Icos-/ CD3 Icos Y181F OT-2 T cells doi:1.138/nture158 IgD Supplementry Figure 1. is required for folliculr locliztion of ctivted helper T cells. Icos nd Icos-/- OT-2 T cells were retrovirlly trnsduced with, s indicted, or or mutnt Y181F tht is incple of signling ut expressed normlly on the cell surfce, nd then intrvenously trnsferred into B6 mice tht were susequently immunized s.c. with OVA. Four dys fter immuniztion, sections of drining lymph nodes were stined for indicted mrkers. Dt re representtive of more thn sections collected from t lest two independent experiments. Br = 1 μm. W W W. N A T U R E. C O M / N A T U R E 1
Stin c 1 Stin -/- Isotype % of Mx 8 6 4 Isotype -/- Isotype 1 Stin d 1 1 2 1 3 1 4 1 5 Stin 8 8 % of Mx 6 4 -/- Isotype % of Mx 6 4 -/- Isotype -/- Isotype 1 3 1 4 1 5 1 2 1 3 1 4 1 5 Supplementry Figure 2. Typicl stining ptterns of on T cells used in the study., Resting T cells stined for y iotinylted primry A nd reveled y streptvidin-pe., T cells ctivted in vitro y nti-cd3 nd nti-cd28 or y splenic APC + ntigen, retrovirlly trnsduced or not, nd rested for 3 dys. Stined s in (). c, Nive OT-2 T cells were ctivted in doptive hosts y OVA protein for 3 to 4 dys. Stined s in (). d, OT-2 T cells ctivted in vitro s in (), trnsferred nd rested in recipient mice, nd then ctivted y NP-OVA immuniztion for 4 dys in vivo. Stined with PE-leled nti-. 2 WWW.NATURE.COM/NATURE
Icos Icosl MD4 Icosl-/- MD4 Icos CD3 OT-2 B2 Icos-/- Icos-/- Supplementry Figure 3. Cognte B cells do not progrm folliculr locliztion of ctivted helper T cells. Nïve dsred-expressing Icos-/- or Icos OT-2 T cells (2 15 per mouse) nd nïve MD4 B cells of indicted Icosl genotypes (8 15 per mouse) were co-trnsferred into B6 mice tht were then sucutneously immunized with HEL-OVA conjugted ntigen. The distriution pttern of OT-2 T cells in the drining lymph nodes ws nlyzed on dy 4. One of two experiments with similr results is shown (t lest 2 mice per condition per experiment). T cells re pseudo-colored s green for consistent presenttion. (Scle r = 1μm) W W W. N A T U R E. C O M / N A T U R E 3
mrna quntity 8 6 4 1 1 Vector Bcl-6 Vector: Bcl-6 Bcl-6 -/- Bcl-6 Bcl-6..5 1. MD4 GC B cells (%) 1.5 *** * ns ns 5 1 15 25 CXCR5 hi PD-1 hi OT-2 cells (%) Supplementry Figure 4. Bcl6 overexpression fils to rescue defective T FH nd GC development due to the deficiency in T cells., Typicl Bcl6 expression profile s mesured t the mrna level y quntittive PCR (left) or t the protein level y intrcellulr stining (right) on Bcl6- nd vector control-trnsduced T cells., OT-2 T cells of indicted genotypes were ctivted in vitro, trnsduced with Bcl6-expressing or empty control vector, sorted to >9% + purity, nd co-trnsferred with nïve MD4 B cells into B6 mice tht were susequently immunized s.c. with HEL-OVA. Five dys lter, the MD4 GC B cell (GL7 hi Fs hi IgM + ) frequency in the CD19 + comprtment nd the T FH cell (CXCR5 hi PD-1 hi + ) frequency in the totl OT-2 popultion were mesured. Dt re pooled from 2 seprte experiments with t lest 7 recipient mice per condition. (*p<.5, *** p<.1; ns = not significnt). 4 WWW.NATURE.COM/NATURE
-trnsduced CXCR5-trnsduced CXCR5 CCR7 CXCR5 CCR7 Isotype Icos -/- Icos Supplementry Figure 5. CXCR5 nd CCR7 expression on in vitro ctivted nd trnsduced T cells. CXCR5 nd CCR7 expression on Icos -/- or Icos OT-2 T cells recovered from the spleens tht were ssyed for T cell distriution pttern s in Fig. 2c. One of two experiments is shown. WWW.NATURE.COM/NATURE 5
Icos Icos -/- OT-2 IgD CXCR5 CXCR5 CD3 Supplementry Figure 6. -dependent, costimultion-independent control of folliculr recruitment of CXCR5 + T cells. Icos -/- or Icos OT-2 T cells were ctivted in vitro y nti-cd3 nd nti-cd28 ntiodies, trnsduced with CXCR5 or -expressing retrovirl vector, nd then trnsferred into nïve B6 mice (3 1 6 sorted + cells per mouse). The distriution pttern (ech + cell identified with circle) of these T cells in the spleen ws nlyzed 24 hours lter. Dt represent 2 experiments involving 2~3 mice per condition per experiment. (Scle r = 1μm). 6 WWW.NATURE.COM/NATURE
Icosl Icosl -/- Fo T % of Mx 1 8 6 4 B2 1 2 1 3 1 4 1 5 L L c L Lethl irrdition injection with OVA-pulsed DCs >8 weeks Merge i.v. trnsfer of 8: mixed BM cells 8 : µmt : WT µmt : Icosl -/- µmt : I-A -/- i.v. trnsfer of OT-2 T cells L Supplementry Figure 7. L expression on B cells nd the strtegy to test its role y BM chimer., Typicl L stining pttern of wildtype lymph node sections co-stined for B2 (left) nd the ckground pttern reveled using Icosl -/- mice (right; lymph node tissue edge nd T-B order dotted to indicte the folliculr re the T zone)., Typicl histogrms of L nd isotype stining on CD11c + DCs (green nd lck) nd CD19 + B cells (red nd grey) y flow cytometry. Note tht while DCs nd B cells express similr levels of L on the individul cell sis, folliculr B cells re numericlly fr more undnt nd ntomiclly much densely pcked in the follicle to give the field-like stining intensity nd pttern. c, A schemtic digrm for the protocol to generte follicles tht re devoid of L or clss II MHC expression s used in Fig. 3. WWW.NATURE.COM/NATURE 7
1 1 Migrtion % 5 Migrtion % 5 Isotype α-.5 2.5.1.5 1. CXCL13 (μg/ml) CCL21 (μg/ml) Supplementry Figure 8. does not modulte CXCR5 or CCR7 receptor sensitivities. OT-2 T cells were ctivted in vitro, trnsduced with CXCR5-expressing vector or not, nd then sujected to the trnswell migrtion ssy. The lower chmer ws filled with recominnt CXCL13 or CCL21 of indicted concentrtions. Cells migrted down from the top chmer over 3-hour period were enumerted y flow cytometry. Antiody tretment (1 μg/ml) ws mintined throughout the ssy. For ssying CXCL13, CXCR5-trnsduced cells were used, ecuse non-trnsduced T cells showed <5% specific migrtion in response to CXCL13. For ssying CCL21, non-trnsduced T cells were used. Dt re men±sem of triplicte wells, nd results re representtive of more thn 3 independent experiments. 8 WWW.NATURE.COM/NATURE
Control * * * α- Frction of cells p <.1 1..8 *.6.4 *.2. Control α- Supplementry Figure 9. Anti- tretment leds to T cell polriztion. Phlloidin stining of F-ctin on OT-2 T cells tht were treted or not with 5 μg/ml nti- t 37 C for 1 minutes. Typicl cell morphologies of the two conditions re shown. The F-ctin-rich, polrized cells (leled with *) nd non-polrized cells were enumerted in the r grph on the right. The p vlue is from the Fisher s exct test on totl of 93 treted nd 66 untreted cells pooled from two independent experiments. WWW.NATURE.COM/NATURE 9
- α- + CAL-11 -CD28 25 n=44 25 n=31 25 n=34-25 25-25 25-25 25-25 -25-25 Centroid velocity (μm/s) 3 2 1 CAL-11 - + α- c *** *** * 1. *** ns ns Persistence.5. - CAL-11 - + - α-cd28 α- α-cd28 Supplementry Figure 1. -triggered persistent motility depends on PI3K signling nd is not feture shred y CD28. LifeAct-mRuy-leled OT-2 T cells were imged y TIRF microscopy s in Figure 4 on lipid ilyers tht disply nti- or nti-cd28 ntiody. Some cells were pre-treted y 1 μm p11δ-specific PI3K inhiitor CAL-11 nd ssyed on the nti- surfce. Numers of cells nlyzed for ech group re s indicted., XY displcement (in micrometer) plots of individul cells with strting positions re-ligned t the sme origin., T cell centroid velocities. c, T cell directionl persistence s mesured y pth length-normlized displcement. Lines denote the mens. (* p<.5, *** p<.1, ns = not significnt). Also see corresponding Supplementry Movie 5-7. 1 WWW.NATURE.COM/NATURE
Imging with IR lser t 8 nm to visulize T cells nd CMF 2 HCleled B cells. Polrized Choosing folliculr order populted y -expressing T cells to zoom in Polrized Imging with the IR lser t 9 nm to visulize oth nd dsred T cells Depolrized Supplementry Figure 11. Strtegies to identify the T-B order for intrvitl imging nd exmples of cell polriztion nlysis., To locte the T-B order, we took dvntge of the fct tht, t dy 3 post ctivtion in vivo, mny of the -expressing OT-2 T cells re congregted t the T-B order s cn e scertined on tissue sections (not shown). We lso took dvntge of the fct tht expressed y the Jx 4353 trnsgenic line nd the CMF 2 HC dye used to leled folliculr B cells cn e simultneously visulized t 8 nm, even though this prticulr wvelength my not e generlly optiml for ll trnsgenic lines. Therefore, y imging t 8 nm first we chose the order of follicles tht ws surrounded y -expressing T cells. We then zoomed into such regions, exemplified here y the squre in red, nd imged t 9 nm so s to simultneously visulize - nd dsred-expressing T cells of vrious genotypes. At this wvelength, CMF 2 HC-leled B cells ecme invisile. To quntitte frequencies of polrized nd depolrized cells t the T-B order, two qudrnts long the pproximte order line from ech imging field, exemplified here y the two squres in white, were cropped for detiled nlysis., Exmples of Icos (1, 3, 5) nd Icos -/- (2, 4, 6) T cells in polrized or depolrized sttes s oserved t the T-B order. Clssifiction s polrized requires shpe indexes (SI) greter thn 2 or, when SI<2, the existence of t lest one pseudopod extension. The SIs for the 6 exmples: (1) 4.8, (2) 4.5, (3) 1.1, (4) 1.4, (5) 1.4, nd (6) 1.3. Arrowheds indicte pseudopod extension. WWW.NATURE.COM/NATURE 11
Cell #2- Cell #1-dsRed Supplementry Figure 12. Pseudopod extension OT-2 T cells t the T-B order. Nïve -expressing nd dsred-expressing OT-2 T cells (1-2 1 6 per mouse) were co-trnsferred into norml B6 hosts. Intrvitl imging of the drining lymph node ws conducted three dys fter sucutneous injection of OVA323-pulsed DCs. Shown re time-lpse imges of migrting T cells t the T-B order, corresponding to Supplementry Movie 8. Arrowheds highlight pseudopod extensions lternting in coordinted left-right fshion for oth - nd dsred-expressing T cells. Scle r = μm. 12 WWW.NATURE.COM/NATURE
Norml B6 hosts dsred f dsred dsred c MSD (μm 2 ) 1:42 4:37 7:22 14:34 15:35 p =.7 depolrized cells (%) 3 1 1 3 1 3 4 5 Time (sec) 4 d e dsred dsred. p <.1 p <.1 3 R 2 =.8,.8.2.4 % of trcks Velocity (μm/sec) p <.1 p <.1 15 R 2 =.7,.8 1 5..2.4.6.8 1..6 % of trcks 1. Persistence.2.4.6..2.4.6.8 1. g (1) (2) (3) Pseudopod & cell polriztion 1 1 Men squred displcement 1 1 Velocity 1 1.15 Directionl persistence 1.95 Velocity -trck Men Velocity Velocity dsred-trck 1.15 Men Velocity Persistence -trck Men Persistence Persistence dsred-trck.95 (4) Men Persistence Normlized Velocity Normlized Velocity dsred Normlized Persistence Normlized Persistence dsred Supplementry Figure 13. Motility chrcteriztion of - nd dsred-trnsgenic OT-2 T cells t the T-B order nd procedures for dt normliztion. -e, Wildtype OT-2 T cells crrying the trnsgene-encoded or dsred were imged t the sme T-B order 3 dys following ctivtion in vivo to control for is potentilly introduced y intrinsic differences in the nd dsred trnsgenic lines. A totl of 6 T cell trcks nd 512 dsred T cell trcks from 3 experiments were nlyzed. () Time-lpse imges showing typicl polrized morphology with evident pseudopods. Circles highlight the few depolrized cells without overt pseudopods. Scle r = μm. () Frequencies of depolrized nd dsred T cells. Ech symol represents one independent experiment, nd symols of the sme shpe represent mtched mesurements mde in the sme experiment (>1 cells counted for ech type of cells in ech experiment). (c) Men squred displcement of migrting cells over period of 7 minutes. (d) Sctter plots of trcked cell velocities pooled from ll experiments (left, line denotes the men, p vlue from t test) nd Gussin fits for velocity distriutions of individul experiments (right, p vlue for comprsion of the men from the extr sum of squres F test, R 2 for goodness of fit). (e) Sctter plots nd Gussin fits for persistence of trcked cells mesured s the pth length-normlized displcement. f, Summry of dt presented in (-e); numers represent reltive differences in men vlues, with eing set s 1. g, Procedure for normliztion. Dt presented in (-e) showed sutle ut significnt differences etween nd dsred trnsgenic lines. Therefore, for ech susequent experiment to compre wildtype nd Icos -/- dsred OT-2 T cells, we normlized individul trck velocity nd persistence dt points to its own group mens (eqution (1) nd (3), essentilly setting the distriution men to unit 1); for dsred trcks, dt points were first modified y the normliztion fctor 1.15 nd.95 otined in (f) for velocity nd persistence, respectively, nd then normlized ginst the men vlue of mtched trcks mesured in the sme imging experiment (eqution (2) nd (4)). This normliztion procedure does not lter properties of Gussin distriutions, which the velocity nd persistence dt essentilly conform to (see d, e, nd Fig. 5e, 5f). As result of this normliztion, velocity nd persistence dt presented in Fig. 5 re without unit ut comprle cross experiments conducted on different hosts (e.g. norml B6 versus chimer). Note: the verge velocity for T cells oserved here (.25-.28 µm/sec or 15-18 µm/min) is higher thn the commonly reported 1-12 µm/min (Chln nd Prker, Annu. Rev. Immunol. 26:585, 8), minly ecuse centroid trcking t n incresed sptiotemporl resolution y necessity tke into ccount more of cell displcement due to morphologicl chnge nd virtionl motility on shorter time scle, which would otherwise e verged out y slower smpling rte. WWW.NATURE.COM/NATURE 13
Lethl irrdition i.v. trnsfer of 8: mixed BM cells Reconstitution for >8 weeks 8 : μmt : Icosl μmt : Icosl -/- s.c. immuniztion with HEL-OVA in lum i.v. trnsfer of 1 4 OT-2 T cells nd 8 1 4 MD4 B cells µmt:icosl -/- c p <.5 p <.1 µmt:icosl..2.4.6.8 MD4 GC B cells (%) CXCR5 hi PD-1 hi OT-2 cells (%) 1 3 4 µmt:icosl -/-.7 97 16 µmt:icosl CD19.36 Fs CXCR5 97 26 MD4 GL7 PD-1 Supplementry Figure 14. Impired GC nd T FH formtion when ystnder B cells do not express L., A schemtic digrm for the ssy protocol. All mesurements were done on dy 5 fter immuniztion., Frequencies (men±sem) of GL7 hi Fs hi IgM + MD4 GC B cells (or GL7 hi Fs hi + when -trnsgenic MD4 line ws used) in the CD19 + comprtment. c, Frequencies (men±sem) of CXCR5 hi PD-1 hi cells in the CFP + OT-2 comprtment. Contour plots show typicl gting with numers indicting % MD4 in CD19, % GL7 hi Fs hi in MD4, nd % CXCR5 hi PD-1 hi in OT-2. MD4 dt represent 4 independent experiments employing 4 independent sets of BM chimer. OT-2 dt represent 2 of these experiments in which CFP-trnsgenic OT-2 T cells were used. Non-fluorescent OT-2 cells were used in the other two nd could not e trcked. For ech experiment, 3~5 recipient mice per group were used. 14 WWW.NATURE.COM/NATURE
Lethl irrdition i.v. trnsfer of 8: mixed BM cells Reconstitution for >8 weeks 8 : WT : Icosl WT : Icosl -/- s.c. immuniztion with HEL-OVA in lum i.v. trnsfer of 1 4 OT-2 T cells nd 8 1 4 MD4 B cells c WT:Icosl -/- WT:Icosl..2.4.6.8 MD4 GC B cells (%) CXCR5 hi PD-1 hi OT-2 cells (%) 1 3 4 WT:Icosl -/-.34 91 3 WT:Icosl CD19.37 Fs 95 CXCR5 28 MD4 GL7 PD-1 Supplementry Figure 15. Norml GC formtion y MD4 nd OT-2 T cells in WT:Icosl -/- 8: mixed BM chimer. The protocol nd dt presenttion were in essentilly the sme mnner s in Supplementry Fig. 14, except tht the chimeric host involved ws 8: WT:WT or WT:Icosl -/-. Five mice per group were nlyzed. WWW.NATURE.COM/NATURE 15
1. n=682 n=699 n=2524 n=1698 1. Velocity (μm/min) Persistence.5. 15 1 5 Velocity (μm/min) Persistence.5. 15 1 5 Icos Icos -/- Icos Icos -/- Supplementry Figure 16. is not required for T cell motility in the T cell zone. Nïve () or in vitro ctivted () CD4 T cells of the indicted genotypes were leled with CMF 2 HC or CMFDA nd injected into nïve B6 mice. Between 24 nd 48 hours post trnsfer, cell migrtion in the T cell zone ws nlyzed y intrvitl microscopy. The whisker ox plots show medin, qurtile, nd 5-95% rnge. Dt were pooled from 2 independent experiments in which dye lels for the two types of cells were swpped. Numers t the top indicte the totl numers of trcks nlyzed for ech group. Of note, recently ctivted Icos -/- T cells were slightly fster thn Icos T cells (men velocity: 1.1±.7 vs. 9.6±.8 μm/min). 16 WWW.NATURE.COM/NATURE
Supplementry Note 1: T cell motility t the T-B order Our results revel requirement for triggering to drive persistent T cell motility in the T-B order, or n re tht cn e pproximtely defined s etween the outer edge of the T cell zone nd the outer edge of the B cell follicle (s in Fig. 1c). On the other hnd, nïve T cells, which express very low ut detectle level of (Supplementry Fig. 2), nd recently ctivted T cells, which express high levels of, do not require to migrte in the T cell zone (Supplementry Fig. 13, Supplementry Movies 13 nd 14). While it is currently uncler s to wht underlies the differentil requirement of for norml T cell motility in the T cell zone versus round the folliculr edge nd eyond, severl possiilities cn e considered. Becuse CCR7 is the min driving force for persistent T cell motility in the T cell zone 1,2, the need for t the T-B order might reflect reduced CCL21 locl concentrtions. Okd et l. specificlly proed CCL21 distriution on lymph node sections 3, nd its intensity ppers to grdully decrese from the T zone towrd the folliculr re. Along the decresing CCL21 grdient into the folliculr re, there would e point t which the locl CCL21 concentrtions re not sufficient to support norml T cell motility, possily coincidentl with the outer edge of the T cell zone s defined in Fig. 1c. The CCL19/CCL21 stining pttern reported y Wors et l. 1 is, leit more punctuted, lso consistent with this ide. When comined with reduced CCR7 expression on T cells reching the T-B order nd possily shllow grdient of CXCL13 loclly t the T-B order, dditionl motility-driving forces my ecome required in the locle. Further, from the perspective of hptokinesis, the T zone stroml FRC cells tht produce nd disply CCL21 nd guide T cell migrtion in the T zone lso stop their fier-like processes t the outer edge of the T cell zone 4,5. Therefore, comined effects of chemokine nd stroml sustrte distriution my crete locl shrp drop-off of the motility-driving force t the outer edge of the T cell zone. In concert with or insted of this lck of motility-driving force, the follicle or B cells my even express surfce-ound or solule suppressive fctors tht downmodulte T cell motility or repulse T cells, therey creting even more cute need for triggering to drive the motility nd overcome the inhiition efore efficient folliculr recruitment is possile. Future studies re needed to ddress these possiilities. 1 Wors, T., Mempel, T. R., Bolter, J., von Andrin, U. H. & Forster, R. CCR7 lignds stimulte the intrnodl motility of T lymphocytes in vivo. J. Exp. Med. 4, 489-495 2 Okd, T. & Cyster, J. G. CC chemokine receptor 7 contriutes to Gi- dependent T cell motility in the lymph node. J. Immunol. 178, 2973-2978 3 Okd, T. et l. Antigen- engged B cells undergo chemotxis towrd the T zone nd form motile conjugtes with helper T cells. PLoS Biol. 3, e15 4 Luther, S. A., Tng, H. L., Hymn, P. L., Frr, A. G. & Cyster, J. G. Coexpression of the chemokines ELC nd SLC y T zone stroml cells nd deletion of the ELC gene in the plt/plt mouse. Proceedings of the Ntionl Acdemy of Sciences of the United Sttes of Americ 97, 12694-12699 5 Bjenoff, M. et l. Stroml cell networks regulte lymphocyte entry, migrtion, nd territorility in lymph nodes. Immunity 25, 989-11 WWW.NATURE.COM/NATURE 17