Data Supplement

Files in this Data Supplement:

• Video 1 (MOV, 2.45 Mb)
  - Indo-1 Ca2+ imaging in basal, inflammatory, and immunized conditions. Montage of Ca2+ imaging records shows DO11.10 CD4+ T cells in a LN from a non-immunized mouse at 2.5 h after adoptive transfer (PBS, left), from a sham-immunized mouse at 1.5 h after adoptive transfer (cyt, middle), and from an antigen-immunized mouse at 2 h after adoptive transfer (OVA + cyt, right). Video shows maximum intensity projections in the x-y (top) and x-z (bottom) directions. Fluctuations between low and high Ca2+i can be seen as color changes between red and green, respectively. DC, if present, are pseudocolored red. Scale bar is 20 um; 75 um z section. Elapsed time indicated in min:s.
• Video 2 (MOV, 9.74 Mb)
  - Relation between Indo-1 fluorescence and Ca2+ signaling. A high-resolution, zoomed view of the right panel from Video S1 illustrate calcium signals of T cells in a LN from an antigen-immunized mouse (OVA + cyt). Colored arrows highlight selected cells in the video (left) and correspond to colored calcium traces (right). Scale bar is 20 um; 75 um z section. Elapsed time indicated in min:s.
• Video 3 (MOV, 1.52 Mb)
  - Early Ca2+ signaling with antigen. T cells begin Ca2+ signaling after entry into LN parenchyma of antigen-immunized mouse (OVA + cyt), < 1 h after adoptive transfer. Video shows maximum intensity projections in the x-y (top) and x-z (bottom) directions. DC are pseudocolored red. A few T cells at the site of homing have low Ca2+i and also appear red. At the beginning, a static image of cumulative fluorescence of cells over the initial 8.5 min illustrates the distribution of signaling cells (green). Scale bar is 20 um; 75 um z section. Elapsed time indicated in min:s.
• Video 4 (MOV, 1.19 Mb)
  - T cell clusters at three hours. Network of T cells clusters undergoing Ca2+ fluctuations at 3 h after adoptive transfer. Video shows maximum intensity projections in the x-y (top) and x-z (bottom) directions. Scale bar is 20 um; 75 um z section. Elapsed time indicated in min:s.
• Video 5 (MOV, 1.25 Mb)
  - Dynamics of Ca2+ signaling in T cell clusters. T cell behavior and Ca2+ signaling in LN during the early development of an immune response (OVA + cyt, 1.5 h after adoptive transfer) is shown by clustered T cells (red circles) and non-motile/solitary T cells (blue arrowheads). Rare motile T cells are tracked by white trailing dots, and a cell that is polarized during transient Ca2+i elevation is marked by an orange arrowhead. Video shows maximum intensity projections in the x-y (top panels) and x-z (bottom panels) directions. Scale bar is 20 um; 75 um z section. Elapsed time indicated in min:s.
• Video 6 (MOV, 2.31 Mb)
  - T cell scanning without antigen. Two examples (left, right) of Ca2+ imaging during T cell scanning of DC under sham-immunized (cyt) condition. DC are pseudocolored red. The T cell of interest in each video is indicated by white arrowhead. Video shows maximum intensity projections in the x-y (top panels) and x-z (bottom panels) directions. Scale bar is 20 um. Elapsed time indicated in min:s.
• Video 7 (MOV, 1.52 Mb)
  - Stable T cell contacts with stationary and migratory DC. Montage of two videos showing Ca2+ signals during T cell clustering around DC (left), and a migratory DC dragging along T cells (right), with maximum intensity projections in the x-y (top panels) and x-z (bottom panels) directions. DC are pseudocolored red. Scale bar is 20 um; 75 um z section. Elapsed time indicated in min:s.
• Video 8 (MOV, 3.17 Mb)
  - Contact termination. Two examples (left, right) of Ca2+ imaging during T cell-DC interaction under antigen-immunized (OVA + cyt) condition. DC are pseudocolored red. The T cell of interest in each video is indicated by white arrowhead. Video shows maximum intensity projections in the x-y (top panels) and x-z (bottom panels) directions. Scale bar is 20 um. Elapsed time indicated in min:s.
• Video 9 (MOV, 2.65 Mb)
  - Serial engagement without antigen. Three-color Ca2+ imaging record shows DO11.10 CD4+ T cells in LN from a mouse injected with LPS-stimulated BMDC (blue) at 1 h after adoptive transfer. Video shows maximum intensity projections in the x-y (top) and x-z (bottom) directions. Fluctuations between low and high Ca2+i can be seen as color changes between red and green, respectively. Scale bar is 20 um; 27.5 um z-section. Elapsed time indicated in min:s.
• Video 10 (MOV, 2.60 Mb)
  - Stable engagement with antigen. Three-color Ca2+ imaging record in LN, as above, but after culturing BMDC with OVA and stimulating with LPS. Scale bar is 20 um; 27.5 um z-section. White arrowheads indicate examples of T cells that continue to undergo Ca2+ signaling in the absence of contact with BMDC.
• Video 11 (MOV, 4.35 Mb)
  - Ca2+ signaling following detachment. Three-color Ca2+ imaging record in LN, as above (after culturing BMDC with OVA and stimulating with LPS), at 4 hours after adoptive transfer of T cells. Scale bar is 10 um; 20 um z-section. Arrowhead indicates a T cell that continues to signal after detaching from BMDC (detached from 03:33 to 13:30). Calcium and velocity traces from this T cell are shown in the last frame of the video; bars under trace indicate duration of contact with BMDC.