Fig. 2. CD45RA/CD276 depletion maintains immune reactivity toward third party and common viral antigens. Remaining alloreactive memory T cells are of T_H17 phenotype.

a CD45RA/CD276 depletion maintains immune response to viral antigens. CD45RA/CD276-depleted, CD45RA-depleted, and undepleted donor PBMCs were exposed for 4 h to a virus peptides mix covering the complete sequence of CMV pp65, EBV consensus, and AdV5 antigens and the frequencies of IFN-γ⁺ and TNF-α⁺ T cells were determined (n = 4). A viral specific response was calculated as follows: (frequencies of IFN-γ- and TNF-α-positive T cells in culture pulsed with viral peptides mix) − (frequencies of IFN-γ- and TNF-α-positive T cells in culture without peptide mix). CD45RA/CD276 depletion maintains immune response to third party. Following CD276 depletion on day 4, remaining cells were stimulated with third party PBMCs and frequencies of IFN-γ⁺ and CD69⁺ T cells were analyzed at day 11 of haplo-MLC by flow cytometry. CD69 coanalysis served as indicator for activation (n = 4). b Transcription factor profile of CD3⁺CD276⁺ and CD3⁺CD276⁻ T cells in CD45RA-depleted haplo-MLC at day 4 was analyzed by qPCR (n = 4). c Transcription factor profile of IFN-γ⁺ T cells from day 11 of CD45RA-depleted haplo-MLC was analyzed by qPCR (n = 2). All experiments were repeated at least twice by using the identical donors with consistent results. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparison test (a, c) or unpaired t tests (b).