ARTICLES https://doi.org/10.1038/s41590-021-00862-z A CD22–Shp1 phosphatase axis controls integrin β7 display and B cell function in mucosal immunity Romain Ballet 1,2 ✉ , Martin Brennan1,2,11, Carolin Brandl3,11, Ningguo Feng1,4,5, Jeremy Berri1,2, Julian Cheng1,2, Borja Ocón1,2, Amin Alborzian Deh Sheikh 6, Alex Marki7, Yuhan Bi1,2, Clare L. Abram8, Clifford A. Lowell8, Takeshi Tsubata 6, Harry B. Greenberg1,4,5, Matthew S. Macauley 9,10, Klaus Ley 7, Lars Nitschke3 and Eugene C. Butcher 1,2 ✉ 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 spe- cialized role of α4β7 in intestinal immunity, CD22 deficiency selectively inhibited intestinal antibody and pathogen responses. he integrin α4β7 functions as a B and T cell adhesion recep- inhibitory motif (ITIM) sequences in membrane receptors, often tor for the mucosal vascular addressin (mucosal addressin cell acting in conjunction with ITIM-bearing molecules to inhibit sig- Tadhesion molecule 1 (MAdCAM-1)) expressed by postcapil- naling pathways, including those involved in integrin activity. In lary high endothelial venules (HEVs) in gut-associated lymphoid T cells, for example, Shp1 controls deactivation of the lymphocyte tissue (GALT), by lamina propria venule sites of effector cell recruit- function-associated antigen 1 integrin (αLβ2) to prevent aberrant 1–3 ment and by stromal cells in GALT . GALT is the major site of B adhesion of leukocytes to β2 integrin ligands or T cell adhesion cell activation and humoral immune induction for intestinal immu- to antigen-presenting cells11. In B cells, upon antigen stimulation, nity. Activated B cells undergo isotype class switching in Peyer’s the phosphorylated ITIM sequences of sialic acid-binding Ig-like patches (PPs) and differentiate into migratory immunoglobulin lectin 2 (CD22; also known as Siglec-2) recruit Shp1 to inhibit 2+ A (IgA)-secreting plasmablasts that use α4β7 to home to mucosal downstream components of B cell antigen receptor-induced Ca surfaces4 where local production of secretory IgA provides immune signaling12. However, Shp1 has not been implicated in the control of protection. Presumably in support of this role, B cells home pref- integrin endocytosis or cell-surface expression. erentially to and predominate in murine PPs, contrasting with Here, we report that the pair Shp1/CD22 acts in a cell-intrinsic 5 peripheral lymph nodes (PLNs) where T cells are the majority . This manner to enhance α4β7 surface abundance, with profound organo- homing preference correlates with higher surface expression of α4β7 typic effects on mucosal immune responses. The findings uncover 6 on B cells than on T cells , but the mechanisms responsible for this a selective role for Shp1 in α4β7 endocytosis and surface expression, differential α4β7 surface expression and its essential role in intestinal define CD22 ITIM- and lectin/carbohydrate-dependent mecha- immunity have not been defined. nisms targeting Shp1 to β7 integrin in B cells, and support the The tyrosine phosphatase Src homology region 2 domain- significance of these mechanisms to efficient intestinal antibody containing phosphatase 1 (Shp1; encoded by the gene Ptpn6) has responses. essential roles in regulating immune homeostasis. Homozygous gene depletion of Ptpn6, as described in motheathen mice (Ptpn6me/me), Results 7 results in severe systemic inflammation and death within weeks . Shp1 augments α4β7 cell-surface display in lymphocytes. In stud- Closely related motheathen viable mutant mice (Ptpn6meV/meV) ies of motheaten viable mutant mice (Ptpn6meV/meV) lacking Shp1 express wild-type (WT) levels of Shp1 but the catalytic activity of activity, immunofluorescence staining with antibodies to the α4β7 the enzyme is greatly reduced, resulting in a similar yet slightly less heterodimer or to the β7 subunit revealed a substantial (~90%) severe phenotype8. Shp1 plays essential roles in the regulation of reduction in the median fluorescence intensity (MFI) of Ptpn6meV/ 9,10 meV tyrosine phosphorylation in T and B cells . Shp1 can be targeted splenic naive B cells compared with wild-type controls. The α4 by its binding to phosphorylated immunoreceptor tyrosine-based subunit, which forms heterodimers with integrin β1 as well as β7, 1Palo Alto Veterans Institute for Research, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA, USA. 2Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA. 3Division of Genetics, Department of Biology, University of Erlangen, Erlangen, Germany. 4Department of Medicine, Division of Gastroenterology and Hepatology, Stanford University School of Medicine, Stanford, CA, USA. 5Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA. 6Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan. 7La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA. 8Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA, USA. 9Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada. 10Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada. 11These authors contributed equally: Martin Brennan, Carolin Brandl. ✉e-mail: [email protected]; [email protected] NatURE IMMUnology | VOL 22 | March 2021 | 381–390 | www.nature.com/natureimmunology 381 ARTICLES NATURE IMMUNOLOGY a 750 600 * * WT Isotype control 450 ** 300 * * Ptpn6 meV/meV WT 150 meV/meV 150 ** Ptpn6 *** 0 103 104 105 0 104 105 0 103 104 105 * **** **** ** *** 100 αL β2 β1 50 MFI (percentage of WT) 0 0 103 104 105 0 103 104 105 0 103 104 105 L 2 1 4 7 7 L 2 1 4 7 7 L 2 1 4 7 7 α β β α β β α β β α β β α β β α β β α4 β7 α4β7 α 4 α 4 α 4 Naive B cells CD4+ T cells CD8+ T cells b 200 WT +/meV ** Ptpn6 150 Isotype control ******** **** *** *** **** ******** **** WT Ptpn6 +/meV 100 0 103 104 105 0 104 105 0 103 104 105 αL β2 β1 50 MFI (percentage of WT) 0 0 103 104 105 0 103 104 105 0 103 104 105 α L β 2 β 1 α 4 β 7 β 7 α L β 2 β 1 α 4 β 7 β 7 α L β 2 β 1 α 4 β 7 β 7 4 4 4 α α α α4 β7 α4β7 Naive B cells CD4+ T cells CD8+ T cells meV/meV Fig. 1 | Selective reduction of integrin α4β7 on motheathen viable B and T cells. a,b, Left, flow cytometry of WT versus motheathen viable (Ptpn6 ) (a) +/meV + + or Ptpn6 (b) splenic naive B cells, CD4 T cells and CD8 T cells stained for αL, β2, β1, α4, β7 or α4β7. Shown are pooled data (means ± s.e.m.) from n = 4 experiments with 4–6 (a) or 6–9 (b) animals per group in total. For each animal within one experiment, the MFI of the integrin staining is expressed as a percentage of the mean MFI of the WT group. Right, representative histogram overlays gated in naive B cells. All groups were compared by two-tailed Student’s t-test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. was reduced ~20% in Ptpn6meV/meV B cells, reflecting the reduction CD22 deficiency. To address this, we used transgenic animals Y2,5,6F in α4β7. In contrast, we found no reduction for integrins αL, β1 and (CD22 ) in which the CD22 ITIM signaling domains were + + 16 β2 (Fig. 1a). β7 was also reduced on CD4 and CD8 T cells (Fig. 1a). mutated to prevent Shp1 binding and downstream CD22 signaling Since the Ptpn6meV/meV phenotype induces profound changes in the (Fig. 2a). B cells in CD22Y2,5,6F animals express normal amounts of 13 14,15 16 Y2,5,6F phenotype and homeostasis of mature B cells and T cells , we CD22 (ref. ). CD22 B cells displayed surface abundances of α4, –/– repeated the above experiments with heterozygous motheaten via- β7 and α4β7 as severely reduced as on Cd22 B cells, again with no +/meV ble mice (Ptpn6 ). α4, β7 and α4β7 integrins were also reduced on effect on the αL or β1 integrin surface expression (Fig. 2b). +/meV heterozygous Ptpn6 B and T cells, while αL, β1 and β2 were not CD22 is a lectin that interacts in cis with α2-6-sialic acid affected (Fig. 1b). Thus, Shp1 positively regulates the cell-surface (α2-6-Sia)-decorated glycoproteins such as CD22 (ref. 17) or CD45 18 19 abundance of integrin α4β7 on lymphocytes without affecting other (ref. ), affecting its distribution and motility on the B cell surface . integrins. To assess a potential role for the CD22 lectin–carbohydrate interac- tions in cell-autonomous α4β7 regulation, we assessed the integrin CD22 mediates Shp1-dependent α4β7 effects in B cells. The cell-surface levels of B cells expressing a mutated lectin domain selective effect of the Ptpn6+/meV phenotype on B cells suggested (CD22R130E) that prevents α2-6-Sia binding16 (Fig. 2a). CD22R130E B potential involvement of CD22, a B cell-specific lectin known cells expressed CD22 at wild-type levels16 and displayed a significant to recruit Shp1 to the plasma membrane through interactions reduction in α4β7 levels (Fig.