Inefficient T-cell homing to tissues limits adoptive T-cell immunotherapy of solid tumors. αLβ2 and α4β1 integrins mediate trafficking of T cells into tissues via engagement of ICAM-1 and VCAM-1, respectively. Inhibiting protein kinase A (PKA)-mediated phosphorylation of α4 integrin in cells results in an increase in αLβ2-mediated migration on mixed ICAM-1-VCAM-1 substrates in vitro, a phenomenon termed "integrin trans-regulation." Here, we created an α4(S988A)-bearing mouse, which precludes PKA-mediated α4 phosphorylation, to examine the effect of integrin trans-regulation in vivo. The α4(S988A) mouse exhibited a dramatic and selective increase in migration of lymphocytes, but not myeloid cells, to sites of inflammation. Importantly, we found that the α4(S988A) mice exhibited a marked increase in T-cell entry into and reduced growth of B16 melanomas, consistent with antitumor roles of infiltrating T cells and progrowth functions of tumor-associated macrophages. Thus, increased α4 trans-regulation of αLβ2 integrin function biases leukocyte emigration toward lymphocytes relative to myeloid cells and enhances tumor immunity.

Rap1-interacting adaptor molecule (RIAM) is a Rap1 effector that mediates the recruitment of talin to integrins, thereby supporting their activation. In this study, we investigated the role of RIAM in an adoptive transfer model for type I diabetes and report that RIAM expression in T cells is necessary for diabetes development. Loss of RIAM did not prevent lymphocyte recruitment to draining lymph nodes 24 h after transfer, but it was required for Ag-driven proliferation and cytotoxic killing. RIAM is recruited to immune synapses along with talin and LFA-1, and loss of RIAM profoundly suppresses Ag-dependent conjugate formation in primary naive and effector T cells. These data identify the requirement of RIAM for formation of immunological synapses and in resulting T cell functions in autoimmunity. Moreover, because RIAM-null mice are healthy, fertile, and display no bleeding abnormalities, our results identify RIAM and its regulators as potential targets for therapies of T cell-mediated autoimmunity.

CD98, which is required for the rapid proliferation of both normal and cancer cells, and MET, the hepatocyte growth factor receptor, are potential targets for therapeutic antitumor Abs. In this study, we report that the antiproliferative activity of a prototype anti-CD98 Ab, UM7F8, is due to Ab-induced membrane-associated ring CH (MARCH) E3 ubiquitin ligase-mediated ubiquitination and downregulation of cell surface CD98. MARCH1-mediated ubiquitination of CD98 is required for UM7F8's capacity to reduce CD98 surface expression and its capacity to inhibit the proliferation of murine T cells. Similarly, CD98 ubiquitination is required for UM7F8's capacity to block the colony-forming ability of murine leukemia-initiating cells. To test the potential generality of the paradigm that MARCH E3 ligases can mediate the antiproliferative response to antitumor Abs, we examined the potential effects of MARCH proteins on responses to emibetuzumab, an anti-MET Ab currently in clinical trials for various cancers. We report that MET surface expression is reduced by MARCH1, 4, or 8-mediated ubiquitination and that emibetuzumab-induced MET ubiquitination contributes to its capacity to downregulate MET and inhibit human tumor cell proliferation. Thus, MARCH E3 ligases can act as cofactors for antitumor Abs that target cell surface proteins, suggesting that the MARCH protein repertoire of cells is a determinant of their response to such Abs.

Diabetes is associated with a deficit of circulating endothelial progenitor cells (EPCs), which has been attributed to their defective mobilization from the bone marrow. The basis for this mobilization defect is not completely understood, and we sought to determine if hyperglycemic conditions enhanced EPC adhesion. We found that culturing EPCs in high glucose media increased adhesion to bone marrow stromal cells. This enhanced adhesion was associated with decreased expression of protein kinase A regulatory subunit 1β (PRKAR1β), activation of protein kinase A (PKA), and phosphorylation of α4-integrin on serine 988. This potentiated adhesion was reversed by treatment with a PKA inhibitor, overexpression of PRKAR1β, or expression of a phosphorylation-defective α4-integrin variant (α4[S988A]). Using a model of type 1 diabetes, we showed that α4(S988A)-expressing mice have more circulating EPCs than their wild-type counterparts. Moreover, diabetic α4(S988A) mice demonstrate enhanced revascularization after hind limb ischemia. Thus, we have identified a novel signaling mechanism activating PKA in diabetes (downregulation of an inhibitory regulatory subunit) that leads to deficits of circulating EPCs and impaired vascular repair, which could be reversed by α4-integrin mutation.

Acute myelogenous leukemia (AML) is an aggressive disease associated with drug resistance and relapse. To improve therapeutic strategies, it is critical to better understand the mechanisms that underlie AML progression. Here we show that the integrin binding glycoprotein CD98 plays a central role in AML. CD98 promotes AML propagation and lethality by driving engagement of leukemia cells with their microenvironment and maintaining leukemic stem cells. Further, delivery of a humanized anti-CD98 antibody blocks growth of patient-derived AML, highlighting the importance of this pathway in human disease. These findings indicate that microenvironmental interactions are key regulators of AML and that disrupting these signals with targeted inhibitors such as CD98 antibodies may be a valuable therapeutic approach for adults and children with this disease.

CD98 is expressed on several tissue types and specifically upregulated on fast-cycling cells undergoing clonal expansion. Various solid (e.g., nonsmall cell lung carcinoma) as well as hematological malignancies (e.g., acute myeloid leukemia) overexpress CD98. We have identified a CD98-specific mouse monoclonal antibody that exhibits potent preclinical antitumor activity against established lymphoma tumor xenografts. Additionally, the humanized antibody designated IGN523 demonstrated robust tumor growth inhibition in leukemic cell-line derived xenograft models and was as efficacious as standard of care carboplatin in patient-derived nonsmall lung cancer xenografts. In vitro studies revealed that IGN523 elicited strong ADCC activity, induced lysosomal membrane permeabilization and inhibited essential amino acid transport function, ultimately resulting in caspase-3 and -7-mediated apoptosis of tumor cells. IGN523 is currently being evaluated in a Phase I clinical trial for acute myeloid leukemia (NCT02040506). Furthermore, preclinical data support the therapeutic potential of IGN523 in solid tumors.

Maintenance of the regulatory T (Treg) cell pool is essential for peripheral tolerance and prevention of autoimmunity. Integrins, heterodimeric transmembrane proteins consisting of α and β subunits that mediate cell-to-cell and cell-to-extracellular matrix interactions, play an important role in facilitating Treg cell contact-mediated suppression. In this article, we show that integrin activation plays an essential, previously unappreciated role in maintaining murine Treg cell function. Treg cell-specific loss of talin, a β integrin-binding protein, or expression of talin(L325R), a mutant that selectively abrogates integrin activation, resulted in lethal systemic autoimmunity. This dysfunction could be attributed, in part, to a global dysregulation of the Treg cell transcriptome. Activation of integrin α₄β₁ led to increased suppressive capacity of the Treg cell pool, suggesting that modulating integrin activation on Treg cells may be a useful therapeutic strategy for autoimmune and inflammatory disorders. Taken together, these results reveal a critical role for integrin-mediated signals in controlling peripheral tolerance by virtue of maintaining Treg cell function.