Cyclin-dependent kinase inhibitor cdkn2c deficiency promotes b1a cell expansion and autoimmunity in a mouse model of lupus

The lupus-prone NZM2410 mice present an expanded B1a cell population that we have mapped to the Sle2c1 lupus susceptibility locus. The expression of Cdkn2c, a gene encoding for cyclin-dependent kinase inhibitor p18(Ink4c) and located within Sle2c1, is significantly lower in B6.Sle2c1 B cells than in B6 B cells. To test the hypothesis that the B1a cell expansion in B6.Sle2c1 mice was due to a defective p18 expression, we analyzed the B1a cell phenotypes of p18-deficient C57BL/6 mice. We found a dose-dependent negative correlation between the number of B1a cells and p18 expression in B cells, with p18-deficient mice showing an early expansion of the peritoneal B1a cell pool. p18 deficiency enhanced the homeostatic expansion of B1a cells but not of splenic conventional B cells, and the elevated number of B6.Sle2c1 B1a cells was normalized by cyclin D2 deficiency. These data demonstrated that p18 is a key regulator of the size of the B1a cell pool. B6.p18(-/-) mice produced significant amounts of anti-DNA IgM and IgG, indicating that p18 deficiency contributes to humoral autoimmunity. Finally, we have shown that Sle2c1 increases lpr-associated lymphadenopathy and T cell-mediated pathology. B6.p18(-/-).lpr mice showed a greater lymphadenopathy than B6.Sle2c1.lpr mice, but their renal pathology was intermediate between that of B6.lpr and B6.Sle2c1.lpr mice. This indicated that p18-deficiency synergizes, at least partially, with lpr-mediated pathology. These results show that Cdkn2c contributes to lupus susceptibility by regulating the size of the B1a cell compartment and hence their contribution to autoimmunity.     

Genetic dissection of the murine lupus susceptibility locus Sle2: contributions to increased peritoneal B-1a cells and lupus nephritis map to different loci

Lupus pathogenesis in the NZM2410 mouse model results from the expression of multiple interacting susceptibility loci. Sle2 on chromosome 4 was significantly linked to glomerulonephritis in a linkage analysis of a NZM2410 x B6 cross. Yet, Sle2 expression alone on a C57BL/6 background did not result in any clinical manifestation, but in an abnormal B cell development, including the accumulation of B-1a cells in the peritoneal cavity and spleen. Analysis of B6.Sle2 congenic recombinants showed that at least three independent loci, New Zealand White-derived Sle2a and Sle2b, and New Zealand Black-derived Sle2c, contribute to an elevated number of B-1a cells, with Sle2c contribution being the strongest of the three. To determine the contribution of these three Sle2 loci to lupus pathogenesis, we used a mapping by genetic interaction strategy, in which we bred them to B6.Sle1.Sle3 mice. We then compared the phenotypes of these triple congenic mice with that of previously characterized B6.Sle1.Sle2.Sle3, which express the entire Sle2 interval in combination with Sle1 and Sle3. Sle2a and Sle2b, but not Sle2c, contributed significantly to lupus pathogenesis in terms of survival rate, lymphocytic expansion, and kidney pathology. These results show that the Sle2 locus contains several loci affecting B cell development, with only the two NZW-derived loci having the least effect of B-1a cell accumulation significantly contributing to lupus pathogenesis.     

Murine lupus susceptibility locus Sle1c2 mediates CD4+ T cell activation and maps to estrogen-related receptor γ

Sle1c is a sublocus of the NZM2410-derived Sle1 major lupus susceptibility locus. We have shown previously that Sle1c contributes to lupus pathogenesis by conferring increased CD4(+) T cell activation and increased susceptibility to chronic graft-versus-host disease (cGVHD), which mapped to the centromeric portion of the locus. In this study, we have refined the centromeric sublocus to a 675-kb interval, termed Sle1c2. Mice from recombinant congenic strains expressing Sle1c2 exhibited increased CD4(+) T cell intrinsic activation and cGVHD susceptibility, similar to mice with the parental Sle1c. In addition, B6.Sle1c2 mice displayed a robust expansion of IFN-γ-expressing T cells. NZB complementation studies showed that Sle1c2 expression exacerbated B cell activation, autoantibody production, and renal pathology, verifying that Sle1c2 contributes to lupus pathogenesis. The Sle1c2 interval contains two genes, only one of which, Esrrg, is expressed in T cells. B6.Sle1c2 CD4(+) T cells expressed less Esrrg than B6 CD4(+) T cells, and Esrrg expression was correlated negatively with CD4(+) T cell activation. Esrrg encodes an orphan nuclear receptor that regulates oxidative metabolism and mitochondrial functions. In accordance with reduced Esrrg expression, B6.Sle1c2 CD4(+) T cells present reduced mitochondrial mass and altered mitochondrial functions as well as altered metabolic pathway utilization when compared with B6 CD4(+) T cells. Taken together, we propose Esrrg as a novel lupus susceptibility gene regulating CD4(+) T cell function through their mitochondrial metabolism.     

Augmentation of NZB autoimmune phenotypes by the Sle1c murine lupus susceptibility interval

The Sle1c lupus susceptibility interval spans a 7-Mb region on distal murine chromosome 1. Cr2 is the strongest candidate gene for lupus susceptibility in this interval, as its protein products are structurally and functionally altered. B6.Sle1c congenic mice develop Abs to chromatin by 9 mo of age with a 30% penetrance and do not develop GN. To determine whether the New Zealand White (NZW)-derived Sle1c interval would interact with New Zealand Black (NZB) genes to result in enhanced autoimmune phenotypes, NZB mice were bred with B6 or B6.Sle1c congenic mice and approximately 20 female offspring were selected from each breeding for longitudinal study. These mice differ only at the Sle1c locus at which they have either a NZB/B6 or NZB/NZW genotype. NZB x B6.Sle1c mice had an accelerated onset of anti-chromatin Abs (100 vs 68% at 6 mo, p = 0.006) and anti-dsDNA Abs (45 vs 5% at 9 mo, p = 0.0048). Furthermore, median titers of anti-chromatin and anti-dsDNA Abs were significantly higher in the NZB x B6.Sle1c group compared with the NZB x B6 group. This corresponded with a higher prevalence of proliferative GN at 12 mo (55 vs 16%, p = 0.0214) as well as increased glomerular deposition of C3 (p = 0.0272) and IgG (p = 0.032), although blood urea nitrogen remained normal and significant proteinuria was not identified in either group. These data show that the Sle1c interval accelerates and augments the loss of tolerance to chromatin and dsDNA induced by NZB genes and induces significantly greater end-organ damage.     

Mechanisms of peritoneal B-1a cells accumulation induced by murine lupus susceptibility locus Sle2

The abundance of B-1a cells found in the peritoneal cavity of mice is under genetic control. The lupus-prone mouse New Zealand Black and New Zealand White (NZB x NZW)F(1) and its derivative NZM2410 are among the strains with the highest numbers of peritoneal B1-a cells. We have previously identified an NZM2410 genetic locus, Sle2, which is associated with the production of large numbers of B-1a cells. In this paper, we examined the mechanisms responsible for this phenotype by comparing congenic C57BL/6 mice with or without Sle2. Fetal livers generated more B-1a cells in B6.Sle2 mice, providing them with a greater starting number of B-1a cells early in life. Sle2-expressing B1-a cells proliferated significantly more in vivo than their B6 counterparts, and reciprocal adoptive transfers showed that this phenotype is intrinsic to Sle2 peritoneal B cells. The rate of apoptosis detected was significantly lower in B6.Sle2 peritoneal cavity B-1a cells than in B6, with or without exogenous B cell receptor cross-linking. Increased proliferation and decreased apoptosis did not affect Sle2 peritoneal B-2 cells. In addition, a significant number of peritoneal cavity B-1a cells were recovered in lethally irradiated B6.Sle2 mice reconstituted with B6.Igh(a) bone marrow, showing radiation resistance in Sle2 B-1a cells or its precursors. Finally, B6.Sle2 adult bone marrow and spleen were a significant source of peritoneal B-1a cells when transferred into B6.Rag2(-/-) mice. This suggests that peritoneal B-1a cells are replenished throughout the animal life span in B6.Sle2 mice. These results show that Sle2 regulates the size of the B-1a cell compartment at multiple developmental checkpoints.     

Genetic dissection of Sle pathogenesis: Sle3 on murine chromosome 7 impacts T cell activation, differentiation, and cell death.

Polyclonal, generalized T cell defects, as well as Ag-specific Th clones, are likely to contribute to pathology in murine lupus, but the genetic bases for these mechanisms remain unknown. Mapping studies indicate that loci on chromosomes 1 (Sle1), 4 (Sle2), 7 (Sle3), and 17 (Sle4) confer disease susceptibility in the NZM2410 lupus strain. B6.NZMc7 mice are C57BL/6 (B6) mice congenic for the NZM2410-derived chromosome 7 susceptibility interval, bearing Sle3. Compared with B6 controls, B6.NZMc7 mice exhibit elevated CD4:CD8 ratios (2.0 vs 1.34 in 1- to 3-mo-old spleens); an age-dependent accumulation of activated CD4+ T cells (33.4% vs 21.9% in 9- to 12-mo-old spleens); a more diffuse splenic architecture; and a stronger immune response to T-dependent, but not T-independent, Ags. In vitro, Sle3-bearing T cells show stronger proliferation, increased expansion of CD4+ T cells, and reduced apoptosis (with or without anti-Fas) following stimulation with anti-CD3. With age, the B cells in this strain acquire an activated phenotype. Thus, the NZM2410 allele of Sle3 appears to impact generalized T cell activation, and this may be causally related to the low grade, polyclonal serum autoantibodies seen in this strain. Epistatic interactions with other loci may be required to transform this relatively benign phenotype into overt autoimmunity, as seen in the NZM2410 strain.     

Genetic determination of T cell help in loss of tolerance to nuclear antigens

Sle1 is a major lupus susceptibility locus in NZM2410 lupus model that is associated with a loss of tolerance to nuclear Ags. At least three genes, Sle1a, Sle1b, and Sle1c contribute to Sle1, and their relative role in lupus pathogenesis is unknown. We show here that Sle1-expressing CD4(+) T cells present an activated phenotype associated with increased proliferation and cytokine production. In addition, Sle1 CD4(+) T cells provide help to anti-chromatin B cells to produce anti-nuclear antibodies, whether or not these B cells express Sle1. The Sle1a locus alone accounts for all these Sle1 phenotypes, implying that a specific genetic defect in Sle1a is necessary and sufficient to produce autoreactive T cells. However, Sle1c induces intermediate T cell activation and only provides help to Sle1-expressing anti-chromatin-producing B cells, demonstrating the synergic interactions between Sle1c T and Sle1 B cells. Moreover, Sle1a and Sle1c were associated with a significantly reduced level of CD4(+)CD25(+) regulatory T cells that precedes autoantibody production, suggesting a causal relationship with the generation of autoreactive T cells. Our study identifies for the first time that a specific genetic defect is responsible for lupus pathogenesis by inducing autoreactive T cells to break self-tolerance and that this genetic defect is also associated with a decreased number of regulatory T cells.     

A New Zealand Black-derived locus suppresses chronic graft-versus-host disease and autoantibody production through nonlymphoid bone marrow-derived cells

The development of lupus pathogenesis results from the integration of susceptibility and resistance genes. We have used a chronic graft-versus-host disease (cGVHD) model to characterize a suppressive locus at the telomeric end of the NZM2410-derived Sle2 susceptibility locus, which we named Sle2c2. cGVHD is induced normally in Sle2c2-expressing mice, but it is not sustained. The analysis of mixed bone marrow chimeras revealed that cGVHD resistance was eliminated by non-B non-T hematopoietic cells expressing the B6 allele, suggesting that resistance is mediated by this same cell type. Furthermore, Sle2c2 expression was associated with an increased number and activation of the CD11b(+) GR-1(+) subset of granulocytes before and in the early stage of cGVHD induction. We have mapped the Sle2c2 critical interval to a 6-Mb region that contains the Cfs3r gene, which encodes for the G-CSFR, and its NZM2410 allele carries a nonsynonymous mutation. The G-CSFR-G-CSF pathway has been previously implicated in the regulation of GVHD, and our functional data on Sle2c2 suppression suggest a novel regulation of T cell-induced systemic autoimmunity through myeloid-derived suppressor cells. The validation of Csf3r as the causative gene for Sle2c2 and the further characterization of the Sle2c2 MDSCs promise to unveil new mechanisms by which lupus pathogenesis is regulated.     

The granulocyte colony stimulating factor pathway regulates autoantibody production in a murine induced model of systemic lupus erythematosus

Introduction

An NZB-derived genetic locus (Sle2c2) that suppresses autoantibody production in a mouse model of induced systemic lupus erythematosus contains a polymorphism in the gene encoding the G-CSF receptor. This study was designed to test the hypothesis that the Sle2c2 suppression is associated with an impaired G-CSF receptor function that can be overcome by exogenous G-CSF.

Methods

Leukocytes from B6.Sle2c2 and B6 congenic mice, which carry a different allele of the G-CSF receptor, were compared for their responses to G-CSF. Autoantibody production was induced with the chronic graft-versus-host-disease (cGVHD) model by adoptive transfer of B6.bm12 splenocytes. Different treatment regimens varying the amount and frequency of G-CSF (Neulasta^®) or carrier control were tested on cGVHD outcomes. Autoantibody production, immune cell activation, and reactive oxygen species (ROS) production were compared between the two strains with the various treatments. In addition, the effect of G-CSF treatment was examined on the production autoantibodies in the B6.Sle1.Sle2.Sle3 (B6.TC) spontaneous model of lupus.

Results

B6.Sle2c2 and B6 leukocytes responded differently to G-CSF. G-CSF binding by B6.Sle2c2 leukocytes was reduced as compared to B6, which was associated with a reduced expansion in response to in vivo G-CSF treatment. G-CSF in vivo treatment also failed to mobilize bone-marrow B6.Sle2c2 neutrophils as it did for B6 neutrophils. In contrast, the expression of G-CSF responsive genes indicated a higher G-CSF receptor signaling in B6.Sle2c2 cells. G-CSF treatment restored the ability of B6.Sle2c2 mice to produce autoantibodies in a dose-dependent manner upon cGVHD induction, which correlated with restored CD4⁺ T cells activation, as well as dendritic cell and granulocyte expansion. Steady-state ROS production was higher in B6.Sle2c2 than in B6 mice. cGVHD induction resulted in a larger increase in ROS production in B6 than in B6.Sle2c2 mice, and this difference was eliminated with G-CSF treatment. Finally, a low dose G-CSF treatment accelerated the production of anti-dsDNA IgG in young B6.TC mice.

Conclusion

The different in vivo and in vitro responses of B6.Sle2c2 leukocytes are consistent with the mutation in the G-CSFR having functional consequences. The elimination of Sle2c2 suppression of autoantibody production by exogenous G-CSF indicates that Sle2c2 corresponds to a loss of function of G-CSF receptor. This result was corroborated by the increased anti-dsDNA IgG production in G-CSF-treated B6.TC mice, which also carry the Sle2c2 locus. Overall, these results suggest that the G-CSF pathway regulates the production of autoantibodies in murine models of lupus.     

Pre-B cell leukemia homeobox 1 is associated with lupus susceptibility in mice and humans

Sle1a.1 is part of the Sle1 susceptibility locus, which has the strongest association with lupus nephritis in the NZM2410 mouse model. In this study, we show that Sle1a.1 results in the production of activated and autoreactive CD4(+) T cells. Additionally, Sle1a.1 expression reduces the peripheral regulatory T cell pool, as well as induces a defective response of CD4(+) T cells to the retinoic acid expansion of TGF-β-induced regulatory T cells. At the molecular level, Sle1a.1 corresponds to an increased expression of a novel splice isoform of Pbx1, Pbx1-d. Pbx1-d overexpression is sufficient to induce an activated/inflammatory phenotype in Jurkat T cells and to decrease their apoptotic response to retinoic acid. PBX1-d is expressed more frequently in the CD4(+) T cells from lupus patients than from healthy controls, and its presence correlates with an increased central memory T cell population. These findings indicate that Pbx1 is a novel lupus susceptibility gene that regulates T cell activation and tolerance.     

Lupus susceptibility genes may breach tolerance to DNA by impairing receptor editing of nuclear antigen-reactive B cells

An NZM2410-derived lupus susceptibility locus on murine chromosome 4, Sle2(z), has previously been noted to engender generalized B cell hyperactivity. To study how Sle2(z) impacts B cell tolerance, two Ig H chain site-directed transgenes, 3H9 and 56R, with specificity for DNA were backcrossed onto the C57BL/6 background with or without Sle2(z). Interestingly, the presence of the NZM2410 "z" allele of Sle2 on the C57BL/6 background profoundly breached B cell tolerance to DNA, apparently by thwarting receptor editing. Whereas mAbs isolated from the spleens of B6.56R control mice demonstrated significant usage of the endogenous (i.e., nontargeted) H chain locus and evidence of vigorous L chain editing; Abs isolated from B6.Sle2(z).56R spleens were largely composed of the transgenic H chain paired with a spectrum of L chains, predominantly recombined to J(k)1 or J(k)2. In addition, Sle2(z)-bearing B cells adopted divergent phenotypes depending on their Ag specificity. Whereas Sle2(z)-bearing anti-DNA transgenic B cells were skewed toward marginal zone B cells and preplasmablasts, B cells from the same mice that did not express the transgene were skewed toward the B1a phenotype. This work illustrates that genetic loci that confer lupus susceptibility may influence B cell differentiation depending on their Ag specificity and potentially contribute to antinuclear autoantibody formation by infringing upon B cell receptor editing. Taken together with a recent report on Sle1(z), these studies suggest that dysregulated receptor-editing of nuclear Ag-reactive B cells may be a major mechanism through which antinuclear Abs arise in lupus.     

Sle1ab mediates the aberrant activation of STAT3 and Ras-ERK signaling pathways in B lymphocytes

The Sle1ab genomic interval on murine chromosome 1 mediates the loss of immune tolerance to chromatin resulting in antinuclear Abs (ANA) production in the lupus-prone NZM2410 mouse. Global gene expression analysis was used to identify the molecular pathways that are dysregulated at the initiation of B lymphocyte autoimmunity in B6.Sle1ab mice. This analysis identified that STAT3 and ras-ERK signaling pathways are aberrantly activated in Sle1ab B lymphocytes, consistent with increased production of IL-6 by splenic B lymphocytes and monocytes in B6.Sle1ab mice. In vitro treatment of splenic mononuclear cells isolated from ANA-positive Sle1ab mice with anti-IL-6 Ab or AG490, an inhibitor of STAT3 signaling pathway, suppressed ANA production in short-term culture, indicating that this pathway was essential to the production of autoantibodies. In vivo treatment of ANA-positive B6.Sle1ab mice with the ras pathway inhibitor, perillyl alcohol, suppressed the increase of ANA. These findings identify IL-6 as a early key cytokine in Sle1ab-mediated disease development and indicate that the STAT3 and ras-ERK signaling pathways are potential therapeutic targets for treating systemic lupus erythematosus.     

Genetic control of the B cell response to LPS: opposing effects in peritoneal versus splenic B cell populations

Lipopolysaccharide (LPS) from gram-negative bacteria activates B cells, enabling them to proliferate and differentiate into plasma cells. This response is critically dependent on the expression of TLR4; but other genes, such as RP105 and MHC class II, have also been shown to contribute to B cell LPS response. Here, we have evaluated the role of genetic control of the B cell response to LPS at the single cell level. We compared the response to LPS of peritoneal cavity (PEC) and splenic B cells on the BALB/c genetic background (LPS-low responder) to those on the C57BL/6J background (LPS-high responder) and their F1 progeny (CB6F1). Both PEC and splenic B cells from B6 exhibited 100% clonal growth in the presence of LPS; whereas, BALB/c PEC and splenic B cells achieved only 50% and 23% clonal growth, respectively. Adding CpG to the LPS stimulus pushed PEC B cell clonal growth in the low responder strain BALB/c up to 90%, showing that the nonresponse to LPS is a specific effect. Surprisingly, PEC B cells on the F1 background behaved as high responders, while splenic B cells behaved as low responders to LPS. The data presented here reveals a previous unsuspected behavior in the genetic control of the B cell response to LPS with an opposing impact in splenic versus peritoneal cavity B cells. These results suggest the existence of an, as yet, unidentified genetic factor exclusively expressed by coelomic B cells that contributes to the control of the LPS signaling pathway in the B lymphocyte.     

Defective B1 cell homing to the peritoneal cavity and preferential recruitment of B1 cells in the target organs in a murine model for systemic lupus erythematosus

We previously reported that B lymphocyte chemoattractant (BLC; CXCL13) was highly and ectopically expressed in aged (NZB x NZW)F1 (BWF1) mice developing lupus nephritis, and that B1 cells were preferentially chemoattracted toward BLC. We demonstrate in this study that B1 cells fail to home to the peritoneal cavity in aged BWF1 mice developing lupus nephritis, and that they are preferentially recruited to the target organs including the kidney, lung, and thymus when injected i.v. In contrast, B1 cells homed to the peritoneal cavity in aged BALB/c mice as effectively as in young mice. Accumulation of B1 cells to the omentum milky spots was also impaired in aged BWF1 mice compared with young mice. CD11bhighF4/80high cells with macrophage morphology were confirmed to be a major cell source for BLC in the peritoneal cavity both in young and aged BWF1 mice. However, the number of BLC-producing peritoneal macrophages was markedly decreased in aged BWF1 mice. These results suggest that the decreased number of BLC-producing peritoneal macrophages together with ectopic high expression of BLC in aged BWF1 mice result in abnormal B1 cell trafficking during the development of murine lupus.     

Enhanced IgA class switching in marginal zone and B1 B cells relative to follicular/B2 B cells

Mouse splenic marginal zone (MZ) B cells and B1 B cells enriched in the peritoneal cavity respond preferentially to T cell-independent Ags compared with follicular (FO)/B2 B cells. Despite the differential responses of B cell subsets to various stimuli, and despite the need for multiple stimuli to induce IgA class switching, the relative contribution of B cell subpopulations to IgA production is unknown. By culturing purified B cell populations, we find that MZ and peritoneal B1 cells switch more readily to IgA than do splenic FO or peritoneal B2 cells in BLyS/LPS/TGF-beta. Addition of IL-4, IL-5, and anti-IgD dextran to the cultures enhances IgA switching in FO/B2 and MZ B cells to a similar frequency, but this treatment suppresses IgA class switching in B1 cells. Thus, IgA switching differs among purified B cell subsets, suggesting that individual B cell populations could contribute differentially to IgA expression in vivo, depending on available stimuli.     

Rapid induction of splenic and peritoneal B-1a cells in adult mice by thymus-independent type-2 antigen

We have produced a transgenic mouse (PV1TgL) that can only generate B lymphocytes with an Ig receptor specific for the synthetic polymer polyvinyl pyrrolidinone. Before immunization, bone marrow B cell numbers are very low, and peripheral lymphoid organs are almost devoid of B cells, confirming the role of positive selection by Ag in the development of mature B cell populations. The predominant population of B cells in the spleens of naive adult PV1TgL mice have most of the characteristics of marginal zone B cells, including anatomical location in the peripheral areas of the splenic white pulp. After immunization, a new population of B cells appears in the spleen with the characteristics of B-1 cells. Similar cells also appear somewhat later in the peritoneal cavity. Our findings suggest that immunization with a thymus-independent Ag can lead to the appearance and expansion of Ag-reactive B-1 cells in an adult mouse.     

Programmed cell death 1 suppresses B-1b cell expansion and long-lived IgG production in response to T cell-independent type 2 antigens

B-1b cells play a key role in producing Abs against T cell-independent type 2 Ags. However, the factors regulating Ab production by this unique B cell subset are not well understood. In this study, a detailed analysis of the B cell response to 2,4,6-trinitrophenol (TNP)-Ficoll was performed using normal mice. TNP-Ficoll delivered i.p. or i.v. induced rapid Ag-specific B-1b cell activation, expansion, isotype switching, and plasmablast/plasma cell differentiation. Ag-specific B-1b cell numbers peaked at day 5 and then gradually declined in the spleen but remained elevated in the peritoneal cavity beyond 40 d postimmunization. In addition to expressing CD43, CD44, and CD86, Ag-activated B-1b cells transiently expressed programmed cell death 1 (PD-1), which functionally suppressed BCR-induced B-1b cell in vitro proliferation when additional costimulatory signals were lacking. Inhibiting PD-1:PD-1 ligand interactions during TNP-Ficoll immunization significantly enhanced Ag-specific B-1b cell expansion and the frequency of IgG isotype switching and plasmablast/plasma cell differentiation. Remarkably, PD-1 mAb blockade during the first week following immunization resulted in significantly increased numbers of both splenic and bone marrow Ag-specific IgG3-secreting cells, but not IgM-secreting cells, at both early (day 5) and late (week 6) time points. Moreover, Ag-specific serum IgG3 levels, as well as IgG2c, IgG2b, and IgA levels, remained significantly elevated in PD-1 mAb-treated mice relative to control Ab-treated mice for ≥6 wk postimmunization. Thus, PD-1:PD-1 ligand interactions occurring shortly after initial T cell-independent type 2 Ag encounter play a critical role in suppressing Ag-specific B-1b cell expansion and the development of long-term IgG-producing bone marrow and spleen cells.