Human Fc Receptor-like 3 Inhibits Regulatory T Cell Function and Binds Secretory IgA

Report

Human Fc Receptor-like 3 Inhibits Regulatory T Cell Function and Binds Secretory IgA Graphical Abstract

Authors Stuti Agarwal, Zachary Kraus, Jessica Dement-Brown, Oyeleye Alabi, Kyle Starost, Mate Tolnay

Correspondence [email protected]

In Brief The key role of secretory IgA in neutralizing pathogens in mucosal surfaces is well established. Agarwal et al. propose that secretory IgA entering the body through breached mucosa engages FCRL3 on regulatory T cells and suppresses their inhibitory function. FCRL3 could be therapeutically targeted to modulate regulatory T cell activity.

Highlights d

Secretory IgA binds FCRL3, whereas dimeric IgA and monomeric IgA do not bind

d

FCRL3 stimulation with an antibody or ligand inhibits regulatory T cell functionality

d

FCRL3-stimulated regulatory T cells express RORgt and produce IL-17, IL-26, and IFNg

Agarwal et al., 2020, Cell Reports 30, 1292–1299 February 4, 2020 https://doi.org/10.1016/j.celrep.2019.12.099

Cell Reports

Report Human Fc Receptor-like 3 Inhibits Regulatory T Cell Function and Binds Secretory IgA Stuti Agarwal,1,2 Zachary Kraus,1,2 Jessica Dement-Brown,1 Oyeleye Alabi,1,3 Kyle Starost,1,4 and Mate Tolnay1,5,* 1Office

of Biotechnology Products, CDER, US Food and Drug Administration, 10903 New Hampshire Ave., Silver Spring, MD 20993, USA authors contributed equally 3Present address: PPDM Discovery Bioanalytics, Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115, USA 4Present address: Harvard T.H. Chan School of Public Health, 665 Huntington Avenue, Boston, MA 02115, USA 5Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.celrep.2019.12.099 2These

SUMMARY

Human Fc receptor-like 3 (FCRL3) is an orphan receptor expressed by lymphocytes, including regulatory T cells. FCRL3 is implicated in several autoimmune diseases; however, its function on regulatory T cells is unknown. We discovered that FCRL3 stimulation of regulatory T cells inhibited their suppressive function. Moreover, FCRL3 stimulation induced IL-17, IL-26, and IFNg production and promoted expression of the Th17-defining transcription factor RORgt without affecting FOXP3 expression. We suggest that FCRL3 engagement mediates a transition of regulatory T cells to a pro-inflammatory Th17-like phenotype. In addition, we identified secretory IgA as a specific FCRL3 ligand. Secretory IgA could serve as an environmental cue for mucosal breaches and locally drive regulatory T cell plasticity to help control infection. Our findings define a mechanism that explains the recognized association of FCRL3 with autoimmune diseases. Targeting FCRL3 to modulate regulatory T cell activity could be exploited to treat both malignancies and autoimmune diseases. INTRODUCTION Regulatory T (Treg) cells are critical for the maintenance of immune homeostasis, whereas dysregulated Treg cell function results in autoimmunity (Josefowicz et al., 2012; Sakaguchi et al., 2008). Thymus-derived Treg cells are positively selected in the thymus based on recognition of self-peptides and function to limit immune responses against the host. Treg cells, like other T cell subsets, have considerable phenotypic plasticity (DuPage and Bluestone, 2016). Treg cells can express FOXP3, the master regulator of Treg cell function, together with the master transcription factor of each T helper cell subset, possibly to adapt to the local inflammatory environment (Wing and Sakaguchi, 2012). Particularly, a small subset of human Treg cells expresses the Th17-defining transcription factor RORgt (retinoic acid-related orphan receptor gamma t) and produce interleukin-17 (IL-17) and IL-22 (Ayyoub et al., 2009). Furthermore, human thymus-derived Treg cells can be induced by the cytokines IL-6, IL-1b, and IL-23 to produce

IL-17 (Koenen et al., 2008). The functional activity of Treg cells is subject to regulation by external signals; Toll-like receptor (TLR) agonists, particularly those to TLR2 and TLR8, act directly on Treg cells and reduce their suppressive function (Peng et al., 2005; Sutmuller et al., 2006; Voo et al., 2014). Similarly, C3a and C5a, binding to their corresponding receptors on Treg cells, diminish suppressive activity (Kwan et al., 2013). TLR2 stimulation promotes transition of human Treg cells into a Th17-like phenotype, characterized by IL-17, IL-22, and RORgt expression, denoting a link between Treg cell functionality and phenotype (Nyirenda et al., 2011). Down-modulation of Treg cell suppressive function through TLR and complement receptors in an inflammatory environment could promote pathogen clearance but at a cost of increased risk of autoimmune responses. The mechanisms that promote homeostatic tolerance to commensals and self-antigens but allow immune responses against pathogens remain ambiguous. Importantly, Treg cellular therapy has the potential to promote tolerance in autoimmune patients and transplant recipients (Kasagi et al., 2014). A complementary therapeutic approach is blocking Treg cell function to set free the anti-tumor immune responses of cancer patients (Sharabi et al., 2018). Fc receptor-like (FCRL) in humans is a family of six transmembrane and two cytoplasmic proteins that are emerging regulators of B cell function (Davis, 2007). A more complete understanding of the functional roles of FCRL family members is hampered by a lack of animal models because of differences in gene organization and protein structures in humans versus mice. Human FCRL3 is expressed on a subset of Treg, B, natural killer (NK), CD8+ T, and gdT cells (Bajpai et al., 2012; Davis et al., 2001; Polson et al., 2006; Nagata et al., 2009). FCRL3 is expressed on Treg cells from fetal thymus but not on in vitro-induced Treg cells (Swainson et al., 2010), suggesting the thymic origin of these cells while not excluding possible expression of FCRL3 by induced Treg cells in mucosal tissues. FCRL3 contains both positive and negative signaling motifs and has dual signaling capacity in B cells (Li et al., 2013); however, no studies to date have addressed FCRL3 function in other cell types. A single-nucleotide polymorphism in the FCRL3 promoter that affects an nuclear factor kB (NF-kB) binding site, which results in higher protein levels, predisposes to a wide range of autoimmune conditions, including rheumatoid arthritis, systemic lupus erythematosus, and autoimmune thyroid diseases, whereas the same polymorphic variant appears to confer protection against multiple sclerosis (Kochi et al., 2005; Matesanz et al., 2008). FCRL3 is one proposed causal gene in rheumatoid arthritis,

1292 Cell Reports 30, 1292–1299, February 4, 2020 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

based on data from patients with early disease (Thalayasingam et al., 2018). Importantly, FCRL3 expression on Treg cells positively correlates with rheumatoid arthritis disease activity (Bajpai et al., 2012). Although it is clear that FCRL3 is a marker for many autoimmune diseases, its ligand remains unknown. Three members of the family, FCRL3–FCRL5, contain homologs of the two domains that confer immunoglobulin-binding competence for Fc receptors (Davis, 2007). FCRL4 binds immunoglobulin A (IgA), and FCRL5 binds IgG (Wilson et al., 2012; Franco et al., 2013), raising the prospect of FCRL3 binding an immunoglobulin. Most pathogens enter through mucosal surfaces. Secretory IgA (SIgA), the most abundant immunoglobulin, protects mucosal surfaces primarily by passively neutralizing pathogens (Brandtzaeg, 2013). Pathogenic bacteria are marked by high SIgA coating, and SIgA-coated bacteria are associated with inflammatory conditions driven by Th17 cells (Palm et al., 2014; Viladomiu et al., 2017). SIgA is composed of dimeric IgA covalently linked to a short J chain and a heavily glycosylated secretory component (SC) that itself is a proteolytic fragment of the polymeric Ig receptor (pIgR) (Bonner et al., 2007; Stadtmueller et al., 2016). Dimeric IgA on epithelial cells binds to pIgR, which then transports the bound IgA through the cell to the mucosal side, where SIgA is released upon proteolytic cleavage of the pIgR, which remains attached to IgA as an SC (Braathen et al., 2007). Active and passive mechanisms exist that retrieve SIgA back from the mucus into tissues, where it can engage immune cells (Rochereau et al., 2013; Matysiak-Budnik et al., 2008). A potential major route of SIgA transport from the mucus into tissues is via mucosal breaches because of physical damage or other causes, resulting in localized microbial translocation. We established a previously unreported communication pathway through FCRL3, allowing SIgA-containing immune complexes direct access to Treg cells, inhibiting Treg cell suppressive function.

40.1% ± 14.5% (mean ± SD, n = 7) of tonsil Treg cells expressed FCRL3 (Figure S2). We conclude that FCRL3 inhibits the suppressive activity of Treg cells. FCRL3 Promotes a Th17-like Phenotype of Treg Cells The transition of Treg cells into a Th17-like phenotype is well documented and could explain the reduced suppressive activity of Treg cells upon FCRL3 stimulation. Therefore, we assessed four cytokines produced by Th17 cells: IL-17, IL-22, IL-26, and interferon g (IFNg) (Wilson et al., 2007). FCRL3-stimulated Treg cells and responder cells were cultured together under the same conditions as employed in our Treg suppression assays. FCRL3 stimulation significantly increased the fraction of Treg cells staining positive for intracellular IL-17 and IL-26 protein after 15 and 24 h. IFNg protein expression was moderately but significantly increased, whereas IL-22 protein was not induced (Figure 2A). To substantiate the findings, we measured secreted IL-26 and IL-17 in Treg and responder cell co-cultures. We found that FCRL3 stimulation of Treg cells, with and without CD3 costimulation, promoted IL-26 secretion, whereas TLR2 or CD3 stimulation alone had no effect (Figure 2B). FCRL3 stimulation also promoted IL-17 secretion (Figure 2B). RORgt is the master regulator of the Th17 phenotype; therefore, RORgt and IL-17 protein levels were assessed in FCRL3stimulated Treg cells together with FOXP3. We found that FOXP3 expression remained stable following FCRL3 stimulation, indicating that diminished Treg functionality was not due to reduced FOXP3 expression (Figure 2C). Moreover, by 24 h after FCRL3 stimulation, 54% of Treg cells co-expressed FOXP3+ and RORgt, indicating that the cells maintained Treg identity while acquiring a Th17-like phenotype. A significant portion of FOXP3+ cells also contained IL-17 protein. We conclude that FCRL3 stimulation promotes the development of FOXP3+ Treg cells that co-express RORgt and produce the pro-inflammatory cytokines IL-17, IL-26, and IFNg.

RESULTS FCRL3 Stimulation Inhibits Treg Cell Suppressive Function The functional role of FCRL3 on Treg cells is unknown. We stimulated sorted Treg cells with a specific FCRL3 monoclonal antibody (Ab) to test its effect on Treg cell function in in vitro suppression assays. Sorted CD4+CD25highCD127 Treg cells were loaded with violet dye to allow distinction from responder cells in culture (Figure S1). Treg cells were then pre-incubated with FCRL3 Ab, CD3 Ab, or TLR2 ligands prior to being added to carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous responder cells stimulated with CD3/CD28 beads. We found that FCRL3 stimulation of Treg cells blocked their ability to inhibit proliferation of both CD4+ and CD8+ responder T cells (Figures 1A–1C). The effect of FCRL3 stimulation was comparable with that of TLR2 ligands, used as a positive control. CD3 stimulation had no effect, as expected, whereas CD3 and FCRL3 co-stimulation was comparable with FCRL3 stimulation alone. Similar results were obtained using sorted CD4+ responder cells (Figures 1D and 1E). To support the physiological relevance of our findings in mucosal tissues, we assessed FCRL3 expression on Treg cells obtained from tonsils, a mucosa-associated lymphoid tissue. We found that

SIgA Binds FCRL3 and Inhibits Treg Cell Suppressive Function Inhibition of Treg cell suppressive function by engaging FCRL3 with an agonistic Ab implies that a physiological FCRL3 ligand would regulate Treg function. However, FCRL3 has been reported not to bind IgG, IgA, or IgM (Wilson et al., 2012). We evaluated whether SIgA binds FCRL3. Recombinant FCRL3 was immobilized on the sensor, and SIgA or plasma IgA was run in solution. We detected SIgA binding to FCRL3, whereas plasma IgA did not bind. SIgA bound to FCRL3 with 0.45 ± 0.32 mM affinity (mean ± SD, n = 3) and followed simple 1:1 kinetics, indicative of a single interaction site (Figure 3A). The interaction consisted of slow association (Ka = 570 ± 69 1/Ms) and slow dissociation (Kd = 2.6 3 10 4 ± 1.8 3 10 4 1/s). To further define the FCRL3 ligand, we size-fractionated the IgA samples and assessed each protein peak. The SIgA sample consisted of one protein peak of 99% SigA or more, based on the presence of IgA heavy chain, J chain, and SC (Figure 3B, top panel). The plasma IgA sample contained 20.5% dimeric IgA with J chain and 77.8% monomeric IgA lacking J chain. We found that SIgA had full FCRL3-binding activity, whereas dimeric IgA and monomeric IgA did not bind at all (Figure 3B, bottom panel).

Cell Reports 30, 1292–1299, February 4, 2020 1293

Treg pre-stimulation

A No stim.

No Treg

None

TLR2

FCRL3

CD3

FCRL3+CD3

CFSE Treg pre-stimulation

B No stim.

No Treg

None

TLR2

FCRL3

CD3

FCRL3+CD3

Figure 1. FCRL3 Stimulation Inhibits Treg Cell Suppressive Function (A–E) Treg cells were incubated with biotinylated FCRL3 Ab, CD3 Ab, or TLR2 ligands, as indicated, and then with streptavidin. Autologous peripheral blood mononuclear cells (PBMCs; A–C) or CD4+ responder cells (D and E) were labeled with CFSE, combined with the pre-stimulated Treg cells, and stimulated with CD3/CD28 beads. Proliferation of CD4+ (A and D) and CD8+ (B) responder cells are shown. In (C) and (E), cell division indices normalized to the no-Treg control are summarized. Mean ± SD (n = 8 for A, n = 9 for B, n = 3 for E); unpaired t test; *p < 0.05, **p < 0.01, ***p < 0.0001.

CFSE 1.5

CD4+ responder cells

*** *** ***

1.0

Cell division index

Cell division index

C

0.5

0 CD3/CD28: Treg: -

+ -

+ +

+ +

+ +

+ +

CD8+ responder cells

1.5

*** *** ***

1.0 0.5

0 CD3/CD28: Treg: -

+ +

+ -

+ +

+ +

+ +

+ +

Treg pre-stimulation

Treg pre-stimulation

Treg pre-stimulation

D No stim.

No Treg

None

FCRL3

CD3

CFSE

Sorted CD4+ responder cells Cell division index

E

1.5

**

*

1.0 0.5

0 CD3/CD28: Treg:

-

+ -

+ +

+ +

+ +

+ +

+ +

FCRL3+CD3

(Wilson et al., 2012). Subsequently, we assessed whether SIgA binds endogenous FCRL3 on primary lymphocytes. We observed significant binding of SIgA to B cells and to Treg cells, where SIgA binding intensity correlated with FCRL3 expression level. However, no significant binding was detected to conventional CD4+FOXP3 T cells, where FCRL3 expression was weakest (Figure 3D). In some samples, cells were pre-incubated with an FCRL3 Ab that blocked SIgA binding to recombinant FCRL3 (Figure S3). The FCRL3-blocking Ab inhibited SIgA binding to both B cells and Treg cells, indicating that SIgA binding was FCRL3 dependent (Figure 3D). As expected, we did not detect binding of plasma IgA to any examined cell type. We conclude that FCRL3 specifically binds SIgA. Finally, we assessed whether SIgA itself alters Treg cell functionality. Biotinylated SIgA and streptavidin treatment impaired Treg-mediated inhibition of responder cell proliferation comparable with biotinylated FCRL3 Ab and streptavidin (Figure 3E). As an additional control, we treated Treg cells with FCRL3 Ab in the absence of further crosslinking; this stimulation produced a consistent but small effect approaching statistical significance (p = 0.055). We conclude that SIgA ligand inhibits Treg cell suppressive function. DISCUSSION

Treg pre-stimulation

Next we assessed whether FCRL3 expressed by cells bound SIgA. We found SIgA bound to FCRL3-transfected but not mock-transfected 293T cells (Figure 3C). In contrast, plasma IgA did not bind to transfected FCRL3, as reported previously

1294 Cell Reports 30, 1292–1299, February 4, 2020

Treg cells are master regulators of peripheral immune tolerance, whereas SIgA is an essential component of mucosal immunity. We established FCRL3 regulates Treg cell function and propose SIgA to be a functional FCRL3 ligand. Our studies identified FCRL3 as a potential target of therapeutic intervention in both malignancies and autoimmune conditions.

No stim. 19.7

FCRL3 43.5

IL-17+ (%)

A

1.9

2.2

IL-22+ (%)

IL-17

0.2

18.3

IL-26+ (%)

IL-22

IL-26

60 40

IFNγ+ (%)

CD4

23.1

IFNγ

0

ng/ml

8

24 h

15 h

24 h

20 10 0 40

*

20

IL-26

* * *

*

0 15 h

20

24 h

*

10 0 15 h

24 h

1000 IL-17

4 0

15 h 30

ng/ml

B

**

20

30 2.2

**

*

500

0

Treg pre-stimulation

73.3

85.1

100

FOXP3+ (%)

FCRL3

CD4

C

No stim.

Double+ (%)

55.1

RORγt

17.6

80

30

FOXP3

IL-17

5.5

FOXP3

27.5

0

Double+ (%)

FOXP3

50

40

15 h

*

24 h

*

0

20 10

15 h

24 h

*

0 15 h

24 h

Figure 2. FCRL3 Stimulation of Treg Cells Promotes a Th17-like Phenotype Treg cells and responder cells were treated as in Figure 1. (A) Intracellular expression of the indicated cytokines in Treg cells was assessed. Representative experiments are shown at 15 h stimulation. Mean ± SD (n = 8 for IL-17, n = 5 for IL-22 and IL-26, n = 6 for IFNg); paired t test; *p < 0.05, **p < 0.01. (B) Secreted IL-26 and IL-17 was measured after 3.5 days. Mean ± SD (n = 3); asterisk indicates that IL-26 was below the level of quantitation. (C) Intracellular expression of the indicated cytokines and transcription factors in Treg cells. Representative experiments are shown at 24 h stimulation. Mean ± SD (n = 3); paired t test; *p < 0.05, **p < 0.01. In (A) and (C), gates were set based on single-Ab unstained control samples (data not shown). Gray bars represent unstimulated and black bars FCRL3stimulated Treg cells.

We found that FCRL3 engagement rendered Treg cells unable to effectively suppress the proliferation of CD4+ and CD8+ responder T cells. Inhibition of Treg-mediated suppression by both FCRL3 Ab and SIgA in co-cultures containing only sorted purified Treg cells and CD4+CD25 responder cells indicates that no other cell type is required as part of the mechanism. Critically, we demonstrated that the SIgA ligand itself inhibited Treg suppressive function, establishing it as a functional FCRL3 ligand. In addition, we established that FCRL3 stimulation promoted the transition of Treg cells toward a Th17-like phenotype, marked by elevated RORgt, IL-17, IL-26, and IFNg expression. Similar phenotypic transition, marked by RORgt, IL-17, and IL22 expression, has been reported following TLR2-induced downregulation of human Treg cell function (Nyirenda et al., 2011). Several studies have established that human Treg cells can co-express FOXP3 and RORgt and produce IL-17 (Ayyoub et al., 2009; Koenen et al., 2008). IL-17 secretion by Treg cells at mucosal sites has been proposed to contribute to early response to infection (Ayyoub et al., 2009). To our knowledge, IL-26 production by Treg cells has not been reported previously. IL-26 directly kills bacteria and promotes innate immunity (Meller et al., 2015); therefore, its production by Treg cells could help control invading bacteria at sites of mucosal breach. Moreover, IL-26 promotes generation of Th17 cells and is overexpressed in rheumatoid arthritis, indicating a potential role for IL-26 in driving inflammatory diseases (Corvaisier et al., 2012). We found that transfected, endogenous, and recombinant FCRL3 binds SIgA. In contrast, FCRL3 did not bind plasma IgA. SIgA bound to transfected FCRL3 on 293T cells. Additionally, SIgA, but not plasma IgA, bound to primary B and Treg cells in an FCRL3-dependent manner. IgA from plasma is a mixture of mostly monomeric IgA and 15% dimeric IgA linked to J chain but lacking SCs (Longet et al., 2013). We found that neither dimeric IgA nor monomeric IgA bound FCRL3, indicating that the specific FCRL3 ligand is SIgA. The interaction of SIgA with recombinant FCRL3 had 0.45 mM affinity and followed 1:1 kinetics, indicative of a single interaction site. We predict that the SC, which in SIgA assumes a conformation distinctly different from that of a free SC (Bonner et al., 2009), is part of the FCRL3 interaction surface because this is the only component not present in dimeric IgA. The SIgA-FCRL3 interaction consisted of slow association and slow dissociation, possibly denoting a biological requirement for the sustained presence of the ligand to initiate

Cell Reports 30, 1292–1299, February 4, 2020 1295

(legend on next page)

1296 Cell Reports 30, 1292–1299, February 4, 2020

Homeostasis:

Inflammation: FCRL3 Ab or SIgA

FCRL3

FCRL3

FOXP3

FOXP3 RORγt

Treg

Th17-like Treg

IL-17 IL-26 IFNγ

? Tolerance to self & commensals

Pathogen & tumor clearance?

Figure 4. The Phenotypic and Corresponding Functional Plasticity of Treg Cells Balances Immune Homeostasis and Inflammation Under homeostatic conditions, Treg cells express FOXP3 and promote tolerance. In inflammatory environments, such as at sites of mucosal breach signaled by SIgA through FCRL3, Treg cells can transition to co-express RORgt and produce IL-17, IL-26, and IFNg. Such cytokine-producing Th17like Treg cells have diminished capacity to restrain immune responses; instead, they might actively contribute to pathogen and tumor clearance. It is unclear whether the transition process is two way and whether Th17-like Treg cells can revert to Treg cells.

a signal. If FCRL3 interacts with the SC as predicted, then the slow association could be due to conformational flexibility of SC when part of SIgA (Bonner et al., 2009), with only some of the SC conformations permitting FCRL3 binding. SIgA is an ideal molecule to signal breach of the mucosal barrier because it is specifically secreted into the mucus while normally excluded from tissues. The most ancient genes in the extended Fc receptor family encode the pIgR, FCRL, and the common g chain, which all appear in bony fish (Akula et al., 2014). Therefore, the finding that SIgA, which contains a large portion of the pIgR, serves as a FCRL3 ligand suggests an ancient evolutionary origin

for this interaction. Strong support of SIgA interacting with human Treg cells in vivo comes from a study that identified the IgA heavy chain as the most over-represented protein in the membraneassociated fraction of human Treg cells compared with conventional T cells (Procaccini et al., 2016). In the same study, the IgA heavy chain protein was also detected within the cytoplasm of Treg cells, suggesting that it is internalized. The presence of IgA on the surface and in human Treg cells cannot be explained by any established mechanism other than SIgA binding to FCRL3. In the present study, we employed both FCRL3 Ab and SIgA ligand to establish the functional role of FCRL3 on Treg cells. In most experiments, the FCRL3 Ab and SIgA were cross-linked with streptavidin, providing robust Treg cell stimulation. Such extensive stimulation could mimic a physiological immune complex assembled in the mucus, containing SIgA, antigen, complement fragments, and TLR ligands. C3a, C5a, and TLR ligands have been reported to reduce Treg cell suppressive function (Kwan et al., 2013; Peng et al., 2005; Sutmuller et al., 2006; Voo et al., 2014; Nyirenda et al., 2011). Therefore, FCRL3 stimulation in vivo may occur in combination with complement receptor and TLR stimulation. Notably, FCRL3 Ab without crosslinking had a small inhibitory effect on Treg suppressive function, raising the possibility that it may be effective as a therapeutic agent. Regardless of the nature of the physiological FCRL3 ligand, we demonstrated the potential of FCRL3 Abs as therapeutic agents. Regulation of Treg cell function by SIgA, alone or as part of an immune complex, could promote a localized inflammatory environment, contributing to elimination of invading pathogens (Figure 4). Such a mechanism may also enable immune responses against pathogens that are cross-reactive with self-antigens (Weissler and Caton, 2014). The 0.45 mM affinity of FCRL3 is insufficient to bind SIgA in serum, which averages 10.9 mg/mL (0.03 mM) (Delacroix and Vaerman, 1981). The requirement of higher-than-systemic SIgA concentrations could therefore limit inhibition of Treg cell function to sites of acute mucosal breach. We propose that SIgA is a physiological FCRL3 ligand that restrains Treg cell activity at sites of mucosal breaches to control infection. Conversely, this mechanism, under certain conditions, could disrupt the balance between self-tolerance and immune responses against pathogens and explain the established association of FCRL3 with autoimmune diseases (Kochi et al., 2005; Matesanz et al., 2008). Our insight that FCRL3 inhibits Treg cell function provides a reasonable explanation why autoimmune

Figure 3. SIgA Binds FCRL3 and Inhibits Treg Cell Suppressive Function (A) Association and dissociation of soluble SIgA (top) and plasma IgA (bottom) two-fold diluted from 10 mM to recombinant FCRL3 by surface plasmon resonance. Blue lines represent actual interactions; black lines represent fits using the 1:1 kinetic model. (B) SIgA and plasma IgA samples were size-fractionated (left panel) and assessed for IgA heavy chain, J chain, and SC. Comparable fractions were combined and re-tested (bottom of right panel). SIgA (peak A), dimeric IgA (dIgA, peak B), monomeric IgA (mIgA, peak C), unfractionated plasma IgA (pIgA), and SIgA were assessed at 4 mM for FCRL3 binding (mean ± SD, n = 3) (right panel). (C) FCRL3-transfected 293T cells or control cells were incubated with biotinylated SIgA or plasma IgA and then with streptavidin. Left: FCRL3 expression (red) is compared with the control (shaded). Center: binding of SIgA (solid red) and plasma IgA (dashed blue) to FCRL3-transfected cells compared with SIgA binding to control cells (shaded). On the right, relative binding compared with the control; mean ± SD (n = 4); unpaired t test; *p < 0.05. (D) Primary lymphocytes were incubated with biotinylated SIgA or plasma IgA and then with streptavidin. Top: FCRL3 expression (red) compared with the isotype control (shaded). Middle: binding of SIgA (solid red), SIgA after FCRL3-blocking Ab (dashed blue) and plasma IgA (dotted orange) compared with the streptavidin control (shaded). Bottom: summary of relative binding compared with the streptavidin control; mean ± SD (n = 6); unpaired t test; *p < 0.05, **p < 0.01. (E) Treg cells were incubated with biotinylated SIgA or FCRL3 Ab with or without streptavidin (SA), combined with sorted CD4+ responder cells, and stimulated with CD3/CD28 beads. A representative experiment is shown at the top. Cell division indexes normalized to the no-Treg control are summarized at the bottom. Mean ± SD (n = 6), unpaired t test, ***p < 0.001.

Cell Reports 30, 1292–1299, February 4, 2020 1297

diseases are more prevalent in individuals with higher FCRL3 expression, clearly pointing to the in vivo significance of this pathway. Phenotypic transition of T cells from a regulatory to an inflammatory program can help control infections and cancer but exacerbate autoimmune conditions. Insights into physiological modulation of Treg cell function offer distinct tools for therapeutic intervention. Modulation of Treg cell function through FCRL3 has therapeutic potential because FCRL3 is a surface protein expressed only on a subset of lymphocytes. Blocking FCRL3 signaling may represent a therapeutic approach to treat autoimmune conditions, whereas promoting FCRL3 signaling could be useful to boost cancer immunotherapy and immune responses to pathogens. Our studies highlight the potential use of FCRL3 Abs to inhibit Treg cell functionality, offering a therapeutic strategy to promote immune responses. This approach could also be combined with established immune checkpoint inhibitors, which act on effector T cells and have revolutionized cancer immunotherapy, to provide more efficient anti-tumor immunity. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d d

d d

KEY RESOURCES TABLE LEAD CONTACT AND MATERIALS AVAILABILITY EXPERIMENTAL MODEL AND SUBJECT DETAILS B Primary Human Cell Cultures METHOD DETAILS B Treg Cell Purification B Treg Cell Suppression Assays B Measurement of Secreted IL-26 and IL-17 B Intracellular Flow Cytometry B Cell Binding Assays B Surface Plasmon Resonance B Size Exclusion Chromatography B Western Blotting QUANTIFICATION AND STATISTICAL ANALYSIS DATA AND CODE AVAILIBILITY

interpretation, and they also edited the manuscript. M.T. conceived the study and wrote the manuscript. DECLARATION OF INTERESTS The authors declare no competing interests. Received: April 8, 2019 Revised: December 18, 2019 Accepted: December 27, 2019 Published: February 4, 2020 REFERENCES Akula, S., Mohammadamin, S., and Hellman, L. (2014). Fc receptors for immunoglobulins and their appearance during vertebrate evolution. PLoS ONE 9, e96903. Alabi, O., Dement-Brown, J., and Tolnay, M. (2017). Human Fc receptor-like 5 distinguishes IgG2 disulfide isoforms and deamidated charge variants. Mol. Immunol. 92, 161–168. Ayyoub, M., Deknuydt, F., Raimbaud, I., Dousset, C., Leveque, L., Bioley, G., and Valmori, D. (2009). Human memory FOXP3+ Tregs secrete IL-17 ex vivo and constitutively express the T(H)17 lineage-specific transcription factor RORgamma t. Proc. Natl. Acad. Sci. USA 106, 8635–8640. Bajpai, U.D., Swainson, L.A., Mold, J.E., Graf, J.D., Imboden, J.B., and McCune, J.M. (2012). A functional variant in FCRL3 is associated with higher Fc receptor-like 3 expression on T cell subsets and rheumatoid arthritis disease activity. Arthritis Rheum. 64, 2451–2459. Bonner, A., Perrier, C., Corthe´sy, B., and Perkins, S.J. (2007). Solution structure of human secretory component and implications for biological function. J. Biol. Chem. 282, 16969–16980. Bonner, A., Almogren, A., Furtado, P.B., Kerr, M.A., and Perkins, S.J. (2009). Location of secretory component on the Fc edge of dimeric IgA1 reveals insight into the role of secretory IgA1 in mucosal immunity. Mucosal Immunol. 2, 74–84. Braathen, R., Hohman, V.S., Brandtzaeg, P., and Johansen, F.E. (2007). Secretory antibody formation: conserved binding interactions between J chain and polymeric Ig receptor from humans and amphibians. J. Immunol. 178, 1589–1597. Brandtzaeg, P. (2013). Secretory IgA: Designed for Anti-Microbial Defense. Front. Immunol. 4, 222. Corvaisier, M., Delneste, Y., Jeanvoine, H., Preisser, L., Blanchard, S., Garo, E., Hoppe, E., Barre´, B., Audran, M., Bouvard, B., et al. (2012). IL-26 is overexpressed in rheumatoid arthritis and induces proinflammatory cytokine production and Th17 cell generation. PLoS Biol. 10, e1001395.

SUPPLEMENTAL INFORMATION

Davis, R.S. (2007). Fc receptor-like molecules. Annu. Rev. Immunol. 25, 525–560.

Supplemental Information can be found online at https://doi.org/10.1016/j. celrep.2019.12.099.

Davis, R.S., Wang, Y.H., Kubagawa, H., and Cooper, M.D. (2001). Identification of a family of Fc receptor homologs with preferential B cell expression. Proc. Natl. Acad. Sci. USA 98, 9772–9777.

ACKNOWLEDGMENTS

Delacroix, D., and Vaerman, J.P. (1981). Reassessment of levels of secretory IgA in pathological sera using a quantitative radioimmunoassay. Clin. Exp. Immunol. 43, 633–640.

We are grateful to Genentech for providing the FCRL3 Ab. We acknowledge the Mid-Atlantic CHTN for providing human tonsils. We thank Adovi Akue, Mark Kukuruga, Scott Lute, Satoshi Nagata, and Weiming Ouyang. This work was supported by the Intramural Research Program of CDER/FDA. S.A., O.A., and K.S. were supported through a Research Fellowship Program administered by Oak Ridge Associated Universities. This article reflects the views of the authors and should not be construed to represent the FDA’s views or policies. AUTHOR CONTRIBUTIONS S.A., Z.K., J.D.-B., O.A., K.S., and M.T. performed experimental work. S.A., Z.K., and M.T. were involved in experimental design and data analysis and

1298 Cell Reports 30, 1292–1299, February 4, 2020

DuPage, M., and Bluestone, J.A. (2016). Harnessing the plasticity of CD4(+) T cells to treat immune-mediated disease. Nat. Rev. Immunol. 16, 149–163. Franco, A., Damdinsuren, B., Ise, T., Dement-Brown, J., Li, H., Nagata, S., and Tolnay, M. (2013). Human Fc receptor-like 5 binds intact IgG via mechanisms distinct from those of Fc receptors. J. Immunol. 190, 5739–5746. Ise, T., Maeda, H., Santora, K., Xiang, L., Kreitman, R.J., Pastan, I., and Nagata, S. (2005). Immunoglobulin superfamily receptor translocation associated 2 protein on lymphoma cell lines and hairy cell leukemia cells detected by novel monoclonal antibodies. Clin. Cancer Res. 11, 87–96. Josefowicz, S.Z., Lu, L.F., and Rudensky, A.Y. (2012). Regulatory T cells: mechanisms of differentiation and function. Annu. Rev. Immunol. 30, 531–564.

Kasagi, S., Zhang, P., Che, L., Abbatiello, B., Maruyama, T., Nakatsukasa, H., Zanvit, P., Jin, W., Konkel, J.E., and Chen, W. (2014). In vivo-generated antigen-specific regulatory T cells treat autoimmunity without compromising antibacterial immune response. Sci. Transl. Med. 6, 241ra78.

Procaccini, C., Carbone, F., Di Silvestre, D., Brambilla, F., De Rosa, V., Galgani, M., Faicchia, D., Marone, G., Tramontano, D., Corona, M., et al. (2016). The Proteomic Landscape of Human Ex Vivo Regulatory and Conventional T Cells Reveals Specific Metabolic Requirements. Immunity 44, 406–421.

Kochi, Y., Yamada, R., Suzuki, A., Harley, J.B., Shirasawa, S., Sawada, T., Bae, S.C., Tokuhiro, S., Chang, X., Sekine, A., et al. (2005). A functional variant in FCRL3, encoding Fc receptor-like 3, is associated with rheumatoid arthritis and several autoimmunities. Nat. Genet. 37, 478–485.

Rochereau, N., Drocourt, D., Perouzel, E., Pavot, V., Redelinghuys, P., Brown, G.D., Tiraby, G., Roblin, X., Verrier, B., Genin, C., et al. (2013). Dectin-1 is essential for reverse transcytosis of glycosylated SIgA-antigen complexes by intestinal M cells. PLoS Biol. 11, e1001658.

Koenen, H.J., Smeets, R.L., Vink, P.M., van Rijssen, E., Boots, A.M., and Joosten, I. (2008). Human CD25highFoxp3pos regulatory T cells differentiate into IL-17-producing cells. Blood 112, 2340–2352.

Sakaguchi, S., Yamaguchi, T., Nomura, T., and Ono, M. (2008). Regulatory T cells and immune tolerance. Cell 133, 775–787.

Kwan, W.H., van der Touw, W., Paz-Artal, E., Li, M.O., and Heeger, P.S. (2013). Signaling through C5a receptor and C3a receptor diminishes function of murine natural regulatory T cells. J. Exp. Med. 210, 257–268.

Sharabi, A., Tsokos, M.G., Ding, Y., Malek, T.R., Klatzmann, D., and Tsokos, G.C. (2018). Regulatory T cells in the treatment of disease. Nat. Rev. Drug Discov. 17, 823–844.

Li, F.J., Schreeder, D.M., Li, R., Wu, J., and Davis, R.S. (2013). FCRL3 promotes TLR9-induced B-cell activation and suppresses plasma cell differentiation. Eur. J. Immunol. 43, 2980–2992.

Stadtmueller, B.M., Huey-Tubman, K.E., Lo´pez, C.J., Yang, Z., Hubbell, W.L., and Bjorkman, P.J. (2016). The structure and dynamics of secretory component and its interactions with polymeric immunoglobulins. eLife 5, e10640.

Longet, S., Miled, S., Lo¨tscher, M., Miescher, S.M., Zuercher, A.W., and Corthe´sy, B. (2013). Human plasma-derived polymeric IgA and IgM antibodies associate with secretory component to yield biologically active secretory-like antibodies. J. Biol. Chem. 288, 4085–4094.

Sutmuller, R.P., den Brok, M.H., Kramer, M., Bennink, E.J., Toonen, L.W., Kullberg, B.J., Joosten, L.A., Akira, S., Netea, M.G., and Adema, G.J. (2006). Tolllike receptor 2 controls expansion and function of regulatory T cells. J. Clin. Invest. 116, 485–494.

Matesanz, F., Ferna´ndez, O., Milne, R.L., Fedetz, M., Leyva, L., Guerrero, M., Delgado, C., Lucas, M., Izquierdo, G., and Alcina, A. (2008). The high producer variant of the Fc-receptor like-3 (FCRL3) gene is involved in protection against multiple sclerosis. J. Neuroimmunol. 195, 146–150. Matysiak-Budnik, T., Moura, I.C., Arcos-Fajardo, M., Lebreton, C., Me´nard, S., Candalh, C., Ben-Khalifa, K., Dugave, C., Tamouza, H., van Niel, G., et al. (2008). Secretory IgA mediates retrotranscytosis of intact gliadin peptides via the transferrin receptor in celiac disease. J. Exp. Med. 205, 143–154. Meller, S., Di Domizio, J., Voo, K.S., Friedrich, H.C., Chamilos, G., Ganguly, D., Conrad, C., Gregorio, J., Le Roy, D., Roger, T., et al. (2015). T(H)17 cells promote microbial killing and innate immune sensing of DNA via interleukin 26. Nat. Immunol. 16, 970–979. Nagata, S., Ise, T., and Pastan, I. (2009). Fc receptor-like 3 protein expressed on IL-2 nonresponsive subset of human regulatory T cells. J. Immunol. 182, 7518–7526. Nyirenda, M.H., Sanvito, L., Darlington, P.J., O’Brien, K., Zhang, G.X., Constantinescu, C.S., Bar-Or, A., and Gran, B. (2011). TLR2 stimulation drives human naive and effector regulatory T cells into a Th17-like phenotype with reduced suppressive function. J. Immunol. 187, 2278–2290. Palm, N.W., de Zoete, M.R., Cullen, T.W., Barry, N.A., Stefanowski, J., Hao, L., Degnan, P.H., Hu, J., Peter, I., Zhang, W., et al. (2014). Immunoglobulin A coating identifies colitogenic bacteria in inflammatory bowel disease. Cell 158, 1000–1010. Peng, G., Guo, Z., Kiniwa, Y., Voo, K.S., Peng, W., Fu, T., Wang, D.Y., Li, Y., Wang, H.Y., and Wang, R.F. (2005). Toll-like receptor 8-mediated reversal of CD4+ regulatory T cell function. Science 309, 1380–1384. Polson, A.G., Zheng, B., Elkins, K., Chang, W., Du, C., Dowd, P., Yen, L., Tan, C., Hongo, J.A., Koeppen, H., and Ebens, A. (2006). Expression pattern of the human FcRH/IRTA receptors in normal tissue and in B-chronic lymphocytic leukemia. Int. Immunol. 18, 1363–1373.

Swainson, L.A., Mold, J.E., Bajpai, U.D., and McCune, J.M. (2010). Expression of the autoimmune susceptibility gene FcRL3 on human regulatory T cells is associated with dysfunction and high levels of programmed cell death-1. J. Immunol. 184, 3639–3647. Thalayasingam, N., Nair, N., Skelton, A.J., Massey, J., Anderson, A.E., Clark, A.D., Diboll, J., Lendrem, D.W., Reynard, L.N., Cordell, H.J., et al. (2018). CD4+ and B Lymphocyte Expression Quantitative Traits at Rheumatoid Arthritis Risk Loci in Patients With Untreated Early Arthritis: Implications for Causal Gene Identification. Arthritis Rheumatol. 70, 361–370. Viladomiu, M., Kivolowitz, C., Abdulhamid, A., Dogan, B., Victorio, D., Castellanos, J.G., Woo, V., Teng, F., Tran, N.L., Sczesnak, A., et al. (2017). IgAcoated E. coli enriched in Crohn’s disease spondyloarthritis promote TH17dependent inflammation. Sci. Transl. Med. 9, eaaf9655. Voo, K.S., Bover, L., Harline, M.L., Weng, J., Sugimoto, N., and Liu, Y.J. (2014). Targeting of TLRs inhibits CD4+ regulatory T cell function and activates lymphocytes in human peripheral blood mononuclear cells. J. Immunol. 193, 627–634. Weissler, K.A., and Caton, A.J. (2014). The role of T-cell receptor recognition of peptide:MHC complexes in the formation and activity of Foxp3+ regulatory T cells. Immunol. Rev. 259, 11–22. Wilson, N.J., Boniface, K., Chan, J.R., McKenzie, B.S., Blumenschein, W.M., Mattson, J.D., Basham, B., Smith, K., Chen, T., Morel, F., et al. (2007). Development, cytokine profile and function of human interleukin 17-producing helper T cells. Nat. Immunol. 8, 950–957. Wilson, T.J., Fuchs, A., and Colonna, M. (2012). Cutting edge: human FcRL4 and FcRL5 are receptors for IgA and IgG. J. Immunol. 188, 4741–4745. Wing, J.B., and Sakaguchi, S. (2012). Multiple treg suppressive modules and their adaptability. Front. Immunol. 3, 178.

Cell Reports 30, 1292–1299, February 4, 2020 1299

STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Secretory IgA, human colostrum

Athens Research

Cat# 16-13-090701

IgA, normal human plasma

Athens Research

Cat# 16-16-090701

Anti-Human CD3 e450

eBioscience

Cat#48-0037-42; RRID: AB_1272055

Antibodies

Anti-Human Cy5.5 CD4 PerCp

eBioscience

Cat# 45-0048-42; RRID: AB_10804390

Anti-Human CD4 PE

eBioscience

Cat#12-0048-42; RRID: AB_2016675

Anti-Human CD4 APC

eBioscience

Cat#17-0048-42; RRID: AB_1963580

Anti-Human CD8 PE

eBioscience

Cat#12-0086-42; RRID: AB_10732344

Anti-Human CD8 APC

eBioscience

Cat#17-0086-42; RRID: AB_10667892

Anti-Human CD25 PE

eBioscience

Cat#12-0259-42; RRID: AB_1659682

Anti-Human CD12 APC

eBioscience

Cat#; 17-1278-42; RRID: AB_1659670

Anti-Human FOXP3 PE

eBioscience

Cat# 12-4774-42; RRID: AB_10670338

Anti-Human FOXP3 Alexafluor

eBioscience

Cat# 53-4774-42; RRID: AB_2043860

Anti-Human RORɣt PE

eBioscience

Cat# 12-6988-82; RRID: AB_1834470 Cat# 563563; RRID: AB_2738277

Anti-Human IFNɣ BV395

BD Bioscience

Anti-Human IL-17A PE-Cyanine 7

eBioscience

Cat# 25-7179-42; RRID: AB_11063994

Anti-Human IL-26 APC

Invitrogen

Cat# MA5-23643; RRID: AB_2577101

Anti-Human IL-22 PE

eBioscience

Cat# 12-7229-42; RRID: AB_1834463

Anti-Human FCRL3 clone 6F2

Genentech

N/A

Anti-Human FCRL3 Biotin

BD Bioscience

Cat# 565056; RRID: AB_565056

Anti-Human CD3

Biolegend

Cat# 300304; RRID: AB 314040

Anti-Human J chain

LifeSpan Biosciences

Cat # LS-C666347

Anti-Human secretory component

Abcam

Cat # ab3924; RRID: AB_2261963

Anti-Human IgA heavy chain

Nordic-MUbio

MAHu/IgAc

Anti-mouse IgG HRP

Cell Signaling Technology

Cat # 7076P2; RRID: AB_330924

Anti-rabbit IgG HRP

Cell Signaling Technology

Cat # 7074P2; RRID:AB_2099233

Chemicals, Peptides, and Recombinant Proteins Human FCRL3 protein (NCBI # NP_443171)

R&D Systems

Cat# 3126-FC

APC streptavidin

Biolegend

Cat# 405207

Streptavidin

Southern Biot

Cat# 7011-101

Cell Trace CFSE

Invitrogen

Cat# C34554

Cell Trace Violet

Invitrogen

Cat# C34557

X-VIVO 20 media

Lonza

Cat# 04-448Q

CD3/CD28 beads

GIBCO

Cat#11131D

Pam3

Biolegend

Cat# Tlr1-bpms

FSL-1

Invitrogen

Cat# Tlr1-fsl1

PGN

Invitrogen

Cat# Tlr1-pgns2

Human CD4+ T cell isolation kit

Miltenyi Biotec

Cat# 130-096-533

FOXP3/Transcription Factor Staining Buffer set

eBioscience

Cat#005523-00

ATCC

Cat# CRL-3216

Satoshi Nagata (Ise et al., 2005)

N/A

Critical Commercial Assays

Experimental Models: Cell Lines 293T cells Recombinant DNA Human FCRL3 (NCBI # NP_443171) expression plasmid

(Continued on next page)

e1 Cell Reports 30, 1292–1299.e1–e3, February 4, 2020

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

FlowJo 7.6.5 and 10.1

FlowJo, LLC

N/A

GraphPad Prism 6

GraphPad Software

N/A

Microsoft Excel

Microsoft Corp.

N/A

BD Bioscience

N/A

Software and Algorithms

Other BD FACSAria II

LEAD CONTACT AND MATERIALS AVAILABILITY Further information and requests for resources and reagents should be directed and will be fulfilled by the Lead Contact, Mate Tolnay ([email protected]) EXPERIMENTAL MODEL AND SUBJECT DETAILS Primary Human Cell Cultures Buffy coats of anonymous healthy human donors were obtained from the NIH Blood Bank and Biological Specialty Corp. (Colmar, PA) following institutionally approved protocols. Tonsils of anonymous human donors were obtained from the Mid-Atlantic CHTN following institutionally approved protocols. METHOD DETAILS Treg Cell Purification Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll gradient centrifugation. CD4+ T cells were enriched using the human CD4+ T cell isolation kit (Miltenyi) and then stained for sorting with CD4-PerCP Cy5.5, CD25-PE and CD127-APC Abs (eBioscience). CD4+CD25hi CD127dim Treg were sorted using FACSAria II. Sorted Treg cells had >95% purity and >90% viability. Treg Cell Suppression Assays Sorted CD4+CD25hi CD127dim Treg cells were labeled with Cell Trace Violet (Invitrogen) and rested in X-vivo 20 medium (Lonza) for 30 min at 37 C. Treg cells at 1 million cells/ml were incubated with 20 mg/ml biotinylated FCRL3 Ab clone 6F2 (Polson et al., 2006), 150 mg/ml biotinylated SIgA, 6 mg/ml biotinylated CD3 Ab (Biolegend) or biotinylated TLR2 ligand mix (1 mg/ml Pam3, 1 mg/ml FSL-1, 0.1 mg/ml PGN; Invivogen) for 10-15 min at 4 C. Biotinylated reagents were crosslinked for 15 min at 37 C using 50 mg/ml streptavidin (Southern Biotech); except 19 mg/ml streptavidin was used with SIgA-stimulation. Samples were washed twice with 10 mL medium. Pre-stimulated Treg cells were added at 1:1 ratio to PBMC labeled with CFSE (Invitrogen). Responder PBMC or purified CD4+ cells were stimulated with CD3/CD28 beads (GIBCO) at 1:2 bead per cell ratio. Samples were incubated for 3.5 days in X-vivo 20 media, when cell division was assessed by flow cytometry. Cell division indexes of responder cells were calculated using the proliferation analysis tool from FlowJo. Figure S1 shows the gating strategy. Measurement of Secreted IL-26 and IL-17 Supernatants of Treg cell suppression assays were collected after 3.5 days and analyzed using Bio-Plex Pro human 12-Plex Treg cytokine assay for IL-26, and human 17-plex cytokine assay for IL-17 (both from Bio-Rad). Intracellular Flow Cytometry Samples were set up exactly as for Treg cell suppression assays. Protein transport inhibitor cocktail (eBioscience) was added 4 hours later. Cells were stained for surface CD4-PerCP Cy5.5 and CD8-FITC, fixed, permeabilized, and stained intracellularly with either IL17A-PeCy7, IL-22-PE (eBioscience), IL-26-APC (Invitrogen) and IFNg-BUV395 (BD Horizon) Abs, or with the indicated CD8, IL-22 and IL-26 Abs replaced with CD8-APC, FOXP3-FITC and RORgt-PE (eBioscience) Abs. Cell Binding Assays 293T cells were transfected with pcDNA3 plasmid expressing FCRL3 using Lipofectamine 3000 (Invitrogen) or were mock transfected. FCRL3 expression was confirmed by FCRL3-biotin (BD PharMingen) and streptavidin staining two days post-transfection. 2x106 transfected cells were incubated with 50 mg biotinylated SIgA or biotinylated plasma IgA (Athens Research) in 0.2 mL for 30 min on ice. Cells were washed once, incubated in 0.2 mL buffer with 6.25 mg of streptavidin-APC (Biolegend) for 15 min on ice, washed twice, and fixed before analysis by flow cytometry. For binding studies with primary cells, 5x106 PBMC were incubated

Cell Reports 30, 1292–1299.e1–e3, February 4, 2020 e2

with CD4-PerCP, CD19-ef488, CD3-ef450 Abs (eBioscience) and then incubated with biotinylated SIgA or biotinylated plasma IgA, then with streptavidin-APC, as described for 293T cells. Some samples were pre-incubated with 10 mg/mL human FCRL3 Ab clone 6F2 for 5 min on ice before incubation with SIgA. Following binding, cells were fixed, permeabilized, and stained with FOXP3-PE Ab using FOXP3 staining kit (eBioscience). IgA binding was assessed by flow cytometry. Surface Plasmon Resonance Experiments were performed on Biacore T200 (GE Healthcare) similarly as described (Alabi et al., 2017). The negative charge of the sensor was reduced by a blank immobilization cycle. 10,000 RU anti-His Ab was equally immobilized on both the sample and reference surfaces of CM5 sensors using amine coupling. 100-140 RU recombinant human FCRL3 (R&D Systems) containing the entire extracellular part with a His-tag was captured from 1 mg/ml solution. Samples were injected at six concentrations (one in duplicate), two-fold diluted from 10 mM in HBS-P buffer with 1 mg/ml nonspecific binding reducer. Association for 8 min and dissociation for 10 min was assessed at 25 C. The sensor was regenerated after each analyte injection with two 1 min injections of 10 mM glycine-HCl, pH 1.5. Data were analyzed using Biacore T200 Evaluation software 3.0, subtracting the reference surface and buffer control signals from each curve. Data were globally fitted using the 1:1 kinetic model. Size Exclusion Chromatography Secretory IgA from human colostrum and IgA from human plasma (both from Athens Research), 10 mg each in 3-4 ml, were sizefractionated on an AKTA Avant system using a HiLoad 16/600 Superdex column (both from GE Life Sciences). Plasma IgA (10 mg total) was size-fractionated in three aliquots to enhance separation. Size fractionation was achieved using 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.2 buffer at a maximum flow rate of 1.6 ml/min. Western Blotting Reduced SIgA and plasma IgA samples (0.5 mg for SC and IgA heavy chain; 0.1 mg for J chain) were resolved by SDS-PAGE, transferred to membranes and then blotted with 1.5 mg/ml anti-J chain, 2 mg/ml anti-SC, or 0.5 mg/ml anti-IgA heavy chain Abs. Secondary Abs with HRP were used at a 1:3000 dilution. QUANTIFICATION AND STATISTICAL ANALYSIS Results were expressed as the mean ± standard deviation (SD). For statistical analysis Microsoft Excel and Prism (GraphPad Software) was used. Statistical significance was determined using two-tailed Student’s t test, paired or unpaired, as indicated. DATA AND CODE AVAILIBILITY The published article includes all datasets generated and analyzed during this study.

e3 Cell Reports 30, 1292–1299.e1–e3, February 4, 2020