Ibrutinib-A double-edge sword in cancer and autoimmune disorders

Targeted therapies have appeared as new treatment options for several disease types, including cancer and autoimmune disorders. Of several targets, tyrosine kinases (TKs) are among the most promising. Overexpression of TKs provides a target for novel therapeutic agents, including small molecule inhibitors of tyrosine kinases (TKI). Ibrutinib (PCI-32765) is a TKI of Bruton’s tyrosine kinase (Btk), a key kinase of the B-cell receptor signaling pathway that plays a significant role in the proliferation, differentiation and survival of B cells. In addition to inhibitory effects, recent studies have shown that ibrutinib has multiple immunomodulatory effects. It binds covalently to IL-2 inducible tyrosine kinase (Itk) in T lymphocytes and suppresses the survival of T-helper (Th) 2 cells. This changes the balance of Th1/Th2 cells toward Th1 subset, which are the main immune cells targeting tumor cells. The dual activity of ibrutinib has paid a great attention and several studies are evaluating the anti-tumor and immunomodulatory effects in cancer, autoimmune disorders and infectious diseases. In this article we review the inhibitory and immunomodulatory effects of ibrutinib in B-cell malignancies, autoimmune diseases and infections, as well as the communication between the Ror1 receptor tyrosine kinase and BCR and effects of ibrutinib on this crosstalk.CLL Global Research FoundationManuscrip

Preclinical therapeutic vaccination strategies in malignancies with focus on B-cell chronic lymphocytic leukemia

A therapeutic strategy that may potentially delay progression or prevent relapse of B-cell chronic lymphocytic leukemia (B-CLL), is antileukemic vaccine therapy. This thesis focuses on comparing different strategies for loading antigen on dendritic cells (DC) with the aim of delineating the most effective antigen presentation platform for stimulating anti-leukemic T cells. We have studied different methods of loading DC with tumor preparations i.e. DCtumor cell fusion, apoptotic tumor cells (Apo-DC), tumor cell lysate (DC-lysate) and total tumor RNA (DC-RNA). A defined, synthetic peptide derived from telomerase has also been investigated as an alternative to the whole tumor cell antigen approach. We examined autologous T cell activation by Apo-DC and compared it to the T-cell stimulatory capacity of DC that were fused with tumor cells. Following overnight incubation 22.6 ± 6.2% (mean ± SEM) of DC had endocytosed apoptotic leukemic cells while the frequency of DC-Ieukemic cell hybrids was 10.5 ± 2.6%. A T cell proliferative response was observed in four out of five CIL patients when using Apo-DC whereas fusion hybrids lacked the ability to elicit a proliferative response. Both preparations were able to induce an IFN-gamma response. In the second study the T cell stimulation capability of Apo-DC was compared with that of DC-RNA and DClysate. In all six CIL patients tested, Apo-DC induced greater HLA-restricted T-cell responses than DC pulsed with tumor lysate or RNA. ELISpot assay revealed high IFN-gamma secretion by T-cells when Apo-DC was used to stimulate autologous T cells. Real-time PCR confirmed higher expression of IFN-gamma and IL-2 mRNA in T-cells stimulated with Apo-DC. Our data suggest that cellular vaccines based on DC loaded with apoptotic bodies may be suitable for vaccination trial in patients with B-CLL. In the third study we pulsed monocyte-derived DC with lysate from the melanoma cell line, A-375 and used it for proliferation assay and to repeatedly stimulate T-cells from a HLA-A2 positive normal donor. The resultant T-cells were examined for cytotoxic activity against A-375 targets as well as the HLA A2positive melanoma cell line DFW. We also examined the ability of lysate-pulsed DC to present melanomaassociated antigens to T-cells. The results demonstrate that lysate from allogeneic tumor cells may be used as source of antigens to stimulate tumor-specific Tcells in melanoma. In the fourth study we investigated the feasibility of large-scale generation of monocyte-derived DC from B-CLL patients as well as the effects of freezing and thawing on the function of DC loaded with autologous tumor cell preparations. Based on the results obtained from eight B-CLL patients, it appeared that the total yield of monocytic precursors, as well as the purity, was higher with immunomagnetic separation compared to counterflow elutriation. DC pulsed with autologous apoptotic tumor cells before cryopreservation retained their morphology, surface phenotype, as well as ability to stimulate T cell proliferation and cytokine production (IFN-gamma). Our results demonstrate that monocyte precursors can be successfully isolated and that an adequate number of DC required for clinical therapy can be generated from blood. In the fifth study, human telomerase reverse transcriptase (hTERT) was identified using RT-PCR in 19 out of 25 patients with B-CLL. DC were generated from PBMC of 7 telomerase-positive and three telomerase-negative B-CLL patients as well as three healthy donors and used for hTERT-specific CTL expansion by pulsing DC with a 16 amino acid long peptide from hTERT and with a Ras peptide of comparable length as control. In six out of seven patients, DC pulsed with the hTERT peptide could generate cytotoxic T lymphocytes (CTLs) against the autologous leukemic cells upon two rounds of restimulation which was not noted with telomerase negative patients or with the Ras control peptide as the antigen. In conclusion, the data obtained in this study showed that B-CLL patients with telomerase-positive leukemic cells have spontaneously occuring telomerase-specific T cells which can be expanded in vitro to lyse the leukemic cells. Telomerase might therefore be a valid target structure for vaccine development in B-CLL

Chronic lymphocytic leukemia, biology, new diagnosis and treatment: review article

Chronic lymphocytic leukemia (CLL) is a malignancy of B CD5+cells and is the most common type of leukemia in adults. The disease is more common in men over 50 years in western countries. CLL is associated with defective apoptosis in B cells. CLL was traditionally regarded as a disease that occurs before naïve B cells meet the antigen in the lymph nodes. Laboratory diagnosis requires white blood cell count, blood smear and immunophenotyping of lymphoid cells by flow cytometry. The disease most often associated with the accumulation of CD5+ CD19+ and CD23+ B cell with reduced number of surface membrane immunoglobulin in peripheral blood, bone marrow, and lymph nodes. Clinical progression of CLL is heterogeneous, some patients need treatment immediately after diagnosis, and others do not require treatment for many years after diagnosis. Over the past decades, considerable effort has been made to understanding the molecular mechanisms underlying the heterogeneous clinical course of the disease and finding prognostic markers for clinical classification. Patients with advanced Binet or Rai stages of disease require treatment. In addition to the interactions that exist between CLL cells, number of non-tumor cell types such as bone marrow stromal cells (BMSCs), nurse like cells (NLCs), follicular dendritic cells (FDCs), T cells, and some cytokines like IL-4 in tumor microenvironment play an important role in the CLL pathogenesis. Various factors including: IGVH mutation status, genetic variation, patient age and presence of other disorders are important for disease management and the type of treatment. CLL patients carrying p53 pathway dysfunction have poor prognosis and poor responses to therapy and very short survival. Available treatments include chemotherapy, chemoimmunotherapy, or drugs targeting B cell receptor signaling, Bruton's tyrosine kinase (BTK) or inhibitors of apoptosis, such as BCL2 and new class of small molecules. Understanding the CLL biology is important in identifying high-risk patients as well as the drug and relevant therapeutic methods for better management of patients. In this review paper, the microenvironment and genetic abnormalities in the CLL as well as new diagnostic and therapeutic approaches based on the new understanding of molecular biology of CLL are discussed

Immunopathology of multiple sclerosis

Multiple sclerosis (MS), an inflammatory disorder of central nervous system (CNS), is the most common cause of neurological disability especially in young adults. The disorder results from interplay between unidentified genetic and environmental factors. In MS, cells of immune system attack myelin, progressive loss of certain body function and physical ability occur. In severe cases, the progression of disease leads to permanent damage. The auto-reactive peripheral CD4+ T cells recognize auto-antigen within CNS parenchyma and polarize toward Th1 phenotype. Activated Th1 cells cause myelin disruption and release of new potential CNS auto- antigen. Pro-inflammatory cytokines such as interferon-, TNF- and chemokines which is secreted by Th1 cells induce additional unspecific inflammatory cells and specific anti myelin antibody-forming B cells that amplify tissue injury. Finally, the apoptotic death of the T cells or conversion of the T cells toward Th2 phenotype positively modulates the outcome of the lesions in CNS.Clinical manifestation of the disease is classified in three types, primary: direct damage (weakness, tremors, tingling,etc); secondary: result of primary (paralysis lead to bedsores and bladder/urinary incontinence problems); tertiary: social, psychological and vocational complication (depression is very common).The aims of treatment are: reduction of sickness attack (by corticosteroid, interferon beta 1b and 1a, glatriamer acetate, natalizumab, etc) and reduction of the disorder. The prospect of the potential tools to prevent MS is tempting, yet challenging to investigate studies about roll of vitamin D in reduction of development of disease. Intensive research on MS provides a promising prospective of the disease management in the future

IL-25 Impact on Malignant B Cells Survival and T Cells Activation in Chronic Lymphocytic Leukemia

T cell dysregulation and shift to T helper 2 responses, boosting tumor microenvironment support, contributes to the survival of leukemic B cells in Chronic Lymphocytic Leukemia. Interleukin (IL)-25 is involved in the initiation of T helper 2 cell responses. Signal transduction of IL-25 begins with the heterodimer receptor (IL-17RA/IL-17RB). The presence of IL-25 in the tumor microenvironment may affect the supportive effects of T cells in the surrounding tumor cell environment. The purpose of this study was to evaluate the role of IL-25 in the biology of CLL. IL-17RB expression in CD3+ and CD19+ cells was assessed in isolated peripheral blood mononuclear cells (PBMCs) of nine CLL patients and nine healthy subjects by real-time polymerase chain reaction and flow cytometry. B cells were positively enriched from PBMCs using magnetic activated cell sorting (MACS). PBMCs and purified leukemic B cells were cultured with recombinant human IL-25 (20ng/ml) for 72 hours, then the viability and apoptosis of cultured cells were measured by MTT assay and AnnexinV/7AAD. Furthermore, the levels of CD69 expression on T lymphocytes and IL-17RB in T and B cells were determined by flow cytometry. The basal level of IL-17RB expression in CLL patients was significantly higher than that in control individuals. In addition, the percentage of IL-17RB+/CD3+, IL-17RB+/CD19+ cells and CD69+/CD3+ cells increased after 72 hours of culture with IL-25 in CLL patients compared to healthy subjects. IL-25 also reduces the apoptosis rate of tumor cells. We found that IL-25 could stimulate T cells in CLL patients and lower B cell death. This suggests that IL-25 might have a role in enhancing the survival of tumor cell by expressing receptors for inflammation, such as IL-17RB, and might be involved in the development of CLL