Although many factors are known to limit CD4 T-cell recovery during HIV infection, the impact of alteration in T-cell lymphopoiesis remains poorly understood. We hypothesize that deficient T-cell differentiation from CD34 hematopoietic progenitors (HP) could, at least partially, explain poor CD4 levels restoration under c-ART in HIV+ patients.
PBMC were obtained from HIV+ immune responders under c-ART (IR, n=16, CD4=1086/mm3, CD4/CD8=1.7) and HIV+ c-ART nonresponders (INR, n=16, CD4=380/mm3, CD4/CD8=0.58) followed in CHU Henri Mondor (Creteil, France) and from HIV- controls (n=18, CTS, Creteil). Ethical committee approval and written informed consent from all subjects were obtained before study initiation. T-cell precursor’s frequency was determined using limiting dilution assay (LDA) on coculture of HP on OP9-DLL1 cell line. LDA results were generated using ELDA webtool. Flow cytometry analysis was performed on LSR2 cytometer, and FlowJo v8.2. Statistical analyses were performed using appropriate test in Prism software.
We have previously shown a specific T-cell, but not B-cell differentiation impairment in CD34 HP from INR patients (see table 1). Now we extended our knowledge by demonstrating that CD34 HP from INR appeared extremely sensitive in regard of extracellular ATP known to induce caspase-1 mediated pytoptosis. We found abnormally high P2X7 levels (ATP recognition) and absent CD73 (ATP hydrolysis) expression. Importantly, caspase-1 inhibitor, VX-765, disturbed normal lymphopoiesis in vitro, thus P2X7 antagonist, PPAD, restored T-cell lymphopoiesis in INR: T-cell frequency of 1/314.7 (1/186.3-532.3) without PPAD vs 1/145.3 (1/98.3-214.7) with PPAD (p<0.05). Transcriptomic analysis revealed a general alteration in cellular death pathway in INR as compared with IR individuals.
Our results suggest that CD34 HP from INR are more prone to cellular death via extracellular ATP that affects normal T-cell lymphopoiesis and impacts CD4 T-cell restoration under c-ART. Currently we are evaluating reasons of this dysregulation and possible ways to interfere with it in therapeutic perspective. Such complementary to c-ART strategies might dramatically improve outcomes in treated immune nonresponders.