Lymph nodes (LN) harbor cells chronically infected by HIV/SIV, especially CD4+ follicular helper lymphocytes which are located within the B cell areas. LN are characterized by low frequency of effector memory CD8 T cells that are, in general, excluded from the follicles. Using a vaccination protocol that combines intramuscular DNA delivery followed by in vivo electroporation, we have analyzed the trafficking of virus-specific CD8+ T lymphocytes.
Eight MamuA01+ rhesus macaques were immunized with plasmid DNA encoding p57gag. Two weeks after the fourth vaccination, the animals were sacrificed, and the dissemination of vaccine-induced T cell responses was monitored throughout the body by immunophenotyping combined with CM9gag tetramer staining, followed by flow cytometry. Chemokine production, including CXCL9, CXCL10 and CXCL11, by lymph node mononuclear cells (LNMC) from the vaccinated animals stimulated ex vivo with SEB and IFNγ was measured by ELISA, multiplex chemiluminescence detection assays (MSD) and intracellular staining.
High magnitude (up to 15% of total CD8+ T cells) of vaccine-induced virus-specific CD8+ T cells were found in peripheral blood from all the immunized macaques. These cells were actively dividing (ki67), expressed high levels of CXCR3 and efficiently migrated into central and peripheral LN. The CM9-specific CD8+ T cells within the LN also expressed CXCR3 and had an effector phenotype (CD95+CD28lowCD45RAlowCD127-CCR6-CCR4-) with no significant CCR7 expression, suggesting that these cells could be located outside the T cell areas. LNMC from these vaccinated macaques stimulated ex vivo with IFN-γ or SEB released high levels of IP-10 (CXCL10) and CXCL11. A combination of surface and intracellular staining with anti-CXCL9 and IP-10 antibodies revealed that these chemokines were produced by HLA-DR+ B lymphocytes, CD11c+CD14- dendritic cells and a subset of cells with the phenotype (CD3-CD20-CD14-CD11c-HLA-DR-CD21+) of follicular dendritic cells (FDC).
Expression of CXCR3 by the vaccine-induced virus-specific CD8+ T cells indicates that these cells can migrate into areas where the chemokines CXCL9, CXCL10 and CXCL11 are produced. Because these chemokines are produced within the LN by cells located in the B cell areas (B lymphocytes and FDC) the data suggest that a CXCR3-dependent and CXCR5-independent pathway of effector cells entry into the follicles exist.