Nature Immunology Supplementary Video s1.mov (8MB)
Phase one with high dose C-peptide. Antigen-specific P14 T cells (green) and control OT-I T cells (blue) interacted with 10μM C-peptide pulsed CD11c+ DCs (red). The peptide-pulsed DCs (20μM CMTMR) were injected with 10ng LPS into the footpad of a recipient C57BL6 mouse. 18 hours later, P14 CD8+ T cells (2.5μM CMFDA, green) and control, OT-I CD8+ T cells (10μM CMAC, blue) were injected i.v. followed 2h later by 100ug Mel-14 antibody i.v. Images were collected from 2.5-3.5h after T cell transfer by MPM-IVM of the right popliteal LN in an anesthetized mouse in the z dimension over a distance of 40μm. Bar = 10μm.
Nature Immunology Supplementary Video s2.mov (9MB)
Early phase two with high dose M-peptide.
Antigen-specific P14 T cells (green) and control OT-I T cells (blue) interacted with 10μM M-peptide pulsed CD11c+ DCs (red). The peptide-pulsed DCs (20μM CMTMR) were injected with 10ng LPS into the footpad of a recipient C57BL6 mouse. 18 hours later, P14 CD8+ T cells (2.5μM CMFDA, green) and control, OT-I CD8+ T cells (10μM CMAC, blue) were injected i.v. followed 2h later by 100ug Mel-14 antibody i.v.. Images were collected from 2.5-3.5h after T cell transfer by MPM-IVM of the right popliteal LN in an anesthetized mouse in the z dimension over a distance of 40μm. Bar = 10μm.
Nature Immunology Supplementary Video s3.mov (8MB)
Phase one with a sub-threshold dose of M-peptide.
Antigen-specific P14 T cells (green) and control OT-I T cells (blue) interacted with 100pM M-peptide pulsed CD11c+ DCs (red). The peptide-pulsed DCs (20μM CMTMR) were injected with 10ng LPS into the footpad of a recipient C57BL6 mouse. 18 hours later, P14 CD8+ T cells (2.5μM CMFDA, green) and control, OT-I CD8+ T cells (10μM CMAC, blue) were injected i.v. followed 2h later by 100μg Mel-14 antibody i.v.. Images were collected from 2.5-3.5h after T cell transfer by MPM-IVM of the right popliteal LN in an anesthetized mouse in the z dimension over a distance of 40μm. Bar = 10μm.
Prolonged phase one with a threshold dose of M-peptide.
Antigen-specific P14 T cells (green) and control OT-I T cells (blue) interacted with 200pM M-peptide pulsed CD11c+ DCs (red). The peptide-pulsed DCs (20μM CMTMR) were injected with 10ng LPS into the footpad of a recipient C57BL6 mouse. 18 hours later, P14 CD8+ T cells (2.5μM CMFDA, green) and control, OT-I CD8+ T cells (10μM CMAC, blue) were injected i.v. followed 2h later by 100mg Mel-14 antibody i.v.. Images were collected from 2.5-3.5h after T cell transfer by MPM-IVM of the right popliteal LN in an anesthetized mouse in the z dimension over a distance of 40μm. Bar = 10μm.
Nature Immunology Supplementary Video s5.mov (9MB)
Late phase two with a threshold dose of M-peptide.
Antigen-specific P14 T cells (green) and control OT-I T cells (blue) interacted with 200pM M-peptide pulsed CD11c+ DCs (red). The peptide-pulsed DCs (20μM CMTMR) were injected with 10ng LPS into the footpad of a recipient C57BL6 mouse. 18 hours later, P14 CD8+ T cells (2.5μM CMFDA, green) and control, OT-I CD8+ T cells (10μM CMAC, blue) were injected i.v. followed 2h later by 100mg Mel-14 antibody i.v.. Images were collected from 6.5-7.5h after T cell transfer by MPM-IVM of the right popliteal LN in an anesthetized mouse in the z dimension over a distance of 40μm. Bar = 10μm.
Nature Immunology Supplementary Video s6.mov (8MB)
Early phase two with very high dose C-peptide.Antigen-specific P14 T cells (green) and control OT-I T cells (blue) interacted with 100μM C-peptide pulsed CD11c+ DCs (red). The peptide-pulsed DCs (20μM CMTMR) were injected with 10ng LPS into the footpad of a recipient C57BL6 mouse. 18 hours later, P14 CD8+ T cells (2.5μM CMFDA, green) and control, OT-I CD8+ T cells (10μM CMAC, blue) were injected i.v. followed 2h later by 100mg Mel-14 antibody i.v.. Images were collected from 4-5h after T cell transfer by MPM-IVM of the right popliteal LN in an anesthetized mouse in the z dimension over a distance of 40μm. Bar = 10μm.
Nature Immunology Supplementary Video s7.mov (7MB)
Earlier phase two for P14 interacting with two populations of DCs pulsed with different levels of M-peptide.
Antigen-specific P14 T cells (green) interacted with two populations of peptide pulsed CD11c+ DCs (red: 200pM M-peptide, blue: 10μM M-peptide). The peptide-pulsed DCs (red, 20μM CMTMR and blue, 10μM CMAC) were injected with 10ng LPS into the footpad of a recipient C57BL6 mouse. 18 hours later, P14 CD8+ T cells (2.5μM CMFDA) were injected i.v. followed 2h later by 100mg Mel-14 antibody i.v.. Images were collected from 4-5h after T cell transfer by MPM-IVM of the right popliteal LN in an anesthetized mouse in the z dimension over a distance of 40μm. Bar = 10μm.
Phase one for P14 T cells and phase two for OT-I T cells interacting with dually peptide pulsed DCs.
Antigen-specific P14 T cells (green) and control OT-I T cells (blue) interacted with dually peptide pulsed CD11c+ DCs (red, 200pM M-peptide and 10μM SIINFEKL). The peptide-pulsed DCs (20μM CMTMR) were injected with 10ng LPS into the footpad of a recipient C57BL6 mouse. 18 hours later, P14 CD8+ T cells (2.5μM CMFDA, green) and control, OT-I CD8+ T cells (10μM CMAC, blue) were injected i.v. followed 2h later by 100mg Mel-14 antibody i.v.. Images were collected from 3.5-4.5h after T cell transfer by MPM-IVM of the right popliteal LN in an anesthetized mouse in the z dimension over a distance of 40 μm. Bar = 10μm.
Nature Immunology Supplementary Video s9.mov (9MB)
Phase two for both P14 T cells and OT-I T cells interacting with dually peptide pulsed DCs.
Antigen-specific P14 T cells (green) and control OT-I T cells (blue) interacted with dually peptide pulsed CD11c+ DCs (red, 10nM M-peptide and 10μM SIINFEKL). The peptide-pulsed DCs (20μM CMTMR) were injected with 10ng LPS into the footpad of a recipient C57BL6 mouse. 18 hours later, P14 CD8+ T cells (2.5μM CMFDA, green) and control, OT-I CD8+ T cells (10μM CMAC, blue) were injected i.v. followed 2h later by 100mg Mel-14 antibody i.v.. Images were collected from 2.5-3.5h after T cell transfer by MPM-IVM of the right popliteal LN in an anesthetized mouse in the z dimension over a distance of 40μm. Bar = 10μm.
Henrickson, S.E., Mempel, T.R., Mazo, I.B., Liu, B., Flynn, M., Artomov, M., Junt, T., Wong, H.C., Chakraborty, A.K. and von Andrian, U.H. T cell sensing of antigen dose governs interactive behavior with dendritic cells and sets a threshold for T cell activation. Nature Immunology, 9(3):282-291, 2008. [Full Text PDF][Supplementary Materials][Supplementary Videos]
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