Autoantibodies frequently detected in patients with aplastic anemia

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Prepublished online August 28, 2003; doi:10.1182/blood-2002-11-3409

Autoantibodies frequently detected in patients with aplastic anemia Naoto Hirano, Marcus O Butler, Michael S von Bergwelt-Baildon, Britta Maecker, Joachim L Schultze, Kevin C O'Connor, Peter H Schur, Seiji Kojima, Eva C Guinan and Lee M Nadler

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bloodjournal.hematologylibrary.org by guest on June28, 5, 2013. For personal use only. Blood FirstFrom Edition Paper, prepublished online August 2003; DOI 10.1182/blood-2002-11-3409

N. Hirano et al Autoantibodies frequently detected in patients with aplastic anemia Naoto Hirano1,

3, 5

, Marcus O. Butler1,

Maecker1, Joachim L. Schultze1,

3, 5

3, 5

, Michael S. von Bergwelt-Baildon1, Britta

, Kevin C. O’Connor4, 5, Peter H. Schur3, 5, Seiji

Kojima6, Eva C. Guinan2, 3, 5, and Lee M. Nadler1, 3, 5

1

Department of Medical Oncology, Dana-Farber Cancer Institute; 2Department of Pediatric

Oncology, Dana-Farber Cancer Institute; 3Department of Medicine, Brigham and Women’s Hospital;

4

Laboratory of Molecular Immunology, Center for Neurologic Disease,

Department of Neurology, Brigham and Women’s Hospital; 5Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115; 6Department of Pediatrics, Nagoya University, Nagoya, Japan 466-8550

Running Title: Autoantigens in aplastic anemia Keywords: aplastic anemia, autoantigen, autoantibody, CTL, HSPC

Address correspondence to: Naoto Hirano, MD, Ph.D. Dana-Farber Cancer Institute Department of Medical Oncology 44 Binney Street, Boston Massachusetts 02115, USA

1 Copyright (c) 2003 American Society of Hematology


N. Hirano et al Phone: (617) 632-6186 Fax: (617) 632-2255 E-mail: [email protected]

2


N. Hirano et al Footnotes: 1

N.H. was supported by the Sankyo Foundation of Life Science. M.O.B. is supported by

K08-CA-87720. M.S.v.B.-B. was supported by the Mildred Scheel Stiftung der Deutschen Krebshilfe e.V. B.M. was supported by the Deutsche Forschungsgemeinschaft. J.L.S. is a recipient of a Special Fellowship of the Leukemia & Lymphoma Society. K.C.O. is a National Multiple Sclerosis Society fellow. E.C.G. is a recipient of a Burroughs Wellcome Clinical Scientist Award. This work was supported by a Translational Research Award by the Leukemia & Lymphoma Society (J.L.S.), P50-HL-54785 and P01-AI-41584 (E.C.G.), and P01-CA-66996 and P01-CA-78378 (L.M N.). 2

To whom requests for reprints should be addressed at Dr. Naoto Hirano, Dana-Farber

Cancer Institute, Department of Medical Oncology, 44 Binney Street, Boston, Massachusetts 02115, USA, Phone: +1-(617) 632-6186, Fax: +1-(617) 632-2255, E-mail: [email protected] 3

The abbreviations used in this paper are: AA, aplastic anemia; HSPC, hematopietic

stem/progenitor cell; SEREX, serologic identification of antigens by recombinant expression cloning; CTL, cytotoxic T cell; MS, multiple sclerosis; RA, rheumatoid arthritis; SORD, sorbitol dehydrogenase; BM, bone marrow; PBMC, peripheral blood mononuclear cell; PBPMC, peripheral blood polymorphonuclear cell; CFU-GM, colony-forming unitgranulocyte macrophage; BFU-E, burst-forming unit-erythroid; MoAb, monoclonal antibody; GST, glutathione S-transferase; aa, amino acid; EST, expressed sequence tag

3


N. Hirano et al Abstract Although accumulating evidence strongly suggests that aplastic anemia (AA) is a T cellmediated autoimmune disease, no target antigens have yet been described for AA. In autoimmune diseases, target autoantigens frequently induce not only cellular T cell responses but also humoral B cell responses. We hypothesized that the presence of antigen specific autoantibodies could be used as a “surrogate marker” for the identification of target T cell autoantigens in AA patients. We screened a human fetal liver library for serologic reactivity against hematopoietic stem/progenitor cell antigens and isolated 32 genes. In 7 out of 18 AA patients, an IgG antibody response was detected to one of the genes, kinectin, which is expressed in all hematopoietic cell lineages tested including CD34+ cells. No response to kinectin was detected in healthy volunteers, multiply transfused non-AA patients, or patients with other autoimmune diseases. Epitope mapping of IgG autoantibodies against kinectin revealed that the responses to several of the epitopes were shared by different AA patients. Moreover, CD8+ cytotoxic T cells raised against kinectinderived peptides suppressed the colony formation of CFU-GM in an HLA class I-restricted fashion. These results suggest that kinectin may be a candidate autoantigen that is involved in the pathophysiology of AA. E-mail: [email protected]

4


N. Hirano et al Introduction Three decades ago, an immune mechanism was first implicated in the pathogenesis of aplastic anemia (AA) 1. Since then, accumulating evidence supports the hypothesis that immune mechanisms contribute to the pathogenesis of AA

2-4

. Immunosuppressive

therapies incorporating anti-thymocyte globulin, corticosteroids, cyclosporine, and/or cyclophosphamide have been successfully used in the treatment of patients with AA with response rates ranging from 50 to 80%

5-9

, suggesting that pancytopenia and bone marrow

failure in at least some AA patients are immunologically mediated. Furthermore, in vitro studies have also supplied supportive evidence for an immune-mediated suppression of hematopoiesis in AA. These include inhibitory effects of AA patient lymphocytes on hematopoietic

stem/progenitor

cell

(HSPC)

growth

10,11

,

overproduction

of

myelosuppressive cytokines such as IFN-gamma and TNF-alpha by patient bone marrow cells

12-16

, and an increased population of activated suppressor T cells

17-19

. Taken together,

these data suggest that at least some cases of AA involve autoimmune phenomena that targets hematopoietic tissue, probably HSPCs.

By establishing T cell lines or clones from an involved organ and analyzing their specificity, investigators have successfully identified unknown target antigens in organ-specific

5


N. Hirano et al autoimmune diseases and neoplasms

20,21

. Previous studies with T cells from AA patients

have identified a pathogenic role for both CD8+ and CD4+ T cells. Peripheral blood T cells capable of suppressing in vitro growth of HSPCs belong mainly to the CD8+ fraction

22,23

.

However, the importance of CD4+ cytotoxic T cells in the pathogenesis of AA has been also described. Several CD4+ T cell clones, that can lyse autologous hematopoietic cells in an HLA class II-restricted fashion, have been isolated from AA patients 24.

In organ-specific autoimmune diseases, autoreactive T cell responses against target autoantigens play a major role in the disease pathogenesis. At the same time, these autoantigens can also evoke humoral responses, i.e. IgG autoantibody production in many cases. For example, target autoantigens such as myelin basic protein in multiple sclerosis and mitochondrial branched chain keto acid dehydrogenase complexes in primary biliary cirrhosis, have been shown to induce both IgG production and CD4+ T cell responses 20. In fact, even though a role for antibody mediated tissue destruction may not be confirmed, in some cases investigators have first identified the humoral response to antigens that serve as the targets of pathologic autoreactive T cells 25. This approach is currently being explored in patients with cancer where serologic identification of antigens by recombinant expression cloning (SEREX) has been applied to the identification of tumor-associated antigens, and

6


N. Hirano et al hundreds of candidate genes have been isolated 26-29. Importantly, some of the antigens like NY-ESO-1 originally identified by antibody reactivity have subsequently been shown to be targets of CD8+ and CD4+ T cell responses in vivo 30,31.

Based on these observations, we hypothesized that the target autoantigens recognized by CD4+ and/or CD8+ T cells in patients with AA might also produce humoral response, i.e. autoantibody production. The characterization of the autoantibodies to hematopoietic tissue, especially HSPCs, by SEREX technology could serve as a surrogate marker for autoantigen specific T cells and might yield novel insights into the T cell-mediated pathophysiology of AA. Here, we describe the identification of novel autoantigens expressed by HSPCs in association with AA.

7


N. Hirano et al Materials and Methods Preparation of blood samples. Blood and bone marrow (BM) samples were obtained from 18 patients with AA, 20 multiply transfused patients with thalassemia and sickle cell anemia, 19 patients with systemic lupus erythematosus (SLE), 24 patients with rheumatoid arthritis (RA), 22 patients with multiple sclerosis (MS), and 35 normal volunteers enrolled in research protocols at Dana-Farber Cancer Institute, Brigham and Women’s Hospital, and Children’s Hospital, Boston, and were immediately stored in aliquots at -80°C until use. All protocols were approved by the Institutional Review Boards of the above institutions.

Identification of immunoreactive cDNA clones. Screening for candidate antigens was performed using a human fetal liver cDNA expression library (Clontech, Palo Alto, CA) as described previously 32. Briefly, XL1-Blue Escherichia coli (Stratagene, La Jolla, CA) were transfected with recombinant phages, plated on agar plates, and cultured at 42°C. Expression of recombinant proteins was induced by incubating the bacterial lawns with an overlay of iso-propyl ß-D-thiogalactoside (IPTG)–impregnated nitrocellulose filters (Hybond C-Extra, Amersham Pharmacia Biotech, Piscataway, NJ). Transfer of released proteins was allowed to proceed at 37°C. In order to increase the

8


N. Hirano et al solubility of the induced proteins, some of the filters were first treated with 8 M urea and subsequently with serially diluted urea to renature recombinant proteins. Filters were then washed in TBST (10 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 8.0) and blocked overnight with blocking buffer (5% wt/vol nonfat dry milk (Nestle, Solon, OH) in TBST). Filters were then incubated with patient serum at 1:500. Specific binding of antibody to recombinant protein was detected by incubation with alkaline phosphatase–conjugated goat anti-human IgG antibody (Promega, Madison, WI)) diluted at 1:7,500 or goat anti-human Fc gamma antibody (Jackson ImmunoResearch, West Grove, PN) at 1:3,000. Visualization of the antigen-antibody complex was accomplished by staining with 5-bromo-4-chloro-3indolyl phosphate and nitro blue tetrazolium (Promega). Complementary DNA inserts from positive clones were subcloned, purified, and in vivo excised to plasmid forms (Clontech) according to the manufacturer’s instructions. The DNA inserts were subsequently sequenced with appropriate sequencing primers.

Phage-plate assay. Each clone of interest was screened as described above by mixing the positive clone of interest with nonreactive phages from the cDNA library as an internal negative control at a ratio of approximately 1:10.

9


N. Hirano et al

Expression and purification of bacterially expressed fusion proteins. Isolated cDNA inserts were subcloned in-frame into the pQE30 (Qiagen) and pGEX-5X3 (Amersham Pharmacia Biotech) vectors for His-tagged and GST (glutathione S-transferase) fusion protein expression, respectively. His-tagged or GST kinectin were constructed using PCR-based subcloning, and the sequence was verified. The induction and affinitypurification of fusion proteins were performed according to the manufacturer’s instructions. Appropriate size and specificity of the expressed protein products were confirmed by Western blot analysis using mouse anti-GST monoclonal antibody (1:3,000; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-His monoclonal antibody (1:3,000; Sigma, St. Louis, MO), or mouse anti-kinectin monoclonal antibody (1:10,000) (a gift from Dr. Toyoshima) as described below.

Western blot analysis. Both purified proteins and bacterial lysates were prepared in SDS-sample buffer. Equal amounts of protein were analyzed by SDS-PAGE, transferred onto PVDF membranes (Millipore, Bedford, MA), and incubated with blocking buffer overnight. Immunoblots were performed with 1:500 or indicated dilution of patient serum, mouse anti-GST

10


N. Hirano et al monoclonal antibody, mouse anti-His monoclonal antibody, or mouse anti-kinectin monoclonal antibody. Immunodetection was performed by incubation with horseradish peroxidase-conjugated anti-human IgG (1:10,000; Promega) or anti-mouse IgG secondary antibody (1:5,000; Promega) as indicated by the host origin of the primary antibody and developed by chemiluminescence (NEN Life Science Products, Boston, MA).

ELISA assay. Recombinant His-tagged kinectin protein encoding aa 434-1,008 was produced and purified as described above. ELISA plates were coated with purified recombinant protein at 1 µg/ml in coating buffer (50 mM carbonate/bicarbonate buffer, pH 9.6) overnight at 4 °C. Plates were washed and blocked overnight at 4°C with 5% nonfat dry milk in TBST. Patient sera was added to a final dilution of 1:500 to 1:5,000 and incubated at room temperature. After wash, the plates were incubated with alkaline phosphatase–conjugated goat anti-human IgG antibody (1:7,500; Promega) at room temperature. Finally, the plates were washed and incubated with PNPP substrate (Pierce, Rockford, IL) at room temperature, and the OD (405 nm) was read (Spectramax 190 Microplate Reader; Molecular Devices, Sunnyvale, CA). A positive reaction was defined as an absorbance value exceeding the mean OD absorbance value of sera from normals by three standard deviations.

11


N. Hirano et al

HLA-A*0201-binding assay. Peptide prediction was performed using publicly available algorithms

33

. TAP-deficient T2

cells (American Type Culture Collection, Manassas, VA) were pulsed with 50 µg/ml of peptide and 5 µg/ml of ß2-microglobulin (CalBiochem, San Diego, CA) for 18 hours at 37°C. HLA-A*0201 expression was then measured by flow cytometry using HLA-A2 specific monoclonal antibody (MoAb) BB7.2 (American Type Culture Collection) followed by incubation with FITC-conjugated F(ab’)2

goat anti-mouse Ig (Zymed, South San

Francisco, CA).

Establishment of cytotoxic T cells against kinectin-derived peptides. CTLs against kinectin-derived peptides were established according to a protocol described previously

34

. Briefly, purified CD8+ T cells from HLA-A2-positive normal donors were

repeatedly stimulated with autologous peptide-pulsed dendritic cells and CD40L-activated B cells, and the standard cytotoxicity assay was performed using peptide-pulsed T2 cells.

Hematopoietic progenitor cell assay.

12


N. Hirano et al CD34+ cells were purified from HLA-A2 positive BM cells using a CD34+ cell isolation kit (Dynal, Lake Success, NY) according to the manufacturer’s instructions. Freshly isolated CD34+ cells were mixed with CTLs against kinectin-derived peptides, 645F and 794S, at 50:1 ratio and incubated at 37°C in a 96 well U-bottomed plate. Ten µg/ml of HLA-A2 specific BB7.2 MoAb (mouse IgG2b) was preincubated with CD34+ cells at 37°C for 30 min to block cytotoxicity by T cells as indicated. After 16 hours, the cell suspension was added with the methylcellulose medium (StemCell Technologies, Vancouver, British Colombia, Canada) supplemented with colony-stimulating factors and 1 x 103 of CD34+ cells were plated on a 35-mm culture dish. After 12-14 days, colony-forming unitgranulocyte macrophage (CFU-GM) derived colonies and burst-forming unit-erythroid (BFU-E) derived colonies were counted under an inverted microscope. Similar experiments were performed three times in an at least sextuplicated manner.

13


N. Hirano et al Results Serologic identification of autoantigens in AA patient sera We performed a serologic screen in order to identify candidate target autoantigens in AA. We screened for HSPC antigens by using a human fetal liver cDNA library, because this organ contains a high proportion of cells derived from HSPCs. Also, we limited our screening to those genes that elicit IgG responses, since our aim was to identify potential autoantigens that are targets of T cells and production of IgG usually requires CD4+ T cell driven class-switching. We screened more than 2 million phage plaques with diluted sera from each of 8 AA patients and identified 32 candidate genes including 10 expressed sequence tags (ESTs) and previously unknown genes (Table 1). Lineage specific expression was determined by searching several available databases, including the NCBI UniGene database (http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs), the NIDDK hembase database (http://hembase.niddk.nih.gov/), the Kazusa Human cDNA Project HUGE database

(http://www.kazusa.or.jp/huge/)

and

the

Blood

SAGE

database

(http://bloodsage.gi.k.u-tokyo.ac.jp/)35-37. Our search revealed that 17 of the 22 immunoreactive candidate genes are expressed by hematopoietic cells and their predominant lineage expression are also listed in Table 1. Also, the expression of these genes

by

CD34+

cells

was

indicated

14

by

searching

the

UniGene

databases


N. Hirano et al (http://www.ncbi.nlm.nih.gov:80/UniGene/library.cgi?ORG=Hs&LID=933/

and

http://www.ncbi.nlm.nih.gov:80/UniGene/library.cgi?ORG=Hs&LID=1537), the Westbrook lab

at

UIC,

Chicago,

hematopoietic

(http://westsun.hema.uic.edu/html/expression.html/),

stem the

cell rzdp

database database

(http://www.rzpd.de/cgi-bin/services/exp/viewExpressionData.pl.cgi/), and published data by Mao et al.

38-41

The remaining 5 genes were expressed by liver and are listed as “Genes

not reported to be expressed by hematopoietic cells.” Of the 32 total genes identified, we focused our analysis on the 22 genes previously described. Using the phage plate assay, we confirmed the reactivity of eight AA patients sera against these genes (Table 1).

In order to confirm patient sera reactivity with protein products of candidate AA genes, we produced GST fusion proteins from isolated clones and performed Western blot analysis using AA patient sera. Figure 1 shows representative results of the Western blot analysis. Here, serum from patient 1AA specifically detects kinectin but not GST alone, sorbitol dehydrogenase (SORD), or H-ferritin. On the other hand, the serum from patient 3AA does not detect kinectin or GST alone, though it does detect H-ferritin and SORD.

Kinectin is expressed by hematopoietic cells at the protein level.

15


N. Hirano et al Initial screening using both the phage plate assay and Western blot analysis demonstrated a kinectin induced IgG antibody response in 4 of 8 AA patients (Table 1). Given this high frequency, we next focused our studies on the association of anti-kinectin autoantibodies with AA. Kinectin is an intracellular protein of 160 kDa, expressed in brain, testis, ovary, fetus, liver, and hematopoietic cells. Little is known about the function of kinectin other than that it is a kinesin-binding protein required for kinesin-based motility 42. Expression of kinectin by hematopoietic cells at the protein level was confirmed by Western blot analysis using a mouse anti-kinectin antibody (Figure 2). As shown, purified CD34+ cells, bone marrow cells, peripheral blood mononuclear cells (PBMCs), and peripheral blood polymorphonuclear cells (PBPMCs) express kinectin protein. Cell lines derived from myeloid (KG1a), erythroid (K562), monocytoid (U937), megakaryocytoid (Meg01), and lymphoid cells (Jurkat and EBV-LBL) are also all positive for kinectin at the protein level (Figure 2). These results confirmed that kinectin is expressed by hematopoietic cells, and suggested that kinectin could serve as an AA-associated antigen.

IgG antibody against kinectin can be detected by ELISA and Western blot analysis In order to determine the prevalence of IgG antibodies directed against kinectin in AA patients, we performed an ELISA using a recombinant kinectin protein. Our initial serologic

16


N. Hirano et al screen identified two partial kinectin cDNAs encoding different regions of the kinectin protein, amino acids (aa) 434-927 and 535-1,008. We therefore expressed and purified a His-tagged partial kinectin cDNA encoding aa 434-1,008. Representative ELISA results with sera (1:500 dilution) from twelve AA patients and 16 normal donors are shown in Figure 3A. A positive reaction is defined as an absorbance value exceeding the mean absorbance value of sera from normals by three standard deviations. Five of twelve AA patients showed a higher titer for IgG antibody against kinectin than did normal volunteers. We also performed serial dilution experiments and two of the five positive sera remain positive even at the dilution of 1 to 2,500. In Figure 3B, we show the representative titration curves of serially diluted sera from eight AA patients.

In order to explore the possibility that antibodies present in sera from AA patients recognize other regions of the kinectin protein, we produced a recombinant full-length kinectin protein fused to GST and then performed Western blot analysis using sera from all AA patients and normal donors (Figure 4). Lysates expressing GST alone were loaded in each lane marked 1, while GST-kinectin fusion protein lysates were loaded in each lane marked 2. Anti-GST monoclonal antibody detected both the 29 kDa GST protein and the 190 kDa GST-kinectin fusion protein. The ladder pattern was probably due to alternative translation,

17


N. Hirano et al initiation, and premature termination of translation rather than protein degradation as the ladder pattern was reproducible with different preparations of the fusion proteins (data not shown). Also, we demonstrated that the anti-kinectin monoclonal antibody recognized the GST-kinectin fusion protein but not the GST moiety. Likewise, ELISA positive AA sera, but not normal donor sera, detected the recombinant kinectin protein, confirming the results obtained by ELISA. The positive pattern indicated by the arrows in Figure 4 differed somewhat between patients suggesting that the antigenic epitopes recognized by AA patients might differ (see below).

Anti-kinectin autoantibody is frequently detected in AA patients but not in normal donors, multiply transfused non-AA patients, or patients with other autoimmune diseases. We extended our screening to a total of 18 AA patients and 35 normal volunteers for antikinectin autoantibody production. We found that 7 of 18 AA patients had IgG antibodies directed against kinectin by ELISA or Western blot analysis, whereas all normal volunteers were negative (Table 2). Results of ELISA and Western blot analysis were concordant in all cases except one AA patient, in whom the discrepancy may be due to differences in the antigenicity of the recombinant kinectin proteins used in the two assays. We also

18


N. Hirano et al investigated the possibility that anti-kinectin autoantibody production was a consequence of transfusion-related allogeneic immune responses by testing sera from patients who had received multiple transfusions. We did not detect any anti-kinectin antibody in all of the 20 multiply transfused patients who had either thalassemia or sickle cell anemia (Table 2). Furthermore, sera from 65 patients with other autoimmune diseases, SLE, RA, and MS, lacked the presence of anti-kinectin antibody, suggesting the possible specificity of antikinectin autoantibody production in AA patients (Table 2).

Lack of association of preceding hepatitis or transfusion with anti-kinectin autoantibody positivity in patients with AA. In Table 3 we provide a summary of the characteristics of AA patients studied for kinectin IgG autoantibodies. The data here does not support that autoantibody production is a result of transfusions or hepatitis. It is well known that, in some cases, AA is preceded by an episode of hepatitis. Potential induction of an anti-kinectin antibody response to hepatitis is unlikely given the fact that 5/6 positive patients did not have a history of preceding hepatitis (Table 3). Furthermore, while most AA patients had been transfused prior to enrollment on this study, one anti-kinectin positive AA patient did not have a history of transfusion, and another anti-kinectin positive patient received the first transfusion immediately prior to

19


N. Hirano et al blood sampling. Therefore, it is unlikely that either of these patients developed anti-kinectin antibodies as a result of transfusion. These facts further support the hypothesis that the production of anti-kinectin autoantibodies in AA patients is not due to preceding hepatitis or an allogeneic reaction to transfused blood products.

Some of the B cell epitope regions of kinectin IgG autoantibodies were shared by AA patients. We postulated that kinectin may contain immunogenic “hotspots” that are recognized by multiple patient sera. We felt that such information might help identify potential T cell epitopes since IgG autoantibodies sometimes recognize amino acid sequences that are also recognized by T cells in some autoimmune diseases

43,44

. To do this, we determined the

kinectin epitope sequences recognized by patient sera. We investigated the antigenic epitopes of kinectin IgG antibodies using a GST-fusion system. Employing a PCR-based subcloning method, we made a series of 8 GST-partial kinectin fusion proteins. Fusion proteins were separated by SDS-PAGE, and Western blot analysis was performed. A blot with anti-GST antibody (Figure 6A) verified the presence of equal molar amounts of 8 different GST-kinectin fusion proteins. A blot with mouse anti-kinectin antibody (Figure 6B) indicated that this antibody could recognize two regions of the kinectin protein present

20


N. Hirano et al on aa 535-735 and aa 1,002-1,300. To the right, blots performed with 2 different AA patient sera show that aa 923-1,008 was recognized by both patient 1AA (Figure 6C) and 7AA (Figure 6D) sera, while fusion protein aa 263-439 was recognized by patient 1AA but not patient 7AA. Figure 6E summarizes the results of partial IgG epitope mapping of kinectin IgG antibodies in seven AA patients. The boxes with the same pattern indicate regions that were recognized by more than one patient. As illustrated, several epitopes are shared by multiple patients while others are not.

Kinectin-derived peptides bind HLA-A2 molecules. Since the antibody response against kinectin in AA patients is an IgG response, CD4+ T cell involvement was strongly suggested. We were also interested in exploring whether a CD8+ T cell response against kinectin could be generated. First, we attempted to determine if kinectin-derived peptides could be presented by HLA-class I molecules. A peptide-motif scoring system (http://bimas.dcrt.nih.gov/molbio/hla_bind/) was employed to predict possible kinectin-derived peptides for HLA-A2. Four kinetin-derived peptide sequences recorded high scores for A2 binding compared with the peptides known to bind A2, consistent with the possibility that these predicted peptides might bind HLA-A2 (Table 4). These peptides were pulsed with ß2-microglobulin onto the HLA-A*0201+ T2 hybridoma

21


N. Hirano et al in a standard functional peptide binding assay. Based on increased expression of HLAA*0201 on pulsed T2 cells, 2 of the 4 kinectin-derived peptides were found to bind strongly to HLA-A*0201 (Table 4). A positive control peptide derived from the tumor antigen MAGE-3 also bound strongly, but two negative control peptides did not.

T cell repertoire against kinectin-derived peptides is preserved in normal donors. In order to investigate whether the peripheral blood from normal donors contain cytotoxic T cell precursors against kinectin, we established CD8+ cytotoxic T lymphocytes (CTLs) against kinectin-derived peptides from normal donors. Purified CD8+ T cells from A2positive normal donors were repeatedly stimulated with autologous peptide-pulsed dendritic cells and CD40L-acivated B cells, and the standard cytolysis assay was performed using T2 cells. CTLs against kinectin-derived peptides 794S (Figure 7A) and 645F (Figure 7B) specifically killed the target cells pulsed with each of the peptides. We succeeded in raising CTLs against kinectin-derived peptides from the peripheral blood of two of three HLA-A2 positive normal donors tested. These results support the assertion that T cell repertoire against kinectin-derived peptides is present in normal individuals.

22


N. Hirano et al Inhibition of CFU-GM, but not BFU-E derived colony formation by CTLs against kinectin-derived peptide 645F. If a CD8+ T cell response against a candidate autoantigen is involved in the pathogenesis of AA, the autoantigen-derived peptides should be presented by HLA class I molecules on target cells, namely HSPCs in AA. Therefore, we employed the cytotoxicity assay to study whether HSPCs can naturally process and present kinectin-derived peptides 645F and 794S via HLA-A2 molecules. HLA-A2 positive CD34+ cells were incubated with CTLs raised against kinectin-derived peptides, 645F and 794S, and were then cultured in methylcellulose medium supplemented with colony stimulating factors. CTLs against 645F inhibited CFU-GM derived colony formation in an HLA-A2 specific fashion but not BFU-E derived colony formation (Table 5). In contrast, CTLs against 794S did not suppress either CFU-GM or BFU-E derived colony formation. CTLs were simultaneously and similarly made from the same donor and inhibited CFU-GM colony formation only when raised against 645F but not against 794S. Therefore, these results are peptide/HLA specific and are not due to allogeneic effects. Since these data show that kinectin-derived 645F is naturally processed and presented by CFU-GM via HLA-A2 molecules, we propose that kinectinderived peptide may be involved in the suppression of CFU-GM colony formation seen in some AA patients. It should be also noted that 645F, but not 794S, is located in one of the B

23


N. Hirano et al cell epitope hotspots identified. Intriguingly, this is analogous to other autoimmune diseases, where IgG autoantibodies recognize amino acid sequences that are also recognized by target T cells 20,43,44.

24


N. Hirano et al Discussion Organ-specific autoantigens in T cell-mediated autoimmune diseases have been shown to be targets of not only pathologic cellular responses but also humoral responses, i.e. autoantibody production 20. The initial identification of a humoral immune response to the target antigen sometimes precedes the confirmation of a pathologic T cell immune response to the same antigen. Since, in some cases, AA is a T cell-mediated autoimmune disease, we reasoned that the pathologic immune response in AA might include both antigen specific T cell and autoantibody production. Although this humoral immune response itself may not be directly involved in the pathophysiology of AA, the presence of antigen specific autoantibodies can be used as a “surrogate marker” to identify T cell target autoantigens in AA patients. In this study we demonstrated that some AA patients have a robust humoral immune response that recognizes multiple autoantigenic proteins expressed by hematopoietic and/or liver cells.

Serologic screening of a fetal liver library with sera from eight AA patients revealed more than 30 potential AA specific candidate autoantigens. The choice of a human fetal liver cDNA library, which is highly enriched for CD34+ cells, compared to peripheral blood or bone marrow, significantly increased the likelihood that we would detect possible HSPC

25


N. Hirano et al autoantigens. We also reasoned that the selection of fetal liver for our cDNA library might also increase our capacity to detect antigens that could be shared by hepatocytes and HSPCs. This concept was particularly attractive given published evidence that some cases of AA are preceded by hepatitis 2,45,46. Theoretically, AA-associated liver inflammation and tissue destruction might evoke a strong immune response directed against antigens shared by both liver and hematopoietic cells. Further investigation is needed to determine whether any of the identified candidate AA autoantigens are involved in the hepatitis associated AA.

Of the multiple candidate autoantigens identified, ELISA and Western Blot analysis revealed that an IgG antibody response to one of the candidate autoantigens, kinectin, was present in a significant number of AA patients (39%). In contrast, no antibody was detected in 35 normal volunteers or in 20 patients with thalassemia or sickle cell anemia who had a history of multiple transfusions. Also, antibody was detected in both transfused and transfusion naïve AA patients, suggesting that anti-kinectin autoantibody development was not simply due to transfusion related alloreactivity. Moreover, the clinical histories of these patients do not support an association of anti-kinectin reactivity with AA-associated hepatitis. Negative studies of sera from patients with the autoimmune diseases, SLE, RA, and MS, further defined the specific association of anti-kinectin responses with AA. These

26


N. Hirano et al results support the hypothesis that an immune response to kinectin is restricted to patients with AA and may be involved in the pathophysiology of this disease.

We postulated that at least some of the identified candidate autoantigens are the targets of a cytolytic T cell response in patients with AA. Since kinectin is a large molecule comprising 1,300 aa 42, it is likely that several kinectin-derived peptides are processed and presented in the context of HLA molecules to T cells. It has been shown that other examples of immunologic antigens, such as the H-Y minor histocompatibility antigens, can be processed and presented by multiple HLA class I alleles. One of the H-Y antigens, SMCY, is a large molecule with 1,539 aa and has been demonstrated to be processed and presented via both HLA-A2 and B7 molecules

47,48

. Another H-Y antigen, known as UTY, is also a large

molecule with 1,186 aa and its peptide epitopes can be presented via both HLA-B8 and B60 49,50

. These examples show that large molecules, like kinectin, can be naturally processed

and presented via multiple HLA class I alleles and can induce multiple antigen specific CD8 T cell responses. In this report, we demonstrated that kinectin is expressed by CD34+ cells and that kinectin-derived 645F peptide can be naturally processed and presented by HLAA2 molecules on CFU-GM but not on BFU-E. Since kinectin is likely to contain multiple peptide epitopes that are processed and presented to T cells, it is possible that epitopes other

27


N. Hirano et al than 645F or 794S are presented by BFU-E and are involved in the suppression of BFU-E specific colony formation in vivo. Attempts to identify such epitopes are currently ongoing. The probability that kinectin-derived peptide epitopes can be processed and presented by multiple HLA alleles provides insight into why kinectin may serve as a target autoantigen in many AA patients of various different HLA types.

In addition to its expression in human hematopoietic cells, kinectin transcripts have also been found in human liver, ovary, testis, and brain

42

. If human HSPCs are the target of

kinectin specific autoreactive T cells in AA patients, why then are these other tissues spared from attack by anti-kinectin specific autoreactive T cells? For T cells to recognize specific peptides derived from an antigen, that antigen must be expressed at the protein level and then must be processed and presented via HLA molecules. We suggest that kinectin-derived peptides may be processed and presented by HLA complexes present on human HSPCs but not by those expressed in other kinectin positive tissue. It has been shown that antigen processing systems in different tissues may differ in what peptides are processed and presented. An example is again provided by the H-Y antigens encoded by the SMCY and UTY genes. Though transcripts of these genes are detected in a wide variety of human tissues, only human hematopoietic cells such as PHA-blasts and AML cells serve as targets

28


N. Hirano et al of T cells directed against H-Y antigens

50

. An additional explanation for the protection of

other kinectin positive tissues from damage by autoreactive T cells also might include expression of kinectin by tissues such as testes and brain that are privileged sanctuaries that exclude T cell migration.

This study confirms that humoral immune responses to hematopoietic antigens can be detected in AA patients. As in other autoimmune diseases, these humoral immune responses may be useful in unraveling the disease pathophysiology of AA. Therefore, we are embarking on a prospective study to help delineate the role of these immune responses in the pathophysiology of AA and to examine whether these findings can translate into improvements in the diagnosis and treatment of AA.

29


N. Hirano et al Acknowledgements We thank Dr. Toyoshima for providing us with the mouse anti-kinectin antibody. We also thank Drs. Nagase and Kronke for providing us with full-length cDNAs encoding kinectin.

30


N. Hirano et al Figure legends Figure 1. Representative Western blot analysis of cloned gene protein products using anti-GST monoclonal antibody and sera from two AA patients. GST-fusion proteins were induced by IPTG, separated by 10% SDS-PAGE, and transferred to PVDF membranes. Triplicates of membranes were prepared and probed with mouse anti-GST monoclonal antibody, patient 1AA, or 3AA serum. Immunoreactive proteins were detected with horseradish peroxidase-conjugated goat anti-mouse or human IgG second antibody.

Figure 2. Expression of human kinectin in hematopoietic cells and cell lines. Equal amounts (25 µg) of total lysates were separated by 7.5% SDS-PAGE, transferred to PVDF filters, and probed with mouse anti-kinectin monoclonal antibody. Immunoreactive proteins were detected with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody.

Figure 3. (A), (B) Representative results of ELISA reactivity of AA patients sera with purified His-tagged kinectin. A positive reaction is defined as an absorbance value exceeding the mean OD absorbance value of sera from normals by three standard deviations.

31


N. Hirano et al

Figure 4. Sera from AA patients show different positive patterns for recombinant GST-full-length kinectin protein on Western blot analysis. Equal amounts of bacterial lysates expressing GST (lane 1) and GST-full-length kinectin fusion protein (lane 2) were separated by 9% SDS-PAGE, transferred to PVDF filters, and probed with mouse anti-GST monoclonal antibody, anti-kinectin monoclonal antibody, AA patients or normal sera. Immunoreactive proteins were detected with horseradish peroxidase-conjugated goat antimouse or anti-human IgG secondary antibody. Arrows indicate the immunoreactive GSTkinectin fusion proteins.

Figure 5. Sensitivity of 2AA patient serum reactivity against GST-kinectin fusion protein. Equal amounts of bacterial lysates containing GST-full-length kinectin fusion proteins were separated by 7.5% SDS-PAGE, transferred to PVDF filters, and probed with patient 2AA sera (diluted at 1:250 to 1:5,000) or normal serum diluted at 1:250. Immunoreactive proteins were detected with horseradish peroxidase-conjugated goat antihuman secondary antibody.

32


N. Hirano et al Figure 6. Partial epitope mapping of kinectin IgG autoantibody using GST-kinectin fusion proteins. We made a series of GST-partial kinectin fusion proteins using PCRbased subcloning. Equal amounts of lysates expressing various different fusion proteins were separated by 9% SDS-PAGE, and Western blot analysis was performed. Blots were probed with mouse anti-GST monoclonal antibody (A), anti-kinectin monoclonal antibody (B), patient 1AA serum (C), or patient 7AA serum (D). Immunoreactive proteins were detected as described in previous figures. (E) Results of partial epitope mapping of kinectin IgG autoantibody in 6 AA patients sera were shown. Regions of the kinectin sequence shaded with identical patterns represent areas that are immunoreactive with multiple AA patient sera.

Figure 7. Standard cytotoxicity assay. We raised CTLs against kinectin-derived peptides 794S (A) and 645F (B) from normal donors. Peptide-pulsed T2 cells were incubated with differing E:T ratios in a 4-hour cytotoxicity assay. (A) CTLs raised against the 794S peptide specifically lyse 794S pulsed ( ), but not T2 cells pulsed with an irrelevant peptide ( , F271 from Mage-3). (B) Similarly, CTLs generated against the 945F peptide recognize only T2 cells pulsed with 645F ( ) but not control T2 cells ( , pulsed with F271; unpulsed).

33

,


N. Hirano et al References 1. Mathe G, Amiel JL, Schwarzenberg L, Choay J, Trolard P, Schneider M, Hayat M, Schlumberger JR, Jasmin C. Bone marrow graft in man after conditioning by antilymphocytic serum. Transplant Proc. 1971;3:325-332 2. Young NS. Acquired aplastic anemia [clinical conference]. JAMA. 1999;282:271-278 3. Maciejewski JP, Risitano A, Kook H, Zeng W, Chen G, Young NS. Immune pathophysiology of aplastic anemia. Int J Hematol. 2002;76 Suppl 1:207-214 4. Young NS. Acquired aplastic anemia. Annals of Internal Medicine. 2002;136:534-546 5. Bacigalupo A, Broccia G, Corda G, Arcese W, Carotenuto M, Gallamini A, Locatelli F, Mori PG, Saracco P, Todeschini G, et al. Antilymphocyte globulin, cyclosporin, and granulocyte colony- stimulating factor in patients with acquired severe aplastic anemia (SAA): a pilot study of the EBMT SAA Working Party. Blood. 1995;85:1348-1353 6. Rosenfeld SJ, Kimball J, Vining D, Young NS. Intensive immunosuppression with antithymocyte globulin and cyclosporine as treatment for severe acquired aplastic anemia. Blood. 1995;85:3058-3065 7. Brodsky RA. High-dose cyclophosphamide for aplastic anemia and autoimmunity. Curr Opin Oncol. 2002;14:143-146 8. Young NS. Immunosuppressive treatment of acquired aplastic anemia and immunemediated bone marrow failure syndromes. Int J Hematol. 2002;75:129-140 9. Rosenfeld S, Follmann D, Nunez O, Young NS. Antithymocyte globulin and cyclosporine for severe aplastic anemia: association between hematologic response and long-term outcome. JAMA. 2003;289:1130-1135

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N. Hirano et al 10. Kagan WA, Ascensao JA, Pahwa RN, Hansen JA, Goldstein G, Valera EB, Incefy GS, Moore MA, Good RA. Aplastic anemia: presence in human bone marrow of cells that suppress myelopoiesis. Proc Natl Acad Sci U S A. 1976;73:2890-2894 11. Hoffman R, Zanjani ED, Lutton JD, Zalusky R, Wasserman LR. Suppression of erythroid-colony formation by lymphocytes from patients with aplastic anemia. N Engl J Med. 1977;296:10-13 12. Zoumbos NC, Gascon P, Djeu JY, Young NS. Interferon is a mediator of hematopoietic suppression in aplastic anemia in vitro and possibly in vivo. Proc Natl Acad Sci U S A. 1985;82:188-192 13. Hinterberger W, Adolf G, Aichinger G, Dudczak R, Geissler K, Hocker P, Huber C, Kalhs P, Knapp W, Koller U, et al. Further evidence for lymphokine overproduction in severe aplastic anemia [see comments]. Blood. 1988;72:266-272 14. Dufour C, Corcione A, Svahn J, Haupt R, Battilana N, Pistoia V. Interferon gamma and tumour necrosis factor alpha are overexpressed in bone marrow T lymphocytes from paediatric patients with aplastic anaemia. Br J Haematol. 2001;115:1023-1031 15. Nakao S, Yamaguchi M, Shiobara S, Yokoi T, Miyawaki T, Taniguchi T, Matsuda T. Interferon-gamma gene expression in unstimulated bone marrow mononuclear cells predicts a good response to cyclosporine therapy in aplastic anemia. Blood. 1992;79:2532-2535 16. Sloand E, Kim S, Maciejewski JP, Tisdale J, Follmann D, Young NS. Intracellular interferon-gamma in circulating and marrow T cells detected by flow cytometry and the response to immunosuppressive therapy in patients with aplastic anemia. Blood. 2002;100:1185-1191

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N. Hirano et al 17. Zoumbos NC, Gascon P, Djeu JY, Trost SR, Young NS. Circulating activated suppressor T lymphocytes in aplastic anemia. N Engl J Med. 1985;312:257-265 18. Herrmann F, Griffin JD, Meuer SG, Meyer zum Buschenfelde KH. Establishment of an interleukin 2-dependent T cell line derived from a patient with severe aplastic anemia, which inhibits in vitro hematopoiesis. J Immunol. 1986;136:1629-1634 19. Maciejewski JP, Hibbs JR, Anderson S, Katevas P, Young NS. Bone marrow and peripheral blood lymphocyte phenotype in patients with bone marrow failure. Exp Hematol. 1994;22:1102-1110 20. Steinman L. A few autoreactive cells in an autoimmune infiltrate control a vast population of nonspecific cells: a tale of smart bombs and the infantry. Proc Natl Acad Sci U S A. 1996;93:2253-2256 21. Kawakami Y, Rosenberg SA. Human tumor antigens recognized by T-cells. Immunol Res. 1997;16:313-339 22. Bacigalupo A, Podesta M, Mingari MC, Moretta L, Van Lint MT, Marmont A. Immune suppression of hematopoiesis in aplastic anemia: activity of T- gamma lymphocytes. J Immunol. 1980;125:1449-1453 23. Nakao S, Harada M, Kondo K, Odaka K, Ueda M, Matsue K, Mori T, Hattori K. Effect of activated lymphocytes on the regulation of hematopoiesis: suppression of in vitro granulopoiesis by OKT8+Ia+T cells induced by alloantigen stimulation. J Immunol. 1984;132:160-164. 24. Nakao S, Takami A, Takamatsu H, Zeng W, Sugimori N, Yamazaki H, Miura Y, Ueda M, Shiobara S, Yoshioka T, Kaneshige T, Yasukawa M, Matsuda T. Isolation of a T-cell

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N. Hirano et al clone showing HLA-DRB1*0405-restricted cytotoxicity for hematopoietic cells in a patient with aplastic anemia. Blood. 1997;89:3691-3699 25. Albert ML, Austin LM, Darnell RB. Detection and treatment of activated T cells in the cerebrospinal fluid of patients with paraneoplastic cerebellar degeneration. Ann Neurol. 2000;47:9-17 26. Sahin U, Tureci O, Schmitt H, Cochlovius B, Johannes T, Schmits R, Stenner F, Luo G, Schobert I, Pfreundschuh M. Human neoplasms elicit multiple specific immune responses in the autologous host. Proc Natl Acad Sci U S A. 1995;92:11810-11813 27. Sahin U, Tureci O, Pfreundschuh M. Serological identification of human tumor antigens. Curr Opin Immunol. 1997;9:709-716 28. Old LJ, Chen YT. New paths in human cancer serology [comment]. J Exp Med. 1998;187:1163-1167 29. Tureci O, Sahin U, Zwick C, Neumann F, Pfreundschuh M. Exploitation of the antibody repertoire of cancer patients for the identification of human tumor antigens. Hybridoma. 1999;18:23-28 30. Jager E, Chen YT, Drijfhout JW, Karbach J, Ringhoffer M, Jager D, Arand M, Wada H, Noguchi Y, Stockert E, Old LJ, Knuth A. Simultaneous humoral and cellular immune response against cancer-testis antigen NY-ESO-1: definition of human histocompatibility leukocyte antigen (HLA)-A2-binding peptide epitopes. J Exp Med. 1998;187:265-270 31. Jager E, Jager D, Karbach J, Chen YT, Ritter G, Nagata Y, Gnjatic S, Stockert E, Arand M, Old LJ, Knuth A. Identification of NY-ESO-1 epitopes presented by human

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N. Hirano et al histocompatibility antigen (HLA)-DRB4*0101-0103 and recognized by CD4(+) T lymphocytes of patients with NY-ESO-1-expressing melanoma. J Exp Med. 2000;191:625630 32. Hirano N, Shibasaki F, Kato H, Sakai R, Tanaka T, Nishida J, Yazaki Y, Takenawa T, Hirai H. Molecular cloning and characterization of a cDNA for bovine phospholipase Calpha: proposal of redesignation of phospholipase C- alpha. Biochem Biophys Res Commun. 1994;204:375-382 33. Parker KC, Bednarek MA, Coligan JE. Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. J Immunol. 1994;152:163-175. 34. Vonderheide RH, Hahn WC, Schultze JL, Nadler LM. The telomerase catalytic subunit is a widely expressed tumor-associated antigen recognized by cytotoxic T lymphocytes. Immunity. 1999;10:673-679. 35. Gubin AN, Njoroge JM, Bouffard GG, Miller JL. Gene expression in proliferating human erythroid cells. Genomics. 1999;59:168-177 36. Lee S, Zhou G, Clark T, Chen J, Rowley JD, Wang SM. The pattern of gene expression in human CD15+ myeloid progenitor cells. Proc Natl Acad Sci U S A. 2001;98:3340-3345 37. Hashimoto S, Nagai S, Sese J, Suzuki T, Obata A, Sato T, Toyoda N, Dong HY, Kurachi M, Nagahata T, Shizuno K, Morishita S, Matsushima K. Gene expression profile in human leukocytes. Blood. 2003;101:3509-3513

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N. Hirano et al 38. Mao M, Fu G, Wu JS, Zhang QH, Zhou J, Kan LX, Huang QH, He KL, Gu BW, Han ZG, Shen Y, Gu J, Yu YP, Xu SH, Wang YX, Chen SJ, Chen Z. Identification of genes expressed in human CD34(+) hematopoietic stem/progenitor cells by expressed sequence tags and efficient full- length cDNA cloning. Proc Natl Acad Sci U S A. 1998;95:81758180 39. Gomes I, Sharma TT, Mahmud N, Kapp JD, Edassery S, Fulton N, Liang J, Hoffman R, Westbrook CA. Highly abundant genes in the transcriptosome of human and baboon CD34 antigen-positive bone marrow cells. Blood. 2001;98:93-99 40. Zhou G, Chen J, Lee S, Clark T, Rowley JD, Wang SM. The pattern of gene expression in human CD34(+) stem/progenitor cells. Proc Natl Acad Sci U S A. 2001;98:13966-13971 41. Steidl U, Kronenwett R, Rohr UP, Fenk R, Kliszewski S, Maercker C, Neubert P, Aivado M, Koch J, Modlich O, Bojar H, Gattermann N, Haas R. Gene expression profiling identifies significant differences between the molecular phenotypes of bone marrowderived and circulating human CD34+ hematopoietic stem cells. Blood. 2002;99:2037-2044 42. Toyoshima I, Yu H, Steuer ER, Sheetz MP. Kinectin, a major kinesin-binding protein on ER. J Cell Biol. 1992;118:1121-1131 43. Coppel RL, Gershwin ME. Primary biliary cirrhosis: the molecule and the mimic. Immunol Rev. 1995;144:17-49 44. Warren KG, Catz I, Steinman L. Fine specificity of the antibody response to myelin basic protein in the central nervous system in multiple sclerosis: the minimal B-cell epitope and a model of its features. Proc Natl Acad Sci U S A. 1995;92:11061-11065

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N. Hirano et al 45. Young NS, Maciejewski J. The pathophysiology of acquired aplastic anemia. N Engl J Med. 1997;336:1365-1372 46. Brown KE, Tisdale J, Barrett AJ, Dunbar CE, Young NS. Hepatitis-associated aplastic anemia [see comments]. N Engl J Med. 1997;336:1059-1064 47. Wang W, Meadows LR, den Haan JM, Sherman NE, Chen Y, Blokland E, Shabanowitz J, Agulnik AI, Hendrickson RC, Bishop CE, et al. Human H-Y: a malespecific histocompatibility antigen derived from the SMCY protein. Science. 1995;269:1588-1590. 48. Meadows L, Wang W, den Haan JM, Blokland E, Reinhardus C, Drijfhout JW, Shabanowitz J, Pierce R, Agulnik AI, Bishop CE, Hunt DF, Goulmy E, Engelhard VH. The HLA-A*0201-restricted H-Y antigen contains a posttranslationally modified cysteine that significantly affects T cell recognition. Immunity. 1997;6:273-281. 49. Vogt MH, Goulmy E, Kloosterboer FM, Blokland E, de Paus RA, Willemze R, Falkenburg JH. UTY gene codes for an HLA-B60-restricted human male-specific minor histocompatibility antigen involved in stem cell graft rejection: characterization of the critical polymorphic amino acid residues for T- cell recognition. Blood. 2000;96:3126-3132. 50. Warren EH, Gavin MA, Simpson E, Chandler P, Page DC, Disteche C, Stankey KA, Greenberg PD, Riddell SR. The human UTY gene encodes a novel HLA-B8-restricted H-Y antigen. J Immunol. 2000;164:2807-2814.

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N. Hirano et al Table 1. Genes isolated by serologic screening with 8 AA patient sera

Genes expressed by hematopoietic cells Positivity by Lineage (UniGene Cluster ID) phage plaque assay distribution Kinectin (Hs.418467) RhoB (Hs.406064) Sorbitol dehydrogenase (Hs.878) Ribosomal protein S3A (Hs.77039) Tropomyosin 2 (Hs.300772) H-ferritin (Hs.418650) PGR 1 (ND) Hsp90-alpha (Hs.356531) S100A9, MRP14 (Hs.112405) Alpha-2-HS-glycoprotein (Hs.324746) Erythrocyte membrane protein band 4.2 (Hs.733) Globin gamma A (Hs.283108) Globin beta (Hs.155376) RBQ-1 (Hs.91065) Nuclease sensitive element binding protein 1 (Hs. 74497) Methionine aminopeptidase 1 (Hs.82007) Hook1 (Hs.250752)

4/8 2/8 2/8 2/8 2/8 1/8 1/8 1/8 1/8 1/8 1/8

M, E, L M, L E M, E, L L M, E ND M, L M E E

+ – + + + – – + + – –

1/8 1/8 1/8 1/8

E E L E

+ + + +

1/8 1/8

M, L E

+ –

Genes not reported to be expressed by hematopoietic cells Alcohol dehydrogenase alpha subunit Aldolase B Group-specific component Apolipoprotein A-II Serum albumin

1/8 1/8 1/8 1/8 1/8

ESTs, unknown genes

ND

ND: not determined; M: myeloid; E: erythroid; L: lymphoid

41

Expression in CD34+ cells


N. Hirano et al

Table 2. Survey of sera from 35 normal donors, 18 AA, 20 multiply transfused, 65 nonAA autoimmune disease patients: reactivity with recombinant kinectin protein

Western blot analysis

ELISA

Normal donors

0/35*

0/35

AA patients

7/18*

6/18

Multiply transfused patients with thalassemia and sickle cell anemia

0/20

0/20

Non-AA autoimmune disease patients (19 SLE, 24 RA, and 22 MS)

NT

0/65

NT: not tested; *P < .001 compared with AA patients by the

42

2

method.


N. Hirano et al Table 3. Characteristics of AA patients studied Age/Sex

Anti-kinectin IgG antibody

Anti-kinectin IgG antibody

+

–

Type of AA

Preceding hepatitis

18/F 48/M 12/M 18/F 9/M 13/M 57/M

moderate severe severe moderate moderate/severe severe moderate/severe

– + – – – – –

3/F 15/F 11/M 24/F 9/M 56/F 0/M 3/F 15/M 33/M 38/M

moderate/severe severe moderate/severe moderate/severe moderate/severe moderate moderate/severe moderate moderate/severe severe moderate/severe

– – – – – – – – – + +

Blood IS therapy transfusion before sampling –

+

±

–

+ + + + +

+ + + + +

– – – + + + + + + + +

– – + – + – – + + – +

±: This patient received his first blood transfusion immediately prior to blood sampling.

43


N. Hirano et al Table 4. Binding of kinectin-derived peptides and controls to HLA-A*0201 Name

Sequence

Protein

Position

Scorea

Fluorescence indexb

794S 645F 600V 553Q F271

SLVEELKKV FLLKAEVQKL VLAEELHKV ALMESEQKV FLWGPRALV (positive control) AHTKDGFNF (negative control)

kinectin kinectin kinectin kinectin MAGE-3

aa794 aa645 aa600 aa552 aa271

656 836 1115 1055 2655

2.8 1.5 0.6 0.3 2.0

Idiotype

aa98

0

0.1

A98-Idc a

Calculated score in arbitrary units. (Mean fluorescence with peptide – mean fluorescence without peptide)/(mean fluorescence without peptide). c Peptide sequence obtained from the idiotypic sequence of a patient with plasma cell leukemia and predicted to bind to HLA-B38. b

44


N. Hirano et al Table 5. Effect of CTLs against kinectin-derived peptide on Colony Formation by CD34+ Cells Experiment

CTLs against Kinectin peptide

Blocking MoAb

CFU-GM

BFU-E

1

none 645F 645F 645F 794S 794S 794S

none none mIgG2b BB7.2 none mIgG2b BB7.2

48 ± 4 26 ± 2 25 ± 3 48 ± 3 44 ± 3 48 ± 3 49 ± 4

22 ± 2 24 ± 4 26 ± 3 27 ± 2 25 ± 2 25 ± 3 24 ± 3

2

none 645F 645F 645F 794S 794S 794S

none none mIgG2b BB7.2 none mIgG2b BB7.2

64 ± 5 36 ± 5 38 ± 2 68 ± 3 68 ± 3 69 ± 5 71 ± 5

22 ± 2 25 ± 2 25 ± 3 25 ± 3 24 ± 5 24 ± 3 24 ± 4

BB7.2: HLA-A2 specific MoAb; mIgG2b: isotype control. Representative results of three similar experiments performed in an at least sextuplicated manner. Data are represented as means ± SD.

45


N. Hirano et al Figures

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53