Triphasic production of IFNγ by innate and adaptive lymphocytes following influenza A virus infection.

Interferon gamma (IFNγ) is a potent antiviral cytokine that can be produced by many innate and adaptive immune cells during infection. Currently, our understanding of which cells produce IFNγ and where they are located at different stages of an infection is limited. We have used reporter mice to investigate in vivo expression of Ifnγ mRNA in the lung and secondary lymphoid organs during and following influenza A virus (IAV) infection. We observed a triphasic production of Ifnγ expression. Unconventional T cells and innate lymphoid cells, particularly NK cells, were the dominant producers of early Ifnγ, while CD4 and CD8 T cells were the main producers by day 10 post-infection. Following viral clearance, some memory CD4 and CD8 T cells continued to express Ifnγ in the lungs and draining lymph node. Interestingly, Ifnγ production by lymph node natural killer (NK), NKT, and innate lymphoid type 1 cells also continued to be above naïve levels, suggesting memory-like phenotypes for these cells. Analysis of the localization of Ifnγ+ memory CD4 and CD8 T cells demonstrated that cytokine+ T cells were located near airways and in the lung parenchyma. Following a second IAV challenge, lung IAV-specific CD8 T cells rapidly increased their expression of Ifnγ while CD4 T cells in the draining lymph node increased their Ifnγ response. Together, these data suggest that Ifnγ production fluctuates based on cellular source and location, both of which could impact subsequent immune responses.

Multifunctional cytokine production marks influenza A virus-specific CD4 T cells with high expression of survival molecules.

Cytokine production by memory T cells is a key mechanism of T cell mediated protection. However, we have limited understanding of the persistence of cytokine producing T cells during memory cell maintenance and secondary responses. We interrogated antigen-specific CD4 T cells using a mouse influenza A virus infection model. Although CD4 T cells detected using MHCII tetramers declined in lymphoid and non-lymphoid organs, we found similar numbers of cytokine+ CD4 T cells at days 9 and 30 in the lymphoid organs. CD4 T cells with the capacity to produce cytokines expressed higher levels of pro-survival molecules, CD127 and Bcl2, than non-cytokine+ cells. Transcriptomic analysis revealed a heterogeneous population of memory CD4 T cells with three clusters of cytokine+ cells. These clusters match flow cytometry data and reveal an enhanced survival signature in cells capable of producing multiple cytokines. Following re-infection, multifunctional T cells expressed low levels of the proliferation marker, Ki67, whereas cells that only produce the anti-viral cytokine, interferon-γ, were more likely to be Ki67+ . Despite this, multifunctional memory T cells formed a substantial fraction of the secondary memory pool. Together these data indicate that survival rather than proliferation may dictate which populations persist within the memory pool.

BTN3A3 evasion promotes the zoonotic potential of influenza A viruses.

Spillover events of avian influenza A viruses (IAVs) to humans could represent the first step in a future pandemic1. Several factors that limit the transmission and replication of avian IAVs in mammals have been identified. There are several gaps in our understanding to predict which virus lineages are more likely to cross the species barrier and cause disease in humans1. Here, we identified human BTN3A3 (butyrophilin subfamily 3 member A3)2 as a potent inhibitor of avian IAVs but not human IAVs. We determined that BTN3A3 is expressed in human airways and its antiviral activity evolved in primates. We show that BTN3A3 restriction acts primarily at the early stages of the virus life cycle by inhibiting avian IAV RNA replication. We identified residue 313 in the viral nucleoprotein (NP) as the genetic determinant of BTN3A3 sensitivity (313F or, rarely, 313L in avian viruses) or evasion (313Y or 313V in human viruses). However, avian IAV serotypes, such as H7 and H9, that spilled over into humans also evade BTN3A3 restriction. In these cases, BTN3A3 evasion is due to substitutions (N, H or Q) in NP residue 52 that is adjacent to residue 313 in the NP structure3. Thus, sensitivity or resistance to BTN3A3 is another factor to consider in the risk assessment of the zoonotic potential of avian influenza viruses.

Antigen presenting cells: Professionals, amateurs, and spectators in the 'long game' of lung immunity.

The lung is frequently and repeatedly exposed to invading pathogens and thus requires constant immunosurveillance. Professional antigen presenting cells (APCs), including dendritic cells, engulf invading pathogens and present their peptides via major histocompatibility complexes (MHC) I and II, to CD8 or CD4 T cells. Epithelial cells and stromal cells (including fibroblasts) provide more than structural support, they are increasingly recognised as key players in the immune response, acting as non-professional APCs through interactions with antigen experienced T cells that migrate to the lung. The importance of the contributions of non-professional and professional APCs to T cell function in vivo, is currently unclear. This review summarises the roles of professional and non-professional APCs in lung immunity, at the steady state and following viral insult, with particular emphasis on their ability to interact with and influence T cells.

Low-Dose Lung Radiation Therapy for COVID-19 Lung Disease: A Preclinical Efficacy Study in a Bleomycin Model of Pneumonitis.

PURPOSE: Low-dose whole lung radiation therapy (LDLR) has been proposed as a treatment for patients with acute respiratory distress syndrome associated with SARS-CoV-2 infection, and clinical trials are underway. There is an urgent need for preclinical evidence to justify this approach and inform dose, scheduling, and mechanisms of action. METHODS AND MATERIALS: Female C57BL/6 mice were treated with intranasal bleomycin sulfate (7.5 or 11.25 units/kg, day 0) and then exposed to whole lung radiation therapy (0.5, 1.0, or 1.5 Gy, or sham; day 3). Bodyweight was measured daily, and lung tissue was harvested for histology and flow cytometry on day 10. Computed tomography lung imaging was performed before radiation (day 3) and pre-endpoint (day 10). RESULTS: Bleomycin caused pneumonitis of variable severity, which correlated with weight loss. LDLR at 1.0 Gy was associated with a significant increase in the proportion of mice recovering to 98% of initial bodyweight, and a proportion of these mice exhibited less severe histopathologic lung changes. Mice experiencing moderate initial weight loss were more likely to respond to LDLR than those experiencing severe initial weight loss. In addition, LDLR (1.0 Gy) significantly reduced bleomycin-induced increases in interstitial macrophages, CD103+ dendritic cells (DCs), and neutrophil-DC hybrids. Overall, bleomycin-treated mice exhibited significantly higher percentages of nonaerated lung in left than right lungs, and LDLR (1.0 Gy) limited further reductions in aerated lung volume in right but not left lungs. LDLR at 0.5 and 1.5 Gy did not improve bodyweight, flow cytometric, or radiologic readouts of bleomycin-induced pneumonitis. CONCLUSIONS: Our data support the concept that LDLR can ameliorate acute inflammatory lung injury, identify 1.0 Gy as the most effective dose, and provide evidence that it is more effective in the context of moderate than severe pneumonitis. Mechanistically, LDLR at 1.0 Gy significantly suppressed bleomycin-induced accumulation of pulmonary interstitial macrophages, CD103+ DCs, and neutrophil-DC hybrids.

Stromal-immune cell crosstalk fundamentally alters the lung microenvironment following tissue insult.

Communication between stromal and immune cells is essential to maintain tissue homeostasis, mount an effective immune response and promote tissue repair. This 'crosstalk' occurs in both the steady state and following a variety of insults, for example, in response to local injury, at sites of infection or cancer. What do we mean by crosstalk between cells? Reciprocal activation and/or regulation occurs between immune and stromal cells, by direct cell contact and indirect mechanisms, including the release of soluble cytokines. Moving beyond cell-to-cell contact, this review investigates the complexity of 'cross-space' cellular communication. We highlight different examples of cellular communication by a variety of lung stromal and immune cells following tissue insults. This review examines how the 'geography of the lung microenvironment' is altered in various disease states; more specifically, we investigate how this influences lung epithelial cells and fibroblasts via their communication with immune cells and each other.

Tolerance induction in memory CD4 T cells is partial and reversible.

Memory T cells respond rapidly in part because they are less reliant on a heightened levels of costimulatory molecules. This enables rapid control of secondary infecting pathogens but presents challenges to efforts to control or silence memory CD4 T cells, for example in antigen-specific tolerance strategies for autoimmunity. We have examined the transcriptional and functional consequences of reactivating memory CD4 T cells in the absence of an adjuvant. We find that memory CD4 T cells generated by infection or immunisation survive secondary activation with antigen delivered without adjuvant, regardless of their location in secondary lymphoid organs or peripheral tissues. These cells were, however, functionally altered following a tertiary immunisation with antigen and adjuvant, proliferating poorly but maintaining their ability to produce inflammatory cytokines. Transcriptional and cell cycle analysis of these memory CD4 T cells suggests they are unable to commit fully to cell division potentially because of low expression of DNA repair enzymes. In contrast, these memory CD4 T cells could proliferate following tertiary reactivation by viral re-infection. These data indicate that antigen-specific tolerogenic strategies must examine multiple parameters of Tcell function, and provide insight into the molecular mechanisms that may lead to deletional tolerance of memory CD4 T cells.

Regulatory T cells control the dynamic and site-specific polarization of total CD4 T cells following Salmonella infection.

FoxP3+ regulatory T cells (Tregs) control inflammation and maintain mucosal homeostasis, but their functions during infection are poorly understood. Th1, Th2, and Th17 cells can be identified by master transcription factors (TFs) T-bet, GATA3, and RORγT; Tregs also express these TFs. While T-bet+ Tregs can selectively suppress Th1 cells, it is unclear whether distinct Treg populations can alter Th bias. To address this, we used Salmonella enterica serotype Typhimurium to induce nonlethal colitis. Following infection, we observed an early colonic Th17 response within total CD4 T cells, followed by a Th1 bias. The early Th17 response, which contains both Salmonella-specific and non-Salmonella-specific cells, parallels an increase in T-bet+ Tregs. Later, Th1 cells and RORγT+ Tregs dominate. This reciprocal dynamic may indicate that Tregs selectively suppress Th cells, shaping the immune response. Treg depletion 1-2 days post-infection shifted the early Th17 response to a Th1 bias; however, Treg depletion 6-7 days post-infection abrogated the Th1 bias. Thus, Tregs are necessary for the early Th17 response, and for a maximal Th1 response later. These data show that Tregs shape the overall tissue CD4 T cell response and highlight the potential for subpopulations of Tregs to be used in targeted therapeutic approaches.

Chemokine Receptor Redundancy and Specificity Are Context Dependent.

Currently, we lack an understanding of the individual and combinatorial roles for chemokine receptors in the inflammatory process. We report studies on mice with a compound deletion of Ccr1, Ccr2, Ccr3, and Ccr5, which together control monocytic and eosinophilic recruitment to resting and inflamed sites. Analysis of resting tissues from these mice, and mice deficient in each individual receptor, provides clear evidence for redundant use of these receptors in establishing tissue-resident monocytic cell populations. In contrast, analysis of cellular recruitment to inflamed sites provides evidence of specificity of receptor use for distinct leukocyte subtypes and no indication of comprehensive redundancy. We find no evidence of involvement of any of these receptors in the recruitment of neutrophils or lymphocytes to resting or acutely inflamed tissues. Our data shed important light on combinatorial inflammatory chemokine receptor function and highlight Ccr2 as the primary driver of myelomonocytic cell recruitment in acutely inflamed contexts.

The Atypical Chemokine Receptor Ackr2 Constrains NK Cell Migratory Activity and Promotes Metastasis.

Chemokines have been shown to be essential players in a range of cancer contexts. In this study, we demonstrate that mice deficient in the atypical chemokine receptor Ackr2 display impaired development of metastasis in vivo in both cell line and spontaneous models. Further analysis reveals that this relates to increased expression of the chemokine receptor CCR2, specifically by KLRG1+ NK cells from the Ackr2-/- mice. This leads to increased recruitment of KLRG1+ NK cells to CCL2-expressing tumors and enhanced tumor killing. Together, these data indicate that Ackr2 limits the expression of CCR2 on NK cells and restricts their tumoricidal activity. Our data have important implications for our understanding of the roles for chemokines in the metastatic process and highlight Ackr2 and CCR2 as potentially manipulable therapeutic targets in metastasis.

The roles of resident, central and effector memory CD4 T-cells in protective immunity following infection or vaccination.

Immunological memory provides rapid protection to pathogens previously encountered through infection or vaccination. CD4 T-cells play a central role in all adaptive immune responses. Vaccines must, therefore, activate CD4 T-cells if they are to generate protective immunity. For many diseases, we do not have effective vaccines. These include human immunodeficiency virus (HIV), tuberculosis and malaria, which are responsible for many millions of deaths each year across the globe. CD4 T-cells play many different roles during the immune response coordinating the actions of many other cells. In order to harness the diverse protective effects of memory CD4 T-cells, we need to understand how memory CD4 T-cells are generated and how they protect the host. Here we review recent findings on the location of different subsets of memory CD4 T-cells that are found in peripheral tissues (tissue resident memory T-cells) and in the circulation (central and effector memory T-cells). We discuss the generation of these cells, and the evidence that demonstrates how they provide immune protection in animal and human challenge models.

Sphingosine-1-Phosphate Promotes the Persistence of Activated CD4 T Cells in Inflamed Sites.

Inflammation can be protective or pathogenic depending on context and timeframe. Acute inflammation, including the accumulation of CD4 T cells, accompanies protective immune responses to pathogens, but the presence of activated CD4 T cells at sites of inflammation is associated with chronic inflammatory disease. While significant progress has been made in understanding the migration of CD4 T cells into inflamed sites, the signals that lead to their persistence are poorly characterized. Using a murine ear model of acute inflammation and intravital two-photon imaging, we have dissected the signals that mediate CD4 T cell persistence. We report the unexpected finding that the bioactive lipid, sphingosine-1-phosphate (S1P), is both necessary and sufficient for the persistence of activated CD4 T cells at peripheral tissues in acute inflammation. S1P mediated the enhanced motility of CD4 T cells at inflamed tissues but did not affect their migration to the downstream draining lymph node. We found that sphingosine kinase-1, which regulates S1P production is increased at inflamed sites in mice and in patients with the chronic inflammatory disease, rheumatoid arthritis. Together, these data suggest that S1P, or its regulators, may be key targets to promote or disrupt accumulation of CD4 T cells at inflamed tissues.

T Cell Response in the Lung Following Influenza Virus Infection.

Methods that enable the identification of virus-specific CD4 and CD8 T cells are key to our understanding of how the adaptive immune response controls viral infection. Here we describe two distinct methods to evaluate the T cell response to influenza A virus (IAV). The number and phenotype of T cells that respond to natural IAV epitopes can be assessed by flow cytometry using MHC class I and class II tetramers. Using this system, IAV-specific T cells can be tracked in various organs within the same animal, or, in different cohorts, the response can be evaluated at several time points following infection. While providing clear quantitative data, flow cytometry cannot provide any information about T cell location within the lung or interactions between responding T cells and other cell types. Here we also describe a method to examine activated CD4 T cells in the lungs of living animals using multiphoton intravital microscopy, thus providing real-time analysis of T cell behavior during an infection.

Development and Function of Protective and Pathologic Memory CD4 T Cells.

Immunological memory is one of the defining features of the adaptive immune system. As key orchestrators and mediators of immunity, CD4 T cells are central to the vast majority of adaptive immune responses. Generated following an immune response, memory CD4 T cells retain pertinent information about their activation environment enabling them to make rapid effector responses upon reactivation. These responses can either benefit the host by hastening the control of pathogens or cause damaging immunopathology. Here, we will discuss the diversity of the memory CD4 T cell pool, the signals that influence the transition of activated T cells into that pool, and highlight how activation requirements differ between naïve and memory CD4 T cells. A greater understanding of these factors has the potential to aid the design of more effective vaccines and to improve regulation of pathologic CD4 T cells, such as in the context of autoimmunity and allergy.

Antigen presentation kinetics control T cell/dendritic cell interactions and follicular helper T cell generation in vivo.

The production of high affinity, class switched antibodies produced by B cells hinges on the effective differentiation of T follicular helper (Tfh) cells. Here we define conditions specifically enhancing Tfh differentiation and providing protection in a model of influenza infection. Tfh responses were associated with prolonged antigen presentation by dendritic cells (DCs), which maintained T cell/DC interactions into stage 3 (>72 hr) of activation. Blocking stage 3 interactions ablated Tfh generation, demonstrating a causal link between T cell-DC behaviour and functional outcomes. The current data therefore explain how duration of antigen presentation affects the dynamics of T cell-DC interactions and consequently determine Tfh cell differentiation in the developing immune response.

Antigen-based immunotherapy (AIT) for autoimmune and allergic disease.

Autoimmune and allergic diseases are major causes of morbidity. Antigen-based immunotherapy (AIT) is immunologically the most satisfying means of specifically targeting only those T cells driving disease, thereby inducing antigen-specific immune tolerance, with the lowest adverse risk profile. AIT is highly effective in rodent models of T cell-driven inflammation and is now in clinical trials. The range of approaches to applying AIT in the clinic prevents a consensus on the molecular basis for this form of tolerance. In particular, there has been a paucity of information on how pre-activated effector and memory T cells respond to AIT. New, advanced murine models of AIT are beginning to deliver such information at the cellular, biochemical, transcriptional and epigenetic levels.

Influenza nucleoprotein delivered with aluminium salts protects mice from an influenza A virus that expresses an altered nucleoprotein sequence.

Influenza virus poses a difficult challenge for protective immunity. This virus is adept at altering its surface proteins, the proteins that are the targets of neutralizing antibody. Consequently, each year a new vaccine must be developed to combat the current recirculating strains. A universal influenza vaccine that primes specific memory cells that recognise conserved parts of the virus could prove to be effective against both annual influenza variants and newly emergent potentially pandemic strains. Such a vaccine will have to contain a safe and effective adjuvant that can be used in individuals of all ages. We examine protection from viral challenge in mice vaccinated with the nucleoprotein from the PR8 strain of influenza A, a protein that is highly conserved across viral subtypes. Vaccination with nucleoprotein delivered with a universally used and safe adjuvant, composed of insoluble aluminium salts, provides protection against viruses that either express the same or an altered version of nucleoprotein. This protection correlated with the presence of nucleoprotein specific CD8 T cells in the lungs of infected animals at early time points after infection. In contrast, immunization with NP delivered with alum and the detoxified LPS adjuvant, monophosphoryl lipid A, provided some protection to the homologous viral strain but no protection against infection by influenza expressing a variant nucleoprotein. Together, these data point towards a vaccine solution for all influenza A subtypes.

Antigen-specific suppression of humoral immunity by anergic Ars/A1 B cells.

Autoreactive anergic B lymphocytes are considered to be dangerous because of their potential for activation and recruitment into autoimmune responses. However, they persist for days and constitute ∼5% of the B cell pool. We assessed their functional potential in the Ars/A1 transgene model, where anergic B cells express a dual-reactive Ag receptor that binds, in addition to a self-Ag, the hapten p-azophenylarsonate (Ars). When Ars/A1 B cells were transferred into adoptive recipients that were immunized with foreign proteins covalently conjugated with Ars, endogenous IgG immune responses to both were selectively and severely diminished, and the development of T helper cells was impaired. Approximately 95% inhibition of the anti-Ars response was attained with ∼4000 transferred Ars/A1 B cells through redundant mechanisms, one of which depended on their expression of MHC class II but not upon secretion of IL-10 or IgM. This Ag-specific suppressive activity implicates the autoreactive anergic B cell as an enforcer of immunological tolerance to self-Ags.

Vaccine adjuvants aluminum and monophosphoryl lipid A provide distinct signals to generate protective cytotoxic memory CD8 T cells.

Vaccines can greatly reduce the spread of and deaths from many infectious diseases. However, many infections have no successful vaccines. Better understanding of the generation of protective CD8 memory T cells by vaccination is essential for the rational design of new vaccines that aim to prime cellular immune responses. Here we demonstrate that the combination of two adjuvants that are currently licensed for use in humans can be used to prime long-lived memory CD8 T cells that protect mice from viral challenge. The universally used adjuvant, aluminum salts, primed long-lived memory CD8 T cells; however, effective cytotoxic T-cell differentiation occurred only in the presence of an additional adjuvant, monophosphoryl lipid A (MPL). MPL-induced IL-6 was required for cytotoxic differentiation. The IL-6 acted by inducing granzyme B production and reducing expression of inhibitory molecule PD1 on the surface of the primed CD8 T cells. CD8 memory T cells generated by antigen delivered with both aluminum salts and MPL provided significant protection from influenza A challenge. These adjuvants could be used in human vaccines to prime protective memory CD8 T cells.

Memory CD4 T cells that express CXCR5 provide accelerated help to B cells.

CD4 T cell help for B cells is critical for effective Ab responses. Although many of the molecules involved in helper functions of naive CD4 T cells have been characterized, much less is known about the helper capabilities of memory CD4 T cells, an important consideration for the design of vaccines that aim to prime protective memory CD4 T cells. In this study, we demonstrate that memory CD4 T cells enable B cells to expand more rapidly and class switch earlier than do primary responding CD4 T cells. This accelerated response does not require large numbers of memory cells, and similar numbers of primary responding cells provide less effective help than do memory cells. However, only memory CD4 T cells that express the B cell follicle homing molecule, CXCR5, are able to accelerate the response, suggesting that the rapidity of the Ab response depends on the ability of CD4 memory T cells to migrate quickly toward B cells.