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Nuclear hormone receptors including the estrogen receptor (ERα) and the retinoic acid receptor regulate a plethora of biological functions including reproduction, circulation and immunity. To understand how estrogen and other nuclear hormones influence antibody production, we characterized total serum antibody isotypes in female and male mice of C57BL/6J, BALB/cJ and C3H/HeJ mouse strains. Antibody levels were higher in females compared to males in all strains and there was a female preference for IgG2b production. Sex-biased patterns were influenced by vitamin levels, and by antigen specificity toward influenza virus or pneumococcus antigens. To help explain sex biases, we examined the direct effects of estrogen on immunoglobulin heavy chain sterile transcript production among purified, lipopolysaccharide-stimulated B cells. Supplemental estrogen in B-cell cultures significantly increased immunoglobulin heavy chain sterile transcripts. Chromatin immunoprecipitation analyses of activated B cells identified significant ERα binding to estrogen response elements (EREs) centered within enhancer elements of the immunoglobulin heavy chain locus, including the Eµ enhancer and hypersensitive site 1,2 (HS1,2) in the 3' regulatory region. The ERE in HS1,2 was conserved across animal species, and in humans marked a site of polymorphism associated with the estrogen-augmented autoimmune disease, lupus. Taken together, the results highlight: (i) the important targets of ERα in regulatory regions of the immunoglobulin heavy chain locus that influence antibody production, and (ii) the complexity of mechanisms by which estrogen instructs sex-biased antibody production profiles., (© The Japanese Society for Immunology. 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

High-affinity memory B cells are preferentially selected during secondary responses and rapidly differentiate into antibody-producing cells. However, it remains unknown whether only high-affinity, mutated memory B cells simply expand to dominate the secondary response or if in fact memory B cells with a diverse VH repertoire, including those with no mutations, accumulate somatic mutations to create a new repertoire through the process of affinity maturation. In this report, we took a new approach to address this question by analyzing the VH gene repertoire of IgG1(+) memory B cells before and after antigen re-exposure in a host unable to generate IgG(+) B cells. We show here that both mutated and unmutated IgG1(+) memory B cells respond to secondary challenge and expand while accumulating somatic mutations in their VH genes in a stepwise manner. Both types of memory cells subsequently established a VH gene repertoire dominated by two major clonotypes, which are distinct from the original repertoire before antigen re-exposure. In addition, heavily mutated memory B cells were excluded from the secondary repertoire. Thus, both mutated and unmutated IgG1(+) memory cells equally contribute to establish a new antibody repertoire through a dynamic process of mutation and selection, becoming optimally adapted to the recall challenge.

Interferon regulatory factor (IRF)-2 is a transcription factor involved in type I (IFN- α/β) signaling. It has been reported that IRF-2 deficiency results in various immune dysfunctions. However, the role of IRF-2 in B-cell functions needs to be elucidated. Unlike wild-type (WT) B cells, IRF-2(-/-) B2 cells were refractory to anti-IgM, but not LPS. Such a defect in proliferation was dependent on IFN- α/β receptor (IFNAR). Marginal zone B cells increased in the proportion relative to B2 cells in IRF-2(-/-) mice produced IgM normally to LPS stimulation. However, IRF-2(-/-) B2 cells were defective in IgM production in an IFNAR-independent manner, although both B-cell subsets differentiated phenotypically to plasma cells at elevated efficiencies. Class switch recombination of IRF-2(-/-) B2 cells by LPS plus IL-4 was also impaired. Their reduced IgM production was conceivably due to an inefficient up-regulation of Blimp-1. Consistent with these in vitro observations, specific antibody production in vivo to a T-dependent antigen by B2 cells was severely impaired in IRF-2(-/- )mice. However, a low, but significant, level of IgG was detected at a late time point, and this IgG exhibited comparable binding affinity to that in WT mice. Follicular helper T-cell development and germinal center formation were normal. A similar tendency was observed when µ chain(-/-) mice were reconstituted with IRF-2(-/- )B cells. These results revealed a multi-faceted role of IRF-2 in the function of B cells, particularly B2 cells, through regulating proliferation in an IFNAR-dependent manner and antibody production via up-regulation of Blimp-1.

The CD1d-binding glycolipid α-galactosylceramide exerts potent adjuvant effects on T-dependent humoral immunity. The mechanism is driven by cognate interaction between CD1d-expressing B cells and TCR-expressing type I CD1d-restricted NKT cells. Thus, far positive effects of alpha-galactosylceramide have been observed on initial and sustained antibody titers as well as B-cell memory. Following vaccination, each of these features is desirable, but good B-cell memory is of paramount importance for long-lived immunity. We therefore tested the hypothesis that CD1d expression in vivo differentially affects initial antibody titers versus B-cell memory responses. CD1d(+/+) and CD1d(+/-) mice were generated and immunized with antigen plus CD1d ligand before analysis of cytokine expression, CD40L expression, initial and longer term antibody responses and B-cell memory. As compared with CD1d(+/+) controls, CD1d(+/-) mice had equivalent numbers of total NKT cells, lower cytokine production, fewer CD40L-expressing NKT cells, lower initial antibody responses, similar long-term antibody responses and higher B-cell memory. Our data indicate that weak CD1d antigen presentation may facilitate good B-cell memory without compromising antibody responses. This work may impact vaccine design since over-stimulation of NKT cells at the time of vaccination may not lead to optimal B-cell memory.

1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and the vitamin D receptor (VDR) are important regulators of autoimmunity. The effect of the VDR on the ability of mice to fight a primary or secondary infection has not been determined. Young and old VDR knockout (KO) mice were able to clear both primary and secondary infections with Listeria monocytogenes. However, the kinetics of clearance was somewhat delayed in the absence of the VDR. Memory T cell development was not different in young VDR KO and wild-type (WT) mice; however, old VDR KO mice had significantly less memory T cells than their WT counterparts but still mounted an adequate immune response as determined by the complete clearance of L. monocytogenes. Although the primary and secondary immune responses were largely intact in the VDR KO mice, the old VDR KO mice had increased cytokines and antibody responses compared with the old WT mice. In particular, old VDR KO mice had elevated antigen non-specific antibodies; however, these magnified immune responses did not correspond to more effective Listeria clearance. The increased antibody and cytokine responses in the old VDR KO mice are consistent with the increased susceptibility of these mice to autoimmunity.

The MRL-lpr/lpr mouse strain is a commonly used model of the human autoimmune disease systemic lupus erythematosus (SLE). Although much is known about the contribution of the lpr Fas mutation to B cell tolerance breakdown, the role of the genetic background of the MRL strain itself is less well explored. In this study, we use the MD4 anti-hen egg lysozyme Ig (IgHEL) transgenic system to explore B cell function in MRL+/+ and non-autoimmune mice. We demonstrate that MRL IgHEL B cells show spontaneous hyperactivity in the absence of self-antigen, which is associated with low total B cell numbers but an expansion of the marginal zone B cell population. However, B cell anergy is normal in the presence of soluble lysozyme [soluble hen egg lysozyme (sHEL)], and MRL IgHEL B cells undergo normal elimination in the presence of sHEL when competing with a polyclonal C57BL/6 B cell repertoire. We conclude that B cell hyperactivity may contribute to the autoimmune phenotype of MRL+/+ and MRL-lpr/lpr strains when it initiates antibody responses to rare or sequestered antigens that are below the threshold for tolerance induction, but that there is no B cell intrinsic defect in anergy in MRL mice.

We have used a transgenic mouse system to examine how differing reactivities of TCRs expressed by naive versus effector cells can shape the functional potential of autoreactive CD4+ T cells. Transgenic mice expressing TCRs that exhibit either high (TS1) or low [TS1(SW)] reactivity toward the I-Ed-restricted determinant S1 from the influenza virus PR8 hemagglutinin (HA) were mated with transgenic mice expressing HA under the control of different promoters. HACII mice express HA driven by an MHC class II promoter, and both the TS1 and TS1(SW) TCRs underwent substantial deletion in this background. HA104 mice express HA driven by an SV40 promoter, and the highly reactive TS1 TCR was substantially deleted. By contrast, the less reactive TS1(SW) TCR underwent little or no deletion in TS1(SW) x HA104 mice, although CD5 up-regulation indicated that they had interacted with the S1 self-peptide. In adoptive transfer studies, naive CD4+ T cells expressing the TS1(SW) TCR failed to proliferate in response to the S1 peptide in HA104 mice, and were inefficient at providing help for HA-specific antibody responses. However, effector CD4+ T cells generated from TS1(SW) x HA104 mice acquired the ability to proliferate in response to the S1 peptide in HA104 mice, and were as efficient as CD4+ T cells expressing the high reactivity TS1 TCR in helping HA-specific antibody responses. Collectively, these studies demonstrate a basis by which CD4+ T cells expressing TCRs with low reactivity toward self-peptides can evade negative selection and acquire enhanced autoreactivity following activation by a cross-reactive antigen.

Contact hypersensitivity (CHS) is a T cell-mediated cellular immune response caused by epicutaneous exposure to contact allergens. In this reaction, after the first epicutaneous allergen sensitization, Langerhans cells (LC) catch allergens and migrate from the skin to draining lymph nodes (LN) and activate naive T cells. Although IL-1 is suggested to be involved in these processes, the mechanisms have not been elucidated completely. In this report, to elucidate roles of IL-1alpha and IL-1beta in CHS, we analyzed ear swelling in 2,4,6-trinitrochlorobenzene (TNCB)-induced CHS using gene-targeted mice. We found that ear swelling was suppressed in IL-1alpha-deficient (IL-1alpha(-/-)) mice but not in IL-1beta(-/-) mice. LC migration from the skin into LN was delayed in both IL-1alpha(-/-) and IL-1beta(-/-) mice, suggesting that this defect was not the direct cause for the reduced CHS in these mice. However, we found that the proliferative response of trinitrophenyl (TNP)-specific T cells after sensitization with TNCB was specifically reduced in IL-1alpha(-/-) mice. Furthermore, adoptive transfer of TNP-conjugated IL-1-deficient epidermal cells (EC) into wild-type mice indicated that only IL-1alpha, but not IL-1beta, produced by antigen-presenting cells in EC could prime allergen-specific T cells. These observations indicate that IL-1alpha, but not IL-1beta, plays a crucial role in TNCB-induced CHS by sensitizing TNP-specific T cells.

NKT cells are CD4(+) or CD4(-)CD8(-) CD1d-restricted lymphocytes, characterized by the property to rapidly produce IL-4 and IFN-gamma in response to TCR ligation. This IL-4 burst is lacking in autoimmunity-prone SJL and NOD strains of mice, which suggests an immunoregulatory role for NKT cells. The NKT cell status was thus investigated in the genetically selected high (H) and low (L) antibody-producer mice. The results show that (i) the frequency of cells expressing the NKT cell markers is 3- to 4-fold lower in thymus and spleen from L than H mice, (ii) L mice spleen cells did not produce IL-4 following injection of anti-TCR alpha beta antibody, and (iii) L mice thymus and spleen cells failed to produce IL-4 after in vitro stimulation by anti-TCR alpha beta antibody or alpha-galactosylceramide, a newly described NKT cell ligand. These parameters were investigated in six interval-specific congenic strains raised for the quantitative trait loci which contain the immunomodulatory genes responsible for the high/low antibody production phenotypes. IL-4 production recovery occurred only in the congenic strain in which the H origin chromosome 4 segment was introgressed on the L background. This finding was not due to increased NKT cell frequency but appeared dependent of antigen-presenting cells in co-culture experiments. This result strongly suggests the presence of gene(s) modulating NKT function on chromosome 4, close to several genes predisposing to autoimmunity.

The alymphoplasia (aly) mutation of mice causes the systemic absence of lymph nodes, Peyer's patches and well-defined lymphoid follicles in the spleen. We found that antibody responses are elicited, albeit weakly, to either T cell-dependent or T cell-independent antigen by aly/aly mutants. However, isotype switching was defective. The T cell-dependent immune response was not elicited in splenectomized aly/aly mice. Neither hypermutation nor germinal center formation was observed in aly/aly mice. These results suggest that T-B collaboration requires either lymph nodes or spleen, and that hypermutation and affinity maturation depend on germinal center formation.

The HLA multigene family consists of HLA class I (HLA-A, B and C) and class II (HLA-DR, DQ and DP) genes, and plays a central role in the regulation of immune response. To investigate how each HLA gene and each HLA allele contribute to the human immune response, we immunized 339 healthy Japanese medical students with recombinant hepatitis B surface antigen (rHBsAg) and determined the HLA types of all vaccinated subjects at the DNA level. The anti-HBs antibody titers showed a log-normal distribution, implying that the immune response to HBsAg in humans is a multifactorial and continuous trait. A stepwise multiple regression analysis demonstrated the alleles at the HLA-class I (HLA-A and B) and class II (HLA-DRB1, DQA1, DQB1, DPA1 and DPB1) loci significantly contributed to antibody production to HBsAg. The predicting equation of anti-HBs antibody levels for individuals with any HLA phenotype was proposed based on a multiple regression analysis. The multiple correlation coefficient of antibody production to HBsAg with the HLA-DRB1 locus was highest (0.34) among all of the HLA loci, whereas those with whole HLA class I or class II loci were 0.36 or 0.44 respectively. The incorporated correlation coefficient of the presence of all HLA gene families with antibody production became 0.50, suggesting that HLA class I and class II loci within the HLA multigene family are dynamically involved in regulation of the immune response to HBsAg.