Antigen-specific monoclonal antibodies isolated from B cells expressing constitutively active STAT5.

BACKGROUND: Fully human monoclonal antibodies directed against specific pathogens have a high therapeutic potential, but are difficult to generate. METHODOLOGY/PRINCIPAL FINDINGS: Memory B cells were immortalized by expressing an inducible active mutant of the transcription factor Signal Transducer and Activator of Transcription 5 (STAT5). Active STAT5 inhibits the differentiation of B cells while increasing their replicative life span. We obtained cloned B cell lines, which produced antibodies in the presence of interleukin 21 after turning off STAT5. We used this method to obtain monoclonal antibodies against the model antigen tetanus toxin. CONCLUSIONS/SIGNIFICANCE: Here we describe a novel and relatively simple method of immortalizing antigen-specific human B cells for isolation of human monoclonal antibodies. These results show that STAT5 overexpression can be employed to isolate antigen specific antibodies from human memory B cells.

Downregulation of active caspase 8 as a mechanism of acquired TRAIL resistance in mismatch repair-proficient colon carcinoma cell lines.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers the apoptotic cascade in various colon cancer cell lines after binding to the membrane receptors DR4 and DR5. However, not all cancer cell lines are sensitive to the therapeutic recombinant human TRAIL (rhTRAIL). To investigate the causes of TRAIL resistance in colon cancer cell lines, models have been developed, mostly in mismatch repair-deficient cells. These cells are prone to mutations in genes containing tandem repeat, including pro-apoptotic protein Bax. We therefore investigated the mechanism underlying TRAIL resistance acquisition in a mismatch repair-proficient colon carcinoma cell line. The TRAIL-resistant cell line SW948-TR was established from the TRAIL-sensitive cell line SW948 by continuous exposure to rhTRAIL, and exhibited 140-fold less sensitivity to rhTRAIL in a cell viability assay. Resistance was stable for over a year in the absence of rhTRAIL. Both cell lines had similar TRAIL receptor cell membrane expression levels. Treatment with the protein synthesis inhibitor cycloheximide sensitized SW948-TR to rhTRAIL-induced apoptosis, indicating that the functionality of the TRAIL receptors was maintained. In SW948-TR, procaspase 8 protein levels but not mRNA levels were notably lower than in SW948. Downregulation of c-FLIP with short interfering RNA (siRNA) sensitized SW948-TR cells to rhTRAIL while caspase 8 siRNA decreased rhTRAIL sensitivity in SW948, indicating the importance of the caspase 8/c-FLIP ratio. Proteasome inhibition with MG132 did not restore basic procaspase 8 levels but stabilized cleaved caspase 8 in rhTRAIL-treated SW948-TR cells. Altogether, our results suggest that colon cancer cells can acquire rhTRAIL resistance by primarily reducing the basal procaspase 8/c-FLIP ratio and by increasing active caspase 8 degradation after rhTRAIL treatment. Proteasome inhibitors can effectively overcome acquired rhTRAIL resistance in mismatch repair-proficient colon cancer cells.

Generation of stable monoclonal antibody-producing B cell receptor-positive human memory B cells by genetic programming.

The B cell lymphoma-6 (Bcl-6) and Bcl-xL proteins are expressed in germinal center B cells and enable them to endure the proliferative and mutagenic environment of the germinal center. By introducing these genes into peripheral blood memory B cells and culturing these cells with two factors produced by follicular helper T cells, CD40 ligand (CD40L) and interleukin-21 (IL-21), we convert them to highly proliferating, cell surface B cell receptor (BCR)-positive, immunoglobulin-secreting B cells with features of germinal center B cells, including expression of activation-induced cytidine deaminase (AID). We generated cloned lines of B cells specific for respiratory syncytial virus and used these cells as a source of antibodies that effectively neutralized this virus in vivo. This method provides a new tool to study B cell biology and signal transduction through antigen-specific B cell receptors and for the rapid generation of high-affinity human monoclonal antibodies.

Generation of human antigen-specific monoclonal IgM antibodies using vaccinated "human immune system" mice.

BACKGROUND: Passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. However, the 'humanization' of murine monoclonal antibodies (mAbs) is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. The immortalization of human B cells represents an alternative for obtaining human mAbs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. In this work we describe a novel approach to generate fully human mAbs by combining a humanized mouse model with a new B cell immortalization technique. METHODOLOGY/PRINCIPAL FINDINGS: After transplantation with CD34+CD38⁻ human hematopoietic progenitor cells, BALB/c Rag2⁻/⁻IL-2Rγc⁻/⁻ mice acquire a human immune system and harbor B cells with a diverse IgM repertoire. "Human Immune System" mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis B surface antigen. Sorted human CD19+CD27+ B cells were retrovirally transduced with the human B cell lymphoma (BCL)-6 and BCL-XL genes, and subsequently cultured in the presence of CD40-ligand and IL-21. This procedure allows generating stable B cell receptor-positive B cells that secrete immunoglobulins. We recovered stable B cell clones that produced IgM specific for tetanus toxoid and the hepatitis B surface antigen, respectively. CONCLUSION/SIGNIFICANCE: This work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mAbs against a wide range of antigens.

Lessons from TRAIL-resistance mechanisms in colorectal cancer cells: paving the road to patient-tailored therapy.

Colorectal cancer is one of the leading causes of cancer-related deaths worldwide. Intrinsic, as well as acquired, resistance to chemotherapy remains a major problem in the treatment of this disease. It is, therefore, of great importance to develop new, patient-tailored, treatment strategies for colorectal cancer patients. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) acts through the pro-apoptotic DR4 and DR5 receptors in tumor cells without harming normal cells and will soon be tested in clinical trials as a novel anti-cancer agent. However, not all human colon cancer cell lines are sensitive to TRAIL due to intrinsic or acquired TRAIL-resistance. This review discusses the mechanisms and modulation of TRAIL-resistance in colon cancer cells. Cell sensitivity to TRAIL can be affected by TRAIL-receptor expression at the cell membrane, DR4/DR5 ratio and functionality of TRAIL-receptors. Additional intracellular factors leading to TRAIL-resistance affect the caspase 8/c-FLIP ratio, such as loss of caspase 8 and caspase 10 due to mutations or gene methylation, CARP-dependent degradation of active caspase 8 and changes in caspase 8 or c-FLIP expression levels. Further downstream in the TRAIL apoptotic pathway, Bax mutations, or increased expression of IAP family members, in particularly XIAP and survivin, also cause resistance. Chemotherapeutic drugs, NSAIDs, interferon-gamma and proteasome inhibitors can overcome TRAIL-resistance by acting on TRAIL-receptor expression or changing the expression of pro- or anti-apoptotic proteins.

Expression of TRAIL (TNF-related apoptosis-inducing ligand) and its receptors in normal colonic mucosa, adenomas, and carcinomas.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in tumour cell lines. Four membrane-bound receptors for TRAIL have been identified, two apoptosis-mediating receptors, DR4 and DR5, and two apoptosis-inhibiting receptors, DcR1 and DcR2. The aim of this study was to examine the role of TRAIL and its receptors in colorectal cancer development. The immunohistochemical expression and localization of TRAIL and its receptors were investigated in normal mucosa (n=10), adenomas (n=19), and carcinomas (n=21). Correlations between the expression of TRAIL and its receptors and the degree of apoptosis (assessed by M30 expression) and histopathological characteristics were explored. TRAIL and its receptors were expressed in normal mucosal epithelium. Expression of the receptors was seen in adenomas and carcinomas. TRAIL expression was lost in a subset of colorectal tumours, more frequently in carcinomas than in adenomas (p<0.05). DR4 and DR5 staining was stronger in neoplastic cells than in normal cells and was accompanied by a higher degree of apoptosis. No differences were found between tumour and normal cells regarding DcR1 and DcR2 expression. No correlations were found between TRAIL or TRAIL receptor expression and histopathological characteristics. In conclusion, marked changes were seen in the course of the adenoma-carcinoma sequence with respect to the expression of TRAIL and TRAIL receptors DR4 and DR5. The stronger expression of DR4 and DR5 in neoplastic cells than in normal cells, together with a higher degree of apoptosis, suggests a possible functional role for these receptors in apoptosis induction in neoplastic colorectal cells.