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Supplementary Figure 1

Upper panel: Western blot analysis of the serums(S) and intestinal fluids (IF) from three distinct mice (1-3) bearing an hybridoma secreting anti-OVA hzIgA using HRP-conjugated goat-anti-human  chain (non reducing conditions). No signal was observed with intestinal fluids and serums from control mice without backpack tumor (not shown). Lower left panel: Western blot analysis with rabbit anti-mouse J chain or control rabbit immunoglobulins of two batches of polymeric anti-OVA hzIgA purified from the hybridoma supernatant (reducing conditions). Lower right panel: Western blot analysis with rabbit anti-mouse SC of anti-OVA hzIgA purified from the pooled intestinal fluids (IF) or serums (S) from 5 OVA-BP mice. For comparison,reactivity of one non-purified intestinal fluid (IF1) containing both anti-OVA hzIgA and endogenous murine SIgA is shown.

Supplementary Figure 2: Unmodified OVA total fluxes andtransepithelial electrical resistance in tyrphostin A8-treated mice carrying 1KI-derived IgA-secreting backpacktumors

Left panel: Total fluxes of 3H-OVA (expressed as pmol/90min.cm2) transported across duodenal fragments mounted in Ussing chambers in OVA-immunized 1KI mice treated or not with TyrA8, or in Balb/c mice carrying or not OVA-BP or -lactoglobulin-BP tumor and treated or not with TyrA8. Right panel: Transepithelial electrical resistance of intestinal segmentsmountedin Ussing chambers in the same groups of mice. Results are presented as means in Ohms.cm² ±SD (n= 8 to 19 mice per group). No significant difference was found among the different groups of mice (non-parametric Mann-Whitney U test).

Supplementary Figure 3: Experimental protocol for stimulation of T cell responses by dietary OVA in mice with IgA-producing backpack tumors

Supplementary Figure 4: Enhanced activation of specific MLN CD4+ T cells by IgA-dependent intestinal transport of OVA

Multicolor flow cytometry analysis of CD44 and CD62L membrane expression (left panel) and Foxp3 intracellular expression in OVA-specific (KJ1-26+) MLN CD4+ T cells (middle panel), and ELISA determination of IL-10 secretion after a 72-hour stimulation by OVA in MLN cells (right panel) from DO11.10 transgenic mice daily gavaged with bicarbonate buffer (Control) or with 25 mg OVA for 4 days (OVA gav), bearing or not an OVA- or β-lactoglobulin backpack tumor (BP), and treated or not with TyrA8. Results are expressed as medians and ranges.

* significantly different from control group, p<0.01 in left panel, p<0.03 in middle panel.

Supplementary movies: Simultaneous mucosal-to-serosal transport of transferrin and of human or humanized IgAin the gut of tyrphostin A8-treated mice

Analysis bymultiphotonic videomicroscopy of transcellular transport of transferrin (Tf, green) and human SIgA (red) or polymeric humanized IgA (red) in duodenal segments of Balb/c mice either untreated (control) or 48 hours after TyrA8 treatment. No transport is observed in the control untreated mouse (movie 1), while both transferrinand human SIgA (movie 2) or humanized IgA (movie 3) are transported from the apical to basal side of enterocytes in TyrA8-treated mice. Extensive co-localization of transferrin and IgA during transport is underlinedby the appearance of yellow/orange areas.

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