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

Nature Immunology volume 22, pages 381–390 (2021)Cite this article

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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.

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Fig. 1: Selective reduction of integrin α4β7 on motheathen viable B and T cells.
Fig. 2: CD22 mediates Shp1-dependent α4β7 augmentation in B cells.
Fig. 3: Direct physical association of CD22 and β7.
Fig. 4: CD22 limits integrin β7 endocytosis in B cells via Shp1 and ligand recognition.
Fig. 5: CD22 restrains tyrosine phosphorylation of cell-surface β7 integrin in B cells.
Fig. 6: Functional assays reveal defective PP homing and altered endothelial interactions of Ptpn6+/meV and Cd22–/– B cells.
Fig. 7: Defects in intestinal responses to oral antigen in CD22-deficient mice.
Fig. 8: Delayed protective immune response to RV infection in CD22-deficient animals.

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Data availability

The data that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank J. Paulson from The Scripps Research Institute for the Cd22–/– and St6gal1–/– mice, the members of the Butcher laboratory for discussions, J. Pan for help with designing the primers, H. Hadeiba and A. Scholz for helpful discussions, C. Garzon-Coral for designing and making the re-usable dishes used for positioning animals in the intravital imaging studies, J. L. Jang for production of the home-made antibodies used in these studies, M. Bscheider for helping to implement the imaging software programs in the Butcher laboratory that were used to record and analyze the video microscopy and M. Lajevic for sharing protocols and expertise. This work was supported by NIH grants R37AI047822 and R01AI130471 and award I01BX002919 (from the Department of Veterans Affairs) to E.C.B., Swiss National Sciences Foundation grants P2GEP3_162055 and P300PA_174365 to R.B., DFG-funded TRR130 (project 04) to L.N., JSPS Grants-in-Aid for Scientific Research 18H02610 and 19H04804 to T.T., grants 1R01 AI125249 (NIH/NIAID) and 1IO 1BX000158-01A1 (Veterans Affairs) to H.B.G., and the Ramón Areces Foundation (Madrid, Spain) Postdoctoral Fellowship and Research Fellow Award (Crohn’s and Colitis Foundation) to B.O. M.S.M. acknowledges funding provided through NIAID (AI118842).

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Author notes

  1. These authors contributed equally: Martin Brennan, Carolin Brandl.

Authors and Affiliations

  1. Palo Alto Veterans Institute for Research, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA, USA

    Romain Ballet, Martin Brennan, Ningguo Feng, Jeremy Berri, Julian Cheng, Borja Ocón, Yuhan Bi, Harry B. Greenberg & Eugene C. Butcher

  2. Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA

    Romain Ballet, Martin Brennan, Jeremy Berri, Julian Cheng, Borja Ocón, Yuhan Bi & Eugene C. Butcher

  3. Division of Genetics, Department of Biology, University of Erlangen, Erlangen, Germany

    Carolin Brandl & Lars Nitschke

  4. Department of Medicine, Division of Gastroenterology and Hepatology, Stanford University School of Medicine, Stanford, CA, USA

    Ningguo Feng & Harry B. Greenberg

  5. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA

    Ningguo Feng & Harry B. Greenberg

  6. Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan

    Amin Alborzian Deh Sheikh & Takeshi Tsubata

  7. La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA

    Alex Marki & Klaus Ley

  8. Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA, USA

    Clare L. Abram & Clifford A. Lowell

  9. Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada

    Matthew S. Macauley

  10. Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada

    Matthew S. Macauley

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Contributions

R.B. conceptualized the study; designed, performed and analyzed the majority of the experiments; and wrote the manuscript. M.B. performed and analyzed the PLA experiments and confocal microscopy experiments. C.B. performed the experiments involving the Cd22Y2,5,6F and Cd22R130E transgenic animals. N.F. performed the oral RV infections and helped to conceptualize and design the RV studies. J.B. and J.C. contributed to analysis of the video microscopy experiments. B.O. performed the gut preparations in the RV studies and helped with the small intestine fragment cultures. A.M. shared intravital microscopy expertise with R.B. Y.B. helped with the RT-qPCR studies. A.A.D.S. and T.T. helped to conceptualize the PLA studies. C.A.L. and C.L.A. provided the motheathen viable mice. H.B.G. contributed to conceptualizing and designing the RV studies. M.S.M., K.L. and L.N. contributed to conceptualizing the study and provided intellectual input. E.C.B. guided, conceptualized and supervised the study and wrote the manuscript.

Corresponding authors

Correspondence to Romain Ballet or Eugene C. Butcher.

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The authors declare no competing interests.

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Peer reviewer information Nature Immunology thanks Dietmar Vestweber and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

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 αL, 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 Fig. 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 αL, β1, α4, β7, or α4β7. Data represent the mean ± SEM of one representative experiment with n = 3 mice per group presented as in Fig. 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–/– (that is n = 8 animals total for the two experimental replicates).

Source data

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–/–, CD22Y2,5,6F, and CD22R130E 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 (<1 s) with the HEVs (namely Brief roller), or one that interacts and rolls on the HEVs for >1 sec (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 (that is 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 α4β7 availability 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 αL, β1, α4, β7, or α4β7 presented as in Fig. 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 Fig. 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 Fig. 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 Fig. 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.

Supplementary information

Supplementary Information

Supplementary Fig. 1.

Reporting Summary

Supplementary Tables

Supplementary Tables 1 and 2.

Supplementary Video 1

Example of a flyer visualized by in situ video microscopy of PPs. The same example of a free-flowing cell shown frame per frame in Extended Data Fig. 8 is shown here as a video (five frames per second; 0.125-s video). Scale bar, 10 µm.

Supplementary Video 2

Example of a brief roller visualized by in situ video microscopy of PPs. The same example of a brief roller shown frame per frame in Extended Data Fig. 8 is shown here as a video (five frames per second; 0.325-s video). Scale bar, 10 µm.

Supplementary Video 3

Example of a roller visualized by in situ video microscopy of PPs. The same example of a roller shown every ten frames in Extended Data Fig. 8 is shown here as a video (20 frames per second; 3.275-s video). Scale bar, 10 µm.

Supplementary Video 4

In situ video microscopy revealing defective arrest of Ptpn6+/meV B cells on PP HEVs (related to Fig. 6). A representative real-time video microscopy experiment was performed showing purified WT (green) and Ptpn6+/meV B cells (red) interacting with PP HEVs (40 frames per second; 32.23-s video). Scale bar, 50 µm.

Supplementary Video 5

In situ video microscopy revealing defective arrest of Cd22–/– B cells on PP HEVs (related to Fig. 6). A representative real-time video microscopy experiment was performed showing purified WT (red) and Cd22–/– B cells (green) interacting with PP HEVs (40 frames per second; 41.628-s video). Scale bar, 50 µm.

Supplementary Video 6

In situ video microscopy reveals increased rolling velocity of Ptpn6+/meV B cells on PP HEVs (Related to Fig 6). Representative wild-type B cell roller (green) and Ptpn6+/meV B cell roller (red) interacting with PP HEVs. (40 frames per second; 20.70 sec movie). Scale bar, 10 µm.

Supplementary Video 7

In situ video microscopy revealing the increased rolling velocity of Cd22–/– B cells on PP HEVs (related to Fig. 6). A representative WT B cell roller (red) and Cd22–/– B cell roller (green) are shown interacting with PP HEVs (40 frames per second; 12,715-s video). Scale bar, 10 µm.

Source data

Source Data Fig. 5

Uncropped western blots.

Source Data Extended Data Fig. 4

Uncropped western blots.

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Ballet, R., Brennan, M., Brandl, C. et al. A CD22–Shp1 phosphatase axis controls integrin β7 display and B cell function in mucosal immunity. Nat Immunol 22, 381–390 (2021). https://doi.org/10.1038/s41590-021-00862-z

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