Yumiko Nishikawa

Biphasic Aire expression in early embryos and in medullary thymic epithelial cells before end-stage terminal differentiation

The roles of autoimmune regulator (Aire)–expressing medullary thymic epithelial cells (mTECs) in the organization of the thymic microenvironment for establishing self-tolerance are enigmatic. We sought to monitor the production and maintenance of Aire-expressing mTECs by a fate-mapping strategy in which bacterial artificial chromosome transgenic (Tg) mice expressing Cre recombinase under the control of the Aire regulatory element were crossed with a GFP reporter strain. We found that, in addition to its well recognized expression within mature mTECs, Aire was expressed in the early embryo before emergence of the three germ cell layers. This observation may help to explain the development of ectodermal dystrophy often seen in patients with AIRE deficiency. With the use of one Tg line in which Cre recombinase expression was confined to mTECs, we found that Aire+CD80high mTECs further progressed to an Aire−CD80intermediate stage, suggesting that Aire expression is not constitutive from after its induction until cell death but instead is down-regulated at the beginning of terminal differentiation. We also demonstrated that many mTECs of Aire-expressing lineage are in close contact with thymic dendritic cells. This close proximity may contribute to transfer of tissue-restricted self-antigens expressed by mTECs to professional antigen-presenting cells.

Lymphotoxin Signal Promotes Thymic Organogenesis by Eliciting RANK Expression in the Embryonic Thymic Stroma

It has recently become clear that signals mediated by members of the TNFR superfamily, including lymphotoxin-β receptor (LTβR), receptor activator for NF-κB (RANK), and CD40, play essential roles in organizing the integrity of medullary thymic epithelial cells (mTECs) required for the establishment of self-tolerance. However, details of the mechanism responsible for the unique and cooperative action of individual and multiple TNFR superfamily members during mTEC differentiation still remain enigmatic. In this study, we show that the LTβR signal upregulates expression of RANK in the thymic stroma, thereby promoting accessibility to the RANK ligand necessary for mTEC differentiation. Cooperation between the LTβR and RANK signals for optimal mTEC differentiation was underscored by the exaggerated defect of thymic organogenesis observed in mice doubly deficient for these signals. In contrast, we observed little cooperation between the LTβR and CD40 signals. Thus, the LTβR signal exhibits a novel and unique function in promoting RANK activity for mTEC organization, indicating a link between thymic organogenesis mediated by multiple cytokine signals and the control of autoimmunity.

Temporal Lineage Tracing of Aire-Expressing Cells Reveals a Requirement for Aire in Their Maturation Program

Understanding the cellular dynamics of Aire-expressing lineage(s) among medullary thymic epithelial cells (AEL-mTECs) is essential for gaining insight into the roles of Aire in establishment of self-tolerance. In this study, we monitored the maturation program of AEL-mTECs by temporal lineage tracing, in which bacterial artificial chromosome transgenic mice expressing tamoxifen-inducible Cre recombinase under control of the Aire regulatory element were crossed with reporter strains. We estimated that the half-life of AEL-mTECs subsequent to Aire expression was ∼7–8 d, which was much longer than that reported previously, owing to the existence of a post-Aire stage. We found that loss of Aire did not alter the overall lifespan of AEL-mTECs, inconsistent with the previous notion that Aire expression in medullary thymic epithelial cells (mTECs) might result in their apoptosis for efficient cross-presentation of self-antigens expressed by AEL-mTECs. In contrast, Aire was required for the full maturation program of AEL-mTECs, as exemplified by the lack of physiological downregulation of CD80 during the post-Aire stage in Aire-deficient mice, thus accounting for the abnormally increased CD80high mTECs seen in such mice. Of interest, increased CD80high mTECs in Aire-deficient mice were not mTEC autonomous and were dependent on cross-talk with thymocytes. These results further support the roles of Aire in the differentiation program of AEL-mTECs.

Establishment of Lymphotoxin β Receptor Signaling-Dependent Cell Lines with Follicular Dendritic Cell Phenotypes from Mouse Lymph Nodes

Follicular dendritic cells (FDCs) have been shown to play a crucial role in the positive selection of high-affinity B cells that are generated by somatic hypermutation in germinal center (GC). Because of technical difficulties in preparing and maintaining pure FDCs, a role for FDCs in this complicated process has not been fully elucidated. In this study, we established a cell line designated as pFL that retained major FDC phenotypes from a three-dimensional culture of mouse lymph node cells. pFL cells proliferated slowly in response to an agonistic anti-lymphotoxin β receptor mAb and TNF-α. A more rapidly growing clone, named FL-Y, with similar requirements for growth was isolated from a long-term culture of pFL. Analysis of surface markers in these two cell lines by immunostaining, flow cytometry, and DNA microarray revealed the expression of genes, including those of CD21, FcγRIIB, lymphotoxin β receptor, ICAM-1, VCAM-1, IL-6, and C4, which have been shown to be characteristic of FDCs. In addition, B cell-activating factor was expressed in these two cell lines. At the pFL or FL-Y:B cell ratio of 1:100, the cell lines markedly sustained B cell survival and Ab production during 2 wk of culture, while most B cells collapsed within 1 wk in the absence of the FDC-like cells. Interestingly, expression of typical GC markers, Fas and GL-7, was notably augmented in B cells that were cocultured with Th cells on these two cell lines. Thus, pFL and FL-Y cells may be useful for providing insight into the functional role for FDCs in GC.

Which Model Better Fits the Role of Aire in the Establishment of Self-Tolerance: The Transcription Model or the Maturation Model?

The discovery of Aire-dependent transcriptional control of many tissue-restricted self-antigen (TRA) genes in thymic epithelial cells in the medulla (medullary thymic epithelial cells, mTECs) has raised the intriguing question of how the single Aire gene can influence the transcription of such a large number of TRA genes within mTECs. From a mechanistic viewpoint, there are two possible models to explain the function of Aire in this action. In the first model, TRAs are considered to be the direct target genes of Aire’s transcriptional activity. In this scenario, the lack of Aire protein within cells would result in the defective TRA gene expression, while the maturation program of mTECs would be unaffected in principle. The second model hypothesizes that Aire is necessary for the maturation program of mTECs. In this case, we assume that the mTEC compartment does not mature normally in the absence of Aire. If acquisition of the properties of TRA gene expression depends on the maturation status of mTECs, a defect of such an Aire-dependent maturation program in Aire-deficient mTECs can also result in impaired TRA gene expression. In this brief review, we will focus on these two contrasting models for the roles of Aire in controlling the expression of TRAs within mTECs.

B Cell Selection and Affinity Maturation During an Antibody Response in the Mouse with Limited B Cell Diversity

The quasi-monoclonal mouse has limited B cell diversity, whose major (∼80%) B cell Ag receptors are comprised of the knockin VH 17.2.25 (VHT)-encoded H chain and the λ1 or λ2 L chain, thereby being specific for 4-hydroxy-3-nitrophenylacetyl. The p-nitrophenylacetyl (pNP) was found to be a low affinity analog of nitrophenylacetyl. We examined affinity maturation of anti-pNP IgG by analyzing mAbs obtained from quasi-monoclonal mice that were immunized with this low affinity Ag. The results are: 1) Although VHT/λ1 and VHT/λ2 IgM were equally produced, VHT/λ2 IgG almost exclusively underwent affinity maturation toward pNP. 2) A common mutation in complementarity-determining region 3 of VHT (T313A) mainly contributed to generating the specificity for pNP. 3) Because mutated VHT-encoded γ-chains could form λ1-bearing IgG in Chinese hamster ovary cells, apparent absence of VHT/λ1 anti-pNP IgG may not be due to the incompatibility between the γ-chains and the λ1-chain, but may be explained by the fact that VHT/λ1 B cells showed 50- to 100-fold lower affinity for pNP than VHT/λ2 B cells. 4) Interestingly, a pNP-specific IgM mAb that shared common mutations including T313A with high affinity anti-pNP IgG was isolated, suggesting that a part of hypermutation coupled with positive selection can occur before isotype switching. Thus, even weak B cell receptor engagement can elicit an IgM response, whereas only B cells that received signals stronger than a threshold may be committed to an affinity maturation process.

Rab13 Small G Protein and Junctional Rab13-binding Protein (JRAB) Orchestrate Actin Cytoskeletal Organization during Epithelial Junctional Development

Background: The Rab13-JRAB system transports cell adhesion molecules. Results: JRAB interacts with actinins and F-actin and spatiotemporally regulates actin dynamics via a conformational change that is dependent upon Rab13. Conclusion: Rab13 and JRAB regulate reorganization of the actin cytoskeleton throughout epithelial junctional development from establishment to maturation of cell-cell adhesion. Significance: The Rab13-JRAB system may simultaneously coordinate vesicle transport and actin cytoskeletal organization. During epithelial junctional development, both vesicle transport and reorganization of the actin cytoskeleton must be spatiotemporally regulated. Coordination of these cellular functions is especially important, but the precise mechanism remains elusive. Previously, we identified junctional Rab13-binding protein (JRAB)/molecules interacting with CasL-like 2 (MICAL-L2) as an effector of the Rab13 small G protein, and we found that the Rab13-JRAB system may be involved in the formation of cell-cell adhesions via transport of adhesion molecules. Here, we showed that JRAB interacts with two actin-binding proteins, actinin-1 and -4, and filamentous actin via different domains and regulates actin cross-linking and stabilization through these interactions. During epithelial junctional development, JRAB is prominently enriched in the actin bundle at the free border; subsequently, JRAB undergoes a Rab13-dependent conformational change that is required for maturation of cell-cell adhesion sites. These results suggest that Rab13 and JRAB regulate reorganization of the actin cytoskeleton throughout epithelial junctional development from establishment to maturation of cell-cell adhesion.

CSF-1 receptor-mediated differentiation of a new type of monocytic cell with B cell-stimulating activity: its selective dependence on IL-34.

With the use of a mouse FDC line, FL‐Y, we have been analyzing roles for FDCs in controlling B cell fate in GCs. Beside these regulatory functions, we fortuitously found that FL‐Y cells induced a new type of CD11b+ monocytic cells (F4/80+, Gr‐1−, Ly6C−, I‐A/E−/lo, CD11c−, CD115+, CXCR4+, CCR2+, CX3CR1−) when cultured with a Lin−c‐kit+ population from mouse spleen cells. The developed CD11b+ cells shared a similar gene‐expression profile to mononuclear phagocytes and were designated as FDMCs. Here, we describe characteristic immunological functions and the induction mechanism of FDMCs. Proliferation of anti‐CD40 antibody‐stimulated B cells was markedly accelerated in the presence of FDMCs. In addition, the FDMC‐activated B cells efficiently acquired GC B cell‐associated markers (Fas and GL‐7). We observed an increase of FDMC‐like cells in mice after immunization. On the other hand, FL‐Y cells were found to produce CSF‐1 as well as IL‐34, both of which are known to induce development of macrophages and monocytes by binding to the common receptor, CSF‐1R, expressed on the progenitors. However, we show that FL‐Y‐derived IL‐34, but not CSF‐1, was selectively responsible for FDMC generation using neutralizing antibodies and RNAi. We also confirmed that FDMC generation was strictly dependent on CSF‐1R. To our knowledge, a CSF‐1R‐mediated differentiation process that is intrinsically specific for IL‐34 has not been reported. Our results provide new insights into understanding the diversity of IL‐34 and CSF‐1 signaling pathways through CSF‐1R.

IL-21–Dependent B Cell Death Driven by Prostaglandin E2, a Product Secreted from Follicular Dendritic Cells

In germinal centers (GCs), B cells are selected through interaction with follicular dendritic cells bearing immune complexes and follicular helper T (Tfh) cells secreting Tfh cytokines, including IL-21. To analyze these cellular interactions, we have explored culture conditions that can simulate GC B cell selection in vitro using a mouse follicular dendritic cell line, FL-YB. FL-YB cells efficiently enhanced viability of cocultured mouse B cells in a BAFF-dependent fashion. Interestingly, we found that addition of IL-21, a major Tfh cytokine, readily induced death of B cells that were cocultured with FL-YB cells, whereas IL-21 alone sustained viability of B cells in the absence of FL-YB cells. The IL-21–induced death was dependent on a low m.w. soluble factor that was released from FL-YB cells, which was finally identified as PGE2. Treatment of B cells with IL-21 plus PGE2, but not either alone, resulted in enhanced expression of a proapoptotic protein Bim and the upstream transcription factor Foxo1. A PGE2 receptor isoform, EP4, was responsible for IL-21/PGE2–induced B cell death. Thus, PGE2 is an endogenous chemical mediator that can switch pleiotropic actions of IL-21 on B cells. IL-21/PGE2–induced B cell death was rescued if B cells were costimulated via CD40. In immunized mice, deficiency of IL-21R in B cells led to a significant decrease in the frequency of activated caspase-3–positive GC B cells concomitant with impaired affinity maturation of Abs. Taken together, results implicate a physiological role of IL-21/PGE2–induced B cell death in GC B cell selection.

50 Expression of ly6c/6g defines a novel aire-dependent subset of medullary thymic epithelial cells with tolerogenic function

Medullary thymic epithelial cells (mTECs) are a heterogeneous population in terms of the spectrum of tissue-restricted Ags (TRAs) expressed from each cell for ensuring the elimination of autoreactive T-cells. Additionally, mTECs comprise cells at different developmental stages and/or in various activation conditions. Because of these heterogeneities, it is unclear whether mTECs are composed of any particular subsets possessing unique properties for their developmental pathway and/or immunological function. Here, we report a distinct mTEC subset characterised by expression of Ly6 family protein prior to and concomitant with Aire expression during its differentiation. Ly6C/6G+ mTECs, constituting 5%–15% of mature mTECs, were preferentially localised at the cortico-medullary junction, and expressed high levels of TRAs and thymocyte-attracting chemokines. Remarkably, Ly6C/6G+ mTECs were absent in Aire-deficient mice, suggesting that this subset requires Aire and/or Aire+ mTECs for its production. Uniquely, Ly6C/6G+ mTECs lack a post-Aire stage because of a tendency to die after Aire had been expressed. With a TCR-transgenic model in mice, we found that in vivo depletion of Ly6C/6G+ mTECs frequently induced organ-specific autoimmunity. We suggest that Ly6C/6G+ mTECs serve as an important source of TRAs for efficient cross-presentation during establishment of self-tolerance.