Premature Replacement of Μ with Α Immunoglobulin Chains Impairs Lymphopoiesis and Mucosal Homing but Promotes Plasma Cell Maturation

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Premature replacement of μ with α immunoglobulin chains impairs lymphopoiesis and mucosal homing but promotes plasma cell maturation

Sophie Ducheza, Rada Amina, Nadine Cognéa, Laurent Delpya, Christophe Siraca, Virginie Pascala, Blaise Corthésyb, and Michel Cognéa,1

aLaboratoire d'Immunologie, Université de Limoges, Centre National de la Recherche Scientiﬁque Unité Mixte 6101, F-87025 Limoges, France; and bR&D Laboratory of the Division of Immunology and Allergy, State University Hospital (Centre Hospitalier Universitaire Vaudois), 1011 Lausanne, Switzerland

Edited by Frederick W. Alt, Howard Hughes Medical Institute, Boston, MA, and approved December 22, 2009 (received for review October 29, 2009) Sequentially along B cell differentiation, the different classes of concentrations so that mIgA+ B cells residing in mucosa-associated membrane Ig heavy chains associate with the Igα/Igβ heterodimer lymphoid tissues (MALT) mediate IgA responses to local immuni- within the B cell receptor (BCR). Whether each Ig class conveys zation (17, 18). Questions about a role of mIgA in B cell develop- specific signals adapted to the corresponding differentiation stage ment arose from observations of the leakiness of the μMT mutation remains debated. We investigated the impact of the forced expres- removing Cμ membrane exons and initially reported to block B cell sion of an IgA-class receptor throughout murine B cell differentia- development (19, 20). This mutation led to the accumulation of IgA tion by knocking in the human Cα Ig gene in place of the Sμ region. ASCs in MALT, in the absence of mIgM/mIgD. It suggested that B Despite expression of a functional BCR, homozygous mutant mice cell progenitors can undergo class switch recombination (CSR), showed a partial developmental blockade at the pro-B/pre-BI and express mIgA, and then differentiate into ASCs (19, 20). In humans, large pre-BII cell stages, with decreased numbers of small pre-BII early CSR to IgA has also been reported in B cell progenitors (21), cells. Beyond this stage, peripheral B cell compartments of reduced but the efficiency and physiological relevance of this potential size developed and allowed specificantibodyresponses,whereas alternate pathway are unknown. mature cells showed constitutive activation and a strong commit- We thus intended to check whether, similarly to δ and γ1 HC, ment to plasma cell differentiation. Secreted IgA correctly α HC would be able to substitute for μ and support lympho- assembled into polymers, associated with the murine J chain, and poiesis, and whether it would modify B cell fate and homing. We was transported into secretions. In heterozygous mutants, cells generated mice in which the first gene downstream of joining expressing the IgA allele competed poorly with those expressing genes (JH) is a knock-in human Cα1(α1KI mice). Using a IgM from the wild-type allele and were almost undetectable among human Cα allowed us to tag the mutated allele and follow its peripheral B lymphocytes, notably in gut-associated lymphoid tis- expression. Because membrane-anchoring and cytoplasmic sues. Our data indicate that the IgM BCR is more efficient in driving domains of human and mouse IgA are 85% identical (four early B cell education and in mucosal site targeting, whereas the IgA conservative replacements and two mismatches), they are BCR appears particularly suited to promoting activation and differ- expected to provide similar signals in a context where association entiation of effector plasma cells. with transducing modules is known to rely on mIg trans- membrane segments (22). B cell receptor | differentiation | immunoglobulin A Homozygous mutants expressing α Ig HC instead of μ/δ showed how an α-class receptor can support the functions mediated by gA is the most abundantly synthesized immunoglobulin in membrane μ HC, both in B cell progenitors and during antigen- Imammals and plays a key role at mucosal surfaces (1). IgA driven B cell maturation in peripheral lymphoid organs. plasma cells differentiate from lymphocytes expressing membrane IgA (mIgA), with α heavy chains (HC) featuring a highly Results conserved membrane-anchoring domain and an intracellular tail Expression of the α1KI IgH Allele. To force expression of mIgA of unknown function (2–5). IgM-to-IgA switching and IgA throughout B cell differentiation, a gene encoding the secreted plasma cell differentiation are modulated by interactions with T and membrane forms of human α1 HC was inserted into the Iμ- r cells and by multiple soluble factors (6). CH intron replacing Sμ (Fig. 1A). A neo cassette flanked by loxP Although all membrane Ig associate with the disulfide-linked Igα/ sites was inserted downstream of the α gene and removed by Igβ (or CD79a/CD79b) complex to constitute the B cell receptor mating with Cre transgenic mice (Fig. 1B). Northern blots com- (BCR), signaling from the BCR was mostly studied for IgM (7, 8). paring mutant to wild-type (WT) B cells confirmed that the The membrane μ HC has a dual role by both providing differ- amount of Ig HC transcripts was not altered by the α1KI entiation signals during early development and activation signals mutation (Fig. S1A). during peripheral antigen-dependent maturation. Replacement of μ Mutant mice were first analyzed by flow cytometry to test by δ HC expression only results in delayed affinity maturation (9), as expression of α1 HC. In heterozygous α1KI/+ mice, mIgA1+ for mice lacking the secreted form of IgM (10). Surface γ1HC(orμ cells were barely detected in lymphoid organs (representing less HC with a γ1 membrane-anchoring/cytoplasmic domain) can also than 1% of B220+ cells), thus indicating that during early substitute for mIgM and support B cell development (11). The mIgG cytoplasmic tail increases extrafollicular antibody-secreting cell (ASC) formation in transgenics and was shown to mediate increased Author contributions: M.C. designed research; S.D., R.A., N.C., L.D., C.S., V.P., B.C., and BCR signal due to specific recruitment of the Grb2 adaptor (12, 13). M.C. performed research; S.D., L.D., C.S., B.C., and M.C. analyzed data; and S.D. and M.C. In addition, transfection experiments showed mIgG to be less sen- wrote the paper. sitive than mIgM to CD22-mediated feedback (12, 14), a feature not The authors declare no conflict of interest. observed in knock-in models in which Cμ was replaced by Cγ1(15, This article is a PNAS Direct Submission. 16). To date, little is known regarding a role for mIgA in conferring 1To whom correspondence should be addressed. E-mail: [email protected]. fi speci c properties to memory mIgA+ cells by comparison with naïve This article contains supporting information online at www.pnas.org/cgi/content/full/ mIgM+ cells. Cross-linking of mIgA raises intracellular calcium 0912393107/DCSupplemental.

3064–3069 | PNAS | February 16, 2010 | vol. 107 | no. 7 www.pnas.org/cgi/doi/10.1073/pnas.0912393107 Downloaded by guest on September 26, 2021 B Cell Development Is Impaired in Homozygous Mutant Animals. Early B cell development analyzed by ﬂow cytometry showed no alteration in heterozygous mutant animals where it overwhelm- ingly relied on the WT IgH allele, yielding lymphocytes with conventional mouse mIg and ﬁnally resulting in very low production of serum human IgA1. In α1KI/α1KI mice lacking any competition with WT μ HC, the α1KI allele supported early B cell differentiation but with a 3-fold reduction in the total number of bone marrow B-lineage cells and with phenotypic alterations (Table 1). Normal absolute values were observed for CD117+B220+ pro-B and CD43+B220+ pro- B/pre-BI/large pre-BII cells (with apparent expansion in relative percentages). By contrast, the CD25+B220+ pre-BII population underwent an 8-fold decrease in absolute value (a roughly 3-fold decrease in percentage) (Table 1 and Fig. S2A). We used 24 h in vitro cultures of sorted B220+ bone marrow cells to improve staining for surface surrogate light chains (SLC) and showed that α1KI/α1KI mice had three times more abundant VpreB+ cells than WT mice (Fig. S2B). In both WT and mutant mice, these cells were large and included pro-B/preBI cells (negative for HC staining) and large pre-BII cells (coexpressing HC and surrogate LC). By contrast, the proportion of CD25+ VpreB− cells (small pre-BII cells in which expression of the pre- BCR is “diluted” after cell proliferation) was severely reduced (Fig. S2B). Pre-B cell staining showed the association of α HC and surrogate light chains within pre-BCR molecules (Fig. S2B). A similar blockade at the transition from large pre-BII (BP1+)

to small pre-BII (BP1−) was observed when sorted progenitor IMMUNOLOGY B cells were first cultured for 5 days in vitro with stromal cells and high doses of IL-7, and then switched for 2 days to low doses of IL- 7 to stimulate pre-B cell differentiation (Fig. S2C). In line with the early differentiation defect, numbers of peripheral mature B cells were severely reduced in the spleen (Table 1). Lymph nodes and Peyer’s patches also were small-sized, and the latter were reduced in number or even occasionally absent. The reduction of spleen B cell numbers similarly affected the high int Fig. 1. IgA1 expression instead of IgM in α1KI mice. (A)(Top) Structure of AA4.1+B220+ (transitional), CD23 CD21 (follicular), CD23 the targeted locus (not to scale) showing an unrearranged IgH locus and the −CD21high (marginal zone), and CD5+B220+ (B1) compart- extent of the deletion within the Iμ-Cμ intron. (Middle) Structure of the ments (Table 1 and Fig. S3A). In α1KI/α1KI mice, B cell lym- r targeting vector in which Cα1 and a neo cassette flanked by loxP sites were phopenia resulted in the apparent relative increase of a peculiar μ α inserted in place of S .(Bottom) The 1KI locus is able to undergo V(D)J population of B220+ BCR-less cells (LC−). In absolute numbers, rearrangement after insertion of Cα1, whereas the neo gene can be deleted by cre-mediated recombination. (B) Southern blot analysis of tail DNA from these unconventional B220+ cells were equally abundant in representative WT, heterozygous, homozygous, and neo-deleted mutant WT controls and carried a similar phenotype (only expressing mice. (C) Splenocytes from WT (Left)orα1KI/α1KI mice (Right) were labeled CD19 on part of them and including mostly non-B cells) (Table 1 with PC5-conjugated anti-B220 and with either FITC-conjugated anti-IgM and Fig. S3B). Abs showing the blockade of the IgM BCR expression in mutant animals Whereas staining for different HC cannot allow comparison of (Top) or FITC-conjugated anti-human IgA1 Abs (Bottom). (D) Total endoge- BCR densities between WT and mutant mice, staining for LC clearly nous Ig production was estimated by ELISA in sera from 6-week-old WT, indicated a decreased BCR density in α1KI/α1KI mice (Fig. S3A). α1KI/+, or α1KI/α1KI mice. Asterisks mark statistically significant differences In the peritoneum, α1KI/α1KI and WT mice also did not sig- ’ < < with controls (Student s t test, **P 0.01; ***P 0.001). The vertical axis is nificantly differ for the proportions of B1 (Mac1+B220+) (47.7 ± logarithmic; values are indicated as μg/mL. 1.6% vs. 46.7 ± 7%) and B2 (Mac1− B220high) cells (31.2 ± 2.3% vs. 43.5 ± 7.9%) (Fig. S3C).

maturation, cells expressing IgM from the WT allele out- Functionality of the IgA1-Based Immune System in Homozygous α1KI competed those cells expressing IgA1 from the α1KI allele. By Mice. As mentioned above, Ig HC mRNA amounts from the contrast, homozygous mutant mice lacked surface IgM and IgD α1KI allele were at a normal level and thus did not account for expression (Fig. S1B) and rather expressed mIgA1 on B220+ the decreased BCR density in mutant mice (Fig. S1A). Regard- cells additionally displaying a strong reduction in number (Table 1, ing membrane-anchored IgA1, immunoprecipitation with anti- Fig. 1C,andFig. S1C). mouse κ LC Abs followed by western blot with anti-CD79a/ Heterozygous α1KI/+ mice had normal serum levels of all CD79b indicated association of the BCR with the Igα/Igβ het- mouse Ig, including IgM, and a low level of circulating human erodimer (Fig. S4A). IgA1 (5–10 μg/mL). Consistent with the absence of mIgM+ cells, We next investigated whether the antibodies produced in α1KI/ serum IgM was undetectable in homozygous α1KI/α1KI mutants α1KI mice had a normal biochemical structure. We evidenced and was replaced by IgA1 (mean titer: 600 μg/mL); murine IgG mostly monomeric IgA1 in serum (Fig. S4B) and polymers and IgA production was not abolished in α1KI/α1KI mice, but exported into secretions. IgA1 level reached 60 μg/mL in the showed a 10- to 100-fold reduction for all isotypes (Fig. 1D). jejunal ﬂuid (55.8 ± 10.3 μg/mL), a level similar to that of The α1KI mutation thus supported the development ofa humoral secretory IgA in secretions of normal mice or humans. We immune system, albeit with reduced numbers of B lymphocytes. analyzed a monoclonal IgA1 from an α1KI/α1KI hybridoma by

Duchez et al. PNAS | February 16, 2010 | vol. 107 | no. 7 | 3065 Downloaded by guest on September 26, 2021 Table 1. Relative values of the various lymphoid populations in bone marrow and spleen n WT α1KI/α1KI

Bone marrow B cells (B220+) 4/8 30.75 ± 4.04% (6.41 ± 2.95) 12.26 ± 1.89%*** (2.15 ± 0.66) Gated on B cells (B220+) Pro-B cells (CD117+B220+) 3/7 7.87 ± 2.25% (0.39 ± 0.21) 24.88 ± 3.26%* (0.44 ± 0.16) Pro-B/pre-B cells (CD43+B220+) 4/8 33.90 ± 4.57% (2.33 ± 1.24) 77.75 ± 3.32%* (1.55 ± 0.38) Pre-B cells (CD25+B220+) 4/8 36.94 ± 3.85% (2.16 ± 0.8) 11.13 ± 1.73%*** (0.3 ± 0.15**) Immature B cells (B220lowIgMlow) 3/7 11.40 ± 3.59% (0.48 ± 0.2) ND Recirculating B cells (B220highIgMhigh) 3/7 4.95 ± 1.85% (0.17 ± 0.06) ND Spleen Macrophages (F480+CD11b+CD19−) 3/5 3.52 ± 0.50% (2.20 ± 0.57) 6.44 ± 0.71%* (2.66 ± 0.47) NK (DX5+CD3−CD19−) 3/5 3.91 ± 0.12% (2.36 ± 0.40) 4.50 ± 0.52% (1.76 ± 0.18) NKT (DX5+CD3+CD19−) 3/5 1.05 ± 0.05% (0.64 ± 0.11) 2.47 ± 0.36%* (1.03 ± 0.20) T cells (DX5−CD3+CD19−) 3/5 39.81 ± 2.49% (23.77 ± 3.45) 48.94 ± 3.71% (19.90 ± 2.65) B220+ cells 7/7 34.07 ± 0.87% (22.17 ± 2.32) 7.29 ± 1.08%*** (2.16 ± 0.47***) Plasma cells CD138+ K intra 7/7 0.50 ± 0.07% (0.31 ± 0.03) 1.06 ± 0.23%* (0.34 ± 0.08) Gated on B220+ cells Resting B cells (IgD+IgM+B220+) 7/7 90.66 ± 0.86% (20.15 ± 2.14) ND Transitional B cells (AA4.1+B220+) 3/3 12.37 ± 2.6% (2.98 ± 0.75) 12.97 ± 1.78% (0.26 ± 0.13*) FO B cells (CD23+CD21+B220+) 7/7 67.27 ± 3.72% (15.12 ± 1.92) 30.31 ± 4.74%*** (0.66 ± 0.21***) − MZ B cells (CD23 /low CD21+B220+) 7/7 13.16 ± 2.67% (2.86 ± 0.62) 7.71 ± 2.29% (0.16 ± 0.05***) B1 cells (CD5lowB220+) 3/3 8.71 ± 0.05% (1.32 ± 0.11) 13.94 ± 2.82% (0.33 ± 0.07*) Non-B cells expressing B220 (B220+LC−) 7/7 6.14 ± 0.96% (1.36 ± 0.28) 34.01 ± 4.16%*** (0.70 ± 0.14) Total B lymphocytes (B220+LC+) 7/7 93.86 ± 0.96% (20.81 ± 2.19) 65.99 ± 4.156%*** (1.46 ± 0.38***) κ+ cells 7/7 90.54 ± 1.12% (20.07 ± 2.12) 63.24 ± 4.03%*** (1.4 ± 0.36***) λ+ cells 7/7 5.10 ± 0.26% (1.13 ± 0.15) 1.77 ± 0.22%*** (0.036 ± 0.009***) CD19+ B cells (B220+CD19+) 7/7 95.46 ± 0.26% (21.15 ± 2.2) 64.96 ± 5.41%*** (1.44 ± 0.36***) B220+mIgM+ cells 7/7 93.17 ± 0.14% (20.65 ± 2.2) ND B220+mIgA+ cells 7/7 ND 62.23 ± 5.00% (1.39 ± 0.39)

Second column indicates numbers (n) of WT/mutant animals used. Third and fourth columns indicate percentages of cells (and absolute values in millions of cells in parentheses). ND, not detectable. Asterisks indicate statistically signiﬁcant differences between values observed in the group of α1KI/α1KI mice and the control group: *P < 0.05; **P < 0.01; ***P < 0.001.

SDS/PAGE under reducing conditions and found it was com- than in WT mice, most of them producing IgA1. Plasma cells had posed of normal-sized α1 Ig HC plus mouse LC (Fig. S4C). To a normal location in the splenic marginal zone and red pulp, in test whether the chimeric IgA assembled correctly with endog- the enteric lamina propria along the intestinal crypts, and around enous mouse J chain, we performed immunoprecipitation of IgA rare Peyer’s patches (Fig. 2 A–C). Flow cytometry also showed an from mice feces and intestinal contents with a monoclonal anti- increased proportion of splenocytes engaged in ASC differ- human IgA1-specific antibody followed by immunodetection entiation and brightly stained intracellularly for κ LC. These using an anti-mouse J-chain antiserum. In WT mice no signal ASCs represented more than one seventh of all κ-expressing B- was detected, whereas J-chain-positive chimeric IgA1 was pres- lineage cells in α1KI/α1KI mice, that is, 6-fold more than among ent in jejunal and caecal content, as well as in feces of α1KI/α1KI WT B cells (Fig. 2D and Table 1). To check that this plasma cell animals (Fig. S4D). accumulation would not be observed in any mouse model carrying Immunization of α1KI/α1KI mice with ovalbumin raised a dysfunctional BCR, we compared α1KI/α1KI mice not only with serum IgA1 antibodies, showing that functional mIgA1+ cells WT but also with SLP65-deficient mice, but the latter rather yielded antigen-specific ASCs. Low levels of class-switched showed increased numbers of mature B cells with no increase in antigen-specific mouse Ig (mostly IgG2a) were also detected plasma cell differentiation (Fig. 2E). (Fig. S4E). Immunizations with various bacterial or viral antigens In vivo BrdU labeling showed a normal ratio of long-lived confirmed that α1KI/α1KI mice responded to a repertoire of versus short-lived plasma cells in α1KI/α1KI mice, indicating not foreign antigens (staphylococcus, respiratory syncytial virus, and only accumulation but also increased ongoing differentiation of HIV gp120 were successfully used to obtain hybridomas). They these cells (Fig. 2F). also appeared to tolerate self-antigens normally, and both eval- A quantitative assessment of plasma cells versus B lympho- uations of anti-nuclear antibodies and rheumatoid factors were cytes was additionally obtained by measuring the ratio of Blimp-1 below the threshold of detection in 10 α1KI/α1KI mice assayed. to CD79a transcripts, which was 7- to 10-fold higher in mutant Altogether, α1KI/α1KI mice developed roughly normal humoral mouse lymph nodes, bone marrow, and spleen than in WT ani- responses, in agreement with their normal longevity in a conven- mals. Three days after intraperitoneal immunization, the plasma tional environment challenging them with multiple pathogens. cell burst still increased in the spleen, with the Blimp-1:CD79a ratio being roughly 20-fold higher in α1KI/α1KI than in control Increased Plasma Cell Differentiation in α1KI/α1KI Mice. Because mice (Fig. 2G). specific responses to immunization and the presence of follicular B cells in peripheral lymphoid organs indicated that mutant B α1KI/α1KI B Cells Are Constitutively Activated and Committed to lymphocytes could be activated, we wished to appraise their CD138+ Differentiation. Given the B cell lymphopenia and re- ability to differentiate into plasma cells. Tissues analyzed by sulting increased proportion of B220+BCR− cells, we restricted immunohistochemistry for the presence of intracellular Ig showed the B lymphocyte phenotype analysis to true B220+LC+ cells much more abundant ASCs in lymphoid tissues of mutant animals (Table 1 and Fig. S3A). Mutant B lymphocytes included de-

3066 | www.pnas.org/cgi/doi/10.1073/pnas.0912393107 Duchez et al. Downloaded by guest on September 26, 2021 creased numbers of cells expressing CD21, CD22, and CD23 and Discussion increased numbers of cells expressing the CD69 activation marker We wished to force expression of mIgA throughout B cell or having lost MHC class II expression (Fig. S5A). differentiation by replacing the Sμ region with a knock-in human B cells from α1KI/α1KI mice featured constitutive phosphor- Cα Ig gene. The use of a Cα gene from a different species ylation of multiple intracellular targets as compared to WT cells. unambiguously distinguished transgenic IgA expression from Phosphorylation of the Akt and ERK kinases was further endogenous switching. increased by BCR cross-linking (Fig. S5B). Mature α1KI/+ B cells showed a strong bias toward expression CD19+ α1KI/α1KI splenocytes proliferated less than CD19+ of the WT rather than the mutated allele. Cells expressing the WT controls after lipopolysaccharide (LPS) stimulation and still knock-in α gene were only detectable within the splenic plasma more markedly after BCR cross-linking by anti-κ LC Abs (Fig. 3A). cell pool and were virtually absent from the MALT, suggesting Among freshly isolated splenocytes, the ratio of CD138+ to CD19 that in physiology, IgM+ cells are preferentially recruited to the + cells showed a bias toward plasma cell differentiation in mutant MALT and locally undergo CSR to IgA under the influence of compared with WT mice. This bias increased after in vitro stim- the mucosal microenvironment. ulation with LPS, anti-CD40 mAb plus IL4, or anti-κ Ab plus IL4 In α1KI/α1KI animals, the most dramatic B-lineage alterations (Fig. 3B). Among such cultures, ongoing apoptosis was similar for included delayed pre-B differentiation contrasting with increased WT and α1KI/α1KI cells and was higher for CD138+ cells than for terminal plasma cell differentiation. The reduced size of the CD25+ CD19+ cells (48.7 ± 7.9% and 16.6 ± 6.7% of annexinV+ 7AAD− small pre-BII cell compartment and of the total B220+ compart- cells, respectively). These conditions thus measured newly formed ment in the bone marrow, together with the expanded proportion of plasma cells and not in vitro accumulated surviving plasma cells. pro-B/pre-BI and large pre-BII cells, indicated that the α-class pre- Cells from α1KI/α1KI mice thus altogether appear as con- BCR was less efficient than a μ-class pre-BCR for turning off CD43 stitutively activated, with an increased tendency to yield CD138+ expression and signaling the completion of V(D)J rearrangement cells and a decreased proliferation in response to BCR cross-linking. upon assembly of a functional pre-BCR or BCR complex. These features are reminiscent of those in mice with premature expression Heterozygous Cells Expressing mIgA1 from the Mutant Allele Poorly of membrane γ HC substituting for μ/δ (16). Compete with mIgM Cells, Even When CSR Is Blocked. Because Despite this partial early blockade reducing their number, B mIgA1+ cells were too rare in α1KI/+ mice for detection by flow cells accumulated in all peripheral lymphoid organs of α1KI/ α cytometry whereas IgA1 was readily measurable in sera, we 1KI mice including spleen follicles, marginal zone, lymph IMMUNOLOGY evaluated IgA1− ASCs by confocal microscopy. Competition nodes, Peyer’s patches, and the peritoneum B1 compartment. between cells expressing only mIgA1 from the α1KI allele and Splenic marginal zone and follicular B cell numbers were af- cells expressing only mIgM from the WT allele was assessed in fected in the same proportion, in contrast with the increased heterozygous α1KI/+ animals on an activation-induced deami- marginal zone differentiation reported in mice expressing an IgG nase (AID)-deficient background (to avoid competition with BCR (15, 16). BCR density appeared lower in α1KI/α1KI than in cells producing mouse IgA after CSR). In this background, the WT animals, although mIgA1 was normally associated with the proportion of peripheral mIgA1+ cells remained undetectable, Igα/Igβ heterodimer. Expression of mIgA1 in peripheral B cells whereas some cells expressing IgA1 reached the plasma cell did not alter their ability to produce specific antibodies after stage and accounted for 7.4% of spleen plasma cells (Fig. 4). immunization and even to undergo CSR at low levels in the Even for entry into MALT, mIgA1+ cells poorly competed with absence of any Sμ repeat. This partial CSR defect was similar to mIgM+ cells and no IgA1 ASCs were detected in the gut lamina that reported for the core Sμ or the complete Sμ deletions (23, propria of these α1KI/+ mice, contrasting with the situation in 24). These data confirm that neither the pentameric repeats α1KI/α1KI mice (Fig. 4). featuring the Sμ region nor expression of μ/δ are mandatory for

Fig. 2. Global in vivo increase of the plasma cell compartment in mutant mice. (A and B) Staining of tissues from WT (Left)andα1KI/α1KI mice (Right)with hemalun and revelation of human IgA1 with HRP-conjugated Ig show the presence of plasma cells (arrows) producing IgA1 and accumulating in (A) the splenic marginal zone and red pulp or (B) around Peyer's patches and in the lamina propria along intestinal crypts of the jejunum. (Original magnification, 100×.) (C) Staining of all plasma cells with anti-mouse Ig (HC + LC) shows accumulation of plasma cells in the spleen of a mutant mouse by comparison with WT. (Original magnification, 200×.) (D) Staining for CD138 and intracellular κ LC shows ASCs as CD138+ cells brightly staining for κ.(E) Staining for CD138+ and intracellular κ LC+ ASCs in splenocytes from SLP65-deficient mice. (F) Absence of significant difference by cell flow cytometry between proportions of recently differentiated (CD138+BrdU+) versus long-lived plasma cells (CD138+BrdU−)fromα1KI/α1KI and WT spleens (means from three and four animals fed with BrdU, respectively). (G) Expression of Blimp-1 transcript in lymphoid tissues by qPCR evaluates the amount of cells engaged in plasma cell differentiation among all of the cells present in tissue samples (reference to 18S RNA) or by comparison with the pool of B lymphocytes (CD79a as a reference transcript). Total RNA was prepared without prior immunization of (▲)WTand(•) α1KI/α1KI mice or 3 days after intraperitoneal immunization of (Δ)WTand(○) α1KI/α1KI mice with BSA.

Duchez et al. PNAS | February 16, 2010 | vol. 107 | no. 7 | 3067 Downloaded by guest on September 26, 2021 More severe reported dysfunctions of the BCR resulted in a complete block at the pro-B stage with neither B cells nor plasma cells, as in the case of Igα or Igβ deficiency (28, 29). The α1KI/ α1KI mice plasma cell expansion was remarkable in view of the reduced mature B cell number and might partly result from homeostatic accumulation, as reported both for mature B cells in response to reduced lymphopoiesis and for plasma cells which can be more preserved than mature B cells in lymphopenic animals (19, 20, 30). However, commitment to plasma cell differentiation was emphasized in vitro by the lower proliferation but higher differentiation into CD138+ cells of α1KI/α1KI cells activated by BCR cross-linking or mitogens, and in vivo by the increased spontaneous Blimp-1 expression in all lymphoid tissues or still more in spleen after immunization. In vivo, the commitment of B lymphocytes toward ASC differentiation also man- ifested in their more frequent expression of the CD69 activation marker and reduced expression of MHC class II, which is repressed by Blimp-1 and silenced early during ASC maturation. The presence of a significant number of IgA1 ASCs in spleen of α1KI/+ mice, where virtually no mIgA1+ B cells were detected, also showed this increased commitment. Interestingly, normal mIgA+ cells purified from human blood include a high proportion of cells expressing CD38, a marker shared by immature cells and cells engaged to plasma cell differentiation (31). Whereas the IgA BCR has been shown to be subjected to CD22 inhibition as for IgM (32), other aspects may differ in signals provided both by classes of BCR and by impact Fig. 3. Mutant B cells are precommitted to plasma cell differentiation. (A) on the behavior of B cells during primary or secondary responses Proliferation of sorted CD43− splenocytes (standardized on the numbers of to antigens. CD19+ B cells) from α1KI/α1KI mice compared to WT (gray) after 4 days in Finally, we show that an IgA-class BCR can support all of the vitro stimulation by LPS (Top), anti-CD40 plus IL-4 (Middle), or anti-κ Abs plus functions mediated by membrane IgM/IgD during B cell differ- IL-4 (Bottom). (B) Ratios of CD138- versus CD19-expressing cells compared entiation and immune responses but with reduced efficiency at between WT (gray) and α1KI/α1KI mice within splenocytes stimulated for 2 early stages, although we cannot exclude that expression of a days by LPS, anti-CD40 plus IL-4, or anti-κ LC plus IL-4 (*P < 0.05; **P < 0.01; human heavy chain in mouse might by itself participate in altering ***P < 0.001 significant difference). B cell development. Once in a mature compartment, mIgA+ lymphocytes show a strong ability to be activated, to switch at a low level despite the absence of Sμ, and to terminally differentiate CSR in the JH-C intron, in contrast to the Sγ1, whose deletion into plasma cells. The sum of these observations supports the virtually abrogates CSR to Cγ1 (25). α β α concept that although associated with the same Ig /Ig hetero- Another feature of mIgA-driven B cell differentiation in 1KI/ dimer, all membrane Ig HCs are not equivalent in terms of con- α 1KI mice was the abundance of plasma cells, affecting to the veying specific signals emanating from their extracellular domains. same extent long-lived (accumulated) and short-lived (recently Rather, cytoplasmic tails and/or extracellular C domains of the differentiated) plasma cells. Such a phenotype has not been various classes of Ig are likely tailored to deliver signals fitting the reported as a general consequence of BCR dysfunction and was maturation stage at which they are usually expressed. Such dif- neither present in mice reconstituted with Syk-deficient B cells ferences also likely account for the implications of different Ig nor in Lyn-deficient mice (26, 27), and was not found either in classes in specialized immune responses and in physically distinct the SLP65-deficient mice used in this study for comparison. compartments of the immune system. In this view, membrane IgM may stand as an “affector BCR” shaping the preimmune repertoire, whereas both the IgG and IgA switched isotypes would constitute “effector BCR” shaping the immune repertoire and favoring the generation of ASCs (33). In human pathology, the frequency of IgG/IgA myeloma and plasmacytoma cases, compared to the paucity of IgM myeloma cases, fits adequately with these observations and suggests that the BCR by itself likely contributes to the plasma cell burst or to the accumulation of a long-lived plasma cell pool in such malignant proliferations. Methods − − Mice. Experiments followed international guidelines. AID / mice were a gift of Dr. T. Honjo (Kyoto, Japan). EIIa-cre transgenics were a gift of Dr. H. Westphal (Bethesda, MD). SLP65-deficient mice were a gift of Dr. H. Jumaa Fig. 4. Confocal imaging of antibody-secreting cells in WT and mutant (Freiburg, Germany). tissues. Evaluation of the number of ASCs secreting human IgA1 (green) versus IgM (red) by confocal microscopy in WT (Left), α1KI/α1KI (Center), or Gene Targeting. The α1KI construct included a 6-kb α1 genomic fragment − − α1KI/+ heterozygous mice bred in an AID / background (Right). In the latter (from a SacI site 200 bp upstream of CH1 to a BamHI site downstream of the animals, counting of plasma cells in several microscopic fields showed that 24 last polyadenylation signal) flanked by an ∼5-kb long 5′ arm (a StuI-SpeI out of 350 plasma cells (i.e., 7.4%) in the spleen were producing IgA1, fragment located upstream of mouse Sμ) and an ∼5-kb long 3′ arm (the Cμ whereas there was no IgA1-positive cell detectable in gut-associated lym- gene cloned as an XhoI fragment). A 1.3-kb ClaI-SalI neor gene flanked by phoid tissues. (Original magnification, 630×.) loxP sites was also stuck in-between the α1 gene and the 3′ arm. E14 ES cells

3068 | www.pnas.org/cgi/doi/10.1073/pnas.0912393107 Duchez et al. Downloaded by guest on September 26, 2021 were transfected with linearized vector and selected using G418 (400 μg/mL). μg/mL anti-CD40 (R&D Systems) and 40 ng/mL murine IL-4 (PeproTech) or Recombinant clones were identified by Southern blot of EcoRI digests with with 10 μg/mL goat anti-mouse κ (Southern Biotechnologies) and 40 ng/mL an external 3′ probe (0.6-kb XhoI-XbaI fragment). Chimeras obtained from murine IL-4 in DMEM supplemented with 10% heat-inactivated FCS. Cells C57BL/6 blastocysts were mated with C57BL/6 females and their progeny was were analyzed each day for staining by rat anti-CD138 or -CD19, with checked by Southern blot. Mutant α1KI/α1KI Neo mice were mated with EIIa- Annexin V and 7AAD to evaluate apoptosis and exclude dead cells cre transgenic mice to yield the cre-deleted α1KI allele. (BD Pharmingen). Proliferation of sorted CD43− splenocytes (on standardized numbers of Cell Flow Cytometry. Cells from 6- to 8-week-old mice were stained with Abs CD19+ B cells) was measured by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-car- conjugated to either FITC, PE, Alexa 633, allophycocyanin, phycoerythrin- boxymethonyphenol)-2-(4-sulfophenyl)-2H (MTS) tetrazolium nonradioactive cyanin 7 (PC7), or phycoerythrin-cyanin 5 (PC5). Abs are listed in SI Methods. cell proliferation assay (Promega) in triplicate and expressed as the mean For surface staining, cells were labeled in the presence of rat anti-mouse optical density (OD) ± SEM. CD16/CD32 (BD Biosciences) to block Fc receptor binding. For intracellular fi staining, cells were xed in PBS, 4% paraformaldehyde and permeabilized in BrdU Labeling. Mice were given drinking water containing 1 mg/mL BrdU PBS, 0.1% saponin (Sigma-Aldrich). Cells were analyzed on a Beckman (Sigma) for 3 weeks. Splenocytes were stained with rat anti-mouse CD138-PE, Coulter FC500 apparatus. made permeable, and stained with FITC-labeled anti-BrdU (BD Biosciences). Newly formed (short-lived) plasma cells stained positive for BrdU, whereas Ig Analysis in Biologic Samples and Autoantibody Screening. ELISAs for mouse long-lived plasma cells remained BrdU negative. Ig were done using plates coated and revealed with 1 μg/mL isotype-specific μ goat Abs (Southern Biotechnologies). ELISA for human IgA was with 1 g/mL Real-Time PCR. Real-time PCR was performed in duplicate using 20 ng of cDNA ′ goat anti-IgA F(ab )2 Abs for coating and with alkaline phosphatase-con- foreachsample.RelativeamountsoftranscriptsweredeterminedusingTaqman jugated goat anti-IgA Abs (Dako). Mouse sera were assayed at 1:100 and assays specific for Blimp-1 (Mm00476128_m1), CD79a (Mm00432423_m1), and 1:1000 dilutions. 18S rRNA (Hs99999901_s1) on an ABI PRISM 7700 cycler (Applied Biosystems). Search for anti-nuclear Abs was done by classical indirect immuno- fluorescence on Hep2 cells (Biomedical Diagnostics) on microscope slides first Confocal Microscopy. Tissue cryosections of 8 μm or magnetically sorted CD19+ incubated with mouse sera at a 1:20 dilution and further incubated with a FITC- splenocytes (Miltenyi Biotech) were fixed with methanol and permeabilized in labeled anti-mouse κ-chain antiserum (Southern Biotechnologies). Rheumatoid 0.15% Triton X-100. Unspecific and FcR binding was blocked with PBS/3% BSA factors were quantified with the N-latex RF kit II (Behring) using a Behring and rat anti-mouse CD16/CD32 (BD Biosciences). Ig staining used Alexa 488- Nephelometer II analyzer. Assays for anti-nuclear Abs and rheumatoid factors goat anti-human IgA or Alexa 546-rat anti-mouse IgM (1B4B1). Slides were were strongly positive with control sera from autoimmune MRL mice. observed on an LSM 510 confocal microscope (Carl Zeiss).

Immunohistochemistry. Paraffin-embedded samples were cut in 4-μm thick IMMUNOLOGY Statistical Analysis. Results are expressed as mean ± SEM, and overall dif- slices and counterstained with Mayer’s hemalun solution. To stain ASCs and ferences between variables were evaluated by a two-tailed unpaired Stu- specifically check human IgA1, tissue sections were incubated with either dent’s t test. unconjugated goat anti-mouse Ig (heavy + light chains) or rabbit anti-human IgA (Dako). Slides were then washed and incubated with HRP-conjugated secondary Abs specific either for goat or rabbit Ig (Dako). HRP staining was ACKNOWLEDGMENTS. The expertise of Micaël Bardel, Claire Carrion, and Christelle Oblet are greatly acknowledged. We thank Salvatore Valitutti and done with diaminobenzidine and hydrogen peroxide. Loïc Dupré for critical reading of the manuscript. This work was supported by grants from Agence Nationale de la Recherche, Ligue Nationale Contre le Proliferation and Activation Assays. Total splenocytes were stimulated for 2–4 Cancer, and Région Limousin. S.D. was supported by a fellowship from days with 20 μg/mL LPS from Salmonella typhimurium (Sigma) or with 5 Région Limousin and by Fondation pour la Recherche Médicale.

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