{'fraction_non_alphanumeric': 0.036818255381129636, 'fraction_numerical': 0.02670335005664347, 'mean_word_length': 4.527057245080501, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
EBI2 augments Tfh cell fate by promoting interaction with IL2-quenching dendritic cells

T follicular helper (Tfh) cells are a CD4 T cell subset that is important for supporting plasma cell and germinal center (GC) responses1,2. The initial induction of Tfh cell properties occurs within the first few days following activation by antigen recognition on dendritic cells (DCs), though how DCs promote this cell-fate decision is not fully understood1,2. Moreover, although Tfh cells are uniquely defined by expression of the follicle-homing receptor CXCR51,2, the guidance receptor promoting the earlier localization of activated T cells at the B cell follicle–T zone interface has been unclear3–5. Here we show that the G-protein coupled receptor EBI2 (GPR183) and its ligand 7α,25-dihydroxycholesterol (7α,25-OHC) mediate positioning of activated CD4 T cells at the follicle–T zone interface. In this location they interact with activated DCs and are exposed to Tfh cell-promoting ICOS ligand. IL2 is a cytokine that has multiple influences on T cell fate, including negative regulation of Tfh cell differentiation6–10. We demonstrate that activated DCs in the outer T zone further augment Tfh cell differentiation by producing membrane and soluble forms of CD25, the IL2 receptor α chain, and quenching T cell-derived IL2. Mice lacking EBI2 in T cells or CD25 in DCs have reduced Tfh cells and mount defective T cell-dependent plasma cell and GC responses. These findings demonstrate that distinct niches within the lymphoid organ T zone support distinct cell fate decisions, and they establish a function for DC-derived CD25 in controlling IL2 availability and T cell differentiation.

immunization with a particulate form of OVA (sheep red blood cell (SRBC) conjugated), and it remained high at day 2 (Extended Data Fig. 1a). Similar EBI2 induction occurred following immunization with OVA in LPS, on lymph node (LN) T cells after immunization with OVA in alum, and in vitro following T cell activation by anti-CD3 and -CD28 (Extended Data Fig. 1b-e). Migration to 7α,25-OHC was augmented at these time points (Extended Data Fig. 1f). Analysis of spleen sections showed that transferred WT T cells accumulated in the outer T zone at 12 hours and day 1 of the SRBC-OVA response and the cells remained enriched in this location at day 2 (Fig. 1a). EBI2 knockout (KO) T cells, by contrast, failed to accumulate in the outer T zone at either time point and instead remained dispersed throughout the T zone (Fig. 1a). Quantitative analysis using a mixed transfer system confirmed that the activated EBI2 KO cells had less access than control cells to the outer T zone ( Fig. 1b and Extended Data Fig. 1g). Similar findings were made at day 2 after immunization with OVA-expressing Listeria monocytogenes (Fig. 1c) and with OVA in LPS (Extended Data Fig. 1h). WT OTII T cells also moved to the B-T zone interface in LNs following immunization with alum-OVA, but EBI2-deficient T cells failed to relocalize ( Fig.  1d and Extended Data Fig. 1i). Activated T cell positioning in the outer T zone was directed by 7α,25-OHC as it was dependent on the enzymes needed for its synthesis (Cyp7b1 and Ch25h) and catabolism (Hsd3b7) (Extended Data Fig. 1j).
Flow cytometric analysis for the early activation marker, CD69, showed that co-transferred EBI2 KO and WT T cells were comparably activated at day 2 of the SRBC-OVA response (Fig. 2a) indicating similar initial exposure to cognate MHC class II-peptide complexes. Upregulation of the costimulatory molecules ICOS and OX40 also occurred to an equivalent extent (Extended Data Fig. 2a). Proliferation began by day 2 and at this time point the WT and EBI2 KO cells responded similarly (Fig. 2b, c). However, by day 3, the EBI2-deficient cells were undergoing less proliferation and their numbers increased more slowly (Fig. 2b,  c). This was not due to a direct effect of 7α,25-OHC on T cell proliferation (Extended Data Fig. 2b, c). Tracking of differentiation markers on the in vivo activated T cells revealed that EBI2 KO cells were compromised in their induction of a Tfh cell phenotype, as assessed by CXCR5, PD-1 (Fig. 2d, e), Bcl6, and Il21 expression (Extended Data Fig. 2d-f). EBI2deficient OTII T cells also differentiated less efficiently into Tfh cells in LNs (Fig. 2f). We also observed reduced Tfh cell responses to Listeria-OVA, reduced polyclonal EBI2 KO Tfh cell responses to SRBCs, and reduced GC and plasma cell responses to these antigens (Supplemental Information and Extended Data Fig. 3a-j).
Tfh cell differentiation is promoted by interaction with both DCs and B cells, and time course studies indicate that DCs are critical early while B cells play a later role 1,2 . Consistent with these requirements, WT OTII T cells showed a partial reduction in Tfh cell differentiation at day 3 of the response in MD4 Ig-transgenic mice that lack cognate B cells capable of OVA antigen presentation to OTII T cells (Fig. 3a). Importantly, however, EBI2 KO OTII T cells formed less Tfh cells than WT OTII T cells in the MD4 recipients, indicating that T cell EBI2 expression augmented Tfh cell development at early time points in a B cell independent manner. Similar observations were made in B cell-deficient μMT mice (Extended Data Fig. 4a). These findings led us to test whether EBI2 was required in T cells for some type of interaction with DCs. Ablation of DCs using Zbtb46-DTR mice 15 caused a complete block in Tfh cell generation (Extended Data Fig. 4b, c), consistent with previous studies using other DC ablation approaches 16 . Splenic CD4 and DCIR2 coexpressing DCs relocalize from bridging channels to the outer T zone within 6 hours of immunization with SRBCs 17,18 , and the cells remain in this region for at least 2 days ( Fig.  3b and Extended Data Fig. 4d). At 12 hour, day 1 and day 2 time points, almost the entire population of activated WT OTII T cells colocalized with the activated DCIR2 + DCs (Fig.  3b, c and Extended Data Fig. 4e). By contrast, activated EBI2 KO T cells were broadly distributed and only partially overlapped with the DCIR2 + DCs (Fig. 3b, c and Extended Data Fig. 4e). Using mice with deficiencies in DCs we established that CD4 + but not CD8 + DCs were important for Tfh cell induction by SRBC-OVA (Supplementary Information and Extended Data Fig. 4f-k).
ICOS signaling is important for Tfh cell differentiation 1,2 and splenic DCs upregulated ICOSL mRNA following activation by SRBCs, with the extent of upregulation being greater in CD4 + than CD8 + DCs (Fig. 3d). However, flow cytometric analysis showed 12 hour activated CD4 + DCs had low surface ICOSL staining ( Fig. 3e and Extended Data Fig. 4l). Since ICOSL undergoes rapid ectodomain shedding following ICOS engagement 19,20 , we considered the possibility that surface levels were reduced due to interactions with ICOShigh activated T cells. Consistent with this idea, when SRBC-immunized mice were also treated with an ICOS blocking antibody to prevent ICOS-induced ICOSL shedding 19,20 , CD4 + but not CD8 + DCs showed increased ICOSL surface abundance compared to unimmunized mice ( Fig. 3e and Extended Data Fig. 4l). We took advantage of the sensitivity of ICOSL to ICOS-induced shedding as a method to measure the amount of interaction between DCs and cognate T cells. Twelve hours following SRBC-OVA immunization, ICOSL levels were higher on CD4 + DCs in mice harboring EBI2-deficient OTII T cells than WT OTII T cells (Fig. 3f). These data suggest that the reduced Tfh differentiation of EBI2 KO OTII T cells occurs at least in part due to lower ICOS engagement with ICOSL on CD4 + DCs. However, in mice treated with an ICOS blocking antibody, though Tfh differentiation of control OTII T cells was reduced, EBI2-deficient OTII T cells were still more defective (Fig. 3g), indicating an ICOS independent influence of EBI2 in augmenting Tfh cell fate. An assessment of mRNA levels of other factors established to have an effect on Tfh differentiation (IL6, TGFβ) showed that they were similarly expressed in CD4 + and CD8 + DCs and they were therefore not considered likely factors accounting for the EBI2dependence of Tfh cell differentiation (Extended Data Fig. 4n).
To search for surface or secreted DC-derived factors that might augment Tfh cell differentiation we performed RNAseq analysis on CD4 + DCs from the spleens of saline or SRBC immunized mice. This analysis revealed CD25, the high affinity IL2 receptor αchain, as one of the most strongly induced genes in SRBC-activated DCs (Extended Data Fig. 5a). CD25 mRNA induction occurred rapidly after immunization and remained elevated for at least 2 days (Fig. 4a) and many CD4 + DCs were surface positive for CD25 over this time frame (Fig. 4b). CD8 + DCs showed little induction of CD25 mRNA or protein under these immunization conditions (Fig. 4a, b). Analysis of LN DCs following OVA plus alum immunization revealed upregulation of CD25 on migratory CD11b + DCs at day 1 and 2 (Extended Data Fig. 5b). Staining of spleen sections identified CD25 + cells in the unstimulated T zone that are likely CD25 hi regulatory T cells, but also showed broad induction of CD25 in the outer T zone within 12 hours of SRBC immunization in a pattern resembling the DCIR2 + DC distribution (Fig. 4c). Similar appearance of CD25 staining in the T zone was seen in T cell-deficient mice, providing evidence that expression by activated DCs was being detected (Extended Data Fig. 5c). CD25 needs to associate with CD122 (IL2Rβ) and IL2Rγ to transmit signals in response to IL2 10 . Despite expression of CD25, activated CD4 + DCs showed minimal CD122 mRNA and protein expression and they did not respond to IL2 as assessed by intracellular pSTAT5 staining (Extended Data Fig. 5d-f). These data led us to consider the possibility that activated DCs express CD25 to alter IL2 availability in the outer T zone.
IL2 has pleiotropic roles in directing T cell fate and this includes a negative influence on Tfh cell differentiation [6][7][8][9][10] . Despite equivalent IL2 production and receptor expression (Supplementary Information and Extended Data Fig. 5g-i), EBI2 KO T cells showed more IL2R signaling than WT T cells at day 1 after immunization as evidenced by higher pSTAT5 levels (Fig. 4d), suggesting that the EBI2 KO T cells were being exposed to more IL2. The elevated induction of pSTAT5 in EBI2 KO T cells was also seen in LNs following immunization with alum-OVA (Fig. 4e). Blimp1, encoded by Prdm1, is induced by IL2 and negatively regulates expression of Bcl6, a factor essential for Tfh cell development 2 . In agreement with higher IL2 exposure, the EBI2 KO T cells showed greater Prdm1 expression at day 3 (Extended Data Fig. 5j).
The above findings led us to test whether DCs antagonize IL2 availability to activated T cells in the outer T zone. Consistent with this possibility, when in vivo activated DCs were incubated briefly in vitro they were found to release soluble CD25 (sCD25) into the culture supernatant (Fig. 4f). DC production of sCD25 was not dependent on interaction with T cells (Fig. 4f). Analysis of spleen tissue extracts showed elevated sCD25 production at 6 hours after SRBC immunization and this was maintained at 24 hours (Fig. 4g) and occurred in a T cell-independent and DC-dependent manner (Fig. 4h). To determine whether the sCD25 functioned as an IL2 antagonist, supernatants from cultures of activated CD4 + DCs were tested in a bioassay for their ability to inhibit IL2R signaling. Regulatory T cells were used as the reporter cells in this bioassay since they were more sensitive to low dose IL2 than Tfh cells (unpubl. obs.). Supernatants from cultured SRBC-activated WT but not CD25 KO CD4 + DCs were able to antagonize IL2R signaling in T cells (Extended Data Fig. 5l).
To determine whether DCs were regulating T cell differentiation in vivo by production of CD25 we generated BM chimeric mice that lacked CD25 in DCs (Supplementary Information and Extended Data Figs. 6a-e). In these recipients, control (EBI2 Het) OTII T cells were compromised in their ability to take on a Tfh cell fate whereas EBI2 KO T cells were only mildly affected (Fig. 4i). Staining of tissue sections from the DT-treated BM chimeras established that the high CD25 abundance in the outer T zone was dependent on CD25 expression by DCs (Extended Data Fig. 6f). Splenic Tfh cell responses to Listeria-OVA and LN Tfh cell responses to alum-OVA were also diminished in mice lacking CD25 on DCs (Extended Data Fig. 6g, h). Moreover, mice lacking CD25 in DCs and harboring transgenic B cells specific for hen egg lysozyme (HEL) mounted reduced plasma cell and GC responses to HEL-conjugated SRBCs ( Fig. 4j and Extended Data Fig. 6i, j) and this was associated with reduced serum anti-HEL IgG1 and IgG2b antibody levels (Fig. 4k). To further test whether reduced exposure of EBI2 KO T cells to sCD25 could account for the defective Tfh cell induction, we treated mice with recombinant sCD25 at day 0 and 1 of the OTII T cell response to SRBC-OVA. This treatment was sufficient to elevate sCD25 levels in tissue extracts (Extended Data Fig. 6k), to antagonize pSTAT5 over-induction in the EBI2 KO T cells (Fig. 4l) and to partially rescue Tfh cell differentiation (Fig. 4m). A full restoration of the Tfh response was not expected given that EBI2-deficient cells are also compromised in accessing ICOSL and possibly other Tfh cell-promoting signals from cells in the outer T zone.
This work establishes a role for EBI2 and 7α,25-OHC in positioning of activated T cells at the follicle-T zone interface, promoting contact with Tfh cell-priming ICOSL hi CD25 + DCs (Supplementary Information and Extended Data Fig. 7). Given that interactions with both DCs and B cells are important for full Tfh cell differentiation 1,2 , we suggest that T cell EBI2 upregulation initially acts to favor interaction with Tfh cell-promoting DCs and subsequently with activated B cells. While EBI2 upregulation is important for Tfh cell induction, the receptor is downregulated at week 2 of the response and this may facilitate Tfh cell retention in GCs 14 . Soluble CD25 was first detected in human serum ~30 years ago and it has since been reported in human and mouse serum in a large number of studies and it has been correlated with various disease conditions [21][22][23] . While there has been evidence that sCD25 can antagonize certain IL2 functions 10,22,24 the significance of sCD25 in vivo has been unclear. Moreover, the function of CD25 in myeloid cells has been mysterious 10,25,26 , with some in vitro studies suggesting it suppresses 27 and others that it augments 28,29 T cell responses. We show that DC production of CD25 plays an important role in quenching IL2 in the outer T zone and it thereby cooperates with other factors, including ICOSL, to facilitate Tfh cell differentiation (Extended Data Fig. 7). While our findings show DCs produce sCD25, we do not exclude the possibility that membrane associated CD25 on DCs also has a regulatory role. Strengths of IL2 signaling influence Treg cell activity, Th17, Th1 and Th2 cell development, and CD8 T cell proliferation and differentiation 10,30 . We suggest that CD25-mediated IL2-quenching by DCs will be a general mechanism acting to guide a range of IL2-sensitive cell activation and differentiation processes.

Mice and bone marrow chimeras
Wild-type C57BL/6NCr and C57BL/6-cBrd/cBrd/Cr (B6-Ly5.2) mice of 7-9 weeks of age were purchased from the National Cancer Institute (Frederick, MD, USA). Ebi2 −/− (containing a GFP reporter in place of the Ebi2 coding exon), Cyp7b1 −/− , Ch25h −/− , Hsd3b7 −/− , Ccr7 −/− , HEL-specific MD4 Ig-transgenic, HEL-specific Hy10 mice, and OVAspecific OTII TCR-transgenic mice have been described ( 11,17,31 and references therein). B cell-deficient μMT mice were kindly provided by Tony Defranco and Christopher Allen (University of California, San Francisco, CA, USA). Cd28 −/− mice were kindly provided by K. Mark Ansel (University of California, San Francisco). TCRβδ −/− , Cd47 −/− , Batf3 −/− and Zbtb46-diphtheria toxin receptor (DTR) mice were from Jackson laboratories (Jax, Bar Harbor, ME, USA). Irf4 fl/fl CD11c-Cre + mice were from Jax and kindly provided by Shomyseh Sanjabi (Gladstone Institutes, San Francisco). Cd25 −/− mice were from Jax and were kindly provided by Markus Muschen (University of California, San Francisco). Bone marrow (BM) chimeras were generated as described 11 and analyzed after 6-12 weeks. Mixed BM chimeras were made by mixing equal amounts of the two types of BM prior to transfer. The sample sizes were guided by previous studies in our laboratory. No animals were excluded from analysis, and sample size estimates were not used. The mouse genotype was not blinded from the investigator. Mice of a given genotype were randomly assigned to groups. However, littermate mice were evenly distributed into control or treatment groups and mice of both groups were co-caged whenever possible. In experiments involving transfers of OTII T cells, since the TCR transgene is on the Y chromosome, male mice were used as donors and recipients. In other experiments, similar numbers of male and female mice were used. All mice were adult and were studied between 7 and 20 weeks of age. Animals were housed in a specific-pathogen free environment in the Laboratory Animal Research Center at the University of California, San Francisco, and all experiments conformed to ethical principles and guidelines approved by the Institutional Animal Care and Use Committee.

Adoptive transfer, immunizations, DC ablation and treatments
For analysis of CD4 + T cell position, activation or Tfh cell differentiation in spleens, 1-5 × 10 6 WT and/or EBI2 KO OTII cells were adoptively transferred into mice. One day after cell transfer, recipients were immunized intraperitoneally (i.p.) with 2×10 8 SRBCs (Colorado Serum Company, Denver, CO) conjugated with OVA (Sigma-Aldrich, St. Louis, MO, USA) as described 32 with minor modifications detailed below, with 25 μg OVA plus 25 μg LPS (E. coli 0111:B4, Sigma-Aldrich), intravenously (i.v.) with 2×10 9 heat-killed Listeria-OVA as described 33 , or subcutaneously with 25 μg OVA in 200 μl Alum (InvivoGen, San Diego, CA, USA). For conjugation of OVA with SRBCs, 1 ml of SRBCs were washed with PBS three times, incubated with 4 ml of 30 mg/ml ice cold OVA in PBS and crosslinked with 1 ml of 100 mg/ml EDCI (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, Sigma-Aldrich) for 1 hour on ice with occasional mixing, followed by washing four times in PBS to remove the free OVA and confirmation of the conjugation by flow cytometry. For HEL-specific antibody responses, 1×10 5 Hy10 B cells 34 were adoptively transferred into desired recipients. One day after cell transfer, recipients were i.p. immunized with SRBCs conjugated with a low affinity mutant of HEL termed HEL2× (ref 35 ) as described 17 . To visualize cell proliferation, cells were labeled with CellTrace ™ violet tracer (Molecular Probes, invitrogen, Carlsbad, CA, USA) according to manufacturer instructions. For DC ablation, Zbtb46-DTR full or mixed chimeras were injected i.p. with 20 ng DT (Sigma-Aldrich) per gram of body weight 3 days before cell transfer and received 4 ng DT per gram of body weight on the third day after the initial DT injection and in some cases again 3 days later. To block ICOS, mice were injected i.v. with anti-mouse ICOS antibody (rat IgG2b, clone 7E.17G9, BioXCell, West Lebanon, NH, USA) or rat IgG2b isotype control one day before and 2 days after cell transfer (0.5 mg/mouse/injection). To block IL2, two doses of recombinant CD25 protein (25 μg/mouse/dose, Sino Biological Inc., Beijing, China) were injected into mice at the same time with immunization or one day after immunization.

Image Quantification
Immunofluorescence images were analyzed using IMARIS (ver 7.3.0). White-pulp cords containing circular T zones were used to quantify outer T-zone positioning of co-transferred WT (red) and EBI2 HET or KO cells (green). OTII cells were defined using the Spots function in IMARIS and coordinates for each OTII cell were exported into R. The center and average radius of the T zone was measured using the Measurement Points function in IMARIS. The "outer T-zone" was defined as the area further than 3/4ths of the average radius from the center of the T zone. The distance of each OTII cell from the center of the T zone and the proportion of cells in the outer T-zone was calculated using R. An average of 70 cells were present for each co-transferred group per T-zone (average of 140 total). IHC images were analyzed manually using the Cell Counter Plugin in ImageJ (ver 1.49). OTII cells that were in contact with DCIR2+ DC were distinguished from lone OTII cells using separate counters.

Soluble CD25 ELISA and bioassay
To test the production of sCD25 by DCs in vitro, mice were first i.p. immunized with SRBCs. At the time of analysis, spleen CD4 + DCs were enriched by depletion of T, B and NK cells with a cocktail of biotin-conjugated antibodies and isolated by positive selection using biotin-conjugated anti-DCIR2 to purities of over 90% (Miltenyi Biotec). The purified DCs were cultured in vitro for 8 hours and the presence of soluble CD25 in culture supernatants was detected using a CD25 ELISA kit (R&D Systems Inc, Minneapolis, MN, USA). To detect sCD25 in spleen tissue, each spleen was mashed into 1 ml of medium through a 70μm cell strainer and centrifuged at 300 g for 10 min and 3,000 g for 15 min at 4°C. The cell-free supernatant was subjected to the ELISA assay. To test for antagonism of IL2 mediated signaling, DC culture supernatants were mixed with different dosages of recombinant mouse IL2 (Biolegend) for 2 hours and added to splenocytes at 37°C for 30 minutes. pSTAT5 levels in CD25 + CD4 + T cells were analyzed as described above.

Detection of SRBC or HEL-specific antibody responses
To assay for anti-SRBC or anti-HEL IgM and IgG from mouse serum, 50 μl of SRBCs or HEL-conjugated mouse RBCs (5 × 10 7 cells/ml in PBS) was incubated with 2 μl of serum for 1 hour at room temperature. After washing, the RBCs were incubated with fluorescent antibodies against mouse IgM, IgG1 and IgG2b for flow cytometric analysis.

Quantitative RT-PCR
Total RNA from sorted cells was isolated and reverse-transcribed, and quantitative PCR was performed as described 31 . Data were analyzed using the comparative C T (2 −ΔΔCt ) method using Hprt as the reference.

RNA-Seq Analysis
Spleens were taken one hour after saline or SRBC immunization. CD4 + DCs were preenriched using MACS manual cell separation columns with anti-CD11c microbeads (Miltenyi Biotec) and further sorted based on surface markers of CD11c + I-Ab + CD4 + CD8 -. Cells were sorted twice on a FACSAira III to purities of over 99%. Sorted DCs (10 6 ) were snap frozen and then RNA was extracted with the QIAGEN RNeasy Kit. RNA quality was checked with the Agilent 2100 Bioanalyzer (RNA integrity number >9 for all samples). Barcoded sequencing libraries were generated with 100 ng of RNA with the Ovation RNA-Seq System V2 and Encore Rapid Library System. Sequencing was performed on an Illumina HiSeq 2500 (UCSF Human Genetics Core) with 100-bp paired-end reads. Sequences were reported as FASTQ files, which were aligned to the mm9 mouse genome with STAR (Spliced Transcript Alignment to a Reference). Generation of Log 2 FC values and further analyses were performed with a Bioconductor package on RStudio. The RNAseq data have been deposited in the Gene Expression Omnibus (NCBI) data repository under accession code GEO: GSE71165.

Statistical analysis
Prism software (GraphPad) was used for all statistical analysis. Statistical comparisons were performed using an ANOVA or a two-tailed Student's t-test. P values were considered significant when less than 0.05.

Extended Data Figure 7. Model of how EBI2-dependent positioning of activated T cells in association with CD25 + DCs in the outer T zone favors Tfh cell differentiation
Initially, cognate T cells throughout the T zone are activated by antigen recognition and promptly start upregulating EBI2 and making IL2. EBI2 guides cells to the 7α,25-OHC high outer T zone and in this location they interact with activated DCs producing membrane and shed CD25 that binds and quenches IL2. This limits IL2R signaling on the T cell via pSTAT5 and allows induction of Bcl6 by other inputs such as ICOSL. T cells that lack EBI2 or remain in the inner T zone for other reasons are exposed to autocrine IL2 and this induces Blimp1, a repressor of Bcl6 2 , disfavoring the Tfh cell fate.