The mechanisms by which a T cell detects antigen using its T cell antigen receptor (TCR) are crucial to our understanding of immunity and the harnessing of T cells therapeutically. A hallmark of the T cell response is the ability of T cells to quantitatively respond to antigenic ligands derived from pathogens while remaining inert to similar ligands derived from host tissues. Recent studies have revealed exciting properties of the TCR and the behaviors of its signaling effectors that are used to detect and discriminate between antigens. Here we highlight these recent findings, focusing on the proximal TCR signaling molecules Zap70, Lck, and LAT, to provide mechanistic models and insights into the exquisite sensitivity and specificity of the TCR.

CD22 is an inhibitory coreceptor on the surface of B cells that attenuates B cell antigen receptor (BCR) signaling and, therefore, B cell activation. Elucidating the molecular mechanisms underlying the inhibitory activity of CD22 is complicated by the ubiquity of CD22 ligands. Although antigens can display CD22 ligands, the receptor is known to bind to sialylated glycoproteins on the cell surface. The propinquity of CD22 and cell-surface glycoprotein ligands has led to the conclusion that the inhibitory properties of the receptor are due to cis interactions. Here, we examine the functional consequences of trans interactions by employing sialylated multivalent antigens that can engage both CD22 and the BCR. Exposure of B cells to sialylated antigens results in the inhibition of key steps in BCR signaling. These results reveal that antigens bearing CD22 ligands are powerful suppressors of B cell activation. The ability of sialylated antigens to inhibit BCR signaling through trans CD22 interactions reveals a previously unrecognized role for the Siglec-family of receptors as modulators of immune signaling.B cell antigen receptor ͉ multivalency ͉ sialic acid ͉ siglec ͉ autoimmunity T he initiation of an immune response or the prevention of autoimmunity depends upon the ability of the B cell antigen receptor (BCR) to transmit signals that positively or negatively regulate B lymphocyte survival, proliferation, and differentiation (1). To avoid detrimental autoimmune responses, a means of differentiating between foreign and self-antigens is required; coreceptors that modulate BCR signaling can ensure that these distinctions are made. CD22 is an inhibitory coreceptor that can attenuate BCR signaling (2, 3). CD22 null mice possess hyperresponsive B cells (4), illustrating a role for CD22 in establishing a threshold for B cell activation. Specifically, an increase in intracellular Ca 2ϩ ion concentration is a hallmark of B cell activation (5, 6), and B cells isolated from CD22 null mice display increased Ca 2ϩ flux in response to antigen (4, 7). Thus, loss of CD22 results in a lowering of the threshold for B cell activation. Other data also support this conclusion: CD22 null mice exhibit increased serum IgM concentrations, decreased surface IgM levels on peripheral B cells, increased induction of apoptosis in response to BCR crosslinking, and increased serum autoantibody titers (8). These observations are consistent with the loss of CD22 leading to increased sensitivity and chronic B cell activation.The process of B cell activation ensues upon binding of multivalent antigen to the BCR. Antigen-induced clustering elicits phosphorylation of the cytoplasmic immunoreceptor tyrosinebased activation motifs (ITAMs), which are present in the BCRassociated signaling proteins Ig␣/␤. The phosphorylation reaction is catalyzed by Src-family kinases such as Lyn. Upon phosphorylation of the BCR components, Syk kinase is recruited to the BCR signaling complex (9). Syk is essential for propagating BCR signaling (10, 11). It acts along with...

Efficacious vaccines require antigens that elicit productive immune system activation. Antigens that afford robust antibody production activate both B and T cells. Elucidating the antigen properties that enhance B–T cell communication is difficult with traditional antigens. We therefore used ring-opening metathesis polymerization to access chemically defined, multivalent antigens containing both B and T cell epitopes to explore how antigen structure impacts B cell and T cell activation and communication. The bifunctional antigens were designed so that the backbone substitution level of each antigenic epitope could be quantified using 19F NMR. The T cell peptide epitope was appended so that it could be liberated in B cells via the action of the endosomal protease cathepsin D, and this design feature was critical for T cell activation. Antigens with high BCR epitope valency induce greater BCR-mediated internalization and T cell activation than did low valency antigens, and these high-valency polymeric antigens were superior to protein antigens. We anticipate that these findings can guide the design of more effective vaccines.

T cells require the protein tyrosine phosphatase CD45 to detect and respond to antigen because it activates the Src family kinase Lck, which phosphorylates the T cell antigen receptor (TCR) complex. CD45 activates Lck by opposing the negative regulatory kinase Csk. Paradoxically, CD45 has also been implicated in suppressing TCR signaling by dephosphorylating the same signaling motifs within the TCR complex upon which Lck acts. We sought to reconcile these observations using chemical and genetic perturbations of the Csk/CD45 regulatory axis incorporated with computational analyses. Specifically, we titrated the activities of Csk and CD45 and assessed their influence on Lck activation, TCR-associated ζ-chain phosphorylation, and more downstream signaling events. Acute inhibition of Csk revealed that CD45 suppressed ζ-chain phosphorylation and was necessary for a regulatable pool of active Lck, thereby interconnecting the activating and suppressive roles of CD45 that tune antigen discrimination. CD45 suppressed signaling events that were antigen independent or induced by low-affinity antigen but not those initiated by high-affinity antigen. Together, our findings reveal that CD45 acts as a signaling “gatekeeper,” enabling graded signaling outputs while filtering weak or spurious signaling events.

SUMMARY The Src Family kinase Lck sets a critical threshold for T cell activation because it phosphorylates the TCR complex and the Zap70 kinase. How a T cell controls the abundance of active Lck molecules remains poorly understood. We have identified an unappreciated role for a phosphosite, Y192, within the Lck SH2 domain which profoundly affects the amount of active Lck in cells. Notably, mutation of Y192 blocks critical TCR proximal signaling events and impairs thymocyte development in retrogenic mice. We determined that these defects are caused by hyperphosphorylation of the inhibitory C-terminal tail of Lck. Our findings reveal that modification of Y192 inhibits the ability of CD45 to associate with Lck in cells and dephosphorylate the C-terminal tail of Lck which prevents its adoption of an active open conformation. These results suggest a negative feedback loop which responds to signaling events that tune active Lck amounts and TCR sensitivity.

In organisms, cell-fate decisions result from external cues presented by the extracellular microenvironment or the niche. In principle, synthetic niches can be engineered to give rise to patterned cell signaling, an advance that would transform the fields of tissue engineering and regenerative medicine. Biomaterials that display adhesive motifs are critical steps in this direction, but promoting localized signaling remains a major obstacle. We sought to exert precise spatial control over activation of TGF-β signaling. TGF-β signaling, which plays fundamental roles in development, tissue homeostasis, and cancer, is initiated by receptor oligomerization. We therefore hypothesized that preorganizing the transmembrane receptors would potentiate local TGF-β signaling. To generate surfaces that would nucleate the signaling complex, we employed defined self-assembled monolayers that present peptide ligands to TGF-β receptors. These displays of nondiffusible ligands do not compete with the growth factor but rather sensitize bound cells to subpicomolar concentrations of endogenous TGF-β. Cells adhering to the surfaces undergo TGF-β-mediated growth arrest and the epithelial to mesenchymal transition. Gene expression profiles reveal that the surfaces selectively regulate TGF-β responsive genes. This strategy provides access to tailored surfaces that can deliver signals with spatial control.

The C-terminal Src kinase (Csk), the primary negative regulator of Src-family kinases (SFK), plays a crucial role in controlling basal and inducible receptor signaling. To investigate how Csk activity regulates T cell antigen receptor (TCR) signaling, we utilized a mouse expressing mutated Csk (CskAS) whose catalytic activity is specifically and rapidly inhibited by a small molecule. Inhibition of CskAS during TCR stimulation led to stronger and more prolonged TCR signaling and to increased proliferation. Inhibition of CskAS enhanced activation by weak but strictly cognate agonists. Titration of Csk inhibition revealed that a very small increase in SFK activity was sufficient to potentiate T cell responses to weak agonists. Csk plays an important role, not only in basal signaling, but also in setting the TCR signaling threshold and affinity recognition.DOI: http://dx.doi.org/10.7554/eLife.08088.001

B cells detect foreign antigens through their B cell antigen receptor (BCR). The BCR, when engaged by antigen, initiates a signaling cascade. Concurrent with signaling is endocytosis of the BCR complex, which acts to downregulate signaling and facilitate uptake of antigen for processing and display on the cell surface. The relationship between signaling and BCR endocytosis is poorly defined. Here, we explore the interplay between BCR endocytosis and antigens that either promote or inhibit B cell activation. Specifically, synthetic antigens were generated that engage the BCR alone or both the BCR and the inhibitory co-receptor CD22. The lectin CD22, a member of the Siglec family, binds sialic acid-containing glycoconjugates found on host tissues, inhibiting BCR signaling to prevent erroneous B cell activation. At low concentrations, antigens that can co-cluster the BCR and CD22 promote rapid BCR endocytosis; whereas, slower endocytosis occurs with antigens that bind only the BCR. At higher antigen concentrations, rapid BCR endocytosis occurs upon treatment with either stimulatory or inhibitory antigens. Endocytosis of the BCR, in response to synthetic antigens, results in its entry into early endocytic compartments. Although the CD22-binding antigens fail to activate key regulators of antigen presentation (e.g., Syk), they also promote BCR endocytosis, indicating that inhibitory antigens can be internalized. Indeed, at low concentrations inhibitory antigens induce more rapid BCR uptake than do stimulatory antigens. Together, our observations support a functional role for BCR endocytosis in downregulating BCR signaling. The reduction of cell surface BCR levels in the absence of B cell activation should raise the threshold for BCR activation. The ability of the activating synthetic antigens to trigger both signalling and entry of the BCR into early endosomes suggests strategies for targeted antigen delivery.