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Aberrant Expression of CD27 and Soluble CD27 (sCD27) in HIV Infection and in AIDS-Associated Lymphoma
Clinical Immunology Vol. 93, No. 2, November, pp. 114 –123, 1999 Article ID clim.1999.4782, available online at http://www.idealibrary.com on
Aberrant Expression of CD27 and Soluble CD27 (sCD27) in HIV Infection and in AIDS-Associated Lymphoma Daniel Widney,* Girija Gundapp,† Jonathan W. Said,† ,‡ ,§ Meta van der Meijden, ¶ Benjamin Bonavida,* Aisha Demidem,* Cristina Trevisan,\ Jeremy Taylor,‡ ,§ ,\ Roger Detels,‡ ,** and Otoniel Martı´nez-Maza* ,‡ ,§ ,¶,1 *Departments of Microbiology, Immunology, and Molecular Genetics, ¶Obstetrics and Gynecology, and †Pathology, and the §Jonsson Comprehensive Cancer Center, University of California at Los Angeles School of Medicine; **Departments of Epidemiology and \Biostatistics, University of California at Los Angeles School of Public Health; and the ‡Los Angeles Center of the Multicenter AIDS Cohort Study (MACS), Los Angeles, California
INTRODUCTION
CD27 is a member of the tumor necrosis factor receptor superfamily that is expressed primarily on T cells, as well as on subsets of B cells and NK cells. CD70, which is expressed on activated B and T cells, but not on resting lymphocytes, is a ligand for CD27. Cell surface CD27 can be proteolytically cleaved to produce a 32-kDa soluble CD27 (sCD27) molecule. Elevated levels of sCD27 are seen in a number of disease states and malignancies. Although it has been reported that cerebrospinal fluid sCD27 levels were elevated in people who had AIDS dementia, little is known about CD27 expression in HIV disease. To determine if sCD27 levels were elevated in those with HIV infection, and/or in those with AIDS-associated non-Hodgkin’s lymphoma (AIDS–NHL), sCD27 levels were measured in HIV-negative and HIV-positive subjects as well as in people who developed AIDS–NHL. Serum sCD27 levels were seen to be elevated in HIV1 subjects. Furthermore, sCD27 levels were particularly elevated in those subjects who went on to develop AIDS–NHL, with serum sCD27 levels in AIDS–NHL subjects being significantly higher than those in HIV1 subjects who did not develop lymphoma. Most AIDS– NHL cell lines and primary AIDS–NHL tumor specimens expressed both CD27 and its ligand, CD70. The proportion of circulating B cells that expressed cell surface CD27 was substantially reduced in those with HIV infection, and B cells from HIV-infected subjects produced decreased levels of sCD27 in culture. Together, these results indicate that CD27/sCD27 expression is abnormal in HIV infection and suggest that this molecule merits further examination as a potential marker for AIDS–NHL. © 1999 Academic Press Key Words: CD27; lymphoma; HIV; AIDS; B cell.
1 To whom correspondence should be addressed at the Department of Microbiology, Immunology, and Molecular Genetics, UCLA School of Medicine, Los Angeles, CA 90095-1747. Fax: (310) 206-5387. Email: [email protected].
1521-6616/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
CD27 is a 55-kDa transmembrane disulfide-linked homodimer and a member of the tumor necrosis factor receptor (TNF-R) superfamily. This family includes, in addition to CD27, TNF-R type I, and TNF-R type II, the OX-40 and 4-1BB molecules, CD30, CD40, and the FAS/APO-1 molecule (CD95) (1–3). CD27, like many of these molecules, is expressed primarily on lymphocytes. In the peripheral blood, it is found on T cells (3), as well as on small subsets of B cells and NK cells (2– 6). In both T and B cells, CD27 appears to be related to the differentiation/activation stage: naive T cells (CD45RA) express CD27, with the expression of this molecule being strongly upregulated after stimulation of the TCR complex. After antigen stimulation and upregulation of the CD45RO marker, the ability to express CD27 is greatly reduced (2, 7, 8). For B cells, Maurer et al. determined that only CD271 cells could be stimulated to secrete immunoglobulin (Ig) in vitro following stimulation with Staphylococcus aureus Cowan (SAC) and interleukin-2 (IL-2) (4). Recently, the ligand for CD27 was shown to be CD70, a type II transmembrane protein that has homology to the TNF family of proteins, which includes TNFa, TNFB, 4-1BBL, CD30L, CD40L, and FAS-ligand (2, 9 –11). CD70 appears on activated B and T cells, but not on resting lymphocytes (12). CD70 appears to costimulate the activation of T lymphocytes through CD27 (10). Although little is known about the function of the CD27/CD70 interaction in B cells, it is known that SAC/IL-2-stimulated B cell cultures produced much higher levels of IgM and IgG when costimulated through CD27 (13). Cell surface CD27 can be proteolytically cleaved to produce a 32-kDa soluble CD27 (sCD27) molecule, which can be detected in a number of body fluids, including serum and urine (14, 15). Elevated serum sCD27 levels are seen in a number of infectious and autoimmune disease states and are believed to be a
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marker of T cell activation (2). Also, various types of B cell malignancies express the CD27 molecule and elevated serum levels of sCD27 were noted in patients with B cell tumors not associated with HIV infection (16). There appeared to be a correlation between tumor load and sCD27 levels, and, while not shown directly, it seemed likely that the tumors themselves were the source of the sCD27 detected in serum. In the 64 patients examined in this study who had low-, intermediate-, or high-grade non-Hodgkin’s lymphoma (NHL), a majority showed a significant elevation in serum sCD27. In HIV infection, a progressive loss of T cell function is seen. Specific B cell response to antigens is also diminished in HIV infection, although, paradoxically, a general polyclonal activation of B cells and accompanying hypergammaglobulinemia is seen (17). Also, a greatly increased frequency of B cell NHL is seen in people infected with HIV (18). These NHL, which are typically of high or intermediate grade, fall into several categories: (i) CNS lymphomas, which are generally immunoblastic and Epstein–Barr virus-positive; (ii) small noncleaved cell lymphoma; (iii) large cell immunoblastic lymphoma (non-CNS); and (iv) intermediate grade-diffuse large noncleaved cell lymphoma (nonCNS) (18, 19). Although little is known about CD27 and sCD27 in HIV infection, it does appear that the ability of CD27 to costimulate T cell activation is not lost (20) and that infection of T cells with HIV does not modulate the expression of CD27 on infected cells (21). Also, elevated sCD27 levels were noted in cerebrospinal fluid from patients with AIDS dementia complex (22). In this study, we show that there is an increase in serum sCD27 levels in subjects with HIV infection. Also, we show that serum sCD27 levels were markedly increased prior to the development of AIDS–NHL, with sCD27 levels in subjects who developed AIDS–NHL being even higher than those seen in subjects with AIDS who did not develop NHL. In fact, elevated sCD27 levels were associated with a statistically significant relative risk for the development of AIDS– NHL. Furthermore, substantial changes in CD27 surface expression and sCD27 production by circulating B cells in HIV infection suggest that CD27 could be involved in the B cell dysfunction and dysregulation which is seen in HIV infection. MATERIALS AND METHODS
Study Subjects Three studies using blood/serum from human subjects were performed: (i) a study of serum sCD27 levels, (ii) a study of cell surface CD27 expression, and (iii) a study of spontaneous sCD27 production in vitro.
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(1) Serum sCD27 study. Serum samples were obtained from subjects in the UCLA Multicenter AIDS Cohort Study (UCLA-MACS). The UCLA-MACS cohort, established in 1984, is a natural history study of HIV composed of adult homosexual men and had 1637 original participants (23). This is an ongoing study, and the participants continue to be followed. At 3- to 6-month intervals, serum is collected from study participants and stored frozen at 270°C. Information on the clinical status of subjects is also collected. Serum from four groups of men was used. Group I comprised men who developed AIDS-associated NHL (AIDS-NHL group). Only 2 of 36 subjects in this group had an AIDS-defining opportunistic infection at the date at which serum was collected for this study. The mean age of this group was 40 years, and the mean CD4 number was 202 cells/mm 3 (ranging from 2 to 923). Group II comprised men with AIDS but without detectable AIDS-related cancers (AIDS, nonlymphoma group), who were matched by CD4 T cell number and age to subjects in Group I (mean age of 39 years, mean CD4 number 186 cells/mm 3, ranging from 0 to 785). Group III men were HIV-positive and asymptomatic with no AIDS-defining illnesses (HIV1 group) (mean age of 36 years, mean CD4 number 445 cells/mm 3). Group IV members were HIV-seronegative men (HIVnegative group) (mean age of 35 years, mean CD4 number 872 cells/mm 3). Groups III and IV were not matched by age or CD4 number to each other or to any other group. Subjects in the control groups (Groups II, III, and IV) were chosen to exclude individuals who had NHL or Kaposi’s sarcoma. Also, any subject in the control groups who later developed NHL was excluded from study. HIV-infected persons were placed in Group II (AIDS, nonlymphoma), as opposed to Group III (HIV1, nonAIDS), according to the 1993 Centers for Disease Control and Prevention Clinical AIDS definition, excluding the criterion that persons with CD4 T cell numbers less than 200 cells/mm 3 be considered to have AIDS. Thus, HIV1 persons who had CD4 T cell numbers less than 200 cells/mm 3 and no other AIDS-defining condition were placed in Group III (HIV1, non-AIDS). Lymphomas were subtyped according to the working formulation for classification of NHL. Group I (AIDS–NHL group) included all available AIDS–NHL cases in the UCLA-MACS at the time that this study was carried out. For 22 subjects in this group, the diagnosis of lymphoma was also the initial AIDS-defining condition. Serum sCD27 levels were measured for each subject using serum drawn at one study visit within the 18 months preceding the AIDS–NHL diagnosis; the average time prior to diagnosis was 6 months. Sera from all other subjects (in the three control groups) represent a serum sample collected at a single MACS visit. All sera tested were frozen and then thawed before measure-
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ment of sCD27 levels by enzyme-linked immunosorbent assay (ELISA). (2) Cell surface CD27 study. Blood was obtained in EDTA tubes by venous puncture from subjects seen at clinics at UCLA who had AIDS (nonlymphoma), who were HIV1 (non-AIDS), or who were HIV-negative. These subjects were not necessarily the same subjects as were used in the serum sCD27 study described above, nor was any attempt made to match the groups by age or CD4 T cell number. Blood was used immediately (not frozen) for these experiments. Serum sCD27 was determined for most of the subjects tested in these studies using stored, frozen specimens. (3) Spontaneous sCD27 production study. Specimens for this study were obtained by venous collection in EDTA tubes from participants in the UCLA-MACS, or from various clinics at UCLA, who had AIDS (nonlymphoma), who were HIV1 (non-AIDS), or who were HIV-negative. This blood was used immediately (not frozen). These subjects were not necessarily the same subjects as were used in the serum sCD27 study described above, nor was any attempt made to match groups by age or CD4 T cell number. Determination of Serum sCD27 Levels Serum levels of sCD27 were measured by ELISA (compact soluble CD27 ELISA kit, Central Laboratory of the Netherlands Red Cross, Amsterdam, The Netherlands), a sandwich type immunoassay which can be used to measure sCD27 in serum. Briefly, this assay utilizes an anti-CD27 monoclonal antibody coated to polystyrene microtiter wells. After addition of samples, or standards, and rinsing, a biotinylated second monoclonal CD27 antibody was added and allowed to bind to sCD27, captured by the coating antibody. After rinsing, horseradish–peroxidase-conjugated streptavidin was added, followed by a biotinylated second antibody and addition of a chromogen, resulting in the formation of a colored product. Absorbance was measured at 450 nm to quantify color development. Levels of sCD27 in samples are determined by comparison with a standard curve prepared from seven sCD27 standards, ranging from 1.56 to 100 units (U)/ml. According to the manufacturer, the lower level of detection of this assay is 1 U/ml, and up to three freeze–thaw cycles have no effect on measured sCD27 levels. Isolation of PBMC Peripheral blood mononuclear cells (PBMC) were separated from EDTA peripheral blood by dilution of the blood 1:1 with saline and centrifugation over FicollPaque (Pharmacia LKB Biotechnology, Piscataway, NJ).
Determination of Cell Surface CD4, CD19, CD27, and CD70 Expression by Flow Cytometry CD4 (T helper/inducer cell) or CD19 (B lymphocyte) percentage was determined by flow cytometry, as described (24). For detection of cell surface CD27, a direct staining protocol was used, utilizing PE-conjugated anti-CD27 monoclonal antibody (clone M-T271, mouse IgG 1, PharMingen, San Diego, CA). For CD70, an indirect staining protocol was used, with the anti-CD70 antibody (clone HNE51, mouse IgG 1, Immunotech, Inc., Westbrook, ME) being followed by FITC-conjugated goat anti-mouse IgG. Appropriate isotype controls were also included. Next, the cells were stained with 7-actinomycin-D (7-AAD) (1 mg/ml, Calbiochem, San Diego, CA) for 30 min as described by Schmid et al. (25) and then fixed in 1% paraformaldehyde plus actinomycin-D (3 mg/ml) (26). To examine the expression of CD27 or CD70 on circulating B cells, two-color flow cytometry was performed, with anti-CD19-FITC (Becton-Dickinson Immunocytometry Systems, Mountain View, CA) being used as a pan-B cell marker. All samples were analyzed within 24 h on a FACScan flow cytometer (Becton-Dickinson), with dead cells being gated out using 7-AAD staining. At least 5000 events were collected for each sample. Data were analyzed with C30 FACScan Research Software. Immunostaining for Determination of CD27 and CD70 Expression on AIDS-NHL Tissue Lymph nodes from 20 patients with lymphoma, 10 of whom were seropositive for HIV-1, were evaluated. Tissues were obtained at the time of surgery and snapfrozen. Four-micrometer sections were mounted on Super Frost slides (Fisher Scientific Princeton, NJ) fixed in cold acetone. Slides were then washed in tap water, transferred to phosphate-buffered saline containing 0.02% Tween 20 (PBST), and then incubated with normal horse serum (Dako, Carpinteria, CA) 1:20 in PBST for 20 min. Immunohistochemical staining was performed with the Shandon Sequenza immunostaining apparatus (Pittsburgh, PA). The slides were incubated with primary antibody, which was either anti-CD27 monoclonal antibody (clone LT-27, mouse IgG 2a, Biodesign International, Kennebunk, ME) at 1:50 dilution or anti-CD70 monoclonal antibody (Immunotech, Inc., Westbrook, ME) at 1:30 dilution, for 45 min. Slides were then treated sequentially with rabbit anti-mouse antibodies conjugated with horseradish peroxidase and swine anti-rabbit antibodies conjugated with horseradish peroxidase (Dako), each at 1:50 dilution containing human AB serum. Antibody localization was performed with the diaminobenzidene reaction and liquid DAB chromagen system (Dako). Slides were counter-
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stained in hematoxylin. Controls included omission of the primary antibody as well as the use of unrelated antibodies in place of the regular primary antibody. Cell Separation and Culture: Spontaneous sCD27 Production Experiments To separate cells into B cell and non-B cell fractions, a magnetic immunobead separation procedure was used. Briefly, PBMC were incubated in cold PBS 1 2% FCS with magnetic beads coated with anti-CD19 antibodies (Dynal, Inc., Lake Success, NY) for 0.5 h. The bead concentration was 10 7/ml, and the ratio of beads to estimated number of B cells present was 5:1. The CD19-positive fraction was separated from the CD19negative fraction using a magnet. The beads were then detached from the CD191 cell fraction by using Dynal’s DETACHaBEAD system for CD19. In all steps of the separation and detachment protocol, the cells were kept at 4°C to prevent nonspecific attachment of the beads to macrophages. This resulted in a CD191 fraction which was .97% pure, as assessed by flow cytometry. This CD191 fraction was cultured in flat-bottom 96-well plates (Costar, Cambridge, MA) in 200 ml total volume, with a cell concentration of 5 3 10 5/ml. Cells were cultured in RPMI 1640 medium supplemented with 10% FCS and 1% penicillin/streptomycin, at 37°C and 5% CO 2 in 25-cm 2 tissue culture flasks (Corning Glass Works, Corning, NY). At 2 and 5 days, supernatant was removed from wells and frozen prior to assessment of sCD27 by ELISA. Statistical Analysis Statistical significance for differences between groups was examined using the two-tailed Wilcoxon signed rank test for paired/matched observations, and the two-tailed Wilcoxon rank sum test for unmatched observations. In some instances, the x 2 test was used. In the serum sCD27 study, logistic regression for matched data was performed to obtain an estimate of the odds ratio to develop lymphoma for subjects who have AIDS given different values of serum sCD27 for the AIDS (non-lymphoma) and AIDS–NHL groups (27). The odds ratio is a good approximation of the relative risk because the incidence of AIDS–NHL in the population of persons with AIDS is relatively small (,10%). Another logistic regression for unmatched data was used to estimate the odds ratio, and thus the relative risk, for AIDS–NHL for subjects who have HIV infection (non-AIDS). Correlations between variables of interest were determined by Spearman’s rank correlation analysis. Repeated-measures models were utilized to investigate differences in sCD27 levels in the various groups and
time points in the spontaneous sCD27 production experiments. In these models, main effects for HIV status and day as well as interaction effects were studied. The different models including different main or interaction effects were compared by using the likelihood ratio test. RESULTS
Elevated Levels of Serum sCD27 Were Seen in HIV Infection, AIDS, and AIDS–NHL Serum sCD27 levels were determined in four groups of study subjects: (i) subjects who developed AIDS– NHL (in sera collected in the 18 months prior to diagnosis of lymphoma), (ii) subjects with AIDS (nonlymphoma), (iii) HIV1 subjects (non-AIDS), and (iv) HIVnegative subjects (Fig. 1). Subjects in the AIDS–NHL and AIDS (nonlymphoma) groups were matched by CD4 T cell number and age. Subjects who developed AIDS–NHL (n 5 36) were seen to have a median serum sCD27 level of 468 U/ml (mean 5 496 U/ml), while those in the AIDS (nonlymphoma) group (n 5 36) had a median serum sCD27 level of 245 U/ml (mean 5 290 U/ml); subjects in the HIV1 group (n 5 48) had a median serum sCD27 level of 211 U/ml (mean 5 248 U/ml), and those in the HIV-negative group (n 5 49) had a median serum sCD27 of 138 U/ml (mean 5 153 U/ml). Therefore, the lowest levels of serum sCD27 were seen in HIV-negative subjects, with increasing levels seen in HIV1 subjects and AIDS patients, and the highest levels seen in those who developed AIDS–NHL. Medians, as opposed to means, were used in the analysis of this data due to the presence of outliers. The median serum sCD27 level for the AIDS– NHL group was approximately twice as high as the median level seen in the AIDS (nonlymphoma) group; serum sCD27 levels were significantly different between the two groups (P , 0.001). A two-tailed Wilcoxon signed rank test was used for this comparison because the points in these two groups were paired. All other comparisons discussed below were made using a two-tailed Wilcoxon rank sum test because the data points were not paired. The serum sCD27 levels in the AIDS–NHL group also were significantly higher than those in the HIV1 group (P , 0.001), while the biggest difference noted was between the AIDS–NHL group and the HIV-negative group (P , 0.001). The difference between the levels of serum sCD27 in the AIDS (nonlymphoma) and HIV1 groups was not statistically significant. However, both groups showed an approximately 1.7-fold increase over the HIV-negative group (P , 0.001 for both comparisons). Thus, statistically significant differences in sCD27 serum levels were seen among these four groups, except for the HIV1 (non-AIDS) vs AIDS (nonlymphoma) compari-
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tween any of these AIDS-NHL subtype groups, using a two-tailed Wilcoxon Rank Sum test. Most AIDS- and Non-AIDS-Lymphomas Express CD27 and/or Its Ligand, CD70
FIG. 1. Elevated levels of serum sCD27 were seen in HIV infection and in subjects who developed AIDS–NHL. Serum from each person in the AIDS-lymphoma group was obtained for one time point in the 18 months prior to the diagnosis of lymphoma; subjects in the AIDS (non-lymphoma) group were matched to those in the AIDSlymphoma group by CD4 T cell number and by age. The horizontal lines represent the medians for each group.
son. Identical conclusions for statistical significance for all the comparisons are seen after Bonferroni correction for multiple comparisons. A further analysis was performed for the AIDS (nonlymphoma) and AIDS–NHL groups to give a measure of the importance of sCD27 levels in predicting AIDS– NHL. A logistic regression for matched cases was used to give an odds ratio for AIDS–NHL compared to AIDS (nonlymphoma) for those subjects with sCD27 values greater than 500 U/ml. The odds ratio estimate was 13 (95% CI 1.70 –99.36) for the cutoff point of 500 U/ml. Because the proportion of AIDS patients who will develop lymphoma is relatively small, the odds ratio is a good estimate of the relative risk for AIDS patients who have serum sCD27 levels above the cutoff point used to develop AIDS–NHL. Thus, the estimate of the odds ratio of 13 means that the proportion of lymphoma cases is approximately 13 times higher among those AIDS patients whose serum sCD27 values were greater than 500 U/ml than among those AIDS patients whose sCD27 values were less than 500 U/ml. For persons who were HIV1 (non-AIDS), another logistic regression (or unmatched case) was performed to give an odds ratio of AIDS–NHL compared to HIV1 (non-AIDS). For subjects with sCD27 greater than 500 U/ml, the odds ratio (and thus relative risk) estimate was 7.9 (95% CI 2.32–26.59). In order to assess the relationship of serum sCD27 level and lymphoma subtypes, the lymphomas were classified as CNS (n 5 11), large cell (non-CNS) (n 5 6), immunoblastic (non-CNS) (n 5 8), and small noncleaved (non-CNS) (n 5 11) subtypes. There was no significant difference seen in serum sCD27 level be-
Seven of 10 high-grade lymphomas from HIV1 subjects were positive for CD27, by immunostaining, including 3/6 cases of B cell immunoblastic lymphoma and 4/4 cases of Burkitt-like lymphoma (not shown). Staining was finely granular and limited to the cytoplasmic membrane of the neoplastic cells. Similar localization was found with antibodies to CD70, with 4/6 cases of immunoblastic lymphoma and 2/4 cases of Burkitt-like lymphoma from HIV1 subjects being immunoreactive. Ten cases of lymphoma from HIV-negative individuals were evaluated. Staining for CD27 occurred in 4/5 cases of B cell large cell lymphoma, 1 case of B cell immunoblastic lymphoma, and 1 case of marginal zone lymphoma. The remaining cases, including 1 case of Burkitt-like lymphoma and 1 case of immunoblastic lymphoma arising posttransplantation, were negative. Staining for CD70 was found in 1 case of immunoblastic lymphoma and 2/5 cases of large cell lymphoma. B Cell Surface CD27 Expression Was Decreased in HIV Infection Lymphoid cell surface expression of CD27 has been shown to positively correlate with sCD27 production (14, 28). Because B cells are known to be hyperactivated in HIV infection, and because the experiments described above showed increased levels of serum sCD27 in people who were HIV1, expression of cell surface CD27 was determined on circulating B cells in HIV infection. To do this, surface expression of CD27 was determined on CD191 cells in HIV1 (non-AIDS) (n 5 36), AIDS (n 5 8), and HIV-negative (n 5 25) persons by flow cytometry. As shown in Fig. 2, the mean percentage B cells positive for surface CD27 expression in the HIV-negative controls was 29.8%. This result is very close to previously reported figures (21). A much lower fraction of B cells were seen to be CD27-positive in the HIV1 group (mean 5 12.8%) and in the AIDS group (mean 5 9.7%). These differences were statistically significant (for HIV-negative vs HIV1, P , 0.001; for HIV-negative vs AIDS, P , 0.001) using Wilcoxon’s rank sum test. Thus, HIV infection was associated with a lower fraction of circulating B cells that were CD27-positive. No significant difference was noted in B cell CD27 expression between the HIV1 and AIDS groups. Serum sCD27 levels were determined in these subjects, as described earlier, to see if there was an asso-
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FIG. 2. A decreased percentage of circulating B cells in HIV infection were CD27-positive. PBMC from persons in the respective groups were examined for CD19 and CD27 expression by flow cytometry. The bars in the figure represent the SEM.
ciation between the decreased B cell surface expression of CD27 and increased serum sCD27 in HIV1 subjects. As expected, serum sCD27 levels were elevated in the HIV1 subjects (mean serum sCD27 5 332 6 38 U/ml, n 5 29 HIV1 subjects, including 3 subjects with AIDS) compared to the HIV-negative subjects (mean 5 144 6 48 U/ml, n 5 9 HIV-negative subjects). Significantly higher serum sCD27 levels were seen in those HIV1 subjects who had the lowest levels of B cell surface CD27 expression (P , 0.025, by x 2 analysis) (Fig. 3). Interestingly, this association between elevated serum sCD27 and relatively decreased B cell surface CD27 expression also was noted in HIV-negative subjects (P # 0.01) and when results from both HIV1 and HIV-negative subjects were combined (P # 0.005). Also, a positive correlation was seen between the percentage of B cells expressing cell surface CD27 and CD4 T cell number among HIV1 subjects, using a Spearman’s rank correlation analysis (correlational coefficient 5 0.307; P 5 0.043) (not shown). A correlational analysis could not be done for the HIV-negative group because CD4 T cell numbers were not available for many of these subjects. Therefore, the decreased cell surface expression of CD27 on B cells was associated with decreased CD4 number, as well as with relatively increased levels of serum sCD27. Spontaneous Production of sCD27 in HIV Infection Because serum sCD27 levels were higher in people who had HIV infection, and because a decreased fraction of circulating CD27-positive B cells was seen in HIV infection, an attempt was made to determine if circulating B cells from HIV1 subjects produced significantly different amounts of sCD27 compared to
those isolated from HIV-negative subjects. For this purpose, the spontaneous production of sCD27 by CD19-positive cells isolated from subjects with and without HIV infection was examined in vitro after 2 and 5 days in culture (Fig. 4). The HIV1 subjects utilized for these studies did not have AIDS. The mean CD4 T cell number for the HIV1 subjects tested (n 5 5 for both Day 2 and Day 5) was 340/mm 3, ranging from 28 to 813/mm 3; CD4 T cell numbers for the HIVnegative group (n 5 4 for Day 2, n 5 7 for Day 5) ranged from 589 to 1700/mm 3. There was no notable difference seen between HIV1 and HIV-negative subjects in the percentage of B cells in PBMC (9.5 6 1.8 and 8.6 6 2.2% CD19-positive cells were seen in PBMC in HIV-negative and HIV1 subjects, respectively). To determine the statistical significance for comparisons of levels of sCD27 seen in these experiments, a repeated-measures model was used. The use of such a model was possible because cells from the same subjects (and drawn at the same time point) were used for both the 2-day and 5-day cultures for many of the cultures in these experiments. Three repeated measures models were fitted one for each different kind of culture: PBMC, CD19-depleted PBMC, and CD19-enriched PBMC. In these three models, time and group main effects, as well as their interactions, were studied. Of relevance to this study, there was no statistically significant difference in sCD27 levels in comparing the HIV1 and HIV-negative groups for either the PBMC or the CD19-depleted cultures (Fig. 4). However, for the CD19-enriched cultures, sCD27 levels were typically 2.5–33 higher, at both 2 and 5 days, in the HIV-negative cultures compared to the HIV1 cultures (Fig. 4). This difference (studied as a group effect in the repeated-measures model) is statistically significant (P 5 0.005). As expected, serum sCD27 levels were seen to be elevated in the HIV1 subjects examined in these experiments (mean serum sCD27 5 120 6 9.0 and 294 6 27 U/ml, HIV-negative and HIV1 subjects, respectively). Unfortunately, the number of subjects tested was too small to allow the determination of any correlation between spontaneous sCD27 production in vitro and serum levels of sCD27. DISCUSSION
Recently, increased levels of sCD27, a soluble cleaved form of CD27, were seen in people who had B cell malignancies (16). CD27, which generally has been considered to be a T cell activation molecule (2), also has been seen to be important in the activation of B cells (5, 13). It is believed that the chronic hyperactivation of B cells seen in HIV infection (29 –31) may contribute to lymphomagenesis, perhaps by increasing the risk for a genetic error, such as a chromosomal
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FIG. 3. Comparison of cell surface CD27 expression on circulating B cells and serum sCD27 levels in HIV1 or HIV-negative subjects. PBMC from HIV-negative subjects (represented by the open circles), those who were HIV1 but did not have AIDS (represented by the solid diamonds), and those who had AIDS (represented by the solid squares) were examined for CD19 and CD27 expression by flow cytometry and for serum sCD27 level by ELISA.
translocation involving a proto-oncogene, which promotes the formation of a lymphoma (32, 33). Because of this association with B cell activation and B cell malignancies, we have examined the expression of CD27 and sCD27 in HIV infection and in AIDS–NHL, finding
FIG. 4. Comparison of spontaneous production of sCD27 by PBMC, B-cell-depleted PBMC, or B cells from HIV1 or HIV-negative subjects. d2 represents results (mean values 6 SEM) seen after 2 days in culture and d5 results seen after 5 days in culture. PBMC, CD19(2), and CD19(1) represent the results seen in cultures of unseparated PBMC, CD19-depleted PBMC, and in CD19-enriched cellular preparations, respectively. Solid bars represent results seen in HIV-negative subjects, open bars results seen in HIV1 subjects.
elevated levels of serum sCD27 preceding the appearance of AIDS–NHL (Fig. 1). This elevation in serum sCD27 is of a magnitude similar to the elevation seen in non-AIDS–NHL (16), suggesting that AIDS–NHL may be similar to other intermediate- and high-grade B cell lymphomas in this aspect of their biology. Further statistical analysis revealed a significant relative risk for the development of AIDS–NHL in HIV-infected subjects with elevated sCD27. While these results suggest that sCD27 may be a marker for AIDS–NHL, more comprehensive epidemiological studies are needed to more precisely define the value of serum sCD27 as a prognostic marker in AIDS–NHL. It has been speculated that the different subtypes of AIDS–NHL actually represent different diseases with different etiologies (19, 34). However, there was no statistically significant difference seen in serum sCD27 levels between the four subtypes of AIDS–NHL as classified in this study (CNS, large cell (non-CNS), immunoblastic (non-CNS), and small, noncleaved (non-CNS)). It is possible that with larger sample sizes a difference in sCD27 serum levels among different AIDS–NHL subtypes will become apparent. The results presented here also indicate that, in addition to the elevated sCD27 levels seen in AIDS– NHL, there is an almost twofold increase in median serum sCD27 levels in subjects with HIV infection (in both the AIDS, nonlymphoma and the HIV1, nonAIDS groups) (Fig. 1), compared to those in the HIV-
CD27 IN HIV INFECTION AND AIDS LYMPHOMA
negative controls. It has been reported that elevated sCD27 levels reflect T cell activation (2). Therefore, it is possible that activated T cells in HIV infection are a source of the elevated sCD27 levels seen. However, such a possibility was not readily apparent from the results of the spontaneous sCD27 production experiments (Fig. 4), since there did not appear to be much difference in spontaneous CD27 secretion by PBMC from HIV1 and HIV-negative subjects. Furthermore, circulating B cells did not appear to be a major source of sCD27 in vitro. In fact, production of sCD27 by circulating B cells was decreased in HIV1 subjects (Fig. 4). This suggests that the major source of the elevated serum sCD27 levels seen in HIV infection and AIDS is not circulating B or T lymphocytes, but instead may be lymphocytes in the thymus, spleen, or lymph nodes, which are known to be major sites of HIV infection and viral replication (35). It has been speculated that the tumor cells were the source of the sCD27 detected in NHL not associated with HIV infection (16). This also may be true for AIDS–NHL, given that most of the primary AIDS-lymphoma tumor tissue specimens examined were seen to express CD27. Certainly, further studies are needed to define the source of serum sCD27 in subjects who develop AIDS–NHL. A substantial decrease in the proportion of circulating B cells that were CD27-positive was seen in HIV infection and AIDS (Fig. 2): while 30% of B cells in the HIV-negative subjects were CD27-positive, consistent with what has been reported by others (16), HIV1 subjects showed an up to threefold decrease in CD27positive B cells, with ,10% of B cells from subjects who had AIDS being CD27-positive. It is interesting to note that the subjects who had the greatest decrease in B cell CD27 expression had relatively higher levels of serum sCD27 (Fig. 3). While this observation is consistent with the possibility that sCD27 is shed by activated cells, a correlation also was seen between decreased CD4 number and elevated serum sCD27, suggesting that the observed increase in serum sCD27 may reflect the extent of HIV disease progression. Further studies are needed to determine whether shedding of surface CD27 by activated B or T lymphocytes contributes to the elevated levels of serum sCD27 seen in HIV infection. CD27 appears to be expressed on B cells that have reached some level of activation (4, 5). Therefore, the low level of CD27-positive B cells seen in HIV1 subjects may reflect a greater proportion of immature B cells. This would be consistent with a previous report that circulating B cells in HIV infection show increased markers of immaturity, such as CALLA (CD10) (31). Alternatively, it is possible that CD27 expression on circulating B cells in HIV infection is decreased because B cells have differentiated past the stage of CD27 expression, because of constant stimulation by
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antigens or B cell stimulatory factors (such as interleukin-6), which are known to be elevated in HIV infection (36, 37). This would be consistent with the polyclonal B cell activation and hypergammaglobulinemia that are seen in HIV infection (36, 37). Either possibility would be consistent with the impaired B cell response to specific antigens that is seen in HIV infection (17). The decreased expression of CD27 by B cells in HIV infection also might be due to excessive crosslinking of CD27 on B cells with its ligand, CD70. It has been seen that the cross-linking of CD27 by CD70 is associated with the down-regulation of CD27 (38, 39). Consistent with this, elevated levels of CD70-positive T cells were seen in HIV infection (40, 41). Furthermore, these T cells showed a marked increase in CD70 expression following activation in vitro (40) compared to T cells from HIV-negative subjects, while B cells from HIV1 subjects had decreased levels of CD70 expression (42). Possibly, elevated T cell expression of CD70 in HIV infection is causing not only a down-regulation/ loss of cell surface CD27, but also may be contributing to the polyclonal B cell activation that accompanies HIV infection. It is also known that signaling through CD27 in T cells is not impaired in HIV infection (20). Certainly, much work needs to be done to test these possibilities. Last, the positive correlation between B cell surface CD27 expression and CD4 T cell number in HIV1 subjects suggests that whatever process results in decreased numbers of CD27-positive B cells is associated with HIV disease progression. A final question arising from, although not answered by, this study concerns the function of elevated sCD27 levels in HIV infection and in the pathogenesis of AIDS–NHL. Recombinant sCD27 can block signaling by CD70 through CD27 in vitro (43). If this occurs in vivo, there is the possibility that the increased sCD27 levels seen may contribute to the immune system impairment seen in HIV infection. The CD70/CD27 interaction provides important co-stimulatory signaling in the activation of both T and B cells to proliferate and produce cytokines or immunoglobulins (13, 44). In AIDS–NHL, it is also possible that high local concentrations of sCD27 surrounding these tumors may specifically impair anti-tumor immune responses. Additionally, sCD27 might promote tumor growth by binding to, and signaling, through its ligand, CD70, which is expressed on many AIDS–NHL patients. Further experiments need to be done to test these possibilities. ACKNOWLEDGMENTS The authors thank Liz Breen, Julie Gage, Reba Knox, Steve Alas, and David Waller for technical advice and assistance, and Janis Giorgi and co-workers, particularly Negoita Neagos and Ingrid Schmid, of the Jonsson Comprehensive Cancer Center Flow Cytometry Core Laboratory for expert technical assistance with the flow
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cytometry. Also, the authors thank Steve Miles for providing patient blood specimens. Last, the authors thank Kevin Barrett, Max Hechter, and Susan Stehn for the provision of data from the Multicenter AIDS Cohort Study (MACS) at UCLA. This work was supported by NIH Grants CA73475, CA57152, AI35040, and CA66533 and by an Amgen Fellowship of the UCLA AIDS Institute to D. W. Flow cytometry and biostatistical analysis were supported by the Jonsson Comprehensive Cancer Center Core Grant (CA16042) and UCLA Center for AIDS Research (CFAR) (AI28697).
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14. Hintzen, R. Q., de Jong, R., Hack, C. E., Chamuleau, M., de Vries, E. F. R., ten Berge, R. J. M., Borst, J., and van Lier, R. A. W., A soluble form of the human T cell differentiation antigen CD27 is released after triggering of the TCR/CD3 complex. J. Immunol. 147, 29 –35, 1991. 15. Loenen, W. A., de Vries, E., Gravestein, L. A., Hintzen, R. Q., van Lier, R. A., and Borst, J., The CD27 membrane receptor, a lymphocyte-specific member of the nerve growth factor receptor family, gives rise to a soluble form by protein processing that does not involve receptor endocytosis. Eur. J. Immunol. 22, 447– 455, 1992. 16. Van Oers, M. H. J., Pals, S. T., Evers, L. M., van der Schoot, C. E., Koopman, G., Bonfrer, J. M. G., Hintzen, R. Q., von dem Borne, A. E. G., and van Lier, R. A. W., Expression and release of CD27 in human B-cell malignancies. Blood 82, 3430 –3436, 1993. 17. Rosenberg, Z., and Fauci, A., Immunology of HIV infection. In “Fundamental Immunology” (W. E. Paul, Ed.), pp. 1375–1397, Raven Press, New York, 1993. 18. Broder, S., and Karp, J. E., Acquired immunodeficiency syndrome and non-Hodgkin’s lymphomas. Cancer Res. 51, 4743– 4756, 1991. 19. Ballerini, P., Gaidano, G., Gong, J. Z., Tassi, V., Saglio, G., Knowles, D. M., and Dalla-Favera, R., Multiple genetic lesions in acquired immunodeficiency syndrome-related NHL. Blood 81, 166 –176, 1993. 20. Meyaard, L., Kuiper, H., Otto, S. A., Wolthers, K. C., van Lier, F. A., and Miedema, F., Evidence for intact costimulation via CD28 and CD27 molecules in hyporesponsive T cells from human immunodeficiency virus-infected individuals. Eur. J. Immunol. 25, 232–237, 1995. 21. Marodon, G., Landau, N. R., and Posnett, D. N., Altered expression of CD4, CD54, CD62L, and CCR5 in primary lymphocytes productively infected with the human immunodeficiency virus. AIDS Res. Hum. Retroviruses 15, 161–171, 1999. 22. Portegies, P., Godfried, M. H., Hintzen, R. Q., Stam, J., van der Poll, T., Vakker, M., van Deventer, S. J. H., van Lier, R. A. W., and Goudsmit, J., Low levels of specific T cell activation marker CD27 accompanied by elevated levels of markers for non-specific immune activation in the cerebrospinal fluid of patients with AIDS dementia complex. J. Neuroimmunol. 48, 241–247, 1993. 23. Kaslow, R. A., Ostrow, D. G., Detels, R., Phair, J. P., Polk, B. F., and Rinaldo, C., The Multicenter AIDS Cohort Study: Rationale, organization and selected characteristics of the participants. Am. J. Epidemiol. 126, 310 –318, 1987. 24. Giorgi, J. V., Cheng, H., Margolick, J. B., Bauer, K. D., Ferbas, J., Waxdal, M., Schmid, I., Hultin, L. E., Jackson, A. L., Park, L., and Taylor, J. M. G., Quality control in the flow cytometric measurement of T-lymphocyte subsets: The Multicenter AIDS Cohort Study experience. Clin. Immunol. Immunopathol. 55, 173–186, 1990. 25. Schmid, I., Uittenbogaart, C. H., Krall, W. J., Braun, J., and Giorgi, J. V., Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence in single laser flow cytometry. Cytometry 13, 204 –208, 1992. 26. Fetterhoff, T. J., Holland, S. P., and Wile, K. J., Fluorescent detection of non-viable cells in fixed cell preparations. Cytometry 14(Suppl. 6), 27, 1993. 27. Hosmer, D. W., and Lemeshow, S., “Applied Logistic Regression,” pp. 187–215, John Wiley & Sons, Inc., New York. 28. Kurosawa, K., Kobata, T., Tachibana, K., Agematsu, K., Hirose, T., Schlossman, S. F., and Morimoto, C., Differential regulation of CD27 expression on subsets of CD4 T cells. Cell. Immunol. 158, 365–375, 1994.
CD27 IN HIV INFECTION AND AIDS LYMPHOMA 29. Mizuma, H., Litwin, S., and Zolla-Pazner, S., B-cell activation in HIV infection: Relationship of spontaneous immunoglobulin secretion to various immunological parameters. Clin. Exp. Immunol. 71, 410 – 416, 1988. 30. Lane, H. C., Masur, H., Edgar, L. C., Whalen, G., Rook, A. H., and Fauci, A. S., Abnormalities of B-cell activation and immunoregulation in patients with the acquired immunodeficiency syndrome. N. Engl. J. Med. 309, 253–258, 1983. 31. Martinez-Maza, O., Crabb, E., Mitsuyasu, R. T., Fahey, J. L., and Giorgi, J. V., Infection with human immunodeficiency is associated with an in vivo increase in B lymphocyte activation and immaturity. J. Immunol. 138, 3720 –3724, 1987. 32. Shiramizu, B., and McGrath, M. S., Molecular pathogenesis of AIDS-associated non-Hodgkin’s lymphoma. Hematol. Oncol. Clin. N. Amer. 5, 323–330, 1991. 33. Klein, G., Multiple phenotypic consequences of the Ig/Myc translocation in B-cell-derived tumors. Genes Chromosomes Cancer 1, 3– 8, 1989. 34. Herndier, B. G., Kaplan, L. D., and McGrath, M. S., Pathogenesis of AIDS lymphomas. AIDS (London) 8, 1025–1049, 1994. 35. Patel, D. D., Hale, L. P., and Haynes, B. F., HIV in lymph node and thymus. In “Immunology of HIV Infection” (S. Gupta, Ed.), pp. 95–121, Plenum, New York, 1996. 36. Breen, E. C., Rezai, A. R., Nakajima, K., Beall, G. N., Mitsuyasu, R. M., Hirano, T., Kishimoto, T., and Martinez-Maza, O., Infection with HIV is associated with elevated IL-6 levels and production. J. Immunol. 144, 480 – 484, 1990. 37. van der Meijden, M., Gage, J., Breen, E. C., Taga, T., Kishimoto, T., and Martı´nez-Maza, O., Upregulation of IL-6 receptor (CD126) expression on monocytes, B lymphocytes, and CD4 T lymphocytes in HIV infection. Cell. Immunol. 190, 156 –166, 1998. Received May 19, 1999; accepted with revision August 6, 1999
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38. Agematsu, K., Kobata, T., Sugita, K., Hirose, T., Schlossman, S. F., and Morimoto, C., Direct cellular communications between CD45RO and CD45RA T cell subsets via CD27/CD70. J. Immunol. 154, 3627–3635, 1995. 39. Ranheim, E. A., Cantwell, M. J., and Kipps, T. J., Expression of CD27 and its ligand, CD70, on chronic lymphocytic leukemia B cells. Blood 85, 3556 –3565, 1995. 40. Wothers, K. C., Otto, S. A., Lens, S. M. A., Kolbach, D. N., van Lier, R. A. W., Miedema, F., and Meyaard, L., Increased expression of CD80, CD86 and CD70 on T cells from HIV-infected individuals upon activation in vitro: Regulation by CD41 T cells. Eur. J. Immunol. 26, 1700 –1706, 1996. 41. Giorgi, J. V., Boumsell, L., and Autran, B., Reactivity of Workshop T-cell Section mAb with circulating CD41 and CD81 T cells in HIV disease and following in vitro activation. In “Leukocyte Typing V: White Cell Differentiation Antigens” (S. Schlossman, L. Boumsell, W. Gilks, J. Harlan, T. Kishimoto, C. Morimoto, J. Ritz, J. Shaw, R. Silverstein, T. Springer, T. Tedder, and R. Todd, Eds.), p. 446, Oxford University Press, Oxford, 1995. 42. Wolthers, K. C., Otto, S. A., Lens, S. M., Van Lier, R. A., Miedema, F., and Meyaard, L., Functional B cell abnormalities in HIV type 1 infection: Role of CD40L and CD70. Aids Res. Hum. Retroviruses 13, 1023–1029, 1997. 43. Agematsu, K., Kobata, T., Sugita, K., Freeman, G. J., Beckmann, M. P., Schlossman, S. F., and Morimoto, C., Role of CD27 in T cell immune response. Analysis by recombinant soluble CD27. J. Immunol. 153, 1421–1429, 1994. 44. Hintzen, R. Q., Lens, S. M. A., Lammers, K., Kuiper, H., Beckmann, M. P., and van Lier, R. A. W., Engagement of CD27 with its ligand CD70 provides a second signal for T cell activation. J. Immunol. 154, 2612–2623, 1995.
Aberrant Expression of CD27 and Soluble CD27 (sCD27) in HIV Infection and in AIDS-Associated Lymphoma Daniel Widney,* Girija Gundapp,† Jonathan W. Said,† ,‡ ,§ Meta van der Meijden, ¶ Benjamin Bonavida,* Aisha Demidem,* Cristina Trevisan,\ Jeremy Taylor,‡ ,§ ,\ Roger Detels,‡ ,** and Otoniel Martı´nez-Maza* ,‡ ,§ ,¶,1 *Departments of Microbiology, Immunology, and Molecular Genetics, ¶Obstetrics and Gynecology, and †Pathology, and the §Jonsson Comprehensive Cancer Center, University of California at Los Angeles School of Medicine; **Departments of Epidemiology and \Biostatistics, University of California at Los Angeles School of Public Health; and the ‡Los Angeles Center of the Multicenter AIDS Cohort Study (MACS), Los Angeles, California
INTRODUCTION
CD27 is a member of the tumor necrosis factor receptor superfamily that is expressed primarily on T cells, as well as on subsets of B cells and NK cells. CD70, which is expressed on activated B and T cells, but not on resting lymphocytes, is a ligand for CD27. Cell surface CD27 can be proteolytically cleaved to produce a 32-kDa soluble CD27 (sCD27) molecule. Elevated levels of sCD27 are seen in a number of disease states and malignancies. Although it has been reported that cerebrospinal fluid sCD27 levels were elevated in people who had AIDS dementia, little is known about CD27 expression in HIV disease. To determine if sCD27 levels were elevated in those with HIV infection, and/or in those with AIDS-associated non-Hodgkin’s lymphoma (AIDS–NHL), sCD27 levels were measured in HIV-negative and HIV-positive subjects as well as in people who developed AIDS–NHL. Serum sCD27 levels were seen to be elevated in HIV1 subjects. Furthermore, sCD27 levels were particularly elevated in those subjects who went on to develop AIDS–NHL, with serum sCD27 levels in AIDS–NHL subjects being significantly higher than those in HIV1 subjects who did not develop lymphoma. Most AIDS– NHL cell lines and primary AIDS–NHL tumor specimens expressed both CD27 and its ligand, CD70. The proportion of circulating B cells that expressed cell surface CD27 was substantially reduced in those with HIV infection, and B cells from HIV-infected subjects produced decreased levels of sCD27 in culture. Together, these results indicate that CD27/sCD27 expression is abnormal in HIV infection and suggest that this molecule merits further examination as a potential marker for AIDS–NHL. © 1999 Academic Press Key Words: CD27; lymphoma; HIV; AIDS; B cell.
1 To whom correspondence should be addressed at the Department of Microbiology, Immunology, and Molecular Genetics, UCLA School of Medicine, Los Angeles, CA 90095-1747. Fax: (310) 206-5387. Email: [email protected].
1521-6616/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
CD27 is a 55-kDa transmembrane disulfide-linked homodimer and a member of the tumor necrosis factor receptor (TNF-R) superfamily. This family includes, in addition to CD27, TNF-R type I, and TNF-R type II, the OX-40 and 4-1BB molecules, CD30, CD40, and the FAS/APO-1 molecule (CD95) (1–3). CD27, like many of these molecules, is expressed primarily on lymphocytes. In the peripheral blood, it is found on T cells (3), as well as on small subsets of B cells and NK cells (2– 6). In both T and B cells, CD27 appears to be related to the differentiation/activation stage: naive T cells (CD45RA) express CD27, with the expression of this molecule being strongly upregulated after stimulation of the TCR complex. After antigen stimulation and upregulation of the CD45RO marker, the ability to express CD27 is greatly reduced (2, 7, 8). For B cells, Maurer et al. determined that only CD271 cells could be stimulated to secrete immunoglobulin (Ig) in vitro following stimulation with Staphylococcus aureus Cowan (SAC) and interleukin-2 (IL-2) (4). Recently, the ligand for CD27 was shown to be CD70, a type II transmembrane protein that has homology to the TNF family of proteins, which includes TNFa, TNFB, 4-1BBL, CD30L, CD40L, and FAS-ligand (2, 9 –11). CD70 appears on activated B and T cells, but not on resting lymphocytes (12). CD70 appears to costimulate the activation of T lymphocytes through CD27 (10). Although little is known about the function of the CD27/CD70 interaction in B cells, it is known that SAC/IL-2-stimulated B cell cultures produced much higher levels of IgM and IgG when costimulated through CD27 (13). Cell surface CD27 can be proteolytically cleaved to produce a 32-kDa soluble CD27 (sCD27) molecule, which can be detected in a number of body fluids, including serum and urine (14, 15). Elevated serum sCD27 levels are seen in a number of infectious and autoimmune disease states and are believed to be a
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marker of T cell activation (2). Also, various types of B cell malignancies express the CD27 molecule and elevated serum levels of sCD27 were noted in patients with B cell tumors not associated with HIV infection (16). There appeared to be a correlation between tumor load and sCD27 levels, and, while not shown directly, it seemed likely that the tumors themselves were the source of the sCD27 detected in serum. In the 64 patients examined in this study who had low-, intermediate-, or high-grade non-Hodgkin’s lymphoma (NHL), a majority showed a significant elevation in serum sCD27. In HIV infection, a progressive loss of T cell function is seen. Specific B cell response to antigens is also diminished in HIV infection, although, paradoxically, a general polyclonal activation of B cells and accompanying hypergammaglobulinemia is seen (17). Also, a greatly increased frequency of B cell NHL is seen in people infected with HIV (18). These NHL, which are typically of high or intermediate grade, fall into several categories: (i) CNS lymphomas, which are generally immunoblastic and Epstein–Barr virus-positive; (ii) small noncleaved cell lymphoma; (iii) large cell immunoblastic lymphoma (non-CNS); and (iv) intermediate grade-diffuse large noncleaved cell lymphoma (nonCNS) (18, 19). Although little is known about CD27 and sCD27 in HIV infection, it does appear that the ability of CD27 to costimulate T cell activation is not lost (20) and that infection of T cells with HIV does not modulate the expression of CD27 on infected cells (21). Also, elevated sCD27 levels were noted in cerebrospinal fluid from patients with AIDS dementia complex (22). In this study, we show that there is an increase in serum sCD27 levels in subjects with HIV infection. Also, we show that serum sCD27 levels were markedly increased prior to the development of AIDS–NHL, with sCD27 levels in subjects who developed AIDS–NHL being even higher than those seen in subjects with AIDS who did not develop NHL. In fact, elevated sCD27 levels were associated with a statistically significant relative risk for the development of AIDS– NHL. Furthermore, substantial changes in CD27 surface expression and sCD27 production by circulating B cells in HIV infection suggest that CD27 could be involved in the B cell dysfunction and dysregulation which is seen in HIV infection. MATERIALS AND METHODS
Study Subjects Three studies using blood/serum from human subjects were performed: (i) a study of serum sCD27 levels, (ii) a study of cell surface CD27 expression, and (iii) a study of spontaneous sCD27 production in vitro.
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(1) Serum sCD27 study. Serum samples were obtained from subjects in the UCLA Multicenter AIDS Cohort Study (UCLA-MACS). The UCLA-MACS cohort, established in 1984, is a natural history study of HIV composed of adult homosexual men and had 1637 original participants (23). This is an ongoing study, and the participants continue to be followed. At 3- to 6-month intervals, serum is collected from study participants and stored frozen at 270°C. Information on the clinical status of subjects is also collected. Serum from four groups of men was used. Group I comprised men who developed AIDS-associated NHL (AIDS-NHL group). Only 2 of 36 subjects in this group had an AIDS-defining opportunistic infection at the date at which serum was collected for this study. The mean age of this group was 40 years, and the mean CD4 number was 202 cells/mm 3 (ranging from 2 to 923). Group II comprised men with AIDS but without detectable AIDS-related cancers (AIDS, nonlymphoma group), who were matched by CD4 T cell number and age to subjects in Group I (mean age of 39 years, mean CD4 number 186 cells/mm 3, ranging from 0 to 785). Group III men were HIV-positive and asymptomatic with no AIDS-defining illnesses (HIV1 group) (mean age of 36 years, mean CD4 number 445 cells/mm 3). Group IV members were HIV-seronegative men (HIVnegative group) (mean age of 35 years, mean CD4 number 872 cells/mm 3). Groups III and IV were not matched by age or CD4 number to each other or to any other group. Subjects in the control groups (Groups II, III, and IV) were chosen to exclude individuals who had NHL or Kaposi’s sarcoma. Also, any subject in the control groups who later developed NHL was excluded from study. HIV-infected persons were placed in Group II (AIDS, nonlymphoma), as opposed to Group III (HIV1, nonAIDS), according to the 1993 Centers for Disease Control and Prevention Clinical AIDS definition, excluding the criterion that persons with CD4 T cell numbers less than 200 cells/mm 3 be considered to have AIDS. Thus, HIV1 persons who had CD4 T cell numbers less than 200 cells/mm 3 and no other AIDS-defining condition were placed in Group III (HIV1, non-AIDS). Lymphomas were subtyped according to the working formulation for classification of NHL. Group I (AIDS–NHL group) included all available AIDS–NHL cases in the UCLA-MACS at the time that this study was carried out. For 22 subjects in this group, the diagnosis of lymphoma was also the initial AIDS-defining condition. Serum sCD27 levels were measured for each subject using serum drawn at one study visit within the 18 months preceding the AIDS–NHL diagnosis; the average time prior to diagnosis was 6 months. Sera from all other subjects (in the three control groups) represent a serum sample collected at a single MACS visit. All sera tested were frozen and then thawed before measure-
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ment of sCD27 levels by enzyme-linked immunosorbent assay (ELISA). (2) Cell surface CD27 study. Blood was obtained in EDTA tubes by venous puncture from subjects seen at clinics at UCLA who had AIDS (nonlymphoma), who were HIV1 (non-AIDS), or who were HIV-negative. These subjects were not necessarily the same subjects as were used in the serum sCD27 study described above, nor was any attempt made to match the groups by age or CD4 T cell number. Blood was used immediately (not frozen) for these experiments. Serum sCD27 was determined for most of the subjects tested in these studies using stored, frozen specimens. (3) Spontaneous sCD27 production study. Specimens for this study were obtained by venous collection in EDTA tubes from participants in the UCLA-MACS, or from various clinics at UCLA, who had AIDS (nonlymphoma), who were HIV1 (non-AIDS), or who were HIV-negative. This blood was used immediately (not frozen). These subjects were not necessarily the same subjects as were used in the serum sCD27 study described above, nor was any attempt made to match groups by age or CD4 T cell number. Determination of Serum sCD27 Levels Serum levels of sCD27 were measured by ELISA (compact soluble CD27 ELISA kit, Central Laboratory of the Netherlands Red Cross, Amsterdam, The Netherlands), a sandwich type immunoassay which can be used to measure sCD27 in serum. Briefly, this assay utilizes an anti-CD27 monoclonal antibody coated to polystyrene microtiter wells. After addition of samples, or standards, and rinsing, a biotinylated second monoclonal CD27 antibody was added and allowed to bind to sCD27, captured by the coating antibody. After rinsing, horseradish–peroxidase-conjugated streptavidin was added, followed by a biotinylated second antibody and addition of a chromogen, resulting in the formation of a colored product. Absorbance was measured at 450 nm to quantify color development. Levels of sCD27 in samples are determined by comparison with a standard curve prepared from seven sCD27 standards, ranging from 1.56 to 100 units (U)/ml. According to the manufacturer, the lower level of detection of this assay is 1 U/ml, and up to three freeze–thaw cycles have no effect on measured sCD27 levels. Isolation of PBMC Peripheral blood mononuclear cells (PBMC) were separated from EDTA peripheral blood by dilution of the blood 1:1 with saline and centrifugation over FicollPaque (Pharmacia LKB Biotechnology, Piscataway, NJ).
Determination of Cell Surface CD4, CD19, CD27, and CD70 Expression by Flow Cytometry CD4 (T helper/inducer cell) or CD19 (B lymphocyte) percentage was determined by flow cytometry, as described (24). For detection of cell surface CD27, a direct staining protocol was used, utilizing PE-conjugated anti-CD27 monoclonal antibody (clone M-T271, mouse IgG 1, PharMingen, San Diego, CA). For CD70, an indirect staining protocol was used, with the anti-CD70 antibody (clone HNE51, mouse IgG 1, Immunotech, Inc., Westbrook, ME) being followed by FITC-conjugated goat anti-mouse IgG. Appropriate isotype controls were also included. Next, the cells were stained with 7-actinomycin-D (7-AAD) (1 mg/ml, Calbiochem, San Diego, CA) for 30 min as described by Schmid et al. (25) and then fixed in 1% paraformaldehyde plus actinomycin-D (3 mg/ml) (26). To examine the expression of CD27 or CD70 on circulating B cells, two-color flow cytometry was performed, with anti-CD19-FITC (Becton-Dickinson Immunocytometry Systems, Mountain View, CA) being used as a pan-B cell marker. All samples were analyzed within 24 h on a FACScan flow cytometer (Becton-Dickinson), with dead cells being gated out using 7-AAD staining. At least 5000 events were collected for each sample. Data were analyzed with C30 FACScan Research Software. Immunostaining for Determination of CD27 and CD70 Expression on AIDS-NHL Tissue Lymph nodes from 20 patients with lymphoma, 10 of whom were seropositive for HIV-1, were evaluated. Tissues were obtained at the time of surgery and snapfrozen. Four-micrometer sections were mounted on Super Frost slides (Fisher Scientific Princeton, NJ) fixed in cold acetone. Slides were then washed in tap water, transferred to phosphate-buffered saline containing 0.02% Tween 20 (PBST), and then incubated with normal horse serum (Dako, Carpinteria, CA) 1:20 in PBST for 20 min. Immunohistochemical staining was performed with the Shandon Sequenza immunostaining apparatus (Pittsburgh, PA). The slides were incubated with primary antibody, which was either anti-CD27 monoclonal antibody (clone LT-27, mouse IgG 2a, Biodesign International, Kennebunk, ME) at 1:50 dilution or anti-CD70 monoclonal antibody (Immunotech, Inc., Westbrook, ME) at 1:30 dilution, for 45 min. Slides were then treated sequentially with rabbit anti-mouse antibodies conjugated with horseradish peroxidase and swine anti-rabbit antibodies conjugated with horseradish peroxidase (Dako), each at 1:50 dilution containing human AB serum. Antibody localization was performed with the diaminobenzidene reaction and liquid DAB chromagen system (Dako). Slides were counter-
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stained in hematoxylin. Controls included omission of the primary antibody as well as the use of unrelated antibodies in place of the regular primary antibody. Cell Separation and Culture: Spontaneous sCD27 Production Experiments To separate cells into B cell and non-B cell fractions, a magnetic immunobead separation procedure was used. Briefly, PBMC were incubated in cold PBS 1 2% FCS with magnetic beads coated with anti-CD19 antibodies (Dynal, Inc., Lake Success, NY) for 0.5 h. The bead concentration was 10 7/ml, and the ratio of beads to estimated number of B cells present was 5:1. The CD19-positive fraction was separated from the CD19negative fraction using a magnet. The beads were then detached from the CD191 cell fraction by using Dynal’s DETACHaBEAD system for CD19. In all steps of the separation and detachment protocol, the cells were kept at 4°C to prevent nonspecific attachment of the beads to macrophages. This resulted in a CD191 fraction which was .97% pure, as assessed by flow cytometry. This CD191 fraction was cultured in flat-bottom 96-well plates (Costar, Cambridge, MA) in 200 ml total volume, with a cell concentration of 5 3 10 5/ml. Cells were cultured in RPMI 1640 medium supplemented with 10% FCS and 1% penicillin/streptomycin, at 37°C and 5% CO 2 in 25-cm 2 tissue culture flasks (Corning Glass Works, Corning, NY). At 2 and 5 days, supernatant was removed from wells and frozen prior to assessment of sCD27 by ELISA. Statistical Analysis Statistical significance for differences between groups was examined using the two-tailed Wilcoxon signed rank test for paired/matched observations, and the two-tailed Wilcoxon rank sum test for unmatched observations. In some instances, the x 2 test was used. In the serum sCD27 study, logistic regression for matched data was performed to obtain an estimate of the odds ratio to develop lymphoma for subjects who have AIDS given different values of serum sCD27 for the AIDS (non-lymphoma) and AIDS–NHL groups (27). The odds ratio is a good approximation of the relative risk because the incidence of AIDS–NHL in the population of persons with AIDS is relatively small (,10%). Another logistic regression for unmatched data was used to estimate the odds ratio, and thus the relative risk, for AIDS–NHL for subjects who have HIV infection (non-AIDS). Correlations between variables of interest were determined by Spearman’s rank correlation analysis. Repeated-measures models were utilized to investigate differences in sCD27 levels in the various groups and
time points in the spontaneous sCD27 production experiments. In these models, main effects for HIV status and day as well as interaction effects were studied. The different models including different main or interaction effects were compared by using the likelihood ratio test. RESULTS
Elevated Levels of Serum sCD27 Were Seen in HIV Infection, AIDS, and AIDS–NHL Serum sCD27 levels were determined in four groups of study subjects: (i) subjects who developed AIDS– NHL (in sera collected in the 18 months prior to diagnosis of lymphoma), (ii) subjects with AIDS (nonlymphoma), (iii) HIV1 subjects (non-AIDS), and (iv) HIVnegative subjects (Fig. 1). Subjects in the AIDS–NHL and AIDS (nonlymphoma) groups were matched by CD4 T cell number and age. Subjects who developed AIDS–NHL (n 5 36) were seen to have a median serum sCD27 level of 468 U/ml (mean 5 496 U/ml), while those in the AIDS (nonlymphoma) group (n 5 36) had a median serum sCD27 level of 245 U/ml (mean 5 290 U/ml); subjects in the HIV1 group (n 5 48) had a median serum sCD27 level of 211 U/ml (mean 5 248 U/ml), and those in the HIV-negative group (n 5 49) had a median serum sCD27 of 138 U/ml (mean 5 153 U/ml). Therefore, the lowest levels of serum sCD27 were seen in HIV-negative subjects, with increasing levels seen in HIV1 subjects and AIDS patients, and the highest levels seen in those who developed AIDS–NHL. Medians, as opposed to means, were used in the analysis of this data due to the presence of outliers. The median serum sCD27 level for the AIDS– NHL group was approximately twice as high as the median level seen in the AIDS (nonlymphoma) group; serum sCD27 levels were significantly different between the two groups (P , 0.001). A two-tailed Wilcoxon signed rank test was used for this comparison because the points in these two groups were paired. All other comparisons discussed below were made using a two-tailed Wilcoxon rank sum test because the data points were not paired. The serum sCD27 levels in the AIDS–NHL group also were significantly higher than those in the HIV1 group (P , 0.001), while the biggest difference noted was between the AIDS–NHL group and the HIV-negative group (P , 0.001). The difference between the levels of serum sCD27 in the AIDS (nonlymphoma) and HIV1 groups was not statistically significant. However, both groups showed an approximately 1.7-fold increase over the HIV-negative group (P , 0.001 for both comparisons). Thus, statistically significant differences in sCD27 serum levels were seen among these four groups, except for the HIV1 (non-AIDS) vs AIDS (nonlymphoma) compari-
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tween any of these AIDS-NHL subtype groups, using a two-tailed Wilcoxon Rank Sum test. Most AIDS- and Non-AIDS-Lymphomas Express CD27 and/or Its Ligand, CD70
FIG. 1. Elevated levels of serum sCD27 were seen in HIV infection and in subjects who developed AIDS–NHL. Serum from each person in the AIDS-lymphoma group was obtained for one time point in the 18 months prior to the diagnosis of lymphoma; subjects in the AIDS (non-lymphoma) group were matched to those in the AIDSlymphoma group by CD4 T cell number and by age. The horizontal lines represent the medians for each group.
son. Identical conclusions for statistical significance for all the comparisons are seen after Bonferroni correction for multiple comparisons. A further analysis was performed for the AIDS (nonlymphoma) and AIDS–NHL groups to give a measure of the importance of sCD27 levels in predicting AIDS– NHL. A logistic regression for matched cases was used to give an odds ratio for AIDS–NHL compared to AIDS (nonlymphoma) for those subjects with sCD27 values greater than 500 U/ml. The odds ratio estimate was 13 (95% CI 1.70 –99.36) for the cutoff point of 500 U/ml. Because the proportion of AIDS patients who will develop lymphoma is relatively small, the odds ratio is a good estimate of the relative risk for AIDS patients who have serum sCD27 levels above the cutoff point used to develop AIDS–NHL. Thus, the estimate of the odds ratio of 13 means that the proportion of lymphoma cases is approximately 13 times higher among those AIDS patients whose serum sCD27 values were greater than 500 U/ml than among those AIDS patients whose sCD27 values were less than 500 U/ml. For persons who were HIV1 (non-AIDS), another logistic regression (or unmatched case) was performed to give an odds ratio of AIDS–NHL compared to HIV1 (non-AIDS). For subjects with sCD27 greater than 500 U/ml, the odds ratio (and thus relative risk) estimate was 7.9 (95% CI 2.32–26.59). In order to assess the relationship of serum sCD27 level and lymphoma subtypes, the lymphomas were classified as CNS (n 5 11), large cell (non-CNS) (n 5 6), immunoblastic (non-CNS) (n 5 8), and small noncleaved (non-CNS) (n 5 11) subtypes. There was no significant difference seen in serum sCD27 level be-
Seven of 10 high-grade lymphomas from HIV1 subjects were positive for CD27, by immunostaining, including 3/6 cases of B cell immunoblastic lymphoma and 4/4 cases of Burkitt-like lymphoma (not shown). Staining was finely granular and limited to the cytoplasmic membrane of the neoplastic cells. Similar localization was found with antibodies to CD70, with 4/6 cases of immunoblastic lymphoma and 2/4 cases of Burkitt-like lymphoma from HIV1 subjects being immunoreactive. Ten cases of lymphoma from HIV-negative individuals were evaluated. Staining for CD27 occurred in 4/5 cases of B cell large cell lymphoma, 1 case of B cell immunoblastic lymphoma, and 1 case of marginal zone lymphoma. The remaining cases, including 1 case of Burkitt-like lymphoma and 1 case of immunoblastic lymphoma arising posttransplantation, were negative. Staining for CD70 was found in 1 case of immunoblastic lymphoma and 2/5 cases of large cell lymphoma. B Cell Surface CD27 Expression Was Decreased in HIV Infection Lymphoid cell surface expression of CD27 has been shown to positively correlate with sCD27 production (14, 28). Because B cells are known to be hyperactivated in HIV infection, and because the experiments described above showed increased levels of serum sCD27 in people who were HIV1, expression of cell surface CD27 was determined on circulating B cells in HIV infection. To do this, surface expression of CD27 was determined on CD191 cells in HIV1 (non-AIDS) (n 5 36), AIDS (n 5 8), and HIV-negative (n 5 25) persons by flow cytometry. As shown in Fig. 2, the mean percentage B cells positive for surface CD27 expression in the HIV-negative controls was 29.8%. This result is very close to previously reported figures (21). A much lower fraction of B cells were seen to be CD27-positive in the HIV1 group (mean 5 12.8%) and in the AIDS group (mean 5 9.7%). These differences were statistically significant (for HIV-negative vs HIV1, P , 0.001; for HIV-negative vs AIDS, P , 0.001) using Wilcoxon’s rank sum test. Thus, HIV infection was associated with a lower fraction of circulating B cells that were CD27-positive. No significant difference was noted in B cell CD27 expression between the HIV1 and AIDS groups. Serum sCD27 levels were determined in these subjects, as described earlier, to see if there was an asso-
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FIG. 2. A decreased percentage of circulating B cells in HIV infection were CD27-positive. PBMC from persons in the respective groups were examined for CD19 and CD27 expression by flow cytometry. The bars in the figure represent the SEM.
ciation between the decreased B cell surface expression of CD27 and increased serum sCD27 in HIV1 subjects. As expected, serum sCD27 levels were elevated in the HIV1 subjects (mean serum sCD27 5 332 6 38 U/ml, n 5 29 HIV1 subjects, including 3 subjects with AIDS) compared to the HIV-negative subjects (mean 5 144 6 48 U/ml, n 5 9 HIV-negative subjects). Significantly higher serum sCD27 levels were seen in those HIV1 subjects who had the lowest levels of B cell surface CD27 expression (P , 0.025, by x 2 analysis) (Fig. 3). Interestingly, this association between elevated serum sCD27 and relatively decreased B cell surface CD27 expression also was noted in HIV-negative subjects (P # 0.01) and when results from both HIV1 and HIV-negative subjects were combined (P # 0.005). Also, a positive correlation was seen between the percentage of B cells expressing cell surface CD27 and CD4 T cell number among HIV1 subjects, using a Spearman’s rank correlation analysis (correlational coefficient 5 0.307; P 5 0.043) (not shown). A correlational analysis could not be done for the HIV-negative group because CD4 T cell numbers were not available for many of these subjects. Therefore, the decreased cell surface expression of CD27 on B cells was associated with decreased CD4 number, as well as with relatively increased levels of serum sCD27. Spontaneous Production of sCD27 in HIV Infection Because serum sCD27 levels were higher in people who had HIV infection, and because a decreased fraction of circulating CD27-positive B cells was seen in HIV infection, an attempt was made to determine if circulating B cells from HIV1 subjects produced significantly different amounts of sCD27 compared to
those isolated from HIV-negative subjects. For this purpose, the spontaneous production of sCD27 by CD19-positive cells isolated from subjects with and without HIV infection was examined in vitro after 2 and 5 days in culture (Fig. 4). The HIV1 subjects utilized for these studies did not have AIDS. The mean CD4 T cell number for the HIV1 subjects tested (n 5 5 for both Day 2 and Day 5) was 340/mm 3, ranging from 28 to 813/mm 3; CD4 T cell numbers for the HIVnegative group (n 5 4 for Day 2, n 5 7 for Day 5) ranged from 589 to 1700/mm 3. There was no notable difference seen between HIV1 and HIV-negative subjects in the percentage of B cells in PBMC (9.5 6 1.8 and 8.6 6 2.2% CD19-positive cells were seen in PBMC in HIV-negative and HIV1 subjects, respectively). To determine the statistical significance for comparisons of levels of sCD27 seen in these experiments, a repeated-measures model was used. The use of such a model was possible because cells from the same subjects (and drawn at the same time point) were used for both the 2-day and 5-day cultures for many of the cultures in these experiments. Three repeated measures models were fitted one for each different kind of culture: PBMC, CD19-depleted PBMC, and CD19-enriched PBMC. In these three models, time and group main effects, as well as their interactions, were studied. Of relevance to this study, there was no statistically significant difference in sCD27 levels in comparing the HIV1 and HIV-negative groups for either the PBMC or the CD19-depleted cultures (Fig. 4). However, for the CD19-enriched cultures, sCD27 levels were typically 2.5–33 higher, at both 2 and 5 days, in the HIV-negative cultures compared to the HIV1 cultures (Fig. 4). This difference (studied as a group effect in the repeated-measures model) is statistically significant (P 5 0.005). As expected, serum sCD27 levels were seen to be elevated in the HIV1 subjects examined in these experiments (mean serum sCD27 5 120 6 9.0 and 294 6 27 U/ml, HIV-negative and HIV1 subjects, respectively). Unfortunately, the number of subjects tested was too small to allow the determination of any correlation between spontaneous sCD27 production in vitro and serum levels of sCD27. DISCUSSION
Recently, increased levels of sCD27, a soluble cleaved form of CD27, were seen in people who had B cell malignancies (16). CD27, which generally has been considered to be a T cell activation molecule (2), also has been seen to be important in the activation of B cells (5, 13). It is believed that the chronic hyperactivation of B cells seen in HIV infection (29 –31) may contribute to lymphomagenesis, perhaps by increasing the risk for a genetic error, such as a chromosomal
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FIG. 3. Comparison of cell surface CD27 expression on circulating B cells and serum sCD27 levels in HIV1 or HIV-negative subjects. PBMC from HIV-negative subjects (represented by the open circles), those who were HIV1 but did not have AIDS (represented by the solid diamonds), and those who had AIDS (represented by the solid squares) were examined for CD19 and CD27 expression by flow cytometry and for serum sCD27 level by ELISA.
translocation involving a proto-oncogene, which promotes the formation of a lymphoma (32, 33). Because of this association with B cell activation and B cell malignancies, we have examined the expression of CD27 and sCD27 in HIV infection and in AIDS–NHL, finding
FIG. 4. Comparison of spontaneous production of sCD27 by PBMC, B-cell-depleted PBMC, or B cells from HIV1 or HIV-negative subjects. d2 represents results (mean values 6 SEM) seen after 2 days in culture and d5 results seen after 5 days in culture. PBMC, CD19(2), and CD19(1) represent the results seen in cultures of unseparated PBMC, CD19-depleted PBMC, and in CD19-enriched cellular preparations, respectively. Solid bars represent results seen in HIV-negative subjects, open bars results seen in HIV1 subjects.
elevated levels of serum sCD27 preceding the appearance of AIDS–NHL (Fig. 1). This elevation in serum sCD27 is of a magnitude similar to the elevation seen in non-AIDS–NHL (16), suggesting that AIDS–NHL may be similar to other intermediate- and high-grade B cell lymphomas in this aspect of their biology. Further statistical analysis revealed a significant relative risk for the development of AIDS–NHL in HIV-infected subjects with elevated sCD27. While these results suggest that sCD27 may be a marker for AIDS–NHL, more comprehensive epidemiological studies are needed to more precisely define the value of serum sCD27 as a prognostic marker in AIDS–NHL. It has been speculated that the different subtypes of AIDS–NHL actually represent different diseases with different etiologies (19, 34). However, there was no statistically significant difference seen in serum sCD27 levels between the four subtypes of AIDS–NHL as classified in this study (CNS, large cell (non-CNS), immunoblastic (non-CNS), and small, noncleaved (non-CNS)). It is possible that with larger sample sizes a difference in sCD27 serum levels among different AIDS–NHL subtypes will become apparent. The results presented here also indicate that, in addition to the elevated sCD27 levels seen in AIDS– NHL, there is an almost twofold increase in median serum sCD27 levels in subjects with HIV infection (in both the AIDS, nonlymphoma and the HIV1, nonAIDS groups) (Fig. 1), compared to those in the HIV-
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negative controls. It has been reported that elevated sCD27 levels reflect T cell activation (2). Therefore, it is possible that activated T cells in HIV infection are a source of the elevated sCD27 levels seen. However, such a possibility was not readily apparent from the results of the spontaneous sCD27 production experiments (Fig. 4), since there did not appear to be much difference in spontaneous CD27 secretion by PBMC from HIV1 and HIV-negative subjects. Furthermore, circulating B cells did not appear to be a major source of sCD27 in vitro. In fact, production of sCD27 by circulating B cells was decreased in HIV1 subjects (Fig. 4). This suggests that the major source of the elevated serum sCD27 levels seen in HIV infection and AIDS is not circulating B or T lymphocytes, but instead may be lymphocytes in the thymus, spleen, or lymph nodes, which are known to be major sites of HIV infection and viral replication (35). It has been speculated that the tumor cells were the source of the sCD27 detected in NHL not associated with HIV infection (16). This also may be true for AIDS–NHL, given that most of the primary AIDS-lymphoma tumor tissue specimens examined were seen to express CD27. Certainly, further studies are needed to define the source of serum sCD27 in subjects who develop AIDS–NHL. A substantial decrease in the proportion of circulating B cells that were CD27-positive was seen in HIV infection and AIDS (Fig. 2): while 30% of B cells in the HIV-negative subjects were CD27-positive, consistent with what has been reported by others (16), HIV1 subjects showed an up to threefold decrease in CD27positive B cells, with ,10% of B cells from subjects who had AIDS being CD27-positive. It is interesting to note that the subjects who had the greatest decrease in B cell CD27 expression had relatively higher levels of serum sCD27 (Fig. 3). While this observation is consistent with the possibility that sCD27 is shed by activated cells, a correlation also was seen between decreased CD4 number and elevated serum sCD27, suggesting that the observed increase in serum sCD27 may reflect the extent of HIV disease progression. Further studies are needed to determine whether shedding of surface CD27 by activated B or T lymphocytes contributes to the elevated levels of serum sCD27 seen in HIV infection. CD27 appears to be expressed on B cells that have reached some level of activation (4, 5). Therefore, the low level of CD27-positive B cells seen in HIV1 subjects may reflect a greater proportion of immature B cells. This would be consistent with a previous report that circulating B cells in HIV infection show increased markers of immaturity, such as CALLA (CD10) (31). Alternatively, it is possible that CD27 expression on circulating B cells in HIV infection is decreased because B cells have differentiated past the stage of CD27 expression, because of constant stimulation by
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antigens or B cell stimulatory factors (such as interleukin-6), which are known to be elevated in HIV infection (36, 37). This would be consistent with the polyclonal B cell activation and hypergammaglobulinemia that are seen in HIV infection (36, 37). Either possibility would be consistent with the impaired B cell response to specific antigens that is seen in HIV infection (17). The decreased expression of CD27 by B cells in HIV infection also might be due to excessive crosslinking of CD27 on B cells with its ligand, CD70. It has been seen that the cross-linking of CD27 by CD70 is associated with the down-regulation of CD27 (38, 39). Consistent with this, elevated levels of CD70-positive T cells were seen in HIV infection (40, 41). Furthermore, these T cells showed a marked increase in CD70 expression following activation in vitro (40) compared to T cells from HIV-negative subjects, while B cells from HIV1 subjects had decreased levels of CD70 expression (42). Possibly, elevated T cell expression of CD70 in HIV infection is causing not only a down-regulation/ loss of cell surface CD27, but also may be contributing to the polyclonal B cell activation that accompanies HIV infection. It is also known that signaling through CD27 in T cells is not impaired in HIV infection (20). Certainly, much work needs to be done to test these possibilities. Last, the positive correlation between B cell surface CD27 expression and CD4 T cell number in HIV1 subjects suggests that whatever process results in decreased numbers of CD27-positive B cells is associated with HIV disease progression. A final question arising from, although not answered by, this study concerns the function of elevated sCD27 levels in HIV infection and in the pathogenesis of AIDS–NHL. Recombinant sCD27 can block signaling by CD70 through CD27 in vitro (43). If this occurs in vivo, there is the possibility that the increased sCD27 levels seen may contribute to the immune system impairment seen in HIV infection. The CD70/CD27 interaction provides important co-stimulatory signaling in the activation of both T and B cells to proliferate and produce cytokines or immunoglobulins (13, 44). In AIDS–NHL, it is also possible that high local concentrations of sCD27 surrounding these tumors may specifically impair anti-tumor immune responses. Additionally, sCD27 might promote tumor growth by binding to, and signaling, through its ligand, CD70, which is expressed on many AIDS–NHL patients. Further experiments need to be done to test these possibilities. ACKNOWLEDGMENTS The authors thank Liz Breen, Julie Gage, Reba Knox, Steve Alas, and David Waller for technical advice and assistance, and Janis Giorgi and co-workers, particularly Negoita Neagos and Ingrid Schmid, of the Jonsson Comprehensive Cancer Center Flow Cytometry Core Laboratory for expert technical assistance with the flow
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cytometry. Also, the authors thank Steve Miles for providing patient blood specimens. Last, the authors thank Kevin Barrett, Max Hechter, and Susan Stehn for the provision of data from the Multicenter AIDS Cohort Study (MACS) at UCLA. This work was supported by NIH Grants CA73475, CA57152, AI35040, and CA66533 and by an Amgen Fellowship of the UCLA AIDS Institute to D. W. Flow cytometry and biostatistical analysis were supported by the Jonsson Comprehensive Cancer Center Core Grant (CA16042) and UCLA Center for AIDS Research (CFAR) (AI28697).
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