Blocking Virus Replication during Acute Murine Cytomegalovirus Infection Paradoxically Prolongs Antigen Presentation and Increases the CD8+ T Cell Response by Preventing Type I IFN-Dependent Depletion of Dendritic Cells.

Increasing amounts of pathogen replication usually lead to a proportionate increase in size and effector differentiation of the CD8+ T cell response, which is attributed to increased Ag and inflammation. Using a murine CMV that is highly sensitive to the antiviral drug famciclovir to modulate virus replication, we found that increased virus replication drove increased effector CD8+ T cell differentiation, as expected. Paradoxically, however, increased virus replication dramatically decreased the size of the CD8+ T cell response to two immunodominant epitopes. The decreased response was due to type I IFN-dependent depletion of conventional dendritic cells and could be reproduced by specific depletion of dendritic cells from day 2 postinfection or by sterile induction of type I IFN. Increased virus replication and type I IFN specifically inhibited the response to two immunodominant epitopes that are known to be dependent on Ag cross-presented by DCs, but they did not inhibit the response to "inflationary" epitopes whose responses can be sustained by infected nonhematopoietic cells. Our results show that type I IFN can suppress CD8+ T cell responses to cross-presented Ag by depleting cross-presenting conventional dendritic cells.

IL-12/IL23 blockade reveals patterns of asynchronous inflammation in pyoderma gangrenosum.

Pyoderma gangrenosum (PG) is a rare neutrophilic dermatosis causing chronic and recalcitrant painful ulcerations. Pathogenic mechanisms are yet poorly understood limiting therapeutic options, however, IL-12/IL-23 inhibition via ustekinumab has previously been associated with positive outcomes. We aimed to elucidate the dysregulated immune landscape of PG and lesional skin changes associated with IL-12/IL-23 blockade. We applied spatial transcriptomics and comparative computation analysis on lesional biopsies from two patients obtained before and after IL-12/IL-23 blockade with ustekinumab. Our data indicate lesional PG skin exhibits complex patterns of inflammation, including a not previously described major infiltration of B cells and establishment of tertiary lymphoid structures. In both patients, IL-12/IL-23 blockade led to marked clinical improvement but was associated with amelioration of contrasting inflammatory pathways. Notably, plasma cell markers and tertiary structures were recalcitrant to the treatment regime suggesting that B cells might play a role in the refractory nature of PG.

Randomized Placebo-Controlled, Biomarker-Stratified Phase Ib Microbiome Modulation in Melanoma: Impact of Antibiotic Preconditioning on Microbiome and Immunity.

Gut-microbiota modulation shows promise in improving immune-checkpoint blockade (ICB) response; however, precision biomarker-driven, placebo-controlled trials are lacking. We performed a multicenter, randomized placebo-controlled, biomarker-stratified phase I trial in patients with ICB-naïve metastatic melanoma using SER-401, an orally delivered Firmicutesenriched spore formulation. Fecal microbiota signatures were characterized at baseline; patients were stratified by high versus low Ruminococcaceae abundance prior to randomization to the SER-401 arm (oral vancomycin-preconditioning/SER-401 alone/nivolumab + SER-401), versus the placebo arm [placebo antibiotic/placebo microbiome modulation (PMM)/nivolumab + PMM (NCT03817125)]. Analysis of 14 accrued patients demonstrated that treatment with SER-401 + nivolumab was safe, with an overall response rate of 25% in the SER-401 arm and 67% in the placebo arm (though the study was underpowered related to poor accrual during the COVID-19 pandemic). Translational analyses demonstrated that vancomycin preconditioning was associated with the disruption of the gut microbiota and impaired immunity, with incomplete recovery at ICB administration (particularly in patients with high baseline Ruminococcaceae). These results have important implications for future microbiome modulation trials. Significance: This first-of-its-kind, placebo-controlled, randomized biomarker-driven microbiome modulation trial demonstrated that vancomycin + SER-401 and anti-PD-1 are safe in melanoma patients. Although limited by poor accrual during the pandemic, important insights were gained via translational analyses, suggesting that antibiotic preconditioning and interventional drug dosing regimens should be carefully considered when designing such trials.

BET inhibitors rescue anti-PD1 resistance by enhancing TCF7 accessibility in leukemia-derived terminally exhausted CD8+ T cells.

Many acute myeloid leukemia (AML) patients exhibit hallmarks of immune exhaustion, such as increased myeloid-derived suppressor cells, suppressive regulatory T cells and dysfunctional T cells. Similarly, we have identified the same immune-related features, including exhausted CD8+ T cells (TEx) in a mouse model of AML. Here we show that inhibitors that target bromodomain and extra-terminal domain (BET) proteins affect tumor-intrinsic factors but also rescue T cell exhaustion and ICB resistance. Ex vivo treatment of cells from AML mice and AML patients with BET inhibitors (BETi) reversed CD8+ T cell exhaustion by restoring proliferative capacity and expansion of the more functional precursor-exhausted T cells. This reversal was enhanced by combined BETi and anti-PD1 treatment. BETi synergized with anti-PD1 in vivo, resulting in the reduction of circulating leukemia cells, enrichment of CD8+ T cells in the bone marrow, and increase in expression of Tcf7, Slamf6, and Cxcr5 in CD8+ T cells. Finally, we profiled the epigenomes of in vivo JQ1-treated AML-derived CD8+ T cells by single-cell ATAC-seq and found that JQ1 increases Tcf7 accessibility specifically in Tex cells, suggesting that BETi likely acts mechanistically by relieving repression of progenitor programs in Tex CD8+ T cells and maintaining a pool of anti-PD1 responsive CD8+ T cells.

FOXP3 expressing CD127lo CD4+ T cells inversely correlate with CD38+ CD8+ T cell activation levels in primary HIV-1 infection.

During the course of HIV-1 infection, the status of immune activation has been determined to be a powerful indicator of disease progression. The immune system has adopted self-regulatory mechanisms to counterbalance undesirable immune responses. CD25+CD4+ T regulatory (Treg) cells that express the transcription regulator, forkhead box P3 (FOXP3), play an important role in this immunosuppression. Using a combination of Treg cell discriminatory markers (FOXP3, CD25, CD127), we predicted that an expansion of Treg cell subsets would negatively correlate with immune activation during the early stages of HIV-1 infection. We report that FOXP3+CD127lo expressing CD4+ T cells increases in primary HIV-1 infection over time. Furthermore, the FOXP3+CD127lo CD4+ T cells may, in fact, reduce the levels of T cell activation following primary infection. It is interesting that the positive correlation between FOXP3+CD127lo CD4+ and CD25+CD127lo CD4+ T cells noted in HIV-uninfected persons is not only lost but may also be reversed in early, chronic HIV-1 infection. Unlike FOXP3+CD127lo CD4+, the level of FOXP3+CD25+CD127lo CD4+ T cells did not correlate with T cell activation, suggesting that these cells were not effective in reducing T cell activation. These observations suggest that different Treg populations may have different effects on reducing immune activation in HIV-1 infection and that the FOXP3+CD127lo CD4+ T cell population may be particularly important in limiting immune activation.

IFNγ-activated dermal lymphatic vessels inhibit cytotoxic T cells in melanoma and inflamed skin.

Mechanisms of immune suppression in peripheral tissues counteract protective immunity to prevent immunopathology and are coopted by tumors for immune evasion. While lymphatic vessels facilitate T cell priming, they also exert immune suppressive effects in lymph nodes at steady-state. Therefore, we hypothesized that peripheral lymphatic vessels acquire suppressive mechanisms to limit local effector CD8+ T cell accumulation in murine skin. We demonstrate that nonhematopoietic PD-L1 is largely expressed by lymphatic and blood endothelial cells and limits CD8+ T cell accumulation in tumor microenvironments. IFNγ produced by tissue-infiltrating, antigen-specific CD8+ T cells, which are in close proximity to tumor-associated lymphatic vessels, is sufficient to induce lymphatic vessel PD-L1 expression. Disruption of IFNγ-dependent crosstalk through lymphatic-specific loss of IFNγR boosts T cell accumulation in infected and malignant skin leading to increased viral pathology and tumor control, respectively. Consequently, we identify IFNγR as an immunological switch in lymphatic vessels that balances protective immunity and immunopathology leading to adaptive immune resistance in melanoma.

Lymphatic Vessels Balance Viral Dissemination and Immune Activation following Cutaneous Viral Infection.

Lymphatic vessels lie at the interface between peripheral sites of pathogen entry, adaptive immunity, and the systemic host. Though the paradigm is that their open structure allows for passive flow of infectious particles from peripheral tissues to lymphoid organs, virus applied to skin by scarification does not spread to draining lymph nodes. Using cutaneous infection by scarification, we analyzed the effect of viral infection on lymphatic transport and evaluated its role at the host-pathogen interface. We found that, in the absence of lymphatic vessels, canonical lymph-node-dependent immune induction was impaired, resulting in exacerbated pathology and compensatory, systemic priming. Furthermore, lymphatic vessels decouple fluid and cellular transport in an interferon-dependent manner, leading to viral sequestration while maintaining dendritic cell transport for immune induction. In conclusion, we found that lymphatic vessels balance immune activation and viral dissemination and act as an "innate-like" component of tissue host viral defense.

HIV-1-specific T Cell-dependent natural killer (NK) cell activation: major contribution by NK cells to interferon-gamma production in response to HIV-1 antigens.

Natural killer (NK) cells can directly recognize virus-infected cells. Here, we demonstrate that NK cells also produce interferon (IFN)-gamma in an HIV-1-specific, T cell-dependent manner. After stimulation of peripheral blood mononuclear cells (PBMCs) from HIV-1-infected individuals with HIV-1-derived peptides, up to half of the IFN-gamma-producing PBMCs are NK cells. These results indicate that T cell-dependent NK cell IFN-gamma production can be important for immune control of HIV-1, and have implications for the interpretation of data from vaccine trials using ELISPOT and ELISA.

Lower cytokine secretion ex vivo by natural killer T cells in HIV-infected individuals is associated with higher CD161 expression.

OBJECTIVE: Natural killer T (NKT) cells are efficiently targeted by HIV and severely reduced in numbers in the circulation of infected individuals. The functional capacity of the remaining NKT cells in HIV-infected individuals is poorly characterized. This study measured NKT cell cytokine production directly ex vivo and compared these responses with both the disease status and NKT subset distribution of individual patients. METHODS: NKT cell frequencies, subsets, and ex-vivo effector functions were measured in the peripheral blood mononuclear cells of HIV-infected patients and healthy controls by flow cytometry. We measured cytokines from NKT cells after stimulation with either alpha-galactosyl ceramide-loaded CD1d dimers (DimerX-alphaGalCer) or phorbol myristate acetate and ionomycin. RESULTS: The frequencies of NKT cells secreting interferon-gamma and tumor necrosis factor-alpha were significantly lower in HIV-infected patients than healthy controls after DimerX-alphaGalCer treatment, but responses were similar after treatment with phorbol myristate acetate and ionomycin. The magnitude of the interferon-gamma response to DimerX-alphaGalCer correlated inversely with the number of years of infection. Both interferon-gamma and tumor necrosis factor-alpha production in response to DimerX-alphaGalCer correlated inversely with CD161 expression. CONCLUSION: The ex-vivo Th1 responses of circulating NKT cells to CD1d-glycolipid complexes are impaired in HIV-infected patients. NKT cell functions may be progressively lost over time in HIV infection, and CD161 is implicated in the regulation of NKT cell responsiveness.

Immune reconstitution of CD56(dim) NK cells in individuals with primary HIV-1 infection treated with interleukin-2.

Natural killer (NK) cells are believed to play a role in human immunodeficiency virus type 1 (HIV-1) disease progression, and NK cell levels are reduced in individuals with chronic HIV-1 infection. Interleukin (IL)-2 therapy results in an expansion of CD4(+) T cells as well as NK cells; however, little is known about the detailed effects of IL-2 therapy on NK cells in HIV-1 infection in general and in early infection in particular. Here, we investigated the effects of combined IL-2 therapy and antiretroviral therapy (ART) on the number, frequency, phenotype, and interferon (IFN)-gamma production of NK cells in individuals with early HIV-1 infection. Patients randomized to receive combined ART and IL-2 therapy predominantly expanded CD56(dim) NK cells, and the expansion was greater than in patients randomized to receive ART alone. Importantly, NK cell receptor expression and IFN-gamma production were maintained over time. This reconstitution of NK cells may be useful in helping contain viremia if patients discontinue therapy or develop drug resistance.

Elevated frequency of gamma interferon-producing NK cells in healthy adults vaccinated against influenza virus.

Recent studies indicate that innate immunity in influenza virus infection is an area of substantial importance for our understanding of influenza virus pathogenesis, yet our knowledge of the mechanisms controlling innate immunity remains limited. Further delineation of the roles of NK cells and innate immunity in viral infection may have important implications for the development of improved influenza virus vaccines. In this study, we evaluated the phenotype and function of NK and T lymphocytes, as well as influenza virus-specific immunoglobulin G production, prior to and following vaccination with the routinely administered trivalent influenza virus vaccine. We demonstrate influenza virus antigen-specific innate and adaptive cellular responses and evaluate changes in NK cell receptor expression over time. Our results demonstrate increased innate and adaptive cellular immune responses and show that NK cells are a significant source of gamma interferon (IFN-gamma) following influenza virus vaccination. An increase in the frequency of IFN-gamma-producing NK cells was observed in many subjects postvaccination. The subset distribution with respect to CD56(dim) and CD56(bright) NK cell subsets remained stable, as did the NK cell phenotype with respect to expression of cell surface activating and inhibitory receptors. These results may form the basis for further investigations of the role of NK cells in immunity to influenza.

Human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T(EMRA) cells in early infection are linked to control of HIV-1 viremia and predict the subsequent viral load set point.

CD8(+) T cells are believed to play an important role in the control of human immunodeficiency virus type 1 (HIV-1) infection. However, despite intensive efforts, it has not been possible to consistently link the overall magnitude of the CD8(+) T-cell response with control of HIV-1. Here, we have investigated the association of different CD8(+) memory T-cell subsets responding to HIV-1 in early infection with future control of HIV-1 viremia. Our results demonstrate that both a larger proportion and an absolute number of HIV-1-specific CD8(+) CCR7(-) CD45RA(+) effector memory T cells (T(EMRA) cells) were associated with a lower future viral load set point. In contrast, a larger absolute number of HIV-1-specific CD8(+) CCR7(-) CD45RA(-) effector memory T cells (T(EM)) was not related to the viral load set point. Overall, the findings suggest that CD8(+) T(EMRA) cells have superior antiviral activity and indicate that both qualitative and quantitative aspects of the CD8(+) T-cell response need to be considered when defining the characteristics of protective immunity to HIV-1.

Natural killer cells in perinatally HIV-1-infected children exhibit less degranulation compared to HIV-1-exposed uninfected children and their expression of KIR2DL3, NKG2C, and NKp46 correlates with disease severity.

NK cells play an integral role in the innate immune response by targeting virally infected and transformed cells with direct killing and providing help to adaptive responses through cytokine secretion. Whereas recent studies have focused on NK cells in HIV-1-infected adults, the role of NK cells in perinatally HIV-1-infected children is less studied. Using multiparametric flow cytometric analysis, we assessed the number, phenotype, and function of NK cell subsets in the peripheral blood of perinatally HIV-1-infected children on highly active antiretroviral therapy and compared them to perinatally exposed but uninfected children. We observed an increased frequency of NK cells expressing inhibitory killer Ig-like receptors in infected children. This difference existed despite comparable levels of total NK cells and NK cell subpopulations between the two groups. Additionally, NK cell subsets from infected children expressed, with and without stimulation, significantly lower levels of the degranulation marker CD107, which correlates with NK cell cytotoxicity. Lastly, increased expression of KIR2DL3, NKG2C, and NKp46 on NK cells correlated with decreased CD4+ T-lymphocyte percentage, an indicator of disease severity in HIV-1- infected children. Taken together, these results show that HIV-1-infected children retain a large population of cytotoxically dysfunctional NK cells relative to perinatally exposed uninfected children. This reduced function appears concurrently with distinct NK cell surface receptor expression and is associated with a loss of CD4+ T cells. This finding suggests that NK cells may have an important role in HIV-1 disease pathogenesis in HIV-1-infected children.

Individuals with pulmonary tuberculosis have lower levels of circulating CD1d-restricted NKT cells.

Mycobacterium tuberculosis (MTB) is a leading cause of mortality worldwide from an infectious agent. Natural killer T (NKT) cells recognize mycobacterial antigens and contribute to anti-MTB immunity in mouse models. NKT cells were measured in subjects with pulmonary tuberculosis, MTB-exposed individuals, and healthy controls. NKT cell levels are selectively lower in peripheral blood mononuclear cells from individuals with pulmonary tuberculosis than in both MTB-exposed subjects and healthy control subjects. This apparent loss of NKT cells from the peripheral blood is sustained during the 6 months after the initiation of MTB treatment. These findings indicate that NKT cells may be an important component of antituberculosis immunity.

Strong HIV-1-specific T cell responses in HIV-1-exposed uninfected infants and neonates revealed after regulatory T cell removal.

BACKGROUND: In utero transmission of HIV-1 occurs on average in only 3%-15% of HIV-1-exposed neonates born to mothers not on antiretroviral drug therapy. Thus, despite potential exposure, the majority of infants remain uninfected. Weak HIV-1-specific T-cell responses have been detected in children exposed to HIV-1, and potentially contribute to protection against infection. We, and others, have recently shown that the removal of CD4(+) CD25(+) T-regulatory (Treg) cells can reveal strong HIV-1 specific T-cell responses in some HIV-1 infected adults. Here, we hypothesized that Treg cells could suppress HIV-1-specific immune responses in young children. METHODOLOGY/PRINCIPAL FINDINGS: We studied two cohorts of children. The first group included HIV-1-exposed-uninfected (EU) as well as unexposed (UNEX) neonates. The second group comprised HIV-1-infected and HIV-1-EU children. We quantified the frequency of Treg cells, T-cell activation, and cell-mediated immune responses. We detected high levels of CD4(+) CD25(+) CD127(-) Treg cells and low levels of CD4(+) and CD8(+) T cell activation in the cord blood of the EU neonates. We observed HIV-1-specific T cell immune responses in all of the children exposed to the virus. These T-cell responses were not seen in the cord blood of control HIV-1 unexposed neonates. Moreover, the depletion of CD4(+) CD25(+) Treg cells from the cord blood of EU newborns strikingly augmented both CD4(+) and CD8(+) HIV-1-specific immune responses. CONCLUSIONS/SIGNIFICANCE: This study provides new evidence that EU infants can mount strong HIV-1-specific T cell responses, and that in utero CD4(+) CD25(+) T-regulatory cells may be contributing to the lack of vertical transmission by reducing T cell activation.

Exploring sponge-derived terpenoids for their potency and selectivity against 12-human, 15-human, and 15-soybean lipoxygenases.

To sharpen the search for new lipoxygenase inhibitors, we designed a screen to probe for both potency and selectivity. The assay utilized 12-human (12-HLO), 15-human (15-HLO), and 15-soybean (15-SLO) lipoxygenases. The IC(50) value data obtained provided new insights about structure-activity relationships (SAR) for redox and nonredox inhibitors. All of the compounds tested were isolated from sponges and consisted of a novel terpenoid, hyrtenone A (1), and 12 known terpenoids. Potent compounds were defined as those having IC(50) values < 1 microM, and selectivity was assessed from the three possible IC(50) value ratios. One of the four terpenoid redox inhibitors studied, puupehenone (2), was equivalent to or better in potency than the well-known redox inhibitor nordihydroguarierate acid (NDGA, 14). However, none of the terpene redox inhibitors exhibited a selectivity ratio on a par with that of 14. Several potent nonredox inhibitors were identified, and one, dimethoxypuupehenol (5), exhibited notable selectivity. The structural elucidation of 1 and the SAR results for 13 natural products are reported. This study suggests that sponge-derived terpenes are a promising source for new lipoxygenase inhibitors.