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Concept: Lck

297

Sunlight has important biological effects in human skin. Ultraviolet (UV) light striking the epidermis catalyzes the synthesis of Vitamin D and triggers melanin production. Although a causative element in skin cancers, sunlight is also associated with positive health outcomes including reduced incidences of autoimmune diseases and cancers. The mechanisms, however, by which light affects immune function remain unclear. Here we describe direct photon sensing in human and mouse T lymphocytes, a cell-type highly abundant in skin. Blue light irradiation at low doses (<300 mJ cm(-2)) triggers synthesis of hydrogen peroxide (H2O2) in T cells revealed by the genetically encoded reporter HyPerRed. In turn, H2O2 activates a Src kinase/phospholipase C-γ1 (PLC-γ1) signaling pathway and Ca(2+) mobilization. Pharmacologic inhibition or genetic disruption of Lck kinase, PLC-γ1 or the T cell receptor complex inhibits light-evoked Ca(2+) transients. Notably, both light and H2O2 enhance T-cell motility in a Lck-dependent manner. Thus, T lymphocytes possess intrinsic photosensitivity and this property may enhance their motility in skin.

Concepts: Thymus, Lck, Major histocompatibility complex, T cell, Vitamin D, Ultraviolet, T cell receptor, Immune system

164

Distinct signaling pathways producing diverse cellular outcomes can utilize similar subsets of proteins. For example, proteins from the T cell receptor (TCR) early signaling complex (ESC) are also involved in interferon-α receptor signaling. Defining the mechanism for how these proteins function within a given pathway is important in understanding the integration and communication of signaling networks with one another. We investigated the contributions of the TCR ESC proteins Lck, ZAP-70, Vav1, SLP-76, and LAT to integrin outside-in signaling in human T cells. Lck, ZAP-70, SLP-76, Vav1, and LAT were activated by α4β1 outside-in signaling but in a manner different from TCR signaling. TCR stimulation recruits ESC proteins to activate the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK). α4β1 outside-in-mediated ERK activation did not require TCR ESC proteins. However, α4β1 outside-in signaling induced CD25 and costimulated CD69 and this was dependent upon TCR ESC proteins. TCR and α4β1 outside-in signaling are integrated through the common use of TCR ESC proteins; however, these proteins display functionally distinct roles in these pathways. These novel insights into the crosstalk between integrin outside-in and TCR signaling pathways are highly relevant to the development of therapeutic strategies to overcome disease associated with T cell deregulation.

Concepts: MAPK/ERK pathway, Lck, Extracellular signal-regulated kinases, Mitogen-activated protein kinase, T cell receptor, T cell, Protein, Signal transduction

24

Direct treatments of cancer such as chemotherapy, radiotherapy and targeted therapy have been shown to depend on recruitment of the immune system for their effectiveness. Recent studies have shown that development of resistance to direct therapies such as BRAF inhibitors in melanoma is associated with suppression of immune responses. We point to emerging data that implicates activation of the polycomb repressive complex 2 (PRC2) and its catalytic component - enhancer of zeste homolog 2 (EZH2) - in progression of melanoma and suppression of immune responses. EZH2 appears to have an important role in differentiation of CD4 T cells and particularly in the function of T regulatory cells, which suppress immune responses to melanoma. We review mechanisms of EZH2 activation at genomic level and from activation of the MAP Kinase, E2F or NF-kB2 pathways. These studies are consistent with activation of EZH2 as a common mechanism for induction of immune suppression in patients failing direct therapies and suggest EZH2 inhibitors may have a role in combination with immunotherapy and targeted therapies to prevent development of immunosuppression. This article is protected by copyright. All rights reserved.

Concepts: Lck, Signal transduction, Immunosuppressive drug, T helper cell, Cancer, Immunology, Immune system, Chemotherapy

17

Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell.

Concepts: T cell, Lck, Major histocompatibility complex, T cell receptor, Immune system, Cell signaling, Protein, Signal transduction

8

Highly potent and broadly neutralizing anti-HIV-1 antibodies (bNAbs) have been used to prevent and treat lentivirus infections in humanized mice, macaques, and humans. In immunotherapy experiments, administration of bNAbs to chronically infected animals transiently suppresses virus replication, which invariably returns to pre-treatment levels and results in progression to clinical disease. Here we show that early administration of bNAbs in a macaque simian/human immunodeficiency virus (SHIV) model is associated with very low levels of persistent viraemia, which leads to the establishment of T-cell immunity and resultant long-term infection control. Animals challenged with SHIVAD8-EO by mucosal or intravenous routes received a single 2-week course of two potent passively transferred bNAbs (3BNC117 and 10-1074 (refs 13, 14)). Viraemia remained undetectable for 56-177 days, depending on bNAb half-life in vivo. Moreover, in the 13 treated monkeys, plasma virus loads subsequently declined to undetectable levels in 6 controller macaques. Four additional animals maintained their counts of T cells carrying the CD4 antigen (CD4(+)) and very low levels of viraemia persisted for over 2 years. The frequency of cells carrying replication-competent virus was less than 1 per 10(6) circulating CD4(+) T cells in the six controller macaques. Infusion of a T-cell-depleting anti-CD8β monoclonal antibody to the controller animals led to a specific decline in levels of CD8(+) T cells and the rapid reappearance of plasma viraemia. In contrast, macaques treated for 15 weeks with combination anti-retroviral therapy, beginning on day 3 after infection, experienced sustained rebound plasma viraemia when treatment was interrupted. Our results show that passive immunotherapy during acute SHIV infection differs from combination anti-retroviral therapy in that it facilitates the emergence of potent CD8(+) T-cell immunity able to durably suppress virus replication.

Concepts: Bacteria, Lck, T cell receptor, Antibody, Infection, Immunology, Monoclonal antibodies, Immune system

7

Ibrutinib has previously been shown to inhibit Bruton’s tyrosine kinase (BTK) and interleukin-2-inducible T-cell kinase (ITK), which mediate B-cell and T-cell receptor signaling, respectively. BTK inhibition with ibrutinib has demonstrated impressive clinical responses in a variety of B-cell malignancies. Whether ibrutinib inhibition of ITK can lead to clinical response in T-cell malignancies is unknown. We hypothesized that ibrutinib-mediated ITK inhibition in T-cell lymphoma would result in decreased signaling through the T-cell receptor pathway and promote antitumor immune response by driving selective cytotoxic Th1 CD4 effector T-cell differentiation. This pilot clinical trial evaluated 2 dose levels of ibrutinib: 560 and 840 mg orally daily. Fourteen patients with relapsed, refractory peripheral T-cell lymphoma and cutaneous T-cell lymphoma were enrolled. Both dose levels were safe and well tolerated, and no dose-limiting toxicities were observed. One patient achieved a partial response (overall response rate, 8% [1/13]). ITK occupancy studies demonstrated a mean occupancy of 50% (range, 15%-80%). Higher ITK occupancy of more than 50% correlated with higher serum levels of tumor necrosis factor-α and interferon-γ and favored a Th1 phenotype. Our data suggest that ibrutinib inhibition of ITK has limited clinical activity in T-cell lymphoma. This study is registered at www.clinicaltrials.gov as #NCT02309580.

Concepts: T cell receptor, Lymphoma, B cell, Lck, T helper cell, Immune system, Cancer, Signal transduction

7

Attempts to eradicate HIV have been thwarted by the persistence of a small pool of quiescent memory CD4 T cells that harbor a transcriptionally silent, integrated form of the virus that can produce infectious virions following an anamnestic immune response. Transcription factors downstream of T-cell receptor activation, such as NF-κB/Rel and nuclear factor of activated T cells (NFAT) transcription members, are considered important regulators of HIV transcription during acute HIV infection. We now report studies exploring their precise role as antagonists of HIV latency using cell and primary CD4 T cell models of HIV-1 latency. Surprisingly, RNA interference studies performed in J-Lat CD4 T cells suggested that none of the NFATs, including NFATc1, NFATc2, NFATc3, and NFAT5, played a key role in the reactivation of latent HIV. However, cyclosporin A markedly inhibited the reactivation response. These results were reconciled when calcium signaling through calcineurin was shown to potentiate prostratin induced activation of NF-κB that in turn stimulated the latent HIV long terminal repeat (LTR). Similar effects of calcineurin were confirmed in a primary CD4 T cell model of HIV latency. These findings highlight an important role for calcineurin in NF-κB-dependent induction of latent HIV transcription. Innovative approaches exploiting the synergistic actions of calcineurin and prostratin in the absence of generalized T-cell activation merit exploration as a means to attack the latent viral reservoir.

Concepts: Lck, T cells, Gene, Virus, NFAT, Immune system, T cell, HIV

5

The rare patients who are able to spontaneously control HIV replication in the absence of therapy show signs of a particularly efficient cellular immune response. To identify the molecular determinants that underlie this response, we characterized the T cell receptor (TCR) repertoire directed at Gag293, the most immunoprevalent CD4 epitope in the HIV-1 capsid. HIV controllers from the ANRS CODEX cohort showed a highly skewed TCR repertoire that was characterized by a predominance of TRAV24 and TRBV2 variable genes, shared CDR3 motifs, and a high frequency of public clonotypes. The most prevalent public clonotypes generated TCRs with affinities at the higher end of values reported for naturally occurring TCRs. The high-affinity Gag293-specific TCRs were cross-restricted by up to 5 distinct HLA-DR alleles, accounting for the expression of these TCRs in HIV controllers of diverse genetic backgrounds. Transfer of these TCRs to healthy donor CD4+ T cells conferred high antigen sensitivity and polyfunctionality, thus recapitulating key features of the controller CD4 response. Transfer of a high-affinity Gag293-specific TCR also redirected CD8+ T cells to target HIV-1 capsid via nonconventional MHC II restriction. Together, these findings indicate that TCR clonotypes with superior functions are associated with HIV control. Amplification or transfer of such clonotypes may contribute to immunotherapeutic approaches aiming at a functional HIV cure.

Concepts: Lck, Antigen, HIV, Protein, Gene, Major histocompatibility complex, T cell receptor, Immune system

4

Postnatal thymic epithelial cell (TEC) homeostatic defect- or natural aging-induced thymic atrophy results in a decline in central T-cell tolerance establishment, which is constituted by thymocyte negative selection and cluster of differentiation (CD) 4+ thymic regulatory T (tTreg) cell generation. Emerging evidence shows this decline mainly results from defects in negative selection, but there is insufficient evidence regarding whether tTreg cell generation is also impaired. We mechanistically studied tTreg cell generation in the atrophied thymus by utilizing both postnatal TEC-defective (resulting from FoxN1-floxed conditional knockout [cKO]) and naturally aged mouse models. We found that the capacity of tTreg cell generation was not impaired compared to CD4+ thymic conventional T cells, suggesting thymic atrophy positively influences tTreg cell generation. This is potentially attributed to decreased T cell receptor (TCR) signaling strength due to inefficiency in promiscuous expression of self-antigens or presenting a neo-self-antigen by medullary TECs, displaying decreased negative selection-related marker genes (Nur77 and CD5high) in CD4 single positive (SP) thymocytes. Our results provide evidence that the atrophied thymus attempts to balance the defective negative selection by enhancing tTreg cell generation to maintain central T-cell tolerance in the elderly. Once the balance is broken, age-related diseases could take place.

Concepts: Atrophy, Lck, Thymocyte, B cell, Major histocompatibility complex, T cell receptor, T cell, Thymus

4

During immune surveillance, T cells survey the surface of antigen-presenting cells. In searching for peptide-loaded major histocompatibility complexes (pMHCs), they must solve a classic trade-off between speed and sensitivity. It has long been supposed that microvilli on T cells act as sensory organs to enable search, but their strategy has been unknown. We used lattice light-sheet and quantum dot-enabled synaptic contact mapping microscopy to show that anomalous diffusion and fractal organization of microvilli survey the majority of opposing surfaces within 1 minute. Individual dwell times were long enough to discriminate pMHC half-lives and T cell receptor (TCR) accumulation selectively stabilized microvilli. Stabilization was independent of tyrosine kinase signaling and the actin cytoskeleton, suggesting selection for avid TCR microclusters. This work defines the efficient cellular search process against which ligand detection takes place.

Concepts: Receptor, T cell, Lck, Cell signaling, T cell receptor, Signal transduction, Protein, Major histocompatibility complex