Long-lived antigen-induced IgM plasma cells demonstrate somatic mutations and contribute to long-term protection

Abstract

Long-lived plasma cells are critical to humoral immunity as a lifelong source of protective antibodies. Antigen-activated B cells-with T-cell help-undergo affinity maturation within germinal centres and persist as long-lived IgG plasma cells in the bone marrow. Here we show that antigen-specific, induced IgM plasma cells also persist for a lifetime. Unlike long-lived IgG plasma cells, which develop in germinal centres and then home to the bone marrow, IgM plasma cells are primarily retained within the spleen and can develop even in the absence of germinal centres. Interestingly, their expressed IgV loci exhibit somatic mutations introduced by the activation-induced cytidine deaminase (AID). However, these IgM plasma cells are probably not antigen-selected, as replacement mutations are spread through the variable segment and not enriched within the CDRs. Finally, antibodies from long-lived IgM plasma cells provide protective host immunity against a lethal virus challenge.

Figure 1. Expanded populations of IgM PCs persist in the spleen in response to diverse immunogens and pathogens.

Cohorts of mice were immunized or infected with NP₂₂CGG, influenza or LCMV virus, and IgG (black circles) and IgM (red circles) ASCs from immunized and unimmunized (black squares, IgG; grey squares, IgM) were measured via ELISPOT over time. (a,b) NP₂₂CGG-specific IgG and IgM PCs at 2 months, 6 months, 1 year and 2 years post immunization (n=6) or in naive controls (n=3) were quantified in the bone marrow and spleen, respectively. (c,d) A/PR/8/34 influenza virus-specific PCs at 3, 7 and 11 months post infection with 0.01xLD₅₀ of virus (n=4) or in naive controls (n=3) within the bone marrow and spleen, respectively. (e,f) A/PR/8/34-specific PCs at 3, 7 and 11 months post immunization with 1,400 HA of virus (n=4) or in naive controls (n=3) within the bone marrow and spleen, respectively. (g,h) LCMV-specific IgG and IgM PCs in the bone marrow and spleen, respectively, at 6 months, 1 year and 2 years post infection with 2 × 10⁵ p.f.u. of LCMV Armstrong virus compared with a 6-month naive control (n=3). The mean (±s.e.m.) is shown for each timepoint and an asterisk (*) indicates time points where IgM ASCs are significantly higher (P<0.05, Student's t-test) than in naive animals.

Figure 2. Antigen-specific, IgM LLPCs persist post-adoptive transfer and preferentially localize in the spleen.

(a) Representative CD138⁺B220⁻ PC gating strategy for FACS. (b) NP₂₂CGG-specific IgG (filled black square) and IgM (filled red square) ASCs in 10⁴ sorted PCs, 2 months post-NP₂₂CGG immunization (n=3) or in age-matched controls (open black square, n=3). (c) Nonspecific (A/PR/8/34 binding) ASCs in 10⁴ sorted PCs, 2 months post-NP₂₂CGG immunization (n=3) or in age-matched controls (n=3). (d) Antigen-specific serum IgG titres found in recipient μMT mice that have been adoptively transferred with bone marrow PCs (black circles) or splenic PCs (red circles) as measured by ELISA at days 7 through 60 (n=5). (e) Antigen-specific serum IgM titres found in recipient μMT mice that have been adoptively transferred with bone marrow PCs (black circles) or splenic PCs (red circles) as measured by ELISA at days 7 through 60 (n=5). (f) Localization of donor, NP₂₂CGG-specific IgG (filled black square) and IgM (filled red square) ASCs, adoptively in bone marrow PC recipient mice. (g) Localization of donor, NP₂₂CGG-specific IgG (filled black square) and IgM (filled red square) ASCs, adoptively in splenic PC recipient mice. The mean (±s.e.m.) of five immunized mice or three naive controls is shown, with ** indicating Student's t-test P<0.005. (h) The half-life of transferred cells was determined by fitting the time-course data of either IgG or IgM PCs in splenic recipient mice (n=4) as a function of their ratio (y/y_initial) with its linear regression.

Figure 3. IgM PCs show evidence of SHM even when GCs are ablated.

(a,b) NPCGG-specific IgG (filled black square) and IgM (filled red square) ASCs in mouse cohorts treated with either control IgG (n=5), αCD40L antibody to deplete GCs (n=7), or in age-matched naive controls (filled red square, n=3), as measured via ELISPOT at day 45 post immunization. The mean (±s.e.m.) is shown with ** indicating P<0.005. (c) IgG (black circles) and IgM IgHV186.2 sequence mutations in cohorts of C57/BL/6 mice at 1 and 3 months post-NP₂₂CGG immunization (pooled), with (red open circles) or without (red circles) αCD40L treatment, compared with total heavy chain sequences from pre-B-cell unmutated controls (open grey circles) isolated from immunized C57/BL/6 cohorts (n=4). Spleen and bone marrow PCs are pooled; Student's t-test *P<0.05 and ***P<0.001. Refer to Supplementary Table 1 for sequence numbers. (d) Representative clonality of C57/BL/6 bone marrow IgG PCs post immunization—each section represents a shared CDR3 junction as a percentage of total sequences as listed in the centre. Representative clonality of C57/BL/6 IgM PCs in the spleen and bone marrow, treated with control Ig (e) or with αCD40L (f). Ig lineage trees for IgG (g) and IgM PCs, treated with with control Ig (h) or with αCD40L (i). Filled nodes represent germline or sample sequences; empty nodes indicate inferred precursors. Each line denotes either a single mutation between parent and daughter if no number is given.

Figure 4. IgM heavy chain mutations are AID-induced and AID-dependent.

(a) Frequency of transition and transversion mutations for IgG (filled black square) and IgM PCs, with (open black square) or without (filled red square) αCD40L GC depletion. (b,c) Individual 5-mer mutabilities estimated from IgG and IgM sequences, respectively. Bars represent mutabilities of the central base as a function of the surrounding bases, read 5′–3′. Green (WA/TW) and red (WRC/GYW) bars indicate hotspot motifs, grey indicates the neutral sites and blue indicates the coldspots (SYC/GRS). Analysis was performed on total IgHV186.2 sequences for each population. (d) Percentage of total mutations found hotspot motifs (WRC/GYW or WA/TW) in red, neutral sites in grey, or coldspot motifs (SYC/GRS) in blue for IgG and IgM sequences. (e) IgM IgHV186.2 sequence mutations in cohorts of C57/BL/6 (filled red circles, n=4) or AID^−/− (open grey circles, n=3) mice post-NP₂₂CGG immunization, compared with total heavy chain sequences from pre-B-cell unmutated controls (open grey circles) isolated from immunized C57/BL/6 cohorts. Spleen and bone marrow PCs are pooled; and Student's t-test *P<0.05 and ***P<0.001. Refer to Supplementary Table 1 for sequence numbers.

Figure 5. IgM heavy chain mutations are not enriched in CDR regions and show no evidence of antigen selection.

Mutational frequency or replacement (in red) and silent mutations are plotted for IgHV186.2 regions of IgG (a) and IgM (b) PCs, as well IgM PCs with αCD40L blockade (c) and AID^−/− IgM PCs (d), with replacement to silent amino acid changes (R/S ratios) given to the right.

Figure 6. GC independent IgM LLPCs are capable of neutralizing influenza virus in vitro and protecting the animal against infection in vivo.

(a,b) PR8-specific IgG (filled black square) and IgM (filled red square) ASCs, respectively, as measured by ELISPOT from cohorts 2 months post immunization with 1,400 HA PR8 (n=5)—treated with control Ig or αCD40L at days 6, 8 and 10 to inhibit GCs—or age-matched naive control mice (filled grey square open square n=3). (c) Antigen-specific IgG (filled black square) and IgM (filled red square) titres as measured by serum ELISA at 150 days post immunization, with or without αCD40L treatment. (d) Neutralization of PR8 virus by day 150 sera from PR8-immunized cohorts (n=5), with (filled red square) or without (filled black square) αCD40L treatment, or age-matched naive controls (filled grey square open square n=3), with or without complement; Student's t-test **P<0.005. (e,f) Mice immunized with A/PR/8/34 virus, with or without αCD40L treatment, were challenged with 10xLD₅₀ of virus 1 year post immunization, with morbidity (body weight loss) and survival rate measured, respectively. The mean (±s.e.m.) of five mice in each cohort is shown.

Figure 7. IgM LLPCs are capable of protecting the animal against infection in vivo in the absence of IgG PCs, memory B cells, and T cell help.

(a) PR8-specific IgG (filled black square) and IgM (filled red square) titres, respectively, as measured via serum ELISA from cohorts at 1 month post infection with 0.1xLD₅₀ PR8 virus—treated with control Ig or αCD40L at days 6, 8 and 10 to inhibit GCs—or age-matched naive control mice (filled grey square open square n=4). (b) Neutralization of PR8 virus by 1 months sera from PR8-immunized cohorts, with (filled red square) or without (filled black square) αCD40L treatment, or age-matched naive controls (filled grey square open square n=4), with or without complement. Mice immunized with A/PR/8/34 virus, with or without αCD40L treatment, were then treated with anti-CD4/CD8 at 1 and 3 days before infection and challenged with 2xLD₅₀ of virus at 2 months post immunization. (c,d), Morbidity (body weight loss) and survival rate measured, respectively, post-PR8 challenge. The mean (±s.e.m.) of four mice in each cohort is shown. (e) PR8-specific IgG (filled black square) and IgM (filled red square) titres, respectively, as measured via ELISA 14 days post-PR8 challenge. *P<0.05, **P<0.005, and ***P<0.001, Student's t-test.