IgA deficiency alters systemic immune response to commensal gut microbes

Peyton E. Conrey1*, Lidiya Denu1*, Kaitlin C. O'Boyle1*, Jean-Bernard Lubin PhD1, Tereza Duranova1, Lauren Gianchetti1, Brittany L. Haltzman1, Laura A Vella, MD/PhD1,Jason Xu2, Kyle Bittinger, PhD1, E. John Wherry PhD2,

Sarah E. Henrickson, MD/PhD1, # and Michael Silverman MD/PhD1, #

(1)The Children's Hospital of Philadelphia, Philadelphia, PA, (2) University of Pennsylvania, Philadelphia, PA. *, # = contributed equally.

Rationale: Selective IgA deficiency (SIgAD) is the most common primary immune deficiency. Symptomatic patients can experience increased atopy, recurrent infections or autoimmunity, though patients are frequently asymptomatic. Since IgA promotes homeostasis with commensal microbes, we investigated whether SIgAD impaired commensal microbe compartmentalization and altered systemic immune responses.

Blood and fecal samples were collected from 13 pairs of pediatric SIgAD patients and IgA sufficient siblings. Deep immunoprofiling using flow cytometry, CyTOF, cytokine analysis and ELISAs for Ig binding to fecal microbes was combined with metagenomic analysis of fecal microbiomes and microbial flow cytometry (mFLOW) of the IgA, IgG and IgM bound fecal microbiomes. mFLOW was performed by applying patient’s serum antibodies to their fecal microbes, assessing binding of immunoglobulin isotypes and performing metagenomic sequencing of Ig-bound microbes.

Figure 1: Experimental workflow.

Abstract

Metagenomic Analysis

Conclusions

Figure 8: Fecal microbiome in SIgAD patients and sibling controls. Microbiome diversity similar among SIgAD patients

and controls.

Study Design

MethodsFigure 3: Elevated serum inflammatory cytokines in SIgAD.

Significant differences are indicated by **p ≤ 0.007 and ***p ≤ 0.005.

Figure 4: Elevated serum IgG in SIgAD. Significant differences are indicated by ***p ≤ 0.005.

• Developed cohort to study immune system and microbiome in SIgAD patients

• Similar bulk fecal microbiome diversity in SIgAD and controls

• Increased systemic inflammation observed in SIgAD patients

• SIgAD has significant impacts on immunophenotype and access of the systemic immune response to commensal gut microbes

• Increased commensal bound IgG+ in SIgAD patients• These findings provide novel strategies for developing

prognostic markers.

Figure 7: Microbial flow (mflow) analysis to characterize the antibody response to the microbiota.

Control

Case

0

20

40

60

All samples IgG stool plus serum (serum definitions only)

**Isotypecontrol Bacteria+secondaryAbs

Bacteria+serum+secondaryAbs

IgA

IgG

IgA deficient

Healthy sibling

mFLOW-Seq: Microbial Flow Cytometry and Metagenomic Sequencing

Flow cytometry

Collect stooland serum

Isolate bacteria Add serum antibodies Add secondary antibodies

Control IgA deficient

% m

icro

bes b

ound

by

IgG

control case0

20

40

60

80

100

% M

icro

bes

Po

siti

ve

mflow Stool neg vs Stool pos-Stool + serum-IgG

control

case

n=17 n=15

* (0.0307)

Figure 5: Deep immunoprofiling of CD3 T cells to identify potentially different cellular populations between conditions.

PBMCs were labeled with metal-conjugated antibodies and detected by Mass Cytometry (CyTOF). A) Dimensionality reduction was

performed using Phenograph and displayed as SIgAD on the left and control on the right. Each cell is represented by a dot and the

cells contributing to a given cluster are a unique color. B) tSNE plot showing the topology of each cluster in SIgAD (left) and Controls (right). Yellow indicates an area of more cells and black or purple

indicates fewer cells in each given cluster.

Observed Shannon

Control Case Control Case

2

3

4

50

75

100

125

150

study_group

Alpha

Divers

ity Me

asure

Alpha DiversityAlpha Diversity

Control SIgAD Control SIgAD

Figure 2: Elevated IgG-memory B cells in SIgAD.Immunoglobulin (Ig) expressing memory B cells

(CD19+CD27+) were detected by flow cytometry surface staining. Significant differences are indicated by *p < 0.05

and ****p ≤ 0.01.

Immunoprofiling

Results• Higher frequency of fecal microbes targeted by serum

IgG in SIgAD patients• Many inflammatory cytokines including IL-4, IL-5, IL13

and IL-17a are elevated in SIgAD• Elevated serum IgG in SIgAD patients• Elevated IgG+ circulating memory B cells in SIgAD• Similar fecal microbiomes between SIgAD and control• Sorted and sequenced IgG, IgA and IgM targeted

microbes from healthy patients and IgG and IgM targeted microbes from SIgAD

Control SIgAD0

200

400

600

IL-1

7a (p

g/m

l)

✱✱✱

Control SIgAD0

200

400

600

800

IL-1

3 (p

g/m

l)

✱✱

Control SIgAD

20

40

60

80

IL-5

(pg/

ml)

✱✱

Control SIgAD0

200

400

600

800

1000

IL-4

(pg

/ml)

✱✱

SIgAD Control

co n tro l s Ig Ad0

500

1000

1500

2000

IgA

ug/m

L

<0.0001

co n tro l s Ig Ad0

2000

4000

6000

Ig M

ug/m

L

control SIgAd0

5000

10000

15000

20000

µg/

mL

***

IgG

𝑢 g/m

L

𝑢 g/m

L

𝑢 g/m

L

IgA IgM IgG

Control SIgAD0

10

20

30

40

% Ig

A+I

gG- o

f Mem

ory

B C

ells ✱✱✱✱

Control SIgAD0

5

10

15

20

% Ig

M+I

gD- o

f Mem

ory

B c

ells

Control SIgAD0

20

40

60

80

% Ig

G+I

gA- o

f Mem

ory

B C

ells ✱

SIgAD Control

Microbial Flow Cytometry

Control

Case

0

20

40

60

All samples IgG stool plus serum (serum definitions only)

**Isotypecontrol Bacteria+secondaryAbs

Bacteria+serum+secondaryAbs

IgA

IgG

IgA deficient

Healthy sibling

mFLOW-Seq: Microbial Flow Cytometry and Metagenomic Sequencing

Flow cytometry

Collect stooland serum

Isolate bacteria Add serum antibodies Add secondary antibodies

Control IgA deficient