unraveling-key-players-humoral-immunity-advanced-optimized-lymphocyte

Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense ...

Harvard Medical School

Rap1-guanosine triphosphate (GTP)-interacting adaptor molecule (RIAM) plays a critical role in actin reorganization and inside-out activation of integrins in lymphocytes and platelets. We investigated the role of RIAM in T cell receptor (TCR)-mediated signaling. Although phosphorylation of the kinase ZAP-70 and formation of a signalosome recruited to the adaptor protein LAT were unaffected, elimination of endogenous RIAM by short hairpin RNA impaired generation of inositol 1,4,5-trisphosphate, mobilization of intracellular calcium ions (Ca(2+)), and translocation of the transcription factor NFAT to the nucleus. The activation of Ras guanine nucleotide-releasing protein 1 was also impaired, which led to the diminished expression of the gene encoding interleukin-2. These events were associated with the impaired translocation of phosphorylated phospholipase C-gamma1 (PLC-gamma1) to the actin cytoskeleton, which was required to bring PLC-gamma1 close to its substrate phosphatidylinositol 4,5-bisphosphate, and were reversed by reconstitution of cells with RIAM. Thus, by regulating the localization of PLC-gamma1, RIAM plays a central role in TCR signaling and the transcription of target genes.

RIAM regulates the cytoskeletal distribution and activation of PLC-gamma1 in T cells.

The receptor programmed death 1 (PD-1) inhibits T cell proliferation and plays a critical role in suppressing self-reactive T cells, and it also compromises antiviral and antitumor responses. To determine how PD-1 signaling inhibits T cell proliferation, we used human CD4(+) T cells to examine the effects of PD-1 signaling on the molecular control of the cell cycle. The ubiquitin ligase SCF(Skp2) degrades p27(kip1), an inhibitor of cyclin-dependent kinases (Cdks), and PD-1 blocked cell cycle progression through the G(1) phase by suppressing transcription of SKP2, which encodes a component of this ubiquitin ligase. Thus, in T cells stimulated through PD-1, Cdks were not activated, and two critical Cdk substrates were not phosphorylated. Activation of PD-1 inhibited phosphorylation of the retinoblastoma gene product, which suppressed expression of E2F target genes. PD-1 also inhibited phosphorylation of the transcription factor Smad3, which increased its activity. These events induced additional inhibitory checkpoints in the cell cycle by increasing the abundance of the G(1) phase inhibitor p15(INK4) and repressing the Cdk-activating phosphatase Cdc25A. PD-1 suppressed SKP2 transcription by inhibiting phosphoinositide 3-kinase-Akt and Ras-mitogen-activated and extracellular signal-regulated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) signaling. Exposure of cells to the proliferation-promoting cytokine interleukin-2 restored activation of MEK-ERK signaling, but not Akt signaling, and only partially restored SKP2 expression. Thus, PD-1 blocks cell cycle progression and proliferation of T lymphocytes by affecting multiple regulators of the cell cycle.

Selective effects of PD-1 on Akt and Ras pathways regulate molecular components of the cell cycle and inhibit T cell proliferation.

CD4(+)Foxp3(+) T regulatory cells (Tregs) display phenotypic and functional plasticity that is regulated by cytokines and other immune cells. Previously, we determined that during co-culture with CD4(+)CD25(-) T cells and antigen presenting cells, Tregs produced IL-17. Here, we investigated the mechanisms underlying the differentiation of IL-17-producing Treg (Tr17) cells and their molecular and functional properties. We determined that during stimulation via TCR/CD3 and CD28, the combination of IL-1β and IL-2 was necessary and sufficient for the generation of Tr17 cells. Tr17 cells expressed Runx1 transcription factor, which was required for sustained expression of Foxp3 and RORγt and for production of IL-17. Surprisingly, Tr17 cells also expressed Runx3, which regulated transcription of perforin and granzyme B thereby mediating cytotoxic activity. Our studies indicate that Tr17 cells concomitantly express Foxp3, RORγt, Runx1 and Runx3 and are capable of producing IL-17 while mediating potent suppressive and cytotoxic function.

Runx1 and Runx3 are involved in the generation and function of highly suppressive IL-17-producing T regulatory cells.

The programmed cell death-1 (PD)-1 receptor (CD279) is a potent T cell inhibitor with a critical role in peripheral tolerance, but it can also compromise anti-viral and antitumor T cell responses. The effects of PD-1 on the cell cycle leading to inhibition of T cell expansion are poorly understood. Recently, we examined the effects of PD-1 on the molecular control of the cell cycle machinery and on TCR-activated signaling pathways that regulate these downstream outcomes. Our studies showed that PD-1 blocks cell cycle progression in the G 1 phase. PD-1 did not alter the expression of G 1 phase cyclins or cyclin-dependent kinases (Cdks) but, instead, suppressed the transcription of SKP2, the substrate recognition component of the SCF (Skp2)  ubiquitin ligase that leads p27 (kip1)  to degradation and resulted in accumulation of p27 (kip1) . Subsequently, T cells receiving PD-1 signals displayed impaired Cdk2 activation and failed to phosphorylate two critical Cdk2 substrates, the retinoblastoma gene product (Rb) and the TGFβ-specific transcription factor Smad3, leading to suppression of E2F target genes but enhanced Smad3 transactivation. These events resulted in upregulation of the Cdk4/6 inhibitor p15 (INK4B)  and repression of the Cdk-activating phosphatase Cdc25A. The suppressive effect of PD-1 on Skp2 expression was mediated by inhibition of both PI3K/Akt and Ras/MEK/Erk pathways and was only partially reversed by IL-2, which restored activation of MEK/Erk but not Akt. Thus, PD-1 targets Ras and PI3K/Akt signaling to inhibit transcription of Skp2 and to activate Smad3 as an integral component of a pathway that regulates blockade of cell cycle progression in T lymphocytes. Here, we discuss the detailed sequence of these signaling events and their implications in mediating cell-intrinsic and -extrinsic mechanisms that inhibit proliferation of T effector cells in response to PD-1-mediated signaling.

PD-1 inhibits T cell proliferation by upregulating p27 and p15 and suppressing Cdc25A.

Adaptive immunity depends on lymphocyte adhesion that is mediated by the integrin lymphocyte functional antigen 1 (LFA-1). The small guanosine triphosphatase Rap1 regulates LFA-1 adhesiveness through one of its effectors, Rap1-interacting adapter molecule (RIAM). We show that RIAM was recruited to the lymphocyte plasma membrane (PM) through its Ras association (RA) and pleckstrin homology (PH) domains, both of which were required for lymphocyte adhesion. The N terminus of RIAM inhibited membrane translocation. In vitro, the RA domain bound both Rap1 and H-Ras with equal but relatively low affinity, whereas in vivo only Rap1 was required for PM association. The PH domain bound phosphoinositol 4,5-bisphosphate (PI(4,5)P(2)) and was responsible for the spatial distribution of RIAM only at the PM of activated T cells. We determined the crystal structure of the RA and PH domains and found that, despite an intervening linker of 50 aa, the two domains were integrated into a single structural unit, which was critical for proper localization to the PM. Thus, the RA-PH domains of RIAM function as a proximity detector for activated Rap1 and PI(4,5)P(2).

Rap1-interacting adapter molecule (RIAM) associates with the plasma membrane via a proximity detector.

Programmed death 1 (PD-1) is a potent inhibitor of T cell responses. PD-1 abrogates activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, but the mechanism remains unclear. We determined that during T cell receptor (TCR)/CD3- and CD28-mediated stimulation, PTEN is phosphorylated by casein kinase 2 (CK2) in the Ser380-Thr382-Thr383 cluster within the C-terminal regulatory domain, which stabilizes PTEN, resulting in increased protein abundance but suppressed PTEN phosphatase activity. PD-1 inhibited the stabilizing phosphorylation of the Ser380-Thr382-Thr383 cluster within the C-terminal domain of PTEN, thereby resulting in ubiquitin-dependent degradation and diminished abundance of PTEN protein but increased PTEN phosphatase activity. These effects on PTEN were secondary to PD-1-mediated inhibition of CK2 and were recapitulated by pharmacologic inhibition of CK2 during TCR/CD3- and CD28-mediated stimulation without PD-1. Furthermore, PD-1-mediated diminished abundance of PTEN was reversed by inhibition of ubiquitin-dependent proteasomal degradation. Our results identify CK2 as a new target of PD-1 and reveal an unexpected mechanism by which PD-1 decreases PTEN protein expression while increasing PTEN activity, thereby inhibiting the PI3K/Akt signaling axis. 

PD-1 increases PTEN phosphatase activity while decreasing PTEN protein stability by inhibiting casein kinase 2.

Graft versus host disease (GvHD), mediated by donor T cells, remains the primary cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation and novel therapeutic approaches are required. Cdk2 is a critical node of signal integration and programming of T cell responses towards immunity versus anergy but is dispensable for hematopoiesis and thymocyte development. We examined the effects of pharmacologic Cdk2 inhibition on alloreactive human T cells. Inhibition of Cdk2 blocked expansion of alloreactive T cells upon culture with HLA-mismatched dendritic cells and prevented generation of IFN-γ-producing alloantigen-specific effectors. In contrast, Cdk2 inhibition preserved effectors specific for Wilms' tumor 1 (WT1) leukemia antigen and for CMV as determined by WT1-specific and CMV-specific pentamers. Cdk2 inhibition preserved Treg cells, which have the ability to prevent GvHD while maintaining GvL. Thus, Cdk inhibitors may improve allogeneic HSCT by reducing alloreactivity and GvHD without loss of pathogen-specific and leukemia-specific immunity.

The cyclin dependent kinase inhibitor (R)-roscovitine mediates selective suppression of alloreactive human T cells but preserves pathogen-specific and leukemia-specific effectors.

During activation, T cells undergo metabolic reprogramming, which imprints distinct functional fates. We determined that on PD-1 ligation, activated T cells are unable to engage in glycolysis or amino acid metabolism but have an increased rate of fatty acid β-oxidation (FAO). PD-1 promotes FAO of endogenous lipids by increasing expression of CPT1A, and inducing lipolysis as indicated by elevation of the lipase ATGL, the lipolysis marker glycerol and release of fatty acids. Conversely, CTLA-4 inhibits glycolysis without augmenting FAO, suggesting that CTLA-4 sustains the metabolic profile of non-activated cells. Because T cells utilize glycolysis during differentiation to effectors, our findings reveal a metabolic mechanism responsible for PD-1-mediated blockade of T-effector cell differentiation. The enhancement of FAO provides a mechanistic explanation for the longevity of T cells receiving PD-1 signals in patients with chronic infections and cancer, and for their capacity to be reinvigorated by PD-1 blockade. 

PD-1 alters T-cell metabolic reprogramming by inhibiting glycolysis and promoting lipolysis and fatty acid oxidation.

Conversion of normal cells to cancer is accompanied with changes in their metabolism. During this conversion, cell metabolism undergoes a shift from oxidative phosphorylation to aerobic glycolysis, also known as Warburg effect, which is a hallmark for cancer cell metabolism. In cancer cells, glycolysis functions in parallel with the TCA cycle and other metabolic pathways to enhance biosynthetic processes and thus support proliferation and growth. Similar metabolic features are observed in T cells during activation but, in contrast to cancer, metabolic transitions in T cells are part of a physiological process. Currently, there is intense interest in understanding the cause and effect relationship between metabolic reprogramming and T cell differentiation. After the recent success of cancer immunotherapy, the crosstalk between immune system and cancer has come to the forefront of clinical and basic research. One of the key goals is to delineate how metabolic alterations of cancer influence metabolism-regulated function and differentiation of tumor resident T cells and how such effects might be altered by immunotherapy. Here, we review the unique metabolic features of cancer, the implications of cancer metabolism on T cell metabolic reprogramming during antigen encounters, and the translational prospective of harnessing metabolism in cancer and T cells for cancer therapy. 

Clinical significance of T cell metabolic reprogramming in cancer.

T lymphocytes undergo extensive changes in their metabolic properties during their transition through various differentiation states, from naïve to effector to memory or regulatory roles. The cause and effect relationship between metabolism and differentiation is a field of intense investigation. Many recent studies demonstrate the dependency of T cell functional outcomes on metabolic pathways and the possibility of metabolic intervention to modify these functions. In this review, we describe the basic metabolic features of T cells and new findings on how these correlate with various differentiation fates and functions. We also highlight the latest information regarding the main factors that affect T cell metabolic reprogramming.

The role of metabolic reprogramming in T cell fate and function.

Host immunity provides wide spectrum protection that serves to eradicate pathogens and cancer cells, while maintaining self-tolerance and immunological homeostasis. Ligation of the T cell receptor (TCR) by antigen activates signaling pathways that coordinately induce aerobic glycolysis, mitochondrial activity, anabolic metabolism, and T effector cell differentiation. Activation of PI3K, Akt, and mTOR triggers the switch to anabolic metabolism by inducing transcription factors such as Myc and HIF1, and the glucose transporter Glut1, which is pivotal for the increase of glucose uptake after T cell activation. Activation of MAPK signaling is required for glucose and glutamine utilization, whereas activation of AMPK is critical for energy balance and metabolic fitness of T effector and memory cells. Coinhibitory receptors target TCR-proximal signaling and generation of second messengers. Imbalanced activation of such signaling pathways leads to diminished rates of aerobic glycolysis and impaired mitochondrial function resulting in defective anabolic metabolism and altered T cell differentiation. The coinhibitory receptors mediate distinct and synergistic effects on the activation of signaling pathways thereby modifying metabolic programs of activated T cells and resulting in altered immune functions. Understanding and therapeutic targeting of metabolic programs impacted by coinhibitory receptors might have significant clinical implications for the treatment of chronic infections, cancer, and autoimmune diseases.

Immunometabolic Regulations Mediated by Coinhibitory Receptors and Their Impact on T Cell Immune Responses.

Lymphocyte activation requires adhesion to antigen-presenting cells. This is a critical event linking innate and adaptive immunity. Lymphocyte adhesion is accomplished through LFA-1, which must be activated by a process referred to as inside-out integrin signaling. Among the few signaling molecules that have been implicated in inside-out integrin activation in hematopoietic cells are the small guanosine triphosphatase (GTPase) Rap1 and its downstream effector Rap1-interacting molecule (RIAM), a multidomain protein that defined the Mig10-RIAM-lamellipodin (MRL) class of adaptor molecules. Through its various domains, RIAM is a critical node of signal integration for activation of T cells, recruits monomeric and polymerized actin to drive actin remodeling and cytoskeletal reorganization, and promotes inside-out integrin signaling in T cells. As a regulator of inside-out integrin activation, RIAM affects multiple functions of innate and adaptive immunity. The effects of RIAM on cytoskeletal reorganization and integrin activation have implications in cell migration and trafficking of cancer cells. We provide an overview of the structure and interactions of RIAM, and we discuss the implications of RIAM functions in innate and adaptive immunity and cancer.

The adaptor molecule RIAM integrates signaling events critical for integrin-mediated control of immune function and cancer progression.

Feeling stressed? It might be your T cells.

In the version of this article initially published, the author surname citing the linked article (Miyama) was incorrect in the seventh and eighth paragraphs. The correct name is Miyajima.

Publisher Correction: Feeling stressed? It might be your T cells.

There has been significant progress in utilizing our immune system against cancer, mainly by checkpoint blockade and T cell-mediated therapies. The field of cancer immunotherapy is growing rapidly but durable clinical benefits occur only in a small subset of responding patients. It is currently recognized that cancer creates a suppressive metabolic microenvironment, which contributes to ineffective immune function. Metabolism is a common cellular feature, and although there has been significant progress in understanding the detrimental role of metabolic changes of the tumor microenvironment (TEM) in immune cells, there is still much to be learned regarding unique targetable pathways. Elucidation of cancer and immune cell metabolic profiles is critical for identifying mechanisms that regulate metabolic reprogramming within the TEM. Metabolic targets that mediate immunosuppression and are fundamental in sustaining tumor growth can be exploited therapeutically for the development of approaches to increase the efficacy of immunotherapies. Here, we will highlight the importance of metabolism on the function of tumor-associated immune cells and will address the role of key metabolic determinants that might be targets of therapeutic intervention for improvement of tumor immunotherapies.

Article	Year
RIAM regulates the cytoskeletal distribution and activation of PLC-gamma1 in T cells. Science signaling\| PubMed ID: 19952372	2009
Selective effects of PD-1 on Akt and Ras pathways regulate molecular components of the cell cycle and inhibit T cell proliferation. Science signaling\| PubMed ID: 22740686	2012
Runx1 and Runx3 are involved in the generation and function of highly suppressive IL-17-producing T regulatory cells. PloS one\| PubMed ID: 22984619	2012
PD-1 inhibits T cell proliferation by upregulating p27 and p15 and suppressing Cdc25A. Cell cycle (Georgetown, Tex.)\| PubMed ID: 23032366	2012
Rap1-interacting adapter molecule (RIAM) associates with the plasma membrane via a proximity detector. The Journal of cell biology\| PubMed ID: 23045549	2012
PD-1 increases PTEN phosphatase activity while decreasing PTEN protein stability by inhibiting casein kinase 2. Molecular and cellular biology\| PubMed ID: 23732914	2013
The cyclin dependent kinase inhibitor (R)-roscovitine mediates selective suppression of alloreactive human T cells but preserves pathogen-specific and leukemia-specific effectors. Clinical immunology (Orlando, Fla.)\| PubMed ID: 24631965	2014
PD-1 alters T-cell metabolic reprogramming by inhibiting glycolysis and promoting lipolysis and fatty acid oxidation. Nature communications\| PubMed ID: 25809635	2015
Clinical significance of T cell metabolic reprogramming in cancer. Clinical and translational medicine\| PubMed ID: 27510264	2016
The role of metabolic reprogramming in T cell fate and function. Current trends in immunology\| PubMed ID: 28356677	2016
Immunometabolic Regulations Mediated by Coinhibitory Receptors and Their Impact on T Cell Immune Responses. Frontiers in immunology\| PubMed ID: 28443090	2017
The adaptor molecule RIAM integrates signaling events critical for integrin-mediated control of immune function and cancer progression. Science signaling\| PubMed ID: 28831022	2017
Feeling stressed? It might be your T cells. Nature immunology\| PubMed ID: 29144505	2017
Publisher Correction: Feeling stressed? It might be your T cells. Nature immunology\| PubMed ID: 29955108	2018
Targeting T Cell Metabolism for Improvement of Cancer Immunotherapy. Frontiers in oncology\| PubMed ID: 30123774	2018

Nikolaos Patsoukis

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