SUPPLEMENTARY INFORMATION

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1 Supplementary Text Results In vitro activation experiments established that treatment with anti-cd3 plus anti- CD28 was sufficient to promote EBI2 transcript on CD4 T cells (Extended Data Fig. 1d, e). CCR7 is slightly downregulated on activated T cells 4 and this change occurred normally on the EBI2 KO T cells (Extended Data Fig. 1k) indicating that the defective positioning was not due to altered regulation of CCR7 expression. WT and EBI2 KO T cells underwent equivalent amounts of proliferation to anti-cd3 and anti-cd28 in the presence or absence of 7α,25-OHC (Extended Data Fig. 2b, c). These data suggest that the oxysterol is most likely not directly promoting T cell proliferation in vivo, but rather is leading to enhanced proliferation due to effects on cell position. In addition to the findings in mixed transfer recipients, a defect in EBI2 KO Tfh cell induction was seen when control and EBI2-deficient OTII T cells were transferred to separate hosts indicating it was not dependent on competition (Extended Data Fig. 2g). To test whether the defect in Tfh cell induction extended to a different antigen type, the response to Listeria-OVA was measured. The EBI2 KO OTII Tfh cell response in mice immunized with Listeria-OVA was reduced (Extended Data Fig. 3a). Polyclonal Tfh cell development was analyzed by transferring control and EBI2 KO CD4 T cells into OTII recipients and immunizing with unconjugated SRBCs such that only the transferred T cells could participate in the response. Analysis at day 8 revealed a strong Tfh cell-induction defect for cells lacking EBI2 (Extended Data Fig. 3b, c). In accord with defective Tfh cell development, the day 12 GC response was 2-fold reduced (Extended Data Fig. 3d, e). The IgM and IgG plasma cell response was also 1

2 reduced (Extended Data Fig. 3f, g). The defective plasma cell response was associated with a reduced serum anti-srbc response (Extended Data Fig. 3h). To test the impact of T cell EBI2-deficiency on the B cell response to Listeria-OVA the OTII T cells were transferred into Cd28 -/- recipient mice that are defective in generating endogenous Tfh cells 1,2. At day 5 after transfer there was a 4-5-fold reduction in the GC and plasma cell response in recipients of EBI2 KO OTII T cells versus control OTII T cells (Extended Data Figs. 3i, j). Consistent with DCIR2 + DC movement to the outer T zone being important for early Tfh cell induction, when DCs lacked CCR7 the DCIR2 + cells failed to relocate to the outer T zone after SRBC immunization (Extended Data Fig. 4f) and few Tfh cells were induced (Extended Data Fig. 4g). Moreover, in Cd47 -/- mice that have a 4-5 fold deficiency in CD4 + DCIR2 + DCs but normal numbers of CD8 + DCs 18, 37,38, Tfh induction in response to SRBC-OVA was strongly impaired (Extended Data Fig. 4h). Irf4 f/f CD11c-Cre mice that had a 2-3-fold CD4 + DC deficiency (Extended Data Fig. 4i and 39 ) also showed a reduction in Tfh cell induction following SRBC immunization (Extended Data Fig. 4j). In contrast, in Batf3 -/- mice that are deficient in CD8 + DCs 39, Tfh cell induction by SRBC-OVA occurred normally and the response of EBI2- deficient T cells remained defective (Extended Data Fig. 4k). In addition to observing elevated ICOSL on DCs in mice where engagement with ICOS on activated T cells was blocked by anti-icos treatment, upregulation of surface ICOSL on CD4 + DCs was observed in SRBC-immunized Cd28 -/- mice that lack ICOS hi activated T cells (Extended Data Fig. 4m). Cd28 -/- recipients were therefore used for the experiments testing the activity of WT and EBI2 KO OTII T cells in causing DC ICOSL shedding to ensure that activated ICOS hi endogenous OVA- or SRBC-specific T cells did not contribute to ICOSL shedding. Il2 mrna was strongly induced in OTII T cells within 6-12 h of SRBC-OVA immunization and had declined by day 1 (Extended Data Fig.5g), similar to findings after other types of immunization 40,41. Intracellular staining showed IL2-expressing T cells were detectable at 12 hours and in increased frequency at day 1 (Extended 2

3 Data Fig. 5h). Il2 mrna and protein induction occurred to equivalent extents in EBI2 het and KO OTII T cells (Extended Data Fig. 5g, h). Induction of CD25 on the control and EBI2 KO OTII T cells also occurred to similar extents (Extended Data Fig. 5i). The higher pstat5 levels observed in EBI2 KO OTII T cells following immunization did not appear to be due to a direct inhibitory effect of EBI2 on IL2 signaling since incubation of WT T cells with 7α,25-OHC in vitro did not alter their sensitivity to IL2 (Extended Data Fig. 5k). Generation of mice lacking CD25 in DCs was achieved by reconstituting WT mice with an equal mixture of CD25 KO and Zbtb46-DTR BM and then treating the mice with DT at days -3, -1 and 2 of the experiment (Extended Data Fig. 6a). This treatment ablates the CD25 WT DCs (Extended Data Fig. 4b) leaving only CD25 KO DCs. Control mice included WT:Zbtb46-DTR chimeras treated with DT to control for the effects of DT-mediated DC ablation, and saline treated CD25 KO:Zbtb46 DTR chimeras to control for effects of having half CD25 KO non-dcs. In the DT-treated CD25 KO:Zbtb46-DTR chimeras the CD25 KO DCs were present in similar numbers to the WT DCs in DT-treated control chimeras, and they were activated comparably to WT DCs following SRBC immunization as assessed by CD86, CCR7 and MHCII expression and relocation to the outer T zone (Extended Data Fig. 6b-d). The numbers of endogenous FoxP3 + Treg cells in DT treated mixed chimeras were not altered by DC CD25-deficiency at the day 3 time point after SRBC-OVA immunization (Extended Data Fig. 6e). Discussion 7α,25-OHC is produced by lymphoid stromal cells and it is metabolized in both stromal cells and CD8 + DCs 31, and we speculate that it is more abundant in the outer T zone than the central T zone. We suggest that gradients of this oxysterol guide activated T cell positioning in the outer T zone. 3

4 Our studies do not rule out that the central T zone could serve as a site of Tfh cell induction under different immunization conditions, and some work has shown that antigen targeting to CD8 + DCs or monocyte-derived DCs can induce Tfh cell responses 42,43. The mode of scd25-mediated IL2 inhibition needs investigation, though one possibility is that it competes with target cell CD25 for IL2, a mechanism that may limit the negative effect to CD25-expressing IL2-responsive cells. Activated B cells upregulate CD25 and in vitro studies indicate they can respond to IL2 (ref. 44) making it possible that DC CD25 will have direct modulatory influences on IL2- driven B cell responses. Regulatory T cells can deplete IL2 through a mechanism involving IL2R-mediated internalization of the cytokine 45. Further work is needed to determine whether DCs inhibit IL2 function by CD25-mediated internalization and degradation as well as by releasing scd25. Certain CD25 disease susceptibility alleles have been correlated with increased levels of scd25 22,23. It will be important to see if these alleles increase scd25 production by activated DCs and if their presence is associated with increased generation of autoreactive Tfh cell responses. References 37 Van, V. Q., Lesage, S., Bouguermouh, S., Gautier, P., Rubio, M., Levesque, M., Nguyen, S., Galibert, L. & Sarfati, M. Expression of the self-marker CD47 on dendritic cells governs their trafficking to secondary lymphoid organs. EMBO J. 25, (2006). 38 Saito, Y., Iwamura, H., Kaneko, T., Ohnishi, H., Murata, Y., Okazawa, H., Kanazawa, Y., Sato-Hashimoto, M., Kobayashi, H., Oldenborg, P. A., Naito, M., Kaneko, Y., Nojima, Y. & Matozaki, T. Regulation by SIRPalpha of dendritic cell homeostasis in lymphoid tissues. Blood 116, (2010). 39 Murphy, K. M. Transcriptional control of dendritic cell development. Adv. Immunol. 120, (2013). 40 Khoruts, A., Mondino, A., Pape, K. A., Reiner, S. L. & Jenkins, M. K. A natural immunological adjuvant enhances T cell clonal expansion through a CD28- dependent, interleukin (IL)-2-independent mechanism. J. Exp. Med. 187, (1998). 4

5 41 O'Gorman, W. E., Dooms, H., Thorne, S. H., Kuswanto, W. F., Simonds, E. F., Krutzik, P. O., Nolan, G. P. & Abbas, A. K. The initial phase of an immune response functions to activate regulatory T cells. J. Immunol. 183, (2009). 42 Kato, Y., Zaid, A., Davey, G. M., Mueller, S. N., Nutt, S. L., Zotos, D., Tarlinton, D. M., Shortman, K., Lahoud, M. H., Heath, W. R. & Caminschi, I. Targeting Antigen to Clec9A Primes Follicular Th Cell Memory Responses Capable of Robust Recall. J. Immunol. 195, (2015). 43 Chakarov, S. & Fazilleau, N. Monocyte-derived dendritic cells promote T follicular helper cell differentiation. EMBO molecular medicine 6, (2014). 44 Mingari, M. C., Gerosa, F., Carra, G., Accolla, R. S., Moretta, A., Zubler, R. H., Waldmann, T. A. & Moretta, L. Human interleukin-2 promotes proliferation of activated B cells via surface receptors similar to those of activated T cells. Nature 312, (1984). 45 Pandiyan, P., Zheng, L., Ishihara, S., Reed, J. & Lenardo, M. J. CD4+CD25+Foxp3+ regulatory T cells induce cytokine deprivation-mediated apoptosis of effector CD4+ T cells. Nat. Immunol. 8, (2007). 5