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The Journal of Immunology, 2001, 166: 4473-4480.
Copyright © 2001 by The American Association of Immunologists

GIF Inhibits Th Effector Generation by Acting on Antigen-Presenting B Cells1 ,2

Katsuji Sugie3,* and Jianyong Huang{dagger}

Divisions of * Allergy and {dagger} Immunochemistry, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glycosylation-inhibiting factor (GIF) is a 13-kDa cytokine secreted from T cells. Administration of bioactive recombinant GIF inhibits IgG1 and IgE Ab responses in vivo. Treatment of B cells with the cytokine reduces the secretion of IgG1 and IgE induced by LPS and IL-4. To examine the effect on cognate T-B interaction, GIF was added to low-density B cells from MD4 transgenic (Tg) mice, which express B cell receptor specific for hen egg lysozyme (HEL). The B cells were subsequently pulsed with HEL-OVA conjugate and cultured with OVA-specific naive CD4 T cells from DO11.10 Tg mice. Treatment of Ag-presenting B cells with GIF reduced expansion and IL-2 secretion of naive T cells and rendered them hyporesponsive to antigenic restimulation, resulting in 50–95% reduction of IL-4 and IFN-{gamma} secretion upon restimulation with Ag. GIF dramatically inhibited Th effector generation when it was added to B cells before pulsing with HEL-OVA, whereas it showed little to no effect when added after B cells were pulsed with Ag. GIF was more effective when B cells from MD4 Tg mice were pulsed with HEL-OVA than when they were pulsed with OVA. This cytokine did not affect Th effector generation when B cells or irradiated splenocytes pulsed with OVA323–339 peptide stimulated naive DO11.10 T cells. Confocal microscopy revealed that GIF inhibited internalization of HEL by B cells from MD4 Tg mice. Therefore, the cytokine may regulate early steps of Ag presentation involving B cell receptors to diminish Th effector generation from naive CD4 T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The mechanisms by which a cytokine regulates the humoral immune response involve differentiation and expansion of Th effector cells and Ab-secreting B cells. In terms of Th differentiation, IL-12 and IFN-{gamma} amplify Th1 development and inhibit generation of Th2 effector cells. IL-4 has an opposite effect (1, 2). Besides determining the differentiation in this Th1/Th2 dichotomy, some cytokines are known to reduce the responsiveness of T cells to Ag. For instance, experiments using TCR-transgenic (TCR-Tg) mice demonstrated that IFN-{gamma} (3), IL-10, and TGF-{beta} (4) are involved in inducing anergy in peripheral CD4 T cells. However, these reports unanimously acknowledged that the cytokines listed above are not likely to be entirely responsible for regulating the T cell responsiveness to Ag and that some other cytokine(s) may be involved in this phenomenon.

Glycosylation-inhibiting factor (GIF)4 was first documented almost 20 years ago as a T cell-derived cytokine that inhibits IgE and IgG1 Ab formation (5). The molecular structure of this cytokine was elucidated (6, 7). It was recently found that T cell-secreted GIF that can inhibit IgE Ab formation is cysteinylated at Cys-60 (8, 9). Significantly, not only the bioactivity of GIF (8) but also its capability to bind to the receptors on target cells (10) is dependent on the posttranslational modification of Cys-60. However, the original hypothesis on the mechanism by which it inhibits the Ab response (11) has been quite controversial. Therefore, a new experimental approach was initiated by delineating target cells for GIF by using Cys-60-modified recombinant human GIF (rhGIF). Among normal lymphoid cells, activated T and B cells expressed 1000–5000 sites per cell of the high-affinity GIF receptors, whereas resting T and B cells, macrophages, and dendritic cells did not express detectable levels (>50 sites per cell) of the receptors (10, 12). Freshly isolated NK T cells expressed GIF receptors, whereas fresh conventional NK cells did not (12).

Functional studies have been initiated by using one of the potential target cells for GIF, i.e., B cells. Cys-60-modified rhGIF inhibited the secretion of IgG1 and IgE from purified B cells stimulated with LPS and IL-4, whereas it did not affect IgM secretion (12). In addition to Ab synthesis, B cells play a regulatory role in the humoral immune response by presenting Ag to CD4 T cells, which in turn provide help as Th effector cells in the formation of Ab by the B cells (13). The importance of B cells as APCs in the Th2 type immune response has been debated for a number of years. Recent experiments using the MD4 Tg mice that express the B cell Ag receptor (BCR) specific for hen egg lysozyme (HEL) (14) demonstrated that Ag-specific B cells are more efficient than splenic adherent cells in inducing Th2 effector cells (15). Consistent with this finding, it was subsequently reported that Ag-primed T cells from B cell-deficient JHD mice (16) fail to provide help for B cell Ig switch to IgG1 (17). These observations point to the possibility that B cell Ag presentation is also involved in the mechanism by which GIF inhibits IgE and IgG1 Ab formation. Present experiments were undertaken to examine the effect of GIF on the generation of Ag-specific Th effector cells by using B cells as APCs. Evidence was obtained that treatment of Ag-presenting B cells with GIF inhibits the generation of Th effector cells from naive T cells. B cells stand out by being extremely efficient in taking up specific Ag through BCR as compared with nonspecific pinocytosis (18). Functional experiments and microscopic observations demonstrate that GIF inhibits early steps of Ag presentation by B cells including Ag uptake through BCR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

DO11.10 Tg mice, which express a TCR specific for OVA (19), were provided by K. M. Murphy (Washington University, St. Louis, MO). MD4 Tg mice, which express IgM and IgD specific for HEL (14), were provided by D. T. Umetsu (Stanford University, Stanford, CA) with permission from C. C. Goodnow (Australian National University, Canberra, Australia). BALB/cByJ and C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). DO11.10 mice on a (BALB/c x C57BL/6)F1 genetic background were generated by crossing DO11.10 (BALB/c genetic background) and C57BL/6J mice. Likewise, MD4 Tg (BALB/c x C57BL/6)F1 mice were generated by crossing MD4 (C57BL/6 background) and BALB/cByJ mice. DO11.10 Tg mice were screened by staining PBLs with the KJ1-26 anti-clonotypic Ab (obtained from J. Kappler, National Jewish Medical and Research Center, Denver, CO) and analyzing them by flow cytometry on a FACSCalibur (BD Biosciences, Mountain View, CA). Forty to 60% of T cells from DO11.10 Tg (BALB/c x C57BL/6)F1 mice were KJ1-26+. MD4 Tg mice were screened by measuring serum anti-HEL IgM Ab by ELISA, as reported previously (15). Flow cytometry demonstrated that 60–90% of B cells from MD4 Tg (BALB/c x C57BL/6)F1 mice bound biotinylated HEL.

Cytokines and Ags

Wild-type and C57A rhGIF were provided by Y. Ishii at Kirin Pharmaceutical Laboratory, Takasaki, Japan. C57A rhGIF was modified with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), as described previously (10). C57A rhGIF that is cysteinylated at Cys-60 (C57A/C60-Cys) was also provided by Y. Ishii. Recombinant mouse IL-12 and IL-4 were purchased from Genzyme (Cambridge, MA). OVA323–339 peptide was synthesized in the peptide facility at La Jolla Institute for Allergy and Immunology (San Diego, CA), as described (20). HEL and OVA were purchased from Sigma (St. Louis, MO) and ICN Pharmaceuticals (Costa Mesa, CA), respectively. HEL-OVA conjugate was prepared as previously reported (15).

T cells

CD4+ T cells were purified from spleens and lymph nodes of DO11.10 Tg (BALB/c x C57BL/6)F1 mice, using the method described previously (21). Briefly, cells were incubated with mAbs to CD8 (3.155), heat-stable Ag (J11D), class II MHC (M5/114 and CA-4.A12), CD45R/B220 (RA3.6B2), CD11b (M1/70), NK1.1 (PK136), and CD11c (33D1), cross-linked with mouse anti-rat {kappa} (MAR 18.5), and treated with complement. All of the Abs were provided by P. R. Rogers and M. Croft (La Jolla Institute for Allergy and Immunology). Residual accessory cells and any in vivo-activated T cells were removed by isolating high-density cells from Percoll (Sigma) gradient (45, 53, 62, 80%) centrifugation. The resultant cells were >95% CD4+, and >95% of the CD4 cells possessed a naive phenotype (CD45RB+, CD62high, CD44low).

APCs

To isolate B cells from MD4 Tg (BALB/c x C57BL/6)F1 mice, total splenocytes were centrifuged through a Percoll gradient (45, 53, 62, 80%). Low- and high-density lymphocytes were isolated from 53/62% and 62/80% interface, respectively. B cells were purified from each fraction by depleting CD43+ cells using anti-CD43 MicroBeads (Miltenyi Biotec, Auburn, CA), as described (22). The resultant cells were >98% B220+. Before incubation with T cells, B cells were treated with 50 µg/ml of mitomycin C (Sigma) for 40 min at 37°C and extensively washed. B cells were not treated with mitomycin C when they were used for fluorescence microscopy. As another source of APCs, spleen cells were obtained from (BALB/c x C57BL/6)F1 mice and irradiated at 3000 rad.

Cell cultures

Cells were cultured in RPMI 1640 (Irvine Scientific, Santa Ana, CA) supplemented with 2 mM L-glutamine, 50 µM 2-ME, nonessential amino acids (Life Technologies/BRL, Gaithersburg, MD), 1 mM sodium pyruvate, 10% FCS (Harlan Bioproducts for Science, Indianapolis, IN), and antibiotics. B cells (2.5 x 105) or 5.0 x 106 irradiated spleen cells were pulsed with varying concentrations of Ag for 2 h at 37°C, washed two times, and added to 1.5 x 105 naive CD4 T cells. Cells were cultured in 2 ml/sample in 24-well plates (Falcon, Franklin Lakes, NJ) for 6 days, after which T cells were recovered and washed three times. All the T cells that were recovered were replated without readjustment for cell number in 24-well plates with 5.0 x 106 irradiated spleen cells and 0.6 µM OVA323–339 peptide in 2 ml. Supernatants were collected between 24 and 48 h of culture for cytokine analyses.

Cytokine secretion

IL-2, IL-4, and IFN-{gamma} were measured by sandwich ELISA using combinations of Abs; JES6-1A12 and biotinylated JES6-5H4, BVD4-1D11 and biotinylated BVD6-24G2, and R4-6A2 and biotinylated XMG1.2, respectively. All the Abs for ELISA were purchased from PharMingen (San Diego, CA). Standard mouse recombinant IL-2 was purchased from PharMingen and mouse recombinant IL-4 and IFN-{gamma} from Genzyme. The levels of detection were 31 pg/ml for IL-2, 20 pg/ml for IL-4, and 14 pg/ml for IFN-{gamma}.

Proliferation

Ag-pulsed low-density B cells from MD4 mice were plated at 2.5 x 104 cells per well with 2 times as many naive CD4 T cells from DO11.10 mice in 0.2 ml per well in 96-well flat-bottom plates (Falcon). At 80 h of culture, wells were labeled with 1 µCi tritiated thymidine (ICN) and harvested 12 h later.

Fluorescence microscopy

HEL and OVA were biotinylated with N-hydroxysuccinimidyl biotin (Vector Laboratories, Burlingame, CA) in 0.1 M NaHCO3 and 1 mM EDTA. After extensive dialysis against PBS containing 1 mM EDTA, aggregates were removed by filtration through a 0.45-µm filter.

Confocal microscopy was performed as previously described (23, 24). Briefly, B cells from MD4 Tg mice were placed on glass slides (Superfrost Plus; Fisher, Pittsburgh, PA) and fixed in 3% paraformaldehyde for 10 min. Cells were permeabilized with 0.2% Triton X-100 (Sigma) for 2 min, washed, and incubated with Texas Red-conjugated streptavidin (Molecular Probes, Eugene, OR). After washing, the cells were mounted in FluoroGuard antifade reagent (Bio-Rad, Hercules, CA) and examined using an Eclipse TE300 microscope (Nikon, Tokyo, Japan) coupled to a MRC-1024 laser scanning confocal imaging system (Bio-Rad). At least 200 cells per sample were analyzed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GIF inhibits Th effector generation from naive CD4 T cells

Recent reports indicated that the interaction of Ag-presenting B cells and CD4 T cells plays an important role in the differentiation and expansion of Th2 effector cells (15, 17). In those experiments, a model Ag of HEL-OVA conjugate was added to HEL-specific B cells freshly isolated from MD4 Tg mice. The Ag was efficiently taken up through BCR for HEL and presented to OVA-specific T cells (15). The present work used this experimental system to determine whether GIF regulates the generation of Th effectors from naive T cells.

B cells from MD4 mice were incubated at 37°C with Cys-modified (C57A/C60-DTNB) rhGIF for 2 h, pulsed with HEL-OVA or OVA, washed, and added to naive CD4 T cells from DO11.10 Tg mice. Additional GIF was added to the culture containing T and B cells. After 6 days of culture, generation of Th effectors from naive T cells was determined by assessing cytokine secretion (IL-4 and IFN-{gamma}) by T cells following their restimulation with OVA323–339 peptide in the presence of splenic APCs. When low-density B cells from MD4 mice were used to present Ag to naive T cells, 1 µg/ml of HEL-OVA was sufficient to generate Th effector cells capable of secreting high amounts of IL-4 and IFN-{gamma} (Fig. 1Go, left panels), whereas 1000 µg/ml of OVA was required to achieve secretion of cytokines equivalent to that induced by 1–10 µg/ml of HEL-OVA (Fig. 1Go, right panels and data not shown). In contrast to low-density B cells, high-density B cells were unable to activate naive CD4 T cells even when >10 µg/ml of HEL-OVA or 1 mg/ml of OVA was used. This is consistent with the earlier finding by Grey and Chesnut that B cells isolated from unimmunized mice are heterogeneous in size and that the low-density fraction of fresh B cells is capable of efficiently presenting Ag (25).



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  		FIGURE 1. GIF inhibits the generation of Th1 and Th2 effector cells. Low-density B cells purified from MD4 Tg mice were incubated for 2 h with or without 500 ng/ml C57A/C60-DTNB rhGIF, pulsed with varying doses of HEL-OVA or OVA, and washed. The B cells were then added to naive CD4 cells from DO11.10 Tg mice. GIF was also added at the initiation of culture at 500 ng/ml. After 6 days, all of the T cells that were recovered were washed and restimulated with 0.6 µM OVA323–339 peptide in the presence of irradiated spleen cells. Supernatants were recovered 48 h later and assayed for cytokine content. The levels of cytokines are plotted on a logarithmic scale. *, Cytokines undetectable (IL-4 <20 pg/ml; IFN-{gamma} <14 pg/ml).

 
When low-density B cells were pulsed with HEL-OVA, GIF dramatically reduced the secretion of cytokines upon restimulation of T cells. In the experiment shown in Fig. 1Go, there was a >90% reduction of both IL-4 and IFN-{gamma}. In contrast to HEL-OVA-induced activation of T cells, when unconjugated OVA was used as Ag, GIF did not inhibit the secretion of cytokines in the experiment of Fig. 1Go (right panels). To assess the validity of this result, data of 10 independent experiments conducted in the same design as Fig. 1Go are summarized in Table IGo. There was considerable variation in the amounts of IL-4 and IFN-{gamma} secreted from effector T cells. However, levels of IL-4 and IFN-{gamma} in the group treated with GIF were significantly lower than those in the untreated group (p < 0.0001 for IL-4 and p = 0.0015 for IFN-{gamma}) when Ag-presenting B cells were pulsed with HEL-OVA. Therefore, GIF seems to consistently inhibit the generation of Th effectors irrespective of the absolute amounts of cytokines. The secretion of IL-4 and IFN-{gamma} was reduced to a similar extent (79.0 ± 19.2 vs 83.9 ± 18.0%). In contrast, when unconjugated OVA was used as Ag, levels of cytokines were not significantly different between the groups treated with and without GIF (p = 0.4848 for IL-4 and p = 0.3939 for IFN-{gamma}). This result suggests that GIF preferentially inhibits Ag presentation mediated by HEL-specific BCR expressed on B cells from MD4 Tg mice.


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  		Table I. Inhibition of Th effector generation by GIF1

 
GIF acts mainly on B cells

In the experiments shown in Fig. 1Go and Table IGo, GIF was added two times, i.e., before B cells were pulsed with Ag and after Ag-pulsed B cells were mixed with T cells. To determine the optimum timing of treatment with GIF, the following experiment was performed. In one group, MD4 B cells were first incubated with GIF at 37°C for 4 h, with Ag being present for the last 2 h. Cells were then washed and added to DO11.10 T cells. In another group, MD4 B cells were first pulsed with Ag, washed, and added to DO11.10 T cells together with GIF. The cells were then cultured and restimulated with Ag as above. When GIF was present before and during the time that the B cells were pulsed with Ag, secretion of both IL-4 and IFN-{gamma} was inhibited by >90% (Fig. 2GoA). However, when GIF was added to T and B cells after the Ag pulse, secretion of IL-4 was not inhibited at all, and that of IFN-{gamma} was only modestly inhibited as compared with the extent of inhibition seen when GIF was added to B cells before Ag. This result suggests that GIF acts mainly on B cells rather than naive T cells.



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  		FIGURE 2. GIF acts mainly on B cells. A, In the group represented by shaded columns, low-density MD4 B cells were incubated with 500 ng/ml C57A/C60-DTNB rhGIF for 4 h, with HEL-OVA being present for the last 2 h. B cells were then washed and added to DO11.10 T cells. In the group represented by hatched columns, B cells were incubated with HEL-OVA for 2 h, washed, and added to T cells with 500 ng/ml C57A/C60-DTNB rhGIF. In the group represented by filled columns, B cells were incubated with HEL-OVA for 2 h, washed, and added to T cells without GIF. After 6 days of culture, all of the T cells that were recovered were washed and restimulated with 0.6 µM OVA323–339 peptide in the presence of irradiated spleen cells. Supernatants were recovered 48 h later and assayed for cytokine content. Levels of cytokines are plotted on a logarithmic scale. Similar results were seen in two other experiments of the same design. B, Irradiated spleen cells were pulsed with 0.3 µM OVA323–339 peptide and added to DO11.10 T cells with no cytokine (filled columns), 500 ng/ml C57A/C60-DTNB rhGIF (hatched columns), or 2 ng/ml recombinant mouse IL-12 (shaded columns). After 6 days of culture, T cells were restimulated with 0.6 µM OVA323–339 peptide in the presence of irradiated spleen cells. Levels of secreted IL-4 and IFN-{gamma} at 48 h of culture were determined and plotted on a logarithmic scale. Similar results were obtained in three other experiments of the same design.

 
Experiments were performed to examine the possibility that GIF directly acts on naive T cells. It has been established that cytokines including IL-4 and IL-12 directly regulate naive T cells to polarize Th differentiation when peptide Ags were presented by irradiated spleen cells (1, 2). Thus, naive T cells from DO11.10 Tg mice were stimulated in the presence or absence of GIF with irradiated spleen cells pulsed with OVA323–339 peptide. In contrast to the experiments using B cell APCs and protein Ags, GIF showed no effect on the generation of Th effectors from naive T cells, whereas IL-12 induced Th1 and suppressed Th2 differentiation (Fig. 2GoB). Therefore, it is unlikely that GIF acts directly on naive T cells. In addition, the results shown in Fig. 2Go, A and B, provide strong evidence that the inhibition of Th effector generation was not due to a nonspecific toxic effect because GIF had little to no effect when present during the 6-day culture period of T cells.

Cys-modification of rhGIF is required for inhibition of B cell Ag presentation

GIF contained in the cytosol of T cells is unmodified, whereas GIF secreted from T cells is cysteinylated at Cys-60 (9). Previous experiments indicated that unmodified wild-type rhGIF cannot bind to GIF receptors on cells and that Cys modification of rhGIF enables the cytokine to bind to the receptors (10). The capability to bind to target cells appears to be relevant to the function of GIF because Cys-modified rhGIF inhibited IgG1 and IgE Ab responses in vivo, whereas unmodified rhGIF did not (8). The requirement for the Cys-60 modification was recapitulated in vitro in inhibiting secretion of IgG1 and IgE induced by LPS and IL-4 (12).

To examine whether Cys-modification is important for inhibition of Ag presentation, B cell APCs from MD4 mice were treated with various derivatives of rhGIF. Unmodified wild-type rhGIF had no effect on cytokine secretion, whereas Cys-60-modified rhGIF (C57A/C60-DTNB) inhibited the secretion of both IL-4 and IFN-{gamma} by ~60% (Fig. 3GoA). Next, a derivative of rhGIF that was cysteinylated at Cys-60 (C57A/C60-Cys) was compared with C57A/C60-DTNB rhGIF. As shown in Fig. 3GoB, both of Cys-60-modified rhGIF inhibited the secretion of cytokines to a similar extent. Fig. 3GoC shows that inhibition of IL-4 required considerably less GIF than inhibition of IFN-{gamma}; >50% inhibition of IL-4 secretion was observed when >=4 ng/ml C57A/C60-Cys rhGIF was added to B cells, whereas >50% inhibition of IFN-{gamma} required >=100 ng/ml of GIF.



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  		FIGURE 3. Modification of Cys-60 is required for GIF to inhibit the generation of Th effector cells. A, Unmodified wild-type (WT) or C57A/C60-DTNB rhGIF was added to low-density MD4 B cells at 500 ng/ml. After 2 h of incubation, B cells were pulsed with 1 µg/ml HEL-OVA and added to DO11.10 T cells. Cells were cultured as in Fig. 1Go, and secretion of cytokines upon antigenic restimulation was measured. B, C57A/C60-DTNB or C57A/C60-Cys rhGIF was added to low-density MD4 B cells at 500 ng/ml. After a 2-h incubation, B cells were pulsed with 1 µg/ml HEL-OVA and added to T cells as in A. Cytokine secretion following antigenic restimulation was measured. C, B cells were incubated for 2 h with varying concentrations of C57A/C60-Cys rhGIF, pulsed with 1 µg/ml HEL-OVA, and added to T cells. The levels of cytokines secreted after restimulation are plotted on a linear scale. The results in A through C are representative of three experiments performed.

 
GIF inhibits both growth and differentiation of naive CD4 T cells

The decrease of Th effector generation raised several possibilities as to the mechanism of action of GIF. First, inhibition of T cell growth in the primary stimulation could contribute to the diminished cytokine secretion following secondary stimulation because cytokines per culture were measured uncorrected for the cell recovery at the time of restimulation. Second, T cell death after the primary stimulation could be enhanced by treatment of APCs with GIF. Third, treatment of APCs with GIF could inhibit the differentiation of naive T cells to effector Th cells.

To address the first possibility, the effect of GIF on the proliferative response of naive CD4 T cells to HEL-OVA was determined (Fig. 4GoA). Without GIF, the proliferation peaked at 0.1 µg/ml of HEL-OVA. Treatment of MD4 B cells with GIF shifted the peak to >=1 µg/ml and diminished [3H]thymidine incorporation at 0.1 µg/ml of HEL-OVA by 60%. IL-2, a major cytokine secreted from naive T cells, is critical for their proliferation. Treatment with GIF reduced by 50% the secretion of IL-2 induced by 1 µg/ml HEL-OVA as determined at 33 h of culture (Fig. 4GoB). As expected, unmodified rhGIF had no effect on the secretion of IL-2 by naive T cells. To evaluate the effect of GIF on T cell proliferation and death, cell recovery on day 6 was determined by a trypan blue dye exclusion test. Treatment of B cells with GIF diminished the number of viable cells recovered on day 6 by 90% (Fig. 4GoC).



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  		FIGURE 4. Treatment of B cells with GIF inhibits expansion of naive T cells and renders them hyporesponsive to antigenic restimulation. A, Low-density MD4 B cells were incubated with 500 ng/ml C57A/C60-DTNB rhGIF for 2 h, pulsed with varying concentrations of HEL-OVA for 2 h, washed, and added to DO11.10 T cells. Cultures were pulsed with [3H]thymidine from 80 to 92 h. Similar results were obtained in three independent experiments. B, Low-density MD4 B cells were incubated for 2 h with 500 ng/ml of either wild-type or C57A/C60-DTNB rhGIF, pulsed with 1 µg/ml of HEL-OVA, washed, and added to DO11.10 T cells. After 33 h of culture, supernatants were recovered, and secretion of IL-2 was determined by ELISA. Similar results were obtained in three independent experiments. C, Low-density MD4 B cells were incubated for 2 h with 500 ng/ml C57A/C60-DTNB, pulsed with 1 µg/ml of HEL-OVA, washed, and added to DO11.10 T cells. After 6 days of culture, numbers of T cells were counted. All of the T cells that were recovered were washed and restimulated with 0.6 µM OVA323–339 peptide, and secretion of cytokines was measured at 48 h. Upper graphs, Levels of IL-4 and IFN-{gamma} per culture. Lower graphs, Concentrations of cytokines per 1.0 x 105 T cells that were restimulated. Similar results were obtained in three independent experiments.

 
To determine the effect of GIF on the differentiation of T cells into effectors, the secretion of cytokines was normalized to the number of viable cells restimulated on day 6. Total secretion of IL-4 and IFN-{gamma} following restimulation of T cells was reduced by 95% when MD4 B cells were treated with GIF (Fig. 4GoC, upper panels). When normalized for the number of viable cells per culture, it was found that GIF still inhibited both IL-4 and IFN-{gamma} secretion by ~70% (Fig. 4GoC, lower panels). These data indicate that treatment of B cell APCs with GIF inhibits the expansion of T cells during the initial culture period and the differentiation of naive T cells to Th effector cells.

GIF does not inhibit B cell presentation of peptide Ag

GIF appears to be more effective in inhibiting Ag presentation when B cells take up Ag preferentially through BCR than via nonspecific pinocytosis (Fig. 1Go and Table IGo). To gain more insight into the site of action of GIF, its effect on presentation of the peptide epitope recognized by the DO11.10 TCR, i.e., OVA323–339, was determined. Low-density B cells from MD4 mice were preincubated with GIF for 2 h and then pulsed with either HEL-OVA conjugate or OVA323–339 peptide for a subsequent 2 h. Cells were then washed and added to naive T cells from DO11.10 mice. On day 6 of culture, T cells were restimulated with Ag, and cytokine secretion measured. Greater than or equal to 10 µM OVA323–339 peptide was required to achieve secretion of cytokines comparable with that induced by 1 µg/ml HEL-OVA conjugate (Fig. 5Go). When MD4 B cells were pulsed with HEL-OVA, GIF inhibited the secretion of both IL-4 and IFN-{gamma} by 60%. In contrast, GIF did not affect the secretion of cytokines when B cells were pulsed with OVA323–339 peptide. This result suggests that GIF inhibits early steps of Ag presentation mediated by BCR, but fails to inhibit presentation of a peptide that does not require processing for the interaction with MHC and its subsequent recognition by T cells.



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  		FIGURE 5. GIF does not affect B cell presentation of processed Ag. Low-density MD4 B cells were incubated with or without 500 ng/ml C57A/C60-DTNB rhGIF for 2 h, followed by addition of either 1 µg/ml HEL-OVA or 10 µM OVA323–339 peptide and incubation for a subsequent 2 h. Cells were washed and added to naive DO11.10 T cells. After 6 days of culture, T cells were recovered and restimulated. Levels of secreted cytokines at 24 h were measured. Similar data were obtained in three independent experiments.

 
GIF inhibits internalization of Ag through BCR

To directly determine whether GIF inhibits Ag uptake by B cells, fluorescence microscopy was performed. HEL bound to MD4 B cells was clearly detectable when the cells were incubated with >=0.25 µg/ml of biotinylated HEL. In contrast, OVA was undetectable even when up to 250 µg/ml of biotinylated OVA was incubated with MD4 B cells (data not shown). To safely assume that Ag uptake through BCR is observed, 1 µg/ml biotinylated HEL was added to B cells from MD4 Tg mice. At 10 min of incubation at 37°C, HEL Ag was uniformly distributed on the surface of B cells (Fig. 6Goa). At 60 min, the Ag was aggregated on the cell surface, and in 29 ± 2% of B cells it was also found inside the cell (Fig. 6Gob). At 120 min, 83 ± 5% of B cells contained the Ag inside the cell (Fig. 6Goc). When GIF was added to MD4 B cells 2 h before Ag, the Ag was homogeneously seen on the cell surface at 10 min (Fig. 6God). At 60 min, HEL Ag was condensed on the cell surface. However, no cell internalized Ag at this time point when B cells were treated with GIF (Fig. 6Goe). At 120 min, 26 ± 4% of B cells that were treated with GIF internalized the Ag, but the remainder of MD4 B cells retained the Ag condensed on the cell surface (Fig. 6Gof). Therefore, treatment of B cells with GIF diminished the uptake of Ag through BCR.



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  		FIGURE 6. GIF inhibits internalization of HEL Ag through BCR. Low-density MD4 B cells were incubated with (d–f) or without (a–c) 500 ng/ml C57A/C60-DTNB rhGIF for 2 h at 37°C. Then, biotinylated HEL was added at 1 µg/ml, and cells were further incubated at the same temperature for 10 (a and d), 60 (b and e), or 120 min (c and f). Cells were recovered, fixed, permeabilized, and stained with Texas Red-conjugated streptavidin. Representative cells of each sample are illustrated. See Results for percentage of cells that internalized HEL. Similar results were obtained in two other experiments.

 
GIF inhibits B cell Ag presentation to Th effectors to a limited extent

Ag uptake by BCR is associated with signal transduction, which induces expression of costimulatory molecules on B cells. The finding that treatment of Ag-presenting B cells with GIF inhibited proliferation (Fig. 4GoA) and IL-2 secretion (Fig. 4GoB) of naive T cells suggests two possibilities: 1) GIF reduces the amount of Ag presented on class II MHC molecules on B cells; and 2) GIF diminishes signal transduction through BCR, resulting in a reduced induction of costimulatory molecules on B cells. To address these possibilities, the ability of GIF to modulate B cell Ag presentation to effector Th cells was examined because activation of Th effector cells, especially secretion of IL-4 from Th2 effectors, is relatively independent of costimulation (26, 27). Th2 effectors were generated in vitro from naive CD4 T cells of DO11.10 Tg mice in the presence of rIL-4. Low-density B cells from MD4 Tg mice were treated with or without GIF, pulsed with HEL-OVA, washed, and added to effector Th2 cells. Fig. 7Go summarizes the results of three independent experiments. Pretreatment of B cells with GIF reduced the secretion of IFN-{gamma} from T cells by 71% (p = 0.011). However, GIF inhibited the secretion of IL-4 only by 30% (p = 0.117). Relative ineffectiveness of GIF to inhibit IL-4 secretion from Th2 effectors suggests that this cytokine may rely on inhibition of BCR-mediated signaling rather than reduction of antigenic peptide/MHC class II complex to inhibit the expansion and differentiation of naive T cells.



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  		FIGURE 7. GIF inhibits B cell Ag presentation to Th effectors to a limited extent. Th2 effector cells, generated by culturing naive CD4 T cells of DO11.10 Tg mice with 0.6 µM OVA323–339 peptide and irradiated splenocytes for 6 days in the presence of 10 ng/ml rIL-4, were washed and plated at 1.5 x 105 cells per well. Mitomycin C-treated, low-density B cells from MD4 Tg mice were incubated with or without 500 ng/ml of C57A/C60-DTNB rhGIF for 2 h at 37°C followed by the addition of 1 µg/ml HEL-OVA and incubation for a subsequent 2 h. B cells were then washed two times and added to Th2 effector cells at 3.0 x 105 cells per well. At 24 and 36 h of cell culture, supernatants were collected and assayed for IL-4 and IFN-{gamma}, respectively. Each point represents the mean ± SE of three independent experiments (treated vs untreated with GIF; *, p = 0.117; **, p = 0.011 as determined by paired Student’s t test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recent experiments showed that GIF reduced the secretion of IgG1 and IgE from purified B cells induced by LPS and IL-4, with the secretion of IgM unaffected (12). The present study demonstrates that this cytokine also regulates B cells in their function as APCs in cognate T-B interaction, resulting in reduced expansion of naive CD4 T cells accompanied by diminished differentiation to Th effector cells. GIF inhibited BCR-dependent Ag presentation to naive CD4 T cells but failed to affect BCR-independent presentation, suggesting that the cytokine may act on BCR-associated events of Ag presentation. GIF inhibited internalization of Ag through BCR. However, B cell Ag presentation to Th2 effector cells was less sensitive to GIF than that to naive T cells, suggesting that the reduction of antigenic peptide/MHC class II complex may not solely be the mechanism by which the cytokine regulates APC function of B cells. Thus it may be possible that inhibition of expression and/or function of costimulatory molecules is involved in the regulation of naive CD4 T cells by this cytokine.

Unlike cytokines that determine the differentiation of Th effectors, e.g., IL-4 and IL-12, GIF had no effect on the generation of Th effectors when it was added to naive T cells from TCR-Tg mice with irradiated spleen cells pulsed with an Ag peptide (Fig. 2GoB). Therefore, it seemed unlikely that GIF directly regulates naive T cells in their expansion or differentiation into effector Th cells. Recent reports indicated that Ag presentation by B cells is important for Th2 effector generation (15, 17). When B cell APCs and a protein Ag specifically recognized by the B cells were used, GIF dramatically inhibited the generation of Th effectors (Fig. 1Go and Table IGo).

Because it had been unknown whether GIF directly regulates T cells or indirectly does so through acting on B cells, the cytokine was added two times, i.e., before B cells were pulsed with Ag and after Ag-pulsed B cells were mixed with T cells. The issue of whether it acts on T or B cells was addressed as follows. First, the optimum timing of adding GIF to the culture was before, but not after, B cells were pulsed with Ag (Fig. 2GoA). Second, as stated above, GIF showed no effect on Th effector generation when irradiated spleen cells pulsed with OVA323–339 peptide were used to stimulate naive DO11.10 T cells (Fig. 2GoB). Third, GIF was more efficient in inhibiting Th effector generation when protein Ag specifically recognized by BCR was used than when protein Ag irrelevant to BCR (Table IGo) or peptide Ag (Fig. 5Go) was used. These results collectively support the notion that GIF acts on B cells rather than naive T cells.

Although GIF regulates APC function of B cells, generation of Th effectors is a relatively distal readout of Ag presentation. The amounts of IL-4 and IFN-{gamma} secreted from Th effectors were inevitably quite variable. Nevertheless, GIF treatment of Ag-presenting B cells in the stimulation of naive T cells consistently inhibited the secretion of cytokines from Th effectors in 10 independent experiments of the same design (Table IGo). Accordingly, it is reasonable to conclude that GIF inhibits Th effector generation from naive T cells.

B cells take up Ag via two pathways, i.e., BCR-mediated and nonspecific pinocytosis. Low-density B cells isolated from MD4 Tg mice induced Th effector cells when pulsed with 1 µg/ml HEL-OVA, whereas two to three orders of magnitude higher concentrations of OVA was required to achieve equivalent stimulation of T cells (Fig. 1Go). This dose response is in line with that for proliferation of naive T cells induced by HEL-OVA and OVA (15), quantitatively reflecting the efficiencies of taking up Ag through Ag-specific BCR and nonspecific pinocytosis (18). In repeated experiments, GIF was significantly more effective in reducing the generation of Th effectors when HEL-OVA was added to HEL-specific B cells than when OVA was added to the same cells (Table IGo). Therefore, the cytokine seems to preferentially inhibit BCR-dependent Ag presentation. Furthermore, it is unlikely that GIF inhibits binding of antigenic peptide to MHC class II because it was unable to affect Ag presentation when OVA323–339 peptide instead of the whole antigenic protein was used (Fig. 5Go). These functional data suggest the possibility that this cytokine inhibits events associated with BCR, including Ag internalization, signal transduction, and/or early processing in the B cell.

The internalization of HEL Ag by MD4 B cells was directly investigated by fluorescence microscopy. Biotinylated HEL was added to MD4 B cells at 1 µg/ml, a concentration at which HEL is preferentially taken up through BCR specific for the Ag. When MD4 B cells were incubated with GIF, binding of HEL to MD4 B cells at 10 min of incubation was unaffected, but internalization of HEL after 60 min was clearly reduced (Fig. 6Go). These data indicate that GIF inhibits Ag uptake via BCR.

Although fluorescence microscopy demonstrated that the cytokine inhibited Ag uptake through BCR, treatment of Ag-presenting B cells with GIF modestly inhibited secretion of IFN-{gamma} from Th effectors and even less efficiently that of IL-4 (Fig. 7Go). Thus, GIF may be inefficient in reducing the amount of antigenic peptide presented on class II MHC. It is possible that this cytokine inhibits Ag uptake to a limited extent and that the slight reduction of internalized Ag generated a remarkable difference in fluorescence microscopy. Different sensitivities of naive and effector T cells to GIF treatment of B cells may point to the possibility that regulation of costimulation is critical for this cytokine to modulate the APC function of B cells. A number of costimulatory molecules have been described, and some of them are induced by signal transduction through BCR (28, 29, 30). It was reported that cognate interaction of MD4 B cells with CD4 T cells up-regulated B7 molecules and CD44 on B cells (31). Preliminary experiments showed that GIF inhibits induction of CD44 on MD4 B cells, whereas it has little effect on that of B7 (K. Sugie, unpublished observations). Although the role of CD44 in cognate T-B interaction needs to be established, the data may suggest that GIF inhibits BCR signaling.

Lastly, GIF is an unusual cytokine in that a cysteine residue, Cys-60, becomes cysteinylated in the course of secretion from T cells by a posttranslational modification (9). This modification is required for the capability to bind to GIF receptors on target cells (10), to inhibit IgE Ab formation in vivo (8), and to inhibit IgG1 and IgE secretion from purified B cells induced by LPS and IL-4 (12). The present data indicate that modification of Cys-60 is also essential for this cytokine to regulate Ag presentation by B cells. To date, no cytokine except GIF has been known to be cysteinylated in the course of secretion. However, recent studies have unraveled that posttranslational cysteinylation of proteins including H-Y Ag (32) and a viral peptide (33) is required for such determinants to be recognized by specific T cells. The mechanism by which GIF and these antigenic peptides undergo cysteinylation awaits clarification in the future.

In conclusion, the results presented in this work and published elsewhere support the following scenario. In the productive interaction of cognate CD4 T and B cells, B cells present Ag bound to class II MHC and express increased levels of costimulatory molecules, both of which are critical to differentiation and clonal expansion of CD4 T cells. GIF, posttranslationally modified and secreted from T cells, acts on B cells to inhibit BCR-mediated Ag uptake and signal transduction, leading to a combined reduction of Ag/MHC class II complex and costimulatory molecules. Thus the cytokine suppresses generation of effector Th cells. The inhibition of cognate T-B interaction and that of isotype switch to IgG1 and IgE may contribute together to the mechanism by which GIF regulates the humoral immune response. Further work is necessary to determine the costimulatory molecules GIF regulates and the biochemical mechanism by which this cytokine modulates B cell function. It also remains unknown whether the cytokine directly acts on effector T cells, which express higher numbers of GIF receptors than naive T cells (12).


   Acknowledgments
 
We thank Drs. Howard M. Grey, Ralph Kubo, Michael Croft, Paul R. Rogers, Fu-Tong Liu, Alessandra Franco, and Susanne Schneider for helpful discussions and critical reading of the manuscript; Dr. Yasuyuki Ishii for various derivatives of rhGIF; Drs. Christopher C. Goodnow, Dale T. Umetsu, and Kenneth M. Murphy for Tg mice; and Drs. John Kappler, Paul R. Rogers, and Michael Croft for Abs.


   Footnotes
 
1 This paper is publication 359 from the La Jolla Institute for Allergy and Immunology. Back

2 K.S. dedicates this paper to Dr. Kimishige Ishizaka. Back

3 Address correspondence and reprint requests to Dr. Katsuji Sugie, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121. Back

4 Abbreviations used in this paper: GIF, glycosylation-inhibiting factor; HEL, hen egg lysozyme; Tg, transgenic; rhGIF, recombinant human GIF; BCR, B cell Ag receptor; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid). Back

Received for publication August 15, 2000. Accepted for publication January 30, 2001.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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