A Rodriguez-Pozo(1) (2)*, D Pejoski(1) (2), H Abdelouahab(1) (2), N Tchitchek(1) (2), N Dereuddre-Bosquet(1) (2), Y Lévy(2) (3), A Cosma(1) (2),
R Le Grand(1) (2), A-S Beignon(1) (2)
(1)Université Paris Sud, CEA, Inserm UMR 1184, IDMIT, France. (2)Vaccine Research Institute, France. (3)Inserm U955, France
*firstname.lastname@example.org, Tel: +33658338090
The deep characterization of the abundance, phenotype and compartmentalization of Ag specific-B cell subsets and their correlation with the Ab response will improve vaccine research. Mass cytometry and associated analytical tools allow for the comprehensive interrogation of cellular phenotypes, which may advance the discovery of essential cellular subsets.
Here, we studied B cell subpopulations after homologous prime-boost immunization in peripheral blood over time and across five different lymphoid tissues (tonsil, bone marrow, spleen, inguinal and axillary lymph nodes) a year after the last immunization. We used cynomolgus macaques (n=5) and MVA, as model of immunogenicity. Isolated cells were stained with a 27 parameters Ab panel, acquired using CyTOF and analyzed using SPADE algorithm for automatic clustering of CD3–CD14– cell populations.
Our data confirmed the high diversity and complexity of circulating B cells subsets expressing unique patterns of activation/exhaustion, homing, BCR-signaling and B-T cell interaction markers. In addition, we identified B cells clusters specific to and/or shared between tissues microenvironments. When comparing a week and a month post-boost with baseline (before any immunization), we observed statistically significant vaccine-induced changes in the proportions of some B cell clusters in blood. The expansion and contraction of one resting memory B cells cluster correlated with serum anti-vaccine Ab titers. We could also track vaccine-specific B cells. They peaked a week post-boost in blood and displayed an activated memory B cells phenotype. MVA Ag-specific cells were still detectable a year post-boost, albeit at very low frequency in peripheral blood, bone marrow, lymph nodes and spleen.
In conclusion, using mass cytometry and multiparametric analysis, we could phenotype macaque B cells from blood and lymphoid tissues at an unprecedented level. In blood, some subphenotypes changed in proportion after vaccination, one correlated in frequency with Ab titers and others were enriched in vaccine-specific B cells. Future work will include functional assays which require cell sorting of complex subphenotypes and the study of TFH across lymphoid tissues.
This study is currently supported by ANR-10-LABX-77, ANR-10-EQPX-02-01, ANR-11-INBS-0008, FP7-HEALTH-2011-280873, VRI ImMemory, CONCYTEC/CIENCIACTIVA del Gobierno del Perú