Figure 1

Signalling defects in highly differentiated CD8⁺ T cells. Buffy Coat samples were obtained from the Transfusion Center of University of Rome ‘Tor Vergata’. T-cell subsets were isolated according to the expression of the co-stimulatory molecules CD28 and CD27 using Miltenyi Biotec microbeads (Auburn, CA, USA) and activated with anti-CD3 antibody, Ab (purified OKT3 0.5 μg/ml), in the presence or absence of rhIL-2 (5 ng/ml, R&D systems, Minneapolis, MN, USA) at 37 °C in a humidified 5% CO₂ incubator. CD45RA/27-based populations were obtained by labeling CD8⁺ T cells with conjugated anti-CD27 and CD45RA Abs (BD Biosciences, San Jose, CA, USA) and sorting populations using a FACSAria (BD Biosciences). This separation gives four populations with progressive features in terms of differentiation and ageing, with naive cells being CD27⁺/CD45RA⁺, central memory CD27⁺/CD45RA⁻, effector memory CD27⁻/CD45RA⁻ and highly differentiated effector memory (EM)-like phenotype (EMRA) that re-express the CD45RA molecule. Abs used for western blot analysis were from Cell Signalling (Danvers, MA, USA) and Santa Cruz Biotechnology (Dallas, TX, USA). The quality of extracts and loading control was tested by probing membranes with anti-GAPDH and anti-Histone H1 Abs for cytoplasmic and nuclear extracts, respectively. Cytoplasmic extracts were negative for Histone H1 (data not shown). For co-immunoprecipitation analysis HNGT-cell lysates were immunoprecipitated with anti-AKT1 Ab, separated by SDS-PAGE, and probed with RICTOR, HSP90 and hTERT Abs. (a) Representative dot plots of the phenotypic analysis of CD28/27 and CD45RA/27 CD8⁺ T-cell populations. (b) To achieve full activation, AKT has to be phosphorylated at Ser473 by mammalian target of rapamycin (mTOR) complex 2 (mTORC2). CD28⁻/27⁻ subpopulation showed impaired RICTOR-AKT signaling activation and HSP90 expression partially reversed by rhIL-2. This result was confirmed by reduced levels of AKT-co-immunoprecipitated RICTOR, HSP90 and hTERT. Improper function of HSP90 client proteins can be induced by the HSP90 inhibitor geldanamycin (100 nM), which reduced pSer473-AKT and hTERT nuclear import in naive CD28⁺ T cells without affecting T-cell viability. (c) Reduced HSP90 and pSer473-AKT levels were found also in highly differentiated CD8⁺ T cells separated by CD45 RA and CD27-based isolation