In situ staining techniques were used to visualize physical interactions between dendritic cell subsets and naive Ag-specific CD4 T cells in the lymph node. Before injection of Ag, CD8+ dendritic cells and naive OVA-specific CD4 T cells were uniformly distributed throughout the T cell-rich paracortex, whereas CD11b+ dendritic cells were located mainly in the outer edges of the paracortex near the B cell-rich follicles. Many OVA-specific CD4 T cells were in contact with CD8+ dendritic cells in the absence of OVA. Within 24 h after s.c. injection of soluble OVA, the OVA-specific CD4 T cells redistributed to the outer paracortex and interacted with CD11b+, but not CD8+ dendritic cells. This behavior correlated with the uptake of OVA and the presence of peptide-MHC complexes on the surface of CD11b+ dendritic cells, and subsequent IL-2 production by the Ag-specific CD4 T cells. These results are consistent with the possibility that CD11b+ dendritic cells play a central role in the activation of CD4 T cells in response to s.c. Ag.

TCR reactivity is tuned during thymic development. Immature thymocytes respond to low-affinity self-ligands resulting in positive selection. Following differentiation, T cells no longer respond to low-affinity ligands, but respond well to high-affinity (foreign) ligands. We show in this study that this response includes integrin activation, supramolecular activation cluster formation, Ca2+ flux, and CD69 expression. Because glycosylation patterns are known to change during T cell development, we tested whether alterations in sialylation influence CD8 T cell sensitivity to low affinity TCR ligands. Using neuraminidase treatment or genetic deficiency in the ST3Gal-I sialyltransferase, we show that desialylation of mature CD8 T cells enhances their sensitivity to low-affinity ligands, although these treatments do not completely recapitulate the dynamic range of immature T cells. These studies identify sialylation as one of the factors that regulate CD8 T cell tuning during development.

Heat shock protein 70 (hsp70) is a potent adjuvant that links innate and adaptive immune responses. To study how hsp70 activates naive CD8+ T cells in vivo, we tracked Ag-specific CD8+ T cells in mice immunized with a fusion protein containing chicken OVA linked to hsp70 derived from Mycobacterium tuberculosis (OVA.TBhsp70). On a molar basis, OVA.TBhsp70 was several hundred times more effective than OVA peptide plus CFA in eliciting specific CD8+ T cell responses. Immunization with OVA.TBhsp70 activated >90% of detectable OVA-specific CD8+ T cells within 3 days and led to the persistence of cytotoxic effectors for at least 17 days. These studies demonstrate that the potent adjuvant effect of M. tuberculosis hsp70 results from the relatively complete, rapid, and durable activation of Ag-specific CD8+ T cells.