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Blood, Vol. 95 No. 9 (May 1), 2000:
pp. 2890-2896
IMMUNOBIOLOGY
From the Medical and Natural Sciences Research Center, Section for
Transplantation Immunology and Immunohaematology, University of
Tübingen, Tübingen, Germany; Institute of Physiological
Chemistry, University of Bochum, Bochum, Germany.
The HLA-DR-associated peptides from peripheral blood mononuclear
cells of 2 patients with plasmacytoma and 1 with chronic myeloid
leukemia were isolated, identified, and compared. Several were
identified as derivatives of the defensin family. Defensins (or human
neutrophil peptides [HNP]) are antimicrobial, cationic peptides of 29 to 35 amino acids in length and are the major constituents of the
azurophilic granules of human neutrophils. Using peripheral blood cells
from leukapheresis, containing about 90% of polymorphonuclear cells,
we could identify HNP-1, -2, and -4 and propeptides of up to 49 amino
acids in length, eluted from HLA class II molecules. Binding of
isolated and synthetic defensin peptides to various HLA-DR alleles
using an in vitro binding/competition assay based on size exclusion
chromatography revealed that defensin may bind into the peptide-binding
groove. In a T-cell competition assay, defensins were able to reduce
the proliferation of an HLA-DR-restricted T-cell line after
preincubation of stimulating cells (CHO-DRB1*0401 transfectants) with
defensin. Therefore, binding of defensins might prevent T-cell
recognition of HLA class II molecules expressed on different blood
precursor cells (all of which are "nonprofessional" antigen-presenting cells) by blocking the HLA peptide-binding groove
or, alternatively, might protect defensin-expressing cells from
self-destruction.
(Blood. 2000;95:2890-2896)
Only antigen-presenting cells (APC), which express
major histocompatibility complex (MHC) class II molecules, are able to present a great variety of peptide antigens derived from proteins entering the endocytic pathway to CD4+ T cells.
Constitutive MHC class II gene expression is not only tightly
restricted to APC but is also under developmental control. Cells of the
B-cell lineage acquire the capacity to express MHC class II genes early
during ontogeny but normally lose this property during terminal
differentiation into plasma cells.1 This phenomenon is due
to a silencing of the transactivator gene CIITA.2
Little is known about the function of these temporarily expressed MHC class II molecules. Recently, Harris et al3 reported on MHC class II-associated self-peptides from the hematopoietic progenitor cell line KG-1. This cell line, originating from a patient with acute
myelogenous leukemia, is believed to be derived from an oligopotent
myeloid progenitor cell and is morphologically heterogeneous, containing myeloblasts and, to a lesser extent, promyelocytes, myelocytes, granulocytes, macrophages, and eosinophils. A number of
HLA-DR-associated peptides, mainly derived from intracellular rather
than from exogenous or transmembrane protein sources, was identified.3 Furthermore, KG-1 showed a much lower
frequency of class II-associated invariant chain peptides (CLIP) on the plasma membrane, compared to professional APCs. Therefore, the authors
suggested that exogenous antigen processing may be a developmentally acquired characteristic in the myeloid lineage. Another paper from the
same group4 describes naturally processed peptides bound by
HLA-DR1 or 3 (or both) from the CD34+ blast cells of a
patient with chronic myeloid leukemia (CML). The authors identified a
panel of peptides from different protein sources stemming from
membrane-associated, luminal, but also cytoplasmic proteins. The
peptide lengths varied between 13 and 18 amino acids and 3 of them
seemed to be tissue specific. Two of them, namely MRP14 and
leukotriene-B4-omega-hydrolase, were derived from proteins specific for
myeloid cells; another one, granzyme H, was specific for T lymphocytes
and natural killer cells.
To characterize HLA class II-associated peptides stemming directly from
patient material, we studied polymorphonuclear cells (PMNs) from a
patient with CML and from 2 patients with plasmacytoma. Surprisingly,
we found a very high amount of peptides derived from the defensin
family associated with HLA-DR molecules from these cell sources.
Defensins or human neutrophil peptides (HNP) are highly potent
antimicrobial peptides of about 30 amino acids in length, effective against a wide variety of bacteria, many fungi, some enveloped viruses,
and even a wide range of normal and malignant mammalian target cells
(for reviews, see Lehrer et al5, Ganz and
Lehrer6, and Hancock7). They function by
inserting into various cell membranes due to their ability to
aggregate, forming voltage-regulated channels.8,9
Defensins, which are released from stimulated neutrophils, show further
cytotoxic activity to various autologous cells,10
induction of histamine release by mast cells,11 and chemotactic activity for monocytes and T cells.12,13
Recently, it has been shown that defensins may influence complement
activation by binding to C1q.14,15 Defensin has also been
shown to stimulate the binding of lipoprotein (a) to human vascular
endothelial and smooth muscle cells.16 Very recently, it
was reported that defensins are able to enhance serum IgG antibody
responses in mice after intranasal delivery.17 The authors
showed further that defensins enhanced both proliferative responses and
T-helper cytokine secretion profiles of naive CD4+ T cells.
Peptides of the defensin family are highly expressed in promyeloic
cells and constitute more than 5% of the total cellular protein in
human and rabbit PMN.6 Furthermore, defensins are frequently found in Paneth cells, platelets, and macrophages, as well
as in mammalian trachea, intestine, and tongue.7 Therefore, members of the defensin family seem to be important components of the
innate immune system of a large number of mammals (and also of some
plants), active against microbial infections. These findings support
the hypothesis that different classes of antimicrobial peptides play an
important role in the natural defense against microbes.
Leukapheresis and cell preparation
High-performance liquid chromatography (HPLC) Nanospray electrospray ionization mass spectrometry (ESI-MS) of defensin For exact mass determination, ESI-MS spectrometry was performed with a TSQ 7000 mass spectrometer (Finnigan, La Jolla, CA) equipped with a nano electrospray (nano ESI) ion source. Nanospray capillaries were obtained from PROTANA (Odense, Denmark). An HPLC fraction (0.5 µL) was used for nano ESI mass spectrometry.Defensin isolation from the promyelocytic human cell line HL60 Defensin isolation was performed initially as described elsewhere,19 using some modifications. Briefly, HL60 cells were cultured in RPMI 1640 with 5% fetal calf serum (FCS) in suspension to collect a total of 1 × 109cells. After harvesting the cells, 10 mL of extraction medium (1 mol/L HCl, 5% [v/v] formic acid, 1% [w/v] NaCl, 1% TFA) was added. Cells were homogenized and pelleted (2000g, 15 minutes, 4°C), and the pellet re-extracted with 3 mL extraction medium. The collected extraction supernatants were then concentrated using a Sep Pac C18 column (Pharmacia, Uppsala, Sweden). After washing with 0.1% TFA, the elution of the bound material was performed stepwise by increasing acetonitrile concentrations: 20% acetonitrile/0.1% TFA, 40% acetonitrile/0.1% TFA, 60% acetonitrile/0.1% TFA, and 80% acetonitrile/0.1% TFA. Each fraction was collected, lyophilized, and subsequently tested for its defensin content by dot blot using the anti-HNP-1,- 2, -3-specific mouse mAb Def-3 (BMA Biomedicals, Augst, Switzerland). Only the fraction eluted with 80% acetonitrile contained defensins. This fraction was further purified by RP-HPLC (column: Vydac C4, 2 × 150 mm, gradient: 0-15% B in 10 minutes, 15-45% B in 20 minutes, 45-70% B in 50 minutes, and 70-100% B in 60 minutes.). All peaks detected at 214 nm were collected and tested for their defensin content by Western blot after running a 16% SDS-PAGE. The detection was performed using the enhanced chemiluminescence method (ECL, Amersham Buchler, Braunschweig, Germany). Most of the HPLC fractions tested were positive for defensin, but different molecular weights corresponding to different precursor forms of defensins were detected. Each fraction containing defensin was analyzed by mass spectrometry (Finnigan MAT), but none of the fractions proved to be homogeneous for one single defensin peptide. However, the fractions contained no other protein contaminants and were more than 95% pure defensin molecules.In vitro binding/competition assay For reasons related to solubility of the material, we were unable to label defensins directly with a fluorescence marker (7-amino-4-methylcoumarin-3-acetic acid [AMCA]). Therefore, to investigate binding/competition of defensins to different solubilized HLA class II alleles, an in vitro binding/competition assay based on gel filtration was used, as described previously.18,20 Briefly, we used fluorescent AMCA-labeled allele-specific binding peptides and immunoaffinity-purified HLA-DR isolates from different homozygous Epstein-Barr virus (EBV)-transformed cell lines (B-LCLs). As competitors, the defensin-containing fraction from HL60 or a synthetic HNP-2 (Bachem, Heidelberg, Germany) was used in different concentrations.Defensin competition assay To determine the competition capacity of isolated defensins on a preformed MHC class II/peptide complex, HLA-DR1 isolate (0.2 µmol/L) was incubated with AMCA-HA 306-18 (1.5 µmol/L) for 24 hours at 37°C in binding buffer. Then defensins (10 and 20 µg, respectively), or unlabeled self-peptides (HA 306-18, IM 19-31, CLIP 80-104, each at a concentration of 30 µmol/L) were added and the competition kinetics were determined using a gel filtration-based competition assay.Association kinetics assay To detect the association kinetics of hemagglutinin 306-18 to HLA-DR1 in the presence of defensin, HLA-DR1 (0.16 µmol/L) was preincubated with 10 µg defensin isolate in binding buffer (0.15 mol/L sodium phosphate, 0.1% [w/v] Zwittergent 3-12, protease inhibitor mix, 15% acetonitrile, pH 5.2) at 37°C for 1 hour or overnight. Then, 1.5 µmol/L AMCA-hemagglutinin 306-318 was added to start the association kinetics. The relative protein-bound AMCA fluorescence intensity was detected at different time points. As a control, the same assay was performed without defensin and in the presence of defensin and HLA-DM.Cell lines and antibodies Epstein-Barr virus-transformed, homozygous B-LCLs WT100BIS (HLA-DRB1*0101), LD2B (HLA-DRB1*1501/DRB5*0101), COX (HLA-DRB1*0301/DRB3*0101), BSM (HLA-DRB1*0401/DRB4*0101), and DBB (HLA-DRB1*0701/DRB4*0101) served as sources for the HLA-DR alleles. The expression of the HLA-DRB3 and DRB4 alleles was low compared to that of the DRB1 alleles. Cells were cultured in RPMI (Life Technologies, Eggenstein, Germany) supplemented with 5% FCS (Life Technologies), 20 mmol/L HEPES, 2 mmol/L glutamine, and antibiotics and propagated in roller bottles to 1 to 2 × 109 cells. The mAb L243, which recognizes a nonpolymorphic determinant present on HLA-DR dimers, was obtained from ATCC. The HNP1-3 recognizing monoclonal mouse antibody Def-3 was obtained from BMA Biomedicals.T-cell proliferation assay The CHO K1 cells were transfected with the genes for HLA-DRB1*0401 and HLA-DRA and cotransfected with the gene coding for CD80 as described previously (kind gifts of Dr D. Sansom, Bath, UK).21 The expression of the human genes was checked by FACS analysis before the cells were used as APCs. For priming of T cells, 1 × 106 PBMC of healthy donors, carrying the HLA-DRB1*0401 allele, were cocultured with 5 × 105 CHO cells (fixed with 0.025% glutaraldehyde for 2 minutes) in 16-mm diameter Costar wells in 2 mL RPMI 1640 supplemented with 10% heat-inactivated human male serum. After 7 days of culture, cells were harvested, washed, and plated at 2 × 104 cells/well into round-bottomed microtiter plates, to which 2.5 × 104 CHO cells as stimulators were added. To be used as stimulators, 5 × 105 CHO cells were preincubated with different concentrations of defensin isolate (none, 10, 20, or 30 µmol/L, respectively) in 500 µL RPMI 1640 for 2 hours at 37°C. Pulsed CHO cells were washed twice, fixed with glutaraldehyde (0.025%, 2 minutes), and washed again. Then they were cocultured with the T cells for 1 or 2 days, respectively, at 37°C. After 24 and 48 hours, 37 kBq of tritiated thymidine (3H-TdR; Amersham-Buchler) was added and cells were harvested after 16 hours on glass fiber filtermats using a semiautomatic cell harvester. T-cell proliferation was determined by 3H-TdR incorporation by liquid scintillation counting. The anti DR-specific mAb L243 was used to block the HLA-DR molecules in this stimulation system. For 2 different kinetics, 2 identical plates were set up, containing all cultures performed in triplicate. The results are expressed as mean counts per minute (cpm).
HLA-DR-bound self-peptides From 9.6 × 109 PBMC (plasmacytoma patient 1; P1) and 1.4 × 1010 PBMC (plasmacytoma patient 2; P2) 690 and 900µg of HLA-DR was isolated, corresponding to 1.15 and 1.5 nmol of MHC protein. In the case of P2, for example, 12 of the most prominent HPLC fractions were directly submitted to the Edman sequencer. A further 14 fractions were sequenced after re-chromatography. The peptide yield of these sequencing runs differed from about 10 to 100 pmol/run for the directly sequenced fractions and from 5 to 20 pmol for the re-chromatographed fractions. Altogether, these 26 fractions contained about 450 pmol of peptides. From these 26 sequencing runs 9 sequences could be clearly identified. From these 9 sequences, 6 were derived from the defensin family. These 6 defensins yielded 110 pmol, which corresponded to 0.24% of the total yield of protein in all 26 fractions. This means that within the high-copy peptides occupying the binding groove of the HLA-DR molecules of this patient, about one fourth were defensins. Table 1 summarizes the peptides identified from the 2 patients with plasmacytoma and the single patient with CML. The most striking feature of the HLA-DR-associated peptides identified from these sources was the predominance of peptides from the defensin family. HNP-1, -2, and -4 and several HNP precursors were identified. HNP-1 and -2 differ only in their N-terminal residue, having an alanine in HNP-1 and no residue in HNP-2. The peptides from the defensin family had lengths from 29 to 49 amino acids with masses in the range of 3400 to 5600 d. This is very unusual for HLA-DR-associated self-peptides, which are usually 13 to 25 amino acids in length.22 Ten of 18 identified peptides were derived from the defensin family; 4 of the remaining sequences stemmed from the CLIP region of the invariant chain. One of the sequences of P2 proved to be the N-terminus of the hemoglobin -chain. Apart from defensin and CLIP, the usual type of
MHC class II-binding peptide of 13 to 25 amino acids was almost completely absent from these isolates. There may possibly be 3 additional peptide sequences in these isolates, but their lengths were
not determined and the database alignment with known sequences was not
perfect. The potential self-peptide candidates may represent sequences
derived from the ribosomal protein S26, from the cell cycle protein
CDC27HS, and from an unknown source.
HLA-DR bound autologous peptides with unusual lengths The length of most of the sequenced peptides could be determined by mass spectrometry. Figure 1 shows an ESI-MS spectrum of HNP from a representative HPLC fraction. The spectrum shows the nested set of HNP-1 and HNP-2 in 3 different charge states [M+2H]2+, [M+3H]3+, and [M+4H]4+. The mean masses deduced from the charge states were 3440.8 d for HNP-1 and 3370.4 d for HNP-2. The theoretical average mass of HNP-1 and HNP-2 are 3439.1 d and 3368.4 d calculated from the deduced sequence including 3 disulfide bonds. The mass difference of both defensins is 70.4 d corresponding just to the N-terminal alanine residue (theoretical average mass: 71 d).
Competition of defensins with allele-specific binding peptides Next, the binding of synthetic HNP-2 and isolated defensins to different solubilized HLA-DR molecules in vitro was investigated. For this purpose, an in vitro binding/competition assay based on gel filtration was used. To be able to use unmodified, native defensin peptides, competition experiments against allele-specific, fluorescently AMCA-labeled binding peptides were performed (Figure 2). The competition curves for the synthetic HNP-2 (Figure 2A) revealed allelic differences, showing that HLA-DR3 possessed highest affinity (IC50 5.2 µmol/L), followed by HLA-DR7 (IC50 16.3 µmol/L). HLA-DR1, 2, and 4, on the other hand, showed nearly identical, but markedly decreased affinities (IC50 about 92.5 µmol/L). Isolated defensins, however, proved to be about 10-fold less effective in their ability to compete on the different HLA-DR alleles (Figure 2B). In this case, the allele specificity was less marked, but still detectable. The results were again consistent with best binding to the HLA-DR3 allele. The IC50 values could not be calculated here because a nonhomogeneous mixture of defensins with no defined mass was used.
Defensins have a slow binding/competition kinetics The competition kinetics of defensins to HLA-DR1-bound AMCA-labeled hemagglutinin (HA) 306-18 peptide were investigated next. After incubating HLA-DR1 with AMCA-HA 306-18 for 24 hours at 37°C, isolated defensins or self-peptides (HA 306-18, influenza matrix protein [IM] 19-31, invariant chain-derived CLIP 80-104) were added and the competition kinetics were determined. As shown in Figure 3, the defensin isolate proved to be an effective competitor for the HLA-DR1 binder AMCA-HA 306-18, with a clear dose-dependent effect. After 70 hours, 45% competition using 10 µg defensin isolate versus 64% competition with 20 µg defensin isolate was determined. Using HA 306-18 as the competitor, 86% competition after 70 hours of incubation was observed. Another known HLA-DR-restricted T-cell epitope, IM 19-31,23 however, showed only 40% competition after 70 hours. CLIP 80-104, which has been shown to be a good HLA-DR1 binder,24 gave 73% competition after 70 hours. Interestingly, defensin showed an even slower competition kinetics compared to the other peptides tested and showed no clear saturation even after 70 hours. Half-maximal competition was achieved after about 16 hours for HA 306-18, 12 hours for CLIP 80-104, 13 hours for IM 19-31, but only after 25 or 29 hours for defensin (10 µmol/L; 20 µmol/L). This phenomenon might point to a complicated binding mechanism due to the unusual structure and length of these peptides.
Decreased binding capacity of peptide antigens to HLA-DR1 after preincubation with defensins Reciprocally, the binding kinetics of AMCA-HA 306-18 to HLA-DR1 after preincubation with defensin isolate for 1 hour or overnight and in the presence of 1.5 µmol/L solubilized HLA-DM were determined. Figure 4 shows that although the absolute amount of bound AMCA-HA peptide was clearly influenced by preincubation with defensins, the binding kinetics were not affected (all samples had the same half-maximal association time [t1/2]). Without preincubation with defensins, the relative protein-bound fluorescence (rF), corresponding to the absolute fluorescence divided by the UV signal at 214 nm (protein) concentration was about 8 after 4.5 days. The preincubation with defensin for 1 hour resulted in a 25% decrease of rF to about 6. The overnight preincubation with defensin, however, resulted in a 37% decrease of rF to about 5. After overnight incubation with defensin and HLA-DM, however, an increased rF by 25% of up to nearly 10 after 4.5 days was found. Interestingly, the t1/2 was identical in all cases, being about 18 hours.
Defensins are able to decrease the T-cell response of an HLA-DR-restricted T-cell clone To investigate the functional effects, if any, of defensin binding to class II molecules at the cell surface, HLA-DR-restricted T cells were used. These T cells were generated against CHO cells transfected with HLA-DRA, HLA-DRB1*0401, and CD80. After having shown that defensins alone do not markedly influence T-cell proliferation (data not shown), we used CHO cells pulsed with different defensin concentrations (none, 10, 20, or 30 µmol/L) to stimulate the T cells. After 12 and 24 hours of coculture, 3H-TdR was added and incorporated radioactivity was measured 16 hours later.
Here we report for the first time that endogenously bound natural
peptide antibiotics from the defensin family can be eluted from MHC
class II molecules of PBMC of 2 patients with plasmacytoma and 1 with
CML. In the 2 plasmacytoma patients defensins were the dominant
endogenous HLA-DR-associated peptides and corresponded to about 25% of
the high-frequency peptides. Furthermore, in plasmacytoma patient 1, 4 different CLIP peptides with lengths of up to 43 amino acids were
found. Interestingly, Harris et al,3 who examined HLA-DR-associated endogenous peptides from the hematopoietic precursor cell line KG-1, found a decreased frequency of CLIP. The finding of the
N-terminus of the hemoglobin We thank Drs Robert Busch and Elizabeth Mellins, Stanford University,
Palo Alto, CA, for providing us with a HLA-DM isolate. We also thank Dr
Martin Deeg for mass spectrometric analyzes, Manuela Braun for defensin
isolation, and Tanja Bauer for expert technical assistance. Thanks also
to Dr Rupert Handgretinger, University Childrens Hospital,
Tübingen for providing us with plasmacytoma patient material and
Prof C. A. Müller, Dr Thomas Flad, and Jutta Gamper, Medical
Hospital, Tübingen, for providing us with CML cells and helpful discussions.
Submitted July 26, 1999; accepted January 5, 2000.
Supported by grants from the Interdisciplinary Clinical Research Center
(IKFZ), University of Tübingen (T.M.H.), DFG grant Pa 361/5-1,
E.U. grant BMH4-CT98-3058 (G.P.), and the University of Tübingen
Medical Faculty Fortüne grant no. 399 (S.H.).
Reprints: Hubert Kalbacher, Medical and Natural Sciences
Research Center (MNF), University of Tübingen, Ob dem Himmelreich 7, D-72074 Tübingen, Germany; e-mail:
hubert.kalbacher{at}uni-tuebingen.de.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
1.
Accolla RS, Scupoli MT, Cambiaggi C, Tosi G, Sartoris S.
Cell lineage-specific and developmental stage-specific controls of MHC class-II-antigen expression.
Int J Cancer Suppl.
1991;6:20[Medline]
[Order article via Infotrieve].
2.
Silacci P, Mottet A, Steimle V, Reith W, Mach B.
Developmental extinction of major histocompatibility complex class II gene expression in plasmocytes is mediated by silencing of the transactivator gene CIITA.
J Exp Med.
1994;180:1329
3.
Harris PE, Maffei A, Colovai AI, Kinne J, Tugulea S, Suciu-Foca N.
Predominant HLA-class II bound self-peptides of a hematopoietic progenitor cell line are derived from intracellular proteins.
Blood
1996;87:5104
4.
Papadopoulos KP, Suciu-Foca N, Hesdorffer CS, Tugulea S, Maffei A, Harris PE.
Naturally processed tissue- and differentiation stage-specific autologous peptides bound by HLA class I and II molecules of chronic myeloid leukemia blasts.
Blood.
1997;90:4938
5.
Lehrer RI, Lichtenstein AK, Ganz T.
Defensins: antimicrobial and cytotoxic peptides of mammalian cells.
Annu Rev Immunol.
1993;11:105[Medline]
[Order article via Infotrieve].
6.
Ganz T, Lehrer RI.
Defensins.
Curr Op Immunol.
1994;6:584[Medline]
[Order article via Infotrieve].
7.
Hancock REW.
Peptide antibiotics.
Lancet.
1997;349:418[Medline]
[Order article via Infotrieve].
8.
Hill CP, Yee J, Selsted ME, Eisenberg D.
Crystal structure of defensin HNP-3, an amphiphilic dimer: mechanisms of membrane permeabilization.
Science.
1991;251:1481
9.
White SH, Wimley WC, Selsted ME.
Structure, function, and membrane integration of defensins.
Curr Opin Struct Biol.
1995;5:521[Medline]
[Order article via Infotrieve].
10.
Okrent DG, Lichtenstein AK, Ganz T.
Direct cytotoxicity of polymorphonuclear leukocyte granule proteins to human lung-derived cells and endothelial cells.
Am J Respir Cell Mol Biol.
1991;5:101.
11.
Yamashita T, Saito K.
Purification, primary structure, and biological activity of guinea pig neutrophil cationic peptides.
Infect Immun.
1989;57:2405
12.
Territo MC, Ganz T, Selsted ME, Lehrer RI.
Monocyte chemotactic activity of defensins from human neutrophils.
J Clin Invest.
1989;84:2017.
13.
Chertov O, Michiel DF, Xu L, et al.
Identification of defensin-1, defensin-2, and CAP37/azurocidin as T-cell chemoattractant proteins released from interleukin-8-stimulated neutrophils.
J Biol Chem.
1997;271:2935
14.
Prohaszka Z, Nemet K, Csermely P, Hudecz F, Mezo G, Füst G.
Defensins purified from human granulocytes bind C1q and activate the classical complement pathway like the transmembrane glycoprotein gp41 of HIV-1.
Mol Immunol.
1997;34:809[Medline]
[Order article via Infotrieve].
15.
Van den Berg RH, Faber-Krol MC, von Wetering S, Hiemstra PS, Daha MR.
Inhibition of activation of the classical pathway of complement by human neutrophil defensins.
Blood.
1998;92:3898
16.
Higazi AAR, Lavi E, Bdeir K, et al.
Defensins stimulates the binding of lipoprotein (a) to human vascular endothelial and smooth muscle cells.
Blood.
1997;89:4290
17.
Lillard JW, Boyaka PN, Chertov O, Oppenheim JJ, McGhee JR.
Mechanisms for induction of acquired host immunity by neutrophil peptide defensins.
Proc Natl Acad Sci U S A.
1999;96:651
18.
Halder T, Pawelec G, Kirkin AF, Zeuthen J, Meyer HE, Kalbacher H.
Isolation of novel HLA-DR-restricted potential tumor antigens from the melanoma cell line FM3.
Cancer Res.
1997;57:3238
19.
Bateman A, Singh A, Shustik C, Mars WM, Solomon S.
The isolation and identification of multiple forms of the neutrophil granule peptides from human leukemic cells.
J Biol Chem.
1991;266:7524
20.
Max H, Halder T, Kalbus M, Gnau V, Jung G, Kalbacher H.
A 16mer peptide of the human autoantigen calreticulin is a most prominent HLA-DR4Dw4-associated self-peptide.
Hum Immunol.
1994;41:39[Medline]
[Order article via Infotrieve].
21.
Sansom DM, Wilson A, Boshell M, Lewis J, Hall ND.
B7/CD28 but not LFA-3/CD2 interactions can provide 3rd-party co-stimulation for human T-cell activation.
Immunology
1993;80:242[Medline]
[Order article via Infotrieve].
22.
Chicz RM, Urban RG, Gorga JC, Vignali DAA, Lane WS, Strominger JL.
Specificity and promiscuity among naturally processed peptides bound to HLA-DR alleles.
J Exp Med.
1993;178:27
23.
Busch R, Strang G, Howland K, Rothbard JB.
Degenerate binding of immunogenic peptides to HLA-DR proteins on B cell surfaces.
Int Immunol.
1990;2:443
24.
Sette A, Southwood S, Miller J, Appella E.
Binding of major histocompatibility complex class II to the invariant chain-derived peptide, CLIP, is regulated by allelic polymorphism in class II.
J Exp Med.
1995;181:677
25.
Gosselin EJ, Wardwell K, Rigby WF, Guyre PM.
Induction of MHC class II on human polymorphonuclear neutrophils by granulocyte/macrophage colony-stimulating factor, IFN-gamma, and IL-3.
J Immunol.
1993;151:1482[Abstract].
26.
Mudzinski SP, Christian TP, Guo E, Cirenza E, Hazlett KR, Gosselin EJ.
Expression of HLA-DR (major histocompatibility complex class II) on neutrophils from patients treated with granulocyte-macrophage colony-stimulating factor for mobilization of stem cells [letter].
Blood
1995;86:2452
27.
Smith WB, Guida L, Sun Q, et al.
Neutrophils activated by granulocyte-macrophage colony-stimulating factor express receptors for interleukin-3 which mediate class II expression.
Blood.
1995;86:3938
28.
Reinisch W, Tillinger W, Lichtenberger C, et al.
In vivo induction of HLA-DR on human neutrophils in patients treated with interferon-
29.
Fanger NA, Liu C, Guyre PM, et al.
Activation of human T cells by major histocompatibility complex class II expressing neutrophils: proliferation in the presence of superantigen, but not tetanus toxoid.
Blood.
1997;89:4128
30.
Brown JH, Jardetzky TS, Gorga JC, et al.
Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1.
Nature.
1993;364:33[Medline]
[Order article via Infotrieve].
31.
Kropshofer H, Vogt AB, Stern LJ, Hämmerling GJ.
Self release of CLIP in peptide loading of HLA-DR molecules.
Science.
1995;270:1357
32.
Cunningham AC, Zhang JG, Moy JV, Ali S, Kirby JA.
A comparison of class II MHC-expressing human lung epithelial and endothelial cells.
Immunology
1997;91:458[Medline]
[Order article via Infotrieve].
33.
Panja A, Barone A, Mayer L.
Stimulation of lamina propria lymphocytes by intestinal epithelial cells: evidence for recognition of nonclassical restriction elements.
J Exp Med.
1994;179:943
34.
Hoyne GF, Callow MG, Kuo MC, Thomas WR.
Presentation of peptides and proteins by intestinal epithelial cells.
Immunology.
1993;80:204[Medline]
[Order article via Infotrieve].
35.
Hershberg RM, Cho DH, Youakim A, et al.
Highly polarized HLA class II antigen processing and presentation by human intestinal epithelial cells.
J Clin Invest.
1998;102:792[Medline]
[Order article via Infotrieve].
36.
Wilson JL, Cunningham AC, Kirby JA.
Alloantigen presentation by B cells: analysis of the requirement for B-cell activation.
Immunology.
1995;86:325[Medline]
[Order article via Infotrieve].
37.
Leon MP, Bassendine MF, Wilson JL, Ali S, Thick M, Kirby JA.
Immunogenicity of biliary epithelium: investigation of antigen presentation to CD4+ T cells.
Hepatology.
1996;24:561[Medline]
[Order article via Infotrieve].
38.
Rammensee HG, Friede T, Stevanovic S.
MHC ligands and peptide motif: first listing.
Immunogenetics.
1995;41:178[Medline]
[Order article via Infotrieve].
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peripheral blood mononuclear cells of different
tumor patients (plasmacytoma, chronic myeloid leukemia)
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Abstract
Introduction
purification
and identification of eluted peptides
), HLA-DRB1*
HLA-DRB1*1501/DRB5*0101 (
), HLA-DRB1*0301 (×), HLA-DRB1*0401
(
) and HLA-DRB1*0701 (
) against fluorescently (AMCA)-labeled
binding peptides using a gel filtration assay. As binding peptides, HA
306-18 (PKYVKQNTLKLAT; 1.5 µmol/L) was used for HLA-DR1, -2 and -4. For HLA-DR3, heat shock protein 65, 3-13 (KTIAYDEEARR; 1.5 µmol/L)
and for HLA-DR7, heat shock protein 70, 38-52 (TPSYVAFTDTERLIG; 1.5 µmol/L) was used. For (A), the concentration unit µg/40 µL can be
transferred to µmol/L by multiplication with the factor 7.4. For (B),
however, the µmol/L concentration of the used defensin isolate cannot
be determined due to inhomogeneous masses of different defensin
molecules.
(IFN-
