Tumor necrosis factor receptor superfamily member 25 (TNFRSF25) agonists in islet transplantation: Endogenous in vivo regulatory T cell expansion promotes prolonged allograft survival.

Regulatory T cells (Tregs) modulate alloimmune responses and may facilitate minimization or withdrawal of immunosuppression posttransplant. Current approaches, however, rely on complex ex vivo Treg expansion protocols. Herein, we explore endogenous in vivo Treg expansion through antibody-mediated agonistic stimulation of the tumor necrosis factor receptor superfamily member 25 (TNFRSF25) pathway and its potential to prolong graft survival in a mouse model of islet allotransplantation. C57BL/6 male mice were treated with a single dose of TNFRSF25 agonistic antibodies (4C12 or mPTX-35) or IgG control. Diabetes was induced using streptozotocin. Four days later, flow cytometry was completed to corroborate Treg expansion, and 500 islets (CBA/J male mice) were transplanted. Glycemia was assessed thrice weekly until rejection/endpoint. Early intra-graft Treg infiltration was assessed 36 h posttransplant. TNFRSF25 antibodies enabled pronounced Treg expansion and treated mice had significantly prolonged graft survival compared with controls (p < .001). Additionally, the degree of Treg expansion significantly correlated with graft survival (p < .001). Immunohistochemistry demonstrated marked Treg infiltration in long-term surviving grafts; intra-graft Treg infiltration occurred early posttransplant. In conclusion, a single dose of TNFRSF25 antibodies enabled in vivo Treg expansion, which promotes prolonged graft survival. TNFRSF25-mediated in vivo Treg expansion could contribute to achieving lasting immunological tolerance in organ transplantation.

Lack of B Lymphocytes Enhances CD8 T Cell-Mediated Resistance against Respiratory Viral Infection but Compromises Memory Cell Formation.

Following a respiratory virus infection, CXCR3hi CX3CR1lo and CXCR3lo CX3CR1hi CD8 T cells localize to different compartments within the lung and play an important role in host resistance, but mechanisms governing their optimal generation are poorly defined. We serendipitously found that B cell-deficient (µMT-/-) mice were highly resistant to lethal infection with a virulent poxvirus strain and that depletion of CD8 T cells rendered these mice susceptible to infection. B cells were not required for the expansion of virus-specific CD8 T cells, but a greater proportion of activated CD8 T cells acquired an effector-like CXCR3lo CX3CR1hi phenotype in the absence of B cells. After recovery from infection, CD8 T cells in µMT-/- mice contracted normally but failed to survive and seed the memory cell pool in both the lungs and spleen. These findings reveal a previously unappreciated role for B cells in regulating the balance between CD8 T cell-mediated resistance against respiratory viral infection and memory cell development.IMPORTANCE B cells play critical role in host resistance against many respiratory viral infections. However, the role of B cells beyond antibody-producing cells is less well defined. In this study, we made a surprising observation that mice lacking B cells were more resistant to respiratory infection with vaccinia virus than wild-type mice. This enhanced resistance was mediated by CD8 T cells because when we depleted CD8 T cells in B cell-deficient mice, these mice were unable to survive the infection. Interestingly, CD8 T cells in B cell-deficient mice were skewed more toward effector phenotype and less toward memory phenotype, which resulted in severely compromised memory CD8 T cell development. Thus, our study shows a novel role of B cells as regulators of CD8 T cell-mediated host resistance and memory CD8 T cell formation during respiratory viral infection.

Batf3-Dependent Dendritic Cells Promote Optimal CD8 T Cell Responses against Respiratory Poxvirus Infection.

Respiratory infection with vaccinia virus (VacV) elicits robust CD8+ T cell responses that play an important role in host resistance. In the lung, VacV encounters multiple tissue-resident antigen-presenting cell (APC) populations, but which cell plays a dominant role in priming of virus-specific CD8+ effector T cell responses remains poorly defined. We used Batf3-/- mice to investigate the impact of CD103+ and CD8α+ dendritic cell (DC) deficiency on anti-VacV CD8+ T cell responses. We found that Batf3-/- mice were more susceptible to VacV infection, exhibiting profound weight loss, which correlated with impaired accumulation of gamma interferon (IFN-γ)-producing CD8+ T cells in the lungs. This was largely due to defective priming since early in the response, antigen-specific CD8+ T cells in the draining lymph nodes of Batf3-/- mice expressed significantly reduced levels of Ki67, CD25, and T-bet. These results underscore a specific role for Batf3-dependent DCs in regulating priming and expansion of effector CD8+ T cells necessary for host resistance against acute respiratory VacV infection.IMPORTANCE During respiratory infection with vaccinia virus (VacV), a member of Poxviridae family, CD8+ T cells play important role in resolving the primary infection. Effector CD8+ T cells clear the virus by accumulating in the infected lungs in large numbers and secreting molecules such as IFN-γ that kill virally infected cells. However, precise cell types that regulate the generation of effector CD8+ T cells in the lungs are not well defined. Dendritic cells (DCs) are a heterogeneous population of immune cells that are recognized as key initiators and regulators of T-cell-mediated immunity. In this study, we reveal that a specific subset of DCs that are dependent on the transcription factor Batf3 for their development regulate the magnitude of CD8+ T cell effector responses in the lungs, thereby providing protection during pulmonary VacV infection.

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.

Inflammatory monocytes contribute to the persistence of CXCR3hi CX3CR1lo circulating and lung-resident memory CD8+ T cells following respiratory virus infection.

Phenotypically diverse memory CD8+ T cells are present in the lungs that either re-circulate or reside within the tissue. Understanding the key cellular interactions that regulate the generation and then persistence of these different subsets is of great interest. Recently, DNGR-1+ dendritic cell (DC) mediated priming was reported to control the generation of lung-resident but not circulating memory cells following respiratory viral infection. Here, we report an important role for Ly6C+ inflammatory monocytes (IMs) in contributing to the persistence of memory CD8+ T cells but not their generation. Effector CD8+ T cells expanded and contracted normally in the absence of IMs, but the memory compartment declined significantly over time. Quite unexpectedly, this defect was confined to tissue resident and circulating CXCR3hi CX3CR1lo memory cells but not CXCR3hi CX3CR1int and CXCR3lo CX3CR1hi subsets. Thus, two developmentally distinct innate cells orchestrate the generation and persistence of memory T cell subsets following a respiratory virus infection. See also: News and Commentary by Lafouresse & Groom.

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.

Tissue-specific programming of memory CD8 T cell subsets impacts protection against lethal respiratory virus infection.

How tissue-specific anatomical distribution and phenotypic specialization are linked to protective efficacy of memory T cells against reinfection is unclear. Here, we show that lung environmental cues program recently recruited central-like memory cells with migratory potentials for their tissue-specific functions during lethal respiratory virus infection. After entering the lung, some central-like cells retain their original CD27hiCXCR3hi phenotype, enabling them to localize near the infected bronchiolar epithelium and airway lumen to function as the first line of defense against pathogen encounter. Others, in response to local cytokine triggers, undergo a secondary program of differentiation that leads to the loss of CXCR3, migration arrest, and clustering within peribronchoarterial areas and in interalveolar septa. Here, the immune system adapts its response to prevent systemic viral dissemination and mortality. These results reveal the striking and unexpected spatial organization of central- versus effector-like memory cells within the lung and how cooperation between these two subsets contributes to host defense.

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.

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

UNLABELLED: 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.

Vaccine-induced protection against orthopoxvirus infection is mediated through the combined functions of CD4 T cell-dependent antibody and CD8 T cell responses.

UNLABELLED: Antibody production by B cells in the absence of CD4 T cell help has been shown to be necessary and sufficient for protection against secondary orthopoxvirus (OPV) infections. This conclusion is based on short-term depletion of leukocyte subsets in vaccinated animals, in addition to passive transfer of immune serum to naive hosts that are subsequently protected from lethal orthopoxvirus infection. Here, we show that CD4 T cell help is necessary for neutralizing antibody production and virus control during a secondary ectromelia virus (ECTV) infection. A crucial role for CD4 T cells was revealed when depletion of this subset was extended beyond the acute phase of infection. Sustained depletion of CD4 T cells over several weeks in vaccinated animals during a secondary infection resulted in gradual diminution of B cell responses, including neutralizing antibody, contemporaneous with a corresponding increase in the viral load. Long-term elimination of CD8 T cells alone delayed virus clearance, but prolonged depletion of both CD4 and CD8 T cells resulted in death associated with uncontrolled virus replication. In the absence of CD4 T cells, perforin- and granzyme A- and B-dependent effector functions of CD8 T cells became critical. Our data therefore show that both CD4 T cell help for antibody production and CD8 T cell effector function are critical for protection against secondary OPV infection. These results are consistent with the notion that the effectiveness of the smallpox vaccine is related to its capacity to induce both B and T cell memory. IMPORTANCE: Smallpox eradication through vaccination is one of the most successful public health endeavors of modern medicine. The use of various orthopoxvirus (OPV) models to elucidate correlates of vaccine-induced protective immunity showed that antibody is critical for protection against secondary infection, whereas the role of T cells is unclear. Short-term leukocyte subset depletion in vaccinated animals or transfer of immune serum to naive, immunocompetent hosts indicates that antibody alone is necessary and sufficient for protection. We show here that long-term depletion of CD4 T cells over several weeks in vaccinated animals during secondary OPV challenge reveals an important role for CD4 T cell-dependent antibody responses in effective virus control. Prolonged elimination of CD8 T cells alone delayed virus clearance, but depletion of both T cell subsets resulted in death associated with uncontrolled virus replication. Thus, vaccinated individuals who subsequently acquire T cell deficiencies may not be protected against secondary OPV infection.

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.

The orchestrated functions of innate leukocytes and T cell subsets contribute to humoral immunity, virus control, and recovery from secondary poxvirus challenge.

A pivotal role for antigen-specific recall responses to secondary virus infection is well established, but the contribution of innate immune cells to this process is unknown. Recovery of mice from a primary orthopoxvirus (ectromelia virus [ECTV]) infection requires the function of natural killer (NK) cells, granulocytes, plasmacytoid dendritic cells (pDC), T cells, and B cells. However, during a secondary challenge, resolution of infection is thought to be dependent on antibody but not T cell function. We investigated the contribution of NK cells, granulocytes, and pDC to virus control during a secondary virus challenge in mice that had been primed with an avirulent, mutant strain of ECTV. Mice depleted of NK cells, granulocytes, or pDC effectively controlled virus, as did mice depleted of both CD4 and CD8 T cell subsets. However, mice concurrently depleted of all three innate cell subsets had elevated virus load, but this was significantly exacerbated in mice also depleted of CD4 and/or CD8 T cells. Increased viral replication in mice lacking innate cells plus CD4 T cells was associated with a significant reduction in neutralizing antibody. Importantly, in addition to T-dependent neutralizing antibody responses, the function of CD8 T cells was also clearly important for virus control. The data indicate that in the absence of innate cell subsets, a critical role for both CD4 and CD8 T cells becomes apparent and, conversely, in the absence of T cell subsets, innate immune cells help contain infection.

Targeting 4-1BB (CD137) to enhance CD8 T cell responses with poxviruses and viral antigens.

Attenuated vaccinia virus (VACV) vectors are considered prime vaccine candidates for use in immunotherapy of infectious disease. In spite of this, recent data show that the level of attenuation may hamper the efficient generation of protective CD8 T cells. This suggests that additional adjuvant-like activities may need to be combined with attenuated VACV for optimal vaccination. Stimulatory reagents to the TNFR family molecule 4-1BB (CD137) may represent such an adjuvant for vaccination. Previous murine studies have found that 4-1BB can participate in optimal priming of effector and memory CD8 T cells in response to several virus infections, and concordantly direct stimulation of 4-1BB with agonist reagents effectively boosts the CD8 T cell response against those viruses. In contrast, we recently reported that 4-1BB plays no role in the response to a virulent strain of VACV, questioning whether agonists of 4-1BB will be useful adjuvants for vaccination with VACV vectors. Here we show that agonist anti-4-1BB strongly enhanced the primary viral-specific effector CD8 T cell response during infection with live virulent VACV and attenuated VACV, and during immunization with VACV peptides given in IFA. However, accumulation of memory CD8 T cells was enhanced only following infection with virulent VACV or with peptide vaccination, but not with attenuated VACV, correlating in part with more transient expression of 4-1BB on CD8 T cells with attenuated virus. Our data therefore suggest that 4-1BB may be a promising candidate for targeting as an adjuvant for short-term enhancement of CD8 T cell responses with VACV vaccine strategies, but additional receptors may need to be engaged with 4-1BB to allow long-term CD8 T cell immunity with attenuated VACV vectors.

CD8 T cells are essential for recovery from a respiratory vaccinia virus infection.

The precise immune components required for protection against a respiratory Orthopoxvirus infection, such as human smallpox or monkeypox, remain to be fully identified. In this study, we used the virulent Western Reserve strain of vaccinia virus (VACV-WR) to model a primary respiratory Orthopoxvirus infection. Naive mice infected with VACV-WR mounted an early CD8 T cell response directed against dominant and subdominant VACV-WR Ags, followed by a CD4 T cell and Ig response. In contrast to other VACV-WR infection models that highlight the critical requirement for CD4 T cells and Ig, we found that only mice deficient in CD8 T cells presented with severe cachexia, pulmonary inflammation, viral dissemination, and 100% mortality. Depletion of CD8 T cells at specified times throughout infection highlighted that they perform their critical function between days 4 and 6 postinfection and that their protective requirement is critically dictated by initial viral load and virulence. Finally, the ability of adoptively transferred naive CD8 T cells to protect RAG⁻/⁻ mice against a lethal VACV-WR infection demonstrated that they are both necessary and sufficient in protecting against a primary VACV-WR infection of the respiratory tract.

OX40:OX40L axis: emerging targets for improving poxvirus-based CD8(+) T-cell vaccines against respiratory viruses.

The human respiratory tract is an entry point for over 200 known viruses that collectively contribute to millions of annual deaths worldwide. Consequently, the World Health Organization has designated respiratory viral infections as a priority for vaccine development. Despite enormous advances in understanding the attributes of a protective mucosal antiviral immune response, current vaccines continue to fail in effectively generating long-lived protective CD8(+) T-cell immunity. To date, the majority of licensed human vaccines afford protection against infectious pathogens through the generation of specific immunoglobulin responses. In recent years, the selective manipulation of specific costimulatory pathways, which are critical in regulating T cell-mediated immune responses, has generated increasing interest. Impressive results in animal models have shown that the tumor necrosis factor receptor (TNFR) family member OX40 (CD134) and its binding partner OX40L (CD252) are key costimulatory molecules involved in the generation of protective CD8(+) T-cell responses at mucosal surfaces, such as the lung. In this review, we highlight these new findings with a particular emphasis on their potential as immunological adjuvants to enhance poxvirus-based CD8(+) T-cell vaccines.

Topology and identification of critical residues of the O-acetyltransferase of serotype-converting bacteriophage, SF6, of Shigella flexneri.

The modification of the LPS O-antigen, seen in the diverse serotypes of Shigella flexneri is brought about by the glucosyltransferases (Gtr) and the O-acetyltransferase (Oac). In this study, we establish the membrane topology of Oac using the dual reporter PhoA-LacZalpha. We have determined that Oac is an integral membrane protein with 10 transmembrane regions. The hydrophilic N- and C-termini are oriented in the cytoplasm. Functionally important cytoplasmic and periplasmic loops have also been identified. Furthermore, cytoplasmic residues R73 and R75R76 were found to be critical to Oac function.