Volume 33, Issue 3, Pages 412-423 (September 2010) Visualizing the Functional Diversification of CD8+ T Cell Responses in Lymph Nodes  Hélène Beuneu, Fabrice Lemaître, Jacques Deguine, Hélène D. Moreau, Isabelle Bouvier, Zacarias Garcia, Matthew L. Albert, Philippe Bousso  Immunity  Volume 33, Issue 3, Pages 412-423 (September 2010) DOI: 10.1016/j.immuni.2010.08.016 Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 Detection of IFN-γ Gene Activation during T Cell Activation In Vitro Naive CD44lo CD8+ T cells from OT-I Rag1−/− or from OT-I Rag1−/− Yeti mice were cultured with DCs pulsed with the indicated concentration of OVA257-264 peptide for 2 days. (A) Phenotype of CD8+ T cells from OT-I Rag1−/− (black circles) or from OT-I Rag1−/−Yeti (white squares) after in vitro activation by DCs. Graphs represent the total number of CD8+ T cells recovered, the percentages of CD8+ T cells displaying a blastic phenotype or expressing the activation markers CD44 or CD25, and the percentage of CD8+ T cells producing IFN-γ or TNF-α (in the absence of restimulation). (B) YFP reporter activity induced by DCs presenting varying densities of pMHC. Representative FACS plots are shown together with graphs displaying the YFP MFI in the total CD8+ T cell population (black lines) or the YFP+ CD8+ T cell population (yellow lines). Control cells correspond to OT-I Rag1−/− T cells (not on the Yeti background) stimulated with DCs pulsed with a peptide concentration of 1 × 10−7 M. Immunity 2010 33, 412-423DOI: (10.1016/j.immuni.2010.08.016) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 IFN-γ Gene Activation during T Cell Activation by DCs In Vivo DCs (2 × 106 cells) were pulsed with 1 × 10−7 or 1 × 10−9 M OVA257-264 peptide and injected in the right footpad of CD45.1 C57BL/6 mice. Eighteen hours later, CD44lo OT-I Yeti (CD45.2) T cells (5 × 106 cells) were adoptively transferred. 24 or 48 hr after T cell injection, CD45.2+ CD8+ T cells from draining and nondraining lymph nodes (LNs) were analyzed by flow cytometry. (A) YFP expression induced in vivo by DCs presenting varying densities of pMHC. Flow cytometry profiles showing YFP expression in CD8+ CD45.2+ cells at 24 hr and 48 hr. Graphs show the mean fluorescence intensity (MFI) of YFP fluorescence in total or in YFP+ OT-I Yeti T cells. (B) Distinct T cell phenotypes after in vivo activation by DCs presenting different levels of pMHC. For the indicated peptide concentration, FACS profiles show CD25 and CD44 expression levels and intracellular content of TNF-α, IL-2, and IFN-γ after 4 hr of culture in the presence of Brefeldin A (without restimulation) as a function of YFP levels. Data are representative of three independent experiments. Immunity 2010 33, 412-423DOI: (10.1016/j.immuni.2010.08.016) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 Rapid Formation of Stable CD8+ T Cell-DC Contacts in the Lymph Nodes DCs (2 × 106 cells) isolated from CD11c-YFP mice were pulsed with 1 × 10−7 or 1 × 10−9 M OVA257-264 peptide or left unpulsed and injected in the right footpad of recipient mice. SNARF-labeled CD44lo OT-I Rag1−/− T cells (5 × 106 cells) were injected intravenously 18 hr later. Three hours after T cell transfer, lymph nodes were subjected to two-photon imaging. (A) Representative T cell tracks are overlayed with an image from the corresponding movie. Trajectories are shown corresponding to 5 min of imaging in mice immunized with unpulsed DCs (left), DCs pulsed with 1 × 10−9 M peptide (center), and DCs pulsed with 1 × 10−7 M peptide (right). DCs (green), T cells (red). Scale bars represent 10 μm. (B) The duration of T cell-DC contacts and the arrest coefficient of individual T cells are plotted for the different immunizing conditions. (C) Contact histories for individual OT-I CD8+ T cells. Red bars correspond to time points at which the analyzed T cell contacted a DC. Black bars correspond to time points at which the T cell showed no apparent interaction with DCs. Each line corresponds to an individual T cell. (D) Representative T cell tracks corresponding to 5 min of imaging are plotted with their initial coordinates being set at the origin. Results are representative of at least four movies obtained in two independent experiments. Immunity 2010 33, 412-423DOI: (10.1016/j.immuni.2010.08.016) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Kinetics of IFN-γ Gene Activation during Stable T Cell-DC Interactions Visualized by Two-Photon Imaging (A–C) Measuring IFN-γ gene activation in lymph node T cells by two-photon imaging. CD44lo OT-I Yeti T cells were labeled with SNARF and adoptively transferred in recipient mice. DCs isolated from CD11c-YFP mice pulsed with 1 × 10−8 M OVA257-264 peptide were injected in the footpad. After 24 hr, LNs were imaged by two-photon microscopy. (A) Cropped images of individual T cells in the LN draining the DCs injection site showing extensive variability in the YFP/SNARF ratio. (B) Representative images from time-lapse movies obtained in the draining and nondraining lymph nodes. DCs (green), YFP-negative T cells (red), YFP-positive T cells (orange to yellow). Scale bars represent 10 μm. (C) For individual T cells, SNARF fluorescence was plotted against the YFP levels measured by two-photon imaging (left). The same LNs were dissociated and analyzed by flow cytometry. Flow cytometry profiles showing YFP and SNARF fluorescence are gated on SNARF+ cells. (D and E) Different kinetics of gene activation during the course of stable interactions. DCs pulsed with either 1 × 10−7 or 1 × 10−9 M OVA257-264 peptide were injected in the footpads of recipient mice. SNARF-labeled naive OT-I Yeti T cells were injected 18 hr later and two-photon imaging was performed 24 hr after T cell transfer. (D) Representative images showing T cells establishing stable interactions with DCs pulsed with 1 × 10−7 or 1 × 10−9 M peptide. The YFP/SNARF ratio for individual T cells found to form a long-lasting interaction with a peptide-pulsed DC is graphed for both peptide concentrations tested. Note that only the highest peptide concentration induced YFP upregulation in a fraction of T cells forming stable contacts with DCs. Scale bars represent 10 μm. (E) Flow cytometry profile showing the YFP and FSC values in T cells recovered from the draining lymph node at 24 hr. Note that the highest peptide concentration induces YFP expression prior to blastogenesis. Immunity 2010 33, 412-423DOI: (10.1016/j.immuni.2010.08.016) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 Functional T Cell Diversification Is Initiated Prior to Cell Division CD44lo OT-I Yeti T cells were labeled with SNARF and adoptively transferred in recipient mice. DCs isolated from CD11c-YFP mice pulsed with 1 × 10−8 M OVA257-264 peptide were injected in the footpad. Two-photon imaging was performed at 40 hr and time-lapse movies were analyzed for the occurrence of T cell division. (A) Diversity of YFP fluorescence at the timing of the first T cell division. Three examples of dividing T cells with variable reporter activity are shown. (B) Graph shows the YFP and SNARF fluorescence measured for the dividing T cells just prior to mitosis. Each dot corresponds to one cell. (C) YFP/SNARF ratio was plotted for nondividing and dividing T cells. (D) Example of two T cells dividing at the same time but exhibiting distinct fluorescent reporter activities (left). The SNARF (red line) and YFP (green line) fluorescent signals were monitored over time for each of these cells and their two daughter cells (right). (E) Histogram showing the various YFP/SNARF ratios of all dividing cells and their daughter cells. Scale bars represent 10 μm. Representative of at least five movies in three independent experiments. Immunity 2010 33, 412-423DOI: (10.1016/j.immuni.2010.08.016) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 Analyzing the Early Progeny of Single Activated T Cells CD44lo CD8+ T cells purified from Rag1−/− OT-I Yeti mice were activated by DCs pulsed with 1 × 10−8 M OVA257-264 peptide for 24 hr and then cloned in minitray culture plates. After 72 hr, individual T cell progenies were analyzed by flow cytometry. (A) Representative flow cytometry profiles corresponding to four individual T cell progenies. The YFP MFI is indicated for each clonal progeny. (B) Graph representing the distribution of YFP levels for each cells of 13 clonal progenies analyzed in a representative experiment. Each column corresponds to one progeny and each dot represents the YFP intensity of one cell. (C) Statistical analysis showing that the intraclonal variability in YFP expression is significantly lower than that of the pooled T cell population. For each clone, the ratio of the standard deviations for YFP levels in the progeny to the standard deviation of the pooled populations is indicated by a dot. (D and E) CD8+ T cells from OT-I Yeti mice were stimulated with peptide-pulsed DCs for 24 hr and sorted based on their YFP expression. YFPlo and YFPhi T cells were then subjected to short-term progeny analysis. (D) Representative flow cytometry profiles corresponding to four representative T cell progenies derived from YFPlo or YFPhi T cell populations. (E) The mean intensity of YFP fluorescence is plotted for individual progenies derived from sorted YFPlo or YFPhi T cells (p < 0.0001). (F) Correlation between IFN-γ-YFP reporter activity and CD8+ T cell multifunctional potential. CD44lo OT-I Rag1−/− Yeti CD8+ T cells were activated by peptide-pulsed dendritic cells and cloned according to our early T cell progeny assay. After 72 hr, cells were recovered from the various wells, pooled, and subjected to intracellular cytokine staining. Flow cytometry profiles (left) showing cytokine production for gated YFPlo and YFPhi effector CD8+ T cells. On the right, pie charts represent the fraction of T cells producing 0, 1, 2, or 3 of the cytokines tested in the YFPhi or YFPlo populations. Results are representative of two or three independent experiments. ∗∗∗p < 0.0001. Immunity 2010 33, 412-423DOI: (10.1016/j.immuni.2010.08.016) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 7 Interclonal Diversification during CD8+ T Cell Activation by DCs In Vivo Naive OT-I Yeti CD8+ T cells (CD45.2) were adoptively transferred in CD45.1 C57BL/6 recipient mice that were further immunized with peptide-pulsed dendritic cells in the footpad. After 40 hr, CD45.2 CD8+ T cells were purified from the draining lymph node and subjected to early T cell progeny analysis. (A) Flow cytometry profile showing the YFP levels for five representative individual T cell progenies. (B) Graph compiling the YFP intensity of the different cells constituting a given progeny. Each column corresponds to a distinct progeny and each dot represents one cell. (C) Statistical analysis showing the limited intraclonal diversity for YFP levels as described in Figure 6F. ∗∗p < 0.005. Similar results were obtained in two independent experiments. Immunity 2010 33, 412-423DOI: (10.1016/j.immuni.2010.08.016) Copyright © 2010 Elsevier Inc. Terms and Conditions