Dissection of suboptimal immune responses in pediatric patients through new approaches: multiplex RT-PCR and age-specific tissue engineered human model

During my PhD program I have studied the characteristics of the immune response in perinatally HIV infected children, treated early with the ART therapy, with the aim to explore the suboptimal mechanisms of their immune system. The early initiation of the therapy not only reduce morbidity and mortality by controlling the replication of the virus, but also helps in preserving the immune T and B cells compartments. Nonetheless, some ET children show an absence, in periphery, of specific antibodies that let question us about the effective ability of the memory compartments to properly fight the infection during a virus re-exposure. These seronegative (SN) patients represent the optimal candidates in order to achieve HIV remission thank to their smaller size and variability of HIV reservoir. Thus, understanding the molecular mechanisms that stand behind HIV+ populations either in response to HIV re-challenge or in their ability to mount an effective response upon immunization may help in improving future immune therapies for these immunocompromised individuals. It is for this reason that in the present thesis I have dissected quantitatively and qualitatively 8 the characteristics of specific T and B cell populations in two different study designs, aiming at identifying biomarkers that could be informative. Firstly, we investigated the differences that SN and Seropositive (SP) patients have in HIV-specific cells using flow cytometry and transcriptomic assays upon HIV peptides stimulation. Secondarily, using the same methodologies, we investigated phenotypic and transcriptional signatures of H1N1-specific B cell in HIV+ study participants differently responding to trivalent-influenza vaccine (TIV), and in healthy controls. Our results suggested that SN and SP are distinguished by HIVspecific T cell function and by distinct transcriptional signatures of HIV-specific B cells after in vitro stimulation, presumably due to a different antigen exposure. Moreover, we highlight the presence of responders (R) and non-responders (NR) to TIV among the HIV+ patients and the fact that higher PIK3C2B expression in H1N1-specific B cells of NR can be associated with lower H1N1 immunogenicity. During the last year of my PhD, I had the opportunity to study in the laboratory of the Precision Vaccine Program under the guidance of professor O. Levy, developing a new model able to reproduce the microanatomy and physiology of a human 9 capillary vein. We used this tissue engineering approach to compare the autonomous extravasation of newborn and adult monocytes under more natural circumstances, in order to see if such differences could contribute to the divergent immune responsiveness and susceptibility to infections observed during early life. Our data suggest that neonatal monocytes are naturally impaired to colonize quiescent tissues, a cell-mediated phenomenon that may require epigenetic changes acquired as aging. Given the broad applicability of this model depending on whose donor’s monocytes are used, the TC will be likely used in other study designs involving different immunocompromised participants, to test their responses after the exposition to adjuvants or vaccines.