Characterization of a CD4 T cell population enriched in T follicular helper cells in macaques during chronic SIV infection
Blackburn, Matthew James
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A preventive vaccine for HIV infection is urgently needed to curb the HIV/AIDS pandemic. To date only one human trial testing the combination of an ALVAC-HIV/gp120 protein strategy (Thai trail) has resulted in some protection from HIV infection. The correlate of protection elicited by this vaccine strategy was non-neutralizing antibodies to the gp120 protein. Nevertheless, the overall efficacy of the Thai trial was limited (31.2%); indicating that more work needs to be performed to ameliorate the Thai trial vaccine efficacy. T Follicular Helper (TFH) cells are subset of CD4 T cells that localize within the follicular region of lymph nodes, are required for the formation and maintenance of the germinal center, and provide help to B cells. TFH may therefore be critical for the development of effective antibodies to HIV/SIV. Here, we characterize TFH in different lymphoid compartments of naïve and infected rhesus macaques, the preferred animal model to assess the efficacy of candidate vaccines for HIV. First, we looked at the frequency of TFH within the various lymphoid compartments. TFH, characterized as PD-1++, ICOS++ and CCR7-, were higher within the spleen, the lamina propria of the rectal mucosa, and the tonsils than the lymph nodes. Interestingly, during chronic SIV infection, the frequency of TFH significantly increased in the lymph nodes while remaining fairly constant in the spleen. We then functionally characterized TFH in the lymph nodes of infected and non-infected macaques by performing an intracellular cytokine staining to measure the production of IFN-γ, TNF-α, IL-17, and IL-21 after in vitro stimulation with PMA-ionomycin and SIV-env and SIV-gag overlapping peptides. Interestingly, while TFH (CCR7-/PD-1++) and non-TFH were capable of producing IFN-γ, TNF-α and IL-21 after stimulation with PMA- ionomycin, in chronically infected animals, we observed an impaired production of IL-21. As localization in the germinal center is believed to be relevant for TFH functionality, we established a migrational assay as a way to better discriminate TFH from non-TFH in macaques. The aim was to mimic the in vivo migration of non-TFH to the T cell zone and of TFH to the B cell zone of the lymph nodes, induced by CCL19/CCL21, and CXCL13, respectively, using a two-step assay. We obtained an enrichment of phenotypic defined non-TFH (first migration: CCL19/CCL21) and TFH cells (second migration: CXCL13) from lymph nodes from both naïve and infected macaques. We show that CD4 T cells from naïve macaques that migrated to the CXCL13 had higher levels of Bcl-6 expression and were capable of producing higher levels of IL-21 and lower levels of IFN-γ than cells that migrated to the CCL19 and CCL21-T zone chemokines. Additionally, in SIV infected macaques, CD4 T cells that migrated to CXCL13 were impaired in the production of IL-21 following stimulation with PMA- ionomycin. These results validate our two-step migration assay as an innovative way to study TFH in macaques.
Thesis (M.A.)--Boston University