Antitumor immunity induced by antibody-based natural killer cell engager therapeutics armed with not-alpha IL-2 variant.

Harnessing innate immunity is emerging as a promising therapeutic approach in cancer. We report here the design of tetraspecific molecules engaging natural killer (NK) cell-activating receptors NKp46 and CD16a, the ß-chain of the interleukin-2 receptor (IL-2R), and a tumor-associated antigen (TAA). In vitro, these tetraspecific antibody-based natural killer cell engager therapeutics (ANKETs) induce a preferential activation and proliferation of NK cells, and the binding to the targeted TAA triggers NK cell cytotoxicity and cytokine and chemokine production. In vivo, tetraspecific ANKETs induce NK cell proliferation and their accumulation at the tumor bed, as well as the control of local and disseminated tumors. Treatment of non-human primates with CD20-directed tetraspecific ANKET leads to CD20+ circulating B cell depletion, with minimal systemic cytokine release and no sign of toxicity. Tetraspecific ANKETs, thus, constitute a technological platform for harnessing NK cells as next-generation cancer immunotherapies.

SLAT promotes TCR-mediated, Rap1-dependent LFA-1 activation and adhesion through interaction of its PH domain with Rap1.

SLAT (also known as DEF6) promotes T cell activation and differentiation by regulating NFAT-Ca(2+) signaling. However, its role in TCR-mediated inside-out signaling, which induces integrin activation and T cell adhesion, a central process in T cell immunity and inflammation, has not been explored. Here, we show that SLAT is crucial for TCR-induced adhesion to ICAM-1 and affinity maturation of LFA-1 in CD4(+) T cells. Mechanistic studies revealed that SLAT interacts, through its PH domain, with a key component of inside-out signaling, namely the active form of the small GTPase Rap1 (which has two isoforms, Rap1A and Rap1B). This interaction has been further shown to facilitate the interdependent recruitment of Rap1 and SLAT to the T cell immunological synapse upon TCR engagement. Furthermore, a SLAT mutant lacking its PH domain drastically inhibited LFA-1 activation and CD4(+) T cell adhesion. Finally, we established that a constitutively active form of Rap1, which is present at the plasma membrane, rescues the defective LFA-1 activation and ICAM-1 adhesion in SLAT-deficient (Def6(-/-)) T cells. These findings ascribe a new function to SLAT, and identify Rap1 as a target of SLAT function in TCR-mediated inside-out signaling.

Targeting phosphatase-dependent proteoglycan switch for rheumatoid arthritis therapy.

Despite the availability of several therapies for rheumatoid arthritis (RA) that target the immune system, a large number of RA patients fail to achieve remission. Joint-lining cells, called fibroblast-like synoviocytes (FLS), become activated during RA and mediate joint inflammation and destruction of cartilage and bone. We identify RPTPσ, a transmembrane tyrosine phosphatase, as a therapeutic target for FLS-directed therapy. RPTPσ is reciprocally regulated by interactions with chondroitin sulfate or heparan sulfate containing extracellular proteoglycans in a mechanism called the proteoglycan switch. We show that the proteoglycan switch regulates FLS function. Incubation of FLS with a proteoglycan-binding RPTPσ decoy protein inhibited cell invasiveness and attachment to cartilage by disrupting a constitutive interaction between RPTPσ and the heparan sulfate proteoglycan syndecan-4. RPTPσ mediated the effect of proteoglycans on FLS signaling by regulating the phosphorylation and cytoskeletal localization of ezrin. Furthermore, administration of the RPTPσ decoy protein ameliorated in vivo human FLS invasiveness and arthritis severity in the K/BxN serum transfer model of RA. Our data demonstrate that FLS are regulated by an RPTPσ-dependent proteoglycan switch in vivo, which can be targeted for RA therapy. We envision that therapies targeting the proteoglycan switch or its intracellular pathway in FLS could be effective as a monotherapy or in combination with currently available immune-targeted agents to improve control of disease activity in RA patients.

Association of the EF-hand and PH domains of the guanine nucleotide exchange factor SLAT with IP3 receptor 1 promotes Ca²âº signaling in T cells.

The guanine nucleotide exchange factor SLAT (SWAP-70-like adaptor of T cells) regulates T cell activation and differentiation by enabling Ca(2+) release from intracellular stores in response to stimulation of the T cell receptor (TCR). We found a TCR-induced association between SLAT and inositol 1,4,5-trisphosphate (IP3) receptor type 1 (IP3R1). The N-terminal region of SLAT, which contains two EF-hand motifs that we determined bound Ca(2+), and the SLAT pleckstrin homology (PH) domain independently bound to IP3R1 by associating with a conserved motif within the IP3R1 ligand-binding domain. Disruption of the SLAT-IP3R1 interaction with cell-permeable, IP3R1-based fusion peptides inhibited TCR-stimulated Ca(2+) signaling, activation of the transcription factor NFAT (nuclear factor of activated T cells), and production of cytokines, suggesting that this interaction is required for optimal T cell activation. The finding that SLAT is an IP3R1-interacting protein required for T cell activation suggests that this interaction could be a potential target for a selective immunosuppressive drug.

SLAT/Def6 plays a critical role in the development of Th17 cell-mediated experimental autoimmune encephalomyelitis.

SWAP-70-like adapter of T cells (SLAT; also known as Def6) is a novel guanine nucleotide exchange factor for Rho GTPases that has been previously shown to play a role in CD4+ T cell activation and Th1/Th2 differentiation. However, the role of SLAT/Def6 in autoimmunity and its associated Th1- and Th17-specific responses has not yet been clearly elucidated. We used a prototypical and pathologically relevant Th1/Th17-mediated autoimmune model, that is, experimental autoimmune encephalomyelitis, to assess the role of SLAT/Def6 in autoantigen-specific T cell response. We found that T cell-expressed SLAT/Def6 was critical for experimental autoimmune encephalomyelitis development and pathogenesis, as evidenced by the resistance of Def6-deficient (Def6(-/-)) mice to clinical signs of the disease associated with a lack of CNS inflammation and demyelination in myelin oligodendrocyte glycoprotein-immunized Def6(-/-) mice. Moreover, Def6 deficiency resulted in a severely diminished myelin oligodendrocyte glycoprotein-specific CD4+ T cell proliferation as well as a defect in IFN-gamma and IL-17 production in secondary lymphoid organs and the CNS. Lastly, Def6(-/-) CD4+ T cells were grossly deficient in their ability to differentiate into Th17 cells both in vitro and in vivo in a T cell-intrinsic manner. Therefore, our study establishes T cell-expressed SLAT/Def6 as a pivotal positive regulator of Th17 inflammatory responses and, thus, essential in controlling autoimmune and inflammatory diseases.

Dok-4 is a novel negative regulator of T cell activation.

Dok-4 (downstream of tyrosine kinase-4) is a recently identified member of the Dok family of adaptor proteins, which are characterized by an amino-terminal pleckstrin homology domain, a phosphotyrosine-binding domain, and a carboxyl-terminal region containing several tyrosines and poly-proline-rich motifs. Two members of the Dok family, Dok-1 and Dok-2, have already been described as negative regulators in T cells. However, the function of Dok-4, which is also expressed in T cells, remains unknown. In this study, we report that Dok-4 is phosphorylated after TCR engagement and shuttled within the cytoplasm of T cells before being recruited to the polarized microtubule organizing center after the formation of the immunological synapse. Loss-of-function experiments using RNA interference constructs show that Dok-4 is a negative regulator of ERK phosphorylation, IL-2 promoter activity, and T cell proliferation. Exogenous expression of wild-type Dok-4 induces a significant activation of Rap1, which is involved in the regulation of ERK. The pleckstrin homology domain of Dok-4 is required both for its cytoplasmic shuttling and relocalization as well as for its inhibitory properties on T cell activation. Thus, Dok-4 represents a novel negative regulator of T cells.

ICOS ligation recruits the p50alpha PI3K regulatory subunit to the immunological synapse.

ICOS ligation in concert with TCR stimulation results in strong PI3K activation in T lymphocytes. The ICOS cytoplasmic tail contains an YMFM motif that binds the p85alpha subunit of class IA PI3K, similar to the YMNM motif of CD28, suggesting a redundant function of the two receptors in PI3K signaling. However, ICOS costimulation shows greater PI3K activity than CD28 in T cells. We show in this report that ICOS expression in activated T cells triggers the participation of p50alpha, one of the regulatory subunits of class IA PI3Ks. Using different T-APC cell conjugate systems, we report that p50alpha accumulates at the immunological synapse in activated but not in resting T cells. Our results demonstrate that ICOS membrane expression is involved in this process and that p50alpha plasma membrane accumulation requires a functional YMFM Src homology 2 domain-binding motif in ICOS. We also show that ICOS triggering with its ligand, ICOSL, induces the recruitment of p50alpha at the synapse of T cell/APC conjugates. In association with the p110 catalytic subunit, p50alpha is known to carry a stronger lipid kinase activity compared with p85alpha. Accordingly, we observed that ICOS engagement results in a stronger activation of PI3K. Together, these findings provide evidence that p50alpha is likely a determining factor in ICOS-mediated PI3K activity in T cells. These results also suggest that a differential recruitment and activity of class IA PI3K subunits represents a novel mechanism in the control of PI3K signaling by costimulatory molecules.