Volume 22, Issue 4, Pages 849-859 (January 2018) ICAMs Are Not Obligatory for Functional Immune Synapses between Naive CD4 T Cells and Lymph Node DCs  Sara W. Feigelson, Adam Solomon, Adi Biram, Miki Hatzav, Moria Lichtenstein, Ofer Regev, Stav Kozlovski, Diana Varol, Caterina Curato, Dena Leshkowitz, Steffen Jung, Ziv Shulman, Ronen Alon  Cell Reports  Volume 22, Issue 4, Pages 849-859 (January 2018) DOI: 10.1016/j.celrep.2017.12.103 Copyright © 2018 The Authors Terms and Conditions

Cell Reports 2018 22, 849-859DOI: (10.1016/j.celrep.2017.12.103) Copyright © 2018 The Authors Terms and Conditions

Figure 1 Normal T Cell Homing to Lymph Nodes Reconstituted with ICAM-1 and ICAM-2 Double Deficient APCs (A) CMTMR-labeled T lymphocytes recovered from skin-draining popliteal LNs of either WT or ICAM-1/2 DKO mice 2 hr after adoptive transfer. n = 3, 4, representative of two experiments. ∗∗∗p < 0.0002. (B) CFSE-labeled OT-II CD4 lymphocytes recovered from popliteal LNs 18 hr after adoptive transfer. n = 3, representative of three experiments. ∗∗p < 0.007. (C) Schematic representation of the generation of WT/WT and ICAM-1/2 DKO/WT chimera. (D) ICAM-1 and ICAM-2 expression in CD45 cells derived from the popliteal LNs of either WT/WT (red) or DKO/WT (blue) chimeric mice. See also Figure S1. (E) Immunohistochemical staining for ICAM-1 (top) and ICAM-2 (bottom) of LNs within the indicated mice. Scale bar: 100 μm. (F) CMTMR-labeled T lymphocytes recovered from popliteal LNs of either WT/WT or DKO/WT chimeric mice 2 hr after adoptive transfer. n = 3, representative of two experiments. n.s. = not significant. (G) CFSE-labeled OT-II CD4 lymphocytes recovered from popliteal LNs of the indicated chimeric mice 18 hr after adoptive transfer. n = 2, representative of four experiments. Error bars indicate +/- SEM. Cell Reports 2018 22, 849-859DOI: (10.1016/j.celrep.2017.12.103) Copyright © 2018 The Authors Terms and Conditions

Figure 2 Normal Antigen-Triggered Priming and Differentiation of OT-II CD4 T Cells in CFA-Immunized Lymph Nodes Devoid of APC ICAMs (A) A scheme depicting adoptive transfer of CFSE-labeled CD45.1 OT-II cells into chimeric mice (CD45.2), followed by the indicated immunization protocols. (B) Induction of CD69 on transferred OT-II in popliteal LNs of either WT/WT or DKO/WT chimeric mice 24 hr following subcutaneous immunization with CFA/OVA (20 μg, solid line) or PBS control (dotted line). Representative of three experiments. (C) Proliferation histograms of CFSE-labeled OT-II CD4 T cells adoptively transferred into the indicated chimeric mice, and primed 18 hr later by intra-footpad immunization with CFA/OVA (20 μg). CSFE levels (numbers indicate the percentage of CFSE-diluted cells) were determined 72 hr post immunization. (D and E) The percentage of the OT-II CD4 T lymphocytes shown in (C) that divided at least once (D), and their division index (E) 72 hr following intra-footpad immunization with CFA/OVA. n = 6–8; ∗p < 0.03; ∗∗∗p < 0.001. (F) Intracellular IFN-γ staining of the OT-II CD4 cells isolated from the LNs, as described in (C). n = 4, n.s. Representative of three experiments. (G and H) The numbers of CD4 T cells (G) and OT-II Th1 effectors (H) (stained positive for IFN-γ) generated in vitro when incubated for 72 hr with either WT (W) or DKO irradiated splenocytes in the presence of the indicated concentrations of OVA. Error bars indicate +/- SEM. Cell Reports 2018 22, 849-859DOI: (10.1016/j.celrep.2017.12.103) Copyright © 2018 The Authors Terms and Conditions

Figure 3 Endogenous OVA-Loaded CD40-Stimulated DCs Deficient in ICAMs Elicit Normal Proliferation and Differentiation of OT-II CD4 T Cells in Lymph Nodes (A) A scheme depicting adoptive transfer of CFSE-labeled CD45.1 OT-II cells into chimeric mice, followed by the indicated immunization protocols. (B) ICAM-1 and ICAM-2 expression on APCs (CD45+MHC-IIhi) derived from WT/WT popliteal LNs at resting state and 24 hr after intra-footpad immunization with αCD40 mAb (10 μg). See also Figure S2. (C) Two photon (2P) intravital micrographs of the popliteal LN of WT/WT (left) or ICAM-1/2 DKO/WT chimera (right), each injected with DsRed OT-II and GFP B cells, followed by immunization with OVA and αCD40 mAb (10 μg) after 18 hr. The popliteal LNs were imaged 4–6 hr after OVA immunization. Follicles are outlined with a dashed white line. (D) Proliferation histograms of CFSE-labeled OT-II CD4 cells adoptively transferred into the indicated chimeric mice and primed 18 hr later by intra-footpad injections of the indicated doses of OVA peptide with or without co-injected αCD40 mAb. CSFE (numbers indicate the percentage of CFSE-diluted cells) levels were determined 72 hr post immunization. (E and F) The percentage of OT-II CD4 lymphocytes shown in (D) that divided at least once (E) and their division index (F) (n = 3 or 4, n.s.). (G) Intracellular IFN-γ staining of the OT-II cells isolated from the lymph nodes examined in (D) (n = 3, n.s.). Error bars indicate +/- SEM. Cell Reports 2018 22, 849-859DOI: (10.1016/j.celrep.2017.12.103) Copyright © 2018 The Authors Terms and Conditions

Figure 4 Endogenous Stimulated DCs Deficient in ICAMs Loaded with αDEC-205-OVA Drive Normal Proliferation and Differentiation of OT-II CD4 T Cells in Lymph Nodes (A) Induction of CD69 and CD25 on transferred OT-II isolated from popliteal LNs of either WT/WT or ICAM-1/2 DKO/WT chimeric mice 24 and 48 hr, respectively, following intra-footpad injection of αCD40 mAb (10 μg) and low-dose (0.05 μg) αDEC-205-OVA. (B) Proliferation histograms of CFSE-labeled OT-II CD4 cells injected into the indicated chimeric mice and primed 18 hr later by intra-footpad injections of αCD40 mAb and the indicated doses of αDEC-205-OVA. CSFE levels were determined 72 hr post immunization. (C and D) The percentage of OT-II lymphocytes shown in (B) that divided at least once (C) and their division index (D) (n = 3 to 4, n.s.). (E) Intracellular IFN-γ staining of the OT-II cells isolated from the lymph nodes examined in (B). (F) Percentage of OT-II Tfh (CD45.1+, CD4+, CD44hi, CD62Llo, PD-1+, and CXCR5+) effectors generated and accumulated in the popliteal LNs of the indicated chimera 7 days after intra-footpad immunization with αCD40 mAb and αDEC-205-OVA (0.05 μg) (n = 3, n.s.). The experiments described in (A–F) are representative of 2 to 3 independent experiments. (G) Statistical analysis of MARS-seq data (see database GSE108341) of DCs isolated from the indicated chimeric mice 24 hr after intra-footpad injection of αCD40 mAb (10 μg). Shown is a volcano plot for the indicated experimental groups comparison depicting the significance (−log10 p values) versus the log2 fold change on the y and x axes, respectively. The orange dots depict the differentially expressed genes. See also Figure S4. Error bars indicate +/- SEM. Cell Reports 2018 22, 849-859DOI: (10.1016/j.celrep.2017.12.103) Copyright © 2018 The Authors Terms and Conditions

Figure 5 DC ICAMs Are Dispensable for Antigen-Driven CD4 T Cell Arrests on Endogenous Dendritic Cells inside Lymph Nodes (A) A scheme depicting adoptive transfer of fluorescently labeled OT-II CD4+ cells (dsRed, red) and polyclonal CD4+ T cells (GFP, green), followed by the indicated immunization protocol and intravital imaging. (B) Representative 2P images of dsRed OT-II (red) and GFP-polyclonal (green) CD4+ T cells migrating inside the T zone of WT/WT (top) or DKO/WT (bottom) chimeric mice loaded with αDEC-205-OVA (0.05 μg) and αCD40 mAb (10 μg). Scale bar, 50 μm. (C) Mean velocity (Vmean) of individual dsRed OT-II and GFP polyclonal T cells (indicated by red and green dots, respectively) tracked inside the T zones of either WT/WT or ICAM-1/2 DKO/WT chimeric lymph nodes, immunized as above. (D) T cell arrest coefficient (i.e. >4 min) of the same OT-II and polyclonal groups analyzed in (C). See also Movies S1 and S2. (E) Scheme depicting the generation of chimeric mice reconstituted with bone marrow from DTA-CD11C (90%), WT-CFP (5%), and ICAM-1/2-DKO-GFP (5%) mice. DsRed CD4+ OT-II cells were injected into the mice as in previous experiments. (F) Representative 2PM images of a dsRed OT-II CD4+ cell (red) interacting with either a WT (CFP, cyan, top) or ICAM-1/2-DKO (GFP, green, bottom) DC in the T zone of the chimeric lymph nodes described in (D), immunized with αDEC-205-OVA (1 μg) and αCD40 mAb (10 μg). The yellow arrow points to the T cell-DC contact. Scale bar, 10 μm. (G and H) OT-II-cell-DC contact duration times monitored by the 2PM imaging experiment described in (E) and (F) in mice immunized with αCD40 mAb and either low (G) (0.05 μg) or high (H) (1 μg) αDEC-205-OVA. Data are pooled from two independent experiments and are representative of 4 to 5. See also Movies S3 and S4. Error bars indicate +/- SEM. Cell Reports 2018 22, 849-859DOI: (10.1016/j.celrep.2017.12.103) Copyright © 2018 The Authors Terms and Conditions