Matthew Schrage

B Cell Developmental Requirement for the Gαi2 Gene

Null mutation of the Gαi2 trimeric G protein results in a discrete and profound mucosal disorder, including inflammatory bowel disease (IBD), attenuation of IL-10 expression, and immune function polarized to Th1 activity. Genetic and adoptive transfer experiments have established a role for B cells and IL-10 in mucosal immunologic homeostasis and IBD resistance. In this study, we addressed the hypothesis that Gαi2 is required for the development of IL-10-producing B cells. Gαi2−/− mice were reduced in the relative abundance of marginal zone (MZ), transitional type 2 (T2), and B-1a B cells and significantly increased in follicular mature and B-1b B cells. Reconstitution of RAG2−/− mice with Gαi2−/− bone marrow induced an IBD-like colitis and a deficiency in absolute numbers of MZ, T2, and B-1 B cells. Thus, the Gαi2−/− genotype in colitis susceptibility and B cell development involved a cis effect within the hemopoietic compartment. In vitro, the B cell population of Gαi2−/− mice was functionally deficient in LPS-induced proliferation and IL-10 production, consistent with the exclusive capacity of T2 and MZ cell subpopulations for LPS responsiveness. In vivo, Gαi2−/− mice were selectively impaired for the IgM response to T-independent type II, consistent with the relative depletion of MZ and peritoneal B-1 subpopulations. Collectively, these results reveal a selective role for Gαi2 in MZ and B-1 B cell development. Disorders of this Gαi2-dependent process in B cell development may represent a mechanism for IBD susceptibility.

Formation of B and T cell subsets require the cannabinoid receptor CB2

A recent and surprising body of research has linked changes in immune function to biologic and therapeutic targeting of cannabinoid receptors, which prototypically respond to delta-9 tetrahydrocannabinol. The peripheral cannabinoid receptor CB2 is highly expressed in immune cell types (macrophages, dendritic cells, and B cells), and pharmacologically alters their cytokine production and responsiveness. Accordingly, cannabinoid agonists can powerfully alter susceptibility to certain microbial infections, atherosclerosis, and cancer immunotherapy. What is unknown is the physiologic role of natural levels of endocannabinoids and their receptors in normal immune homeostasis. Gαi2−/− mice are deficient in the formation of certain B and T cell subsets and are susceptible to immune dysregulation, notably developing inflammatory bowel disease. A key issue is the identity of the Gi-coupled receptors relevant to this Gαi2-signaling pathway. We find that mice deficient in CB2, the Gi-coupled peripheral endocannabinoid receptor, have profound deficiencies in splenic marginal zone, peritoneal B1a cells, splenic memory CD4+ T cells, and intestinal natural killer cells and natural killer T cells. These findings partially phenocopy and extend the lymphocyte developmental disorder associated with the Gαi2−/− genotype, and suggest that the endocannabinoid system is required for the formation of T and B cell subsets involved in immune homeostasis. This noncompensatable requirement for physiologic function of the endocannabinoid system is novel. Because levels of endocannabinoids are highly restricted microanatomically, local regulation of their production and receptor expression offers a new principle for regional immune homeostasis and disease susceptibility, and extends and refines the rationale for CB2-targeted immunotherapy in immune and inflammatory diseases.

Antibody-Based Profiling of the Phosphoinositide 3-Kinase Pathway in Clinical Prostate Cancer

Purpose: As kinase inhibitors transition from the laboratory to patients, it is imperative to develop biomarkers that can be used in the clinic. The primary objectives are to identify patients most likely to benefit from molecularly targeted therapies and to document modulation of the drug target. Constitutive activation of the phosphoinositide 3-kinase (PI3K) pathway and its downstream effectors, as a result of PTEN loss or by other mechanisms, occurs in a high proportion of prostate cancers, making it an ideal template for the design of clinical trials involving PI3K pathway inhibitors. Prostate cancers also present unique organ-specific challenges, in that tumors are heterogeneous and diagnostic tissue is extremely limited. Experimental Design: Working within these limitations, we have developed a set of immunohistochemical assays that define activation of the PI3K pathway in clinical samples. Results and Conclusions: Using both univariate and multivariate analyses, we show that loss of PTEN is highly correlated with the activation of AKT, and this, in turn, is associated with the phosphorylation of S6, one of its main effectors. These three antibodies are potentially able to define a molecular signature of PTEN loss and/or AKT pathway activation in prostate cancer.

PREOPERATIVE PROSTATE NEEDLE BIOPSY p27 CORRELATES WITH SUBSEQUENT RADICAL PROSTATECTOMY p27, GLEASON GRADE AND PATHOLOGICAL STAGE

PURPOSE Loss of p27 protein expression in radical prostatectomy specimens has been shown to be an adverse prognostic factor in patients with clinically localized prostate cancer. To our knowledge no studies have examined p27 expression in prostate needle biopsies. To test the potential predictive power of p27 in prostate biopsies we compared p27 expression in preoperative biopsies and matched prostatectomy specimens of patients with clinically localized prostate cancer. MATERIALS AND METHODS Matched biopsies and radical prostatectomy specimens from 44 patients were examined. Mean followup was 22.7 months (range 1 to 46). Tumors expressing less than 30% positive nuclei were classified as low expressors and tumors expressing greater than 30% positive nuclei were classified as high expressors of p27 protein. RESULTS Expression of p27 in prostate biopsies correlated significantly with subsequent p27 expression in radical prostatectomy specimens (p = 0.002). Sensitivity and specificity of biopsy p27 for predicting subsequent prostatectomy p27 were 87.5% and 88.9%, respectively (p <0.001). Univariate analysis showed that low expression of p27 in the biopsy correlated significantly with biopsy and prostatectomy Gleason score (p = 0.000 and 0.001, respectively), and final pathological stage (p = 0.028). Despite the small sample size and short followup, 36.4% of patients with low p27 expression had a biochemical recurrence compared to only 12.1% with high expression (hazards ratio 3.56). In addition, Kaplan-Meier analysis suggested that low p27 expression in prostate biopsies may be associated with a shorter time to recurrence, although this did not reach statistical significance (p = 0.081). CONCLUSIONS Expression of p27 in prostate biopsies can be used to predict the degree of expression in radical prostatectomy specimens. As loss of p27 protein expression in prostatectomy specimens has been shown to correlate with biochemical recurrence and shortened prostate specific survival, these results suggest that biopsy p27 may help identify high risk patients preoperatively.

Villous B Cells of the Small Intestine Are Specialized for Invariant NK T Cell Dependence

B cells are important in mucosal microbial homeostasis through their well-known role in secretory IgA production and their emerging role in mucosal immunoregulation. Several specialized intraintestinal B cell compartments have been characterized, but the nature of conventional B cells in the lamina propria is poorly understood. In this study, we identify a B cell population predominantly composed of surface IgM+ IgD+ cells residing in villi of the small intestine and superficial lamina propria of the large intestine, but distinct from the intraepithelial compartment or organized intestinal lymphoid structures. Small intestinal (villous) B cells are diminished in genotypes that alter the strength of BCR signaling (Bruton tyrosine kinasexid, Gαi2−/−), and in mice lacking cognate BCR specificity. They are not dependent on enteric microbial sensing, because they are abundant in mice that are germfree or genetically deficient in TLR signaling. However, villous B cells are reduced in the absence of invariant NK T cells (Jα18−/− or CD1d−/− mice). These findings define a distinct population of conventional B cells in small intestinal villi, and suggest an immunologic link between CD1-restricted invariant NK T cells and this B cell population.