The Kinase PDK1 Is Essential for B-Cell Receptor Mediated Survival Signaling

Phosphoinositide-dependent kinase 1 (PDK1) plays an important role in integrating the T cell antigen receptor (TCR) and CD28 signals to achieve efficient NF-κB activation. PDK1 is also an important regulator of T cell development, mediating pre-TCR induced proliferation signals. However, the role of PDK1 in B cell antigen receptor (BCR) signaling and B cell development remains largely unknown. In this study we provide genetic evidence supporting the role of PDK1 in B cell survival. We found PDK1 is required for BCR mediated survival in resting B cells, likely through regulation of Foxo activation. PDK1-dependent signaling to NF-κB is not crucial to resting B cell viability. However, PDK1 is necessary for triggering NF-κB during B cell activation and is required for activated B cell survival. Together these studies demonstrate that PDK1 is essential for BCR-induced signal transduction to Foxo and NF-κB and is indispensable for both resting and activated B cell survival

Androgen ablation mitigates tolerance to a prostate/prostate cancer-restricted antigen

SummaryTo understand the T cell response to prostate cancer, we created transgenic mice that express a model antigen in a prostate-restricted pattern and crossed these animals to TRAMP mice that develop spontaneous prostate cancer. Adoptive transfer of prostate-specific CD4 T cells shows that, in the absence of prostate cancer, the prostate gland is mostly ignored. Tumorigenesis allows T cell recognition of the prostate gland—but this recognition is tolerogenic, resulting in abortive proliferation and ultimately in hyporesponsiveness at the systemic level. Androgen ablation (the most common treatment for metastatic prostate cancer) was able to mitigate this tolerance—allowing prostate-specific T cells to expand and develop effector function after vaccination. These results suggest that immunotherapy for prostate cancer may be most efficacious when administered after androgen ablation

Prognostic significance of translocations in the presence of mutated IGHV and of cytogenetic complexity at diagnosis of chronic lymphocytic leukemia

Mutations of the IGH variable region in patients with chronic lymphocytic leukemia (CLL) are associated with a favorable prognosis. Cytogenetic complexity (>3 unrelated aberrations) and translocations have been associated with an unfavorable prognosis. While mutational status of IGHV is stable, cytogenetic aberrations frequently evolve. However, the relationships of these features as prognosticators at diagnosis are unknown. We examined the CpG-stimulated metaphase cytogenetic features detected within one year of diagnosis of CLL and correlated these features with outcome and other clinical features including IGHV. Of 329 untreated patients, 53 (16.1%) had a complex karyotype (16.1%), and 85 (25.8%) had a translocation. Median time to first treatment (TFT) was 47 months. In univariable analyses, significant risk factors for shorter TFT (p3.5, log-transformed WBC, unmutated IGHV, complex karyotype, translocation, and FISH for trisomy 8, del(11q) and del(17p). In multivariable analysis, there was significant effect modification of IGHV status on the relationship between translocation and TFT (p=0.002). In IGHV mutated patients, those with a translocation had over 3.5 times higher risk of starting treatment than those without a translocation (

IκBβ acts to inhibit and activate gene expression during the inflammatory response

The activation of pro-inflammatory gene programs by nuclear factor-κB (NF-κB) is primarily regulated through cytoplasmic sequestration of NF-κB by the inhibitor of κB (IκB) family of proteins1. IκBβ, a major isoform of IκB, can sequester NF-κB in the cytoplasm2, although its biological role remains unclear. Although cells lacking IκBβ have been reported3, 4, in vivo studies have been limited and suggested redundancy between IκBα and IκBβ5. Like IκBα, IκBβ is also inducibly degraded; however, upon stimulation by lipopolysaccharide (LPS), it is degraded slowly and re-synthesized as a hypophosphorylated form that can be detected in the nucleus6, 7, 8, 9, 10, 11. The crystal structure of IκBβ bound to p65 suggested this complex might bind DNA12. In vitro, hypophosphorylated IκBβ can bind DNA with p65 and c-Rel, and the DNA-bound NF-κB:IκBβ complexes are resistant to IκBα, suggesting hypophosphorylated, nuclear IκBβ may prolong the expression of certain genes9, 10, 11. Here we report that in vivo IκBβ serves both to inhibit and facilitate the inflammatory response. IκBβ degradation releases NF-κB dimers which upregulate pro-inflammatory target genes such as tumour necrosis factor-α (TNF-α). Surprisingly, absence of IκBβ results in a dramatic reduction of TNF-α in response to LPS even though activation of NF-κB is normal. The inhibition of TNF-α messenger RNA (mRNA) expression correlates with the absence of nuclear, hypophosphorylated-IκBβ bound to p65:c-Rel heterodimers at a specific κB site on the TNF-α promoter. Therefore IκBβ acts through p65:c-Rel dimers to maintain prolonged expression of TNF-α. As a result, IκBβ^(−/−) mice are resistant to LPS-induced septic shock and collagen-induced arthritis. Blocking IκBβ might be a promising new strategy for selectively inhibiting the chronic phase of TNF-α production during the inflammatory response

Cellular and molecular mechanisms underlying naive and effector CD4 cell tolerization

The induction of antigen-specific T cell tolerance in the periphery is essential to prevent autoimmune diseases and also helps tumors to evade immune neutralization. Dissecting the mechansims that mediate peripheral T cell tolerance will therefore aid in the development of therapeutic strategies to manage these diseases. ^ Previous data from our lab and other groups has shown that not only naïve, but also effector/memory T cells that are initially primed by viral antigens can undergo peripheral tolerization in response to self-antigen. This thesis describes cellular and molecular mechanisms that regulate the tolerization of both naïve and Th1 effector CD4 cells. Interestingly, during the tolerization of Th1 effectors, functions such as the ability to produce the effector cytokines IFN-γ and TNF-α are lost more rapidly than non-effector functions such as the ability to proliferate and to produce IL-2. These functional loses are conferred primarily through the development of cell-intrinsic signaling defects that are located relative to the T cell receptor (TCR) both proximally (which impair the expression of all cytokines) and distally (which selectively impairs the expression of IFN-γ and TNF-α). Furthermore, the down-modulation of T-bet, a key transcription factor that directs the differentiation of naïve CD4 cells into IFN-γ-expressing Th1 effectors, was identified as a TCR-distal defect that selectively impairs IFN-γ, but not TNF-α, expression. Although T-bet controls IFN-γ expression in part through programming remodeling of the IFN-γ gene locus from an closed/repressed to open/transcriptionally competent configuration, analysis of histone acetylation at the IFN-γ promoter indicated that down-modulation of T-bet expression during Th1 effector CD4 cell tolerization does not impair IFN-γ expression potential through alterations in chromatin structure. ^ Although naïve CD4 cells undergoing tolerization only develop weak capacity to express IFN-γ, we made the surprising finding that the IFN-γ gene locus nevertheless undergoes remodeling, and that the inability to express IFN-γ in response to antigenic stimulation is primarily due to the development of a TCR-proximal signaling defect. Interestingly, it appears that IL-2 (a cytokine whose absence is generally associated with tolerance) produced by naïve CD4 cells undergoing tolerization actually drives the remodeling of the IFN-γ gene. During these studies we also made the surprising finding that in naïve CD4 cells undergoing both virally-induced Th1 differentiation and self-antigen-induced tolerization that IL-2 expression and IL-2 signaling (as measured by STAT5 activation) were largely mutually-exclusive. Thus, paracrine, rather than autocrine, IL-2 signaling appears to be the dominant signaling mode during early CD4 cell response.

Cellular and molecular mechanisms underlying naive and effector CD4 cell tolerization

The induction of antigen-specific T cell tolerance in the periphery is essential to prevent autoimmune diseases and also helps tumors to evade immune neutralization. Dissecting the mechansims that mediate peripheral T cell tolerance will therefore aid in the development of therapeutic strategies to manage these diseases. ^ Previous data from our lab and other groups has shown that not only naïve, but also effector/memory T cells that are initially primed by viral antigens can undergo peripheral tolerization in response to self-antigen. This thesis describes cellular and molecular mechanisms that regulate the tolerization of both naïve and Th1 effector CD4 cells. Interestingly, during the tolerization of Th1 effectors, functions such as the ability to produce the effector cytokines IFN-γ and TNF-α are lost more rapidly than non-effector functions such as the ability to proliferate and to produce IL-2. These functional loses are conferred primarily through the development of cell-intrinsic signaling defects that are located relative to the T cell receptor (TCR) both proximally (which impair the expression of all cytokines) and distally (which selectively impairs the expression of IFN-γ and TNF-α). Furthermore, the down-modulation of T-bet, a key transcription factor that directs the differentiation of naïve CD4 cells into IFN-γ-expressing Th1 effectors, was identified as a TCR-distal defect that selectively impairs IFN-γ, but not TNF-α, expression. Although T-bet controls IFN-γ expression in part through programming remodeling of the IFN-γ gene locus from an closed/repressed to open/transcriptionally competent configuration, analysis of histone acetylation at the IFN-γ promoter indicated that down-modulation of T-bet expression during Th1 effector CD4 cell tolerization does not impair IFN-γ expression potential through alterations in chromatin structure. ^ Although naïve CD4 cells undergoing tolerization only develop weak capacity to express IFN-γ, we made the surprising finding that the IFN-γ gene locus nevertheless undergoes remodeling, and that the inability to express IFN-γ in response to antigenic stimulation is primarily due to the development of a TCR-proximal signaling defect. Interestingly, it appears that IL-2 (a cytokine whose absence is generally associated with tolerance) produced by naïve CD4 cells undergoing tolerization actually drives the remodeling of the IFN-γ gene. During these studies we also made the surprising finding that in naïve CD4 cells undergoing both virally-induced Th1 differentiation and self-antigen-induced tolerization that IL-2 expression and IL-2 signaling (as measured by STAT5 activation) were largely mutually-exclusive. Thus, paracrine, rather than autocrine, IL-2 signaling appears to be the dominant signaling mode during early CD4 cell response.

Differential Role of the Transcription Factor NF-κB in Selection and Survival of CD4+ and CD8+ Thymocytes

SummaryInhibition of the transcription factor nuclear factor (NF)-κB activity leads to a reduction in numbers of CD8+ single-positive (SP) thymocytes, suggesting a selective role for NF-κB in these cells. To further explore the role of NF-κB in SP thymocytes, we utilized transgenic models that allowed either inhibition or activation of NF-κB. We showed that activation of NF-κB played an important role in the selection of major histocompatibility complex (MHC) class I-restricted CD8+ T cells. Surprisingly, NF-κB was not activated in positively selected CD4+ thymocytes, and inhibition of NF-κB did not perturb positive or negative selection of CD4+ cells. However, enforced activation of NF-κB via a constitutively active inhibitor of κB (IκB) kinase transgene led to a nearly complete deletion of CD4 cells by pushing positively selecting CD4+ cells into negative selection. These studies therefore revealed a surprising difference of NF-κB activation in CD4+ and CD8+ thymocytes and suggested that NF-κB contributes to the establishment of thresholds of signaling that determine positive or negative selection of thymocytes