| Clinical research:Part 1 Correlation analysis between vitamin D level and immune checkpoint inhibitor efficacy and immune-related adverse events severityBackground:Immune checkpoint inhibitor(ICI)has brought landmark progress to tumor therapy.However,immune related adverse events(irAEs)are the main difficult problem in tumor immunotherapy.Vitamin D(VitD)plays an immunomodulatory role,and vitamin D receptor(VDR)is widely expressed on immune cells.Objective:We aimed to explore the relationship between serum VitD levels and ICIs efficacy and the occurrence/severity of irAEs.Methods:The patients with advanced thoracic cancers initially treated with anti-PD-1 therapy with or without irAEs were enrolled.We collected the clinical data and measured the serum 25-hydroxyvitamin D[25(OH)D]levels by liquid chromatography tandem mass spectrometry(LC-MS/MS)before and after ICIs treatment.Results:A total of 77 patients were enrolled,including 29 patients with irAE and 48 patients without.Among the 29 patients with irAEs,12 had single irAE and 17 had multiple irAEs.There were 42 irAEs in total.The baseline 25(OH)D level of partial response(PR)group was significantly higher than non-PR group(19.39±7.16 ng/ml vs.16.28±5.99 ng/ml,P=0.04).The median OS in VitD sufficient group,VitD insufficient group and VitD deficient group were 429 days,372 days,and 379 days and the differences were statistically significant(P=0.02).In irAE patients,the baseline 25(OH)D levels in mild irAE(grade 1)group was higher than moderate/severe irAE(grade 2/3/4)group(20.07±8.64 ng/ml vs.15.22±2.30 ng/ml,P=0.02).While,the 25(OH)D levels had no significant differences before and after ICIs treatment(P>0.05).Conclusions:The baseline 25(OH)D levels related to the ICI efficacy and irAE.While VitD levels could not improve correspondingly after ICI treatment with tumor control.Hence,the assessment of baseline VitD status and improvement of VitD insufficiency/deficiency is needed,in order to enhance the ICIs efficacy and attenuate irAEs with its excellent security.Clinical research:Part 2 Expression of vitamin D receptor in immune checkpoint inhibitors related colitisBackground:Vitamin D receptor(VDR)is a member of the steroid hormone receptor family,which is widely expressed in a variety of cells,including colonic epithelial cells and immune cells,and is involved in the pathogenesis and progression of cancer,immune diseases,infectious diseases,and endocrine and metabolic diseases.Vitamin D levels have been shown to be associated with immune checkpoint inhibitors related colitis.Objective:To compare the expression of VDR in colonic mucosa between patients with irAE colitis and healthy controls.Methods:7 patients with immune checkpoint inhibitors related colitis and 9 healthy controls admitted from June 2018 to December 2021 at the Endoscopy Center of Peking Union Medical College Hospital were enrolled.Expression of VDR in intestinal mucosa was detected by immunohistochemistry(IHC),and the difference was compared by semiquantitative analysis.Results:The expression of VDR in colonic mucosa was abundant in healthy controls and was widely distributed in colonic epithelial cells.Positivity rate of VDR in colon tissue of immune checkpoint inhibitors related colitis was significantly lower than that of healthy controls(6.0±0.3 vs.8.4±0.2,P<0.01).Conclusion:Expression of VDR in immune checkpoint inhibitors related colitis is downregulated.Part 3 The protective effects and mechanisms of Lactobacillus rhamnosus GG/Vitamin D3 on immune checkpoint inhibitors related colitis in miceBackground:Patients with VitD deficiency are more likely to develop high-grade irAEs after ICI therapy,and the expression of VDR in colonic mucosa is down-regulated in patients with irAE colitis.Lactobacillus rhamnosus GG(LGG)and its supernatant secretions have been reported to up-regulate the expression of VDR.Objective:This study was aimed to evaluate the protective effects and mechanisms of LGG and VitD3 alone and in combination treatment on immune checkpoint inhibitors related colitis in mice.Methods:25 C57BL/6 male mice were randomly divided into 5 groups(n=5)including DSS group,irAE colitis group,LGG group,VitD3 group and LGG combined with VitD3 group.DSS group was intraperitoneally injected 0.9%sodium chloride solution every other day from day 0 to day 9,and irAE colitis group was intraperitoneally injected antiPD-1 and anti-CTLA-4 every other day from day 0 to day 9.From day 4 to day 9,each group drank drinking water containing 2.5%DSS.LGG 2.5 × 10^8 CFU/d,VitD3 100IU/d,and LGG 2.5 × 10^8 CFU/d combined with VitD3 100IU/d were given to the intervention groups correspondingly by gavage.The severity of colitis was assessed by body weight loss,disease activity index(DAI)score,colon length and histopathological score.Colon samples were collected for evaluating the relative expression of cytokines by qRT-PCR and VDR expression in colon were detected by qRT-PCR and Western blot.The number of Treg cells in colon was evaluated by IHC semi-quantitative score.Results:Compared to the mice in DSS group,the body weight loss and DAI scores increased in mice from irAE colitis group(P<0.05),indicating that modeling is successful,and the expression of VDR protein decreased in irAE colitis group(P<0.05).Compared to irAE colitis group.the mice treated with LGG had lower body weight loss,colon length and histopathological scores(P<0.05).Body weight loss and histopathological scores decreased in the mice treated with VitD3(P<0.05).While the mice in LGG combined with VitD3 group displayed decreasing histopathological scores(P<0.05)and tended to have lower body weight loss(P=0.05).Compared with DSS group,the relative expression of IL-10 mRNA in colonic mucosa of irAE colitis group was increased(P=0.04),while there were no significant differences in the relative expression of IL-6 mRNA.IL-10 mRNA and TNF-α mRNA among other groups(P>0.05).There was no significant difference in the expression of VDR in colonic mucosa between the intervention groups and irAE colitis group(P>0.05).Compared with DSS group,there was no significant difference in the number of Treg cells in irAE colitis group.The number of Treg cells in LGG group(P<0.01)was significantly increased,and VitD3 group(P=0.07)showed an increasing trend,while there was no significant difference in LGG+VitD3 group.Conclusion:LGG and VitD3 alone and in combination alleviate the irAE colitis in mice.Part 4 Effect of Lactobacillus rhamnosus GG on differentiation of Treg cellsBackground:LGG has a protective effect on irAE colitis,but its mechanism is still unclear.Other experiments found that after adjusting the intestinal flora,the number of Treg cells in irAE colitis animal models and patients with irAE colitis increased and their metabolic function was enhanced.Objective:This study aimed to explore the effect of LGG on differentiation of Treg cells in vitro.Methods:CD4+naive T cells from human peripheral blood mononuclear cells were isolated by MACS and cells were cultured in 24 well plate which was in 4℃ incubation of plate-bound anti-CD3mAb and anti-CD28mAb for one night.Na?ve T cells were cocultured in vitro by different concentrations of LGG supernatant and cell culture medium respectively.All cells were cultured in 37℃ for 5 days,and the ratio of CD4+CD25+Foxp3+Treg cells was analyzed by flow cytometry.Results:Compared with culture medium,LGG concentration of 107 CFU/mL(47.83%±5.21%vs.40.47%± 3.96%,P=0.02),108 CFU/mL(53.00%± 3.78%vs.41.90%± 1.66%,P=0.04)and 5 × 108 CFU/mL(68.60%± 1.42%vs.33.00%± 3.28%,P<0.01)could significantly promote the differentiation of naive T cells to Treg cells,and the effect of LGG on differentiation of Treg cells was in a concentration-dependent manner.The proportion of CD4+CD25+Foxp3+Treg cells was highest when the concentration of LGG was 5 × 108 CFU/mL.Conclusion:LGG supernatant can induce peripheral blood CD4+CD25+Foxp3+Treg cells differentiation. |
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