Cell-Intrinsic CD38 Expression Sustains Exhausted CD8+ T Cells by Regulating Their Survival and Metabolism during Chronic Viral Infection.

Acute and chronic viral infections result in the differentiation of effector and exhausted T cells with functional and phenotypic differences that dictate whether the infection is cleared or progresses to chronicity. High CD38 expression has been observed on CD8+ T cells across various viral infections and tumors in patients, suggesting an important regulatory function for CD38 on responding T cells. Here, we show that CD38 expression was increased and sustained on exhausted CD8+ T cells following chronic lymphocytic choriomeningitis virus (LCMV) infection, with lower levels observed on T cells from acute LCMV infection. We uncovered a cell-intrinsic role for CD38 expression in regulating the survival of effector and exhausted CD8+ T cells. We observed increased proliferation and function of Cd38-/- CD8+ progenitor exhausted T cells compared to those of wild-type (WT) cells. Furthermore, decreased oxidative phosphorylation and glycolytic potential were observed in Cd38-/- CD8+ T cells during chronic but not acute LCMV infection. Our studies reveal that CD38 has a dual cell-intrinsic function in CD8+ T cells, where it decreases proliferation and function yet supports their survival and metabolism. These findings show that CD38 is not only a marker of T cell activation but also has regulatory functions on effector and exhausted CD8+ T cells. IMPORTANCE Our study shows how CD38 expression is regulated on CD8+ T cells responding during acute and chronic viral infection. We observed higher CD38 levels on CD8+ T cells during chronic viral infection compared to levels during acute viral infection. Deleting CD38 had an important cell-intrinsic function in ensuring the survival of virus-specific CD8+ T cells throughout the course of viral infection. We found defective metabolism in Cd38-/- CD8+ T cells arising during chronic infection and changes in their progenitor T cell phenotype. Our studies revealed a dual cell-intrinsic role for CD38 in limiting proliferation and granzyme B production in virus-specific exhausted T cells while also promoting their survival. These data highlight new avenues for research into the mechanisms through which CD38 regulates the survival and metabolism of CD8+ T cell responses to viral infections.

Deconvolution of pro- and antiviral genomic responses in Zika virus-infected and bystander macrophages.

Genome-wide investigations of host-pathogen interactions are often limited by analyses of mixed populations of infected and uninfected cells, which lower sensitivity and accuracy. To overcome these obstacles and identify key mechanisms by which Zika virus (ZIKV) manipulates host responses, we developed a system that enables simultaneous characterization of genome-wide transcriptional and epigenetic changes in ZIKV-infected and neighboring uninfected primary human macrophages. We demonstrate that transcriptional responses in ZIKV-infected macrophages differed radically from those in uninfected neighbors and that studying the cell population as a whole produces misleading results. Notably, the uninfected population of macrophages exhibits the most rapid and extensive changes in gene expression, related to type I IFN signaling. In contrast, infected macrophages exhibit a delayed and attenuated transcriptional response distinguished by preferential expression of IFNB1 at late time points. Biochemical and genomic studies of infected macrophages indicate that ZIKV infection causes both a targeted defect in the type I IFN response due to degradation of STAT2 and reduces RNA polymerase II protein levels and DNA occupancy, particularly at genes required for macrophage identity. Simultaneous evaluation of transcriptomic and epigenetic features of infected and uninfected macrophages thereby reveals the coincident evolution of dominant proviral or antiviral mechanisms, respectively, that determine the outcome of ZIKV exposure.

HMGB2 regulates the differentiation and stemness of exhausted CD8+ T cells during chronic viral infection and cancer.

Chronic infections and cancers evade the host immune system through mechanisms that induce T cell exhaustion. The heterogeneity within the exhausted CD8+ T cell pool has revealed the importance of stem-like progenitor (Tpex) and terminal (Tex) exhausted T cells, although the mechanisms underlying their development are not fully known. Here we report High Mobility Group Box 2 (HMGB2) protein expression is upregulated and sustained in exhausted CD8+ T cells, and HMGB2 expression is critical for their differentiation. Through epigenetic and transcriptional programming, we identify HMGB2 as a cell-intrinsic regulator of the differentiation and maintenance of Tpex cells during chronic viral infection and in tumors. Despite Hmgb2-/- CD8+ T cells expressing TCF-1 and TOX, these master regulators were unable to sustain Tpex differentiation and long-term survival during persistent antigen. Furthermore, HMGB2 also had a cell-intrinsic function in the differentiation and function of memory CD8+ T cells after acute viral infection. Our findings show that HMGB2 is a key regulator of CD8+ T cells and may be an important molecular target for future T cell-based immunotherapies.

PD-1 Immune Checkpoint Blockade and PSGL-1 Inhibition Synergize to Reinvigorate Exhausted T Cells.

Chronic viral infections where the antigen persists long-term, induces an exhaustion phenotype in responding T cells. It is now evident that immune checkpoints on T cells including PD-1, CTLA-4, and PSGL-1 (Selplg) are linked with the differentiation of exhausted cells. Chronic T cell receptor signaling induces transcriptional signatures that result in the development of various exhausted T cell subsets, including the stem-like T cell precursor exhausted (Tpex) cells, which can be reinvigorated by immune checkpoint inhibitors (ICIs). While PSGL-1 has been shown to inhibit T cell responses in various disease models, the cell-intrinsic function of PSGL-1 in the differentiation, maintenance, and reinvigoration of exhausted T cells is unknown. We found Selplg-/- T cells had increased expansion in melanoma tumors and in early stages of chronic viral infection. Despite their increase, both WT and Selplg-/- T cells eventually became phenotypically and functionally exhausted. Even though virus-specific Selplg-/- CD4+ and CD8+ T cells were increased at the peak of T cell expansion, they decreased to lower levels than WT T cells at later stages of chronic infection. We found that Selplg-/- CD8+ Tpex (SLAMF6hiTIM3lo, PD-1+TIM3+, TOX+, TCF-1+) cell frequencies and numbers were decreased compared to WT T cells. Importantly, even though virus-specific Selplg-/- CD4+ and CD8+ T cells were lower, they were reinvigorated more effectively than WT T cells after anti-PD-L1 treatment. We found increased SELPLG expression in Hepatitis C-specific CD8+ T cells in patients with chronic infection, whereas these levels were decreased in patients that resolved the infection. Together, our findings showed multiple PSGL-1 regulatory functions in exhausted T cells. We found that PSGL-1 is a cell-intrinsic inhibitor that limits T cells in tumors and in persistently infected hosts. Additionally, while PSGL-1 is linked with T cell exhaustion, its expression was required for their long-term maintenance and optimal differentiation into Tpex cells. Finally, PSGL-1 restrained the reinvigoration potential of exhausted CD4+ and CD8+ T cells during ICI therapy. Our findings highlight that targeting PSGL-1 may have therapeutic potential alone or in combination with other ICIs to reinvigorate exhausted T cells in patients with chronic infections or cancer.

Targeting the PSGL-1 Immune Checkpoint Promotes Immunity to PD-1-Resistant Melanoma.

Immune-checkpoint inhibitors have had impressive efficacy in some patients with cancer, reinvigorating long-term durable immune responses against tumors. Despite the clinical success of these therapies, most patients with cancer continue to be unresponsive to these treatments, highlighting the need for novel therapeutic options. Although P-selectin glycoprotein ligand-1 (PSGL-1) has been shown to inhibit immune responses in a variety of disease models, previous work has yet to address whether PSGL-1 can be targeted therapeutically to promote antitumor immunity. Using an aggressive melanoma tumor model, we targeted PSGL-1 in tumor-bearing mice and found increased effector CD4+ and CD8+ T-cell responses and decreased regulatory T cells (Treg) in tumors. T cells exhibited increased effector function, activation, and proliferation, which delayed tumor growth in mice after anti-PSGL-1 treatment. Targeting PD-1 in PSGL-1-deficient, tumor-bearing mice led to an increased frequency of mice with complete tumor eradication. Targeting both PSGL-1 and PD-1 in wild-type tumor-bearing mice also showed enhanced antitumor immunity and slowed melanoma tumor growth. Our findings showed that therapeutically targeting the PSGL-1 immune checkpoint can reinvigorate antitumor immunity and suggest that targeting PSGL-1 may represent a new therapeutic strategy for cancer treatment.

A Zika virus mutation enhances transmission potential and confers escape from protective dengue virus immunity.

Zika virus (ZIKV) and dengue virus (DENV) are arthropod-borne pathogenic flaviviruses that co-circulate in many countries. To understand some of the pressures that influence ZIKV evolution, we mimic the natural transmission cycle by repeating serial passaging of ZIKV through cultured mosquito cells and either DENV-naive or DENV-immune mice. Compared with wild-type ZIKV, the strains passaged under both conditions exhibit increased pathogenesis in DENV-immune mice. Application of reverse genetics identifies an isoleucine-to-valine mutation (I39V) in the NS2B proteins of both passaged strains that confers enhanced fitness and escape from pre-existing DENV immunity. Introduction of I39V or I39T, a naturally occurring homologous mutation detected in recent ZIKV isolates, increases the replication of wild-type ZIKV in human neuronal precursor cells and laboratory-raised mosquitoes. Our data indicate that ZIKV strains with enhanced transmissibility and pathogenicity can emerge in DENV-naive or -immune settings, and that NS2B-I39 mutants may represent ZIKV variants of interest.

SREBP2-dependent lipid gene transcription enhances the infection of human dendritic cells by Zika virus.

The emergence of Zika virus (ZIKV) as a global health threat has highlighted the unmet need for ZIKV-specific vaccines and antiviral treatments. ZIKV infects dendritic cells (DC), which have pivotal functions in activating innate and adaptive antiviral responses; however, the mechanisms by which DC function is subverted to establish ZIKV infection are unclear. Here we develop a genomics profiling method that enables discrete analysis of ZIKV-infected versus neighboring, uninfected primary human DCs to increase the sensitivity and specificity with which ZIKV-modulated pathways can be identified. The results show that ZIKV infection specifically increases the expression of genes enriched for lipid metabolism-related functions. ZIKV infection also increases the recruitment of sterol regulatory element-binding protein (SREBP) transcription factors to lipid gene promoters, while pharmacologic inhibition or genetic silencing of SREBP2 suppresses ZIKV infection of DCs. Our data thus identify SREBP2-activated transcription as a mechanism for promoting ZIKV infection amenable to therapeutic targeting.

PSGL-1 Immune Checkpoint Inhibition for CD4+ T Cell Cancer Immunotherapy.

Immune checkpoint inhibition targeting T cells has shown tremendous promise in the treatment of many cancer types and are now standard therapies for patients. While standard therapies have focused on PD-1 and CTLA-4 blockade, additional immune checkpoints have shown promise in promoting anti-tumor immunity. PSGL-1, primarily known for its role in cellular migration, has also been shown to function as a negative regulator of CD4+ T cells in numerous disease settings including cancer. PSGL-1 is highly expressed on T cells and can engage numerous ligands that impact signaling pathways, which may modulate CD4+ T cell differentiation and function. PSGL-1 engagement in the tumor microenvironment may promote CD4+ T cell exhaustion pathways that favor tumor growth. Here we highlight that blocking the PSGL-1 pathway on CD4+ T cells may represent a new cancer therapy approach to eradicate tumors.

The Ability of Zika virus Intravenous Immunoglobulin to Protect From or Enhance Zika Virus Disease.

The closely related flaviviruses, dengue and Zika, cause significant human disease throughout the world. While cross-reactive antibodies have been demonstrated to have the capacity to potentiate disease or mediate protection during flavivirus infection, the mechanisms responsible for this dichotomy are still poorly understood. To understand how the human polyclonal antibody response can protect against, and potentiate the disease in the context of dengue and Zika virus infection we used intravenous hyperimmunoglobulin (IVIG) preparations in a mouse model of the disease. Three IVIGs (ZIKV-IG, Control-Ig and Gamunex®) were evaluated for their ability to neutralize and/or enhance Zika, dengue 2 and 3 viruses in vitro. The balance between virus neutralization and enhancement provided by the in vitro neutralization data was used to predict the IVIG concentrations which could protect or enhance Zika, and dengue 2 disease in vivo. Using this approach, we were able to define the unique in vivo dynamics of complex polyclonal antibodies, allowing for both enhancement and protection from flavivirus infection. Our results provide a novel understanding of how polyclonal antibodies interact with viruses with implications for the use of polyclonal antibody therapeutics and the development and evaluation of the next generation flavivirus vaccines.

Detection of Zika virus in mouse mammary gland and breast milk.

Clinical reports of Zika Virus (ZIKV) RNA detection in breast milk have been described, but evidence conflicts as to whether this RNA represents infectious virus. We infected post-parturient AG129 murine dams deficient in type I and II interferon receptors with ZIKV. ZIKV RNA was detected in pup stomach milk clots (SMC) as early as 1 day post maternal infection (dpi) and persisted as late as 7 dpi. In mammary tissues, ZIKV replication was demonstrated by immunohistochemistry in multiple cell types including cells morphologically consistent with myoepithelial cells. No mastitis was seen histopathologically. In the SMC and tissues of the nursing pups, no infectious virus was detected via focus forming assay. However, serial passages of fresh milk supernatant yielded infectious virus, and immunohistochemistry showed ZIKV replication protein associated with degraded cells in SMC. These results suggest that breast milk may contain infectious ZIKV. However, breast milk transmission (BMT) does not occur in this mouse strain that is highly sensitive to ZIKV infection. These results suggest a low risk for breast milk transmission of ZIKV, and provide a platform for investigating ZIKV entry into milk and mechanisms which may prevent or permit BMT.

Human Polyclonal Antibodies Prevent Lethal Zika Virus Infection in Mice.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that represents a major threat to global health. ZIKV infections in adults are generally asymptomatic or present with mild symptoms. However, recent outbreaks of ZIKV have revealed that it can cause Congenital Zika Syndrome in neonates and Guillain-Barré syndrome in adults. Currently, no ZIKV-specific vaccines or antiviral treatments are available. In this study, we tested the efficacy of convalescent plasma IgG hyperimmune product (ZIKV-IG) isolated from individuals with high neutralizing anti-ZIKV titers as a therapeutic candidate against ZIKV infection using a model of ZIKV infection in Ifnar1-/- mice. ZIKV-IG successfully protected mice from lethal ZIKV challenge. In particular, ZIKV-IG treatment at 24 hours after lethal ZIKV infection improved survival by reducing weight loss and tissue viral burden and improving clinical score. Additionally, ZIKV-IG eliminated ZIKV-induced tissue damage and inflammation in the brain and liver. These results indicate that ZIKV-IG is efficacious against ZIKV, suggesting this human polyclonal antibody is a viable candidate for further development as a treatment against human ZIKV infection.

Synergism between the tyrosine kinase inhibitor sunitinib and Anti-TNF antibody protects against lethal dengue infection.

Dengue virus (DENV) currently circulates in more than 100 countries and causes an estimated 390 million infections per year. While most cases manifest as a self-resolving fever, ∼1.5% of infections develop into a more severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), which causes ∼20,000 deaths annually. The underlying pathological feature of DHF/DSS, also known as Severe Dengue, is an acute increase in vascular permeability leading to hypovolemia and shock. Angiogenic factors and cytokines, such as vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF), have been implicated in the increased vascular permeability, suggesting a potential therapeutic strategy for Severe Dengue. Here, we employed a mouse model of antibody-dependent enhancement of DENV infection, which recapitulates the fatal capillary leakage and shock of human Severe Dengue, to investigate the effects of approved VEGF- and TNF-targeting drugs. DENV infection caused a significant increase in serum VEGF levels within 2 days and resulted in ∼80% mortality within 8 days of infection. Treatment of mice with sunitinib, a VEGF receptor tyrosine kinase inhibitor, once (day 2) or twice (days 1 and 2) post-infection reduced mortality by 50-80% compared with untreated mice. Notably, sunitinib treatment decreased serum TNF levels, white blood cell counts, and hematocrit levels relative to untreated mice, but had only marginal effects on tissue viral burden. Combination therapy with anti-TNF antibody and sunitinib significantly reduced vascular leakage and synergized to provide superior protection from lethal DENV infection compared with either agent alone. These data suggest that a two-pronged anti-angiogenic and anti-inflammatory approach may be useful for the rapid treatment of DHF/DSS.

Maternally Acquired Zika Antibodies Enhance Dengue Disease Severity in Mice.

Antibody (Ab)-dependent enhancement can exacerbate dengue virus (DENV) infection due to cross-reactive Abs from an initial DENV infection, facilitating replication of a second DENV. Zika virus (ZIKV) emerged in DENV-endemic areas, raising questions about whether existing immunity could affect these related flaviviruses. We show that mice born with circulating maternal Abs against ZIKV develop severe disease upon DENV infection. Compared with pups of naive mothers, those born to ZIKV-immune mice lacking type I interferon receptor in myeloid cells (LysMCre+Ifnar1fl/fl) exhibit heightened disease and viremia upon DENV infection. Passive transfer of IgG isolated from mice born to ZIKV-immune mothers resulted in increased viremia in naive recipient mice. Treatment with Abs blocking inflammatory cytokine tumor necrosis factor linked to DENV disease or Abs blocking DENV entry improved survival of DENV-infected mice born to ZIKV-immune mothers. Thus, the maternal Ab response to ZIKV infection or vaccination might predispose to severe dengue disease in infants.

Cross-reactive Dengue virus-specific CD8+ T cells protect against Zika virus during pregnancy.

As Zika virus (ZIKV) emerges into Dengue virus (DENV)-endemic areas, cases of ZIKV infection in DENV-immune pregnant women may rise. Here we show that prior DENV immunity affects maternal and fetal ZIKV infection in pregnancy using sequential DENV and ZIKV infection models. Fetuses in ZIKV-infected DENV-immune dams were normal sized, whereas fetal demise occurred in non-immune dams. Moreover, reduced ZIKV RNA is present in the placenta and fetuses of ZIKV-infected DENV-immune dams. DENV cross-reactive CD8+ T cells expand in the maternal spleen and decidua of ZIKV-infected dams, their depletion increases ZIKV infection in the placenta and fetus, and results in fetal demise. The inducement of cross-reactive CD8+ T cells via peptide immunization or adoptive transfer results in decreased ZIKV infection in the placenta. Prior DENV immunity can protect against ZIKV infection during pregnancy in mice, and CD8+ T cells are sufficient for this cross-protection. This has implications for understanding the natural history of ZIKV in DENV-endemic areas and the development of optimal ZIKV vaccines.