Jeffrey C. Nolz

Regulation of T-cell activation by the cytoskeleton

To become activated, T cells must efficiently recognize antigen-presenting cells or target cells through several complex cytoskeleton-dependent processes, including integrin-mediated adhesion, immunological-synapse formation, cellular polarization, receptor sequestration and signalling. The actin and microtubule systems provide the dynamic cellular framework that is required to orchestrate these processes and ultimately contol T-cell activation. Here, we discuss recent advances that have furthered our understanding of the crucial importance of the T-cell cytoskeleton in controlling these aspects of T-cell immune recognition.

The WAVE2 Complex Regulates Actin Cytoskeletal Reorganization and CRAC-Mediated Calcium Entry during T Cell Activation

BACKGROUND The engagement of the T cell receptor results in actin cytoskeletal reorganization at the immune synapse (IS) and the triggering of biochemical signaling cascades leading to gene regulation and, ultimately, cellular activation. Recent studies have identified the WAVE family of proteins as critical mediators of Rac1-induced actin reorganization in other cell types. However, whether these proteins participate in actin reorganization at the IS or signaling pathways in T cells has not been investigated. RESULTS By using a combination of biochemical, genetic, and cell biology approaches, we provide evidence that WAVE2 is recruited to the IS, is biochemically modified, and is required for actin reorganization and beta-integrin-mediated adhesion after TCR crosslinking. Moreover, we show that WAVE2 regulates calcium entry at a point distal to PLCgamma1 activation and IP(3)-mediated store release. CONCLUSIONS These data reveal a role for WAVE2 in regulating multiple pathways leading to T cell activation. In particular, this work shows that WAVE2 is a key component of the actin regulatory machinery in T cells and that it also participates in linking intracellular calcium store depletion to calcium release-activated calcium (CRAC) channel activation.

WAVE2 Regulates High-Affinity Integrin Binding by Recruiting Vinculin and Talin to the Immunological Synapse

ABSTRACT T-cell-receptor (TCR)-mediated integrin activation is required for T-cell-antigen-presenting cell conjugation and adhesion to extracellular matrix components. While it has been demonstrated that the actin cytoskeleton and its regulators play an essential role in this process, no mechanism has been established which directly links TCR-induced actin polymerization to the activation of integrins. Here, we demonstrate that TCR stimulation results in WAVE2-ARP2/3-dependent F-actin nucleation and the formation of a complex containing WAVE2, ARP2/3, vinculin, and talin. The verprolin-connecting-acidic (VCA) domain of WAVE2 mediates the formation of the ARP2/3-vinculin-talin signaling complex and talin recruitment to the immunological synapse (IS). Interestingly, although vinculin is not required for F-actin or integrin accumulation at the IS, it is required for the recruitment of talin. In addition, RNA interference of either WAVE2 or vinculin inhibits activation-dependent induction of high-affinity integrin binding to VCAM-1. Overall, these findings demonstrate a mechanism in which signals from the TCR produce WAVE2-ARP2/3-mediated de novo actin polymerization, leading to integrin clustering and high-affinity binding through the recruitment of vinculin and talin.

Protective Capacity of Memory CD8+ T Cells Is Dictated by Antigen Exposure History and Nature of the Infection

Infection or vaccination confers heightened resistance to pathogen rechallenge because of quantitative and qualitative differences between naive and primary memory T cells. Herein, we show that secondary (boosted) memory CD8+ T cells were better than primary memory CD8+ T cells in controlling some, but not all acute infections with diverse pathogens. However, secondary memory CD8+ T cells were less efficient than an equal number of primary memory cells at preventing chronic LCMV infection and are more susceptible to functional exhaustion. Importantly, localization of memory CD8+ T cells within lymph nodes, which is reduced by antigen restimulation, was critical for both viral control in lymph nodes and for the sustained CD8+ T cell response required to prevent chronic LCMV infection. Thus, repeated antigen stimulation shapes memory CD8+ T cell populations to either enhance or decrease per cell protective immunity in a pathogen-specific manner, a concept of importance in vaccine design against specific diseases.

The WAVE2 complex regulates T cell receptor signaling to integrins via Abl- and CrkL–C3G-mediated activation of Rap1

WAVE2 regulates T cell receptor (TCR)–stimulated actin cytoskeletal dynamics leading to both integrin clustering and affinity maturation. Although WAVE2 mediates integrin affinity maturation by recruiting vinculin and talin to the immunological synapse in an Arp2/3-dependent manner, the mechanism by which it regulates integrin clustering is unclear. We show that the Abl tyrosine kinase associates with the WAVE2 complex and TCR ligation induces WAVE2-dependent membrane recruitment of Abl. Furthermore, we show that WAVE2 regulates TCR-mediated activation of the integrin regulatory guanosine triphosphatase Rap1 via the recruitment and activation of the CrkL–C3G exchange complex. Moreover, we demonstrate that although Abl does not regulate the recruitment of CrkL–C3G into the membrane, it does affect the tyrosine phosphorylation of C3G, which is required for its guanine nucleotide exchange factor activity toward Rap1. This signaling node regulates not only TCR-stimulated integrin clustering but also affinity maturation. These findings identify a previously unknown mechanism by which the WAVE2 complex regulates TCR signaling to Rap1 and integrin activation.

Local antigen in nonlymphoid tissue promotes resident memory CD8+ T cell formation during viral infection

CD8+ T cells activated during viral infection migrate to infected skin in an antigen-independent manner. Local recognition of antigens drives the differentiation into Trm CD8+ T cells.

Naive, effector and memory CD8 T-cell trafficking: parallels and distinctions.

Trafficking of CD8 T cells, in both the steady-state and during episodes of infection or inflammation, is a highly dynamic process and involves a variety of receptor-ligand interactions. A thorough, mechanistic understanding of how this process is regulated could potentially lead to disease prevention strategies, through either enhancing (for infectious diseases or tumors) or limiting (for autoimmunity) recruitment of antigen-specific CD8 T cells to areas of tissue inflammation. As CD8 T cells transition from naive to effector to memory cells, changes in gene expression will ultimately dictate anatomical localization of these cells in vivo. In this article, we discuss recent advances in understanding how antigenic stimulation influences expression of various trafficking receptors and ligands, and how this determines the tissue localization of CD8 T cells.

IL-15 regulates memory CD8 + T cell O-glycan synthesis and affects trafficking

Memory and naive CD8+ T cells exhibit distinct trafficking patterns. Specifically, memory but not naive CD8+ T cells are recruited to inflamed tissues in an antigen-independent manner. However, the molecular mechanisms that regulate memory CD8+ T cell trafficking are largely unknown. Here, using murine models of infection and T cell transfer, we found that memory but not naive CD8+ T cells dynamically regulate expression of core 2 O-glycans, which interact with P- and E-selectins to modulate trafficking to inflamed tissues. Following infection, antigen-specific effector CD8+ T cells strongly expressed core 2 O-glycans, but this glycosylation pattern was lost by most memory CD8+ T cells. After unrelated infection or inflammatory challenge, memory CD8+ T cells synthesized core 2 O-glycans independently of antigen restimulation. The presence of core 2 O-glycans subsequently directed these cells to inflamed tissue. Memory and naive CD8+ T cells exhibited the opposite pattern of epigenetic modifications at the Gcnt1 locus, which encodes the enzyme that initiates core 2 O-glycan synthesis. The open chromatin configuration in memory CD8+ T cells permitted de novo generation of core 2 O-glycans in a TCR-independent, but IL-15-dependent, manner. Thus, IL-15 stimulation promotes antigen-experienced memory CD8+ T cells to generate core 2 O-glycans, which subsequently localize them to inflamed tissues. These findings suggest that CD8+ memory T cell trafficking potentially can be manipulated to improve host defense and immunotherapy.

Immunologic considerations for generating memory CD8 T cells through vaccination

Following infection or vaccination, naïve CD8 T cells that receive the appropriate integration of antigenic, co‐stimulatory and inflammatory signals undergo a programmed series of biological changes that ultimately results in the generation of memory cells. Memory CD8 T cells, in contrast to naïve cells, more effectively limit or prevent pathogen re‐infection because of both qualitative and quantitative changes that occur following their induction. Unlike vaccination strategies aimed at generating antibody production, the ability to generate protective memory CD8 T cells has proven more complicated and problematic. However, recent experimental results have revealed important principles regarding the molecular and genetic basis for memory CD8 T cell formation, as well as identified ways to manipulate their development through vaccination, resulting in potential new avenues to enhance protective immunity.

Strategies and Implications for Prime-Boost Vaccination to Generate Memory CD8 T Cells

Generating a large population of memory CD8 T cells is an appealing goal for vaccine design against a variety of human diseases. Indeed, experimental models have demonstrated that the overall number of memory CD8 T cells present at the time of infection correlates strongly with the ability to confer host protection against a range of different pathogens. Currently, the most conceivable approach to rapidly generate a large population of memory CD8 T cells is through the use of prime-boost vaccination. In addition, recent experimental findings have uncovered important principles that govern both the rate and magnitude of memory CD8 T cell formation. Thus, this has resulted in novel prime-boost vaccination strategies that could potentially be used in humans to generate protective populations of memory CD8 T cells.