A CD22-Shp1 phosphatase axis controls integrin β7 display and B cell function in mucosal immunity

Abstract

The integrin α₄β₇ selectively regulates lymphocyte trafficking and adhesion in the gut and gut-associated lymphoid tissue (GALT). Here, we describe unexpected involvement of the tyrosine phosphatase Shp1 and the B cell lectin CD22 (Siglec-2) in the regulation of α₄β₇ surface expression and gut immunity. Shp1 selectively inhibited β₇ endocytosis, enhancing surface α₄β₇ display and lymphocyte homing to GALT. In B cells, CD22 associated in a sialic acid-dependent manner with integrin β₇ on the cell surface to target intracellular Shp1 to β₇. Shp1 restrained plasma membrane β₇ phosphorylation and inhibited β₇ endocytosis without affecting β₁ integrin. B cells with reduced Shp1 activity, lacking CD22 or expressing CD22 with mutated Shp1-binding or carbohydrate-binding domains displayed parallel reductions in surface α₄β₇ and in homing to GALT. Consistent with the specialized role of α₄β₇ in intestinal immunity, CD22 deficiency selectively inhibited intestinal antibody and pathogen responses.

Extended Data Fig. 1. CD22-deficient T cells display normal cell surface levels of α₄β₇.

Flow cytometry of WT or Cd22 ^–/– live CD3⁺ CD4⁺ T cells isolated from spleens and stained with antibodies against the integrins α_L, β₁, α₄, β₇, or α₄β₇. Shown are pooled data (mean ± SEM) from n=3 independent experiments with 7 animals per group total presented as in Figure 1. Representative histogram overlays gated in CD4⁺ T cells are shown.

Extended Data Fig. 2. B cell expression of St6gal1-dependent CD22-binding carbohydrates controls α₄β₇ expression.

Flow cytometry of WT, Cd22 ^–/–, or St6Gal1 ^–/– naïve B cell (CD19⁺ IgD⁺) isolated from spleen and stained with antibodies against the integrins α_L, β₁, α₄, β₇, or α₄β₇. Data represent the mean ± SEM of one representative experiment with n=3 mice per group presented as in Figure 1. Representative histogram overlays gated in naïve B cells are shown. Groups were compared using One-way ANOVA with Dunnett’s multiple comparisons test. **P ≤ 0.01, and ****P≤0.0001

Extended Data Fig. 3. Removal of α2-6 Sia linkages on B cells with Arthrobacter Uereafaciens sialidase.

Purified wild type B cells purified from spleen were incubated for one hour at 37 °C with Arthrobacter ureafaciens sialidase or with vehicle control (PBS, Vehicle Ctr). a,b, Flow cytometry of Arthrobacter ureafaciens-treated or vehicle control-treated WT naïve B cells (CD19+ IgD+), isolated from spleen and stained for DAPI and SNA-FITC. a, The percentage of viable cells is shown. b, The MFI of the SNA staining was expressed as a percentage of the mean MFI of the vehicle ctr-treated WT B cell group. Representative histogram overlay gated in live naïve B cells is shown.

Extended Data Fig. 4. CD22-deficient T cells display wild-type levels of tyrosine phosphorylation in cell surface β₇.

Left panel: detection of β₇ and phosphotyrosine (pTyr) levels in the cell surface and intracellular β₇ fractions of wild-type (WT) and CD22-deficient (Cd22 ^–/–) T cells after the double IP as shown in Fig. 5a. Right panel: quantification of pTyr levels normalized to β₇ levels (pTyr/β₇ ratio). Within each experiment, the pTyr/β₇ of the cell surface β₇ of the WT group was set to 100, and data expressed as a percentage of this total. Each dot represents one independent experiment with n=4 animals pooled for WT and Cd22 ^–/– (i.e. n=8 animals total for the two experimental replicates).

Extended Data Fig. 5. Normal T cell numbers in CD22-deficient Peyer’s patches and lymph nodes.

Numbers of CD4⁺ T cells (CD3⁺ CD4⁺) in MLN, PLN, and PP of WT and Cd22 ^–/– shown as a percentage of the mean of the WT group. Shown are pooled data (mean ± SEM) of n=2 experiments with n=7-8 mice per group total.

Extended Data Fig. 6. Functional assays reveal normal homing of CD22 mutant B cells to the spleen and bone marrow.

Localization of WT, Cd22 ^–/–, CD22^Y2,5,6F, and CD22^R130E B cells in blood, spleen and bone marrow (BM) after homing assays as illustrated in Fig. 6c. Data are shown as a percentage of the mean localization ratio of the WT group. Shown are pooled data (mean ± SEM) of n=3-5 experiments with 11-16 mice per group total.

Extended Data Fig. 7. Localization of WT, Cd22 ^–/–, and Ptpn6 ^+/meV T cells in PLN and PP after short-term homing assays.

WT, Cd22 ^–/–, or Ptpn6 ^+/meV splenocytes labeled with CFSE, or CellTracker Violet (CTV) or both were injected i.v. into a recipient WT mouse. PLN, MLN or PP cells isolated from the recipient were stained with anti-CD3 and anti-CD4 for quantification of short-term (90 min) homing of CD4 T cells. For each donor and each organ, the number of isolated CD4 T cells (Output) was normalized to the number of injected CD4 T cells (Input) to yield a T cell localization ratio. Shown is the mean ± SEM from three independent experiments with n=11 mice per group total. Representative dot plots gated in live CD3⁺ CD4⁺ T cells are shown, including the number of cells within each gate. Groups were compared using One-way ANOVA with Dunnett’s multiple comparison test. **P ≤ 0.01, and ns: not significant.

Extended Data Fig. 8. Definition of flyer, brief roller and roller cells visualized by in situ video microscopy of Peyer’s patches.

a, The mean velocity of wild-type (WT) and CD22-deficient (Cd22 ^–/–) B cells free flowing through the vessels without any interactions (namely flyer) was calculated and shown for each individual cell. Shown are pooled results (~ 50 cells per group) analyzed from 3–4 representative HEVs and 3 independent experiments. b,c, The instant velocity (b) and displacement (c) of a representative free flowing cell (Flyer), of one that interacts very briefly (<1s) with the HEVs (namely Brief roller), or one that interacts and rolls on the HEVs for >1sec (namely Roller) is shown together with frame-per-frame tracking of the cells (identified with *). Scale bars: 10 μm. d, In three independent in situ experiments with 1:1 ratio of WT B cells donor versus Cd22 ^–/– B cells donor, the total number of events (i.e. flyer, brief roller, or roller) was counted for each donor in 3–4 representative HEVs for the total duration of the movie (~ 250–300 total cells analyzed per group). The percentage of WT and Cd22 ^–/– B cells experiment per experiment is shown. ns: not significant.

Extended Data Fig. 9. WT B cells with reduced α₄β₇availability mimic the behavior of defective CD22-deficient B cell homing to PP.

a, WT B cells were pre-incubated with inhibitory anti-α₄β₇ Ab DATK32 (50 μg/mL) and washed extensively. DATK32-pretreated B cells were either counter-stained with Phyco-erythrin(PE)-conjugated DATK32 for flow cytometry analyses (b), or use in functional assays (c-f). b, Flow cytometry of WT + DATK32 vs. WT + Vehicle B cells stained for α_L, β₁, α₄, β₇, or α₄β₇ presented as in Figure 1. Shown are pooled data (mean ± SEM) from n=2 independent experiments with 4 animals per group total. (c) Localization of WT, Cd22 ^–/–, and WT + DATK32 B cells in PLN and PP after homing assays analyzed and presented as in Figure 6. Shown are pooled data (mean ± SEM) of n=3 experiments with 11 mice per group total. Representative dot plots gated in live naïve B cells are also shown including the number of cells within each gate. d-f, In situ video microscopy analyses of WT B cells + DATK32 vs. Cd22 ^–/– B cells interactions with PP-HEVs analyzed and presented as in Figure 6. Data represent the mean ± SEM of three independent experiments (d,f) and representative cells from all 3 experiments (e). Groups were compared using One-way ANOVA with Dunnett’s multiple comparisons test (b,c), unpaired two-tailed Student’s t-test (d,e), and paired two-tailed Student’s t-test (f). *P ≤ 0.05, **P ≤ 0.01, and ****P≤0.0001. ns: not significant.

Extended Data Fig. 10. Defective homing of CD22-deficient B cells in St6gal1-deficient recipient mice.

Localization of WT and Cd22 ^–/– B cells in the PPs of wild-type (WT) or ligand-deficient (St6Gal1 ^–/–) mice after short-term (1.5 hr) homing assays designed as in Figure 6a. For each donor, the number of B cells isolated (Output) was normalized to the number of injected B cells (Input) and shown as a percentage of the WT → WT group mean. Shown are pooled data (mean ± SEM) from three experiments with n=7 mice per group total. Groups were compared using two-tailed Student’s t-test. ***P ≤ 0.001.

Figure 1. Selective reduction of integrin α₄β₇ on motheathen viable B and T cells.

a, Flow cytometry of WT or motheathen viable (Ptpn6 ^meV/meV) splenic naïve B cells (left panel), CD4⁺ T cells (mid-panel), and CD8⁺ T cells (right panel) stained for α_L, β₂, β₁, α₄, β₇, or α₄β₇. Shown are pooled data (mean ± SEM) from n=4 experiments with 1-2 animals per group per experiment (i.e. 4-6 animals per group total). For each animal within one experiment, the MFI of the integrin staining was expressed as a percentage of the mean MFI of the WT group. Representative histogram overlays gated in naïve B cells are shown (Iso Ctr: Isotype control). b, Flow cytometry of WT or Ptpn6 ^+/meV splenic naïve B cells (left panel), CD4⁺ T cells (mid-panel), and CD8⁺ T cells (right panel) stained for α_L, β₂, β₁, α₄, β₇, or α₄β₇. Shown are pooled data (mean ± SEM) from n=4 experiments with 4-9 animals per group total. Groups were compared using two-tailed Student’s t-test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P≤0.0001.

Figure 2. CD22 mediates the Shp1-dependent α₄β₇ augmentation in B cells.

a, Scheme of wild-type CD22, CD22^Y2,5,6F, and CD22^R130E mutants. b, Flow cytometry of WT and CD22 mutant splenic B cells stained for α_L, β₁, α₄, β₇, or α₄β₇. Shown are pooled data (mean ± SEM) from n=3 experiments with 7 animals per group total analyzed and presented as in Fig. 1. Representative histogram overlays gated in naïve B cells are shown (Iso Ctr: Isotype control). c-d, Immunofluorescent staining of cell surface (c) and intracellular (d) β₇ and CD22 in B cells with nucleus (DAPI, blue); cytoplasm (MitoTracker™ Deep Red, Red); β₇ (green) and CD22 (magenta). Scale bars: 2 μM. e, For each cell as shown in (c,d), we quantified the intensity of the β₇ fluorescence. Shown are pooled data (mean ± SEM) from three independent experiments with ~100 cells total analyzed per condition. The mean of the β₇ fluorescence intensity for the WT Cell Surface group was set to 100, and data shown as a percentage of this total. f, Flow cytometry of WT or Cd22 ^–/– B cells isolated from blood, bone marrow (BM), peripheral lymph nodes (PLN), and Peyer’s patches (PP) and stained for α₄β₇ or isotype-matched control. Shown are pooled data (mean ± SEM) from n=2 independent experiments with five animal per group total. g, mRNA expression of α_L, β₁, α₄, and β₇ integrins in B cells from WT and Cd22 ^–/– mice. Shown are pooled data (mean ± SEM) from two independent experiments with three to four animals per group. h, Immunofluorescent staining of intracellular Shp1 and cell surface β₇ and CD22 in B cells with nucleus (DAPI, blue); Shp1 (red); β₇ (green); and CD22 (magenta). Arrow heads: Shp1– β₇ colocalization. Arrows: CD22–Shp1–β₇ colocalization. i,j, For each cell as shown in (h), we calculated Shp1–β₇ (i) and CD22 – Shp1/ β₇ (j) proximity indexes (see Methods). Shown are pooled data (mean ± SEM) from two independent experiments with 130 cells total (i) and 40 cells total (j) analyzed per condition. Groups were compared using One-way ANOVA with Dunnett’s multiple comparison test (b,e), and two-tailed Student’s t-test (f,g,i). N/A: not applicable. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P≤0.0001.

Figure 3. Direct physical association of CD22 and β₇.

a, Association between CD22 and integrin β₇ was assessed by proximity ligation assay (PLA) using purified B cells from wild-type either untreated (WT) or sialidase-treated (WT+ Sialidase), or Cd22 ^–/– animals. b, Association between CD22 and integrin β₇ was assessed as a negative integrin control. c, Association between α₄ and β₁ were analyzed as a positive control for the β₁ integrin. Shown are pooled data (mean ± SEM) from two independent experiments with ~400 cells analyzed per condition. Scale bars: 2 μM. Groups were compared using One-way ANOVA with Dunnett’s multiple comparison test (a) and two-tailed Student’s t-test (c). ****P≤0.0001. ns: not significant. n.d.: non detectable.

Figure 4. CD22 limits integrin β₇ endocytosis in B cells via Shp1 and ligand-recognition.

a, Splenocyte staining with anti-CD19 and anti-IgD (to identify B cells) and pHrodo-Red-conjugated tranferrin (Tf), HMβ1-1 (anti-β₇), or FIB504 (anti-β₇) antibodies at either 4°C (no endocytosis) or 37°C (endocytosis) was used to calculate endocytosis levels (i.e. MFI pHrodo-Red staining at 37°C – MFI pHrodo-Red staining at 4°C). Staining at 4°C with Phycoerythrin (PE)-conjugated RI7217 (anti-Transferrin Receptor 1, TfR1), HMβ1-1, FIB504 or the matching PE-conjugated isotype control antibodies was used to calculate extra-cellular TfR1, β₁, and β₇ levels (i.e. MFI PE staining at 4°C – MFI Isotype Control staining at 4°C). For each molecule (illustrated with β₇ in panel (a)) and each experiment, the Relative Endocytosis Ratio (RER) was calculated by normalizing endocytosis levels to extra-cellular levels. b, RER of Tf, β₁, and β₇ in WT, St6Gal1 ^–/–, Ptpn6 ^+/meV, and Cd22 ^–/– B cells with (100μM) or without primaquine (PQ) treatment. The mean RER of the WT B cells group without PQ (No Tx) was set to 1, and all data were normalized to that mean value. Shown are pooled data (mean ± SEM) from n=3 independent experiments with 6 animals per group total. Groups were compared using Two-way ANOVA with Sidak’s multiple comparison test. *P ≤ 0.05, **P ≤ 0.01, and ****P≤0.0001.

Figure 5. CD22 restrains tyrosine phosphorylation of cell surface β₇ integrin in B cells.

a, Scheme of the double immunoprecipitation experiment. Cell surface proteins of purified naïve B cells were biotinylated with Sulfo-NHS-biotin at 4˚C for 1hr. Biotinylation was confirmed by flow cytometry using a streptavidin (SA)-conjugated probe. Biotinylated B cells lysate was used for immunoprecipitation (IP) of β₇ integrin (including biotinylated cell surface β₇ and biotin-free intracellular β₇). Following elution for β₇, a second IP with SA beads was used to recover cell surface β₇ (eluate of the SA-IP) from intracellular β₇ (flow-through of the SA-IP) prior to immunoblot studies. b, Left panel: detection of β₇ and phosphotyrosine (pTyr) levels in the cell surface and intracellular β₇ fractions (130 kDa) of wild-type (WT), CD22-deficient (Cd22 ^–/–), and Ptpn6 ^+/meV B cells after the double IP as shown in panel (a). Right panel: quantification of pTyr levels normalized to β₇ levels (pTyr/β₇ ratio). Within each experiment, the pTyr/β₇ of the cell surface β₇ of the WT group was set to 100, and data expressed as a percentage of this total. Shown are pooled data (mean ± SEM) from n=2-3 independent experiments. Each dot represents one independent experiment with n=8 pooled animals for WT and Cd22 ^–/– (i.e. n=24 animals total for n=3 independent experimental replicates) and n=4 pooled animals for Ptpn6 ^+/meV (i.e. n=8 animals total for n=2 experimental replicates). Data were analyzed using two-tailed Student’s t-test. **P ≤ 0.01. ns: not significant.

Figure 6. Functional assays reveal defective PP homing and altered endothelial interactions of Ptpn6 ^+/meV B cells and Cd22 ^–/– B cells

a, Numbers of PP in WT and Cd22 ^–/– B cells. Representative PP images from WT and Cd22 ^–/– indicated with arrowhead. Scale bars: 2 mm b, Numbers of germinal center/activated B cells (GC/A, CD19⁺ IgD^-) and naïve B cells (CD19⁺ IgD⁺) in MLN, PLN, and PP of WT and Cd22 ^–/– shown as a percentage of the mean of the WT group. Shown are pooled data (mean ± SEM) of n=2 experiments with 11-12 mice per group total. c, Scheme of short-term homing assay. d,e, Localization of WT, Cd22 ^–/– and Ptpn6 ^+/meV B cells (d) or WT, Cd22 ^–/–, CD22^Y2,5,6F, and CD22^R130E B cells (e) in PLN, MLN and PP after homing assays. Data are shown as a percentage of the mean localization ratio of the WT group. Shown are pooled data (mean ± SEM) of n=3-5 experiments with 11-16 mice per group total. Representative dot plots gated on naive B cells are shown. f, Scheme of in situ video microscopy experiment. Representative movie pictures are shown with WT B cells (green) and Ptpn6 ^+/meV (red). Scale bar: 100 mm. g-l, Data in panels (g,h,j,k) represent the mean ± SEM of three independent experiments. (g,j) Number of WT vs. Ptpn6 ^+/meV (g) or WT vs. Cd22 ^–/– (j) arresters on PP HEVs shown second per second from the first cell entering the HEVs. The total amount of WT B cells arresters at the end each experiment (i.e. WT B cells max; ~ 40–80 cells in average) was set to 100, and the data expressed as a percentage of this total. h,k, The behavior (i.e. flyer, brief roller, or roller as defined in Extended Data Fig. 7) of each cell entering the HEVs was analyzed in 3-4 representative PP-HEVs per experiment. Results are shown as a percentage of the total numbers of cells entering HEVs (~ 250–300 cells analyzed per group) (i,l) Average rolling velocity (mean ± SEM) of representative rollers from n=3 experiments. Groups were compared using two-tailed Student’s t-test (a,b,h,i,k,l), paired two-tailed Student’s t-test (g,j), and One-way ANOVA with Dunnett’s multiple comparison test (d,e). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P≤0.0001.

Figure 7. Defects in intestinal responses to oral antigen in CD22-deficient mice.

Cohorts of WT or Cd22 ^−/− mice were immunized with Cholera Toxin B (CTB) via the Oral, intra-nasal (Nasal), or intra-muscular (Muscular) routes for two weeks. a-d, The serum levels of CTB-specific IgA (a), total IgA (b), CTB-specific IgG (c), total IgG (d) were measured by ELISA and expressed as net OD450 or percentage of the mean OD450 measured in the lowest dilution factor of the WT group. e,f, SI segments were cultured ex vivo for three days to titer the quantity of secreted CTB-specific IgA, total IgA, and CTB-specific IgA normalized to total IgA (e) and CTB-specific IgG, total IgG, and CTB-specific IgG normalized to total IgG (f) produced by ELISA and expressed as a percentage of the WT group mean. Shown are pooled data (mean ± SEM) from n=2 experiments with 5-6 mice per group total. Groups were compared using Two-way ANOVA with Sidak’s multiple comparison test (a-d), and two-tailed Student’s t-test (e,f). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P≤0.0001. ns: not significant.

Figure 8. Delayed protective immune response to RV infection in CD22-deficient animals.

a-c, Five days old WT or Cd22 ^−/− pups were orally gavaged with the RV strain EW. The production of RV-specific IgA, total IgA, and RV IgA normalized to total IgA (a) or RV-specific IgG, total IgG, and RV IgG normalized to total IgG (b) in fecal samples was measured by ELISA up to twelve days post-infection (D12 p.i.). The mean of the OD450 for the WT group at D12 p.i. was set to 100, and data expressed as a percentage of this total. c, Fecal RV Ag shedding as measured by ELISA and expressed as net OD450, normalized to the sample weight. Shown are pooled data (mean ± SEM) from n=4 independent experiments with 10-15 animals per group total. (d-h) In separate experiments, five days old WT and Cd22 ^−/− pups were orally inoculated with RV EW and sacrificed at day 12 p.i. d,e, The serum levels of RV-specific IgA and total IgA (d) and RV-specific IgG and total IgG (e) were measured by ELISA. The mean of the maximal OD450 for the WT group was set to 100, and data expressed as a percentage of this total. (f) The numbers of RV-specific IgA antibody secreting B cells (ASC) were measured in the spleen, MLN, and SI lamina propria (SI LP) by ELISPOT. Shown are numbers of RV IgA ASC per 10⁶ total cells expressed as a percentage of the WT group mean. g,h, SI fragment cultures. Data are displayed and presented as in Fig. 7e,f to show RV IgA, total IgA, ratios of RV IgA/total IgA (g) and RV IgG, total IgG, ratios of RV IgG/total IgG (h) measured by ELISA. Shown are pooled data (mean ± SEM) from n=2 experiments with 10-11 mice per group total. Groups were compared using Two-way ANOVA with Sidak’s multiple comparison test (a,b,d,e), and unpaired two-tailed Student’s t-test (c,f,g,h). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P≤0.0001. ns: not significant.