Supplementary Methods
For immunohistochemistry, 17-µm frozen sections of lymph node were processed and
incubated with biotinylated anti-mouse TCR -chain mAb H57-597 (PharMingen, San
Diego, CA) followed by Streptavidin-FITC (Southern Biotechnology) or StreptavidinTexas-Red (Jackson ImmunoResearch) and anti-I-Ek-FITC (PharMingen). For
immunofluorescence staining, T-cells were first fixed in 4% paraformaldehyde in PBS
for 5 min at 37˚C, then incubated with biotinylated anti-mouse TCR -chain mAb H57597 (PharMingen) followed by Streptavidin-FITC (Southern Biotechnology) or
Streptavidin-Texas-Red (Jackson ImmunoResearch). As a control for non-specific
staining, T-cells were incubated with biotinylated hamster IgG isotype standard
(PharMingen) followed by Streptavidin-FITC (Southern Biotechnology). The pattern of
staining seen with the anti-TCR -chain mAb was verified by using an anti-V11-chain
mAb (PharMingen). In some experiments, T-cells were permeabilized after staining with
anti-TCR antibody and incubated with anti-ZAP-70 1 or anti-SHP-1 rabbit antibodies
(Upstate Biotechnology, Lake Placid, NY) followed by anti-rabbit antibody conjugated to
Texas-Red. Stained sections or isolated T-cells were photographed on a Leica compound
microscope (Leica Camera AG, Solms, Germany) with a 63x or 100x oil immersion
objective NA1.32 connected to a digital video camera and analyzed using Openlab
software (Improvision Inc, Lexington, MA), Adobe Photoshop 6.0 (Adobe Systems, San
Jose, CA), and the public domain NIH Image program. For confocal microscopy, images
were collected on a Leica TCS-NT/SP confocal microscope (Leica Microsystems, Exon,
PA USA) using a 100x oil immersion objective NA 1.4, zoom 2.8 and an argon laser at
488 nm. Z stacks were collected using 0.203 µm optical section thickness. DIC
(differential interference contrast) images were collected using the transmitted light
detector. Images were processed using the Leica TCS-NT/SP software (version 1.6.551)
and Imaris 3.0.4 (Bitplane AG, Zurich, Switzerland), followed by final formatting using
Adobe Photoshop 6.0 (Adobe Systems, San Jose, CA). A TCR polarization index was
calculated using Photoshop software according to ref. 2, with some modifications.
Briefly, each cell was divided into quadrants. The Image/Histogram function was then
used to obtain the mean pixel intensity in each quadrant. The TCR polarization index was
calculated by the dividing the highest quadrant value by the lowest.
1.
Burkhardt, A. L. et al. Temporal regulation of non-transmembrane protein
tyrosine kinase enzyme activity following T cell antigen receptor engagement.
J Biol Chem 269, 23642-7 (1994).
2.
Reichert, P., Reinhardt, R. L., Ingulli, E. & Jenkins, M. K. Cutting edge: in
vivo identification of TCR redistribution and polarized IL-2 production by
naive CD4 T cells. J Immunol 166, 4278-81 (2001).
Supplementary Figure 1
Single cell analyses of T-cells isolated from 5C.C7-H2k mice and rested in in vitro
culture.
(a-b) T-cells were kept on ice or incubated for 30 min. at 37oC before being placed in the
culture with DCEK pre-pulsed with the indicated concentrations of PCC peptide. The Tcells were analyzed after 5.5 hr of stimulation for surface expression of CD69 (a) and
intracellular IL-2 synthesis (b).
(c-d) Forward scatter analysis as an indicator of cell size was performed after 16 and 28
hr of stimulation.
(e) Cell viability was assayed by FACS analysis at the indicated times using 7aminoactinomycin D staining.
Supplementary Figure 2
Proliferation and forward scatter (size) analysis of T-cells isolated from mock injected
and anti-I-Ab antibody (Y3P) injected AND-H2b mice.
(a) T-cells were placed in culture with DCEK pre-pulsed with indicated concentrations of
PCC peptide and analyzed after 48 hr. for 3H-incorporation.
(b-c) T-cells were placed in culture with DCEK pre-pulsed with indicated concentrations
of PCC peptide and analyzed for forward scatter after 16 and 31 hr. The data in (a – c)
come from the same experiment that generated the data shown in Fig. 2e-g.
Supplementary Figure 3
T-cells isolated from H2b AND mice were placed in culture with lymph node cells from
either wild-type H-2b or wild-type H-2q mice. The ratio used was the same as that of
CFSE-labeled donor H-2b AND present in isolated lymph node cells from wild-type H-2b
or wild-type H-2q recipients analyzed in the experiments presented in Figure 2. These
cultures were supplemented with I-Ek expressing DCEK pre-pulsed with various
concentrations of PCC peptide. 3H-thymidine incorporation as an indicator of cell
proliferation was measured 80 hrs. later. The Figure displays the responses of naïve H-2b
AND T-cells cultured separately with cells isolated from each of two wild-type H-2b and
two wild-type H-2q mice (labeled as B6 or H-2q 1 and B6 or H-2q 2).