20 min 40 min 60 min. Occlusion duration

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1 Infrt volume (mm 3 ) min 4 min 6 min Olusion durtion Shm 2 min 4 min 6 min Supplementry Figure 1 () Quntifition of infrt volume 48h fter different durtions of trnsient MCAO (n=7). Dt show men ± SD () Cresyl violet stining of rin setions illustrtes the typil extent nd distriution of rin dmge indued y eh durtion of MCAO. 2 min MCAO uses injury lrgely restrited to the dorsolterl stritum wheres 4 min nd 6 min MCAO use moderte to extensive dmge in oth ortil nd suortil regions. No dmge is evident in the rins of shm-operted mie. Sle r, 1mm.

2 CD21/35 CD21/35 CD21/35 Totl splenoyte numer 1.x1 7 8.x1 7 6.x1 7 4.x1 7 2.x1 7 Splenoytes Shm MCAO + 2d Live ells B22 + ells MZ FO FSC B22 CD23 Bone mrrow ells Live ells B22 + ells Imm Mt FSC B22 CD23 Live ells CD138 + ells Mem Plsm CD138 B22 Supplementry Figure 2 () Totl splenoyte ounts show 65% redution 2 d fter MCAO in omprison to shm surgery (shm n=4, MCAO n=6). Dt show men + SD; P<.1; unpired t-test. () Gting strtegy for detetion of spleni mrginl zone (MZ, B22 + CD23 - CD21/35 hi ) nd Folliulr (FO, B22 + CD23 + CD21/35 int ) B ells. () Gting strtegy for detetion of one mrrow B ell susets. Immture B ells (Imm) were identified s B22 + CD23 - CD21 + nd mture reirulting nive B ells (Mt) s B22 + CD23 + CD21 +. Memory B ells (Mem) were identified s CD138 + B22 - CD21 -/lo nd plsm B ells were identified s CD138 + B22 - CD21 -/lo.

3 Fold hnge (2 (ΔΔCt) ) BAFFR B22 Fold hnge (2 (ΔΔCt) ) CD4 B22 Fold hnge (2 (ΔΔCt) ) CD21/35 F4/8 Cr2 Mirorry RNA Protein 3 Shm MCAO Cd Shm MCAO * Shm MCAO Tnfsfr13 3 Shm MCAO. 1.5 Shm MCAO * d Shm MCAO Shm MCAO Term P Vlue Hemtopoieti ell linege 1.x1-1 Primry immunodefiieny 3.3x1-8 B ell reeptor signlling pthwy 2.1x1-7 Toll-like reeptor signlling pthwy 7.6x1-6 Nturl killer ell medited ell ytotoxiity 1.8x1-5 Cell dhesion moleules 3.2x1-5 T ell reeptor signlling pthwy 1.1x1-4 Intestinl immune network for IgA prodution 2.x1-4 Cytokine-ytokine reeptor intertion 2.x1-4 Chemokine signlling pthwy 2.x1-4. Supplementry Figure 3 Mirorry dt for seleted B ell-relted genes were vlidted t RNA level y qpcr. Redued expression of Cr2 (), Cd4 () nd Tnfsfr13 () RNA ws onfirmed y qpcr. A loss of CD21/35 (enoded y Cr2 gene) immunolelling () in the MZ (rrows) nd redued FDC immunolelling in the follile nd redued o-loliztion of CD4 (red, ) nd BAFFR (red, ) with B22 + B ell immunolelling (green,, ) in mie 5 d fter MCAO in omprison to shm surgery vlidted these hnges t protein level (Shm n=4, MCAO + 5d n=5). (d) Trnsripts identified s downregulted fter MCAO were nlysed in KEGG to identify ffeted pthwys. Pthwys ssoited with the development nd funtion of vrious spets of the immune system were identified s defiient fter MCAO. (e) Mirorry expression levels for trnsripts of the integrin LFA-1 (Itgl nd Itg2), importnt for retention of B ells in the MZ, re deresed in spleens fter MCAO in omprison to shm-operted ontrols. (f) In ontrst, trnsripts for the dhesion moleules these integrins ind to, expressed y stroml ells (Vm1 nd Im1) re inresed/ unhnged fter MCAO (shm n=2, MCAO n=3). Sle rs,, 2 mm. Dt show men ± SD;,,, e, f unpired t-test; ns not signifint, *P<.5, P<.1, *P<.1. e f Itgl Shm MCAO Vm1 Shm MCAO Itg2 ns Shm MCAO Im1 ns Shm MCAO

4 Dextrn-FITC Nive Shm MCAO + 2d Supplementry Figure 4 Fluoresently lelled model ntigen (green, Dextrn-FITC) is trpped in the spleens of nïve nimls nd nimls reovered 2 d fter MCAO or shm-surgery, 1 h fter i. v. injetion.

5 1/ΔCT 1/ΔCT CD93 CD21/35 Live lymphoytes B22 + CD93 - ells MZ FO FSC B22 CD * MZ FO. MZ FO Supplementry Figure 5 The expression levels of Adr2 were determined y qpcr nlysis of RNA extrted from mrginl zone (MZ) nd folliulr (FO) B ell popultions sorted y FACS from mixed splenoyte suspension. () MZ (B22 + CD93 - CD23 - CD21/35 hi ) nd FO (B22 + CD93 - CD23 + CD21/35 + ) B ells were otined y ell sorting. () levels of Adr2 were slightly higher in FO B ells in omprison to MZ B ells. () of Cd1d, enoding non-lssil MHC protein expressed y MZ B ells, ws expressed t signifintly higher level in RNA extrted from MZ ompred to FO B ells onfirming the enrihment of MZ B ell RNA. n=9. Dt show men ± SD; unpired t-test, P<.1, *P<.1

6 B22 % 2-AR + ells B22 IgD Live lymphoytes FSC Irrelevnt Primry Seondry only 2-AR Live lymphoytes B22 + CD43 lo/- B22 + CD43 lo/- Mt Pro Pre Imm CD43 IgM d Live lymphoytes CD138 + e 8 Mem 6 CD Plsm 2 CD138 CD93 Pro Pre Imm Mt Plsm Mem Supplementry Figure 6 A more stringent sudivision of one mrrow B ell susets ws used to onfirm 2-drenergi reeptor (2-AR) expression on B ells t different developmentl stges. () Live lymphoytes were gted. () The threshold for positive 2-drenergi reeptor stining ws set sed on irrelevnt primry nd seondry only ontrols. () Live lymphoytes were plotted on CD43 nd B22, Pro B ells were identified s CD43 + B22 lo. CD43 lo/- B22 + ells were gted nd plotted ginst IgM nd IgD immunolelling. Mture reirulting B ells (Mt) were identified s IgM + IgD +, immture B ells (Imm) were identified s IgM + IgD - nd pre-b ells were identified s IgM - IgD -. (d) CD138 + ells were gted nd plotted ginst CD93 nd B22 immunolelling. CD93 + ells were exluded s immture B ells. CD138 + CD93 - B22 + ells were identified s memory B ells (Mem) nd CD138 + CD93-B22- ells were identified s plsm ells. (e) of 2-AR on distint one mrrow B ell susets s identified in () nd (d). The expression pttern ws equivlent to tht oserved using n lterntive gting strtegy (see Fig 6e). n=4. Dt show men ± SD.

7 IgM onentrtion [log(mg/ml)] Bteril lod [log(cfu/ml)] Infrt volume (mm 3 ) 8 P= PBS Proprnolol r = -.46 P = r = -.65 P = Infrt volume (mm 3 ) IgM onentrtion [log(mg/ml)] Supplementry Figure 7 () Infrt volume in the rins of nimls treted with PBS or proprnolol prior to nd 4 h fter MCAO. (shm n=4, MCAO n=6). () Reltionship etween plsm IgM onentrtion (log trnsformed) nd infrt volume. () Reltionship etween plsm IgM onentrtion (log trnsformed) nd lung teril lod. Dt show men + SD; () unpired t-test.

8 Ptients Controls (n=36) Infetion (n=12) No infetion (n=22) Medin ge (min, mx), yers 7 (37, 91) 7 (53, 91) 68.5 (37, 86) Mle 24 (66.7) 9 (75.) 14 (63.6) Signifint theroslerosis 17 (47.2) 8 (66.7) 8 (36.4) Current or former smoker 23 (63.9) 8 (66.7) 17 (77.3) Hypertension 11 (3.6) 9 (75.) 14 (63.6) Atril firilltion 2 (5.6) 2 (16.7) 4 (18.2) Dietes mellitus 6 (16.7) 3 (13.6) Ishemi hert disese 7 (19.4) 2 (16.7) 5 (22.7) Previous stroke or trnsient ishemi ttk 1 (8.3) 6 (27.3) Meditions Sttin 11 (3.6) 2 (16.7) 9 (4.9) Β-loker 7 (19.4) 7 (58.3) 8 (36.3) Angiotensin Converting Enzyme Inhiitor 3 (8.3) 1 (8.3) 2 (9.1) Stroke hrteristis Medin NIHSS (min, mx) 17.5 (9, 23) 12.5 (3, 12.5) Left hemisphere 6 (5.) 15 (68.2) TACI 8 (66.7) 6 (27.3) PACI 4 (33.3) 9 (4.9) LACI 5 (22.7) POCI 2 (9.1) mrs t presenttion.5 (, 3) (, 4) Supplementry Tle 1 Bseline hrteristis of ptients with or without infetions. All dt s n (%), unless speified. defined s 5% stenosis of t lest one internl rotid rtery, nd/or lowest nkle rhil pressure index of <.92. reeiving prior to dmission or strted during the first week. NIHSS, Ntionl Institutes of Helth Stroke Sle; TACI, Totl Anterior Cirultion Infrtion; PACI, Prtil Anterior Cirultion Infrtion; POCI, Posterior Cirultion Infrtion; LACI, Lunr infrtion; mrs, modified Rnkin Sle