Isotype Class Research Articles

Mechanisms of costimulation

The immune system has the remarkable ability to defend against a diversity of microbial pathogens, yet not respond to self. The discovery of the T-cell antigen receptor (TCR) provided an explanation for the specificity and diversity of T-cell responses. Yet, functional studies of Lafferty and colleagues (1) suggested that antigen alone was not sufficient to drive the activation of naive T cells and led to the two signal concept of T-cell activation. According to this model, productive T-cell activation requires a first signal provided by the interaction of antigenic peptide/major histocompatibility complex (MHC) with the TCR and a second, antigen-independent co-signal, the ‘costimulatory signal’. In addition, seminal studies of Jenkins and Schwartz (2, 3) demonstrated that TCR-mediated activation of T cells in the absence of costimulation resulted in antigen-specific unresponsiveness (termed T-cell anergy), rendering the T cells unable to respond to subsequent exposure to antigen. Thus, costimulation was postulated to have a pivotal role in determining whether the outcome of T-cell encounter with antigen would be activation or anergy. The critical role of costimulation in regulating the immune response has given impetus to the study of costimulation and resulted in incredible growth of this field, since an understanding of costimulation is of fundamental and therapeutic interest. Early studies defined the function of the CD28 receptor as a stimulatory receptor for the activation of naive T cells (2), and identified the B7 family members, B7-1 (CD80) and B7-2 (CD86) as its ligands (5–7). The interaction between CD28 and B7-1/B7-2 fulfilled many of the requirements for the costimulatory signal postulated by Lafferty, Schwarz, and colleagues (1). The CD28 homolog cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) (3) was found to be a higher affinity binding partner for B7-1 and B7-2, as compared to CD28 (4). It was presumed that CTLA-4 would also be a stimulatory receptor; however, the dramatic fatal inflammatory phenotype of CTLA-4 deficient (−/−) mice revealed the critical inhibitory function of CTLA-4 (10, 11). Furthermore, the phenotype of CTLA-4−/− mice demonstrated that T-cell costimulatory receptors could deliver inhibitory, as well as stimulatory, second signals for T-cell responses, and provided the first indication that inhibitory second signals (co-inhibitory) could regulate T-cell tolerance. While the B7-1/B7-2:CD28/CTLA-4 pathway is the best characterized T-cell costimulatory pathway, there are now many additional costimulatory pathways. These pathways fall into two major families: the immunoglobulin (Ig) superfamily, which includes the B7/CD28, CD2, and T-cell Ig and mucin domain (TIM) families, and the tumor necrosis family (TNF)/TNF receptor (TNFR) family. Reviews in this issue of Immunol Rev discuss the functions of pathways within all of these families. These articles not only review the current understanding of costimulation on the fundamental level, but also the therapeutic opportunities and challenges of targeting costimulatory pathways for treating autoimmunity, graft rejection, cancer and infectious diseases. In this introduction, I provide perspectives on key functions of costimulatory pathways, highlighting recent advances, and discuss how this progress is changing our view of the functions of T-cell costimulation.

The role of costimulation in antibody deficiencies: ICOS and common variable immunodeficiency

The identification of mutations in the inducible costimulator (ICOS) gene in nine patients with common variable immunodeficiency (CVID) was a major breakthrough. CVID is a complex, highly heterogeneous primary immunodeficiency disease, and the discovery of these mutations revealed a molecular basis. ICOS belongs to the CD28 family of costimulatory molecules and is expressed exclusively on activated T cells. It has at least three critical functions: germinal center formation, isotype class switching, and the development of memory B cells. The discovery of human ICOS deficiency showed that a monogenic disorder could account for the full spectrum of manifestations seen in childhood and adulthood-onset CVID, including autoimmune, inflammatory, and malignant disease complications, as well as recurrent infections. Moreover, this discovery showed that a disorder which had previously been perceived as a B-cell disease might in fact have its genetic origin in human T cells. In this article, we review the role of ICOS in the mammalian immune system and human disease, as well as the discovery and characteristics of patients with ICOS deficiency. Finally, we also discuss how these 'human knockouts' have contributed to our understanding of ICOS functions and have suggested potential avenues for using therapeutic ICOS manipulation to treat other diseases.

Blockade of the CD30/CD30L pathway inhibits renal disease in young, SLE-prone NZB/W F1 mice (50.41)

Abstract CD30 and CD30L are interacting members of the TNFR and TNF family, respectively. CD30 is expressed by subsets of activated T and B cells, whereas CD30L is expressed by subsets of activated T cells, macrophages and dendritic cells. The CD30 / CD30L interaction modulates T cell activation, germinal center formation and antibody isotype class switching. Given the importance of this pathway to B cell function, we tested the hypothesis that blockade of the CD30 / CD30L pathway would decrease or abrogate autoantibody formation and renal disease in the NZB/W F1 murine model of systemic lupus erythematosis (SLE). Young (5 mo) NZB/W F1 mice were treated with 300 μg CD30L blocking (M15-N297A) or blocking / depleting (M15-IgG2a) antibody weekly for 3 months during the onset of autoimmunity. Both treatments a) reduced the onset and severity of proteinuria, b) significantly decreased the severity of renal histologic scores, c) decreased glomerular immune complex and complement deposition and d) these effects were associated with reduced production of anti-DNA antibodies of the IgG, but not IgM isotype. CD30 / CD30L blockade may be a viable approach for the treatment of SLE.

Cytokine-secreting follicular T cells shape the antibody repertoire (34.19)

Abstract High-affinity antibodies are critical for host protection against pathogens and toxins, and underlie successful vaccines. Generation of such antibodies requires T cell-dependent help, which mediates germinal center (GC) reactions where mutation and selection of B cells expressing receptors with high-affinity for antigen occurs. Follicular CD4 T (TFH) cells, which comprise only 5-10% of GC cells, have been imaged in stable conjugates with GC B cells, but no functional analysis of B/TFH cell interactions in GC has been performed. Using an IL-4 reporter system, we show that TFH cells comprise essentially all of the cytokine-secreting T cells in lymph nodes and are functionally distinct from T cells secreting the same cytokine in peripheral tissues. TFH cells with different cytokine profiles could be visualized in the same GC and could be isolated as conjugates with B cells undergoing cytokine-specific immunoglobulin isotype class switching, expressing high levels of AID, and evidence of somatic hypermutation. These findings support a model wherein GC B cells compete for cytokines produced by rare antigen-specific GC TFH cells that mediate highly localized effects and regulate the size, affinity and isotype of the B cell antibody response.

CD30 / CD30L interactions promote class-switched antibody responses to T-dependent antigens (34.3)

Abstract The cell-surface glycoproteins CD30 and CD30L are members of the TNFR and TNF superfamilies, respectively. CD30 is expressed on subpopulations of activated T and B cells. CD30L is expressed on activated T cells, macrophages, and dendritic cells, as well as germinal center B cells. CD30 / CD30L interactions provide activation-induced costimulatory signals that sustain T-cell responses, mediate B-cell activity, and generate long-lived memory T cells for chronic B-cell help. Although other activation-induced costimulatory molecules induce antibody isotype class switching, it is less clear what the function is of CD30 signaling on class switching and antibody production. Therefore, we tested the effect of in vivo blockade of the CD30 / CD30L pathway on antibody class switching in response to T-dependent antigens using two systems that require activated-T cells to stimulate B-cell responses. First, BALB/c mice immunized with a T-dependent antigen and treated with anti-CD30L antibodies produced less antigen-specific antibodies of IgG1 and IgE isotypes as compared to control-treated mice, whereas antigen-specific antibodies of the IgM isotype were similar in all groups. Likewise, NZB/W F1 lupus-prone mice treated with anti-CD30L antibodies produced less anti-dsDNA antibodies of the IgG1 and IgG2a isotypes as compared to control-treated mice, whereas anti-dsDNA antibodies of the IgM isotype were similar in all groups. In both systems, specific subpopulations of lymphocytes necessary for responses to T-dependent antigens were reduced by blockade of the CD30 / CD30L pathway. Our results demonstrate that CD30 / CD30L interactions positively regulate T-cell dependent B-cell responses necessary for class-switched antibody responses.

Tiul1 and TGIF are Involved in Downregulation of TGFβ1-induced IgA Isotype Expression

TGF-β1 is well known to induce Ig germ-line α (GLα) transcription and subsequent IgA isotype class switching recombination (CSR). Homeodomain protein TG-interacting factor (TGIF) and E3-ubiquitin ligases TGIF interacting ubiquitin ligase 1 (Tiul1) are implicated in the negative regulation of TGF-β signaling. In the present study, we investigated the roles of Tiul1 and TGIF in TGFβ1-induced IgA CSR. We found that over-expression of Tiul1 decreased TGFβ1-induced GLα promoter activity and strengthened the inhibitory effect of Smad7 on the promoter activity. Likewise, overexpression of TGIF also diminished GLα promoter activity and further strengthened the inhibitory effect of Tiul1, suggesting that Tiul1 and TGIF can down-regulate TGFβ1-induced GLα expression. In parallel, overexpression of Tiul1 decreased the expression of endogenous IgA CSR-predicitive transcripts (GLTα, PSTα, and CTα) and TGFβ1-induced IgA secretion, but not GLTγ3 and IgG3 secretion. Here, over-expressed TGIF further strengthened the inhibitory effect of Tiul1. These results suggest that Tiul1 and TGIF act as negatively regulators in TGFβ1-induced IgA isotype expression.

Characterization of B cells in synovial fluid and tissue from patients with JIA

Results SF B cells were found to be significantly enriched in CD27+ switch memory (sm) 1 cells and in the recently identified isotype class switch memory (CD19+CD27IgG+IgA+) B cells (sm2) compared to paired and healthy PB (P < 0.0001). CCR5, CCR8, and CCR9 expression was significantly higher on SF sm1 and sm2 B cells than on correspondent paired PB B cells (P < 0.001). Naive (IgD+, CD27-) B cells were significantly reduced in SF compared to paired and control PB (P < 0.0001). Similarly, transitional B cells (CD19+CD24highCD38highIgMhighIgDhigh) were significantly less numerous in SF than in paired PB from JIA patients (P < 0.0001).

Cutting Edge: Langerin+ Dendritic Cells in the Mesenteric Lymph Node Set the Stage for Skin and Gut Immune System Cross-Talk

Topical transcutaneous immunization (TCI) presents many clinical advantages, but its underlying mechanism remains unknown. TCI induced Ag-specific IgA Ab-secreting cells expressing CCR9 and CCR10 in the small intestine in a retinoic acid-dependent manner. These intestinal IgA Abs were maintained in Peyer's patch-null mice but abolished in the Peyer's patch- and lymph node-null mice. The mesenteric lymph node (MLN) was shown to be the site of IgA isotype class switching after TCI. Unexpectedly, langerin(+)CD8alpha(-) dendritic cells emerged in the MLN after TCI; they did not migrate from the skin but rather differentiated rapidly from bone marrow precursors. Depletion of langerin(+) cells impaired intestinal IgA Ab responses after TCI. Taken together, these findings suggest that MLN is indispensable for the induction of intestinal IgA Abs following skin immunization and that cross-talk between the skin and gut immune systems might be mediated by langerin(+) dendritic cells in the MLN.

IgM+ B-Cells Lacking CD27 Expression Display Somatically Mutated Ig VH Genes and Define a New Memory Population.

Somatic hypermutation (SHM) and isotype class switch recombination are critical mechanisms that diversify normal B-cells in response to antigen, and are generally confined to the germinal center (GC). Post-GC B-cells differentiate either into memory B-cells or antibody forming cells maturing as plasma cells. Memory B-cells which exit the GC have been regarded as invariably expressing CD27 and this population comprises both IgM+ and switched B-cells. It is, however, the presence of SHM that remains the defining feature of memory status in B-cells. Recently, it has been reported that dye extruding membrane ATP-binding transporters expressed by naive B-cells can distinguish them from memory B-cells which lack transporters and this, notably, led to the identification of IgG+ memory B-cells that did not express CD27. However, these observations were not correlated with SHM status. Here, we have examined these findings further and focused specifically on IgM+D+ B-cells in relation to our interest in mapping origins of B-cell tumors. Using dye-based transporter assays, we identified a substantial IgM+CD27− population in PBMNC from healthy individuals which lacked transporter activity. These cells were next isolated using a different strategy, employing IgD and CD27 expression, and a highly purified (>99%) IgD+CD27− fraction obtained by FACS sorting. There was virtually nil contamination by CD27+ B-cells. IgM transcripts were then specifically analyzed in this CD27− population for VH gene mutations by using Cmu downstream primers. We examined VH3 and VH5 gene transcripts from 2 separate sorts from a healthy donor following amplification by primary and nested RT-PCR and cloning products for sequence analysis. Of 104 VH3 clones from this CD27− fraction, 38 (37%) were germline and the remainder mutated (55% with 98–99.9% homology to germline, 9% <97.9%). Of these, 27% displayed 3–5 or more nucleotide mutations. Analysis of 124 VH5 clones from the same fraction showed 50% to be germline and the remainder mutated (45% with 98–99.9% homology, 5% <97.9%), of which 16% exhibited 3–5 or more mutations. Only 1 VH5 donor germline gene exists in the IgH repertoire, but 4 allelic variants which differ by 1 nucleotide are known. We mapped the VH5 germline gene in our donor using recombination sequence primers and only 1 allele was identified, identical to the donor gene mapped by database alignment, eliminating polymorphisms as a source of apparent base changes. Mutational load was also over and above a low level of PCR error which was quantified in the analysis. This confirmed that the levels of mutation detected in the rearranged VH5 transcripts in the IgM+D+CD27− population were generated by SHM. In contrast, between 74–90% of VH3 and VH5 transcripts from the IgM+D−CD27+ population were mutated, with a higher frequency of sequences (65–87%) displaying 3–5 or more mutations. Our data reveal the existence of a novel IgM+D+ population of normal B-cells which are somatically mutated but lack CD27. Unmutated IgM+D+ B-cells in this population point to heterogeneity in the CD27− B-cell pool. As yet, the clonal derivation of these mutated CD27− cells is undefined. Importantly, they have relevance for understanding the cell of origin in Hairy Cell leukaemia and Waldenstrom's macroglobulinemia, where tumor cells are mutated yet lack CD27 expression.

Molecular Mechanisms of Antibody Somatic Hypermutation

Functional antibody genes are assembled by V-D-J joining and then diversified by somatic hypermutation. This hypermutation results from stepwise incorporation of single nucleotide substitutions into the V gene, underpinning much of antibody diversity and affinity maturation. Hypermutation is triggered by activation-induced deaminase (AID), an enzyme which catalyzes targeted deamination of deoxycytidine residues in DNA. The pathways used for processing the AID-generated U:G lesions determine the variety of base substitutions observed during somatic hypermutation. Thus, DNA replication across the uracil yields transition mutations at C:G pairs, whereas uracil excision by UNG uracil-DNA glycosylase creates abasic sites that can also yield transversions. Recognition of the U:G mismatch by MSH2/MSH6 triggers a mutagenic patch repair in which polymerase eta plays a major role and leads to mutations at A:T pairs. AID-triggered DNA deamination also underpins immunoglobulin variable (IgV) gene conversion, isotype class switching, and some oncogenic translocations in B cell tumors.

The germinal center response is impaired in the absence of T cell‐expressed CXCR5

Germinal centers support the differentiation of memory B cells and long-lived antibody-secreting cells during infection or upon vaccination. Here, we constructed mice with T cells that selectively lack the chemokine receptor CXCR5 to determine if expression of this receptor by T cells is mandatory for germinal center formation and function. In these animals, germinal centers that are properly localized in B cell follicles and contain T cells do form after immunization with a thymus-dependent antigen. However, fewer and smaller germinal centers form, resulting in a significant reduction in the frequency of germinal center B cells. The defect in germinal center formation is paralleled by decreased frequencies of isotype-switched antibody-secreting cells in the spleen and bone marrow and reduced serum concentrations of total and high-affinity hapten-specific IgG1. The results demonstrate that although CXCR5-dependent T cell positioning is important for maximal induction and expansion of germinal centers, stimulation of isotype class switching, and development of antibody-secreting cells that seed the spleen and bone marrow, it is not absolutely required for the formation and function of follicular germinal centers.

A Waldenström's Macroglobulinemia Case with Functional Class Switch Recombination.

Waldenström Macroglobulinemia (WM) is characterized by monoclonal IgM paraprotein and bone marrow (BM) infiltration by lymphoplasmacytic lymphoma. The normal counterpart of WM malignant cell seems to be a post germinal centre IgM B-cell, which tumoral transformation occurs after cessation of somatic mutation (SM) but prior to Class switch recombination (CSR). However, recently has been reported that CSR can be possible “ex-vivo”, since clonotypic transcripts encoding post-switch isotypes have been observed in some WM cells cultured with CD40L/IL-40. However, this process has not been shown to occur “in vivo” until now.#3754, a 51-year-old woman, was diagnosed in 2001 of WM with a M-IgM spike (46 g/L), anemia (Hb 9·6 g/dL), 76% lymphoplasmacytic monoclonal B-cells in BM and normal cytogenetics. In April 2005, an important IgG increase was observed (23 g/L). Immunofixation demonstrated an IgG-k paraprotein in the mid g-region and a monoclonal IgM-k paraprotein at the b-region corresponding to the two monoclonal peaks detected on serum electrophoresis. After 6-mercaptoethanol treatment, a single band was seen at the line stained with kappa, suggesting the presence of a single clone. Other causes of IgG monoclonal components were excluded considering clinical factors, immunophenotype analyses (San Miguel et al, 2003), quantity of DNA and cell cycle analyses (Ocio et al, 2005). However, the definitive proof for a unique monoclonal population was provided through molecular analysis. A single clonotypic rearrangement was detected by amplifying the complete VDJH fragment at diagnosis moment, according to the protocol describes in Biomed II (Leukemia 2003; 17.2257–2317). Method describes from Billadeu et al (Billadeau et al, 1993) was used for isotype identification. So, cDNA monoclonal amplification was observed at tubes corresponding to Cm, Cd and Cg. All monoclonal PCR products were directly sequenced in an automated ABI 377 DNA sequencer. VH, DH & JH segments identification, as well as SH recognition was made using the V-BASE sequence directory alignment program, and the CH regions were compared at BLAST. All sequences obtained showed the same clonotypic CDR3 sequence (VH4-59/JH6) as well as the same SH (10,75%) pattern that monoclonal amplification at diagnosis, indicating the presence of the same clone at that moment (Figure 1).In conclusion, we report for the first time a WM case in which tumor cells were able to carry out CSR, showing IgG and IgM clonotypic amplification, as well as producing both paraprotein components. This constitutes the first in vivo demonstration that CSR is possible in WM cells, and are able to develop a fully functional isotype class switch recombination not only in vitro but also in vivo. [Display omitted]

Protein Mapping and Characterization of Post-Translational Modifications of Ku86 Protein Variants in Multiple Myeloma Cell Lines.

A single biological event, immunoglobulin heavy chain (IgH) isotype class switch recombination (CSR) is thought to mark the onset of multiple myeloma (MM). The DNA repair enzyme, DNA-dependent protein kinase (DNA-PK), which consists of a heterodimeric regulatory subunit (Ku70/Ku86) and its catalytic subunit (DNA-PKcs), is the principal mediator of IgH isotype CSR. Studies in Ku70 and Ku86 knockout mice have suggested that Ku proteins could have a role to play in carcinogenesis. Since we have previously detected truncated variants of Ku86 (Ku86v) in 86% to 100% of freshly isolated patient MM cells, we therefore hypothesize that Ku86v could be a putative oncoprotein. Two variants of Ku86 are predominantly found in MM cells: a 69 kDa variant truncated at the C-terminus (Ku86v-N), which has lost its DNA repair function; and a 56 kDa N-terminus truncated variant (Ku86v-C), which is not translocated into the nucleus and aberrantly expressed on the cell membrane. Our prior studies using Northern blotting, whole cell polyacrylamide gel electrophoresis (PAGE) and electrophoretic mobility shift assays (EMSA) have demonstrated that Ku86vs in MM cells are not the result of alternative RNA splicing; or in vitro degradation, as has been described in human lymphocytes. Moreover, since many proteins in MM cells are known to be heavily glycosylated, and deglycosylation could lead to shorter forms of the protein, we now show using Endo H digestion that Ku86vs are also not the result of N-linked deglycosylation. Rather, Ku86vs are formed by in vivo proteolytic cleavage via a trypsin-like serine protease. Since the proteasome degradation pathway is highly active in MM cells; and protease digestion of DNA-PK holoenzyme is enhanced by proteasome inhibition (i.e. bortezomib treatment), these data support the notion that in vivo activation of putative Ku86v oncoproteins within MM cells could arise via a constitutively active post-translational proteolytic process. To define a functional role for Ku86v, we first demonstrate that Ku86v is physically associated with the most abundant anti-apoptotic protein in MM cells, BCL2. We then used cyanogen bromide immunoprecipitation to purify BCL2-bound Ku86vs followed by 2-dimensional gel electrophoresis (2DGE) to resolve these purified proteins, and demonstrate that only the 56 kDa Ku86v-C (pI 5.0) bound to BCL2. Both full length Ku86 and Ku86v-N were not detected. We then isolated Ku86v-C from the 2D gel and preliminary mass spectrometric analyses suggest that the N-terminus truncation retains the Ku autoantigen related protein 1 (KARP1) motif of Ku86. The significance of this is still under investigation. In conclusion, we have found that Ku86v-C (56 kDa pI 5.0) is formed by post-translational proteolytic cleavage. Since, Ku86v-C is significantly associated with BCL2 oncoprotein, we therefore speculate that Ku86v-C could possibly potentiate BCL2's anti-apoptotic function. Accordingly, Ku86v-C could be considered as a potential target for therapeutic intervention in MM.

Immunoglobulins—Basic considerations

Immunoglobulins (Igs) or antibodies (Abs) are the principal operators of the adaptive humoral immune response. For optimum functional activity they acquire an optimized structure for antigen (Ag) recognition, precipitation, agglutination, phagocytosis (IgG1/3 and IgA), cytotoxicity (IgG1/3), transport through mucosa (IgA and IgM) and placenta (IgG1/3), complement activation (IgG1/3 and IgM) and release of inflammatory mediators (IgE). A diversity with potentially up to 10(15) different Ab specificities is generated during Ag-independent B cell development in the bone marrow by combinatorial V-D-J joining, creation of junctional diversity, and combinatorial association of L and H chains. Furthermore,Ab variety is created during Ag-dependent B cell maturation in peripheral lymphatic tissues by isotype class switching and somatic hypermutation. Two types of enzymes play a key role in Ab diverseness, i. e., the products of recombination-activating genes RAG1 and RAG2 and the affinity induced deaminase (AID). The prevailing adult-type B2 cells provide the basis for the acquired humoral immune response characterized by Ab production,Ag processing and presentation, immunological memory and tolerance along with the generation of the anti-idiotype network,whereas the fetal-type B1 cells may play a role in innate immunity and autoimmunity. Impairment of B cell immunity includes immunodeficiency (agammaglobulinemia), malignant transformation (leukemia, lymphoma, plasmocytoma) and immune dysregulation (allergy, autoimmunity). The diagnostic relevance of Abs comprises classical serology (immunoprecipitation, agglutination, complement binding, RIA, ELISA), immunocytochemistry and immunohistochemistry, immunofluorescence (microscopic and flow cytometric), cytotoxicity tests, immunoblots, immunospot assays and immunoabsorption (affinity chromatography). Therapeutic application of Abs (passive immunization) is directed against infections, intoxications, solid tumors, leukemias and lymphomas, graft rejection and graft-versus-host reaction, hemolytic anemia, and autoimmune diseases. The generation of genetically engineered monoclonal Abs (mAbs) has revolutionized the diagnostic and therapeutic potential of Abs in almost all disciplines of modern medicine.

Enhancement of the Immunogenicity of Synthetic Carbohydrates by Conjugation to Virosomes: A Leishmaniasis Vaccine Candidate

Novel virosomal formulations of a synthetic oligosaccharide were prepared and evaluated as vaccine candidates against leishmaniasis. A lipophosphoglycan-related synthetic tetrasaccharide antigen was conjugated to a phospholipid and to the influenza virus coat protein hemagglutinin. These glycan conjugates were embedded into the lipid membrane of reconstituted influenza virus virosomes. The virosomal formulations elicited both IgM and IgG anti-glycan antibodies in mice, indicating an antibody isotype class switch to IgG. The antisera cross-reacted in vitro with the corresponding natural carbohydrate antigens expressed by leishmania cells. These findings support the concept of using virosomes as universal antigen delivery platform for synthetic carbohydrate vaccines.

T-bet Binding to Newly Identified Target Gene Promoters Is Cell Type-independent but Results in Variable Context-dependent Functional Effects

Recently developed target gene identification strategies based upon the chromatin immunoprecipitation assay provide a powerful method to determine the localization of transcription factor binding within mammalian genomes. However, in many cases, it is unclear if the binding capacity of a transcription factor correlates with an obligate role in gene regulation in diverse contexts. It is therefore important to carefully examine the relationship between transcription factor binding and its ability to functionally regulate gene expression. T-bet is a T-box transcription factor expressed in several hematopoietic cell types. By utilizing a chromatin immunoprecipitation assay coupled to genomic microarray technology approach, we identified numerous promoters, including CXCR3, IL2Rbeta, and CCL3, that are bound by T-bet in B cells. Most surprisingly, the ability of T-bet to associate with the target promoters is not dependent upon the cell type background. Several of the promoters appear to be functionally regulated by T-bet. However, we could not detect a functional consequence for T-bet association with many of the identified promoters in overexpression studies or an examination of wild type and T-bet-/- primary B, CD4+, and CD8+ T cells. Thus, there is a high variability in the functional consequences, if any, that result from the association of T-bet with individual target promoters.

Impaired class switch recombination (CSR) in Waldenström macroglobulinemia (WM) despite apparently normal CSR machinery

AbstractAnalysis of clonotypic isotype class switching (CSR) in Waldenström macroglobulinemia (WM) and IgM monoclonal gammopathy of undetermined significance (MGUS) reveals a normal initial phase of B-cell activation as determined by constitutive and inducible expression of activation-induced cytidine deaminase (AID). Switch μ (Sμ) analysis shows that large deletions are not common in WM or IgM MGUS. In CD40L/IL-4-stimulated WM cultures from 2 patients, we observed clonotypic IgG exhibiting intraclonal homogeneity associated with multiple hybrid Sμ/Sγ junctions. This suggests CSR had occurred within WM cells. Nevertheless, the estimated IgG/IgM-cell frequency was relatively low (1/1600 cells). Thus, for the majority of WM B cells, CSR does not occur even when stimulated in vitro, suggesting that the WM cell is constitutively unable to or being prevented from carrying out CSR. In contrast to WM, the majority of IgM MGUS clones exhibit intraclonal heterogeneity of IgH VDJ. Furthermore, most IgM MGUS accumulate more mutations in the upstream Sμ region than do WM, making them unlikely WM progenitors. These observations suggest that switch sequence analysis may identify the subset of patients with IgM MGUS who are at risk of progression to WM.

Decoupling of normal CD40/interleukin-4 immunoglobulin heavy chain switch signal leads to genomic instability in SGH-MM5 and RPMI 8226 multiple myeloma cell lines

The processes mediating genomic instability and clonal evolution are obscure in multiple myeloma (MM). Acquisition of new chromosomal translocations into the switch region of the immunoglobulin heavy chain (IgH) gene (chromosome 14q32) in MM, often heralds transformation to more aggressive disease. Since the combined effects of CD40 plus interleukin-4 (IL-4) mediate IgH isotype class switch recombination (CSR), and this process involves DNA double strand break repair (DSBR), we hypothesized that CD40 and/or IL-4 activation of MM cells could induce abnormal DNA DSBR and lead to genomic instability and clonal evolution. In this study, we show that MM cell lines that are optimally triggered via CD40 and/or IL-4 demonstrate abnormal decoupling of IL-4 signal transduction from CD40. Specifically, CD40 alone was sufficient to trigger maximal growth of tumor cells. We further demonstrate that CD40 triggering induced both DNA DSBs as well as newly acquired karyotypic abnormalities in MM cell lines. Importantly, these observations were accompanied by induction of activation induced cytidine deaminase expression, but not gross apoptosis. These data support the role of abnormal CD40 signal transduction in mediating genomic instability, suggesting a role for the CD40 pathway and intermediates in myelomagenesis and clonal evolution in vivo.

PKA-mediated phosphorylation regulates the function of activation-induced deaminase (AID) in B cells

During humoral immune responses, two distinct genetic modification events diversify the Ig genes in germinal center (GC) B cells: somatic hypermutation and class switch recombination (CSR). Both processes require the activity of activation-induced cytidine deaminase (AID), an enzyme expressed specifically in GC B cells. However, the mechanisms that regulate AID activity are largely unknown. Here we report that protein kinase A (PKA) phosphorylates AID and regulates its activity in GC B cells. AID physically interacts with the PKA holoenzyme in the cytoplasm and is phosphorylated by the PKA catalytic subunit at specific residues. AID phosphorylation is required for CSR, because substitution of the two phosphorylation targets impairs its ability to rescue CSR in AID-deficient B cells. Pharmacologic inhibition of PKA prevents isotype class switching in a murine B-cell lymphoma cell line; conversely, B cells from mice where PKA activity is made constitutive by conditional deletion of the PKA regulatory subunit gene display enhanced CSR. These findings implicate PKA in the regulation of AID function and suggest that the control of T cell-dependent immune responses may be modulated, via AID, by signals that activate PKA.

Toxoplasma gondii-Specific Immunoglobulin M Limits Parasite Dissemination by Preventing Host Cell Invasion

An important role for immunoglobulin M (IgM) during early acute virulent Toxoplasma gondii infection was identified using IgM-/- mice that lack surface and secretory IgM but maintain normal B-cell functionality and isotype class switching. Following intraperitoneal inoculation with the virulent RH strain, IgM-/- mice displayed significantly fewer peritoneal parasites than wild-type (WT) mice, which correlated with increased tachyzoite dissemination to the liver, lung, and spleen in IgM-/- mice compared with WT mice. Early splenic T-cell activation, as measured by CD69 expression, was augmented in IgM-/- mice, and serum and peritoneal cavity gamma interferon levels were also elevated in IgM-/- mice compared with WT controls. Consequently, the difference in parasite dissemination was not attributable to an impaired proinflammatory immune response in the IgM-/- mice. Specific IgM was found to bind to tachyzoites in vivo in WT mice, and this correlated with an increased ability of antiserum collected from WT mice at day 6 postinfection to block tachyzoite cell invasion, compared with comparable serum collected from IgM-/- mice at the same time point. Tachyzoite invasion of host cells was similar if parasites were incubated with WT or IgM-/- nonimmune serum, suggesting that natural IgM does not function to limit parasite dissemination during early T. gondii infection. Our results highlight an important role for parasite-specific IgM in limiting systemic dissemination of tachyzoites during early acute T. gondii infection.