Local T/B cooperation in inflamed tissues is supported by T follicular helper-like cells

Transcription

1 Received 16 Nov 15 Accepted 8 Jn 16 Pulished 6 Fe 16 DOI: 1.138/ncomms1875 Locl T/B coopertion in inflmed tissues is supported y T folliculr helper-like cells OPEN Dn Vu Vn 1,, Ktj C. Beier 3, Le-Jen Pietzke 1,, Mysun S. Al Bz 1,, Rndi K. Feist 1,, Stephnie Gurk, Eckrd Hmelmnn, Richrd A. Kroczek & Andres Hutloff 1, Autoimmune diseses nd other inflmmtory conditions re chrcterized y lrge lymphocytic tissue infiltrtes in which T nd B cells cn e found in close contct. Here, using murine irwy inflmmtion model, we compre ntigen-specific T nd B cells in lung tissue versus lung-drining lymph node. In the lung we identify B-cell popultion exhiiting clssicl germinl centre phenotype without eing orgnized into ectopic lymphoid tissue. By contrst, clssicl CXCR5 þ Bcl-6 þ T folliculr helper cells re not present. Nevertheless, lung-infiltrting T cells exhiit folliculr helper-like properties including the potentil to provide help to nive B cells. The lung tissue is lso survivl niche for memory T nd B cells remining in residul perironchil infiltrtes fter resolution of inflmmtion. Collectively, this study shows the importnce of T/B coopertion not only in lymph nodes ut lso in inflmed peripherl tissues for locl ntiody responses to infection nd utoimmunity. 1 Chronic Immune Rections, Germn Rheumtism Reserch Centre, A Leiniz Institute, Chritépltz 1, 1117 Berlin, Germny. Moleculr Immunology, Roert Koch Institute, Nordufer, Berlin, Germny. 3 Peditric Pneumology nd Immunology, Chrité University Medicine, Augustenurger Pltz 1, Berlin, Germny. Children s Hospitl of Ruhr University, Alexndrinenstre 5, 791 Bochum, Germny. Correspondence nd requests for mterils should e ddressed to A.H. (emil: hutloff@drfz.de). NATURE COMMUNICATIONS 7:1875 DOI: 1.138/ncomms

2 NATURE COMMUNICATIONS DOI: 1.138/ncomms1875 Leukocytic infiltrtes in peripherl tissues re frequently found in utoimmune conditions like rheumtoid rthritis, systemic lupus erythemtosus, Sjögren syndrome nd multiple sclerosis, ut lso in the lungs of sthm ptients. These infiltrtes typiclly contin ntigen-specific T nd B cells, s well s different cell popultions of the innte immune system, nd contriute sustntilly to tissue destruction nd immunopthology. B cells produce (uto-) ntiodies loclly nd lso seem to ply role s ntigen-presenting cells for T lymphocytes in the periphery. T cells in turn produce proinflmmtory cytokines, which ttrct tissue-destructive grnulocytes nd lso ffect non-lymphoid cells, for exmple, golet cells to produce mucus in the cse of irwy inflmmtion. However, n often neglected function of T cells in inflmed tissues is their potentil to provide help to ntigen-specific B cells. This helper function of T cells is the mjor role of T folliculr helper (TFH) cells, specilized popultion of T cells in secondry lymphoid orgns (SLOs) 1. This T-cell popultion is crucil for B-cell mturtion nd differentition in the germinl centre (GC) response. Without TFH cells, no ffinity-mtured long-lived plsm cells nd memory B cells re generted. These two terminlly differentited B-cell popultions re the sis for protective immunity; however, they cn pose mjor prolem when producing utorective ntiodies. Therefore, TFH cells re n ttrctive trget for the tretment of utoimmune nd other inflmmtory diseses. Under certin conditions, SLO-like structures cn develop in inflmed tissues. They re known s ectopic lymphoid tissue or s induced ronchus-ssocited lymphoid tissue in the lungs 3,. Ectopic lymphoid tissues represent highly ordered structures with seprte T- nd B-cell zones. Another importnt chrcteristic is the presence of folliculr dendritic cells (FDCs) similr to follicles in SLO. Ectopic lymphoid tissues exhiit mny fetures of SLO, including formtion of germinl centres in which T nd B cells cooperte 5. However, the development of ectopic lymphoid tissue in inflmed tissues is n exceptionl cse, which requires experimentl settings with strong stimuli or other fcilittors like virl infection 3. In humn utoimmune conditions, fully differentited ectopic follicles re only rrely oserved 6,7. Nevertheless, FDC-negtive lymphocytic infiltrtes contin T nd B cells in very close contct rising the question whether T/B coopertion cn lso tke plce in infiltrtes not exhiiting the fetures of ectopic lymphoid tissue. To nlyse the coopertion of ntigen-specific T nd B cells in inflmed tissues in more detil, we use novel lung inflmmtion mouse model, which mkes it possile to nlyse nd compre the interction of ntigen-specific T nd B cell simultneously in inflmed lung tissue, s well s in the lung-drining lymph nodes. With this model we identify the inflmed lung tissue s the mjor reservoir of ntigen-specific T nd B cells. The lung tissue does not only contin ntigen-specific plsm cells ut lso popultion of GC-like B cells. In contrst, no clssicl CXCR5 þ Bcl-6 þ TFH cells re present in the lung. However, we identify popultion of lung-infiltrting helper T cells, which seem to tke over the functions of TFH cells. Finlly, we show tht the lung tissue is n importnt survivl niche for ntigen-specific memory T nd B cells, which might e importnt for fst locl secondry responses. Results Antigen-specific T nd B cells ccumulte in lung tissue. The low nturl frequency of ntigen-specific T nd B cells mkes it difficult to nlyse their interction in n inflmmtory rection. Therefore, we developed n in vivo T/B coopertion system, in which ovlumin (OVA)-specific T cells were co-trnsferred with nitrophenol (NP)-specific B cells into immunocompetent recipient mice (Fig. 1). After doptive trnsfer, recipient mice were chllenged intrnslly (i.n.) with n NP OVA conjugte s ntigen nd lipopolyscchride (LPS) s n djuvnt. This system mkes it possile to nlyse ntigenspecific T nd B cells simultneously in lung tissue nd lungdrining lymph nodes, using the congenic mrkers Thy-1.1 nd CD5.1 (in ll susequent figures, ntigen-specific refers to cells gted on either CD þ Lin Thy-1.1 þ or CD19 þ Lin CD5. CD5.1 þ ). One to two dys fter ntigen chllenge, ntigen-specific T nd B cells ccumulted in the lung-drining lymph nodes where they proliferted vigorously. Although cells evenly distriuted to ll SLOs on trnsfer, no ctivted ntigenspecific T nd B cells were found in non-drining orgns like the spleen. In the lungs, ctivted ntigen-specific T nd B cells ppered round dy 5 (Supplementry Fig. 1). After repeted ntigen chllenges, stle popultion of lung-infiltrting T nd B cells developed, which did not chnge much over time. Therefore, we routinely nlysed recipient mice on dy 17 for ntigen-specific T nd B cells in the drining lymph nodes versus the inflmed lung. At tht time, more thn 8% of ll ntigenspecific T nd 6% of B cells were found in the lungs, identifying the inflmed tissue s the mjor reservoir of ntigen-specific T nd B cells (Fig. 1). Infiltrtes hrour T nd B cells in intimte contct. After repeted i.n. chllenge with ntigen, we oserved the typicl signs of chronic lung inflmmtion, such s ppernce of lrge lymphocytic infiltrtes nd irwy nrrowing with incresed mucus production (Fig. ). Immunohistologicl exmintion on dy 17 reveled lrge lymphocytic clusters contining eosinophils nd ntigen-specific T cells locted djcent to ntigen-specific B cells. Under chronic inflmmtory conditions, lymphocytic infiltrtes cn develop further into ectopic lymphoid tissue, chrcterized y the presence of FDC 3,. Therefore, we stined the lung infiltrtes for CD1/35 (complement receptor ), which is expressed on FDC t high density. More thn 96% of infiltrtes were negtive for CD1/35 (Fig. c nd Supplementry Fig. ). Only some very lrge infiltrtes locted ner the centrl ronchus stined positive for FDC mrkers (Fig. nd Supplementry Fig. ). In contrst to the FDC-negtive infiltrtes composed of loose ggregtes of T nd B cells, these CD1/35-positive infiltrtes showed highly orgnized seprte T- nd B-cell zones (Fig., lower pnel). It is well estlished tht T/B coopertion cn tke plce in these SLO-like structures 3,. However, the close contct of ntigen-specific T nd B cells in the predominting FDC-negtive loose ggregtes rised the question whether locl T/B coopertion might tke plce there s well. Lung tissue contins B cells with GC-like phenotype. To find indictions for T/B coopertion in the inflmed tissue, we compred the phenotype of lung-infiltrting B cells with tht of lymph node B cells. First, we looked for ntiody-secreting cells, which re generted oth in the extrfolliculr nd GC B-cell responses. On dy 17, up to 3% of ntigen-specific B cells in the drining lymph nodes expressed CD138 nd hd downregulted CD19, chrcteristic feture of plsm cells (Fig. 3). The lung tissue contined very similr frction of ntigen-specific plsm cells. However, when considering the higher solute numers of ntigen-specific B cells (compre Fig. 1), the lung tissue turned out to contin eight times more plsm cells thn the lungdrining lymph node. Intrcellulr stining of immunogloulin suclsses reveled tht ntigen-specific plsm cells from oth sites were minly switched towrds IgG1 nd IgA, with few cells producing IgG nd IgE (Supplementry Fig. 3). Histologicl nlysis showed tht CD138-positive plsm cells were eqully NATURE COMMUNICATIONS 7:1875 DOI: 1.138/ncomms1875

3 NATURE COMMUNICATIONS DOI: 1.138/ncomms1875 ARTICLE found in FDC-positive nd -negtive infiltrtes (Supplementry Fig. ). Next, we nlysed the genertion of GC B cells. Around 6% of the trnsgenic B cells in the lymph node displyed GC phenotype, defined y stining with penut gglutinin (PNA) nd the monoclonl ntiody GL7 (Fig. 3). Looking for these clssicl GC B-cell mrkers in the lungs, we surprisingly found prominent popultion of PNA/GL7 doule-positive cells, which only differed y slightly reduced inding of PNA (Fig. 3). Although their frequency in the lung ws lower, their solute numer ws similr to tht in the lymph node. GC B cells hve een detected in ectopic lymphoid structures of inflmed tissues 5. However, the high percentge of PNA þ /GL7 þ cells in our inflmmtion model ws unexpected given the fct tht the vst mjority of lung infiltrtes consisted of loose T/B ggregtes nd not ectopic lymphoid tissue. Therefore, we nlysed dditionl mrkers tht re more specific for GC B cells. The GL7 þ B cells in the lung expressed the trnscription fctor Bcl-6, which is key regultor of GC B-cell differentition, nd hd downregulted CD38, nother ttriute of GC B cells 8. Moreover, PNA/GL7 doule-positive cells in lymph node nd lung expressed the ctivtion-induced cytidine deminse, which is involved in somtic hypermuttion of GC B cells (Fig. 3). To define the locliztion of the GC-like B cells, we nlysed lung section ner the centrl ronchus, which contined oth ectopic lymphoid tissue nd unstructured infiltrtes (Fig. 3c). As expected, some GL7 þ GC B cells were present in ectopic lymphoid tissue contining CD1/35 þ FDC. However, most GL7 þ B cells were found in infiltrtes without FDC, demonstrting tht GC-like B cells cn exist outside GC structures (Fig. 3c). This explins how up to % of lung-infiltrting B cells displyed GC phenotype lthough 96% of ll lung infiltrtes in our model were composed of loose T/B ggregtes. Stining for the cell cycle mrker Ki-67 reveled tht B cells within these infiltrtes were highly proliferting (Fig. 3d). This further indictes tht ctive T/B coopertion tkes plce within these loose lung infiltrtes. Finlly, we sequenced the trnsgenic B-cell receptor hevy chin from FDC-positive nd -negtive infiltrtes otined y lser microdissection. Both types of infiltrtes contined hypermutted B cells. The rtio of replcement versus silent muttions (1. versus 1.33) nd lso the percentge of clones with the ffinity-determining tryptophn to leucine muttion t codon 33 (6% versus 5%) were similr in FDC-positive nd -negtive infiltrtes. Tken together, our dt show tht B cells in lymph nodes nd lung re similr in their phenotype. However, in contrst to lymph nodes, where PNA þ /GL7 þ cells re present only in GC, we identified GC-like B cells outside of ectopic lymphoid structures in inflmed lung tissue. Lung-infiltrting T cells re no clssicl TFH cells. As the genertion of GC B cells in lymph nodes criticlly depends on the presence of TFH cells, we nlysed the proportion of trnsgenic T cells expressing the TFH mrkers CXCR5 nd PD-1. In the Trnsgenic T cells Live/B Trnsgenic B cells Live/CD3 /CD8 /CD OT-II T B1-8i B CD CD OVAspecific NPspecific Thy-1.1 CD5.1 Intrnsl immuniztion: NP-OVA + LPS Anlysis B6 recipient Time (d) # OT-II T cells ( 1 6 ) OVA-specific T cells * # B1-8i B cells ( 1 6 ) NP-specific B cells * Rtio ln versus lung (%) Ln Lung Ln Lung T cells B cells Figure 1 Inflmed lung tissue is the mjor reservoir of ntigen-specific T nd B cells. () Generl set-up for the irwy inflmmtion model. Prectivted OT-II T cells nd nive B1-8i/kpp light chin knockout B cells were trnsferred into syngeneic C57BL/6 mice. OT-II cells express T-cell receptor specific for OVA, B1-8i cells hve knock-in for the germline VH186 hevy chin, which recognizes the hpten NP in comintion with ny lmd 1 light chin 31. On the indicted dys, recipient mice were immunized i.n. with n NP OVA conjugte nd LPS. Cells from lungs nd lung-drining lymph nodes were typiclly nlysed on dy 17 when irwy inflmmtion ws fully estlished. Antigen-specific T nd B cells were identified y flow cytometry using the congenic mrkers Thy-1.1 nd CD5.1, respectively. () Totl numers of ntigen-specific T nd B cells in lung-drining lymph node (Ln) nd lung tissue on dy 17. Shown re solute cell numers of representtive experiment nd the rtio of ntigen-specific T nd B cells in lymph node versus lung (pooled dt from 6 experiments with 3 nimls). Error rs show s.e.m. *Po.1 y Mnn Witney U-test. NATURE COMMUNICATIONS 7:1875 DOI: 1.138/ncomms

4 NATURE COMMUNICATIONS DOI: 1.138/ncomms1875 Non-inflmed lung Trnsgenic T cells (Thy-1.1) Trnsgenic B cells (CD5.1) Inflmed lung Eosinophils (MBP-1) Mucus (PAS) Folliculr dendritic cells (CD1/35) c 8 Trnsgenic T cells (Thy-1.1) Trnsgenic B cells (CD5.1) % FDC + re of totl lymphocytic infiltrtes 6 Figure Lung infiltrtes contin ntigen-specific T nd B cells in close contct. () Cryosections from non-inflmed nd inflmed lung tissues (dy 17) were stined with hemtoxylin to visulize cellulr infiltrtes. The imges on the right show seril sections of the highlighted perivsculr infiltrte or the nery ronchus in higher mgnifiction. Immunostining (red) for OVA-specific T cells, NP-specific B cells nd eosinophils ws performed using the indicted mrkers. Production of mucus ws visulized y periodic cid Schiff stining. Scles rs, mm for the low-resolution imges (originl mgnifiction 5) nd 5 mm for the high-resolution imges (originl mgnifiction ). () Ectopic lymphoid tissue ws identified y stining of seril cryosections for FDC nd ntigen-specific T nd B cells using the indicted mrkers. Scle rs, 1 mm for the low-resolution imges nd mm for the high-resolution imge (originl mgnifiction ). Imges re representtive of independent experiments with 1 18 nimls totl. (c) Are of FDC-contining regions s percentge of totl infiltrte re, quntified s shown in Supplementry Fig.. Ech dot represents dt from single pulmonry loe. lung-drining lymph nodes out % of T cells displyed TFH phenotype (Fig. ). In contrst, this popultion ws missing completely in the inflmed lung tissue. Interestingly, only CXCR5 ws sent on lung T cells, wheres PD-1 ws eqully expressed on lung s well s on lymph node T cells (Fig. ). To define TFH cells more precisely we stined their mster trnscription fctor Bcl-6. As expected, ntigen-specific CXCR5 PD-1 T cells in the lymph node were negtive for Bcl-6, wheres significnt Bcl-6 expression ws found in the CXCR5 þ PD-1 þ suset. In contrst, lung-infiltrting T cells did not express Bcl-6 t ll (Fig., right pnel). To further compre lung-infiltrting nd lymph node T cells, we nlysed dditionl mrkers tht re typiclly expressed y TFH cells: the chemokine receptor CCR7, which is lso involved in the positioning of TFH cells in the B-cell zones of SLO, nd CDL nd ICOS, which re importnt effector molecules. Interestingly, lung-infiltrting T cells hd downregulted CCR7 to n even stronger extent thn ntigen-specific T cells in the lymph node (Fig. ). At the sme time, expression levels of CDL nd ICOS were higher on lung-infiltrting T cells compred with lymph node T cells. T-cell help to B cells is medited y CDL nd secretion of interleukin (IL)- nd IL-1. To ssess the cytokine-producing potentil of lung-infiltrting nd lymph node T cells, these cell popultions were re-stimulted with ntigen in vitro. IL- nd IL-1 were the two dominting cytokines, produced y more thn % of ntigen-specific T cells (Fig. c). While IL- ws produced to similr mount y lymph node nd lung T cells, IL-1 production y lung-infiltrting T cells ws in ll experiments significntly higher thn y ntigen-specific T cells isolted from the drining lymph nodes. Interestingly, expression of dditionl effector cytokines such s IL-5, IL-1, IL-13, IL-17 nd interferon (IFN)-g ws lrgely confined to lung T cells with n t lest threefold higher expression thn ntigen-specific T cells from the drining lymph nodes. The higher expression of Th effector cytokines y lung T cells ws ssocited with significntly incresed expression of the mster trnscription fctor GATA-3 (Fig. d). In contrst to the clssicl TFH cells in the lymph node, there ws no cell surfce mrker on lung-infiltrting T cells identifying suset with prticulr B helper cpcity. Therefore, we nlysed co-expression of different cytokines. IL-1 turned out to e led NATURE COMMUNICATIONS 7:1875 DOI: 1.138/ncomms1875

5 NATURE COMMUNICATIONS DOI: 1.138/ncomms1875 ARTICLE CD NP-specific B cells Lymph node 31% CD19 Lung 7% % CD19 low CD # CD19 low CD138 + cell ( 1 5 ) NS 8 6 Rtio ln versus lung (%) PNA NP-specific B cells Lymph node Lung 59% GL7 % 59% 38% % GL7 + PNA + 8 * 6 Ln Lung # GL7 + PNA + ( 1 5 ) NS Ln Lung Rtio ln versus lung (%) GL Bcl-6 c GL7 d CD38 GC CD1/35 MERGE CD5.1 Ki-67 Non-GC AID Figure 3 Similr phenotype of B cells in lymph node nd lung tissue. Flow cytometric nlysis (dy 17) of ntigen-specific (CD5.1 þ ) B cells from lung-drining lymph nodes nd lung tissue. () Comprison of plsm cells, defined s CD19 low CD138 þ, from lymph nodes nd lungs. Shown re flow cytometric plots, nd the frequency nd solute numers of NP-specific B cells with plsm cell phenotype from representtive experiment nd the rtio of plsm cells in lung-drining lymph nodes s pooled dt from 3 experiments with nimls totl. () Comprison of GC/GC-like B cells from lymph nodes (Ln) nd lung tissue. Frequency nd solute numers from representtive experiment, nd reltive distriution of GL7 þ PNA þ B cells etween lymph node nd lung s pooled dt from experiments with 6 nimls. For further chrcteriztion, cells were co-stined for CD38 nd intrcellulr Bcl-6 or ctivtion-induced cytidine deminse (AID). CD38 expression is shown s histogrms on GL7/Bcl-6 doule-negtive (lck) versus doule-positive (ornge) cells. AID expression is shown on GL7/PNA-positive (red line) nd -negtive (lck line) cells reltive to n isotype control (filled grey histogrm; conctented flow cytometry dt from five nimls). (c) Stining of lung tissue contining ectopic lymphoid tissue defined y the presence of FDC (CD1/35 þ, yellow) nd greter numer of unstructured infiltrtes. Stining with the GC B-cell mrker GL7 is shown in mgent. (d) Co-stining of trnsgenic B cells (mgent) nd the cell prolifertion mrker Ki-67 (yellow) in n FDC-negtive infiltrte. Scle rs, 1 mm, originl mgnifiction. Dt re representtive for t lest two independent experiments. Error rs show s.e.m. NS, not significnt: PZ.5, *Po.5, Po.1 y Mnn Witney U-test. cytokine for lung effector T cells. IL--, IL-1-, IL-13- nd IFN-gproducing cells were highly enriched in the IL-1-positive compred with the IL-1-negtive suset (Supplementry Fig. 5). As second cytokine, the IL-1/IL- doule-positive versus the IL-1 þ IL- suset demrcted cells with highest expression of IL-1 nd IL-13. To demonstrte tht the B helper potentil of lung-infiltrting T cells ws not consequence of the prectivtion of OT-II T cells under Th-polrizing conditions, we rn our irwy inflmmtion model lso with the trnsfer of nive T cells. Under these conditions, ll previous oservtions could e reproduced (Supplementry Fig. 6). We found GC-like B-cell popultion in the lung ut no CXCR5 þ TFH cells. The cytokine profile prtly chnged with lmost no IL-- nd IL-13-producing cells. Insted, popultion of up to 3% IL-17-producing cells ws generted in the lung, which is typicl rection for ntigens tken up vi n intrnsl route 9. Importntly, production of IL-1 ws unchnged with round 3% IL-1 þ cells. Agin, NATURE COMMUNICATIONS 7:1875 DOI: 1.138/ncomms

6 NATURE COMMUNICATIONS DOI: 1.138/ncomms1875 PD OVA-specific T cells 6 Lymph node Lung % CXCR5 + PD-1 + * CXCR5 Ln Lung OVA-specific T cells (lymph node versus lung) TFH 171±8 Lung 98± Non-TFH 88± Bcl-6 IL-1 turned out to e led cytokine for lung T cells, with higher IL-17 nd IFN-g production in the IL-1-positive versus -negtive suset. Collectively, in striking contrst to the presence of GC-like B cells in the lung, no clssicl CXCR5 þ Bcl-6 þ TFH cells could e found. However, regrding other mrkers, lung-infiltrting T cells shred mny similrities with TFH cells nd hd high B helper potentil y expressing CDL, IL- nd especilly IL-1. Lung-infiltrting T cells produced dditionl effector cytokines like IL-5, which functions not only s chemottrctnt for eosinophils ut is lso importnt for the clss-switch of B cells towrds IgA 1. MFI PD-1 c CD % IL- + CD IL- IL-5 IL % % IL PD-1 CCR7 CDL ICOS Ln NS * Lung % % IL-1 + % IL-5 + MFI CCR % 8% IL-17 IL-1 IFN-γ GATA-3 6 * MFI CDL Ln Lung Ln Lung Ln Lung % IL % 3.% IL-13 Ln Lung Ln Lung Ln Lung Ln Lung * % IFN-γ Ln Lung Ln Lung Ln Lung Ln Lung 6 OVA-specific T cells (lung) OVA-specific T cells (lung) 3 1 * 5 * MFI ICOS d MFI GATA-3 % IL * * 1% 1 5 OVA-specific T cells Figure Lung tissue does not contin clssicl TFH cells. Comprison of ntigen-specific (Thy-1.1 þ ) T cells from lymph nodes (Ln) nd lung tissue. () Anlysis of CXCR5/PD-1 expression. The right pnel shows intrcellulr stining for Bcl-6 in lymph node TFH cells (CXCR5 þ /PD-1 þ ) nd non-tfh cells (CXCR5 /PD-1 ), nd in the whole popultion of lung-infiltrting T cells. The inset numers indicte the men fluorescence intensity (MFI)± s.e.m. () Expression of PD-1, CCR7, CDL (intrcellulr stining, no restimultion) nd ICOS on lymph node (grey-filled histogrms) versus lung-infiltrting (lck open histogrms) T cells. (c) Intrcellulr stining for IL-, IL-5, IL-1, IL-13, IL-1 nd IFN-g fter shortterm restimultion with OVA peptide in vitro. A representtive stining from lung-infiltrting T cells is shown for ll cytokines. (d) Intrcellulr stining for GATA-3 compring the MFI etween lymph node nd lung T cells (dotted line ¼ GATA-3 stining on non-ntigen-specific (endogenous) lymph node CD þ T cells). Dt re representtive of 3 independent experiments with 7 9 nimls per experiment. NS, not significnt: PZ.5, *Po.5, Po.1, *Po.1 y Mnn Witney U-test. Lung-infiltrting T cells provide help for B cells. To functionlly test their cpcity for B-cell help, ntigen-specific T cells from lung-drining lymph nodes or lung tissue were sorted nd co-cultured with nive NP-specific B cells in the presence of cognte ntigen. After 7 dys, B cells co-cultured with lung T cells hd produced lrge mounts of IgG1, lthough not quite to the extent s B cells co-cultured with T cells from lymph nodes (Fig. 5, upper pnel). However, production of IgA ws similrly high in oth groups (Fig. 5, lower pnel). Moreover, lung T cells induced the lignds for GL7 nd PNA, nd the ctivtion mrker CD69 on the co-cultured B cells to the sme extent s lymph node T cells (Fig. 5). Tken together, lung-infiltrting T cells re fully cple to provide B-cell help, lthough they lck the prototypic TFH cell mrkers CXCR5 nd Bcl-6. The lung is mjor reservoir for memory B cells. After looking t T/B coopertion during the effector phse of irwy inflmmtion, we nlysed ntigen-specific T nd B cells during the memory phse. Even s lte s dy 1, weeks fter lst ntigen ppliction, perivsculr lymphocytic infiltrtes could still e found in the lung (Fig. 6). These infiltrtes contined sustntil numers of ntigen-specific B cells. At the sme time, rely ny NP-specific B cells were detectle in the lymph node (Fig. 6). In contrst to the effector phse, where ctivted ntigen-specific cells were exclusively found in the lung nd lung-drining lymph node, memory B cells were now lso present in spleen nd one mrrow. While the highest frequency of memory B cells ws found in the lung, the mjority of memory B cells in solute numers resided in the spleen tking into ccount the higher cellulrity of this orgn (Fig. 6). We nlysed dditionl mrkers to chrcterize these lungresident ntigen-specific B cells. While during the effector phse lmost ll ntigen-specific B cells in the lungs displyed either GC or plsm cell phenotype, CD138 þ, intrcellulr Ig þ or GL7 þ, PNA þ nd CD38 low cells were no longer present t this lte time (Fig. 6c nd Supplementry Fig. 7). Hlf of the ntigenspecific B cells in the lungs expressed surfce IgM, wheres the others were clss-switched towrds either IgG1 or IgA (Fig. 6c). Around % of ll B cells expressed CD8, which is regrded s mrker for highly mutted memory B cells 11. In ddition, we nlysed the expression of PD-L nd CD73, which re lso expressed y susets of memory B cells 1. Altogether, more thn 85% of the B cells expressed PD-L, CD73 or CD8, nd cn therefore e regrded s typicl memory B cells, identifying the lung s n importnt niche for ntigen-specific memory B cells. Since o1% of the ntigen-specific B cell numers found during the effector phse remined t dy 1, we hd to tke into ccount the possiility tht they might e exclusively locted in the FDC-positive infiltrtes ner the centrl ronchus. However, like in the effector phse, the vst mjority of infiltrtes were FDC-negtive. Co-stining of CD5.1 nd CD1/35 reveled tht 6 NATURE COMMUNICATIONS 7:1875 DOI: 1.138/ncomms1875

7 NATURE COMMUNICATIONS DOI: 1.138/ncomms1875 ARTICLE OD NP-specific IgG1 % GL7 + PNA + B cells NP-specific IgA 5 NS OD Lymph node T cells Lung T cells Nive T cells MFI CD Dilution 1 Ln Lung Nive Figure 5 Lung-infiltrting T cells provide B-cell help. Sorted ntigen-specific (Thy-1.1 þ ) effector T cells (dy 17) from lungs or lymph nodes (Ln), or nive OT-II cells were co-cultured with nive NP-specific B cells in the presence of NP OVA for 7 dys. Therefter, () NP-specific IgG1 nd IgA were mesured y ELISA nd () B cells were stined with PNA, GL7 or nti-cd69, nd nlysed y flow cytometry. Shown re representtive results from three independent experiments with six replictes ech. Error rs show s.e.m. NS, not significnt: PZ.5, Po.1 y Mnn Witney U-test. lso in the memory phse the gret mjority of ntigen-specific B cells resided in FDC-negtive infiltrtes (Fig. 6d). Memory T nd B cells re in close contct in the lung. To dditionlly nlyse ntigen-specific memory T cells, we switched our system to the doptive trnsfer of Smrt T-cell receptortrnsgenic insted of OT-II T cells, which llows for long-term trcking in vivo 13 (Fig. 7). We co-stined lung sections for ntigen-specific T nd B cells on dy 6 (7 dys fter the lst ntigen chllenge). Like in the effector phse (compre Fig. ) oth cell types were still found in close contct within the residul infiltrtes (Fig. 7). Antigen-specific memory T cells were recovered not only from lung tissue ut lso from lungdrining lymph node, spleen nd one mrrow (Fig. 7c). Consistent with the distriution of the memory B cells, the highest frequency of memory T cells ws found in the lung, however, the highest solute numer ws in the spleen. Discussion The interction of T nd B cells in SLO s well s in ectopic lymphoid tissue, which re structurlly nd functionlly similr, hs een investigted in gret detil in the pst. However, it is still uncler whether T/B coopertion cn lso tke plce in unordered T/B infiltrtes lcking the chrcteristics of SLO, such s clerly seprted T- nd B-cell zones nd the presence of FDC. This question is of mjor interest, ecuse these unstructured T/B ggregtes represent the vst mjority of infiltrtes in inflmed tissues in utoimmune nd llergic diseses. While erly studies reported the presence of FDC-positive ectopic lymphoid tissue in round 5% of ll synovil iopsies from rheumtoid rthritis ptients 1, more recent study found fully differentited follicles in only 6% of the iopsies 6. The predominnt type of infiltrtes ws chrcterized s either diffuse T- nd B-cell infiltrtes or T/B ggregtes lcking FDC structures. Similr oservtions were mde in studies nlysing T/B infiltrtes in kidneys of systemic lupus erythemtosus ptients 7,15. To study T/B coopertion in inflmed tissues, we complemented the well-estlished murine irwy inflmmtion model sed on the doptive trnsfer of OVA-specific T cells 16 y n dditionl trnsfer of ntigen-specific B cells. This novel irwy inflmmtion model llowed us the prllel nlysis of ntigen-specific T nd B cells during erly nd lte phses of the immune response, thus providing new insights into the phenotype nd function of T nd B cells in peripherl inflmed tissues. The doptive trnsfer system enled us to trck ntigen-specific T nd B cells, nd showed tht in the chronic phse of the immune response the vst mjority of ntigenspecific T nd B cells were not locted in the drining lymph nodes ut in the inflmed tissue. Similr to the sitution in utoimmune ptients, T/B infiltrtes in the lung minly consisted of loose ggregtes lcking FDC nd ny ordered structures. Nevertheless, not only plsm cells ut lso GC-like B cells were found in these infiltrtes. This shows tht SLO-like structures nd FDC re not solutely necessry for the development of GC-like B cells. So fr, this only hs een shown for the very specil cse of the so clled milky spots in the omentum 17. These structures, importnt for peritonel cvity immune responses, completely lck FDC ut develop GC B cells on ntigen exposure. Despite the presence of GC-like B cells, no clssicl TFH cells expressing CXCR5 nd Bcl-6 were found in the inflmed lung tissue. Three recent pulictions demonstrted the presence of CXCR5-positive TFH cells in inflmed lung tissue. However, in one study CXCR5 þ T cells were directly shown to reside in ectopic lymphoid structures 18 ; in the other two studies 19, this spect ws not nlysed, ut oth used infection models, where the development of ectopic lymphoid tissue cn e presumed. We, however, focused on ntigen-specific T cells in loose lymphocytic ggregtes, nd not in ectopic lymphoid tissue. These lung-infiltrting T cells lcked clssicl TFH cell mrkers, ut still hd potent B-cell helper cpcities nd were present in close contct with strongly proliferting GC-like B cells. NATURE COMMUNICATIONS 7:1875 DOI: 1.138/ncomms

8 NATURE COMMUNICATIONS DOI: 1.138/ncomms1875 CD5.1 Br V Br V % NP-specific B cells (of live) Ln Lung Spl Bm # NP-specific B cells ( 1 5 ) 8 6 Ln Lung Spl Bm c NP-specific B cells (lung) % 5% % % 39% % 5% CD38 d CD5.1 CD1/35 IgM IgG IgA 3% CD PD-L CD5.1 CD1/35 35% CD PD-L 5% Figure 6 Lung tissue is mjor reservoir for ntigen-specific memory B cells. Anlysis of lung-resident B cells 8 dys fter the lst ntigen chllenge (dy 1). () Histologicl nlysis of lung tissue showing perironchil infiltrte. Antigen-specific B cells (CD5.1 þ, green) were preferentilly found in the proximity of vessels. Br, ronchus, V, vessels. Scle r, 5 mm, originl mgnifiction ws. () Frequencies nd solute numers of ntigenspecific (CD5.1 þ ) B cells isolted from lymph node (Ln), lung, spleen (Spl) nd one mrrow (Bm) on dy 1. (c) Chrcteriztion of lung-resident ntigen-specific B cells showing representtive stining for CD38, IgM, IgG1, IgA nd the memory mrkers PD-L, CD8 nd CD73. Representtive dt from two independent experiments with six mice per experiment re shown. (d) Co-stining of ntigen-specific B cells (CD5.1) nd FDC (CD1/35) in lung tissue on dy. Scle rs, 5 mm in the overview nd 1 mm in the detil enlrgement. The high frequency of Ki-67-positive B cells indictes tht ctive T/B coopertion tkes plce. Indeed, recent detiled clonl tree nlysis of B cells locted in Ki-67 þ ut FDC-negtive T/B ggregtes from lupus nephritis ptients reveled ongoing somtic hypermuttion 7. These FDC-negtive T/B infiltrtes in the kidney were first descried y our group 15. Alredy in this erly study we hd mny indictions tht ctive T/B coopertion tkes plce in the infiltrtes nd leds to the locl genertion of plsm cells. The CXCR5-negtive B helper T cells from the lung exhiit some fetures of other recently descried T-cell popultions with B helper potentil, such s splenic extrfolliculr helper T cells found in certin utoimmune mouse models 1. Despite their non-tfh phenotype with very low CXCR5 expression, these extrfolliculr T cells exhiited strong potentil for B-cell help through expression of IL-, IL-1 nd CDL, nd might e importnt for the extrfolliculr plsm cell rection. The second exmple is one mrrow-resident CD þ memory T cells tht lso lck typicl TFH fetures, ut re, nevertheless, cple of providing help to B cells for ntiody production nd ffinity mturtion. In the rheumtoid joint CXCR5- nd Bcl-6-negtive T cells were shown to e source of CXCL13, which might e importnt for the recruitment of B cells 3. A very recent puliction descried CXCR5-negtive ut IL-1-positive T cells in the inflmed lung. So why re lung-infiltrting B helper T cells negtive for CXCR5? Possily, ecuse they simply do not need this chemokine receptor to get into contct with B cells. In the unordered infiltrtes with interspersed T nd B cells s oserved in our model, T cells do not hve to migrte from T-cell re into B-cell follicle s in the lymph node. In ddition, expression of the CXCR5 lignd CXCL13 is unlikely in these FDC-negtive lung infiltrtes, since in the mouse this chemokine is minly produced y FDC 5. Our study did not only identify the lung tissue s mjor reservoir for ntigen-specific effector cells ut lso s niche for long-lived memory B cells. Tissue-resident memory T cells were recently recognized s discrete memory T-cell suset, which fcilittes rpid secondry responses directly t the site of ntigen entry 6. Our study now shows tht not only ntigen-specific T ut lso B cells re found in close contct in the lung tissue. So fr, only three studies descried the presence of memory B cells in the lung 7 9, nd ll were virl infection models, which (different to our model) typiclly include the development of ectopic lymphoid tissue. The persisting T/B infiltrtes in the lung might e importnt in secondry immune responses for the rpid genertion of ntigen-specific plsm cells directly t the site of ntigen entry. Interestingly, ntigen-specific memory T nd B cells dditionlly distriuted to other clssicl memory cell niches like spleen nd one mrrow. Therefore, immune responses in 8 NATURE COMMUNICATIONS 7:1875 DOI: 1.138/ncomms1875

9 NATURE COMMUNICATIONS DOI: 1.138/ncomms1875 ARTICLE SM T P13- specific B NPspecific B1-8i Intrnsl immuniztion NP-SMA + LPS Anlysis the ddition of OVA peptide, recominnt IL-, nti-ifn-g nd nti-il OT-II T nd 1 6 B1-8i B cells were doptively trnsferred into C57BL/6 mice y intrvenous injection, 16 h lter mice were chllenged i.n. with 5 mg NP-OVA conjugte nd 5 mg LPS (Sigm) s djuvnt. Chllenge with sme mounts of ntigen ws repeted on dy 1, 1 nd 13. For nlysis of memory T cells, nive T cells from Smrt mice were directly co-trnsferred into recipients. Mice were chllenged i.n. with NP nd the cognte peptide (P13 GP 61 8 ) chemiclly coupled to mouse serum lumin ( mg on dy nd 1, nd 5 mg on dys 1, 13 nd 17) nd 5 mg LPS. CD5.1 Thy-1.1 B6 recipient Time (d) Cell isoltion. Lungs were perfused with PBS. Bronchil lymph nodes were dissected from the lung tissue under microscope nd prepred seprtely for flow cytometric nlysis y meshing through 7-mm sieve. Single-cell suspensions from lung tissue were prepred using the gentle MACS dissocitor (Miltenyi Biotec) nd digestion with.5 mg ml 1 collgense D nd mgml 1 DNse I (Roche) for 5 min t 37 C. Spleens were meshed through sieve, nd one mrrow ws flushed out of femur nd tii of hind legs. All cells were counted using Guv EsyCyte cpillry flow cytometer nd ViCount solution (Millipore). c % Tg T cells (of live) # Tg T cells ( 1 5 ) Ln Lung Spl Bm Ln Lung Spl Bm Figure 7 Clusters of ntigen-specific memory T nd B cells remin in the non-inflmed lung. () Smrt T-cell receptor trnsgenic (Tg) T cells were co-trnsferred with B1-8i B cells into C57BL/6 recipients. Mice were repetedly chllenged i.n. with NP nd Smrt peptide coupled to mouse serum lumin s non-immunogenic crrier nd nlysed 7 dys (d) fter the lst chllenge. () Histologicl nlysis of lung tissue showing ntigenspecific T cells (Thy-1.1 þ ) in yellow nd B cells (CD5.1 þ ) in mgent. Scle rs, 5 mm in the overview nd 5 mm in the detil enlrgement. (c) Frequencies nd solute numers of ntigen-specific T cells isolted from lymph node (Ln), lung, spleen (Spl) nd one mrrow (Bm). Representtive dt from independent experiments with 1 mice totl. the lung cn give rise not only to locl ut lso systemic protection. Our detiled nlysis of T/B coopertion in inflmed tissues is lso highly relevnt from clinicl perspective. With improved therpeutic options for utoimmune diseses preventing long phses of chronic disese, the development of ectopic lymphoid tissue s finl stge of tissue infiltrtion is nowdys only rrely oserved. However, FDC-negtive, unstructured lymphocytic tissue infiltrtes re frequent pthologicl finding, nd our study shed more light on the interction of utorective T nd B cells in these infiltrtes. Methods Mice. OT-II (Jx stock 19) nd Smrt 3 T-cell receptor trnsgenic mice or B1-8i mice 31 (dditionlly crossed with kpp light chin knockout mice 3 to ensure NP-specificity of B cells) were crossed to B6PL (Thy-1.1 þ, Jx stock 6) or Ly-5.1 (CD5.1 þ, Jx stock 1) mice, respectively. Trnsgenic strins nd wild-type C57BL/6 mice were red under specific pthogen-free conditions in the niml fcility of the Federl Institute for Risk Assessment, Berlin. For experiments femle mice in n ge etween 8 nd 16 weeks were used. Animl experiments were conducted ccording to the Germn niml protection lws, nd pproved y the responsile governmentl uthority (LAGeSo). Airwy inflmmtion model. OT-II T cells from spleens were sorted mgneticlly for CD6L high cells (Miltenyi Biotec), nd cultured with splenocytes for 5 dys with T/B co-cultures. To compre the B-cell helper qulities of effector T cells from lymph nodes nd lung, cells from oth orgns were enriched with nti-thy-1.1 microeds (Miltenyi Biotec) followed y flow-sorting on FACS ARIA II (BD Biosciences) for CD þ Thy-1.1 þ Thy-1. CD high CD8 B cells. Nive T cells from spleens of OT-II mice were sorted s CD þ Thy-1.1 þ CD6L þ B cells. Nive B cells were sorted from spleens of B1-8i mice s CD19 þ CD cells. T nd B cells were co-cultured in 96-well round-ottom pltes ( 6 individul wells) t 1: rtio in the presence of 1 mgml 1 NP OVA for 7 dys. Flow cytometry. Single-cell suspensions from lymph node, lung, spleen or one mrrow were stined with different comintions of fluorophore-conjugted ntiodies (Supplementry Tle I). Streptvidin-PE-Cy7 (.5 mgml 1 )or -PerCP (.5 mgml 1 ) were used s secondry regents for iotinylted A. Fc receptors were locked with mgml 1.G (nti-cd16/3). For nlysis of cytokine production, cells were re-stimulted in vitro with OVA peptide for.5 h with Brefeldin A dded fter 1 h. Following stining of cell surfce ntigens, cells were fixed with formldehyde, permeilized with sponin nd stined intrcellulrly with nti-cytokine ntiodies (Supplementry Tle I). For detection of IL-1, n IL-1R-Fc chimer, followed y Cy5-conjugted got nti-humn Ig (Jckson ImmunoReserch, 1:8), ws used. For intrcellulr stining of trnscription fctors (Supplementry Tle I) the FoxP3 Stining Buffer Set from ebioscience ws used. For ctivtion-induced cytidine deminse stining, cells were stined intrcellulrly efore surfce stining, nd Alex Fluor 67-conjugted got nti-rt Ig (Invitrogen,.5 mgml 1 ) ws used s secondry regent. For intrcellulr stining of Ig, cells were fixed with formldehyde, permeilized with sponin uffer nd stined with fluorescein isothiocynte (FITC)-conjugted nti-ig ntiodies (Supplementry Tle I). To discriminte ded cells, either,6-dimidino--phenylindole () ws dded to live cells immeditely efore nlysis or cells were stined with 1.3 mm Pcific Ornge succinimidyl ester (Invitrogen) efore fixtion. Smples were nlysed on n LSR II or LSR Fortess flow cytometer (BD Biosciences). Anlysis gtes were set on live cells defined y sctter chrcteristics nd exclusion of doulets nd ded cells. In ll figures, ntigen-specific T cells re defined s B CD þ Thy-1.1 þ nd B cells s CD3/CD8 CD19 þ CD5. CD5.1 þ. Dt were nlysed with FlowJo Softwre (Treestr). Enzyme-linked immunosorent ssy. NP-specific IgG1 nd IgA ws mesured y enzyme-linked immunosorent ssy. Pltes were precoted with NP-coupled BSA, nd ound ntiodies were detected y using horserdish peroxidse-coupled ntiodies to mouse IgG1 or IgA (Southern Biotechnologies) nd tetrmethylenzidine s sustrte. Histology. Lungs were filled with 5% TissueTek OCT compound vi the trche nd snp frozen in ice-cold isopentne. The 8-mm cryostt sections of the right lower loe were cut nd fixed with cetone. To reduce unspecific signls, peroxidse inctivtion nd locking with Csein Solution (Vector Lortories) ws performed. For light microscopy, tissue section ws stined with FITC-coupled OX-7 (nti-thy-1.1, 1.5 mgml 1 ), A (nti-cd5.1, 5 mgml 1 ), 7G6 (nti-cd1/35, 1 mgml 1 ), unconjugted 81- (nti-cd138, 5 mgml 1 ) or MBP-1 (nti-mbp, kindly provided y J. Lee, Scottsdle, 1:,5). As secondry regents, peroxidse-conjugted ntiser ginst FITC, digoxygenin (Roche, 1:3) or rt- IgG (Jckson ImmunoReserch, 1:,) were used. Slides were developed using 3-mino-9-ethylcrzole s sustrte, nuclei were counterstined with hemtoxyline. A periodic cid Schiff-stining kit (Crl Roth) ws used to stin mucopolyscchrides. Slides were nlysed on n Axioskop light microscope equipped with n Axiocm (Crl Zeiss). NATURE COMMUNICATIONS 7:1875 DOI: 1.138/ncomms

10 NATURE COMMUNICATIONS DOI: 1.138/ncomms1875 For immunofluorescence microscopy, sections were stined with FITC- or Alex Fluor 56-coupled 7G6 (nti-cd1/35, 1 mgml 1 ), FITC-coupled A (nti-cd5.1,.5 mgml 1 ), digoxygenin-conjugted OX-7 (nti-thy-1.1,.5 mgml 1 ), digoxygenin-conjugted GL7 (nti-gc B cells, 1 mgml 1 )or hmster nti-ki-67 (Acm, 1:1), followed y Alex Fluor 59-coupled nti-hmster Ig (Invitrogen, 1:5). Signls were mplified with peroxidseconjugted nti-fitc followed y FITC-coupled tyrmide (Invitrogen). After inctivtion of peroxidse, the second ntiody ws detected with nti-digoxygenin-peroxidse (Roche, 1:3) nd Alex Fluor 67-coupled tyrmide. Nuclei were counterstined with. Imges were cptured on n LSM 78 with ZEN1 imging softwre (Crl Zeiss). B-cell receptor sequencing. Four FDC-positive nd six negtive infiltrtes (determined y CD1/35 stining on consecutive section) were isolted from frozen lung sections using n Arcturus Verits lser cpture microdissection system. RNA ws isolted using the Arcturus PicoPure RNA isoltion kit. The VH186. sequence of the trnsgenic B1-8i B-cell receptor ws mplified from cdna using universl primer in the signl peptide (5 -GATGGAGCTGTATCA TGCTCTTCTTGGCAG-3 ) nd specific primers for the hevy chin of IgA (5 -CACTGGGTCACTTGACAGAGCTTGTG-3 ), IgG1 (5 -GCTGCTCAGAGTG TAGAGGTCAGACTGC-3 ) nd IgM (5 -TGAAGGAAATGGTGCTGGGC AGG-3 ). PCR products were cloned into pjet1.. Sequences from 1 single clones were nlysed for somtic hypermuttion. Sttisticl nlysis. Experimentl groups contined t lest five nimls (rndomly ssigned), which routinely provides the sttisticl power to detect iologicl significnt differences in our experimentl system. Dt were nlysed using GrphPd Prism 5. No dt exclusion criteri were used. References 1. Crotty, S. Folliculr helper CD T cells (TFH). Annu. Rev. Immunol. 9, (11).. McHeyzer-Willims, M., Okitsu, S., Wng, N. & McHeyzer-Willims, L. Moleculr progrmming of B cell memory. Nt. Rev. Immunol. 1, 3 (1). 3. Rndll, T. D. Bronchus-ssocited lymphoid tissue (BALT) structure nd function. Adv. Immunol. 17, (1).. Neyt, K., Perros, F., GeurtsvnKessel, C. H., Hmmd, H. & Lmrecht, B. N. Tertiry lymphoid orgns in infection nd utoimmunity. Trends Immunol. 33, (1). 5. Moyron-Quiroz, J. E. et l. Role of inducile ronchus ssocited lymphoid tissue (ibalt) in respirtory immunity. Nt. Med. 1, (). 6. Cntert, T. et l. B lymphocyte utoimmunity in rheumtoid synovitis is independent of ectopic lymphoid neogenesis. J. Immunol. 181, (8). 7. Chng, A. et l. In situ B cell-medited immune responses nd tuulointerstitil inflmmtion in humn lupus nephritis. J. Immunol. 186, (11). 8. Ridderstd, A. & Trlinton, D. M. Kinetics of estlishing the memory B cell popultion s reveled y CD38 expression. J. Immunol. 16, (1998). 9. Zygmunt, B. M., Rhroui, F., Groee, L. & Guzmn, C. A. Intrnsl immuniztion promotes th17 immune responses. J. Immunol. 183, (9). 1. Sonod, E. et l. Differentil regultion of IgA production y TGF-et nd IL-5: TGF-et induces surfce IgA-positive cells ering IL-5 receptor, wheres IL-5 promotes their survivl nd mturtion into IgA-secreting cells. Cell Immunol. 1, (199). 11. Anderson, S. M., Tomyko, M. M., Ahuj, A., Hermn, A. M. & Shlomchik, M. J. New mrkers for murine memory B cells tht define mutted nd unmutted susets. J. Exp. Med., (7). 1. Tomyko, M. M., Steinel, N. C., Anderson, S. M. & Shlomchik, M. J. Cutting edge: hierrchy of mturity of murine memory B cell susets. J. Immunol. 185, (1). 13. Weer, J. P., Fuhrmnn, F. & Hutloff, A. T-folliculr helper cells survive s long-term memory cells. Eur. J. Immunol., (1). 1. Tkemur, S. et l. Lymphoid neogenesis in rheumtoid synovitis. J. Immunol. 167, (1). 15. Hutloff, A. et l. Involvement of inducile costimultor in the exggerted memory B cell nd plsm cell genertion in systemic lupus erythemtosus. Arthritis Rheum. 5, (). 16. Lloyd, C. M., Gonzlo, J. A., Coyle, A. J. & Gutierrez-Rmos, J. C. Mouse models of llergic irwy disese. Adv. Immunol. 77, (1). 17. Rngel-Moreno, J. et l. Omentl milky spots develop in the sence of lymphoid tissue-inducer cells nd support B nd T cell responses to peritonel ntigens. Immunity 3, (9). 18. Slight, S. R. et l. CXCR5( þ ) T helper cells medite protective immunity ginst tuerculosis. J. Clin. Invest. 13, (13). 19. Strutt, T. M., McKinstry, K. K., Kung, Y., Brdley, L. M. & Swin, S. L. Memory CD þ T-cell-medited protection depends on secondry effectors tht re distinct from nd superior to primry effectors. Proc. Ntl Acd. Sci. USA 19, E551 E56 (1).. Moguche, A. O. et l. ICOS nd Bcl6-dependent pthwys mintin CD T cell popultion with memory-like properties during tuerculosis. J. Exp. Med. 1, (15). 1. Odegrd, J. M. et l. ICOS-dependent extrfolliculr helper T cells elicit IgG production vi IL-1 in systemic utoimmunity. J. Exp. Med. 5, (8).. Tokoyod, K. et l. Professionl memory CD þ T lymphocytes preferentilly reside nd rest in the one mrrow. Immunity 3, (9). 3. Mnzo, A. et l. Mture ntigen-experienced T helper cells synthesize nd secrete the B cell chemottrctnt CXCL13 in the inflmmtory environment of the rheumtoid joint. Arthritis Rheum. 58, (8).. Coquet, J. M. et l. Interleukin-1-producing CD( þ ) T cells promote type immunity to house dust mites. Immunity 3, (15). 5. Ansel, K. M. et l. A chemokine-driven positive feedck loop orgnizes lymphoid follicles. Nture 6, (). 6. Mueller, S. N., Gehrdt, T., Crone, F. R. & Heth, W. R. Memory T cell susets, migrtion ptterns, nd tissue residence. Annu. Rev. Immunol. 31, (13). 7. Joo, H. M., He, Y. & Sngster, M. Y. Brod dispersion nd lung locliztion of virus-specific memory B cells induced y influenz pneumoni. Proc. Ntl Acd. Sci. USA 15, (8). 8. Onoder, T. et l. Memory B cells in the lung prticipte in protective humorl immune responses to pulmonry influenz virus reinfection. Proc. Ntl Acd. Sci. USA 19, 85 9 (1). 9. Adchi, Y. et l. Distinct germinl center selection t locl sites shpes memory B cell response to virl escpe. J. Exp. Med. 1, (15). 3. Oxenius, A., Bchmnn, M. F., Zinkerngel, R. M. & Hengrtner, H. Virus-specific MHC-clss II-restricted TCR-trnsgenic mice: effects on humorl nd cellulr immune responses fter virl infection. Eur. J. Immunol. 8, 39 (1998). 31. Sonod, E. et l. B cell development under the condition of llelic inclusion. Immunity 6, 5 33 (1997). 3. Zou, Y. R., Tked, S. & Rjewsky, K. Gene trgeting in the Ig kpp locus: efficient genertion of lmd chin-expressing B cells, independent of gene rerrngements in Ig kpp. EMBO J. 1, (1993). Acknowledgements This study ws supported y grnts from the Germn Reserch Council (DFG) nd the Fritz Thyssen Foundtion to A.H. We thnk Gudrun Steinhuser for help with lser microdissection. Klus Rjewsky generously provided B1-8i nd Mx Löhning Smrt mice. Author contriutions D.V. designed nd performed experiments nd nlysed dt; K.B., L.J.P., M.S.A., R.K.F. nd S.G. performed experiments; E.H. nd R.A.K. designed experiments; A.H. designed experiments, performed experiments, nlysed dt nd wrote the mnuscript. Additionl informtion Accession codes: Unique B cell receptor sequences from FDC-negtive nd -positive infiltrtes hve een deposited t the Europen Nucleotide Archive ( under the ccession codes LT159851, LT15985, LT159853, LT15985, LT159855, LT159856, LT159857, LT159858, LT159859, LT15986, LT159861, LT15986, LT159863, LT15986 nd LT Supplementry Informtion ccompnies this pper t nturecommunictions Competing finncil interests: The uthors declre no competing finncil interests. Reprints nd permission informtion is ville online t reprintsndpermissions/ How to cite this rticle: Vu Vn, D. et l. Locl T/B coopertion in inflmed tissues is supported y T folliculr helper-like cells. Nt. Commun. 7:1875 doi: 1.138/ncomms1875 (16). This work is licensed under Cretive Commons Attriution. Interntionl License. The imges or other third prty mteril in this rticle re included in the rticle s Cretive Commons license, unless indicted otherwise in the credit line; if the mteril is not included under the Cretive Commons license, users will need to otin permission from the license holder to reproduce the mteril. To view copy of this license, visit 1 NATURE COMMUNICATIONS 7:1875 DOI: 1.138/ncomms1875