Integrins are essential for all the T cell functions. Integrins can enable T cells to migrate in and out of lymph nodes and other tissues and mediate cell-to-cell interactions. Integrin activation also plays a critical role in maintaining regulatory T (Treg) cell function. Rap1 is a small GTPase playing an essential role in recruitment and tethering of talin1 to the integrin cytoplasmic domain, which results in integrin activation. Rap1 can bind to talin1 directly. Previous work showed that the Rap1–talin1 F0 domain interaction has a minimum effect on platelet activation. Our lab recently identified an additional Rap1 binding site in the talin1 F1 domain, which is critical for integrin activation in platelets. However, the relative roles of these two binding sites for integrin activation on lymphocytes are still obscure. Here, by testing talin1 mice bearing point mutations, which block Rap1 binding without affecting talin1 expression, in talin1 F0 and F1 domains (R35E, R118E), we found that the binding between talin1 and Rap1 plays a key role in T cell adhesion and migration. The loss of direct interaction between talin1and Rap1 also leads to defects in the suppressive function of Treg cells specifically. Taken together, talin1 is a direct effector of Rap1 GTPase that mediates integrin activation in both CD4+ T cells and Treg cells.

Integrins have been known for its role in facilitating cell migration. Recent studies have revealed that integrin activation also plays important roles in maintaining T cell homeostasis and peripheral tolerance, but the mechanism of integrin activation in regulatory T cells, a subset of T cells that maintains peripheral tolerance, remains obscure. Here, by using various Treg-specific gene knocked out mice, we found talin and Rap1 are critical for integrin activation in regulatory T cells. The connection between Rap1 and talin is mediated by an adaptor, RIAM. Surprisingly, RIAM knockout did not affect integrin activation in Tregs. Using mutant mice, we found another adaptor, Lamellipodin, can provide some of the connection between Rap1 and talin. In addition, we found that in lymphocytes, direct binding of Rap1 to two sites in talin makes another contribution to integrin activation in Tregs. Taken together, distinct pathways are involved in Tregs and enable their ability to maintain immune homeostasis through their suppression function.

Upon vascular injury, platelet stimulation with agonists permits αIIbβ3 integrin to bind its cognate ligands to enable stable adherences to the vessel wall and platelet aggregation. Integrins remain in a low-affinity state until intracellular signaling pathways induce a high-affinity form, a process defined as integrin activation. Binding of the cytosolic adapter protein talin1 to the integrin β cytoplasmic tail is a critical, final step in integrin activation. Previous studies implicated GTPases, Rap1a and Rap1b, in regulating talin1 binding to integrins in platelets. Structural and biochemical evidence showed a direct interaction between Rap1 and talin1 F0 domain, further confirmed in Dictyostelium. We hypothesized that Rap1 may directly interact with talin1 in platelets leading to its association with the β3 cytoplasmic tail to activate αIIbβ3. A mouse strain harboring the R35E mutation in talin1 F0 domain that blocks binding to Rap1 was generated. There was no significant defect in the ability of the Tln1R35E/R35E platelets to activate αIIbβ3 integrins. RIAM, a known Rap1 effector, and upregulations of talin2 were excluded as compensatory mechanisms. Results indicated that Rap1b binding to talin1 F0 domain makes a minimal contribution in activating platelet αIIbβ3. Remarkably, Rap1 also binds to talin1 F1 domain, a structural homolog to the F0 domain. Disrupting this interaction prevents αIIbβ3 integrin activation in a CHO cell model system. These findings suggest that talin1 may function as a direct effector of Rap1 through its F1 domain. Future studies will investigate the Rap1-talin1 F1 interaction in murine platelets by mutating the F1 domain.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract including two major categories, Crohn’s disease (CD) and ulcerative colitis (UC). Rapid recruitment of lymphocytes from peripheral circulation to the gut mucosa is a key feature of IBD. The process of lymphocytes homing to the gut is a multiple-step adhesion

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cascade that is mediated by adhesion molecules such as integrin α4β7. Integrin α4β7 is expressed on lymphocytes and interacts with its ligand, mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1), which is primarily expressed in the high endothelial venules in the small intestine, the colon, and Peyer’s Patches. Integrin α4β7 is a therapeutic target in IBD. Clinical studies report that vedolizumab, a humanized antibody against integrin α4β7, effectively maintains clinical remission in IBD patients. However, aggravated colitis was observed in a small percentage of patients treated with high dose of vedolizumab, which is probably due to aberrant innate immune response to intestinal mucosal injury and infection. Here, the role of integrin β7 on recruiting conventional and regulatory T lymphocytes during intestinal inflammation is further investigated by using two IBD mouse models. Conventional/effector T cells activate other effector immune cells whereas regulatory T cells suppress inflammatory activities and maintain peripheral tolerance. The results demonstrated that deficiency in integrin β7 exacerbates induced spontaneous colitis. The suppression function of β7-deficient Treg cells remains intact. However, integrin β7-deficiency impairs the homing capacity of both regulatory T cells and effector T cells to the GALT. These findings possibly explain the adverse effects seen in high dose vedolizumab treatment, suggesting that excessive blocking of integrin β7 function should be avoided to prevent potential adverse effects of this medication.