a) Representative immunoblot indicating loss of talin1 in OVA peptide expanded FACS sorted CD4+ T cells from Talin1^Loxp/Loxp:CD4-Cre or control mice b) Single cell suspensions from the thymus, lymph nodes, spleen and blood were stained with CD3, CD4, and CD8 antibodies. Plots show CD3+ subset. c) The total # of CD3+/CD4+ cells in lymph node and spleen d) # CD3+/CD4+ cells in 2 cervical and inguinal lymph nodes e) # of CD3+/CD4+ cells in spleen. f) # CD3+/CD4+ cells per µl blood. Bar graph represents mean +/− standard error from 3 independent experiments using 6 mice. *=p<0.05 g) PKH26 and CFSE labeled splenocytes from Talin^+/+:CD4-Cre and Talin1^Loxp/Loxp:CD4-Cre mice were transferred into recipient mice in equal proportion. One hour after injection, the ratio of CD4+ talin-deficient to control cells was determined by flow cytometry. Data represent mean +/− standard error. Data are from two independent experiments using 10 mice total.

a/b) OVA peptide expanded T cells were stained with CFSE and stimulated with irradiated splenocytes in the presence of 0, 0.01, 0.1, or 1 µg/ml OVA peptide. a) Representative histograms of CFSE dilution b) Average proliferative index +/− standard error *=p<0.05. c/d) in vivo proliferation of CFSE labeled naïve CD4+ cells injected into recipient mice 18 hours prior to stimulation with LPS or LPS and ovalbumin. d) Representative histograms of CFSE dilution e) average proliferative index +/− standard error. *=p<0.05. e/f/g) cytokine production from OVA peptide expanded T cells restimulated with anti-CD3/CD28 coated plates in the presence of brefeldin A prior to intracellular staining and flow cytometry analysis a/b) representative scatter plots of cytokine staining g) average % of CD4+ T cell producting ctyokine +/− SEM. *=P<0−.05. All plots and graphs are representative of at least three independent experiments

a/b) OVA peptide expanded T cells were stained with CFSE and stimulated with anti-CD3/CD28 coated beads or PMA and ionomycin. a) Representative histograms of CFSE dilution b) Average proliferative index +/− standard error. c) Immunoblotting of control and talin1-deficient lysates following stimulation with anti-CD3 crosslinking for the indicated times. d) Band density quantification of phospho-protein relative to total. Graph represents average +/− standard error from 4 independent experiments e) OVA peptide expanded T cells loaded with Indo-1 were coated with biotinylated anti-CD3. After acquiring baseline ratio of bound:unbound calcium, streptavadin was added to induce TCR crosslinking and change in the ratio of 405 to 495 was measured. Plot is representative of 3 independent experiments

a) OVA peptide expanded T cells were fluorescently labeled and adhered to ICAM-1 coated plates untreated or following stimulation with anti-CD3 crosslinking, PMA or MnCl₂. Following washing step, the percentage of cells adherent to the plate was determined. b) OVA peptide expanded CD4+ T cells were calcein labeled and incubated with PKH-26 labeled LB27.4 cells loaded with OVA peptide +/− MnCl₂. The percentage of OVA-dependent conjugation was determined by flow cytometry. c) OVA peptide expanded CD4+ T cells were imaged during interactions with red labeled LB27.4 cells +/− OVA peptide. Representative still images of T cell:APC contact are shown. White arrow follows a single T cell over time. d) Average time of T cell:APC contact e) Graphs depicting the number and duration of APC contacts made by 26 control and talin1-deficient T cells during 45 minutes of imaging. Data are representative or pooled from 3 independent experiments *=p<0.05.

OVA peptide expanded Talin1^Loxp/Loxp:Rosa26-CreER^T2:OTII cells were retrovirally transduced with mCherry vector and GFP-PH AKT. Prior to analysis, cells were treated with 4-OH tamoxifen or vehicle to induce genomic deletion of talin1. a) Immunoblotting shows >97% loss of talin with tamoxifen treatment. b) Ratiometric images of PH-AKT signaling relative to mCherry. White star depicts antigen presenting cell. Blots and images are representative of three independent experiments including 15 conjugation events. Scale bar represents 10 µm.

a/b) OVA peptide expanded cells were conjugated with OVA peptide loaded LB27.4 cells and fixed on coverslips and stained with the indicated antibodies. Graph is line scan through conjugate: X axis corresponds to fluorescent intensity while Y axis corresponds to µm position through conjugate. Blue line represents APC, green line represents LFA-1, and red line represents indicated antibody. Scale bar represented 10 µm. Images are representative of three independent experiments and at least 30 conjugates. c) Brightfield images of an equal number of unstimulated cells in round- and flat-bottomed plates. Scale bar represents 100 µm. d) Bar graph depicting proliferative index of OVA peptide expanded T cells in response to OVA peptide in round-bottomed plates. Data are mean +/− standard error from three independent experiments, *=p<0.05.

a-d) OVA peptide expanded control and talin1-deficient T cells were conjugated with LB27.4 B cells in the presence or absence of antigen, fixed on poly-L-lysine coverslips, and stained with the indicated antibodies. Graph is line scan through conjugate: X axis corresponds to fluorescent intensity while Y axis corresponds to µm position through conjugate. Blue line represents APC, green line represents LFA-1, and red line represents indicated antibody. Images are representative of 10 conjugation events from 3 independent experiments. Scale bar represents 10 µm.

a) OVA peptide expanded Talin1^Loxp/Loxp:Rosa26-CreER^T2:OTII T cells were retrovirally transduced with Lifeact-mRUBY and UtrCH-GFP and imaged during T cell contact with LB27.4 cells in the presence or absence of OVA peptide. Prior to the experiment, T cells were treated with ethanol or 4-OH tamoxifen to deplete talin1. Still images of time-lapse movie are representative of 10 contacts observed from 2 independent experiments. Scale bar represents 10 µm. * corresponds to APC