Antibodies and IL-3 support helminth-induced basophil expansion

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Supporting Information

Herbst et al.

SI Materials and Methods

ELISAs. Total and Heligmosomoides polygyrus bakeri excretory–

secretory (HES)-speciﬁc antibody ELISAs were performed as previously described (1). IL-3 ELISAs were performed using the BioLegend antibodies MP2-8F8 and MP2-43D11 according to manufacturer instructions. Circulating HES was detected in the serum of infected mice by sandwich ELISA using a previously described mAb (clone 13.1 at 2μg/mL) (2) labeled with EZ-link NHS–LC–biotin (Thermo Scientiﬁc); the nominal concentration of circulating HES was calculated by reference to standard curve using a stock concentration of culture-derived HES.

Analysis of Helminth-Induced Cytokine Production. MesLN from infected mice were cultured in medium Iscove’s modiﬁed Dul- becco’s medium (Lonza) plus 7% FCS (Lonza) and 5 μg/mL HES (excretory/secretary products collected from adult L5 H.

polygyrus bakeri) for 72 h then restimulated with phorbol–12- myristat–13-acetate (PMA) (Sigma-Aldrich) and ionomycin (Sigma-Aldrich,) for 4 h with Brefeldin A (10μg/mL) added for theﬁnal 2 h. Permeabilized cells were stained with CD4–PercP, anti–IL-4–APC (11B11), IFN–γ-FITC (XMG1.2), or anti–IL-3–

PE (M12-SF8) (Biolegend). Alternatively, bone marrow or spleen cells were incubated with PMA/ionomycin for 24 h and IL-3 levels in the supernatant was determined by ELISA. For some samples, NK1.1⁺ cells were depleted by magnetic cell sorting via Nk1.1 biotin and streptavidin microbeads (MACS) (Milteny Biotech). For intracellular cytokine staining of stimu-

lated bone marrow or spleen cells, cells were additionally incubated with monensin, permeabilzed, and stained with anti–IL- 3 PE (M12-SF8) (Biolegend).

Quantiﬁcation of IL-3 mRNA Expression. Basophils were puriﬁed from in vitro bone marrow cultures by MACs sorting of CD49b⁺ (DX5 Microbeads; Miltenyi Biotech) cells and incubated with 1μg/mL IgE (TIB141) or 1μg/mL anti-FcγRIII (24.G2, rat Ig- G2a) for 60 min at 37 °C. Antibodies were cross-linked with 1μg/

mL antimouse IgE (6HD5) or 1μg/mL antirat IgG2a (2A 8F4;

Southern Biotech) for 60 min. Total RNA was isolated from the indicated cells using TRI reagent (Molecular Research Center) and reverse transcribed using Fast Lane kit (Qiagen). Real-time RT-PCR was performed using Brilliant SYBR Green (Stra- tagene) and an iCycler (Bio-Rad Laboratories). Expression was normalized according to expression of the housekeeping gene β-actin. Sequences of primers used were IL-3, 5′-TTA GCA CTG TCT CCA GAT C-3′and 5′-ACT GAT GAT GAA GGA CC-3′; and β-actin, 5′-CTT TTC ACG GTT GGC CTT AG-3 and 5′-CCC TGA AGT ACC CCA TTG AAC-3′.

Statistical Analysis.For all data, significant differences were determined between gene-deficient or treatment groups and wild- type mice by a one-tailed Studentttest with a confidence interval of 95%. SignificantPvalues are shown at *P<0.05, **P<0.01, or ***P<0.001.

1. McCoy KD, et al. (2008) Polyclonal and speciﬁc antibodies mediate protective immunity against enteric helminth infection.Cell Host Microbe4:362–373.

2. Hewitson JP, et al. (2011) Heligmosomoides polygyrus elicits a dominant nonprotective antibody response directed against restricted glycan and peptide epitopes.J Immunol 187:4764–4777.

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0 200 400 600 800 1000 0

200 400 600 800 1000

10⁰ 10¹ 10² 10³ 10⁴ 100

10¹ 10² 103 10⁴

7.13

0 200 400 600 800 1000 0

200 400 600 800 1000

10⁰ 10¹ 10² 10³ 10⁴ 10⁰

10¹ 10² 103 10⁴

1.37 IgE

Side scatter CD49b C57BL/6

AID-/-

E:eosinophil B:basophil N:neutrophil

blood smear 100x B

E

N

100 μm blood smear 20x

A

B

Fig. S1. Identification of basophils in C57BL/6 and antibody-deficient mice. (A) Pictures represent blood smears fromH. polygyrus bakeri-infected mice stained with Diff-Quik. The eosinophils, basophils, and neutrophils were determined on the basis of morphology and staining as illustrated. (B) Representative FACS plots of basophils using CD49b and IgE or FcεR1 as markers for C57BL/6 or antibody-deficient mice, respectively. Backgated basophils are shown in red to indicate their forward versus side scatter properties. In all experiments, basophils from C57BL/6 mice were additionally stained for FcεR1. However, as a high degree of IgE binding in infected mice was observed to interfere with the efficiency of anti-FcεR1 staining, surface IgE was generally determined as a more reliable marker of FcεR1 expression in these mice.

C

0 1000 2000 3000

Day 0 Day 7

*

0 10 20 30 40 50 60 70 80 90

ng/ml (HES equivalent)

native C57BL/6

boiled C57BL/6 Days following infection

C57BL/6μMT C57BL/6μMT B

A

Day 0 Day 7 Days following infection

Day 10 105

106 107

103

HES -specific IgM (Unirts/ml)

C57BL/6 AID-/-

*

***

Fig. S2. B-cell–deficient mice exhibit increased levels of circulating helminth antigens. (A)H. polygyrus bakeri-specific IgM was quantified for C57BL/6 and AID^−/−mice following primary infection. (B) Presence ofH. polygyrus bakeri-derived antigens (HES) in the serum of C57BL/6 or B-cell–deficient (μMT^−/−) mice was determined by ELISA as described inMaterials and Methods. (C) In a separate experiment, the presence of HES in the pooled serum of C57BL/6 (n= 5) was determined before and after boiling.

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FSC Lineage (CD19, Nk1.1, Gr-1, CD3) IgE IgE

SSC

Live

baso progenitors

mature baso Lineage negative

Sca-1

Sca-1 negative ckit negative Bone marrow

0 50K 100K 150K 200K 250K 0

50K 100K 150K 200K 250K

78.6

0 10³ 10⁴ 10⁵

0 500 1000 1500 2000 2500

31.9 68.1

0 10³ 10⁴ 10⁵

0 1000 2000 3000

4.1 95.9

0 10² 10³ 10⁴ 10⁵ 0

10² 10³ 10⁴ 10⁵

97.7 1.9

0 10² 10³ 10⁴ 10⁵ 0

10² 10³ 10⁴

10⁵ 6.26 0.46

91.2 2.04

FSC Lineage (CD19, Nk1.1, Gr-1, CD3) IgE IgE

SSC c-kit

Live

Sca-1 c-kit negative

baso progenitors

mature baso Lineage negative

0 50K 100K 150K 200K 250K 0

50K 100K 150K 200K 250K

72

0 10³ 10⁴ 10⁵

0 500 1000 1500 2000 2500

52.8 47.2

0 10³ 10⁴ 10⁵

0 500 1000 1500

82.9 17.1

0 10² 10³ 10⁴ 10⁵ 0

10² 10³ 10⁴ 10⁵

97.7 1.88

0 10² 10³ 10⁴ 10⁵ 0

10² 10³ 10⁴

10⁵ 1.57 0.46

96.7 1.29 Sca-1

Sca-1 negative ckit negative

CD34

Spleen

c-kit CD34

A

B

Fig. S3. Gating strategy of mature and progenitor basophils. Basophils from the bone marrow (A) or spleen (B) were deﬁned by a lack of expression of the markers CD3, CD19, NK1.1, Ly6G, Sca-1, and c-kit and a positive expression for CD16/CD32⁺and FcεRI or IgE. Basophils were then further deﬁned as progenitors or mature cells by the presence or absence of CD34. Gating strategies are shown in representative plots fromH. polygyrus bakeri-infected C57BL/6 mice.

A

C57BL/6 IL-3-/-

10

⁴

T otal IgG1 (ng/ml) 10

⁵

10

⁶

10

⁷

10

⁸

10

⁰

T otal IgE (ng/ml) 10

¹

10

²

10

³

10

⁵

10

⁴

0 10 20 30

Days following infection

0 10 20 30

Days following infection

B

Fig. S4. IL-3 is not necessary for antibody production following helminth infection. (A) Total IgG1 and (B) IgE levels present in the serum of C57BL/6 or IL-3^−/−

mice are shown for the indicated time points following primary infection withH. polygyrus backeri.

Bone Marrow Spleen

IL-3 [ng/ml]

whole organ C57BL/6

NK1.1 depleted

C57BL/6 whole organ C57BL/6

NK1.1 depleted C57BL/6 0

0.1 0.2 0.3 0.4 0.5 0.6

0 2 4 6 8 10 12

14 0.079

IL-3 [ng/ml]

Fig. S5. NK1.1⁺cells do not contribute to ex vivo IL-3 production following helminth infection. Bone marrow and spleen cells were isolated fromH. polygyrus bakeriinfected C57BL/6 mice at day 10 postinfection and restimulated with PMA/ionomycin for 24 h. Levels of IL-3 in supernatant of whole organ culture or NK1.1-depleted cell fractions were measured by ELISA.

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A

C57BL/6 IL-4-/-

10⁴

0 10 40 60

Total IgG1 (ng/ml)

Days following infection 10⁵

10⁶ 10⁷ 10⁸

10⁰

Total IgE (ng/ml) 10¹

10² 10³ 10⁵ 10⁴

20 30 50 0 10 20 30 40 50 60

Days following infection

10⁰

HES specific Ig (U/ml)

10⁶

IgG1 IgE

C57BL/6 IL-4-/-

10¹ 10² 10³ 10⁵ 10⁴

C

***

**

10¹

HES specific IgG1 (absorbance OD)

Dilution factor 10² 10³ 10⁴ 0

0.5 1.0 1.5

B

C57BL/6

10⁴

0 10

Total IgG1 (ng/ml)

Days following infection 10⁵

10⁶ 10⁷

20

10⁰

Total IgE (ng/ml)

10¹ 10² 10³

D E

0 150

100

50

**

C57BL/6 IL4Rα-/- C57BL/6

Adult worm numbers

0 10

Days following infection 20

Fig. S6. IL-4–IL-4Rαinteractions are required for helminth-induced antibodies and protective immunity. (A) Total IgG1 and IgE levels present in the serum of C57BL/6, IL-4^−/−, or TCRβδ^−/−mice are shown for the indicated time points following primary infection withH. polygyrus backeri. (B) Antibodies exhibiting speciﬁcity for L5 HES products were determined for C57BL/6, IL-4^−/−, or TCRβδ^−/−mice at day 13 following secondary infection withH. polygyrus backeri. (C) Total IgG1 and IgE levels present in the serum of C57BL/6 or IL-4Rα^−/−mice are shown for the indicated time points following primary infection withH.

polygyrus backeri. (D) Antibodies exhibiting speciﬁcity for L5 HES products and (E) numbers of adult worms were determined for C57BL/6 or IL-4Rα^−/−mice at day 13 following secondary infection withH. polygyrus backeri.

- IgG

+anti-IgG

IgE +anti-IgE 10

1000 100

fold increase in IL-3 mRNA 1

Fig. S7. IgG or IgE cross-linking can elicit IL-3 mRNA expression by bone-marrow–derived basophils in vitro. Relative expression of IL-3 mRNA was determined by quantitative real-time RT-PCR for bone-marrow–derived basophils stimulated by IgE or IgG cross-linking. Data are expressed as fold change of activated versus control basophils. All data are derived from one experiment and are representative of least two independent experiments.

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CD49b

IL-3

0 100 1000 10000 1x10⁵

1.57

0 100 1000 10000 1x10⁵

0 100 1000 10000 1x105

0.16

A

B

% of Max

NK1.1

0 100 1000 10000 1x10⁵

0 20 40 60 80 100

0 100 1000 10000 1x10⁵ 0

20 40 60 80 100

CD4

0 100 1000 10000 1x10⁵

0 20 40 60 80 100

IgE

H. polygyrus bakeri Naive

Fig. S8. CD49⁺CD4⁺NK1.1⁻T cells are the main ex vivo producers of IL-3 following helminth infection. Spleen cells were isolated fromH. polygyrus bakeri- infected C57BL/6 mice at day 10 postinfection, enriched for CD49⁺cells by positive selection using DX5 MACs beads, and restimulated with PMA/ionomycin + monensin for 24 h. (A) IL-3 production was determined for cells isolated from infected or naïve mice by intracellular cytokine staining. (B) IL-3⁺cells (all CD49b^int-hi) from infected mice were gated and their expression of CD4, NK1.1, and IgE was determined (solid lines). Shaded histograms indicate staining for CD4 and NK1.1 in IL-3^-vecells or for IgE in IgE^+veIL-3^-vebasophils.

A

0 1 2 3 4 5 6

0 5 10 15

% CD4 cells producing IL-4

Days following infection C57BL/6

AID-/-

C

0 1 2 3 4

0 5 10

AID-/-

15 B

0 5 10 15 20

0 5 10 15

% CD4 cells producing IL-3

AID-/-

1o Hp 2o Hp

C57BL/6 C57BL/6 + isotype

C57BL/6 + Ba103 D

104

103 105

Fig. S9. AID^−/−mice exhibit normal T-cell cytokine production followingH. polygyrus bakeriinfection. At the indicated time points followingH. polygyrus bakeriinfection, mesenteric lymph node cells from C57BL/6 or AID^−/−mice were cultured with L5 excretory/secretory (HES) antigens and (A) IL-4, (B) IL-3, and (C) IFNγproduction was determined by intracellular cytokine staining andﬂow cytometry. (D) C57BL/6 mice were subjected to primary or secondary infection with H. polygyrus bakeri. Secondary infected mice additionally received 10μg of an isotype control or basophil-depleting antibody (Ba103) on days−2, 0, 5, and 8 postinfection and total IgE levels were determined by ELISA at day 12 postinfection.