Ex Pluribus Unum: The CD4 T Cell Response against Influenza A Virus.

Current Influenza A virus (IAV) vaccines, which primarily aim to generate neutralizing antibodies against the major surface proteins of specific IAV strains predicted to circulate during the annual 'flu' season, are suboptimal and are characterized by relatively low annual vaccine efficacy. One approach to improve protection is for vaccines to also target the priming of virus-specific T cells that can protect against IAV even in the absence of preexisting neutralizing antibodies. CD4 T cells represent a particularly attractive target as they help to promote responses by other innate and adaptive lymphocyte populations and can also directly mediate potent effector functions. Studies in murine models of IAV infection have been instrumental in moving this goal forward. Here, we will review these findings, focusing on distinct subsets of CD4 T cell effectors that have been shown to impact outcomes. This body of work suggests that a major challenge for next-generation vaccines will be to prime a CD4 T cell population with the same spectrum of functional diversity generated by IAV infection. This goal is encapsulated well by the motto 'ex pluribus unum': that an optimal CD4 T cell response comprises many individual specialized subsets responding together.

STAT1 Controls the Functionality of Influenza-Primed CD4 T Cells but Therapeutic STAT4 Engagement Maximizes Their Antiviral Impact.

It is generally accepted that influenza A virus (IAV) infection promotes a Th1-like CD4 T cell response and that this effector program underlies its protective impact. Canonical Th1 polarization requires cytokine-mediated activation of the transcription factors STAT1 and STAT4 that synergize to maximize the induction of the "master regulator" Th1 transcription factor, T-bet. Here, we determine the individual requirements for these transcription factors in directing the Th1 imprint primed by influenza infection in mice by tracking virus-specific wild-type or T-bet-deficient CD4 T cells in which STAT1 or STAT4 is knocked out. We find that STAT1 is required to protect influenza-primed CD4 T cells from NK cell-mediated deletion and for their expression of hallmark Th1 attributes. STAT1 is also required to prevent type I IFN signals from inhibiting the induction of the Th17 master regulator, Rorγt, in Th17-prone T-bet-/- cells responding to IAV. In contrast, STAT4 expression does not appreciably impact the phenotypic or functional attributes of wild-type or T-bet-/- CD4 T cell responses. However, cytokine-mediated STAT4 activation in virus-specific CD4 T cells enhances their Th1 identity in a T-bet-dependent manner, indicating that influenza infection does not promote maximal Th1 induction. Finally, we show that the T-bet-dependent protective capacity of CD4 T cell effectors against IAV is optimized by engaging both STAT1 and STAT4 during Th1 priming, with important implications for vaccine strategies aiming to generate T cell immunity.

Intermediate Levels of Pre-Existing Protective Antibody Allow Priming of Protective T Cell Immunity against Influenza.

Overcoming interfering impacts of pre-existing immunity to generate universally protective influenza A virus (IAV)-specific T cell immunity through vaccination is a high priority. In this study, we passively transfer varied amounts of H1N1-IAV-specific immune serum before H1N1-IAV infection to determine how different levels of pre-existing Ab influence the generation and protective potential of heterosubtypic T cell responses in a murine model. Surprisingly, IAV nucleoprotein-specific CD4 and CD8 T cell responses are readily detected in infected recipients of IAV-specific immune serum regardless of the amount transferred. When compared with responses in control groups and recipients of low and intermediate levels of convalescent serum, nucleoprotein-specific T cell responses in recipients of high levels of IAV-specific serum, which prevent overt weight loss and reduce peak viral titers in the lungs, are, however, markedly reduced. Although detectable at priming, this response recalls poorly and is unable to mediate protection against a lethal heterotypic (H3N2) virus challenge at later memory time points. A similar failure to generate protective heterosubtypic T cell immunity during IAV priming is seen in offspring of IAV-primed mothers that naturally receive high titers of IAV-specific Ab through maternal transfer. Our findings support that priming of protective heterosubtypic T cell responses can occur in the presence of intermediate levels of pre-existing Ab. These results have high relevance to vaccine approaches aiming to incorporate and evaluate cellular and humoral immunity towards IAV and other viral pathogens against which T cells can protect against variants escaping Ab-mediated protection.

Regulation of CD4 T Cell Responses by the Transcription Factor Eomesodermin.

Central to the impacts of CD4 T cells, both positive in settings of infectious disease and cancer and negative in the settings of autoimmunity and allergy, is their ability to differentiate into distinct effector subsets with specialized functions. The programming required to support such responses is largely dictated by lineage-specifying transcription factors, often called 'master regulators'. However, it is increasingly clear that many aspects of CD4 T cell immunobiology that can determine the outcomes of disease states involve a broader transcriptional network. Eomesodermin (Eomes) is emerging as an important member of this class of transcription factors. While best studied in CD8 T cells and NK cells, an increasing body of work has focused on impacts of Eomes expression in CD4 T cell responses in an array of different settings. Here, we focus on the varied impacts reported in these studies that, together, indicate the potential of targeting Eomes expression in CD4 T cells as a strategy to improve a variety of clinical outcomes.

Prostasin regulates PD-L1 expression in human lung cancer cells.

The serine protease prostasin is a negative regulator of lipopolysaccharide-induced inflammation and has a role in the regulation of cellular immunity. Prostasin expression in cancer cells inhibits migration and metastasis, and reduces epithelial-mesenchymal transition. Programmed death-ligand 1 (PD-L1) is a negative regulator of the immune response and its expression in cancer cells interferes with immune surveillance. The aim of the present study was to investigate if prostasin regulates PD-L1 expression. We established sublines overexpressing various forms of prostasin as well as a subline deficient for the prostasin gene from the Calu-3 human lung cancer cells. We report here that PD-L1 expression induced by interferon-γ (IFNγ) is further enhanced in cells overexpressing the wildtype membrane-anchored prostasin. The PD-L1 protein was localized on the cell surface and released into the culture medium in extracellular vesicles (EVs) with the protease-active prostasin. The epidermal growth factor-epidermal growth factor receptor (EGF-EGFR), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) participated in the prostasin-mediated up-regulation of PD-L1 expression. A Gene Set Enrichment Analysis (GSEA) of patient lung tumors in The Cancer Genome Atlas (TCGA) database revealed that prostasin and PD-L1 regulate common signaling pathways during tumorigenesis and tumor progression.

Prostasin regulates PD-L1 expression in human lung cancer cells.

The serine protease prostasin is a negative regulator of lipopolysaccharide-induced inflammation and has a role in the regulation of cellular immunity.  Prostasin expression in cancer cells inhibits migration and metastasis, and reduces epithelial-mesenchymal transition.  Programmed death-ligand 1 (PD-L1) is a negative regulator of the immune response and its expression in cancer cells interferes with immune surveillance.  The aim of this study was to investigate if prostasin regulates PD-L1 expression.  We established sublines over-expressing various forms of prostasin as well as a subline deficient for the prostasin gene from the Calu-3 human lung cancer cells.  We report here that PD-L1 expression induced by interferon-gamma (IFNg) is further enhanced in cells over-expressing the wild-type membrane-anchored prostasin.  The PD-L1 protein was localized on the cell surface and released into the culture medium in extracellular vesicles (EVs) with the protease-active prostasin.  The epidermal growth factor-epidermal growth factor receptor (EGF-EGFR), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) participated in the prostasin-mediated up-regulation of PD-L1 expression.  A Gene Set Enrichment Analysis (GSEA) of patient lung tumors in The Cancer Genome Atlas (TCGA) database revealed that prostasin and PD-L1 regulate common signaling pathways during tumorigenesis and tumor progression.

Durable CD4 T-Cell Memory Generation Depends on Persistence of High Levels of Infection at an Effector Checkpoint that Determines Multiple Fates.

We have discovered that the determination of CD4 effector and memory fates after infection is regulated not only by initial signals from antigen and pathogen recognition, but also by a second round of such signals at a checkpoint during the effector response. Signals to effectors determine their subsequent fate, inducing further progression to tissue-restricted follicular helpers, cytotoxic CD4 effectors, and long-lived memory cells. The follicular helpers help the germinal center B-cell responses that give rise to high-affinity long-lived antibody responses and memory B cells that synergize with T-cell memory to provide robust long-lived protection. We postulate that inactivated vaccines do not provide extended signals from antigen and pathogen beyond a few days, and thus elicit ineffective CD4 T- and B-cell effector responses and memory. Defining the mechanisms that underlie effective responses should provide insights necessary to develop vaccine strategies that induce more effective and durable immunity.

Polymeric Nanoparticles with a Sera-Derived Coating for Efficient Cancer Cell Uptake and Killing.

Nanoparticle-mediated cancer drug delivery remains an inefficient process. The protein corona formed on nanoparticles (NPs) controls their biological identity and, if optimized, could enhance cancer cell uptake. In this study, a hyperbranched polyester polymer (HBPE) was synthesized from diethyl malonate and used to generate NPs that were subsequently coated with normal sera (NS) collected from mice. Cellular uptake of NS-treated HBPE-NPs was compared to PEGylated HBPE-NPs and was assessed using MDA-MB-231 triple-negative breast cancer (TNBC) cells as well as endothelial and monocytic cell lines. NS-treated HBPE-NPs were taken up by TNBC cells more efficiently than PEGylated HBPE-NPs, while evasion of monocyte uptake was comparable. NS coatings facilitated cancer cell uptake of HBPE-NPs, even after prior interaction of the particles with an endothelial layer. NS-treated HBPE-NPs were not inherently toxic, did not induce the migration of endothelial cells that could lead to angiogenesis, and could efficiently deliver cytotoxic doses of paclitaxel (taxol) to TNBC cells. These findings suggest that HBPE-NPs may adsorb select sera proteins that improve uptake by cancer cells, and such NPs could be used to advance the discovery of novel factors that improve the bioavailability and tissue distribution of drug-loaded polymeric NPs.

CD122-targetted IL-2 signals cause acute and selective apoptosis of B cells in Peyer's Patches.

Interleukin-2 (IL-2) has both pro- and anti-inflammatory properties that have been harnessed clinically and that are used experimentally to modulate leukocyte subsets in vivo. In mice, the bioavailability and half-life of IL-2 in vivo can be increased by complexing recombinant IL-2 with different clones of anti-IL-2 monoclonal antibodies that differentially target the cytokine to cells expressing different kinds of IL-2 receptors. While the impacts of systemic IL-2: anti-IL-2 antibody complex (IL-2C) administration are well-defined in the spleen and peripheral lymph nodes, how immune cells in the gut and gut-associated lymphoid tissues respond to IL-2C is not well characterized. Here, we analyze how major leukocyte populations in these tissues respond to IL-2C. We find that IL-2C targeting cells expressing IL-2 receptor beta cause an acute decrease in cellularity of Peyer's Patches while cell numbers in the lamina propria and intraepithelial lymphocytes are unaffected. Cell contraction in Peyer's Patches is associated with the apoptosis of multiple B cell subsets. Our results are important to consider for understanding off-target impacts of IL-2C regimes in experimental models and for considering how IL-2 may contribute to the etiology or severity of gut-associated conditions such as Crohn's Disease.

CD25-Targeted IL-2 Signals Promote Improved Outcomes of Influenza Infection and Boost Memory CD4 T Cell Formation.

IL-2 is a pleotropic cytokine with potent pro- and anti-inflammatory effects. These divergent impacts can be directed in vivo by forming complexes of IL-2 and anti-IL-2 mAbs (IL-2C) to target IL-2 to distinct subsets of cells based on their expression of subunits of the IL-2R. In this study, we show that treatment of mice with a prototypical anti-inflammatory IL-2C, JES6-1-IL-2C, best known to induce CD25+ regulatory CD4 T cell expansion, surprisingly causes robust induction of a suite of inflammatory factors. However, treating mice infected with influenza A virus with this IL-2C reduces lung immunopathology. We compare the spectrum of inflammatory proteins upregulated by pro- and anti-inflammatory IL-2C treatment and uncover a pattern of expression that reveals potentially beneficial versus detrimental aspects of the influenza-associated cytokine storm. Moreover, we show that anti-inflammatory IL-2C can deliver survival signals to CD4 T cells responding to influenza A virus that improve their memory fitness, indicating a novel application of IL-2 to boost pathogen-specific T cell memory while simultaneously reducing immunopathology.

Virus-induced natural killer cell lysis of T cell subsets.

In addition to direct anti-viral activity, NK cells regulate viral pathogenesis by virtue of their cytolytic attack on activated CD4 and CD8 T cells. To gain insight into which differentiated T cell subsets are preferred NK targets, transgenic T cells were differentiated in vitro into Th0, Th1, Th2, Th17, Treg, Tc1, and Tc2 effector cells and then tested for lysis by enriched populations of lymphocytic choriomeningitis virus (LCMV)-induced activated NK cells. There was a distinct hierarchy of cytotoxicity in vitro and in vivo, with Treg, Th17, and Th2 cells being more sensitive and Th0 and Th1 cells more resistant. Some distinctions between in vitro vs in vivo generated T cells were explainable by type 1 interferon induction of class 1 histocompatibility antigens on the effector T cell subsets. NK receptor (NKR)-deficient mice and anti-NKR antibody studies identified no one essential NKR for killing, though there could be redundancies.

Memory CD4 T cell-derived IL-2 synergizes with viral infection to exacerbate lung inflammation.

Defining the most penetrating correlates of protective memory T cells is key for designing improved vaccines and T cell therapies. Here, we evaluate how interleukin (IL-2) production by memory CD4 T cells, a widely held indicator of their protective potential, impacts immune responses against murine influenza A virus (IAV). Unexpectedly, we show that IL-2-deficient memory CD4 T cells are more effective on a per cell basis at combating IAV than wild-type memory cells that produce IL-2. Improved outcomes orchestrated by IL-2-deficient cells include reduced weight loss and improved respiratory function that correlate with reduced levels of a broad array of inflammatory factors in the infected lung. Blocking CD70-CD27 signals to reduce CD4 T cell IL-2 production tempers the inflammation induced by wild-type memory CD4 T cells and improves the outcome of IAV infection in vaccinated mice. Finally, we show that IL-2 administration drives rapid and extremely potent lung inflammation involving NK cells, which can synergize with sublethal IAV infection to promote acute death. These results suggest that IL-2 production is not necessarily an indicator of protective CD4 T cells, and that the lung environment is particularly sensitive to IL-2-induced inflammation during viral infection.

T-bet optimizes CD4 T-cell responses against influenza through CXCR3-dependent lung trafficking but not functional programming.

Although clearance of many intracellular pathogens requires T-bet-dependent CD4 T cell programming, the extent to which T-bet is needed to direct protective CD4 responses against influenza is not known. Here, we characterize wild-type and T-bet-deficient CD4 cells during murine influenza infection. Surprisingly, although T-bet expression has broad impacts on cytokine production by virus-specific CD4 cells, the protective efficacy of T-bet-deficient effector cells is only marginally reduced. This reduction is due to lower CXCR3 expression, leading to suboptimal accumulation of activated T-bet-deficient cells in the infected lung. However, T-bet-deficient cells outcompete wild-type cells to form lung-resident and circulating memory populations following viral clearance, and primed T-bet-deficient mice efficiently clear supralethal heterosubtypic influenza challenges even when depleted of CD8 T cells. These results are relevant to the identification of more incisive correlates of protective T cells and for vaccines that aim to induce durable cellular immunity against influenza.

Cigarette smoke extract acts directly on CD4 T cells to enhance Th1 polarization and reduce memory potential.

Although cigarette smoke is known to alter immune responses, whether and how CD4 T cells are affected is not well-described. We aimed to characterize how exposure to cigarette smoke extract impacts CD4 T cell effector generation in vitro under Th1-polarizing conditions. Our results demonstrate that cigarette smoke directly acts on CD4 T cells to impair effector expansion by decreasing division and increasing apoptosis. Furthermore, cigarette smoke enhances Th1-associated cytokine production and increases expression of the transcription factor T-bet, the master regulator of Th1 differentiation. Finally, we show that exposure to cigarette smoke extract during priming impairs the ability of effectors to form memory cells. Our findings thus demonstrate that cigarette smoke simultaneously enhances effector functions but promotes terminal differentiation of CD4 T cell effectors. This study may be relevant to understanding how smoking can both aggravate autoimmune symptoms and reduce vaccine efficacy.

Short-Lived Antigen Recognition but Not Viral Infection at a Defined Checkpoint Programs Effector CD4 T Cells To Become Protective Memory.

Although memory CD4 T cells are critical for effective immunity to pathogens, the mechanisms underlying their generation are still poorly defined. We find that following murine influenza infection, most effector CD4 T cells undergo apoptosis unless they encounter cognate Ag at a defined stage near the peak of effector generation. Ag recognition at this memory checkpoint blocks default apoptosis and programs their transition to long-lived memory. Strikingly, we find that viral infection is not required, because memory formation can be restored by the addition of short-lived, Ag-pulsed APC at this checkpoint. The resulting memory CD4 T cells express an enhanced memory phenotype, have increased cytokine production, and provide protection against lethal influenza infection. Finally, we find that memory CD4 T cell formation following cold-adapted influenza vaccination is boosted when Ag is administered during this checkpoint. These findings imply that persistence of viral Ag presentation into the effector phase is the key factor that determines the efficiency of memory generation. We also suggest that administering Ag at this checkpoint may improve vaccine efficacy.

Interleukin 27R regulates CD4+ T cell phenotype and impacts protective immunity during Mycobacterium tuberculosis infection.

CD4+ T cells mediate protection against Mycobacterium tuberculosis (Mtb); however, the phenotype of protective T cells is undefined, thereby confounding vaccination efforts. IL-27 is highly expressed during human tuberculosis (TB), and absence of IL-27R (Il27ra) specifically on T cells results in increased protection. IL-27R deficiency during chronic Mtb infection does not impact antigen-specific CD4+ T cell number but maintains programmed death-1 (PD-1), CD69, and CD127 expression while reducing T-bet and killer cell lectin-like receptor G1 (KLRG1) expression. Furthermore, T-bet haploinsufficiency results in failure to generate KLRG1+, antigen-specific CD4+ T cells, and in improved protection. T cells in Il27ra(-/-) mice accumulate preferentially in the lung parenchyma within close proximity to Mtb, and antigen-specific CD4+ T cells lacking IL-27R are intrinsically more fit than intact T cells and maintain IL-2 production. Improved fitness of IL-27R-deficient T cells is not associated with increased proliferation but with decreased expression of cell death-associated markers. Therefore, during Mtb infection, IL-27R acts intrinsically on T cells to limit protection and reduce fitness, whereas the IL-27R-deficient environment alters the phenotype and location of T cells. The significant expression of IL-27 in TB and the negative influence of IL-27R on T cell function demonstrate the pathway by which this cytokine/receptor pair is detrimental in TB.

Intraepithelial T-cell cytotoxicity, induced bronchus-associated lymphoid tissue, and proliferation of pneumocytes in experimental mouse models of influenza.

Immunopathologic examination of the lungs of mice with experimental influenza virus infection reveals three prominent findings. (i) There is rapidly developing perivascular (arterial) and peribronchial infiltration with T-cells and invasion of T-cells into the bronchiolar epithelium, separation of epithelial cells from each other and from the basement membrane, leading to defoliation of the bronchial epithelium. This reaction is analogous to a viral exanthema of the skin, such as measles and smallpox. This previously described but unappreciated reaction most likely is an effective way to eliminate virus-infected cells, but may contribute to acute toxicity and mortality. (ii) After this, there is formation of dense collections of lymphocytes adjacent to bronchi consisting mainly of B-cells, with a scattering of T-cells and macrophages. This is known as induced bronchial-associated lymphoid tissue (iBALT) and correlates with increased interleukin (IL)-17 in the lung. iBALT provides sites for a local immune reaction in the lung to both the original infection and related viral infections (heterologous immunity). (iii) Within the first 2-3 weeks, there is proliferation of type II pneumocytes and/or terminal bronchial epithelial cells extending from the terminal bronchioles into the adjacent alveoli, eventually leading to large zones of the lung filled with tumor-like epithelial cells with squamous metaplasia. The proliferation correlates with IL-17 and IL-22 expression, and the extent of this reaction appears to be determined by the availability of T-regulatory cells.

Effector CD4 T-cell transition to memory requires late cognate interactions that induce autocrine IL-2.

It is unclear how CD4 T-cell memory formation is regulated following pathogen challenge, and when critical mechanisms act to determine effector T-cell fate. Here, we report that following influenza infection most effectors require signals from major histocompatibility complex class II molecules and CD70 during a late window well after initial priming to become memory. During this timeframe, effector cells must produce IL-2 or be exposed to high levels of paracrine or exogenously added IL-2 to survive an otherwise rapid default contraction phase. Late IL-2 promotes survival through acute downregulation of apoptotic pathways in effector T cells and by permanently upregulating their IL-7 receptor expression, enabling IL-7 to sustain them as memory T cells. This new paradigm defines a late checkpoint during the effector phase at which cognate interactions direct CD4 T-cell memory generation.

The number of responding CD4 T cells and the dose of antigen conjointly determine the TH1/TH2 phenotype by modulating B7/CD28 interactions.

Our previous in vivo studies show that both the amount of Ag and the number of available naive CD4 T cells affect the Th1/Th2 phenotype of the effector CD4 T cells generated. We examined how the number of OVA-specific CD4 TCR transgenic T cells affects the Th1/Th2 phenotype of anti-SRBC CD4 T cells generated in vivo upon immunization with different amounts of OVA-SRBC. Our observations show that a greater number of Ag-dependent CD4 T cell interactions are required to generate Th2 than Th1 cells. We established an in vitro system that recapitulates our main in vivo findings to more readily analyze the underlying mechanism. The in vitro generation of Th2 cells depends, as in vivo, upon both the number of responding CD4 T cells and the amount of Ag. We demonstrate, using agonostic/antagonistic Abs to various costimulatory molecules or their receptors, that the greater number of CD4 T cell interactions, required to generate Th2 over Th1 cells, does not involve CD40, OX40, or ICOS costimulation, but does involve B7/CD28 interactions. A comparison of the level of expression of B7 molecules by APC and CD4 T cells, under different conditions resulting in the substantial generation of Th1 and Th2 cells, leads us to propose that the critical CD28/B7 interactions, required to generate Th2 cells, may directly occur between CD4 T cells engaged with the same B cell acting as an APC.

Multipronged CD4(+) T-cell effector and memory responses cooperate to provide potent immunity against respiratory virus.

Over the last decade, the known spectrum of CD4(+) T-cell effector subsets has become much broader, and it has become clear that there are multiple dimensions by which subsets with a particular cytokine commitment can be further defined, including their stage of differentiation, their location, and, most importantly, their ability to carry out discrete functions. Here, we focus on our studies that highlight the synergy among discrete subsets, especially those defined by helper and cytotoxic function, in mediating viral protection, and on distinctions between CD4(+) T-cell effectors located in spleen, draining lymph node, and in tissue sites of infection. What emerges is a surprising multiplicity of CD4(+) T-cell functions that indicate a large arsenal of mechanisms by which CD4(+) T cells act to combat viruses.