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

Abstract

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 (T_FH) cells. Remarkably, immature B cells were sufficient for the maturation of T_FH cells after CD180 targeting: T_FH cells were induced in BAFFR^-/- mice (with only transitional 1 B cells) and not in μMT 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 T_FH 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 T_FH cells, providing insights toward vaccination of mature B cell-deficient individuals and implications in treating autoimmune disorders.

Figure 1.. The Ag-specific BCR is internalized rapidly following targeting to CD180.

B1-8^hi mice were inoculated with 50μg NP-Iso, NP-Iso + αCD180 or NP-αCD180. Three, six or 25 h later, spleens were harvested and processed for flow cytometry. (A) Representative flow plots of total live, single, B220⁺ cells three h after inoculation. The top panel demonstrates the gating strategy we 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. Transitional (T)1 B cells were defined as CD24^hi, CD21^lo; T2 as CD24^hi, CD21^mid; follicular (FO) as CD24^mid, CD21^mid; marginal zone (MZ) as CD24^hi, CD21^hi. The bottom panel demonstrates the change in NP-BCR expression following inoculation. (B) NP-BCR^hi expressing cells per spleen following inoculation in different B220⁺ splenic B cell subsets. Data is the mean ± SEM combined of two to three experiments per time point with n=4-8 per group. (C,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 is representative of three individual experiments. * p<0.05 and ** p<0.01 NP-Iso vs. NP-αCD180. † p<0.05, ‡ p<0.01 NP-Iso + αCD180 vs. NP-αCD180 by Kruskal-Wallis test with Dunn’s multiple comparison test (B) or 2-way ANOVA with Tukey’s multiple comparison test (D).

Figure 2.. Targeting Ags to CD180 results in Ag processing and presentation by B cells.

(A) B1-8^hi mice were inoculated with 50μg NP-Iso, NP-Iso + αCD180 or NP-αCD180. 24 h later, the expression of MHC II and CD86 was evaluated on NP-BCR⁻ and NP-BCR⁺ splenic B cells by flow cytometry. Data is the mean ± SEM representative of two independent experiments with n=3-4 per group. (B) B10.A mice were inoculated with 35μg HEL-Iso, HEL-Iso + αCD180 or HEL-αCD180. Three or 24 h later HEL-peptide bound-MHC II I-A^k, represented as peptide bound MHC II over total MHC II expression (HEL-I-A^k/I-A^k MFI) was evaluated on splenic B cell subsets and conventional (c)DCs by flow cytometry. cDCs were defined as B220⁻, CD3⁻, NK1.1⁻, CD11b^−/lo, CD11c^hi. Data is the mean ± SEM representative of two independent experiments per time point with n=3-4 per group. (C) C57BL/6 mice were inoculated with 35μg OVA-Iso, OVA-Iso + αCD180 or OVA-αCD180. Three or 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. 1×10⁵ APCs were co-cultured with 1×10^5 CFSE-labeled OT-II T cells. Three days later, T cell proliferation was evaluated as CFSE dilution by flow cytometry. Data is the mean ± SEM combined of three experiments per time point with n=1 (Mock, OVA-Iso) or n=6 (OVA-Iso + αCD180 and OVA-αCD180). * p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001 by 2-way ANOVA with Sidak’s multiple comparison test (A), Kruskal-Wallis test with Dunn’s multiple comparison test (B) or Mann-Whitney test (C).

Figure 3.. CD180 Ag targeting induces Ag-specific B cells to migrate to the T-B border region of the spleen.

(A) B1-8^hi mice were inoculated with 50μg NP-Iso, NP-Iso + αCD180 or NP-αCD180. 24 h later, the expression of CXCR5 was evaluated on NP-BCR⁻ and NP-BCR⁺ splenic B cells by flow cytometry. Data is the mean ± SEM representative of two independent experiments with n=3-4 per group. * p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001 by 2-way ANOVA with Sidak’s multiple comparison test. (B-E) B cells, enriched from B1-8^hi mice, were transferred into WT mice, which were rested overnight and then inoculated with 50μg NP-Iso + αCD180 or NP-αCD180. Spleens were isolated from uninoculated mice (Day 0) or inoculated mice every day for three days. Spleens were cryopreserved, sectioned and stained for NP-BCR, B220, CD3 and Ly6G. Images were acquired by confocal microscopy at 20x objective and are representative of 5 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 4 sections representing 2 different mice per group.

Figure 4.. CD180 targeting induces rapid, T cell-dependent Ag-specific B cell proliferation.

Splenic B cells were isolated from B1-8^hi (Ly5.1⁺) mice, labeled with CFSE and transferred into C57BL/6 mice. The next day the mice were inoculated with 50μg NP-Iso, NP-Iso + αCD180 or NP-αCD180 and spleens were harvested three days later for analysis by flow cytometry. (A) Representative flow plots of B220⁺, Ly5.1⁺ cells, and data (mean ± SEM) of CFSE diluted, NP-BCR⁺ cells per spleen. (B) Representative flow plots of B220⁺, Ly5.1⁺ cells showing their CD21, CD24 along the axes and either CFSE or NP-BCR expression as a heat map overlay. (C) Total B220⁺, Ly5.1⁺ B cell subsets from the spleen. (D) C57BL/6 mice were inoculated with anti-CD4 or an isotype control Ab 5h before B cell adoptive transfer and 24h before NP-Ab inoculation (as above). Three days later B220⁺, Ly5.1⁺, NP-BCR⁺, CFSE diluted cells were examined in the spleen by flow cytometry. Data is mean ± SEM combined from two independent experiments, n=5 per group. * p<0.05, ** p<0.01 by Kruskal-Wallis test with Dunn’s multiple comparison test.

Figure 5.. CD180 targeting induces rapid T_FH cell maturation.

(A) C57BL/6 (WT) mice were inoculated with 25μg OVA-Iso + αCD180 or OVA-αCD180; 3d later OVA-tetramer-specific cells were isolated from splenocytes by magnetic bead enrichment. Total B220⁻, CD11c⁻, CD11b⁻, CD3⁺, CD8⁻, CD4⁺, CD44⁺, tetramer⁺ cells per spleen were analyzed by flow cytometry. (B,C) WT, BAFFR^−/− and μMT mice were inoculated with 25μg OVA-αCD180 and T_FH cells were analyzed by flow cytometry 3d later following OVA-tetramer enrichment. (B) Representative flow plots of CXCR5 and PD-1 expression on the CD44⁺, tetramer⁺ populations. (C) Total Tetramer⁺, CXCR5⁺, PD-1⁺ T_FH cells per spleen (left) and T_FH cells as a % of CD44⁺ Tetramer⁺ cells (right). Data is mean ± SEM combined from three independent experiments (A,C). n=6 (Mock), 9 (OVA-Iso + αCD180, OVA-αCD180 in BAFFR^−/−, OVA-αCD180 in μMT) or 12(OVA-αCD180 in WT) per group. * p<0.05, ** p<0.01 by Kruskal-Wallis test with Dunn’s multiple comparison test. T_FH: T follicular helper cell.

Figure 6.. B cell proliferation differs as a result of CD40 vs. CD180 targeting.

Splenic B cells were isolated from B1-8^hi (Ly5.1⁺) mice, labeled with CFSE and transferred into C57BL/6 mice. The next day the mice were inoculated with 50μg NP-Iso, NP-αCD40 or NP-αCD180 and spleens were harvested three days later for analysis by flow cytometry. (A) Representative flow plots of B220⁺, Ly5.1⁺ cells showing their CD21, CD24 expression along the axes and CFSE expression as a heatmap overlay. (B) Splenic B cell subsets as a percent of total B220⁺, Ly5.1⁺ cells. Data is mean ± SEM combined from two experiments, n=6 per group. (C) Representative flow plots of B220⁺, Ly5.1⁺ cells showing their CD21, CD24 along the axes and NP-BCR expression as a heat map overlay. (D) CFSE diluted NP-BCR⁻ (left) or NP-BCR⁺ (right) B220⁺, Ly5.1⁺ cells per spleen. (E) Division index of NP-BCR⁻ (left) or NP-BCR⁺ (right) cells. Data (D,F) is mean ± SEM combined from three experiments, n=9 per group. * p<0.05, *** p<0.001 by Kruskal-Wallis test with Dunn’s multiple comparison test (B), or Mann-Whitney test (D,E).

Figure 7.. CD40 targeting does not recapitulate key events following CD180 targeting.

(A,B) C57BL/6 mice were inoculated with 35μg OVA-αCD40 or OVA-αCD180; 24h 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. 1×10⁵ APCs were co-cultured with 1×10⁵ 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 is the mean ± SEM combined of three experiments with n=4 (OVA-αCD40) or n=6 (OVA-αCD180). (C) C57BL/6 mice were inoculated with 25μg OVA-αCD40 or OVA-αCD180; 3d later OVA-tetramer-specific cells were isolated from splenocytes by magnetic bead enrichment. Total CD44⁺, Tetramer⁺ (left) and Tetramer⁺, CXCR5⁺, PD-1⁺ T_FH cells (right) were analyzed by flow cytometry. Data is mean ± SEM combined from three independent experiments, n=12 per group. (D,E) C57BL/6 mice were inoculated with 50μg NP-Iso, NP-αCD40 or NP-αCD180. Once a week, mice were bled and NP-specific IgG (D, left) and the ratio of high affinity NP-specific IgG (NP2) to total NP-specific IgG (NP20) (D, right) was quantitated by ELISA. Nine weeks after inoculation, spleens and bone marrow were harvested and NP-specific IgG secreting antibody secreting cells (ASCs) were evaluated by ELISPOT (E). Data is mean ± SEM combined from two independent experiments, n=8 per group. * p<0.05, ** p<0.01, ***p<0.001 by Mann-Whitney test (A, B, C, D right), Kruskal-Wallis test with Dunn’s multiple comparison test (E) or 2-way ANOVA with Sidak’s multiple comparison test (F left).

Figure 8.. Comparing CD180 vs. CD40 targeting.

(1) Upon interaction with a CD180 targeting construct, cognate T1 and FO B cells internalize, process and present Ag on MHCII 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 towards the T cell zone. (2) Once at the T-B border, CD180 targeted activated cognate B cells require cognate interactions and begin to proliferate. (3) These clonal offspring seed germinal centers. (4) Cognate T cells that interact with CD180 targeted B cells, either mature or immature, expand and rapidly mature into T_FH with upregulation of CXCR5 and PD-1. (5) The end result of these interactions is the production of high affinity Ag-specific IgG and long-lived plasma cells. (6) Non-cognate B cells that interact with the Ag-αCD180 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 due to 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 T_FH 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 germinal centers. (10) Non-cognate B cells, however, robustly proliferate due to CD40 signaling.