Strength in Numbers: Visualizing CTL-Mediated Killing In Vivo.

Cytotoxic CD8+ T lymphocytes (CTLs) have long been believed to be extremely efficient killers. Forster and colleagues (Halle et al., 2016) used in vivo imaging to tell a different story, in which each CTL killed only 2-16 targets a day, and several CTLs per target were needed to get the job done.

The secreted protein Cowpox Virus 14 contributes to viral virulence and immune evasion by engaging Fc-gamma-receptors.

The genome of cowpoxvirus (CPXV) could be considered prototypical for orthopoxviridae (OXPV) since it contains many open reading frames (ORFs) absent or lost in other OPXV, including vaccinia virus (VACV). These additional ORFs are non-essential for growth in vitro but are expected to contribute to the broad host range, virulence and immune evasion characteristics of CPXV. For instance, unlike VACV, CPXV encodes proteins that interfere with T cell stimulation, either directly or by preventing antigen presentation or co-stimulation. When studying the priming of naïve T cells, we discovered that CPXV, but not VACV, encodes a secreted factor that interferes with activation and proliferation of naïve CD8+ and CD4+ T cells, respectively, in response to anti-CD3 antibodies, but not to other stimuli. Deletion mapping revealed that the inhibitory protein is encoded by CPXV14, a small secreted glycoprotein belonging to the poxvirus immune evasion (PIE) family and containing a smallpoxvirus encoded chemokine receptor (SECRET) domain that mediates binding to chemokines. We demonstrate that CPXV14 inhibition of antibody-mediated T cell activation depends on the presence of Fc-gamma receptors (FcγRs) on bystander cells. In vitro, CPXV14 inhibits FcγR-activation by antigen/antibody complexes by binding to FcγRs with high affinity and immobilized CPXV14 can trigger signaling through FcγRs, particularly the inhibitory FcγRIIB. In vivo, CPXV14-deleted virus showed reduced viremia and virulence resulting in reduced weight loss and death compared to wildtype virus whereas both antibody and CD8+ T cell responses were increased in the absence of CPXV14. Furthermore, no impact of CPXV14-deletion on virulence was observed in mice lacking the inhibitory FcγRIIB. Taken together our results suggest that CPXV14 contributes to virulence and immune evasion by binding to host FcγRs.

Cellular and Humoral Immune Responses in Mice Immunized with Vaccinia Virus Expressing the SARS-CoV-2 Spike Protein.

The COVID-19 pandemic is a global health emergency, and the development of a successful vaccine will ultimately be required to prevent the continued spread and seasonal recurrence of this disease within the human population. However, very little is known about either the quality of the adaptive immune response or the viral Ag targets that will be necessary to prevent the spread of the infection. In this study, we generated recombinant Vaccinia virus expressing the full-length spike protein from SARS-CoV-2 (VacV-S) to evaluate the cellular and humoral immune response mounted against this viral Ag in mice. Both CD8+ and CD4+ T cells specific to the SARS-CoV-2 spike protein underwent robust expansion, contraction, and persisted for at least 40 d following a single immunization with VacV-S. Vaccination also caused the rapid emergence of spike-specific IgG-neutralizing Abs. Interestingly, both the cellular and humoral immune responses strongly targeted the S1 domain of spike following VacV-S immunization. Notably, immunization with VacV-expressing spike conjugated to the MHC class II invariant chain, a strategy previously reported by us and others to enhance the immunogenicity of antigenic peptides, did not promote stronger spike-specific T cell or Ab responses in vivo. Overall, these findings demonstrate that an immunization approach using VacV or attenuated versions of VacV expressing the native, full-length SARS-CoV-2 spike protein could be used for further vaccine development to prevent the spread of COVID-19.

One bug or another: promiscuous T cells form lifelong memory.

As humans age, their T cell repertoire becomes dominated by cells exhibiting "memory" characteristics. In this issue of Immunity, Su et al. (2013) demonstrate that a large percentage of virus-specific CD4(+) T cells also exhibit a memory phenotype in unexposed adults.

Pathogen-specific inflammatory milieux tune the antigen sensitivity of CD8(+) T cells by enhancing T cell receptor signaling.

CD8(+) T cells confer host protection through T-cell-receptor (TCR)-mediated recognition of foreign antigens presented by infected cells. Thus, generation of CD8(+) T cell populations with high antigen sensitivity is critical for efficient pathogen clearance. Besides selection of high-affinity TCRs, the molecular mechanisms regulating the antigen sensitivity of CD8(+) T cells remain poorly defined. Herein, we have demonstrated that the antigen sensitivity of effector and memory CD8(+) T cells is dynamically regulated and can be tuned by pathogen-induced inflammatory milieux independently of the selection of cells with higher TCR affinity. Mechanistically, we have demonstrated that the signal-transduction capacity of key TCR proximal molecules is enhanced by inflammatory cytokines, which reduced the antigen density required to trigger antimicrobial functions. Dynamic tuning of CD8(+) T cell antigen sensitivity by inflammatory cytokines most likely optimizes immunity to specific pathogens while minimizing the risk of immunopathology at steady state.

Central memory CD8+ T cells become CD69+ tissue-residents during viral skin infection independent of CD62L-mediated lymph node surveillance.

Memory CD8+ T cells in the circulation rapidly infiltrate non-lymphoid tissues following infection and provide protective immunity in an antigen-specific manner. However, the subsequent fate of memory CD8+ T cells after entering non-lymphoid tissues such as the skin during a secondary infection is largely unknown. Furthermore, because expression of CD62L is often used to identify the central memory (TCM) CD8+ T cell subset, uncoupling the physical requirement for CD62L-mediated lymph node homing versus other functional attributes of TCM CD8+ T cells remains unresolved. Here, we show that in contrast to naïve CD8+ T cells, memory CD8+ T cells traffic into the skin independent of CD62L-mediated lymph node re-activation and provide robust protective immunity against Vaccinia virus (VacV) infection. TCM, but not effector memory (TEM), CD8+ T cells differentiated into functional CD69+/CD103- tissue residents following viral clearance, which was also dependent on local recognition of antigen in the skin microenvironment. Finally, we found that memory CD8+ T cells expressed granzyme B after trafficking into the skin and utilized cytolysis to provide protective immunity against VacV infection. Collectively, these findings demonstrate that TCM CD8+ T cells become cytolytic following rapid infiltration of the skin to protect against viral infection and subsequently differentiate into functional CD69+ tissue-residents.

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.

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.

Molecular mechanisms of CD8(+) T cell trafficking and localization.

Cytotoxic CD8(+) T cells are potent mediators of host protection against disease due to their ability to directly kill cells infected with intracellular pathogens and produce inflammatory cytokines at the site of infection. To fully achieve this objective, naïve CD8(+) T cells must be able to survey the entire body for the presence of foreign or "non-self" antigen that is delivered to draining lymph nodes following infection or tissue injury. Once activated, CD8(+) T cells undergo many rounds of cell division, acquire effector functions, and are no longer restricted to the circulation and lymphoid compartments like their naïve counterparts, but rather are drawn to inflamed tissues to combat infection. As CD8(+) T cells transition from naïve to effector to memory populations, this is accompanied by dynamic changes in the expression of adhesion molecules and chemokine receptors that ultimately dictate their localization in vivo. Thus, an understanding of the molecular mechanisms regulating CD8(+) T cell trafficking and localization is critical for vaccine design, control of infectious diseases, treatment of autoimmune disorders, and cancer immunotherapy.

Division-linked generation of death-intermediates regulates the numerical stability of memory CD8 T cells.

Infection or successful vaccination results in the formation of long-lived memory CD8 T-cell populations. Despite their numerical stability, memory CD8 T-cell populations are thought to completely turn over through proliferation within a 2- to 3-mo period. Therefore, steady-state memory cell proliferation must be balanced by a precisely regulated and equivalent death rate. However, the mechanisms regulating this balancing process remain completely undefined. Herein, we provide evidence for "death-intermediate memory cells" (T(DIM)) within memory CD8 T-cell populations generated by infection. Importantly, CD62L(Lo)/CD27(Lo) T(DIM)s are functionally characterized by an inability to produce cytokines, the failure to internalize T-cell receptor following antigenic stimulation, and signatures of apoptotic death. Furthermore, we demonstrate that, mechanistically, T(DIM) are directly generated from dividing "central memory" T-cell populations undergoing memory turnover in vivo. Collectively, these results demonstrate that as central memory CD8 T cells proliferate, they continuously generate a population of CD8 T cells that are nonfunctional and apoptotic; thus, our data support a model wherein division-linked generation of T(DIM) contributes to numerically stable CD8 T-cell memory.

N-linked glycans: an underappreciated key determinant of T cell development, activation, and function.

N-linked glycosylation is a post-translational modification that results in the decoration of newly synthesized proteins with diverse types of oligosaccharides that originate from the amide group of the amino acid asparagine. The sequential and collective action of multiple glycosidases and glycosyltransferases are responsible for determining the overall size, composition, and location of N-linked glycans that become covalently linked to an asparagine during and after protein translation. A growing body of evidence supports the critical role of N-linked glycan synthesis in regulating many features of T cell biology, including thymocyte development and tolerance, as well as T cell activation and differentiation. Here, we provide an overview of how specific glycosidases and glycosyltransferases contribute to the generation of different types of N-linked glycans and how these post-translational modifications ultimately regulate multiple facets of T cell biology.

T cell receptor signaling strength establishes the chemotactic properties of effector CD8+ T cells that control tissue-residency.

Tissue-resident memory (TRM) CD8+ T cells are largely derived from recently activated effector T cells, but the mechanisms that control the extent of TRM differentiation within tissue microenvironments remain unresolved. Here, using an IFNγ-YFP reporter system to identify CD8+ T cells executing antigen-dependent effector functions, we define the transcriptional consequences and functional mechanisms controlled by TCR-signaling strength that occur within the skin during viral infection to promote TRM differentiation. TCR-signaling both enhances CXCR6-mediated migration and suppresses migration toward sphingosine-1-phosphate, indicating the programming of a 'chemotactic switch' following secondary antigen encounter within non-lymphoid tissues. Blimp1 was identified as the critical target of TCR re-stimulation that is necessary to establish this chemotactic switch and for TRM differentiation to efficiently occur. Collectively, our findings show that access to antigen presentation and strength of TCR-signaling required for Blimp1 expression establishes the chemotactic properties of effector CD8+ T cells to promote residency within non-lymphoid tissues.

T helper 1 effector memory CD4+ T cells protect the skin from poxvirus infection.

Poxvirus infections of the skin are a recent emerging public health concern, yet the mechanisms that mediate protective immunity against these viral infections remain largely unknown. Here, we show that T helper 1 (Th1) memory CD4+ T cells are necessary and sufficient to provide complete and broad protection against poxvirus skin infections, whereas memory CD8+ T cells are dispensable. Core 2 O-glycan-synthesizing Th1 effector memory CD4+ T cells rapidly infiltrate the poxvirus-infected skin microenvironment and produce interferon γ (IFNγ) in an antigen-dependent manner, causing global changes in gene expression to promote anti-viral immunity. Keratinocytes express IFN-stimulated genes, upregulate both major histocompatibility complex (MHC) class I and MHC class II antigen presentation in an IFNγ-dependent manner, and require IFNγ receptor (IFNγR) signaling and MHC class II expression for memory CD4+ T cells to protect the skin from poxvirus infection. Thus, Th1 effector memory CD4+ T cells exhibit potent anti-viral activity within the skin, and keratinocytes are the key targets of IFNγ necessary for preventing poxvirus infection of the epidermis.

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.

Infection-induced lymphatic zippering restricts fluid transport and viral dissemination from skin.

Lymphatic vessels are often considered passive conduits that flush antigenic material, pathogens, and cells to draining lymph nodes. Recent evidence, however, suggests that lymphatic vessels actively regulate diverse processes from antigen transport to leukocyte trafficking and dietary lipid absorption. Here we tested the hypothesis that infection-induced changes in lymphatic transport actively contribute to innate host defense. We demonstrate that cutaneous vaccinia virus infection by scarification activates dermal lymphatic capillary junction tightening (zippering) and lymph node lymphangiogenesis, which are associated with reduced fluid transport and cutaneous viral sequestration. Lymphatic-specific deletion of VEGFR2 prevented infection-induced lymphatic capillary zippering, increased fluid flux out of tissue, and allowed lymphatic dissemination of virus. Further, a reduction in dendritic cell migration to lymph nodes in the absence of lymphatic VEGFR2 associated with reduced antiviral CD8+ T cell expansion. These data indicate that VEGFR2-driven lymphatic remodeling is a context-dependent, active mechanism of innate host defense that limits viral dissemination and facilitates protective, antiviral CD8+ T cell responses.

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.

The Ezh2 methyltransferase complex: actin up in the cytosol.

Ezh2, a polycomb group protein, is known to function in histone methylation, thereby regulating gene expression. However, in a recent study by Su et al., the Ezh2-containing complex has been given an additional role in cellular regulation. Cytosolic Ezh2 methyltransferase complexes were shown to associate with Vav1 and control receptor-induced actin polymerization and proliferation in a methylation-dependent manner. Overall, these findings implicate lysine methylation as a posttranslational modification crucial for receptor-mediated signal transduction events.

WAVE2 regulates high-affinity integrin binding by recruiting vinculin and talin to the immunological synapse.

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.

P2X7R: The Achilles heel of follicular helper memory T cells.

Protecting TFH memory CD4+ T cells from NAD-induced cell death reveals both their longevity and plasticity (see related Research Article by Künzli et al.).

Control of memory CD8+ T cell longevity and effector functions by IL-15.

IL-15 is a member of the common gamma chain family of cytokines and plays important roles in regulating several aspects of innate and adaptive immunity. Besides its established role in controlling homeostatic proliferation and survival of memory CD8+ T cells and natural killer cells, recent findings demonstrate that inflammatory IL-15 can also stimulate a variety of effector functions, such as enhanced cytotoxicity, entry into the cell cycle, and trafficking into non-lymphoid tissues. Here, we discuss how IL-15 is critical in regulating many functions of memory CD8+ T cells and how these processes act collectively to ensure optimal protective cellular immunity against re-infections.