Chronic lymphocytic leukemia (CLL) involves a profound humoral immune defect and tumor-specific humoral tolerance that directly contribute to disease morbidity and mortality. CD154 gene therapy can reverse this immune defect, but attempts to do this pharmacologically have been unsuccessful. The immunemodulatory agent lenalidomide shows clinical activity in CLL, but its mechanism is poorly understood. Here, we demonstrate that lenalidomide induces expression of functional CD154 antigen on CLL IntroductionChronic lymphocytic leukemia (CLL) is the most common adult leukemia, and is characterized by an elevated frequency of infections, secondary malignancy, and autoimmune complications compared with the general population. Current treatment options for CLL are palliative and further exacerbate the immune deficiency seen in this disease. Nonetheless, CLL represents an "immunoresponsive" disease as evidenced by extended disease remission and potential cure with reduced intensity allogeneic stem cell transplantation (reviewed in Gribben 1 ). This suggests that strategies that restore immune function have potential to effectively eliminate CLL.The immune defect in CLL is characterized by both humoral and cellular immune defects. Although detailed studies of normal B cells in CLL patients have not been performed due to the difficulty in isolating these cells, hypogammaglobulinemia is often present at diagnosis and becomes worse with disease progression. A profound cellular immune defect 2-4 is present in CLL with significant alterations in genes involved in differentiation, cytoskeleton formation, vesicle trafficking, and cell death. 4 Coculture of CLL cells with normal T cells produces the same T-cell defects observed in CLL patients, 4 suggesting a direct role of the leukemia cells in contributing to the T cell-dependent cellular immune deficiency. The clinical manifestation of the humoral and cellular immune defects in CLL patients includes hypogammaglobulinemia, 5,6 poor response to both polysaccharide-based 7-9 and proteinbased 10 vaccines, and a high predisposition to infections 11,12 that represents a leading cause of death.To date, attempts to reverse the immune defects in CLL have been limited. Most promising has been adenovirus-delivered CD154 gene therapy that in small numbers of patients reversed cellular and humoral tumor tolerance. CD154 is the surface ligand of CD40 and is expressed on activated T cells, natural killer cells, and dendritic cells, but not normal B cells. Activation of T cells promotes increased surface expression of CD154, thereby promoting both activation and antigen presentation in normal and transformed B cells. Congenital mutations in the CD154 gene promote profound cellular and humoral immune deficiency. Although mutations of the CD154 gene have not been described in CLL, these patients have diminished CD154 expression on T cells after CD3 ligation. 13 Transduction of murine or human CD154 into primary CLL cells ex vivo with adenovirus gene therapy vectors, followed by systemic reintr...

TCL-1 expression is variable in CLL, and no study has examined its association with treatment response. We measured TCL-1 protein in CLL cells from 51 patients who then received pentostatin, cyclophosphamide, and rituximab. TCL-1 expression did not correlate with any pre-treatment characteristics. Lower TCL-1 levels were associated with higher probability of attaining flow cytometry-negative status post-treatment (52% versus 17%, p=0.046). Trends toward improved complete remission rate (49% versus 19%, p=0.064) and progression-free survival (medians: 33 versus 20 months, p=0.199) were noted with lower TCL-1 expression. These data suggest TCL-1 expression may help predict treatment outcome in CLL patients following chemoimmunotherapy, and examination in larger studies is warranted.

The immunomodulatory drug lenalidomide has demonstrated efficacy in chronic lymphocytic leukemia (CLL) patients, despite a lack of direct cytotoxic effect in vitro. The mechanism of lenalidomide efficacy in vivo is thought to occur via a combination of enhanced immune activity and an alteration of tumor cell–microenvironment interactions. We demonstrate in whole blood from CLL patients that lenalidomide significantly depletes malignant B cells. Lenalidomide also induced production of interleukin-21 (IL21) and its mRNA in T cells from CLL patients. Lenalidomide also enhanced upregulation of functional IL21 receptor (IL21R) on the cell surface and increased receptor mRNA in vitro. The in vitro combination of IL21 and lenalidomide enhanced IL21-mediated cytotoxicity toward CLL cells through a variety of mechanisms. We show association of cell death with upregulation of Bid by IL21, enhanced upregulation of Bid by the combination therapy, and diminished Lck and downstream BCR signaling activation of Syk and PLCG2. Collectively, we demonstrated an immune cell–tumor cell interaction through lenalidomide-mediated induction of IL21 and IL21R, with enhanced IL21-mediated cytotoxicity, which provides justification for this combination in clinical trials for CLL patients.

CpG oligodeoxynucleotides (ODNs) upregulate the interleukin-21 receptor (IL21R) and enhance IL-21-mediated cytotoxicity in chronic lymphocytic leukemia (CLL) B cells. We demonstrate that treatment of CLL B cells with the ODN CpG-685 leads to increased IL21R expression, and that this increased expression enhances the effects of IL-21 treatment as evidenced by increased phosphorylation of JAK1, STAT1, and STAT3, as compared to IL-21 treatment without prior CpG stimulation. Induction of IL21R by CpG-685 also enhanced IL-21-mediated cytotoxicity. The mechanism by which CpG ODNs upregulate IL21R has not been elucidated, although IL21R regulation in T cells has been shown to be linked to T cell receptor-induced Sp1 binding to the IL21R promoter. Here, we demonstrate that luciferase reporter constructs containing the Sp1 binding site have increased basal luciferase activity compared to constructs lacking the Sp1 binding site, but fail to increase luciferase activity with CpG-685 stimulation in CLL B cells. By treating CLL cells with an NF-κB inhibitor, we inhibit the CpG ODN-mediated induction of IL21R, thus demonstrating that CpG-685 upregulates IL21R through an NF-κB mediated pathway. These findings suggest an alternative mechanism for induction of IL-21 receptor in CLL B cells and provide a basis for creation of future combination therapies.

Adequate dosing of lenalidomide in Chronic Lymphocytic Leukemia (CLL) remains unclear. This study determined maximum tolerated dose (MTD) in relapsed CLL patients (Cohort A) and patients achieving a partial response (PR) or better to recent therapy (Cohort B). Thirty-seven patients were enrolled. MTD was 2.5 mg followed by 5.0 mg continuous. In Cohort A, tumor flare grade 1–2 occurred in 15 patients (50%) and grade 3 in 1 patient (3%). Cohort A had 19 of 23 evaluable (83%) patients, 4 PR (17%) and 15 (65%) stable disease (SD), Cohort B had 6 of 7 patients (86%) with SD. Despite overall response rate not being high, many patients remained on therapy several months with SD.