Engineered Antigen- Presenting Cells Expressing Human HLA Class-II DR Alleles Support Efficient Enrichment and Expansion of Antigen Specific Th1 CD4[+] T-Cells Which Are Functionally Augmented by IL-15Rα/IL-15 Complexes

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1905-1905
Author(s):  
Aisha N. Hasan ◽  
Rosanna J. Ricafort ◽  
Annamalai Selvakumar ◽  
Ekaterina Doubrovina ◽  
Isabelle Riviere ◽  
...  
Keyword(s):  
T Cells ◽  
Peptide Pool ◽  
Antigen Presenting Cells ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Class I ◽  
Invariant Chain ◽  
Antigen Presenting ◽  
Nih 3T3

Abstract Abstract 1905 Previous studies have affirmed the therapeutic efficacy of adoptively transferred antigen specific CD8+ and CD4+ T-cells (TC) against viral infections and tumors. A major challenge in optimizing this approach is to develop strategies to permit generation of CD4+ and long lived CD8+ TC of defined antigen specificity. We previously described a panel of NIH 3T3 based artifical antigen presenting cells (AAPC) for the immediate generation of HLA class-I restricted CMVpp65 specific CD8+ TC. We now describe a panel of NIH 3T3 based AAPC, each transduced to express a shared HLA DRA 0101 alpha chain and one of the following β chains of the human HLA class-II alleles DRB1 0301, 0701, 1501, 0401 and 1101. At least one of these alleles is inherited by 61% and 59% of caucasians and blacks respectively. These AAPCs were also transduced to co-express the human TC costimulatory molecules B7.1, ICAM-1 and LFA-3. Sensitization of TC from seropositive donors in the presence of IL-2 with AAPCs sharing one of these alleles, either loaded with a CMVpp65 peptide pool or transduced to express the CMV pp65 protein, resulted in 33–71 fold expansion of CMVpp65 specific CD4+ TC that exhibited a Th1 cytokine profile, producing TNF-α and IFNγ in response to the same CMVpp65 epitopes. These TC were also cytotoxic against peptide loaded HLA class-II sharing targets. Epitope mapping demonstrated that the HLA DRB1 0301 restricted TC responded to a CMVpp65 epitope known to be presented by this allele QEFFWDANDIY (aa 509–527) and to an unreported epitope DVEEDLTMTRN (aa 245–263). The DRB1 0701 restricted CD4+ TC responded to 4 different epitopes. Two of these also included nonamer peptide sequences previously reported to be presented by HLA class-I alleles; Q IFLEV QAIRE and PQYSEH PTFTS presented by HLA B44, and a third AGILARNLVPM, contained an epitope presented by HLA B0801. The fourth epitope, KYQEFFWDANDIY is known to be also presented by HLA DRB1 0301. The DRB1 1501 restricted CD4+ TC were also responsive to a known class-II epitope LLQTGIHVRVS (aa 37–55) as well as a new epitope LVSQYTPDSTP (aa 53–71). CD4+ TC from 3 donors also responded to CMVpp65 when sensitized with autologous DCs loaded with CMVpp65 peptide pool, and each recognized the same epitopes as TC sensitized with the class-II AAPCs. Supplementation of TC cultures with soluble IL-15Rα/ IL-15 complexes markedly augmented the proportion of IFNg+ CD4+ TC, while increasing concentrations of IL-2 resulted in generation of Th2 type CD4+ TC generating IL-4, IL-5 and IL-2 in response to re-stimulation with CMVpp65 peptides. This system can therefore be harnessed by cytokine modulation to selectively generate CD4+ TC with a Th1, or Th2 cytokine profile. The fact that the class-II AAPC transduced to express the full sequence of CMVpp65 are able to process and present antigenic epitopes on the surface of the expressed HLA class-II allele in the absence of the human invariant chain and HLA-DM suggests that the mouse 3T3 cells contain sufficiently homologous proteins to permit the transfer of processed peptides to human Class-II alleles for presentation. Alternatively, invariant chain independent pathways could permit delivery of certain immunogenic epitopes to the expressed class II HLA alleles. The repertoire of epitopes presented by the Class-II AAPCs with or without the invariant chain is currently under study. Our results suggest that the panel of AAPCs expressing these HLA DRB1 alleles provides a novel and rapid approach for the generation of Th1 CD4+ virus-specific TC of desired HLA class-II restriction for adoptive therapy of CMV disease to foster lasting immunity with co-infused CMVpp65-specific CD8+ TC. Class-II AAPCs used with different concentrations or types of cytokines may also be useful to generate other functional subsets of CD4+ TC to promote tolerance or enhance tumor- specific immunity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mapping the HLA Ligandome Landscape of Chronic Myeloid Leukemia (CML)—Towards Peptide Based Immunotherapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4518-4518 ◽  
Author(s):  
Daniel J. Kowalewski ◽  
Mirle Schemionek ◽  
Lothar Kanz ◽  
Helmut R. Salih ◽  
Tim H. Brümmendorf ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Myeloid Leukemia ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Class I ◽  
Peptide Vaccination ◽  
Hla Ligands ◽  
Hla Ligandome

Abstract Despite the success of targeted therapy with tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) remains largely incurable. Immunotherapy, and in particular multi-peptide vaccination, may be a promising approach to eliminate residual CML cells. As of now, a multitude of potential vaccine targets have been proposed based on reverse immunology and functional genomic approaches focusing either on BCR-ABL junction peptides, which represent CML-specific neo-antigens, or on aberrantly expressed self-proteins such as WT-1, PR and hTERT. However, the results of clinical studies employing such antigens have so far not been encouraging. This might in part be due to the inherent limitations of the above mentioned approaches: evidence of natural presentation of the predicted epitopes is lacking and the correlation of transcript abundance and HLA restricted presentation of the corresponding gene product has been shown to be skewed. Modern mass spectrometry, on the other hand, enables the comprehensive analysis of the entirety of naturally presented HLA ligands on tissues of interest, termed the HLA ligandome. Here we implemented this direct approach and comparatively mapped the HLA ligandome landscape of 16 primary CML samples and 40 healthy volunteer (HV) controls (30 blood and 10 bone marrow samples). We identified more than 30,000 different naturally presented HLA class I ligands representing ~10,000 source proteins. Regression analysis suggests source protein identifications on CML (4,337 different proteins) to attain >95% of maximum achievable coverage with the implemented analytical setup. Based on this extensive dataset, we investigated the HLA restricted presentation of established CML-associated/specific antigens and applied a novel approach defining tumor-associated antigens strictly based on exclusive and frequent representation in CML ligandomes. Strikingly, we found the vast majority of previously described antigens including wild-type BCR protein (6% CML, 5% HV), Myeloperoxidase (56% CML, 15% HV) and Proteinase 3 (38% CML, 11% HV) to be (also) represented on normal PBMC or BMNC. No evidence of naturally presented BCR-ABL junction peptides was found. However, we identified a panel of 7 LiTAAs (ligandome-derived tumor-associated antigens) represented by 16 different HLA ligands, showing CML-exclusive representation in ≥25% of CML patient ligandomes. As CD4+ T cells mediate important indirect and direct effects in anti-tumor immunity, we further applied our approach to HLA class II ligandomes of 15 CML patients and 18 HV (13 blood and 5 bone marrow samples), identifying more than 9,000 different naturally presented HLA class II ligands (1,900 source proteins). Applying the same antigen-ranking strategy as described for HLA class I, we identified 7 additional HLA class II LiTAAs represented by 50 corresponding LiTAPs (ligandome-derived tumor-associated peptides). Overlap analysis of CML-exclusive source proteins revealed 6 proteins to be represented both in HLA class I and II ligandomes. Notably, for Galectin-1, which shows CML-exclusive representation in 19% of HLA class I and 13% of HLA class II ligandomes, one of the HLA class II ligands was found to contain a complete, embedded HLA class I peptide. Such naturally presented embedded HLA ligands might present optimal vaccine candidates that are recognized by both, CD4+ and CD8+ T cells. Functional analysis of the here defined HLA class I and II LiTAPs with regard to induction of T cell responses is presently ongoing and serves to validate them as prime targets for the development of an off-the-shelf peptide vaccination in CML patients. Disclosures No relevant conflicts of interest to declare.

Identification of Four New HLA Class II Restricted Minor Histocompatibility Antigens Contributing to Graft Versus Leukemia Reactivity.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3247-3247
Author(s):  
Anita N. Stumpf ◽  
Edith D. van der Meijden ◽  
Cornelis A.M. van Bergen ◽  
Roelof Willemze ◽  
J.H. Frederik Falkenburg ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Cd4 T Cell ◽  
Class I ◽  
T Cell Clones ◽  
Cell Clones

Abstract Patients with relapsed hematological malignancies after HLA-matched hematopoietic stem cell transplantation (HSCT) can be effectively treated with donor lymphocyte infusion (DLI). Donor-derived T cells mediate beneficial graft-versus-leukemia (GvL) effect but may also induce detrimental graft-versus-host disease (GvHD). These T cell responses are directed against polymorphic peptides which differ between patient and donor due to single nucleotide polymorphisms (SNPs). These so called minor histocompatibility antigens (mHag) are presented by HLA class I or II, thereby activating CD8+ and CD4+ T cells, respectively. Although a broad range of different HLA class I restricted mHags have been identified, we only recently characterized the first autosomal HLA class II restricted mHag phosphatidylinositol 4-kinase type 2 beta (LB-PI4K2B-1S; PNAS, 2008, 105 (10), p.3837). As HLA class II is predominantly expressed on hematopoietic cells, CD4+ T cells may selectively confer GvL effect without GvHD. Here, we present the molecular identification of four new autosomal HLA class II restricted mHags recognized by CD4+ T cells induced in a patient with relapsed chronic myeloid leukemia (CML) after HLAmatched HSCT who experienced long-term complete remission after DLI with only mild GvHD of the skin. By sorting activated CD4+ T cells from bone marrow mononuclear cells obtained 5 weeks after DLI, 17 highly reactive mHag specific CD4+ T cell clones were isolated. Nine of these T cell clones recognized the previously described HLADQ restricted mHag LB-PI4K2B-1S. The eight remaining T cell clones were shown to exhibit five different new specificities. To determine the recognized T cell epitopes, we used our recently described recombinant bacteria cDNA library. This method proved to be extremely efficient, since four out of five different specificities could be identified as new HLA-class II restricted autosomal mHags. The newly identified mHags were restricted by different HLA-DR molecules of the patient. Two mHags were restricted by HLA-DRB1 and were found to be encoded by the methylene-tetrahydrofolate dehydrogenase 1 (LBMTHFD1- 1Q; DRB1*0301) and lymphocyte antigen 75 (LB-LY75-1K; DRB1*1301) genes. An HLA-DRB3*0101 restricted mHag was identified as LB-PTK2B-1T, which is encoded by the protein tyrosine kinase 2 beta gene. The fourth mHag LB-MR1-1R was restricted by HLA-DRB3*0202 and encoded by the major histocompatibility complex, class I related gene. All newly identified HLA class II restricted mHags exhibit high population frequencies of 25% (LB-MR1-1R), 33% (LB-LY75-1K), 68% (LB-MTHFD1- 1Q), and 70% (LB-PTK2B-1T) and the genes encoding these mHags show selective (LY- 75) or predominant (MR1, MTHFD1, PTK2B) expression in cells of hematopoietic origin as determined by public microarray databases. All T cell clones directed against the newly identified mHags recognized high HLA class II-expressing B-cells, mature dendritic cells (DC) and in vitro cultured leukemic cells with antigen-presenting phenotype. The clone recognizing LB-MTHFD1-1Q also showed direct recognition of CD34+ CML precursor cells from the patient. In conclusion, we molecularly characterized the specificity of the CD4+ T cell response in a patient with CML after HLA-matched HSCT who went into long-term complete remission after DLI. By screening a recombinant bacteria cDNA library, four new different CD4+ T cell specificities were characterized. Our screening method and results open the possibility to identify the role of CD4+ T cells in human GvL and GvHD, and to explore the use of hematopoiesis- and HLA class II-restricted mHag specific T cells in the treatment of hematological malignancies.

scholarly journals Uptake of HLA Alloantigens via CD89 and CD206 Does Not Enhance Antigen Presentation by Indirect Allorecognition

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Eytan Breman ◽  
Jurjen M. Ruben ◽  
Kees L. Franken ◽  
Mirjam H. M. Heemskerk ◽  
Dave L. Roelen ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Antigen Presentation ◽  
Antigen Presenting Cells ◽  
Mannose Receptor ◽  
Class Ii ◽  
Hla Class Ii ◽  
Indirect Allorecognition ◽  
Antigen Presenting

In organ transplantation, alloantigens are taken up by antigen presenting cells and presented via the indirect pathway to T-cells which in turn can induce allograft rejection. Monitoring of these T-cells is of major importance; however no reliable assay is available to routinely monitor indirect allorecognition. Recently we showed that HLA monomers can be successfully used to monitor indirect allorecognition. Targeting antigens to endocytic receptors on antigen presenting cells may further enhance the presentation of antigens via HLA class II and improve the efficiency of this assay. In the current study we explored targeting of HLA monomers to either CD89 expressing monocytes or mannose receptor expressing dendritic cells. Monomer-antibody complexes were generated using biotin-labeled monomers and avidin labeling of the antibodies. We demonstrate that targeting the complexes to these receptors resulted in a dose-dependent HLA class II mediated presentation to a T-cell clone. The immune-complexes were efficiently taken up and presented to T-cells. However, the level of T-cell reactivity was similar to that when only exogenous antigen was added. We conclude that HLA-A2 monomers targeted for presentation through CD89 on monocytes or mannose receptor on dendritic cells lead to proper antigen presentation but do not enhance indirect allorecognition via HLA-DR.

scholarly journals Invariant chain modulates HLA class II protein recycling and peptide presentation in nonprofessional antigen presenting cells

2007 ◽  
Vol 249 (1) ◽  
pp. 20-29 ◽  
Author(s):  
Azizul Haque ◽  
Laela M. Hajiaghamohseni ◽  
Ping Li ◽  
Katherine Toomy ◽  
Janice S. Blum
Keyword(s):  
Antigen Presenting Cells ◽  
Class Ii ◽  
Hla Class Ii ◽  
Invariant Chain ◽  
Peptide Presentation ◽  
Antigen Presenting

scholarly journals Antigen identification for HLA class I– and HLA class II–restricted T cell receptors using cytokine-capturing antigen-presenting cells

2021 ◽  
Vol 6 (55) ◽  
pp. eabf4001
Author(s):  
Mark N. Lee ◽  
Matthew Meyerson
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Human Leukocyte ◽  
Antigen Presenting Cells ◽  
Hla Class I ◽  
Class Ii ◽  
Class I ◽  
Identification System ◽  
Antigen Presenting

A major limitation to understanding the associations of human leukocyte antigen (HLA) and CD8+ and CD4+ T cell receptor (TCR) genes with disease pathophysiology is the technological barrier of identifying which HLA molecules, epitopes, and TCRs form functional complexes. Here, we present a high-throughput epitope identification system that combines capture of T cell–secreted cytokines by barcoded antigen-presenting cells (APCs), cell sorting, and next-generation sequencing to identify class I– and class II–restricted epitopes starting from highly complex peptide-encoding oligonucleotide pools. We engineered APCs to express anti-cytokine antibodies, a library of DNA-encoded peptides, and multiple HLA class I or II molecules. We demonstrate that these engineered APCs link T cell activation–dependent cytokines with the DNA that encodes the presented peptide. We validated this technology by showing that we could select known targets of viral epitope–, neoepitope-, and autoimmune epitope–specific TCRs, starting from mixtures of peptide-encoding oligonucleotides. Then, starting from 10 TCRβ sequences that are found commonly in humans but lack known targets, we identified seven CD8+ or CD4+ TCR-targeted epitopes encoded by the human cytomegalovirus (CMV) genome. These included known epitopes, as well as a class I and a class II CMV epitope that have not been previously described. Thus, our cytokine capture–based assay makes use of a signal secreted by both CD8+ and CD4+ T cells and allows pooled screening of thousands of encoded peptides to enable epitope discovery for orphan TCRs. Our technology may enable identification of HLA-epitope-TCR complexes relevant to disease control, etiology, or treatment.

scholarly journals Impaired HLA-class-I stability in a sarcoma cell line which stimulates exclusively HLA-class-II-restricted autologous T cells

1996 ◽  
Vol 67 (5) ◽  
pp. 743-748 ◽  
Author(s):  
Michael Heike ◽  
Ute Schmitt ◽  
Alexandra Höhne ◽  
Christoph Huber ◽  
Karl-Hermann Meyer zum Büschenfelde ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Line ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Class I ◽  
Sarcoma Cell ◽  
Sarcoma Cell Line

Processing of Filamentous Bacteriophage Virions in Antigen-Presenting Cells Targets Both HLA Class I and Class II Peptide Loading Compartments

2003 ◽  
Vol 22 (1) ◽  
pp. 11-18 ◽  
Author(s):  
Muriel Gaubin ◽  
Cristina Fanutti ◽  
Zohar Mishal ◽  
Antoine Durrbach ◽  
Piergiuseppe De Berardinis ◽  
...  
Keyword(s):  
Antigen Presenting Cells ◽  
Hla Class I ◽  
Class Ii ◽  
Class I ◽  
Filamentous Bacteriophage ◽  
Peptide Loading ◽  
Antigen Presenting

scholarly journals The HLA Ligandome Landscape of Chronic Myeloid Leukemia Delineates Novel T-Cell Epitopes for Immunotherapy

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4243-4243
Author(s):  
Tatjana Bilich ◽  
Annika Nelde ◽  
Leon Bichmann ◽  
Helmut R. Salih ◽  
Daniel Johannes Kowalewski ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Class I ◽  
Cell Responses ◽  
Tki Treatment

Abstract Chronic myeloid leukemia (CML) is characterized by the translocation t(9;22), which leads to the formation of the BCR-ABL fusion transcript. Several approved tyrosine kinase inhibitors (TKIs) target the resulting fusion protein, leading to an improved prognosis of CML patients. Currently, the main treatment goal is the achievement of a deep molecular response (MR), in which TKI therapy can be terminated. Several studies provide evidence that immunological control plays a major role for the course of CML and contributes to the achievement of deep MR in CML patients under TKI treatment (CMLTKI). This implies that reinforcing these immune responses might sustain long-term TKI-free survival or even cure for CML patients. Besides unspecific immunotherapy, such as interferon or immune checkpoint blocking antibodies, a more specific and minor side effect targeting of CML cells might be achieved by antigen-specific immunotherapy approaches. The prerequisite for such strategies is the identification of T-cell targets represented by tumor-associated human leukocyte antigen (HLA)-presented peptides on malignant cells. In this study, we used a mass spectrometry-based approach to identify naturally presented, CML-associated peptides in primary CML samples (HLA class I, n=21, 11,945 peptides, 5,478 source proteins; class II, n=20, 5,991 peptides, 1,302 proteins). Comparative HLA peptidome profiling using a comprehensive dataset of various benign tissues (e.g. blood, bone marrow, spleen, and lung) revealed frequently presented and strictly CML-associated antigens. In detail, the benign tissue dataset comprises hematological benign samples (class I, n=108, 51,233 peptides, 11,437 proteins; class II, n=88, 42,753 peptides, 4,877 proteins) and non-hematological benign tissues (28 tissues, n=166; class I, 128,590 peptides, 16,405 proteins; class II, 143,652 peptides, 13,410 proteins). We identified 50 CML-associated, HLA class I-restricted peptides with HLA allotype adjusted representation frequencies of ≥38% presented on HLA-A*02, -A*03, -A*11, and -B*07. HLA class II comparative profiling delineated 36 peptides exclusively and frequently presented in the HLA peptidome of ≥20% analyzed CML patients. For immunological characterization, we selected 8 HLA class I- and 6 class II-restricted highly CML-associated antigens. These peptides were analyzed in IFNγ ELISPOT assays using PBMCs from CMLTKI patients and healthy volunteers (HVs). Peptide-specific immune recognition was detected for 1/8 (13%) HLA class I peptides in 2/17 (12%) of CMLTKI patients. We hypothesized that this weak immune response might be due to an impaired CD8+ T cell function that reportedly is caused by TKI treatment. Thus, we compared T-cell responses against viral epitopes in IFNγ ELISPOT assays of CMLTKI patients with that of HVs and chronic lymphocytic leukemia (CLL) patients: in line with our hypothesis, we observed significantly reduced IFNγ release of T cells from CMLTKI patients compared to HVs and CLL patients (p<0.001), whereas CD8+ T-cell counts were not reduced. In contrast, no reduced IFNγ production was observed for HLA class II-restricted viral epitopes. These results were confirmed by memory T-cell responses detected for 6/6 (100%) HLA class II CML-associated peptides with frequencies up to 24% (4/17) of analyzed CMLTKI patients. To assess the immunogenicity of all HLA class I peptides, we performed in vitro artificial antigen presenting cell-based priming experiments using CD8+ T cells of HVs and CML patients. Effective priming of T cells was observed for 8/8 CML-associated peptides in ≥70% of analyzed HVs with frequencies of 0.1-33.9% (mean 2.2%) of CD8+ peptide-specific T cells. Notably, peptide-specific CD8+ T cells with frequencies of 0.1-2.2% (mean 0.4%) could also be induced in samples of CMLTKI patients that had not displayed preexisting immune responses. For 6/8 peptides, we observed multifunctionality of peptide-specific T cells by IFNγ and TNF production as well as upregulation of the degranulation marker CD107a. Cytotoxicity assays with polyclonal peptide-specific effector T cells confirmed the capacity to induce antigen-specific lysis for 3/4 analyzed peptides. Taken together, we here identified novel, naturally presented, CML-associated antigens and validated them as promising targets for tailored T cell-based immunotherapeutic approaches for CML patients. Disclosures Salih: Several patent applications: Patents & Royalties: e.g. EP3064507A1. Kowalewski:Immatics Biotechnologies GmbH: Employment. Schuster:Immatics Biotechnologies GmbH: Employment. Brümmendorf:Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Takeda: Consultancy. Niederwieser:Miltenyi: Speakers Bureau; Novartis: Research Funding.

scholarly journals Predominant HLA-class II bound self-peptides of a hematopoietic progenitor cell line are derived from intracellular proteins

Blood ◽  
1996 ◽  
Vol 87 (12) ◽  
pp. 5104-5112 ◽  
Author(s):  
PE Harris ◽  
A Maffei ◽  
AI Colovai ◽  
J Kinne ◽  
S Tugulea ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Mhc Class Ii ◽  
Antigen Processing ◽  
Antigen Presenting Cells ◽  
Class Ii ◽  
Hla Class Ii ◽  
Invariant Chain ◽  
Hla Dr ◽  
Antigen Presenting ◽  
Self Peptides

Human myeloid progenitor cells temporarily express HLA class II molecules during the differentiation pathway to granulocytes and macrophages. The significance of major histocompatibility complex (MHC) class II molecules at this stage of development is unknown. As a first stop of inquiry into their function, we have characterized the profile of major self-peptides bound to the HLA-DR molecules expressed by KG-1 cells, a line that shares many of the phenotypic characteristics of colony-forming unit-granulocyte-macrophage progenitors. Searches of protein data bases showed that all matching peptides bound to the HLA- DR molecules of KG-1 cells corresponded to intracellular, rather than exogenous or transmembrane, precursor proteins. Because the absence of a conventional self-peptide repertoire could be related to altered trafficking of class II molecules, the biosynthesis of HLA-DR and the invariant chain proteins was determined. The MHC class II associated invariant chain protein is synthesized normally in KG-1 cells, but processed fragments of invariant chain, class II-associated invariant chain peptides (CLIPs), occupy the antigen-binding groove of KG-1 class II molecules at a much lower frequency compared with that of mature antigen-presenting cells. Low CLIP occupancy of HLA-DR is a characteristic shared by KG-1 cells, normal CD34+ progenitor cells, and HLA-DR+ breast carcinoma cells. The unusual profile of MHC class II bound peptides and the low level of CLIP bound to HLA-DR suggest that the antigen-processing pathway of KG-1 is different from that characterized in professional antigen-presenting cells and that exogenous antigen-processing may be a developmentally acquired characteristic in the myeloid lineage.

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