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Metabolic programming controls immune cell lineages and functions, but little is known about γδ T cell metabolism. Here, we found that γδ T cell subsets making either interferon-γ (IFN-γ) or interleukin (IL)-17 have intrinsically distinct metabolic requirements. Whereas IFN-γ + γδ T cells were almost exclusively dependent on glycolysis, IL-17 + γδ T cells strongly engaged oxidative metabolism, with increased mitochondrial mass and activity. These distinct metabolic signatures were surprisingly imprinted early during thymic development and were stably maintained in the periphery and within tumors. Moreover, pro-tumoral IL-17 + γδ T cells selectively showed high lipid uptake and intracellular lipid storage and were expanded in obesity and in tumors of obese mice. Conversely, glucose supplementation enhanced the antitumor functions of IFN-γ + γδ T cells and reduced tumor growth upon adoptive transfer. These findings have important implications for the differentiation of effector γδ T cells and their manipulation in cancer immunotherapy. γδ T cells have potent effector functions through their production of IFN-γ or IL-17. Pennington and colleagues demonstrate that IFN-γ +  γδ T and IL-17 + γδ T cells have distinct metabolic requirements that can be independently targeted to elicit specific immune responses.

Initiation of T cell antigen receptor (TCR) signaling involves phosphorylation of CD3 cytoplasmic tails by the tyrosine kinase Lck. How Lck is recruited to the TCR to initiate signaling is not well known. We report a previously unknown binding motif in the CD3ε cytoplasmic tail that interacts in a noncanonical mode with the Lck SH3 domain: the receptor kinase (RK) motif. The RK motif is accessible only upon TCR ligation, demonstrating how ligand binding leads to Lck recruitment. Binding of the Lck SH3 domain to the exposed RK motif resulted in local augmentation of Lck activity, CD3 phosphorylation, T cell activation and thymocyte development. Introducing the RK motif into a well-characterized 41BB-based chimeric antigen receptor enhanced its antitumor function in vitro and in vivo. Our findings underscore how a better understanding of the functioning of the TCR might promote rational improvement of chimeric antigen receptor design for the treatment of cancer. How Lck is recruited to the TCR to initiate signaling is not well known. Here Minguet and colleagues report a previously unknown binding motif in the CD3ε cytoplasmic tail that interacts in a noncanonical mode with the Lck SH3 domain that may help to improve TCR activation and the antitumor activity of a clinically approved CAR.

The contribution of γδ T cells to immune responses is associated with rapid secretion of interferon-γ (IFN-γ). Here, we show a perinatal thymic wave of innate IFN-γ-producing γδ T cells that express CD8αβ heterodimers and expand in preclinical models of infection and cancer. Optimal CD8αβ + γδ T cell development is directed by low T cell receptor signaling and through provision of interleukin (IL)-4 and IL-7. This population is pathologically relevant as overactive, or constitutive, IL-7R–STAT5B signaling promotes a supraphysiological accumulation of CD8αβ + γδ T cells in the thymus and peripheral lymphoid organs in two mouse models of T cell neoplasia. Likewise, CD8αβ + γδ T cells define a distinct subset of human T cell acute lymphoblastic leukemia pediatric patients. This work characterizes the normal and malignant development of CD8αβ + γδ T cells that are enriched in early life and contribute to innate IFN-γ responses to infection and cancer. Here the authors identify and characterize the development and function of an IFN-γ-producing CD8αβ + subset of γδ T cells that contributes to malignancy.

The B cell antigen receptor (BCR) plays a crucial role in adaptive immunity, since antigen-induced signaling by the BCR leads to the activation of the B cell and production of antibodies during an immune response. However, the spatial nano-scale organization of the BCR on the cell surface prior to antigen encounter is still controversial. Here, we fixed murine B cells, stained the BCRs on the cell surface with immuno-gold and visualized the distribution of the gold particles by transmission electron microscopy. Approximately 30% of the gold particles were clustered. However the low staining efficiency of 15% precluded a quantitative conclusion concerning the oligomerization state of the BCRs. To overcome this limitation, we used Monte-Carlo simulations to include or to exclude possible distributions of the BCRs. Our combined experimental-modeling approach assuming the lowest number of different BCR sizes to explain the observed gold distribution suggests that 40% of the surface IgD-BCR was present in dimers and 60% formed large laminar clusters of about 18 receptors. In contrast, a transmembrane mutant of the mIgD molecule only formed IgD-BCR dimers. Our approach complements high resolution fluorescence imaging and clearly demonstrates the existence of pre-formed BCR clusters on resting B cells, questioning the classical cross-linking model of BCR activation.

Expression of a competent Igh heavy chain initiates a pre-BCR checkpoint during B cell development. Reth and colleagues show that protein arginine methylation by a BTG2-PRMT1 complex is required to inactivate CDK4 and thereby establish pre-B cell arrest. Developing pre-B cells in the bone marrow alternate between proliferation and differentiation phases. We found that protein arginine methyl transferase 1 (PRMT1) and B cell translocation gene 2 (BTG2) are critical components of the pre-B cell differentiation program. The BTG2-PRMT1 module induced a cell-cycle arrest of pre-B cells that was accompanied by re-expression of Rag1 and Rag2 and the onset of immunoglobulin light chain gene rearrangements. We found that PRMT1 methylated cyclin-dependent kinase 4 (CDK4), thereby preventing the formation of a CDK4-Cyclin-D3 complex and cell cycle progression. Moreover, BTG2 in concert with PRMT1 efficiently blocked the proliferation of BCR-ABL1-transformed pre-B cells in vitro and in vivo . Our results identify a key molecular mechanism by which the BTG2-PRMT1 module regulates pre-B cell differentiation and inhibits pre-B cell leukemogenesis.

Fiala & Silva-Santos discuss the molecular determinants of the \"developmental preprogramming\" of ... T cells. They provide novel insights on the integration of different receptor signals during ... T cell development and functional precommitment. They established an in vitro system allowing the manipulation of signals encountered by early T cell progenitors, and addressed three major layers of receptor signaling in developing ... thymocytes.(ProQuest: ... denotes formulae/symbols omitted.)

Metabolic programming controls immune cell lineages and functions, but little is known about [gamma][delta] T cell metabolism. Here, we found that [gamma][delta] T cell subsets making either interferon-[gamma] (IFN-[gamma]) or interleukin (IL)-17 have intrinsically distinct metabolic requirements. Whereas IFN-[gamma].sup.+ [gamma][delta] T cells were almost exclusively dependent on glycolysis, IL-17.sup.+ [gamma][delta] T cells strongly engaged oxidative metabolism, with increased mitochondrial mass and activity. These distinct metabolic signatures were surprisingly imprinted early during thymic development and were stably maintained in the periphery and within tumors. Moreover, pro-tumoral IL-17.sup.+ [gamma][delta] T cells selectively showed high lipid uptake and intracellular lipid storage and were expanded in obesity and in tumors of obese mice. Conversely, glucose supplementation enhanced the antitumor functions of IFN-[gamma].sup.+ [gamma][delta] T cells and reduced tumor growth upon adoptive transfer. These findings have important implications for the differentiation of effector [gamma][delta] T cells and their manipulation in cancer immunotherapy.

The dimerisation of Raf kinases involves a central cluster within the kinase domain, the dimer interface (DIF). Yet, the importance of the DIF for the signalling potential of wild‐type B‐Raf (B‐Raf wt ) and its oncogenic counterparts remains unknown. Here, we show that the DIF plays a pivotal role for the activity of B‐Raf wt and several of its gain‐of‐function (g‐o‐f) mutants. In contrast, the B‐Raf V600E , B‐Raf insT and B‐Raf G469A oncoproteins are remarkably resistant to mutations in the DIF. However, compared with B‐Raf wt , B‐Raf V600E displays extended protomer contacts, increased homodimerisation and incorporation into larger protein complexes. In contrast, B‐Raf wt and Raf‐1 wt mediated signalling triggered by oncogenic Ras as well as the paradoxical activation of Raf‐1 by kinase‐inactivated B‐Raf require an intact DIF. Surprisingly, the B‐Raf DIF is not required for dimerisation between Raf‐1 and B‐Raf, which was inactivated by the D594A mutation, sorafenib or PLX4720. This suggests that paradoxical MEK/ERK activation represents a two‐step mechanism consisting of dimerisation and DIF‐dependent transactivation. Our data further implicate the Raf DIF as a potential target against Ras‐driven Raf‐mediated (paradoxical) ERK activation. An intact dimer interface (DIF) is required for both signalling and dimerisation in wild‐type Raf, but surprisingly for neither in certain oncogenic Raf versions. Paradoxical Raf activation reveals an additional layer of complexity, with the DIF now dispensable for dimerisation but required for signalling.

Immature B cells are subject to tolerance mechanisms that prevent the expression of self-reactive BCRs. Minguet and colleagues identify the membrane protein caveolin-1 as a regulator of BCR spatial organization and signaling that enforces B cell tolerance. Caveolin-1 (Cav1) regulates the nanoscale organization and compartmentalization of the plasma membrane. Here we found that Cav1 controlled the distribution of nanoclusters of isotype-specific B cell antigen receptors (BCRs) on the surface of B cells. In mature B cells stimulated with antigen, the immunoglobulin M BCR (IgM-BCR) gained access to lipid domains enriched for GM1 glycolipids, by a process that was dependent on the phosphorylation of Cav1 by the Src family of kinases. Antigen-induced reorganization of nanoclusters of IgM-BCRs and IgD-BCRs regulated BCR signaling in vivo . In immature Cav1-deficient B cells, altered nanoscale organization of IgM-BCRs resulted in a failure of receptor editing and a skewed repertoire of B cells expressing immunoglobulin-μ heavy chains with hallmarks of poly- and auto-reactivity, which ultimately led to autoimmunity in mice. Thus, Cav1 emerges as a cell-intrinsic regulator that prevents B cell–induced autoimmunity by means of its role in plasma-membrane organization.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.