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. 2015 May 7;145(2):202–212. doi: 10.1111/imm.12436

Basophils inhibit proliferation of CD4+ T cells in autologous and allogeneic mixed lymphocyte reactions and limit disease activity in a murine model of graft versus host disease

Fabian J Hermann 1, Manuel Rodriguez Gomez 1, Kristina Doser 2, Matthias Edinger 2,3, Petra Hoffmann 2,3, Gabriela Schiechl 1, Yvonne Talke 1, Nicole Göbel 1, Kathrin Schmidbauer 1,*, Shahzad N Syed 1, Hilke Brühl 4, Matthias Mack 1,3,✉
PMCID: PMC4427385  PMID: 25545131

Abstract

Basophils are known to modulate the phenotype of CD4+ T cells and to enhance T helper type 2 responses in vitro and in vivo. In this study, we demonstrate that murine basophils inhibit proliferation of CD4+ T cells in autologous and allogeneic mixed lymphocyte reactions. The inhibition is independent of Fas and MHC class II, but dependent on activation of basophils with subsequent release of interleukin-4 (IL-4) and IL-6. The inhibitory effect of basophils on T-cell proliferation can be blocked with antibodies against IL-4 and IL-6 and is absent in IL-4/IL-6 double-deficient mice. In addition, we show that basophils and IL-4 have beneficial effects on disease activity in a murine model of acute graft-versus-host disease (GvHD). When basophils were depleted with the antibody MAR-1 before induction of GvHD, weight loss, GvHD score, mortality and plasma tumour necrosis factor levels were increased while injection of IL-4 improved GvHD. Basophil-depleted mice with GvHD also have increased numbers of CD4+ T cells in the mesenteric lymph nodes. Our data show for the first time that basophils suppress autologous and allogeneic CD4+ T-cell proliferation in an IL-4-dependent manner.

Keywords: basophils, CD4+ T cells, graft-versus-host disease, interleukin-4, interleukin-6, mixed lymphocyte reaction

Introduction

For many years basophils have mainly been linked to allergic reactions and parasite infections.1–4 However, a number of recent publications also found important regulatory functions of basophils in differentiation of T helper type 2 (Th2) cells, humoral immune responses and plasma cell survival.5–10 Basophils also contribute to the development of lupus nephritis11,12 and collagen-induced arthritis.13,14 The effects of basophils on T and B cells are mainly mediated by basophil-derived interleukin-4 (IL-4). Basophils can be activated to release IL-4 and IL-6 by a variety of stimuli, like cross-linkage of surface IgE, chemokines, proteases (e.g. papain), but also by cytokines such as IL-3 and thymic stromal lymphopoietin.2,6,15–20

On the other hand, basophils can also have a beneficial role in autoimmune diseases. In a model of murine colitis, basophils are activated by T-cell-derived factors such as IL-3, release IL-4 and suppress disease-inducing Th1 T cells.21 B cells and humoral immunity are not involved in this model.

Murine basophils can easily be identified by their surface expression of CD49b and the high-affinity IgE receptor FcεR-1, as well as surface-bound IgE.6

In this study we investigated the influence of basophils on proliferation of CD4+ T cells in autologous and allogeneic mixed lymphocyte reactions (MLR). Autologous (or syngeneic) T-cell proliferation was first described by Von Boehmer and Adams22 in 1973, and constitutes the proliferation of CD4+ T cells in response to stimulation by autologous non-T cells. In this setting, dendritic cells present peptides from self proteins and apoptotic cells to syngeneic CD4+ T cells.23,24 In addition, dendritic cells can also activate T cells independent of antigen.25–27 Autologous T-cell proliferation is thought to induce or perpetuate autoimmunity. In allogeneic MLR CD4+ T cells recognize mainly intact allo-MHC class II molecules on allogeneic antigen-presenting cells and undergo a strong polyclonal proliferation.

We show that basophils inhibit the proliferation of CD4+ T cells in autologous and allogeneic MLR. This effect was independent of cell contact, but dependent on activation of basophils with release of the soluble factors IL-4 and IL-6. Basophils also led to a shift of the T-cell phenotype from Th1 to Th2 in allogeneic MLR. In mice with acute graft-versus-host disease (GvHD) basophils decreased the expansion of CD4+ T cells and reduced the development of GvHD. Injection of recombinant IL-4 also ameliorated the development of GvHD.

Materials and methods

Mice

Female BALB/c and C57BL/6N mice, 12–14 weeks old, were purchased from Charles River (Sulzfeld, Germany). B6MLR-Faslpr/J, B6.129S2-H2dlAb1−Ea/J (MHC II−/−) and BALB/c-IL-4ratm1Sz/J (IL-4Rα−/−) mice were obtained from Jackson Laboratories (Bar Harbor, ME). B6.129P2-Il4tm1Cgn/J and B6.129S6-Il6tm1Kopf/J mice were purchased from Jackson Laboratories and cross-bred to create IL-4/IL-6 double knockout mice. All mice were held under specific pathogen-free conditions at a 12-hr day/night cycle in the animal facilities of the University Hospital Regensburg. Autoclaved food and water was provided ad libitum. All animal studies were approved by the local authorities.

Autologous mixed lymphocyte reactions

BALB/c splenocytes (8 × 105 cells/well) were depleted of basophils using anti-CD49b MicroBeads (Miltenyi Biotech, Bergisch Gladbach, Germany), labelled with carboxyfluorescein succinimidyl ester (CFSE; 5 μm) and cultured with the indicated number of IgE+ or CD49+ basophils for 5 days. CFSE is a fluorescent molecule that binds to proteins within the cell, so with every cell division the fluorescence intensity of the cells is reduced by 50%.28 Interleukin-3 was added at a concentration of 10 ng/ml, IL-4 and IL-6 (Peprotech, Rocky Hill, NJ) were added at a concentration of 20 ng/ml each. Unless otherwise specified, cells were cultured in 200 μl RPMI-1640 medium with 10% (vol/vol) fetal calf serum, penicillin (100 U/ml), streptomycin (100 U/ml), non-essential amino acids (0·1 mm), 1 mm sodium pyruvate and 50 μm β-mercaptoethanol in triplicates.

Allogeneic mixed lymphocyte reactions

BALB/c splenocytes were depleted of T cells using anti-CD4 and anti-CD8 MicroBeads (Miltenyi Biotech) and of basophils using phycoerythrin-conjugated (-PE) anti-IgE (R35-72; BD Biosciences, San Jose, CA) and anti-PE MicroBeads; Miltenyi Biotech). C57BL/6N splenocytes were depleted of CD25+ cells using anti-CD25-PE (PC61; BD Biosciences) and anti-PE MicroBeads (Miltenyi Biotech) with LD columns. Subsequently CD4+ T cells were isolated with anti-CD4 MicroBeads (Miltenyi Biotech) and MS-columns (Miltenyi Biotech). The isolated CD4+ CD25− T cells were labelled with CFSE (5 μm). Then, 5 × 105 BALB/c splenocytes depleted of T cells and basophils were cultured with 1 × 105 CD4+ CD25− T cells from C57BL/6N mice and 2 × 104 basophils from BALB/c mice for 5 days in 96-well round-bottom plates in triplicates. Unless otherwise specified, cells were cultured in 200 μl RPMI-1640 medium with 10% (vol/vol) fetal calf serum, penicillin–streptomycin, 0·1 mm non-essential amino acids, 1 mm sodium pyruvate and 50 μm β-mercaptoethanol. Interleukin-3 was added at a concentration of 10 ng/ml, neutralizing antibodies against IL-4 (clone 30340; R&D Systems, Minneapolis, MN) or IL-6 (clone MP5-20F3; R&D Systems) at a concentration of 20 μg/ml.

Isolation of IgE+ and CD49b+ basophils from the bone marrow

CD49b+ or IgE+ basophils were enriched from the bone marrow with MS-columns and anti-CD49b MicroBeads (Miltenyi Biotech) or anti-IgE-PE antibody (R35-72; BD, Franklin Lakes, NJ) followed by anti-PE MicroBeads (Miltenyi Biotech). For FACS-sorting of basophils from the bone marrow, cells were labelled with anti-CD45-FITC and anti-IgE-PE (R35-72; BD), or anti-CD45-FITC and anti-CD49b-PE (DX5; eBioscience, San Diego, CA), as previously described.6 CD45low IgE+ or CD45low CD49b+ basophils were sorted with a BD FACSAria II in the FACS core facility of the University Hospital Regensburg.

Production of IL-4 complex

Two hundred nanograms recombinant murine IL-4 (Peprotech) was pre-incubated with 2 μg anti-IL-4 antibody (clone 30340; R&D Systems) in 50 μl PBS for 5 min at room temperature followed by intraperitoneal injection in a volume of 200 μl/mouse.

Flow cytometry

Cells were incubated with Fc-Block (CD16/32, Clone 93; BioLegend, San Diego, CA) for 10 min followed by directly labelled antibodies according to the manufacturer's instructions.

The following antibodies and reagents were used in flow cytometry: anti-CD45-FITC (30-F11; eBioscience), anti-CD3-PE-Cy7 (145-2C11; eBioscience), anti-CD4-V500 (GK1.5; BD), anti-CD8 eFluor780 (53-6.7; eBioscience), anti-CD49b-PE (DX5; eBioscience), anti-FcεR1α-allophycocyanin (Mar-1; eBioscience), anti-H2Kb-PE (AF6-88.5; BD), anti-H2Kd-FITC (SF1-1.1; BD) and anti-CD19-Peridinin chlorophyll protein-Cy5.5 (eBio1D3; eBioscience); CFSE and AccuCheck counting beads were from Molecular Probes (Eugene, OR), Life Technologies (Carlsbad, CA). Cells were acquired with a BD FACSCanto II (BD) using the facsdiva software (BD) and analysed with flowjo x (Treestar software, Ashland, OR).

Cytokine analysis

Concentrations of interferon-γ (IFN-γ), IL-4, IL-6, IL-13 and IL-3 were determined in the supernatant of cell cultures by ELISA (R&D Systems) in accordance with manufacturer's instructions. Concentrations of IL-6, IFN-γ, IL-17A and tumour necrosis factor were determined in the plasma of GvHD mice with the CBA Enhanced Sensitivity Flex Set System (BD Biosciences). Interleukin-27 was measured by ELISA (eBioscience) in accordance with the manufacturer's instructions.

Splenocytes or lymph node cells were incubated for 4 hr with PMA (10 ng/ml; Sigma Aldrich, St Louis, MO), ionomycin (1 μg/ml; Sigma Aldrich) and brefeldin A (1 μg/ml; Sigma Aldrich), followed by extracellular staining for 20 min at 4° and permeabilization with BD Cytofix/Cytoperm. Cells were washed in BD Permwash solution and intracellularly stained for 20 min at 4° with the following antibodies: anti-IFN-γ-FITC (XMG1.2; BD), anti-IL-4-PE-Cy7 (11B11; BD), anti-IL-17-PE (TC11-18H10; BD). FoxP3 staining was performed with a kit using anti-FoxP3-allophycocyanin (150D/E4; eBioscience). After washing with permeabilization buffer, cells were resuspended in staining buffer and analysed with a BD FACSCanto II.

Isolation of bone marrow cells for transplantation

Bone marrow of C57BL/6 mice was flushed out from the femur and tibia with RPMI-1640 containing 10% fetal calf serum in a 10-ml syringe. The bone marrow cells were depleted of CD90+ cells with anti-CD90.2 MicroBeads (Miltenyi Biotech) and LS-columns in accordance with the manufacturer's instructions. Subsequently, cells were depleted of red blood cells using ACK-lysing buffer (Lonza, Basel Switzerland) and washed twice with RPMI-1640.

In vivo depletion of basophils

Mice were intraperitoneally injected twice daily with 5 μg of anti-FcεR1α antibody (MAR-1; eBioscience) or the appropriate isotype control antibody (hamster IgG; eBioscience) for the indicated period of time.

GvHD experiments

BALB/c mice with a weight of about 20 g were irradiated with 9 Gy using a linear accelerator Lineac 2 (Siemens, Kemnath, Germany) of the University Hospital Regensburg, Germany. After 6–8 hr the mice were transplanted with 2·5 × 106 CD90.2-depleted C57BL6/N bone marrow cells and 2·5 × 105 C57BL6/N CD4+ CD25− T cells (ratio 10 : 1). The mice (n = 8 to n = 10/group) were scored as described previously.29 Mice were killed, if they reached a GvHD score of 6 or lost 35% of their starting weight on two consecutive scoring days.

Statistical analysis

Statistical significance was calculated with a one-sided Students t-test, a one-way analysis of variance test or with a Wilcoxon Log-rank test for survival. Error bars show the standard error of the mean (SEM).

Results

Basophils inhibit autologous T-cell proliferation

To investigate the influence of basophils on murine CD4+ T cells we first studied proliferation of CD4+ T cells in autologous MLR. Using CFSE-labelled splenocytes we determined the time course of autologous CD4+ T-cell proliferation in vitro. As shown in Fig.1(a), the percentage of proliferating T cells markedly increased over a 5-day period. Using 5-day cultures we then analysed the influence of basophils on autologous T-cell proliferation (Fig.1b). Splenocytes were depleted of basophils and cultured in the absence or presence of basophils. Basophils were purified from the bone marrow of syngeneic mice by surface-bound IgE. As a control we used IgE+ cells of in vivo basophil-depleted mice. We and others have shown that the majority of IgE+ cells from the bone marrow are basophils.6 Addition of IgE+ basophils markedly inhibited the autologous CD4+ T-cell proliferation. When IL-3 was added to activate basophils, the suppression of T-cell proliferation was further increased, while IL-3 alone had no effect on the autologous proliferation in basophil-depleted splenocytes. IgE+ cells isolated from mice that were depleted of basophils by injection of the antibody MAR-1 did not suppress autologous CD4+ T-cell proliferation, indicating that basophils but not other IgE+ cells are responsible for the suppression of T-cell proliferation (Fig.1b).

Figure 1.

Figure 1

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Basophils inhibit the autologous proliferation of CD4+ T cells. (a) CFSE-labelled splenocytes (8 × 105/well) were cultured in triplicates for 2·5–5 days in medium. Gating scheme to identify proliferating CD4+ T cells (left) and quantitative analysis of CD4+ T-cell proliferation (right). The proliferation of CD4+ T cells was analysed by CFSE dilution. (b) FACS plots and quantitative analysis showing the influence of activated and non-activated basophils on autologous proliferation of CD4+ T cells. 8 × 105 basophil-depleted CFSE-labelled BALB/c splenocytes were cultured for 5 days with medium alone (Ø), with 1 × 105 IgE+ basophils (IgE+) or with IgE+ cells isolated from the bone marrow of in vivo basophil-depleted BALB/c mice (IgE+ Baso−) (n = 3). Where indicated, interleukin-3 ( IL-3; 10 ng/ml) was added. CD4+ T-cell proliferation was analysed by flow cytometry using CFSE dilution. (c) Basophils, but not other CD49+ cells, inhibit autologous CD4+ T-cell proliferation. 8 × 105 basophil-depleted CFSE-labelled BALB/c splenocytes were cultured for 5 days with different numbers (as indicated on the axis) of CD49+ cells or CD49+ cells depleted of IgE+ basophils (CD49+ IgE−) from BALB/c bone marrow (n = 3). CD4+ T-cell proliferation was analysed by flow cytometry and CFSE dilution. (d) Basophil-depleted CFSE-labelled splenocytes (8 × 105) were cultured for 5 days with or without CD49b+ basophils from wild-type (WT) or MHC class II−/− mice (MHCII−/−) (n = 3). Where indicated, IL-3 (10 ng/ml) was added into the culture. CD4+ T-cell proliferation was assessed by CFSE dilution. (e) Basophil-depleted CFSE-labelled splenocytes (8 × 105) from WT or Fas-deficient (Fas−/−) mice were cultured for 5 days with CD49b+ basophils from WT mice (CD49b+ WT) in the presence or absence of IL-3 (10 ng/ml) (n = 3). Proliferation of CD4+ T was quantified by CFSE dilution. Data are mean ±SEM. One out of three representative experiments is shown. *P < 0·05; **P < 0·01; ***P < 0·001.

As isolation of basophils with antibodies against IgE leads to IgE cross-linkage and hence to activation of basophils, we also used antibodies against CD49b to obtain non-activated basophils from the bone marrow. In this case only approximately 10% of the CD49b+ cells are basophils, while the remaining cells are a mixture of other CD49b-expressing cells.21 To distinguish between effects mediated by basophils and other CD49b+ cells, we used total CD49b+ cells containing basophils or depleted basophils from CD49b+ cells with magnetic beads against IgE (CD49b+ IgE−). Both cell types were cultured with CFSE-labelled basophil-depleted splenocytes at ratios between 1 : 20 and 1 : 320 (Fig.1c). Total CD49b+ cells containing basophils reduced the autologous proliferation of CD4+ T cells much more effectively than CD49b+ IgE− cells, which are devoid of basophils.

Interestingly, non-activated basophils purified by expression of CD49b also suppressed the autologous proliferation of CD4+ T cells (Fig.1c). To get more insight into how basophils could be activated in autologous or allogeneic MLR we measured the release of IL-3. In autologous and allogeneic MLR, CD4+ T cells release substantial amounts of IL-3, as shown by ELISA of culture supernatants (see Supporting information, Fig. S1a,b). Interleukin-3 released from CD4+ T cells is one possible mechanism by which basophils (even those purified via CD49b) become activated during autologous and allogeneic MLR. In addition, we show that during GvHD substantial amounts of IL-3 are released from donor-derived CD4+ T cells (Fig. S1c). If only T-cell-depleted bone marrow was transplanted, no IL-3 was detectable in the plasma of the recipients, whereas high plasma IL-3 levels occurred if CD4+ T cells were co-transplanted.

Basophil-induced inhibition of autologous T cells is independent of MHC class II and Fas

As some groups described expression of MHC II by basophils,30–32 we analysed if MHC class II is involved in the basophil-mediated suppression of CD4+ T cells. Basophils were isolated from the bone marrow of wild-type (WT) or MHCII-deficient (MHCII−/−) mice and cultured with basophil-depleted CFSE-labelled splenocytes from WT mice. Both WT and MHCII−/− basophils inhibited the autologous proliferation of CD4+ T cells to a similar degree. Activation of basophils with IL-3 increased the inhibition in both cases (Fig.1d). To investigate whether Fas is involved in basophil-mediated suppression of T-cell proliferation, we cultured WT basophils with basophil-depleted CFSE-labelled splenocytes from Fas−/− or WT mice. CD4+ T cells from Fas−/− mice showed a somewhat reduced proliferation compared with WT mice; however, basophils still suppressed the autologous proliferation of Fas-deficient CD4+ T cells (Fig.1e).

Inhibitory effects of basophils are dependent on soluble factors

To determine whether direct cell–cell contact is involved in the inhibitory effects of basophils, we examined if the supernatant of FACS-sorted basophils is able to inhibit autologous CD4+ T-cell proliferation. For this purpose, basophils were FACS-sorted from bone marrow using antibodies against surface-bound IgE (IgE+ basophils), or the surface marker CD49b (CD49b+ basophils). The sorted cells were cultured with or without IL-3 and the supernatant was collected after 24 hr. The supernatant was added to CFSE-labelled basophil-depleted splenocytes and autologous T-cell proliferation was measured after 5 days (Fig.2a). The supernatant of IgE+ basophils significantly inhibited autologous CD4+ T-cell proliferation. The supernatant of IL-3-activated IgE+ basophils had an even stronger suppressive effect on T-cell proliferation. In contrast, the supernatant of CD49b+ basophils induced little suppression of CD4+ T-cell proliferation. However, when the CD49b+ basophils were activated by IL-3, the supernatant of these cells was strongly suppressive (Fig.2a). These data indicate that basophils need to be activated either by IL-3 or by cross-linkage of surface IgE, which occurs during isolation of IgE+ basophils, to become suppressive.

Figure 2.

Figure 2

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Basophils inhibit autologous CD4+ T-cell proliferation by soluble factors. (a) Basophils FACS-sorted by surface IgE (IgE+) or by expression of CD49b (CD49b+) were cultured for 24 hr with or without interleukin-3 (IL-3) (1 × 105 cells/ml) and the supernatant (SN) of these cells was collected. Basophil-depleted CFSE-labelled splenocytes (8 × 105 cells/well) were cultured for 5 days with medium alone, with IL-3 (10 ng/ml) or with the supernatant (SN) of the cultured basophils (50%) (n = 3). CD4+ T-cell proliferation was quantified by CFSE dilution. (b) IL-4 and IL-6 were measured in the supernatant (SN) of the cultured basophils used for the experiment described in (a). (c) FACS plots and quantitative analysis of basophil-depleted splenocytes (8 × 105) cultured for 5 days with IL-4, IL-6 or a combination of IL-4 and IL-6 (n = 3). CD4+ T-cell proliferation was analysed by CFSE dilution. (d) FACS plots and quantitative analysis of total splenocytes (8 × 105) from wild-type (WT) or IL-4/6−/− mice cultured for 5 days in the presence of absence of IL-3 (n = 3). CD4+ T-cell proliferation was quantified by CFSE dilution. Data are mean ± SEM. One out of three representative experiments is shown. *P < 0·05; ***P < 0·001.

As basophils are known to release IL-4 and IL-6 after activation6, we quantified both cytokines in the supernatant of IgE+ and CD49b+ basophils. In the absence of IL-3 basophils isolated by surface-bound IgE but not by expression of CD49 released IL-6 and some IL-4. After activation with IL-3 both IgE+ and CD49b+ basophils released substantial amounts of IL-4 and IL-6 (Fig.2b). These results led us to the assumption, that IL-4 and IL-6 could be responsible for basophil-mediated suppression of autologous CD4+ T-cell proliferation. To prove this, we pre-incubated the supernatant of IL-3-activated basophils with neutralizing antibodies against IL-4, IL-6 or both (20 μg/ml) for 1 hr and added the supernatant at a concentration of 50% to basophil-depleted CFSE-labelled BALB/c splenocytes. After 5 days the autologous proliferation of CD4+ T cells was measured (see Supporting information, Fig. S2). The untreated supernatant of activated basophils almost completely suppressed the proliferation of CD4+ T cells, whereas the supernatant pre-incubated with anti-IL-4 antibodies or a combination of anti-IL-4 and anti-IL-6 antibodies was much less effective, indicating that IL-4 and IL-6 are the predominant suppressive factors in the supernatant of basophils.

To directly show that IL-4 and IL-6 are able to suppress autologous CD4+ T-cell proliferation, we cultured basophil-depleted splenocytes with recombinant IL-4, IL-6 or a combination of both cytokines (IL-4 + IL-6) (Fig.2c). Both IL-4 and IL-6 suppressed the autologous proliferation of CD4+ T cells in basophil-depleted splenocytes. Interleukin-4 and the combination of IL-4 and IL-6 were more potent in terms of T-cell suppression than IL-6. Addition of recombinant IL-3 to basophil-depleted splenocytes did not significantly suppress autologous T-cell proliferation (Figs1b,d,e, and 2a).

We next investigated, whether basophils present at low numbers in the spleen are also able to suppress autologous T-cell proliferation. For this purpose, we cultured total CFSE-labelled splenocytes without depleting basophils, activated the basophils with IL-3 and used splenocytes from WT or IL-4/IL-6−/− mice to demonstrate that IL-4 and IL-6 are involved in T-cell suppression. Activation of basophil-containing WT splenocytes with IL-3 led to a significant reduction of autologous CD4+ T-cell proliferation, whereas addition of IL-3 to basophil-containing IL-4/IL-6−/− splenocytes did not reduce autologous CD4+ T-cell proliferation (Fig.2d).

To investigate whether basophils directly interfere with the proliferation of CD4+ T cells we purified CD4+ T cells and activated them with immobilized anti-CD3 antibodies in the presence or absence of basophils for 3 days (see Supporting information, Fig. S3a). Under these conditions, basophils did not suppress T-cell proliferation, suggesting that basophil-derived cytokines like IL-4 do not directly act on T cells but on other cells involved in activation of T cells, e.g. antigen-presenting cells, which are known to be required for autologous proliferation of CD4+ T cells. To answer this question, we cultured purified CD4+ T cells from WT or IL-4-receptor-deficient BALB/c mice with antigen-presenting cells from WT or IL-4-receptor-deficient BALB/c mice for 5 days. As antigen-presenting cells we used T-cell-depleted BALB/c splenocytes. Interleukin-4 markedly suppressed the proliferation of both WT and IL-4-receptor-deficient CD4+ T cells, if the antigen-presenting cells were WT. In contrast, IL-4 completely failed to suppress the proliferation of CD4+ T cells, when the antigen-presenting cells were derived from IL-4-receptor-deficient mice (Fig. S3b). These data demonstrate that IL-4 suppresses autologous proliferation of CD4+ T cells by acting on the antigen-presenting cells but not on the CD4+ T cells.

Basophils suppress T-cell proliferation in allogeneic mixed lymphocyte reactions

To analyse if basophils are also able to inhibit CD4+ T-cell proliferation in allogeneic MLR, BALB/c splenocytes were depleted of T cells and basophils and co-cultured with a defined number of CD4+ CD25− T cells from C57BL/6 mice and basophils from BALB/c mice (Fig.3a). Interleukin-3-activated basophils were able to suppress CD4+ T-cell proliferation in the MLR setting by about 35%. This inhibitory effect was completely dependent on IL-4 but not IL-6, as inhibition of IL-4 but not IL-6 with monoclonal antibodies completely abrogated the suppressive effect of basophils.

Figure 3.

Figure 3

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Basophils inhibit CD4+ T-cell proliferation in allogeneic mixed lymphocyte reactions. (a) BALB/c splenocytes (8 × 105) depleted of T cells and basophils were incubated for 5 days with 2 × 105 CD4+ CD25− T cells from C57BL/6 mice. Where indicated, 2 × 104 IgE+ basophils from BALB/c mice (Baso) were added (n = 3). Interleukin-3 (IL-3) or blocking antibodies against IL-4 and IL-6 (αIL4, αIL6) were added as indicated. CD4+ T-cell proliferation was quantified by flow cytometry and CFSE dilution. (b) The concentration of IL-4, IL-6, IL-13 and interferon-γ (IFN-γ) was measured by ELISA in the supernatant of the experiment described in (a). Data are mean ± SEM. One out of three representative experiments is shown. *P < 0·05; **P < 0·01; ***P < 0·001.

In allogeneic MLR basophils not only reduced CD4+ T-cell proliferation, but also induced a shift of the T-cell phenotype, as measured by the release of cytokines in the supernatant (Fig.3b). In the presence of basophils the supernatant of MLR contained increased levels of IL-13 and reduced levels of IFN-γ. The increased levels of IL-13 and the decreased levels of IFN-γ point towards a T-cell shift from Th1 towards Th2. Murine basophils do not release IL-13 or IFN-γ.6

Basophils limit the acute graft-versus-host disease

Based on the reduced proliferation of CD4+ T cells and the shift towards Th2 in MLR in the presence of basophils, we studied the impact of basophils in a mouse model of acute GvHD. As T cells are the main effector cells during acute GvHD, reduced T-cell expansion could ameliorate GvHD symptoms.33,34 In addition, Th2 cells are regarded as having less potential in initiating GvHD.35,36

BALB/c recipients and C57BL/6 donor mice were treated with the basophil-depleting antibody MAR-1 or the appropriate isotype control antibody on days –4, –3 and –2. On day 0 BALB/c recipients (n = 8/group) received a lethal dose of irradiation and were transplanted with T-cell-depleted bone marrow cells and CD4+ CD25− T cells from C57BL/6 mice. The depletion of basophils before irradiation led to an increased GvHD score and more severe weight loss, as well as a decreased survival, compared with the isotype-treated control group (Fig.4a). To investigate the influence of basophils on an established GvHD, we induced GvHD in mice and depleted the basophils on days 10, 11 and 12 after transplantation. In contrast to the early depletion of basophils, the late depletion of basophils did not increase the severity of GvHD. Only a weak transient deterioration of the GvHD score and weight was observed (Fig.4b).

Figure 4.

Figure 4

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Depletion of basophils aggravates development of graft-versus-host disease (GvHD). (a) BALB/c recipients and C57BL/6 donor mice were depleted of basophils by twice daily injection of the antibody MAR-1 (5 μg) from day –4 to –2. Control mice were treated with the same amount of an isotype control antibody (Armenian hamster IgG). On day 0 recipients (n = 10/group) were irradiated and 6–8 hr later transplanted with 2·5 × 106 T-cell-depleted C57BL/6 bone marrow cells and 2·5 × 105 C57BL/6 CD4+ CD25− T cells. GvHD score, weight loss and survival were monitored. (b) BALB/c recipients (n = 10/group) were irradiated on day 0 and transplanted 6–8 hr later with 2·5 × 106 T-cell-depleted C57BL/6 bone marrow cells and 2·5 × 105 C57BL/6 CD4+ CD25− T cells. In the recipients, basophils were depleted from day 10 to 12 after transplantation. GvHD score, weight loss and survival were monitored. Data are mean ± SEM. One out of three representative experiments is shown. (c) BALB/c recipients (n = 10/group) were injected with interleukin-4 (IL-4) complex (200 ng IL-4 + 2 μg anti IL-4) or PBS on days –3 and –2. Mice were irradiated on day 0 and 6–8 hr later were transplanted with 2·5 × 106 T-cell-depleted C57BL/6 bone marrow cells and 2·5 × 105 C57BL/6 CD4+ CD25− T cells. GvHD score, weight loss and survival were monitored. Data are mean ± SEM. One out of two representative experiments is shown. *P < 0·05.

Based on our finding that basophils suppress MLR mainly by releasing IL-4, we tested if the injection of IL-4 complex on days –3 and –2 before irradiation has a positive effect on the outcome of GvHD (Fig.4c). The IL-4 complex, consisting of 200 ng IL-4 and 2 μg anti-IL-4 antibody, was injected because it is known that the complexed IL-4 has a much higher biological activity in mice than recombinant IL-4.37 Mice (n = 10/group) that were injected with IL-4 complex showed a significantly reduced GvHD score and weight loss compared with the control group.

Depletion of basophils leads to increased expansion of CD4+ T cells in the lymph nodes during GvHD

As we observed a decrease in the proliferation of CD4+ T cells in the MLR experiments in the presence of basophils, we depleted basophils before GvHD induction and analysed T-cell numbers and T-cell phenotypes in the spleen and mesenteric lymph nodes 14 days after GvHD induction. At this time-point > 99% of total leucocytes and > 95% of CD4+ T cells in the spleen and peripheral blood were H-2Kb positive and so were derived from the donor (see Supporting information, Fig. S4). The high degree of chimerism and the almost complete lack of B cells indicate GvHD induction and successful engraftment.38 Depletion of basophils before GvHD induction did not change the number of CD45+ leucocytes or CD4+ T cells in the spleen. Only the numbers of basophils were reduced in the MAR-1-treated mice, as expected (Fig.5a). However, in the mesenteric lymph nodes depletion of basophils led to an increase of CD45+ leucocytes and also increased numbers of CD4+ T cells (Fig.5b). Following depletion of basophils we did not detect significant changes of IL-4, IFN-γ and IL-17 expression in CD4+ T cells or a significantly altered frequency of FoxP3+ regulatory T cells (Fig.5c). Consistent with the increased GvHD, basophil-depleted mice showed significantly elevated levels of the pro-inflammatory cytokine tumour necrosis factor in the plasma (Fig.5d). The plasma levels of other cytokines were not significantly altered. These experiments suggest that the GvHD limiting effects of basophils are primarily mediated by their ability to limit the expansion of CD4+ T cells. The impact on plasma tumour necrosis factor levels probably reflects the severity of GvHD.

Figure 5.

Figure 5

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Depletion of basophils increases the number of CD4+ T cells in lymph nodes during graft-versus-host disease (GvHD). As described in Fig.4(a), basophils were depleted from day –4 to –2 before transplantation in BALB/c recipients (n = 8/group) and in C57BL/6 donors. Transplantation was performed on day 0. The recipients were killed on day 14 and leucocyte subpopulations were quantified by flow cytometry in the spleen (a) and mesenteric lymph nodes (b). (c) Intracellular staining of splenic CD4+ T cells for expression of cytokines and FoxP3. Data are mean ±SEM. One out of two representative experiments is shown. (d) interferon-γ (IFN-γ), interleukin-17 (IL-17), IL-6, tumour necrosis factor (TNF) and IL-27 levels in plasma of the GvHD mice, measured on day 14 after transplantation (n = 8/group). *P < 0·05; **P < 0·01; ***P < 0·001.

Discussion

We have shown that basophils are able to inhibit autologous as well as allogeneic proliferation of CD4+ T cells. The inhibitory effect of basophils on autologous T-cell proliferation was independent of MHC class II and Fas, but dependent on the soluble factors IL-4 and IL-6. The basophil-mediated inhibition of allogeneic T-cell proliferation was exclusively dependent on IL-4. In MLR experiments the addition of basophils led to a decrease of the Th1 cytokine IFN-γ and an increase of the Th2 cytokines IL-4 and IL-13. In vivo, the depletion of basophils before induction of GvHD led to a more severe disease with higher GvHD scores, increased weight loss and reduced survival, whereas the injection of IL-4 significantly ameliorated the severity of GvHD. Consistent with our in vitro data basophil-depleted mice showed higher numbers of CD45+ and CD4+ T cells in the mesenteric lymph nodes compared with the control group. However, depletion of basophils in mice with GvHD did not alter the Th1/Th2 phenotype of the CD4+ T cells or the frequency of regulatory T cells.

Our experiments with transfer of supernatant demonstrate that the inhibition of autologous CD4+ T-cell proliferation is mediated by basophil-derived soluble factors and that IL-4 and IL-6 are critically involved. Experiments with recombinant cytokines confirmed these results and showed greater inhibitory properties for IL-4 compared with IL-6. So far, it was reported that the cytokines IL-15 and IL-2 support autologous T-cell proliferation, but no inhibitory cytokines have been described.39 In allogeneic MLR neutralization of IL-4 but not IL-6 abolished the inhibitory effects of basophils, suggesting that IL-4 is mainly responsible for the suppression of T-cell proliferation in this setting. These results were surprising as IL-4 and IL-6 have been described to support proliferation and to prevent apoptosis of isolated T cells.40,41 In contrast to these studies, our experiments were performed with whole splenocytes containing a variety of cells that are required for induction of autologous or allogeneic T-cell proliferation (e.g. dendritic cells). To show that IL-4 does not directly act on CD4+ T cells we performed experiments with purified CD4+ T cells and with CD4+ T cells and co-stimulatory cells isolated from IL-4-receptor-deficient mice. Our results clearly show that IL-4 suppresses autologous T-cell proliferation by acting on the co-stimulatory cells but not on the CD4+ T cells. The numbers of CD4+ Foxp3+ regulatory T cells were not decreased by depletion of basophils in the GvHD experiments, indicating that regulatory T cells do not play a role in the basophil-mediated inhibition of CD4+ T cells.

Our data also suggest that basophils need to be activated to inhibit CD4+ T-cell proliferation. Activation of basophils occurs if cross-linking antibodies against IgE are used for isolation of basophils or if recombinant IL-3 is added into the culture. However, basophils also inhibit CD4+ T-cell proliferation without artificial activation. One possible mechanism could be the release of substantial amounts of IL-3 from CD4+ T cells during autologous or allogeneic MLR and in the initial phase of GvHD, as shown in the culture supernatant and in the plasma of mice.

In an earlier publication we have shown beneficial effects of basophils in a murine model of colitis.21 In this model adoptively transferred CD4+ T cells release IL-3 and activate basophils. Depletion of basophils led to an increase in CD4+ T cells in the mesenteric lymph nodes and aggravation of colitis. In the colitis model basophils suppressed the development of Th1 cells expressing IFN-γ, IL-2 and tumour necrosis factor, while in the GvHD model we did not observe increased expression of IFN-γ following depletion of basophils. Therefore, the positive effects of basophils on the outcome of GvHD cannot be explained by a shift in T-cell phenotypes but seem to be primarily the result of inhibition of T-cell expansion.

The impact of basophils on GvHD was previously studied by Tawara et al.42 Similar to our results, depletion of basophils did not change the number or phenotype of CD4+ T cells in the spleen. Lymph nodes, however, were not analysed by Tawara et al., but turned out in our studies to be a critical site where depletion of basophils results in increased expansion of CD4+ T cells. In contrast to the study by Tawara et al., we observed an exacerbation of GvHD when basophils were depleted before transplantation of bone marrow cells. This difference may be explained by the fact that we depleted basophils not only in the recipients but also in the donor mice, to avoid the adoptive transfer of donor basophils by the bone marrow transplantation. In addition, depletion of basophils was performed in our study from day –4 to –2 pre-transplantation, while Tawara et al. depleted basophils from day –1 to +1. From previous experiments6 we know that injection of the antibody MAR-1 initially results in activation of basophils with release of IL-4 and IL-6. We therefore rested the mice for 2 days after the last injection of MAR-1. This avoids opposing effects of basophil activation and basophil depletion during transplantation, and may also explain why Tawara et al. found no effect of basophil depletion on development of GvHD. Moreover, Tawara et al. induced GvHD by injection of CD90+ T cells that contain not only CD4+ T cells but also CD8+ T cells. GvHD induced by CD8+ T cells may not be suppressed effectively by basophils.

In summary, basophils have a beneficial role in the induction phase of acute GvHD, mainly by limiting the expansion of allogeneic CD4+ T cells. Hence, basophils or basophil-derived factors may be a new strategy for prevention of GvHD.

Acknowledgments

This study was supported by a grant from the Deutsche Forschungsgemeinschaft.

Glossary

Abbreviations:

CFSE

carboxyfluorescein succinimidyl ester

GvHD

graft-versus-host disease

IFN-γ

interferon-γ

IL

interleukin

MLR

mixed lymphocyte reaction

Th2

T helper type 2

WT

wild-type

Author contributions

FJH, MRG, GS, HB, YT, NG, KS, SNS and MM performed the experiments and/or analysed data. FJH, KD, ME, PH and MM designed the study. FJH, HB and MM wrote the paper.

Disclosures

The authors declare no financial or commercial conflicts of interest associated with this study.

Supporting Information

Additional Supporting Information may be found in the online version of this article:

Figure S1. Release of interleukin-3 during autologous and allogeneic mixed lymphocyte reactions and graft-versus-host disease.

Figure S2. Basophils inhibit autologous CD4+ T-cell proliferation by release of interleukin-4 and interleukin-6.

Figure S3. Impact of basophils and interleukin-4 on proliferation of CD4+ T cells.

Figure S4. Analysis of host/donor chimerism.

imm0145-0202-sd1.pdf (249.9KB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. Release of interleukin-3 during autologous and allogeneic mixed lymphocyte reactions and graft-versus-host disease.

Figure S2. Basophils inhibit autologous CD4+ T-cell proliferation by release of interleukin-4 and interleukin-6.

Figure S3. Impact of basophils and interleukin-4 on proliferation of CD4+ T cells.

Figure S4. Analysis of host/donor chimerism.

imm0145-0202-sd1.pdf (249.9KB, pdf)

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