Dynamics of T cell, antigen-presenting cell, and pathogen interactions during recall responses in the lymph node

Abstract

Memory T cells circulate through lymph nodes where they are poised to respond rapidly upon re-exposure to a pathogen; however, the dynamics of memory T cell, antigen-presenting cell, and pathogen interactions during recall responses are largely unknown. We used a mouse model of infection with the intracellular protozoan parasite, Toxoplasma gondii, in conjunction with two-photon microscopy, to address this question. After challenge, memory T cells migrated more rapidly than naive T cells, relocalized toward the subcapsular sinus (SCS) near invaded macrophages, and engaged in prolonged interactions with infected cells. Parasite invasion of T cells occurred by direct transfer of the parasite from the target cell into the T cell and corresponded to an antigen-specific increase in the rate of T cell invasion. Our results provide insight into cellular interactions during recall responses and suggest a mechanism of pathogen subversion of the immune response.

Figure 1. Memory T cells localize to sites of infection near the lymph node capsule

Confocal analysis of lymph nodes from immunized mice containing GFP-labeled OT1 memory T cells (green) either resting or 5 hours after ear-flap challenge with T. gondii. A) Confocal images of resting lymph nodes (top panel) and lymph nodes after ear-flap challenge with T. gondii+OVA (red) (bottom panel). Middle panels show memory T cell distribution as density maps with dotted white lines to indicate the lymph node circumference. Right panels show T cell density as a function of distance to lymph node capsule. B) Higher magnification confocal images showing naïve (red) and memory (green) T cell distribution in resting lymph nodes (left panel), and lymph nodes 5 hours after challenge with T. gondii+OVA (yellow, middle panel), or after challenge with T. gondii without OVA (yellow, right panel). B cell areas were visualized with B220 staining (blue). C) Naïve and memory T cell distribution in sections shown in (B). The distance from a T cell to lymph node capsule was divided by the lymph node circumference and expressed as normalized distance. Each point on the graph represents an individual T cell and are compiled from 1–2 lymph nodes each. Data for individual quadrants of lymph nodes are provided in Fig. S3

Figure 2. Memory T cells form stable conjugates with T. gondii- infected cells

A) Naïve and memory T cell migration before and after challenge with T. gondii. Draining lymph nodes from mice containing GFP-labeled OT1 memory T cells and SNARF-labeled OT1 naïve T cells were imaged using 2-photon microscopy either in resting state or at indicated times after challenge. The graph on the left shows the average speed of naïve (red) and memory (green) T cells under the indicated conditions (***p<0.0001, **p=0.0064). Middle graph shows average speed of memory T cells from a lymph node 5 hours after challenge with T. gondii+OVA with T cells categorized based on types of contacts formed. The pie graph summarizes the types of T cell clusters observed in 42 datasets. B–C) Mice containing GFP-labeled OT1 memory T cells (green) were challenged with T. gondii+OVA parasites (red) 5 hours prior to analysis. In some experiments mice were transferred with naive OT1 CFP lymphocytes 24 hours before challenge. B) Examples of T cell clusters from a lymph node 5 hours after challenge. Top panel shows an example of a memory T cell cluster around a cell infected with parasites that persists for the length of the imaging run (31 min). Bottom panel shows a T cell cluster that disperses during the imaging run. Right hand images show the paths for parasites (red) and T cells (various colors). The graphs on the right show displacement from origin for parasite and T cells. Black arrow indicates the time point at which cluster dispersal was first observed. Corresponds to video 2B–C. C) A cluster containing both memory (green) and naïve T cells (magenta, white arrow). 2-D projections of 3-D two-photon imaging volumes are shown at time frames indicated, and colored lines represent tracks of individual T cells or parasite. Plot shows the number of naive or memory T cells in clusters as a percentage of the total number of naive or memory T cells in the imaging volume for 4 different imaging runs. Numbers under bars indicate the total number of naïve or memory T cells present in each imaging volume. Corresponds to video 2B–C. D) Mice containing GFP-labeled OT1 memory T cells (cyan) were challenged via earflap with one-to-one mixtures of T. gondii+OVA expressing RFP (red) and control -OVA parasites expressing YFP (blue) and lymph nodes were imaged 5 hours later. Left panel show representative memory T cell clusters surrounding OVA only (left panels) or surrounding mixed + and − OVA parasites infected cells (right panels). Corresponds to video 2D. Graph shows the total number of T cell clusters scored in each category for 12 separate runs.

Figure 3. T cells form long-lasting interactions with SCS macrophages and DCs after challenge

A) OT1 memory T cells form long-lasting interactions with CD11c YFP high infected (top panel) and uninfected (middle panel) DCs, and YFP low cells (bottom panel). Arrows show memory OT1 T cells (blue) engaged in long-lasting contacts with APCs (green). 2-D projections of 3-D two-photon imaging volumes are shown at time frames indicated. Corresponds to video 3A. Right hand images show the paths for T cells (various colors) in contact with YFP high (shown in green, top and middle panels) or YFP low (indicated by red tracks, circled in green, bottom panel) cells. Pie charts summarize the number of infected (left) and uninfected (right) CD11cYFPhigh DCs that supported T cell clusters in 11 datasets. The graph shows the numbers of parasites in YFP high and low cells. CD11c YFP reporter mice containing GFP-labeled OT1 memory T cells were challenged with T. gondii+OVA 5 hours before explanting the draining lymph node for imaging. B) OT1 T cells form clusters around infected CD169 cells. Mice containing GFP-labeled OT1 memory T cells were challenged with T.gondii+OVA (red) for 5 hours and injected with CD169-Alexa 532 5 min prior to explanting of draining lymph nodes for imaging. In some experiments mice were also transferred with CFP-labeled OT1 naive T cells 24 hours prior to challenge. Two-photon images of OT1 memory T cells (green) in stable interactions with CD169 (yellow), with second harmonic signal from the collagen-rich capsule shown in blue. Right hand images show the tracks for T. gondii (red) and OT1 T cells (various colors). Z-projections of an imaging volume are shown at indicated times. Corresponds to video 3Bi. Middle panels show higher magnification images of OT1 memory T cells interacting with an infected CD169 cell. Single optical sections of an imaging volume are shown at indicated times. The graph shows the distribution of T cell cluster sizes around CD169 cells. Bottom panels show a cluster consisting of a naïve (blue) and a memory (green) OT1 T cells forming a cluster around an infected CD169 cell (yellow). Corresponds to video 3Bii.

Figure 4. Direct invasion of T cells during contacts with infected host cells

A) Two examples of T cell invasion during interactions with infected APCs in draining lymph nodes following earflap infection. Single optical sections from two-photon imaging runs showing memory OT1 GFP T cells (green) being invaded by parasites (red). In the top panel, yellow arrows point to the parasite prior T cell invasion while white arrows indicate the parasite inside the T cells. Corresponds to video 4A. Right hand panel shows average speeds of individual T cells containing parasite fluorescence signal (infected T cells) compared to T cells without parasite signal (uninfected T cells) from the same imaging volumes. B, C) Immunofluorescence images of infected T cells stained for the parasite dense granule protein, GRA6 as a marker for the parasitophorous vacuole. RFP-labeled parasites are shown in red, GFP labeled T cells are in green, and GRA6 immunostaining is shown in white. B) Confocal analysis of the lymph node samples described in (A). C) In vitro infected T cells with nuclei labeled with DAPI (blue). D) Naïve mice containing GFP-labeled OT1 T cells were orally infected with T. gondii+OVA cysts for 5–7 days prior to explanting of mesenteric lymph nodes for imaging. Average speed of infected and uninfected OT1 T cells in the mesenteric lymph nodes determined by analysis of two-photon imaging data. Right panels show OT1 T cell (green) invasion by T. gondii+OVA (red) in the mesenteric lymph nodes 5 days after oral infection with 75 cysts. Single optical sections of two-photon imaging volumes are shown. E) Dynamic imaging of OT1 T cells containing parasites in vibratome-cut brain slices from chronically infected mice. Left panels show projections of imaging volumes from time-lapse series obtained by two-photon microscopy. Dashed boxes indicate invaded T cells, with enlarged views to show the parasite fluorescence (arrowhead). The paths of infected OT1 GFP cells are depicted by color-coded lines to depict time (blue-red-yellow-white). Right most panel shows a plot of displacement rate, versus the average speed for individual T cells. Red circles are infected OT1 T cells and black circles are OT1 T cells without visible parasite fluorescence from the same runs. Data was generated 4 different samples, 10 to 39 days p.i. F) An example of an OT1 cell being invaded by a parasite in the brain. Three frames from a time-lapse sequence are shown before (left panel), during (center panel), and after (right panel) invasion. The arrow indicates the position of the OT1 GFP T cell at each time point. Plot on right shows the instantaneous speed versus time of the T cell. Shaded area corresponds to the time the T cell stops near the group of isolated parasites. Red arrow indicates the time point at which the parasite is first seen inside the T cell. Corresponds to Video 4D–F.

Figure 5. Antigen recognition enhances T cell invasion by T. gondii

A) Flow cytometric analyses showing the % of infected cells as a proportion of gated OT1 T cells (circles) or endogenous, polyclonal T cells (triangles) in draining lymph nodes of mice challenged via earflap with OVA-expressing parasites (red) or parasites without OVA (blue) 24 hours before analysis. Graph shows the % of gated T cells containing parasite fluorescence (% infected T cells) plotted as a function of infection rate. Each point represents the indicated T cell population from a single lymph node.. B) Flow cytometric analysis comparing the infection rates of naive and memory OT1 T cells for 4 individual lymph nodes. Mice were challenged with OVA expressing parasites 24 hours before analysis. C, D) Naïve mice were infected orally with 50- 75 T. gondii cysts and analyzed 5–7 days later. C) Flow cytometric analysis of mesenteric lymph nodes. Pie chart shows the proportions of parasites contained in different leukocyte subsets in the mesenteric lymph nodes, and plot on right shows CD69 expression in uninfected or infected CD3+ cells. **p=0.0027 D) Flow cytometric analysis of CD3 and parasite fluorescence in blood leukocytes. Left panels show compiled data and right panels show representative FACS plots. Uninfected mice are shown for comparison. The numbers indicate % of infected cells out of total live gate, the numbers in brackets are % of infected cells out of CD3- and CD3+ populations respectively.

Figure 6. Evidence for parasite spread via invaded T cells

A) Parasite-containing CD8 T cells were isolated from the mesenteric lymph nodes of mice that had been infected orally (7 days earlier) and 10,000 FACS-sorted infected T cells were injected via the earflap of a naive mouse. Five days later mice draining lymph nodes and spleens were analyzed by flow cytometry. Mice that did not receive infected T cells are shown for comparison. B) Mice were orally infected with 50–75 cysts from RFP parasites (day 0) and treated on days 1, 3, 5 with FTY720, and on day 7, infection rates were determined by by flow cytometry of collagenase-dissociated spleen and lymph nodes. *p=0.0018, **p=0.0001. Peripheral blood of treated mice showed a 4 fold decrease in the number of T cells in treated compared to control mice, (data not shown) indicating an efficient and selective block in T cell egress from lymph nodes.