Fig. 7: Antigen acquisition and archiving after chikungunya virus (CHIKV) inoculation and in human lymph nodes (LN)s.

A Mice were infected with 10³ PFU of WT chikungunya virus (CHIKV) or mock inoculated (PBS) 1 day prior to administration of 10 μg OVA-488 or OVA-488/polyI:C/αCD40 (10 μg/5 μg/5 μg). 17 days later mice were euthanized and dLNs were extracted and processed for flow cytometry. Shown are representative flow plots gated on LECs (CD45-CD31 + PDPN + ). Intracellular adhesion molecule 1 (ICAM1)xovalbumin alexa fluor 488 (OVA-488) is shown. B Quantification of mouse groups in A showing percent of OVA-488 positive lymphatic endothelial cells (LEC)s and number of OVA-488 positive LECs. Each dot represents 2 dLNs from 2 pooled mice (n = 6/group) except OVA-488/polyI:C/αCD40 which is 2 dLNs from 1 mouse injected in both footpads (n = 5) and naïve n = 4. Experiment was repeated on two independent occasions. OVA only vs WT CHIKV OVA *p = 0.0441, OVA only vs naïve *p = 0.0307, ****p < 0.0001, ns is p > 0.05 by one-way ANOVA with Tukey’s two-sided multiple comparison test. Data are presented as mean values +/- SEM. C Violet proliferation dye (VPD) labeled OT1 CD8 + T cells were transferred into mice described in A at day 14. At day 17 transferred OT1 T cells in dLNs were gated as (CD8 + CD45.1 + ). Proliferation (percent divided OT1) was calculated based on VPD dilution. Each dot represents 2 dLNs from 2 pooled mice (n = 6/group) except OVA-488/polyI:C/αCD40 which is 2 dLNs from 1 mouse injected in both footpads (n = 5) and naïve n = 4. All data points are shown. Experiment is combined data from two or more experiments. Adjusted p-values are as follows: OVA/polyI:C/αCD40 vs OVA only ***p = 0.0002; OVA only vs WT CHIKV OVA *p = 0.0111; OVA only vs naïve *p = 0.0403, ****p < 0.0001, by one-way ANOVA with Tukey’s two-sided multiple comparison test. D The fraction of ceiling LECs predicted to be antigen competent is shown for each metastasis-free lymph node (MFLN-teal) and follicular lymphoma (FL-red) LN sample with at least 20 identified cLECs. E The median fraction of predicted antigen-competent cells is shown for samples from (D). Points show values for each sample. P-values were calculated using a two-sided Wilcoxon rank sum test with Benjamini-Hochberg correction. F The median fraction of predicted antigen competent cells is shown for a second independent model as in (E). Points show values for each sample. P-values were calculated using a two-sided Wilcoxon rank sum test with Benjamini-Hochberg correction. G Human pancreatic LN pieces were treated overnight with 5 μg/mL of OVA-488 and 2.5 μg/mL Poly I:C, 5 μg/mL OVA-488 alone, or PBS (no treatment). LNs were digested, stained, and gated on LECs (CD45-PDPN + CD31 + ). Shown are representative flow cytometry plots as in (A). H Quantification of percent OVA-488 + LN LECs 1 or 3 days after overnight treatment described in (G). Each dot represents 1 LN piece. All data points are shown. no treatment (NT) d1: n = 12, ova488 d1: n = 9, OVA-488 polyI:C d1: n = 11, NT d3: n = 13, OVA-488 d3: n = 11, OVA-488 polyI:C d3: n = 14. Experiment was repeated on 2 independent occasions for OVA-488 d1 and 3 independent occasions for all other samples except NT and OVA-488 polyI:C d3 which were performed on 4 occasions. Each occasion represents 1 deceased donor. ns is p > 0.05, OVA vs NT d3 adjusted *p = 0.0116, OVA polyI:C vs NT d3 adjusted ***p = 0.0003, ****p < 0.0001 by one-way ANOVA with Tukey’s two-sided multiple comparison test. Data are presented as mean values +/- SEM. I Quantification of percent OVA-488 + LN BECs (CD45-PDPN-CD31 + ) 1 or 3 days after overnight treatment described in (G). Each dot represents 1 LN piece. All data points are shown. Sample numbers and replicates are exactly as in (H). ns is p > 0.05, OVA polyI:C vs NT d3 adjusted **p = 0.0048 and OVA polyI:C vs NT d3 adjusted **p = 0.0014 by one-way ANOVA with Tukey’s two-sided multiple comparison test. Data are presented as mean values +/- SEM. J Quantification of percent OVA-488 + LN fibroblastic reticular cells (FRC)s (CD45-PDPN + CD31-) 1 or 3 days after overnight treatment as described in (I). Each dot represents 1 LN piece. All data points are shown. Sample numbers and replicates are exactly as in (H). Ns= p > 0.5 where NT vs OVA-488 d3 p = 0.0705 by one-way ANOVA with Tukey’s two-sided multiple comparison test. Error bars indicate standard error of the mean. K Quantification of percent OVA-488 + LN classical monocytes (CD45 + CD19-HLA-DR + CD14 + ) 1 or 3 days after overnight treatment as described in (I). Each dot represents 1 LN piece. All data points are shown. Sample numbers and replicates are exactly as in (H). ns is p > 0.05, NT vs OVA-488 d1 adjusted *p = 0.0361, NT vs OVA polyI:C d3 adjusted **p = 0.0047, adjusted ****p < 0.0001 by one-way ANOVA with Tukey’s two-sided multiple comparison test. Data are presented as mean values +/− SEM.