SUPPLEMENTARY INFORMATION

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Aubrey Jackson
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propri-derived TNFα cells in wildtype, Rg2 nd µmt mice., Intestinl lmin propri cells (LPC) of wild-type (), Rg2 nd µmt mice were isolted nd nlyzed y flow cytometry.

NB: The residul ppernce of some -producing cells in µmt mice my e ssocited with the smll numers of immture B cells in their intestinl LP 9.

1 SUPPLEMENTARY INFORMATION doi: /nture10698 Rg1 µmt LPLs CD3 LPLs CD11c hi LPLs CD3 splenocytes CD19 LPLs CD19 splenocytes 2 Ocontrol J 4 CD11c Supplementry Figure 1 Chrcteriztion of intestinl lmin propri-derived TNFα cells in wildtype, Rg2 nd µmt mice., Intestinl lmin propri cells (LPC) of wild-type (), Rg2 nd µmt mice were isolted nd nlyzed y flow cytometry. Representtive flow cytometry plots of LPC stined for CD11c nd re shown. A significnt reduction in cells ws oserved in the smll intestinl lmin propri (n = 4 per group). NB: The residul ppernce of some -producing cells in µmt mice my e ssocited with the smll numers of immture B cells in their intestinl LP 9., Splenocytes nd intestinl lmin propri cells were stined nd sorted y flow cytometry. Genomic DNAs from sorted cells were mplified y PCR for hevy chin V(D)J-rerrnged products using the V ll primer nd the J 4-C intronic primer to mplify joints, seprted on n grose gel, southern lotted nd proed with n internl (coding region) J 4-specific proe. 1

RESEARC SUPPLEMENTARY INFORMATION c Supplementry Figure 2 Chrcteriztion of

were isolted, cytospins prepred nd stined nd nlyzed y , nucleic cid stin

As depicted y the rrows, the expression of is restricted to plsm cells.

declining upon cell surfce locliztion of.

2 RESEARC SUPPLEMENTARY INFORMATION c Supplementry Figure 2 Chrcteriztion of expression in smll intestinl lmin propri-derived cells y cytospin nd immune fluorescence microscopy. Smll intestinl lmin propri cells (LPC) of wild-type mixed chimeric mice were isolted, cytospins prepred nd stined nd nlyzed y fluorescence microscopy., nucleic cid stin (lue) nd (green),, (lue) nd (red), c, (lue), (green) nd (red). As depicted y the rrows, the expression of is restricted to plsm cells. Cytoplsmic locliztion of ppers to positively correlte with high expression, declining upon cell surfce locliztion of. Emedded in the pictures re mgnifictions of single cells. Representtive pictures re shown following imge cquisition under oil t 630x. 2

SUPPLEMENTARY INFORMATION RESEARC d e TNFα TNFα c f TNFα TNFα Supplementry Figure 3 Chrcteriztion of nd TNFα expression in smll intestinl lmin propriderived cells y cytospin nd immune fluorescence

3 SUPPLEMENTARY INFORMATION RESEARC d e TNFα TNFα c f TNFα TNFα Supplementry Figure 3 Chrcteriztion of nd TNFα expression in smll intestinl lmin propriderived cells y cytospin nd immune fluorescence microscopy. Smll intestinl lmin propri cells (LPC) of wild-type mixed chimeric mice were isolted, cytospins prepred nd stined nd nlyzed y fluorescence microscopy., nucleic cid stin (lue) nd (green),, (lue) nd TNFα (red), c, (lue), (green) nd TNFα (red), d, (lue), e, (lue) nd TNFα (red), f, (lue), (green) nd TNFα (red). On top of the pictures mgnifictions of single cells re shown. As depicted y the rrows, the expression of nd/or TNFα is restricted to plsm cells, indicting the presence of TNFα, - TNFα s well s TNFα - plsm cells. Representtive pictures re shown following imge cquisition under oil t 630x. 3

4 RESEARC SUPPLEMENTARY INFORMATION Longitudinl Trnsverse Merge YFP EpCAM Supplementry Figure 4 Chrcteriztion nd locliztion of intestinl plsm cells in AID YFP nimls. Longitudinl nd trnsverse sections of smll intestines of AID YFP nimls were stined with specific fluorochrome-tgged ntiodies for EpCAM (lue) which stins the epithelil lyer, nd (red), nd nlyzed y fluorescence microscopy. The rrows highlight AID plsm cells, which pper in yellow. Representtive pictures re shown from totl of 2 seprte experiments, n=3 mice per experiment. 4

SUPPLEMENTARY INFORMATION RESEARC S17 one mrrow strom B220 BM cells (CD45.2) IL-7/TGFβ/IL-21 Gut strom (CD45.1) B220 BM cells (CD45.2) IL-7/TGFβ/IL-21 gted on CD45.

1) BM cells (CD45.2) IL-7,TGFβ,IL-21,αCD40 Gut strom (LTβR ) BM cells (CD45.2) IL-7,TGFβ,IL-21,αCD40 gted on CD45.1-0.0 0.0 0.172 0.

1 intestinl lmin propri cells ( Gut strom ) or the S17 one mrrow stroml cell line, in the presence of IL-7, TGFβ, IL-21 nd αcd40 ntiody. The expression of, nd YFP ws nlyzed y flow cytometry.

, B220 one mrrow (BM) cells from CD45.2 wild type mice were co-cultured for 7 dys in the presence of IL-7, TGFβ, IL-21 nd αcd40 ntiody with: the BM-derived stroml cell line S17, CD45.

5 SUPPLEMENTARY INFORMATION RESEARC S17 one mrrow strom B220 BM cells (CD45.2) IL-7/TGFβ/IL-21 Gut strom (CD45.1) B220 BM cells (CD45.2) IL-7/TGFβ/IL-21 gted on CD isotype αcd40 YFP (AID) gted on CD isotype αcd40 YFP (AID) S17 BM cells (CD45.2) IL-7,TGFβ,IL-21,αCD40 BM strom (CD45.1) BM cells (CD45.2) IL-7,TGFβ,IL-21,αCD40 Gut strom (CD45.1) BM cells (CD45.2) IL-7,TGFβ,IL-21,αCD40 Gut strom (LTβR ) BM cells (CD45.2) IL-7,TGFβ,IL-21,αCD40 gted on CD Supplementry Figure 5 In vitro genertion of -producing plsm cells., B220 one mrrow (BM) cells derived from AID-YFP mice were co-cultured for up to 7 dys with CD45.1 intestinl lmin propri cells ( Gut strom ) or the S17 one mrrow stroml cell line, in the presence of IL-7, TGFβ, IL-21 nd αcd40 ntiody. The expression of, nd YFP ws nlyzed y flow cytometry. cells = green rectngle, - cells = lue rectngle nd - - cells = red rectngle. Dt re representtive of t lest three independent experiments. Representtive flow cytometry plots re shown., B220 one mrrow (BM) cells from CD45.2 wild type mice were co-cultured for 7 dys in the presence of IL-7, TGFβ, IL-21 nd αcd40 ntiody with: the BM-derived stroml cell line S17, CD45.1 BM-derived cells ( BM strom ), CD45.1 intestinl lmin propri cells ( Gut strom ), CD45.1 intestinl lmin propri cells ( Gut strom ) from LTβR nimls (NB, LTβR mice hve een ck-crossed to the CD45.1 congenic ckground). To ensure selective nlysis of BMderived precursors, cells were pre-gted on the CD popultion. Representtive flow cytometry plots of cells nlyzed for nd. cells re depicted y the red rectngle. Dt re representtive of t lest three independent experiments. 5

6 RESEARC SUPPLEMENTARY INFORMATION mixed chimers: All cells cn mke TNF/ Control mixed chimers: 60% of ll cells cn mke TNF/ dko mixed chimers: B cells cnnot mke TNF/ J B cell- deficient vs TNFα X B cell- deficient vs TNFα X J B cell- deficient Supplementry Figure 6 Genertion of mixed one mrrow chimeric mice Schemtic representtion of mixed one mrrow chimer genertion. Rg2 or J mice were irrdited to deplete rdio-sensitive cells nd nimls were reconstituted with mixture of 1) one mrrow from wild-type () J nimls (yielding mice where ll B cells re TNFα ), 2) one mrrow from TNFα - - doule-deficient nimls (yielding reconstituted mice where most B cells nd other rdio-sensitive cells re TNFα ) nd 3) one mrrow from TNFα - - doule-deficient J nimls (yielding reconstituted mixed chimers where ll B cells re TNFα - - ). 6

SUPPLEMENTARY INFORMATION RESEARC dko 04 03 1.0E04 Schemtic representtion of mixed one mrrow chimer genertion. Rg2 or J mice 02 1.

0E01 B cells re TNFα), 2) one mrrow from TNFα-- doule-deficient 1.

05 B cells Numer/spleen dko 1.0E05 06 Neutrophils 1.0E06 CD8 T cells CD35 B220 CD3ε Genertion of mixed one mrrow chimeric mice Supplementry Figure 6 07 CD4 T cells 1.

Supplementry Figure 7 Anlysis of splenic rchitecture of mixed chimeric mice.

Sec>ons of spleens from nd dko mixed one chimeric miceone weremrrow stined with speciﬁc ﬂuorochrome tgged n>odies for, Sections of recons>tuted spleens from nd dkomrrow

igh levels of CD35 re expressed y Folliculrwere Dendri>c Cells (FDC), nd MdCAM1 is expressedntiodies in the mrginl (MZ) sinus.

Dt re represent>ve of t lest four independent mixed chimeric nimls nlyzed. Represent>ve pictures re shown.

(green), (red)cell nd B220 (lue), nd nlyzed susets were determined y ﬂow cytometry.

igh levels of CD35 re expressed y Folliculr Dendritic Cells (FDC), nd MdCAM1 is expressed in the mrginl zone (MZ) sinus.

7 SUPPLEMENTARY INFORMATION RESEARC dko E04 Schemtic representtion of mixed one mrrow chimer genertion. Rg2 or J mice E03 were irrdited to deplete rdio-sensitive cells nd nimls were reconstituted with E02 mixture of 1) one mrrow from wild-type () J nimls (yielding mice 00 where ll 1.0E01 B cells re TNFα), 2) one mrrow from TNFα-- doule-deficient 1.0E00 CD3ε nimls (yielding reconstituted mice where most B cells nd other rdio-sensitive cells MdCAM1 B220 re TNFα ) nd 3) one mrrow from TNFα-- doule-deficient J CD11 mdc 05 B cells Numer/spleen dko 1.0E05 06 Neutrophils 1.0E06 CD8 T cells CD35 B220 CD3ε Genertion of mixed one mrrow chimeric mice Supplementry Figure 6 07 CD4 T cells 1.0E07 pdc CD8 DC c cells/spleen (1.0E) nimls (yielding reconstituted mixed chimers where ll B cells re TNFα--). Supplementry Figure 7 Anlysis of splenic rchitecture of mixed chimeric mice. Anlysis of splenic rchitecture of mixed chimeric mice. Sec>ons of spleens from nd dko mixed one chimeric miceone weremrrow stined with speciﬁc ﬂuorochrome tgged n>odies for, Sections of recons>tuted spleens from nd dkomrrow reconstituted mixed chimeric mice CD35 (green), B220 (red) nd CD3e (lue), CD3ε (green), MdCAM1 (red) nd B220 (lue), nd nlyzed y ﬂuorescence microscopy. igh levels of CD35 re expressed y Folliculrwere Dendri>c Cells (FDC), nd MdCAM1 is expressedntiodies in the mrginl (MZ) sinus. In mice tht lck TNFα in B cells, FDC network stined with specific fluorochrome-tgged for,zone CD35 (green), B220 nd the MZ re disrupted10. Dt re represent>ve of t lest four independent mixed chimeric nimls nlyzed. Represent>ve pictures re shown. c, Spleens from nd dko(red) recons>tuted were MdCAM1 isolted, single suspensions prepred nd the totl counts of the indicted cell nd CD3εmixed (lue)chimeric, CD3 mice ε (green), (red)cell nd B220 (lue), nd nlyzed susets were determined y ﬂow cytometry. Asolute cell suset counts (logrithmic scle) in spleen re not diﬀerent etween nd dko mice (lck versus grey rs respec>vely) in non infected mice. y fluorescence microscopy. igh levels of CD35 re expressed y Folliculr Dendritic Cells (FDC), nd MdCAM1 is expressed in the mrginl zone (MZ) sinus. In mice tht lck TNFα in B cells, FDC networks nd the MZ re disrupted1. Dt re representtive of t lest four independent mixed chimeric nimls nlyzed. Representtive pictures re Supplementry Figure 7 shown. c, Spleens from nd dko reconstituted mixed chimeric mice were isolted, single cell suspensions prepred nd the totl counts of the indicted cell susets were determined y flow cytometry. Asolute cell suset counts (logrithmic scle) in spleen re not different etween nd dko mice (lck rs versus grey rs respectively) in non-infected mice. 5 Supplementry Figure 8 /TNFα doule-deficient mixed chimers re more susceptile to infection with Citrocter rodentium., Lrge intestines (LI) from nd dko mixed chimers mice were hrvested 11 dys post-infection nd the pthologicl scores were nlyzed y stndrd histologicl stining W W W. N A T U R E. C O M / N A T U R E 7 procedures using hemtoxylin nd eosin (&E). LI s from dko nimls show more

RESEARC SUPPLEMENTARY INFORMATION dko 5 Dys post infection Dys post infection % Body weight loss 0-5 -10-15 1 2 3 4 5 6 7 8 9 10 11 J DKO Jh J Jh J DKO Jh dko J -20 * c CFU spleen liver cecum Spleen

Jh Jh DKO!Jh DKO!Jh Jh DKO!Jh DKO!Jh Jh DKO!Jh dko J J dko J dko J J dko J dko J J dko J Supplementry Figure 8 7.0x10 08 6.0x10 08 5.0x10 08 4.0x10 08 3.0x10 08 2.0x10 08 1.0x10 08 0.

, Lrge intestines (LI) from nd dko mixed chimers mice were hrvested 11 dys post-infection nd the pthologicl scores were nlyzed y stndrd histologicl stining procedures using hemtoxylin nd eosin (&E).

The pnel shows the originl mgnifiction of 10x nd scle rs represent 250 µm., The percentge of ody weight loss in dko J versus J dko J mixed chimers fter C. rodentium infection over time is depicted.

8 RESEARC SUPPLEMENTARY INFORMATION dko 5 Dys post infection Dys post infection % Body weight loss J DKO Jh J Jh J DKO Jh dko J -20 * c CFU spleen liver cecum Spleen Liver 3.5x10 5 ** 5.0x10 5 ** 3.0x x x x x x x x x x10 4 * 6.0x x x x x x10 08 Cecum DKO!Jh Jh DKO!Jh DKO!Jh Jh DKO!Jh DKO!Jh Jh DKO!Jh dko J J dko J dko J J dko J dko J J dko J Supplementry Figure 8 7.0x x x x x x x /TNFα doule-deficient mixed chimers re more susceptile to infection with Citrocter rodentium., Lrge intestines (LI) from nd dko mixed chimers mice were hrvested 11 dys post-infection nd the pthologicl scores were nlyzed y stndrd histologicl stining procedures using hemtoxylin nd eosin (&E). LI s from dko nimls show more severe pthology. &E stining of two representtive dko mice nd mice re shown. Note the infiltrtes in the dko mice (rrows) nd the shortened crypt length (scle rs). The pnel shows the originl mgnifiction of 10x nd scle rs represent 250 µm., The percentge of ody weight loss in dko J versus J dko J mixed chimers fter C. rodentium infection over time is depicted. Significntly higher ody weight loss in J dko J mice ws oserved from dy 7 to dy 11 post-infection (n = 7 per group). NB: One J dko J mouse ws found ded t dy 10 post-infection. Also note tht in this experiment, C. rodentium ws somewht ttenuted compred to prior experiments, thus resulting in lter onset of weight loss nd lrger vriility in weight loss. c, The coloniztion y C. rodentium ws determined 11 dys fter infection y homogenizing spleens, livers nd cecums followed y seril dilution plting on nlidixic cid-contining LB pltes nd counting of colonies. C. rodentium coloniztion of ll 3 orgns is significntly enhnced in dko mice t dy 11 post-infection. * p < 0.05, ** p <

9 SUPPLEMENTARY INFORMATION RESEARC Tle I Frequency of CD11c lo cells (n=20) LTβ (n=6) LTβR (n=6) J (n=6) dko (n=5) % CD11c lo 0.50** Men (SEM) & (0.05) 0.03 (0.01) 0.01 (0.00) (0.00) 0.03 (0.03) All vlues indicte Men nd (SEM) nd represent t lest 3 different experiments. Frequency of CD11c lo from totl live lymphocytes. **Significnt difference when is compred with the other four groups, p< Tle II Frequency of /TNFα cells (n=14) dko (n=10) % Men (SEM) % TNFα Men (SEM) 1.27 (0.42)* 0.73 (0.26) 6.50 (1.51)* 3.72 (1.09)) % TNFα 0.76 (0.17)* 0.37 (0.13) All vlues indicte Men nd (SEM) nd represent t lest 3 different experiments. Cells re gted previously on B220 - live cells. * Significnt difference when is compred with dko in ech condition, p<0.05. cells represented 14.5% /- 2.1% of the totl lmin propri preprtion from the cohort of mice. 9

10 RESEARC SUPPLEMENTARY INFORMATION Tle III Frequency of different popultions of YFP vs YFP - cells (n=14) - AID - AID - AID Men (SEM) 1.18 (0.19) (0.78) 1.70 (0.32) All vlues indicte Men nd (SEM) nd represent t lest 2 different experiments using AID-cre x YFP mice. Tle IV Frequency of /TNFα cells AID (n=9) - AID (n=9) - AID - (n=7) % Men (SEM) % TNF Men (SEM) 3.37 (0.94)** 0.31 (0.11) 0.05 (0.02) 4.55 (0.47)*** 0.72 (0.41) (0.005) % TNF 1.50 (0.68)* 0.03 (0.01) (0.004) All vlues indicte Men nd (SEM) nd represent t lest 2 different experiments using AID-cre x YFP mice. Significnt difference when AID is compred with the other two groups, *p<0.05, **p<0.005, ***p<

11 SUPPLEMENTARY INFORMATION RESEARC References 1 Tumnov, A. V. et l. Cellulr source nd moleculr form of TNF specify its distinct functions in orgniztion of secondry lymphoid orgns. Blood 116, , (2010). 11

Lineage-specific functions of Bcl6 in immunity and inflammation are mediated through distinct biochemical mechanisms

Lineage-specific functions of Bcl6 in immunity and inflammation are mediated through distinct biochemical mechanisms Supplementry informtion for: Linege-specific functions of Bcl6 in immunity nd inflmmtion re medited through distinct iochemicl mechnisms Chunxin Hung, Kterin Htzi & Ari Melnick Division of Hemtology nd