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ot-2 t cells ( 510/well ) were incubated for 30 min at 37c in 96-well u - bottom plates with dcs ( 10/well ) or lps - activated b cells ( 210 per well ) that had been pulsed with antigen .
conjugates were enumerated by flow cytometry after the cell mixture was stained at 4c for cd4 , cd11c , and cd19 and repeatedly washed . to visualize t - dc interactions ( fig .
1 ) , ova323-pulsed dcs were injected subcutaneously at 210 per mouse 24 hours prior to intravenous transfer of sap and sap t cells ( 310 each ) .
imaging was conducted 12 to 24 hours later . to examine activation phenotypes , 10 dcs and 210 gfp - expressing t cells per mouse
2 ) , 310 ot-2 t cells of each genotype were co - transferred into mice together with 510 wild - type b cells .
immunization was done 12 hours prior to cell transfer , and imaging was conducted 24 to 36 hours thereafter . to visualize t - b interactions under non - competitive conditions and to assay t and b cell expansion ( fig .
3 ) , 610 gfp - expressing ot-2 t cells were co - transferred together with 310 cfp - expressing b cells 24 hours prior to immunization .
imaging and cytometric analyses were conducted 6072 and 96 hours later , respectively . to visualize gc recruitment and retention of t cells ( fig .
4 ) , 310 cfp - expressing sap and 310 gfp - expressing sap ot-2 t cells were co - transferred with 310 non - fluorescent md4 b cells .
dye - labelled nave b cells ( 2410 ) were given 1 day before imaging to provide follicle / gc landmarks .
longer than 2 hours , the animal s hydration was maintained by lactated ringer s solution given via a catheter .
the typical x - y - z dimension was 0.51.10.51.13 m , and the time resolution was 3045s . for experiments involving co - transfer of two types of dye - labelled t cells ,
the cells were always reciprocally labelled to control for potential dye - induced behavioural differences .
the mann - whitney rank sum test was used to calculate p values for highly skewed distributions . for gaussian - like distributions ,
ot-2 t cells ( 510/well ) were incubated for 30 min at 37c in 96-well u - bottom plates with dcs ( 10/well ) or lps - activated b cells ( 210 per well ) that had been pulsed with antigen .
conjugates were enumerated by flow cytometry after the cell mixture was stained at 4c for cd4 , cd11c , and cd19 and repeatedly washed .
1 ) , ova323-pulsed dcs were injected subcutaneously at 210 per mouse 24 hours prior to intravenous transfer of sap and sap t cells ( 310 each ) .
imaging was conducted 12 to 24 hours later . to examine activation phenotypes , 10 dcs and 210 gfp - expressing t cells per mouse
2 ) , 310 ot-2 t cells of each genotype were co - transferred into mice together with 510 wild - type b cells .
immunization was done 12 hours prior to cell transfer , and imaging was conducted 24 to 36 hours thereafter . to visualize t - b interactions under non - competitive conditions and to assay t and b cell expansion ( fig .
3 ) , 610 gfp - expressing ot-2 t cells were co - transferred together with 310 cfp - expressing b cells 24 hours prior to immunization .
imaging and cytometric analyses were conducted 6072 and 96 hours later , respectively . to visualize gc recruitment and retention of t cells ( fig .
4 ) , 310 cfp - expressing sap and 310 gfp - expressing sap ot-2 t cells were co - transferred with 310 non - fluorescent md4 b cells .
dye - labelled nave b cells ( 2410 ) were given 1 day before imaging to provide follicle / gc landmarks .
imaging sessions longer than 2 hours , the animal s hydration was maintained by lactated ringer s solution given via a catheter .
the typical x - y - z dimension was 0.51.10.51.13 m , and the time resolution was 3045s . for experiments involving co - transfer of two types of dye - labelled t cells ,
the cells were always reciprocally labelled to control for potential dye - induced behavioural differences .
the mann - whitney rank sum test was used to calculate p values for highly skewed distributions . for gaussian - like distributions ,
generation of long - term antibody - mediated immunity depends on the germinal centre ( gc ) reaction , which requires cooperation between antigen - specific t and b lymphocytes . in the human x - linked lymphoproliferative disease and its gene - targeted mouse model , loss - of - function mutations in signalling lymphocyte activation molecule - associated protein ( sap , encoded by sh2d1a ) 
 cause a profound defect in gc formation by an as yet unknown mechanism . using two - photon intravital imaging , here we show that sap deficiency selectively impairs the ability of cd4 + t cells to stably interact with cognate b cells but not antigen - presenting dendritic cells . 
 this selective defect results in a failure of antigen - specific b cells to receive adequate levels of contact - dependent t cell help to expand normally , despite sap/ t cells exhibiting the known characteristics of otherwise competent helper t cells . 
 furthermore , lack of stable interactions with b cells renders sap/ t cells unable to be efficiently recruited to and retained in a nascent gc to sustain the gc reaction . 
 these results offer a compelling explanation for the gc defect due to sap deficiency and provide novel insights into the bi - directional communication between cognate t and b cells in vivo .