Kazuhito Honjo

Altered Ig levels and antibody responses in mice deficient for the Fc receptor for IgM (FcμR)

Cell surface Fc receptor for IgM antibody (FcμR) is the most recently identified member among FcRs. We determined the cellular distribution of mouse FcμR and the functional consequences of Fcmr disruption. Surface FcμR expression was restricted to B-lineage cells, from immature B to plasma cells, except for a transient down-modulation during germinal center reactions. Fcmr ablation had no significant effect on overall B- and T-cell development, but led to a reduction of marginal zone B cells and an increase in splenic B1 B cells. Preimmune serum IgM in mutant mice was significantly elevated as were natural autoantibodies. When immunized with live attenuated pneumococci, mutant mice mounted robust antibody responses against phosphorylcholine, but not protein, determinants compared with wild-type mice. By contrast, upon immunization with a hapten-carrier conjugate, nitrophenyl-coupled chicken γ-globulin (NP-CGG), the mutant mice had a diminished primary IgG1 response to both NP and CGG. These findings suggest that FcμR has an important role in IgM homeostasis and regulation of humoral immune responses.

Cd4+ T-cell receptor transgenic T cells alone can reject vascularized heart transplants through the indirect pathway of alloantigen recognition

The vast array of epitopes presented by allografts and the diversity of T cells responding to them complicates mechanistic studies of rejection. To minimize these problems, we developed a transgenic (Tg) model system limited to a single T-cell receptor (TCR)/peptide/major histocompatibility complex molecule. Two alloantigen-specific CD4 T-cell clones were used to isolate cDNA encoding the TCRalpha and TCRbeta chains that recognize the Kd54-68/I-Ab epitope. Two different TCR Tg lines were produced in C57BL/6 (B6) mice and crossed onto the B6.Rag1-/- background. B6.Rag1-/- recipients of T cells from TCR Tg Rag1-/-mice promptly rejected B10.D2, but not irrelevant B10.BR, cardiac grafts. Thus, a single allogeneic epitope presented by self-major histocompatibility complex class II is sufficient to activate TCR Tg T cells and serve as a target for rejection.

Heterogeneity of T cell clones specific for a single indirect alloantigenic epitope (I-Ab/H-2Kd54-68) that mediate transplant rejection.

Background. One of the complexities of solid organ allograft rejection is the inherent diversity of the specific T cell antigenic epitopes that participate in this response, including the role of direct alloantigen recognition and indirect recognition of donor-derived peptides in recipient antigen-presenting cells.To probe the role of distinct T cell receptor (TCR) avidity differences and the role of cytokine expression patterns of different effector T cells that may participate in allograft rejections, we have identified a dominant allopeptide derived from the H-2Kd molecule, recognized by H-2b CD4 T cells in the context of syngeneic I-Ab. Methods. To identify a stimulatory peptide derived from the H-2Kd molecule, a panel of synthetic overlapping peptides was screened for immunogenicity and a panel of T cell clones established. These clones were characterized for TCR V&bgr; usage by mAb staining and/or reverse transcribed-polymerase chain reaction analysis, peptide dose sensitivity as a marker of TCR avidity, cytokine expression phenotype in vitro, and their ability to mediate rejection of a vascularized cardiac allograft after adoptive transfer to immunodeficient mice. Results. The H-2Kd54–68 peptide was identified as a dominant stimulatory peptide by the ability of T cells from C57BL/6 (H-2b) mice primed by a combination of allogeneic spleen cell injection and mixed peptide immunization to mount an in vitro proliferative response and interferon-&ggr; production by peptide stimulation. Furthermore, direct immunization with synthetic H-2Kd54–68 peptide of normal C57BL/6 mice resulted in accelerated rejection of both skin and cardiac allografts from B10.D2 (H-2d) mice, but not 3rd party B10.BR (H-2k) grafts. A panel of 15 distinct CD4+ clones specific for H-2Kd54–68 peptide were established and shown to utilize a variety of TCR V&bgr; and different apparent TCR avidities to H-2Kd54–68 peptide when stimulated in vitro. To characterize these clones further, two clones were chosen based on the difference of avidity to H-2Kd54–68 peptide. The cytokine expression pattern was determined and indirect alloantigen specificity confirmed by analysis of responses to purified peptide and B10.D2 spleen cells using normal H-2b and I-A&bgr; chain knockout mice as APC donors. Both of these T cell clones were able to mediate rejection of B10.D2, but not B10.BR hearts, in immunodeficient mice, but the morphological pattern of T cell infiltration was distinct. Conclusions. These results demonstrate the potential importance of fine dissection of the alloantigeneic response to solid organ transplants and provide unique insights into the role of TCR avidity and cytokine expression patterns in different morphological patterns of transplant rejection.

Evidence for Cooperativity in the Rejection of Cardiac Grafts Mediated by CD4+ TCR Tg T Cells Specific for a Defined Allopeptide

Understanding the mechanisms of rejection of organs transplanted between unrelated individuals is confounded by the complexity of the alloantigens and the diversity of T cells responding to these alloantigens. To circumvent these problems, we developed a transgenic (Tg) C57BL/6 model system in which the T‐cell receptor (TCR) expressed by CD4 T cells is specific for a defined allogeneic H‐2Kd peptide and the cardiac donor expressed H‐2Kd as a transgene on the C57BL/6 background (B6.Kd). These TCR Tg T cells were previously shown to mediate rapid rejection of a B10.D2 cardiac allograft when transferred to Rag1 recipients, demonstrating that the “indirect” pathway of allorecognition is sufficient for complete rejection in the absence of other T cells or antibody. Here, we report that B6.Kd hearts were rejected in an accelerated fashion by Rag1−/− TCR Tg T cells adoptively transferred to normal B6 recipients. Rejection in this model was associated with large myocardial infarcts and significant coronary artery inflammation. Moreover, transferred TCR Tg CD4+ cells mediated allograft injury without the requirement for cytotoxic function from recipient‐derived CD8 T cells. A non‐linear relationship was observed between the initial precursor frequency of the antigen‐specific TCR Tg cells and the ultimate tempo of acute rejection, which is taken as evidence for cooperativity between components of the system.

Is Toso/IgM Fc receptor (FcμR) expressed by innate immune cells?

Lang et al. have recently published that innate immune cells, including bone-marrow granulocytes and splenic macrophages, express the plasma-membrane protein Toso as a unique regulator of innate immune responses during bacterial infection and septic shock (1). In contrast, we and others have previously reported that the IgM Fc receptor (FcμR), originally designated as Toso or Fas apoptosis inhibitory molecule 3, is expressed only by B-lineage cells in mice and is involved in IgM homeostasis, B-cell survival, and humoral immune responses (2, 3). Because of these conflicting results, we have extensively reexamined the cellular distribution of Toso/FcμR and wish to provide some new results and comments.

Is Toso an antiapoptotic protein or an Fc receptor for IgM

To the editor: We have read with great interest the publication by Nguyen et al entitled “Toso regulates the balance between apoptotic and nonapoptotic death receptor signaling by facilitating RIP1 ubiquitination.”[1][1] Because there are important discrepancies between their results[1][1] and

Emerging Roles for the FCRL Family Members in Lymphocyte Biology and Disease

Members of the extended Fc receptor-like (FCRL) family in humans and mice are preferentially expressed by B cells and possess tyrosine-based immunoregulatory function. Although the majority of these proteins repress B cell receptor-mediated activation, there is an emerging evidence for their bifunctionality and capacity to counter-regulate adaptive and innate signaling pathways. In light of these findings, the recent discovery of ligands for several of these molecules has begun to reveal exciting potential for them in normal lymphocyte biology and is launching a new phase of FCRL investigation. Importantly, these fundamental developments are also setting the stage for defining their altered roles in the pathogenesis of a growing number of immune-mediated diseases. Here we review recent advances in the FCRL field and highlight the significance of these intriguing receptors in normal and perturbed immunobiology.

The Long Elusive IgM Fc Receptor, FcμR

IgM exists as both a monomer on the surface of B cells and a pentamer secreted by plasma cells. Both pre-immune “natural” and antigen-induced “immune” IgM antibodies are important for protective immunity and for immune regulation of autoimmune processes by recognizing pathogens and self-antigens. Effector proteins interacting with the Fc portion of IgM, such as complement and complement receptors, have thus far been proposed but fail to fully account for the IgM-mediated protection and regulation. A major reason for this deficit in our understanding of IgM function seems to be lack of data on a long elusive Fc receptor for IgM (FcμR). We have recently identified a bona fide FcμR in both humans and mice. In this article we briefly review what we have learned so far about FcμR.

Enhanced auto-antibody production and Mott cell formation in FcμR-deficient autoimmune mice

The IgM-Fc receptor (FcμR) is involved in IgM homeostasis as evidenced by increased pre-immune serum IgM and natural auto-antibodies of both IgM and IgG isotypes in Fcmr-deficient C57BL/6 (B6) mice. To determine the impact of Fcmr-ablation on autoimmunity, we introduced the Fcmr null mutation onto the Fas-deficient autoimmune-prone B6.MRL Fas (lpr/lpr) mouse background (B6/lpr). Both IgM and IgG auto-antibodies against dsDNA or chromatin appeared earlier in FcμR(-) B6/lpr than FcμR(+) B6/lpr mice, but this difference became less pronounced with age. Splenic B2 cells, which were 2-fold elevated in FcμR(+) B6/lpr mice, were reduced to normal B6 levels in FcμR(-) B6/lpr mice, whereas splenic B1 cells were comparable in both groups of B6/lpr mice. By contrast, marginal zone (MZ) B cells were markedly reduced in FcμR(-) B6/lpr mice compared with either FcμR(+) B6/lpr or wild type (WT) B6 mice. This reduction appeared to result from rapid differentiation of MZ B cells into plasma cells in the absence of FcμR, as IgM antibody to a Smith (Sm) antigen, to which MZ B cells are known to preferentially respond, was greatly increased in both groups (B6/lpr and B6) of FcμR(-) mice compared with FcμR(+) B6/lpr or B6 mice. Mott cells, aberrant plasma cells with intra-cytoplasmic inclusions, were also increased in the absence of FcμR. Despite these abnormalities, the severity of renal pathology and function and survival were all indistinguishable between FcμR(-) and FcμR(+) B6/lpr mice. Collectively, these findings suggest that FcμR plays important roles in the regulation of auto-antibody production, Mott cell formation and the differentiation of MZ B cells into plasma cells in B6.MRL Fas (lpr/lpr) mice.

Unique Ligand-Binding Property of the Human IgM Fc Receptor

The IgM Fc receptor (FcμR) is the newest FcR, and coligation of FcμR and Fas/CD95 on Jurkat cells with agonistic IgM anti-Fas mAb was shown to inhibit Fas-induced apoptosis. The ligand-binding activity of human FcμR was further examined. FcμR-mediated protection from apoptosis was partially blocked by addition of 104 molar excess of IgM or its soluble immune complexes, but it could be inhibited by addition of 10-fold excess of IgM anti-CD2 mAb. This suggests that FcμR binds more efficiently to the Fc portion of IgM reactive with plasma-membrane proteins than to the Fc portion of IgM in solution. The former interaction occurred in cis on the same cell surface, but not in trans between neighboring cells. This cis engagement of FcμR resulted in modulation of Ca2+ mobilization via CD2 on Jurkat cells or BCRs on blood B cells upon cross-linkage with the corresponding IgM mAbs. Several functional changes were observed with FcμR mutants: 1) significant increase in IgM ligand binding in the cytoplasmic tail-deletion mutant, 2) enhanced cap formation in FcμR upon IgM binding at 4°C with a point mutation of the transmembrane His to Phe, and 3) less protective activity of FcμR in IgM anti-Fas mAb-mediated apoptosis assays with a point mutation of the membrane-proximal Tyr to Phe. These findings show the importance of the cis engagement of FcμR and its critical role in receptor function. Hence, FcμR on B, T, and NK cells may modulate the function of surface proteins recognized by natural or immune IgM Abs on the shared membrane cell surface.