Themis controls T cell activation, effector functions, and metabolism of peripheral CD8+ T cells.

Themis is important in regulating positive selection of thymocytes during T cell development, but its role in peripheral T cells is less understood. Here, we investigated T cell activation and its sequelae using a tamoxifen-mediated, acute Themis deletion mouse model. We find that proliferation, effector functions including anti-tumor killing, and up-regulation of energy metabolism are severely compromised. This study reveals the phenomenon of peripheral adaptation to loss of Themis, by demonstrating direct TCR-induced defects after acute deletion of Themis that were not evident in peripheral T cells chronically deprived of Themis in dLck-Cre deletion model. Peripheral adaptation to long-term loss was compared using chronic versus acute tamoxifen-mediated deletion and with the (chronic) dLck-Cre deletion model. We found that upon chronic tamoxifen-mediated Themis deletion, there was modulation in the gene expression profile for both TCR and cytokine signaling pathways. This profile overlapped with (chronic) dLck-Cre deletion model. Hence, we found that peripheral adaptation induced changes to both TCR and cytokine signaling modules. Our data highlight the importance of Themis in the activation of CD8+ T cells.

CD28-CAR-T cell activation through FYN kinase signaling rather than LCK enhances therapeutic performance.

Signal transduction induced by chimeric antigen receptors (CARs) is generally believed to rely on the activity of the SRC family kinase (SFK) LCK, as is the case with T cell receptor (TCR) signaling. Here, we show that CAR signaling occurs in the absence of LCK. This LCK-independent signaling requires the related SFK FYN and a CD28 intracellular domain within the CAR. LCK-deficient CAR-T cells are strongly signaled through CAR and have better in vivo efficacy with reduced exhaustion phenotype and enhanced induction of memory and proliferation. These distinctions can be attributed to the fact that FYN signaling tends to promote proliferation and survival, whereas LCK signaling promotes strong signaling that tends to lead to exhaustion. This non-canonical signaling of CAR-T cells provides insight into the initiation of both TCR and CAR signaling and has important clinical implications for improvement of CAR function.

Themis is indispensable for IL-2 and IL-15 signaling in T cells.

To perform their antiviral and antitumor functions, T cells must integrate signals both from the T cell receptor (TCR), which instruct the cell to remain quiescent or become activated, and from cytokines that guide cellular proliferation and differentiation. In mature CD8+ T cells, Themis has been implicated in integrating TCR and cytokine signals. We investigated whether Themis plays a direct role in cytokine signaling in mature T cells. Themis was required for IL-2- and IL-15-driven CD8+ T cell proliferation both in mice and in vitro. Mechanistically, we found that Themis promoted the activation of the transcription factor Stat and mechanistic target of rapamycin signaling downstream of cytokine receptors. Metabolomics and stable isotope tracing analyses revealed that Themis deficiency reduced glycolysis and serine and nucleotide biosynthesis, demonstrating a receptor-proximal requirement for Themis in triggering the metabolic changes that enable T cell proliferation. The cellular, metabolic, and biochemical defects caused by Themis deficiency were corrected in mice lacking both Themis and the phosphatase Shp1, suggesting that Themis mediates IL-2 and IL-15 receptor-proximal signaling by restraining the activity of Shp1. Together, these results not only shed light on the mechanisms of cytokine signaling but also provide new clues on manipulating T cells for clinical applications.

Non-Stimulatory pMHC Enhance CD8 T Cell Effector Functions by Recruiting Coreceptor-Bound Lck.

Under physiological conditions, CD8+ T cells need to recognize low numbers of antigenic pMHC class I complexes in the presence of a surplus of non-stimulatory, self pMHC class I on the surface of the APC. Non-stimulatory pMHC have been shown to enhance CD8+ T cell responses to low amounts of antigenic pMHC, in a phenomenon called co-agonism, but the physiological significance and molecular mechanism of this phenomenon are still poorly understood. Our data show that co-agonist pMHC class I complexes recruit CD8-bound Lck to the immune synapse to modulate CD8+ T cell signaling pathways, resulting in enhanced CD8+ T cell effector functions and proliferation, both in vitro and in vivo. Moreover, co-agonism can boost T cell proliferation through an extrinsic mechanism, with co-agonism primed CD8+ T cells enhancing Akt pathway activation and proliferation in neighboring CD8+ T cells primed with low amounts of antigen.

Targeting CAR to the Peptide-MHC Complex Reveals Distinct Signaling Compared to That of TCR in a Jurkat T Cell Model.

Chimeric antigen receptor T cells (CAR-T) utilize T cell receptor (TCR) signaling cascades and the recognition functions of antibodies. This allows T cells, normally restricted by the major histocompatibility complex (MHC), to be redirected to target cells by their surface antigens, such as tumor associated antigens (TAAs). CAR-T technology has achieved significant successes in treatment of certain cancers, primarily liquid cancers. Nonetheless, many challenges hinder development of this therapy, such as cytokine release syndrome (CRS) and the efficacy of CAR-T treatments for solid tumors. These challenges show our inadequate understanding of this technology, particularly regarding CAR signaling, which has been less studied. To dissect CAR signaling, we designed a CAR that targets an epitope from latent membrane protein 2 A (LMP2 A) of the Epstein-Barr virus (EBV) presented on HLA*A02:01. Because of this, CAR and TCR signaling can be compared directly, allowing us to study the involvement of other signaling molecules, such as coreceptors. This comparison revealed that CAR was sufficient to bind monomeric antigens due to its high affinity but required oligomeric antigens for its activation. CAR sustained the transduced signal significantly longer, but at a lower magnitude, than did TCR. CD8 coreceptor was recruited to the CAR synapse but played a negligible role in signaling, unlike for TCR signaling. The distinct CAR signaling processes could provide explanations for clinical behavior of CAR-T therapy and suggest ways to improve the technology.

Lck bound to coreceptor is less active than free Lck.

Src family kinase Lck plays critical roles during T cell development and activation, as it phosphorylates the TCR/CD3 complex to initiate TCR signaling. Lck is present either in coreceptor-bound or coreceptor-unbound (free) forms, and we here present evidence that the two pools of Lck have different molecular properties. We discovered that the free Lck fraction exhibited higher mobility than CD8α-bound Lck in OT-I T hybridoma cells. The free Lck pool showed more activating Y394 phosphorylation than the coreceptor-bound Lck pool. Consistent with this, free Lck also had higher kinase activity, and free Lck mediated higher T cell activation as compared to coreceptor-bound Lck. Furthermore, the coreceptor-Lck coupling was independent of TCR activation. These findings give insights into the initiation of TCR signaling, suggesting that changes in coreceptor-Lck coupling constitute a mechanism for regulation of T cell sensitivity.

A Dual Inhibitor of Cdc7/Cdk9 Potently Suppresses T Cell Activation.

T cell activation is mediated by signaling pathways originating from the T cell receptor (TCR). Propagation of signals downstream of the TCR involves a cascade of numerous kinases, some of which have yet to be identified. Through a screening strategy that we have previously introduced, PHA-767491, an inhibitor of the kinases Cdc7 and Cdk9, was identified to impede TCR signaling. PHA-767491 suppressed several T cell activation phenomena, including the expression of activation markers, proliferation, and effector functions. We also observed a defect in TCR signaling pathways upon PHA-767491 treatment. Inhibition of Cdc7/Cdk9 impairs T cell responses, which could potentially be detrimental for the immune response to tumors, and also compromises the ability to resist infections. The Cdc7/Cdk9 inhibitor is a strong candidate as a cancer therapeutic, but its effect on the immune system poses a problem for clinical applications.

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation.

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.

Themis-associated phosphatase activity controls signaling in T cell development.

Thymocyte-expressed molecule involved in selection (Themis) has been shown to be important for T cell selection by setting the threshold for positive versus negative selection. Themis interacts with the protein tyrosine phosphatase (PTP) Src-homology domain containing phosphatase-1 (Shp1), a negative regulator of the T cell receptor (TCR) signaling cascade. However, how Themis regulates Shp1 is still not clear. Here, using a very sensitive phosphatase assay on ex vivo thymocytes, we have found that Themis enhances Shp1 phosphatase activity by increasing its phosphorylation. This positive regulation of Shp1 activity by Themis is found in thymocytes, but not in peripheral T cells. Shp1 activity is modulated by different affinity peptide MHC ligand binding in thymocytes. Themis is also associated with phosphatase activity, due to its constitutive interaction with Shp1. In the absence of Shp1 in thymocytes, Themis interacts with Shp2, which leads to almost normal thymic development in Shp1 conditional knockout (cKO) mice. Double deletion of both Themis and Shp1 leads to a thymic phenotype similar to that of Themis KO. These findings demonstrate unequivocally that Themis positively regulates Shp1 phosphatase activity in TCR-mediated signaling in developing thymocytes.

Nonstimulatory peptide-MHC enhances human T-cell antigen-specific responses by amplifying proximal TCR signaling.

Foreign antigens are presented by antigen-presenting cells in the presence of abundant endogenous peptides that are nonstimulatory to the T cell. In mouse T cells, endogenous, nonstimulatory peptides have been shown to enhance responses to specific peptide antigens, a phenomenon termed coagonism. However, whether coagonism also occurs in human T cells is unclear, and the molecular mechanism of coagonism is still under debate since CD4 and CD8 coagonism requires different interactions. Here we show that the nonstimulatory, HIV-derived peptide GAG enhances a specific human cytotoxic T lymphocyte response to HBV-derived epitopes presented by HLA-A*02:01. Coagonism in human T cells requires the CD8 coreceptor, but not T-cell receptor (TCR) binding to the nonstimulatory peptide-MHC. Coagonists enhance the phosphorylation and recruitment of several molecules involved in the TCR-proximal signaling pathway, suggesting that coagonists promote T-cell responses to antigenic pMHC by amplifying TCR-proximal signaling.

Cell Type-Specific Regulation of Immunological Synapse Dynamics by B7 Ligand Recognition.

B7 proteins CD80 (B7-1) and CD86 (B7-2) are expressed on most antigen-presenting cells and provide critical co-stimulatory or inhibitory input to T cells via their T-cell-expressed receptors: CD28 and CTLA-4. CD28 is expressed on effector T cells and regulatory T cells (Tregs), and CD28-dependent signals are required for optimum activation of effector T cell functions. CD28 ligation on effector T cells leads to formation of distinct molecular patterns and induction of cytoskeletal rearrangements at the immunological synapse (IS). CD28 plays a critical role in recruitment of protein kinase C (PKC)-θ to the effector T cell IS. CTLA-4 is constitutively expressed on the surface of Tregs, but it is expressed on effector T cells only after activation. As CTLA-4 binds to B7 proteins with significantly higher affinity than CD28, B7 ligand recognition by cells expressing both receptors leads to displacement of CD28 and PKC-θ from the IS. In Tregs, B7 ligand recognition leads to recruitment of CTLA-4 and PKC-η to the IS. CTLA-4 plays a role in regulation of T effector and Treg IS stability and cell motility. Due to their important roles in regulating T-cell-mediated responses, B7 receptors are emerging as important drug targets in oncology. In this review, we present an integrated summary of current knowledge about the role of B7 family receptor-ligand interactions in the regulation of spatial and temporal IS dynamics in effector and Tregs.

Ligand-engaged TCR is triggered by Lck not associated with CD8 coreceptor.

The earliest molecular events in T-cell recognition have not yet been fully described, and the initial T-cell receptor (TCR)-triggering mechanism remains a subject of controversy. Here, using total internal reflection/Forster resonance energy transfer microscopy, we observe a two-stage interaction between TCR, CD8 and major histocompatibility complex (MHC)-peptide. There is an early (within seconds) interaction between CD3ζ and the coreceptor CD8 that is independent of the binding of CD8 to MHC, but that requires CD8 association with Lck. Later (several minutes) CD3ζ-CD8 interactions require CD8-MHC binding. Lck can be found free or bound to the coreceptor. This work indicates that the initial TCR-triggering event is induced by free Lck.

Fine-tuning T cell receptor signaling to control T cell development.

T cell development from immature CD4(+)CD8(+) double-positive (DP) thymocytes to the mature CD4 or CD8 single-positive (SP) stage requires proper T cell receptor (TCR) signaling. The current working model of thymocyte development is that the strength of the TCR-mediated signal - from little-or-none, through intermediate, to strong - received by the immature cells determines whether they will undergo death by neglect, positive selection, or negative selection, respectively. In recent years, several developmentally regulated, stage-specifically expressed proteins and miRNAs have been found that act like fine-tuners for signal transduction and propagation downstream of the TCR. This allows them to govern thymocyte positive selection. Here, we summarize recent findings on these molecules and suggest new concepts of TCR positive-selection signaling.

Themis sets the signal threshold for positive and negative selection in T-cell development.

Development of a self-tolerant T-cell receptor (TCR) repertoire with the potential to recognize the universe of infectious agents depends on proper regulation of TCR signalling. The repertoire is whittled down during T-cell development in the thymus by the ability of quasi-randomly generated TCRs to interact with self-peptides presented by major histocompatibility complex (MHC) proteins. Low-affinity TCR interactions with self-MHC proteins generate weak signals that initiate 'positive selection', causing maturation of CD4- or CD8αß-expressing 'single-positive' thymocytes from CD4(+)CD8αß(+) 'double-positive' precursors. These develop into mature naive T cells of the secondary lymphoid organs. TCR interaction with high-affinity agonist self-ligands results in 'negative selection' by activation-induced apoptosis or 'agonist selection' of functionally differentiated self-antigen-experienced T cells. Here we show that positive selection is enabled by the ability of the T-cell-specific protein Themis to specifically attenuate TCR signal strength via SHP1 recruitment and activation in response to low- but not high-affinity TCR engagement. Themis acts as an analog-to-digital converter translating graded TCR affinity into clear-cut selection outcome. By dampening mild TCR signals Themis increases the affinity threshold for activation, enabling positive selection of T cells with a naive phenotype in response to low-affinity self-antigens.

Coreceptor affinity for MHC defines peptide specificity requirements for TCR interaction with coagonist peptide-MHC.

Recent work has demonstrated that nonstimulatory endogenous peptides can enhance T cell recognition of antigen, but MHCI- and MHCII-restricted systems have generated very different results. MHCII-restricted TCRs need to interact with the nonstimulatory peptide-MHC (pMHC), showing peptide specificity for activation enhancers or coagonists. In contrast, the MHCI-restricted cells studied to date show no such peptide specificity for coagonists, suggesting that CD8 binding to noncognate MHCI is more important. Here we show how this dichotomy can be resolved by varying CD8 and TCR binding to agonist and coagonists coupled with computer simulations, and we identify two distinct mechanisms by which CD8 influences the peptide specificity of coagonism. Mechanism 1 identifies the requirement of CD8 binding to noncognate ligand and suggests a direct relationship between the magnitude of coagonism and CD8 affinity for coagonist pMHCI. Mechanism 2 describes how the affinity of CD8 for agonist pMHCI changes the requirement for specific coagonist peptides. MHCs that bind CD8 strongly were tolerant of all or most peptides as coagonists, but weaker CD8-binding MHCs required stronger TCR binding to coagonist, limiting the potential coagonist peptides. These findings in MHCI systems also explain peptide-specific coagonism in MHCII-restricted cells, as CD4-MHCII interaction is generally weaker than CD8-MHCI.

Properties and applications of single-chain major histocompatibility complex class I molecules.

Stable major histocompatibility complex (MHC) class I molecules at the cell surface consist of three separate, noncovalently associated components: the class I heavy chain, the ß(2)-microglobulin light chain, and a presented peptide. These three components are assembled inside cells via complex pathways involving many other proteins that have been studied extensively. Correct formation of disulfide bonds in the endoplasmic reticulum is central to this process of MHC class I assembly. For a single specific peptide to be presented at the cell surface for possible immune recognition, between hundreds and thousands of peptide-containing precursor polypeptides are required, so the overall process is relatively inefficient. To increase the efficiency of antigen presentation by MHC class I molecules, and for possible therapeutic purposes, single-chain molecules have been developed in which the three, normally separate components have been joined together via flexible linker sequences in a single polypeptide chain. Remarkably, these single-chain MHC class I molecules fold up correctly, as judged by functional recognition by cells of the immune system, and more recently by X-ray crystallographic structural data. This review focuses on the interesting properties and potential of this new type of engineered MHC class I molecule.

Dimerization of soluble disulfide trap single-chain major histocompatibility complex class I molecules dependent on peptide binding affinity.

Stable presentation of peptide epitope by major histocompatibility complex (MHC) class I molecules is a prerequisite for the efficient expansion of CD8(+) T cells. The construction of single-chain MHC class I molecules in which the peptide, ß(2)-microglobulin, and MHC heavy chain are all joined together via flexible linkers increases peptide-MHC stability. We have expressed two T cell epitopes that may be useful in leukemia treatment as single-chain MHC class I molecules, aiming to develop a system for the expansion of antigen-specific CD8(+) T cells in vitro. Disulfide trap versions of these single-chain MHC molecules were also created to improve anchoring of the peptides in the MHC molecule. Unexpectedly, we observed that soluble disulfide trap single-chain molecules expressed in eukaryotic cells were prone to homodimerization, depending on the binding affinity of the peptide epitope. The dimers were remarkably stable and efficiently recognized by conformation-specific antibodies, suggesting that they consisted of largely correctly folded molecules. However, dimerization was not observed when the disulfide trap molecules were expressed as full-length, transmembrane-anchored molecules. Our results further emphasize the importance of peptide binding affinity for the efficient folding of MHC class I molecules.

Ligand dimensions are important in controlling NK-cell responses.

Size-dependent protein segregation at the cell-cell contact interface has been suggested to be critical for regulation of lymphocyte function. We investigated the role of ligand dimensions in regulation of mouse NK-cell activation and inhibition. Elongated forms of H60a, a mouse NKG2D ligand, were generated and expressed stably in the RMA cell line. RMA cells expressing the normal size H60a were lysed efficiently by both freshly isolated and IL-2 stimulated C57BL/6 mouse-derived NK cells; however the level of lysis decreased as the H60a ligand size increased. Importantly, H60a elongation did not affect NKG2D binding, as determined by soluble NKG2D tetramer staining, and by examining NK-cell target cell conjugate formation. CHO cells are efficient at activating NK cells from C57BL/6 mice, and expression of a single chain form of H-2K(b), a ligand for the mouse inhibitory receptor Ly49C, strongly inhibited such activation of Ly49C/I positive NK cells. Elongation of H-2K(b) resulted in decreased inhibition of both lysis and IFN-gamma production by NK cells. These results establish that small ligand dimensions are important for both NK-cell activation and inhibition, and suggest that there are shared features between the mechanisms of receptor triggering on different types of lymphocytes.

Membranous structures transfer cell surface proteins across NK cell immune synapses.

Intercellular transfer of cell surface proteins is widespread and facilitates several recently discovered means for immune cell communication. Here, we examined the molecular mechanism for intercellular exchange of the natural killer (NK) cell receptor KIR2DL1 and HLA-C, prototypical proteins that swap between NK cells and target cells. Transfer was contact dependent and enhanced for cells expressing cognate receptor/ligand pairs but did not depend on KIR2DL1 signaling. To a lesser extent, proteins transferred independent from specific recognition. Intracellular domains of transferred proteins were not exposed to the extracellular environment and transferred proteins were removed by brief exposure to low pH. By fluorescence microscopy, transferred proteins localized to discrete regions on the recipient cell surface. Higher resolution scanning electron micrographs revealed that transferred proteins were located within specific membranous structures. Transmission electron microscopy of the immune synapse revealed that membrane protrusions from one cell interacted with the apposing cell surface within the synaptic cleft. These data, coupled with previous observations, lead us to propose that intercellular protein transfer is mediated by membrane protrusions within and surrounding the immunological synapse.