Pro- and antitumorigenic functions of γδ T cells.

γδ T cells are a subset of T cells that are characterized by the expression of a TCR-γδ instead of a TCR-αß. Despite being outnumbered by their αß T cell counterpart in many tissues, studies from the last 20 years underline their important and non-redundant roles in tumor and metastasis development. However, whether a γδ T cell exerts pro- or antitumorigenic effects seems to depend on a variety of factors, many of them still incompletely understood today. In this review, we summarize mechanisms by which γδ T cells exert these seemingly contradictory effector functions in mice and humans. Furthermore, we discuss the current view on inducing and inhibiting factors of γδ T cells during cancer development.

Perinatal thymic-derived CD8αß-expressing γδ T cells are innate IFN-γ producers that expand in IL-7R-STAT5B-driven neoplasms.

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 CD8αß heterodimers and expand in preclinical models of infection and cancer. Optimal CD8αß+ 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 CD8αß+ γδ T cells in the thymus and peripheral lymphoid organs in two mouse models of T cell neoplasia. Likewise, CD8αß+ 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 CD8αß+ γδ T cells that are enriched in early life and contribute to innate IFN-γ responses to infection and cancer.

Kidins220 and Aiolos promote thymic iNKT cell development by reducing TCR signals.

Development of T cells is controlled by the signal strength of the TCR. The scaffold protein kinase D-interacting substrate of 220 kilodalton (Kidins220) binds to the TCR; however, its role in T cell development was unknown. Here, we show that T cell-specific Kidins220 knockout (T-KO) mice have strongly reduced invariant natural killer T (iNKT) cell numbers and modest decreases in conventional T cells. Enhanced apoptosis due to increased TCR signaling in T-KO iNKT thymocytes of developmental stages 2 and 3 shows that Kidins220 down-regulates TCR signaling at these stages. scRNA-seq indicated that the transcription factor Aiolos is down-regulated in Kidins220-deficient iNKT cells. Analysis of an Aiolos KO demonstrated that Aiolos is a downstream effector of Kidins220 during iNKT cell development. In the periphery, T-KO iNKT cells show reduced TCR signaling upon stimulation with α-galactosylceramide, suggesting that Kidins220 promotes TCR signaling in peripheral iNKT cells. Thus, Kidins220 reduces or promotes signaling dependent on the iNKT cell developmental stage.

Maternal γδ T cells shape offspring pulmonary type 2 immunity in a microbiota-dependent manner.

Immune development is profoundly influenced by vertically transferred cues. However, little is known about how maternal innate-like lymphocytes regulate offspring immunity. Here, we show that mice born from γδ T cell-deficient (TCRδ-/-) dams display an increase in first-breath-induced inflammation, with a pulmonary milieu selectively enriched in type 2 cytokines and type 2-polarized immune cells, when compared with the progeny of γδ T cell-sufficient dams. Upon helminth infection, mice born from TCRδ-/- dams sustain an increased type 2 inflammatory response. This is independent of the genotype of the pups. Instead, the offspring of TCRδ-/- dams harbors a distinct intestinal microbiota, acquired during birth and fostering, and decreased levels of intestinal short-chain fatty acids (SCFAs), such as pentanoate and hexanoate. Importantly, exogenous SCFA supplementation inhibits type 2 innate lymphoid cell function and suppresses first-breath- and infection-induced inflammation. Taken together, our findings unravel a maternal γδ T cell-microbiota-SCFA axis regulating neonatal lung immunity.

Distinct metabolic programs established in the thymus control effector functions of γδ T cell subsets in tumor microenvironments.

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.

From thymus to periphery: Molecular basis of effector γδ-T cell differentiation.

The contributions of γδ T cells to immune (patho)physiology in many pre-clinical mouse models have been associated with their rapid and abundant provision of two critical cytokines, interferon-γ (IFN-γ) and interleukin-17A (IL-17). These are typically produced by distinct effector γδ T cell subsets that can be segregated on the basis of surface expression levels of receptors such as CD27, CD44 or CD45RB, among others. Unlike conventional T cells that egress the thymus as naïve lymphocytes awaiting further differentiation upon activation, a large fraction of murine γδ T cells commits to either IFN-γ or IL-17 expression during thymic development. However, extrathymic signals can both regulate pre-programmed γδ T cells; and induce peripheral differentiation of naïve γδ T cells into effectors. Here we review the key cellular events of "developmental pre-programming" in the mouse thymus; and the molecular basis for effector function maintenance vs plasticity in the periphery. We highlight some of our contributions towards elucidating the role of T cell receptor, co-receptors (like CD27 and CD28) and cytokine signals (such as IL-1ß and IL-23) in these processes, and the various levels of gene regulation involved, from the chromatin landscape to microRNA-based post-transcriptional control of γδ T cell functional plasticity.

Noncanonical binding of Lck to CD3ε promotes TCR signaling and CAR function.

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.

Proximal Lck Promoter-Driven Cre Function Is Limited in Neonatal and Ineffective in Adult γδ T Cell Development.

During T cell development, Lck gene expression is temporally controlled by its proximal and distal promoters. The pLckCre transgenic mouse available from The Jackson Laboratory, in which the proximal promoter of Lck drives Cre expression, is a commonly used Cre driver line to recombine genes flanked by loxP sites in T cells. pLckCre drives recombination early in thymocyte development and is frequently used to delete genes in αß and γδ T cells. We found that pLckCre failed to efficiently delete floxed genes in γδ T cells in contrast to a complete deletion in conventional as well as unconventional αß T cells. Mechanistically, γδ T cells inefficiently transcribed the endogenous proximal Lck promoter compared with αß T cells during adult thymic development. A small population of γδ T cells that had activated pLckCre was detected, many of which were located in nonlymphoid organs as well as precommitted IL-17- or IFN-γ-producing γδ T effector cells. In newborn thymi, both pLckCre and endogenous Lck proximal promoter expression were substantially enhanced, giving rise to an elevated fraction of γδ T cells with recombined floxed genes that were increased in unique γδ T subsets, such as the IL-17-producing γδ T cells. Our data point out striking differences in Lck transcription between perinatal and adult γδ T cell development. Taken together, the data presented in this study shed new light on γδ T cell development and stimulate a reanalysis of data generated using the pLckCre transgenic mice.

Caveolin-1: The Unnoticed Player in TCR and BCR Signaling.

T and B lymphocytes are key players of the adaptive immune system. They recognize pathogenic cues via the T cell antigen receptor (TCR) and the B cell antigen receptor (BCR) to get activated and execute their protective function. TCR and BCR signaling are initiated at the plasma membrane and subsequently propagated into the cell, ultimately leading to cell activation and a protective immune response. However, inappropriate activation of T and B cells can be detrimental to the host resulting in autoimmune disorders, immunodeficiencies, and cancer. The TCR and BCR are located at the plasma membrane, which composition is highly heterogenic. Membrane compartmentalization based on specific lipid-lipid and protein-lipid interactions has raised the interest of the scientific community, converting the plasma membrane into an active player in the initiation of signaling and adding an additional layer of regulation to our current understanding of the functioning of antigen receptors. Caveolin-1 is an integral membrane protein and a crucial component of caveolae. It has been long thought that lymphocytes lack Caveolin-1 expression, due to the absence of detectable caveolae in lymphocytes and the failure to detect Caveolin-1 in T and B cell lines. However, Caveolin-1 is expressed at low levels in primary lymphocytes, and recent studies have shown the importance of Caveolin-1 for the basal membrane organization of the BCR and the TCR as well as their reorganization upon activation. Here, we review our current understanding of the initial signaling events of TCR and BCR activation with respect to receptor compartmentalization on the plasma membrane and with special emphasis on the previously unnoticed role of Caveolin-1.

Caveolin-1-dependent nanoscale organization of the BCR regulates 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.

The BTG2-PRMT1 module limits pre-B cell expansion by regulating the CDK4-Cyclin-D3 complex.

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.

Activation loop phosphorylation regulates B-Raf in vivo and transformation by B-Raf mutants.

Despite being mutated in cancer and RASopathies, the role of the activation segment (AS) has not been addressed for B-Raf signaling in vivo. Here, we generated a conditional knock-in mouse allowing the expression of the B-Raf(AVKA) mutant in which the AS phosphoacceptor sites T599 and S602 are replaced by alanine residues. Surprisingly, despite producing a kinase-impaired protein, the Braf(AVKA) allele does not phenocopy the lethality of Braf-knockout or paradoxically acting knock-in alleles. However, Braf(AVKA) mice display abnormalities in the hematopoietic system, a distinct facial morphology, reduced ERK pathway activity in the brain, and an abnormal gait. This phenotype suggests that maximum B-Raf activity is required for the proper development, function, and maintenance of certain cell populations. By establishing conditional murine embryonic fibroblast cultures, we further show that MEK/ERK phosphorylation and the immediate early gene response toward growth factors are impaired in the presence of B-Raf(AVKA). Importantly, alanine substitution of T599/S602 impairs the transformation potential of oncogenic non-V600E B-Raf mutants and a fusion protein, suggesting that blocking their phosphorylation could represent an alternative strategy to ATP-competitive inhibitors.

Kidins220/ARMS binds to the B cell antigen receptor and regulates B cell development and activation.

B cell antigen receptor (BCR) signaling is critical for B cell development and activation. Using mass spectrometry, we identified a protein kinase D-interacting substrate of 220 kD (Kidins220)/ankyrin repeat-rich membrane-spanning protein (ARMS) as a novel interaction partner of resting and stimulated BCR. Upon BCR stimulation, the interaction increases in a Src kinase-independent manner. By knocking down Kidins220 in a B cell line and generating a conditional B cell-specific Kidins220 knockout (B-KO) mouse strain, we show that Kidins220 couples the BCR to PLCγ2, Ca(2+), and extracellular signal-regulated kinase (Erk) signaling. Consequently, BCR-mediated B cell activation was reduced in vitro and in vivo upon Kidins220 deletion. Furthermore, B cell development was impaired at stages where pre-BCR or BCR signaling is required. Most strikingly, λ light chain-positive B cells were reduced sixfold in the B-KO mice, genetically placing Kidins220 in the PLCγ2 pathway. Thus, our data indicate that Kidins220 positively regulates pre-BCR and BCR functioning.

The CD3 conformational change in the γδ T cell receptor is not triggered by antigens but can be enforced to enhance tumor killing.

Activation of the T cell receptor (TCR) by antigen is the key step in adaptive immunity. In the αßTCR, antigen induces a conformational change at the CD3 subunits (CD3 CC) that is absolutely required for αßTCR activation. Here, we demonstrate that the CD3 CC is not induced by antigen stimulation of the mouse G8 or the human Vγ9Vδ2 γδTCR. We find that there is a fundamental difference between the activation mechanisms of the αßTCR and γδTCR that map to the constant regions of the TCRαß/γδ heterodimers. Enforced induction of CD3 CC with a less commonly used monoclonal anti-CD3 promoted proximal γδTCR signaling but inhibited cytokine secretion. Utilizing this knowledge, we could dramatically improve in vitro tumor cell lysis by activated human γδ T cells. Thus, manipulation of the CD3 CC might be exploited to improve clinical γδ T cell-based immunotherapies.

Activation of the TCR complex by peptide-MHC and superantigens.

Drug hypersensitivity reactions are immune mediated, with T lymphocytes being stimulated by the drugs via their T-cell antigen receptor (TCR). In the nonpathogenic state, the TCR is activated by foreign peptides presented by major histocompatibility complex molecules (pMHC). Foreign pMHC binds with sufficient affinity to TCRαß and thereby elicits phosphorylation of the cytoplasmic tails of the TCRαß-associated CD3 subunits. The process is called TCR triggering. In this review, we discuss the current models of TCR triggering and which drug properties are crucial for TCR stimulation. The underlying molecular mechanisms mostly include pMHC-induced exposure of the CD3 cytoplasmic tails or alterations of the kinase-phosphatase equilibrium in the vicinity of CD3. In this review, we also discuss triggering of the TCR by small chemical compounds in context of these general mechanisms.

Pre-clustering of the B cell antigen receptor demonstrated by mathematically extended electron microscopy.

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.

Visualization of TCR nanoclusters via immunogold labeling, freeze-etching, and surface replication.

T cells show high sensitivity for antigen, even though their T-cell antigen receptor (TCR) has a low affinity for its ligand, a major histocompatibility complex molecule presenting a short pathogen-derived peptide. Over the past few years, it has become clear that these paradoxical properties rely at least in part on the organization of cell surface-expressed TCRs in TCR nanoclusters. We describe a protocol, comprising immunogold labeling, cell surface replica generation, and electron microscopy (EM) analysis that allows nanoscale resolution of the distribution of TCRs and other cell surface molecules of cells grown in suspension. Unlike most of the light microscopy-based single-molecule resolution techniques, this technique permits visualization of these molecules on cell surfaces that do not adhere to an experimental support. Given the potential of adhesion-induced receptor redistributions, our technique is a relevant complement to the substrate adherence-dependent techniques. Furthermore, it does not rely on introduction of fluorescently labeled recombinant molecules and therefore allows direct analysis of nonmanipulated primary cells.