JOURNAL OF INTERFERON RESEARCH 12:267-274 (1992) Mary Ann Liebert, Inc., Publishers
Interferon-a
Suppresses the Capacity of T Cells to Help Antibody Production by Human B Cells
VOLKER BRINKMANN, CHRISTOPH H. HEUSSER, JACQUES BAER, ERICH KILCHHERR, and FRANCOIS ERARD
ABSTRACT This study was designed to analyze the effect of interferon-a (IFN-a) on the potential of T cells to help B-cell differentiation in vitro. Human splenic T cells preactivated via the T-cell receptor (TCR)/CD3 complex, as well as inurine EL4 thymoma T cells preactivated with phorbol esters, stimulated human B cells via a species cross-reactive physical interaction to differentiate into antibody-producing cells. If the human or murine T cells were activated in the presence of IFN-a, normal proliferation and interIeukin-2 (IL-2) production occurred, but the cells did not acquire any B-cell helper potential. Therefore, IFN-a modulates the B-cell stimulatory potential of T cells by interfering with the T-cell activation process. In contrast, IFN-a neither acted on B cells directly nor on already activated T cells, because it did not suppress B-cell differentiation induced by T cells preactivated in the absence of IFN-a. IFN-a did not induce the production of inhibitory T-cell factor(s), since T cells preactivated in the presence of IFN-a did not inhibit the interaction of B cells with T cells optimally preactivated in the absence of IFN-a. Taken together the data indicate that IFN-a suppresses the potential of T cells to stimulate B-cell differentiation by interfering with the T-cell activation process, but acts neither on B cells directly nor on already activated T cells.
INTRODUCTION (IFN-a) reported INTERFERON-a immunoregulatory activities."-2' has been
to have a wide Due to its antiviral activity,'3 5) IFN-a has been used successfully for the treatment of viral infections,'5"' including AIDS,'7"' and also displays beneficial effects in several human tumor-associated dis-
range of
eases.I9"'2' Recent studies have proposed the use of IFN-a in severe atopic disorders, because the cytokine induces an isotype-specific reduction of immunoglobulin E (IgE) serum titers in hyper IgE patients,"3' and suppresses IgE production by IL-4-stimulated human peripheral blood mononuclear cells (PBMC) in v/fro."4' These findings may indicate a regulatory role of IFN-a in the T-cell-dependent B-cell differentiation and isotype selection process that forms the basis of immune responses in vivo. However, using different in vitro B-cell differentiation models, either enhancement,"5 l6> suppression,"7' ora lack of IFN-a activity"8' were reported. These contradictory results
Ciba-Geigy Limited,
Pharmaceuticals Research Division.
may be related to the use of unfractionated cell populations and different activation principles, which trigger different subpopulations of cells."'2,4~19' Despite this divergence, several in vivo studies indicate that IFN-a modulates Ig production during the early phase of an immune response."' If IFN-a was given to mice before an immunization with sheep red blood cells (SRBC), the generation of antibody-producing cells was completely inhibited, whereas administration of IFN-a several days after immunization had no effect or slightly increased antibody titers.'2""22' Taken together, the above data point to a central role of IFN-a in immunoregulation, but neither define the target cell nor the mode of IFN-a action. Only recently, efficient and well-defined in vitro systems have become available,'23 3" where purified human B cells can be driven efficiently into Ig-secreting cells via a physical interaction with preactivated T cells. The cellular interaction in these models was found to be major histocompatibility complex (MHC)-unrestricted, antigen-nonspecific,'23'27'29"36' and
Allergy/Immunology, CH-4002 Basel. 267
Switzerland.
BRINKMANN ET AL.
268
cross-species reactive, because human B cells could be trig-" gered in vitro by either human anti-CD3-activated T cells'27-3 or by PMA-activated murine EL4 thymoma cells.'23-26' We have now taken advantage of the above systems and have defined the effect of IFN-a on the T-cell activation process during which T cells acquire the potential to help antibody production by B cells in vitro.
overnight to allow preactivation. After 24 h, B cells (10,000/ml), IL-2 (10 U/ml), and IL-4 (10 ng/ml) were added. After 10 days, culture supematants were analyzed for Ig.
tured
Determination
of Anti-CD3-Stimulated T-Cell Growth:
Ninety-six-well roundbottomed culture plates were coated with anti-CD3 antibody as described above. T cells (2 x 105/ml) were
added in
a
total volume of 0.2 ml and cultured at 37°C in
C02. On day 3, cells were pulsed for 6 h with [3H]TdR ( I p-Ci/well).
5%
MATERIALS AND METHODS Interleukin-4: Recombinant human interleukin-4 (IL-4) was purified by gel filtration from extracts of Escherichia coli cells transfected with the human IL-4 gene as described elsewhere.'37' Judging from silver-stained polyacrylamide gel electrophoresis, a purity of >95% was achieved. One unit of activity was defined as the amount of IL-4 required to cause halfmaximal [3H]TdR uptake by 104/0.2 ml of T cells preactivated for 6 days with 1 p-g/ml PHA and then washed extensively. Specific activities (2.5 x 107 U/mg) were comparable to that of preparations commercially available from Genzyme. IFN-a: Recombinant human IFN-a B (= a8) was produced described elsewhere.I3X> The specific activity was 3 x 107 U/mg. Antiviral activity on human and murine cells has been reported with this IFN-a subtype.i39'40'
as
B-Cell and T-Cell Preparation: Human splenic mononuclear cells were isolated by Ficoll-Hypaque centrifugation'4" and stored in liquid nitrogen. Total B cells were negatively selected by depleting T cells with 2-aminoethylisothiouronium bromide-treated sheep red blood cells'42' and monocytes by incubating the cells in culture flasks for 1 h (>88% CD19+,
Interferon-a
Suppresses the Capacity of T Cells to Help Antibody Production by Human B Cells
VOLKER BRINKMANN, CHRISTOPH H. HEUSSER, JACQUES BAER, ERICH KILCHHERR, and FRANCOIS ERARD
ABSTRACT This study was designed to analyze the effect of interferon-a (IFN-a) on the potential of T cells to help B-cell differentiation in vitro. Human splenic T cells preactivated via the T-cell receptor (TCR)/CD3 complex, as well as inurine EL4 thymoma T cells preactivated with phorbol esters, stimulated human B cells via a species cross-reactive physical interaction to differentiate into antibody-producing cells. If the human or murine T cells were activated in the presence of IFN-a, normal proliferation and interIeukin-2 (IL-2) production occurred, but the cells did not acquire any B-cell helper potential. Therefore, IFN-a modulates the B-cell stimulatory potential of T cells by interfering with the T-cell activation process. In contrast, IFN-a neither acted on B cells directly nor on already activated T cells, because it did not suppress B-cell differentiation induced by T cells preactivated in the absence of IFN-a. IFN-a did not induce the production of inhibitory T-cell factor(s), since T cells preactivated in the presence of IFN-a did not inhibit the interaction of B cells with T cells optimally preactivated in the absence of IFN-a. Taken together the data indicate that IFN-a suppresses the potential of T cells to stimulate B-cell differentiation by interfering with the T-cell activation process, but acts neither on B cells directly nor on already activated T cells.
INTRODUCTION (IFN-a) reported INTERFERON-a immunoregulatory activities."-2' has been
to have a wide Due to its antiviral activity,'3 5) IFN-a has been used successfully for the treatment of viral infections,'5"' including AIDS,'7"' and also displays beneficial effects in several human tumor-associated dis-
range of
eases.I9"'2' Recent studies have proposed the use of IFN-a in severe atopic disorders, because the cytokine induces an isotype-specific reduction of immunoglobulin E (IgE) serum titers in hyper IgE patients,"3' and suppresses IgE production by IL-4-stimulated human peripheral blood mononuclear cells (PBMC) in v/fro."4' These findings may indicate a regulatory role of IFN-a in the T-cell-dependent B-cell differentiation and isotype selection process that forms the basis of immune responses in vivo. However, using different in vitro B-cell differentiation models, either enhancement,"5 l6> suppression,"7' ora lack of IFN-a activity"8' were reported. These contradictory results
Ciba-Geigy Limited,
Pharmaceuticals Research Division.
may be related to the use of unfractionated cell populations and different activation principles, which trigger different subpopulations of cells."'2,4~19' Despite this divergence, several in vivo studies indicate that IFN-a modulates Ig production during the early phase of an immune response."' If IFN-a was given to mice before an immunization with sheep red blood cells (SRBC), the generation of antibody-producing cells was completely inhibited, whereas administration of IFN-a several days after immunization had no effect or slightly increased antibody titers.'2""22' Taken together, the above data point to a central role of IFN-a in immunoregulation, but neither define the target cell nor the mode of IFN-a action. Only recently, efficient and well-defined in vitro systems have become available,'23 3" where purified human B cells can be driven efficiently into Ig-secreting cells via a physical interaction with preactivated T cells. The cellular interaction in these models was found to be major histocompatibility complex (MHC)-unrestricted, antigen-nonspecific,'23'27'29"36' and
Allergy/Immunology, CH-4002 Basel. 267
Switzerland.
BRINKMANN ET AL.
268
cross-species reactive, because human B cells could be trig-" gered in vitro by either human anti-CD3-activated T cells'27-3 or by PMA-activated murine EL4 thymoma cells.'23-26' We have now taken advantage of the above systems and have defined the effect of IFN-a on the T-cell activation process during which T cells acquire the potential to help antibody production by B cells in vitro.
overnight to allow preactivation. After 24 h, B cells (10,000/ml), IL-2 (10 U/ml), and IL-4 (10 ng/ml) were added. After 10 days, culture supematants were analyzed for Ig.
tured
Determination
of Anti-CD3-Stimulated T-Cell Growth:
Ninety-six-well roundbottomed culture plates were coated with anti-CD3 antibody as described above. T cells (2 x 105/ml) were
added in
a
total volume of 0.2 ml and cultured at 37°C in
C02. On day 3, cells were pulsed for 6 h with [3H]TdR ( I p-Ci/well).
5%
MATERIALS AND METHODS Interleukin-4: Recombinant human interleukin-4 (IL-4) was purified by gel filtration from extracts of Escherichia coli cells transfected with the human IL-4 gene as described elsewhere.'37' Judging from silver-stained polyacrylamide gel electrophoresis, a purity of >95% was achieved. One unit of activity was defined as the amount of IL-4 required to cause halfmaximal [3H]TdR uptake by 104/0.2 ml of T cells preactivated for 6 days with 1 p-g/ml PHA and then washed extensively. Specific activities (2.5 x 107 U/mg) were comparable to that of preparations commercially available from Genzyme. IFN-a: Recombinant human IFN-a B (= a8) was produced described elsewhere.I3X> The specific activity was 3 x 107 U/mg. Antiviral activity on human and murine cells has been reported with this IFN-a subtype.i39'40'
as
B-Cell and T-Cell Preparation: Human splenic mononuclear cells were isolated by Ficoll-Hypaque centrifugation'4" and stored in liquid nitrogen. Total B cells were negatively selected by depleting T cells with 2-aminoethylisothiouronium bromide-treated sheep red blood cells'42' and monocytes by incubating the cells in culture flasks for 1 h (>88% CD19+,
