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Promiscuous Binding of Invariant Chain-Derived CLIP Peptide to Distinct HLA-I Molecules Revealed in Leukemic Cells

  • Marvin M. van Luijn ,

    Contributed equally to this work with: Marvin M. van Luijn, Arjan A. van de Loosdrecht, Margit H. Lampen, Peter A. van Veelen

    m.luijn@vumc.nl

    Affiliations Department of Hematology, Cancer Center Amsterdam, VU Institute for Cancer and Immunology, VU University Medical Center, Amsterdam, The Netherlands, Sanquin Research and Landsteiner Laboratory, Department of Immunopathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

  • Arjan A. van de Loosdrecht ,

    Contributed equally to this work with: Marvin M. van Luijn, Arjan A. van de Loosdrecht, Margit H. Lampen, Peter A. van Veelen

    Affiliation Department of Hematology, Cancer Center Amsterdam, VU Institute for Cancer and Immunology, VU University Medical Center, Amsterdam, The Netherlands

  • Margit H. Lampen ,

    Contributed equally to this work with: Marvin M. van Luijn, Arjan A. van de Loosdrecht, Margit H. Lampen, Peter A. van Veelen

    Affiliation Department of Clinical Oncology, Leiden University Medical Center, Leiden, The Netherlands

  • Peter A. van Veelen ,

    Contributed equally to this work with: Marvin M. van Luijn, Arjan A. van de Loosdrecht, Margit H. Lampen, Peter A. van Veelen

    Affiliation Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands

  • Adri Zevenbergen,

    Affiliation Department of Hematology, Cancer Center Amsterdam, VU Institute for Cancer and Immunology, VU University Medical Center, Amsterdam, The Netherlands

  • Michel G. D. Kester,

    Affiliation Laboratory of Experimental Hematology, Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands

  • Arnoud H. de Ru,

    Affiliation Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands

  • Gert J. Ossenkoppele,

    Affiliation Department of Hematology, Cancer Center Amsterdam, VU Institute for Cancer and Immunology, VU University Medical Center, Amsterdam, The Netherlands

  • Thorbald van Hall ,

    These authors also contributed equally to this work.

    Affiliation Department of Clinical Oncology, Leiden University Medical Center, Leiden, The Netherlands

  • S. Marieke van Ham

    These authors also contributed equally to this work.

    Affiliation Sanquin Research and Landsteiner Laboratory, Department of Immunopathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Promiscuous Binding of Invariant Chain-Derived CLIP Peptide to Distinct HLA-I Molecules Revealed in Leukemic Cells

  • Marvin M. van Luijn, 
  • Arjan A. van de Loosdrecht, 
  • Margit H. Lampen, 
  • Peter A. van Veelen, 
  • Adri Zevenbergen, 
  • Michel G. D. Kester, 
  • Arnoud H. de Ru, 
  • Gert J. Ossenkoppele, 
  • Thorbald van Hall, 
  • S. Marieke van Ham
PLOS
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Abstract

Antigen presentation by HLA class I (HLA-I) and HLA class II (HLA-II) complexes is achieved by proteins that are specific for their respective processing pathway. The invariant chain (Ii)-derived peptide CLIP is required for HLA-II-mediated antigen presentation by stabilizing HLA-II molecules before antigen loading through transient and promiscuous binding to different HLA-II peptide grooves. Here, we demonstrate alternative binding of CLIP to surface HLA-I molecules on leukemic cells. In HLA-II-negative AML cells, we found plasma membrane display of the CLIP peptide. Silencing Ii in AML cells resulted in reduced HLA-I cell surface display, which indicated a direct role of CLIP in the HLA-I antigen presentation pathway. In HLA-I-specific peptide eluates from B-LCLs, five Ii-derived peptides were identified, of which two were from the CLIP region. In vitro peptide binding assays strikingly revealed that the eluted CLIP peptide RMATPLLMQALPM efficiently bound to four distinct HLA-I supertypes (-A2, -B7, -A3, -B40). Furthermore, shorter length variants of this CLIP peptide also bound to these four supertypes, although in silico algorithms only predicted binding to HLA-A2 or -B7. Immunization of HLA-A2 transgenic mice with these peptides did not induce CTL responses. Together these data show a remarkable promiscuity of CLIP for binding to a wide variety of HLA-I molecules. The found participation of CLIP in the HLA-I antigen presentation pathway could reflect an aberrant mechanism in leukemic cells, but might also lead to elucidation of novel processing pathways or immune escape mechanisms.

Introduction

In immune surveillance against invading pathogens and tumor cells, antigen processing and presentation by HLA molecules is essential for induction of potent T cell-mediated immunity.

Classically, exogenously derived antigens, such as bacterial components, are processed in the endosomal/lysosomal system for loading onto HLA class II (HLA-II) complexes. After synthesis in the endoplasmic reticulum (ER), the HLA-II heterodimer binds to the invariant chain (Ii) for transport to late endosomes [1]. Here, Ii is cleaved until only a small fragment, the class II-associated invariant chain peptide (CLIP) remains bound to the class II peptide-binding groove [2]. In MHC class II loading compartments (MIICs), CLIP is exchanged for an antigenic peptide with aid of HLA-DM [3], [4], and HLA-II/peptide complexes are exported to the plasma membrane and presented to CD4+ T cells. In tumor cells that have APC function, efficient processing of endogenous, potentially tumor-associated antigens (TAAs) is pivotal for T cell priming of and/or recognition by specific effector T cells. We and others previously showed that such endogenous antigen presentation can also involve HLA-II complexes [5], [6]. Ii silencing in certain tumor cells downmodulates CLIP, but not HLA-II expression levels [7] and results in increased presentation of endogenous antigens and tumor-specific CD4+ T cell activation [5], [8]. These studies contradict with the proposed requirement of Ii for HLA-II stabilization and transport [9], but agree with its function in preventing binding of endogenous peptides to HLA-II complexes in the ER [10].

For HLA-I antigen presentation, endogenous proteins, e.g. tumor- and virus-associated proteins, are normally degraded by the cytoplasmic proteasome followed by translocation of peptides into the ER via the transporter associated with antigen processing (TAP) molecule. Here, peptides with the appropriate binding motif associate with newly formed HLA class I (HLA-I) heavy chain/β2m heterodimers and are transported to the plasma membrane for presentation to CTLs (reviewed in [11]). Professional APCs, including macrophages, dendritic cells (DCs) and B cells, have well-equipped machinery to detect, internalize and process exogenous antigens. These antigens are processed for HLA-II-mediated presentation, but can also be routed for presentation by HLA-I, resulting in cross-priming of CTLs (reviewed in [12]). Two general routes for this so-called cross-presentation have been described: exogenous antigens are degraded and directly loaded onto HLA-I molecules in the endo-lysosomal pathway [13] or, alternatively, gain access to the cytoplasm for proteasome-dependent processing and are directed either back into endosomes or the ER via TAP [14], [15]. The precise mechanism by which HLA-I molecules may enter the endo-lysosomal pathway is poorly defined.

Ii is a type II transmembrane protein that exists in different isoforms and contains one or more internal targeting signals for specific transport of newly synthesized HLA-II complexes to the MIICs [16], [17]. In addition to its role in HLA-II transport, the role of Ii as chaperone seems to be more versatile. Ii binds to the actin-based motor protein myosin-II to negatively affect DC migration [18], to adhesion molecule CD44 to activate T cells [19] as well as to costimulatory molecule CD70 for targeting to the MIICs [20]. In the present study, we show an accessory role for Ii and CLIP in HLA-I processing and antigen presentation by leukemic cells.

Methods

Patient Material

Bone marrow samples from nine newly diagnosed acute myeloid leukemia (AML) patients were collected after obtaining written informed consent and according to the Declaration of Helsinki. This was approved by the review board (‘Medisch Ethische Toetsingscommissie, METc’) of the VU University Medical Center, Amsterdam, The Netherlands. Classification of acute promyelocytic leukemia (APL) was based on standard genetic and molecular detection of t(15;17), as part of routine diagnostic procedures at our department. HLA-DR-negative AML patients contained high numbers of myeloid cells (>80% of the total WBC count), which were defined as CD45dim/SSClow-int by flow cytometry. Mononuclear cells were isolated using Ficoll-PaquePLUS (Amersham Biosciences, Freiburg, Germany) and directly used for protein analysis or cryopreserved in liquid nitrogen.

Cell Lines and Culturing

Human leukemic cell lines KG-1, THP-1 and Kasumi-1 were purchased from the American Type Culture Collection (ATTC). KG-1 cells were maintained in IMDM (Gibco, Paisley, UK) supplemented with 20% FBS (Greiner, Alphen a/d Rijn, The Netherlands), 25 mM Hepes (Sigma-Aldrich, St Louis, MO, USA), 1% L-glutamine and 50 µM 2-ME (both Gibco). THP-1 and Kasumi-1 were cultured in RPMI 1640 medium (Gibco) containing 10% and 15% FBS, respectively. For peptide elutions, Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (B-LCLs) were generated from peripheral blood mononuclear cells (PBMC) from healthy blood donors or patients. This was performed in approval with the Leiden University Medical Center review board. EBV-transformed B-LCL JY, PHEB and 5544 were cultured in IMDM containing 8% FBS, 100 IU/ml penicillin and 2 mM L-glutamine. For the competition-based peptide binding assays, B-LCLs expressing HLA-I alleles of interest were a generous gift from Dr. J. Kessler (Leiden University Medical Center; [21]). T2 cells were obtained from Dr. P. Cresswell (Yale University School of Medicine, New Haven, CT, USA).

Antibodies and Immunofluorescence Stainings

We used the following mouse anti-human monoclonal antibodies (MoAbs): PE-labeled anti-CLIP (clone cerCLIP.1; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-HLA-ABC (clone W6/32; Dako, Glostrup, Denmark); FITC-labeled CD74, anti-HLA-DR (clone L243) and anti-HLA-DRPQ; PerCP-labeled CD45 (all BD Biosciences, San Jose, CA, USA). Anti-Ii MoAb (clone PIN1.1) was kindly provided by Dr. P. Cresswell. For stainings of murine cells, PE-labeled CD8 (clone 53–6.7; Biolegend, San Diego, CA, USA) and APC-labeled anti-IFN-γ (clone XMG1.2; BD Biosciences) antibodies were used.

For immunofluorescence, cells were incubated with 10% human γ-globulin (60 mg/ml; Sanquin, Amsterdam, The Netherlands) before the MoAb of interest was added. Moabs were added during 15 min at room temperature (RT). Intracellular staining for Ii was performed on cells fixed with PBS 1% paraformaldehyde and permeabilized with PBS 0.1% saponin (Sigma-Aldrich). After incubation with PIN1.1 for 30 min at RT, cells were stained for 20 min with a secondary, PE-labeled rabbit anti-mouse (RAM) IgG (Dako). Each incubation step was followed by two washing steps in PBS with 0.1% HSA and 0.05% sodium azide. We used a FACSCalibur flow cytometer and CellQuestPro software (BD Biosciences) or Flowjo software (Treestar Inc, Ashland, OR, USA) to analyze the percentage of positive cells. Leukemic cells of AML patients were defined by CD45dim/SSClow expression.

Co-immunoprecipitations and Western Blotting

See File S1 in the Supporting Information section.

Retrovirus Production and Transduction

Ii expression was silenced using the retroviral pSIREN-RetroQ vector (Clontech, Palo Alto, CA, USA) consisting of a puromycin resistance gene together with a cloned Ii siRNA insert (sequence no. 53; [22]) Retrovirus, a kind gift from Dr. S. Ostrand-Rosenberg (University of Maryland, Baltimore, USA), was produced as reported [23]. For retroviral transduction, 1×106 cells/ml were cultured until 40% confluency. Following washing with PBS, cells were resuspended in DMEM (Gibco) containing 10% FBS, 10 mM Hepes and polybrene, followed by the drop-wise addition of retroviral supernatant to attain a polybrene concentration of 4 µg/ml. Cells were incubated at 37°C for 6 h, washed three times with excess PBS and kept in culture medium for three days. Subsequently, 0.5 µg/ml puromycin was added and increased gradually during a period of two weeks to a final concentration of 1.0 µg/ml in order to select Ii siRNA-transduced cells. Ii expression in transduced cells was checked by flow cytometric analysis. The process of siRNA formation and retroviral transduction itself was validated not to affect Ii expression [6], [22].

HLA Class I Peptide Isolation, HPLC and Mass Spectrometry

HLA-I/peptide complexes were purified from >1010 EBV-transformed B-LCLs by affinity chromatography using protein A beads (GE Healthcare) covalently bound to MoAbs against HLA-I (clone W6/32; used for B-LCL JY) or HLA-A2 (clone BB7.2; [24]; used for B-LCL PHEB and 5544). Peptides were eluted from isolated HLA-I molecules and separated from class I heavy chains and β2m by passage through Centriprep filtration units with a 10 kD, and the complex peptide pool was fractionated on a 15 cm×200 µm RP-C18 (Reprosil-Pur C18-AQ 3 µm) column, packed in house. The gradient was run from 0% to 50% solvent B (10/90/0.1 v/v/v water/acetonitrile/formic acid) in 45 min.

Peptide fractions from the first dimension separation were reduced to near dryness and resuspended in 95/3/0.1 v/v/v water/acetonitrile/formic acid. These resuspended fractions were analyzed by on-line nano-HPLC mass spectrometry using a system earlier described [25]. Fractions were injected onto a precolumn (100 µm×15 mm; Reprosil-Pur C18-AQ 3 µm, 5 µm, Phenomenex) and eluted via an analytical nano-HPLC column (15 cm×50 µm; Reprosil-Pur C18-AQ 3 µm). The gradient was run from 0% to 50% solvent B (10/90/0.1 v/v/v water/acetonitrile/formic acid) in 90 min. The nano-HPLC column was drawn to a tip of approximately 5 µm and acted as the electrospray needle of the MS source.

For mass spectrometry, we used a LTQ-FT Ultra mass spectrometer (Thermo, Bremen, Germany) that was operated in data-dependent mode, automatically switching between MS and MS/MS acquisition. Full scan mass spectra were acquired in the Fourier-transform ion cyclotron resonance (FT-ICR) with a resolution (m/Δm at full width half maximum) of 25,000 at a target value of 5,000,000. The two most intense ions were then isolated for accurate mass measurements by a selected ion monitoring scan in FT-ICR with a resolution of 50,000 at a target accumulation value of 50,000. The selected ions were then fragmented in the linear ion trap using collision-induced dissociation at a target value of 10,000. In a post analysis process, raw data were converted to peak lists using Bioworks Browser software, Version 3.1. For peptide identification, MS/MS data were submitted to the human IPI database using Mascot Version 2.2.04 (Matrix Science) with the following settings: 2 ppm and 0.8-Da deviation for precursor and fragment masses, respectively; no enzyme was specified. Mascot output files were loaded into Scaffold (http://www.proteomesoftware.com) and exported to Excel.

HLA Class I Binding Prediction and Synthesis of Peptides

The capability of peptides to bind to which HLA-I molecules was predicted via the netMHC server (http://www.cbs.dtu.dk/services/NetMHC; [26], [27]), which makes use of approximation algorithms via artificial neural networks (ANNs) and is trained on 9- to 11-mer peptides to predict binding to HLA-I antigen binding pockets. Predicted peptides were synthesized by standard Fmoc chemistry and using a Syro II peptide synthesizer (MultiSyntech, Witten, Germany), as described previously [28]. The integrity of each peptide was routinely validated by HPLC and mass spectrometry.

Competition-based Cellular Peptide Binding Assay

To test binding affinity of eluted and predicted peptides to HLA-I, competition-based cellular peptide binding assays were performed as described earlier [21]. In short, B-LCLs were treated with a mild acid (1∶1 mixture of 0.263M C6H8O7*H2O and 0.126M Na2HPO4*2H2O) for 1 min to remove the naturally HLA-I bound peptides. Cells were buffered with cold IMDM containing 2% FCS immediately thereafter and resuspended at a concentration of 4×105 cells/ml in 2% FCS and 1.5 µg/ml human β2m (Sigma-Aldrich). Then, 4×104 cells/well were incubated with 150 nM of fluorescently labeled reference peptide and a serial dilution of one of the eluted or predicted peptides. The following HLA-I allele-restricted reference peptides were used: FLPSDCFPSV (for HLA-A0201), KVFPCALINK (for HLA-A0301), APAPAPCWPL (for HLA-B0702) and GEFGGCGSV (for HLA-B4002), each containing a fluorescent label bound to the cysteine residue [21]. After overnight incubation at 4°C, cells were washed twice in PBS supplemented with 1% BSA and fixed in 0.5% paraformaldehyde. Cells were analyzed with CellQuestPro or FlowJo software (Tree Star, Ashland, OR, USA). IC50 values were defined with GraphPad Prism 4.02 (GraphPad Software Inc., La Jolla, USA) using the following formula: Y = Bmax*X/(IC50+X), in which Bmax is the maximal binding capacity of the positive control peptide, X the concentration of peptide tested and IC50 the concentration of peptide needed to reach half-maximal binding.

Mouse Immunizations

HLA-A2 transgenic mice (B6 background HLA-A2/H2-D; [29]) were immunized subcutaneously with 50 µg of invariant chain peptides and 150 µg HBV T helper peptide (TPPAYRPPNAPIL) in PBS. The injection side was covered with 60 mg of Aldara cream containing 5% imiquimod (3 M Pharma Nederland BV). Immunization was repeated on day 7 combined with two intraperitoneal injections of 600.000 IU human recombinant IL-2 (Novartis) on day 7 and 8 [30]. The predicted invariant chain peptides were pooled to a total of 50 µg each. As positive control, mice were injected with 50 µg of MLIVYVRFWWL. At day 13, blood samples were taken and tested for CD8+ T cell reactivity by overnight incubation with the corresponding peptide and intracellular staining for IFN-γ.

Results

CLIP Presented on HLA-DR-negative Leukemic Cells from Patients is not Explained by Plasma Membrane Expression of HLA-II or CD74

We found that patients with acute promyelocytic leukemia (APL; [31]), a genetically determined subtype of HLA-DR-negative acute myeloid leukemia (AML), contained a high frequency of leukemic cells with surface display of the CLIP epitope. Since CLIP expression was not observed on normal promyelocytes from healthy individuals, this remarkable observation may suggest that CLIP presentation at the plasma membrane of these leukemic cells is leukemia-specific and occurs in the context of other types of HLA-II proteins or as unprocessed Ii. To examine the underlying mechanism of CLIP presentation, we analyzed protein expression of total HLA-II and CD74 (i.e. unprocessed Ii) at the plasma membrane. Flow cytometric analysis however showed a lack of both proteins on HLA-DRCLIP+ leukemic cells (Figure 1A and B), which shows that the expression of CLIP can neither be attributed to its presentation by HLA-DP or HLA-DQ molecules, nor to the expression of unprocessed Ii.

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Figure 1. Surface display of the CLIP epitope on HLA-II-negative leukemic cells and influence of the invariant chain on HLA-I expression.

(A) Surface expression of CLIP, HLA-DR, total HLA-II (‘HLA-DRPQ’) and invariant chain (‘CD74’) of myeloid cells from an acute promyelocytic leukemia (APL) patient, as determined by flow cytometry. Myeloid cells were defined as CD45dim/SSClow/int and expression thresholds were set according to unstained myeloid cells. (B) Quantitative analysis on frequencies of myeloid cells from APL patients that express CLIP (n = 9), HLA-DR (n = 9), total HLA-II (n = 6) and CD74 (n = 6). Frequencies indicate percentage tumor cells that reach threshold expression based on unstained leukemic cells. (C) The effect of invariant chain Ii down-modulation in KG-1 (CLIP-) and THP-1 (CLIP+) leukemic cells on HLA-I expression at the cell surface. Intracellular staining (ICS) of Ii (PIN1.1) and surface staining of HLA-I (W6/32) were compared between Ii siRNA-transduced and non-transduced cells.

https://doi.org/10.1371/journal.pone.0034649.g001

Invariant Chain Silencing in Myeloid Leukemia Cell Lines Differentially Influences HLA-I Surface Expression

In the mutant TAP- and HLA-II-deficient T2 cell line, the precursor of CLIP, Ii, was shown to interact with H-2Db alleles [32]. In addition, Ii can transport HLA-I molecules to endosomal compartments where exogenous peptides are present [33]. We next investigated whether there was a relation between Ii and HLA-I cell surface expression in CLIP-presenting and non-presenting leukemic cells. Retroviral introduction of Ii-specific siRNAs strongly downmodulates CLIP expression on several myeloid leukemia cell lines, as we recently reported [7]. When these cell lines were assessed for HLA-I protein expression by flow cytometry, KG-1 cells, which present low surface levels of CLIP [34], revealed hardly any effect of Ii silencing (Figure 1C). In THP-1 cells, which highly express CLIP [34], however, silencing of Ii induced a strong reduction in HLA-I surface expression (Figure 1C). In addition, we found indications that Ii was indeed able to associate with HLA-I-related products in leukemic cells, since Ii isoforms were present in HLA-I and Ii immunoprecipitates (Figure S1A), respectively. This was mostly seen in Ii-overexpressing Kasumi-1 cells (Figure S1A and S2), which also showed an increase in free-form β2m after Ii silencing (Figure S1B). The involvement of Ii in HLA-I plasma membrane expression in CLIP-presenting leukemic cells suggests a relation between surface expression of CLIP and HLA-I molecules. Also TAP expression was limited in CLIP+ THP-1 cells, in contrast to CLIP KG-1 cells (Figure S2), indicating that the involvement may rely on the ability to process endogenous antigens for loading in the ER.

Peptides Derived from the Invariant Chain are Presented at the Cell Surface by HLA-I Molecules

We next assessed whether HLA-I molecules can indeed present Ii-derived peptides at the plasma membrane. The peptide repertoires of HLA-I molecules on EBV-transformed B cells, including CLIP-positive JY cells (Figure S3) were evaluated through biochemical purification. Out of the total characterized peptide repertoire, five peptides were derived from the Ii protein (Figure 2 and Table 1), the sequences of which were validated by mass spectrometry according to their synthetic counterparts. Remarkably, two peptides were located within or near the CLIP region, known for its association with HLA-II peptide-binding grooves. Isolation of the 5 identified peptides was restricted to HLA-A0201 and HLA-B0702 alleles presented on EBV-transformed B cells. To examine the HLA-I allele specificity and binding affinity of these peptides, competition-based peptide binding assays were performed (Table 1). Four out of five peptides had binding capacity to the B0702 allele and three of these were even categorized as high affinity binders. Notably, peptide 1 and peptide 3, the peptide located within the CLIP region, also bound to the A0201 allele, which might suggest a promiscuous binding capacity of these peptides to HLA-I. Altogether, these data demonstrate that surface HLA-I molecules are able to present a variety of peptides generated from Ii.

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Figure 2. Invariant chain-derived peptides identified in isolated HLA-I molecules of B-LCLs.

Peptide elutions of purified HLA-I molecules from EBV-transformed B-LCLs resulted in the identification of five peptides originating from the invariant chain. HLA-I purification and subsequent mass spectrometry analysis are described in Materials and Methods. Of note, peptide 3 and 4 are located in the CLIP region, known for universal binding to HLA-II molecules.

https://doi.org/10.1371/journal.pone.0034649.g002

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Table 1. HLA-I binding affinity of eluted peptides derived from the invariant chain.

https://doi.org/10.1371/journal.pone.0034649.t001

Invariant Chain-derived Peptides Located at the CLIP Region Show Promiscuous Binding to Various HLA-I Alleles

As CLIP is known to bind promiscuously to the binding groove of a broad range of HLA-II molecules [35], we further explored the HLA-I binding specificity of the eluted peptide located at the CLIP region of Ii (peptide no. 3; Figure 2 and Table 1). This naturally presented peptide (RMATPLLMQALPM) demonstrated a high affinity for both HLA-A0201 and -B0702 (Table 1). To determine if peptide RMATPLLMQALPM could bind promiscuously to HLA-I molecules, we addressed its binding capacity to HLA-I molecules bearing structurally different binding grooves. HLA-I molecules are classified according to overlapping binding repertoires and consensus structures in the main peptide binding pockets, so-called supertypes [36]. HLA-A0201 and -B0702 are well known representatives of the A2 and B7 supertype, respectively. The same accounts for HLA-A0301 and -B4002, which represent the common supertypes A3 and B40 [36]. Interestingly, peptide RMATPLLMQALPM bound with a relatively high affinity to all four HLA-I alleles (Figure 3), which have completely different peptide-binding grooves, suggesting that the CLIP sequence involved in promiscuous HLA-II binding also underlies a promiscuous binding to HLA-I molecules.

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Figure 3. Promiscuous binding of CLIP-peptide to distinct HLA-I alleles, representing four supertypes.

The CLIP peptide RMATPLLMQALPM (peptide 3) was tested for binding affinity in a competition-based cellular peptide binding assay. The four tested HLA-I alleles (HLA-A0201, -A0301, -B0702 and -B4002) harbor a completely distinct binding pocket and bind different peptide ligands. CLIP peptide shows intermediate to high binding affinity to all of these. Separate positive control peptides efficiently bind to their respective HLA allele: GILGFVFTL (A0201 peptide), QVPLRPMTYK (A0301 peptide), SPSVDKARAEL (B0702 peptide) and GEFGGFGSV (B4002 peptide) with IC50 values of 3.7, 0.2, 0.7 and 0.2, respectively [21]. The peptide concentration started at 100 µg/ml for HLA-A0201, -A0301 and -B4002 and 25 µg/ml for HLA-B0702, followed by a serial dilution of a factor two. Exact IC50 values of the CLIP peptides are depicted in Table 2.

https://doi.org/10.1371/journal.pone.0034649.g003

The unexpected promiscuous binding of peptide RMATPLLMQALPM to distinct HLA-I molecules prompted us to systematically assess length variants of peptides located in the CLIP region of Ii for such binding feature. The CLIP amino acid sequence of Ii was subjected to standard HLA-I binding prediction algorithms for 9- to 11–mer peptides [26], [27], which resulted in highly predictive values of three length variants (9-, 10- and 11-mer) of the naturally presented 13-mer RMATPLLMQALPM (Table 2). The three peptides were predicted to selectively bind to HLA-A2 subtypes and, in case of the 11-mer, also to -B7 subtypes. Subsequently, we tested these predicted peptides for actual binding to our selected alleles representing four different supertypes, HLA-A0201, -B0702, -A0301 and -B4002. In contrast to their predictions, all the length variants bound to each of the four HLA-I alleles with reasonably high affinity (Table 2), indicating that peptides located in the CLIP region of Ii have universal binding abilities to polymorphic HLA-I molecules. Finally, we tested whether the identified Ii-derived peptides were immunogenic in the context of HLA-I, as reported for self peptides termed T-cell epitopes associated with impaired peptide processing (TEIPPs; [37][39]), but immunizations of HLA-A2 transgenic mice with these peptides showed no CD8+ CTL responses (Figure S4). From these data, we conclude that the CLIP region of Ii harbors a unique peptide sequence that can accommodate binding to structurally completely different grooves of both HLA-I and -II.

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Table 2. HLA-I binding affinity of peptide length variants located in the CLIP region of the invariant chain.

https://doi.org/10.1371/journal.pone.0034649.t002

Discussion

Processing and presentation of antigens via HLA molecules by APCs is the key mechanism for generating a specific immune response against pathogens and TAAs. In normal APCs, Ii and CLIP have essential roles in the HLA-II antigen presentation pathway. We and others previously described that in tumor cells that are able to function as APC, expression of Ii and CLIP could serve as immune escape mechanism by interference with HLA-II-mediated TAA presentation [5], [8], [40]. Here, we reveal that Ii can also be associated with HLA-I antigen presentation in malignant cells. This study points at an alternative HLA-I antigen presentation pathway that may affect current concepts of antigen cross-presentation and tumor immune escape.

One of the early events during HLA-I processing is the binding of heavy chains to β2m in the ER. This results in HLA-I stabilization, appropriate folding and incorporation into the class I peptide-loading complex, consisting of TAP and other ER-resident chaperones important for efficient HLA-I peptide loading. Our observation that Ii is involved in HLA-I processing in leukemic cells (Figure 1C and Figure S1) indicates that it may take part of this process as well. This agrees with earlier studies using HLA-I-transfected T2 cells [32], [41], [42] and suggests that like newly synthesized HLA-II αβ complexes, also HLA-I/β2m heterodimers can interact with Ii in the ER lumen until peptides are loaded. As Ii inhibits premature peptide binding to HLA-II complexes in the ER [10], the question rises how the association of HLA-I with Ii is related to the function of the class I peptide-loading complex in this compartment. Ii-silenced THP-1 cells lacking the TAP molecule (Figure S2) revealed a strong reduction in HLA-I surface expression level, in contrast to TAP-expressing KG-1 cells (Figure 1C). Additionally, in transfected TAP-deficient T2 cells, HLA-I/Ii complexes relied on the absence of TAP-dependent HLA-I peptides for their stability [32], [41] and were also part of the class I peptide-loading complex [43]. For this, we propose that two different types of competition mechanisms can be active in APCs: one involving the binding of Ii to either HLA-I or HLA-II and one involving the binding of either Ii or peptide loading complex peptides to HLA. In this model, the balance between TAP and Ii expression might be of major importance. The likelihood of Ii to interact with HLA-I molecules increases in the absence of TAP, as has been described for T2 cells [43], but likely also in the situation of Ii abundance, as we demonstrate for Ii-high Kasumi-1 cells (Figure S1 and S2). Our finding that peptides derived from Ii were predicted and able to bind to HLA-A0201 and -B0702 (Table 1 and Figure 2) further suggests that the peptide-binding groove is the HLA-I site with which Ii associates. Indeed, mutations in the binding groove of HLA-B7 molecules affected their interaction with Ii and expression at the plasma membrane in transfected T2 cells [42]. It remains to be determined which specific HLA-I alleles are prone to bind to Ii, but the promiscuous binding of CLIP-related Ii peptides to the four tested HLA-I superfamilies (Table 2 and Figure 3) indicates great similarity to the promiscuous binding capacity of the CLIP region in the context of HLA-II [44]. However, this contradicts with the large differences in key anchor motifs between each HLA-I superfamily. The shallow binding grooves of HLA-II binds peptides based on favorable interactions rather than need of specific amino acids at each position. Even one amino acid with high affinity to the HLA-II binding groove is sufficient for peptide binding, which could also be the case for HLA-I, as the methionine at position 99 of the CLIP region served as crucial anchor residue for H2-Kb molecules [43].

The observed involvement of Ii and CLIP in HLA-I processing in leukemic cells, as the result of a potentially imbalanced TAP/Ii ratio, can have different outcomes on tumor immune escape. In Ii overexpressing leukemias, such as AML (e.g. Kasumi-1 cells; Figure S2) and chronic lymphocytic leukemia (CLL; [45]), but also colorectal carcinoma [46], Ii instead of TAA-derived peptides may be bound to HLA-I molecules after synthesis in the ER, thereby preventing TAA presentation at the plasma membrane and recognition by TAA-specific CTLs. In TAP-deficient tumors, self peptides termed TEIPPs have been identified that are specifically detected by CTLs [37], [39]. In addition, we observed a possible relation of TAP and CLIP expression in leukemia, with CLIP- leukemic cells positive and CLIP+ leukemic cells negative for TAP (including APL cells in Figure 1A; Figure S2; [6]). Therefore, the formation of HLA-I/Ii complexes may lead to processing of Ii into CLIP and surface presentation of this self-peptide as a TEIPP. In immunized HLA-A2 transgenic mice however, we could not detect CTL responses against CLIP-related Ii peptides (Figure S4). Another possibility is that HLA-I/Ii complexes in leukemic cells are transported from the ER into the endo-lysosomal pathway to be loaded with TAA-derived peptides obtained from authophagy and lysosomal processing. In normal APCs, Ii is classically known for its role in trafficking HLA-II molecules to the MIICs. It was recently demonstrated that in DCs Ii is also able to direct CD70 molecules to these compartments [20], indicating that the interaction of Ii with HLA-I molecules could account for such a transporting function as well and represent a novel cross-presentation pathway. Sugita and colleagues already demonstrated a role for Ii in the transport of HLA-I molecules to endosomes [33], which might explain the accumulation of newly synthesized HLA-I molecules in endosomal storage compartments [13]. Indeed very recently, in murine DCs, Ii was shown to be critical in MHC class I trafficking from the ER to late endosomes for antigen loading, serving as a mediator of cross-presentation [47]. Although further HLA-I/Ii processing for peptide loading in such endosomal compartments remains undefined, we detected CLIP-related Ii peptides in HLA-I-specific eluates of B-LCLs (Table 1 and Figure 2), indicating that Ii can be processed to CLIP in HLA-I. These peptides were not derived from HLA-II molecules, as validations for contamination with HLA-II during each step of peptide elution showed HLA-I heavy chains and β2m, but no HLA-II monomers. It is thus appealing to further examine HLA-I-mediated exchange of CLIP for antigenic peptides in the endo-lysosomal pathway as well as presentation of CLIP at the plasma membrane for the effect on CTL activation.

In this report, we present important data showing the promiscuous involvement of Ii and CLIP in the HLA-I antigen presentation pathway of leukemic APCs. To define the similarity with HLA-II processing, further exploration of their role in intracellular transport and peptide loading of HLA-I molecules is necessary. Since Ii and CLIP are involved in both HLA-I and HLA-II antigen presentation, it will be attractive to design immunotherapeutic strategies that modulate their expression, thereby controlling antigen presentation with the purpose to target immune surveillance against leukemias and possibly prevent autoimmunity.

Supporting Information

Figure S1.

Association of Ii with the HLA-I complex in leukemic cells. (A) Immunoblotting of Ii in both HLA-I (W6/32) and Ii (PIN1.1) immunoprecipitates of KG-1, THP-1 and Kasumi-1 cells. IgG immunoprecipiates were used as negative controls. (B) The presence of free-form β2m (12 kD) in total lysates of leukemic cells derived from the Ii-overexpressing Kasumi-1 cell line. Immunoblots for Ii and β2m were performed under SDS conditions and antibodies used for staining were against Ii (PIN1.1) or β2m (rabbit polyclonal antibody, kindly provided by Dr. J.J. Neefjes, NKI, Amsterdam, The Netherlands).

https://doi.org/10.1371/journal.pone.0034649.s002

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Figure S2.

Immunoblot analysis of Ii and TAP expression in the KG-1 (CLIP), THP-1 (CLIP+) and Kasumi-1 (CLIP+) leukemic cell line. Blots were loaded with total cell lysates and stained with primary anti-Ii (clone PIN1.1) and anti-TAP1 (clone 148.3, a kind gift from Dr. E.J. Wiertz and Dr. M.E. Ressing, University Medical Center Utrecht, The Netherlands) MoAb, demonstrating specific bands of 33 kD and 74 kD, respectively.

https://doi.org/10.1371/journal.pone.0034649.s003

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Figure S3.

CLIP expression on the surface of T2 and EBV-transformed JY cells, as determined by flow cytometry using a PE-labeled cerCLIP.1 MoAb.

https://doi.org/10.1371/journal.pone.0034649.s004

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Figure S4.

The in vivo effect of Ii-derived peptides on CD8+ T cell activation. HLA-A2 transgenic mice (n = 3 per group) were immunized with Ii-derived peptides identified in peptide elution studies (see Table 1 for numbering), or with a pool of Ii-derived peptides identified on basis of an in silico prediction algorithm (see Table 2). After 13 days, peptide-reactive CD8+ T cells from blood were stained with PE-labeled CD8 and APC-labeled anti-IFN-γ antibodies and analyzed by flow cytometry. IFN-γ-positive CD8+ T cell frequencies are expressed as the percentage within the total pool of CD8+ T cells.

https://doi.org/10.1371/journal.pone.0034649.s005

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Acknowledgments

We want to thank Dr. James Thompson for generating the retroviral Ii siRNA construct and Marjolein Sluijter for performing the mouse experiments.

Author Contributions

Conceived and designed the experiments: AAL GO TH SMH. Performed the experiments: MML MHL PAV AZ AHR MGK. Analyzed the data: MML MHL PAV AZ AHR MGK. Contributed reagents/materials/analysis tools: AAL PAV GO TH SMH. Wrote the paper: MML. Assisted in writing the manuscript: TH SMH.

References

  1. 1. Elliott EA, Drake JR, Amigorena S, Elsemore J, Webster P, et al. (1994) The invariant chain is required for intracellular transport and function of major histocompatibility complex class II molecules. J Exp Med 179: 681–694.EA ElliottJR DrakeS. AmigorenaJ. ElsemoreP. Webster1994The invariant chain is required for intracellular transport and function of major histocompatibility complex class II molecules.J Exp Med179681694
  2. 2. Romagnoli P, Germain RN (1994) The CLIP region of invariant chain plays a critical role in regulating major histocompatibility complex class II folding, transport, and peptide occupancy. J Exp Med 180: 1107–1113.P. RomagnoliRN Germain1994The CLIP region of invariant chain plays a critical role in regulating major histocompatibility complex class II folding, transport, and peptide occupancy.J Exp Med18011071113
  3. 3. Fling SP, Arp B, Pious D (1994) HLA-DMA and -DMB genes are both required for MHC class II/peptide complex formation in antigen-presenting cells. Nature 368: 554–558.SP FlingB. ArpD. Pious1994HLA-DMA and -DMB genes are both required for MHC class II/peptide complex formation in antigen-presenting cells.Nature368554558
  4. 4. Sloan VS, Cameron P, Porter G, Gammon M, Amaya M, et al. (1995) Mediation by HLA-DM of dissociation of peptides from HLA-DR. Nature 375: 802–806.VS SloanP. CameronG. PorterM. GammonM. Amaya1995Mediation by HLA-DM of dissociation of peptides from HLA-DR.Nature375802806
  5. 5. Armstrong TD, Clements VK, Martin BK, Ting JP, Ostrand-Rosenberg S (1997) Major histocompatibility complex class II-transfected tumor cells present endogenous antigen and are potent inducers of tumor-specific immunity. Proc Natl Acad Sci U S A 94: 6886–6891.TD ArmstrongVK ClementsBK MartinJP TingS. Ostrand-Rosenberg1997Major histocompatibility complex class II-transfected tumor cells present endogenous antigen and are potent inducers of tumor-specific immunity.Proc Natl Acad Sci U S A9468866891
  6. 6. van Luijn MM, Chamuleau ME, Ressing ME, Wiertz EJ, Ostrand-Rosenberg S, et al. (2010) Alternative Ii-independent antigen-processing pathway in leukemic blasts involves TAP-dependent peptide loading of HLA class II complexes. Cancer Immunol Immunother 59: 1825–1838.MM van LuijnME ChamuleauME RessingEJ WiertzS. Ostrand-Rosenberg2010Alternative Ii-independent antigen-processing pathway in leukemic blasts involves TAP-dependent peptide loading of HLA class II complexes.Cancer Immunol Immunother5918251838
  7. 7. van Luijn MM, Chamuleau ME, Thompson JA, Ostrand-Rosenberg S, Westers TM, et al. (2010) Class II-associated invariant chain peptide down-modulation enhances the immunogenicity of myeloid leukemic blasts resulting in increased CD4+ T-cell responses. Haematologica 95: 485–493.MM van LuijnME ChamuleauJA ThompsonS. Ostrand-RosenbergTM Westers2010Class II-associated invariant chain peptide down-modulation enhances the immunogenicity of myeloid leukemic blasts resulting in increased CD4+ T-cell responses.Haematologica95485493
  8. 8. Thompson JA, Srivastava MK, Bosch JJ, Clements VK, Ksander BR, et al. (2008) The absence of invariant chain in MHC II cancer vaccines enhances the activation of tumor-reactive type 1 CD4(+) T lymphocytes. Cancer Immunol Immunother 57: 389–398.JA ThompsonMK SrivastavaJJ BoschVK ClementsBR Ksander2008The absence of invariant chain in MHC II cancer vaccines enhances the activation of tumor-reactive type 1 CD4(+) T lymphocytes.Cancer Immunol Immunother57389398
  9. 9. Anderson MS, Miller J (1992) Invariant chain can function as a chaperone protein for class II major histocompatibility complex molecules. Proc Natl Acad Sci U S A 89: 2282–2286.MS AndersonJ. Miller1992Invariant chain can function as a chaperone protein for class II major histocompatibility complex molecules.Proc Natl Acad Sci U S A8922822286
  10. 10. Roche PA, Cresswell P (1990) Invariant chain association with HLA-DR molecules inhibits immunogenic peptide binding. Nature 345: 615–618.PA RocheP. Cresswell1990Invariant chain association with HLA-DR molecules inhibits immunogenic peptide binding.Nature345615618
  11. 11. Koch J, Tampe R (2006) The macromolecular peptide-loading complex in MHC class I-dependent antigen presentation. Cell Mol Life Sci 63: 653–662.J. KochR. Tampe2006The macromolecular peptide-loading complex in MHC class I-dependent antigen presentation.Cell Mol Life Sci63653662
  12. 12. Bevan MJ (2006) Cross-priming. Nat Immunol 7: 363–365.MJ Bevan2006Cross-priming.Nat Immunol7363365
  13. 13. Di Pucchio T, Chatterjee B, Smed-Sorensen A, Clayton S, Palazzo A, et al. (2008) Direct proteasome-independent cross-presentation of viral antigen by plasmacytoid dendritic cells on major histocompatibility complex class I. Nat Immunol 9: 551–557.T. Di PucchioB. ChatterjeeA. Smed-SorensenS. ClaytonA. Palazzo2008Direct proteasome-independent cross-presentation of viral antigen by plasmacytoid dendritic cells on major histocompatibility complex class I. Nat Immunol9551557
  14. 14. Burgdorf S, Scholz C, Kautz A, Tampe R, Kurts C (2008) Spatial and mechanistic separation of cross-presentation and endogenous antigen presentation. Nat Immunol 9: 558–566.S. BurgdorfC. ScholzA. KautzR. TampeC. Kurts2008Spatial and mechanistic separation of cross-presentation and endogenous antigen presentation.Nat Immunol9558566
  15. 15. Guermonprez P, Saveanu L, Kleijmeer M, Davoust J, Van Endert P, et al. (2003) ER-phagosome fusion defines an MHC class I cross-presentation compartment in dendritic cells. Nature 425: 397–402.P. GuermonprezL. SaveanuM. KleijmeerJ. DavoustP. Van Endert2003ER-phagosome fusion defines an MHC class I cross-presentation compartment in dendritic cells.Nature425397402
  16. 16. Arunachalam B, Lamb CA, Cresswell P (1994) Transport properties of free and MHC class II-associated oligomers containing different isoforms of human invariant chain. Int Immunol 6: 439–451.B. ArunachalamCA LambP. Cresswell1994Transport properties of free and MHC class II-associated oligomers containing different isoforms of human invariant chain.Int Immunol6439451
  17. 17. Pieters J, Bakke O, Dobberstein B (1993) The MHC class II-associated invariant chain contains two endosomal targeting signals within its cytoplasmic tail. J Cell Sci 106 (Pt 3): 831–846.J. PietersO. BakkeB. Dobberstein1993The MHC class II-associated invariant chain contains two endosomal targeting signals within its cytoplasmic tail.J Cell Sci 106 (Pt3)831846
  18. 18. Faure-Andre G, Vargas P, Yuseff MI, Heuze M, Diaz J, et al. (2008) Regulation of dendritic cell migration by CD74, the MHC class II-associated invariant chain. Science 322: 1705–1710.G. Faure-AndreP. VargasMI YuseffM. HeuzeJ. Diaz2008Regulation of dendritic cell migration by CD74, the MHC class II-associated invariant chain.Science32217051710
  19. 19. Naujokas MF, Morin M, Anderson MS, Peterson M, Miller J (1993) The chondroitin sulfate form of invariant chain can enhance stimulation of T cell responses through interaction with CD44. Cell 74: 257–268.MF NaujokasM. MorinMS AndersonM. PetersonJ. Miller1993The chondroitin sulfate form of invariant chain can enhance stimulation of T cell responses through interaction with CD44.Cell74257268
  20. 20. Zwart W, Peperzak V, de VE, Keller AM, van der HG, et al. (2010) The invariant chain transports TNF family member CD70 to MHC class II compartments in dendritic cells. J Cell Sci 123: 3817–3827.W. ZwartV. PeperzakVE deAM KellerHG van der2010The invariant chain transports TNF family member CD70 to MHC class II compartments in dendritic cells.J Cell Sci12338173827
  21. 21. Kessler JH, Mommaas B, Mutis T, Huijbers I, Vissers D, et al. (2003) Competition-based cellular peptide binding assays for 13 prevalent HLA class I alleles using fluorescein-labeled synthetic peptides. Hum Immunol 64: 245–255.JH KesslerB. MommaasT. MutisI. HuijbersD. Vissers2003Competition-based cellular peptide binding assays for 13 prevalent HLA class I alleles using fluorescein-labeled synthetic peptides.Hum Immunol64245255
  22. 22. Thompson JA, Dissanayake SK, Ksander BR, Knutson KL, Disis ML, et al. (2006) Tumor cells transduced with the MHC class II Transactivator and CD80 activate tumor-specific CD4+ T cells whether or not they are silenced for invariant chain. Cancer Res 66: 1147–1154.JA ThompsonSK DissanayakeBR KsanderKL KnutsonML Disis2006Tumor cells transduced with the MHC class II Transactivator and CD80 activate tumor-specific CD4+ T cells whether or not they are silenced for invariant chain.Cancer Res6611471154
  23. 23. Dissanayake SK, Thompson JA, Bosch JJ, Clements VK, Chen PW, et al. (2004) Activation of tumor-specific CD4(+) T lymphocytes by major histocompatibility complex class II tumor cell vaccines: a novel cell-based immunotherapy. Cancer Res 64: 1867–1874.SK DissanayakeJA ThompsonJJ BoschVK ClementsPW Chen2004Activation of tumor-specific CD4(+) T lymphocytes by major histocompatibility complex class II tumor cell vaccines: a novel cell-based immunotherapy.Cancer Res6418671874
  24. 24. Parham P, Brodsky FM (1981) Partial purification and some properties of BB7.2. A cytotoxic monoclonal antibody with specificity for HLA-A2 and a variant of HLA-A28. Hum Immunol 3: 277–299.P. ParhamFM Brodsky1981Partial purification and some properties of BB7.2. A cytotoxic monoclonal antibody with specificity for HLA-A2 and a variant of HLA-A28.Hum Immunol3277299
  25. 25. Meiring HD, van der Heeft E, ten Hove GJ, de Jong APJM (2002) Nanoscale LC-MS(n): technical design and applications to peptide and protein analysis. Leuk Res 25: 557–568.HD MeiringE. van der HeeftGJ ten HoveAPJM de Jong2002Nanoscale LC-MS(n): technical design and applications to peptide and protein analysis.Leuk Res25557568
  26. 26. Lundegaard C, Lund O, Nielsen M (2008) Accurate approximation method for prediction of class I MHC affinities for peptides of length 8, 10 and 11 using prediction tools trained on 9mers. Bioinformatics 24: 1397–1398.C. LundegaardO. LundM. Nielsen2008Accurate approximation method for prediction of class I MHC affinities for peptides of length 8, 10 and 11 using prediction tools trained on 9mers.Bioinformatics2413971398
  27. 27. Lundegaard C, Lamberth K, Harndahl M, Buus S, Lund O, et al. (2008) NetMHC-3.0: accurate web accessible predictions of human, mouse and monkey MHC class I affinities for peptides of length 8–11. Nucleic Acids Res 36: W509–W512.C. LundegaardK. LamberthM. HarndahlS. BuusO. Lund2008NetMHC-3.0: accurate web accessible predictions of human, mouse and monkey MHC class I affinities for peptides of length 8–11.Nucleic Acids Res36W509W512
  28. 28. Hiemstra HS, Duinkerken G, Benckhuijsen WE, Amons R, de Vries RR, et al. (1997) The identification of CD4+ T cell epitopes with dedicated synthetic peptide libraries. Proc Natl Acad Sci U S A 94: 10313–10318.HS HiemstraG. DuinkerkenWE BenckhuijsenR. AmonsRR de Vries1997The identification of CD4+ T cell epitopes with dedicated synthetic peptide libraries.Proc Natl Acad Sci U S A941031310318
  29. 29. Newberg MH, Smith DH, Haertel SB, Vining DR, Lacy E, et al. (1996) Importance of MHC class 1 alpha2 and alpha3 domains in the recognition of self and non-self MHC molecules. J Immunol 156: 2473–2480.MH NewbergDH SmithSB HaertelDR ViningE. Lacy1996Importance of MHC class 1 alpha2 and alpha3 domains in the recognition of self and non-self MHC molecules.J Immunol15624732480
  30. 30. Ly LV, Sluijter M, Versluis M, Luyten GP, van Stipdonk MJ, et al. (2010) Peptide vaccination after T-cell transfer causes massive clonal expansion, tumor eradication, and manageable cytokine storm. Cancer Res 70: 8339–8346.LV LyM. SluijterM. VersluisGP LuytenMJ van Stipdonk2010Peptide vaccination after T-cell transfer causes massive clonal expansion, tumor eradication, and manageable cytokine storm.Cancer Res7083398346
  31. 31. Melnick A, Licht JD (1999) Deconstructing a disease: RARalpha, its fusion partners, and their roles in the pathogenesis of acute promyelocytic leukemia. Blood 93: 3167–3215.A. MelnickJD Licht1999Deconstructing a disease: RARalpha, its fusion partners, and their roles in the pathogenesis of acute promyelocytic leukemia.Blood9331673215
  32. 32. Cerundolo V, Elliott T, Elvin J, Bastin J, Townsend A (1992) Association of the human invariant chain with H-2 Db class I molecules. Eur J Immunol 22: 2243–2248.V. CerundoloT. ElliottJ. ElvinJ. BastinA. Townsend1992Association of the human invariant chain with H-2 Db class I molecules.Eur J Immunol2222432248
  33. 33. Sugita M, Brenner MB (1995) Association of the invariant chain with major histocompatibility complex class I molecules directs trafficking to endocytic compartments. J Biol Chem 270: 1443–1448.M. SugitaMB Brenner1995Association of the invariant chain with major histocompatibility complex class I molecules directs trafficking to endocytic compartments.J Biol Chem27014431448
  34. 34. Harris PE, Maffei A, Colovai AI, Kinne J, Tugulea S, et al. (1996) Predominant HLA-class II bound self-peptides of a hematopoietic progenitor cell line are derived from intracellular proteins. Blood 87: 5104–5112.PE HarrisA. MaffeiAI ColovaiJ. KinneS. Tugulea1996Predominant HLA-class II bound self-peptides of a hematopoietic progenitor cell line are derived from intracellular proteins.Blood8751045112
  35. 35. Rammensee HG, Friede T, Stevanoviic S (1995) MHC ligands and peptide motifs: first listing. Immunogenetics 41: 178–228.HG RammenseeT. FriedeS. Stevanoviic1995MHC ligands and peptide motifs: first listing.Immunogenetics41178228
  36. 36. Sette A, Sidney J (1999) Nine major HLA class I supertypes account for the vast preponderance of HLA-A and -B polymorphism. Immunogenetics 50: 201–212.A. SetteJ. Sidney1999Nine major HLA class I supertypes account for the vast preponderance of HLA-A and -B polymorphism.Immunogenetics50201212
  37. 37. Lampen MH, Verweij MC, Querido B, van der Burg SH, Wiertz EJ, et al. (2010) CD8+ T cell responses against TAP-inhibited cells are readily detected in the human population. J Immunol 185: 6508–6517.MH LampenMC VerweijB. QueridoSH van der BurgEJ Wiertz2010CD8+ T cell responses against TAP-inhibited cells are readily detected in the human population.J Immunol18565086517
  38. 38. Seidel UJ, Oliveira CC, Lampen MH, van Hall T (2012) A novel category of antigens enabling CTL immunity to tumor escape variants: Cinderella antigens. Cancer Immunol Immunother 61: 119–125.UJ SeidelCC OliveiraMH LampenT. van Hall2012A novel category of antigens enabling CTL immunity to tumor escape variants: Cinderella antigens.Cancer Immunol Immunother61119125
  39. 39. van Hall T, Wolpert EZ, van Veelen P, Laban S, van der Veer M, et al. (2006) Selective cytotoxic T-lymphocyte targeting of tumor immune escape variants. Nat Med 12: 417–424.T. van HallEZ WolpertP. van VeelenS. LabanM. van der Veer2006Selective cytotoxic T-lymphocyte targeting of tumor immune escape variants.Nat Med12417424
  40. 40. van Luijn MM, van den Ancker W, Chamuleau ME, Zevenbergen A, Westers TM, et al. (2011) Absence of class II-associated invariant chain peptide on leukemic blasts of patients promotes activation of autologous leukemia-reactive CD4+ T cells. Cancer Res 71: 2507–2517.MM van LuijnW. van den AnckerME ChamuleauA. ZevenbergenTM Westers2011Absence of class II-associated invariant chain peptide on leukemic blasts of patients promotes activation of autologous leukemia-reactive CD4+ T cells.Cancer Res7125072517
  41. 41. Reber AJ, Turnquist HR, Thomas HJ, Lutz CT, Solheim JC (2002) Expression of invariant chain can cause an allele-dependent increase in the surface expression of MHC class I molecules. Immunogenetics 54: 74–81.AJ ReberHR TurnquistHJ ThomasCT LutzJC Solheim2002Expression of invariant chain can cause an allele-dependent increase in the surface expression of MHC class I molecules.Immunogenetics547481
  42. 42. Vigna JL, Smith KD, Lutz CT (1996) Invariant chain association with MHC class I: preference for HLA class I/beta 2-microglobulin heterodimers, specificity, and influence of the MHC peptide-binding groove. J Immunol 157: 4503–4510.JL VignaKD SmithCT Lutz1996Invariant chain association with MHC class I: preference for HLA class I/beta 2-microglobulin heterodimers, specificity, and influence of the MHC peptide-binding groove.J Immunol15745034510
  43. 43. Powis SJ (2006) CLIP-region mediated interaction of Invariant chain with MHC class I molecules. FEBS Lett 580: 3112–3116.SJ Powis2006CLIP-region mediated interaction of Invariant chain with MHC class I molecules.FEBS Lett58031123116
  44. 44. Weenink SM, Milburn PJ, Gautam AM (1997) A continuous central motif of invariant chain peptides, CLIP, is essential for binding to various I-A MHC class II molecules. Int Immunol 9: 317–325.SM WeeninkPJ MilburnAM Gautam1997A continuous central motif of invariant chain peptides, CLIP, is essential for binding to various I-A MHC class II molecules.Int Immunol9317325
  45. 45. Veenstra H, Jacobs P, Dowdle EB (1996) Abnormal association between invariant chain and HLA class II alpha and beta chains in chronic lymphocytic leukemia. Cell Immunol 171: 68–73.H. VeenstraP. JacobsEB Dowdle1996Abnormal association between invariant chain and HLA class II alpha and beta chains in chronic lymphocytic leukemia.Cell Immunol1716873
  46. 46. Moller P, Koretz K, Schlag P, Momburg F (1991) Frequency of abnormal expression of HLA-A,B,C and HLA-DR molecules, invariant chain, and LFA-3 (CD58) in colorectal carcinoma and its impact on tumor recurrence. Int J Cancer: Suppl 6155–162.P. MollerK. KoretzP. SchlagF. Momburg1991Frequency of abnormal expression of HLA-A,B,C and HLA-DR molecules, invariant chain, and LFA-3 (CD58) in colorectal carcinoma and its impact on tumor recurrence.Int JCancerSuppl 6155162
  47. 47. Basha G, Omilusik K, Chavez-Steenbock A, Reinicke AT, Lack N, et al. (2012) A CD74-dependent MHC class I endolysosomal cross-presentation pathway. Nat Immunol 13: 237–245.G. BashaK. OmilusikA. Chavez-SteenbockAT ReinickeN. Lack2012A CD74-dependent MHC class I endolysosomal cross-presentation pathway.Nat Immunol13237245