Congenital iRHOM2 deficiency causes ADAM17 dysfunction and environmentally directed immunodysregulatory disease.

We report a pleiotropic disease due to loss-of-function mutations in RHBDF2, the gene encoding iRHOM2, in two kindreds with recurrent infections in different organs. One patient had recurrent pneumonia but no colon involvement, another had recurrent infectious hemorrhagic colitis but no lung involvement and the other two experienced recurrent respiratory infections. Loss of iRHOM2, a rhomboid superfamily member that regulates the ADAM17 metalloproteinase, caused defective ADAM17-dependent cleavage and release of cytokines, including tumor-necrosis factor and amphiregulin. To understand the diverse clinical phenotypes, we challenged Rhbdf2-/- mice with Pseudomonas aeruginosa by nasal gavage and observed more severe pneumonia, whereas infection with Citrobacter rodentium caused worse inflammatory colitis than in wild-type mice. The fecal microbiota in the colitis patient had characteristic oral species that can predispose to colitis. Thus, a human immunodeficiency arising from iRHOM2 deficiency causes divergent disease phenotypes that can involve the local microbial environment.

Local blockade of epithelial PDL-1 in the airways enhances T cell function and viral clearance during influenza virus infection.

In order to maintain the gas exchange function of the lung following influenza virus infection, a delicate orchestration of positive and negative regulatory pathways must be maintained to attain viral eradication while minimizing local inflammation. The programmed death receptor 1 ligand/programmed death receptor 1 (PDL-1/PD-1) pathway plays an important immunoregulatory role, particularly in the context of T cell function. Here, we have shown that influenza virus infection of primary airway epithelial cells strongly enhances PDL-1 expression and does so in an alpha interferon receptor (IFNAR) signaling-dependent manner. PD-1 is expressed primarily on effector T cells in the lung, compared to effector memory and central memory cells, and shortly after influenza virus infection, an increased number of PD-1(+) T cells are recruited to the airways. Using in vitro cocultures of airway epithelial cells and T cells and in vivo models of influenza virus infection, we have demonstrated that blockade of airway epithelial PDL-1 improves CD8 T cell function, defined by increased production of gamma interferon (IFN-γ) and granzyme B and expression of CD107ab. Furthermore, PDL-1 blockade in the airways served to accelerate influenza virus clearance and enhance infection recovery. Our findings suggest that local manipulation of the PDL-1/PD-1 axis in the airways may represent a therapeutic alternative during acute influenza virus infection.

Induction of ICOS+CXCR3+CXCR5+ TH cells correlates with antibody responses to influenza vaccination.

Seasonal influenza vaccine protects 60 to 90% of healthy young adults from influenza infection. The immunological events that lead to the induction of protective antibody responses remain poorly understood in humans. We identified the type of CD4+ T cells associated with protective antibody responses after seasonal influenza vaccinations. The administration of trivalent split-virus influenza vaccines induced a temporary increase of CD4+ T cells expressing ICOS, which peaked at day 7, as did plasmablasts. The induction of ICOS was largely restricted to CD4+ T cells coexpressing the chemokine receptors CXCR3 and CXCR5, a subpopulation of circulating memory T follicular helper cells. Up to 60% of these ICOS+CXCR3+CXCR5+CD4+ T cells were specific for influenza antigens and expressed interleukin-2 (IL-2), IL-10, IL-21, and interferon-γ upon antigen stimulation. The increase of ICOS+CXCR3+CXCR5+CD4+ T cells in blood correlated with the increase of preexisting antibody titers, but not with the induction of primary antibody responses. Consistently, purified ICOS+CXCR3+CXCR5+CD4+ T cells efficiently induced memory B cells, but not naïve B cells, to differentiate into plasma cells that produce influenza-specific antibodies ex vivo. Thus, the emergence of blood ICOS+CXCR3+CXCR5+CD4+ T cells correlates with the development of protective antibody responses generated by memory B cells upon seasonal influenza vaccination.

Toll-like receptor expression and induction of type I and type III interferons in primary airway epithelial cells.

Interferons (IFNs) are a critical component of the first line of antiviral defense. The activation of Toll-like receptors (TLRs) expressed by dendritic cells triggers different signaling cascades that result in the production of large amounts of IFNs. However, the functional consequences of TLR activation and differential IFN production in specific cell populations other than antigen-presenting cells have not yet been fully elucidated. In this study, we investigated TLR expression and polarization in airway epithelial cells (AECs) and the consequences of TLR agonist stimulation for the production of type I (IFN-α/ß) and type III (IFN-λ) IFNs. Our results show that the pattern of expression and polarization of all TLRs in primary AEC cultures mirrors that of the human airways ex vivo and is receptor specific. The antiviral TLRs (TLR3, TLR7, and TLR9) are mostly expressed on the apical cell surfaces of epithelial cells in the human trachea and in primary polarized AECs. Type III IFN is the predominant IFN produced by the airway epithelium, and TLR3 is the only TLR that mediates IFN production by AECs, while all TLR agonists tested are capable of inducing AEC activation and interleukin-8 production. In response to influenza virus infection, AECs can produce IFN-λ in an IFNAR- and STAT1-independent manner. Our results emphasize the importance of using primary well-differentiated AECs to study TLR and antiviral responses and provide further insight into the regulation of IFN production during the antiviral response of the lung epithelium.

Plasticity and virus specificity of the airway epithelial cell immune response during respiratory virus infection.

Airway epithelial cells (AECs) provide the first line of defense in the respiratory tract and are the main target of respiratory viruses. Here, using oligonucleotide and protein arrays, we analyze the infection of primary polarized human AEC cultures with influenza virus and respiratory syncytial virus (RSV), and we show that the immune response of AECs is quantitatively and qualitatively virus specific. Differentially expressed genes (DEGs) specifically induced by influenza virus and not by RSV included those encoding interferon B1 (IFN-B1), type III interferons (interleukin 28A [IL-28A], IL-28B, and IL-29), interleukins (IL-6, IL-1A, IL-1B, IL-23A, IL-17C, and IL-32), and chemokines (CCL2, CCL8, and CXCL5). Lack of type I interferon or STAT1 signaling decreased the expression and secretion of cytokines and chemokines by the airway epithelium. We also observed strong basolateral polarization of the secretion of cytokines and chemokines by human and murine AECs during infection. Importantly, the antiviral response of human AECs to influenza virus or to RSV correlated with the infection signature obtained from peripheral blood mononuclear cells (PBMCs) isolated from patients with acute influenza or RSV bronchiolitis, respectively. IFI27 (also known as ISG12) was identified as a biomarker of respiratory virus infection in both AECs and PBMCs. In addition, the extent of the transcriptional perturbation in PBMCs correlated with the clinical disease severity. Our results demonstrate that the human airway epithelium mounts virus-specific immune responses that are likely to determine the subsequent systemic immune responses and suggest that the absence of epithelial immune mediators after RSV infection may contribute to explaining the inadequacy of systemic immunity to the virus.

KLRG1+NKG2A+ CD8 T cells mediate protection and participate in memory responses during γ-herpesvirus infection.

Functional CD8 T cell effector and memory responses are generated and maintained during murine γ-herpesvirus 68 (γHV68) persistent infection despite continuous presentation of viral lytic Ags. However, the identity of the CD8 T cell subpopulations that mediate effective recall responses and that can participate in the generation of protective memory to a γ-herpesvirus infection remains unknown. During γHV68 persistence, ∼75% of γHV68-specific CD8 T cells coexpress the NK receptors killer cell lectin-like receptor G1 (KLRG1) and NKG2A. In this study, we take advantage of this unique phenotype to analyze the capacity of CD8 T cells expressing or not expressing KLRG1 and NKG2A to mediate effector and memory responses. Our results show that γHV68-specific KLRG1(+)NKG2A(+) CD8 T cells have an effector memory phenotype as well as characteristics of polyfunctional effector cells such us IFN-γ and TNF-α production, killing capacity, and are more efficient at protecting against a γHV68 challenge than their NKG2A(-)KLRG1(-) counterparts. Nevertheless, γHV68-specific NKG2A(+)KLRG1(+) CD8 T cells express IL-7 and IL-15 receptors, can survive long-term without cognate Ag, and subsequently mount a protective response during antigenic recall. These results highlight the plasticity of the immune system to generate protective effector and proliferative memory responses during virus persistence from a pool of KLRG1(+)NKG2A(+) effector memory CD8 T cells.

Lambda interferon is the predominant interferon induced by influenza A virus infection in vivo.

The type I alpha/beta interferons (IFN-α/ß) are known to play an important role in host defense against influenza A virus infection, but we have now discovered that the recently identified type III IFNs (IFN-λ) constitute the major response to intranasal infection with this virus. Type III IFNs were present at much higher levels than type I IFNs in the lungs of infected mice, and the enhanced susceptibility of STAT2-/- animals demonstrated that only signaling through the IFN-α/ß or IFN-λ pathways was sufficient to mediate protection. This finding offers a possible explanation for the similar levels of antiviral protection found in wild-type (WT) mice and in animals lacking a functional type I IFN receptor (IFNAR-/-) but also argues that our current understanding of type III IFN induction is incomplete. While murine IFN-λ production is thought to depend on signaling through the type I IFN receptor, we demonstrate that intranasal influenza A virus infection leads to the robust type III IFN induction in the lungs of both WT and IFNAR-/- mice. This is consistent with previous studies showing that IFNAR-mediated protection is redundant for mucosal influenza virus infection and with data showing that the type III IFN receptor is expressed primarily by epithelial cells. However, the overlapping effects of these two cytokine families are limited by their differential receptor expression, with a requirement for IFN-α/ß signaling in combating systemic disease.

Dendritic cells loaded with tumor B cells elicit broad immunity against murine gammaherpesvirus 68 but fail to prevent long-term latency.

It is still unknown whether a noninfectious gammaherpesvirus vaccine is able to prevent or reduce virus persistence. This led us to use dendritic cells loaded with tumor B cells as a vaccine approach for the murine gammaherpesvirus 68 (gammaHV68) model of infection. Dendritic cells loaded with UV-irradiated latently infected tumor B cells induce broad, strong, and long-lasting immunity against gammaHV68. Dendritic cell vaccination prevents the enlargement of lymph nodes and severely limits acute infection and early latency but does not prevent gammaHV68 from establishing long-term latency. Our findings support the concept that attenuated viruses may be the best vaccine option for preventing gammaherpesvirus persistence.

Persistent gamma-herpesvirus infection induces a CD4 T cell response containing functionally distinct effector populations.

The direct effector mechanisms of CD4 T cells during gamma-herpesvirus 68 (gammaHV68)-persistent infection are less well understood than those of their CD8 T cell counterparts, although there is substantial evidence that CD4 T cells are critical for the control of persistent gamma-herpesvirus infection. Our results show that in gammaHV68-persistently infected mice, CD4 T cells are not cytokine polyfunctional, but there is a division of labor in the CD4 T cell compartment in which CD4 T cells polarize toward two distinct populations with different effector functions: IFN-gamma producers and CD107(+) cytolytic effectors. These two CD4 T cell effector populations degranulate and produce IFN-gamma during steady state without need for exogenous antigenic restimulation, which is fundamentally different from that observed with gammaHV68-specific CD8 T cells. By using anti-IFN-gamma Ab depletions and IFN-gamma-deficient mice, we show that CD4 T cell-mediated cytotoxicity in vivo is not dependent on IFN-gamma activity. In addition, our data show that purified CD4 T cells isolated from gammaHV68-latently infected mice have the capacity to inhibit gammaHV68 reactivation from latency. Our results support the concept that CD4 T cells are critical effectors for the control of gamma-herpesvirus latent infection, and they mediate this effect by two independent mechanisms: IFN-gamma production and cytotoxicity.

CD4 T cells mediate killing during persistent gammaherpesvirus 68 infection.

CD4 T cells are critical for the control of gammaherpesvirus persistence, but their direct effector mechanisms of virus control in vivo are still poorly understood. In this study, we use murine gammaherpesvirus 68 (gammaHV68) in in vitro and in vivo cytotoxicity assays to show CD4-dependent killing of gammaHV68-loaded cells in mice persistently infected with gammaHV68. Our results underscore the cytotoxic capacity of CD4 T cells during gammaHV68 persistence.

Memory generation and maintenance of CD8+ T cell function during viral persistence.

During infection with viruses that establish latency, the immune system needs to maintain lifelong control of the infectious agent in the presence of persistent Ag. By using a gamma-herpesvirus (gammaHV) infection model, we demonstrate that a small number of virus-specific central-memory CD8+ T cells develop early during infection, and that virus-specific CD8+T cells maintain functional and protective capacities during chronic infection despite low-level Ag persistence. During the primary immune response, we show generation of CD8+ memory T cell precursors expressing lymphoid homing molecules (CCR7, L-selectin) and homeostatic cytokine receptors (IL-7alpha, IL-2/IL-15beta). During long-term persistent infection, central-memory cells constitute 20-50% of the virus-specific CD8+ T cell population and maintain the expression of L-selectin, CCR7, and IL-7R molecules. Functional analyses demonstrate that during viral persistence: 1) CD8+ T cells maintain TCR affinity for peptide/MHC complexes, 2) the functional avidity of CD8+ T cells measured as the capacity to produce IFN-gamma is preserved intact, and 3) virus-specific CD8+ T cells have in vivo killing capacity. Next, we demonstrate that at 8 mo post-virus inoculation, long-term CD8+ T cells are capable of mediating a protective recall response against the establishment of gammaHV68 splenic latency. These observations provide evidence that functional CD8+ memory T cells can be generated and maintained during low-load gammaHV68 persistence.

Protection against respiratory syncytial virus by a recombinant Newcastle disease virus vector.

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infants and the elderly, but no safe and effective RSV vaccine is yet available. For reasons that are not well understood, RSV is only weakly immunogenic, and reinfection occurs throughout life. This has complicated the search for an effective live attenuated viral vaccine, and past trials with inactivated virus preparations have led to enhanced immunopathology following natural infection. We have tested the hypothesis that weak stimulation of innate immunity by RSV correlates with ineffective adaptive responses by asking whether expression of the fusion glycoprotein of RSV by Newcastle disease virus (NDV) would stimulate a more robust immune response to RSV than primary RSV infection. NDV is a potent inducer of both alpha/beta interferon (IFN-alpha/beta) production and dendritic cell maturation, while RSV is not. When a recombinant NDV expressing the RSV fusion glycoprotein was administered to BALB/c mice, they were protected from RSV challenge, and this protection correlated with a robust anti-F CD8+ T-cell response. The effectiveness of this vaccine construct reflects the differential abilities of NDV and RSV to promote dendritic cell maturation and is retained even in the absence of a functional IFN-alpha/beta receptor.

Early establishment of gamma-herpesvirus latency: implications for immune control.

The human gamma-herpesviruses, EBV and Kaposi's sarcoma-associated herpesvirus, infect >90% of the population worldwide, and latent infection is associated with numerous malignancies. Rational vaccination and therapeutic strategies require an understanding of virus-host interactions during the initial asymptomatic infection. Primary EBV infection is associated with virus replication at epithelial sites and entry into the circulating B lymphocyte pool. The virus exploits the life cycle of the B cell and latency is maintained long term in resting memory B cells. In this study, using a murine gamma-herpesvirus model, we demonstrate an early dominance of latent virus at the site of infection, with lung B cells harboring virus almost immediately after infection. These data reinforce the central role of the B cell not only in the later phase of infection, but early in the initial infection. Early inhibition of lytic replication does not impact the progression of the latent infection, and latency is established in lymphoid tissues following infection with a replication-deficient mutant virus. These data demonstrate that lytic viral replication is not a requirement for gamma-herpesvirus latency in vivo and suggest that viral latency can be disseminated by cellular proliferation. These observations emphasize that prophylactic vaccination strategies must target latent gamma-herpesvirus at the site of infection.

T cell reactivity during infectious mononucleosis and persistent gammaherpesvirus infection in mice.

Intranasal infection of mice with murine gammaherpesvirus 68 causes a dramatic increase in numbers of activated CD8(+) T cells in the blood, analogous in many respects to EBV-induced infectious mononucleosis in humans. In the mouse model, this lymphocytosis has two distinct components: an early, conventional virus-specific CD8(+) T cell response, and a later response characterized by a dramatic increase among CD8(+) T cells that bear Vbeta4(+) TCRs. We previously demonstrated that Vbeta4(+)CD8(+) T cells recognize an uncharacterized ligand expressed on latently infected B cells in an MHC-independent manner. The frequency of Vbeta4(+)CD8(+) T cells increases dramatically following the peak of viral latency in the spleen. In the current studies, we show that elevated Vbeta4(+)CD8(+) T cell levels are sustained long-term in persistently infected mice, apparently a consequence of continued ligand expression. In addition, we show that Vbeta4(+)CD8(+) T cells can acquire effector functions, including cytotoxicity and the capacity to secrete IFN-gamma, although they have an atypical activation profile compared with well-characterized CD8(+) T cells specific for conventional viral epitopes. The characteristics of Vbeta4(+)CD8(+) T cells (potential effector function, stimulation by latently infected B cells, and kinetics of expansion) suggested that this dominant T cell response plays a key role in the immune control of latent virus. However, Ab depletion and adoptive transfer studies show that Vbeta4(+)CD8(+) T cells are not essential for this function. This murine model of infection may provide insight into the role of unusual populations of activated T cells associated with persistent viral infections.

Murid herpesvirus 4 strain 68 M2 protein is a B-cell-associated antigen important for latency but not lymphocytosis.

This work describes analyses of the function of the murid herpesvirus 4 strain 68 (MHV-68) M2 gene. A frameshift mutation was made in the M2 open reading frame that caused premature termination of translation of M2 after amino acid residue 90. The M2 mutant showed no defect in productive replication in vitro or in lungs after infection of mice. Likewise, the characteristic transient increase in spleen cell number, Vbeta4 T-cell-receptor-positive CD8(+) T-cell mononucleosis, and establishment of latency were unaffected. However, the M2 mutant virus was defective in its ability to cause the transient sharp rise in latently infected cells normally seen in the spleen after infection of mice. We also demonstrate that expression of M2 is restricted to B cells in the spleen and that M2 encodes a 30-kDa protein localizing predominantly in the cytoplasm and plasma membrane of B cells.

Maintenance of long term gamma-herpesvirus B cell latency is dependent on CD40-mediated development of memory B cells.

It has been proposed that the gamma-herpesviruses maintain lifelong latency in B cells by gaining entry into the memory B cell pool and taking advantage of host mechanisms for maintaining these cells. We directly tested this hypothesis by kinetically monitoring viral latency in CD40(+) and CD40(-) B cells from CD40(+)CD40(-) mixed bone marrow chimera mice after infection with a murine gamma-herpesvirus, MHV-68. CD40(+) B cells selectively entered germinal centers and differentiated into memory B cells. Importantly, latency was progressively lost in the CD40(-) B cells and preferentially maintained in the long-lived, isotype-switched CD40(+) B cells. These data directly demonstrate viral exploitation of the normal B cell differentiation pathway to maintain latency.

Differential gamma-herpesvirus distribution in distinct anatomical locations and cell subsets during persistent infection in mice.

Murine gamma-herpesvirus 68 (MHV-68) provides an important experimental model for analyzing gamma-herpesvirus latent infection. After intranasal infection with MHV-68, we analyzed the distribution of the virus in different anatomical locations and purified populations of cells. Our data show that long-term latency is maintained in a variety of anatomical locations and cell populations with different frequencies. Importantly, we demonstrate that although latency in the lung is established in a variety of cell subsets, long-term latency in the lung is only maintained in B cells. In contrast, splenic latency is maintained in macrophages and dendritic cells, as well as in B cells. In blood, isotype-switched B cells constitute the major viral reservoir. These results show that the cell subsets in which latency is established vary within different anatomical sites. Finally, we demonstrate that long-term latency is accompanied by a low level of infectious virus in lung and spleen. These data have important implications for understanding the establishment and maintenance of latency by gamma(2)-herpesviruses.

Gamma-herpesvirus latency is preferentially maintained in splenic germinal center and memory B cells.

The gamma-herpesviruses are oncogenic B cell lymphotrophic viruses that establish life-long latency in the host. Murine gamma-herpesvirus 68 (MHV-68) infection of mice represents a unique system for analyzing gamma-herpesvirus latency in splenic B cells at different stages of infection. After intranasal infection with MHV-68 we analyzed the establishment of latency 14 days after infection, and the maintenance of latency 3 months after infection in different purified subpopulations of B cells in the spleen. The data show that MHV-68 latency is mainly established in germinal center B cells and that long-term latency is preferentially maintained in two different subsets of isotype-switched B cells, germinal center and memory B cells. Cell cycle analysis indicates that MHV-68 is located in both cycling and resting isotype-switched B cells. Analysis of viral gene expression showed that both lytic and latent viral transcripts were differentially expressed in germinal center and memory B cells during long-term latency. Together, these observations suggested that gamma-herpesviruses exploit the B cell life cycle in the spleen.

A mouse model for infectious mononucleosis.

Epstein-Barr virus (EBV) is a ubiquitous human gamma-herpesvirus that establishes life-long latency and is associated with lymphoproliferative disorders and the development of several malignancies. EBV infection is frequently, but not always, associated with the development of a syndrome termed infectious mononucleosis. The recent isolation and characterization of a murine gamma-herpesvirus, MHV-68 (gammaHV-68) has provided the first small animal model for studying immunity and pathogenesis of a gamma-herpesvirus in its natural host. MHV-68 has important biological and genetic similarities with the human gamma-herpesviruses. Following intranasal infection of mice with MHV-68, an acute respiratory infection in the lung develops and is cleared, followed by the establishment of latency. Similar to EBV, MHV-68 latency is largely established in B cells, although other cell types can be latently infected. The establishment of latency correlates with a prominent splenomegaly, polyclonal B cell activation with associated autoantibody production, and CD8+ T cell-dominated peripheral blood lymphocytosis, in many aspects mirroring EBV-induced infectious mononucleosis. There are key differences in the MHV-68- and EBV-induced CD8+ T cell responses however. Whereas the expanded CD8+ T cells associated with EBV-induced mononucleosis are largely the outgrowth of T cells responding to lytic viral epitopes elicited during the acute phase of the response, the CD8+ T cell lymphocytosis associated with MHV-68-induced infectious mononucleosis is dominated by an oligoclonal population of T cells expressing Vbeta4+ T cell receptors that are not reactive to acute viral epitopes. The focus of this article will be to highlight the similarities and differences in the infectious mononucleosis syndrome associated with human and murine gamma-herpesviruses.

Antibody-mediated control of persistent gamma-herpesvirus infection.

The human gamma-herpesviruses, EBV and Kaposi's sarcoma-associated herpesvirus, establish life-long latency and can reactivate in immunocompromised individuals. T cells play an important role in controlling persistent EBV infection, whereas a role for humoral immunity is less clear. The murine gamma-herpesvirus-68 has biological and structural similarities to the human gamma-herpesviruses, and provides an important in vivo experimental model for dissecting mechanisms of immune control. In the current studies, CD28(-/-) mice were used to address the role of Abs in control of persistent murine gamma-herpesvirus-68 infection. Lytic infection was controlled in the lungs of CD28(-/-) mice, and latency was maintained in B cells at normal frequencies. Although class-switched virus-specific Abs were initially generated in the absence of germinal centers, titers and viral neutralizing activity rapidly waned. T cell depletion in CD28(-/-) mice with compromised Ab responses, but not in control mice with intact Ab responses, resulted in significant recrudescence from latency, both in the spleen and the lung. Recrudescence could be prevented by passive transfer of immune serum. These data directly demonstrate an important contribution of humoral immunity to control of gamma-herpesvirus latency, and have significant implications for clinical intervention.