Experimental Study Of Therapeutic Effect Of Agonist Anti-CD40 MAb Combined With CTL In Hu-SCID Mouse B Lymphoma Model

CD40, a member of TNFR superfamily, is a type I membrane glycoprotein with the Mr of 48KD. Human CD40 molecule was found in 1980s and encoded by a gene located at 20q11-20q13.2. Its cDNA with the length of 1.5KD encoded 277 amino acid (AA). Interaction by CD40/CD40L is a main signal during immune response. CD40 is expressed on many types of cells including B cells, dendritic cells, endothelial cells epithelial cells and also many carcinoma cells, such as B lymphoma cells. B lymphoma is malignant tumor mostly derived from B cells in lymphoid tissues. With the significant advance in chemotherapy and radiotherapy, about 50-60% patients suffering from this disease can achieve complete regression (CR). However, in many cases residual tumor cells always cause the recrudescence of the same disease. 5cll is an agonist monoclonal antibody prepared in our department. Treatment with 5c11 influenced biological behavior of B lymphoma cell line Daudi including proliferation inhibition, cell cycle arrest and enhancement of the sensitivity to apoptosis. Experimental results indicated the significant therapeutic potential of 5c11.The SCID mouse is of particular interest because of the absence of mature and functional T and B lymphocytes. Due to a defect in the recombinase system, linked to a nonsense mutation of DNA-dependent protein kinase catalytic subunit, SCID mice cannot successfully rearrange their immune receptor genes and consequently have no mature T or B cells. Thus, SCID mice can tolerate a graft with human cells and particularly those of purified peripheral blood mononuclear cells (PBMC) administered by intraperitonealinjection. The reconstituted SCID mice also called humanized SCID (hu-SCID) mouse, provide a unique model to particularly study tumor immunotherapy in an in vivo animal system.In this study we aimed to establish hu-SCID mouse human B lymphoma model and study the therapeutic effect of 5c 11, CTL or 5c 11 combined with CTL in this model.1. Establishment and identification of hu-SCID mouse medelSCID mice were treated by CTX to inhibit the hemocytopoiesis. With successive 4-day injection, human peripheral blood mononuclear cells (PBMC) were engrafted into SCID mice through intraperitoneal injection. After 4, 8 and 12 weeks of engraftment , peripheral blood, spleen and liver tissues of engrafted SCID mice were harvested. Human CD3+, CD19+ cells in peripheral blood were analyzed by fluorescence microscopy and FCM; human CD3+, CD19+ cells in spleen and liver tissues were detected by immune histochemistry; human IgG level in SCID mouse serum was measured by ELISA. Results showed that after engraftment of 4, 8 and 12 weeks, human CD3+, CD19+ cells in SCID peripheral blood were identified by fluorescence microscopy and the percents were 31%> 10% respectively determined by FCM. Through immune histochemistry human CD3+> ' CD19+ cells were detected in mouse spleen but not in liver tissue. Furthermore the titer of human IgG in mouse serum was 390ug/mh 1 lOOug/ml and 1040|xg/ml at each time point respectively.2. 5cll promoted the cytotoxity of CTL to B lymphoma cell line Daudi cells in vitroHuman B lymphoma cell line, Daudi cells were treated with 5ug/ml CD40 mAb (5C11) for 24 hours. Annexin V/PI binding assay was used to analyze apoptosis, FACS to analyze expression of Fas (CD95). Human PBMC derived DCs was loaded with apoptotic Daudi cells and stimulated by 5c 11 for additional 48 hours to maturation. Tumor specific CTL was generated in vitro by autologous T cells co-cultured with mature DCs. Daudi cells were co-cultured with 5c 11, CTL or 5c 11 combined with CTL. DNA fragmentations were detected by JAM assay. The results showed that 5c 11 did not induce Daudi cells apoptosis, but could up-regulate the expression of CD95. CTL could lyse Daudi cellseffectively after co-cultured for 8 hours and the fragmentation of Daudi cells wassignificantly enhanced by the combination of 5c 11.3. Therapeutic effect of 5cll, CTL and Sell combined with CTL on B lymphomaB lymphoma model was established in humanized SCID mouse (hu-SCID) by subcutaneous injecting Daudi cells. 1 week and 3 weeks after transplantation, the mice were regarded as minimal tumor burden and extensive tumor burden mice respectively. Tumor bearing mice were treated with 5cll, CTL or 5cll combined with CTL respectively by intraperitoneal injection. Results showed that all the treatments could postpone the growth of tumor in vivo and significantly prolonged the survival of tumor bearing mice. Minimal tumor burden mice got the CR of 33% or 67% respectively when received CTL treatment or the combination treatment of 5cl 1 and CTL. Furthermore, 5cl 1 also promoted the secretion of IFN-y in vivo. CD25+ cells could be detected in the tumors of mice treated by CTL or 5c 11 combined with CTL, and the amount in combination treatment mice was more than that of CTL single treatment.