Intrinsic B cell hypo-responsiveness in mice prematurely expressing human CR2/CD21 during B cell development

We previously reported that human CR2 (hCR2) prematurely expressed under a murine Vlambda2 promoter/Vlambda2-4 enhancer minigene during the CD43+ CD25- late pro-B cell stage of development results in peripheral B cells with impaired responses to immunization with T-dependent antigens. Herein, we show that hCR2 transgenic (Tg) mice also demonstrate a severe defect in T-independent antigen responses and are substantially protected from clinical arthritis, synovitis and cartilage/bone destruction in a collagen-induced arthritis model. This outcome is found despite the apparently normal development of autoreactive T cells with equivalent cytokine and proliferative responses to antigen when compared to non-Tg control mice. These data suggest the presence of an intrinsic B cell defect in the hCR2 Tg mice. We also show that an hCR2-dependent Ca2+ influx can be generated in both developing and mature Tg B cells, but with different rates of decay as compared to control wild-type (WT) mice. In addition, although analysis of tyrosine-phosphorylated proteins in WT and Tg B cells following B cell receptor (BCR)-induced activation revealed the presence of distinctly different phosphorylation patterns, no differences were identified in several candidate protein targets. Overall, these data suggest that premature hCR2 expression and the consequences thereof during B cell development intrinsically alters the way mature B cells develop and subsequently respond to antigen through the BCR signaling complex.     

B cell activation leads to shedding of complement receptor type II (CR2/CD21)

Complement receptor type II (CR2/CD21) is the major receptor for C3d fragments on immune complexes. CD21 also serves as the receptor for Epstein-Barr virus in humans. On mature B cells, CD21 reduces the threshold of BCR signaling together with CD81, Leu13 and CD19, but it also occurs on other cells of the immune system where it performs unknown functions. A soluble form of CD21 (sCD21) is shed from the cell surface and is found in human blood plasma. An as-yet-unknown protease is thought to be responsible for this shedding. Altered levels of sCD21 occur in plasma in certain clinical conditions. We show here by mass spectrometry that sCD21 in human plasma of healthy donors is predominantly a short form of CD21 without the exon-11-encoded sequences. Whereas the N terminus of sCD21 was found unmodified, the C terminus is truncated, implying that only the extracellular portion of CD21 is shed. Peripheral blood B cells, but not T cells, contribute to the plasma CD21-pool. CD21 shedding is induced by stimulation with PMA plus Ca(2+) ionophore, or by stimulation of the BCR with anti-IgM+anti-CD40.     

CD21/CR2的研究进展

补体Ⅱ型受体CD21/CR2是B淋巴细胞膜表面C3d/iC3b受体,在免疫应答中起重要的作用。同时人CD21/CR2也是EB病毒膜表面糖蛋白gp350/gp220、HIV-1等的受体,CD21/CR2以不同的结构域与这些配体结合,表现出多种生物学特性。弄清CD21/CR2的结构和功能,可以指导基础免疫研究,也可明确临床许多疾病的发病机理和治疗方向。     

Expression of Epstein-Barr virus nuclear antigen-2 (EBNA2) induces CD21/CR2 on B and T cell lines and shedding of soluble CD21

Stable transfection of Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) expressed as a fusion protein with the hormone-binding domain of the estrogen receptor was used to study expression of CD21 and other surface markers in different cell lines. Special emphasis was placed on cell lines with a normally low expression of CD21, especially on T cell lines. After induction of EBNA2, a substantial increase in CD21 mRNA was observed, as well as increased production of membrane CD21. This was found not only in cell lines of B cell origin, but also in the T cell line Jurkat. The amount of CD21 was quantitated by means of a fluorescence immunoassay, and found to correlate with the presence of EBNA2 protein. A decrease in EBNA2 abundance was associated with complete loss of cell-associated CD21. As we could also detect large amounts of soluble CD21 (sCD21) in the supernatant of the transfected cell lines, which exceeded the total amount contained in the respective cell lysates, this indicates considerable shedding of the newly synthesized receptor molecules induced by EBNA2, comparable to the situation described for CD23. It further provides an explanation of the recent findings of increased sCD21 levels in sera of patients with EBV-associated disease, and suggests a possible additional function of EBNA2 in vivo.     

Defective B cell ontogeny and immune response in human complement receptor 2 (CR2, CD21) transgenic mice is partially recovered in the absence of C3

Mice prematurely expressing human CR2 (hCR2) in the B cell lineage have a defective B cell ontogeny and immune response. Our recent analysis of this phenotype suggested that signaling through hCR2 and presumably mouse CD19 on the B cell surface, during bone marrow development, could result in the observed changes in B cell function in these mice. To test this hypothesis, we back crossed hCR2(high) transgenic mice onto the CD19(-/-) background. CD19(-/-)hCR2(high) mice were found to possess even fewer mature B cells than their CD19(+/+)hCR2(high) littermates, demonstrating that loss of CD19 exacerbated the effects elicited through hCR2. This data suggests that CD19 provides a survival signal during B cell development in this model. Next, we examined if the removal of the main ligand for CR2, namely C3d, through back-crossing onto the C3(-/-) background could restore normal B cell development. However, we found only minor recovery in peripheral B cell numbers and no obvious change in function. This was despite a three-fold increase in the level of hCR2 expression on B cells isolated from the spleen or bone marrow of C3(-/-)hCR2(high) mice when compared with C3 sufficient littermates. These data demonstrate that hCR2 is integrated in mouse B cell signaling and that the downstream effects of hCR2 expression during early B cell development are partially but not completely due to interaction with C3 fragments and signaling through CD19 in the bone marrow environment.     

Origin and properties of soluble CD21 (CR2) in human blood

By analysis with a panel of CD21 MoAbs it is shown that a large part of the soluble CD21 in human blood plasma is of the long isoform (CD21L), as judged by comparison with antigen produced by mouse L cells transfected with CD21L-cDNA and reactivity with the restricted CD21 MoAb R4/23. This is compatible with the hypothesis that soluble CD21 in the blood is mainly derived from follicular dendritic cells (FDC). Cells from a human keratinocyte cell line transfected with cDNA from the Burkitt lymphoma cell line Raji also produced soluble CD21L (sCD21L), whereas the short form of sCD21 (sCD21S) was the major component of sCD21 produced by the B lymphoblastoid cell line LICR-LON-HMy and the T cell line Jurkat. Confocal studies of FDC isolated from human tonsil revealed that CD21 was present in the cytoplasm. On gel filtration sCD21 from untreated serum has an apparent size considerably greater than the 130 kD found by SDS–PAGE analysis. This may be partly accounted for by the non-globular shape of the molecule, but may also indicate, as reported by others, that in its native state sCD21 is complexed with other proteins. However, no evidence of complexing with sCD23 or C3d could be found.     

CD4 and CD8 double‐negative adult T‐cell leukemia/lymphoma with monomorphic cells expressing CD99: A diagnostic challenge in a country non‐endemic for human T‐cell leukemia virus

Adult T‐cell leukemia/lymphoma (ATLL) is a peripheral T‐cell neoplasm caused by human T‐cell lymphotropic virus type I (HTLV‐I). The neoplastic cells are highly pleomorphic and are usually CD4+ and CD8? phenotypically. We reported the case of a 46‐year‐old woman presenting with fever, abdominal distention, lymphadenopathy, leukocytosis and hypercalcemia. Nodal biopsy showed diffuse infiltration by monomorphic small to medium‐sized atypical lymphocytes expressing CD3, CD25, CD30 and CD99, but not CD1a, CD4, CD8, CD34, terminal deoxynucleotidyl transferase or ALK. An initial diagnosis of T‐lymphoblastic leukemia/lymphoma was made based on cytomorphology, CD4 and CD8 double negativity, and the expression of CD99. The diagnosis was later revised to ATLL based on the positive serology study for anti‐HTLV I/II antibody and confirmation by the clonal integration of HTLV‐I proviral DNA into the tumor tissues by Southern blotting analysis. The patient had a stage IVB disease and died of septic shock after 2 courses of chemotherapy 3 months after diagnosis. Immunohistochemical staining for CD99 in archival ATLL tissues showed a positive rate of 67% (4 of 6 tumors). Our case showed that ATLL with atypical morphology and immunophenotype in HTLV non‐endemic areas might pose a diagnostic challenge and CD99 expression is frequent in ATLL.     

Interleukin‐21 (IL‐21) synergizes with IL‐2 to enhance T‐cell receptor‐induced human T‐cell proliferation and counteracts IL‐2/transforming growth factor‐β‐induced regulatory T‐cell development

Interleukin‐2 (IL‐2) is a mainstay for current immunotherapeutic protocols but its usefulness in patients is reduced by severe toxicities and because IL‐2 facilitates regulatory T (Treg) cell development. IL‐21 is a type I cytokine acting as a potent T‐cell co‐mitogen but less efficient than IL‐2 in sustaining T‐cell proliferation. Using various in vitro models for T‐cell receptor (TCR)‐dependent human T‐cell proliferation, we found that IL‐21 synergized with IL‐2 to make CD4⁺ and CD8⁺ T cells attain a level of expansion that was impossible to obtain with IL‐2 alone. Synergy was mostly evident in naive CD4⁺ cells. IL‐2 and tumour‐released transforming growth factor‐β (TGF‐β) are the main environmental cues that cooperate in Treg cell induction in tumour patients. Interleukin‐21 hampered Treg cell expansion induced by IL‐2/TGF‐β combination in naive CD4⁺ cells by facilitating non‐Treg over Treg cell proliferation from the early phases of cell activation. Conversely, IL‐21 did not modulate the conversion of naive activated CD4⁺ cells into Treg cells in the absence of cell division. Treg cell reduction was related to persistent activation of Stat3, a negative regulator of Treg cells associated with down‐modulation of IL‐2/TGF‐β‐induced phosphorylation of Smad2/3, a positive regulator of Treg cells. In contrast to previous studies, IL‐21 was completely ineffective in counteracting the suppressive activity of Treg cells on naive and memory, CD4⁺ and CD8⁺ T cells. Present data provide proof‐of‐concept for evaluating a combinatorial approach that would reduce the IL‐2 needed to sustain T‐cell proliferation efficiently, thereby reducing toxicity and controlling a tolerizing mechanism responsible for the contraction of the T‐cell response.     

The CR2/CD19 complex on human B cells contains the src-family kinase Lyn

The complement receptor 2 (CR2 or CD21) can be found in non-covalentassociation with the Blymphocyte specific CD19 complex at thesurface of mature human B cells. Upon ligation of the B cellantigen receptor complex (BCR), members of the CR2-CD19 complexmay associate with membrane immunoglobulin (mlg). Moreover,CD19 and CD21 ligands, either murine mAb, C3d fragments or Epstein—Barrvirus, are known to have profound effects on B cell activation.We here show that CD19 is tightly linked to the non-receptorsrc kinase Lyn and that the CD19 glycoprotein itself servesas a substrate for a yet undefined serine/threonine kinase presentwithin the complex. In the process of antigen recognition, mlgand the CR2-CD19 complex may bind different sites of a complement-opsonizedantigenic particle. We hypothesize that in this process, approximationto the BCR allows CD19-associated Lyn kinase to phosphorylatepotential substrates within the antigen—receptor complex,thereby effecting its coupling to the intracellular compartment.     

CD21–/low B cells in human blood are memory cells

The complement receptor 2 (CR2, CD21) is part of a complex (CD21/CD19/CD81) acting as a co‐receptor to the B cell receptor (BCR). Simultaneous triggering of the BCR and CD21 lowers the threshold for B cell activation. Although CD21 is important, B cells that express low amounts or lack surface CD21 (CD21^–/low) are increased in conditions with chronic inflammation, e.g. autoimmune diseases. However, little is known about the CD21^–/low B cell subset in peripheral blood from healthy donors. Here, we show that CD21^–/low cells represent approximately 5% of B cells in peripheral blood from adults but are barely detectable in cord blood, after excluding transitional B cells. The CD21^–/low subset can be divided into CD38^–24⁺ and CD38^–24^low cells, where most of the CD38^–24⁺ are CD27⁺immunoglobulin (Ig)M⁺IgD⁺ and the CD38^–24^low are switched CD27^–. Expression levels of additional markers, e.g. CD95 and CD62L, are similar to those on classical memory B cells. In contrast to naive cells, the majority of CD21^–/low cells lack expression of the ABCB1 transporter. Stimulation with a combination of BCR, Toll‐like receptor (TLR)?7/8 and interleukin (IL)?2 induces proliferation and differentiation of the CD21^–/low B cells comparable to CD21⁺CD27⁺ memory B cells. The response excluding BCR agonist is not on par with that of classical memory B cells, although clearly above that of naive B cells. This is ascribed to a weaker response by the CD38^–24^low subset, implying that some memory B cells require not only TLR but also BCR triggering. We conclude that the CD21^–/low cells in healthy donors are memory B cells.     

Mice expressing human CR1/CD35 have an enhanced humoral immune response to T-dependent antigens but fail to correct the effect of premature human CR2 expression

We have previously demonstrated that mice expressing human complement receptor type 2 (CR2/CD21) during the CD43(+)/CD25(-) late pro-B cell stage of B cell development have marked changes in their subsequent B cell ontogeny. Here, we show that the humoral immune response to the T cell dependent antigen, sheep red blood cells (SRBCs) can be moderately enhanced with the addition of human CR1 (driven by the lambda promoter/enhancer transgene) to endogenous mCR1/CR2 expression on the B cell surface but that hCR1 expression alone (on the mouse CR1/2 deficient background) has no effect on the humoral immune response or general B cell development. Furthermore, expression of hCR1 had no recuperative effect on the markedly altered B cell phenotype noted with premature expression of hCR2 (either in the presence or absence of endogenous mCR1/2). We conclude that hCR1 alone cannot replace the role of CR2 in mice and that the effects of premature hCR2 expression during BCR development are not significantly altered by the addition of hCR1 at that developmental stage or beyond; thus hCR2 signaling in the mouse remains dominant over subsequent input from either hCR1 or endogenous receptors.     

Analysis of murine CD22 during B cell development: CD22 is expressed on B cell progenitors prior to IgM

CD22 is a B cell-restricted glycoprotein involved in cell adhesion and signaling. Since CD22 is likely to play an important role in interactions between B cells and other cells, and in regulating signaling thresholds, we characterized the expression of murine CD22 during different stages of B cell development. In contrast to previous reports, we show that CD22 is expressed on B cell progenitors prior to expression of IgM. IL-7-responsive B cell precursors from the fetal liver and early B lineage cells (B220+IgM-) from the bone marrow both express a low density of surface CD22. The majority of the earliest B cell progenitors (B220+IgM-CD43+) in the bone marrow, however, do not express CD22. As B cells mature, the density of CD22 molecules on the cell surface increases. B220brightIgM+ bone marrow cells express high levels of CD22, as do splenic B cells. The correlation of CD22 levels with B cell maturation is replicated in an in vitro culture system, which distinguishes stages of B cell development based on function. Following activation of mature resting splenic B cells with anti-mu mAb or lipopolysaccharide (LPS), levels of CD22 decrease. Finally, we show that the addition of anti-CD22 mAb augments the proliferative response of both anti-mu- and LPS-stimulated B cells, suggesting a role for CD22 in diverse signaling pathways.      

Distribution of the chromatin protein DEK distinguishes active and inactive CD21/CR2 gene in pre- and mature B lymphocytes

DEK is an abundant and ubiquitous chromatin protein that has only recently attracted attention. DEK preferentially binds to cruciform and superhelical DNA and induces positive supercoils into closed circular DNA. It is quite likely therefore that DEK performs an important architectural function in chromatin. However, it is not known how DEK is distributed in chromatin. As the first study of its kind, we investigate the distribution of DEK at the CD21/complement receptor 2 gene regulatory regions in two B lymphocyte lines, namely Ramos, which expresses the CD21 gene, and Nalm-6, which does not. We use a chromatin immunoprecipitation approach and show that DEK appears to be distributed over various regions of the expressed and silent genes, but occurs in 2- to 3-fold higher amounts at a promoter-proximal site of the expressed gene. Moreover, induction of CD21 expression in Nalm-6 cells leads to accumulation of DEK at this site. We propose that the accumulation of DEK is functionally linked to gene expression.     

CD252 regulates mast cell mediated,CD1d‐restricted NKT‐cell activation in mice

The interaction between tissue‐resident mast cells (MCs) and recruited immune cells contributes to tissue immunosurveillance. However, the cells, mechanisms, and receptors involved in this crosstalk remain ill defined. Invariant natural killer T (iNKT) cells are CD1d‐restricted innate lymphocytes that recognize glycolipid antigens and have emerged as critical players in immunity. Here, we show that primary mouse peritoneal MCs express surface CD1d, which is upregulated in vivo following administration of alpha‐galactosylceramide. In contrast, in BM‐derived MCs CD1d was found to be stored intracellularly and to relocate at the cell surface upon IgE‐mediated degranulation. Activated BM‐derived MCs expressing surface CD1d and loaded with alpha‐galactosylceramide were found to induce iNKT‐cell proliferation and the release of IFN‐γ, IL‐13, and IL‐4 in a CD1d‐restricted manner. Moreover, the costimulatory molecules CD48, CD137L, CD252, CD274, and CD275 affected MC‐induced IFN‐γ release and iNKT‐cell proliferation. Interestingly, among the costimulatory molecules, CD48 and CD252 exhibited a distinctly regulatory activity on iNKT‐cell release of both IFN‐γ and IL‐13. In conclusion, we demonstrate that the crosstalk between MCs and iNKT cells may regulate inflammatory immune responses.     

CD4+ T‐cell development in a mouse expressing a transgenic TCR derived from a Treg

CD4⁺Foxp3⁺ Treg maintain peripheral tolerance and influence immune responses to foreign antigens. The thymus is an important source of Treg, but controversy exists as to whether T cells are selected into the Treg lineage based on signals received through TCR specific for self‐peptides. To examine the specificity of TCR expressed by Treg and its effect on CD4⁺ T‐cell development, we generated Treg‐TCR transgenic mice. Deletion of >90% of CD4⁺ T cells in RAG‐sufficient mice, and nearly 100% deletion in RAG^?/? mice expressing this TCR indicate that the TCR is specific for an unknown, naturally expressed peptide in the thymus. Deletion occurs late in development, suggesting this peptide is presented by APC in the thymic medulla. These studies are the first to describe the effects of expressing a Treg‐TCR on CD4⁺ T‐cell development. The implications of our data for models of Treg selection are discussed.     

NKG2D ligation relieves 2B4‐mediated NK‐cell self‐tolerance in mice

Along with MHC class I (MHCI), 2B4 provides nonredundant NK‐cell inhibition in mice. The immunoregulatory role of 2B4 has been increasingly appreciated in models of tumor and viral infection, however, the interactions among 2B4, MHCI, and other activating NK‐cell receptors remain uncertain. Here, we dissect the influence of two distinct inhibitory pathways in modulating NK‐cell‐mediated control of tumors expressing strong activating ligands, including RAE‐1γ. In vitro cytotoxicity and in vivo peritoneal clearance assays using MHCI⁺CD48⁺ (RMA‐neo), MHCI⁺CD48⁺RAE‐1γ (RMA‐RAE‐1γ), MHCI^?CD48⁺ (RMA‐S‐neo), and MHCI^?CD48⁺RAE‐1γ (RMA‐S‐RAE‐1γ) tumor lines demonstrated that NKG2D activation supersedes the inhibitory effect of both 2B4‐ and MHCI‐mediated immune‐tolerance systems. Furthermore, 2B4KO mice subcutaneously challenged with RMA‐neo and RMA‐S‐neo exhibited reduced tumor growth and significantly prolonged survival compared with WT mice, implying that 2B4 is constitutively engaged in the NK‐cell tolerance mechanism in vivo. Nevertheless, the inhibitory effect of 2B4 is significantly attenuated when NK cells encountered highly stressed tumor cells expressing RAE‐1γ, resulting in an immune response shift toward NK‐cell activation and tumor regression. Therefore, our data highlight the importance of the 2B4‐mediated inhibitory system as an alternate self‐tolerance mechanism, whose role can be modulated by the strength of activating receptor signaling within the tumor microenvironment.