Fig 2: Characterization of anti-4-1BB trimerbodies. a Sensorgrams (black curves) and fitting curves for 1D8 antibodies (2 and 4 nM), obtained by biolayer interferometry with surface-immobilized m4-1BB. b The binding to 4-1BB on the cell surface of stimulated mouse CD8a+ T cells measured by FACS. c–e Costimulatory activity of anti-4-1BB antibodies. Mouse CD8a+ T cells were stimulated with immobilized anti-CD3 mAb in the presence of m4-1BBL, 1D8N5, 1D8N18, or 1D8 IgG, and proliferation (c) and secretion of IFN-? (d) were measured after 48 h and cell viability (e) after 72 h. Data are reported as fold change relative to the values obtained from anti-CD3 mAb-stimulated CD8a+ T cells. Rat IgG2a and MFE-23N18 were used as controls. Data are mean ± SD (n = 3), *P = 0.05, **P = 0.01, Student's t test. f RICS analyses performed in living HEK293m4-1BB-S cells at regions containing clusters formed upon 1D8 IgG or 1D8N18 addition and at regions where clusters where not present (insert and zoomed-in regions ii, and i, respectively). Representative maximum intensity projection maps showing the RICS analyzed regions of interest. Values in the zoomed-in regions show the diffusion coefficient of bound antibody. The color heat map indicates in blueish tones the lower intensity and in redder tones the higher intensity. g Statistical analysis of the quantified diffusion coefficient obtained from 5 to 7 independent live cell experiments and 3–5 different regions of interest per cell (N-CR non-clustered region, CR clustered region). Data are presented as median (center line), upper and lower quartiles (boxes), and minimum-maximum (whiskers). h Arrangement of 1D8N18 trimerbody in solution, as determined by SAXS. Rigid-body overlaying of the ab initio-determined SAXS envelope for 1D8N18. The generated model (where each chain is colored in blue, magenta, or cyan) fits into the envelope (colored in pale gray)

Fig 3: Colitis-driven IL-1R1 signaling on CD4+ T cells alters the antigen-specific response to S. epidermidis(A) On day 4 of re-challenge phase, serum and skin were collected from Ctl or DSS WT mice and IL-1ß was measured via multiplexed ELISA.(B) CD4-Cre- and CD4-Cre+ mice were colonized with S. epi-2W on postnatal days 7, 10, and 13 before DSS treatment and re-challenge with S. epi-2W 3 weeks later.(C and D) Percentage of Foxp3+2W+CD44+CD4+ and Foxp3+CD4+ T cells in skin-dLNs.(E and F) Absolute number of Foxp3-2W+CD44+CD4+ and Foxp3-CD4+ T cells in skin-dLNs.(G) Percentage of IL-17A+ cells gated on live CD45+CD3+GL3-TCRß+CD4+Foxp3 in skin.(H) Percentage of Ly6G+CD11b+ cells gated on live CD45+ in skin.(I and J) IL-1R1 fl/fl Foxp3-Cre- and Foxp3-Cre+ (I) or IL-6R fl/fl CD4-Cre- and CD4-Cre+ (J) mice were colonized with S. epi-2W on postnatal days 7, 10, and 13 before DSS treatment and re-challenge with S. epi-2W 3 weeks later. Percentage of Foxp3+2W+CD44+CD4+ in skin-dLNs on day 10.Each point represents an individual mouse. All data above are representative of 2–4 independent experiments with at least three mice per group. p values correlate with symbols as follows: ns, not significant, p > 0.05, *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001.

Fig 4: Colitis alters the cutaneous antigen-specific CD4+ response to S. epidermidis(A) WT mice were colonized with S. epi-2W on postnatal days 7, 10, and 13 before induction of DSS colitis and re-challenge with S. epi-2W plus superficial skin abrasion 3 weeks later.(B and C) Weight loss and reduced colon length in DSS colitis.(D) Representative flow cytometry plots of 2W+CD44+ cells gated on live CD45+DUMP-TCRß+CD4+ from tetramer-enriched fraction in skin-dLNs from control or DSS mice on day 10 of the re-challenge phase.(E and F) Percentage and absolute number of 2W+CD44+CD4+ T cells in skin-dLNs.(G) Representative flow cytometry plots of Foxp3+ cells gated on 2W+CD44+ from the same tetramer-enriched fractions in (D).(H and I) Percentage of Foxp3+2W+CD44+CD4+ T cells in skin-dLNs and skin.(J and K) Percentage of Foxp3+CD4+ T cells in skin-dLNs and skin.(L) Percentage of IL-17A+ cells gated on live CD45+CD3+GL3-TCRß+CD4+Foxp3 in skin.(M) Percentage of Ly6G+CD11b+ cells gated on live CD45+ in skin.(N) Myeloperoxidase (MPO) enzyme activity in skin.(O) Relative abundance of major phyla and select species from skin swabs of DSS versus control mice on day 10 of re-challenge.(P and Q) Villin-Cre+ and Villin-Cre mice were colonized with S. epi-2W on postnatal days 7, 10, and 13 before i.p. tamoxifen injection and re-challenge with S. epi-2W 3 weeks later. Percentage of Foxp3+2W+CD44+CD4+ T cells and Foxp3+CD4+ T cells in skin-dLNs.Each point represents an individual mouse. All data above are representative of 2–4 independent experiments with at least 3 mice per group. p values correlate with symbols as follows: ns, not significant, p > 0.05, *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001.

Fig 5: B cells promote primary CD8 T cell responses to subunit vaccines(A) Total splenic B220+CD19+ B cells from WT C57BL/6 mice 0–7 days after i.v. administration of combined-adjuvant vaccine (anti-CD40, poly(I:C), and OVA).(B–D) 200 CD45.1+ OT1 T cells were transferred into WT or µMT-/- mice 1 day before combined-adjuvant vaccination. Spleens were harvested 7 days later. (B) Representative tetramer and CD45.1 staining. (C) Total OT1 cells (left) and tetramer+ endogenous CD8 T cells (right). (D) Tetramer as a percentage of endogenous CD8 T cells.(E) Representative immunofluorescent staining of 2 naive mice per group with DAPI (blue), CD3 (yellow), B220 (magenta), and CD169 (green). Scale bar represents 50 µm for all images.(F) Total splenic B220+CD19+ B cells from WT or MD4 mice 0–7 days after combined-adjuvant vaccine.(G and H) Total splenic tetramer+ CD8 T cells (top) or tetramer+ cells as a percentage of CD8 T cells (bottom) were measured 7 days after subunit vaccination or infection. (G) Ag-specific CD8 T cell responses after i.v. (left) or s.c. (right) administration of combined-adjuvant + OVA vaccine. (H) Responses after i.v. subunit immunization with the single adjuvants poly(I:C) (left) or Pam3Cys (right).(I) Responses after i.p. administration of OVA emulsified in alum.(J) Responses after infection with LM-OVA.Data shown are means ± SEM; n = 3 mice per group, representative of = 2 experiments, except for (F) and (H), which are from one experiment each. Significance was defined by two-tailed, unpaired Student’s t tests, where *p < 0.05, **p < 0.01, and ***p < 0.001.See also Figure S2.