Limited clonal relatedness between gut IgA plasma cells and memory B cells after oral immunization

Abstract

Understanding how memory B cells are induced and relate to long-lived plasma cells is important for vaccine development. Immunity to oral vaccines has been considered short-lived because of a poor ability to develop IgA B-cell memory. Here we demonstrate that long-lived mucosal IgA memory is readily achieved by oral but not systemic immunization in mouse models with NP hapten conjugated with cholera toxin and transfer of B1-8(high)/GFP(+) NP-specific B cells. Unexpectedly, memory B cells are poorly related to long-lived plasma cells and less affinity-matured. They are α4β7-integrin(+)CD73(+)PD-L2(+)CD80(+) and at systemic sites mostly IgM(+), while 80% are IgA(+) in Peyer's patches. On reactivation, most memory B cells in Peyer's patches are GL7(-), but expand in germinal centres and acquire higher affinity and more mutations, demonstrating strong clonal selection. CCR9 expression is found only in Peyer's patches and appears critical for gut homing. Thus, gut mucosal memory possesses unique features not seen after systemic immunization.

Figure 1. Oral priming immunizations generate clonally related long-lived plasma cells in the gut lamina propria and bone marrow.

(a) Wild-type C57BL/6 mice were orally primed with three doses of 20 μg NP-CT at 10 days apart and serum anti-NP or anti-CT titres of the IgG (diamonds) or IgA (squares) class were determined by enzyme-linked immunosorbent assay at the indicated time points and shown with s.d. error bars. (b,c) After 1 year, lymphocytes were isolated from the small intestinal lamina propria (SI LP), bone marrow (BM) or spleen (Spl), and the total number of anti-NP or anti-CT IgA or IgG antibody-forming cells (AFCs)/10⁶ isolated cells were determined by enzyme-linked immunospot. (b) The number of NP-specific IgA and IgG AFCs in the different tissues and (c) the proportion of NP or CT-specific AFCs of all IgA or IgG AFCs in the LP or BM are shown with s.d. error bars. (d–g) RNA was prepared from the SI or BM from six mice, and NP-binding heavy chain IgG or IgA genes were cloned and sequenced. The average number of IgH V region mutations in unique sequences (d) and the proportion of sequences that carried the affinity-increasing CDR1 W33=>L or CDR3 Y=>G mutations (e) were determined in V_H186.2 gene rearrangements. Mann–Whitney test P values are given. The method used to define NP-binding V_H186.2 gene sequences as opposed to non-NP-binding sequences is described in the Methods section. (f) Clustal Omega analysis was used to determine sequence similarities in individual mice. Clones that share CDR3 VDJ rearrangements are marked with black lines. (g,h) Schematic representation of clones from the SI LP and BM that share IgA V region rearrangements (g) or IgA and IgG1 clones from the BM that share V region gene sequences (h). Point mutations in the V regions are marked in red if shared with other sequences in the group and black if unique to a single sequence. (i) Clonal tree analysis of clonally related NP-binding V_H186.2 sequences from individual mice identified clones that contain both IgA and IgG1 V region gene sequences. The number of mutations between neighbouring nodes is given next to the connecting edge, where no number is given the edge represents a single mutation. Data from five to six mice in each group in one representative experiment (a–c) of three giving similar results (pooled data in d–f).

Figure 2. Re-exposure to oral or systemic antigen after 1 year markedly boosts local and systemic immune responses in orally primed memory B cells.

(a) Wild-type C57BL/6 mice were orally primed with three doses of 20 μg NP-CT at 10 days apart and challenged 1 year later with a single oral challenge dose with NP-CT. (b) After 7 days, the numbers of anti-NP IgA (left) or IgG (right) AFCs/10⁶ cells in different tissues were determined by enzyme-linked immunospot in individual primed-only, primed and challenged or age-matched challenged-only mice. (c) Serum IgA or IgG anti-NP titres log₁₀ titres in individual mice. The dashed line identifies the detection limit for the assay. (d) The proportions of anti-NP or anti-CT IgA (left) or IgG (right) AFCs of total AFCs in indicated tissues are shown with s.d. error bars. (e–g) RNA was prepared from the SI LP, colon (Col), BM or spleen (Spl) from boosted mice, and NP-binding V_H186.2 genes were cloned and analysed: (e) V region mutations, (f) the frequency of the affinity-increasing CDR1 W33=>L or CDR3 Y=>G mutations and (g) a representation of clones from different tissues that share IgA V region rearrangements, with red indicating shared and black unique mutations. (h–l) Mice were either orally or intraperitoneally (i.p.) primed with NP-CT or NP-CGG plus CTA1-DD adjuvant, respectively, and then challenged after 6 months with NP-CT orally or i.p., as indicated (h). The number of anti-NP AFCs/10⁶ cells in the SI LP in individual mice is shown (i). Anti-NP IgA log₁₀ titres in gut lavage (j) or serum (k) or serum anti-NP IgG log₁₀ titres (l) in individual mice are shown. Data from three to six mice in each group in one representative experiment of three giving similar results (b–d). Graphs (e,f,i,i–l) are based on pooled data from three independent experiments. Mann–Whitney test P values are given (*0.01≤P<0.05, **0.005≤P<0.01, ***0.001≤P<0.005, ****P≤0.001).

Figure 3. Orally primed memory B cells express CD80, CD73 and PD-L2 and reside in B-cell follicles in mucosal as well as systemic lymphoid tissues.

(a) An adoptive transfer model with GFP⁺ NP-specific B cells was employed for monitoring memory B-cell development after oral immunization. Wild-type C57BL/6 mice were injected with 100,000 GFP⁺ λ-expressing NP-specific splenic B cells prepared from B1-8^high/GFP mice, as described in the Methods section. (b) One year after oral priming immunizations with NP-CT (left) an oral booster immunization was administered and 7 days later (right) sections of gut LP were analysed for the presence of GFP⁺ NP-specific plasma cells by confocal microscopy. Flow cytometry analysis of the mean (−) and individual (x) frequency of GFP⁺CD138⁺ plasma cells in single-cell suspensions from the BM or SI LP. (c) Before the booster immunization, we determined the frequency of memory GFP⁺ NP-specific CD138⁻B220⁺CD19⁺ cells/10⁶ total B cells in different tissues (spleen (Spl), MLN or PP) of six mice analysed individually by flow cytometry. (d) The proportion of GFP⁺ and GFP⁻ CD138⁻B220⁺CD19⁺ B cells that had an IgM⁻IgD⁻ isotype-switched phenotype in the indicated tissues. (e) The percentage of GFP⁺IgM⁻IgD⁻ isotype-switched memory B cells from PP that expressed IgA in four individual mice. (f) Expression of CD38 on GFP⁺ memory B cells and GL7⁺ and GL7⁻ IgA-expressing GFP⁻ B cells in PP. (g, see also Supplementary Fig. 5b) Representative frozen sections analysed by confocal microscopy for GFP⁺ memory B cells (green) in the PP, MLN and Spl with germinal centres labelled with anti-GL7-eFlour 660 (red) and B-cell follicles with anti-B220-biotin/Streptavidin-Alexa Flour 594 (blue). Below the main panels are channel-separated close-ups to demonstrate that the GFP⁺ cells are B220⁺, but do not express GL7. (h) Flow cytometry analysis for CD73 and PD-L2 expression on CD138⁻B220⁺CD19⁺ GFP⁺ memory B cells isolated from indicated tissues. For comparisons, GFP⁻ B cells are shown. The percentage of CD73⁺PD-L2⁺ memory B cells in three individual memory mice is depicted (right). (i) The frequency of CD80-expressing GFP⁺ memory B cells. (j) The percentage of B cells from the PP, MLN or Spl with a memory IgA⁺IgM⁻IgD⁻CD73⁺CD38⁺GL7⁻ phenotype in 6 months (left) or 2-year-old (right) unimmunized mice. (k) Expression of the gut homing α4β7 receptor (LPAM integrin) in GFP⁺ memory B cells, as opposed to that found in GFP⁻ B cells or non-B cells in the PP and Spl. (l) Lack of CD73⁺PD-L2⁺-expressing CD19⁺ B cells from the PP, MLN or Spl in CD40^−/− mice. Representative data from groups of two to six mice and two to four independent experiments giving similar results. Mann–Whitney test and P values are given (not significant (NS) P>0.05, *0.01≤P<0.05, **0.005≤P<0.01, ***0.001≤P<0.005).

Figure 4. Re-circulating CD80⁺ memory B cells formed after oral immunization generate α4b7-dependent gut IgA responses.

(a) Wild-type C57BL/6 mice were orally primed with three doses of 20 μg NP-CT at 10 days apart and 6–12 months later CD80⁺ and CD80⁻ B cells from the Spl, MLN or PP were sorted using a FACSAria and injected intravenously into μMT recipient mice that had been orally immunized once with 10 μg of CT 10 days earlier. One day after cell transfer μMT mice were orally immunized with 20 μg NP-CT and the anti-NP and anti-CT responses in serum or SI LP (AFC) were monitored. (b) Depicts the frequency of responding mice based on detection of anti-NP (left) or anti-CT (right) IgA or IgG serum antibody responses (as depicted in c) after transfer of CD80⁺ or CD80⁻ B cells using 1 × 10⁵ cells from MLN and PP and 2–10 × 10⁵ from the spleen (Spl). The number of recipient mice in each group (n=) is given in the left panel for NP responses. (c) Specific log₁₀ antibody titres in individual mice that responded to an oral challenge immunization (ND, not detectable). (d) Small intestinal LP anti-NP or anti-CT IgA responses given as AFCs/10⁶ cells in μMT mice injected with CD80⁺ memory B cells from indicated tissues. The data presented in a–d are based on the data pooled from four independent experiments giving similar results. (e) Mice adoptively transferred with NP-specific GFP-expressing B cells were orally or i.p. primed with NP-CT and 6 months later a challenge dose with NP-CT was given orally or i.p. (f–i) The percentage of GFP⁺ CD138⁺/all plasma cells in the SI LP and spleen of responding mice (f) and GFP⁺ B220⁺/all B cells in the PP and spleen in these mice (g). (h) A statistical analysis of the impact of p.o. versus i.p. priming or p.o. versus i.p. challenge immunizations for strong IgA responses (explained in Supplementary Fig. 6b,c). (i) Tissue sections of the PP and spleen showing GC location of responding of GFP⁺ memory B cells after challenge. (j) Orally primed mice were treated with α4β7-blocking DATK32 Mab before and during p.o. or i.p. challenge immunizations with NP-CT and the number of GFP⁺ plasma cells in SI LP sections were determined (explained in Supplementary Fig. 6a). The Mann–Whitney nonparametric test was used for statistical analysis of GFP⁺ gut homing plasma cells in the presence or absence of α4β7 Mab blockade (P=0.0120). Results in e–j are based on the pooled data from three independent experiments.

Figure 5. Memory B cells expand in and around pre-existing germinal centres on reactivation, but most cells do not acquire GL7 expression.

(a) One year after oral priming immunizations, the proportion of NP-specific memory B cells in various tissues was monitored kinetically following a single p.o. challenge immunization with NP-CT. The frequency of NP-specific GFP⁺ cells among total CD19⁺B220⁺CD138⁻ B cells at given time points was determined in individual mice (x; protocol as in Fig. 3a). (b,c) Flow cytometry analysis of GL7 and IgD expression on GFP⁺ memory B cells following the p.o. challenge. (b,c) The proportion of GFP⁺ cells that were GL7⁺ IgD⁻ was determined using the indicated gates. (c) The percentage of GFP⁺ memory B cells that were GL7⁺IgD⁻ was low in the PP, while it was two- to threefold higher in the MLN. (d,e) Antibody-labelled frozen sections of the PP and MLN were prepared 7 days after the challenge and analysed with confocal microscopy for GL7 (red) and B220 (blue) expression on expanding memory GFP⁺ (green) B cells. (e) Close-ups of the PP section marked in the mid panel in d with each individual channel shown in black and white and an overlay in colour in which individual GFP⁺ cells are circled to illustrate that most responding GFP⁺ memory B cells express B220, but not the GL7 marker even if located in a germinal centre. (f) Flow cytometry analysis on day 7 of IgA expression in responding GFP⁺ memory B cells in the PP and MLN. The percentage of IgA⁺ B cells/all GFP⁺ B cells in the PP or MLN in individual mice is given (right panel). (g) Flow cytometry analysis of the expression of CD73 and PD-L2 in responding GFP⁺ CD19⁺B220⁺CD138⁻ memory B cells on different days following a p.o. challenge immunization. Each bar represents the percentage of memory B cells that co-express CD73 and PD-L2 (grey) or only CD73 (black). (h) Mice p.o. primed with NP-CT received a p.o. or i.p. challenge immunization 6 months later in the presence or absence of anti-CD40L Mab (clone MR-1), and the proportion of GFP⁺ cells/total B220⁺ B cells or CD138⁺ plasma cells in the lamina propria (LP), spleen (Spl) or PP in individual mice was determined using flow cytometry. The Mann–Whitney non-parametric test showed that GC disruption with anti-CD40L Mab significantly reduced GFP⁺ plasma cell numbers in the SI LP (P=0.0193) and responding B-cell numbers in the PP (P=0.0053). (i) NP-specific GFP⁺ cells in the PP or spleen in p.o. or i.p. challenged mice were divided into responding memory B220⁺ B cells and GFP^high B220⁻ plasmablasts/plasma cells (shown in Supplementary Fig. 6e,f), and the expression of IgA, CCR9, CCR10 and integrin α4β7 was determined by flow cytometry. As a control, GFP⁻ B cells from the PP are included in each panel. (j) The proportion of mice that express CCR9 or IgA of the indicated GFP⁺ cell populations after an i.p. (open symbols) or p.o. (closed) challenge immunization. Significance was calculated with the Mann–Whitney test (f) and analysis of variance followed by Tukey's multiple comparison correction (j) and P values are given (**0.005≤P<0.01, ***0.001≤P<0.005, ****P<0.001). Data from four to five mice in each group in one representative experiment out of three giving similar results are shown.

Figure 6. NP-binding IgA V_H186.2 gene sequence analysis indicates poor relatedness between gut memory B cells and long-lived plasma cells.

(a) Experimental design. Traditional Sanger sequencing analysis of NP-binding IgA V_H186.2 genes for mutational load was performed in sorted cells 1 year following three oral priming immunizations. We compared IgA V_H186.2 gene sequences in long-lived plasma cells (Long PC) with those of CD80-expressing memory B cells (Long Mem) and IgA V_H186.2 gene sequences in responding memory B cells (Resp Mem) with those of SI LP plasma cells (Boost PC) 10 days after boosting (Figs 1d,e and 2e,f; Supplementary Fig. 7a,b). (b) The number of mutations (individual and mean) in the V_H186.2 gene region (upper panel) and the proportion of gene sequences with high-affinity mutations (lower panel). (c) The number of mutations in the V_H186.2 region of NP-binding sequences was compared with those found in non-NP-binding V_H186.2 sequences. Sorted CD80⁺ memory B cells (left panel) and orally primed IgA plasma cells before (Long) and after booster (Boost) (right panel) are included. P values are given as in b. (d) Distribution of mutations in NP-binding (above the line) and non-NP-binding V_H186.2V regions (below the line) are shown. Affinity-enhancing CDR1 mutations at position 98 and the counter-selected mutation in 102 are marked with one or two stars, respectively. (e,f) Examples of clonal trees generated using the IgTree program from individual mice before (e; n=3) and after (f; n=6) an oral booster with NP-CT are shown. (g) Tree shape statistics were analysed and significant differences were observed in PL_min (minimum tree path length) and trunk length in trees constructed from NP-binding V_H186.2 gene sequences from mice before and after a booster immunization. Pooled data from three independent experiments with three to six mice per group. (h) Targeted NGS sequencing of NP-specific IgA V_H186 genes expressed in sorted long-lived plasma cells in the SI LP and BM or sorted memory CD80⁺ B cells from individual mice (n=3). Figures represent mean mutation rates and frequencies of high-affinity mutations analysed in 86,000 NP-binding sequences. For b,c,g and h, the Mann–Whitney test P values are given (not significant (NS) P>0.05, *0.01≤P<0.05, **0.005≤P<0.01, ***0.001≤P<0.005, ****P≤0.001). (i,j) NGS sequences from IgA long-lived plasma cells and CD80⁺ memory B cells from three individual mice were divided into clones (described in Supplementary Fig. 9). (i) Relative proportion of sequences generated from each mouse that were present in shared clones (for example, two clones from mouse 1 contained sequences from both the LP and BM, and in the first clone 83% of all sequences from the BM and 24% from the LP were present). (j) The distribution of sequences between clones (clones with only one sequence excluded). The actual number of sequences allocated to a clone is indicated by the size of the pie chart, and the distribution of origin of the sequences within the pie chart with colours.