The Role Of Toll-Like Receptors In Mantle Cell Lymphoma

Background and Purpose: MCL（Mantle Cell Lymphoma，MCL）is a kindof B cell malignancies, in2008WHO reclassified lymphatic hematopoieticdiseases, MCL was assigned to be the Mature B cells malignant tumor, whichis accounted for5-10%of non-hodgkin’s lymphoma. MCL tumor cellsoriginally were from primary follicles or secondary follicle by the antigenstimulation of CD5+CD23-naive fringes or peripheral blood memory B cells.The major treatment for MCL is chemotherapy. Although various treatmentshave been improved, but the complete response rates of patients are in differentlevels, overall survival is still poor[1]. Therefore, new therapeutic strategies areneeded to improve the overall survival of patients and decreasetreatment-associated morbidity.TLRs (Toll-like receptors, TLRs) are pattern recognition receptors whichevolute conservative and embryonic encoding. TLRs played important roles inidentification of pathogenic microorganisms[2]. TLRs were first found in thebody of the Fruit flies in1988［3］, scholar found its homologue in the humanbody in1997, called human Toll proteins［4］. Until now, thirteen have beenfound in human and mice. TLRs are transmembrane proteins, extracellularregion rich in leucine, transmembrane region that rich in cysteine,TOLL/IL-1homologous intracellular area in the vast majority of the bloodmalignant tumor. TLRs have different expression levels in B cell lymphoma and myeloma［5-8］, which have biological effects. In order to understand therole of TLRs in MCL patients, we detected the expression of TLRs in MCL,and the effect of TLR ligand to MCL cell lines. The ligand of TLR9is CpGmotif. CpG motif is a set of nucleotide sequences, which is unmethylated andrich in cytosine guanine, extracted from bacterial DNA and first found andapplied by an American scholar［9］. But CpG motif is easily degradated. Sosynthetic CpG-DNAs, which are made of denucleotides, are useful in studies.Many in vitro experiments reported that CpG ODNs invovled in anti-tumortreatment, improving efficiency of vaccines, and allergies［10］. It has beenrepoted in1996that CpG bind to TLR9to activate transcription factors andfaciliated transcription of related genes of cytokines［11］. Such as, CpG ODNsignificantly induced apoptosis of chronic lymphocytic leukemia cells purifiedfrom patients, which is associated with the activation of JAK/STAT pathway［12］.In order to understand the TLR in MCL, we studied the expression ofTLRs and the effects of TLR ligands on MCL cell lines to provide clues forfurther researches. It will also provide new potential therapeutic targets forimproving the therapeutic effect of MCL and survival and prognosis of disease.Methods:1.Detecting the expression of TLRs： （1）RNA was extracted from MCL cell lines in the logarithmic growth andwas reversed to cDNA according to manufactures,instructions. The expressionsof TLRs were detected by PCR and real time PCR;（2）The expression of TLR9in MCL cell lines was detected by intracellularstaining;（3）The expression of TLR9protein in MCL cell lines was detected byWestern Blot;（4）The expression of TLR9in MCL patients,lymph node was identified, andlymph node from health donor was used as control.2. Observing morphology change of MCL cells before and after typeB CpG684stimulation by giemsa staining.3. Detecting the apoptosis induced by type B CpG in MCL cell lines: The MCLcell lines in the logarithmic growth period were tested, typeB CpG684stimulated cells for five days, typeA CpG2216was used as negative control.Results：1. Expression of TLRs in mRNA level：（1）PCR results: TLR1-10expression level of MCL cell lines G519and Minoare different, TLR3,5,9and10showed high expressions;（2）Real time PCR results：in MCL cell lines G519and Mino, TLR1-10havedifferent expressions, among them, TLR9is highest, the fold is1.45×104±168; 2. Expression of TLR9in protein level：（1）Western blot result：The expression of TLR9in MCL cell lines G519andMino have obvious stripe;（2）Flow cytometry result：MCL cell lines G519and Mino showed theexpression of TLR9;（3）The immunohistochemical result: The expression of TLR9in MCLpatients has been detected.3. Giemsa’s staining: After stimulated by typeB CpG684, MCL Cells becomelarger, nuclear is more obvious.4.CpG2216and CpG684stimulate MCL cell lines：TypeB CpG684stimulate MCL cell lines, induce apoptosis of cells, and isdose dependent; typeA CpG2216stimulate MCL has no significant change.Conclusion：1. The expression of TLR1-10in MCL cell lines are different, the higher oneis TLR9. This is confirmed in lymph node of MCL patients.2. After stimulated by typeB CpG684, MCL Cells become larger.3. After stimulated by CpG684, apotosis of MCL cell lines were induced.