and monocytic cell lines and confirmed that HLA-Cw*0401 was poorly expressed on the cell surface relative toHLA-A*0201. To better understand the amino acid sequences gov-erning HLA-C surface expression, we examined the intracellulartrafficking of chimeric molecules that contained the HLA-A*0201extracellular domain and the HLA-C cytoplasmic tail (A2/C) orthe HLA-Cw*0401 extracellular domain and the HLA-A cytoplas-mic tail (Cw4/A). Not surprisingly, the extracellular domain ofHLA-C was responsible for promoting retention in the ER. Re-markably, however, the cytoplasmic tail also had an effect on cellsurface expression by increasing internalization at the cell surfaceand targeting the molecules for degradation in acidic organelles.Mutagenesis studies revealed that aspartic acid at position 333,serine at position 335, and isoleucine at position 337 were keyamino acids that affected the activity of this motif. Finally, wefound that the complex regulation of HLA-C surface expressionallowed the specific up-regulation of HLA-C upon differentiation

*Graduate Program Immunology, †Graduate Program in Cellular and Molecular Bi-ology, ‡Department of Medicine, §Department of Microbiology and Immunology, and¶Masters of Public Health Program, University of Michigan, Ann Arbor, MI 48109

Received for publication July 11, 2007. Accepted for publication April 2, 2008.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institutes of Health Grants RO1 AI051198 andAI046998. M.W. was supported by the University of Michigan Cellular and Molec-ular Biology Training Program. M.R.S. was supported by the University of MichiganResearch Training in Experimental Immunology Training Grant and the Herman andDorothy Miller Award. D.A.K. was supported by an Irvington Institute Fellowship.2 Address correspondence and reprint requests to Dr. Kathleen L. Collins, Universityof Michigan, 3510 Medical Science Research Building I, 1150 West Medical CenterDrive, Ann Arbor, MI 48109. E-mail address: [email protected] Abbreviations used in this paper: MHC-I, MHC class I; KIR, killer cell Ig-likereceptor; HA, hemagglutinin; ER, endoplasmic reticulum; MFI, mean fluorescenceintensity; RIPA, radioimmunoprecipitation assay; endo H, endoglycosidase H; IRES,internal ribosome entry site.

Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00

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of primary monocytes and monocytic cell lines into macrophage-like cells. The specific induction of HLA-C expression with dif-ferentiation strongly suggests that there is a unique role for HLA-Cin APCs. We propose that inhibitory signals sent via HLA-C playa role in down-modulating the normal CD8� cellular immune re-sponse and/or that it functions to specifically limit the lysis ofAPCs that are cross-presenting Ag.

Materials and MethodsDNA constructs

MSCV 2.1 hemagglutinin (HA)-HLA-A*0201 was constructed as previ-ously described (12). For MSCV 2.1 HA-HLA-Cw*0401, the HLA-Cw*0401 open reading frame (Peter Parham, Stanford University) wasamplified with the following primers, 5�-CAATCTCCCCAGACGCCGAGATGCG-3� and 5�-CCGCTCGAGTCAGGCTTTACAAGCGATGAGAGA-3�. The PCR product was digested with NaeI and XhoI and the 3�fragment was gel purified. This fragment was then ligated to the 5� leadersequence plus the HA tag from HA-HLA-A*0201 (isolated by digestingMSCV 2.1 HA-HLA-A*0201 with EcoRI and NaeI) and MSCV 2.1 di-gested with EcoRI and XhoI.

MSCV 2.1 HA-Cw4/A2 was constructed by digesting MSCV 2.1 HA-HLA-Cw*0401 with EcoRI and XhoI, subcloning this fragment into thesame sites in Litmus 29 to generate Litmus 29 HLA-Cw*0401. A three-way ligation was then performed with a EcoRI to SapI fragment fromLitmus 29 HLA-Cw*0401 that encodes the extracellular domain of HLA-Cw*0401, a DNA fragment encoding the HLA-A*0201 cytoplasmic taildigested with Sap I and XhoI (generated by PCR amplification of MSCV2.1 HA-HLA-A*0201 with the following primers 5�-GTGATCACTGGAGCTGTGGTCGCTGCT-3� and 5�-CCGCTCGAGTCACACTTTACAAGCTGTGAGAGACAC-3�), and MSCV 2.1 digested with XhoI and EcoRI.

MSCV A2/C was constructed by first PCR amplifying the Cw*0401 cyto-plasmic tail using the following primers 5�-CAGGTGACCGGTGCTGTGGTCGCTGCTGTGATGTGGAGGAGGAAGAGCTCAGGTGGA-3� and 5�-CACCTGCAGCTGTCAGGCTTTACAAGCGATGAG-3� and thendigesting with AgeI. This fragment was then ligated into pcDNA3.1 HLA-A*0201 AgeI (a plasmid containing an HLA-A*0201 open reading framewith a silent sequence change to introduce an AgeI site (13)) digested withAgeI and EcoRV to generate pcDNA3.1 A2/Cw4. A DNA fragment en-coding part of the HLA-A*0201 extracellular domain and the HLA-Cw*0401 cytoplasmic tail was isolated by digesting pcDNA3.1 A2/Cw4with PmlI and EcoRV. This fragment was then ligated into MSCV2.1HA-HLA-A*0201 digested with PmlI and HpaI.

pcDNA3.1(�) internal ribosome entry site (IRES) GFP was generatedby isolating the IRES GFP cassette from MSCV IRES GFP (14) digestedwith XhoI and SalI. This cassette was then ligated into pcDNA3.1(�) digestedwith XhoI. pcDNA3.1(�) HA-HLA-A*0201 IRES GFP and pcDNA3.1(�)HA-HLA-Cw*0401 were generated by isolating HA-HLA-A*0201 or HA-HLA-Cw*0401 from MSCV 2.1 HA-HLA-A*0201 or MSCV2.1 HA-HLA-Cw*0401 as follows: MSCV 2.1 HA-HLA-A*0201 or MSCV 2.1 HA-HLA-Cw*0401 were digested with EcoRI, filled in using Klenow, and then digestedwith XhoI. These fragments were then ligated into pcDNA3.1(�) IRES GFPdigested with EcoRV and XhoI.

The MSCV 2.1 HA-A2/Cw4 point mutations N327D, E334V, I337T,D333A, SSAA, S335E, and MSCV 2.1 HA-A*0201 T337I were introducedusing PCR. The 5� primer for all the constructs was 5�-CGACCGCCTCGATCCTCC-3�. The 3� primers used are as follows: N327D 5�-CCGCTCGAGTCAGGCTTACAAGCGATGAGAGACTCATCAGAGCCCTGGGCACTGTCGCTGGACGC-3�, E334V 5�-CCGCTCGAGTCAGGCTTTACAAGCGATGAGAGATACATCAGAGCCCTG-3�, I337T 5�-CGGCTCGAGCTGTCAGGCTTTACAAGCTGTGAGAGACTC-3�, D333A 5�-GCCCTCGAGTCAGGCTTTACAAGCGATGAGAGACTCTGCAGAGCCCTGGGCACTGTTGCTGGA-3�, SSAA 5�-CCGCTCGAGCGGTCAGGCTTTACAAGCGATGAGTGCCTCATCTGCGCC-3� and S335E 5�-CCGCTCGAGCGGTCAGGCTTTACAAGCGATGAGCTCCTCATCAGA-3�. The templatefor all the PCR was MSCV 2.1 HA-A2/Cw4. The resulting PCR product wasdigested with EcoRI and XhoI and ligated into MSCV 2.1 digested with thesame enzymes. The C320Y point mutation was generated using a two-roundPCR mutagenesis approach. The first-round PCR consisted of two reactions;reaction 1 contained the primers 5�-CGACCGCCTCGATCCTCC-3� and 5�-AGCCTGAGAGTAGCTCCCTCC-3�, and reaction 2 contained the primers5�-GGAGGGAGCTACTCTCAGGCT-3� and 5�-CCGCTCGAGTCAGGGTTTACAAGCGATGAGAGA-3�. The template for both reactions wasMSCV 2.1 HA-A2/Cw4. The second-round PCR contained primers 5�-CGACCGCCTCGATCCTCC-3� and 5�- CCGCTCGAGTCAGGGTTTACAAGCGATGAGAGA-3�. The template for this reaction was 1 �l from each of the

one-round PCR. The resulting PCR product was digested with EcoRI and XhoIand ligated into MSCV 2.1 digested with the same enzymes.

Cell lines

THP-1 and U937 macrophage cell lines were obtained from the AmericanType Culture Collection. THP-1 cells were cultured in RPMI 1640 sup-plemented with 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyru-vate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, 10% FBS, 0.05 mM2-ME, and 2 mM penicillin, streptomycin, and glutamine. U937 and CEMcells were cultured with RPMI 1640 supplemented with 10% FBS, 10 mMHEPES, and 2 mM penicillin, streptomycin, and glutamine (R10). Celllines expressing various MHC-I molecules were generated using murineretroviral vectors as previously described (14, 15) except that they werepseudotyped with pCMV VSV-G (Dr. Nancy Hopkins, Massachusetts In-stitute of Technology). Cells (1 � 106) were spin infected with the retro-viral supernatants by centrifuging at 2500 rpm in a table top centrifuge for2 h with 8 �g/ml polybrene. The cells were then selected with neomycin(1 mg/ml).

PBMC isolation and electroporation

PBMCs were isolated from buffy coats provided by the Lansing Red Crossby Ficoll-Hypaque centrifugation. Following isolation they were stimu-lated with 10 �g/ml PHA (Sigma-Aldrich); 24 h later IL-2 was added at 50U/ml, and fresh IL-2 was added after 3 days. Five days after isolation, 5 �106 stimulated PBMCs were electroporated using the Amaxa Nucleofectorsystem. Electroporations were performed according to the manufacturer’sprotocol, except following electroporation, the cells were placed in 500 �lof medium in 1.5 ml Eppendorf tubes and incubated for 10 min at 37°Cbefore being placed in a 12-well dish.

Macrophage differentiation

Buffy coats provided by the New York Blood Center were purified byFicoll-Hypaque centrifugation, and CD14� mononuclear cells were iso-lated using the EasySep human CD14-positive selection kit (StemCellTechnologies). Purity of the sorted CD14-positive cells was assessed byflow cytometry using FITC-conjugated mouse anti-human CD14 Ab (cloneM5E2; BD Pharmingen). To assess MHC-I expression levels, freshly pu-rified, undifferentiated cells were preincubated with 10% Fc block (Accu-rate Chemical & Scientific) in FACS buffer (10 mM HEPES, 2% FBS, 1%human serum, 0.02% azide) for 20 min on ice and then stained with Absdirected against HLA-A2 (BB7.2), HLA-C (L31; gift of Patrizio Giaco-mini, Regina Elena Cancer Institute, Italy), Bw4 (One Lambda), and Bw6(One Lambda), depending on the donors MHC-I phenotype. The cells werealso stained with matched, isotype control Abs (protein A purified IgG2bfor BB7.2, IgG1 ascites for L31, and IgM for anti-Bw4 and anti-Bw6). Forstaining with L31, a citrate-phosphate buffer (pH 3.0) was used to release�2-microglobulin and expose the epitope as described previously (16, 17).To induce maturation, the CD14� cells were plated at 1 � 106/ml in R10plus GM-CSF for 5 days. The cells were then harvested and stained againwith anti-MHC-I Abs as described above.

For differentiation of monocytic cell lines, one million THP-1 or U937cells were treated with LPS (100 ng/ml for THP-1 and 10 ng/ml for U937)solubilized in DMSO in 1 ml of medium in a 24-well plate. Twenty fourhours later, an additional 1 ml of medium was added containing PMA (200ng/ml for THP-1 and 10 ng/ml for U937) and LPS. After 72 h at 37°C, cellswere harvested by treatment with cell dissociation solution (Sigma-Al-drich) for 20 min at 37°C.

Western blot analysis

Cells were lysed in PBS, 0.3% CHAPS, 0.1% SDS (pH 8), and 1 mMPMSF. They were then normalized for total protein and separated by SDS-PAGE. Western blot analysis was performed with the following Abs: HA(HA.11, 1:5,000; Covance Research Products), and rat anti-mouse-HRP(1:25,000; Zymed Laboratories).

Immunofluorescence microscopy

CEM T cells were prepared for immunofluorescence microscopy as pre-viously described (12) except that they were permeabilized with 0.1% dig-itonin (Wako Chemicals) diluted in Dulbecco’s PBS with calcium andmagnesium and blocked with equal parts wash buffer and Fc receptorblocker (Accurate Chemical & Scientific). To identify cell surface staining(Fig. 1G), ConA conjugated to AlexaFluor 488 (Molecular Probes) wasdiluted to 40 �g/ml and incubated with cells for 5 min on ice. To identifyacidic compartments (Fig. 4C), CEM cells were pretreated with 100 nmbafilomycin A or DMSO for 4 h at 37°C. Following treatment, CEM cellswere adhered to glass slides, fixed, permeabilized, and stained for indirect

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FIGURE 1. The extracellular and cytoplasmic tail domains of HLA-C influence surface expression. A, The cytoplasmic tail sequences of HLA-A,HLA-B, and HLA-C molecules. Amino acid differences unique to HLA-C are boxed. B, Schematic diagram of HA-tagged MHC-I molecules. Openboxes represent HLA-A*0201 sequences, black boxes represent HLA-Cw*0401 sequences, the gray box represents the HA tag, and L indicates theposition of the leader sequence. C, Expression of HA-tagged chimeric MHC-I molecules. CEM T cells (control) or CEM T cells expressing theindicated MHC-I were pulse-labeled with [35S]methionine and cysteine for 15 min. Lysates from these samples were immunoprecipitated with anAb against HA, and separated by SDS-PAGE. D, Quantitation of pulse-labeling. A phosphor imager and ImageQuant software were used to quantifysamples. Values were corrected for differences in methionine and cysteine content. The counts from the HLA-A*0201 sample were set to 100% andthe values from other samples were expressed relative to it. The results are displayed as the means � SD from three experiments. E, Cell surfaceexpression of HA-A2, HA-Cw4, HA-A2/C, and HA-Cw4/A. CEM T cell lines were generated expressing the indicated MHC-I molecules. The cellswere stained with an Ab directed against the HA tag and analyzed by flow cytometry. The filled gray curve represents the expression of the indicatedmolecules while the filled black curve represents control cells stained with the HA Ab. The MFI � SD from six experiments is shown in the upperright hand corner. F, Cell surface expression of untagged HLA-A2 and A2/C in CEM T cells. The CEM T cell line expressing un-tagged HLA-A2has been previously described (12, 39, 40). Untagged MSCV A2/C was generated from pcDNA3.1 A2/Cw4 (see Materials and Methods) and wasexpressed in CEM cell lines as described in Materials and Methods. The cell surface expression of the indicated untagged MHC-I was assessed byflow cytometry using the HLA-A2-specific Ab, BB7.2 as described in Materials and Methods. The lightly shaded curve represents parental CEMcell lines. The darkly shaded curve represents CEM cells expressing untagged HLA-A2 and the unshaded curve represents CEM cell expressinguntagged A2/C. G. Subcellular distribution of MHC-I molecules. The indicated CEM T cell lines were incubated with ConA to label the cell surface(green). They were then fixed, permeabilized, and stained with HA to examine MHC-I localization (red). Merge panels represent the product of thesetwo overlapping images in which regions of overlap are highlighted in yellow. All images were collected on a Zeiss LSM 510 confocal microscopeand single z-sections are displayed. H, Quantification of ConA and HA colocalization. Quantification was determined as described in Materials andMethods. Quantification is shown for four cells expressing HA-A2 and 4 cells expressing HA-A2/C. I, Cell surface expression of HA-A2 andHA-Cw4 in primary T cells. PBMCs were electroporated with a bicistronic GFP-expressing construct as described in Materials and Methods. Twentyfour hours post electroporation PBMCs were stained using an Ab directed against HA and analyzed by flow cytometry. Cells with equal GFP expression wereselected and their GFP expression is shown in the top panels. The corresponding HA stain is shown in the panels below. In the bottom panels, the filled curveis the background staining on mock-transfected cells and the black line represents HA staining for cells transfected with the indicated construct. As a control, stableCEM T cells expressing the same molecules were also stained in parallel and are shown in the panels on the right.

7806 LYSOSOMAL TARGETING SIGNAL IN HLA-C

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immunofluorescence as previously described (12). MHC-I was visualizedusing BB7.2 (1:100), and AlexaFluor 488 goat anti-mouse IgG2b (1:250;Invitrogen). LAMP-1 was visualized using a anti-LAMP-1 Ab (cloneH4A3, 1:500; BD Pharmingen) and AlexaFluor 546 goat anti-mouse IgG1(1:250; Invitrogen). For identification of the ER (Fig. 2C), HA-tagged mol-ecules and KDEL were stained using mouse mAbs (HA.11, 1:50 and anti-KDEL, 1:200 (Stressgen)) followed by staining with appropriate secondaryAbs (goat anti-mouse IgG1 AlexaFluor 488, and goat anti-mouse IgG2aAlexaFluor 546, 1:250; Molecular Probes). Images were collected using aZeiss LSM 510 confocal microscope and processed with Adobe Photoshopsoftware.

Microscopy quantification

Microscopy images were quantified independently by two individuals whoassessed the relative amount of colocalization and assigned a colocalizationscore between 0 and 3, where 3 was the maximal possible colocalization.For each condition, the samples were averaged and the percentage of max-imum colocalization was determined by dividing each score by the highestscore achieved.

Flow cytometry

Stains were performed as previously described (18) using an anti-HA Ab(HA.11, 1:50) or an anti-HLA-A2 Ab (BB7.2) (19) and goat-anti-mouse-PE (1:250; Biosource or Invitrogen). FACS analysis of the THP-1

and U937 cells was the same except the cells were incubated with Fcreceptor blocker (Accurate Chemical & Scientific) for 20 min at 4°C beforethe anti-HA Ab incubation.

Transport, internalization, and metabolic labeling assays

The transport and endocytosis assays were performed essentially as pre-viously described (20) except that an Ab directed against the HA tag(HA.11) was used. For metabolic labeling of total protein, fifteen millionCEM T cells were pulse-labeled for 15 min with [35S]methionine and cys-teine. For inhibitor studies, one third of the sample was harvested after thepulse while the remaining cells were then chased for 12 h in either RPMI1640 with DMSO or 100 nM bafilomycin A (Sigma-Aldrich). Lysates weregenerated in PBS, 0.3% CHAPS, 0.1% SDS (pH 8), 1 mM PMSF, andprecleared overnight. They were immunoprecipitated for 2 h with an Abagainst HA (HA.11) and washed three times in radioimmunoprecipitationassay (RIPA) buffer (50 mM Tris (pH 8), 150 mM NaCl, 1% Nonidet P-40,0.5% deoxycholate, 0.1% SDS). The immunoprecipitates were then elutedby boiling in 10% SDS, re-precipitated with an Ab against HA, and washedthree times in RIPA buffer. The final immunoprecipitates were then sepa-rated by SDS-PAGE.

For metabolic labeling of phosphorylated protein, five million CEM Tcells were labeled for 4 h with 0.5 mCi/ml [32P]orthophosphate in phos-phate-free medium (RPMI 1640; Specialty Media) supplemented with 10%dialyzed FBS (Invitrogen). The cells were lysed with Nonidet P-40 lysis

FIGURE 2. The HLA-C tail does not affect transport from the ER into the Golgi apparatus. A and B, CEM T cells expressing the indicated MHC-I werepulse-labeled with [35S]methionine and cysteine for 15 min, chased for the indicated time period and harvested. Lysates were immunoprecipitated with anAb against HA, treated with endo H, and separated by SDS-PAGE. C indicates control CEM cells that do not express the HA tag. B, A phosphor imagerand ImageQuant software were used to quantify the gel shown in part A. The percentage of molecules that were resistant to endo H digestion over timewas calculated as follows: [((endo H resistant)/(endo H resistant � endo H sensitive)) � 100]. C, The HLA-C extracellular domain promotes colocalizationwith markers of the ER. CEM T cells expressing the indicated MHC-I were fixed, permeabilized, and stained for HA to examine MHC-I localization (green),or KDEL (red) to mark the ER. The merge panels depict regions of overlap in yellow. All images were collected on a Zeiss LSM 510 confocal microscopeand single z-sections are displayed. D, Quantification of KDEL and MHC-I colocalization. The relative amount of colocalization between KDEL and BB7.2staining was determined as described in Materials and Methods. Quantification is shown for 6 cells expressing HA-A2, five cells expressing HA-A2/C, fourcells expressing HA-Cw4, and five cells expressing HA-Cw4/A.

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buffer (1% Nonidet P-40, 0.15 M NaCl, 0.01 M sodium phosphate, 2 mMEDTA, 50 mM sodium fluoride, 0.2 mM sodium vanadate, and 1 mMPMSF) and precleared overnight. They were immunoprecipitated withBB7.2 Ab for 2 h and washed three times in RIPA buffer (50 mM Tris (pH8), 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS). Theimmunoprecipitates were then separated by SDS-PAGE and the dried gelwas exposed to a phosphor imager screen and analyzed on a Typhoon TrioPhosphorImager (GE Healthcare). Where indicated, cells were treated with100 nM bafilomycin A or DMSO solvent control for 18–24 h.

Recycling assays

A measurement of the rate of recycling of internalized molecules was per-formed as described previously (21), except that washes were performed atroom temperature to avoid inhibiting recycling with cold temperature (22).Briefly, cells were incubated with 150 �g/ml cycloheximide (Sigma-Aldrich) for 2–3 h in RPMI 1640 plus 10% serum. Then cells were har-vested and an aliquot was removed and placed on ice. The remainder of thesamples was stripped of stainable HLA-A2 by washing in 50 mM glycine,100 mM NaCl (pH 3.4), twice, at room temperature for 1 min. The strippedcells were then washed in PBS and incubated at 37°C, 5% CO2 in mediumwithout serum for the indicated period of time, in the presence of 150�g/ml cycloheximide. (Samples were incubated without serum to avoidsubstitution of bovine �2-microglobulin present in serum for the human�2-microglobulin removed with the stripping protocol.) Cells were thenplaced on ice and stained for HLA-A2 with BB7.2, using wash buffers thatincluded BSA instead of serum.

ResultsHLA-C allotypes are classical MHC-I molecules that play a dualrole. They are able to activate CD8� CTLs via their ability topresent Ags. Additionally, HLA-C allotypes have the capacity tosend inhibitory signals to CD8� T cells bearing inhibitory recep-tors. Perhaps as a result of this unique role, HLA-C molecules areknown to be expressed at much lower levels on the cell surfacethan other classical MHC-I molecules (HLA-A and HLA-B allo-

types). However, is unclear what elements determine these differ-ences in expression. The extracellular domains of MHC-I proteinsare known to be highly polymorphic, whereas the cytoplasmic taildomains are generally highly conserved within allotypes (Fig. 1A).A comparison among the three types of classical MHC-I moleculesrevealed that there are four amino acids unique to the HLA-Ccytoplasmic tail domain (Fig. 1A).

To determine which amino acid differences played a role inreducing HLA-C surface expression, we first developed a way toclearly compare expression of these molecules. This was accom-plished by attaching an HA tag to the N terminus of HLA-A*0201and HLA-Cw4*0401 (HA-A2 and HA-Cw4; Fig. 1B). The HA tag,which was inserted just after the leader cleavage site, allowed us tocompare the expression of heterologous proteins using the sameAb so that differences in Ab affinity did not confound our results.In prior publications, we have demonstrated that the presence ofthis tag does not affect the maturation and expression of HLA-A2(12). Additionally, we have demonstrated that this tag does notaffect recognition by the conformationally sensitive anti-HLA-A2Ab, BB7.2 (12, 19).

We also made chimeric molecules in which the cytoplasmic taildomains of HLA-A or HLA-C were fused to the transmembranedomain of HA-Cw4 and HA-A2 to create HA-Cw4/A and HA-A2/C, respectively (Fig. 1B). DNAs encoding each of these pro-teins were cloned into murine retroviral vectors, and viral super-natants were used to transduce CEM T cells at a low multiplicityof infection to limit the number of transductants with multipleintegrated copies. Bulk cell lines were then grown in selectivemedium to obtain a uniform population. To ensure that our resultswere not influenced by arbitrary variations introduced by individual

FIGURE 3. The HLA-C tail disrupts protein transport and accelerates internalization from the cell surface. A and B, CEM T cells expressing the indicatedMHC-I were pulse-labeled with [35S]methionine and cysteine. They were then chased for 1 or 4 h in biotin. Lysates from these samples were immunoprecipitatedwith an Ab against HA and one third of the immunoprecipitate was analyzed by SDS-PAGE (total). The remaining two thirds was reprecipitated with avidin-agarose and then analyzed by SDS-PAGE (surface). NC indicates an immunoprecipitation of control CEM cells that do not express the HA tag. B, Quantitationof cell surface transport. A phosphor imager and ImageQuant software were used to quantify recovered protein. The percentage of MHC-I molecules that hadreached the cell surface at the indicated time point was calculated as follows: [((surface MHC-I)/(total MHC-I*2)) � 100]. The mean from two experiments �SD is shown. C, A2/C is internalized at an accelerated rate. CEM T cells stably expressing the indicated protein were incubated on ice with the anti-HLA-A2 Ab,BB7.2. The cells were shifted to 37°C for the indicated time and then stained with a secondary Ab to determine the percentage of HA-A2 (black square) orHA-A2/C (gray square) remaining on the cell surface over time. The mean � SD for an experiment performed in quadruplicate is shown. D, Internalization ofCD4 by the HIV Nef protein. CEM T cells were transduced with adenoviral vectors as previously described (40, 41) and the flow cytometric internalization assaywas used to measure endocytosis as described for HLA-A2 and A2/C. Briefly, CEM T cells were incubated on ice with the anti-CD4 Ab, OKT4. The cells wereshifted to 37°C for the indicated time and then stained with a secondary Ab goat �-mouse-PE (1:250; Invitrogen). The cells were analyzed by flow cytometry andthe percentage of CD4 remaining on the surface of control cells (black square) or HIV-1 Nef expressing cells (gray square) was calculated. The mean � SD foran experiment performed in duplicate is shown. E, The cytoplasmic tail of HLA-C does not inhibit recycling. The rates of HA-A2/C, HA-A2/C I337T, and HA-A2recycling in CEM T cells were measured as described previously (20) and as outlined in Materials and Methods.

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transfections or transductions, the cell lines used in our investigationswere re-made with new transfections and transductions on three sep-arate occasions and in each case yielded similar relative expressionlevels.

Because, the translation initiation and leader sequences were thesame for each molecule, we were able to measure initial proteinsynthesis as an estimate of the amount of translatable RNA in thecell. As shown in Fig. 1C (and quantified in Fig. 1D), the expres-sion level of HA-A2 and HA-A2/C was not significantly different( p � 0.12). Despite this, we found that there was �3-fold lessHA-A2/C on the cell surface (mean fluorescence intensity(MFI) � 50 � 4 for HA-A2/C, compared with 149 � 18 forHA-A2) (Fig. 1E). This was not an artifact of the presence of theHA tag, as independently constructed and expressed HLA-A2 andA2/C that lacked the tag behaved similarly when stained with the

HLA-A2-specific mAb, BB7.2 (Fig. 1F). In addition, these datawere corroborated by confocal microscopy (Fig. 1, G and H),which confirmed that HA-A2 was largely expressed on the cellsurface, where it colocalized significantly with ConA. In compar-ison, HA-A2/C had a staining pattern that was distinctly differentrelative to that of ConA (Fig. 1G, compare panels 3 and 7, andquantification shown in Fig. 1H).

We also found that molecules with an HLA-C extracellular do-main were expressed poorly on the cell surface relative to thosecontaining HLA-A extracellular domains (MFI � 12 � 3 for HA-Cw4 and 20 � 2 for HA-Cw4/A2) (Fig. 1E). Given that we noteda slightly lower level of initial protein synthesis for these mole-cules relative to HLA-A2 (Fig. 1, C and D), we also verified theseresults in a different system that could better account for potentialdifferences in gene copy number. In this case, HA-A2 and HA-Cw4

FIGURE 4. The HLA-C cytoplasmic tail promotes lysosomal targeting. A and B, CEM T cells expressing the indicated MHC-I molecule were pulse-labeled with [35S]methionine and cysteine for 15 min, and chased for the indicated time. Lysates from these samples were immunoprecipitated with ananti-HA Ab as in Fig. 2A. C results from the parental CEM T cell line that does not express the HA tag. B, Quantitation of pulse-labeling. A phosphor imagerand ImageQuant software was used to determine the amount of MHC-I at each time point. The amount at time 0 was set to 100% and the percentageremaining was then calculated for each time point. The mean � SD from two experiments is shown. C, HA-A2/C is directed to acidic compartments. CEMT cells expressing the indicated MHC-I were treated for 4 h with 100 nM bafilomycin or DMSO and stained with an Ab directed against HLA-A2 andLAMP-1, a marker of lysosomal compartments. The images were collected using a Zeiss 510 LSM and processed using Adobe Photoshop software. Singlez-sections are displayed. D, Quantification of LAMP-1 and MHC-I colocalization in bafilomycin-treated cells. The degree of colocalization of LAMP-1 andBB7.2 staining was determined as described in Materials and Methods. Quantification is shown for 28 cells expressing HA-A2 and 30 cells expressingHA-A2/C. E and F, Bafilomycin stabilizes the degradation of HA-A2/C. Pulse-chase analysis was performed as in A except that the cells were chased ineither 100 nM bafilomycin A or solvent (DMSO). F, A phosphor imager and ImageQuant software was used to determine the percentage of MHC-Iremaining [(value following chase/initial value) � 100]. The mean � SD is shown for three experiments.

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were cloned into a different vector, which allowed expression of bothMHC-I and GFP from the same RNA molecule via an IRES element.Thus, this vector system allowed us to monitor the relative number ofgene copies present in the cell by measuring GFP expression byFACS. These constructs were directly transfected into activated pri-mary T cells and the surface expression levels of HA-A2 and HA-Cw4 in primary T cells expressing equivalent amounts of GFP wasmeasured and compared with what was observed in CEM T cells. Asshown in Fig. 1I, at similar GFP expression levels, the cell surfaceexpression of HA-A2 and HA-Cw4 was comparable to that achievedin stable CEM T cell lines stained in parallel (Fig. 1I, right panels).Moreover, as shown in Fig. 1I, the pattern we observed was notchanged by adding twice as much DNA to the transfection, indicatingthat, the surface expression of HA-Cw4 and HA-A2 was not substan-tially affected by differences in transfection conditions.

The HLA-C cytoplasmic tail contains an internalization andlysosomal trafficking signal

To determine how the cytoplasmic tail affected cell surface ex-pression, we used pulse-chase labeling with endoglycosidase H(endo H) digestion to measure the rate at which molecules har-boring this domain were transported into the Golgi apparatuswhere they become modified such that they were resistant to endoH digestion. We observed that the cytoplasmic tail did not influ-ence the rate molecules acquired resistance to digestion by endo H(Fig. 2, A and B). In contrast, we confirmed prior indications thatthe extracellular domain of HLA-C promoted ER retention and

degradation (Fig. 2A). These data were confirmed by confocal mi-croscopy in which molecules containing the HLA-C extracellulardomain colocalized with KDEL, a marker of the ER compartment(Fig. 2, C and D).

We then used pulse-labeling followed by a chase period in thepresence of a cell impermeable biotinylation reagent, to determinethe rate at which these molecules arrived at the cell surface. Inthese assays, the cell lysates were first immunoprecipitated withanti-HA. Then, one third of the cell lysates was analyzed directly(total), and the remaining two thirds was eluted from the beads andre-precipitated with avidin-agarose to isolate the subset of MHC-Iat the cell surface (Fig. 3, A and B). As determined by phosphorimager analysis, we found that HA-A2/C was transported to thecell surface �2-fold more slowly than HA-A2 ( p � 0.01, n � 2).

We then compared the internalization rate of HA-A2 and HA-A2/C using a flow cytometric assay in which cells were incubatedon ice with a primary Ab directed against an HLA-A2 specificepitope (BB7.2). The cells were then shifted to 37°C for the indi-cated time period after which they were labeled with a secondaryAb. Flow cytometry was then used to quantify the amount of Agremaining on the cell surface (20). As shown, we observed that,HA-A2/C was internalized twice as fast as HA-A2 ( p � 0.01, n �3, Fig. 3C). The magnitude of the rate increase was somewhat lessthan what we observed with the well-documented internalizationof CD4 induced by the HIV Nef protein (23, 24), in which Nefaccelerates internalization �3- to 6-fold (Fig. 3D).

FIGURE 5. Isoleucine at position 337 (I337) isrequired for accelerated internalization and lysoso-mal targeting. A and B, Expression of A2/C cyto-plasmic tail point mutations. CEM T cells (negativecontrol) or CEM T cells expressing the indicatedMHC-I were pulse-labeled as described in Fig. 1C,and analyzed by SDS-PAGE. Bands were quantifiedusing a phosphor imager and ImageQuant software.The results are displayed in B as the means � SDfrom three experiments. C and D, I337 is necessaryfor reduced steady-state cell surface expression ofHA-A2/C. CEM T cell lines were generated ex-pressing the indicated MHC-I. C, CEM T cells ex-pressing the indicated mutant were stained with anAb directed against the HA tag and analyzed by flowcytometry. The filled gray curve represents the ex-pression of the indicated molecule while the filledblack curve represents untagged cells stained withthe HA Ab. D, Quantitation of the HA-A2/C cyto-plasmic tail mutant surface expression. The resultsare depicted as MFI � SD from three experiments.E, I337 is not required for slow export of HA-A2/C.A transport assay was performed as in Fig. 3A, ex-cept cells were chased in biotin for only 1 h. Thetransport assay was quantified as in Fig. 3B, exceptthat percentage of HA-A2 transported to the cellsurface was set to 100%. The mean � SD is shownfor two experiments. F, I337 is necessary for accel-erated internalization of HA-A2/C. A flow cytomet-ric internalization assay was used as in Fig. 3C todetermine the percentage of each molecule remain-ing on the cell surface over time. The mean � SDfor an experiment performed in triplicate is shown.G and H, I337 is necessary for lysosomal targetingof HA-A2/C. A pulse chase was performed as inFig. 4E and was quantified as in Fig. 4F. Themean � SD is shown for two experiments.

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We also examined whether HLA-A2 and A2/C differed withrespect to how rapidly they were recycled to the cell surface fol-lowing internalization. As shown in Fig. 3E, we did not detect anysignificant differences in recycling of HA-A2/C relative toHLA-A2 that would explain its lower expression levels. In fact, aslightly higher percentage of HA-A2/C was recycled to the cellsurface from internal compartments ( p � 0.036, n � 3).

We then examined the ultimate fate of HA-A2/C with longerpulse-chase experiments to determine whether it was targeted tolysosomal compartments after internalization. As shown in Fig. 4Aand quantified in Fig. 4B, we found that mature, endo H resistantHA-A2/C was degraded approximately twice as fast as HA-A2.(HA-A2 had a half-life of 10.5 h, compared with 6 h for HA-A2/C). These data were confirmed by confocal microscopy in whichwe noted that HA-A2/C displayed extensive colocalization withLAMP-1, a marker of lysosomal organelles, when degradation wasinhibited by bafilomycin, an inhibitor of the vacuolar ATPase thatis required for efficient acidification and degradation in lysosomalcompartments (Fig. 4C and quantified in Fig. 4D).

Finally, to provide further evidence that the degradation of HA-A2/C occurred in acidic compartments, such as lysosomes, wetreated CEM T cells with bafilomycin to determine whether it re-versed the degradation observed by pulse-chase analysis. Asshown in Fig. 4E and quantified in Fig. 4F, bafilomycin treatmentresulted in a 6-fold increase of HA-A2/C compared with a 2.2-foldincrease for HA-A2 ( p � 0.015, n � 3). In sum, these data suggestthat the HLA-C cytoplasmic tail contains an internalization andlysosomal targeting signal.

Identification of a trafficking signal in the HLA-C cytoplasmictail that promotes intracellular localization and lysosomaltargeting

To determine which amino acids were responsible for the effects ofthe HLA-C tail, we focused on four amino acid differences be-tween HLA-C and HLA-A/B molecules (Fig. 1A). Each of these

amino acids was mutated in HA-A2/C, and stable CEM T cell lineswere made as described above for HA-A2/C. Initial protein syn-thesis measurements indicated that the expression of each of thesemolecules was not significantly different from that of HA-A2/C,except for HA-A2/C D333A, which was expressed slightly less( p � 0.04, Fig. 5, A and B).

We then examined surface expression via flow cytometric anal-ysis on cells stained with an anti-HA Ab. As shown in Fig. 5, Cand D, only one of the amino acids substitutions reversed the effectof the HLA-C tail. Specifically, changing isoleucine at position337 in the HLA-C tail to the threonine found in HLA-A and B tails(I337T) increased surface expression by 3-fold compared withHA-A2/C ( p � 0.001, n � 3). Conversely, we found that thereciprocal mutation in the HLA-A cytoplasmic tail (A2 T337I),reduced surface expression of HA-A2 � 3-fold ( p � 0.0001, n �3, Fig. 5, C and D).

An analysis of intracellular transport, using the assay describedabove, revealed that HA-A2/C I337T transport was reduced com-pared with wild type HA-A2 ( p � 0.001, n � 3), but was notsignificantly different from A2/C (Fig. 5E). Whereas, the flow cy-tometric internalization assay (described above) revealed that sub-stitution of I337 reduced the internalization rate 15-fold (from3.73% per minute to 0.25% per minute p � 0.01, Fig. 5F).

Finally, we used pulse-chase analysis plus or minus bafilomycinto measure the degree to which wild type and mutant moleculeswere degraded in acidic compartments. As shown in Fig. 5G andquantified in Fig. 5H, substitution of I337 in HA-A2/C increasedthe amount of recovered protein 4-fold in the control, DMSOtreated, sample (compare lanes 5 and 8 in Fig. 5G, and quantifi-cation in Fig. 5H, p � 0.01), resulting in expression that wassimilar to that of HA-A2 (compare lanes 3 and 8 in Fig. 5E). Thus,I337 is a determinant required for accelerated internalization anddegradation of molecules containing an HLA-C cytoplasmic taildomain.

FIGURE 6. Aspartic acid at position 333 (D333) in the HLA-C cytoplasmic tail attenuates the internalization and lysosomal targeting signal. A, D333 is notneeded for reduced HA-A2/C cell surface transport rate. CEM T cells expressing the indicated protein were used for a transport assay performed as described inFig. 3A and quantified as in Fig. 3B. B, Substitution of D333 accelerates internalization. The rate of internalization was determined using CEM T cells stablyexpressing the indicated mutant. A flow cytometric internalization assay was used as in Fig. 3C to determine the percentage of MHC-I remaining of the cell surfaceover time. The mean � SD for an experiment performed in triplicate is shown. C and D, Substitution of D333 enhances lysosomal targeting. To assess thedegradation rate of each construct a pulse chase was performed using CEM T cells expressing the indicated mutant as described in Fig. 2.

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To further define the internalization and lysosomal targetingmotif in HLA-C, the highly conserved aspartic acid (D) at position333 was changed to an alanine. Based on initial protein synthesis,this mutant was expressed slightly less than HA-A2/C (Fig. 5, Aand B, p � 0.04), but its expression was not significantly differentfrom HLA-A2 and most A2/C mutants (C320Y, N327D, I337T,T337I; Fig. 5, A and B). Substitution of D333 resulted in a sub-stantial loss of cell surface expression compared with HA-A2/C,HA-A2 and the other HA-2/C mutants (Fig. 5, C and D, p valuesranged from �1 � 10�4–10�6). The intracellular transport ratewas not the explanation for the reduction in surface expression asthis was similar to HA-A2/C (Fig. 6A). However, we found thatsubstitution of D333 resulted in a 2-fold increase in internalization

rate compared with HA-A2/C ( p � 1 � 10�4) and a 5-fold in-crease relative to wild type HA-A2 ( p � 1 � 10�4, Fig. 6B).Additionally, pulse-chase analysis revealed that mutation of D333also caused an increase in turnover of mature, endo H-resistantmolecules (Fig. 6, C and D). In sum, these data indicate that D333functions to attenuate the downstream dihydrophobic internaliza-tion and lysosomal targeting signal.

Evidence for regulated cell surface expression of HLA-C inAPCs

The data we have acquired indicates that there are multiple mech-anisms by which cells precisely regulate HLA-C expression. Theextracellular domain promotes retention and degradation in the ER

FIGURE 7. Macrophage differentiation up-regulates HLA-C and HA-A2/C. A, Up-regulation of HLA-C upon differentiation of primary CD14� cellsinto macrophages. CD14� mononuclear cells were isolated from the blood of a normal donor. A subset of the cells was stained immediately (untreated)with anti-HLA-C (L31), an Ab against a subset of HLA-B allotypes (HLA-Bw6) or isotype control Abs. The remainder of the cells were cultured for 5days in GM-CSF, harvested and stained with the same Abs. B, Up-regulation of HA-Cw4 and HA-A2/C with differentiation of monocytic cells lines intomacrophage-like cells. U937 (left panels) and THP-1 (right panels) cell lines were derived as described in Materials and Methods. Cell surface expressionwas determined by flow cytometry using an Ab to HA for undifferentiated (DMSO) and differentiated (LPS/PMA) cells. C, Macrophage differentiationstabilizes the endo H-resistant form of HA-A2/C and HA-Cw4. U937 and THP-1 cell lines were treated with LPS and PMA as in B. Lysates were generated,digested with endo H, and analyzed by Western blot using an Ab directed at HA. R, Endo H-resistant band; S, endo H-sensitive band. D, Macrophagedifferentiation inhibits internalization of HA-A2/C. The flow cytometric internalization assay described in Fig. 3C was used to determine the percentageof HA-A2 or HA-A2/C remaining on the cell surface over time in undifferentiated (DMSO) or differentiated (LPS/PMA) U937 cell lines. For theseexperiments an HLA-A2-specific mAb (BB7.2) was used. The mean � SD for an experiment performed in quadruplicate is shown.

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and the cytoplasmic domain promotes a limited amount of inter-nalization and degradation. Thus, it seems that complex mecha-nisms exist to maintain significant intracellular levels of HLA-Cwhile limiting (but not eliminating) surface expression. It makessense that a molecule capable of sending a dominant inhibitorysignal to killer T cells should be tightly regulated, as high expres-sion could result in higher activation thresholds for the detection ofvirally infected or tumorigenic cells. However, the fact that intra-cellular pools of HLA-C are maintained suggests that there may beconditions in which it is advantageous to rapidly up-regulateHLA-C cell surface expression. We were unable to specificallyinduce HLA-C surface expression in T cells with a variety of stim-uli, such as IFNs � and � or with compounds that stimulate T cells,such as IL-2 and PHA. Additionally, we were unable to up-regu-late HLA-C expression by treatment of T cells with specific patho-gens, such as HIV and adenovirus (data not shown).

It remained possible, however, that HLA-C had evolved topresent peptides from certain types of pathogens, or that it func-tioned to inhibit killing under some conditions. For example, whenAPCs activate naive CTLs by cross-presenting exogenous Ag, itwould not be advantageous to lyse the uninfected, cross-presentingAPC. Thus it would make sense for these cells to up-regulate mol-ecules that would send inhibitory signals to effector T cells thatmight otherwise mistake the APC for an infected target cell andlyse it. Indeed, it has recently been reported that coincident withacquisition of killer T cell effector functions, CTLs up-regulate theKIR family of inhibitory receptors (5).

To examine this possibility further, we isolated primary humanCD14� mononuclear cells from a normal, healthy donor’s periph-eral blood. Some of the cells were stained immediately for HLA-Cand HLA-B allotypes and the remainder was incubated in GM-CSF for five days to induce macrophage differentiation. To mea-sure HLA-C surface expression, we obtained an Ab that specifi-cally recognizes most HLA-C allotypes, (17, 25). As shown in Fig.7A, left panel, HLA-C staining, as measured with the L31 Ab, waslow in freshly isolated, undifferentiated CD14� (�2.8-fold abovebackground and more than 40-fold less than Bw6). After 5 days ofculture in GM-CSF, HLA-C surface expression was dramaticallyup-regulated more than 20-fold relative to Bw6.

The data described above confirms that HLA-C expression isnormally much lower than that of HLA-B allotypes in undifferen-tiated primary monocytes using Abs that recognize naturalepitopes. However, this approach does not allow the determinationof which domains of HLA-C are responsible for regulated expres-sion in differentiated macrophages. To further investigate this ob-servation in a more well defined system, we expressed HA-A2,HA-Cw4 and HA-A2/C in cell lines (THP-1 and U937) that werecapable of differentiating into macrophage-like cells by the addition ofLPS and PMA. For comparison, we also expressed HA-taggedB*4405 and B*4402 in these cells. These two HLA-B moleculesdiffer by only a single amino acid, but are known to vary substantiallyin terms of peptide loading and rates of ER egress (26).

In undifferentiated nonadherent solvent (DMSO)-treated cells,HA-A2, HA-A2/C and HA-Cw4 were expressed in a manner thatwas very similar to what we observed in primary T cells and stableCEM T cell lines (Fig. 7B, panels 1 and 3). HA-A2 was expressedat comparably high levels and was largely endo H resistant (Fig.7C, lanes 2 and 6), whereas HA-Cw4 was expressed at very lowlevels at the cell surface (Fig. 7B, panels 1 and 3) and was largelyendo H sensitive (Fig. 7C, lanes 18 and 22). HA-A2/C was ex-pressed at intermediate levels (Fig. 7B, panels 1 and 3) and hadreduced amounts of endo H-resistant material relative to HLA-A2(Fig. 7C, lane 10 and 14), presumably due to increased internal-ization and degradation in acidic compartments as was observed in

other cell types (Figs. 3 and 4). B*4405 was expressed well on thecell surface (Fig. 7B, panels 5 and 7) and was largely endo Hresistant (Fig. 7C, lanes 34 and 38), whereas B*4402 was ex-pressed comparatively less well on the cell surface (Fig. 7B, panels5 and 7) and was largely endo H sensitive due to ER retention (Fig.7C, lanes 26 and 30; see also Ref. 26).

When U937 and THP-1 cell lines were treated with LPS andPMA to induce differentiation into macrophage-like cells, we ob-served no change in the surface expression of HA-A2 or HA-B*4402, and we noted a small decrease in the surface expressionof HA-B*4405 (Fig. 7B). In contrast, we observed that A2/C cellsurface expression was increased to achieve levels that were sim-ilar to wild-type HA-A2 (Fig. 7B, compare panels 1 and 2 or 3 and4). Additionally, as shown in Fig. 7A, we also observed an increasein full-length HA-Cw4 cell surface expression.

The increase in surface expression of A2/C was reflected by anincrease in the amount of endo H-resistant protein detected byWestern blot analysis (Fig. 7C, compare lanes 10 and 12 for U937cells or lanes 14 and 16 for THP-1 cells). We also noted an in-crease in the ratio of endo H resistant: sensitive forms of full lengthHLA-Cw*0401 (Fig. 7C, compare lanes 18 and 20 for U937 orlanes 22 and 24 for THP-1). Albeit, most of the full-length HLA-Cmolecules remained endo H sensitive.

The relative amount of endo H-resistant material for HA-A2 andHA-B*4405 remained unchanged. However, we did note an in-crease in the fraction of HA-B*4402 that became resistant to endoH (Fig. 7C, compare lanes 26 and 28 or 30 and 32). Thus, mac-rophage differentiation resulted in a complex set of effects thatenhanced ER exit of some MHC-I molecules, like B*4402, that arenormally retained in the ER because of problems with protein

FIGURE 8. Serines 332/335 in the HLA-C cytoplasmic tail are phos-phorylated. CEM T cells (A) or U937 cells (B) expressing the indicatedMHC-I were treated with 100 nM bafilomycin or DMSO overnight andmetabolically labeled with [32P]orthophosphate as described in Materialsand Methods. MHC-I molecules were immunoprecipitated from lysateswith an Ab directed against HLA-A2 (BB7.2) and separated by SDS-PAGE. In the bottom panel, Western blots of lysates from cells grown inparallel are shown. C, Treatment of U937 cells with LPS and PMA toinduce differentiation into macrophage-like cells results in a reduction ofphosphorylation and a stabilization of A2/C protein. U937 cells weretreated with DMSO or PMA and LPS as described in Fig. 7. Cells werelabeled with [32P]orthophosphate and immunoprecipitated as describedabove. In the bottom panel, Western blots of lysates from cells grown inparallel are shown. “Control” indicates results obtained with the parentalcell line that lacked expression of HLA-A2.

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loading (26). In addition, macrophage differentiation increased theamount of endo H-resistant HA-A2/C, which is normally low be-cause of lysosomal targeting of mature molecules.

To better understand the striking up-regulation of surface HA-A2/C and the stabilization of mature, endo H-resistant forms ofA2/C in differentiated macrophage-like cells, we first examinedinternalization rate. As shown in Fig. 7D, we found that HA-A2/Cwas internalized 3.6 times more rapidly than HA-A2 in DMSOtreated monocytic cells. However, after treatment with LPS andPMA, HA-A2 and HA-A2/C were internalized at very similarrates, suggesting that the activity of the cytoplasmic tail signal wasmodified with differentiation.

Evidence that HLA-C cell surface expression in macrophages isregulated by phosphorylation

The HLA-C internalization and lysosomal targeting signal is sur-rounded by serine residues (SDXSLI) and thus could be regulated by

phosphorylation (27, 28). To examine this, we mutated these serineresidues to alanine to prevent phosphorylation (A2/C SSAA). Theseconstructs were expressed in both CEM cells and in U937 cells asdescribed above. Then, phosphorylation of HA-A2, HA-A2/C and themutant A2/C SSAA was directly assessed by labeling the cells with32P orthophosphate and immunoprecipitating each molecule with theHLA-A2-specific Ab, BB7.2. As shown in Fig. 8, A and B, we readilydetected phosphorylation of HLA-A2 (Fig. 8, A and B, lane 2). How-ever, A2/C was expressed at reduced levels (Fig. 8, A, lane 10 and B,lane 9) and clear detection of phosphorylated A2/C required stabili-zation of the degraded molecules with the lysosomal inhibitor, bafilo-mycin (Fig. 8, A and B, compare lanes 3 and 4). Mutation of serineresidues 332 and 335 stabilized A2/C protein (Fig. 8B, compare lanes9 and 11) and dramatically reduced recovery of phosphorylated mol-ecules (Fig. 8, A lane 8; B, lane 5). Thus, serines 332 and 335 wereclearly necessary for both degradation and phosphorylation of theHLA-C cytoplasmic tail.

FIGURE 9. Serine at position 335 regulates HLA-Cw*0401 expression in macrophage cell lines. A and B, Mutation of the phosphorylated serines toalanines stabilizes A2/C surface expression. U937 cells expressing the indicated MHC-I molecule were treated with DMSO or LPS/PMA as indicated andwere then stained with the anti-HLA-A2 Ab, BB7.2. B, The fold increase in MFI relative to HA-A2 is shown. The mean � SD for three experiments isshown. C and D, Macrophage differentiation inhibits lysosomal targeting of HA-A2/C through inhibition of Ser335 phosphorylation. The indicated U937cell lines were pulse-labeled with 35S and chased for 12 h plus or minus bafilomycin as in Fig. 4E. The indicated protein was immunoprecipitated withBB7.2 and separated by SDS-PAGE. White lines indicate places where the gel image was cropped and rearranged to place samples in a consistent orderrelative to one another. D, A phosphor imager and ImageQuant software were used to quantify each band. Fold stabilization was calculated as follows: [%remaining for each condition/% remaining for the untreated sample] (% remaining was calculated as in Fig. 4F). E, Macrophage differentiation inhibitsinternalization of HA-A2/C through hypophosphorylation of Ser335. An internalization assay performed as described in Fig. 3C was used to determine thepercentage of the indicated MHC-I molecules remaining on the cell surface over time in DMSO vs LPS/PMA-treated U937 cells. The anti-HLA-A2 mAbBB7.2 was used to detect surface expression in these experiments. The mean � SD for an experiment performed in triplicate is shown.

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To determine whether phosphorylation affected surface expres-sion, we examined these molecules by flow cytometry in undif-ferentiated U937 and CEM cells. As shown in Fig. 9A, we foundthat mutation of both serines to alanines increased HA-A2/C ex-pression to that of wild type HA-A2. Mutation of serine 332 alonehad no effect (data not shown), indicating that serine 335 wassufficient for this phenotype. Mutation of this serine residue toglutamic acid (S335E), to mimic phosphorylation, did not affectexpression of HA-A2/C in undifferentiated cells. Remarkablyhowever, when macrophage differentiation was induced in U937cells with LPS and PMA, we found that mimicking phosphoryla-tion prevented up-regulation, whereas unmodified HA-A2/C wasup-regulated 4.7-fold ( p � 1 � 10�6, Fig. 9, A and B). Thus, thesedata provide confirmation that the phosphorylated form of A2/C isdegraded and that inhibition of A2/C phosphorylation results in astabilization of A2/C protein.

To further examine the mechanism by which S335 affected HA-A2/C expression, we examined degradation by pulse-chase anal-ysis. As shown in Fig. 9C, HA-A2/C expression was reduced by97% by 12 h (compare lanes 15 and 16 with lanes 17 and 18 in Fig.9C). Inhibition of phosphorylation by alanine substitutions(SSAA) resulted in stabilization of the molecule nearly 4-fold(compare lanes 17 and 18 with lanes 29 and 30 in Fig. 9C). Incontrast, mimicking phosphorylation of S335 by glutamic acidsubstitution resulted in degradation similar to HA-A2/C (comparelane 17 and 18 with lanes 41 and 42 in Fig. 9C). In all cases, theobserved degradation was rescued with bafilomycin, indicatingthat it was secondary to lysosomal targeting.

Following treatment with LPS and PMA to induce macrophagedifferentiation, we observed a 4-fold increase in HA-A2/C proteinat the 12 h chase point compared with DMSO-treated cells (com-pare lanes 17 and 18 with lanes 23 and 24 in Fig. 9C). Mimickingphosphorylation at position 335 (S335E) prevented differentiation-induced stabilization of HA-A2/C (compare lanes 23 and 24 withlanes 47 and 48 in Fig. 9C). Conversely, inhibiting phosphoryla-tion by substituting alanine at the same position maintained proteinstability under all conditions (Fig. 9C, lanes 35–38). In sum, thesedata strongly indicate that phosphorylation of S335 in the HLA-Ctail is necessary for lysosomal targeting and that macrophage dif-ferentiation inhibits phosphorylation of this residue.

To examine the effect of mimicking or inhibiting phosphoryla-tion on internalization rate, we used the flow cytometric internal-ization assay described above. As shown in Fig. 9E (left graph),mimicking phosphorylation (S335E) accelerated internalization1.7-fold ( p � 0.01), whereas preventing phosphorylation at thisposition (SSAA) inhibited it 2.0-fold ( p � 0.01). Following treat-ment with LPS and PMA, we again observed a decrease in theinternalization rate of HA-A2/C (Fig. 9E, right graph). However,the phosphorylation mimic (S335E) did not respond, and contin-ued to be internalized at a rapid rate. These data further support themodel that phosphorylation is necessary for the internalization anddegradation of HLA-C, and that macrophage differentiation re-sulted in hypophosphorylation, and increased expression ofHLA-C.

Finally, to directly demonstrate that phosphorylation of A2/C isreduced upon macrophage differentiation, phosphorylation ofHA-A2 and HA-A2/C was directly assessed by labeling the cellswith 32P orthophosphate and each molecule was immunoprecipi-tated with the HLA-A2-specific Ab, BB7.2. As shown in Fig. 8C,induction of differentiation dramatically increased the recovery oftotal A2/C relative to that of HA-A2, as measured by Western blotanalysis (compare Fig. 8C, lanes 5 and 6 with Fig. 8B, lanes 8 and9) without demonstrating a corresponding increase in phosphory-lated forms (Fig. 8C, lanes 2 and 3). Thus, these data support our

model, that differentiation reduced A2/C phosphorylation, whichin turn resulted in stabilization of A2/C protein.

DiscussionIn this study, we demonstrated that HLA-C is a highly regulatedMHC-I molecule and in most cell types its expression is limited atalmost every step in the biosynthetic pathway. Maintenance of alow level of HLA-C surface expression may allow a balance be-tween the signals needed for Ag presentation and appropriate in-hibition of NK cells, without sending strong dominant signals thatmight overly increase activation thresholds. The extracellular do-main of HLA-C reduced expression by promoting ER retentionwhereas the cytoplasmic tail affected expression via the activity ofan internalization and lysosomal targeting signal.

The influence of the extracellular domain of MHC-I on its sur-face expression was not surprising. This region of the moleculedictates the peptides MHC-I will bind and ultimately regulatesrelease from the ER and transport to the cell surface. Indeed, theaddition of HLA-C-specific peptides has been reported to promotethe release of HLA-C from TAP in vitro (29), and thus would beexpected to decrease ER retention. Based on these data, it is tempt-ing to speculate that HLA-C expression would increase with abroadening of intracellular peptides such as would occur with in-fection by viruses or intracellular bacteria. To this end we didexamine several viruses (HIV and adenovirus) without detectingany significant change in HLA-C expression. Obviously, however,we cannot rule out the possibility that specific peptides found inother kinds of pathogens might stimulate the release of HLA-Cfrom the ER.

The bigger surprise was the discovery of an internalization andlysosomal targeting signal within the HLA-C cytoplasmic tail.This motif was identified by the demonstration that mutating iso-leucine at position 337 to a threonine reversed the phenotype con-ferred by swapping the HLA-C cytoplasmic tail for the HLA-A2cytoplasmic tail. Interestingly, the sequence surrounding this res-idue resembles a Golgi localized, �-ear containing, ARF-binding(GGA) consensus binding motif (DXXLL: reviewed in (30)).GGAs are localized to the TGN and endosomal compartments, andare thought to play a role in trafficking between the TGN andendosomes. Thus, it was possible that GGAs played a role in tar-geting HLA-C into the endolysosomal pathway from the cell sur-face or the TGN. However, while we observed some reduction insurface expression and some alteration in intracellular localizationwith knockdown of GGA-2 and -3, we observed no significantchange in the surface transport rate, internalization, recycling ordegradation rates. Also, arguing against a role for the GGAs, wefound that mutation of the required aspartic acid residue at position333 (DXSLI) to an alanine, actually increased the activity of thesignal. Based on these data, the role of this amino acid was not toprovide a GGA binding site, but rather to attenuate the dihydro-phobic signal so as to allow some HLA-C to remain on the cellsurface. Thus, we have defined a set of amino acids in the HLA-Ccytoplasmic tail, which comprise a novel signal that serves tomaintain a precise, low level of HLA-C surface expression. Fur-ther work will be needed to identify the corresponding traffickingprotein that binds it.

Interestingly, the activity of the HLA-C internalization andlysosomal targeting signal also depended on an adjacent serine(DXSLI), which we directly demonstrated was phosphorylated invivo. Changes in this position increased or decreased internaliza-tion and degradation, depending on the substitution that was made.When serine 335 was changed to a glutamic acid residue, which

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mimicked the negative charge provided by phosphorylation, inter-nalization and degradation occurred rapidly. When phosphoryla-tion was prevented by changing the serine to an alanine, internal-ization and degradation were inhibited and cell surface expressionwas increased.

The complex regulation of HLA-C trafficking was puzzling inthat HLA-C cell surface expression was kept low while HLA-Cintracellular levels remained fairly high. These observations sug-gested that under most conditions it is beneficial to keep HLA-Cexpression low to reduce inhibitory signals that might limit im-mune surveillance, but that there might be some circumstances inwhich HLA-C might be rapidly up-regulated, either to increase thecapacity of the cell to present certain types of Ags, or to turn downan immune response by increasing signaling to KIRs.

To examine whether HLA-C might be specifically up-regulatedunder some conditions of immune activation, we treated CEMcells with IFNs (� and �) or with chemicals known to activate Tcells (PHA and IL-2), without success. Additionally, we infectedthe cells with viral pathogens such as adenovirus and HIV, againwithout significant affect. We then turned to APCs, because thesecells have unique roles in Ag presentation (e.g., the capacity topresent exogenous Ags in association with MHC-I).

We found that undifferentiated primary monocytes and mono-cytic cell lines expressed low levels of HLA-C, similar to the othercell types we examined. Upon differentiation, however, we ob-served a reduction in phosphorylation of A2/C, which correlatedwith a reduction in internalization and degradation and a corre-sponding up-regulation of HLA-C and molecules bearing theHLA-C cytoplasmic tail. Under the same conditions, the surfaceexpression of HLA-A and HLA-B molecules remained essentiallyunchanged or was even reduced somewhat. The dependence of thiseffect on the cytoplasmic tail, which we demonstrated governspost-Golgi trafficking, ruled out the possibility that this was solelydue to a change in the peptide loading capacity of the APCs in theER. Up-regulation of expression with differentiation depended onthe serine adjacent to the dihydrophobic motif (DXSLI). When thisserine was modified to a glutamic acid, mimicking phosphoryla-tion, low expression and rapid internalization was maintained uponinduction of differentiation. When phosphorylation was inhibitedby changing the serine to an alanine residue, high surface expres-sion and reduced internalization resulted and was maintained uponinduction of differentiation.

These observations, together with the strong evidence thatHLA-C plays a crucial role as an inhibitor of NK cell lysis byvirtue of its specific binding of KIRs, suggests that HLA-C isup-regulated on macrophages to down-regulate and/or specificallyinhibit lysis of cells bearing these receptors. Interestingly, it hasrecently been demonstrated that CTLs acquire KIRs coincidentwith acquisition of effector functions (5). Thus, HLA-C may beup-regulated to provide feedback inhibition of CTLs, once theyhave fully matured. Alternatively, another, perhaps more intrigu-ing possibility is that HLA-C is specifically up-regulated on APCsto protect them from lysis by mature CTLs while they are cross-presenting exogenous Ags to naive CTLs. The capacity to specif-ically prevent the lysis of cross-presenting APCs would be advan-tageous in the setting of a chronic infection in which it wasnecessary to continuously present Ags over an extended period oftime. In preserving these cells by such a mechanism, the resultingincreased threshold to lysis may inadvertently create a protectedreservoir that aids in the persistence of certain organisms. Indeed,there is a long list of persistent pathogens that can be found inmacrophages, including HIV, leishmania, brucella, salmonella,herpes viruses, tuberculosis, legionella, plus others (31–38). Thus,HLA-C may be precisely regulated to balance the need for con-

tinued immune activation by APCs presenting Ags against the costof allowing some pathogens to persist.

AcknowledgmentsWe are grateful to Patrizio Giacomini for Ab to HLA-C and to the Uni-versity of Microscopy and Image Analysis Laboratory for confocalmicroscope use.

DisclosuresThe authors have no financial conflict of interest.

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