Targeting Antigens to CD180 but Not CD40 Programs Immature and Mature B Cell Subsets to Become Efficient APCs

This information is current as Kelsey Roe, Geraldine L. Shu, Kevin E. Draves, Daniela of September 25, 2021. Giordano, Marion Pepper and Edward A. Clark J Immunol published online 4 September 2019 http://www.jimmunol.org/content/early/2019/09/03/jimmun ol.1900549 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 4, 2019, doi:10.4049/jimmunol.1900549 The Journal of Immunology

Targeting Antigens to CD180 but Not CD40 Programs Immature and Mature B Cell Subsets to Become Efficient APCs

Kelsey Roe,1 Geraldine L. Shu, Kevin E. Draves, Daniela Giordano, Marion Pepper, and Edward A. Clark

Targeting Ags to the CD180 receptor activates both B cells and dendritic cells (DCs) to become potent APCs. After inoculating mice with Ag conjugated to an anti-CD180 Ab, B cell receptors were rapidly internalized. Remarkably, all B cell subsets, including even transitional 1 B cells, were programed to process, present Ag, and stimulate Ag-specific CD4+ T cells. Within 24–48 hours, Ag-specific B cells were detectable at T–B borders in the spleen; there, they proliferated in a T cell–dependent manner and

induced the maturation of T follicular helper (TFH) cells. Remarkably, immature B cells were sufficient for the maturation of TFH Downloaded from 2/2 cells after CD180 targeting: TFH cells were induced in BAFFR mice (with only transitional 1 B cells) and not in mMT mice (lacking all B cells) following CD180 targeting. Unlike CD180 targeting, CD40 targeting only induced DCs but not B cells to

become APCs and thus failed to efficiently induce TFH cell maturation, resulting in slower and lower-affinity IgG Ab responses. CD180 targeting induces a unique program in Ag-specific B cells and to our knowledge, is a novel strategy to induce Ag presentation in both DCs and B cells, especially immature B cells and thus has the potential to produce a broad range of Ab

specificities. This study highlights the ability of immature B cells to present Ag to and induce the maturation of cognate TFH cells, http://www.jimmunol.org/ providing insights toward vaccination of mature B cell–deficient individuals and implications in treating autoimmune disorders. The Journal of Immunology, 2019, 203: 000–000.

argeting Ag directly to APCs is a highly efficient strategy dependent (TD) (2). The direct conjugation of Ag to the anti-CD180 to induce both humoral and cellular immunity (1). Ag Ab is required to induce this Ab response; mice inoculated with anti- T targeting to CD180 (also called RP105), achieved by CD180 conjugated to the hapten 4-hydroxy-3-nitrophenacetyl (NP) coupling an Ag directly to an anti-CD180 Ab (Ag-anti-CD180), plus free OVA generated Ab to NP and not OVA and vice versa (2). has the advantage of targeting both dendritic cells (DCs) and Furthermore, targeting Ag to CD180 can protect immunodeficient B cells and of providing an adjuvant effect by activating CD180. mice from a lethal virus infection. Mice deficient for the B cell– by guest on September 25, 2021 Previously, we reported that this platform induces rapid and high- activating factor receptor (BAFFR) lack mature B cells but do affinity Ag-specific IgG responses, which are predominantly T cell produce transitional 1 (T1) B cells (3). Vaccination of BAFFR2/2 mice with West Nile virus (WNV) E protein conjugated to anti- CD180 was sufficient to protect them from a subsequent lethal Department of Immunology, University of Washington, Seattle, WA 98109 WNV challenge (4). Remarkably, the addition of an adjuvant was 1 Current address: Center for Immunity and Immunotherapies, Seattle Children’s not required to induce protection. Conversely, mMT mice, which Research Institute, Seattle, WA. lack all B cells, were not protected from WNV infection, sug- ORCIDs: 0000-0002-4319-3696 (K.R.); 0000-0001-8362-7017 (D.G.); 0000-0001- 2/2 8061-5475 (E.A.C.). gesting that the T1 B cells present in the BAFFR mice are used by the CD180-targeting vaccine to induce protection. Received for publication May 14, 2019. Accepted for publication July 29, 2019. CD180 is a pattern recognition receptor, expressed on DCs, This work was supported by a grant from the National Institutes of Health (R01AI52203 to E.A.C.). macrophages, and B cells. Whereas it is related to TLR family Conceptualization, K.R. and E.A.C.; data curation, K.R.; formal analysis, K.R.; funding members, unlike TLRs, it lacks a TIR domain and does not use acquisition, E.A.C.; investigation, K.R., K.E.D., and G.L.S.; methodology, K.R., D.G., MyD88 or TRIF for signal propagation (5). Instead, CD180 ap- M.P., and E.A.C.; project administration, K.R.; resources, M.P. and E.A.C.; supervision, pears to act as a regulator of other signaling receptors, especially E.A.C.; visualization, K.R.; writing – original draft, K.R.; writing – review and editing, K.R., D.G., M.P., and E.A.C. TLRs (5). B cells can be activated via stimulation by an anti- Address correspondence and reprint requests to Dr. Kelsey Roe at the current CD180 Ab, resulting in proliferation that is dependent on CD19 address: Center for Immunity and Immunotherapies, Seattle Children’s Research but not MyD88 (6, 7). Indeed, CD180 stimulation by Ab cross- Institute, 1900 9th Avenue, Seattle, WA 98101, or Dr. Edward A. Clark, Depart- linking on B cells activates signaling elements reminiscent of ment of Immunology, University of Washington, 750 Republican Street, Seattle, WA 98109. E-mail addresses: [email protected] (K.R.) or [email protected] BCR signaling, including Btk, Lyn, and Vav (7–9). In addition, (E.A.C.) CD180 regulates B cell TLR signaling; whereas activation of The online version of this article contains supplemental material. B cells via CD180 does not require TLR4, CD180 expression is Abbreviations used in this article: AP-2, adaptor protein 2; ASC, Ab-secreting required for LPS-mediated activation of TLR4 (10). Furthermore, cell; BAFFR, B cell–activating factor receptor; BM, bone marrow; DC, dendritic CD180 signaling synergizes to enhance the activation of several cell; FO, follicular; GC, germinal center; Iso, isotype; LLPC, long-lived plasma cell; MHC II, MHC class II; MZ, marginal zone; NP, 4-hydroxy-3-nitrophenacetyl; TLRs, including TLR-2, -4, -7, and -9 (11). Apparently, during Ag PBST, PBS containing 0.05% Tween 20; PFA, paraformaldehyde; T1, transitional 1; targeting, CD180 synergizes with BCR signaling to improve T2, transitional 2; TD, T cell dependent; TFH, T FO helper; WNV, West Nile virus; B cell activation and resulting Ab responses. WT, wild-type. In this study, we characterized the early events of B cell activa- Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 tion following Ag–anti-CD180 inoculation, comparing responses of

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900549 2 CD180-TARGETED B CELL RESPONSES different splenic B cell subsets. We demonstrate that CD180-targeted tetramer-specific cells. The bound fraction was stained for flow cytometry B cells, including immature T1 cells, are activated to become as described below. Absolute cell counts were analyzed using AccuCheck efficient APCs, which contribute to the development of robust TD Counting Beads (Invitrogen) as per the manufacturer’s instructions. humoral responses. Intriguingly, targeting Ags to CD180 induced Flow cytometry an earlier and higher-affinity Ag-specific IgG response than when RBCs were lysed with RBC lysis buffer (Invitrogen) and isolated Ags were targeted to CD40. Our data suggest that CD180 tar- splenocytes were stained with Live/Dead Aqua (Thermo Fisher Scientific) geting, by capitalizing on the capabilities of newly formed B cells or Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific) for 20 min and the Ag-presenting capacity of multiple B cell subsets as well at 4˚C in the absence of FBS to differentiate live and dead cells. Cells as DCs, may provide a novel avenue with which to effectively were subsequently stained for surface markers (Supplemental Table I) in the presence of Fc block (anti-CD16/32; BioLegend) and 2% FBS. Cells treat patients with immunodeficiencies or cancer. expressing NP-specific BCRs were detected by staining with NP–PE (Biosearch Technologies). HEL peptide–loaded MHC class II (MHC II) Materials and Methods was detected using a mAb clone C3H4 (16) (kindly provided by Dr. Ron Mice Germain, National Institutes of Allergy and Infectious Diseases, Bethesda, MD), which was biotinylated and detected using streptavidin-PE-Texas a a C57BL/6, C57BL/6 knock-in Ly-5.1, and B10.A-H2 -H2-T18 /SgSnJ mice Red (BD Biosciences). After washing, cells were fixed in 1% parafor- were purchased from The Jackson Laboratory (Bar Harbor, ME). B6.SJL- maldehyde (PFA) and stored at 4˚C until analysis. Cells were analyzed on hi B1-8 knock-in Ly-5.1 mice were a gift from Dr. Michel Nussenzweig an LSR II flow cytometer (BD Biosciences), and data were analyzed using (Rockefeller University, New York, NY) (12). Mature B cell–deficient FlowJo (v.10; Tree Star). See Fig. 1A for gating strategies. BAFFR2/2 mice were kindly provided by Dr. Klaus Rajewsky (Harvard Medical School, Boston, MA). B cell–deficient mMT mice were a gift from In vitro internalization assay Dr. David Rawlings (Seattle Children’s Research Institute, Seattle, WA). Downloaded from m OT-II OVA-specific CD4+ TCR transgenic knock-in Ly-5.1 mice were a The K46 M17 (K46) murine B cell lymphoma cell line expressing the gift from Dr. Michael Gerner (University of Washington, Seattle, WA). All B1-8 high-affinity NP-specific BCR (17) was a gift from Dr. Louis strains, except B10.A were on a C57BL/6 background. All mice were age- B. Justement (University of Alabama, Birmingham, AL). Cells were and sex-matched for experiments and used at 8–12 wk of age. Mice were cultured in RPMI 1640 (GenClone), supplemented with 10% FBS (Thermo housed in a specific pathogen–free environment; all procedures were ap- Fisher Scientific), 1 mM sodium pyruvate (GE Healthcare), nonessential proved by the University of Washington Institutional Animal Care and Use amino acids (GenClone), L-glutamate, penicillin, streptomycin (Corning), and 50 mM 2-ME. Alexa Fluor 647 was conjugated to NP–Iso and NP–

Committee. http://www.jimmunol.org/ anti-CD180 using a labeling kit (Invitrogen) according to the manu- Ag-targeting constructs and adjuvants facturer’s instructions. Fluorescent Ag conjugates and aB220–eFluor 450 (RA3-6B2; eBioscience) were incubated with K46 cells at 4˚C for The rat IgG2a anti-CD180 (RP/14) was produced from a hybridoma (gift 20 min to allow for receptor binding. Cells were then incubated at 37˚C to of Dr. Kensuke Miyake, University of Tokyo, Tokyo, Japan) or pur- allow for BCR internalization and, subsequently, fixed with 1% PFA to halt chased from BioLegend (Ultra-LEAF purified). Ultra-LEAF–purified anti- internalization. Cells were analyzed using the ImageStreamX Mark II flow CD40 (1C10, rat IgG2a) and rat IgG2a isotype (Iso) control (RTK2758) cytometer (Amnis), and data were analyzed using IDEAS software were purchased from BioLegend. NP–Ab conjugates were prepared by (Amnis). Internalization was quantified on focused, single cells as follows: conjugation to the succinimidyl ester of NP (Biosearch Technologies) as an internal cellular zone was defined using an adaptive erode mask based previously described (2). Protein Ags OVA (Sigma-Aldrich) or hen egg on surface B220 staining, and internalization was quantitated using the lysozyme (HEL) (Sigma-Aldrich) were conjugated to mAbs via thioether internal zone mask and the fluorescence of the Ag. Normalized inter- by guest on September 25, 2021 linkages as previously described (13). Free NP was removed by dialysis nalization was calculated by dividing the percent of internalized cells with Mr cut-off tubing of 8 kDa (NP–Abs). Molar ratios of NP to mAb at time 3 min over time 0 min. were confirmed by spectrophotometry and ranged from NP5-Ab to NP10-Ab. Molar ratios of OVA or HEL to mAb were quantitated by ELISA and ranged APC isolation and ex vivo T cell stimulation from Ag –AbtoAg–Ab. All Ag–Ab constructs were filter sterilized (0.2 mM) 1 2 + and stored at 4˚C until use. The amount of conjugate administered references B6 mice (Ly-5.2 ) were inoculated with OVA–Ab conjugates. Splenocytes + the weight of the anti-CD180 Ab, and, in all experiments, the moles of were processed to single cells as above. CD11c cells were isolated by Ag inoculated were equivalent between all mice. Because of differences in magnetic bead positive selection (Miltenyi Biotec) according to the man- conjugation ratios, this resulted in slightly higher weights of both the Iso ufacturer’s instructions. From the negative fraction, B cells were isolated and anti-CD40 Ab in all inoculations. All inoculations were prepared to by magnetic bead negative selection (STEMCELL Technologies) accord- 200 ml in pharmaceutical grade PBS and administered by retro-orbital ing to the manufacturer’s instructions. B cells were then FACS sorted into + hi lo + mid mid inoculation. The TLR7 agonist R848 was obtained from Invivogen and, T1 (B220 , CD24 , CD21 ), follicular (FO) (B220 , CD24 , CD21 ) + hi hi when used, mixed with the Ag–Ab preparation. and marginal zone (MZ) (B220 , CD24 , CD21 ) subsets (as in Fig. 1A, with smaller gates to limit subset spillover, as is a common sorting pro- Adoptive transfers cedure), using a FACSAria flow cytometer (BD Biosciences). Meanwhile, + + hi CD4 T cells (Ly-5.1 ) were isolated from splenocytes from OT-II mice by Splenocytes from B1-8 mice were processed by mechanical disruption magnetic bead negative selection (STEMCELL Technologies) and labeled between frosted glass slides. B cells were isolated by magnetic bead with CFSE as above. A total of 1 3 105 CFSE-labeled CD4+ T cells . negative selection (STEMCELL Technologies) and were 95% pure as were combined with 1 3 105 APC (DC or B cell subset) and incubated 3 6 determined by flow cytometry. A total of 50 10 B cells/ml were labeled in a 96-well plate for 3 d, following which cells were stained for surface m with 20 M CFSE (Invitrogen) at 37˚C for 10 min and washed thoroughly. markers and analyzed by flow cytometry as above. Labeled cells were transferred in 200 ml i.v. 16–18 h prior to Ag–Ab in- oculation. Male cells were never transferred into female mice. Immunohistochemistry + CD4 T cell depletion Spleens were isolated from mice and cut in half. One half was fixed in 1% m PFA and then cryoprotected in 30% sucrose. The spleen half was embedded Mice were inoculated with 100 g anti-CD4 (GK1.5, prepared in our 2 m m in OCT and frozen at 80˚C; 6- m sections were cut, and slides were laboratory or purchased from BioLegend) or 100 g Rat IgG2b Iso control 2 ∼ stored at 80˚C until use. Sections were allowed to thaw before equili- (BioLegend) i.p. 24 h prior to Ag–Ab inoculation and 5 h prior to the brating in PBS. Sections were washed with PBS containing 0.05% Tween adoptive transfer of B cells. 20 (PBST), blocked for 30 min (PBST plus 10 mg/ml Fc blocking Ab plus Tetramer-specific cell enrichment 10% normal goat serum), and then stained with B220-BV421, CD3-FITC, NP–PE, and Ly-6G–Alexa Fluor 647 (in PBST plus 3% BSA) for 1 h OVA-specific T cells were identified using OVA–IAb tetramers 2C and 3C, at room temperature. Following several PBST washes, glass slides were prepared as previously described (14). Tetramer-specific cells were mounted over sections with ProLong Diamond (Thermo Fisher Scien- enriched as previously described (15). Briefly, isolated splenocytes were tific) mounting medium. Confocal images were obtained using a 203 incubated with the OVA tetramer followed by anti-PE microbeads objective on a Nikon Eclipse Ti inverted microscope with a Nikon (Miltenyi Biotec). The suspension was run over magnetic LS columns C2 confocal system. Images were processed using Nikon NIS-Elements (Miltenyi Biotec), washed multiple times, and then eluted to enrich AR v5.10.01. The Journal of Immunology 3

ELISA and ELISPOT mice immunized with NP–Iso plus anti-CD180 demonstrated NP-specific ELISA and ELISPOT were performed as described previously similar BCR internalization kinetics to mice immunized with (2). Briefly, for ELISAs, sera were incubated over NP–BSA coated poly- NP–Iso (Fig. 1B). styrene plates, detected using HRP–aIgG plus TMB substrate, and quan- To rule out the possibility that NP in the immunization was titated by comparison with a standard curve of known IgG concentrations. blocking the binding of the NP–PE stain, we confirmed the de- For ELISPOT assays, splenocytes or isolated bone marrow (BM) cells crease of NP–BCR expression by staining for Ig l L chain asso- were incubated at 37˚C overnight over NP–BSA–coated mixed cellulose ester membrane filter plates, and IgG spots were detected by HRP–aIgG ciated with the NP–BCR (12). As expected, the expression of and AEC substrate. Ig l decreased with similar kinetics as NP-binding BCR follow- ing NP–anti-CD180 inoculation (Supplemental Fig. 1D). We also Statistical analyses tested for the expression of the NP–BCR in permeabilized cells Raw experimental data were analyzed using a Mann–Whitney U test, a and, as expected, found that the BCR was being internalized rather Kruskal–Wallis test with Dunn multiple comparison, or a two-way than shed; similar numbers of NP-binding cells were detected in ANOVA with Tukey multiple comparison test, using GraphPad Prism 7. Differences of p , 0.05 were considered significant. mock and immunized mice (Supplemental Fig. 1E). To further assess our in vivo findings, we developed an in vitro internaliza- tion assay using the K46mM17 B cell line that expresses the B1-8 Results NP-specific BCR (17). Alexa Fluor 647–NP–Iso and Alexa Fluor Targeting Ag to CD180 induces rapid BCR internalization 647–NP–anti-CD180 were incubated with K46 cells and allowed CD180 Ag targeting induces an expansion of FO and immature T1 to internalize. Cells were then visualized at different time points and transitional 2 (T2) subsets, whereas MZ B cells decline 1 d after using the ImageStream flow cytometer (Fig. 1C, 1D). The red Ag Downloaded from immunization (2). CD180 Ag targeting also induces T1 B cells in moved from the cell surface to the cytoplasm within 3 min after BAFFR2/2 mice to promote protective immune responses (4). treatment with NP–anti-CD180. This process took at least 5 min if Given these findings, we decided to characterize early phenotypic the Ag was attached to the Iso Ab (NP–Iso), even in the presence changes in different splenic B cell subsets induced by CD180 of unconjugated anti-CD180 (NP–Iso plus anti-CD180). The targeting. B1-8hi mice, with a transgenic BCR that recognizes the results are consistent with the differential kinetics of BCR

hapten NP on 15–20% of splenic B cells, were immunized with internalization observed in vivo. http://www.jimmunol.org/ NP conjugated to anti-CD180 (NP–anti-CD180) or an Iso control (NP–Iso). We examined the expression of the NP-specific BCR by B cells present Ag and stimulate Ag-specific T cells following staining cells with an NP–PE conjugate. The expression of CD21 CD180 targeting and CD24 on B220+ cells was used to define splenic B cell sub- Given the adjusted rate of BCR-Ag internalization when Ag was sets, as seen in Fig. 1A and as previously described (18). There attached to anti-CD180, we hypothesized that Ag processing within was no change to the total number of splenic B cell subsets 3–6 h B cells might also be altered, thus resulting in differential abilities after inoculation, whereas there was a modest increase in T2 and to present Ag. To address potential changes in B cell activation, FO B cells and a decrease in total MZ B cells by 24 h (Supple- we first examined levels of MHC II and CD86 on the surface of mental Fig. 1A), as previously reported (2). However, 3 h after NP–BCR2 or NP–BCR+ B cells 24 h after immunization of B1-8hi by guest on September 25, 2021 inoculation, the number of cells expressing the NP-specific BCR mice (as in Fig. 1). Given that NP–BCR expression is variable at decreased significantly; this corresponded with an increase in this time point depending on the inoculation and subset (Fig. 1B), IgM2 B cells, suggesting that the BCR was being internalized we decided to quantify NP–BCR+ cells based on their expres- following interaction with its cognate Ag (Fig. 1A bottom panel). sion of NP–BCR+ or reduction of surface IgM (see Supplemental Interestingly, when NP was conjugated to anti-CD180, the NP– Fig. 2). When mice were inoculated with either NP–Iso or NP– BCR was internalized more rapidly than when NP was conju- anti-CD180, NP-specific B cells had elevated levels of MHC II on gated to the Iso control (Fig. 1A, 1B). By 3 h after inoculation, the all the B cell subsets compared with NP–BCR2 cells (Fig. 2A). frequency of NP-specific B220+ cells in mice that had received With the exception of FO B cells, inoculation of NP–Iso plus anti- NP–Iso had decreased∼7–8-fold (e.g., from 13 to 1.7%, Fig. 1A), CD180 induced only moderate increases in MHC II expression and many B cells still expressed high levels of the NP–BCR. regardless of BCR specificity. NP–BCR+ B cells also showed in- However, in mice inoculated with NP–anti-CD180, the frequency creased expression of CD86 following BCR engagement, and, of NP–BCR+ cells dropped more than 100-fold, and the few de- additionally, CD86 levels were significantly increased on B cells tectable NP–BCR+ cells had very low BCR expression levels from mice inoculated with NP–anti-CD180 as compared with NP– (Fig. 1A). This rapid internalization occurred in all splenic B Iso+/2 anti-CD180 (Fig. 2A). Remarkably, NP–anti-CD180 in- cell subsets, including immature T1 and T2 B cells (Fig. 1B, oculation even induced increased expression of both MHC II and Supplemental Fig. 1B). We did not observe a corresponding de- CD86 on NP-specific immature T1 B cells. crease in the expression of BCR-associated proteins, such as To assess the ability of different B cell subsets to present Ag, CD21 or CD23 (Supplemental Fig. 1C). By 6 h after inoculation, we used two different assays: identification of peptide/MHC II the expression of the NP–BCR reached its nadir in both immu- complexes on the surface of B cells and a functional readout of nization groups. However, whereas the NP–BCR had returned to Ag presentation via the stimulation of Ag-specific CD4+ T cells. the surface by 24 h postinoculation in NP–Iso immunized mice, B10.A mice (MHC II I-Ak) were immunized with HEL con- it was slower to be re-expressed on FO, T1, and T2 B cells from jugated to anti-CD180 or the Iso control Ab. HEL peptide NP–anti-CD180–treated mice. MZ B cells from mice that received (aa 46–61) expressed in association with MHC II I-Ak was NP–anti-CD180 still had not re-expressed the NP–BCR after 24 h. quantitated by staining cells with the C4H3 mAb (16). To ac- Given the overall reduction of MZ B cells by 24 h (Supplemental count for changes in total MHC II expression levels following Fig. 1A), it is possible that Ag-specific MZ B cells migrated inoculation and any potential nonspecificity of the C4H3 Ab, from the spleen. Nonetheless, the NP-specific BCR is inter- we evaluated Ag presentation by determining the ratio of Ag- nalized more rapidly on all the subsets as compared with NP– bound MHC II to total MHC II (Fig. 2B). HEL peptide–MHC II Iso inoculation. Interestingly, direct conjugation of the Ag to complexes were expressed on all B cell subsets, including T1 anti-CD180 was required for the faster internalization rate, as B cells, as early as 3 h following HEL–anti-CD180 inoculation 4 CD180-TARGETED B CELL RESPONSES Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. The Ag-specific BCR is internalized rapidly following targeting to CD180. B1-8hi mice were inoculated with 50 mg NP–Iso, NP–Iso plus anti-CD180, or NP–anti-CD180. Three, six, or twenty-five hours later, spleens were harvested and processed for flow cytometry. (A) Representative flow plots of total live, single, B220+ cells 3 h after inoculation. The top panel demonstrates the gating strategy used to identify different splenic B cell subsets. After gating out debris, doublets, and dead cells, B220+ B cell subsets were evaluated using CD21 and CD24 expression. T1 B cells were defined as CD24hi, CD21lo; T2 was defined as CD24hi, CD21mid; FO was defined as CD24mid, CD21mid; MZ was defined as CD24hi, CD21hi. The bottom panel demonstrates the change in NP–BCR expression following inoculation. (B) NP–BCRhi–expressing cells per spleen following inoculation in different B220+ splenic B cell subsets. Data are the mean 6 SEM combined of two to three experiments per time point (n = 4–8 per group). (C and D) In vitro internalization assay in the NP-specific BCR-expressing K46 B cell line. Cells were incubated with fluorescently conjugated NP conjugates, and internalization was evaluated using the ImageStream flow cytometer and IDEAS software. Data are representative of three individual experiments. *p , 0.05, **p , 0.01 NP–Iso versus NP–anti-CD180, †p , 0.05, ‡p , 0.01 NP–Iso plus anti-CD180 versus NP–anti-CD180 by Kruskal–Wallis test with Dunn multiple comparison test (B)or two-way ANOVA with Tukey multiple comparison test (D). aCD180, anti-CD180. but not after HEL–Iso+/2 anti-CD180 inoculation; expression was presentation by T1 B cells following CD180 Ag targeting, we tested maintained for at least 24 h. whether these B cells could also stimulate Ag-specific T cells. Immature B cells are not known to stimulate Ag-specific T cells. C57BL/6 (B6) mice were inoculated with OVA–Ab conjugates; However, given the induction of CD86 expression on and Ag splenic CD11c+ DCs and B cells were then isolated; and T1, FO, The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 2. Targeting Ags to CD180 results in Ag processing and presentation by B cells. (A) B1-8hi mice were inoculated with 50 mg NP–Iso, NP–Iso plus anti-CD180, or NP–anti-CD180. Twenty-four hours later, the expression of MHC II and CD86 were evaluated on NP–BCR2 and NP–BCR+ splenic B cells by flow cytometry. Data are the mean 6 SEM representative of two independent experiments with n = 3–4 per group. (B) B10.A mice were inoculated with 35 mg HEL–Iso, HEL–Iso plus anti-CD180 or HEL–anti-CD180. Three or twenty-four hours later, HEL peptide-bound MHC II I-Ak, represented as peptide-bound MHC II over total MHC II expression (HEL–I-Ak/I-Ak mean fluorescence intensity) was evaluated on splenic B cell subsets and con- ventional (c) DCs by flow cytometry. cDCs were defined as B2202, CD32, NK1.12, CD11b2/lo, CD11chi. Data are the mean 6 SEM representative of two independent experiments per time point (n = 3–4 per group). (C) C57BL/6 mice were inoculated with 35 mg OVA–Iso, OVA–Iso plus anti-CD180, or OVA–anti-CD180. Three or twenty-four hours later, DCs were isolated from spleen by positive bead selection, B cells were isolated by negative bead enrichment, and B cell subsets were sorted by FACS. A total of 1 3 105 APCs were cocultured with 1 3 105 CFSE-labeled OT-II T cells. Three days later, T cell proliferation was evaluated as CFSE dilution by flow cytometry. Data are the mean 6 SEM combined of (Figure legend continues) 6 CD180-TARGETED B CELL RESPONSES and MZ B cell subsets were sorted by FACS. The B cells or DCs Taken together, these data indicate that CD180 targeting induced were incubated with CFSE-labeled OT-II T cells specific for OVA. B cells to present Ag, migrate to T–B borders, and proliferate, As expected, DCs from OVA–anti-CD180–inoculated mice ob- possibly in a TD manner. tained at both 3 and 24 h postinoculation were robust stimula- Ag-specific B cells, with the help of T cells, proliferate rapidly tors of OVA-specific T cells (93–96% of CD4+ T cells had diluted following CD180 targeting CFSE; Fig. 2C). All B cell subsets tested were also able to stimulate T cells to various levels. At 3 h post immunization, FO To address further which B cell subsets proliferate in response to and MZ cells demonstrated equivalent T cell stimulatory capac- CD180 targeting, we adoptively transferred CFSE-labeled B1-8hi ities, whereas by 24 h, FO B cells had a slightly reduced capacity B cells into B6 mice 1 d prior to NP–anti-CD180 inoculation. to stimulate T cells compared with MZ B cells. Intriguingly, T1 NP-specific B cells underwent several cell divisions within 3 d B cells were capable of stimulating OVA-specific T cells at both following NP–anti-CD180 inoculation (Fig. 4A). In contrast, unlike time points, although to a lesser extent than the mature B cell in mock control mice, Ag-specific B cells were no longer detected in subsets. As expected, B cells or DCs from mice inoculated with mice that had received NP–Iso or NP–Iso plus anti-CD180 (Fig. 4A). OVA–Iso were unable to stimulate proliferation in OVA-specific In addition, nonspecific B cells proliferated to some degree in T cells. Intriguingly, APCs from mice that had received OVA–Iso response to both NP–anti-CD180 and NP–Iso plus anti-CD180 in- plus anti-CD180 were similarly unable to activate Ag-specific oculation. As both the numbers of nonspecific proliferating B cells T cells. These data demonstrate that when Ag is directly conju- and level of proliferation were the same following both inocula- gated to anti-CD180, both immature and mature B cells are capable tions (Supplemental Fig. 3A, 3B), this nonspecific B cell prolif- of presenting Ag and stimulating Ag-specific T cells. eration was likely because of the activation of CD180, independent Downloaded from of other signals, such as the BCR. Indeed, the division index, the CD180 targeting induces Ag-specific B cells to migrate to the average number of cell divisions that a cell underwent, was sig- splenic T cell zone nificantly higher in Ag-specific B cells following NP–anti-CD180 The ability of B cells to present Ag and activate Ag-specific inoculation than nonspecific B cells (Supplemental Fig. 3C), show- T cells in vitro suggested that CD180 targeting may program ing that Ag-specific cells were more likely to begin to proliferate

B cells to migrate to the T cell zone to interact with cognate and undergo more rounds of proliferation than nonspecific B cells. http://www.jimmunol.org/ T cells. To test this hypothesis, we first examined the surface These data suggest that CD180 and BCR signaling synergizes in expression of CXCR5, the chemokine receptor that binds to Ag-specific cells to boost proliferation and/or there are additional CXCL13, guiding cells to B cell follicles (19). All Ag-specific signals received by the Ag-specific cells that promote activation. FO B cells that encountered Ag decreased their CXCR5 ex- The Ag-specific proliferating B cells induced by NP–anti-CD180 pression 24 h after inoculation (Fig. 3A), consistent with report- comprised a single population that straddled the FO and T1 gates ed events following BCR stimulation (19). However, the levels (Fig. 4B). The overall number of T1 B cells increased after CD180 of CXCR5 on Ag-specific FO B cells from mice inoculated with targeting (Fig. 4C), which is likely reflective of their proliferation NP–anti-CD180 were significantly lower than Ag-specific FO (Fig. 4B) but may also be due to increased survival. These data

B cells from mice inoculated with NP–Iso+/2anti-CD180. This highlight the unique ability of CD180 targeting to activate newly by guest on September 25, 2021 decrease in CXCR5 expression suggested that FO B cells were formed Ag-specific B cells. gaining the ability to exit B cell follicles and might migrate to Given the rapid Ag presentation by B cells and their ability to T cell zones. stimulate Ag-specific T cells in vitro after CD180 targeting, To address this possibility, we isolated B cells from B1-8hi mice we tested whether the in vivo Ag-specific B cell proliferation and adoptively transferred them into B6 mice, rested the mice observed3dafterAgtargetingwasdependentonTcells. for∼18 h, and then inoculated them with NP–Iso plus anti- We transferred CFSE-labeled B1-8hi B cells into B6 mice that CD180 or NP–anti-CD180. We isolated spleens every day after previously had been inoculated with an anti-CD4 mAb to de- immunizationfor3dtoexamineB cell migration by confocal plete CD4+ T cells, or an Iso control mAb. The numbers of microscopy (Fig. 3B–D). NP-specific B cells at the time of in- Ag-specific B cells that proliferated in the absence of CD4+ oculation (∼18 h after transfer) were present in B220+ B cell T cells was significantly reduced compared with the Iso control– follicles and in extra-FO regions, defined by presence of Ly-6G+ treated mice (Fig. 4D). This suggests that the CD180-targeted neutrophils (Fig. 3B). One day following NP–anti-CD180 inocu- B cells are not only capable of stimulating Ag-specific T cells, lation, NP-specific B cells could be found at the border between the but that these T cells are, in turn, then required for Ag-specific B cell follicles and the T cell zone, as defined by the presence of B cell proliferation. CD3+ cells (Fig. 3C top panel). More NP-specific B cells were identified at the T/B border 1 d following NP–anti-CD180 inocula- CD180-targeted B cells contribute to the rapid development of tion than at day 0 or following NP–Iso plus anti-CD180 inoculation Ag-specific T FO helper cells (Fig. 3D), suggesting that NP-specific B cells trafficked to the T/B Given that Ag-specific B cell proliferation 3 d after CD180 tar- border following CD180 targeting. By day 2 after NP–anti-CD180 geting was TD, we next investigated if these T–B interactions led to inoculation (Fig. 3C, middle panel), the numbers of NP-specific T cell activation and maturation as well. Three days following B cells had clearly increased, and they remained near T–B inoculation of mice with OVA–Iso plus anti-CD180 or OVA–anti- borders even at day 3 (Fig. 3C, bottom panel). By contrast, 3 d CD180, OVA-specific CD4+ T cells were analyzed using tetramer after inoculation of mice with NP–Iso plus anti-CD180, very enrichment. Total splenocytes were incubated with a class II few NP-specific B cells could be found, and those that were OVA tetramer and then run over a magnetic column to enrich for detected were located in follicles or extra-FO spaces (Fig. 3E). Ag-specific CD4+ T cells. The resulting fraction was analyzed by

three experiments per time point (n = 1) for Mock, OVA–Iso or (n = 6) for OVA–Iso plus anti-CD180 and OVA–anti-CD180. *p , 0.05, **p , 0.01, ***p , 0.001, by two-way ANOVAwith Sidak multiple comparison test (A), Kruskal–Wallis test with Dunn multiple comparison test (B), or Mann–Whitney U test (C). aCD180, anti-CD180. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. CD180 Ag targeting induces Ag-specific B cells to migrate to the T–B border region of the spleen. (A) B1-8hi mice were inoculated with 50 mg NP–Iso, NP–Iso plus anti-CD180, or NP–anti-CD180. Twenty-four hours later, the expression of CXCR5 was evaluated on NP–BCR2 and NP– BCR+ splenic B cells by flow cytometry. Data are the mean 6 SEM representative of two independent experiments (n = 3–4 per group). *p , 0.05, ****p , 0.0001 by two-way ANOVAwith Sidak multiple comparison test. (B–E) B cells enriched from B1-8hi mice were transferred into WT mice, which were rested overnight and then inoculated with 50 mg NP–Iso plus anti-CD180 or NP–anti-CD180. Spleens were isolated from uninoculated mice (day 0) or inoculated mice every day for 3 d. Spleens were cryopreserved, sectioned, and stained for NP–BCR, B220, CD3 and Ly-6G. Images were acquired by confocal microscopy at 203 objective and are representative of five images taken from two sections and two mice per group. (D) Total NP-specific B cells were counted at the border region between the B220-expressing B cell follicle and the CD3-expressing T cell zone from a total of 20 images from four sections representing two different mice per group. ***p , 0.001 by Kruskal–Wallis test with Dunn multiple comparison test. aCD180, anti-CD180.

flow cytometry for the expression of CXCR5 and PD-1 to identify T cells in vitro, we next investigated whether B cells were required Ag-specific T FO helper (TFH) cells. As seen in Fig. 5A, there was for TFH cell maturation. Following inoculation of NP–anti-CD180, + + a significant increase in OVA-specific CXCR5 , PD-1 TFH cells mMT mice (lacking all B cells) failed to form mature TFH cells by 3 d after OVA–anti-CD180 inoculation as compared with the 3 d (Fig. 5B, 5C). Intriguingly, there was not a significant dif- mock or unconjugated control. Thus, at the same time that Ag- ference in either the total number of TFH cells per spleen or the specific B cells proliferate following CD180 targeting, Ag-specific TFH cells as a percentage of the total OVA-specific population in + 2/2 CD4 T cells are programmed to mature into TFH cells. Given that BAFFR mice (with only T1 B cells) as compared with WT we had observed CD180-targeted B cells presenting Ag to cognate controls following CD180 targeting (Fig. 5C). Furthermore, there 8 CD180-TARGETED B CELL RESPONSES Downloaded from http://www.jimmunol.org/

FIGURE 4. CD180 targeting induces rapid, TD, Ag-specific B cell proliferation. Splenic B cells were isolated from B1-8hi (Ly-5.1+) mice labeled with by guest on September 25, 2021 CFSE and transferred into C57BL/6 mice. The next day, the mice were inoculated with 50 mg NP–Iso, NP–Iso plus anti-CD180, or NP–anti-CD180, and spleens were harvested 3 d later for analysis by flow cytometry. (A) Representative flow plots of B220+, Ly-5.1+ cells, and data (mean 6 SEM) of CFSE- diluted, NP–BCR+ cells per spleen. (B) Representative flow plots of B220+, Ly-5.1+ cells showing their CD21, CD24 along the axes and either CFSE or NP–BCR expression as a heat map overlay. (C) Total B220+, Ly-5.1+ B cell subsets from the spleen. (D) C57BL/6 mice were inoculated with anti-CD4 or an Iso control Ab 5 h before B cell–adoptive transfer and 24 h before NP–Ab inoculation (as above). Three days later, B220+, Ly-5.1+, NP–BCR+, CFSE- diluted cells were examined in the spleen by flow cytometry. Data are mean 6 SEM combined from two independent experiments (n = 5 per group). *p , 0.05, **p , 0.01 by Kruskal–Wallis test with Dunn multiple comparison test. aCD4, anti-CD4; aCD180, anti-CD180.

+ was less difference in the TFH cells as a percentage of tetramer CD180, it is expressed on both B cell and non–B cell APCs and cells in WT and BAFFR2/2 mice (mean 43.38 versus 37.25) because anti-CD40 alone can induce DCs to become efficient + (Fig. 5C, right panel) compared with the total tetramer TFH cell APCs (20). number in the spleen (mean 30.02 versus 18.10) (Fig. 5C, left We first compared the ability of CD40 targeting versus CD180 panel). This would suggest that, whereas the mature B cells in the targeting to induce early Ag-specific B cell proliferation (Fig. 6). + hi WT mice contribute to the maturation of TFH cells and, thus, there B6 mice were inoculated with CFSE-labeled Ly-5.1 B1-8 are more total TFH cells in the WT mice, immature B cells have B cells and then with either NP–anti-CD180 or NP–anti-CD40; an equivalent capacity as mature B cells in providing maturation 3 d later, spleens were isolated, and CFSE dilution was evalu- signals to cognate T cells, resulting in more equal levels as mea- ated. By examining the fluorescent intensity of CFSE as a heatmap sured by the percentage of total Ag-specific T cells. Overall, these overlay of the Ly-5.1+ B cell’s CD21 and CD24 expression (Fig. 6A), data suggest that the rapid maturation of TFH cells following we observed that multiple B cell subsets proliferate in response CD180 targeting requires B cells and that immature B cells are to CD40 targeting, as evident by the green, low-intensity CFSE sufficient for this response. signal. In contrast, CD180 targeting induced proliferation in a more restrictive population, straddling the FO/T1 gates (Fig. 6A). In contrast to CD180-targeted B cells, Ag-specific B cells do Furthermore, CD180 targeting induced proliferation of T1 B cells not proliferate following CD40 targeting (Fig. 6B), and the proportion of surviving transferred B cells that The findings above show that targeting Ag to CD180 induces were T1 B cells was significantly greater than following NP–Iso rapid Ag presentation, movement, and proliferation of Ag-specific inoculation (Fig. 6B). This was not observed following CD40 B cells and that these responses are dependent on the direct targeting; there was no change in the proportion of T1 B cells conjugation of Ag to anti-CD180. We next asked whether these following NP–anti-CD40 inoculation compared with NP–Iso in- responses were unique to CD180 targeting or if we could reca- oculation. To address further whether this phenomenon was re- pitulate the results by targeting Ag to a different B cell–activating stricted to CD180 targeting, we tested the ability of R848, a TLR7 receptor, CD40. We selected CD40 as a target because, like agonist, to improve T1 B cell survival; TLR7 is expressed in T1 The Journal of Immunology 9

FIGURE 5. CD180 targeting induces rapid TFH cell maturation. (A) C57BL/6 WT mice were inoculated with 25 mg OVA–Iso plus anti-CD180 or OVA– anti-CD180; 3 d later, OVA tetramer-specific cells were isolated from splenocytes by magnetic bead enrichment. Total B2202, CD11c2, CD11b2, CD3+, CD82, CD4+, CD44+, tetramer+ cells per spleen were analyzed by flow cytometry. (B and C) WT, BAFFR2/2, and mMT mice were inoculated with 25 mg

OVA–anti-CD180, and TFH cells were analyzed by flow cytometry 3 d later following OVA tetramer enrichment. (B) Representative flow plots of CXCR5 + + + + + Downloaded from and PD-1 expression on the CD44 , tetramer populations. (C) Total tetramer , CXCR5 , PD-1 TFH cells per spleen (left) and TFH cells as a percentage of CD44+ tetramer+ cells (right). Data are mean 6 SEM combined from three independent experiments (A and C)(n = 6, Mock; 9, OVA–Iso plus anti-CD180, OVA–anti-CD180 in BAFFR2/2, OVA–anti-CD180 in mMT; or 12, OVA–anti-CD180 in WT per group). *p , 0.05, **p , 0.01 by Kruskal–Wallis test with Dunn multiple comparison test. aCD180, anti-CD180.

B cells and TLR7 stimulation induces proliferation of T1 B cells vigorous proliferation in Ag-specific B cells, whereas CD40 tar-

(21). We did not observe significant changes in the T1 B cell geting induced more robust proliferation in nonspecific B cells http://www.jimmunol.org/ population following NP–Iso plus R848 inoculation compared (Fig. 6D, 6E). These data suggest that CD180 signaling, in con- with NP–Iso alone (Supplemental Fig. 3D), highlighting the unique trast to CD40 signaling, is able to synergize with BCR signaling to capacity of CD180 targeting to activate this immature subset. program Ag-specific B cell activation. Furthermore, CD40 sig- Despite the robust proliferation of B cells following CD40 naling alone is a stronger inducer of B cell activation than CD180 targeting (Fig. 6A), very few of the proliferating B cells were Ag- alone, demonstrating a greater chance of off-target activation specific (Fig. 6C). Indeed, CD180 but not CD40 targeting induced following CD40 targeting. by guest on September 25, 2021

FIGURE 6. B cell proliferation differs as a result of CD40 versus CD180 targeting. Splenic B cells were isolated from B1-8hi (Ly-5.1+) mice labeled with CFSE and transferred into C57BL/6 mice. The next day, the mice were inoculated with 50 mg of NP–Iso, NP–anti-CD40, or NP–anti-CD180, and spleens were harvested 3 d later for analysis by flow cytometry. (A) Representative flow plots of B220+, Ly-5.1+ cells showing their CD21, CD24 expression along the axes and CFSE expression as a heatmap overlay. (B) Splenic B cell subsets as a percentage of total B220+, Ly-5.1+ cells. Data are mean 6 SEM combined from two experiments (n = 6 per group). (C) Representative flow plots of B220+, Ly-5.1+ cells showing their CD21, CD24 along the axes and NP–BCR expression as a heat map overlay. (D) CFSE-diluted NP–BCR2 (left) or NP–BCR+ (right) B220+, Ly-5.1+ cells per spleen. (E) Division index of NP–BCR2 (left) or NP–BCR+ (right) cells. Data (D and E) are mean 6 SEM combined from three experiments (n = 9 per group). *p , 0.05, ***p , 0.001, ****p , 0.0001 by Kruskal–Wallis test with Dunn multiple comparison test (B) or Mann–Whitney U test (D and E). aCD40, anti-CD40; aCD180, anti-CD180. 10 CD180-TARGETED B CELL RESPONSES

CD40 targeting does not replicate the induction of T–B rapid, robust, high- affinity and long-lasting Ag-specific Ab re- interactions observed following CD180 targeting sponse than targeting Ag to CD40 (Fig. 8). We next compared the ability of B cell subsets and DCs to stimulate Ag-specific CD4+ T cells following CD40 versus CD180 target- Discussion ing. B6 mice were inoculated with OVA–anti-CD40 or OVA– In this study, we have demonstrated how Ag–anti-CD180 robustly anti-CD180; 24 h later, splenic DCs and B cell subsets were activates multiple B cell subsets to induce a rapid, high-affinity isolated and incubated with CFSE-labeled OT-II T cells. Unlike and long-lasting Ag-specific IgG Ab response. Immediately fol- CD180 targeting, inoculating mice with OVA–anti-CD40 did not lowing inoculation of Ag–anti-CD180, the Ag-specific BCR is rapidly internalized on all B cell subsets. Subsequently, Ag is activate either T1 or FO B cells to stimulate Ag-specific T cell processed and presented on MHC II, and the costimulatory ligand proliferation (Fig. 7A). CD40-targeted MZ B cells induced very CD86 is induced. These Ag-specific B cells then migrate to T–B low levels of T cell proliferation, significantly lower than CD180 borders within a day or two and proliferate in a TD manner. targeted MZ B cells. Whereas CD11c+ DCs from CD40 targeted Ag-specific T cells receive signals, leading to their maturation into mice could induce Ag-specific CD4 T cell proliferation, they were T cells, a process that requires B cells and that immature B cells significantly less effective compared with CD180-targeted DCs. FH are sufficient to induce. The initial clonal expansion of B cells More OT-II T cells stimulated by CD180 targeted DCs divided includes, in part, an immature B cell population that most likely and, once they started proliferating, underwent more cell divisions seeds the germinal centers (GCs) evident by day 7 (2). Intriguingly, than OT-II T cells stimulated by CD40-targeted DCs (Fig. 7B). targeting to CD180 induced an earlier and more robust Ag-specific Next, we compared the ability of CD40 targeting versus CD180 Ab response than targeting to CD40. Downloaded from targeting to induce the maturation of T cells. B6 mice were FH Critically, the key events described above are dependent on inoculated with either OVA–anti-CD40 or OVA–anti-CD180. the physical connection between the Ag and the anti-CD180 Ab, Three days later OVA-specific T cells, following tetramer en- as the unconjugated control (Ag–Iso plus anti-CD180) failed to richment of splenocytes, were examined by flow cytometry. induce Ag presentation, Ag-specific T cell activation, or Ag-specific Compared with CD40 targeting, there was an increase in the B cell proliferation. These findings are somewhat counterintuitive, total number of CD4+, CD44+, and tetramer+ cells in the spleens as Ag-specific B cells exposed to Ag–Iso plus anti-CD180 should http://www.jimmunol.org/ of mice following CD180 targeting, but this was NS (Fig. 7C, + still receive signals through both the BCR and CD180. The di- left). However, the phenotype of these CD4 T cells was dif- vergence in response and ultimate inability of separated Ag and ferent; we found that there was a significant increase in the + + anti-CD180 Ab to induce a robust Ab response is most likely numbers of OVA-specific, CXCR5 ,PD-1 TFH cells in mice that because of differences between Ag–anti-CD180 versus Ag plus had received OVA–anti-CD180 compared with OVA–anti-CD40 anti-CD180 in Ag internalization and subsequent processing and (Fig. 7C, right). presentation. After encountering an Ag, the BCR induces a sig- Given the differences in both B cell and T cell activation fol- naling cascade that includes the phosphorylation and activation lowing CD40 versus CD180 targeting, we next compared the of downstream kinases as well as the adaptor protein 2 (AP-2) ability of these two platforms to induce Ag-specific IgG responses. complex, which is critical for the initiation of clathrin-mediated by guest on September 25, 2021 Both the magnitude and kinetics of NP-specific IgG Ab responses endocytosis (22). AP-2 binds to the BCR via an YxxF motif on were different after targeting Ag to CD40 versus CD180 (Fig. 7D, the Igab subunit, F being a bulky hydrophobic amino acid, such left). Following inoculation of mice with NP–anti-CD180, NP- as leucine or isoleucine (23). Intriguingly, the short cytoplasmic m specific IgG production peaked at day 7 (mean 7325 g/ml se- tail of CD180, contains a conserved YxxI sequence (5); thus, this rum), whereas the peak response following CD40 targeting with sequence could potentially provide an additional partner for AP-2 ∼ NP–anti-CD40 occurred at day 14 and was 10-fold lower (mean binding and, thereby, facilitate the more rapid BCR internalization m 748 g/ml serum). Furthermore, the NP-specific IgG titers decreased observed following Ag–anti-CD180 inoculation (Fig. 1). Activa- more rapidly following CD40 targeting than CD180 targeting; by tion of Lyn downstream of the BCR directly phosphorylates the ∼ 49 d postinoculation, the titers were 2-fold higher in mice that clathrin H chain promoting the coupling of clathrin to actin, had received NP–anti-CD180 compared with those that received whereas other elements of BCR signaling, such as Vav, Bam32, NP–anti-CD40. To assess relative affinity of the Abs, we measured and Btk, are also critical for endocytosis (22, 24). The Lyn and Btk the binding of IgG to BSA with low levels of conjugated NP kinases are also activated following the ligation of CD180 on (NP2), which will bind to high-affinity Abs and compared the the surface of B cells (5). Thus, it is conceivable that the bind- binding of IgG to BSA conjugated to high levels of NP (NP20), ing of both the BCR and CD180 through the Ag–anti-CD180 which will bind Ab of both high and low affinity. CD180 targeting complex enhances internalizationbybringingtogethercritical was also better at inducing affinity maturation than targeting to signaling partners. Further studies are required to assess this CD40. At days 14 and 21 following inoculation, the NP2/NP20 possibility. ratio was .2-fold higher in mice that had received NP–anti-CD180 Once the B cells have acquired the Ag via direct delivery from versus NP–anti-CD40 (Fig. 7D, right), indicating a greater pro- Ag–anti-CD180, they downregulate CXCR5, travel to B–T bor- portion of high-affinity Ab in these mice. Finally, we examined the ders within 1 d, and rapidly begin to proliferate (Fig. 3). This levels of NP-specific IgG Ab-secreting cells (ASCs) in the spleen initial proliferation requires T cells (Fig. 4) and may well be in- and BM following targeting of Ag to CD40 or CD180. Nine weeks dependent of DCs. In support of this hypothesis, we previously after inoculation with NP–anti-CD180, there were on average 590 demonstrated that the majority of the Ag-specific IgG response NP-specific IgG ASCs in the spleen compared with more than 10 d after Ag–anti-CD180 inoculation requires the expression of 10-fold fewer ASCs (mean 45 NP-specific IgG ASCs) in mice CD180 on B cells but not DCs (2). Scandella and colleagues inoculated with NP–anti-CD40 (Fig. 7E, left). Similarly, there (25) observed a similar phenomenon following vesicular stoma- were significantly more NP-specific ASCs in the BM of mice titis virus infection, wherein vesicular stomatitis virus–specific inoculated with NP–anti-CD180 compared with NP–anti-CD40 B cells traveled to T–B borders rapidly, proliferated, and produced (18.9 versus 2.1 per 106 BM cells; Fig. 7E, right). Collectively, neutralizing Abs in mice that had been depleted of CD11c+ DCs. these data demonstrate that Ag targeting to CD180 induced a more Furthermore, it has been demonstrated that naive CD4+ T cells are The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/ FIGURE 7. CD40 targeting does not recapitulate key events following CD180 targeting. (A and B) C57BL/6 mice were inoculated with 35 mgofOVA– anti-CD40 or OVA–anti-CD180; 24 h later, DCs were isolated from spleen by positive bead selection, B cells were isolated by negative bead enrichment, and B cell subsets were sorted by FACS. A total of 1 3 105 APCs were cocultured with 1 3 105 CFSE-labeled OT-II T cells. Three days later, T cell proliferation was evaluated as CFSE dilution by flow cytometry. The division index (B, left) is the average number of divisions that a cell has undergone. Data are the mean 6 SEM combined of three experiments (n = 4, OVA–anti-CD40) or (n = 6, OVA–anti-CD180). (C) C57BL/6 mice were inoculated with 25 mg of OVA–anti-CD40 or OVA–anti-CD180; 3 d later, OVA tetramer-specific cells were isolated from splenocytes by magnetic bead enrichment. Total + + + + + CD44 , tetramer (left) and tetramer , CXCR5 , PD-1 TFH cells (right) were analyzed by flow cytometry. Data are mean 6 SEM combined from three independent experiments (n = 12 per group). (D and E) C57BL/6 mice were inoculated with 50 mg of NP–Iso, NP–anti-CD40, or NP–anti-CD180. Once a D D

week, mice were bled, and NP-specific IgG ( , left) and the ratio of high-affinity NP-specific IgG (NP2) to total NP-specific IgG (NP20) ( , right) was by guest on September 25, 2021 quantitated by ELISA. Nine weeks after inoculation, spleens and BM were harvested, and NP-specific IgG-secreting ASCs were evaluated by ELISPOT (E). Data are mean 6 SEM combined from two independent experiments (n = 8 per group). *p , 0.05, **p , 0.01, ***p , 0.001 by Mann–Whitney U test (A–D, right), Kruskal–Wallis test with Dunn multiple comparison test (E), or two-way ANOVA with Sidak multiple comparison test (D, left).

+ predominantly activated by Ag-presenting B cells and not DCs PD-1 TFH cells in WT mice but not B cell–deficient mMT mice following virus-like particle vaccination (26). Regardless, both following Ag–anti-CD180 inoculation (Fig. 5). It is likely that the B cells and DCs from CD180-targeted mice are capable of pre- combined Ag presentation and expression of T cell costimulatory senting Ag to cognate T cells. The consequences of Ag presentation molecules, such as CD86 following CD180 targeting (Fig. 2), are by B cells are multifaceted and distinct from those of DCs. To start driving this reaction. Indeed, prolonged T/B interactions at the with, the peptide/MHC II complexes derived from BCR-associated follicle border, which are necessary for both the production of Ag acquisition are predominantly of the rare M1 MHC II con- extra-FO plasmablasts and the seeding of GCs, are dependent formation, defined by the Ia.2 epitope (27). This MHC epitope is on cognate interactions, suggesting that the B cell MHC II/TCR associated with the robust activation of CD4+ T cells (28). It is interaction is critical (32, 33). This is partly because of TCR- tempting to speculate, therefore, that CD4+ T cells activated by dependent activation of ICOS on the T cell, which is critical for B cells are qualitatively different from those stimulated by DCs. TFH cell development and maintenance (32). However, it is also This may be one potential explanation for the higher-affinity Abs possible that CD180 Ag targeting influences the cytokine profile following Ag–anti-CD180 inoculation than Ag–anti-CD40, given of Ag-specific B cells, which may influence the maturation of TFH the ability of CD180 targeting but not CD40 targeting to induce cells. Additional studies have demonstrated that, whereas ini- B cells to present Ag to T cells. tial CD4+ T cell activation and expansion may rely on DCs, the The differential effects of B cell– versus DC-derived T cell APC function of B cells is critical to the formation of memory activation are not fully understood. The combined APC action T cell populations (34). Furthermore, BCR-related Ag presenta- of B cells and DCs leads to more robust proliferation and cytokine tion by B cells can break CD4+ T cell tolerance (26), a critical step production by CD4+ T cells than when DCs or B cells alone are for many immunotherapeutic vaccination strategies, including the capable of presenting Ag (29). Furthermore, B cells are required treatment of patients chronically infected with hepatitis B virus for the maturation of TFH cells (30). Indeed, whereas the initial (35). Thus, the combined APC action of both B cells and DCs upregulation of CXCR5 on CD4+ cells relies on DC MHC II ex- following Ag–anti-CD180 inoculation highlight a strength of pression, only the combined APC function of both DCs and B cells this vaccine strategy in producing robust and sustained adaptive + hi is sufficient to induce fully mature CXCR5 ,PD-1 TFH cells fol- immune responses. lowing Ag plus alum stimulation (31). This finding corresponds The effect of targeting Ag to APCs has been explored for many with our data, in which we observed Ag-specific CXCR5+, years. The majority of strategies have been to target DCs through a 12 CD180-TARGETED B CELL RESPONSES Downloaded from

FIGURE 8. Comparing CD180 versus CD40 targeting. (1) Upon interaction with a CD180 targeting construct, cognate T1 and FO B cells internalize, process, and present Ag on MHC II along with concomitant CD86 expression. FO B cells also downregulate CXCR5 and migrate to the T–B border. It is unclear whether T1 B cells also migrate toward the T cell zone. (2) Once at the T–B border, CD180-targeted–activated cognate B cells require cognate http://www.jimmunol.org/ interactions and begin to proliferate. (3) These clonal offspring seed GCs. (4) Cognate T cells that interact with CD180-targeted B cells, either mature or immature, expand and rapidly mature into TFH cells with upregulation of CXCR5 and PD-1. (5) The end result of these interactions is the production of high-affinity Ag-specific IgG and LLPCs. (6) Noncognate B cells that interact with the Ag–anti-CD180 are capable of processing and presenting Ag. However, they do not receive a signal through the BCR and therefore do not downregulate CXCR5 and therefore do not travel to the T–B border. These cells undergo limited proliferation because of CD180 signaling. (7) By contrast, CD40-targeted cognate B cells do not present Ag and are not programmed to seek out cognate T cell interactions. There is limited evidence of Ag-specific B cell proliferation, and (8) CD40-targeted B cells do not interact with cognate T cells; thus, by day 3, TFH cell maturation is not evident. (9) The result of CD40 targeting is the production of moderate-affinity, Ag-specific IgG and short-lived plasma cells, which may form independently of GCs (10). Noncognate B cells, however, robustly proliferate because of CD40 signaling. by guest on September 25, 2021 number of different receptors (36–39). Many of these platforms adaptive programming (via CD40) (Fig. 8). CD180 targeting pro- excel at inducing Ag-specific CD8+ T cells, but few are able to grams Ag-specific B cells to seek cognate T cell interactions, elicit robust TD Ab responses without the addition of an adjuvant whereas CD40 targeting itself mimics T cell help, thus removing (37). For example, targeting to DEC-205 is only able to activate the checkpoint of Ag-specific T cell help. Therefore, the early in- Ag-specific CD4+ T cells and induce Ag-specific IgG in combi- duction of Ag presentation in B cells by CD180 targeting results in nation with additional signals, such as anti-CD40 (37). Indeed, Ag rapid maturation of TFH cells not observed following CD40 tar- targeting to DEC-205 without an adjuvant induced Ag-specific geting (Fig. 7C). This may explain the more rapid and higher- tolerance (1, 40). Directing Ag to the DC receptor DCIR2 in- affinity Ag-specific IgG produced in response to CD180 targeting duces DCs to hand-off Ag to and activate B cells, which in turn compared with CD40 (Fig. 7D). In addition, the generation of long- become potent APCs, but are unable to promote GC formation lived plasma cells (LLPCs) relies on TFH cell interactions in GCs without the addition of a TLR7- or TLR9-based adjuvant (41). (44). The lack of LLPCs in the BM following CD40 targeting Park and colleagues (42) demonstrated that targeting influenza Ag (Fig. 7E) suggests that GCs may not form or are severely limited. to Clec9A is able to protect mice from death but not severe disease Although CD40 targeting induced moderate-affinity maturation, this following a lethal influenza challenge; boosting mice with Ag + may not necessarily require formation of GCs, as several studies CpG before challenge lessened morbidity significantly. By com- have found evidence of GC-independent somatic hypermutation parison, our studies show that a single immunization targeting Ag (Refs. 45, 46 and G.H. Pritchard, A.T. Krishnamurthy, J. Netland, to CD180 without an adjuvant is sufficient to induce robust TD E.N. Arroyo, K.K. Takehara, and M. Pepper, manuscript posted on Ag-specific IgG responses and protective Ab responses (4), a bioRxiv). Furthermore, Iso-switched plasmablasts can form with- regimen with the potential to decrease off-target or adverse events. out any specific Th subset, including TFH cells, but require a CD40 Although many studies have focused on targeting to CD40 and signal (47). Thus, mimicking CD40–CD40L interactions follow- CD40-based adjuvants because CD40 signaling can robustly ing CD40 targeting, although capable of inducing Iso-switched activate APCs, there are some concerns of adverse off-target plasmablasts, do not fully recapitulate a cognate interaction effects with this platform, such as cytokine release syndrome and are therefore not sufficient to drive a robust GC reaction and (43). Adding to this concern, our data reveal that targeting to LLPC formation. The reduction in LLPCs in CD40-targeted mice CD40 does not efficiently activate Ag-specific B cells to become compared with CD180-targeted mice suggest that long-term term APCs but, rather, causes robust nonspecific B cell proliferation B cell responses are more robust in response to CD180. Indeed, (Fig. 6D). In contrast, CD180 targeting effectively and specif- we have previously demonstrated secondary Ab responses follow- ically activated Ag-specific B cells to become APCs. This repre- ing CD180 targeting are much higher than Ag plus alum inoculation sents a fundamental difference between these two strategies of (2). However, following a prime-boost vaccination scheme using a targeting innate immune programing (via CD180) versus activating replication-deficient adenovirus expressing CD40L and respiratory The Journal of Immunology 13 syncytial virus L, Ag did lead to a boost in neutralizing Abs Consistently, it has been estimated that, whereas selection of and a reduction in virus titer postvirus challenge (48). Further B cells begins in the BM, approximately one third of peripheral studies are required to define more fully the differences in sec- tolerance occurs at the late transitional stage within the periphery ondary B cell responses would be following CD180 versus CD40 (66). Furthermore, a study of circulating human B cells identified targeting. a unique H chain repertoire in transitional B cells as compared Another important feature of CD180 targeting is its ability to with both immature BM and naive mature B cells (67), suggesting target and activate immature or newly formed B cells. Immature that both positive- and negative-selective pressures may be acting B cells migrating from the BM first travel to the spleen as T1 B cells upon peripheral newly formed B cells. Some of this diversification (49, 50). Typically, T1 B cells are programmed to undergo apo- may occur in the gut; studies in germ-free mice reveal that the ptosis upon BCR stimulation; indeed, this may be a mode of mucosal commensal-reactive Ig repertoire is defined within T1 continued negative selection within the spleen (51). However, T1 B cells (68). Similarly, T2 B cells within human GALT have been B cells can be activated by other signals, including TLR ligation found to be activated and express mutated IGHV genes (69). Thus, or IL-4 stimulation (52–55). Furthermore, BCR-stimulated T1 interaction between T1/T2 B cells with commensal Ags drives B cells can be rescued from apoptosis with the addition of certain diversification of the Ig repertoire. Indeed, T1 B cells, unlike FO signals, such as IL-4 or CD40 (52, 56). CD180 appears to have a B cells, constitutively express activation-induced cytamine de- similar effect on T1 B cells, given the expansion of this population aminase and undergo somatic hypermutation in BM (21, 53, 54, observed following BCR plus CD180 stimulation in the CD180- 70). Thus, T1 B cells are attractive targets in the hunt to generate targeted B cells (Fig. 4C). The TLR-related functions of T1 methods to induce broadly neutralizing Abs by vaccination. It has

B cells are largely T cell independent; there is little evidence of T1 been demonstrated, for example, that germline targeting of human Downloaded from B cells directly interacting with T cells. Indeed, Chung and col- naive B cells can induce the production of broadly neutralizing leagues (57) reported that T1 B cells can present Ag on MHC II Abs against HIV (71). Therefore, the combined ability to activate but that they fail to upregulate CD86 and therefore are poor B cells as APCs and to target immature B cells via CD180 Ag stimulators of Ag-specific T cells. Ag–anti-CD180 activation of targeting make this vaccine platform ideal in forming robust hu- T1 B cells appears to bypass this limitation, as T1 B cells not only moral and cellular immunity, especially in immunodeficient or

upregulate CD86 and MHC II but also are quite potent stimulators immunosuppressed individuals or with the goal of generating http://www.jimmunol.org/ of Ag-specific T cells. Intriguingly, after Ag–anti-CD180 inocu- broadly neutralizing Abs. 2/2 lation, we observed mature TFH cells in BAFFR mice but not mMT mice, suggesting, for the first time, to our knowledge, that Acknowledgments even newly formed B cells are able capable of providing matu- We thank Drs. Natalia Giltiay and Keith Elkon for helpful insights and dis- ration signals to developing TFH cells. Furthermore, the initial cussion. We thank Nicole Arroyo for assistance in preparation of the OVA clonal B cell expansion that occurs following CD180 targeting is tetramer. This research was supported by the Cell and Analysis Flow of a select population of cells at the T1–FO interface (Fig. 4B), a Cytometry and Imaging Core in the Department of Immunology at the population not observed following CD40 targeting (Fig. 6A). Our University of Washington. previous data suggests that these are, in fact, activated immature by guest on September 25, 2021 2/2 B cells. B cells from BAFFR mice, which in the absence of Disclosures BAFF signaling fail to mature beyond the T1 stage, are responsive The authors have no financial conflicts of interest. to Ag–anti-CD180 vaccination, forming Ag-specific plasma cells. This response results in Ag-specific IgG production that is suffi- cient to protect mice from a lethal viral challenge (4). References The ability to achieve protective immunity through targeting 1. Chappell, C. P., N. V. Giltiay, K. E. Draves, C. Chen, M. S. Hayden-Ledbetter, immature B cells demonstrates a significant advantage of this M. J. Shlomchik, D. H. Kaplan, and E. A. Clark. 2014. Targeting antigens through blood dendritic cell antigen 2 on plasmacytoid dendritic cells promotes vaccination platform in situations in which the B cell repertoire is immunologic tolerance. J. 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