B-cell chronic lymphocytic leukemia (B-CLL IntroductionChronic lymphocytic leukemia (CLL) is the most common adult leukemia in the United States and is characterized by progressive accumulation of malignant B cells in the blood, bone marrow, and lymphoid organs. CLL has been considered a prime example of a malignancy involving defects in the regulation of cell death; the slow accumulation of B-cell CLL (B-CLL) cells is presumably the result of a low proliferative index coupled with an intrinsic defect in apoptosis. However, recent clinical data from B-CLL patients given deuterated water have shown that there is a considerably higher turnover of CLL cells than previously recognized, 1 and that rates of B-CLL cell proliferation, as well as cell death, can vary widely among the lymphatic and extralymphatic compartments. Observations from other studies suggest that there are 2 types of malignant cells: quiescent and apoptosis-resistant cells in the blood, and actively dividing cells found in lymphatic aggregates in the lymph nodes and bone marrow. 2 B-CLL cells in peripheral blood are arrested in the G0/G1 phase of the cell cycle 3 and express high levels of the cell-cycle inhibitor p27. 4 In contrast, Survivinand Ki67-positive B-CLL cells, 5 and those with low expression of p27, 6 have been identified in proliferation centers of the lymph nodes and bone marrow. Thus, the goal of developing therapeutics to treat and cure CLL is to disrupt the pathologic conditions that promote malignant cell growth while accelerating tumor cell death and clearance.Until recently, treatment of progressive CLL, with steroids and alkylating agents, was largely palliative, with no impact on the natural history of the disease. 7 Introduction of purine analogs as single-agent therapies (fludarabine [F]) 8 and in combination with alkylators (fludarabine/cyclophosphamide [FC]) 9 has improved clinical responses and complete remission rates. Purine analogues have a high specificity for lymphoid cells and can induce death in both proliferating and resting cells. As a result, these agents are as effective as single agents for treating bulky CLL disease, and substantially reduce tumor burden with little extramedullary toxicity. Severe myelosuppression and immunosuppression are, however, associated with this class of drugs, and despite improvements in clinical responses, an increase in median survival time has not been demonstrated. Addition of the monoclonal antibody rituximab Rituxan; Genentech [South San Francisco, CA] and IDEC Pharmaceuticals [San Diego, CA]) to FC regimens has resulted in significantly higher overall response rates (ORRs), complete responses (CRs), molecular remissions, and importantly, longer median overall survival. 10-12 Grades 3 to 4 myelosuppression, infection, and viral reactivation remain major morbidities.Monoclonal antibodies have demonstrated activity in CLL as single agents, fueling interest in targeted therapeutic agents that engage the immune effector system to kill tumor cells. Alemtuzumab (CamPath; Millenniu...

Targeting interactions between a4b7 integrin and endothelial adhesion molecule MAdCAM-1 to inhibit lymphocyte migration to the gastrointestinal tract is an effective therapy in inflammatory bowel disease (IBD). Following lymphocyte entry into the mucosa, a subset of these cells expresses aEb7 integrin, which is expressed on proinflammatory lymphocytes, to increase cell retention. The factors governing lymphocyte migration into the intestinal mucosa and aE integrin expression in healthy subjects and IBD patients remain incompletely understood. We evaluated changes in factors involved in lymphocyte migration and differentiation within tissues. Both ileal and colonic tissue from active IBD patients showed upregulation of ICAM-1, VCAM-1, and MAdCAM-1 at the gene and protein levels compared with healthy subjects and/or inactive IBD patients. b1 and b7 integrin expression on circulating lymphocytes was similar across groups. TGF-b1 treatment induced expression of aE on both b7 + and b7 2 T cells, suggesting that cells entering the mucosa independently of MAdCAM-1/a4b7 can become aEb7 + . ITGAE gene polymorphisms did not alter protein induction following TGF-b1 stimulation. Increased phospho-SMAD3, which is directly downstream of TGF-b, and increased TGF-bresponsive gene expression were observed in the colonic mucosa of IBD patients. Finally, in vitro stimulation experiments showed that baseline b7 expression had little effect on cytokine, chemokine, transcription factor, and effector molecule gene expression in aE + and aE 2 T cells. These findings suggest cell migration to the gut mucosa may be altered in IBD and a4b7 2 , and a4b7 + T cells may upregulate aEb7 in response to TGF-b once within the gut mucosa.

Background: Anti-integrin therapy is a new frontline strategy in the treatment of inflammatory bowel diseases (IBD). The anti-β7 integrin antibody etrolizumab is currently being investigated for safety and efficacy in Crohn’s disease (CD) and ulcerative colitis (UC) in several phase III trials. Mechanistically, etrolizumab is known to block β7 integrin ligand binding and reduces intestinal trafficking of β7-expressing cells. Etrolizumab blocks β7 integrin ligand binding and reduces β7-positive lymphocyte migration and retention in the inflamed gut mucosa, but the exact mechanisms by which this inhibition occurs are not fully understood.Methods: Cellular effects of etrolizumab or etrolizumab surrogate antibody (etrolizumab-s) were investigated in cell culture models and analyzed by flow cytometry, fluorescence microscopy, ImageStream®, stimulated emission depletion (STED) microscopy and functional dynamic in vitro adhesion assays. Moreover, effects on α4β7 integrin were compared with the pharmacodynamically similar antibody vedolizumab.Results: As demonstrated by several different approaches, etrolizumab and etrolizumab-s treatment led to internalization of β7 integrin. This resulted in impaired dynamic adhesion to MAdCAM-1. Internalized β7 integrin localized in endosomes and re-expression of β7 was dependent on de novo protein synthesis. In vitro etrolizumab treatment did not lead to cellular activation or cytokine secretion and did not induce cytotoxicity. Internalization of α4β7 integrin was increased with etrolizumab compared with vedolizumab.Discussion: Our data suggest that etrolizumab does not elicit secondary effector functions on the single cell level. Integrin internalization may be an important mechanism of action of etrolizumab, which might explain some but not all immunological effects observed with etrolizumab.

The dysregulation of multiple signaling pathways, including those through endosomal Toll-like receptors (TLRs), Fc gamma receptors (FcγR), and antigen receptors in B cells (BCR), promote an autoinflammatory loop in systemic lupus erythematosus (SLE). Here, we used selective small-molecule inhibitors to assess the regulatory roles of interleukin-1 receptor (IL-1R)–associated kinase 4 (IRAK4) and Bruton’s tyrosine kinase (BTK) in these pathways. The inhibition of IRAK4 repressed SLE immune complex– and TLR7-mediated activation of human plasmacytoid dendritic cells (pDCs). Correspondingly, the expression of interferon (IFN)–responsive genes (IRGs) in cells and in mice was positively regulated by the kinase activity of IRAK4. Both IRAK4 and BTK inhibition reduced the TLR7-mediated differentiation of human memory B cells into plasmablasts. TLR7-dependent inflammatory responses were differentially regulated by IRAK4 and BTK by cell type: In pDCs, IRAK4 positively regulated NF-κB and MAPK signaling, whereas in B cells, NF-κB and MAPK pathways were regulated by both BTK and IRAK4. In the pristane-induced lupus mouse model, inhibition of IRAK4 reduced the expression of IRGs during disease onset. Mice engineered to express kinase-deficient IRAK4 were protected from both chemical (pristane-induced) and genetic (NZB/W_F1 hybrid) models of lupus development. Our findings suggest that kinase inhibitors of IRAK4 might be a therapeutic in patients with SLE.

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