Jayajit Das

The Balance between T Cell Receptor Signaling and Degradation at the Center of the Immunological Synapse Is Determined by Antigen Quality

The role of the center of the immunological synapse (the central supramolecular activation cluster or cSMAC) is controversial. One model suggests that the role of the cSMAC depends on antigen quality and can both enhance signaling and receptor downregulation, whereas a second model proposes that the sole function of the cSMAC is to downregulate signaling. An important distinction between the models is whether signaling occurs in the cSMAC. Here, we demonstrate that at early time points, signaling occurs outside the cSMAC, but occurs in the cSMAC at later time points. Additionally, we show that cSMAC formation enhances the stimulatory potency of weak agonists for the TCR. Combined with previous studies showing that cSMAC formation decreases the signaling by strong agonists, our data support a model proposing that signaling and receptor degradation both occur in the cSMAC and that the balance between signaling and degradation in the synapse is determined by antigen quality.

Origin of the sharp boundary that discriminates positive and negative selection of thymocytes

T lymphocytes play a key role in adaptive immunity and are activated by interactions of their T cell receptors (TCR) with peptides (p) derived from antigenic proteins bound to MHC gene products. The repertoire of T lymphocytes available in peripheral organs is tuned in the thymus. Immature T lymphocytes (thymocytes) interact with diverse endogenous peptides bound to MHC in the thymus. TCR expressed on thymocytes must bind weakly to endogenous pMHC (positive selection) but must not bind too strongly to them (negative selection) to emerge from the thymus. Negatively selecting pMHC ligands bind TCR with a binding affinity that exceeds a sharply defined (digital) threshold. In contrast, there is no sharp threshold separating positively selecting ligands from those that bind too weakly to elicit a response. We describe results of computer simulations and experiments, which suggest that the contrast between the characters of the positive and negative selection thresholds originates in differences in the way in which Ras proteins are activated by ligands of varying potency. The molecular mechanism suggested by our studies generates hypotheses for how genetic aberrations may dampen the digital negative selection response, with concomitant escape of autoimmune T lymphocytes from the thymus.

Pairing computation with experimentation: a powerful coupling for understanding T cell signalling

T cells are activated when extracellular stimuli, such as a ligand binding to the T cell receptor, are converted into functional outputs by the T cell signalling network. T cell receptor signalling is a highly complex, stochastic and dynamic process involving many interacting proteins. This complexity often confounds intuition, making it difficult to develop mechanistic principles that underly experimental observations. In this Review, we describe how computational approaches can partner successfully with biological experimentation to help address this challenge, and we illustrate this paradigm by summarizing recent work that shows new aspects of the T cell signalling network.

Sensitivity of T cells to antigen and antagonism emerges from differential regulation of the same molecular signaling module

Activation of T helper cells is necessary for the adaptive immune response to pathogens, and spurious activation can result in organ-specific autoimmunity (e.g., multiple sclerosis). T cell activation is initiated by membrane-proximal signaling that is predicated on the binding of the T cell receptor expressed on the T cell surface to peptide major histocompatibility complex (pMHC) molecules presented on the surface of antigen-presenting cells. These signaling processes regulate diverse outcomes, such as the ability of T cells to discriminate sensitively between stimulatory pMHC molecules and those that are characteristic of “self,” and the phenomenon of antagonism (wherein the presence of certain pMHC molecules impairs T cell receptor signaling). We describe a molecular model for membrane-proximal signaling in T cells from which these disparate observations emerge as two sides of the same coin. This development of a unified mechanism that is consistent with diverse data would not have been possible without explicit consideration of the stochastic nature of the pertinent biochemical events. Our studies also reveal that certain previously proposed concepts are not dueling ideas but rather are different stimuli-dependent manifestations of a unified molecular model for membrane-proximal signaling. This model may provide a conceptual framework for further investigations of early events that regulate T cell activation in response to self and foreign antigens and for the development of intervention protocols to inhibit aberrant signaling.

Decreased Diacylglycerol Metabolism Enhances ERK Activation and Augments CD8+ T Cell Functional Responses

Modulation of T cell receptor signal transduction in CD8+ T cells represents a novel strategy toward enhancing the immune response to tumor. Recently, levels of guanine exchange factors, RasGRP and SOS, within T cells have been shown to represent a key determinant in the regulation of the analog to the digital activation threshold of Ras. One important for regulating activation levels of RasGRP is diacylglycerol (DAG), and its levels are influenced by diacylglycerol kinase-ζ (DGKζ), which metabolizes DAG into phosphatidic acid, terminating DAG-mediated Ras signaling. We sought to determine whether DGKζ-deficient CD8+ T cells demonstrated enhanced in vitro responses in a manner predicted by the current model of Ras activation and to evaluate whether targeting this threshold confers enhanced CD8+ T cell responsiveness to tumor. We observed that DGKζ-deficient CD8+ T cells conform to most predictions of the current model of how RasGRP levels influence Ras activation. But our results differ in that the EC50 value of stimulation is not altered for any T cell receptor stimulus, a finding that suggests a further degree of complexity to how DGKζ deficiency affects signals important for Ras and ERK activation. Additionally, we found that DGKζ-deficient CD8+ T cells demonstrate enhanced responsiveness in a subcutaneous lymphoma model, implicating the analog to a digital conversion threshold as a novel target for potential therapeutic manipulation.

The ζ Isoform of Diacylglycerol Kinase Plays a Predominant Role in Regulatory T Cell Development and TCR-Mediated Ras Signaling

Despite its relatively low abundance, DGKζ exerts a major inhibitory effect on T cell receptor signaling. Getting More Activity for Less DGK Strong antigen-dependent activation of the T cell receptor (TCR) in thymocytes leads to the development of natural regulatory T (nTreg) cells through a pathway involving diacylglycerol (DAG). Metabolism of DAG by diacylglycerol kinases (DGKs) to generate phosphatidic acid (PA) limits DAG signaling. In the second of a pair of papers, Joshi et al. characterized the relative contributions of DGKα and DGKζ to TCR-dependent signaling. Although DGKα was more abundant than DGKζ in the cytosol and at the interface between T cells and antigen-presenting cells, only loss of DGKζ enhanced TCR signaling and increased generation of nTreg cells in mice. Mathematical modeling suggested that these results might be explained by the differential catalytic activities of the DGK isoforms, and experiments in T cells showed that DGKζ produced the greatest amounts of PA. Together, these results suggest that the relatively low abundance of DGKζ in T cells belies its importance in inhibiting TCR signaling. Diacylglycerol (DAG) is a critical second messenger that mediates T cell receptor (TCR)–stimulated signaling. The abundance of DAG is reduced by the diacylglycerol kinases (DGKs), which catalyze the conversion of DAG to phosphatidic acid (PA) and thus inhibit DAG-mediated signaling. In T cells, the predominant DGK isoforms are DGKα and DGKζ, and deletion of the genes encoding either isoform enhances DAG-mediated signaling. We found that DGKζ, but not DGKα, suppressed the development of natural regulatory T (Treg) cells and predominantly mediated Ras and Akt signaling downstream of the TCR. The differential functions of DGKα and DGKζ were not attributable to differences in protein abundance in T cells or in their localization to the contact sites between T cells and antigen-presenting cells. RasGRP1, a key DAG-mediated activator of Ras signaling, associated to a greater extent with DGKζ than with DGKα; however, in silico modeling of TCR-stimulated Ras activation suggested that a difference in RasGRP1 binding affinity was not sufficient to cause differences in the functions of each DGK isoform. Rather, the model suggested that a greater catalytic rate for DGKζ than for DGKα might lead to DGKζ exhibiting increased suppression of Ras-mediated signals compared to DGKα. Consistent with this notion, experimental studies demonstrated that DGKζ was more effective than DGKα at catalyzing the metabolism of DAG to PA after TCR stimulation. The enhanced effective enzymatic production of PA by DGKζ is therefore one possible mechanism underlying the dominant functions of DGKζ in modulating Treg cell development.

Positive feedback regulation results in spatial clustering and fast spreading of active signaling molecules on a cell membrane

Positive feedback regulation is ubiquitous in cell signaling networks, often leading to binary outcomes in response to graded stimuli. However, the role of such feedbacks in clustering, and in spatial spreading of activated molecules, has come to be appreciated only recently. We focus on the latter, using a simple model developed in the context of Ras activation with competing negative and positive feedback mechanisms. We find that positive feedback, in the presence of slow diffusion, results in clustering of activated molecules on the plasma membrane, and rapid spatial spreading as the front of the cluster propagates with a constant velocity (dependent on the feedback strength). The advancing fronts of the clusters of the activated species are rough, with scaling consistent with the Kardar-Parisi-Zhang equation in one dimension. Our minimal model is general enough to describe signal transduction in a wide variety of biological networks where activity in the membrane-proximal region is subject to feedback regulation.

Peptide selectivity discriminates NK cells from KIR2DL2‐ and KIR2DL3‐positive individuals

Natural killer cells are controlled by peptide selective inhibitory receptors for MHC class I, including the killer cell immunoglobulin‐like receptors (KIRs). Despite having similar ligands, KIR2DL2 and KIR2DL3 confer different levels of protection to infectious disease. To investigate how changes in peptide repertoire may differentially affect NK cell reactivity, NK cells from KIR2DL2 and KIR2DL3 homozygous donors were tested for activity against different combinations of strong inhibitory (VAPWNSFAL), weak inhibitory (VAPWNSRAL), and antagonist peptide (VAPWNSDAL). KIR2DL3‐positive NK cells were more sensitive to changes in the peptide content of MHC class I than KIR2DL2‐positive NK cells. These differences were observed for the weakly inhibitory peptide VAPWNSRAL in single peptide and double peptide experiments (p < 0.01 and p < 0.03, respectively). More significant differences were observed in experiments using all three peptides (p < 0.0001). Mathematical modeling of the experimental data demonstrated that VAPWNSRAL was dominant over VAPWNSFAL in distinguishing KIR2DL3‐ from KIR2DL2‐positive donors. Donors with different KIR genotypes have different responses to changes in the peptide bound by MHC class I. Differences in the response to the peptide content of MHC class I may be one mechanism underlying the protective effects of different KIR genes against infectious disease.

Molecular Origin and Functional Consequences of Digital Signaling and Hysteresis During Ras Activation in Lymphocytes

Simulations, theory, and experiments reveal a potential molecular mechanism for digital signaling and short-term molecular memory in lymphocytes. Activation of Ras proteins underlies functional decisions in diverse cell types. Two molecules, Ras-GRP and SOS (Ras–guanine nucleotide–releasing protein and Son of Sevenless, respectively), catalyze Ras activation in lymphocytes. Binding of active Ras to the allosteric pocket of SOS markedly increases the activity of SOS. Thus, there is a positive feedback loop regulating SOS. Combining in silico and in vitro studies, we demonstrate that “digital” signaling in lymphocytes (cells are “on” or “off”) is predicated on this allosteric regulation of SOS. The SOS feedback loop leads to hysteresis in the dose-response curve, which may enable T cells to exhibit “memory” of past encounters with antigen. Ras activation by Ras-GRP alone is “analog” (a graded increase in activation in response to an increase in the amplitude of the stimulus). We describe how the complementary analog (Ras-GRP) and digital (SOS) pathways act on Ras to efficiently convert analog input to digital output and make predictions regarding the importance of digital signaling in lymphocyte function and development.

NK cells: tuned by peptide?

Natural killer cells express multiple receptors for major histocompatibility complex (MHC) class I, including the killer cell immunoglobulin‐like receptors (KIRs) and the C‐type lectin‐like CD94:NKG2 receptors. The KIR locus is extremely polymorphic, paralleling the diversity of its classical MHC class I ligands. Similarly, the conservation of the NKG2 family of receptors parallels the conservation of MHC‐E, the ligand for CD94:NKG2A/C/E. Binding of both CD94:NKG2 heterodimers and KIR to their respective MHC class I ligand is peptide dependent, and despite the evolution of these receptors, they have retained the property of peptide selectivity. Such peptide selectivity affects these two systems in different ways. HLA‐E binding non‐inhibitory peptides augment inhibition at CD94:NKG2A, while HLA‐C binding non‐inhibitory peptides antagonize inhibition at KIR2DL2/3, implying that KIRs are specialized to respond positively to changes in peptide repertoire. Thus, while specific KIRs, such as KIR2DL3, are associated with beneficial outcomes from viral infections, viral peptides augment inhibition at CD94:NKGA. Conversely, NKG2A‐positive NK cells sense MHC class I downregulation more efficiently than KIRs. Thus, these two receptor:ligand systems appear to have complementary functions in recognizing changes in MHC class I.