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The Journal of Immunology, 2001, 166: 5578-5584.
Copyright © 2001 by The American Association of Immunologists

Stimulatory Effects of B7-Related Protein-1 on Cellular and Humoral Immune Responses in Mice

Jane Guo, Marina Stolina, James V. Bready, Songmei Yin, Tom Horan, Steven K. Yoshinaga and Giorgio Senaldi1

Amgen Inc., Thousand Oaks, CA 91320


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inducible costimulator (ICOS) and B7-related protein-1 (B7RP-1) constitute a receptor-ligand pair involved in T cell costimulation. In this study, the stimulatory effects of B7RP-1 on cellular and humoral immune responses were investigated giving mice a construct with the extracellular domain of murine B7RP-1 fused with human IgG1 Fc (B7RP-1-Fc). B7RP-1-Fc stimulated contact hypersensitivity (CH) given near either the time of sensitization or challenge with oxazolone. When given near challenge time, B7RP-1-Fc stimulated CH more than a construct containing the extracellular domain of murine B7.2 and Fc (B7.2-Fc). B7RP-1-Fc increased the number of cells in lymph nodes draining the skin sensitized with oxazolone, especially activated T cells. B7RP-1-Fc also increased the ability of the cells in these lymph nodes to induce CH when transfused into naive mice. B7RP-1-Fc stimulated the production of anti-keyhole limpet hemocyanin (KLH) Ab, increasing anti-KLH IgG, IgG2a, and IgE, whereas B7.2-Fc did not affect this production. B7RP-1-Fc also increased the number of cells in lymph nodes draining the skin immunized with KLH and their production of IFN-{gamma}, IL-4, and IL-10 in response to KLH. Finally, B7RP-1-Fc increased the presence of eosinophils in the bronchoalveolar lavage and lungs of mice sensitized and challenged with OVA so to mount an asthmatic reaction. B7RP-1-Fc stimulates both cellular and humoral immune responses in vivo by increasing number and function of T and B cells reacting to Ag exposure.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
An Ag-specific immune response originates with the activation of T cells (1). For optimal activation, T cells require Ag-mediated stimulation through the TCR as well as costimulation through other receptors (2, 3, 4). The most important of these costimulatory receptors is CD28 (5, 6, 7, 8, 9), for which two ligands have been identified, B7.1 (CD80) and B7.2 (CD86) (10, 11, 12, 13). B7.1 and B7.2 also bind CTLA4 (CD152), a homologue of CD28 that inhibits T cells counterbalancing CD28 (7, 14, 15, 16, 17). B7.1 and B7.2 bind CTLA4 with higher affinity than CD28 (18, 19).

A new receptor-ligand pair has recently been described that costimulates T cells (20, 21, 22, 23). It consists of inducible costimulator (ICOS),2 a CD28 homologue (24), and B7-related protein-1 (B7RP-1), a B7.1 and B7.2 homologue (25). It has been shown that it is possible to affect immune responses in mice using constructs with human IgG1 Fc fused with either the extracellular domain of murine B7RP-1 (B7RP-1-Fc) (20) or murine ICOS (ICOS-Ig) (26). We have reported that B7RP-1-Fc enhances contact hypersensitivity (CH) (20), whereas Coyle et al. (26) have reported that ICOS-Fc blocks Ag-specific IgG production and the development of an asthmatic reaction. These observations are seminal but preliminary in illustrating the in vivo stimulatory properties of soluble B7RP-1 and, conversely, the inhibitory properties of soluble ICOS. Accumulating additional information on the immunomodulatory effects of B7RP-1-Fc in vivo is important in learning about the physiological role of ICOS/B7RP-1 interaction and in evaluating this molecule as a potential therapeutic. The CD28 and B7 families are rapidly growing (2) and now also count PD-1 (27) and B7-H1 (28) as members, respectively, that form another receptor-ligand pair regulating T cell function (29). This gives some sense of urgency as to characterizing the functional significance of these pairs and establishing whether stimulatory or inhibitory manipulation may serve as a therapeutic approach.

The aim of this study is to explore the effects of B7RP-1-Fc on different types of cellular and humoral immune responses in mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and mice

The Fc fusion proteins B7RP-1-Fc, B7.2-Fc, and CTLA4-Fc were prepared by fusing in frame the sequences encoding the amino-terminal 269 aa of B7RP-1 (20), 249 aa of B7.2 (12), and 151 aa of CTLA4 (14) to the sequence encoding the carboxy-terminal 235 aa of human IgG1 Fc. The fused sequences were ligated within the pcDNA3 vector and transfected into 293T cells. The sequence encoding the carboxy-terminal 235 aa of human IgG1 Fc was expressed in Escherichia coli alone to generate nonfused Fc. Fc proteins were purified from the cell culture supernatants by protein A chromatography and found to be >95% pure by SDS-PAGE and endotoxin free by the Limulus amebocyte lysate test.

Mice (BALB/c females 9 to 14 wk old and 19–21 g) were obtained from Charles River Laboratories (Wilmington, MA), housed in rooms at constant temperature and humidity under 12 h light/dark cycles, and fed with standard laboratory diet and water ad libitum.

Induction of CH

Mice were first sensitized by applying a 0.3% solution of oxazolone (Sigma, St. Louis, MO) in acetone and olive oil onto the shaved skin of the abdomen. Seven days after sensitization, mice were challenged (day 0) by applying 1% oxazolone onto the right ear. The acetone and olive oil solvent was applied at the same time onto the left ear as a control. The thickness of the ears was measured daily with a micrometer (Mitutoyo, Kawasaki, Japan) starting immediately before challenge. The difference in ear thickness ({Delta}ET) between right and left ears was used to express the results (30). Mice were given fusion proteins or nonfused Fc (doses as indicated in Results) i.p. 1 day before and 1 and 3 days after either sensitization or challenge.

Counts and analysis of the ability to induce CH of lymph node cells from oxazolone-sensitized mice

Mice were sensitized as above and sacrificed 7 days later to collect axillary (two) and inguinal (one) lymph nodes from each body side. These lymph nodes drain the skin of the abdomen onto which oxazolone was applied. Mice were given B7RP-1-Fc or nonfused Fc (5 mg/kg) i.p. 1 day before and 1, 3, and 5 days after sensitization. Lymph nodes were gently disrupted by manual homogenization to yield a cell suspension. The total cell number was obtained with a H1E counter (Technicon, Tarrytown, NY). Percentages of cell subsets were derived by direct immunofluorescence double staining and flow cytometry using FITC- and PE-conjugated mAb (PharMingen, San Diego, CA) and a FACScan analyzer (Becton Dickinson, Mountain View, CA). Cells were either transfused i.v. from individual to individual into randomly preassigned recipients or pooled first and then transfused into recipients in equal number (5 x 107). Recipient mice were challenged with oxazolone immediately after transfusion (day 0) and monitored for {Delta}ET as above.

Induction of anti-KLH Ab

Mice were immunized on day 0 by s.c. injection of 100 µg keyhole limpet hemocyanin (KLH) (Pierce, Rockford, IL) in CFA along the flanks of the abdomen. Mice were bled to detect serum anti-KLH Ab immediately before and 7, 14, and 21 days after immunization (31). Mice were given fusion proteins or nonfused Fc (5 mg/kg) i.p. 1 day before and 1, 3, and 5 days after immunization. Anti-KLH IgM, IgG, IgG1, IgG2a, IgA, and IgE were measured in serum by ELISA. Briefly, plates were coated with KLH in PBS, blocked, and added with dilutions of test samples. Captured anti-KLH Ab were revealed using biotinylated Ab specific for Ig classes and subclasses (all from Southern Biotechnology Associates (Birmingham, AL), except anti-IgE, which was from PharMingen), neutravidin-conjugated HRP (Pierce), and TMB microwell peroxidase substrate system (Kirkegaard and Perry Laboratories, Gaithersburg, MD). Plates were also coated with standard preparations (IgG and IgM from Calbiochem, San Diego, CA), IgG1 and IgG2a from Southern Biotechnology Associates, and IgA and IgE from PharMingen) in PBS, blocked, and further processed using biotinylated Ab and other reagents as above. OD were quantitated in a Thermomax ELISA reader (Molecular Devices, Menlo Park, CA).

Analysis of proliferation and cytokine production of lymph node cells from KLH-immunized mice

Mice were immunized with KLH as above and sacrificed 7 days later to collect axillary (two) and inguinal (one) lymph nodes from each body side. These lymph nodes drain the skin of the abdomen injected with KLH. Mice were given fusion proteins or nonfused Fc (5 mg/kg) i.p. 1 day before and 1, 3, and 5 days after immunization. Lymph nodes were disrupted, and the total cell number was counted as above. Cells (105 per well) were cultured in the absence or presence of KLH (50 µg/ml) for 4 days, pulsed during the last 18 h with [3H]thymidine (Amersham, Piscataway, NJ), and harvested to count incorporated radioactivity with a 1205 Betaplate counter (Wallac, Gaithersburg, MD). Culture supernatants from four wells were pooled to measure cytokines by ELISA (Biosource International, Camarillo, CA). Cell proliferation was expressed as the stimulation index (SI), i.e., the ratio of the [3H]thymidine incorporation cpm of KLH-exposed cells to the cpm of nonexposed cells. Cell production of cytokines was also expressed as SI, i.e., the ratio of cytokine concentration in the culture supernatant of KLH-exposed cells to concentration in the supernatant of nonexposed cells.

Induction of an asthmatic reaction

Mice were sensitized by two i.p. injections, 6 days apart, of 8 µg OVA (Sigma) in PBS with alum. Twenty-one days after the first injection mice were challenged by exposure for 1 h to an aerosol of 0.5% OVA in PBS. Mice were sacrificed 3 days after challenge to collect bronchoalveolar lavage (BAL) and lungs (32). Mice were given B7RP-1-Fc or nonfused Fc (5 mg/kg) i.p. 1 day before and 1, 3, 5, and 7 days after the first injection of OVA. BAL cells were counted using a Sysmex counter (Toa Medical, Kobe, Japan) and differentiated assessing cytospin preparations stained with the Diff-Quick kit (Dade Behring, Dudingen, Switzerland). Lungs were formalin fixed, paraffin embedded, sectioned, and stained with Congo red to count eosinophils.

Counts of lymph node and spleen cells from normal mice

Mice were given B7RP-1-Fc or nonfused Fc (5 mg/kg) i.p. daily for 7 days and sacrificed 1 day after the last administration to collect the spleen and axillary (two) and inguinal (one) lymph nodes from each body side. Lymph nodes and spleen were then disrupted to yield a cell suspension. The total cell number and percentages were derived as above.

Statistical analysis

Results are expressed as mean ± SE. Comparisons were made using the ANOVA for repeated measures and the Student t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of B7RP-1-Fc, B7.2-Fc, and CTLA4-Fc on CH

Mice were challenged with oxazolone on day 0, i.e., 7 days after sensitization. Fc fusion proteins and nonfused Fc (5 mg/kg) were given near the time of sensitization (1 day before and 1 and 3 days after sensitization) or challenge (1 day before and 1 and 3 days after challenge). Given near sensitization time, B7RP-1-Fc and B7.2-Fc did not change {Delta}ET compared with nonfused Fc from day 1 to 3 but then increased it from day 4 (Fig. 1GoA). B7RP-1-Fc and B7.2-Fc reversed the decrease in {Delta}ET from day 1 peak on day 4 and 5, respectively, reactivating CH (Fig. 1GoA). B7RP-1-Fc and B7.2-Fc similarly affected {Delta}ET at all times (Fig. 1GoA). Given near sensitization time, CTLA4-Fc decreased {Delta}ET compared with Fc at all times tested (Fig. 1GoA). Given near challenge time, B7RP-1-Fc and B7.2-Fc again did not change {Delta}ET compared with Fc from day 1 to 3 but then increased it from day 4 (Fig. 1GoB). B7RP-1-Fc and B7.2-Fc again reversed the decrease in {Delta}ET from day 1 peak on day 4 and 6, respectively (Fig. 1GoB). In this case, B7RP-1-Fc increased {Delta}ET more than B7.2-Fc from day 4 (Fig. 1GoB). The {Delta}ET increase brought about by B7RP-1-Fc was 1.4 (on day 8) to 2.3 (on day 5) times larger than that brought about by B7.2-Fc. Given near challenge time, CTLA4-Fc did not significantly change {Delta}ET compared with Fc (Fig. 1GoB). Nonfused Fc did not change {Delta}ET compared with saline given near either sensitization or challenge time (Fig. 1Go).



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  		FIGURE 1. Effects of B7RP-1-Fc, B7.2-Fc, and CTLA4-Fc on CH. Mice were challenged with oxazolone on day 0, i.e., 7 days after sensitization. Fc fusion proteins (5 mg/kg) were given near the time of sensitization (A) or first challenge (B). A, Compared with Fc, B7RP-1-Fc and B7.2-Fc increased {Delta}ET from day 4 (p < 0.001). CTLA4-Fc decreased {Delta}ET at all times (p < 0.001). B, Compared with Fc, B7RP-1-Fc and B7.2-Fc increased {Delta}ET from day 4 (p < 0.001). B7RP-1-Fc increased {Delta}ET more than B7.2-Fc (p < 0.001). n = 10.

 
To study the combined effects of B7RP-1-Fc and B7.2-Fc on CH, B7RP-1-Fc and B7.2-Fc were given at two different doses either alone or in combination near the time of challenge. At high dose (2 mg/kg B7RP-1-Fc alone, 2 mg/kg B7.2-Fc alone, and 1 mg/kg of each in combination), B7RP-1-Fc, B7.2-Fc, and B7RP-1-Fc + B7.2-Fc increased {Delta}ET compared with Fc (2 mg/kg) from day 3 (Fig. 2GoA). At this dose, B7RP-1-Fc and B7RP-1-Fc + B7.2-Fc increased {Delta}ET more than B7.2-Fc from day 4 (Fig. 2GoA). B7RP-1-Fc + B7.2-Fc also increased {Delta}ET more than B7RP-1-Fc from day 4 (Fig. 2GoA). At low dose (0.4 mg/kg B7RP-1-Fc alone, 0.4 mg/kg B7.2-Fc alone, and 0.2 mg/kg of each in combination), B7RP-1-Fc and B7RP-1-Fc + B7.2-Fc increased {Delta}ET compared with Fc (0.4 mg/kg) from day 4, whereas B7.2-Fc did not significantly change {Delta}ET (Fig. 2GoB). Also at this dose, B7RP-1-Fc and B7RP-1-Fc + B7.2-Fc increased {Delta}ET more than B7.2-Fc, and B7RP-1-Fc + B7.2-Fc more than B7RP-1-Fc (Fig. 2GoB). Thus, one-half of the doses of B7RP-1-Fc alone combined with one-half of the doses of B7.2-Fc alone increased {Delta}ET on days 4 to 7 more than the entire doses of B7RP-1-Fc alone or of B7.2 alone, indicating that B7RP-1-Fc and B7.2-Fc acted additively or synergistically.



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  		FIGURE 2. Combined effects of B7RP-1-Fc and B7.2-Fc on CH. Mice were challenged with oxazolone on day 0, i.e., 7 days after sensitization. B7RP-1-Fc, B7.2-Fc, B7RP-1-Fc + B7.2-Fc, and Fc were given at high (2 or 1 + 1 mg/kg, (A)) and low (0.4 or 0.2 + 0.2 mg/kg (B)) dose near the time of challenge. A, Compared with Fc, B7RP-1-Fc B7.2-Fc, and B7RP-1-Fc + B7.2-Fc increased {Delta}ET from day 3 (p < 0.001). B7RP-1-Fc and B7RP-1-Fc + B7.2-Fc increased {Delta}ET more than B7.2-Fc from day 4 (p < 0.001). B7RP-1-Fc + B7.2-Fc increased {Delta}ET more than B7RP-1-Fc from day 4 (p < 0.001). B, Compared with Fc, B7RP-1-Fc and B7RP-1-Fc + B7.2-Fc increased {Delta}ET from day 4 (p < 0.001). B7RP-1-Fc and B7RP-1-Fc + B7.2-Fc increased {Delta}ET more than B7.2-Fc (p < 0.001).

 
Effects of B7RP-1-Fc on the cells from lymph nodes draining the skin sensitized with oxazolone

B7RP-1-Fc was given 1 day before and 1, 3, and 5 days after sensitization with oxazolone. Axillary and inguinal lymph nodes draining the sensitized skin were taken 7 days after sensitization and homogenized to prepare cells for counting. There were 1.8 times more cells in the lymph nodes from the B7RP-1-Fc-treated mice than from the controls (Table IGo). The percentages of T (CD3+) and B cells (CD3-B220+) in the lymph nodes from the B7RP-1-Fc-treated mice did not significantly differ from Fc-treated controls, but those of activated T cells (either CD3+CD25+ or CD3+CD69+) were higher than controls (Table IGo). Conversely, the percentages of nonactivated T cells (either CD3+CD25- or CD3+CD69-) were lower (Table IGo). The absolute numbers of all T and B cell subsets, however, were higher in the lymph nodes from the B7RP-1-Fc-treated mice than from the control mice (Table IGo).


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  		Table I. Effects of B7RP-1-Fc on lymph node cell counts in oxazolone-sensitized mice

 
In a second study, mice were treated with B7RP-1-Fc and sensitized with oxazolone as above. Axillary and inguinal lymph nodes draining the sensitized skin were also taken as above but this time to prepare cells for transfusion into naive mice. The cells from the donor mice were either transfused from individual to individual into randomly preassigned recipients or instead pooled and then transfused into recipients in equal number. Recipient mice were challenged with oxazolone immediately after transfusion. When cells were transfused from individual to individual, the mice that received cells from the B7RP-1-Fc-treated donors were transfused more cells and showed an increase in {Delta}ET from day 1 compared with the mice that received cells from Fc-treated donors (Fig. 3GoA). When cells were pooled and transfused in equal number, the mice that received cells from the B7RP-1-Fc-treated donors showed an increase in {Delta}ET from day 2 compared with controls (Fig. 3GoB).



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  		FIGURE 3. Effects of B7RP-1-Fc on the ability of the cells from lymph nodes draining the skin sensitized with oxazolone to induce CH. B7RP-1-Fc was given 1 day before and 1, 3, and 5 days after sensitization with oxazolone. Axillary and inguinal lymph nodes were taken 7 days after sensitization. Cells were either transfused into naive recipients from individual to individual (A) or pooled and then transfused in equal number (B). Recipients were challenged with oxazolone on day 0 immediately after transfusion. A, Mice that received cells from the B7RP-1-Fc-treated donors were transfused more cells (inset) and showed an increase in {Delta}ET from day 1 (p < 0.001) compared with those that received cells from Fc-treated donors. B, Mice that received cells from the B7RP-1-Fc-treated donors showed an increase in {Delta}ET from day 2 compared with those that received cells from Fc-treated donors (p < 0.001). n = 7. Nil indicates {Delta}ET in mock-transfused recipient mice.

 
Effects of B7RP-1-Fc, B7.2-Fc, and CTLA4-Fc on Ag-specific Ab production

Mice were immunized with KLH on day 0. Fc fusion proteins were given 1 day before and 1, 3, and 5 days after immunization. Mice treated with B7RP-1-Fc showed an increase in serum anti-KLH IgG (on day 14), IgG2a (on days 7 and 14), and IgE (on day 14) compared with control mice, which were treated with nonfused Fc (Fig. 4Go). Mice treated with B7.2-Fc did not show any significant change in anti-KLH Ab compared with controls. Compared with controls, mice treated with CTLA4-Fc showed a significant decrease in all classes and subclasses of anti-KLH Ab at nearly all time points (Fig. 4Go).



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  		FIGURE 4. Effects of B7RP-1-Fc, B7.2-Fc, and CTLA4-Fc on anti-KLH Ab production. Mice were immunized with KLH on day 0. Fc fusion proteins were given 1 day before and 1, 3, and 5 days after immunization. *, p < 0.05; **, p < 0.01; ***, p < 0.001 refer to comparisons to Fc. n = 10.

 
Effects of B7RP-1-Fc, B7.2-Fc, and CTLA4-Fc on the cells from lymph nodes draining the skin immunized with KLH

Fc fusion proteins were given as above before and after immunization with KLH. Axillary and inguinal lymph nodes draining the immunized skin were taken 7 days after immunization and homogenized to prepare cells for proliferation and cytokine production in response to KLH. The lymph nodes from the B7RP-1-Fc-treated mice contained more cells than those from nonfused Fc-treated control mice, whereas the lymph nodes from the CTLA4-Fc-treated mice contained fewer cells (Fig. 5GoA). The lymph nodes from the B7.2-Fc-treated mice contained a number of cells similar to controls. The proliferative response to KLH of the lymph node cells from the B7RP-1-Fc- and the B7.2-Fc-treated mice was not significantly different from controls, whereas the proliferative response of the lymph node cells from the CTLA4-Fc-treated mice was 5 times lower (Fig. 5GoB). The production of IL-2 in response to KLH of the lymph node cells from the B7RP-1-Fc-treated mice was also not significantly different from controls, but the production of IFN-{gamma}, IL-4, and IL-10 were 3.9-, 2.7-, and 3.1-fold higher, respectively (Fig. 5GoC).



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  		FIGURE 5. Effects of B7RP-1-Fc, B7.2-Fc, and CTLA4-Fc on the cells from lymph nodes draining the skin immunized with KLH. Mice were immunized with KLH on day 0. Fc fusion proteins were given 1 day before and 1, 3, and 5 days after immunization. Axillary and inguinal lymph nodes were taken 7 days after immunization. A, Cell numbers. p values refer to comparisons to Fc. n = 10. B, Cell proliferation in response to KLH expressed as the ratio (SI) of the [3H]thymidine incorporation cpm given by cells exposed to KLH to the cpm given by nonexposed cells. p value refers to comparison to Fc. n = 10. C, Cell production of cytokines in response to KLH expressed as the ratio (SI) of the cytokine concentration in the culture supernatant of cells exposed to KLH to the concentration in the supernatant of nonexposed cells. n = 7.

 
Effects of B7RP-1-Fc on an asthmatic reaction

Mice were sensitized with OVA twice, 6 days apart, challenged 21 days after first sensitization, and sacrificed after another 3 days to collect BAL and lungs. B7RP-1-Fc was given 1 day before and 1, 3, 5, and 7 days after first sensitization. B7RP-1-Fc-treated showed an increase in BAL eosinophils and lymphocytes, but not neutrophils and monocytes, compared with control mice, which were treated with nonfused Fc (Fig. 6GoA). Nonfused Fc did not change BAL cell counts compared with saline (Fig. 6GoA). Mice treated with CTLA4-Fc had virtually no eosinophils in the BAL (data not shown). B7RP-1-Fc-treated mice also had more eosinophils in the lungs than controls (mean (SE) 8.6 (2.5) vs 1.9 (0.6) eosinophils/microscopic field, n = 10, p < 0.02 (Fig. 6Go, B and C)).



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  		FIGURE 6. Effects of B7RP-1-Fc on the cells in the BAL and lungs of mice undergoing an asthmatic reaction. Mice were sensitized with OVA twice, 6 days apart, and challenged 21 days after first sensitization. BAL and lungs were collected 3 days after challenge. B7RP-1-Fc was given 1 day before and 1, 3, 5, and 7 days after first sensitization. A, BAL cell numbers. n = 10. Neutro, neutrophils; Mono, monocytes; Lympho, lymphocytes. B and C, Lung sections from a B7RP-1-Fc- and an Fc-treated mouse stained with Congo red. Arrows indicate eosinophils (Eosino). Bar = 50 µm.

 
Effects of B7RP-1-Fc on the cell counts of lymph nodes and spleen of normal mice

B7RP-1-Fc was given daily for 7 days. Axillary and inguinal lymph nodes and the spleen were taken to count cells the day after the last administration. The lymph nodes from the B7RP-1-Fc-treated mice contained a number of total cells not significantly different from Fc-treated mice. The percentages of T cells (CD3+), activated T cells (either CD3+CD25+ or CD3+CD69+), and B cells (CD3-B220+) did not significantly differ between B7RP-1-Fc-treated and control mice. As to nonactivated T cells, the percentage of CD3+CD25- cells was lower in the B7RP-1-Fc-treated mice than controls, although that of CD3+CD69- cells did not significantly differ between the two groups (Table IIGo). The absolute numbers of all cell subsets were not significantly different between B7RP-1-Fc-treated and control mice. Also the spleens from the B7RP-1-Fc-treated mice contained a number of total cells not significantly different from control mice. The percentages of T cells (CD3+) and CD3+ CD25- nonactivated T cells were lower in B7RP-1-Fc-treated than control mice, whereas the percentages of CD3+CD25+ activated T cells and B cells (CD3-B220+) were higher (Table IIGo). The absolute numbers of T cells and CD3+CD25- nonactivated T cells were lower in B7RP-1-Fc-treated than control mice, whereas the absolute number of CD3+CD25+ activated T cells was higher (Table IIGo). Absolute numbers of B cells and percent and absolute numbers of CD3+CD69+ and CD3+CD69- cells did not significantly differ between B7RP-1-Fc-treated and control mice. The peripheral blood cell counts and the serum levels of IgG and IgM did not significantly differ between B7RP-1-Fc-treated and control mice (data not shown).


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  		Table II. Effects of B7RP-1-Fc on lymph node and spleen cell counts in normal mice

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that B7RP-1-Fc stimulates both cellular and humoral immune responses in vivo by increasing number and function of T and B cells reacting to Ag exposure. B7RP-1-Fc stimulates primary and secondary cellular responses and primary humoral responses of either the Th1 or Th2 type. This study also illustrates the relevance of the ICOS-B7RP-1 interaction to the regulation of immunity and points to the possibility of developing B7RP-1-Fc as an immunostimulatory therapeutic.

B7RP-1-Fc stimulates CH, a prototype cellular immune response (33). B7RP-1-Fc is effective when given near either sensitization or challenge time, indicating that ICOS is involved in the regulation of both primary and secondary immune responses. Whereas CD28 is constitutively expressed by T cells (34), ICOS is expressed de novo on T cell activation (24, 35, 36), an observation that has led to the suggestion that ICOS is involved in secondary rather than primary responses (20, 24). The involvement of ICOS in the regulation of primary responses is especially illustrated in this study by the results of the experiments of cell transfusion, in which sensitization and challenge are induced in different individuals. CTLA4-Fc inhibits CH development when given near the time of sensitization and not challenge. This is consistent with the concept that CD28 has a role during primary and not secondary immune responses (37, 38, 39). The involvement of CD28 in the regulation of secondary responses is indicated in this study by the finding that B7.2-Fc treatment stimulates CH when given near challenge time. Thus, a stimulatory approach with B7.2-Fc reveals a role for CD28 unnoticed by an inhibitory one with CTLA4-Fc.

B7RP-1-Fc also stimulates the production of Ag-specific Ab, i.e., the humoral immune response. Treatment with B7RP-1-Fc at the time of immunization with KLH results in an increase in the production of anti-KLH IgG, especially IgG2a, and IgE. In contrast, treatment with B7.2-Fc does not seem to affect anti-KLH Ab production. CD28-B7 interaction is necessary for the production of Ag-specific Ab by a naive individual (26, 40, 41), consistent with the effects of CTLA4-Fc treatment on anti-KLH Ab production observed in the present study. However, it is possible that ICOS plays a more critical role than CD28 in enhancing B cell help. IgG2a production is mediated by a Th1 response (42), whereas IgE production is mediated by a Th2 response (43). The fact that B7RP-1-Fc treatment stimulates both IgG2a and IgE production indicates that costimulation through ICOS, at least at the time of primary Ag recognition, does not impart an obvious Th1-Th2 decision.

B7RP-1-Fc stimulates the development of an asthmatic reaction, consistently with the ability of B7RP-1-Fc to stimulate Ag-specific Ab production, including the production of IgE. Treatment with B7RP-1-Fc of OVA-sensitized mice results after challenge with an OVA aerosol in an increase in BAL and lung eosinophils. This confirms and expands the indication from the study by Coyle et al. (26) that the ICOS-B7RP-1 interaction is involved in the development of an asthmatic reaction.

Treatment with B7RP-1-Fc increases the absolute number of total, T, and B cells in the lymph nodes draining skin sensitized with oxazolone. B7RP-1-Fc also increases the absolute number of total cells in the lymph nodes draining skin immunized with KLH and, interestingly, the BAL lymphocytes in OVA-sensitized mice after challenge with an OVA aerosol. These observations indicate that B7RP-1-Fc may stimulate cellular and humoral responses by increasing the number of T and B cells reacting to Ag exposure. B7RP-1-Fc treatment also increases the relative numbers of activated T cells and, conversely, decreases the relative numbers of nonactivated T cells in the lymph nodes draining skin sensitized with oxazolone. B7RP-1-Fc was also found to increase cytokine production in response to KLH by cells in the lymph nodes draining skin immunized with KLH. These observations indicate that B7RP-1-Fc may stimulate cellular and humoral responses by also increasing the function of cells reacting to Ag exposure. B7RP-1-Fc concomitantly increases the production of IFN-{gamma}, IL-4, and IL-10 in response to KLH by cells in the lymph nodes draining skin immunized with KLH. Because IFN-{gamma} is a mediator of the Th1 response, whereas IL-4 and IL-10 are mediators of the Th2 response (44), this may explain why B7RP-1-Fc stimulates both IgG2a and IgE production. Interestingly, McAdam et al. (36) have reported that ICOS stimulation concomitantly enhances the production of IFN-{gamma}, IL-4, and IL-10 by CD4+ cells in vitro.

Treatment with B7RP-1-Fc of normal mice brings about nearly no change in the peripheral lymph nodes and blood. This indicates that treatment with B7RP-1-Fc is much more effective when given in conjunction with an Ag than alone, consistently with the costimulatory role of ICOS (23). B7RP-1-Fc treatment of normal mice, however, brings about some changes in the spleen, likely because of the i.p. administration. A decrease in the relative number of T cells and an increase in that of B cells were observed, possibly an indication that ICOS signals in favor of B cell help.

The effects of B7RP-1-Fc on immune responses indicate that this molecule may be developed as an immunostimulatory therapeutic. Soluble proteins including the extracellular regions of B7.1 or B7.2 have been found to be very active in stimulating immunity and treating tumors in mice (45, 46). Interestingly, this study shows that B7RP-1-Fc and B7.2-Fc may have additive or synergistic immunostimulatory effects that could be exploited to elaborate new forms of adjuvant intervention.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. G. Senaldi, Amgen Inc., Amgen Center M/S 15-2-B, 1 Amgen Center Drive, Thousand Oaks, CA 91320. Back

2 Abbreviations used in this paper: ICOS, inducible costimulator; B7RP-1, B7 related protein-1; CH, contact hypersensitivity; {Delta}ET, difference in ear thickness; KLH, keyhole limpet hemocyanin; SI, stimulation index; BAL; bronchoalveolar lavage. Back

Received for publication November 16, 2000. Accepted for publication February 20, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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