Henry H. Wortis

Scanning electron microscopy of human T-cell and B-cell rosettes.

Abstract The surface of human lymphocytes was studied by scanning electron microscopy. Lymphocytes from peripheral blood or tonsils were identified as thymus-derived (T) cells or thymus-independent (B) cells by virtue of the ability of T cells to form rosettes with sheep red cells and for some B cells to form rosettes with complement-coated human red blood cells. The rosettes were gluteraldehyde fixed and subsequently examined by scanning electron microscopy. Lymphocytes, both rosetting and non-rosetting, had multiple surface microvilli. As compared to rosetting B cells, rosetting T cells were generally smaller and smoother, with fewer and shorter microvilli. Microvilli appeared to be the sole cell-cell contact point between T cells and sheep red blood cells; B cells made contact through both villous and non-villous areas. Microvilli are an important mode of primary contact between lymphocytes and the outside world. (N Engl J Med 289:548–551, 1973)

Regulation of Nuclear Localization and Transcriptional Activity of TFII-I by Bruton’s Tyrosine Kinase

ABSTRACT Bruton’s tyrosine kinase (Btk) is required for normal B-cell development, as defects in Btk lead to X-linked immunodeficiency (xid) in mice and X-linked agammaglobulinemia (XLA) in humans. Here we demonstrate a functional interaction between the multifunctional transcription factor TFII-I and Btk. Ectopic expression of wild-type Btk enhances TFII-I-mediated transcriptional activation and its tyrosine phosphorylation in transient-transfection assays. Mutation of Btk in either the PH domain (R28C, as in the murine xid mutation) or the kinase domain (K430E) compromises its ability to enhance both the tyrosine phosphorylation and the transcriptional activity of TFII-I. TFII-I associates constitutively in vivo with wild-type Btk and kinase-inactive Btk but not xid Btk. However, membrane immunoglobulin M cross-linking in B cells leads to dissociation of TFII-I from Btk. We further show that while TFII-I is found in both the nucleus and cytoplasm of wild-type and xid primary resting B cells, nuclear TFII-I is greater in xid B cells. Most strikingly, receptor cross-linking of wild-type (but not xid) B cells results in increased nuclear import of TFII-I. Taken together, these data suggest that although the PH domain of Btk is primarily responsible for its physical interaction with TFII-I, an intact kinase domain of Btk is required to enhance transcriptional activity of TFII-I in the nucleus. Thus, mutations impairing the physical and/or functional association between TFII-I and Btk may result in diminished TFII-I-dependent transcription and contribute to defective B-cell development and/or function.

Sialic Acid Binding Domains of CD22 Are Required For Negative Regulation of B Cell Receptor Signaling

CD22, a negative regulator of B cell antigen receptor signaling, binds glycoconjugates terminating in α2, 6 sialic acid. The physiological ligand(s) for CD22 remain unknown. We asked whether the sialic acid binding domains are necessary for CD22 to function as a negative regulator. We generated two mutants that lack sialic acid binding activity and expressed them in a novel CD22−/− murine B cell line. Anti-IgM activated B cells expressing either CD22 mutant had greater Ca2+ responses than cells expressing wild-type CD22. Each variant also had reduced CD22 tyrosine phosphorylation and Src homology 2 domain–containing protein tyrosine phosphatase-1 association. These data suggest that the α2, 6 sialic acid ligand binding activity of CD22 is critical for its negative regulatory functions.

CD22 attenuates calcium signaling by potentiating plasma membrane calcium-ATPase activity

Binding of antigen to the B cell receptor induces a calcium response, which is required for proliferation and antibody production. CD22, a B cell surface protein, inhibits this signal through mechanisms that have been obscure. We report here that CD22 augments calcium efflux after B cell receptor crosslinking. Inhibition of plasma membrane calcium-ATPase (PMCA) attenuated these effects, as did disruption by homologous recombination of the gene encoding PMCA4a and PMCA4b. PMCA coimmunoprecipitated with CD22 in an activation-dependent way. CD22 cytoplasmic tyrosine residues were required for association with PMCA and enhancement of calcium efflux. Moreover, CD22 regulation of efflux and the calcium response required the tyrosine phosphatase SHP-1. Thus, SHP-1 and PMCA provide a mechanism by which CD22, a tissue-specific negative regulator, can affect calcium responses.

Cutting Edge Commentary: Origins of B-1 Cells

The origin of B-1a cells, a minority population of B cells that express CD5, are abundant in coelomic cavities, and often produce autoantibodies, has been the subject of study for many years. Accumulating evidence demonstrates that the hypothesis that only B cells arising in fetal or neonatal tissues have the potential to become B-1a cells cannot be true. Rather, B cell receptor-mediated signaling initiated by ligation of autoantigen has now been shown to be required for induction of the B-1a phenotype. Furthermore, cells with a functional B-1a phenotype can be induced from adult precursors by appropriate Ag. At the same time, microenvironment-specific events may determine the likelihood that a given B cell, either adult or fetal derived, enters this pathway. CD5 expression and possibly localization to the peritoneum appear to provide some protection to autoreactive cells otherwise slated for elimination.

The ability of CD40L, but not lipopolysaccharide, to initiate immunoglobulin switching to immunoglobulin G1 is explained by differential induction of NF-kappaB/Rel proteins.

ABSTRACT Antibodies of the immunoglobulin G1 class are induced in mice by T-cell-dependent antigens but not by lipopolysaccharide (LPS). CD40 engagement contributes to this preferential isotype production by activating NF-κB/Rel to induce germ line γ1 transcripts, which are essential for class switch recombination. Although LPS also activates NF-κB, it poorly induces germ line γ1 transcripts. Western blot analyses show that CD40 ligand (CD40L) induces all NF-κB/Rel proteins, whereas LPS activates predominantly p50 and c-Rel. Electrophoretic mobility shift assays show that in CD40L-treated cells, p50-RelA and p50-RelB dimers are the major NF-κB complexes binding to the germ line γ1 promoter, whereas in LPS-treated cells, p50–c-Rel and p50-p50 dimers are the major binding complexes. Transfection of expression plasmids for NF-κB/Rel fusion proteins (forced dimers) indicates that p50-RelA and p50-RelB dimers activate the germ line γ1 promoter and that p50–c-Rel and p50-p50 dimers inhibit this activation by competitively binding to the promoter without activating the promoter. Therefore, germ line γ1 transcription depends on the composition of NF-κB/Rel proteins.

Normal B-1a cell development requires B cell-intrinsic NFATc1 activity

B-1a cells, an anatomically, phenotypically, and functionally distinct subset of B cells that produce the bulk of natural serum IgM and much of gut-associated IgA, are an important component of the early response to pathogens. Because the induced expression of CD5, a hallmark of B-1a cells, requires a nuclear factor of activated T cells (NFAT)-dependent enhancer, we examined the role of NFAT transcription factors in B-1a development. Here we show that the B-1a compartment is normal in mice lacking NFATc2 but essentially absent in mice lacking NFATc1. Loss of NFATc1 affects both peritoneal and splenic B-1a cells. Because there is a loss of B-1 cells defined by markers other than CD5, NFATc1 is not required simply for CD5 expression on B-1a cells. Using mixed-allotype chimeras and retroviral-mediated gene transduction we show that the requirement for NFATc1 is B cell-intrinsic. We also demonstrate that NFATc1 protein expression is elevated ≈5-fold in B-1a cells compared with B-2 cells. This is the first definitive demonstration of a B cell-intrinsic function for an NFAT family transcription factor.

Immunological Studies of Nude Mice

Nude mice pose several interesting biological questions. On the one hand they present a variety of pleiotropic effects, ranging from hairlessness to thymic dysgenesis, which have an unknown common property. On the other hand they may very well be a model for the study of immune reactions in which a single major participant—the thymus—is absent.

Two New Isotype-Specific Switching Activities Detected for Ig Class Switching

Ig class switch recombination (CSR) occurs by an intrachromosomal deletional process between switch (S) regions in B cells. To facilitate the study of CSR, we derived a new B cell line, 1.B4.B6, which is uniquely capable of μ→γ3, μ→ε, and μ→α, but not μ→γ1 CSR at its endogenous loci. The 1.B4.B6 cell line was used in combination with plasmid-based isotype-specific S substrates in transient transfection assays to test for the presence of trans-acting switching activities. The 1.B4.B6 cell line supports μ→γ3, but not μ→γ1 recombination, on S substrates. In contrast, normal splenic B cells activated with LPS and IL-4 are capable of plasmid-based μ→γ1 CSR and demonstrate that this S plasmid is active. Activation-induced deaminase (AID) was used as a marker to identify existing B cell lines as possible candidates for supporting CSR. The M12 and A20 cell lines were identified as AID positive and, following activation with CD40L and other activators, were found to differentially support μ→ε and μ→α plasmid-based CSR. These studies provide evidence for two new switching activities for μ→γ1 and μ→ε CSR, which are distinct from μ→γ3 and μ→α switching activities previously described. AID is expressed in all the B cell lines capable of CSR, but cannot account for the isotype specificity defined by the S plasmid assay. These results are consistent with a model in which isotype-specific switching factors are either isotype-specific recombinases or DNA binding proteins with sequence specificity for S DNA.

Surface Markers, Heavy Chain Sequences and B Cell Lineages

A unifying theory of B cell development and lineage commitment is presented. There are two firmly established B lineages: cells which normally arise only from fetal sources and lack N insertions in their rearranged heavy chains; and N-containing cells which arise from adult bone marrow precursors (and perhaps from late fetal sources). Commitment to the expression of CD5 and the capacity for long-life (or self-renewal) are induced as a consequence of sIg cross-linking, typically by a repeating epitope, thymus independent type two antigen. Alternatively, activation resulting from cognate interaction with a helper T cell does not induce CD5 but results in lower expression of J11d. In this case activation occurs in the absence of sIg cross-linking. It is further proposed that differences in the Ig repertoire make it highly likely that fetal/neonatal, but not adult derived B cells will be induced to express CD5. The model offers a plausible explanation for the correlation of CD5 expression and natural autoantibody production by neonatal B cells. Possible sources of pathogenic autoantibody are discussed in the context of this model.