Adhesion Molecules And Htlv Infection

Our group has utilized a variety of approaches in order to identify a novel cell membrane antigen important for HTLV-I and HTLV-II infection and syncytium formation. We reported that a membrane antigen expressed on human B and T cells is involved in HTLV-I and HTLV-II cell-free infection and syncytium formation.45 It was observed that polyclonal monospecific sera generated against the fusogenic subline (WH) of the human B-cell line BJAB recognized an approximately 80- to 90-kDa antigen that was not detectable on the nonfusogenic subline of BJAB-CC/84.47 Importantly, we demonstrated that the BJAB-WH subline could also be infected with cells infected with different HTLV-I and HTLV-II isolates (FLW, MoT, and MT2). Confirming results of others, we demonstrated that infection by cell-free virus was generally not as efficient as cell-contact-mediated infection. Only in half of the experiments were we able to infect target cells with cell-free virus. This process generally required more than 3 weeks of incubation with concentrated cell-free HTLV-I and HTLV-II. As expected, there was no detectable cell-free infection with the nonfusogenic BJAB-CC/84 cells.

We generated monoclonal antibodies against this molecule in an attempt to further characterize this membrane glycoprotein that plays an important role in syncytium formation.47 These mAbs, as determined by FACS, were able to bind to the fusogenic/infectible WH cells, but not to the nonfusogenic CC/84 cells of the BJAB cell line. MAbs not only recognized the 80- to 90-kDa antigen on the surface of target cells, but also inhibited syncytium formation between target cells and HTLV-I- and HTLV-I-infected cells.

Subsequently, our attempts to identify this molecule using screening of human T-cell cDNA libraries as well as purification and sequencing of this molecule were not successful. Therefore, we used FACS analysis, immuno-precipitation with monospecific sera, and Western blotting in order to determine the identity of the 80- to 90-kDa glycoprotein. We focused on a range of molecules that may be relevant in HTLV biology and infection. Studies indicate that different cell surface antigens including receptors and ligands can be altered after HTLV-I and/or HTLV-II infection, including CD9, CD11a, CD13, CD18, CD33, CD40, CD49e, CD54, CD58, and CD80. First, antibodies against a range of known cell surface antigens were tested for their ability to bind BJAB-WH and not BJAB-CC84 cells. FACS analysis showed that anti-ICAM-3 mAbs bound to fusogenic BJAB-WH cells and not to BJAB-CC84 cells (Fig. 3). All other anti-CD mAbs exhibited similar bind-

FIGURE 3. ICAM-3 mAbs bind to WH, not CC84 sublines of BJAB cells. Binding of various anti-CD50 mAbs to BJAB-WH and BJAB-CC84 cells. Binding activity was measured by FACS analysis and is shown as mean fluorescence for one representative experiment. Monospecific antisera and mAbs (3.3.10) against BJAB-WH cells were used as a positive control. For FACS, 10 1^1 of each mAb (1 ^g/ml) or polyclonal sera (1:100 dilution) was incubated with 0.2 x 106 WH or CC84 sublines of BJAB cells for 1 h on ice. After incubation cells were washed and stained with anti-mouse Ig-FITC conjugate as described earlier.47

FIGURE 3. ICAM-3 mAbs bind to WH, not CC84 sublines of BJAB cells. Binding of various anti-CD50 mAbs to BJAB-WH and BJAB-CC84 cells. Binding activity was measured by FACS analysis and is shown as mean fluorescence for one representative experiment. Monospecific antisera and mAbs (3.3.10) against BJAB-WH cells were used as a positive control. For FACS, 10 1^1 of each mAb (1 ^g/ml) or polyclonal sera (1:100 dilution) was incubated with 0.2 x 106 WH or CC84 sublines of BJAB cells for 1 h on ice. After incubation cells were washed and stained with anti-mouse Ig-FITC conjugate as described earlier.47

FIGURE 4. FACS comparison of mAbs to various known cell surface antigens. Binding of various mAbs to BJAB-WH and BJAB-CC84 cells. Results are shown as change in mean fluorescence as determined by FACS analysis. Monospecific antisera and mAbs (3.3.10) against BJAB-WH cells were used as a positive control. Ten of each mAb (1 ^g/ml) or polyclonal sera (1:100 dilution) was incubated with 0.2 x 106 WH or CC84 sublines ofBJAB cells for 1 h on ice. After incubation, cells were washed and stained with anti-mouse Ig-FITC conjugate as described previously.47 Data are shown for one representative experiment.

FIGURE 4. FACS comparison of mAbs to various known cell surface antigens. Binding of various mAbs to BJAB-WH and BJAB-CC84 cells. Results are shown as change in mean fluorescence as determined by FACS analysis. Monospecific antisera and mAbs (3.3.10) against BJAB-WH cells were used as a positive control. Ten of each mAb (1 ^g/ml) or polyclonal sera (1:100 dilution) was incubated with 0.2 x 106 WH or CC84 sublines ofBJAB cells for 1 h on ice. After incubation, cells were washed and stained with anti-mouse Ig-FITC conjugate as described previously.47 Data are shown for one representative experiment.

ing characteristics on both BJAB-WH and BJAB-CC84 cells (Fig. 4). Several anti-ICAM-3 mAbs inhibited over 86% of HTLV-I-induced syncytia formation (Fig. 5). Syncytia inhibition by anti-ICAM-3 was comparable to inhibition by the anti-BJAB-WH monospecific sera as well as mAbs against BJAB-WH. The mAbs against surface antigens did not inhibit syncytia formation as effectively as the anti-ICAM-3 mAbs (Fig. 6). Importantly, anti-BJAB-WH monospecific sera, anti-BJAB-WH mAb, and anti-ICAM-3 mAb immuno-precipitated the same size molecule (80-90 kDa) from the lysates ofmeta-bolically labeled BJAB-WH, but not BJAB-CC/84 cells (Fig. 7). Additionally,

FIGURE 5. Syncytium inhibition with different anti-ICAM-3 mAbs. Syncytium formation inhibition ability ofvarious anti-CD50 mAbs. Syncytium inhibition was performed as described.47A mixture of BJAB-WH target cells (50 x 103/well) and HTLV-I/MT2 infected cells (50 x 103/ well) was used for the fusion assay. For analysis of syncytium inhibition, we incubated a 1:100 dilution of anti-BJAB-WH monospecific polyclonal sera or 1 ^g/ml mAb antibodies first with target cells followed by the addition of infected cells. The quantity of syncytia was calculated and data were presented as Vn/ Vo, where Vn is the number of syncytia in the control and Vois the number of syncytia in experimental wells. Negative control wells contained media only. Data represent the average of two experiments.

FIGURE 5. Syncytium inhibition with different anti-ICAM-3 mAbs. Syncytium formation inhibition ability ofvarious anti-CD50 mAbs. Syncytium inhibition was performed as described.47A mixture of BJAB-WH target cells (50 x 103/well) and HTLV-I/MT2 infected cells (50 x 103/ well) was used for the fusion assay. For analysis of syncytium inhibition, we incubated a 1:100 dilution of anti-BJAB-WH monospecific polyclonal sera or 1 ^g/ml mAb antibodies first with target cells followed by the addition of infected cells. The quantity of syncytia was calculated and data were presented as Vn/ Vo, where Vn is the number of syncytia in the control and Vois the number of syncytia in experimental wells. Negative control wells contained media only. Data represent the average of two experiments.

anti-BJAB-WH sera blocked binding of fluorescein isothiocyanate (FITC)-conjugated anti-ICAM-3 mAb with target cells (Fig. 8). Therefore, we demonstrated that monoclonal anti-ICAM-3, our monospecific antisera, and mAb against BJAB-WH cells bound the same cellular molecule, ICAM-3, which is involved in HTLV infection.

ICAM-3 is a member of the immunoglobulin supergene family. This is a five-immunoglobulindomain, 120-kDa adhesion molecule expressed on a variety of cells including lymphocytes and professional antigen-presenting cells (APC) . ICAM-3 is a costimulatory molecule for both resting and acti-

Antibodies £

FIGURE 6. Syncytium inhibition with mAbs to various known cell surface antigens. Experiments were performed exactly as discussed in Fig. 5. Data represent the average of two experiments.

Antibodies £

FIGURE 6. Syncytium inhibition with mAbs to various known cell surface antigens. Experiments were performed exactly as discussed in Fig. 5. Data represent the average of two experiments.

vated T lymphocytes and plays an essential role in the initiation of the immune response.48 ICAM-3 is closely related to ICAM-1 and binds LFA-1 (lymphocyte function-associated molecule) through its two N-terminal do-mains.49 The constitutive high expression of ICAM-3 on resting leukocytes coupled with the observation that ICAM-3 is the primary LFA-1 ligand on resting T cells suggests that ICAM-3 may be most important LFA-1 ligand in the initiation of the immune/inflammatory response.50 The ability of ICAM-3 to mediate syncytium formation indicates that this molecule could represent a cellular binding/fusogenic receptor or could be an adhesion molecule involved in cell-to-cell spreading of HTLV. As mentioned above, ICAM-3 has molecular weight 120 kDa; interestingly, our identified antigen has a molecular lower weight of 80-90 kDa. It is possible that ICAM-3 exists in a truncated form on BJAB-WH cells, accounting for the lower molecular weight.

FIGURE 7. Gel immunoprecipitation. (a) Anti-BJAB-WH monospecific seraimmunoprecipi-tated 80-90kDa molecule from the lysate ofmetabolically labeled BJAB-WH (lane A), but not BJAB-CC84 (laneB) cells. (b) Both anti-BJAB-WH (laneA) and anti-ICAM-3 (lane C) mAb immunoprecipitated the same size molecule (80-90kDa) from the lysates of metabolically labeled BJAB-WH (lanes A, C), but not BJAB-CC84 (lanes B, D) cells.

FIGURE 7. Gel immunoprecipitation. (a) Anti-BJAB-WH monospecific seraimmunoprecipi-tated 80-90kDa molecule from the lysate ofmetabolically labeled BJAB-WH (lane A), but not BJAB-CC84 (laneB) cells. (b) Both anti-BJAB-WH (laneA) and anti-ICAM-3 (lane C) mAb immunoprecipitated the same size molecule (80-90kDa) from the lysates of metabolically labeled BJAB-WH (lanes A, C), but not BJAB-CC84 (lanes B, D) cells.

Earlier, Sommerfelt et al.35 determined through the use of human-mouse somatic hybrids that HTLV uses a receptor coded by human chromosome 17. In these experiments, the only human chromosome common to all cell hybrids susceptible to HTLV infection was human chromosome 17.35 More recently, researchers have found that the HTLV receptor gene may reside on the long arm of chromosome 17 localized between q21 and q23.4 The gene encoding ICAM-3 has been mapped to chromosome 19. We feel that it is quite possible that chromosome 17 actually encodes binding recep tor, whereas ICAM-3 act as a fusogenic receptor or adhesion molecule for viral binding, fusion, and infection. Alternatively, the gene on chromosome 17 may not encode an actual receptor, but may be important for the elaboration of a cofactor to the primary receptor or possibly the expression of a coreceptor.

There is other evidence that adhesion molecules can play a variety of roles in virus infection. The role of ICAMs as receptors or coreceptors for

FIGURE 8. FACS blocking with labeled anti-ICAM-3. Anti-BJAB-WH sera blocked binding of FITC conjugated anti-ICAM-3 mAb with target cells. Blocking of anti-ICAM-3 binding with anti-WH sera as determined by FACS analysis. Antisera at a dilution up to 1:100 had the ability to block binding of anti-CD50-FITCwith BJAB-WH cells. We used anti-CD50 MoAb labeled with FITC and unlabeled in the blocking experiments. Cells were incubated (30 min, 4°C) firstwith anti-WH monospecific sera (1:5 diluted, 5ul) or with anti-CD50 (1:10 diluted, 5ul) and than with anti-CD50-FITC (1:10 diluted, 5ul). Data is shown for one representative experiment.

FIGURE 8. FACS blocking with labeled anti-ICAM-3. Anti-BJAB-WH sera blocked binding of FITC conjugated anti-ICAM-3 mAb with target cells. Blocking of anti-ICAM-3 binding with anti-WH sera as determined by FACS analysis. Antisera at a dilution up to 1:100 had the ability to block binding of anti-CD50-FITCwith BJAB-WH cells. We used anti-CD50 MoAb labeled with FITC and unlabeled in the blocking experiments. Cells were incubated (30 min, 4°C) firstwith anti-WH monospecific sera (1:5 diluted, 5ul) or with anti-CD50 (1:10 diluted, 5ul) and than with anti-CD50-FITC (1:10 diluted, 5ul). Data is shown for one representative experiment.

other viruses has been documented. It has been shown that ICAM-1, ICAM-2, and ICAM-3 function as ligands for LFA-1 in HIV-1-mediated syncytia formation.52 Results suggest that certain epitopes of ICAM-3 may be involved in mediating HIV-1-specific entry into lymphoid and monocytoid cells.52 Certain Abs to ICAM-3 significantly inhibited HIV-1-specific entry, but not syncytium formation. These data are consistent with our earlier results demonstrating that monospecific antibodies do not inhibit syncytia formation between HIV-1/MN-infected cells and SUPT-1 target cells.47Addi-tionally, it has been shown that antibodies against the cell adhesion molecule LFA-1 can block HIV-1-induced syncytium formation.53 Others have also shown that LFA-1 plays an important role as an accessory protein in the biology of HIV.54,55

Other researchers have indicated that adhesion molecules may be central to HTLV infection. Fukudome et al.56 showed that antibodies against ICAM-1 (CD54) and LFA-I (CD 11a) also partially inhibit fusion of HTLV-infected cells with target cells. In a search for the cellular receptor for HTLV, Hildreth et al.51 found that vascular cell adhesion molecule-1 (VCAM-1), another member of the Ig supergene family, can mediate HTLV-I-induced syncytium formation. VCAM-1 is the major cellular ligand for the integrin VLA-4 (CD29/CD49d).57 Syncytium formation between HTLV-I-expressing cells and VCAM-1-positive cells could be blocked with antiserum against HTLV-I gp46 and with a monoclonal antibody against VCAM-1. Also, antibodies against the VCAM-1 counter-receptor VLA-4 failed to block VCAM-1-mediated HTLV-I syncytium formation.

These data along with our results suggested that several different adhesion molecules (VCAM-1, ICAM-1, and ICAM-3) could be involved in the HTLV infection process. Whether or not they serve as viral binding or fusion receptors or simply accessory molecules for HTLV-I-induced cell fusion remains to be investigated. Molecules such as VCAM-1 and ICAM-3 could increase the binding energy between HTLV-I-infected cells and uninfected cells, thereby increasing fusion efficiency. Since integrins have also been shown to be signaling molecules, adhesion molecules may support HTLV-I syncytium formation through signal transduction after interacting with its ligand.57 Therefore, these adhesion molecules may be important in this type of signaling event. Such events may be necessary to trigger fusion.

Another possible function of adhesion molecules is simply to bring HTLV-I-infected cells and target cells closer together. After the initial association of the adhesion molecules and their ligands, the cellular receptor for HTLV may interact with the viral envelope expressed on the surface of infected cells. Another possibility is that adhesion molecules may function as the viral receptor. In this case, this molecule will interact directly with viral viral viral viral viral

Adhesion molecules bring cells doser together, allowing viral receptor to interact with viral envelope

Adhesion molecules bring cells doser together, allowing viral receptor to interact with viral envelope

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