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Dual targeting of lymphocyte homing and retention through α4β7 and αEβ7 inhibition in inflammatory bowel disease

Summary Anti-integrins are therapeutically effective for inflammatory bowel disease, yet the relative contribution of α4β7 and αEβ7 to gut lymphocyte trafficking is not fully elucidated. Here, we evaluate the effect of α4β7 and αEβ7 blockade using a combination of murine models of gut trafficking and longitudinal gene expression analysis in etrolizumab-treated patients with Crohn's disease (CD). Dual blockade of α4β7 and αEβ7 reduces CD8+ T cell accumulation in the gut to a greater extent than blockade of either integrin alone. Anti-αEβ7 reduces epithelial:T cell interactions and promotes egress of activated T cells from the mucosa into lymphatics. Inflammatory gene expression is greater in human intestinal αEβ7+ T cells. Etrolizumab-treated patients with CD display a treatment-specific reduction in inflammatory and cytotoxic intraepithelial lymphocytes (IEL) genes. Concurrent blockade of α4β7 and αEβ7 promotes reduction of cytotoxic IELs and inflammatory T cells in the gut mucosa through a stepwise inhibition of intestinal tissue entry and retention.

Intestinal biopsies from patients with Crohn disease that has been treated with etrolizumab (anti-b7) show treatmentspecific reductions in gene expression associated with CD8 + cytotoxic T cells.

INTRODUCTION
To mount an efficient immune response, lymphocytes travel between secondary lymphoid organs and mucosal tissues to enable antigen recognition, leading to activation and expansion. The gastrointestinal tract harbors a high antigenic load derived from food and microbiota and is a highly dynamic compartment in terms of immune cell movement. 1 In the mesenteric lymph node (mLN), activated T lymphocytes are imprinted in a specialized microenvironment that results in increased expression of integrin a4b7 and CCR9. 2,3 Elevated a4b7 enables T cells to have an increased capacity to adhere to mucosal addressin cell adhesion molecule 1 (MAdCAM-1) on endothelial venules in the lamina propria (LP). 4 Within the LP, T cells further upregulate aE integrin expression in response to the transforming growth factor b (TGF-b)-rich environment. 5,6 E-cadherin ex-pressed by intestinal epithelial cells serves as a high-affinity ligand for aEb7. [7][8][9] aEb7 is also expressed on a subset of dendritic cells in both mouse and human and are involved in T cell differentiation and imprinting of homing receptors. 10 Gut-resident T cells also express CD69 to repress S1PR1-dependent tissue egress into lymphatics through CD69:S1P1 complex formation, another mechanism of lymphocyte retention in tissue. 11,12 Inflammatory bowel disease (IBD) encompasses both ulcerative colitis (UC) and Crohn's disease (CD) and is characterized by aberrant inflammatory responses in the gastrointestinal (GI) tract. [13][14][15] Patients with IBD have increased activated lymphocytes in gut tissues, and anti-integrin therapies are efficacious. 15,16 Natalizumab, which blocks the a4 subunit of the a4b7 and a4b1 integrin heterodimers, was the first anti-integrin antibody approved for use in CD in 2004. 17 Vedolizumab is an anti-integrin monoclonal antibody that blocks the a4b7 heterodimer while sparing a4b1 integrin and is approved for both UC and CD. 18,19 Etrolizumab, [20][21][22][23] an investigational monoclonal antibody that blocks the b7 subunit of both a4b7 and aEb7, is currently under development for CD. 24 Despite the potential for gut-homing cells to be differentially affected by the integrin heterodimers targeted by these therapeutics, the relative contribution of a4b7 and aEb7 in lymphocyte homing and retention is not yet fully understood. 25 Here, we established a surgical photoconversion system to investigate the integrin dependencies of T cell homing and retention in the gut and tissue egress to the mLN in a mouse model. Single-cell RNA sequencing (RNA-seq) and flow cytometry show that human inflammatory intestinal CD8 + T cell subsets, and not regulatory T cells, have high expression of aEb7. Consistent with that, we demonstrate a significant reduction of genes associated with cytotoxic IELs in patients with CD enrolled in a placebo-controlled study of etrolizumab (ClinicalTrials.gov: NCT02394028), which showed efficacy in an exploratory subcohort analysis. 26 Taken together, these mechanistic mouse model studies and human clinical studies suggest that targeting b7 integrin will effectively reduce inflammatory CD8 + T cells and intestinal inflammation through dual effects on lymphocyte homing and retention.

Migration of CD8 + T cells from mLN to gut mucosa is additively inhibited by combined blockade of a4b7 and aEb7
To evaluate the role of integrins in T cell trafficking from mLN into the gut mucosa, we established a surgical photoconversion procedure in the KikGR transgenic mouse model. 27 KikGR protein changes color from green to red fluorescence upon exposure to violet light, which in these experiments was focused specifically on the mLN, thus ''stamping'' cells with red fluorescence to enable tracking their migration out of the mLN. As a proofof-concept of this model, we evaluated steady-state mucosal immune cell trafficking from the mLN of control or FTY720 (a S1PR1 agonist)-dosed animals after photoconversion. Lymphocyte egress and re-distribution to various tissues was analyzed 16 h after photoconversion. Because of the critical role of S1PR1 in T cell egress from lymph nodes, most KikR + photostamped T cells are confined to the mLN of FTY720-treated animals ( Figure 1A). In contrast, dynamic egress of mLN T cells was observed in vehicle-treated animals, with re-distribution of photo-stamped KikR + T cells to secondary lymphoid organs, including the spleen, inguinal LN, and Peyer's patches, as well as to the small intestine and colon ( Figure 1A). Photo-stamped cells that migrated to the secondary lymphoid organs were mostly CD62L high CD44 low naive T cells, whereas T cells that had migrated into the colon LP were predominantly CD44 hi effector/memory T cells ( Figure S1A).
After the successful proof-of-concept study, this model was used to evaluate anti-a4b7 and anti-b7 effects on T cell trafficking between the mLN and intestine. Anti-a4b7 blockade reduced the frequency of migrated KikR + CD4 + and CD8 + T cells by 50% in the LP compartment of the colon and small intestine ( Figures 1B-1D and S1B). Anti-b7 antibody blockade, which blocks both a4b7 and aEb7, showed greater inhibition of CD8 + T cells in comparison with that of CD4 + T cells ( Figures  1B-1D and S1B). Although most of the mLN CD4 + and CD8 + T cells express a4b7, aE expression is found on more than 60% of CD8 + T cells but less than 10% of CD4 + T cells (Figure S1C). Therefore, increased inhibition of CD8 + T cells by anti-b7 is likely due to the differential expression of aE integrin between CD8 + and CD4 + T cells.
An anti-aE blocking antibody, which only binds to the aEb7 heterodimer, was next used to further ascertain the individual contribution of a4b7 and aEb7 in CD8 + T cell migration from mLN to the gut. 28 Treatment with anti-aE blocking antibody significantly reduced photo-stamped KikR + CD8 + T cells in the colon and small intestine as compared with control antibody (Figures 1E and 1F). As was previously observed with the b7 blockade, combined blockade of a4b7 and aEb7 further reduced KikR + CD8 + T cells $50% more than blockade of either a4b7 or aEb7 alone ( Figures 1E and 1F). Taken together, our data suggest that blockade of a4b7 and aEb7 has an additive effect in reducing CD8 + T cell trafficking to the small and large intestine.
Combined blockade of a4b7 and aEb7 reduced trafficking of antigen-specific T cells in comparison with blockade of a4b7 or aEb7 alone To test the role of a4b7 and aEb7 in antigen-dependent T cell migration to the intestinal LP and epithelium, we used an oral antigen-challenge model to elicit an immune response in the small intestine. 29 Ovalbumin (OVA)-specific T cell receptor (TCR) transgenic MHC I-restricted (OT-1) T cells were adoptively transferred into wild-type (WT) mice before oral immunization with OVA and cholera toxin (CT:OVA). Anti-integrin antibodies were administered to the recipient mice 3 days after CT:OVA immunization when activated T cells began to migrate from the mLN to the small intestine. Treatment with anti-a4b7 or anti-aE blockade led to a reduction in OT-1 T cells in both the LP and intraepithelial compartments, whereas the combination of these blocking antibodies resulted in further reduction of OT-1 T cells in both compartments (Figures 2A and 2B). In this model, blockade of both a4b7 and aEb7 leads to a greater reduction of antigen-specific CD8 + T cells in comparison with blockade of either a4b7 or aEb7 alone.
Blockade of aEb7 or E-cadherin diminishes CD8 + T cell interaction with intestinal epithelium We next asked whether blockade of E-cadherin, the only known ligand of aEb7, also regulates CD8 + T cell accumulation similarly to anti-aE blockade. A blocking antibody against E-cadherin was used to disrupt E-cadherin:aEb7 interactions after CT:OVA oral immunization. 28 OT-1 CD8 + T cell accumulation in the intestinal mucosa was similarly reduced by anti-E-cadherin or anti-aE (Figure 3A), confirming that E-cadherin:aEb7 interaction is critical for T cell accumulation in the gut epithelium.
T cells travel along and through the mesh-like structure of the basement membrane, an extracellular matrix composed of collagen and laminin, and interact with intestinal epithelial cells. 30,31 E-cadherin protein is found on the basolateral side of the intestinal epithelium ( Figure 3B; Video S1). Activated OT-1 T cells expressing red fluorescent protein (OT-1-tdTomato) 2 Cell Reports Medicine 2, 100381, August 17, 2021 Article ll OPEN ACCESS were used to enable live cell imaging. OT-1-tdTomato T cells localized to the basement membrane in close proximity to basolateral surfaces of epithelial cells, with extended cell bodies suggesting activation ( Figure 3B; Video S1). Upon blockade of aEb7 or E-cadherin, OT-1-tdTomato T cells preferentially localize to the LP with a few T cells observed in the epithelium ( Figure 3B).
To follow T cell-epithelial cell interactions over time, OT-1-tdTomato CD8 + T cells were transferred into E-cadherin/cyan fluorescent protein (CFP) reporter mice, and activated T cells were tracked from the luminal side of the small intestine using two-photon intravital imaging ( Figure 3C). As previously re-ported, 30,32 activated T cells migrated toward subepithelial regions and exhibited dynamic interactions with epithelial cells during movement across the basement membrane ( Figure 3C; Video S2). Upon aEb7 blockade, activated T cells were observed to migrate across the basement membrane to reach the subepithelial regions less frequently ( Figure 3C; Video S3). Quantification of individual T cells showed a statistically significant increase in maximum track speed ( Figure 3D) and track length ( Figure 3E) after aEb7 blockade in comparison with the control. Because the blockade of aEb7 led to increased T cell motility and reduced T cell-epithelial cell contact, our findings are consistent with a critical role for aEb7 and E-cadherin in (B-D) Anti-integrin or isotype control antibodies were administered to KikGR transgenic mice before surgical photoconversion of mLN. Colonic CD4 + (top) and CD8 + (bottom) T cell accumulation was reduced by anti-integrin antibody treatment. Data are from a representative sample of six to eight mice analyzed (B). Reduction in KikR + CD45 + TCRb + CD8 + T cells from colon (C) and small intestine (D) after anti-b7 treatment in comparison with anti-a4b7 treatment. (E and F) Isotype control, anti-aE, and/or anti-a4b7 integrin antibodies were administered to KikGR mice before surgical photoconversion of mLN. The percentage of photoconverted KikR + cells among CD45 + TCRb + CD8 + T cells in the colon (E) and small intestine (F) are shown. Bar graph shows means ± SEM. Data are from six to eight animals combined from two to three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Tukey's multiple-comparison post-test. Blockade of aEb7 increases T cell migration from the gut to the mLN Lymphatic vessels serve as conduits for lymphocyte movement out of the gut and into the mLN. 33,34 The decrease in tissue retention after aEb7 blockade may, therefore, directly affect T cell egress through lymphatics. To test that hypothesis, activated KikGR + OT-1 + cells were photo-stamped within the small intestine to enable lymphocyte tracking, and both KikR + OT-1 cells remaining in the small intestine and KikR + OT-1 cells in the mLN were evaluated 16 h later ( Figure S2A). aEb7 or E-cadherin blockade led to an $50% reduction in KikR + OT-1 T cells in the small intestine ( Figures 4A and S2B) and increased KikR + OT-1 cells in the MLN by 3-fold ( Figures 4B and S2C). Treatment with anti-a4b7 antibody did not alter KikR + OT-1 cells in the small intestine or mLN in comparison with the control, but the total number of OT-1 + cells was reduced, making the percentage similar to the FTY720 treatment ( Figures 4A, 4B, S2B, and S2C).

Cell Reports
T cell migration through the lymphatic vessels to the draining LNs is dependent on S1PR1. 11,33 Treatment with FTY720, a S1PR1 agonist, increased T cells in the small intestine and reduced T cells in the mLN in comparison with the control (Figures 4A, 4B, S2B, and S2C). Consistent with an effect on lymphocyte egress from the mucosa, photo-stamped T cells were rare in intestinal lymphatics of FTY720-treated mice. In contrast, aEb7 blockade increased T cell frequency within the lymphatic vessels ( Figures 4C and 4D). Taken together, these findings support a critical role for aEb7, but not a4b7, in T cell retention within gut tissues, and the blockade increases subsequent egress of activated effector T cells from the mucosa to the draining LNs.
aEb7 expression is restricted to cytotoxic CD8 + IELs and proinflammatory CD4 T cell subsets in human colonic tissue Studies in a photoconvertible mouse model demonstrated additive roles for a4b7 and aEb7 integrins in T cell migration and retention, suggesting targeting those complementary mecha-nisms with anti-integrin therapies may be important in IBD. Analysis of aE-expressing pooled IEL and LP immune cells in healthy and IBD human colonic biopsies showed that, similar to the mouse, most CD8 + T cells express aEb7 ($80%), whereas only $10% of the CD4 + T population are aEb7 + (Figures 5A and  S3A). aE + CD4 + T cells produced higher levels of inflammatory cytokines, including interleukin 17 (IL-17) and interferon g (IFN-g) as compared with aE À CD4 + T cells ( Figures 5B and  S3B). Human colonic regulatory T cells (CD4 + CD25 high CD127 low-Foxp3 + ) have low to absent aEb7 expression as compared with non-regulatory T cells, in contrast to the proinflammatory phenotype of aE + CD4 + T cells ( Figures 5C and S3C). Comparison of human and mouse T cells, using the Helicobacter hepaticus anti-IL10R model of colitis, 35 suggested similarities in aE + CD8 + T cells ( Figures 5H, S3D, and S3G), whereas the proinflammatory state of aE + CD4 + human T cells is directly opposite that of murine aE + CD4 + T cells, which have a regulatory T cell phenotype and produce significantly less inflammatory cytokines ( Figures  S3E and S3F).

Blockade of b7 integrin by etrolizumab reduces cytotoxic CD8 + IEL gene expression in intestinal biopsies
Our mechanistic studies suggest b7 blockade may alter CD8 + T cell subset accumulation in intestinal tissue in patients with IBD. We tested the effect of b7 blockade in patients with CD

. aEb7 or E-cadherin blockade inhibits T cell interactions with intestinal epithelium
Adoptive transfer of tdTomato + OT-1 cells into WT or E-cadherin-CFP recipients was followed by oral challenge with cholera toxin:OVA the following day. Three days after immunization, recipient mice were treated with isotype or anti-aE or anti-E-cadherin antibodies before analysis. who were enrolled in a phase III study of etrolizumab. 24 Colonic or ileal biopsies were taken for RNA-seq before randomization to etrolizumab or placebo treatment arms and at 14 weeks after treatment. Etrolizumab-treated patients showed significant decreases in integrin gene expression with reductions in b7 integrin (ITGB7) and a4 integrin (ITGA4) ( Figure 6A) in both the ileum and the colon. Although decreased expression of aE integrin (ITGAE) was observed after etrolizumab treatment, the difference was not statistically significant relative to placebo ( Figures 6A and  S5A).
Significant post-treatment changes in gene expression were observed in etrolizumab-treated patients but not in placebotreated controls (Figures 6B and 6C). Comparison of baseline gene expression between areas with active disease and areas without active disease showed an inverse correlation between genes altered by etrolizumab treatment and genes associated with endoscopic activity (Figures 6D and 6E), suggesting a treatment-specific reduction in active inflammation by etrolizumab. The genes most downregulated by etrolizumab include genes associated with B cells (IGHG1, PAX5, and CD79B), T cells (CD8A, CD5, and CD28), and inflammatory cytokines (IFN-g, IL-23, and TNF) ( Figure 6B).
Because CD can affect the entire gastrointestinal tract, subanalyses by anatomic region were performed. Gene expression changes between active and inactive disease were similar in both the ileum and colon Figure S5 (Figures S6A and S6B), and inflammatory gene expression was also similarly reduced in both locations after etrolizumab treatment Figures S5 (Figure  S6C). These data demonstrate that inflammation-associated genes are downregulated by etrolizumab treatment in the colon and ileum. No correlation was observed in placebo-treated patients ( Figure 6E).
To identify the T cell subsets affected by etrolizumab treatment, gene modules specific for each type of T cell were generated using our single-cell RNA-seq data ( Figure 5D; Data S1). Signature scores were calculated for each of the gene modules in the bulk RNA-seq data from the CD cohort. CD8 + cytotoxic IEL and CD8 + LP T cell signature genes decreased in etrolizumab-treated patients in comparison with those treated with placebo (Figures 6F, S5B, and S7). Other T cell subsets also decreased from baseline in etrolizumab-treated patients, but there was no statistically significant difference between the etrolizumab and the placebo arms ( Figure 6F). CD8 + cytotoxic IEL-specific genes, such as CD8A, NKG7, and CCL5, were strongly downregulated after etrolizumab treatment (Figures 6G and S5C).
Vedolizumab, which blocks a4b7 integrin, has been suggested to inhibit intestinal T cell trafficking, yet a recent study analyzing pre-and post-treatment colonic biopsies did not find any reduction in CD4 + and CD8 + T cells at 14 weeks. 37 A study in a small cohort of HIV-infected patients with IBD showed an increase in total T cells in the terminal ileum, with no change in CD4/CD8 ratio, after vedolizumab treatment. 38 To assess the effect of vedolizumab on cytotoxic IELs, we analyzed publicly available biopsy microarray data from patients with UC before and 12 weeks after treatment with vedolizumab. 39 Similar to Zeissig et al. 37 , we did not observe a significant reduction of CD8 + cytotoxic IELs or CD8 + LP T cell-associated genes (Figure S7). Taken together with the reduction in CD8 + cytotoxic IELs and CD8 + LP T cell-associated genes after etrolizumab treatment, these data suggest that blockade of both a4b7 and aEb7 is required to achieve a reduction in genes associated with CD8 + cytotoxic IELs and CD8 + LP T cells.

DISCUSSION
IBD is a chronic relapsing-remitting intestinal inflammatory disorder associated with an increase in activated lymphocytes in the intestinal mucosa. 13 Etrolizumab, an anti-b7 integrin monoclonal antibody, is being tested for efficacy in CD in phase III (ClinicalTrials.gov: NCT02394028). Previous reports using humanized mouse models have shown that a4b7 is critical for homing of T cells to the colon 40 and that blockade of aEb7 can further decrease accumulation of CD8 + T cells. 41 Here, we applied a surgical photoconversion system to quantify T cell homing and Article ll OPEN ACCESS retention and found that a combined blockade of a4b7 and aEb7 is more effective in reducing CD8 + T cell accumulation in gut tissue than is blocking either integrin alone in an antigen-specific mouse model. Further, aEb7 and E-cadherin mediate interactions of activated CD8 + T cells with the basolateral epithelium and prolong tissue retention time through inhibiting intestinal tissue egress. Our findings confirm that blockade of a4b7 and aEb7 integrins inhibit T cell intestinal accumulation and extend their role to tissue entry and retention in a respective stepwise manner to inhibit T cell accumulation in the gut mucosa in a mouse model. Single-cell RNA-seq and flow cytometry of human colonic T cells show that most aE + T cells are pro-inflammatory. Intestinal biopsy data from a placebo-controlled study of patients with CD 26 demonstrate etrolizumab-specific reduction in genes associated with gut CD8 + cytotoxic IELs, along with a broad decrease of genes associated with active inflammation, consistent with a role for a4b7 and aEb7 integrins in CD8 T cell accumulation in the gut mucosa.
Gastrointestinal immune priming and tissue trafficking is achieved through the induction of integrin and chemokine receptors that mediate gut homing of T cells activated within intestinaldraining lymphoid organs. Integrin-dependent gut trafficking is not all encompassing. T cells can still migrate to the gut during inflammation in the absence of a4b7 expression, 42 and anti-a4b7 blockade only partially reduces T cell trafficking to the gut (Figure 1). Integrins a4b1 and aLb2 may also mediate intestinal homing, a mechanism of particular importance in the ileum. 43 Expression of addressins VCAM-1 and ICAM-1 on intestinal vascular endothelium can mediate adhesion and may be upregulated under inflammatory conditions, 44 although reports in IBD are mixed. 45,46 aEb7 + dendritic cells (DCs) have been shown to have reduced induction of regulatory T cells and increased induction of pro-inflammatory T cell responses in patients with IBD. 47 Once T cells upregulate aE expression, their ability to interact with the epithelium through direct binding between aEb7 and E-cadherin on the basolateral side of the intestinal epithelium is increased, and previous studies have shown aE-dependent accumulation of CD8 + and Th9 T cells in a humanized mouse model. 41 Our data are consistent with a role for aEb7 and E-cadherin interactions in T cell migration across the intestinal basement membrane into the intraepithelial space to enable tissue retention, which could be through direct or indirect effects. In the mechanistic murine studies presented here, inhibition of both a4b7-dependent cell entry and aEb7-dependent retention leads to a greater reduction of T cells in the gut over inhibition of either pathway alone.
There are important differences in aE expression between human and mouse CD4 + T cell lineages, which we have directly assessed here. Human intestinal regulatory T cells have low to absent expression of aEb7, whereas murine regulatory T cells express a high level of aEb7 (Figures 5 and S3E). In addition, human aE + CD4 + T cells are more polyfunctional with a higher proportion of cells that produce proinflammatory cytokines, including IL-17 and IFN-g 48 ( Figure 5). In both human and mouse, most gut CD8 + T cells express high levels of aEb7; however, human CD8 + IELs express much higher levels of cytotoxicity-associated genes (Figures 5 and S3G). These differences highlight the importance of cross-validation of observations between pre-clinical and clinical studies to inform our mechanistic understanding of therapies in IBD. It is also critical to continue to validate observations, such as the relative roles of a4b7 and aEb7 in integrin-mediated homing and retention made in model systems directly in human clinical studies wherever possible, with particular attention to new methodologies that can increase our mechanistic understanding.
Although anti-integrin therapies have been suggested to block trafficking of lymphocytes into the intestinal mucosa, data from patients treated with anti-integrins has been mixed. One study showed a reduction in intestinal CD4 + T cells in patients with CD treated with natalizumab (anti-a4) using immunohistochemistry. 49 More recently, patients with UC and those with CD underwent a cell-labeling and imaging approach before and after vedolizumab (anti-a4b7) treatment, and no change in T cell migration to the intestinal mucosa was observed. 37 In the same study, no change in T cell frequency was observed when evaluated by immune cell phenotyping by flow cytometry after 14 weeks of vedolizumab treatment. 37 Vedolizumab treatment in HIV + patients with IBD resulted in an increase in T cells in the ileum as well as a striking decrease in the size of lymphoid aggregates. Although HIV-1 tropism for mucosal homing cells complicates the interpretation, the decrease in lymphoid aggregate size, which may be more dependent on a4b7, instead of mucosal T cells, is notable. 38 In the phase III cohort of patients with CD assessed here, as well as in a previous phase II study in patients with UC, 23 etrolizumab reduced expression of T cell genes in intestinal mucosa after 14 and 10 weeks, respectively. We applied the same methodology to a second vedolizumab study in patients with UC 39 ( Figure S7) and confirmed that no change of T cell-associated gene sets was observed 12 weeks after treatment. Possible explanations for this negative finding may be the reduced dynamic range for measuring gene-expression changes inherent to microarrays in comparison with RNA-seq 50 or that blockade of a4b7-dependent cell entry by vedolizumab may take longer than 12 weeks to have a noticeable effect on colonic T cells. However, our analysis showing T cell genes do not decrease after vedolizumab treatment is consistent with the Zeissig et al. 37 study and, together with our murine data, suggests the reduction of cytotoxic IEL genes by etrolizumab may be attributable to the additional blockade of aEb7-dependent tissue retention, which may contribute to greater or faster inhibition of inflammation during induction therapy with etrolizumab compared with vedolizumab. Future clinical studies with head-to-head comparison of etrolizumab and vedolizumab will be required to fully evaluate the differences between these therapeutic approaches.
The reduction in CD8 + cytotoxic IELs observed only in etrolizumab-treated patients suggests a potential effect of etrolizumab on the dysregulated mucosal immune response in IBD. Article ll OPEN ACCESS T cells have been associated with prognostic outcomes in IBD, 51 and the role of IELs, which are predominantly CD8 + , in inflammation is beginning to be better appreciated. CD8 + cytotoxic IELs are closely associated with the intestinal epithelium and, under normal conditions, can quickly provide defense against pathogen dissemination 31,52 because of their resting expression of cytotoxic genes, which enables them to respond without proliferation. 53,54 Upon stimulation, rapid IFN-g secretion by CD8 + cytotoxic IELs augments T cell cytotoxicity against infected epithelial cells and increases barrier permeability. 55-58 Additionally, gut-resident CD8 + T cells express high levels of NK receptors, such as NKG2D and KLRD1, which enable recognition of stressed epithelial cells. 59 Two recent studies of CD8 + T cells from healthy volunteers and patients with UC using single-cell (sc)RNA-seq identified aEb7 + effector TNF-a + IFN-g + CD8 + T cells. 60,61 It has long been recognized that the frontline role of cytotoxic IELs is not without risks of aberrant activation. 62 and recent data show that chronic inflammation in celiac disease, a gastrointestinal disorder triggered by immune response to dietary gluten, induces a pathogenic phenotype in IELs characterized by increased IFN-g production and expression of CCL4. 63 Whether similar effects of chronic inflammation on cytotoxic IEL populations occur in IBD will require further investigation but may soon yield to scRNA-seq techniques able to probe CD8 + cellular heterogeneity.
In conclusion, we demonstrate that integrins a4b7 and aEb7 have cooperative roles in CD8 + T cell accumulation in the gut mucosa in a murine model of T cell trafficking. These data highlight an underappreciated role for aEb7 in T cell retention at the epithelial barrier through T cell-epithelial interactions that inhibit S1PR1-dependent tissue egress and migration to mLN. Key differences between human and mouse aEb7 expression on CD4 + T cell lineages, particularly regulatory T cells, were identified. Finally, we observed a significant reduction in expression of gene sets associated with CD8 + cytotoxic IELs after treatment with anti-b7 etrolizumab therapy in a large phase III study of patients with CD. Although recent data from phase III UC studies of etrolizumab showed mixed efficacy in top-line clinical efficacy, 64 clinical studies of etrolizumab in CD are on-going.

Limitations of the study
Recognizing that mouse models have limitations in their ability to recapitulate human biology is important to acknowledge. Here, we report data using mouse-surrogate antibodies to dissect migration to, and retention of, lymphocytes in the intestinal mucosa, which show a role for a4b7 and aEb7 in T cell trafficking, suggesting that the therapeutic potential in IBD may be greater if both integrin heterodimers are blocked. Because of the technical challenges of photo-conversion, a key component of the experimental model, we focused on the small intestine because cell retention in the colon could not be extensively investigated. In addition, possible depletion effects of murine surrogate antibodies have not been rigorously tested and could be mitigated by use of Fcg receptor (FcGR)-deficient mice as recipients in experiments. Patient data from a clinical study of etrolizumab, which blocks both a4b7 and aEb7, provide correlative data on the effectiveness of b7 blockade in reducing intestinal CD8 T cell populations analogous to our observations in murine models. There are limitations in the interpretation of human data based on sample size numbers, particularly given the number of paired samples in subanalyses based on treatment group and anatomic location, which will require validation in on-going etrolizumab trials.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

ACKNOWLEDGMENTS
We thank Amanda Tatro, Oliwia Zurek, Zhiyong Yang, and Andrew Chan for critical review and editorial suggestions for this manuscript. The art work in the graphic abstract was assisted by Apothecom. This research was supported and funded by Genentech, Inc., USA. analysis of sorted aE + and aE -T cells, tissue samples from patients undergoing intestinal resections as part of their clinical care at the Mayo Clinic. into an observational study (IRB 10-006628; Mayo Clinic Institutional Review Board). All patients provided consent in accordance with Institutional Review Board guidance for each institution as noted above. Patient characteristics are shown in Table S1.
Etrolizumab clinical study in CD Moderately to severely active CD patients with a CDAI or 220-480 and a centrally read SES-CD score of R 7 (R4 for isolated ileitis) who had previously been treated with corticosteroids, immunosuppressants or anti-TNF were enrolled in an exploratory cohort of 350 patients within the BERGAMOT etrolizumab study (ClinicalTrials.gov: NCT02394028). Briefly, patients underwent endoscopy as part of their screening visit and baseline biopsies were taken in the ileum or the colon for analysis prior to randomization. Patients were then randomized into placebo or etrolizumab cohorts at a 1:4 ratio with subcutaneous administration at 0, 2, 4, 8 and 12 weeks. Following the induction period of 14 weeks, patients underwent a second ileocolonoscopy and follow-up biopsies were obtained from the ileum or colon and placed directly into RNAlater (QIAGEN). Biopsy samples were scored as being from active or inactive segments using the cut-off of colonic SES-CD R 7 for colonic biopsies and ileal SES-CD R 4 for ileal biopsies. Patient characteristics are shown in Table S2.

METHOD DETAILS
Blocking antibodies for murine studies Anti-b7 (FIB504) and anti-a4b7 (DATK32) antibodies were ordered from ATCC and their sequences were individually cloned from hybridomas. Antigen-binding domain sequences from the parent hybridomas were fused to mouse IgG1 Fc in a pRK expression vector and transfected into HEK293 cells. Transfected supernatants were purified on a HiTrap column (GE healthcare) with Mabselect Sure resin (GE healthcare) with a phosphate-buffered saline (PBS) loading buffer. Antibodies were eluted with 0.1 M citrate (pH 3.0) and neutralized with 3 M Tris, pH 8.0, to a final pH of $7.0 prior to dialysis against PBS, pH 7.2. Each antibody was run on a Superdex S200 10/300 GL size exclusion column (SEC) (GE Healthcare) using PBS, pH 7.2, load buffer at a flow rate of 1 mL/min (30 cm/h) to remove any aggregates. Pooled fractions were filtered using a 0.2 mm filter and the final antibody preparation was assessed by analytical SEC carried out with a TSK-GEL, Super SW3000, 4.6 mm 3 30 cm, 4 mm (Tosoh Bioscience) column using a Dionex Ultimate 3000 system (Thermo Fisher Scientific) to confirm > 95% homogeneity of monomeric antibody. Anti-aE blocking antibody (clone number M290) was from BioXcell and anti-E-cadherin antibody (clone number ECCD-2) was from Invitrogen.

Flow cytometry antibodies and staining
Cell suspension was washed twice in staining buffer (PBS with 2% of fetal bovine serum), blocked with TruStain FcX (anti-mouse CD16/32, Biolegend) for 5mins at room temperature, and stained with indicated fluorescence conjugated antibodies below for 30mins. For intranuclear staining, cells were fixed, permeabilized and stained using the Foxp3/Transcription Factor Staining Kit according to the manufacturer's instructions (eBioscience). For intracellular cytokine staining, cells were stimulated with the Cell Activation Cocktail with Brefeldin A (BioLegend) for 5 hours, followed by fixation, permeabilization and staining using the Cytofix/ Cytoperm kit (BD Bioscience). Fluorochrome-conjugated anti-mouse antibodies used are as follows: CD45 (30-F11), TCRb (H57-597), CD8 (53-6. Murine surgical photoconversion models of T cell trafficking Photoconversion of mLN or specified intestinal segment was performed during survival surgeries carried out in a sterile environment under proper anesthesia. Following shaving of the surgical site, a small abdominal incision of $1 cm allowed the cecum to be gently extruded to localize mLN, colon, and/or small intestine. A piece of sterile foil with a 5-mm hole was used to surround the tissue of interest, e.g., mLN, and limit exposure of surrounding tissue to the photoconverting light. For ileum photoconversion, regions with Peyer's patches (PPs) was avoided. A silver LED (Prizmatix) with an aperture polymer optical fiber (1.5 mm core diameter) and a lens focuser was used as the violet light source (415 nm) to treat the target tissue as indicated. Each exposed area was treated with violet light for a total period of 5 minutes then extruded organs were returned to the abdominal cavity and the incision was sutured closed. Anti-integrin antibodies (250 mg/mouse) were administered intraperitoneally (IP) 3 hours prior to photoconversion and analysis was performed 16 hours post photoconversion.
T cell adoptive transfer and oral antigenic challenge KikGR + OT-1 + green fluorescent CD8 + T cells were enriched from splenocytes using the MACS CD8 + T cells isolation kit (Miltenyi Biotech) according to the manufacturer's instructions. Each C57BL/6 wild-type recipient mouse received 5 3 10 6 KikGR + OT-1 + T cells intravenously (IV) and were then orally challenged with 150 mg Cholera toxin:OVA (Sigma) at a 1:25 molar ratio the following Intra-vital two-photon microscopy live imaging T cell adoptive transfer and oral antigenic challenge was performed as described above but using tdTomato + OT-1 + CD8 + T cells adoptively transferred into E-cadherin-mCFP recipient mice to enable intravital imaging. Intravital microscopy was performed on anesthetized mice with the ileum exposed and opened 1 cm longitudinally along the anti-mesenteric border. The incision site was placed far away from the vasculature to ensure continuous blood supply. The mucosal surface was placed against a customized stage with a moistened PBS pad to prevent dehydration. The tissue was immobilized on a stage using a plastic ring with pinholes and a coverslip was placed on top of the mucosal surface. The luminal side of the ileum was imaged using a two-photon laser-scanning microscope (Ultima In Vivo Multiphoton Microscopy System; Bruker Technologies) using a 16X objective (Olympus) and dual Ti:sapphire lasers (MaiTai and Insight lasers, Spectra-Physics;) tuned to 890 nm and 1020 nm. Analysis was performed using Imaris 9.2.1 (Bitplane; an Oxford Instruments company).

Immunofluorescence and confocal microscopy
For confocal microscopy, T cell adoptive transfer and oral antigenic challenge was performed as described above. Upon sacrifice of recipient mice, the ileal portion of the small intestine was excised and prepared as a ''swiss roll.'' Tissues were fixed in 4% paraformaldehyde for 6-8 hours or in Cytofix/Cytoperm Buffer (BD Bioscience) for 2-4 hours for lymphatics staining, and then dehydrated in 30% sucrose overnight prior to embedding in optimal cutting temperature (OCT) freezing media. Cryosections (8-10 mm) were stained with the following primary antibodies: Laminin (Sigma-Aldrich, L9393), E-cadherin (Thermo fisher, ECCD2), EpCAM (Abcam) and LYVE-1 (R&D systems). All images were acquired using a Leica TCS SPE upright confocal microscope. Frequency of tdTomato + cells in LYVE1 + lymphatics was blindly quantified from 100 villi across tissue sections and a mean percentage of 100 villi was used for each individual mouse.
Helicobacter hepaticus anti-IL10R induced colitis model Female C57BL/6 mice (Taconic) at 5-6 weeks old were treated weekly with anti-IL10R antibody (1 mg/ mouse administered IP) (BioXcell). Oral administration of $1.2 3 10 9 colony-forming units (CFU) Helicobacter hepaticus (ATCC 51448) was performed on two consecutive days as previously described, 35 and mice were monitored three times per week for signs of colitis, such as watery stool. Following onset of disease symptoms, colitic mice were sacrificed and cells were isolated from the intestine as described above.
Preparation of cells from patient tissue samples for flow cytometry Patient resection samples from the Mayo clinic and patient colonic biopsies from UCSF were shipped on ice in RPMI 1640 containing 10% FBS and antibiotics. Frozen biopsies from Stanford were thawed in a 37 C water bath immediately prior to processing. For resection tissue, the mucosa layer was separated from the serosal layer and cut into small pieces. Tissue samples were washed in HBSS (w/o Mg 2+ ) and then transferred to 37 C prewarmed digestion buffer (DMEM/F12 50:50, 10% FBS, 15mM HEPES, PenStrep, 0.35mg/mL Collagenase D, 0.5mM DTT) and incubated in a 37 C water bath for 10 minutes with agitation by shaking every 5 minutes. Cells released from the tissue into the supernatant were removed and transferred to the wash buffer (DMEM/F12 50:50, 10% FBS, 15mM HEPES, PenStrep). Following three rounds of digestion, pooled cells were spun down, counted and stained as described above for flow cytometry analysis or sorting.
Preparation of tissue samples for RNA-seq CD45 + TCRab + TCRg -aE + and aEsingle cells were sorted directly into RLT lysis buffer (QIAGEN) with b-mercaptoethanol. RNA was isolated from sorted cells using the QIAGEN RNeasy Mini Kit (QIAGEN) according to the manufacturer's instructions. Tissues were digested into single cell suspension and sorted by FACS into 3 populations: CD45 + Epcam -, CD45 -Epcam + , and CD45 -Epcam -. Sort time was capped at 2 hours. Sample processing for single-cell RNA-seq was done using Chromium Single Cell 3 0 Library and Gel bead kit v3 following manufacturer's guide (10x Genomics). The cell density and viability of single-cell suspension were determined by Vi-CELL XR cell counter (Beckman Coulter). All of the processed samples had a very high percentage of viable cells. The cell