Jennifer M. Lund

Coordination of Early Protective Immunity to Viral Infection by Regulatory T Cells

Suppression of immune responses by regulatory T cells (Tregs) is thought to limit late stages of pathogen-specific immunity as a means of minimizing associated tissue damage. We examined a role for Tregs during mucosal herpes simplex virus infection in mice, and observed an accelerated fatal infection with increased viral loads in the mucosa and central nervous system after ablation of Tregs. Although augmented interferon production was detected in the draining lymph nodes (dLNs) in Treg-deprived mice, it was profoundly reduced at the infection site. This was associated with a delay in the arrival of natural killer cells, dendritic cells, and T cells to the site of infection and a sharp increase in proinflammatory chemokine levels in the dLNs. Our results suggest that Tregs facilitate early protective responses to local viral infection by allowing a timely entry of immune cells into infected tissue.

IPS-1 Is Essential for the Control of West Nile Virus Infection and Immunity

The innate immune response is essential for controlling West Nile virus (WNV) infection but how this response is propagated and regulates adaptive immunity in vivo are not defined. Herein, we show that IPS-1, the central adaptor protein to RIG-I-like receptor (RLR) signaling, is essential for triggering of innate immunity and for effective development and regulation of adaptive immunity against pathogenic WNV. IPS-1−/− mice exhibited increased susceptibility to WNV infection marked by enhanced viral replication and dissemination with early viral entry into the CNS. Infection of cultured bone-marrow (BM) derived dendritic cells (DCs), macrophages (Macs), and primary cortical neurons showed that the IPS-1-dependent RLR signaling was essential for triggering IFN defenses and controlling virus replication in these key target cells of infection. Intriguingly, infected IPS-1−/− mice displayed uncontrolled inflammation that included elevated systemic type I IFN, proinflammatory cytokine and chemokine responses, increased numbers of inflammatory DCs, enhanced humoral responses marked by complete loss of virus neutralization activity, and increased numbers of virus-specific CD8+ T cells and non-specific immune cell proliferation in the periphery and in the CNS. This uncontrolled inflammatory response was associated with a lack of regulatory T cell expansion that normally occurs during acute WNV infection. Thus, the enhanced inflammatory response in the absence of IPS-1 was coupled with a failure to protect against WNV infection. Our data define an innate/adaptive immune interface mediated through IPS-1-dependent RLR signaling that regulates the quantity, quality, and balance of the immune response to WNV infection.

Differentiation of regulatory Foxp3+ T cells in the thymic cortex.

Regulatory Foxp3+ T cells (TR) are indispensable for preventing autoimmune pathology in multiple organs and tissues. During thymic differentiation T cell receptor (TCR)–ligand interactions within a certain increased affinity range, in conjunction with γc-containing cytokine receptor signals, induce Foxp3 expression and thereby commit developing thymocytes to the TR lineage. The contribution of distinct MHC class II–expressing accessory cell types to the differentiation process of Foxp3+ thymocytes remains controversial, because a unique role in this process has been ascribed to either thymic dendritic cells (tDC) or to medullary thymic epithelial cells (mTEC). Furthermore, it was suggested that the thymic medulla, where the bulk of the negative selection of self-reactive thymocytes takes place, provides a specialized microenvironment supporting TR differentiation. Here, we report that the cortex, as defined by cortical thymic epithelial cells (cTEC), is sufficient for supporting TR differentiation. MHC class II expression restricted to both cTEC and mTEC or to cTEC alone did not significantly affect the numbers of Foxp3+ thymocytes. Furthermore, genetic or pharmacologic blockade of thymocyte migration resulted in a prominent accumulation of Foxp3+ thymocytes in the cortex, demonstrating that secondary signals required for Foxp3 up-regulation exist in the cortex. Our results suggest that mTEC or tDC do not serve as a cell type singularly responsible for TR differentiation and that neither the cortex nor the medulla exclusively provides an environment suitable for Foxp3 induction. Instead, multiple accessory cell types probably contribute to the thymic generation of regulatory Foxp3+ T cells.

Genetic Diversity in the Collaborative Cross Model Recapitulates Human West Nile Virus Disease Outcomes

ABSTRACT West Nile virus (WNV) is an emerging neuroinvasive flavivirus that now causes significant morbidity and mortality worldwide. The innate and adaptive immune responses to WNV infection have been well studied in C57BL/6J inbred mice, but this model lacks the variations in susceptibility, immunity, and outcome to WNV infection that are observed in humans, thus limiting its usefulness to understand the mechanisms of WNV infection and immunity dynamics. To build a model of WNV infection that captures human infection outcomes, we have used the Collaborative Cross (CC) mouse model. We show that this model, which recapitulates the genetic diversity of the human population, demonstrates diversity in susceptibility and outcomes of WNV infection observed in humans. Using multiple F1 crosses of CC mice, we identified a wide range of susceptibilities to infection, as demonstrated through differences in survival, clinical disease score, viral titer, and innate and adaptive immune responses in both peripheral tissues and the central nervous system. Additionally, we examined the Oas1b alleles in the CC mice and confirmed the previous finding that Oas1b plays a role in susceptibility to WNV; however, even within a given Oas1b allele status, we identified a wide range of strain-specific WNV-associated phenotypes. These results confirmed that the CC model is effective for identifying a repertoire of host genes involved in WNV resistance and susceptibility. The CC effectively models a wide range of WNV clinical, virologic, and immune phenotypes, thus overcoming the limitations of the traditional C57BL/6J model, allowing genetic and mechanistic studies of WNV infection and immunity in differently susceptible populations. IMPORTANCE Mouse models of West Nile virus infection have revealed important details regarding the innate and adaptive immune responses to this emerging viral infection. However, traditional mouse models lack the genetic diversity present in human populations and therefore limit our ability to study various disease outcomes and immunologic mechanisms subsequent to West Nile virus infection. In this study, we used the Collaborative Cross mouse model to more effectively model the wide range of clinical, virologic, and immune phenotypes present upon West Nile virus infection in humans. Mouse models of West Nile virus infection have revealed important details regarding the innate and adaptive immune responses to this emerging viral infection. However, traditional mouse models lack the genetic diversity present in human populations and therefore limit our ability to study various disease outcomes and immunologic mechanisms subsequent to West Nile virus infection. In this study, we used the Collaborative Cross mouse model to more effectively model the wide range of clinical, virologic, and immune phenotypes present upon West Nile virus infection in humans.

Regulatory T Cells Shape the Resident Memory T Cell Response to Virus Infection in the Tissues

Regulatory T cells (Tregs) are well known for their role in dampening the immune responses to self-Ags and, thereby, limiting autoimmunity. However, they also must permit immune responses to occur against foreign infectious agents. Using a mouse model of West Nile virus infection, we examined the role of Tregs in the generation of effector and memory T cell responses in the secondary lymphoid organs, as well as the infected tissues. We found that Treg numbers and activation increased in both the secondary lymphoid organs and CNS postinfection. Using Foxp3DTR knock-in mice, we found that Treg-deficient mice had increased Ag-driven production of IFN-γ from both CD4+ and CD8+ T cells in the spleen and CNS during the effector phase. In mice lacking Tregs, there were greater numbers of short-lived effector CD8+ T cells in the spleen during the peak of the immune response, but the memory CD8+ T cell response was impaired. Specifically, we demonstrate that Treg-dependent production of TGF-β results in increased expression of CD103 on CD8+ T cells, thereby allowing for a large pool of resident memory T cells to be maintained in the brain postinfection.

A Mouse Model of Chronic West Nile Virus Disease

Infection with West Nile virus (WNV) leads to a range of disease outcomes, including chronic infection, though lack of a robust mouse model of chronic WNV infection has precluded identification of the immune events contributing to persistent infection. Using the Collaborative Cross, a population of recombinant inbred mouse strains with high levels of standing genetic variation, we have identified a mouse model of persistent WNV disease, with persistence of viral loads within the brain. Compared to lines exhibiting no disease or marked disease, the F1 cross CC(032x013)F1 displays a strong immunoregulatory signature upon infection that correlates with restraint of the WNV-directed cytolytic response. We hypothesize that this regulatory T cell response sufficiently restrains the immune response such that a chronic infection can be maintained in the CNS. Use of this new mouse model of chronic neuroinvasive virus will be critical in developing improved strategies to prevent prolonged disease in humans.

A novel HIV vaccine adjuvanted by IC31 induces robust and persistent humoral and cellular immunity

The HIV vaccine strategy that, to date, generated immune protection consisted of a prime-boost regimen using a canarypox vector and an HIV envelope protein with alum, as shown in the RV144 trial. Since the efficacy was weak, and previous HIV vaccine trials designed to generate antibody responses failed, we hypothesized that generation of T cell responses would result in improved protection. Thus, we tested the immunogenicity of a similar envelope-based vaccine using a mouse model, with two modifications: a clade C CN54gp140 HIV envelope protein was adjuvanted by the TLR9 agonist IC31®, and the viral vector was the vaccinia strain NYVAC-CN54 expressing HIV envelope gp120. The use of IC31® facilitated immunoglobulin isotype switching, leading to the production of Env-specific IgG2a, as compared to protein with alum alone. Boosting with NYVAC-CN54 resulted in the generation of more robust Th1 T cell responses. Moreover, gp140 prime with IC31® and alum followed by NYVAC-CN54 boost resulted in the formation and persistence of central and effector memory populations in the spleen and an effector memory population in the gut. Our data suggest that this regimen is promising and could improve the protection rate by eliciting strong and long-lasting humoral and cellular immune responses.

Antiretroviral Pre-Exposure Prophylaxis Does Not Enhance Immune Responses to HIV in Exposed but Uninfected Persons

BACKGROUND Antiretroviral preexposure prophylaxis (PrEP), using daily oral combination tenofovir disoproxil fumarate plus emtricitabine, is an effective human immunodeficiency virus (HIV) prevention strategy for populations at high risk of HIV acquisition. Although the primary mode of action for the protective effect of PrEP is probably direct antiviral activity, nonhuman primate studies suggest that PrEP may also allow for development of HIV-specific immune responses, hypothesized to result from aborted HIV infections providing a source of immunologic priming. We sought to evaluate whether PrEP affects the development of HIV-specific immune response in humans. METHODS AND RESULTS Within a PrEP clinical trial among high-risk heterosexual African men and women, we detected HIV-specific CD4(+) and CD8(+) peripheral blood T-cell responses in 10%-20% of 247 subjects evaluated. The response rate and magnitude of T-cell responses did not vary significantly between those assigned PrEP versus placebo, and no significant difference between those assigned PrEP and placebo was observed in measures of innate immune function. CONCLUSIONS We found no evidence to support the hypothesis that PrEP alters either the frequency or magnitude of HIV-specific immune responses in HIV-1-exposed seronegative individuals. These results suggest that PrEP is unlikely to serve as an immunologic prime to aid protection by a putative HIV vaccine.

The Immune Fulcrum: Regulatory T Cells Tip the Balance Between Pro- and Anti-inflammatory Outcomes upon Infection

Regulatory T cells (Tregs) are indispensable for immune homeostasis and the prevention of autoimmunity. In the context of infectious diseases, Tregs are multidimensional. Here, we describe how they may potentiate effector responses by assisting in recruitment of T cells into the infection site to resolve infection, facilitate accelerated antigen-specific memory responses, limit pathology, and contribute to disease resolution and healing, to the great benefit of the host. We also explore the villainous functions of Tregs during infection by reviewing several diseases in which the depletion or reduction in Treg frequency allows for better generation of effector memory, and results in acute resolution of infection, as opposed to chronicity or severe long-term outcomes. We describe findings generated using mouse models of infection as well as experiments performed using human cells and tissues. We propose that Tregs represent an immunologic fulcrum, promoting both pathogen clearance and damage control by preventing excessive destruction of infected tissues though unchecked immune responses.

Detection and differentiation of HIV-1 group O sera from HIV-1 group M and HIV-2 using recombinant antigens and peptides

Recombinant antigens and peptides were used to develop an HIV slot immunoblot assay to confirm and differentiate infection by HIV-1 group M, HIV-1 group O or HIV-2. Recombinant antigens from the gag, pol or env regions of HIV-1 and HIV-2, in addition to synthetic peptides from the immunodominant region (IDR) of transmembrane proteins gp41 (HIV-1) or gp36 (HIV-2), were blotted on nitrocellulose strips and used as a substitute for competitive Western blots. Evaluation of a large number of samples (N = 440) from various regions of the world, using the immunoblot, showed effective differentiation of HIV-1 group M, HIV-1 group O and HIV-2. The immunoblot identified correctly all (24/24) HIV-1 group O samples that were confirmed subsequently by PCR and sequence analysis. The immunoblot is a useful tool for identifying HIV-1 group O seropositive samples and has the potential to identify other serological HIV variants that may represent detection problems for HIV screening assays using HIV-1 group M subtype B reagents.