CD28 Superagonist Antibody Proliferating Foxp3-expressing Regulatory T Cells And The Effects On DSS-induced Colitis In Foxp3^EGFP Mice

CD28 Superagonist Antibody proliferating Foxp3-expressing regulatory T cells and the effects on DSS-induced Colitis in Foxp3EGFP miceBackground and Purpose:CD4+CD25+Foxp3+regulatory T cells (Treg) is a class of independent T-cell population accounting of about 5% to 10% in peripheral CD4+T cells. Treg cells are important for immunological homeostasis, self-tolerance and transplantation tolerance. Superagonistic CD28-specific monoclonal antibody(supCD28 MAb, clone:D665)can fully activate T cells and preferentially proliferate CD4+Foxp3+Treg cells that increase the effects of anti-inflammation and regulate autoimmunity.Studies have shown that CD4+CD25+Foxp3+regulatory T cells play an important role in the pathogenesis of IBD, lack of Treg cells induced severe autoimmune colitis because of hyperresponsiveness in the symbiotic intestinal microflora of mice. On the other hand infusion of Treg cells in SCID mice can alleviate inflammation in the CD4+CD45RBHIGHT cells induced colitis. DSS-induced colitis, similar to human ulcerative colitis, is recognized as an animal model of IBD.The aims of this study:(1) investigate the effects of proliferation Treg cells by intraperitoneal injection of supCD28 MAb in Foxp3EGFP mice and influence cytokines related to the differentiation pathway of Foxp3 and Thl, Th2. It is proved that the proliferatied Treg cells not only increase the quantity, but also have the same function. (2) investigate the effects of supCD28 MAb on DSS-induced colitis to provide a new experimental evidence to treat IBD.Methods:Part I:30 Foxp3EGFP mice were randomised divided into 5 groups. Group 1-4 injected with supCD28 MAb (n=6 each group) as treated group and group 5(n=6) as control group. The treated group were given single dose of sup CD28MAb 1mg/mice by intraperitoneal injection. The lymphocytes were collected from spleens, mesenteric lymph nodes, thymus and peripheral blood at day 3, day 7, day 14 and day 30 after injection. The lymphocytes stained by anti-CD4, CD62L, CD103, CD152 antibodys were analysed on flow cytometry to compare Treg cells level in each group. Total RNA was extracted from lymphocytes in spleen and mesenteric lymph nodes,then reverse transcripted to cDNA. Real-time RT-PCR was used to analyse Foxp3, IFN-γ, TNF-α, IL-6, IL-10, TGF-βin each group.PartⅡ:20 Foxp3EGFP mice were randomised divided into control group (n=6), DSS model group (n=6), and the DSS model supCD28MAb treatment group (n=8). Control group were given distilled water, DSS model group were given 3.5% DSS solution for free drink, the treatment group were given supCD28 MAb by intraperitoneal injection at the first day of 3.5% DSS solution feeding. Disease Activity Index(DAI) was evaluated daily. All of the mice were sacrificed on day 8, then total colon specimens were measured and stained by HE, applied for the pathologic score of colon. Spleen and mesenteric lymph nodes cells in model group and treated group were stained with anti-CD4, CD62L, CD 103, CD 152 antibodies, analyzed by FACS, comparing changes of Treg cells in the two groups. Total RNA was extracted from lymphocytes in the specimens of spleen and colon, then reverse transcript to cDNA. Real-time RT-PCR was used to analysis Foxp3, IFN-γ, TNF-α, IL-6, IL-10, TGF-βin each group.Results:PartⅠ:1. Three days after injection with supCD28 MAb, the volume of spleen, mesenteric lymph nodes significantly increased compared with the control group, while the thymus volume has not changed. Foxp3+CD4+Treg cells increased about 3 fold in spleen and mesenteric lymph nodes,2-fold in peripheral blood lymphocytes. Foxp3+CD4+Treg cells in thymus were not statistically different in the two groups.2. In Mesenteric lymph nodes, the proportion of Foxp3+CD4+Treg cells reached a peak at day3 after injection, then decreased gradually, and restored to the original level in about 30 days. In spleen and peripheral blood, the proportion of Foxp3+CD4+Treg cells reached a peak at day7 after injection, then decreased gradually, and return to the original level at day30. In thymus, significant changes in proportion of Foxp3+CD4+Treg cells were not observed.3. In treated group, the proportion of CD103, CD152 and CD62L expression on Foxp3+Treg cells were 5～10 times higher than that in controls in the spleen and mesenteric lymph node 3 days after injection.4. Real-time quantitative PCR results showed that:the expression of Foxp3 in spleen lymphocytes was significantly increased compared with the control group at day3 after injection, reached a peak of about 18.5-flod. The expression of Th1 and Th2 pathway cytokines both increased, and IL-10 increased to 151-fold most significantly 3 days after injection.PartⅡ:1. The general condition of mice in the supCD28 MAb treated group was better than condition of the DSS model group. Total length of colon in supCD28 MAb treated group 8.00±0.44cm was longer than the DSS model group 6.63±0.30cm (p<0.05), DAI score 1.75±0.88 was significantly lower than the DSS model group 3.41±0.57, and was statistically significant (p<0.05). The histological score of colon in supCD28 MAb treated group 8.50±5.07 was significantly lower than the DSS model group 29.00±3.83 (p<0.05).2. In supCD28 MAb treated group, the proportion of CD103, CD152 and CD62L expression on Foxp3+Treg cells were higher than that in the DSS model group in the spleen and mesenteric lymph node.3. Real-time quantitative PCR results showed:In the colon and spleen the expression of anti-inflammatory cytokines, such as Foxp3, IL-10, TGF-β, were significantly reduced in DSS model group, but in supCD28 MAb treated group they remained in high levels. Pro-inflammatory cytokines, such as IL-6, were significantly higher in DSS model group than that in supCD28MAb treated group.Conclusion:1. A single intraperitoneal injection of supCD28 MAb could preferentially increase CD4+ Foxp3+Treg cells in vivo and with the same immunological function.2. CD4+Foxp3+Treg cells proliferated by intraperitoneal injection of supCD28 MAb in mice may influence the differentiation pathways of Foxp3, Th1, Th2, Treg cells, primarily by secreting Foxp3, IL-10.3. SupCD28 MAb can reduce the damage of colon in DSS-induced colitis by increasing the quantity of CD4+Foxp3+Treg cells, the mechanism is probably by secreting considerable amount of anti-inflammatory factors, especially IL-10, to regulate the Th1/Th2 balance.