CD4+ T lymphocytes as a primary cellular target for BAT mAb stimulation

Annat Raiter, Galina Rodionov, Abraham Novogrodsky and Britta Hardy

Felsenstein Medical Research Center, Tel Aviv University, Sackler School of Medicine, Rabin Medical Center, Beilinson Campus, Petah-Tikva 49100, Israel

Correspondence to: B. Hardy


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
BAT is a monoclonal antibody (mAb) produced against membranes of a human Burkitt lymphoma cell line (Daudi) that was selected for its ability to stimulate lymphocyte proliferation. BAT manifests anti-tumor properties in mice bearing a variety of murine tumors. BAT also induced regression of human tumors inoculated into SCID mice that had been engrafted with human lymphocytes. The anti-tumor activity of BAT was related to its immune stimulatory properties. Previous data indicated that T lymphocytes and NK cells mediate in vivo the anti-tumor activity. In order to define the primary target cell for BAT stimulatory activity, the in vitro stimulatory effect of BAT on purified lymphocyte subpopulations was investigated. Human CD4+, CD8+ T cells and CD56+ NK cells were purified and their in vitro response to BAT was investigated. Results indicate that BAT selectively stimulated CD4+ cells as assessed by proliferation and secretion of IFN-{gamma}. FACS analysis has also revealed a selective increase in BAT antigen on CD4+ T cells that were cultured with BAT antibody. The effector cells that mediate BAT-induced tumor eradication may, however, be distinct from those that serve as the primary cellular target of the antibody. Cytokines such as IFN-{gamma} that are produced by CD4+ cells may be involved in activation of additional cell types that may be involved in tumor destruction.

Keywords: agonistic antibody, BAT receptors, IFN-{gamma}, immune stimulation, T cell activation


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have previously described BAT mAb that we have prepared against membranes of Daudi cells, a Burkitt lymphoma cell line, which binds and stimulates human T lymphocytes to proliferate and elicit cytolytic activity (1). BAT also stimulates murine splenocytes and induced regression of a variety of tumors in mice. Tumor regression was related to the immune-stimulatory properties of this antibody. This conclusion was supported by adoptive transfer experiments in which splenocytes from mice treated by BAT induced regression of tumors in mice (2). BAT also induced tumor regression of human melanoma xenografts in SCID mice that were engrafted with human lymphocytes (3). We have previously reported that both NK and T cells mediate the anti-tumor activity of BAT (3). In order to define the primary target cell for BAT stimulatory activity, the in vitro stimulatory effects of BAT on purified CD4+, CD8+ and CD56+ lymphocyte subpopulations was investigated.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
mAb
BAT was generated and purified as described (1). In brief, BALB/c mice were immunized with membranes from Daudi cells. Spleen cells were fused with NS-O myeloma cells. BAT was selected by its ability to bind Daudi cells and to induce proliferation of human peripheral blood mononuclear cells (PBMC). Cells were grown in RPMI 1640 medium supplemented with FCS (10%), sodium pyruvate, glutamine and antibodies, and incubated at 37°C in a humidified atmosphere containing 5% CO2. BAT was purified on a Protein G–Sepharose column according to the manufacturer's instructions (Pharmacia, St Albans, UK). Phycoerythrin (PE)-conjugated anti-CD4 and anti-CD8 mouse anti-human mAb were used for flow cytometry and were purchased from PharMingen (San Diego, CA).

Cells separation and purification
Human PBMC were obtained from blood of healthy donors by Ficoll-Hypaque density centrifugation. Cells were washed and suspended in RPMI 1640 containing 5% human type AB serum (Sigma, Rehovot, Israel).

Selection of CD4+, CD8+ and CD56+ lymphocyte subpopulations was done using MACS magnetic microbeads (Milteny Biotech, Bergisch, Gladbach, Germany). According to the manufacturer's instructions PBMC (108 cells) were suspended in 800 µl PBS supplemented with 0.5% BSA and 2 mM EDTA. Sufficient mouse anti-human CD4-, CD8- or CD56-coated magnetic beads were added to the cell suspension for 15 min at 4°C. After washing, cells were transferred to MACS LS+ separation columns for rapid positive selection of lymphocyte subpopulations. Separated subpopulations were analyzed by flow cytometry.

T cell purification from PBMC was done by nylon wool columns (Uni-Sorb; Novamed, Jerusalem, Israel). Lymphocytes (8x107) were suspended in 2 ml of RPMI 1640 medium with 5% FCS. The suspension was injected slowly into the nylon wool column that was then incubated at 37°C. After 1 h, the nylon wool column was washed 2 times with 10 ml of medium, and the non-adherent T cells were collected and counted.

Activation of lymphocytes by BAT
Human peripheral blood lymphocytes (PBL) or purified CD4+, CD8+ or CD56+ cells (2x106/ml) were incubated in culture medium containing 5% human AB serum. PBL and isolated lymphocyte subpopulations were incubated with BAT (0.1 µg/ml) for 5 days in vitro.

Proliferation assays
Aliquots (200 µl) of 2x106 cells/ml from activated PBL or activated purified T cell subpopulations were incubated in 96-well flat-bottom plates and 3[H]thymidine (1 µCi/well) was added 20 h before harvesting. Cultures were harvested into glass filters and radioactivity was counted using a liquid ß-scintillation counter.

Flow cytometry analysis
Human PBL or purified CD4+ and CD8+ cells before or after BAT activation were washed twice with PBS containing 0.5% BSA and 0.05% Na-azide. Samples containing 0.5x106 cells were used. Cells were incubated with a saturated amount of BAT for 45 min at 4°C followed by anti-mouse–FITC for 30 min (Jackson ImmunoResearch, West Grove, PA). After washing the cells, PE-conjugated antibodies anti-CD4 or anti-CD8 (PharMingen, San Diego, CA) were added for 30 min and incubated at 4°C.

A FACScan (Becton Dickinson, Esembodegem, Belgium) analyzed cell samples by CellQuest program. Side scatter and forward scatter of dot-plots determined the gate of cells. Calibration of the cytometer was done by FITC- and PE-conjugated CaliBRITE beads. Isotype controls for CD4 and CD8 antibodies was PE–IgG1 (PharMingen).

Cytokine profile
The supernatants of human PBL or purified CD4+, CD8+ or CD56+ lymphocytes cultured with or without BAT for 5 days in vitro was tested for release of human IFN-{gamma}, tumor necrosis factor-{alpha}, IL-2, IL-6 and IL-4. Quantitative determination of the cytokines concentration was evaluated by an ELISA procedure using Predicta kits (Genzyme, Cambridge, MA) according to the manufacturer's instructions.

BAT-binding antigen on CD4+ and CD8+ cells
Purified CD4+ and CD8+ cells (4x106 cells) from normal PBL were mixed with 50 µl of lysis buffer (10 mM Tris–HCl buffer, pH 7.6 with 5 mM EDTA, 0.14 M NaCl, 0.1 mM PMSF, 10 mM NaF and 0.5% NP-40) and incubated for 30 min at 0°C. The mixtures were centrifuged and the supernatants were collected. Samples of CD4+ and CD8+ cell lysates contained 50 µg/lane. Daudi cell lysate was used at 10 µg/lane. Proteins were separated by SDS–PAGE (10%) and then transferred to nitrocellulose membranes. Detection of the BAT-binding protein was done by incubation with BAT mAb overnight at 4°C followed by incubation for 45 min with peroxidase-conjugated anti-mouse IgG (Sigma, Munchen, Germany) and by chemiluminescent substrate (Pierce, Rockford, IL).


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
BAT-induced proliferation of CD4+ T cells
We have previously reported the ability of BAT to induce proliferation of human PBL. In order to identify the responding lymphocyte subpopulations, we used magnetic beads purified CD4+, CD8+ and CD56+ cells. Figure 1Go demonstrates a FACS analysis histogram of the three purified subpopulations. Each purified population was incubated with its specific PE-labeled antibody. Non-specific labeling was determined by staining the cells with anti-mouse–PE. Incubation of PBL with BAT (0.1 µg/ml) for 5 days induced a low but significant (P = 0.0227) increase in proliferation. Purified lymphocyte subpopulations were cultured at similar conditions with BAT. BAT selectively stimulated a significant increase in thymidine uptake in CD4+ cells (P = 0.0089) while neither CD8+ nor CD56+ cells responded (Fig. 2Go).



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Fig. 1. FACS analysis histogram of CD4+, CD8+ and CD56+ purified lymphocyte populations as determined by staining with the related antibody labeled with PE. Anti-mouse–PE was used as isotype control. Lymphocytes were purified using anti-CD4, anti-CD8 or anti-CD56 coated magnetic beads.

 


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Fig. 2. 3[H]Thymidine uptake in PBL and purified CD4+, CD8+ and CD56+ lymphocyte subpopulations from normal blood donors (n = 12). Cells were cultured for 5 days in the presence and absence of BAT mAb (0.1 µg/ml). The increase in PBL and CD4+ cultured with BAT was significant (P = 0.022 and P = 0.008).

 
Cytokine profile
In order to elucidate whether CD4+ Th cells, which responded by proliferation to BAT activation, are of the Th1 or Th2 type, we determined the levels of different cytokines in the medium of PBL cultured with BAT for 5 days. As seen in four independent experiments (Fig. 3Go), IFN-{gamma} levels were increased in BAT-treated cells. IL-2, IL-4 and IL-6 levels were not increased in most cases under these experimental conditions. TNF-{alpha} secretion was marginally increased in three out of the four experiments. The amount of IFN-{gamma} in supernatants of purified CD4+, CD8+ and CD56+ cells incubated with BAT was measured. The results depicted in Fig. 4Go demonstrate a significant increase (P = 0.0523) in IFN-{gamma} secretion in cultures of purified CD4+ cells contrary to cultures of purified CD8+ or CD56+ cells that were treated with BAT. BAT induced a significant increase in IFN-{gamma} secretion in PBL (P = 0.0441).



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Fig. 3. Cytokines levels (pg/ml) determined in supernatants of PBL cultured with BAT (0.1 µg/ml) and without BAT for 5 days. Four separate experiments were studied.

 


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Fig. 4. IFN-{gamma} levels (pg/ml) in supernatants from 5 day cultures of human PBL CD4+, CD8+ or CD56+ lymphocyte subpopulations from eight to 10 normal blood donor cells were incubated with or without BAT (0.1 µg/ml). The increase in IFN-{gamma} secretion in PBL and CD4+ cell cultures was significant (P = 0.044 and P = 0.052).

 
BAT antigen on CD4+ and CD8+ T lymphocytes: up-regulation by BAT in CD4+ T cells
The binding of BAT to normal CD4+ and CD8+ T lymphocytes was determined in 10 different normal volunteers. Percent of BAT binding to either CD4+ or CD8+ cells was determined using FACS analysis by double staining with BAT–FITC and either anti-CD4–PE or anti-CD8–PE antibodies. Total numbers of CD4+ or CD8+ cells were also determined. As seen in Fig. 5Go, all CD8+ cells bind BAT antibody (95 ± 5%), whereas the percent of CD4+ cells that bind BAT varies among the different individuals (68 ± 30%). The expression of BAT-binding protein in CD4+ and CD8+ cells was also determined at the molecular level using Western blot analysis. Data indicated (Fig. 6Go) that both T cell subpopulations contain the BAT antigen and that the apparent mol. wt of the BAT antigen in the T lymphocytes is similar to that detected on Daudi cells which were used to raise the BAT mAb .



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Fig. 5. Binding of BAT to CD4+ and CD8+ T cells isolated from PBL of 10 normal individuals. Percent of BAT binding CD4+ cells was determined by double staining with BAT–FITC and anti-CD4–PE antibodies. The total number of CD4+ or CD8+ cells was also determined in the same cell samples using FACS analysis.

 


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Fig. 6. Molecular detection of BAT binding protein by Western blot analysis. Lysates of purified lymphocytes subpopulations (50 µg/lane of CD4+ and CD8+ lysates) and Daudi cells (10 µg/lane) were analyzed as described.

 
The effect of BAT on the expression of BAT-binding sites in CD4+ and CD8+ cells was investigated. Incubation of PBL or purified CD4+ cells with BAT (0.1 µg/ml) resulted in an increase in percent of BAT-binding cells (Fig. 7Go). Moreover, the intensity of BAT receptors, as determined by mean fluorescence, was also increased on purified CD4+ cell populations that were cultured with BAT (from a mean fluorescence of 83 to 160). In contrast, no change in percent of BAT-binding sites and intensity was noted in CD8+ cells cultured with BAT.



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Fig. 7. Effect of incubation with BAT on the expression of BAT-binding protein in PBL and lymphocyte subpopulations. Cells were cultured with or without BAT (0.1 µg/ml) for 5 days followed by double staining with anti-CD4–PE and BAT–FITC or anti-CD8–PE and BAT–FITC. The percent of double-labeled cells was determined using FACS analysis.

 
BAT activation does not increase IL-2 receptors
In order to elucidate whether BAT-induced lymphocyte proliferation is also manifested by an increase in the percentage of IL-2 receptors on T cells, we cultured purified T cells with BAT or anti-CD3. Incubation of T cells with anti-CD3 for 3 days induced proliferation and an increase in IL-2 receptors. In contrast, incubation of T cells with BAT for 5 days induced cell proliferation without an increase in IL-2 receptors (Table 1Go).


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Table 1. Expression of IL-2 receptors (CD25) in T lymphocytes incubated with BAT or anti-CD3
 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
T cell activation has now been shown to require at least two signals. The first is antigen specific, is delivered through the TCR via the peptide–MHC and causes the T cell to enter the cell cycle. The second or co-stimulatory, signal is required for cytokine production and proliferation, and is mediated through ligand interaction on the surface of the T cell. Several such co-stimulatory molecules were described to induce immune activation of lymphocytes and subsequent increase anti-tumor activity (4). The mAb BAT was produced against a 48 kDa monomeric protein on the membranes of a Burkitt lymphoma B cell line. BAT induces stimulation of T cells accompanied by a strong anti-tumor activity. A single i.v. administration of BAT induced regression of a variety of tumors in mice. The anti-tumor activity of BAT was related to its immune stimulatory properties as was also manifested by adoptive transfer of lymphocytes from BAT-treated mice. We have previously demonstrated that T lymphocytes and NK cells mediate the anti-tumor activity of BAT mAb (3). In this study we performed experiments to define the primary cellular target for BAT in human PBL. The in vitro stimulatory effect of BAT on selected human lymphocyte subpopulations was determined. Our findings indicated that BAT selectively stimulated CD4+ T cells. The significance of this finding for the anti-tumor effect of BAT is supported by recent evidence indicating that CD4+ T cells are equally critical components of the anti-tumor immune response (5). It was also reported that secondary anti-CD3/anti-CD28 activation expanded tumor-reactive CD4+ T cell response to human cancers. Cytokine release assays demonstrated that positively selected CD4+ cells activated with anti-CD3/anti-CD28 released greater amounts of cytokine IFN-{gamma} (6). Moreover, CD4+ T cells were found to participate in tumor-eradicating immunity in B7-transfected tumor cells that elicit tumor-eradicating immunity that leads to the regression of tumors (7). Our findings that BAT selectively stimulated the secretion of IFN-{gamma} by CD4+ T cells may also be relevant to the anti-tumor activity of the antibody. It was shown that local secretion of IFN-{gamma} induces an anti-tumor response and that the in vivo efficacy of tumor-infiltrating lymphocytes (TIL) was associated with their ability to secrete IFN-{gamma} (8). The local secretion resulted in recruitment of host immune cells to the tumor and eventually in a successful host anti-tumor immune response. TIL from IFN-{gamma} gene knockout mice failed to induce tumor regression and the therapeutic efficacy of IFN-{gamma}-transfected tumor cells was eliminated when either CD4+ T cells or CD8+ T cells were depleted (8). Data depicted in Figs 5 and 6GoGo indicate that BAT-binding protein is expressed in both CD4+ and CD8+ cells. However, only CD4+ cells are responsive to the antibody. This finding indicates that legation of the cell surface BAT-binding protein is not by itself sufficient to induce activation. It is pertinent to note that T and B cells bind equally concanavalin A, whereas the lectin induces selective activation only of the T cells. Of interest are the findings that BAT induces increase in BAT antigen receptors but failed to increase IL-2 receptors. In contrast to anti-CD3, BAT stimulation seems to be IL-2 independent (Table 1Go). Additional differences between BAT- and anti-CD3-induced anti-tumor activity were recently described (9). BAT stimulation of CD4+ cells was corroborated also by FACS analysis indicating a selective increase in BAT binding to the CD4+ cells (Fig. 7Go). It is possible that stimulation of CD4+ T cells in association with NK cells produces a cascade of events leading to the development of a Th0 or Th1 response or to a shift of a Th2 response towards a Th1 or Th0 profile. Assuming that NK cells can influence CD4+ and that CD4+ T cells are able to facilitate the generation of CD8+ cytotoxic T lymphocytes (CTL) through their elaboration of cytokines, the interaction between NK cells, activated CD4+ T cells and CD8+ CTL precursors may also be considered as contributory to the induction of effector CTL (10).


    Abbreviations
 
mAb monoclonal antibody
CTL cytotoxic T lymphocyte
PBL peripheral blood lymphocyte
PBMC peripheral blood mononuclear cell
PE phycoerythrin
TIL tumor-infiltrating lymphocytes

    Notes
 
Transmitting editor: I. Pecht

Received 27 March 2000, accepted 4 August 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Hardy, B., Galli, M., Rivlin, E., Goren, L. and Novogrodsky, A. 1995. Activation of human lymphocytes by a monoclonal antibody to B lymphoblastoid cells; molecular mass and distribution of binding protein. Cancer Immunol. Immunother. 40:376.[ISI][Medline]
  2. Hardy, B., Yampolski, I., Kovjazin, R., Galli, M. and Novogrodsky, A. 1994. A monoclonal antibody against a human B lymphoblastoid cell line induces tumor regression in mice. Cancer Res. 54:5793.[Abstract]
  3. Hardy, B., Kovjazin, R., Raiter, A., Ganor, N. and Novogrodsky, A. 1997. A lymphocyte-activating monoclonal antibody induces regression of human tumors in severe combined immunodeficient mice. Proc. Natl Acad. Sci. USA 94:5756.[Abstract/Free Full Text]
  4. Schlom, J. and Hodge, J. W. 1999. The diversity of T-cell co-stimulation in the induction of antitumor immunity. Immunol. Rev. 170:73.[ISI][Medline]
  5. Pardoll, D. M. and Topalian, S. L. 1998. The role of CD4 T cell responses in antitumor immunity. Curr. Opin. Immunol. 10:588.[ISI][Medline]
  6. Li, Q., Furman, S. A., Bradford, C. R. and Chang, A. E. 1999. Expanded tumor-reactive CD4 T-cell responses to human cancers induced by secondary anti-CD3/anti-CD28 activation Clin. Cancer Res. 5:461.[Abstract/Free Full Text]
  7. La-Motta, R. N., Sharpe, A. H., Bluestone, J. A. and Mokyr, M. B. 1998. Importance of B7-1 expressing host antigen-presenting cells for the eradication of B7-2 transfected P815 tumor cells. J. Immunol. 161:6552.[Abstract/Free Full Text]
  8. Sadanaga, N., Nagoshi, M., Lederer, J. A., Joo, H. G., Eberlein, T. J. and Goedegebuure, P. S. 1999. Local secretion of IFN-gamma induces an antitumor response: comparison between T cells plus IL-2 and IFN-gamma transfected tumor cells. J. Immunother. 22:315.[ISI][Medline]
  9. Hardy, B., Kovjazin R., Raiter, A., Ganor, N. and Novogrodsky, A. 1997. Immune stimulatory and anti-tumor properties of anti-CD3 and BAT monoclonal antibodies: a comparative study. Hum. Antibodies 8:95.[Medline]
  10. Kos, F. J. and Engelman, E. G. 1966. Immune regulation: a critical link between NK cells and CTLs. Immunol. Today 17:174.