IL-2 produced by HBV-specific T cells as a biomarker of viral control and predictor of response to PD-1 therapy across clinical phases of chronic hepatitis B.

BACKGROUND: There are no immunological biomarkers that predict control of chronic hepatitis B (CHB). The lack of immune biomarkers raises concerns for therapies targeting PD-1/PD-L1 because they have the potential for immune-related adverse events. Defining specific immune functions associated with control of HBV replication could identify patients likely to respond to anti-PD-1/PD-L1 therapies and achieve a durable functional cure. METHODS: We enrolled immunotolerant, HBeAg+ immune-active (IA+), HBeAg- immune-active (IA-), inactive carriers, and functionally cured patients to test ex vivo PD-1 blockade on HBV-specific T cell functionality. Peripheral blood mononuclear cells were stimulated with overlapping peptides covering HBV proteins +/-α-PD-1 blockade. Functional T cells were measured using a 2-color FluoroSpot assay for interferon-γ and IL-2. Ex vivo functional restoration was compared to the interferon response capacity assay, which predicts overall survival in cancer patients receiving checkpoint inhibitors. RESULTS: Ex vivo interferon-γ+ responses did not differ across clinical phases. IL-2+ responses were significantly higher in patients with better viral control and preferentially restored with PD-1 blockade. Inactive carrier patients displayed the greatest increase in IL-2 production, which was dominated by CD4 T cell and response to the HBcAg. The interferon response capacity assay significantly correlated with the degree of HBV-specific T cell restoration. CONCLUSIONS: IL-2 production was associated with better HBV control and superior to interferon-γ as a marker of T cell restoration following ex vivo PD-1 blockade. Our study suggests that responsiveness to ex vivo PD-1 blockade, or the interferon response capacity assay, may support stratification for α-PD-1 therapies.

Safety and tolerability of first-line durvalumab with tremelimumab and chemotherapy in esophageal squamous cell carcinoma.

BACKGROUND: Advanced or metastatic esophageal squamous cell carcinoma (ESCC) is associated with poor prognosis; new first-line systemic treatment options are needed. Combining immuno-oncology therapies with standard chemotherapy may represent a promising approach for the treatment of solid tumors. Results from a Phase Ib study evaluating durvalumab with tremelimumab and chemotherapy in patients with advanced or metastatic ESCC are reported. METHODS: Adults with advanced or metastatic ESCC who were candidates for first-line platinum-based chemotherapy received durvalumab 1500 mg (Day 1), tremelimumab 75 mg (Day 1), cisplatin 80 mg/m2 (Day 1) and 5-fluorouracil (5-FU) 800 mg/m2 (Days 1-5) in 28-day cycles until disease progression or discontinuation due to toxicity. The study consisted of safety run-in (Part A) and expansion (Part B) periods. The primary endpoint was safety. Antitumor activity was an exploratory endpoint. RESULTS: Sixteen patients were enrolled, 6 in Part A and 10 in Part B, and received a median of 4.0 treatment cycles. All patients were Asian; median age was 65.0 years. All patients experienced adverse events (AEs) related to cisplatin and 5-FU, and 8 (50.0%) patients experienced AEs related to durvalumab and tremelimumab. Grade ≥3 treatment-related AEs occurred in 7 (43.8%) patients. There were no deaths associated with AEs. Six (37.5%) patients achieved an objective response. Median progression-free survival was 3.75 months, and median overall survival was 9.69 months. CONCLUSIONS: Durvalumab with tremelimumab and chemotherapy demonstrated manageable safety and antitumor activity in patients with advanced or metastatic ESCC, warranting further investigation in randomized trials. Registered with ClinicalTrials.gov: NCT02658214.

MATTERHORN: phase III study of durvalumab plus FLOT chemotherapy in resectable gastric/gastroesophageal junction cancer.

Standard-of-care for resectable gastric/gastroesophageal junction cancer includes surgery and neoadjuvant-adjuvant 5-fluorouracil-leucovorin-oxaliplatin-docetaxel (FLOT) chemotherapy. Early-phase clinical studies support further clinical development of the immune checkpoint inhibitor (ICI); durvalumab, an anti-PD-L1 antibody, in patients with gastric/gastroesophageal junction cancer. Accumulating evidence indicates that ICIs combined with FLOT chemotherapy improve clinical outcomes in patients with advanced or metastatic cancer. We describe the rationale for and the design of MATTERHORN, a randomized, double-blind, placebo-controlled, phase III study investigating the efficacy and safety of neoadjuvant-adjuvant durvalumab and FLOT chemotherapy followed by adjuvant durvalumab monotherapy in patients with resectable gastric/gastroesophageal junction cancer. The planned sample size is 900 patients, the primary end point is event-free survival and safety and tolerability will be evaluated. Clinical trial registration: NCT04592913 (ClinicalTrials.gov).

Functional Exhaustion of HBV-Specific CD8 T Cells Impedes PD-L1 Blockade Efficacy in Chronic HBV Infection.

Background: A functional cure for chronic HBV could be achieved by boosting HBV-specific immunity. In vitro studies show that immunotherapy could be an effective strategy. However, these studies include strategies to enrich HBV-specific CD8 T cells, which could alter the expression of the anti-PD-1/anti-PD-L1 antibody targets. Our aim was to determine the efficacy of PD-L1 blockade ex vivo. Methods: HBV-specific CD8 T cells were characterized ex vivo by flow cytometry for the simultaneous analysis of six immune populations and 14 activating and inhibitory receptors. Ex vivo functionality was quantified by ELISpot and by combining peptide pool stimulation, dextramers and intracellular flow cytometry staining. Results: The functionality of HBV-specific CD8 T cells is associated with a higher frequency of cells with low exhaustion phenotype (LAG3-TIM3-PD-1+), independently of the clinical parameters. The accumulation of HBV-specific CD8 T cells with a functionally exhausted phenotype (LAG3+TIM3+PD-1+) is associated with lack of ex vivo functionality. PD-L1 blockade enhanced the HBV-specific CD8 T cell response only in patients with lower exhaustion levels, while response to PD-L1 blockade was abrogated in patients with higher frequencies of exhausted HBV-specific CD8 T cells. Conclusion: Higher levels of functionally exhausted HBV-specific CD8 T cells are associated with a lack of response that cannot be restored by blocking the PD-1:PD-L1 axis. This suggests that the clinical effectiveness of blocking the PD-1:PD-L1 axis as a monotherapy may be restricted. Combination strategies, potentially including the combination of anti-LAG-3 with other anti-iR antibodies, will likely be required to elicit a functional cure for patients with high levels of functionally exhausted HBV-specific CD8 T cells.

Evaluation of Newcastle disease virus mediated dendritic cell activation and cross-priming tumor-specific immune responses ex vivo.

We have developed an oncolytic Newcastle disease virus (NDV) that has potent in vitro and in vivo anti-tumor activities and attenuated pathogenicity in chickens. In this ex vivo study using the same recombinant NDV backbone with GFP transgene (NDV-GFP, designated as rNDV), we found that rNDV induces maturation of monocyte-derived immature dendritic cells (iDCs) by both direct and indirect mechanisms, which promote development of antigen-specific T cell responses. Addition of rNDV directly to iDCs culture induced DC maturation, as demonstrated by the increased expression of costimulatory and antigen-presenting molecules as well as the production of type I interferons (IFNs). rNDV infection of the HER-2 positive human breast cancer cell line (SKBR3) resulted in apoptotic cell death, release of proinflammatory cytokines, and danger-associated molecular pattern molecules (DAMPs) including high-mobility group protein B1 (HMGB1) and heat shock protein 70 (HSP70). Addition of rNDV-infected SKBR3 cells to iDC culture resulted in greatly enhanced upregulation of the maturation markers and release of type I IFNs by DCs than rNDV-infected DCs only. When co-cultured with autologous T cells, DCs pre-treated with rNDV-infected SKBR3 cells cross-primed T cells in an antigen-specific manner. Altogether, our data strongly support the potential of oncolytic NDV as efficient therapeutic agent for cancer treatment.

Vaccination with synthetic long peptide formulated with CpG in an oil-in-water emulsion induces robust E7-specific CD8 T cell responses and TC-1 tumor eradication.

BACKGROUND: Despite considerable efforts at developing therapeutic vaccines for cancer, clinical translation of preclinical successes has been challenging, largely due to the difficulty of inducing strong and sustained cytotoxic T lymphocyte (CTL) responses in patients. Several peptide-based cancer vaccines have failed to show sustainable tumor regression in the clinic, possibly because of a lack of optimization of both the adjuvant and antigen components of the preparations. Here, we aimed to develop and optimize a vaccine format utilizing a synthetic long peptide (SLP) containing the human papilloma virus 16 (HPV16) E7 antigen, with a centrally located defined MHC class I epitope, and evaluate its immunogenicity and efficacy in combination with various adjuvant formulations. METHODS: E731-73 SLP was tested alone or in combination with toll-like receptor (TLR)3, TLR4, TLR7/8 and TLR9 agonists and formulated in oil-in-water (o/w) or water-in-oil (w/o) emulsions to determine a vaccine format inducing a robust CD8 T cell response in murine models. Once a lead vaccine format was determined, we examined its ability to inhibit tumor growth in the murine TC-1 model that expresses HPV16 E7 antigen. RESULTS: We identified the TLR9 agonist CpG formulated in a squalene-based o/w emulsion as the most potent adjuvant, inducing the expansion of multifunctional antigen specific CD8 T cells with cytolytic potential. We also demonstrated that SLP E731-73 + CpG + o/w emulsion vaccine can provide prophylactic and more importantly, therapeutic benefit in the TC-1 murine tumor model. CONCLUSIONS: Our results demonstrate that the novel vaccine format E7 SLP + CpG delivered in an o/w emulsion holds potential for the promotion of strong CTL responses and tumor eradication and encourages further development of peptide/adjuvant vaccines in cancer immunotherapy strategies.

HBeAg seroconversion is associated with a more effective PD-L1 blockade during chronic hepatitis B infection.

BACKGROUND & AIMS: Current therapies for chronic hepatitis B virus (HBV) infection control viral replication but do not eliminate the risk of progression to hepatocellular carcinoma. HBV-specific CD8 T cells are necessary for viral control, but they are rare and exhausted during chronic infection. Preclinical studies have shown that blockade of the PD-1:PD-L1 axis can restore HBV-specific T cell functionality. The aim of this study was to analyze how the clinical and treatment status of patients impacts the ability of HBV-specific T cells to respond to PD-L1 blockade. METHODS: Expression patterns of the PD-1:PD-L1/PD-L2 axis were analyzed in healthy donors and chronically infected patients in different clinical phases of disease. A functional assay was performed to quantify baseline HBV-specific T cell responses in chronically infected patients. Baseline responses were then compared to those attained in the presence of an anti-PD-L1 monoclonal antibody (MEDI2790). RESULTS: Chronically infected patients were characterized by the upregulation of PD-1 within the T cell compartment and a concomitant upregulation of PD-L1 on myeloid dendritic cells. The upregulation was maximal in HBV e antigen (HBeAg)-positive patients but persisted after HBeAg negativization and was not restored by long-term treatment. HBV reactivity, measured as frequency of HBV-specific T cells, was significantly higher in HBeAg-negative patients with lower HBV DNA levels, independently of HBV surface antigen or alanine aminotransferase levels. Anti-PD-L1 blockade with MEDI2790 increased both the number of IFN-γ-producing T cells and the amount of IFN-γ produced per cell in 97% of patients with detectable HBV reactivity, independently of patients' clinical or treatment status. CONCLUSION: Patients with lower levels of HBV DNA and the absence of HBeAg have more intact HBV-specific T cell immunity and may benefit the most from PD-L1 blockade as a monotherapy. LAY SUMMARY: Hepatitis B virus (HBV)-specific T cell responses during chronic infection are weak due to the upregulation of inhibitor molecules on the immune cells. In this study we show that the inhibitory PD-1:PD-L1 axis is upregulated during chronic HBV infection and successful antiretroviral therapy does not restore normal levels of PD-1 and PD-L1 expression. However, in HBV e antigen-negative patients, treatment with an anti-PD-L1 antibody can increase the functionality of HBV-specific T cell responses by an average of 2-fold and is a promising new therapy for patients with chronic HBV infection.

GITR ligand fusion protein agonist enhances the tumor antigen-specific CD8 T-cell response and leads to long-lasting memory.

BACKGROUND: The expansion of antigen-specific CD8 T cells is important in generating an effective and long-lasting immune response to tumors and viruses. Glucocorticoid-induced tumor necrosis factor receptor family-related receptor (GITR) is a co-stimulatory receptor that binds the GITR ligand (GITRL). Agonism of GITR can produce important signals that drive expansion of effector T cell populations. METHODS: We explored two separate murine tumor models, CT26 and TC-1, for responsiveness to GITR Ligand Fusion Protein(GITRL-FP) monotherapy. In TC-1, GITRL-FP was also combined with concurrent administration of an E7-SLP vaccine. We evaluated tumor growth inhibition by tumor volume measurements as well as changes in CD8 T cell populations and function including cytokine production using flow cytometry. Additionally, we interrogated how these therapies resulted in tumor antigen-specific responses using MHC-I dextramer staining and antigen-specific restimulations. RESULTS: In this study, we demonstrate that a GITR ligand fusion protein (GITRL-FP) is an effective modulator of antigen-specific CD8 T cells. In a CT26 mouse tumor model, GITRL-FP promoted expansion of antigen-specific T cells, depletion of regulatory T cells (Tregs), and generation of long-lasting CD8 T cell memory. This memory expansion was dependent on the dose of GITRL-FP and resulted in complete tumor clearance and protection from tumor rechallenge. In contrast, in TC-1 tumor-bearing mice, GITRL-FP monotherapy could not prime an antigen-specific CD8 T cell response and was unable to deplete Tregs. However, when combined with a vaccine targeting E7, treatment with GITRL-FP resulted in an augmentation of the vaccine-induced antigen-specific CD8 T cells, the depletion of Tregs, and a potent antitumor immune response. In both model systems, GITR levels on antigen-specific CD8 T cells were higher than on all other CD8 T cells, and GITRL-FP interacted directly with primed antigen-specific CD8 T cells. CONCLUSIONS: When taken together, our results demonstrate that the delivery of GITRL-FP as a therapeutic can promote anti-tumor responses in the presence of tumor-specific CD8 T cells. These findings support further study into combination partners for GITRL-FP that may augment CD8 T-cell priming as well as provide hypotheses that can be tested in human clinical trials exploring GITR agonists including GITRL-FP.

A novel HSV-2 subunit vaccine induces GLA-dependent CD4 and CD8 T cell responses and protective immunity in mice and guinea pigs.

BACKGROUND/OBJECTIVES: There is currently no licensed prophylactic or therapeutic vaccine for HSV-2 infection. METHODS: We developed a novel preclinical vaccine candidate, G103, consisting of three recombinantly expressed HSV-2 proteins (gD and the UL19 and UL25 gene products) adjuvanted with the potent synthetic TLR4 agonist glucopyranosyl lipid A (GLA) formulated in stable emulsion. The vaccine was tested for immunogenicity and efficacy in pre-clinical models for preventative and therapeutic vaccination. RESULTS: Vaccination of mice with G103 elicited antigen-specific binding and neutralizing antibody responses, as well as robust CD4 and CD8 effector and memory T cells. The T cell responses were further boosted by subsequent challenge with live virus. Prophylactic immunization completely protected against lethal intravaginal HSV-2 infection in mice, with only transient replication of virus in the genital mucosa and sterilizing immunity in dorsal root ganglia. Supporting the use of G103 therapeutically, the vaccine expanded both CD4 and CD8 T cells induced in mice by previous infection with HSV-2. In the guinea pig model of recurrent HSV-2 infection, therapeutic immunization with G103 was approximately 50% effective in reducing the number of lesions per animal as well as the overall lesions score. CONCLUSIONS: Taken together, the data show that G103 is a viable candidate for development of a novel prophylactic and therapeutic HSV-2 vaccine.

Development of a replication-competent lentivirus assay for dendritic cell-targeting lentiviral vectors.

It is a current regulatory requirement to demonstrate absence of detectable replication-competent lentivirus (RCL) in lentiviral vector products prior to use in clinical trials. Immune Design previously described an HIV-1-based integration-deficient lentiviral vector for use in cancer immunotherapy (VP02). VP02 is enveloped with E1001, a modified Sindbis virus glycoprotein which targets dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) expressed on dendritic cells in vivo. Vector enveloped with E1001 does not transduce T-cell lines used in standard HIV-1-based RCL assays, making current RCL testing formats unsuitable for testing VP02. We therefore developed a novel assay to test for RCL in clinical lots of VP02. This assay, which utilizes a murine leukemia positive control virus and a 293F cell line expressing the E1001 receptor DC-SIGN, meets a series of evaluation criteria defined in collaboration with US regulatory authorities and demonstrates the ability of the assay format to amplify and detect a hypothetical RCL derived from VP02 vector components. This assay was qualified and used to test six independent GMP production lots of VP02, in which no RCL was detected. We propose that the evaluation criteria used to rationally design this novel method should be considered when developing an RCL assay for any lentiviral vector.

Virological and preclinical characterization of a dendritic cell targeting, integration-deficient lentiviral vector for cancer immunotherapy.

Dendritic cells (DCs) are essential antigen-presenting cells for the initiation of cytotoxic T-cell responses and therefore attractive targets for cancer immunotherapy. We have developed an integration-deficient lentiviral vector termed ID-VP02 that is designed to deliver antigen-encoding nucleic acids selectively to human DCs in vivo. ID-VP02 utilizes a genetically and glycobiologically engineered Sindbis virus glycoprotein to target human DCs through the C-type lectin DC-SIGN (CD209) and also binds to the homologue murine receptor SIGNR1. Specificity of ID-VP02 for antigen-presenting cells in the mouse was confirmed through biodistribution studies showing that following subcutaneous administration, transgene expression was only detectable at the injection site and the draining lymph node. A single immunization with ID-VP02 induced a high level of antigen-specific, polyfunctional effector and memory CD8 T-cell responses that fully protected against vaccinia virus challenge. Upon homologous readministration, ID-VP02 induced a level of high-quality secondary effector and memory cells characterized by stable polyfunctionality and expression of IL-7Rα. Importantly, a single injection of ID-VP02 also induced robust cytotoxic responses against an endogenous rejection antigen of CT26 colon carcinoma cells and conferred both prophylactic and therapeutic antitumor efficacy. ID-VP02 is the first lentiviral vector which combines integration deficiency with DC targeting and is currently being investigated in a phase I trial in cancer patients.

Design of a novel integration-deficient lentivector technology that incorporates genetic and posttranslational elements to target human dendritic cells.

As sentinels of the immune system, dendritic cells (DCs) play an essential role in regulating cellular immune responses. One of the main challenges of developing DC-targeted therapies includes the delivery of antigen to DCs in order to promote the activation of antigen-specific effector CD8 T cells. With the goal of creating antigen-directed immunotherapeutics that can be safely administered directly to patients, Immune Design has developed a platform of novel integration-deficient lentiviral vectors that target and deliver antigen-encoding nucleic acids to human DCs. This platform, termed ID-VP02, utilizes a novel genetic variant of a Sindbis virus envelope glycoprotein with posttranslational carbohydrate modifications in combination with Vpx, a SIVmac viral accessory protein, to achieve efficient targeting and transduction of human DCs. In addition, ID-VP02 incorporates safety features in its design that include two redundant mechanisms to render ID-VP02 integration-deficient. Here, we describe the characteristics that allow ID-VP02 to specifically transduce human DCs, and the advances that ID-VP02 brings to conventional third-generation lentiviral vector design as well as demonstrate upstream production yields that will enable manufacturing feasibility studies to be conducted.

Identification of a small molecule with activity against drug-resistant and persistent tuberculosis.

A cell-based phenotypic screen for inhibitors of biofilm formation in mycobacteria identified the small molecule TCA1, which has bactericidal activity against both drug-susceptible and -resistant Mycobacterium tuberculosis (Mtb) and sterilizes Mtb in vitro combined with rifampicin or isoniazid. In addition, TCA1 has bactericidal activity against nonreplicating Mtb in vitro and is efficacious in acute and chronic Mtb infection mouse models both alone and combined with rifampicin or isoniazid. Transcriptional analysis revealed that TCA1 down-regulates genes known to be involved in Mtb persistence. Genetic and affinity-based methods identified decaprenyl-phosphoryl-ß-D-ribofuranose oxidoreductase DprE1 and MoeW, enzymes involved in cell wall and molybdenum cofactor biosynthesis, respectively, as targets responsible for the activity of TCA1. These in vitro and in vivo results indicate that this compound functions by a unique mechanism and suggest that TCA1 may lead to the development of a class of antituberculosis agents.

A Rev-Independent gag/pol Eliminates Detectable psi-gag Recombination in Lentiviral Vectors.

Lentiviral vectors (LVs) are being developed for clinical use in humans for applications including gene therapy and immunotherapy. A safety concern for use of LVs in humans is the generation of replication-competent lentivirus (RCL), which may arise due to recombination between the split genomes of third-generation LVs. Although no RCL has been detected to date, design optimizations that minimize recombination events between split genome vectors would provide an added safety benefit that may further reduce the risk of RCL formation. Here we describe design elements introduced to the gag/pol plasmid with the intention of eliminating psi-gag recombination between the vector genome and gag/pol. These design changes, consisting of codon optimization of the gag/pol sequence and the deletion of the Rev-responsive element, abrogate the requirement for Rev in expression of Gag protein, thus the resulting gag/pol construct being Rev independent (RI gag/pol). We show that generating vector using the RI gag/pol construct has no effect on particle production or transduction titers. The RI and wild-type gag/pol vectors function equivalently as antigen-specific immunotherapy, potently inducing antigen-specific CD8 T cells that protect against challenge with vaccinia virus. Most importantly, the designed RI gag/pol eliminated detectable psi-gag recombination. Interestingly, we detected recombination between the vector genome and gag/pol from regions without sequence homology. Our findings imply that although unpredictable recombination events may still occur, the RI gag/pol design is sufficient to prevent psi-gag recombination.

Differential responses of immune cells to type I interferon contribute to host resistance to viral infection.

Type I interferons (IFNs) are central to antiviral defense, but how they orchestrate immune cell function is incompletely understood. We determined that IFNs produced during murine cytomegalovirus (MCMV) infection differentially affect dendritic cells (DCs) and natural killer (NK) cells. IFNs induce cell-intrinsic responses in DCs, activating antiproliferative, antiviral, and lymphocyte-activating gene networks, consistent with high activity of the transcription factor STAT1 in these cells. By comparison, NK cells exhibit lower STAT1 expression and reduced IFN responsiveness. Rather, IFNs indirectly affect NK cells by inducing IL-15, which activates the transcription factor E2F and stimulates genes promoting cell expansion. IFN cell-intrinsic responses are necessary in DCs, but not NK cells, for MCMV resistance. Thus, sensitivity to IFN-induced cytokines and differences in IFN receptor signaling program immune cells to mount distinct responses that promote viral control.

Comparing the kinetics of NK cells, CD4, and CD8 T cells in murine cytomegalovirus infection.

NK cells recognize virus-infected cells with germline-encoded activating and inhibitory receptors that do not undergo genetic recombination or mutation. Accordingly, NK cells are often considered part of the innate immune response. The innate response comprises rapid early defenders that do not form immune memory. However, there is increasing evidence that experienced NK cells provide increased protection to secondary infection, a hallmark of the adaptive response. In this study, we compare the dynamics of the innate and adaptive immune responses by examining the kinetic profiles of the NK and T cell response to murine CMV infection. We find that, unexpectedly, the kinetics of NK cell proliferation is neither earlier nor faster than the CD4 or CD8 T cell response. Furthermore, early NK cell contraction after the peak of the response is slower than that of T cells. Finally, unlike T cells, experienced NK cells do not experience biphasic decay after the response peak, a trait associated with memory formation. Rather, NK cell contraction is continuous, constant, and returns to below endogenous preinfection levels. This indicates that the reason why Ag-experienced NK cells remain detectable for a prolonged period after adoptive transfer and infection is in part due to the high precursor frequency, slow decay rate, and low background levels of Ly49H(+) NK cells in recipient DAP12-deficient mice. Thus, the quantitative contribution of Ag-experienced NK cells in an endogenous secondary response, with higher background levels of Ly49H(+) NK cells, may be not be as robust as the secondary response observed in T cells.

Genetic labeling reveals altered turnover and stability of innate lymphocytes in latent mouse cytomegalovirus infection.

Mouse CMV (MCMV) infection rapidly induces the proliferation of NK cells, which correlates with immunological protection. Whether NK cells primed during acute response against MCMV are maintained for the long term is not known. In this study, we used TcrdH2BeGFP mice in which maturing NK cells are genetically labeled with a pulse of very stable histone-2B-eGFP. In this system, we found that the reporter protein was diluted out upon NK cell division during acute MCMV infection. At the same time, mature NK cells in uninfected mice showed only very limited turnover in vivo. Three months after primary infection when MCMV latency was established, the majority of peripheral NK cells still displayed a higher record of proliferation than NK cells in mock-infected controls. This observation included both Ly49H(+) and Ly49H(-) NK cells. Conversely, naive NK cells did not show more proliferation after transfer into latently MCMV-infected mice than that after transfer into mock-infected control mice. This indicated that the observed alterations of the NK cell compartment in MCMV latency were "legacy" (i.e., resulting from prior events during the initial immune response). Together, these results suggest that antiviral immune responses induce sustained alterations of innate lymphocyte populations that extend far beyond the first days of acute infection.

Natural killer cells in immunodefense against infective agents.

Following the discovery of innate immune receptors, the topics of innate immunity and its role in defense against infective agents have recently blossomed into very active research fields, after several decades of neglect. Among innate immune cells, natural killer (NK) cells are endowed with the unique ability to recognize and kill cells infected with a variety of pathogens, irrespective of prior sensitization to these microbes. NK cells have a number of other functions, including cytokine production and immunoregulatory activities. Major advances have recently been made in the understanding of the role of NK cells in the physiopathology of infectious diseases. The cellular and molecular mechanisms regulating the acquisition of effector functions by NK cells and their triggering upon pathogenic encounters are being unraveled. The possibility that the power of NK cells could be harnessed for the design of innovative treatments against infections is a major incentive for biologists to further explore NK cell subset complexity and to identify the ligands that activate NK cell receptors.

Cutting edge: Overlapping functions of TLR7 and TLR9 for innate defense against a herpesvirus infection.

As initially demonstrated with murine cytomegalovirus (MCMV), plasmacytoid dendritic cells (pDCs) are the major source of IFN-alpha/beta in response to a variety of viruses in vivo. However, contradictory results have been obtained pertaining to the mechanisms promoting IFN-alpha/beta production by pDCs in response to MCMV. In this study we show that TLR7 and TLR9 exert redundant functions for IFN-alpha/beta, IL-12p40, and TNF-alpha production by pDCs in vivo during MCMV infection. In contrast, we confirm that systemic production of IL-12p70 strictly depends on TLR9. The combined loss of TLR7 and TLR9 recapitulates critical features of the phenotype of MyD88-deficient mice, including a dramatic decrease in systemic IFN-alpha/beta levels, an increase in viral load, and increased susceptibility to MCMV-induced mortality. This is the first demonstration of the implication of TLR7 in the recognition of a DNA virus.

Novel insights into the relationships between dendritic cell subsets in human and mouse revealed by genome-wide expression profiling.

BACKGROUND: Dendritic cells (DCs) are a complex group of cells that play a critical role in vertebrate immunity. Lymph-node resident DCs (LN-DCs) are subdivided into conventional DC (cDC) subsets (CD11b and CD8alpha in mouse; BDCA1 and BDCA3 in human) and plasmacytoid DCs (pDCs). It is currently unclear if these various DC populations belong to a unique hematopoietic lineage and if the subsets identified in the mouse and human systems are evolutionary homologs. To gain novel insights into these questions, we sought conserved genetic signatures for LN-DCs and in vitro derived granulocyte-macrophage colony stimulating factor (GM-CSF) DCs through the analysis of a compendium of genome-wide expression profiles of mouse or human leukocytes. RESULTS: We show through clustering analysis that all LN-DC subsets form a distinct branch within the leukocyte family tree, and reveal a transcriptomal signature evolutionarily conserved in all LN-DC subsets. Moreover, we identify a large gene expression program shared between mouse and human pDCs, and smaller conserved profiles shared between mouse and human LN-cDC subsets. Importantly, most of these genes have not been previously associated with DC function and many have unknown functions. Finally, we use compendium analysis to re-evaluate the classification of interferon-producing killer DCs, lin-CD16+HLA-DR+ cells and in vitro derived GM-CSF DCs, and show that these cells are more closely linked to natural killer and myeloid cells, respectively. CONCLUSION: Our study provides a unique database resource for future investigation of the evolutionarily conserved molecular pathways governing the ontogeny and functions of leukocyte subsets, especially DCs.