Interactions with Asialo-Glycoprotein Receptors and Platelets Are Dispensable for CD8+ T Cell Localization in the Murine Liver.

Liver-resident CD8+ T cells can play critical roles in the control of pathogens, including Plasmodium and hepatitis B virus. Paradoxically, it has also been proposed that the liver may act as the main place for the elimination of CD8+ T cells at the resolution of immune responses. We hypothesized that different adhesion processes may drive residence versus elimination of T cells in the liver. Specifically, we investigated whether the expression of asialo-glycoproteins (ASGPs) drives the localization and elimination of effector CD8+ T cells in the liver, while interactions with platelets facilitate liver residence and protective function. Using murine CD8+ T cells activated in vitro, or in vivo by immunization with Plasmodium berghei sporozoites, we found that, unexpectedly, inhibition of ASGP receptors did not inhibit the accumulation of effector cells in the liver, but instead prevented these cells from accumulating in the spleen. In addition, enforced expression of ASGP on effector CD8+ T cells using St3GalI-deficient cells lead to their loss from the spleen. We also found, using different mouse models of thrombocytopenia, that severe reduction in platelet concentration in circulation did not strongly influence the residence and protective function of CD8+ T cells in the liver. These data suggest that platelets play a marginal role in CD8+ T cell function in the liver. Furthermore, ASGP-expressing effector CD8+ T cells accumulate in the spleen, not the liver, prior to their destruction.

Splenic Dendritic Cells and Macrophages Drive B Cells to Adopt a Plasmablast Cell Fate.

Upon encountering cognate antigen, B cells can differentiate into short-lived plasmablasts, early memory B cells or germinal center B cells. The factors that determine this fate decision are unclear. Past studies have addressed the role of B cell receptor affinity in this process, but the interplay with other cellular compartments for fate determination is less well understood. Moreover, B cell fate decisions have primarily been studied using model antigens rather than complex pathogen systems, which potentially ignore multifaceted interactions from other cells subsets during infection. Here we address this question using a Plasmodium infection model, examining the response of B cells specific for the immunodominant circumsporozoite protein (CSP). We show that B cell fate is determined in part by the organ environment in which priming occurs, with the majority of the CSP-specific B cell response being derived from splenic plasmablasts. This plasmablast response could occur independent of T cell help, though gamma-delta T cells were required to help with the early isotype switching from IgM to IgG. Interestingly, selective ablation of CD11c+ dendritic cells and macrophages significantly reduced the splenic plasmablast response in a manner independent of the presence of CD4 T cell help. Conversely, immunization approaches that targeted CSP-antigen to dendritic cells enhanced the magnitude of the plasmablast response. Altogether, these data indicate that the early CSP-specific response is predominately primed within the spleen and the plasmablast fate of CSP-specific B cells is driven by macrophages and CD11c+ dendritic cells.

Liver Environment-Imposed Constraints Diversify Movement Strategies of Liver-Localized CD8 T Cells.

Pathogen-specific CD8 T cells face the problem of finding rare cells that present their cognate Ag either in the lymph node or in infected tissue. Although quantitative details of T cell movement strategies in some tissues such as lymph nodes or skin have been relatively well characterized, we still lack quantitative understanding of T cell movement in many other important tissues, such as the spleen, lung, liver, and gut. We developed a protocol to generate stable numbers of liver-located CD8 T cells, used intravital microscopy to record movement patterns of CD8 T cells in livers of live mice, and analyzed these and previously published data using well-established statistical and computational methods. We show that, in most of our experiments, Plasmodium-specific liver-localized CD8 T cells perform correlated random walks characterized by transiently superdiffusive displacement with persistence times of 10-15 min that exceed those observed for T cells in lymph nodes. Liver-localized CD8 T cells typically crawl on the luminal side of liver sinusoids (i.e., are in the blood); simulating T cell movement in digital structures derived from the liver sinusoids illustrates that liver structure alone is sufficient to explain the relatively long superdiffusive displacement of T cells. In experiments when CD8 T cells in the liver poorly attach to the sinusoids (e.g., 1 wk after immunization with radiation-attenuated Plasmodium sporozoites), T cells also undergo Lévy flights: large displacements occurring due to cells detaching from the endothelium, floating with the blood flow, and reattaching at another location. Our analysis thus provides quantitative details of movement patterns of liver-localized CD8 T cells and illustrates how structural and physiological details of the tissue may impact T cell movement patterns.

A New Method Based on the von Mises-Fisher Distribution Shows that a Minority of Liver-Localized CD8 T Cells Display Hard-To-Detect Attraction to Plasmodium-Infected Hepatocytes.

Malaria is a disease caused by Plasmodium parasites, resulting in over 200 million infections and 400,000 deaths every year. A critical step of malaria infection is when sporozoites, injected by mosquitoes, travel to the liver and form liver stages. Malaria vaccine candidates which induce large numbers of malaria-specific CD8 T cells in mice are able to eliminate all liver stages, preventing fulminant malaria. However, how CD8 T cells find all parasites in 48 h of the liver stage lifespan is not well understood. Using intravital microscopy of murine livers, we generated unique data on T cell search for malaria liver stages within a few hours after infection. To detect attraction of T cells to an infection site, we used the von Mises-Fisher distribution in 3D, similar to the 2D von Mises distribution previously used in ecology. Our results suggest that the vast majority (70-95%) of malaria-specific and non-specific liver-localized CD8 T cells did not display attraction towards the infection site, suggesting that the search for malaria liver stages occurs randomly. However, a small fraction (15-20%) displayed weak but detectable attraction towards parasites which already had been surrounded by several T cells. We found that speeds and turning angles correlated with attraction, suggesting that understanding mechanisms that determine the speed of T cell movement in the liver may improve the efficacy of future T cell-based vaccines. Stochastic simulations suggest that a small movement bias towards the parasite dramatically reduces the number of CD8 T cells needed to eliminate all malaria liver stages, but to detect such attraction by individual cells requires data from long imaging experiments which are not currently feasible. Importantly, as far as we know this is the first demonstration of how activated/memory CD8 T cells might search for the pathogen in nonlymphoid tissues a few hours after infection. We have also established a framework for how attraction of individual T cells towards a location in 3D can be rigorously evaluated.