Mapping the SLP76 interactome in T cells lacking each of the GRB2-family adaptors reveals molecular plasticity of the TCR signaling pathway.

The propagation and diversification of signals downstream of the T cell receptor (TCR) involve several adaptor proteins that control the assembly of multimolecular signaling complexes (signalosomes). The global characterization of changes in protein-protein interactions (PPI) following genetic perturbations is critical to understand the resulting phenotypes. Here, by combining genome editing techniques in T cells and interactomics studies based on affinity purification coupled to mass spectrometry (AP-MS) analysis, we determined and quantified the molecular reorganization of the SLP76 interactome resulting from the ablation of each of the three GRB2-family adaptors. Our data showed that the absence of GADS or GRB2 induces a major remodeling of the PPI network associated with SLP76 following TCR engagement. Unexpectedly, this PPI network rewiring minimally affects proximal molecular events of the TCR signaling pathway. Nevertheless, during prolonged TCR stimulation, GRB2- and GADS-deficient cells displayed a reduced level of activation and cytokine secretion capacity. Using the canonical SLP76 signalosome, this analysis highlights the plasticity of PPI networks and their reorganization following specific genetic perturbations.

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.

Quantitative Interactomics in Primary T Cells Provides a Rationale for Concomitant PD-1 and BTLA Coinhibitor Blockade in Cancer Immunotherapy.

Deciphering how TCR signals are modulated by coinhibitory receptors is of fundamental and clinical interest. Using quantitative interactomics, we define the composition and dynamics of the PD-1 and BTLA coinhibitory signalosomes in primary effector T cells and at the T cell-antigen-presenting cell interface. We also solve the existing controversy regarding the role of the SHP-1 and SHP-2 protein-tyrosine phosphatases in mediating PD-1 coinhibition. PD-1 predominantly recruits SHP-2, but when absent, it recruits SHP-1 and remains functional. In contrast, BTLA predominantly recruits SHP-1 and to a lesser extent SHP-2. By separately analyzing the PD-1-SHP-1 and PD-1-SHP-2 complexes, we show that both dampen the TCR and CD28 signaling pathways equally. Therefore, our study illustrates how comparison of coinhibitory receptor signaling via quantitative interactomics in primary T cells unveils their extent of redundancy and provides a rationale for designing combinations of blocking antibodies in cancer immunotherapy on the basis of undisputed modes of action.

The scaffolding function of the RLTPR protein explains its essential role for CD28 co-stimulation in mouse and human T cells.

The RLTPR cytosolic protein, also known as CARMIL2, is essential for CD28 co-stimulation in mice, but its importance in human T cells and mode of action remain elusive. Here, using affinity purification followed by mass spectrometry analysis, we showed that RLTPR acts as a scaffold, bridging CD28 to the CARD11/CARMA1 cytosolic adaptor and to the NF-κB signaling pathway, and identified proteins not found before within the CD28 signaling pathway. We further demonstrated that RLTPR is essential for CD28 co-stimulation in human T cells and that its noncanonical pleckstrin-homology domain, leucine-rich repeat domain, and proline-rich region were mandatory for that task. Although RLTPR is thought to function as an actin-uncapping protein, this property was dispensable for CD28 co-stimulation in both mouse and human. Our findings suggest that the scaffolding role of RLTPR predominates during CD28 co-stimulation and underpins the similar function of RLTPR in human and mouse T cells. Along that line, the lack of functional RLTPR molecules impeded the differentiation toward Th1 and Th17 fates of both human and mouse CD4+ T cells. RLTPR was also expressed in both human and mouse B cells. In the mouse, RLTPR did not play, however, any detectable role in BCR-mediated signaling and T cell-independent B cell responses.