Protein kinase C-η controls CTLA-4-mediated regulatory T cell function.

Regulatory T (Treg) cells, which maintain immune homeostasis and self-tolerance, form an immunological synapse (IS) with antigen-presenting cells (APCs). However, signaling events at the Treg cell IS remain unknown. Here we show that the kinase PKC-η associated with CTLA-4 and was recruited to the Treg cell IS. PKC-η-deficient Treg cells displayed defective suppressive activity, including suppression of tumor immunity but not of autoimmune colitis. Phosphoproteomic and biochemical analysis revealed an association between CTLA-4-PKC-η and the GIT2-αPIX-PAK complex, an IS-localized focal adhesion complex. Defective activation of this complex in PKC-η-deficient Treg cells was associated with reduced depletion of CD86 from APCs by Treg cells. These results reveal a CTLA-4-PKC-η signaling axis required for contact-dependent suppression and implicate this pathway as a potential cancer immunotherapy target.

The lymphoid lineage-specific actin-uncapping protein Rltpr is essential for costimulation via CD28 and the development of regulatory T cells.

Although T cell activation can result from signaling via T cell antigen receptor (TCR) alone, physiological T cell responses require costimulation via the coreceptor CD28. Through the use of an N-ethyl-N-nitrosourea-mutagenesis screen, we identified a mutation in Rltpr. We found that Rltpr was a lymphoid cell-specific, actin-uncapping protein essential for costimulation via CD28 and the development of regulatory T cells. Engagement of TCR-CD28 at the immunological synapse resulted in the colocalization of CD28 with both wild-type and mutant Rltpr proteins. However, the connection between CD28 and protein kinase C-θ and Carma1, two key effectors of CD28 costimulation, was abrogated in T cells expressing mutant Rltpr, and CD28 costimulation did not occur in those cells. Our findings provide a more complete model of CD28 costimulation in which Rltpr has a key role.

Molecular Imaging of PD-1 Unveils Unknown Characteristics of PD-1 Itself by Visualizing "PD-1 Microclusters".

Programmed cell death-1 (PD-1) is one of the most famous coinhibitory receptors that are expressed on effector T cells to regulate their function. The PD-1 ligands, PD-L1 and PD-L2, are expressed by various cells throughout the body at steady state and their expression was further regulated within different pathological conditions such as tumor-bearing and chronic inflammatory diseases. In recent years, immune checkpoint inhibitor (ICI) therapies with anti-PD-1 or anti-PD-L1 has become a standard treatment for various malignancies and has shown remarkable antitumor effects. Since the discovery of PD-1 in 1992, a huge number of studies have been conducted to elucidate the function of PD-1. Herein, this paper provides an overview of PD-1 biological findings and sheds some light on the current technology for molecular imaging of PD-1.

A motif in the V3 domain of the kinase PKC-θ determines its localization in the immunological synapse and functions in T cells via association with CD28.

Protein kinase C-θ (PKC-θ) translocates to the center of the immunological synapse, but the underlying mechanism and its importance in T cell activation are unknown. Here we found that the V3 domain of PKC-θ was necessary and sufficient for localization to the immunological synapse mediated by association with the coreceptor CD28 and dependent on the kinase Lck. We identified a conserved proline-rich motif in V3 required for association with CD28 and immunological synapse localization. We found association with CD28 to be essential for PKC-θ-mediated downstream signaling and the differentiation of T helper type 2 cells (T(H)2 cells) and interleukin 17-producing helper T cells (T(H)17 cells) but not of T helper type 1 cells (T(H)1 cells). Ectopic expression of V3 sequestered PKC-θ from the immunological synapse and interfered with its functions. Our results identify a unique mode of CD28 signaling, establish a molecular basis for the immunological synapse localization of PKC-θ and indicate V3-based 'decoys' may be therapeutic modalities for T cell-mediated inflammatory diseases.

Dynein-driven transport of T cell receptor microclusters regulates immune synapse formation and T cell activation.

When T cells recognize a peptide-major histocompatibility complex on antigen-presenting cells (APCs), T cell receptor microclusters (TCR-MCs) are generated and move to the center of the T cell-APC interface to form the central supramolecular activation cluster (cSMAC). cSMAC formation depends on stimulation strength and regulates T cell activation. We demonstrate that the dynein motor complex colocalized and coimmunoprecipitated with the TCR complex and that TCR-MCs moved along microtubules (MTs) toward the center of the immune synapse in a dynein-dependent manner to form cSMAC. MTs are located in close proximity to the plasma membrane at the activation site. TCR-MC velocity and cSMAC formation were impaired by dynein or MT inhibitors or by ablation of dynein expression. T cells with impaired cSMAC formation exhibited enhanced cellular activation including protein phosphorylation and interleukin-2 production. These results indicate that cSMAC formation by TCR-MC movement depends on dynein and MTs, and the movement regulates T cell activation.

Spatiotemporal basis of CTLA-4 costimulatory molecule-mediated negative regulation of T cell activation.

T cell activation is positively and negatively regulated by a pair of costimulatory receptors, CD28 and CTLA-4, respectively. Because these receptors share common ligands, CD80 and CD86, the expression and behavior of CTLA-4 is critical for T cell costimulation regulation. However, in vivo blocking of CD28-mediated costimulation by CTLA-4 and its mechanisms still remain elusive. Here, we demonstrate the dynamic behavior of CTLA-4 in its real-time competition with CD28 at the central-supramolecular activation cluster (cSMAC), resulting in the dislocalization of protein kinase C-θ and CARMA1 scaffolding protein. CTLA-4 translocation to the T cell receptor microclusters and the cSMAC is tightly regulated by its ectodomain size, and its accumulation at the cSMAC is required for its inhibitory function. The CTLA-4-mediated suppression was demonstrated by the in vitro anergy induction in regulatory T cells constitutively expressing CTLA-4. These results show the dynamic mechanism of CTLA-4-mediated T cell suppression at the cSMAC.

Spatiotemporal regulation of T cell costimulation by TCR-CD28 microclusters and protein kinase C theta translocation.

T cell activation is mediated by microclusters (MCs) containing T cell receptors (TCRs), kinases, and adaptors. Although TCR MCs translocate to form a central supramolecular activation cluster (cSMAC) of the immunological synapse at the interface of a T cell and an antigen-presenting cell, the role of MC translocation in T cell signaling remains unclear. Here, we found that the accumulation of MCs at cSMAC was important for T cell costimulation. Costimulatory receptor CD28 was initially recruited coordinately with TCR to MCs, and its signals were mediated through the assembly with the kinase PKCtheta. The accumulation of MCs at the cSMAC was accompanied by the segregation of CD28 from the TCR, which resulted in the translocation of both CD28 and PKCtheta to a spatially unique subregion of cSMAC. Thus, costimulation is mediated by the generation of a unique costimulatory compartment in the cSMAC via the dynamic regulation of MC translocation.

Dynamic regulation of T-cell costimulation through TCR-CD28 microclusters.

SUMMARY: T-cell activation requires contact between T cells and antigen-presenting cells (APCs) to bring T-cell receptors (TCRs) and major histocompatibility complex peptide (MHCp) together to the same complex. These complexes rearrange to form a concentric circular structure, the immunological synapse (IS). After the discovery of the IS, dynamic imaging technologies have revealed the details of the IS and provided important insights for T-cell activation. We have redefined a minimal unit of T-cell activation, the 'TCR microcluster', which recognizes MHCp, triggers an assembly of assorted molecules downstream of the TCR, and induces effective signaling from TCRs. The relationship between TCR signaling and costimulatory signaling was analyzed in terms of the TCR microcluster. CD28, the most valuable costimulatory receptor, forms TCR-CD28 microclusters in cooperation with TCRs, associates with protein kinase C theta, and effectively induces initial T-cell activation. After mature IS formation, CD28 microclusters accumulate at a particular subregion of the IS, where they continuously assemble with the kinases and not TCRs, and generate sustained T-cell signaling. We propose here a 'TCR-CD28 microcluster' model in which TCR and costimulatory microclusters are spatiotemporally formed at the IS and exhibit fine-tuning of T-cell responses by assembling with specific players downstream of the TCR and CD28.

Evaluation of therapeutic PD-1 antibodies by an advanced single-molecule imaging system detecting human PD-1 microclusters.

With recent advances in immune checkpoint inhibitors (ICIs), immunotherapy has become the standard treatment for various malignant tumors. Their indications and dosages have been determined empirically, taking individually conducted clinical trials into consideration, but without a standard method to evaluate them. Here we establish an advanced imaging system to visualize human PD-1 microclusters, in which a minimal T cell receptor (TCR) signaling unit co-localizes with the inhibitory co-receptor PD-1 in vitro. In these microclusters PD-1 dephosphorylates both the TCR/CD3 complex and its downstream signaling molecules via the recruitment of a phosphatase, SHP2, upon stimulation with the ligand hPD-L1. In this system, blocking antibodies for hPD-1-hPD-L1 binding inhibits hPD-1 microcluster formation, and each therapeutic antibody (pembrolizumab, nivolumab, durvalumab and atezolizumab) is characterized by a proprietary optimal concentration and combinatorial efficiency enhancement. We propose that our imaging system could digitally evaluate PD-1-mediated T cell suppression to evaluate their clinical usefulness and to develop the most suitable combinations among ICIs or between ICIs and conventional cancer treatments.

The immunological synapse, TCR microclusters, and T cell activation.

T cell activation begins with the interaction between an antigen-specific T cell and an antigen-presenting cell (APC). This interaction results in the formation of the immunological synapse, which had been considered to be responsible for antigen recognition and T cell activation. Recent advances in imaging analysis have provided new insights into T cell activation. The T cell receptor (TCR) microclusters, TCRs, kinases, and adaptors are generated upon antigen recognition at the interfaces between the T cells and the APCs and serve as a fundamental signaling unit for T cell activation. CD28-mediated costimulation is also found to be regulated by the formation of microclusters. Therefore, the dynamic regulations of TCR and CD28 microcluster formation, migration, and interaction are the key events for the initiation of T cell-mediated immune responses. Comprehensive analyses of the composition and characteristics of the TCR microcluster have identified its dynamic features. This review will outline new discoveries of the microclusters and its related concept in T cell activation.

CRTAM confers late-stage activation of CD8+ T cells to regulate retention within lymph node.

In vivo immune response is triggered in the lymph node, where lymphocytes for entry into, retention at, and migration to effector sites are dynamically regulated. The molecular mechanism underlying retention regulation is the key to elucidating in vivo regulation of immune response. In this study, we describe the function of the adhesion molecule class I-restricted T cell-associated molecule (CRTAM) in regulating CD8+ T cell retention within the lymph node and eventually effector function. We previously identified CRTAM as a receptor predominantly expressed on activated CD8+ T cells, and nectin-like molecule-2 (Necl2) as its ligand. In vivo function of CRTAM-Necl2 interaction was analyzed by generating CRTAM(-/-) mice. CRTAM(-/-) mice exhibited reduced protective immunity against viral infection and impaired autoimmune diabetes induction in vivo. Although Ag-specific CRTAM(-/-) CD8+ T cells showed normal CTL functions in vitro, their number in the draining lymph node was reduced. Because CRTAM+ T cells bound efficiently to Necl2-expressing CD8+ dendritic cells (DCs) that reside in T cell area of lymph node, CRTAM may induce retention by binding to CD8+ DCs at the late stage of activation before proliferation. The CRTAM-mediated late interaction with DCs induced retention of activated CD8+ T cells in an Ag-independent fashion, and this possibly resulted in effective CTL development in the draining lymph node.

PD-L2 suppresses T cell signaling via coinhibitory microcluster formation and SHP2 phosphatase recruitment.

The coinhibitory receptor, PD-1, is of major importance for the suppression of T cell activation in various types of immune responses. A high-resolution imaging study showed that PD-1 forms a coinhibitory signalosome, "PD-1 microcluster", with the phosphatase, SHP2, to dephosphorylate the TCR/CD3 complex and its downstream signaling molecules. Such a consecutive reaction entirely depended on PD-1-PD-L1/2 binding. PD-L2 is expressed on professional antigen-presenting cells and also on some tumor cells, which possibly explains the discrepant efficacy of immune checkpoint therapy for PD-L1-negative tumors. Here, we performed precise imaging analysis of PD-L2 forming PD-1-PD-L2 clusters associating with SHP2. PD-L2 could compete with PD-L1 for binding to PD-1, occupying the same space at TCR microclusters. The PD-1 microcluster formation was inhibited by certain mAbs with functional consequences. Thus, PD-1 microcluster formation provides a visible index for the effectiveness of anti-PD-1- or anti-PD-L1/2-mediated T cell suppression. PD-L2 may exert immune suppressive responses cooperatively with PD-L1 on the microcluster scale.

Predominant role of T cell receptor (TCR)-alpha chain in forming preimmune TCR repertoire revealed by clonal TCR reconstitution system.

The CDR3 regions of T cell receptor (TCR)-alpha and -beta chains play central roles in the recognition of antigen (Ag)-MHC complex. TCR repertoire is created on the basis of Ag recognition specificity by CDR3s. To analyze the potential spectrum of TCR-alpha and -beta to exhibit Ag specificity and generate TCR repertoire, we established hundreds of TCR transfectants bearing a single TCR-alpha or -beta chain derived from a cytotoxic T cell (CTL) clone, RT-1, specific for HIVgp160 peptide, and randomly picked up TCR-beta or -alpha chains. Surprisingly, one-third of such TCR-beta containing random CDR3 beta from naive T cells of normal mice could reconstitute the antigen-reactive TCR coupling with RT-1 TCR-alpha. A similar dominant function of TCR-alpha in forming Ag-specific TCR, though low-frequency, was obtained for lymphocytic choriomeningitis virus-specific TCR. Subsequently, we generated TCR-alpha and/or -beta transgenic (Tg) mice specific for HIVgp160 peptide, and analyzed the TCR repertoire of Ag-specific CTLs. Similar to the results from TCR reconstitution, TCR-alpha Tg generated CTLs with heterogeneous TCR-beta, whereas TCR-beta Tg-induced CTLs bearing a single TCR-alpha. These findings of Ag recognition with minimum involvement of CDR3 beta expand our understanding regarding the flexibility of the spectrum of TCR and suggest a predominant role of TCR-alpha chain in determining the preimmune repertoire of Ag-specific TCR.

Genetic analysis reveals an intrinsic property of the germinal center B cells to generate A:T mutations.

The immunoglobulin genes undergo a high frequency of point mutations at both C:G and A:T pairs in the germinal center (GC) B cells. This hypermutation process is initiated by the activation-induced cytidine deaminase (AID), which converts cytosine to uracil and generates a U:G lesion. Replication of this lesion, or its repair intermediate the abasic site, could introduce C:G mutations but the mechanisms leading to mutations at non-damaged A:T pairs remain elusive. Using a lacZ-transgenic system in which endogenous genome mutations can be detected with high sensitivity, we found that GC B cells exhibited a much higher ratio of A:T mutations as compared to naïve B, non-GC B, and cells of other tissues. This property does not require AID or active transcription of the target gene, and is dependent on DNA polymerase eta. These in vivo results demonstrate that GC B cells are unique in having an intrinsic propensity to generate A:T mutations during repair of endogenous DNA damage. These findings have important implications in understanding how AID, which can only target C:G base pairs, is able to induce the entire spectrum of mutations observed in immunoglobulin variable region genes in GC B cells.

Immunological synapse and microclusters: the site for recognition and activation of T cells.

An immunological synapse (IS) is formed at the interface between antigen-presenting cells and T cells, and is believed to be the structure responsible for antigen recognition and T-cell activation. However, recent imaging analyses reveal that T-cell receptor microclusters (MCs) formed prior to IS are the site for antigen recognition and T-cell activation. MCs are continuously generated at the periphery of the interface, even after IS formation, and induce sustained activation signals. MC formation is not accompanied by lipid-raft clustering. Central supramolecular activation cluster is considered functional in recycling and degradation of T-cell receptors, directional secretion of cytokines and cytolytic granules, generation of sustained signals, or maintenance of the cell-cell conjugation.

LFA-1 decreases the antigen dose for T cell activation in vivo.

Leukocyte adhesion molecule leukocyte function-associated antigen (LFA)-1 not only mediates intercellular binding but also delivers co-stimulatory signals in T cells. LFA-1 has been shown to decrease the threshold of TCR signal and an antigen dose required for T cell activation and proliferation in vitro. However, physiological significance of the role of LFA-1 in TCR signal has remained unclear. We examined whether LFA-1 decreased the antigen dose for T cell activation in vivo. We showed here that, although collagen-induced arthritis (CIA) could not be induced by immunization and challenge with a standard amount of type-II collagen in LFA-1-deficient mice, a higher dose of the antigen did induce CIA in the absence of LFA-1. We also showed that CD4+ T cells could be primed by immunization with a high, but not low, dose of ovalbumin antigen in LFA-1-deficient mice. These results suggest that LFA-1 decreases the threshold of TCR signal for T cell activation in vivo as well as in vitro. Further studies using TCR-transgenic LFA-1-deficient mice showed that LFA-1 cooperated with TCR in sustained Erk1/2 phosphorylation. Moreover, TCR could induce sustained Erk1/2 phosphorylation in the absence of LFA-1 when T cells were stimulated with a high, but not low, dose of antigen, suggesting that LFA-1 may cooperate with TCR in sustaining Erk1/2 phosphorylation.

Inhibition of T cell activation and function by the adaptor protein CIN85.

T cell activation is initiated by signaling molecules downstream of the T cell receptor (TCR) that are organized by adaptor proteins. CIN85 (Cbl-interacting protein of 85 kDa) is one such adaptor protein. Here, we showed that CIN85 limited T cell responses to TCR stimulation. Compared to activated wild-type (WT) T cells, those that lacked CIN85 produced more IL-2 and exhibited greater proliferation. After stimulation of WT T cells with their cognate antigen, CIN85 was recruited to the TCR signaling complex. Early TCR signaling events, such as phosphorylation of ζ-chain-associated protein kinase 70 (Zap70), Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP76), and extracellular signal-regulated kinase (Erk), were enhanced in CIN85-deficient T cells. The inhibitory function of CIN85 required the SH3 and PR regions of the adaptor, which associated with the phosphatase suppressor of TCR signaling-2 (Sts-2) after TCR stimulation. Together, our data suggest that CIN85 is recruited to the TCR signaling complex and mediates inhibition of T cell activation through its association with Sts-2.

Analyzing the Dynamics of Signaling Microclusters.

T cell antigen receptor (TCR) stimulation induces recruitment and accumulation of various types of signaling molecules and forms signaling microclusters. The dynamics of the microclusters are important for regulating the quality and quantity of T cell activation. We describe here our protocols for analysis of signaling microclusters by using supported planar bilayers.

Blockage of Core Fucosylation Reduces Cell-Surface Expression of PD-1 and Promotes Anti-tumor Immune Responses of T Cells.

Programmed cell death 1 (PD-1) is highly expressed on exhausted T cells and inhibits T cell activation. Antibodies that block the interaction between PD-1 and its ligand prevent this inhibitory signal and reverse T cell dysfunction, providing beneficial anti-tumor responses in a substantial number of patients. Mechanisms for the induction and maintenance of high PD-1 expression on exhausted T cells have not been fully understood. Utilizing a genome-wide loss-of-function screening method based on the CRISPR-Cas9 system, we identified genes involved in the core fucosylation pathway as positive regulators of cell-surface PD-1 expression. Inhibition of Fut8, a core fucosyltransferase, by genetic ablation or pharmacologic inhibition reduced cell-surface expression of PD-1 and enhanced T cell activation, leading to more efficient tumor eradication. Taken together, our findings suggest that blocking core fucosylation of PD-1 can be a promising strategy for improving anti-tumor immune responses.

Role of interleukin-25 in development of spontaneous arthritis in interleukin-1 receptor antagonist-deficient mice.

Interleukin (IL)-25, which is a member of the IL-17 family of cytokines, induces production of such Th2 cytokines as IL-4, IL-5, IL-9 and/or IL-13 by various types of cells, including Th2 cells, Th9 cells and group 2 innate lymphoid cells (ILC2). On the other hand, IL-25 can suppress Th1- and Th17-associated immune responses by enhancing Th2-type immune responses. Supporting this, IL-25 is known to suppress development of experimental autoimmune encephalitis, which is an IL-17-mediated autoimmune disease in mice. However, the role of IL-25 in development of IL-17-mediated arthritis is not fully understood. Therefore, we investigated this using IL-1 receptor antagonist-deficient (IL-1Ra-/-) mice, which spontaneously develop IL-17-dependent arthritis. However, development of spontaneous arthritis (incidence rate, disease severity, proliferation of synovial cells, infiltration of PMNs, and bone erosion in joints) and differentiation of Th17 cells in draining lymph nodes in IL-25-/- IL-1Ra-/- mice were similar to in control IL-25+/+ IL-1Ra-/- mice. These observations indicate that IL-25 does not exert any inhibitory and/or pathogenic effect on development of IL-17-mediated spontaneous arthritis in IL-1Ra-/- mice.