To better understand the initiation of CD8+ T cell responses during infection, the primary response to the intracellular parasite Toxoplasma gondii was characterized using 2-photon microscopy combined with an experimental system that allowed visualization of dendritic cells (DCs) and parasite specific CD8+ T cells. Infection with T. gondii induced localization of both these populations to the sub-capsular/interfollicular region of the draining lymph node and DCs were required for the expansion of the T cells. Consistent with current models, in the presence of cognate antigen, the average velocity of CD8+ T cells decreased. Unexpectedly, infection also resulted in modulation of the behavior of non-parasite specific T cells. This TCR-independent process correlated with the re-modeling of the lymph node micro-architecture and changes in expression of CCL21 and CCL3. Infection also resulted in sustained interactions between the DCs and CD8+ T cells that were visualized only in the presence of cognate antigen and were limited to an early phase in the response. Infected DCs were rare within the lymph node during this time frame; however, DCs presenting the cognate antigen were detected. Together, these data provide novel insights into the earliest interaction between DCs and CD8+ T cells and suggest that cross presentation by bystander DCs rather than infected DCs is an important route of antigen presentation during toxoplasmosis.

Natural infection by Toxoplasma gondii occurs via oral ingestion of tissue cysts that rupture in the small intestine, releasing zoites that infect locally before disseminating throughout the host. The studies presented here used fluorescent parasites combined with flow cytometry and multiphoton microscopy techniques to understand the events associated with parasite replication in the mucosa. At 3 days postinfection with tissue cysts, parasites were localized in small foci and flow cytometry revealed parasites present in macrophages, neutrophils, and monocytes in the lamina propria. By day 6 postinfection, there were large foci of replicating parasites; however, foci unexpectedly varied in the number of villi involved and were associated with the presence of viable tachyzoites within the intestinal lumen. Consistent with the flow cytometry data, neutrophils and monocytes in the lamina propria were preferentially associated with parasite plaques. In contrast, dendritic cells comprised a small fraction of the infected immune cell population and were localized at the periphery of parasite plaques. Together, these findings reveal the formation of localized sites of parasite replication and inflammation early during infection and suggest that sustained replication of T. gondii in the gut may be a function of pathogen luminal spread.

Under normal conditions the immune system has limited access to the brain; however, during toxoplasmic encephalitis (TE), large numbers of T cells and APCs accumulate within this site. A combination of real time imaging, transgenic reporter mice, and recombinant parasites allowed a comprehensive analysis of CD11c+ cells during TE. These studies reveal that the CNS CD11c+ cells consist of a mixture of microglia and dendritic cells (DCs) with distinct behavior associated with their ability to interact with parasites or effector T cells. The CNS DCs upregulated several chemokine receptors during TE, but none of these individual receptors tested was required for migration of DCs into the brain. However, this process was pertussis toxin sensitive and dependent on the integrin LFA-1, suggesting that the synergistic effect of signaling through multiple chemokine receptors, possibly leading to changes in the affinity of LFA-1, is involved in the recruitment/retention of DCs to the CNS and thus provides new insights into how the immune system accesses this unique site.

Summary The innate and adaptive immune responses that confer resistance to the intracellular pathogen Toxoplasma gondii critically depend on IL-12 production, which drives interferon-γ (IFN-γ) expression. Certain cytokines can activate STAT3 and limit IL-12 production to prevent infection-associated immune pathology but T.gondii also directly activates STAT3 to evade host immunity. We show that Suppressor of Cytokine Signaling molecule 3 (SOCS3), a target of STAT3 which limits signaling by the pleiotropic cytokine IL-6, is upregulated in response to infection but is dispensable for the immune-inhibitory effects of T. gondii. Unexpectedly, mice with targeted deletion of SOCS3 in macrophages and neutrophils have reduced IL-12 responses and succumb to toxoplasmosis. Anti–IL-6 administration or IL-12 treatment blocked disease susceptibility suggesting that in the absence of SOCS3, macrophages are hypersensitive to the anti-inflammatory properties of IL-6. Thus, SOCS3 has a critical role in suppressing IL-6 signals and promoting immune responses to control T. gondii infection.