| Introduction:As a common opportunistic infection of HIV infected patients and the main factor leading to the death of HIV patients,TB infection could be accelerating the processes of HIV infection.Although the typical clinical symptoms and signs,imaging features and immunological characteristics of HIV/TB infection were different from TB infection,the sensitivities of the traditional detection methods were greatly reduced as well as the difficulties of those increased.It was resulted that misdiagnosises and delays of the therapy opportunities would be probably occurred to caues the death or/and the spread of the tuberculosis in patients.The aim of this work is to reduce the lymphocyte immunosuppressive effect of HIV infected individuals by antibodies of LAG-3,TIM-3,CTLA-4 and PD-1,to improve the immune responses of F-SPOT.TB to TB specific antigens and further improve the detection rates of TB infection for HIV/TB coinfected patients.Objectives:To increase the detection rates of TB infection for HIV/TB coinfected patients with the improved F-SPOT.TB(refered as T cell spot test of tuberculosis infection),the negative costimulators of LAG-3(Lymphocyte Activation Gene-3),TIM-3(T Cell Immunoglobulin Mucin-3),CTLA-4(Cytotoxic T Lymphocyte-associated Antigen-4)and PD-1(Programmed Cell Death Protein-1)were involved,which were to decrease the immunosuppressive effect of lymphocyte for HIV infected patients.Methods:The frozen peripheral blood mononuclear cells(PBMC)of the 38 HIV/TB coinfected patients(negative values of traditional F-SPOT.TB,the concentrations of CD4~+T cells<200 unit/ml),the 14 HIV/TB coinfected patients(positive values of traditional F-SPOT.TB,the concentrations of CD4~+T cells<200 unit/ml),the 18 HIV infected patients(negative values of traditional F-SPOT.TB,the concentrations of CD4~+T cells<200 unit/ml)and the 12-health people(negative values of traditional F-SPOT.TB)were collected.The immunofluorescence flow cytometry was used to detect the expression levels of the negative costimulators of LAG-3,TIM-3,CTLA-4 and PD-1 on CD4~+T cells and CD8~+T cells in each experimental group.The expression of IFN-?on CD4~+T cells and CD8~+T cells was analyzed.On the basis of the traditional F-SPOT.TB,the corresponding antibodies of the negative costimulators of LAG-3,TIM-3,CTLA-4 and PD-1 were added into the blocking holes of each sample to significantly evaluate the experimental results according to the of spot numbers in the blocking holes.Flow cytometry results were checked by the BD FACS Canto II Diva software.The data were recorded with Microsoft Excel 2016,analyzed with the unilateral analysis(ANOVA,SPSS v11.5 for Windows)at the interval level of p<0.05 and pictured with the Origin v 7.5 Professional software.Results:(1)Screening of Negative Costimulators by Flow Cytometry:The expressions of negative costimulators(LAG-3,TIM-3,CTLA-4,PD-1)on HIV/TB confirmed by flow cytometry were much more significant compared with healthy individuals and single HIV ones,reflacting that whether in CD4~+T or in CD8~+T,the expressions of LAG-3,TIM-3,CTLA-4 and PD-1 of HIV/TB patients were highest,second for HIV patients,final for healthy people(p<0.05).Thus,it is expected to improve TB detection rates for HIV/TB negative patients to further screen out HIV/TB co-infection in by F-SPOT.TB with the combinations of the negative costimulators(LAG-3,TIM-3,CTLA-4 and PD-1)and their antibodies.(2)Immunofluorescence and Flow Cytometry Analysis:It was meant that the secretions and expressions of IFN-?were much significantly reduced in the peripheral blood TIM-3~+CD4~+T lymphocyte and TIM-3~+CD8~+T lymphocyte,PD-1~+CD4~+T lymphocyte and PD-1~+CD8~+T lymphocyte compared with those in TIM-3~-CD4~+T lymphocyte and TIM-3~-CD8~+T lymphocyte,PD-1~-CD4~+T lymphocyte and PD-1~-CD8~+T lymphocyte.Moreover,the secretions and expressions of IFN-?~+CD4~+T cells and IFN-?~+CD8~+T cells of HIV/TB coinfected patients were also decreased at the level of p<0.05.(3)Increase of TB Detection Rates for HIV/TB Patients by Improved F-SPOT.TB:The improved F-SPOT.TB did increase the numbers of the 16 HIV/TB coinfected patients(negative values of traditional F-SPOT.TB)in the 38 HIV/TB coinfected patients(negative values of traditional F-SPOT.TB),meaning that the significantly positive values were of42.1%at the level of p<0.05.The test numbers of the 14 HIV/TB coinfected patients(positive values of traditional F-SPOT.TB)by the improved F-SPOT.TB were not changed,as well as those of the 18 HIV infected patients(negative values of traditional F-SPOT.TB)and the 12-health people(negative values of traditional F-SPOT.TB).It was meant that there were no false positive and/or false negative judgments.Conclusion:Whether in CD4~+T lymphocyte and CD8~+T lymphocyte in the peripheral blood,the expressions of LAG-3,TIM-3,CTLA-4 and PD-1 of HIV/TB patients were higher.The expressions of IFN-?were much significantly reduced in the peripheral blood TIM-3~+CD4~+T lymphocyte and TIM-3~+CD8~+T lymphocyte,PD-1~+CD4~+T lymphocyte and PD-1~+CD8~+T lymphocyte.Moreover,the expressions of IFN-?~+CD4~+T cells and IFN-?~+CD8~+T cells of HIV/TB coinfected patients were also decreased at the level of p<0.05.Comparied with the traditional F-SPOT.TB,the improved F-SPOT.TB could increase the 42.1%points for the 16 HIV/TB coinfected patients in the 38 HIV/TB coinfected patients without checked out by traditional F-SPOT.TB(negative values of traditional F-SPOT.TB,the concentrations of CD4~+T cells<200 unit/ml)who would be recognized to get the treatment on TIMe.The TB detection rates for HIV/TB coinfected patients by the improved F-SPOT.TB were increased with the helps of the antibodies of the negative costimulators of LAG-3,TIM-3,CTLA-4 and PD-1,especially on the HIV/TB coinfected patient groups of the concentrations of CD4~+T cells<200 unit/ml. |
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