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Modulation of Bovine Leukemia Virus-Associated Spontaneous Lymphocyte Proliferation by Monoclonal Antibodies to Lymphocyte Surface Molecules
CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY
Vol. 83, No. 2, May, pp. 156–164, 1997 Article No. II974340
Modulation of Bovine Leukemia Virus-Associated Spontaneous Lymphocyte Proliferation by Monoclonal Antibodies to Lymphocyte Surface Molecules Diana M. Stone, Linda K. Norton, and William C. Davis Department of Veterinary Microbiology and Pathology, Washington State University, P.O. Box 647040, Pullman, Washington 99164-7040
Both human T lymphotropic virus (HTLV) and bovine leukemia virus (BLV) infections are characterized by in vitro proliferation of peripheral blood lymphocytes in the absence of exogenous antigens or mitogens. Differential expression of lymphocyte surface molecules in HTLV and BLV infection suggests that lymphocyte dysregulation may involve signaling through surface molecules involved in immune regulation. We examined the expression of adhesion and major histocompatibility (MHC) molecules on circulating lymphocytes from BLV-infected cows with persistent lymphocytosis and the ability of monoclonal antibodies to these molecules to modulate spontaneous lymphocyte proliferation. The integrin molecule, CD11c, and both MHC class I and MHC class II molecules were upregulated on B and T lymphocytes from PL cows. Anti-CD11c antibody was stimulatory to lymphocyte proliferation regardless of BLV status and had a greater stimulatory effect on spontaneously proliferating lymphocytes from persistently lymphocytotic cows than on normal bovine lymphocytes. Antibodies to bovine class I and class II inhibited spontaneous lymphocyte proliferation. Results suggest that lymphocyte dysregulation in BLV-induced persistent lymphocytosis involves upregulation of and signaling through lymphocyte surface molecules which are involved in immune activation of lymphocytes. q 1997 Academic Press
INTRODUCTION
Bovine leukemia virus (BLV) belongs to a family of oncogenic retroviruses which includes human T lymphotropic virus (HTLV)-I and -II and the simian T cell leukemia virus-I (1). These retroviruses share a common genomic organization (1, 2) and have been associated with preneoplastic lymphocyte dysregulation, lymphoid neoplasia, and/or progressive myelopathies (3–6). BLV is unique in the HTLV family of retroviruses by having a tropism for B lymphocytes instead of T lymphocytes. Approximately 30% of cattle naturally infected with BLV develop a sustained nonmalignant polyclonal expansion of CD5/ B lymphocytes (7) referred to as persistent lymphocytosis (PL), with vari-
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0090-1229/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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able progression to CD5/ B cell leukemia or lymphoma (8). Factors regulating disease development in HTLV and BLV infections remain unclear. One of the hallmarks of HTLV and BLV infection is the in vitro proliferation of peripheral blood lymphocytes when cultured in the absence of exogenous antigen or mitogen (9–13), referred to as spontaneous lymphocyte proliferation (SLP). BLV-induced PL in cattle provides an excellent model system to investigate the mechanisms which promote SLP in these lymphotropic retroviral infections. The transition from asymptomatic infection to PL in BLV-infected cattle is well defined and there is an association between appearance of SLP and BLV-induced PL (13–15). PL in cattle is an early yet persistent event associated with viral infection, thus providing a convenient period of time to study the cellular and molecular events controlling SLP in a naturally infected outbred population of animals. An understanding of the factors controlling SLP in cells from BLV-infected cows may shed light on how the HTLV/BLV family of retroviruses promotes an abnormal state of lymphocyte activation which can progress either to cell transformation, as in HTLV-I-associated adult T cell leukemia and bovine leukosis, or to a heightened inflammatory response as in HTLV-associated myelopathy (3, 4). Because clinical and lymphocyte phenotypic changes in HTLV and BLV infection are associated with SLP, it is suggested that SLP is an in vitro correlate of the disease process (9, 16). Some of the phenotypic changes observed in T lymphocytes from HTLV-infected individuals with SLP include elevated numbers of CD4 and/or CD8 T lymphocytes (11, 17), increased expression of the integrin molecules CD11a and CD18 (18, 19), increased interleukin-2 receptor (IL-2R) expression (11, 12, 20, 21), and increased HLA-DR expression (17, 22–25). In BLV-induced PL there is often an increase in the number of circulating T lymphocytes as well as B lymphocytes (26), enhanced expression of bovine major histocompatibility complex (BoLA) class II DP-like molecules (27), and enhanced induction of IL2Ra expression on B lymphocytes from these cows (26, 27). These observations suggest that SLP associated
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with the HTLV/BLV family of retroviruses may be due to changes in the expression of lymphocyte surface molecules involved in immune regulation of lymphocytes. In the present study we examined the expression of surface molecules involved in lymphocyte regulation on spontaneously proliferating lymphocytes from PL cows and the ability of monoclonal antibodies (mabs) to these molecules to modulate SLP. Results show that a greater proportion of both B and T lymphocytes from PL cows express the integrin molecule CD11c than normal bovine B and T lymphocytes. This molecule is further upregulated on B lymphocytes from PL cows after 3-day unstimulated cultures, correlating with SLP. Anti-CD11c antibody is stimulatory to bovine peripheral blood mononuclear cells (PBMC) regardless of BLV status and has a greater stimulatory effect on spontaneously proliferating lymphocytes from PL cows than on normal bovine lymphocytes. Results show that both class I and class II molecules are upregulated on B and T lymphocytes from PL cows and that mabs to BoLA class I and class II significantly inhibit SLP. These results support the hypothesis that SLP associated with lymphotropic retroviral infections involves signaling of lymphocytes through surface molecules involved in immune regulation.
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IgG2a) (29–33). Mab COLIS205 (IgG2a), which does not recognize bovine antigens, was used as a nonbinding and isotype control. In the blocking studies mabs of the IgM and IgG1 isotypes were further compared to mabs CACT61A and CACT80C, respectively, as these two mabs did not significantly block SLP (Fig. 3). Flow-Cytometric Analysis Single- and dual-color stainings were performed as previously described (26). Labeled cells were enumerated with a FACSort (Becton–Dickinson Immunocytometry Systems Inc., San Jose, CA). Patterns of reactivity of the mabs with lymphocytes were analyzed with CELLQUEST software. Data on 25,000 cells from each PL cow and 50,000 cells from each control cow were analyzed to determine the percentage of bovine B and T lymphocytes expressing surface molecules and the intensity of expression of these molecules on lymphocyte subsets (Figs. 1 and 2). More cells were collected from control cows to ensure sufficient numbers of B lymphocytes for analysis. Data on 3000 cells were collected per sample for phenotyping the lymphocytes from each cow used in the SLP blocking experiments (Table 1). Lymphocyte Culture and Proliferation Assay
MATERIALS AND METHODS
Animals Adult Holstein cows naturally infected with BLV and characterized by persistent lymphocytosis, defined as lymphocyte counts three or more standard deviations above the mean for age-matched, BLV-seronegative cattle (28), were identified as part of a university milking herd. These cows tested seropositive for BLV by two sequential agar gel immunodiffusion tests using gp51 as antigen (Pitman–Moore, Mundelein, IL). Control cows were healthy adult Holstein cows with normal lymphocyte counts, seronegative for BLV, and were housed in the same herd as the infected animals. Monoclonal Antibodies Mouse anti-bovine mabs were obtained from the Washington State University Monoclonal Antibody Center (Pullman, WA). These mabs specifically recognize determinants on molecules expressed on bovine granulocytes and monocytes (DH59B, IgG1), bovine B lymphocytes (sIgM) (BIg73A, IgG1 ; PIg45A, IgG2b), the bovine gd T cell receptor (TCR) (CACT61A, IgM), bovine (Bo) CD4 cells (CACT138A, IgG1), BoCD8 cells (CACT80C, IgG1), BoCD3 cells (MM1A, IgG1), BoCD5 cells (B29A, IgG2a), BoMHC-I cells (H58A, IgG2a), BoMHC-II DP-like cells (H42A, IgG2a), DQ cells (TH81A5, IgG2a), DR cells (TH14B, IgG2a), BoCD11a cells (BAT75A, IgG1), BoCD11c cells (BAQ153A, IgM), BoCD18 cells (BAQ30A, IgG1), and the bovine IL-2 receptor a chain (CACT108A,
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Blood was collected by jugular venipuncture into acid–citrate–dextrose (ACD) at one part ACD to four parts whole blood. PBMC were separated by Accupaque (1.083) (Accurate Chemical and Scientific Corp., Westbury, NY) sedimentation as previously described (26). Viability counts were done using the trypan blue dye exclusion method. The PBMC contained over 95% viable cells and less than 1% contamination with granulocytes. Lymphocyte proliferation was assayed in RPMI 1640 supplemented with 20% Fetal Clone I (Hyclone Labs, Logan, UT), antibiotics, 2 mM L-glutamine, 300 mM 2-mercaptoethanol, 10 mM Hepes with 5 1 105 PBMC/well in 96-well flat-bottom tissue culture plates (Sarstedt Inc., Newton, NC). Mabs were added at the initiation of culture at 10 mg/ml for blocking studies. Cells were cultured at 377C with 5% CO2 for 3 days. [3H]Thymidine (0.5 mCi) was added to each well at 18 hr prior to termination of culture. Cells were harvested on an automated 96-well plate harvester (Tomtec Inc., Orange, CT) and the amount of [3H]thymidine incorporated was determined by liquid scintillation spectroscopy (Wallac Inc., Gaithersburg, MD). Data are expressed as the mean of six replicate samples. The percentage inhibition was calculated as 1 0 (mean cpm of cultures with antibody/mean cpm of cultures without antibody) 1 100. Cell Cycle Analysis Cells cultured for 3 days were stained with antisIgM mab for single fluorescent FITC flow cytometry.
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FIG. 1. Expression of MHC class I/II and CD11c on B and T lymphocytes and expression of CD18, CD11a, CD2, and sIgM on PBMC from BLV-infected PL cows and uninfected controls. Expression of surface molecules was determined by dual-label flow-cytometric analysis on fresh lymphocytes (Day 0) and unstimulated cultured lymphocytes (Day 3). Values are means and standard deviations of 3 BLV-infected PL cows and 3 uninfected controls. Significant comparisons at P £ 0.05 are indicated by brackets and an asterisk.
Stained cells were fixed in 2% formaldehyde in phosphate-buffered saline (PBS) for 60 min on ice. Cells were permeabilized with 0.2% Tween 20 (Sigma Chemical Co., St. Louis, MO) for 15 min at 377C, washed once in PBS, and resuspended in PBS with 10 mg/ml propidium iodide (Calbiochem-Novabiochem Corp., LaJolla, CA) and 22 Kunitz units/ml of RNase A (Sigma Chemical Co.). Samples were kept in the dark at least 30 min before analysis by flow cytometry. G0 samples were uncultured PBMC from a non-PL cow which were fixed, permeabilized, and stained with propidium iodide.
significant differences between SLP and lymphocyte proliferation levels with mabs added (Table 2). [3H]Thymidine incorporation below SLP are reported as percentage inhibition and [3H]thymidine incorporation greater than SLP are reported as percentage stimulation. The Kolmogorov–Smirnov two-sample test was used to compare histograms of fluorescence intensity for surface molecules on lymphocyte subsets (Fig. 2). A 99.9% confidence level of significance was used where the value Dcrit/s(n) Å 1.63 and rejection of the null hypothesis is determined by D/s(n) ú Dcrit/s(n) (34). RESULTS
Statistical Analysis Analysis of variance was used to compare lymphocytes from PL and BLV-negative cows for percentage expression of surface molecules (Fig. 1). P £ 0.05 was considered significant. For each PL cow confidence intervals at the P £ 0.05 level were used to determine
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Modulation of Expression of Cell Surface Molecules on Spontaneously Proliferating Lymphocytes from BLV/ PL Cows Previous studies showed that a greater percentage of B lymphocytes from PL cows expressed the CD5 mol-
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ecule than normal bovine B lymphocytes (7) and that there was enhanced induction of IL-2Ra expression on in vitro stimulated B lymphocytes from PL cows (26,
FIG. 3. Inhibition of spontaneously proliferating PBMC from BLV-infected PL cows. Represented are mean percentage inhibition in the presence of mabs to lymphocyte surface molecules in 4 BLVinfected PL cows. Error bars represent the P Å 0.05 confidence interval.
FIG. 2. Cell surface density of MHC class I and class II molecules on fresh sIgM/ B lymphocytes and CD2/ T lymphocytes from BLVinfected PL cows (solid boldface lines) compared to uninfected controls (solid thin lines). Control stained cells are indicated with stippled lines in the first panels. These data are representative of 3 BLV-infected PL cows and 3 uninfected controls. Histograms are normalized. MHC fluorescence intensity is indicated on the horizontal axis and the number of cells on the vertical axis. Dcrit/s(n) Å 1.63 for a 99.9% confidence level and the D/s(n) for each comparison is as follows: B cells: MHC-I (6.84), DP (10.74), DQ (18.30), DR (18.43); T cells: MHC-I (27.69), DP (17.09), DQ (25.65), DR (13.55).
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27). To more completely characterize bovine lymphocytes for expression of surface molecules involved in immune regulation we evaluated the expression of MHC class I and class II molecules and the adhesion molecules CD11a, CD11c, and CD18 on lymphocyte subsets from freshly isolated and 3-day unstimulated cultures of PBMC from three PL cows and three noninfected control cows by dual-color microfluorimetry (Figs. 1 and 2). As expected, PBMC from PL cows were characterized by a significantly greater percentage of IgM/ lymphocytes and a significantly lower percentage of CD2/ lymphocytes compared to PBMC from BLVnegative cows (Fig. 1). The percentage of B (IgM/) and T (CD2/) lymphocytes did not change significantly from time 0 to Day 3 of culture. Also as expected, all freshly isolated and cultured bovine B and T lymphocytes expressed MHC-I (Fig. 1), but the intensity of MHC-I expression on freshly isolated cells was moderately higher on B and T lymphocytes from PL cows compared to that of normal bovine lymphocytes (Fig. 2). The MHC-II DP-like, DQ, and DR molecules were expressed on greater than 96% of freshly isolated B lymphocytes from PL cows, whereas 58 to 80% of circulating B lymphocytes from BLV-negative cows expressed detectable levels of these molecules (Fig. 1). By Day 3, greater than 90% of all bovine B lymphocytes expressed MHC-II DP-like, DQ, and DR molecules regardless of BLV status (Fig. 1). The intensity of DP-like, DQ, and DR expression on MHC-II-positive B lymphocytes was consistently higher on freshly isolated B lymphocytes from PL cows than on normal bovine B lymphocytes (Fig. 2). A significantly greater percentage of freshly
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TABLE 1 Peripheral Blood B and T Lymphocytes in BLV-Infected, Lymphocytotic Cows and Normal, BLV-Noninfected Cows Cell number BLV status/ cow no.
Total lymphocytes
IgM
IgM/CD5a
CD3
CD4
CD8
g/d
Positive 1379 1580 1555 1583 1570 1570c
14,276b 17,755 12,556 13,452 9768 10,872
ND 5682 3390 6053 2344 6306
ND 4829 2576 5024 2274 5738
ND 6392 4269 2959 5665 2935
ND 3018 1758 2152 2735 1196
ND 3729 6278 807 2344 1305
ND 2841 1256 942 1661 1413
Negative 1529 1485 1467 1315 1651 1651c
3250 2484 2380 2856 4230 4161
ND 174 262 371 677 541
ND 80 89 360 162 395
ND 745 1404 1599 2961 2580
ND 994 643 971 1184 1040
ND 373 476 771 804 915
ND 199 286 400 592 957
a
Number of IgM/ lymphocytes that express CD5. Number of lymphocytes/ml of whole blood. c Repeat sampling 1 week later. b
isolated T lymphocytes from PL cows expressed MHCII DQ compared to normal bovine T lymphocytes (Fig. 1), but there was no significant difference in percentages of DP-like and DR expression. By Day 3, however, a greater percentage of T lymphocytes from PL cows
TABLE 2 Percentage Inhibitiona of Spontaneous Lymphocyte Proliferation by PBMC from BLV PL Cows Cow number Mab
1379
1580
1555
1583
1570
CD11c CD18 CD11a CD5 IL-2Ra CD4 CD8 g/d MHC-I MHC-II/DP MHC-II/DQ MHC-II/DR Iso-IgG2a CD3
/b244c 43 37 29 32 /70 /7 /15 65 66 81 59 /50 /23
/93 35 38 19 29 /9 20 /29 91 18 25 32 /47 /1147
/89 /3 /26 48 11 /62 /9 /54 87 81 84 85 /20 /1420
/66 16 /18 9 4 /65 /23 /25 71 80 50 80 /2 /4090
/12 64 38 5 3 4 4 11 /12 /32 /110 /46 /8 /2410
SLP mean SLP stderr
2505 (165)
3688 (102)
2442 (121)
2419 (230)
6404 (351)
a
Percentage inhibition is calculated as 1 0 (cpm/SLP) 1 100. Positive numbers are percentage stimulation. c Numbers in boldface are significantly different from spontaneous proliferation at P £ 0.05. b
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expressed DP-like, DQ, and DR molecules compared to normal bovine T lymphocytes (Fig. 1). As with B lymphocytes from PL cows, the intensity of MHC-II DP-like, DQ, and DR expression at Day 0 was moderately but significantly higher on T lymphocytes from PL cows compared to that on normal bovine T lymphocytes (Fig. 2). Within individual cows there was a modest decrease in the intensity of expression of all surface molecules evaluated from Day 0 to Day 3 of culture regardless of BLV status or lymphocyte subset (data not shown). Phenotypic analysis of freshly isolated cells showed that all bovine PBMC expressed the b2 CD11a/CD18 integrin regardless of BLV status (Fig. 1) and that the intensity of expression of the integrin as detected with anti-CD11a and CD18 mabs did not significantly differ between lymphocytes from PL cows and normal bovine lymphocytes (data not shown). In contrast, a significantly greater percentage of both freshly isolated B and T lymphocytes from PL cows expressed CD11c compared to normal bovine B and T lymphocytes (Fig. 1). Between 0 and 3 days of culture there was a modest but significant change in the percentage of lymphocytes expressing CD11c in the PL cows. There was a significant decrease in the percentage of T lymphocytes expressing CD11c (P Å 0.0396) and a significant increase in the percentage of B lymphocytes expressing CD11c (P Å 0.0005). Between 0 and 3 days of culture there was no significant change in the percentage of B or T lymphocytes expressing CD11c in BLV-negative cows. There was no consistent change in the intensity of CD11c expression on B or T lymphocytes from 0 to 3 days regardless of BLV status (data not shown).
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Phenotypic Analysis of PBMC from Cows Used in the Mab Blocking Studies Five BLV-infected PL cows and five age-matched BLV-noninfected, clinically normal cows were selected for the mab blocking studies. Analysis of PBMC prior to culture demonstrated the expected differences between BLV-infected PL cows and control cows in phenotype and in absolute numbers within lymphocyte subsets. All BLV-infected PL cows were characterized by elevated total white blood counts and elevated total lymphocyte counts due primarily to markedly increased numbers of CD5-expressing B lymphocytes (Table 1). Most PL cows were also characterized by moderately increased numbers of CD4, CD8, and gd T lymphocytes. B lymphocytes from two control cows (cow 1315 and cow 1651 at repeat sampling) were predominantly CD5/ (97 and 73%, respectively), but the absolute numbers of peripheral blood B lymphocytes and CD5/ B lymphocytes from these cows were within 2 standard deviations of previously reported means for clinically normal cows (26). Transient increases in the percentage of circulating CD5/ B lymphocytes in clinically normal BLV-negative cows with absolute numbers of CD5/ B lymphocytes remaining within normal limits have been reported (26). It is proposed that this reflects transient in vivo B lymphocyte activation because there is also a concomitant increase in B lymphocyte IL-2Ra expression (26). Effect of Antibodies to Lymphocyte Surface Molecules on SLP in PL Cows Differential expression of cell surface molecules on lymphocytes from PL cows which are involved in lymphocyte regulation suggests that signaling through these molecules may be important in SLP. PBMC from five PL cows were cultured alone or with antibodies directed against CD5, IL-2Ra, MHC-I and -II, the integrin molecules CD11a/CD18 and CD11c/CD18, the T lymphocyte subset molecules CD4 and CD8, and the gd TCR (Table 2). The mean [3H]thymidine uptake in lymphocytes from these cows without antibodies added exceeded 2000 cpm (Table 2), which was defined as SLP. Addition of mabs against MHC-I and MHC-II DPlike, DQ, and DR molecules significantly inhibited SLP in four of the five PL cows (Table 2). The effect of varying concentrations of antibodies to MHC-II DP-like, DQ, and DR (0.01 to 10.0 mg/ml) indicated that all concentrations were inhibitory to SLP in the four PL cows and inhibition was dose-dependent (data not shown). In cow 1570 mab to MHC-I had no significant effect on SLP and mabs to all three MHC-II molecules had a significant stimulatory effect on lymphocyte proliferation (Table 2). PBMC from cow 1570 were cultured a week later with mab to MHC-II DR and again this mab was stimulatory (77% stimulation). Mabs to CD11c and CD3 were stimulatory to PBMC in four of the five PL
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cows (Table 2). No consistent differences in proliferative responses to the other mabs were observed (Table 2). When data from the four cows which showed SLP inhibition with mabs to all three MHC-II molecules were analyzed as a group (Fig. 3), the mean percentages of inhibition for DP-like, DQ, and DR mabs were 61, 60, and 64%, respectively. Although other mabs were inhibitory to SLP in individual cows (Table 2), when analyzed as a group mab to CD5 only modestly inhibited SLP and mabs to IL-2Ra, CD11a, CD18, CD4, CD8, and the gd TCR did not have a significant effect (Fig. 3). Mabs to CD11c and CD3 were stimulatory for PBMC from PL cows, resulting in a mean percentage stimulation of 123 and 1670%, respectively. Marked variability among the responses of individual cows to these stimulatory mabs was noted (Table 2). Cell Cycle Analysis of Proliferating Lymphocytes Cultured with Anti-MHC-II Antibody Cell cycle analysis of spontaneously proliferating lymphocytes from PL cows has shown that it is primarily B lymphocytes which enter the cell cycle (unpublished observation). Because of the marked inhibitory effect of all three anti-MHC-II antibodies on BLV-associated SLP, we asked whether this inhibition was correlated with a decrease in B lymphocytes entering the cell cycle. PBMC from one PL cow were cultured with and without anti-MHC-II DQ mab for 3 days. Cell cycle analysis of freshly isolated cells showed that 11% of the B lymphocytes were already in the cell cycle (Fig. 4a). By 3 days 20% of viable B lymphocytes were in the cell cycle (Fig. 4b). The proportion of viable B lymphocytes in the cell cycle decreased to 15% when antiMHC-DQ was added (Fig. 4c). This correlated with a 35% inhibition in SLP. Effect of Antibodies to Lymphocyte Surface Molecules on the Proliferation of Normal Bovine Lymphocytes PBMC from five BLV-noninfected cows were also cultured with mabs against lymphocyte surface molecules. Three-day cultures without antibodies from three of these cows demonstrated the expected low background level of [3H]thymidine incorporation (means of 220– 607 cpm). [3H]Thymidine incorporation of cultured PBMC from two of the noninfected cows (cows 1315 and 1651), however, showed means of 2420 and 3647 cpm, levels which were within the range of SLP observed in lymphocytes from the PL cows in this study. Levels of thymidine incorporation which overlap values for PL cows have been reported in lymphocytes from 5 to 10% of BLV-free cattle (15). The SLP observed in BLV-free cows, however, is intermittent (unpublished observation) and is most likely due to transient antigenic stimulation of PBMC due to a subclinical infection with an unknown microorganism. The SLP intermittently observed in clinically normal BLV-negative
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FIG. 4. Effect of anti-MHC-II DQ antibody on cell cycle progression of sIgM/ B lymphocytes from a BLV-infected PL cow. DNA content of sIgM-labeled B lymphocytes was determined by propidium iodide staining of permeabilized cells at time 0 (a) and after a 3-day culture without (b) and with (c) 10 mg/ml anti-MHC-II DQ antibody. Only viable cells were analyzed. Fluorescence intensity of DNA staining is indicated on the horizontal axis and the number of sIgM/ cells on the vertical axis.
cows, therefore, should be inhibited by mabs which block antigen-mediated signaling of lymphocytes. The mab to MHC-I significantly inhibited the SLP in both of these BLV-negative cows (77 and 88% inhibition). Mabs to MHC-II DP, DR, and DQ were inhibitory at some concentration in both of these cows. The dose response to MHC-II mabs in cow 1651 paralleled the response in PL cows with all concentrations being inhibitory and maximum inhibition being reached at 0.10 mg/ml and above. Maximum inhibition of lymphocyte proliferation for this cow was 88% for DP, 82% for DR, and 72% for DQ. Mabs to MHC-II molecules were stimulatory to PBMC from cow 1315 at 10 mg/ml and inhibitory at concentrations below 10 mg/ml. The maximum percentage inhibition for this cow occurred at 0.10 mg/ ml for all three mabs and was 14% for anti-DP, 50% for anti-DR, and 73% for DQ. Mabs to CD5, IL-2Ra, CD11a, CD18, CD4, CD8, and gd TCR did not have a significant effect on [3H]thymidine incorporation of PBMC from these two cows. As in PL cows, mab to CD11c and CD3 were stimulatory for PBMC from all five BLV-negative cows. The mean percentage stimulation was 81% (range from 13 to 235%) for anti-CD11c mab and 4909% (range from 898 to 16,031%) for antiCD3 mab. DISCUSSION
PBMC from BLV-infected PL cows were investigated to determine the role of lymphocyte surface molecules in the regulation of SLP. Results from this study demonstrate that lymphocyte surface molecules involved in lymphocyte regulation are upregulated on both B and T lymphocytes from PL cows and that mabs to these molecules modulate SLP. Results demonstrate that subpopulations of normal bovine B and T lymphocytes express the integrin molecule CD11c, a molecule normally restricted to myeloid cells (35). This molecule is not only upregulated on B lymphocytes from PL cows
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as previously reported (36), but is also upregulated on T lymphocytes from PL cows. An unexpected finding in this study was the stimulatory effect of anti-CD11c antibody on lymphocyte proliferation regardless of BLV status, including an enhanced stimulatory effect on the already spontaneously proliferating PBMC from PL cows. Although CD11c is absent from normal human resting peripheral blood B and T lymphocytes, it is expressed on several human B cell lines, cytotoxic T lymphocyte clones, and stimulated B cell chronic lymphocytic leukemia cells and is a marker for hairy cell leukemia, a B cell neoplasm (35, 37–41). The function of CD11c expression on a subpopulation of normal bovine lymphocytes or on activated or transformed lymphocytes from any species is not yet known. The fact that anti-CD11c was stimulatory to bovine PBMC suggests that the function of CD11c may include positive signal transduction events when expressed on lymphocytes. Increased expression of CD11c on lymphocytes may not only be a marker of lymphocyte dysregulation but may also have functional relevance for the expansion of B lymphocytes in PL and the transformation of lymphocytes in some human cancers. Mabs to MHC-II significantly decreased SLP in PL cows and the high background proliferation observed in two BLV-negative cows, although the inhibitory concentration of mab differed for one BLV-negative cow. This suggests that the SLP associated with PL reflects a normal mechanism of lymphocyte proliferation, one which is most likely initiated by antigen in the context of MHC-II. Anti-MHC-II mabs may be inhibiting the normal antigen-mediated, MHC-II dependent, B–T lymphocyte interaction. What is abnormal in PL is that SLP is persistent, probably due to the continuous presence of BLV antigen. This suggests that the expanded population of B lymphocytes in PL may be, in part, due to chronic antigenic stimulation. Findings from other studies also support a role for antigen in SLP. PL is associated with increased viral load and increased viral
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gene expression (42–44) and BLV antisera inhibit SLP (15). Importantly, anti-MHC-II antibodies did not completely inhibit SLP. This suggests that other costimulatory signals may be involved in BLV-associated SLP. The participation of costimulatory molecules in HTLVassociated T lymphocyte proliferation was recently demonstrated (45). Anti-MHC-II antibodies failed to inhibit SLP in one PL cow (cow 1570) and, in fact, were stimulatory to PBMC from this cow. In addition, anti-CD11c failed to enhance proliferation of PBMC from this cow. Clearly, signaling events regulating lymphocyte proliferation in this cow were qualitatively different from those of the other four PL cows used in this study. One explanation is that these lymphocyte surface molecules become coupled to different signaling pathways at different stages of lymphocyte dysregulation in BLV-induced PL. Information is not available on when the cows in this study became BLV seropositive or how long they had been PL. The inhibitory effect of anti-MHC-I antibodies on SLP was unexpected. It is unlikely that inhibition is due to blocking of antigen presentation to CD8 T lymphocytes and loss of CD8 cytokines, as preliminary experiments indicate that CD8 T lymphocyte depletion does not significantly inhibit SLP (unpublished observation). Inhibition of SLP by MHC-I mab, however, does suggest the involvement of cellular interactions in SLP. One possibility is that mabs to MHC-I inhibit interactions with monocytes, resulting in a lack of monocyte-derived cytokines required for optimum Tlymphocyte-dependent B lymphocyte activation. Signaling events regulating SLP in HTLV and BLV share some important features but also differ in significant ways. There is evidence that the SLP associated with HTLV and BLV infection involve integrin molecules and MHC-II signaling of lymphocytes. However, the integrin molecules upregulated on the surface of circulating lymphocytes in HTLV- and BLV-infected individuals appear to be different and the effect of mab to these molecules on SLP is in opposite directions. Lymphocytes from HTLV-II-infected individuals show increased density of b2 integrins as detected with antiCD11a and CD18 mabs and SLP is inhibited by these mabs (18). BLV-induced PL is characterized by increased CD11c expression and anti-CD11c mab is stimulatory to SLP. As in SLP associated with PL in cattle, antibodies to HLA-DR inhibit the SLP characterizing HTLV-II-infected persons (12), and HLA class I antibodies inhibit SLP observed in some HTLV-I infected, disease-free individuals. In contrast, anti-HLA class II antibodies had no effect on SLP from HTLV-I-infected individuals (16). Results from the present study provide insights into how oncogenic retroviruses may initially dysregulate lymphocytes. The SLP of both BLV and HTLV infections is associated with changes in the expression of
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lymphocyte surface molecules involved in immune regulation and is modulated by antibodies to these molecules. An understanding of how this family of retroviruses modulate the expression of lymphocyte surface molecules and effect their signaling pathways may reveal a primary mechanism responsible for early stages of lymphocyte dysregulation. This knowledge may subsequently lead to the development of strategies to prevent or control the pathologies of these persistent retroviral infections in animals and humans which can be tested with this animal model. REFERENCES 1. Sagata, N., Yasanuga, T., Tsuzuku-Kawamura, J., Ohihi, K., Ogawa, Y., and Ihawa, I., Complete nucleotide sequence of the genome of bovine leukemia virus: Its evolutionary relationship to other retroviruses. Proc. Natl. Acad. Sci. USA 82, 677–682, 1985. 2. Ciminale, V., and Pavlakis, G. N., Complex splicing in the human T-cell leukemia virus (HTLV) family of retroviruses: Novel mRNAs and proteins produced by HTLV type I. J. Virol. 66, 1737–1745, 1992. 3. Hollsberg, P., and Hafler, D. A., Pathogenesis of diseases induced by human lymphotropic virus type I infection. N. Engl. J. Med. 328, 1173–1182, 1993. 4. Kim, J. H., and Durack, D. T., Manifestations of human Tlymphotropic virus type I infection. Am. J. Med. 84, 919–928, 1988. 5. Mchizuki, M., Watanabe, K., and Yamaguchi, K., Uveitis associated with human T lymphotropic virus type I: Seroepidemiologic, clinical, and virologic studies. J. Infect. Dis. 166, 943–944, 1992. 6. Posenblatt, J. D., Chen, I. S. Y., and Wachsman, W., Infection with HTLV-I and HTLV-II: Evolving concepts. Semin. Hematol. 25, 230–246, 1988. 7. Depelchin, A., Letesson, J. J., Lostrie-Trussart, N., Mammerickx, M., Portetelle, D., and Burny, A., Bovine leukemia virus (BLV)-infected B-cells express a marker similar to the CD5 T cell marker. Immunol. Lett. 20, 69–76, 1989. 8. Burny, A., Bruck, C., Cleuter, Y., Couez, D., Deschamps, J., Ghysdael, J., Bregoire, D., Kettmann, R., Mammerickx, M., Marbaix, G., and Portetelle, D., In ‘‘Bovine Leukemia Virus: A New Mode of Leukemogenesis’’ (G. Klein, Ed.), pp. 35–55, Raven Press, New York, 1985. 9. Itoyama, Y., Minato, S., Kira, J., Goto, I., Sato, H., Okochi, K., and Yamamoto, N., Spontaneous proliferation of peripheral blood lymphocytes increased in patients with HTLV-I-associated myelopathy. Neurology 38, 1302–1307, 1988. 10. Prince, H. E., Lee, H., Jensen, E. R., Swanson, P., Weber, D., Fitzpatrick, L., Doyle, M., and Kleinman, S., Immunologic correlates of spontaneous lymphocyte proliferation in human Tlymphotropic virus infection. Blood 78, 169–174, 1991. 11. Prince, H. E., Weber, D. M., and Jensen, E. R., Spontaneous lymphocyte proliferation in HTLV-I/II infection reflects preferential activation of CD8 and CD16/56 cell subsets. Clin. Immunol. Immunopathol. 58, 419–430, 1991. 12. Lal, R. B., Hjelle, B., and Rudolph, D. L., Spontaneous proliferation of HTLV-II-infected peripheral blood lymphocytes: HLADR-driven, IL-2-dependent response. Microbiol. Immunol. 36, 865–872, 1992. 13. Muscoplat, C. C., Alhaji, I., Johnson, D. W., Pomeroy, K. A., Olson, J. M., Larson, V. L., Stevens, J. B., and Sorensen, D. K., Characteristics of lymphocyte responses to phytomitogens: Com-
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29. Davis, W. C., Oarsen, R. A., and Monagham, M. L., Genetic markers identified by immunogenetic methods. Am. Fish. Soc. Symp. 7, 521–540, 1990. 30. Davis, W. C., Hamilton, M. J., Park, Y., Larsen, R. A., Wyatt, C. R., and Okada, K., Ruminant leukocyte differentiation molecules. In ‘‘MHC Differentiation Antigens, and Cytokines in Animals and Birds. Monographs in Animal Immunology’’ (O. Barta, Ed.), Vol. I, pp. 47–70, BAR-Lab, Inc., Blacksburg, VA, 1990. 31. Naessens, J., Sileghem, M., NacHugh, N., Park, Y. H., Davis, W. C., and Toye, P., Selection of BoCD25 monoclonal antibodies by screening mouse L cells transfected with the bovine p55-interleukin-2 (IL-2) receptor gene. Immunology 76, 305–309, 1992. 32. Davis, W. C., MacHugh, N. D., Park, Y., Hamilton, M. J., and Wyatt, C. R., Identification of a monoclonal antibody reactive with the bovine orthologue of CD3 (BoCD3). Vet. Immunol. Immunopathol. 39, 85–91, 1993. 33. Ababou, A., Goyeneche, J., Davis, W. C., and Levy, D., Evidence for the expression of three different BoLA-class II molecules on the bovine BL-3 cell line: Determination of a non-DR non-DQ gene product. J. Leukocyte Biol. 56, 182–186, 1994. 34. Young, I. T., Proof without prejudice: Use of the Kolmogorov– Smirnov test for the analysis of histograms from flow systems and other sources. J. Histochem. Cytochem. 25, 935–941, 1977. 35. Kishimoto, T. K., Larson, R. S., Corbi, A. L., Dustin, M. L., Staunton, D. E., and Springer, T. A. The leukeocyte integrins: LFA-1, Mac-1, and p150,95. Adv. Immunol. 46, 149–182, 1989. 36. Matheise, J. P., Delcommenne, M., Mager, A., Didembourg, C. H., and Letesson, J. J., CD5/ B cells from bovine leukemia virus infected cows are activated cycling cells responsive to interleukin 2. Leukemia 6, 304–309, 1992. 37. Schwarting, R., Stein, H., and Wang, C. Y., The monoclonal antibodies aS-HCL 1 (aLeu-14) and aS-HCL 3 (aLeu-M5) allow the diagnosis of hairy cell leukemia. Blood 65, 974–983, 1985. 38. Springer, R. A., Miller, L. J., and Anderson, D. C., p150,95, the third member of the Mac-1, LFA-1 human leukocyte adhesion glycoprotein family. J. Immunol. 136, 240–245, 1986. 39. Miller, L. J., Schwarting, R., and Springer, T. A., Regulated expression of the Mac-1, LFA-1, p150,95 glycoprotein family during leukocyte differentiation. J. Immunol. 137, 2891–2900, 1986. 40. Keizer, G. D., te Velda, A. A., Scharting, R., Figdor, C. G., and de Vries, J. I., Role of p150,95 in adhesion, migration, chemotaxis and phagocytosis of human monocytes. Eur. J. Immunol. 17, 1317–1322, 1987. 41. De la Hera, A., Alvarez-Mon, M., Sanchez-Madrid, F., MartinezA, C., and Durantez, A., Co-expression of Mac-1 and p150,95 on CD5/ B cells. Structural and functional characterization in a human chronic lymphocytic leukemia. Eur. J. Immunol. 18, 1131–1134, 1988. 42. Mirsky, M. L., Da, Y., and Lewin, H. A., Detection of bovine leukemia virus proviral DNA in individual cells. PCR Methods Appl. 2, 333–340, 1993. 43. Jensen, W. A., Rovnak, J., and Cockerell, G. L., In vivo transcription of the bovine leukemia virus tax/rex region in normal and neoplastic lymphocytes of cattle and sheep. J. Virol. 65, 2484– 2490, 1991. 44. Haas, L., Divers, T., and Casey, J. W., Bovine leukemia virus gene expression in vivo. J. Virol. 66, 6223–6226, 1992. 45. Lal, R. B., Rudolph, D. L., Dezzutti, C. S., Linsley, P. S., and Prince, H. E., Costimulatory effects of T cell proliferation during infection with human T lymphotropic virus types I and II are mediated through CD80 and CD86 ligands. J. Immunol. 157, 1288–1296, 1996.
Received August 15, 1996; accepted with revision January 28, 1997
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Modulation of Bovine Leukemia Virus-Associated Spontaneous Lymphocyte Proliferation by Monoclonal Antibodies to Lymphocyte Surface Molecules Diana M. Stone, Linda K. Norton, and William C. Davis Department of Veterinary Microbiology and Pathology, Washington State University, P.O. Box 647040, Pullman, Washington 99164-7040
Both human T lymphotropic virus (HTLV) and bovine leukemia virus (BLV) infections are characterized by in vitro proliferation of peripheral blood lymphocytes in the absence of exogenous antigens or mitogens. Differential expression of lymphocyte surface molecules in HTLV and BLV infection suggests that lymphocyte dysregulation may involve signaling through surface molecules involved in immune regulation. We examined the expression of adhesion and major histocompatibility (MHC) molecules on circulating lymphocytes from BLV-infected cows with persistent lymphocytosis and the ability of monoclonal antibodies to these molecules to modulate spontaneous lymphocyte proliferation. The integrin molecule, CD11c, and both MHC class I and MHC class II molecules were upregulated on B and T lymphocytes from PL cows. Anti-CD11c antibody was stimulatory to lymphocyte proliferation regardless of BLV status and had a greater stimulatory effect on spontaneously proliferating lymphocytes from persistently lymphocytotic cows than on normal bovine lymphocytes. Antibodies to bovine class I and class II inhibited spontaneous lymphocyte proliferation. Results suggest that lymphocyte dysregulation in BLV-induced persistent lymphocytosis involves upregulation of and signaling through lymphocyte surface molecules which are involved in immune activation of lymphocytes. q 1997 Academic Press
INTRODUCTION
Bovine leukemia virus (BLV) belongs to a family of oncogenic retroviruses which includes human T lymphotropic virus (HTLV)-I and -II and the simian T cell leukemia virus-I (1). These retroviruses share a common genomic organization (1, 2) and have been associated with preneoplastic lymphocyte dysregulation, lymphoid neoplasia, and/or progressive myelopathies (3–6). BLV is unique in the HTLV family of retroviruses by having a tropism for B lymphocytes instead of T lymphocytes. Approximately 30% of cattle naturally infected with BLV develop a sustained nonmalignant polyclonal expansion of CD5/ B lymphocytes (7) referred to as persistent lymphocytosis (PL), with vari-
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able progression to CD5/ B cell leukemia or lymphoma (8). Factors regulating disease development in HTLV and BLV infections remain unclear. One of the hallmarks of HTLV and BLV infection is the in vitro proliferation of peripheral blood lymphocytes when cultured in the absence of exogenous antigen or mitogen (9–13), referred to as spontaneous lymphocyte proliferation (SLP). BLV-induced PL in cattle provides an excellent model system to investigate the mechanisms which promote SLP in these lymphotropic retroviral infections. The transition from asymptomatic infection to PL in BLV-infected cattle is well defined and there is an association between appearance of SLP and BLV-induced PL (13–15). PL in cattle is an early yet persistent event associated with viral infection, thus providing a convenient period of time to study the cellular and molecular events controlling SLP in a naturally infected outbred population of animals. An understanding of the factors controlling SLP in cells from BLV-infected cows may shed light on how the HTLV/BLV family of retroviruses promotes an abnormal state of lymphocyte activation which can progress either to cell transformation, as in HTLV-I-associated adult T cell leukemia and bovine leukosis, or to a heightened inflammatory response as in HTLV-associated myelopathy (3, 4). Because clinical and lymphocyte phenotypic changes in HTLV and BLV infection are associated with SLP, it is suggested that SLP is an in vitro correlate of the disease process (9, 16). Some of the phenotypic changes observed in T lymphocytes from HTLV-infected individuals with SLP include elevated numbers of CD4 and/or CD8 T lymphocytes (11, 17), increased expression of the integrin molecules CD11a and CD18 (18, 19), increased interleukin-2 receptor (IL-2R) expression (11, 12, 20, 21), and increased HLA-DR expression (17, 22–25). In BLV-induced PL there is often an increase in the number of circulating T lymphocytes as well as B lymphocytes (26), enhanced expression of bovine major histocompatibility complex (BoLA) class II DP-like molecules (27), and enhanced induction of IL2Ra expression on B lymphocytes from these cows (26, 27). These observations suggest that SLP associated
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with the HTLV/BLV family of retroviruses may be due to changes in the expression of lymphocyte surface molecules involved in immune regulation of lymphocytes. In the present study we examined the expression of surface molecules involved in lymphocyte regulation on spontaneously proliferating lymphocytes from PL cows and the ability of monoclonal antibodies (mabs) to these molecules to modulate SLP. Results show that a greater proportion of both B and T lymphocytes from PL cows express the integrin molecule CD11c than normal bovine B and T lymphocytes. This molecule is further upregulated on B lymphocytes from PL cows after 3-day unstimulated cultures, correlating with SLP. Anti-CD11c antibody is stimulatory to bovine peripheral blood mononuclear cells (PBMC) regardless of BLV status and has a greater stimulatory effect on spontaneously proliferating lymphocytes from PL cows than on normal bovine lymphocytes. Results show that both class I and class II molecules are upregulated on B and T lymphocytes from PL cows and that mabs to BoLA class I and class II significantly inhibit SLP. These results support the hypothesis that SLP associated with lymphotropic retroviral infections involves signaling of lymphocytes through surface molecules involved in immune regulation.
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IgG2a) (29–33). Mab COLIS205 (IgG2a), which does not recognize bovine antigens, was used as a nonbinding and isotype control. In the blocking studies mabs of the IgM and IgG1 isotypes were further compared to mabs CACT61A and CACT80C, respectively, as these two mabs did not significantly block SLP (Fig. 3). Flow-Cytometric Analysis Single- and dual-color stainings were performed as previously described (26). Labeled cells were enumerated with a FACSort (Becton–Dickinson Immunocytometry Systems Inc., San Jose, CA). Patterns of reactivity of the mabs with lymphocytes were analyzed with CELLQUEST software. Data on 25,000 cells from each PL cow and 50,000 cells from each control cow were analyzed to determine the percentage of bovine B and T lymphocytes expressing surface molecules and the intensity of expression of these molecules on lymphocyte subsets (Figs. 1 and 2). More cells were collected from control cows to ensure sufficient numbers of B lymphocytes for analysis. Data on 3000 cells were collected per sample for phenotyping the lymphocytes from each cow used in the SLP blocking experiments (Table 1). Lymphocyte Culture and Proliferation Assay
MATERIALS AND METHODS
Animals Adult Holstein cows naturally infected with BLV and characterized by persistent lymphocytosis, defined as lymphocyte counts three or more standard deviations above the mean for age-matched, BLV-seronegative cattle (28), were identified as part of a university milking herd. These cows tested seropositive for BLV by two sequential agar gel immunodiffusion tests using gp51 as antigen (Pitman–Moore, Mundelein, IL). Control cows were healthy adult Holstein cows with normal lymphocyte counts, seronegative for BLV, and were housed in the same herd as the infected animals. Monoclonal Antibodies Mouse anti-bovine mabs were obtained from the Washington State University Monoclonal Antibody Center (Pullman, WA). These mabs specifically recognize determinants on molecules expressed on bovine granulocytes and monocytes (DH59B, IgG1), bovine B lymphocytes (sIgM) (BIg73A, IgG1 ; PIg45A, IgG2b), the bovine gd T cell receptor (TCR) (CACT61A, IgM), bovine (Bo) CD4 cells (CACT138A, IgG1), BoCD8 cells (CACT80C, IgG1), BoCD3 cells (MM1A, IgG1), BoCD5 cells (B29A, IgG2a), BoMHC-I cells (H58A, IgG2a), BoMHC-II DP-like cells (H42A, IgG2a), DQ cells (TH81A5, IgG2a), DR cells (TH14B, IgG2a), BoCD11a cells (BAT75A, IgG1), BoCD11c cells (BAQ153A, IgM), BoCD18 cells (BAQ30A, IgG1), and the bovine IL-2 receptor a chain (CACT108A,
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Blood was collected by jugular venipuncture into acid–citrate–dextrose (ACD) at one part ACD to four parts whole blood. PBMC were separated by Accupaque (1.083) (Accurate Chemical and Scientific Corp., Westbury, NY) sedimentation as previously described (26). Viability counts were done using the trypan blue dye exclusion method. The PBMC contained over 95% viable cells and less than 1% contamination with granulocytes. Lymphocyte proliferation was assayed in RPMI 1640 supplemented with 20% Fetal Clone I (Hyclone Labs, Logan, UT), antibiotics, 2 mM L-glutamine, 300 mM 2-mercaptoethanol, 10 mM Hepes with 5 1 105 PBMC/well in 96-well flat-bottom tissue culture plates (Sarstedt Inc., Newton, NC). Mabs were added at the initiation of culture at 10 mg/ml for blocking studies. Cells were cultured at 377C with 5% CO2 for 3 days. [3H]Thymidine (0.5 mCi) was added to each well at 18 hr prior to termination of culture. Cells were harvested on an automated 96-well plate harvester (Tomtec Inc., Orange, CT) and the amount of [3H]thymidine incorporated was determined by liquid scintillation spectroscopy (Wallac Inc., Gaithersburg, MD). Data are expressed as the mean of six replicate samples. The percentage inhibition was calculated as 1 0 (mean cpm of cultures with antibody/mean cpm of cultures without antibody) 1 100. Cell Cycle Analysis Cells cultured for 3 days were stained with antisIgM mab for single fluorescent FITC flow cytometry.
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FIG. 1. Expression of MHC class I/II and CD11c on B and T lymphocytes and expression of CD18, CD11a, CD2, and sIgM on PBMC from BLV-infected PL cows and uninfected controls. Expression of surface molecules was determined by dual-label flow-cytometric analysis on fresh lymphocytes (Day 0) and unstimulated cultured lymphocytes (Day 3). Values are means and standard deviations of 3 BLV-infected PL cows and 3 uninfected controls. Significant comparisons at P £ 0.05 are indicated by brackets and an asterisk.
Stained cells were fixed in 2% formaldehyde in phosphate-buffered saline (PBS) for 60 min on ice. Cells were permeabilized with 0.2% Tween 20 (Sigma Chemical Co., St. Louis, MO) for 15 min at 377C, washed once in PBS, and resuspended in PBS with 10 mg/ml propidium iodide (Calbiochem-Novabiochem Corp., LaJolla, CA) and 22 Kunitz units/ml of RNase A (Sigma Chemical Co.). Samples were kept in the dark at least 30 min before analysis by flow cytometry. G0 samples were uncultured PBMC from a non-PL cow which were fixed, permeabilized, and stained with propidium iodide.
significant differences between SLP and lymphocyte proliferation levels with mabs added (Table 2). [3H]Thymidine incorporation below SLP are reported as percentage inhibition and [3H]thymidine incorporation greater than SLP are reported as percentage stimulation. The Kolmogorov–Smirnov two-sample test was used to compare histograms of fluorescence intensity for surface molecules on lymphocyte subsets (Fig. 2). A 99.9% confidence level of significance was used where the value Dcrit/s(n) Å 1.63 and rejection of the null hypothesis is determined by D/s(n) ú Dcrit/s(n) (34). RESULTS
Statistical Analysis Analysis of variance was used to compare lymphocytes from PL and BLV-negative cows for percentage expression of surface molecules (Fig. 1). P £ 0.05 was considered significant. For each PL cow confidence intervals at the P £ 0.05 level were used to determine
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Modulation of Expression of Cell Surface Molecules on Spontaneously Proliferating Lymphocytes from BLV/ PL Cows Previous studies showed that a greater percentage of B lymphocytes from PL cows expressed the CD5 mol-
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ecule than normal bovine B lymphocytes (7) and that there was enhanced induction of IL-2Ra expression on in vitro stimulated B lymphocytes from PL cows (26,
FIG. 3. Inhibition of spontaneously proliferating PBMC from BLV-infected PL cows. Represented are mean percentage inhibition in the presence of mabs to lymphocyte surface molecules in 4 BLVinfected PL cows. Error bars represent the P Å 0.05 confidence interval.
FIG. 2. Cell surface density of MHC class I and class II molecules on fresh sIgM/ B lymphocytes and CD2/ T lymphocytes from BLVinfected PL cows (solid boldface lines) compared to uninfected controls (solid thin lines). Control stained cells are indicated with stippled lines in the first panels. These data are representative of 3 BLV-infected PL cows and 3 uninfected controls. Histograms are normalized. MHC fluorescence intensity is indicated on the horizontal axis and the number of cells on the vertical axis. Dcrit/s(n) Å 1.63 for a 99.9% confidence level and the D/s(n) for each comparison is as follows: B cells: MHC-I (6.84), DP (10.74), DQ (18.30), DR (18.43); T cells: MHC-I (27.69), DP (17.09), DQ (25.65), DR (13.55).
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27). To more completely characterize bovine lymphocytes for expression of surface molecules involved in immune regulation we evaluated the expression of MHC class I and class II molecules and the adhesion molecules CD11a, CD11c, and CD18 on lymphocyte subsets from freshly isolated and 3-day unstimulated cultures of PBMC from three PL cows and three noninfected control cows by dual-color microfluorimetry (Figs. 1 and 2). As expected, PBMC from PL cows were characterized by a significantly greater percentage of IgM/ lymphocytes and a significantly lower percentage of CD2/ lymphocytes compared to PBMC from BLVnegative cows (Fig. 1). The percentage of B (IgM/) and T (CD2/) lymphocytes did not change significantly from time 0 to Day 3 of culture. Also as expected, all freshly isolated and cultured bovine B and T lymphocytes expressed MHC-I (Fig. 1), but the intensity of MHC-I expression on freshly isolated cells was moderately higher on B and T lymphocytes from PL cows compared to that of normal bovine lymphocytes (Fig. 2). The MHC-II DP-like, DQ, and DR molecules were expressed on greater than 96% of freshly isolated B lymphocytes from PL cows, whereas 58 to 80% of circulating B lymphocytes from BLV-negative cows expressed detectable levels of these molecules (Fig. 1). By Day 3, greater than 90% of all bovine B lymphocytes expressed MHC-II DP-like, DQ, and DR molecules regardless of BLV status (Fig. 1). The intensity of DP-like, DQ, and DR expression on MHC-II-positive B lymphocytes was consistently higher on freshly isolated B lymphocytes from PL cows than on normal bovine B lymphocytes (Fig. 2). A significantly greater percentage of freshly
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TABLE 1 Peripheral Blood B and T Lymphocytes in BLV-Infected, Lymphocytotic Cows and Normal, BLV-Noninfected Cows Cell number BLV status/ cow no.
Total lymphocytes
IgM
IgM/CD5a
CD3
CD4
CD8
g/d
Positive 1379 1580 1555 1583 1570 1570c
14,276b 17,755 12,556 13,452 9768 10,872
ND 5682 3390 6053 2344 6306
ND 4829 2576 5024 2274 5738
ND 6392 4269 2959 5665 2935
ND 3018 1758 2152 2735 1196
ND 3729 6278 807 2344 1305
ND 2841 1256 942 1661 1413
Negative 1529 1485 1467 1315 1651 1651c
3250 2484 2380 2856 4230 4161
ND 174 262 371 677 541
ND 80 89 360 162 395
ND 745 1404 1599 2961 2580
ND 994 643 971 1184 1040
ND 373 476 771 804 915
ND 199 286 400 592 957
a
Number of IgM/ lymphocytes that express CD5. Number of lymphocytes/ml of whole blood. c Repeat sampling 1 week later. b
isolated T lymphocytes from PL cows expressed MHCII DQ compared to normal bovine T lymphocytes (Fig. 1), but there was no significant difference in percentages of DP-like and DR expression. By Day 3, however, a greater percentage of T lymphocytes from PL cows
TABLE 2 Percentage Inhibitiona of Spontaneous Lymphocyte Proliferation by PBMC from BLV PL Cows Cow number Mab
1379
1580
1555
1583
1570
CD11c CD18 CD11a CD5 IL-2Ra CD4 CD8 g/d MHC-I MHC-II/DP MHC-II/DQ MHC-II/DR Iso-IgG2a CD3
/b244c 43 37 29 32 /70 /7 /15 65 66 81 59 /50 /23
/93 35 38 19 29 /9 20 /29 91 18 25 32 /47 /1147
/89 /3 /26 48 11 /62 /9 /54 87 81 84 85 /20 /1420
/66 16 /18 9 4 /65 /23 /25 71 80 50 80 /2 /4090
/12 64 38 5 3 4 4 11 /12 /32 /110 /46 /8 /2410
SLP mean SLP stderr
2505 (165)
3688 (102)
2442 (121)
2419 (230)
6404 (351)
a
Percentage inhibition is calculated as 1 0 (cpm/SLP) 1 100. Positive numbers are percentage stimulation. c Numbers in boldface are significantly different from spontaneous proliferation at P £ 0.05. b
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expressed DP-like, DQ, and DR molecules compared to normal bovine T lymphocytes (Fig. 1). As with B lymphocytes from PL cows, the intensity of MHC-II DP-like, DQ, and DR expression at Day 0 was moderately but significantly higher on T lymphocytes from PL cows compared to that on normal bovine T lymphocytes (Fig. 2). Within individual cows there was a modest decrease in the intensity of expression of all surface molecules evaluated from Day 0 to Day 3 of culture regardless of BLV status or lymphocyte subset (data not shown). Phenotypic analysis of freshly isolated cells showed that all bovine PBMC expressed the b2 CD11a/CD18 integrin regardless of BLV status (Fig. 1) and that the intensity of expression of the integrin as detected with anti-CD11a and CD18 mabs did not significantly differ between lymphocytes from PL cows and normal bovine lymphocytes (data not shown). In contrast, a significantly greater percentage of both freshly isolated B and T lymphocytes from PL cows expressed CD11c compared to normal bovine B and T lymphocytes (Fig. 1). Between 0 and 3 days of culture there was a modest but significant change in the percentage of lymphocytes expressing CD11c in the PL cows. There was a significant decrease in the percentage of T lymphocytes expressing CD11c (P Å 0.0396) and a significant increase in the percentage of B lymphocytes expressing CD11c (P Å 0.0005). Between 0 and 3 days of culture there was no significant change in the percentage of B or T lymphocytes expressing CD11c in BLV-negative cows. There was no consistent change in the intensity of CD11c expression on B or T lymphocytes from 0 to 3 days regardless of BLV status (data not shown).
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Phenotypic Analysis of PBMC from Cows Used in the Mab Blocking Studies Five BLV-infected PL cows and five age-matched BLV-noninfected, clinically normal cows were selected for the mab blocking studies. Analysis of PBMC prior to culture demonstrated the expected differences between BLV-infected PL cows and control cows in phenotype and in absolute numbers within lymphocyte subsets. All BLV-infected PL cows were characterized by elevated total white blood counts and elevated total lymphocyte counts due primarily to markedly increased numbers of CD5-expressing B lymphocytes (Table 1). Most PL cows were also characterized by moderately increased numbers of CD4, CD8, and gd T lymphocytes. B lymphocytes from two control cows (cow 1315 and cow 1651 at repeat sampling) were predominantly CD5/ (97 and 73%, respectively), but the absolute numbers of peripheral blood B lymphocytes and CD5/ B lymphocytes from these cows were within 2 standard deviations of previously reported means for clinically normal cows (26). Transient increases in the percentage of circulating CD5/ B lymphocytes in clinically normal BLV-negative cows with absolute numbers of CD5/ B lymphocytes remaining within normal limits have been reported (26). It is proposed that this reflects transient in vivo B lymphocyte activation because there is also a concomitant increase in B lymphocyte IL-2Ra expression (26). Effect of Antibodies to Lymphocyte Surface Molecules on SLP in PL Cows Differential expression of cell surface molecules on lymphocytes from PL cows which are involved in lymphocyte regulation suggests that signaling through these molecules may be important in SLP. PBMC from five PL cows were cultured alone or with antibodies directed against CD5, IL-2Ra, MHC-I and -II, the integrin molecules CD11a/CD18 and CD11c/CD18, the T lymphocyte subset molecules CD4 and CD8, and the gd TCR (Table 2). The mean [3H]thymidine uptake in lymphocytes from these cows without antibodies added exceeded 2000 cpm (Table 2), which was defined as SLP. Addition of mabs against MHC-I and MHC-II DPlike, DQ, and DR molecules significantly inhibited SLP in four of the five PL cows (Table 2). The effect of varying concentrations of antibodies to MHC-II DP-like, DQ, and DR (0.01 to 10.0 mg/ml) indicated that all concentrations were inhibitory to SLP in the four PL cows and inhibition was dose-dependent (data not shown). In cow 1570 mab to MHC-I had no significant effect on SLP and mabs to all three MHC-II molecules had a significant stimulatory effect on lymphocyte proliferation (Table 2). PBMC from cow 1570 were cultured a week later with mab to MHC-II DR and again this mab was stimulatory (77% stimulation). Mabs to CD11c and CD3 were stimulatory to PBMC in four of the five PL
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cows (Table 2). No consistent differences in proliferative responses to the other mabs were observed (Table 2). When data from the four cows which showed SLP inhibition with mabs to all three MHC-II molecules were analyzed as a group (Fig. 3), the mean percentages of inhibition for DP-like, DQ, and DR mabs were 61, 60, and 64%, respectively. Although other mabs were inhibitory to SLP in individual cows (Table 2), when analyzed as a group mab to CD5 only modestly inhibited SLP and mabs to IL-2Ra, CD11a, CD18, CD4, CD8, and the gd TCR did not have a significant effect (Fig. 3). Mabs to CD11c and CD3 were stimulatory for PBMC from PL cows, resulting in a mean percentage stimulation of 123 and 1670%, respectively. Marked variability among the responses of individual cows to these stimulatory mabs was noted (Table 2). Cell Cycle Analysis of Proliferating Lymphocytes Cultured with Anti-MHC-II Antibody Cell cycle analysis of spontaneously proliferating lymphocytes from PL cows has shown that it is primarily B lymphocytes which enter the cell cycle (unpublished observation). Because of the marked inhibitory effect of all three anti-MHC-II antibodies on BLV-associated SLP, we asked whether this inhibition was correlated with a decrease in B lymphocytes entering the cell cycle. PBMC from one PL cow were cultured with and without anti-MHC-II DQ mab for 3 days. Cell cycle analysis of freshly isolated cells showed that 11% of the B lymphocytes were already in the cell cycle (Fig. 4a). By 3 days 20% of viable B lymphocytes were in the cell cycle (Fig. 4b). The proportion of viable B lymphocytes in the cell cycle decreased to 15% when antiMHC-DQ was added (Fig. 4c). This correlated with a 35% inhibition in SLP. Effect of Antibodies to Lymphocyte Surface Molecules on the Proliferation of Normal Bovine Lymphocytes PBMC from five BLV-noninfected cows were also cultured with mabs against lymphocyte surface molecules. Three-day cultures without antibodies from three of these cows demonstrated the expected low background level of [3H]thymidine incorporation (means of 220– 607 cpm). [3H]Thymidine incorporation of cultured PBMC from two of the noninfected cows (cows 1315 and 1651), however, showed means of 2420 and 3647 cpm, levels which were within the range of SLP observed in lymphocytes from the PL cows in this study. Levels of thymidine incorporation which overlap values for PL cows have been reported in lymphocytes from 5 to 10% of BLV-free cattle (15). The SLP observed in BLV-free cows, however, is intermittent (unpublished observation) and is most likely due to transient antigenic stimulation of PBMC due to a subclinical infection with an unknown microorganism. The SLP intermittently observed in clinically normal BLV-negative
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FIG. 4. Effect of anti-MHC-II DQ antibody on cell cycle progression of sIgM/ B lymphocytes from a BLV-infected PL cow. DNA content of sIgM-labeled B lymphocytes was determined by propidium iodide staining of permeabilized cells at time 0 (a) and after a 3-day culture without (b) and with (c) 10 mg/ml anti-MHC-II DQ antibody. Only viable cells were analyzed. Fluorescence intensity of DNA staining is indicated on the horizontal axis and the number of sIgM/ cells on the vertical axis.
cows, therefore, should be inhibited by mabs which block antigen-mediated signaling of lymphocytes. The mab to MHC-I significantly inhibited the SLP in both of these BLV-negative cows (77 and 88% inhibition). Mabs to MHC-II DP, DR, and DQ were inhibitory at some concentration in both of these cows. The dose response to MHC-II mabs in cow 1651 paralleled the response in PL cows with all concentrations being inhibitory and maximum inhibition being reached at 0.10 mg/ml and above. Maximum inhibition of lymphocyte proliferation for this cow was 88% for DP, 82% for DR, and 72% for DQ. Mabs to MHC-II molecules were stimulatory to PBMC from cow 1315 at 10 mg/ml and inhibitory at concentrations below 10 mg/ml. The maximum percentage inhibition for this cow occurred at 0.10 mg/ ml for all three mabs and was 14% for anti-DP, 50% for anti-DR, and 73% for DQ. Mabs to CD5, IL-2Ra, CD11a, CD18, CD4, CD8, and gd TCR did not have a significant effect on [3H]thymidine incorporation of PBMC from these two cows. As in PL cows, mab to CD11c and CD3 were stimulatory for PBMC from all five BLV-negative cows. The mean percentage stimulation was 81% (range from 13 to 235%) for anti-CD11c mab and 4909% (range from 898 to 16,031%) for antiCD3 mab. DISCUSSION
PBMC from BLV-infected PL cows were investigated to determine the role of lymphocyte surface molecules in the regulation of SLP. Results from this study demonstrate that lymphocyte surface molecules involved in lymphocyte regulation are upregulated on both B and T lymphocytes from PL cows and that mabs to these molecules modulate SLP. Results demonstrate that subpopulations of normal bovine B and T lymphocytes express the integrin molecule CD11c, a molecule normally restricted to myeloid cells (35). This molecule is not only upregulated on B lymphocytes from PL cows
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as previously reported (36), but is also upregulated on T lymphocytes from PL cows. An unexpected finding in this study was the stimulatory effect of anti-CD11c antibody on lymphocyte proliferation regardless of BLV status, including an enhanced stimulatory effect on the already spontaneously proliferating PBMC from PL cows. Although CD11c is absent from normal human resting peripheral blood B and T lymphocytes, it is expressed on several human B cell lines, cytotoxic T lymphocyte clones, and stimulated B cell chronic lymphocytic leukemia cells and is a marker for hairy cell leukemia, a B cell neoplasm (35, 37–41). The function of CD11c expression on a subpopulation of normal bovine lymphocytes or on activated or transformed lymphocytes from any species is not yet known. The fact that anti-CD11c was stimulatory to bovine PBMC suggests that the function of CD11c may include positive signal transduction events when expressed on lymphocytes. Increased expression of CD11c on lymphocytes may not only be a marker of lymphocyte dysregulation but may also have functional relevance for the expansion of B lymphocytes in PL and the transformation of lymphocytes in some human cancers. Mabs to MHC-II significantly decreased SLP in PL cows and the high background proliferation observed in two BLV-negative cows, although the inhibitory concentration of mab differed for one BLV-negative cow. This suggests that the SLP associated with PL reflects a normal mechanism of lymphocyte proliferation, one which is most likely initiated by antigen in the context of MHC-II. Anti-MHC-II mabs may be inhibiting the normal antigen-mediated, MHC-II dependent, B–T lymphocyte interaction. What is abnormal in PL is that SLP is persistent, probably due to the continuous presence of BLV antigen. This suggests that the expanded population of B lymphocytes in PL may be, in part, due to chronic antigenic stimulation. Findings from other studies also support a role for antigen in SLP. PL is associated with increased viral load and increased viral
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gene expression (42–44) and BLV antisera inhibit SLP (15). Importantly, anti-MHC-II antibodies did not completely inhibit SLP. This suggests that other costimulatory signals may be involved in BLV-associated SLP. The participation of costimulatory molecules in HTLVassociated T lymphocyte proliferation was recently demonstrated (45). Anti-MHC-II antibodies failed to inhibit SLP in one PL cow (cow 1570) and, in fact, were stimulatory to PBMC from this cow. In addition, anti-CD11c failed to enhance proliferation of PBMC from this cow. Clearly, signaling events regulating lymphocyte proliferation in this cow were qualitatively different from those of the other four PL cows used in this study. One explanation is that these lymphocyte surface molecules become coupled to different signaling pathways at different stages of lymphocyte dysregulation in BLV-induced PL. Information is not available on when the cows in this study became BLV seropositive or how long they had been PL. The inhibitory effect of anti-MHC-I antibodies on SLP was unexpected. It is unlikely that inhibition is due to blocking of antigen presentation to CD8 T lymphocytes and loss of CD8 cytokines, as preliminary experiments indicate that CD8 T lymphocyte depletion does not significantly inhibit SLP (unpublished observation). Inhibition of SLP by MHC-I mab, however, does suggest the involvement of cellular interactions in SLP. One possibility is that mabs to MHC-I inhibit interactions with monocytes, resulting in a lack of monocyte-derived cytokines required for optimum Tlymphocyte-dependent B lymphocyte activation. Signaling events regulating SLP in HTLV and BLV share some important features but also differ in significant ways. There is evidence that the SLP associated with HTLV and BLV infection involve integrin molecules and MHC-II signaling of lymphocytes. However, the integrin molecules upregulated on the surface of circulating lymphocytes in HTLV- and BLV-infected individuals appear to be different and the effect of mab to these molecules on SLP is in opposite directions. Lymphocytes from HTLV-II-infected individuals show increased density of b2 integrins as detected with antiCD11a and CD18 mabs and SLP is inhibited by these mabs (18). BLV-induced PL is characterized by increased CD11c expression and anti-CD11c mab is stimulatory to SLP. As in SLP associated with PL in cattle, antibodies to HLA-DR inhibit the SLP characterizing HTLV-II-infected persons (12), and HLA class I antibodies inhibit SLP observed in some HTLV-I infected, disease-free individuals. In contrast, anti-HLA class II antibodies had no effect on SLP from HTLV-I-infected individuals (16). Results from the present study provide insights into how oncogenic retroviruses may initially dysregulate lymphocytes. The SLP of both BLV and HTLV infections is associated with changes in the expression of
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lymphocyte surface molecules involved in immune regulation and is modulated by antibodies to these molecules. An understanding of how this family of retroviruses modulate the expression of lymphocyte surface molecules and effect their signaling pathways may reveal a primary mechanism responsible for early stages of lymphocyte dysregulation. This knowledge may subsequently lead to the development of strategies to prevent or control the pathologies of these persistent retroviral infections in animals and humans which can be tested with this animal model. REFERENCES 1. Sagata, N., Yasanuga, T., Tsuzuku-Kawamura, J., Ohihi, K., Ogawa, Y., and Ihawa, I., Complete nucleotide sequence of the genome of bovine leukemia virus: Its evolutionary relationship to other retroviruses. Proc. Natl. Acad. Sci. USA 82, 677–682, 1985. 2. Ciminale, V., and Pavlakis, G. N., Complex splicing in the human T-cell leukemia virus (HTLV) family of retroviruses: Novel mRNAs and proteins produced by HTLV type I. J. Virol. 66, 1737–1745, 1992. 3. Hollsberg, P., and Hafler, D. A., Pathogenesis of diseases induced by human lymphotropic virus type I infection. N. Engl. J. Med. 328, 1173–1182, 1993. 4. Kim, J. H., and Durack, D. T., Manifestations of human Tlymphotropic virus type I infection. Am. J. Med. 84, 919–928, 1988. 5. Mchizuki, M., Watanabe, K., and Yamaguchi, K., Uveitis associated with human T lymphotropic virus type I: Seroepidemiologic, clinical, and virologic studies. J. Infect. Dis. 166, 943–944, 1992. 6. Posenblatt, J. D., Chen, I. S. Y., and Wachsman, W., Infection with HTLV-I and HTLV-II: Evolving concepts. Semin. Hematol. 25, 230–246, 1988. 7. Depelchin, A., Letesson, J. J., Lostrie-Trussart, N., Mammerickx, M., Portetelle, D., and Burny, A., Bovine leukemia virus (BLV)-infected B-cells express a marker similar to the CD5 T cell marker. Immunol. Lett. 20, 69–76, 1989. 8. Burny, A., Bruck, C., Cleuter, Y., Couez, D., Deschamps, J., Ghysdael, J., Bregoire, D., Kettmann, R., Mammerickx, M., Marbaix, G., and Portetelle, D., In ‘‘Bovine Leukemia Virus: A New Mode of Leukemogenesis’’ (G. Klein, Ed.), pp. 35–55, Raven Press, New York, 1985. 9. Itoyama, Y., Minato, S., Kira, J., Goto, I., Sato, H., Okochi, K., and Yamamoto, N., Spontaneous proliferation of peripheral blood lymphocytes increased in patients with HTLV-I-associated myelopathy. Neurology 38, 1302–1307, 1988. 10. Prince, H. E., Lee, H., Jensen, E. R., Swanson, P., Weber, D., Fitzpatrick, L., Doyle, M., and Kleinman, S., Immunologic correlates of spontaneous lymphocyte proliferation in human Tlymphotropic virus infection. Blood 78, 169–174, 1991. 11. Prince, H. E., Weber, D. M., and Jensen, E. R., Spontaneous lymphocyte proliferation in HTLV-I/II infection reflects preferential activation of CD8 and CD16/56 cell subsets. Clin. Immunol. Immunopathol. 58, 419–430, 1991. 12. Lal, R. B., Hjelle, B., and Rudolph, D. L., Spontaneous proliferation of HTLV-II-infected peripheral blood lymphocytes: HLADR-driven, IL-2-dependent response. Microbiol. Immunol. 36, 865–872, 1992. 13. Muscoplat, C. C., Alhaji, I., Johnson, D. W., Pomeroy, K. A., Olson, J. M., Larson, V. L., Stevens, J. B., and Sorensen, D. K., Characteristics of lymphocyte responses to phytomitogens: Com-
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Received August 15, 1996; accepted with revision January 28, 1997
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