Study Of Change Of The Immune Status Of HCMV Specific T Cell In Patients With Urothelium Cancer

Objective The peripheral blood samples from urothelium cancer patients wererandomly collected and were detected by many methods, such as virus antigendetection and specificity viral nucleic acid testing.To investigate the proportion ofhuman cytomegalovirus（HCMV）specitie CD₃⁺、CD₄⁺and CD₈⁺T cells in peripheralblood samples were detected by flow cytometry assay，and calculated the CD₄⁺CD₈⁺ratio, analysis of congenital HCMV active infection leads to changes in cellularimmunity status,so as to provide an effective means of immunotherapy and todetermine clinical efficacy with urothelium cancer patients.Methods （1）We collected33cases of peripheral blood samples from the urotheliumcancer patients,and were detected by following methods：HCMV IgG and IgM of33blood specimens was detected by indirect enzyme-linked immunosorbentassay（ELISA）.HCMV isolation, observing cytopathic effect （CPE） of HCMV.Theidentification of HCMV isolates:HCMV-DNA was detected by HCMV IE and UL83RT-PCR; HCMV pp65antigen by indirect immunofluorescence assay （IFA）.Theperipheral blood mononuclear cells（PBMCs） were separated from peripheral bloodsamples,then the total DNA was extracted from PBMCs of peripheral bloodsamples.The PBMCs samples of HCMV-DNA was detected by HCMV UL83PCRand HCMV UL55nested-PCR. The PBMCs were separated from peripheral bloodsamples and were detected by HCMV-pp65antigenemia assay. The positive samplesof HCMV isolation, HCMV pp65antigenemia assay,HCMV IgG and IgM, HCMVUL83PCR and HCMV UL55nested-PCR was HCMV active infection, the otherswas without HCMV active infection.（2）0.4ml peripheral blood was taken from33 peripheral blood samples, then the proportion of T lymphocyte subpopulation incluingCD₃⁺, CD₄⁺and CD₈⁺from the peripheral blood samples were detected by flowcytometry assay, and calculated the CD₄⁺CD₈⁺ratio.The proportion of HCMVspecific CD₃, CD₄and CD₈were measured using pentamer folded withHCMV-petide NLVPMVATV, and calculated the CD₄⁺/CD₈⁺ratio.Results （1） Laboratory diagnosis diagnostic criteria of human cytomegalovirusactive infection: Positive rate of ELISA HCMV IgG and IgM were93.9%（31/33） and21.2%（7/33）;Viral isolation rate was3.0%（1/33）; The HCMV isolates was detectedby HCMV IE and UL83RT-PCR,and were sequenced and compared with the genesequence database, The HCMV isolates of HCMV pp65IFA was positive. Positiverate of HCMV UL83PCR and UL55nested-PCR were21.2%（7/33）and27.3%（9/33）, the result were sequenced and compared with the gene sequence database.The positive rate of HCMV pp65antigenemia assay was18.2%（6/33）; HCMV activeinfection rate was18.2%（6/33） in patients with urothelium cancer.According to theHCMV active infection results, it divided into2groups that HCMV active infectiongroup（6cases） and HCMV unactive infection group （27cases）.（2） Results of changesin HCMV non-specific T lymphocytes subsets by flow cytometry assay：The CD₃⁺lymphocyte in the HCMV active infection group and the HCMV unactive infectiongroup by flow cytometry assay was （66.55±3.53）%and （75.68±6.48）%, and there wasdifference between two groups（P﹤0.05）.The CD₄lymphocyte of them was（26.52±4.95）%and （43.54±6.76）%, and there was significant difference between twogroups （P﹤0.01）,. The CD₈lymphocyte in the HCMV active infection group and theHCMV unactive infection group by flow cytometry assay was （37.25±4.01）%and（29.76±8.26）%, and there was difference between two groups（P﹤0.05）. The CD₄⁺CD₈of them was （0.73±0.18） and （1.62±0.63）, respectively, and there wassignificant difference between two groups （P﹤0.01）.Copare with the without HCMVactive infection group,the proportion of CD₃⁺and CD₄⁺T cells and the percentage ofCD₄⁺/CD₈⁺ratio were decreased in HCMV active infection group,CD₈⁺T cells were increased in HCMV active infection group.The HCMV specific CD₃⁺T cells in theHCMV active infection group and the HCMV unactive infection group by flowcytometry assay was （64.67±5.47）%and （73.10±5.34）%, and there was differencebetween two groups（P﹤0.05）. The HCMV specific CD_＋4T cells of them was（24.25±3.90）%and （45.67±6.28）%, and there was significant difference between twogroups （P﹤0.01）.The HCMV specific CD_＋8T cells in the HCMV active infectiongroup and the HCMV unactive infection group by flow cytometry assay was（39.08±5.80）%and （25.17±7.59）%, and there was significant difference between twogroups（P﹤0.01）. The CD_＋4CD_＋8of them was （0.64±0.16）%and （1.95±0.59）,respectively, and there was significant difference between two groups （P﹤0.01）.Copare with the HCMV active infection group,the proportion of HCMVspecific CD₃⁺and CD₄⁺T cells and the percentage of CD₄⁺/CD₈⁺ratio were decreasedin HCMV active infection group,HCMV specific CD₈⁺T cells were increased inHCMV active infection group.Conclusion （1） HCMV active infection rate was18.2%in patients with urotheliumcancer.The urothelium cancer patients has higher the possibility of active HCMVinfectionaged above50years of age, and the patients of HCMV active infection aremore prone to urothelium cancer recurrence or low back pain symptoms.urotheliumcancer patients with higher pathological grade, the greater the probability of HCMVactive infection.（2）Copare with the without HCMV active infection group, the proportion of CD₃⁺andCD₄⁺T cells and the percentage of CD₄⁺/CD₈⁺ratio were decreased in HCMV activeinfection group,the proportion of CD₈⁺T cells were increased in HCMV activeinfection group; the proportion of HCMV specific CD₃⁺and CD₄⁺T cells and thepercentage of CD₄⁺/CD₈⁺ratio were decreased in HCMV active infection group,theproportion of HCMV specific CD₈⁺T cells were increased in HCMV active infectiongroup.Visible,The urothelium cancer patients with HCMV active infection play a significant inhibition of normal function on HCMV specific cellular immune system,It is closely related to obstruct the normal development of the urothelium cancerpatients.（3）The abnormal symptoms of urothelium cancer patients was related with HCMVspecific cellular immune status abnormalities in HCMV active infectiongroup.Especially, the older the recurrence of urothelium cancer was more prone toHCMV active infection, which leads to non-specific and HCMV-specific cellularimmune status abnormalities.The urothelium cancer patients with HCMV activeinfection play a significant inhibition of normal function on HCMV specific cellularimmune system. It is necessary to establish a follow-up file and do medicalexamination to33cases of urothelium cancer patients.