Study Of The Effect Of PD-1 And TIGIT Molecules On TB-specific IFN-γ Secretion

Diagnosis of active TB infection in subects with acquired immunodeficiency virus(HIV)remains a challenge.Interferon gamma release assay(IGRA)is an immunological test for detection of Mycobacterium tuberculosis(MTB)infection,which can effectively detect TB mono-infection.The TB-specific IFN-γ Elispot assay is one type of gamma interferon release assay,but sensitivity of TB-specific IFN-γ Elispot assay to detect active tuberculosis infection in HIV patients is significantly reduced.Published data supported that the up-regulated expression of immune checkpoint molecules including PD-1,LAG3 and TIGIT on T cells among peripheral blood mononuclear cell(PBMCs)of HIV patients or active TB(a TB)patients contributes to the impaired HIV or TB-specific cellular immune response.In this study,we studied the effect of immune checkpoint PD-1 and TIGIT on sensitivity of TB-specific IFN-γ Elispot assay to detect TB mono-infection and HIV/ a TB co-infection.In order to accurately evaluate the effect of PD-1 and TIGIT on secretion of TBspecific IFN-γ by PBMC,a rapid,sensitive and cost-effective method to determine the blocking activity of PD-1 and TIGIT inhibitors(or conferred to as blocking antibodies)was established using micrometer magnetic beads in this study.Compared to the TIGIT stably expressing cells based ligand binding assay,the mag-beads based ligand binding assay showed two times higher in detection sensitivity,and the relative inhibitory activity of two TIGIT inhibitors determined by the cells based binding assay and the beads based binding assay are almost identical.Furthermore,the sensitivity of the beads based binding assay to determine blocking activity of a PD-1 inhibitor,Nivolumab,was at least 8 times higher than that of the published methods.On the other hand,the beads based binding assay was able to rapidly and cost-effectively determine blocking activity of TIGIT and PD-1 inhibitor within 90 minutes using 1/10 reagent by the functional ELISA assay.By this assay,TIGIT inhibitor “A15153G” and PD-1 inhibitor “Nivolumab” was identified showing better blocking activity among two TIGIT inhibitors and two PD-1 inhibitors,respectively.Compared to healthy donors,PD-1 and TIGIT were significantly up-regulated on PBMC T cells of TB patients and HIV/TB co-infected patients.In addition,PBMC T cells from HIV/TB co-infected subjects showed greater expression of PD-1 and TIGIT than PBMC T cells from TB mono-infected subjects.In the TB-specific IFN-γ ELISPOT assay,co-administration of the PD-1 and TIGIT inhibitor(Nivolumab and A15153 G,respectively)significantly increased spot amount for PBMC of both TB patients and HIV/TB co-infected subjects.Notably,rate to detect TB from PBMC of HIV/TB co-infected subjects increased from 33% up to 66% upon co-administration of Nivolumab and A15153 G.These data indicated that co-administration of inhibitors of PD-1 and TIGIT was able to improve rate to detect TB with PBMC of HIV/TB co-infected subjects by the TB-specific IFN-γ ELISPOT assay.