Tarun E. Hutchinson

CD8 T cell memory to a viral pathogen requires trans cosignaling between HVEM and BTLA.

Defining the molecular interactions required to program activated CD8 T cells to survive and become memory cells may allow us to understand how to augment anti-viral immunity. HVEM (herpes virus entry mediator) is a member of the tumor necrosis factor receptor (TNFR) family that interacts with ligands in the TNF family, LIGHT and Lymphotoxin-α, and in the Ig family, B and T lymphocyte attenuator (BTLA) and CD160. The Ig family members initiate inhibitory signaling when engaged with HVEM, but may also activate survival gene expression. Using a model of vaccinia virus infection, we made the unexpected finding that deficiency in HVEM or BTLA profoundly impaired effector CD8 T cell survival and development of protective immune memory. Mixed adoptive transfer experiments indicated that BTLA expressed in CD8α+ dendritic cells functions as a trans-activating ligand that delivers positive co-signals through HVEM expressed in T cells. Our data demonstrate a critical role of HVEM-BTLA bidirectional cosignaling system in antiviral defenses by driving the differentiation of memory CD8 T cells.

CD8 T Cells Use IFN-γ To Protect against the Lethal Effects of a Respiratory Poxvirus Infection

CD8 T cells are a key component of immunity to many viral infections. They achieve this through using an array of effector mechanisms, but precisely which component/s are required for protection against a respiratory orthopox virus infection remains unclear. Using a model of respiratory vaccinia virus infection in mice, we could specifically determine the relative contribution of perforin, TRAIL, and IFN-γ–mediated pathways in protection against virus induced morbidity and mortality. Unexpectedly, we observed that protection against death was mediated by IFN-γ without any involvement of the perforin or TRAIL-dependent pathways. IFN-γ mRNA and protein levels in the lung peaked between days 3 and 6 postinfection. This enhanced response coincided with the emergence of virus-specific CD8 T cells in the lung and the cessation of weight loss. Transfer experiments indicated that CD8 T cell–autonomous expression of IFN-γ restricts virus-induced lung pathology and dissemination to visceral tissues and is necessary for clearance of virus. Most significantly, we show that CD8 T cell–derived IFN-γ is sufficient to protect mice in the absence of CD4 and B-lymphocytes. Thus, our findings reveal a previously unappreciated mechanism by which effector CD8 T cells afford protection against a highly virulent respiratory orthopox virus infection.

OX40 Cooperates with ICOS To Amplify Follicular Th Cell Development and Germinal Center Reactions during Infection

Cognate interactions between T follicular helper (Tfh) cells and B cells are essential for promoting protective Ab responses. Whereas costimulatory receptors such as ICOS are accepted as being important for the induction of Tfh cell fate decision, other molecules may play key roles in amplifying or maintaining the Tfh phenotype. In this study, with vaccinia virus infection in mice, we show that OX40 was expressed on Tfh cells that accumulated at the T/B borders in the white pulp of the spleen and that OX40-dependent signals directly shaped the magnitude and quality of the their response to viral Ags. OX40 deficiency in Tfh cells profoundly impaired the acquisition of germinal center (GC) B cell phenotype, plasma cell generation, and virus-specific Ab responses. Most significantly, we found that sustained interactions between OX40 and its ligand, OX40L, beyond the time of initial encounter with dendritic cells were required for the persistence of high numbers of Tfh and GC B cells. Interestingly, OX40 was coexpressed with ICOS on Tfh cells in and around the GC, and ICOS–ICOSL interactions were similarly crucial at late times for maintenance of the Tfh and GC B cells. Thus, OX40 and ICOS act in a cooperative, nonredundant manner to maximize and prolong the Tfh response that is generated after acute virus infection.

Natural Killer Cells and Innate Interferon Gamma Participate in the Host Defense against Respiratory Vaccinia Virus Infection

ABSTRACT In establishing a respiratory infection, vaccinia virus (VACV) initially replicates in airway epithelial cells before spreading to secondary sites of infection, mainly the draining lymph nodes, spleen, gastrointestinal tract, and reproductive organs. We recently reported that interferon gamma (IFN-γ) produced by CD8 T cells ultimately controls this disseminated infection, but the relative contribution of IFN-γ early in infection is unknown. Investigating the role of innate immune cells, we found that the frequency of natural killer (NK) cells in the lung increased dramatically between days 1 and 4 postinfection with VACV. Lung NK cells displayed an activated cell surface phenotype and were the primary source of IFN-γ prior to the arrival of CD8 T cells. In the presence of an intact CD8 T cell compartment, depletion of NK cells resulted in increased lung viral load at the time of peak disease severity but had no effect on eventual viral clearance, disease symptoms, or survival. In sharp contrast, RAG−/− mice devoid of T cells failed to control VACV and succumbed to infection despite a marked increase in NK cells in the lung. Supporting an innate immune role for NK cell-derived IFN-γ, we found that NK cell-depleted or IFN-γ-depleted RAG−/− mice displayed increased lung VACV titers and dissemination to ovaries and a significantly shorter mean time to death compared to untreated NK cell-competent RAG−/− controls. Together, these findings demonstrate a role for IFN-γ in aspects of both the innate and adaptive immune response to VACV and highlight the importance of NK cells in T cell-independent control of VACV in the respiratory tract. IMPORTANCE Herein, we provide the first systematic evaluation of natural killer (NK) cell function in the lung after infection with vaccinia virus, a member of the Poxviridae family. The respiratory tract is an important mucosal site for entry of many human pathogens, including poxviruses, but precisely how our immune system defends the lung against these invaders remains unclear. Natural killer cells are a type of cytotoxic lymphocyte and part of our innate immune system. In recent years, NK cells have received increasing levels of attention following the discovery that different tissues contain specific subsets of NK cells with distinctive phenotypes and function. They are abundant in the lung, but their role in defense against respiratory viruses is poorly understood. What this study demonstrates is that NK cells are recruited, activated, and contribute to protection of the lung during a severe respiratory infection with vaccinia virus.

Peptide-stimulation enhances compartmentalization and the catalytic activity of lung endothelial NOS.

We reported that an 11 amino acid synthetic peptide (P1) activates lung endothelial cell nitric oxide synthase (eNOS) independent of its change in expression and/or phosphorylation. Since caveolae/eNOS dissociation is known to enhance the catalytic activity of eNOS, we examined whether P1-mediated increase of eNOS activity is associated with caveolae/cholesterol modulation, increased caveolin-1 phosphorylation, and intracellular compartmentalization of eNOS in pulmonary artery endothelial cells (PAEC). PAEC were incubated with or without (control) P1 or cholesterol modulators/caveolae disruptors, cholesterol oxidase (CHOX) and methyl-β-cyclodextrin (CD), for 1 h at 37°C. After incubation cells were used for: i) immunoprecipitation, ii) isolation of plasma membrane (PM)-, Golgi complex (GC)-, and non-Golgi complex (NGC)-enriched fractions, iii) immunofluorescence confocal imaging, and iv) electron microscopy for localization and/or eNOS activity. P1, CHOX, and CD-stimulation caused dissociation of eNOS from PM with increased localization to GC and/or NGC. P1 and CHOX significantly increased eNOS activity in PM and GC and CD-stimulation increased eNOS activity localized only in GC. P1 increased phosphorylation of caveolin-1 in intact cells and GC fraction. Immunofluorescence and/or immunogold labeled imaging/electron microscopy analysis of P1-, CHOX-, and CD-stimulated intact cells confirmed eNOS/caveolae dissociation and translocation of eNOS to GC. These results suggest that: i) P1-stimulation translocates eNOS to GC and enhances the catalytic activity of eNOS in both the PM and GC fractions of PAEC, ii) CHOX- but not CD-mediated caveolae and/or cholesterol modulation mimics the effect of P1-stimulated compartmentalization and activation of eNOS in PAEC, and iii) P1-stimulated caveolae/cholesterol modulation, phosphorylation of caveolin-1, and activation of eNOS is physiologically relevant since P1 is known to enhance NO/cGMP-dependent vasorelaxation in the pulmonary circulation.

Protein tyrosine phosphatase PTPN22 has dual roles in promoting pathogen versus homeostatic-driven CD8 T-cell responses

PTPN22 (protein tyrosine phosphatase non receptor 22) encodes a tyrosine phosphatase that functions as a key regulator of immune homeostasis. In particular, PTPN22 inhibits T‐cell receptor signaling and selectively promotes type I interferon responses in myeloid cells. To date, there is little information on the CD8 T‐cell‐intrinsic role of PTPN22 in response to a viral pathogen. We unexpectedly found that PTPN22‐deficient virus‐specific CD8 T cells failed to accumulate in wild‐type hosts after lymphocytic choriomeningitis virus infection. Lack of PTPN22 expression altered CD8 T‐cell activation and antiviral cytokine production, but did not significantly affect the composition of effector and memory cell precursors. Most significantly, in vivo, PTPN22‐deficient CD8 T cells showed a profound defect in upregulating STAT‐1 after lymphocytic choriomeningitis virus infection and considerably less phosphorylation of STAT‐1 in response to IFN‐α treatment in vitro compared with their wild‐type counterparts. In stark contrast, following transfer into lymphopenic mice, CD8 T‐cell expansion and central‐like phenotype, was considerably increased in the absence of PTPN22. Collectively, our results suggest that PTPN22 has dual roles in T‐cell clonal expansion and effector function; whereas it promotes antigen‐driven responses during acute infection by positively regulating interferon signaling in T cells, PTPN22 inhibits homeostatic‐driven proliferation.

Rapid control of pandemic H1N1 influenza by targeting NKT-cells.

Swine influenza A viruses (IAV) are a major cause of respiratory disease in pigs and humans. Currently approved anti-influenza therapies directly target the virus, but these approaches are losing effectiveness as new viral strains quickly develop drug resistance. To over come this challenge, there is an urgent need for more effective antiviral drugs. Here we tested the anti-influenza efficacy of the invariant natural killer T (NKT) cell superagonist, α-galactosylceramide (α-GalCer), which stimulates a wide array of anti-viral immune responses. We show that intranasal but not systemic administration of α-GalCer to piglets infected with pandemic A/California/04/2009 (CA04) H1N1 IAV ameliorated disease symptoms and resulted in the restoration of weight gain to the level of uninfected pigs. Correspondingly, viral titers in the upper-and lower-respiratory tract were reduced only in piglets that had received intranasal α-GalCer. Most significantly, lung inflammation as a consequence of virus persistence was largely prevented when NKT-cells were targeted via the respiratory route. Thus, targeting mucosal NKT-cells may provide a novel and potent platform for improving the course of disease in swine infected with seasonal and pandemic influenza viruses, and leads to the suggestion that this may also be true in humans and therefore deserves further study.

The Lupus Susceptibility Gene Pbx1 Regulates the Balance between Follicular Helper T Cell and Regulatory T Cell Differentiation

Pbx1 controls chromatin accessibility to a large number of genes and is entirely conserved between mice and humans. The Pbx1-d dominant-negative isoform is more frequent in CD4+ T cells from lupus patients than from healthy controls. Pbx1-d is associated with the production of autoreactive T cells in mice carrying the Sle1a1 lupus-susceptibility locus. Transgenic (Tg) expression of Pbx1-d in CD4+ T cells reproduced the phenotypes of Sle1a1 mice, with increased inflammatory functions of CD4+ T cells and impaired Foxp3+ regulatory T cell (Treg) homeostasis. Pbx1-d–Tg expression also expanded the number of follicular helper T cells (TFHs) in a cell-intrinsic and Ag-specific manner, which was enhanced in recall responses and resulted in Th1-biased Abs. Moreover, Pbx1-d–Tg CD4+ T cells upregulated the expression of miR-10a, miR-21, and miR-155, which were implicated in Treg and follicular helper T cell homeostasis. Our results suggest that Pbx1-d impacts lupus development by regulating effector T cell differentiation and promoting TFHs at the expense of Tregs. In addition, our results identify Pbx1 as a novel regulator of CD4+ T cell effector function.

Transcription Factor Bcl11b Controls Effector and Memory CD8 T cell Fate Decision and Function during Poxvirus Infection

CD8+ T cells play an important role in host resistance to many viral infections, but the underlying transcriptional mechanisms governing their differentiation and functionality remain poorly defined. By using a highly virulent systemic and respiratory poxvirus infection in mice, we show that the transcription factor Bcl11b provides a dual trigger that sustains the clonal expansion of virus-specific effector CD8+ T cells, while simultaneously suppressing the expression of surface markers associated with short-lived effector cell (SLEC) differentiation. Additionally, we demonstrate that Bcl11b supports the acquisition of memory precursor effector cell (MPEC) phenotype and, thus, its absence causes near complete loss of lymphoid and lung-resident memory cells. Interestingly, despite having normal levels of T-bet and Eomesodermin, Bcl11b-deficient CD8+ T cells failed to execute effector differentiation needed for anti-viral cytokine production and degranulation, suggesting a non-redundant role of Bcl11b in regulation of this program. Thus, Bcl11b is a critical player in fate decision of SLECs and MPECs, as well as effector function and memory formation.

The TNF Superfamily Molecule LIGHT Promotes the Generation of Circulating and Lung-Resident Memory CD8 T Cells following an Acute Respiratory Virus Infection

The transition of effector T cells or memory precursors into distinct long-lived memory T cell subsets is not well understood. Although many molecules made by APCs can contribute to clonal expansion and effector cell differentiation, it is not clear if clonal contraction and memory development is passive or active. Using respiratory virus infection, we found that CD8 T cells that cannot express the TNF family molecule lymphotoxin-like, exhibits inducible expression, competes with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes (LIGHT) are unimpaired in their initial response and clonally expand to form effector cell pools. Thereafter, LIGHT-deficient CD8 T cells undergo strikingly enhanced clonal contraction with resultant compromised accumulation of both circulating and tissue-resident memory cells. LIGHT expression at the peak of the effector response regulates the balance of several pro- and antiapoptotic genes, including Akt, and has a preferential impact on the development of the peripheral memory population. These results underscore the importance of LIGHT activity in programming memory CD8 T cell development, and suggest that CD8 effector T cells can dictate their own fate into becoming memory cells by expressing LIGHT.