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Blood, Vol. 95 No. 4 (February 15), 2000:
pp. 1393-1399
IMMUNOBIOLOGY
From the Department of Pathology and Laboratory Medicine, University
of Pennsylvania School of Medicine, Philadelphia, PA, and the Institute
of Human Genetics, Polish Academy of Sciences, Poznan, Poland.
Chronic B-cell stimulation may be a predisposing event in the early
pathogenesis of the acquired immunodeficiency syndrome (AIDS)-related
lymphoma (ARL). ARL-derived immunoglobulin (Ig) genes are significantly
diversified from germline, suggesting that antigenic stimulation via Ig
receptors may occur prior to malignant transformation. We have
evaluated 6 ARL-derived antibodies for binding to human
immunodeficiency virus (HIV) and cell surface epitopes. Five cases
expressed IgM, and 1 case expressed IgG. Expressed V genes were
significantly diversified (3%-15%) from known germline V genes. A
non-Ig producing mouse myeloma cell line was transfected with
expression vectors containing the lymphoma-derived V genes. By
enzyme-linked immunosorbent assay and Western blot assay, the
lymphoma-derived Ig's showed no reactivity against HIV recombinant
proteins. Also, no specific HIV reactivity was observed by flow
cytometry with lymphoma-derived Ig's against the T-cell line infected
with T-tropic HIV-1 or peripheral blood mononuclear cells infected with
M-tropic HIV strains, indicating lack of binding to native HIV
epitopes. However, 2 of the lymphoma-derived Ig's (ARL-7 and ARL-14)
bound strongly to non-HIV-infected cells of various tissue origins.
Thus, these findings suggest that the transformed B cells of
AIDS-associated lymphomas may not arise from the pool of anti-HIV
specific B cells but, rather, may develop from B cells responding to
other antigens, including self-antigens.
(Blood. 2000;95:1393-1399)
Considerable evidence from clinical and experimental
studies supports the notion of chronic stimulation of B cells in human immunodeficiency virus (HIV) infection.1-4 For example,
peripheral blood B cells from HIV-infected individuals display markedly
increased spontaneous proliferation and immunoglobulin secretion,
associated with monoclonal and oligoclonal paraproteins.3,4
The mechanism(s) responsible for this B-cell hyperactivity is not
known. Possibly, alterations in cytokine profiles as a consequence of
decreased CD4-positive T cells could lead to a shift in the balance of
negative and positive modulators of B-cell activation. For example, the induction of cytokines, such as interleukin-6, tumor necrosis factor- Another possibility for the mechanism involved in maintaining the
chronic B-cell hyperactivity is that the virus is a direct stimulator
of B-cell proliferation. In this regard, evidence exists that HIV
itself induces B-cell activation and proliferation.1,4,9,10 For conventional antigen recognition and activation, B cells recognize and bind antigen via surface immunoglobulin generated by variable region gene utilization of the heavy (VH) and light
(V To test the hypothesis that HIV-specific triggering of B cells is
involved in lymphomagenesis, we cloned the AIDS-related lymphoma
(ARL)-derived immunoglobulin genes into a plasmid construct that
allowed us to express these antibodies in a nonimmunoglobulin-secreting mouse myeloma cell line. The secreted antibodies did not bind to
HIV-related epitopes by use of various experimental approaches, including enzyme-linked immunosorbent assays (ELISA) employing recombinant HIV proteins, Western blotting of HIV-infected cell lysates, or flow cytometry of HIV-infected T cells. Interestingly, 2 of
6 ARL-derived immunoglobulins bound strongly to hematopoietic and
nonhematopoietic cell-associated epitopes (for example, HIV-unrelated), which have not been characterized further. Although a lymphoma-derived B-cell line with anti-gp160 reactivity has been reported
previously,26 the results of our current study suggest that
HIV-specific B-cell activation may not play a significant role in
lymphoma development. On the other hand, self-reactivity along with
other factors, such as cytokine imbalances,5,6 concomitant
Epstein-Barr virus (EBV) or Kaposi's sarcoma-associated herpesvirus
infection,27,28 deregulated oncogene expression, and B
cell-endothelial cell interactions, may contribute to lymphoma
development in HIV-infected individuals.
Patient samples
Cells and antibodies for FACS analysis
Polymerase chain reaction
Cloning of PCR products
Transfection of eukaryotic cell line A total of 10 µg of pRTM vector, containing the IgH insert, and 10 µg of pSV2 vector, containing IgL insert, were linearized with 20 U of PvuI (Pharmacia Biotech, Piscataway, NJ). Transfection of these vectors into the P3-X63-Ag8.653, a nonsecreting mouse myeloma cell line,36 was performed by electroporation (960 µF and 0.4 kV) using 0.4-cm cuvettes (Bio-Rad). Transfected cells were incubated in RPMI 1640 containing 20% FBS, 2% Q, and 1% penicillin/streptomycin. After 48 to 72 hours, neomycin (G418; Gibco BRL) was added to a final concentration of 1 mg/mL, and cells were transferred into 96-well tissue culture plates. Cells growing under neomycin selection were checked for immunoglobulin production by ELISA, and the best secretors were further expanded. Table 3 shows characteristics of the obtained transfectomas.
Purification of ARL antibodies ARL-derived antibodies contained in the culture supernatant from each transfectoma were purified using goat anti-human IgM agarose columns (Sigma), eluted with 0.1 mol/L glycine/HCl containing 0.02% NaN3, pH 2.5, and immediately neutralized to pH 7 with 0.5 mol/L phosphate buffer, pH 8. Eluates obtained were further dialyzed with nitrocellulose membranes (Collodion Membranes, Schleicher & Schuell Inc, Keene, NH). The purity of the concentrated antibodies was checked on 12% acrylamide gels under denaturing conditions. Proteins were transferred to 0.2-µm PDVF membranes (Bio-Rad). The Western blots containing the heavy and light chains were incubated with goat F(ab') fragments anti-human IgM/IgG (H&L) biotin-labeled, followed by streptavidin/horseradish peroxidase, and developed by the ECL-Western blotting detection kit (Amersham International, Buckinghamshire, England). The size of the bands was compared to standard molecular weight markers to verify that the transfectomas secreted both heavy and light chains of the appropriate molecular weight.ELISA for detecting immunoglobulin production by the transfectomas MaxiSorp plates (Nunc Inc, Naperville, IL) were coated with 100 µL/well of goat anti-human IgM (Sigma) (2 µg/mL in phosphate-buffered saline (PBS)). After 3 washes with PBS containing 0.1% Tween 20, pH 7.3, wells were blocked with PBS containing 0.5% dry milk and 0.01% thimerosal, pH 7.0, for 1 hour at 37°C. Supernatants (100 µL) were added to the wells and incubated for 1 hour at 37°C. After 3 washes, 100 µL/well of goat anti-human chain labeled with alkaline
phosphatase (Sigma) was added and incubated for 1 hour at 37°C.
Plates were washed, developed with P-Nitrophenyl Phosphate (Sigma), and
read (OD 410 nm) by an ELISA microplate reader (Dynatech Laboratories
Inc, Chantilly, VA). The use of the "sandwich"
anti-µ/ARL-Ab/anti- allowed us to discard clones transfected with
only 1 of the 2 desired V genes.
Anti-HIV reactivity ELISA kit for determining anti-HIV reactivity (Abbott HIV AB HIV-1/HIV-2 3.0) was used as recommended by the manufacturers (Abbott Laboratories, Santa Clara, CA). For the Western blot assays, we used the Novapath HIV-1 Immunoblot Kit (Bio-Rad). The Novapath kit is an in vitro qualitative assay for the detection of antibodies to individual proteins of HIV-1. Specificities of these antibodies can be inferred from the position of bands on the nitrocellulose strips. The kit tested for the presence of the immunoglobulins specific for the following HIV proteins or glycoproteins: gag (p18, p24, p55), pol (p51, p65, p32), env (gp120, gp160, gp 41-43). Because the kit is designed to detect only IgG isotype (the secondary antibody provided is a goat anti-human IgG-AP), we substituted the provided secondary antibody for a goat anti- AP antibody (Sigma) that would also detect IgM antibodies.
Flow cytometric analysis Aliquots of 5 × 105 to 1 × 106 cells were incubated with ARL-derived antibodies and diluted in PBS containing 2% FBS, at 4°C, for 30 minutes. The cells were washed and incubated with goat F(ab') fragments anti-human IgG/IgM (H&L) biotin-labeled, for 30 minutes, and washed before streptavidin-FITC was added. After the final washing, cells were resuspended in PBS containing 2% FCS and 0.9% paraformaldehyde prior to analysis. To differentiate HIV-infected versus noninfected T-cell lines, aliquots of cells were permeabilized using a previously described technique.37 The human mAb 71-31 was used as a positive control for HIV-infected T cells.Autoantibody assays Anti-cardiolipin antibody assay. The ELISA procedure was performed as described previously.38 Cardiolipin (Avanti Polar Lipids Inc, Alabaster, AL) was diluted 1:100 in absolute ethanol. A MaxiSorp plate was coated with the antigen (50 µL/well) and left uncovered at 4°C overnight. The next day, after 3 washes with PBS containing 0.1 g/L CaCl2, plates were blocked with PBS containing 0.1 g/L CaCl2, 3% BSA, and 10% FBS, pH 7.2 (blocking buffer), for 1 hour at 37°C. After washing, ARL-derived antibodies and controls were added to the wells, diluted in PBS containing 0.1 g/L CaCl2 and 10% FBS, pH 7.2 (diluting buffer), and incubated for 1 hour at 37°C. Wells were washed before adding goat anti-human heavy- and light-chain alkaline phosphatase (Southern Biothechnology Associates Inc, Birmingham, AL) (1:500 dilutions) for 1 hour at 37°C. After a final washing, samples were developed and read (O.D. 410 nm) as described above. Anti-ssDNA ELISA. DNA (Spermidine trihydrochloride) (Sigma) stock solution was prepared as previously described.39 Immulon 2 plates (Dynatech) were coated with 2.5 µg/50 µL per well of Poly-L-Lysine (Sigma), diluted in ddH2O, and left at room temperature for 30 minutes. Plates were washed 3 times with PBS and coated with 50 µL of single-stranded (ss)DNA, 2.5 µg/mL in autoclaved triethanolamine-buffered saline (TBS), pH 7.4, and left at room temperature for 2 hours. DNA was denatured (95°C for 5 minutes) and chilled rapidly before use. After 3 more washes, wells were blocked with Poly-L-Glutamine (Sigma) and diluted in ddH2O (2.5 µg/50 µL) for 2 hours at room temperature. Two more washes were performed before coating the plate with TBS (50 µL/well) for storage overnight at 4°C. The following day, plates were washed 3 times with PBS containing 0.1% Tween 20, pH 7.3 (washing solution), and blocked with PBS containing 0.5% dry milk and 0.01% thimerosal, pH 7.3 (blocking buffer), for 1 hour at 37°C. ARL-derived antibodies and controls were incubated for 1 hour at 37°C. After 3 washes, the secondary antibody (the same used for anticardiolipin antibodies [ACLA]-ELISA) was incubated for 1 hour at 37°C. Plates were developed and read (O.D. 410 nm) as described above. Anti-i/I and Pr2 blood cell binding assay. This assay was carried as previously described.40,41
Lack of binding to HIV-1 proteins by ARL-derived immunoglobulin Transfectomas expressing the VH and VL genes of 6 EBV-negative ARL were evaluated for binding to HIV-related epitopes. No binding was detected to recombinant HIV proteins as measured by ELISA (Table 2) or to immunoblotted HIV peptides (data not shown). To evaluate the binding of ARL-derived immunoglobulins to native HIV proteins, we performed flow cytometric assays using a T-cell line infected with HIV-1 III-B or PBMC infected with HIV-1 Ba-L or HIV-1 SF162. To confirm productive HIV infection of cells used for FACS analysis, we used permeabilized cells because the 71-31 mAb with anti-p24 specificity or the rabbit polyclonal anti-p24 antibody only recognize cytoplasmic HIV epitopes. No specific HIV reactivity was observed by flow cytometry with lymphoma-derived immunoglobulins against the T-cell line infected with T-tropic HIV-1 or peripheral blood mononuclear cells infected with M-tropic HIV strains (data not shown). However, 2 of the 6 ARL-derived immunoglobulins (ARL-7 and ARL-14) bound to both infected as well as uninfected nonpermeabilized T cell lines (Figure 1). The binding pattern of ARL-7 and ARL-14 to nonpermeabilized cells indicated that these antibodies bound to surface, non-HIV-related epitopes.
Binding of ARL-7 and ARL-14 to purified T lymphocytes from PBMC To determine binding of ARL-derived immunoglobulin to primary human cells, we performed flow cytometry using purified peripheral T lymphocytes, the purity being more than 95% CD3-positive T cells. We found that ARL-7 bound weakly to peripheral T cells, with 5% of cells binding more strongly to ARL-7 than the isotype control. In contrast, ARL-14 bound to most peripheral T cells, with 86% of cells demonstrating reactivity compared with the isotype control (Figure 2). ARL-7 also bound to 62% of thymocytes isolated from fresh thymus (data not shown).
Binding of ARL-7 and ARL-14 to different cell lines Next, we wished to evaluate whether the cell-surface binding of ARL-7 and ARL-14 was restricted to T cells. Thus, we performed flow cytometric assays with both antibodies using a panel of hematopoietic and nonhematopoietic cells. The binding of these antibodies and their isotype control (expressed as mean channel fluorescence intensity) to the different human cells tested is shown in Table 4. We observed that ARL-7 bound to a broad spectrum of cell types, while ARL-14 bound to a smaller number of cell types. ARL-7 bound most strongly to the K-562 erythroleukemic cell line (mean channel fluorescence 218 ± 4.2), whereas ARL-14 bound most strongly to the REH pro-B cell line (mean channel fluorescence 221 ± 4). Some cell lines were analyzed only once and, thus, no SD is noted. Interestingly, for peripheral blood T lymphocytes we tested multiple donors, and a wide range of binding was observed, suggesting inter-donor variability.
Correlating surface binding of ARL-7 with cell cycle We noted variation in binding of the ARL-7 antibody to T-cell lines (range, 23%-56%), peripheral blood T cells (only 5%), and thymocytes (62%). In addition, significant variation in binding was seen within a T-cell line population. We therefore speculated that the ARL-7 target might be cell-cycle related. After surface staining with ARL-7, cells were permeabilized and incubated with propidium iodide (PI) for nuclear staining to define cell populations in different phases of the cell cycle. Figure 3 shows that cell-surface staining with ARL-7 did not vary between cells of different stages of cell cycle, indicating that the antigen to which ARL-7 bound was not related to a specific phase of the cellular cycle.
Binding of ARL antibodies to commonly tested auto-antigens Because 2 of 6 ARL-derived antibodies showed strong reactivity to cell surface determinants suggestive of autoreactivity, we speculated that perhaps the remaining ARL-derived antibodies might have specificity to self-antigens. However, we could not demonstrate binding of the 6 lymphoma-derived immunoglobulins to cardiolipin (Table 5). Only 1 ARL-derived immunoglobulin had weak reactivity to ssDNA (Table 6). The lack of binding to ssDNA and cardiolipin suggests that these IgM antibodies are less likely to be polyreactive. Additionally, the antibodies did not bind to i/I autoantigens on red blood cells (data not shown).
Several laboratories have carried out in vitro and in vivo analyses to document B-cell hyperactivity during the course of HIV infection. In a previous study, we have provided evidence for early B-cell activation preceding lymphoma development.29 We were able to identify an expanded B-cell clone in the bone marrow of an HIV-infected individual 3 years prior to its progression to clinical lymphoma. These studies suggested that B-cell hyperactivity might predispose certain B cells to lymphomagenesis. In view of the significant degree of somatic diversification of immunoglobulin genes expressed by ARL, we speculated that an antigen-driven B-cell process may play an important role in ARL pathogenesis. Because serum paraproteins with specificity for various HIV epitopes exist in HIV-infected individuals, it has been questioned whether HIV-specific B-cell activation might predispose to lymphoma development. To examine for reactivity to HIV epitopes, we expressed lymphoma-derived immunoglobulin VH and VL genes from 6 ARL in a mammalian-expression system. This approach was necessary because ARL is not associated with paraproteins that are clearly tumor-related, as is the case in plasmacytic B-cell tumors. First, we used ELISA and Western blotting assays to investigate for HIV reactivity. Because none of the tumor-related immunoglobulins bound to HIV proteins in these assays, we used immunocytometry that would detect native HIV epitopes. The tumor-related antibodies were negative for HIV reactivity by this method as well, thus suggesting that HIV reactivity of ARL-derived immunoglobulin is uncommon. This finding therefore does not support our initial hypothesis that interaction between HIV and surface immunoglobulin often predisposes HIV-specific B cells to lymphomagenesis. However, it is possible that HIV may bind to and activate B lymphocytes via nonimmunoglobulin receptors in a CD4-dependent or CD4-independent manner.42 For example, HIV proteins have been shown to bind to cell surface determinants, such as galactosyl ceramides, CD26 antigen, tryptase TL2, and adhesion molecules.40,41,43
To Darlisha Williams, who provided the M-tropic-infected and the uninfected PBMCs.
Submitted August 3, 1999; accepted October 21, 1999.
Supported by National Institutes of Health grant P50HL54 516.
Reprints: Leslie E. Silberstein, Departments of Medicine and Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 284 John Morgan Building, 36th and Hamilton Walk, Philadelphia, PA 19104-6082; e-mail: silbersl{at}mail.med.upenn.edu.
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.
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Top
Abstract
Introduction
, and others, within the course of HIV infection may support the cellular dysregulation and the development of neoplastic
disease.
) genes. By this mechanism, HIV might initiate a proliferative
response by B cells with specificity for viral envelope proteins.
However, several lines of evidence argue against this as the only
mechanism by which HIV stimulates B-cell proliferation. First, while a
significant proportion of immunoglobulin-producing cells from patients
with acquired immunodeficiency syndrome (AIDS) secrete antibodies with specificity for HIV viral proteins, many do not.
, 25 microfarad (µF), and 1.8 kV were applied, and
0.2-cm cuvettes (Bio-Rad) were used. Plasmid DNA was extracted from
single colonies using the QIAGEN-tip 100 kit (QIAGEN, Chatsworth, CA)
as recommended by the manufacturers. All vectors were sequenced to
confirm that the desired inserts were cloned. Nucleotide sequencing was
performed by an automatic sequencer (Perkin Elmer, Norfolk, CT).
