A comparison of Ubiquitin E3 ligases as surrogate markers for T cell Anergy in active TB

Abstract

Background: Globally, there is an estimated 1.2 million TB-related deaths among HIV-negative people with active TB. Previous studies using ex-vivo and animal models indicate that CD4+ T cell unresponsiveness to Mtb, which is characterized by elevated levels of E3 ubiquitin ligases, along with reduced IL-2 cytokine production, might contribute to the immunopathogenesis and progression of TB to its active form. It remains unclear whether of CD4+ T cell anergy seen in in-vitro experiments and animal models are present in humans during active disease. This study therefore, aimed to determine if the expression of the E3 ligases; GRAIL, Cbl-b, and Itch, as well as IL-2, differs between individuals with active TB and those without TB. Study objectives: The study determined and compared expression (RNA: ng/ul) levels of ubiquitin E3 ligases (GRAIL, Cbl-b, and Itch), and IL-2 in CD4+ T cells from individuals with active TB and those with no TB upon ex-vivo stimulation with antiCD3 and anti CD3/antiCD28. Methodology: The study was cross-sectional, with a total of 13 participants; active TB (n=7) and No TB (n=6). Whole blood was collected, Peripheral Blood Mononuclear Cells (PBMCs) and CD4+ T cells were isolated. CD4+ T cells were stimulated with anti-CD3 and anti-CD3/anti-CD28 for 48 hours at 37oC in a 5% CO2 incubator. Cell pellets were lysed and RNA was extracted; Real-time qPCR was performed to measure expression (RNA: ng/ul) levels of E3 ligases Cbl-b, Itch and GRAIL. ELISA was used to measure IL-2 cytokine production (pg/ml) in the supernatant. Data was analyzed using GraphPad Prism 9.1.5. The Unpaired Student’s test was used to compare expression levels of E3 ligases and IL-2 in active TB versus no TB. The level of significance was p < 0.05. Results: Expression levels of E3 ligases (Cbl-b, Itch) was high in individuals with active TB than in individuals with no TB, when CD4+ T cells were stimulated with antiCD3/AntiCD28 and antiCD3 only. IL-2 in individuals with active TB was higher than those with no TB when subjected to the same treatment conditions. Conclusion: These findings provide insight into the immunological changes that may impact immunopathogenesis of TB.