Wasif N. Khan

Defective B cell development and function in Btk-deficient mice

Mutations in the Brutons tyrosine kinase (Btk) gene have been linked to severe early B cell developmental blocks in human X-linked agammaglobulinemia (XLA), and to milder B cell activation deficiencies in murine X-linked immune deficiency (Xid). To elucidate unequivocally potential Btk functions in mice, we generated mutations in embryonic stem cells, which eliminated the ability to encode Btk pleckstrin homology or kinase domains, and assayed their effects by RAG2-deficient blastocyst complementation or introduction into the germline. Both mutations block expression of Btk protein and lead to reduced numbers of mature conventional B cells, severe B1 cell deficiency, serum IgM and IgG3 deficiency, and defective responses in vitro to various B cell activators and in vivo to immunization with thymus-independent type II antigens. These results prove that lack of Btk function results in an Xid phenotype and further suggest a differential requirement for Btk during the early stages of murine versus human B lymphocyte development.

Wiskott-Aldrich Syndrome Protein-Deficient Mice Reveal a Role for WASP in T but Not B Cell Activation

The Wiskott-Aldrich syndrome (WAS) is a human X-linked immunodeficiency resulting from mutations in a gene (WASP) encoding a cytoplasmic protein implicated in regulating the actin cytoskeleton. To elucidate WASP function, we disrupted the WASP gene in mice by gene-targeted mutation. WASP-deficient mice showed apparently normal lymphocyte development, normal serum immunoglobulin levels, and the capacity to respond to both T-dependent and T-independent type II antigens. However, these mice did have decreased peripheral blood lymphocyte and platelet numbers and developed chronic colitis. Moreover, purified WASP-deficient T cells showed markedly impaired proliferation and antigen receptor cap formation in response to anti-CD3epsilon stimulation. Yet, purified WASP-deficient B cells showed normal responses to anti-Ig stimulation. We discuss the implications of our findings regarding WASP function in receptor signaling and cytoskeletal reorganization in T and B cells and compare the effects of WASP deficiency in mice and humans.

B Cell Receptor and BAFF Receptor Signaling Regulation of B Cell Homeostasis

B lymphocyte homeostasis depends on tonic and induced BCR signaling and receptors sensitive to trophic factors, such as B cell-activating factor receptor (BAFF-R or BR3) during development and maintenance. This review will discuss growing evidence suggesting that the signaling mechanisms that maintain B cell survival and metabolic fitness during selection at transitional stages and survival after maturation rely on cross-talk between BCR and BR3 signaling. Recent findings have also begun to unravel the molecular mechanisms underlying this crosstalk. In this review I also propose a model for regulating the amplitude of BCR signaling by a signal amplification loop downstream of the BCR involving Btk and NF-κB that may facilitate BCR-dependent B cell survival as well as its functional coupling to BR3 for the growth and survival of B lymphocytes.

TLR Stimulation Modifies BLyS Receptor Expression in Follicular and Marginal Zone B Cells

Through their differential interactions with B lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL), the three BLyS family receptors play central roles in B cell survival and differentiation. Recent evidence indicates BLyS receptor levels shift following BCR ligation, suggesting that activation cues can alter overall BLyS receptor profiles and thus ligand sensitivity. In this study, we show that TLR stimuli also alter BLyS receptor expression, but in contrast to BCR ligation, TLR9 and TLR4 signals, preferentially increase transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI) expression. Although both of these TLRs act through MyD88-dependent mechanisms to increase TACI expression, they differ in terms of their downstream mediators and the B cell subset affected. Surprisingly, only TLR4 relies on c-Rel and p50 to augment TACI expression, whereas TLR9 does not. Furthermore, although all follicular and marginal zone B cells up-regulate TACI in response to TLR9 stimulation, only marginal zone B cells and a subset of follicular B cells respond to TLR4. Finally, we find that both BLyS and APRIL enhance viability among quiescent and BCR-stimulated B cells. However, although BLyS enhances viability among TLR stimulated B cells, APRIL does not, suggesting that TACI but not BLyS receptor 3 may share survival promoting pathways with TLRs.

Transitional Type 1 and 2 B Lymphocyte Subsets Are Differentially Responsive to Antigen Receptor Signaling

Mature B-lymphocytes develop sequentially from transitional type 1 (T1) and type 2 (T2) precursors in the spleen. To elucidate the mechanisms that regulate the developmental fate of these distinct B cell subsets, we investigated their biochemical and biological responses following stimulation through the B-cell antigen receptor (BCR). As compared with the T1 subset, T2 cells are more responsive to BCR engagement, as evidenced by their robust induction of activation markers, expression of the prosurvival protein Bcl-xL, and enhanced proliferation. BCR stimulation of T2 cells leads to the appearance of B cells with mature phenotypic characteristics, whereas T1 cells die. All of these T2 responses are dependent on the BCR signal transducer Brutons tyrosine kinase, which is dispensable for the T1 to T2 transition. Furthermore, the serine/threonine kinases ERK, p38 MAPK, and Akt are predominantly activated in T2 compared with T1 B cells following BCR cross-linking. We conclude that T1 and T2 B cells respond differentially to BCR engagement via the induction of stage-specific signaling pathways. In turn, these signaling pathways probably govern the development and selection processes that are critical for the formation of the mature B cell compartment.

Regulation of B lymphocyte development and activation by Bruton's tyrosine kinase.

The generation and maintenance of B lymphocytes is controlled by biochemical signals tramsitted by the B cell antigen receptor (BCR) complex. These signals are transduced by multiple cytoplasmic protein tyrosine kinases (PTKs) including Lyn, Syk, and Brutons tyrosine kinase (BTK). Upon BCR engagement, these PTKs activate downstream effectors, including transcription factors that modulate gene expression. In turn activation of down stream effectors is critical for B cell survival, cell cycle progression, and antibody production. Our studies focus on the role of BTK in these biological responses. We have discovered that BTK is required for activation of the BCR-responsive transcription factor, NF-κB. Furthermore, BTK-dependent activation of NF-κB isessential for reprogramming the expression of genes that control B cell survival and proliferation. The biochemical mechanisms by which BTK regulates signaling components that activate NF-κB, and the identification of BTK-responsive genes are under investigation. Elucidation of these regulatory mechanisms is expected to reveal new therapeutic targets for B cell pathologies involving defects in BTK, including X-linked agammaglobulinemia (XLA).

Bruton’s Tyrosine Kinase Mediates NF-κB Activation and B Cell Survival by B Cell-Activating Factor Receptor of the TNF-R Family

Loss of Bruton’s tyrosine kinase (Btk) function results in mouse Xid disease characterized by a reduction in mature B cells and impaired humoral immune responses. These defects have been mainly attributed to impaired BCR signaling including reduced activation of the classical NF-κB pathway. In this study we show that Btk also couples the receptor for B cell-activating factor (BAFF) of the TNF family (BAFF-R) to the NF-κB pathway. Loss of Btk results in defective BAFF-mediated activation of both classical and alternative NF-κB pathways. Btk appears to regulate directly the classical pathway in response to BAFF such that Btk-deficient B cells exhibit reduced kinase activity of IκB kinase γ-containing complexes and defective IκBα degradation. In addition, Btk-deficient B cells produce reduced levels of NF-κB2 (p100) basally and in response to stimulation via the BCR or BAFF-R, resulting in impaired activation of the alternative NF-κB pathway by BAFF. These results suggest that Btk regulates B cell survival by directly regulating the classical NF-κB pathway under both BCR and BAFF-R, as well as by inducing the expression of the components of alternative pathway for sustained NF-κB activation in response BAFF. Thus, impaired BCR- and BAFF-induced signaling to NF-κB may contribute to the observed defects in B cell survival and humoral immune responses in Btk-deficient mice.

B Cell receptor directs the activation of NFAT and NF-κB via distinct molecular mechanisms

BCR engagement initiates intracellular calcium ([Ca2+]i) mobilization which is critical for the activation of multiple transcription factors including NF-kappaB and NFAT. Previously, we showed that Brutons tyrosine kinase (BTK)-deficient (btk-/-) B cells, which display a modestly reduced calcium response to BCR crosslinking, do not activate NF-kappaB. Here we show that BTK is also essential for the activation of NFAT following BCR engagement. Pharmacological mobilization of [Ca2+]i in BTK-deficient DT40 B cells (DT40.BTK) does not rescue BCR directed activation of NF-kappaB and only partially that of NFAT, suggesting existence of additional BTK-signaling pathways in this process. Therefore, we investigated a requirement for BTK in the production of diacylglycerol (DAG). We found that DT40.BTK B cells do not produce DAG in response to BCR engagement. Pharmacological inhibition of PKC isozymes and Ras revealed that the BCR-induced activation of NF-kappaB requires conventional PKCbeta, whereas that of NFAT may involve non-conventional PKCdelta and Ras pathways. Consistent with an essential role for BTK in the regulation of NFAT, B cells from btk-/- mice display defective expression of CD5, a gene under the control of NFAT. Together, these results suggest that BCR employs distinct BTK-dependent molecular mechanisms to regulate the activation of NF-kappaB versus NFAT.

Soluble multi-trimeric TNF superfamily ligand adjuvants enhance immune responses to a HIV-1 Gag DNA vaccine

BACKGROUND DNA vaccines remain an important component of HIV vaccination strategies, typically as part of a prime/boost vaccination strategy with viral vector or protein boost. A number of DNA prime/viral vector boost vaccines are currently being evaluated for both preclinical studies and in Phase I and Phase II clinical trials. These vaccines would benefit from molecular adjuvants that increase correlates of immunity during the DNA prime. While HIV vaccine immune correlates are still not well defined, there are a number of immune assays that have been shown to correlate with protection from viral challenge including CD8+ T cell avidity, antigen-specific proliferation, and polyfunctional cytokine secretion. METHODOLOGY AND PRINCIPAL FINDINGS Recombinant DNA vaccine adjuvants composed of a fusion between Surfactant Protein D (SP-D) and either CD40 Ligand (CD40L) or GITR Ligand (GITRL) were previously shown to enhance HIV-1 Gag DNA vaccines. Here we show that similar fusion constructs composed of the TNF superfamily ligands (TNFSFL) 4-1BBL, OX40L, RANKL, LIGHT, CD70, and BAFF can also enhanced immune responses to a HIV-1 Gag DNA vaccine. BALB/c mice were vaccinated intramuscularly with plasmids expressing secreted Gag and SP-D-TNFSFL fusions. Initially, mice were analyzed 2 weeks or 7 weeks following vaccination to evaluate the relative efficacy of each SP-D-TNFSFL construct. All SP-D-TNFSFL constructs enhanced at least one Gag-specific immune response compared to the parent vaccine. Importantly, the constructs SP-D-4-1BBL, SP-D-OX40L, and SP-D-LIGHT enhanced CD8+ T cell avidity and CD8+/CD4+ T cell proliferation 7 weeks post vaccination. These avidity and proliferation data suggest that 4-1BBL, OX40L, and LIGHT fusion constructs may be particularly effective as vaccine adjuvants. Constructs SP-D-OX40L, SP-D-LIGHT, and SP-D-BAFF enhanced Gag-specific IL-2 secretion in memory T cells, suggesting these adjuvants can increase the number of self-renewing Gag-specific CD8+ and/or CD4+ T cells. Finally adjuvants SP-D-OX40L and SP-D-CD70 increased T(H)1 (IgG2a) but not T(H)2 (IgG1) antibody responses in the vaccinated animals. Surprisingly, the B cell-activating protein BAFF did not enhance anti-Gag antibody responses when given as an SP-D fusion adjuvant, but nonetheless enhanced CD4+ and CD8+ T cell responses. CONCLUSIONS We present evidence that various SP-D-TNFSFL fusion constructs can enhance immune responses following DNA vaccination with HIV-1 Gag expression plasmid. These data support the continued evaluation of SP-D-TNFSFL fusion proteins as molecular adjuvants for DNA and/or viral vector vaccines. Constructs of particular interest included SP-D-OX40L, SP-D-4-1BBL, SP-D-LIGHT, and SP-D-CD70. SP-D-BAFF was surprisingly effective at enhancing T cell responses, despite its inability to enhance anti-Gag antibody secretion.

Requirement of phospholipase C‐γ2 (PLCγ2) for Dectin‐1‐induced antigen presentation and induction of TH1/TH17 polarization

DC recognize microbial components through an array of receptors known as PRR. PRR initiate intracellular signals, which engender DC with the capacity to stimulate T‐cell responses. Dectin‐1 is a PRR that recognizes β‐glucan, a major constituent of many fungis outer cell wall. Here we show that Dectin‐1 activates DC through phospholipase (PLC)γ2 signaling. PLCγ2‐deficient DC were unable to expand antigen‐specific T cells and induce TH1 and TH17 differentiation in response to β‐glucan. Mechanistically, PLCγ2‐deficiency impaired the capacity of DC to secrete polarizing cytokines following exposure to β‐glucan. Dectin‐1 required PLCγ2 to activate MAPK, AP‐1 and NF‐κB, which induce cytokine gene expression. Moreover, PLCγ2 controlled Dectin‐1‐mediated NFAT activation and induction of NFAT‐dependent genes such as IL‐2, cyclooxigenase‐2 and Egr transcription factors. We conclude that PLCγ2 is a crucial signaling mediator that modifies DC gene expression program to activate DC responses to β‐glucan‐containing pathogens.