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Summary: Humanized mice, defined as mice with human immune systems, have become an emerging model to study human hematopoiesis, infectious disease, and cancer. Here, we describe the techniques to generate humanized NSGF6 mice using adult human CD34+ hematopoietic stem and progenitor cells (HSPCs). We describe steps for constructing and monitoring the engraftment of humanized mice. We then detail procedures for tissue processing and immunophenotyping by flow cytometry to evaluate the multilineage hematopoietic differentiation.For complete details on the use and execution of this protocol, please refer to Yu et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract Infants necessitate vaccinations to prevent life-threatening infections. Our understanding of the infant immune responses to routine vaccines remains limited. We analyzed two cohorts of 2-month-old infants before vaccination, one week, and one-month post-vaccination. We report remarkable heterogeneity but limited antibody responses to the different antigens. Whole-blood transcriptome analysis in an initial cohort showed marked overexpression of interferon-stimulated genes (ISGs) and to a lesser extent of inflammation-genes at day 7, which normalized one month post-vaccination. Single-cell RNA sequencing in peripheral blood mononuclear cells from a second cohort identified at baseline a predominantly naive immune landscape including ISGhi cells. On day 7, increased expression of interferon-, inflammation-, and cytotoxicity-related genes were observed in most immune cells, that reverted one month post-vaccination, when a CD8+ ISGhi and cytotoxic cluster and B cells expanded. Antibody responses were associated with baseline frequencies of plasma cells, B-cells, and monocytes, and induction of ISGs at day 7.

Summary: Pre-clinical use of humanized mice transplanted with CD34+ hematopoietic stem and progenitor cells (HSPCs) is limited by insufficient engraftment with adult non-mobilized HSPCs. Here, we developed a novel immunodeficient mice based on NOD-SCID-Il2γc−/− (NSG) mice to support long-term engraftment with human adult HSPCs. As both Flt3L and IL-6 are critical for many aspects of hematopoiesis, we knock-out mouse Flt3 and knock-in human IL6 gene. The resulting mice showed an increase in the availability of mouse Flt3L to human cells and a dose-dependent production of human IL-6 upon activation. Upon transplantation with low number of human HSPCs from adult bone marrow, these humanized mice demonstrated a significantly higher engraftment with multilineage differentiation of human lymphoid and myeloid cells, and tissue colonization at one year after adult HSPC transplant. Thus, these mice enable studies of human hematopoiesis and tissue colonization over time and may facilitate building autologous models for immuno-oncology studies.

Abstract Detecting multiplets in single nucleus (sn)ATAC-seq data is challenging due to data sparsity and limited dynamic range. AMULET (ATAC-seq MULtiplet Estimation Tool) enumerates regions with greater than two uniquely aligned reads across the genome to effectively detect multiplets. We evaluate the method by generating snATAC-seq data in the human blood and pancreatic islet samples. AMULET has high precision, estimated via donor-based multiplexing, and high recall, estimated via simulated multiplets, compared to alternatives and identifies multiplets most effectively when a certain read depth of 25K median valid reads per nucleus is achieved.

The blood transcriptome of human subjects can be profiled on an almost routine basis in translational research settings. Here the authors show that a fixed and well-characterized repertoire of transcriptional modules can be employed as a reusable framework for the analysis, visualization and interpretation of such data

Ov-ASP-1 (rASP-1), a parasite-derived protein secreted by the helminth Onchocerca volvulus, is an adjuvant which enhances the potency of the influenza trivalent vaccine (IIV3), even when used with 40-fold less IIV3. This study is aimed to provide a deeper insight into the molecular networks that underline the adjuvanticity of rASP-1. Here we show that rASP-1 stimulates mouse CD11c+ bone marrow-derived dendritic (BMDCs) to secrete elevated levels of IL-12p40, TNF-α, IP-10 and IFN-β in a TRIF-dependent but MyD88-independent manner. rASP-1-activated BMDCs promoted the differentiation of naïve CD4+ T cells into Th1 cells (IFN-γ+) that was TRIF- and type I interferon receptor (IFNAR)-dependent, and into Tfh-like cells (IL21+) and Tfh1 (IFN-γ+ IL21+) that were TRIF-, MyD88- and IFNAR-dependent. rASP-1-activated BMDCs promoted the differentiation of naïve CD4+ T cells into Th17 (IL-17+) cells only when the MyD88 pathway was inhibited. Importantly, rASP-1-activated human blood cDCs expressed upregulated genes that are associated with DC maturation, type I IFN and type II IFN signaling, as well as TLR4-TRIF dependent signaling. These activated cDCs promoted the differentiation of naïve human CD4+ T cells into Th1, Tfh-like and Th17 cells. Our data thus confirms that the rASP-1 is a potent innate adjuvant that polarizes the adaptive T cell responses to Th1/Tfh1 in both mouse and human DCs. Notably, the rASP-1-adjuvanted IIV3 vaccine elicited protection of mice from a lethal H1N1 infection that is also dependent on the TLR4-TRIF axis and IFNAR signaling pathway, as well as on its ability to induce anti-IIV3 antibody production.

Abstract While Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is disrupting lives across the globe for everyone, it has a more devastating impact on the health of older adults, especially that of older men. This pandemic has highlighted the crucial importance of considering an individual’s age and biological sex in the clinic in addition to other confounding diseases (Kuchel, G.A, J Am Geriatr Soc, 67, 203, 2019, Tannenbaum, C., Nature, 575 451-458, 2009) As an interdisciplinary team of scientists in immunology, hematology, genomics, bioinformatics, and geriatrics, we have been studying how age and sex shape the human immune system. Herein we reflect on how our recent findings on the alterations of the immune system in aging might contribute to our current understanding of COVID-19 infection rate and disease risk.

Whether the immune system aging differs between men and women is barely known. Here the authors characterize gene expression, chromatin state and immune subset composition in the blood of healthy humans 22 to 93 years of age, uncovering shared as well as sex-unique alterations, and create a web resource to interactively explore the data.

Immune cell activation assays have been widely used for immune monitoring and for understanding disease mechanisms. However, these assays are typically limited in scope. A holistic study of circulating immune cell responses to different activators is lacking. Here we developed a cost-effective high-throughput multiplexed single-cell RNA-seq combined with epitope tagging (CITE-seq) to determine how classic activators of T cells (anti-CD3 coupled with anti-CD28) or monocytes (LPS) alter the cell composition and transcriptional profiles of peripheral blood mononuclear cells (PBMCs) from healthy human donors. Anti-CD3/CD28 treatment activated all classes of lymphocytes either directly (T cells) or indirectly (B and NK cells) but reduced monocyte numbers. Activated T and NK cells expressed senescence and effector molecules, whereas activated B cells transcriptionally resembled autoimmune disease- or age-associated B cells (e.g., CD11c, T-bet). In contrast, LPS specifically targeted monocytes and induced two main states: early activation characterized by the expression of chemoattractants and a later pro-inflammatory state characterized by expression of effector molecules. These data provide a foundation for future immune activation studies with single cell technologies (https://czi-pbmc-cite-seq.jax.org/).

Identification and characterization of CD8+ and CD4+ T-cell epitopes elicited by HIV therapeutic vaccination is key for elucidating the nature of protective cellular responses and mechanism of the immune evasion of HIV. Here, we report the characterization of HIV-specific T-cell responses in cART (combination antiretroviral therapy) treated HIV-1 infected patients after vaccination with ex vivo-generated IFNα Dendritic Cells (DCs) loaded with LIPO-5 (HIV-1 Nef 66-97, Nef 116-145, Gag 17-35, Gag 253-284 and Pol 325-355 lipopeptides). Vaccination induced and/or expanded HIV-specific CD8+ T cells producing IFNγ, perforin, granzyme A and granzyme B, and also CD4+ T cells secreting IFNγ, IL-2 and IL-13. These responses were directed against dominant and subdominant epitopes representing all vaccine regions; Gag, Pol and Nef. Interestingly, IL-2 and IL-13 produced by CD4+ T cells were negatively correlated with the peak of viral replication following analytic treatment interruption (ATI). Epitope mapping confirmed that vaccination elicited responses against predicted T-cell epitopes, but also allowed to identify a set of 8 new HIV-1 HLA-DR-restricted CD4+ T-cell epitopes. These results may help to better design future DC therapeutic vaccines and underscore the role of vaccine-elicited CD4+ T-cell responses to achieve control of HIV replication.

The goal of HIV therapeutic vaccination is to induce HIV-specific immune response able to control HIV replication. We previously reported that vaccination with ex vivo generated Dendritic Cells (DC) loaded with HIV-lipopeptides in HIV-infected patients (n = 19) on antiretroviral therapy (ART) was well-tolerated and immunogenic. Vaccine-elicited HIV-specific T cell responses were associated with improved control of viral replication following antiretroviral interruption (ATI from w24 to w48). We show an inverse relationship between HIV-specific responses (production of IL-2, IL-13, IL-21, IFN-g, CD4 polyfunctionality, i.e., production of at least two cytokines) and the peak of viral load during ATI. Here we have performed an integrative systems vaccinology analysis including: (i) post vaccination (w16) immune responses assessed by cytometry, cytokine secretion, and Interferon-γ ELISPOT assays; (ii) whole blood and cellular gene expression measured during vaccination; and (iii) viral parameters following ATI, with the objective to disentangle the relationships between these markers and to identify vaccine signatures. During vaccination, 69 gene expression modules out of 260 varied significantly including (by order of significance) modules related to inflammation (Chaussabel Modules M3.2, M4.13, M4.6, M5.7, M7.1, M4.2), plasma cells (M4.11) and T cells (M4.1, 4.15). Cellular immune responses were positively correlated to genes belonging to T cell functional modules (M4.1, M4.15) at w16 and negatively correlated to genes belonging to inflammation modules (M7.1, M5.7, M3.2, M4.13, M4.2). More specifically, we show that prolonged increased abundance of inflammatory gene pathways related to toll-like receptor signaling (especially TLR4) are associated with both lower vaccine immune responses and control of viral replication post ATI. Further comparison of DC vaccine gene signatures with previously reported non-HIV vaccine signatures, such as flu and pneumococcal vaccines, revealed common pathways across vaccines. Overall, these results show that too long duration and too high intensity of vaccine inflammatory responses hamper the magnitude of effector responses.

Globally, diarrheal diseases are a leading cause of death in children under five and disproportionately affect children in developing countries. Children who contract diarrheal diseases are rarely screened to identify the etiologic agent due to time and cost considerations associated with pathogen-specific screening and hence pathogen-directed therapy is uncommon. The development of biomarkers to rapidly identify underlying pathogens could improve treatment options and clinical outcomes in childhood diarrheal diseases. Here, we perform RNA sequencing on blood samples collected from children evaluated in an emergency room setting with diarrheal disease where the pathogen(s) present are known. We determine host response gene signatures specific to Salmonella, Shigella and rotavirus, but not E. coli, infections that distinguish them from each other and from healthy controls. Specifically, we observed differential expression of genes related to chemokine receptors or inflammasome signaling in Shigella cases, such as CCR3, CXCR8, and NLRC4, and interferon response genes, such as IFI44 and OASL, in rotavirus cases. Our findings add insight into the host peripheral immune response to these pathogens, and suggest strategies and limitations for the use host response transcript signatures for diagnosing the etiologic agent of childhood diarrheal diseases.

Sipuleucel-T is an autologous cellular immunotherapy approved in the US for patients with asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer (mCRPC). This significant advance for mCRPC treatment provides healthcare professionals with another effective therapy to extend survival. As an immunotherapy, sipuleucel-T possesses specific characteristics differentiating it from traditional therapies. At a roundtable meeting of experts, sipuleucel-T data were discussed, focusing on interpretation and clinical implications. Important differences between immunotherapies and traditional therapies were explored, e.g., mode of action, outcomes, data consistency and robustness, timing of sipuleucel-T treatment, and future perspectives in areas such as short-term markers of long-term benefit.

Analysis of the mouse transcriptional response to Listeria monocytogenes infection reveals that a large set of genes are perturbed in both blood and tissue and that these transcriptional responses are enriched for pathways of the immune response. Further we identified enrichment for both type I and type II interferon (IFN) signaling molecules in the blood and tissues upon infection. Since type I IFN signaling has been reported widely to impair bacterial clearance we examined gene expression from blood and tissues of wild type (WT) and type I IFNαβ receptor-deficient (Ifnar1-/-) mice at the basal level and upon infection with L. monocytogenes. Measurement of the fold change response upon infection in the absence of type I IFN signaling demonstrated an upregulation of specific genes at day 1 post infection. A less marked reduction of the global gene expression signature in blood or tissues from infected Ifnar1-/- as compared to WT mice was observed at days 2 and 3 after infection, with marked reduction in key genes such as Oasg1 and Stat2. Moreover, on in depth analysis, changes in gene expression in uninfected mice of key IFN regulatory genes including Irf9, Irf7, Stat1 and others were identified, and although induced by an equivalent degree upon infection this resulted in significantly lower final gene expression levels upon infection of Ifnar1-/- mice. These data highlight how dysregulation of this network in the steady state and temporally upon infection may determine the outcome of this bacterial infection and how basal levels of type I IFN-inducible genes may perturb an optimal host immune response to control intracellular bacterial infections such as L. monocytogenes.

Mycobacterium tuberculosis infection is a leading cause of infectious death worldwide. Gene-expression microarray studies profiling the blood transcriptional response of tuberculosis (TB) patients have been undertaken in order to better understand the host immune response as well as to identify potential biomarkers of disease. To date most of these studies have focused on pulmonary TB patients with gene-expression profiles of extra-pulmonary TB patients yet to be compared to those of patients with pulmonary TB or sarcoidosis.A novel cohort of patients with extra-pulmonary TB and sarcoidosis was recruited and the transcriptional response of these patients compared to those with pulmonary TB using a variety of transcriptomic approaches including testing a previously defined 380 gene meta-signature of active TB.The 380 meta-signature broadly differentiated active TB from healthy controls in this new dataset consisting of pulmonary and extra-pulmonary TB. The top 15 genes from this meta-signature had a lower sensitivity for differentiating extra-pulmonary TB from healthy controls as compared to pulmonary TB. We found the blood transcriptional responses in pulmonary and extra-pulmonary TB to be heterogeneous and to reflect the extent of symptoms of disease.The transcriptional signature in extra-pulmonary TB demonstrated heterogeneity of gene expression reflective of symptom status, while the signature of pulmonary TB was distinct, based on a higher proportion of symptomatic individuals. These findings are of importance for the rational design and implementation of mRNA based TB diagnostics.

The role of regulatory T cells (Tregs) in vaccination has been poorly investigated. We have reported that vaccination with ex vivo-generated dendritic-cells (DC) loaded with HIV-lipopeptides (LIPO-5-DC vaccine) in HIV-infected patients was well tolerated and highly immunogenic. These responses and their relation to viral replication following analytical treatment interruption (ATI) were variable. Here, we investigated whether the presence of HIV-specific Tregs might explain these differences. Co-expression of CD25, CD134, CD39 and FoxP3 was used to delineate both antigen-specific Tregs and effectors T cells (Teffs). Median LIPO-5 specific-CD25+CD134+ polyfunctional T cells increased from 0.1% (IQR 0-0.3) before vaccination (week -4) to 2.1% (IQR 1.1-3.9) at week 16 following 4 immunizations (p=0.001) and were inversely correlated with maximum viral load following ATI (r=-0.77, p=0.001). Vaccinees who displayed lower levels of HIV-specific CD4+CD134+CD25+CD39+FoxP3+ Tregs responded better to the LIPO-5-DC vaccine. After vaccination, the frequency of HIV-specific Tregs decreased (from 69.3 at week -4 to 31.7% at week 16) and inversely correlated with HIV-specific IFN-γ-producing cells (r=-0.64, p=0.002). We show that therapeutic immunization skewed the HIV-specific response from regulatory to effector phenotype which impacts on the magnitude of viral replication following ATI.

Targeting dendritic cell-specific endocytic receptors using monoclonal antibodies fused to desired antigens is an approach widely used in vaccine development to enhance the poor immunogenicity of protein-based vaccines and to induce immune responses. Here, we engineered an anti-human DCIR recombinant antibody, which cross-reacts with the homologous cynomolgous macaque receptor and was fused via the heavy chain C-terminus to HIV Gagp24 protein (αDCIR.Gagp24). In vitro, αDCIR.Gagp24 expanded multifunctional antigen-specific memory CD4+ T cells recognizing multiple Gagp24 peptides from HIV-infected patient peripheral blood mononuclear cells. In non human primates, priming with αDCIR.Gagp24 without adjuvant elicited a strong anti-Gagp24 antibody response after the second immunization, while in the non-targeted HIV Gagp24 protein control groups the titers were weak. The presence of the double-stranded RNA poly(I:C) adjuvant significantly enhanced the anti-Gagp24 antibody response in all the groups and reduced the discrimination between the different vaccine groups. The avidity of the anti-Gagp24 antibody responses was similar with either αDCIR.Gagp24 or Gagp24 immunization, but increased from medium to high avidity in both groups when poly(I:C) was co-administered. This data provides a comparative analysis of DC-targeted and non-targeted proteins for their capacity to induce antigen-specific antibody responses in vivo. This study supports the further development of DCIR-based DC-targeting vaccines for protective durable antibody induction, especially in the absence of adjuvant.

The use of human whole blood for transcriptomic analysis has potential advantages over the use of isolated immune cells for studying the transcriptional response to pathogens and their products. Whole blood stimulation can be carried out in a laboratory without the expertise or equipment to isolate immune cells from blood, with the added advantage of being able to undertake experiments using very small volumes of blood. Toll like receptors (TLRs) are a family of pattern recognition receptors which recognise highly conserved microbial products. Using the TLR2 ligand (Pam3CSK4) and the TLR4 ligand (LPS), human whole blood was stimulated for 0, 1, 3, 6, 12 or 24 hours at which times mRNA was isolated and a comparative microarray was undertaken. A common NFκB transcriptional programme was identified following both TLR2 and TLR4 ligation which peaked at between 3 to 6 hours including upregulation of many of the NFκB family members. In contrast an interferon transcriptional response was observed following TLR4 but not TLR2 ligation as early as 1 hour post stimulation and peaking at 6 hours. These results recapitulate the findings observed in previously published studies using isolated murine and human myeloid cells indicating that in vitro stimulated human whole blood can be used to interrogate the early transcriptional kinetic response of innate cells to TLR ligands. Our study demonstrates that a transcriptomic analysis of mRNA isolated from human whole blood can delineate both the temporal response and the key transcriptional differences following TLR2 and TLR4 ligation.

BackgroundRespiratory syncytial virus (RSV) is the leading cause of viral lower respiratory tract infection (LRTI) and hospitalization in infants. Mostly because of the incomplete understanding of the disease pathogenesis, there is no licensed vaccine, and treatment remains symptomatic. We analyzed whole blood transcriptional profiles to characterize the global host immune response to acute RSV LRTI in infants, to characterize its specificity compared with influenza and human rhinovirus (HRV) LRTI, and to identify biomarkers that can objectively assess RSV disease severity.Methods and findingsThis was a prospective observational study over six respiratory seasons including a cohort of infants hospitalized with RSV (n = 135), HRV (n = 30), and influenza (n = 16) LRTI, and healthy age- and sex-matched controls (n = 39). A specific RSV transcriptional profile was identified in whole blood (training cohort, n = 45 infants; Dallas, Texas, US) and validated in three different cohorts (test cohort, n = 46, Dallas, Texas, US; validation cohort A, n = 16, Turku, Finland; validation cohort B, n = 28, Columbus, Ohio, US) with high sensitivity (94% [95% CI 87%-98%]) and specificity (98% [95% CI 88%-99%]). It classified infants with RSV LRTI versus HRV or influenza LRTI with 95% accuracy. The immune dysregulation induced by RSV (overexpression of neutrophil, inflammation, and interferon genes, and suppression of T and B cell genes) persisted beyond the acute disease, and immune dysregulation was greatly impaired in younger infants (ConclusionsBlood RNA profiles of infants with RSV LRTI allow specific diagnosis, better understanding of disease pathogenesis, and assessment of disease severity. This study opens new avenues for biomarker discovery and identification of potential therapeutic or preventive targets, and demonstrates that large microarray datasets can be translated into a biologically meaningful context and applied to the clinical setting. Please see later in the article for the Editors' Summary.

Determination of antigen-specific T cell repertoires in human blood has been a challenge. Here, we show a novel integrated approach that permits determination of multiple parameters of antigen-specific T cell repertoires. The approach consists of two assays: the Direct assay and the Cytokine-driven assay. Briefly, human PBMCs are first stimulated with overlapping peptides encoding a given antigen for 48 hours to measure cytokine secretion (Direct assay). Peptide-reactive T cells are further expanded by IL-2 for 5 days; and after overnight starvation, expanded cells are stimulated with the same peptides from the initial culture to analyze cytokine secretion (Cytokine-driven assay). We first applied this integrated approach to determine the type of islet-antigen-specific T cells in healthy adults. Out of ten donors, the Direct assay identified GAD65-specific CD4(+) T cells in three adults and zinc transporter 8 (ZnT8)-specific CD4(+) T cells in five adults. The intracytoplasmic cytokine staining assay showed that these islet-antigen-specific CD4(+) T cells belonged to the CD45RO(+) memory compartment. The Cytokine-driven assay further revealed that islet-antigen-specific CD4(+) T cells in healthy adults were capable of secreting various types of cytokines including type 1 and type 2 cytokines as well as IL-10. We next applied our integrated assay to determine whether the type of ZnT8-specific CD4(+) T cells is different between Type 1 diabetes patients and age/gender/HLA-matched healthy adults. We found that ZnT8-specific CD4(+) T cells were skewed towards Th1 cells in T1D patients, while Th2 and IL-10-producing cells were prevalent in healthy adults. In conclusion, the Direct assay and the Cytokine-driven assay complement each other, and the combination of the two assays provides information of antigen-specific T cell repertoires on the breadth, type, and avidity. This strategy is applicable to determine the differences in the quality of antigen-specific T cells between health and disease.

New approaches to define factors underlying the immunopathogenesis of pulmonary diseases including sarcoidosis and tuberculosis are needed to develop new treatments and biomarkers. Comparing the blood transcriptional response of tuberculosis to other similar pulmonary diseases will advance knowledge of disease pathways and help distinguish diseases with similar clinical presentations.To determine the factors underlying the immunopathogenesis of the granulomatous diseases, sarcoidosis and tuberculosis, by comparing the blood transcriptional responses in these and other pulmonary diseases.We compared whole blood genome-wide transcriptional profiles in pulmonary sarcoidosis, pulmonary tuberculosis, to community acquired pneumonia and primary lung cancer and healthy controls, before and after treatment, and in purified leucocyte populations.An Interferon-inducible neutrophil-driven blood transcriptional signature was present in both sarcoidosis and tuberculosis, with a higher abundance and expression in tuberculosis. Heterogeneity of the sarcoidosis signature correlated significantly with disease activity. Transcriptional profiles in pneumonia and lung cancer revealed an over-abundance of inflammatory transcripts. After successful treatment the transcriptional activity in tuberculosis and pneumonia patients was significantly reduced. However the glucocorticoid-responsive sarcoidosis patients showed a significant increase in transcriptional activity. 144-blood transcripts were able to distinguish tuberculosis from other lung diseases and controls.Tuberculosis and sarcoidosis revealed similar blood transcriptional profiles, dominated by interferon-inducible transcripts, while pneumonia and lung cancer showed distinct signatures, dominated by inflammatory genes. There were also significant differences between tuberculosis and sarcoidosis in the degree of their transcriptional activity, the heterogeneity of their profiles and their transcriptional response to treatment.

About half of all subjects with common variable immune deficiency (CVID) are afflicted with inflammatory complications including hematologic autoimmunity, granulomatous infiltrations, interstitial lung disease, lymphoid hyperplasia and/or gastrointestinal inflammatory disease. The pathogenesis of these conditions is poorly understood but singly and in aggregate, these lead to significantly increased (11 fold) morbidity and mortality, not experienced by CVID subjects without these complications. To explore the dysregulated networks in these subjects, we applied whole blood transcriptional profiling to 91 CVID subjects, 47 with inflammatory conditions and 44 without, in comparison to subjects with XLA and healthy controls. As compared to other CVID subjects, males with XLA or healthy controls, the signature of CVID subjects with inflammatory complications was distinguished by a marked up-regulation of IFN responsive genes. Chronic up-regulation of IFN pathways is known to occur in autoimmune disease due to activation of TLRs and other still unclarified cytoplasmic sensors. As subjects with inflammatory complications were also more likely to be lymphopenic, have reduced B cell numbers, and a greater reduction of B, T and plasma cell networks, we suggest that more impaired adaptive immunity in these subjects may lead to chronic activation of innate IFN pathways in response to environmental antigens. The unbiased use of whole blood transcriptome analysis may provides a tool for distinguishing CVID subjects who are at risk for increased morbidity and earlier mortality. As more effective therapeutic options are developed, whole blood transcriptome analyses could also provide an efficient means of monitoring the effects of treatment of the inflammatory phenotype.

Bacterial cyclic glucans are glucose polymers that concentrate within the periplasm of alpha-proteobacteria. These molecules are necessary to maintain the homeostasis of the cell envelope by contributing to the osmolarity of Gram negative bacteria. Here, we demonstrate that Brucella β 1,2 cyclic glucans are potent activators of human and mouse dendritic cells. Dendritic cells activation by Brucella β 1,2 cyclic glucans requires TLR4, MyD88 and TRIF, but not CD14. The Brucella cyclic glucans showed neither toxicity nor immunogenicity compared to LPS and triggered antigen-specific CD8(+) T cell responses in vivo. These cyclic glucans also enhanced antigen-specific CD4(+) and CD8(+) T cell responses including cross-presentation by different human DC subsets. Brucella β 1,2 cyclic glucans increased the memory CD4(+) T cell responses of blood mononuclear cells exposed to recombinant fusion proteins composed of anti-CD40 antibody and antigens from both hepatitis C virus and Mycobacterium tuberculosis. Thus cyclic glucans represent a new class of adjuvants, which might contribute to the development of effective antimicrobial therapies.

Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that persists lifelong in the host. In ∼4% of infected people, HTLV-1 causes a chronic disabling neuroinflammatory disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The pathogenesis of HAM/TSP is unknown and treatment remains ineffective. We used gene expression microarrays followed by flow cytometric and functional assays to investigate global changes in blood transcriptional profiles of HTLV-1-infected and seronegative individuals. We found that perturbations of the p53 signaling pathway were a hallmark of HTLV-1 infection. In contrast, a subset of interferon (IFN)-stimulated genes was over-expressed in patients with HAM/TSP but not in asymptomatic HTLV-1 carriers or patients with the clinically similar disease multiple sclerosis. The IFN-inducible signature was present in all circulating leukocytes and its intensity correlated with the clinical severity of HAM/TSP. Leukocytes from patients with HAM/TSP were primed to respond strongly to stimulation with exogenous IFN. However, while type I IFN suppressed expression of the HTLV-1 structural protein Gag it failed to suppress the highly immunogenic viral transcriptional transactivator Tax. We conclude that over-expression of a subset of IFN-stimulated genes in chronic HTLV-1 infection does not constitute an efficient host response but instead contributes to the development of HAM/TSP.

Staphylococcus aureus infections are associated with diverse clinical manifestations leading to significant morbidity and mortality. To define the role of the host response in the clinical manifestations of the disease, we characterized whole blood transcriptional profiles of children hospitalized with community-acquired S. aureus infection and phenotyped the bacterial strains isolated. The overall transcriptional response to S. aureus infection was characterized by over-expression of innate immunity and hematopoiesis related genes and under-expression of genes related to adaptive immunity. We assessed individual profiles using modular fingerprints combined with the molecular distance to health (MDTH), a numerical score of transcriptional perturbation as compared to healthy controls. We observed significant heterogeneity in the host signatures and MDTH, as they were influenced by the type of clinical presentation, the extent of bacterial dissemination, and time of blood sampling in the course of the infection, but not by the bacterial isolate. System analysis approaches provide a new understanding of disease pathogenesis and the relation/interaction between host response and clinical disease manifestations.

Globally there are approximately 9 million new active tuberculosis cases and 1.4 million deaths annually. Effective antituberculosis treatment monitoring is difficult as there are no existing biomarkers of poor adherence or inadequate treatment earlier than 2 months after treatment initiation. Inadequate treatment leads to worsening disease, disease transmission and drug resistance.To determine if blood transcriptional signatures change in response to antituberculosis treatment and could act as early biomarkers of a successful response.Blood transcriptional profiles of untreated active tuberculosis patients in South Africa were analysed before, during (2 weeks and 2 months), at the end of (6 months) and after (12 months) antituberculosis treatment, and compared to individuals with latent tuberculosis. An active-tuberculosis transcriptional signature and a specific treatment-response transcriptional signature were derived. The specific treatment response transcriptional signature was tested in two independent cohorts. Two quantitative scoring algorithms were applied to measure the changes in the transcriptional response. The most significantly represented pathways were determined using Ingenuity Pathway Analysis.An active tuberculosis 664-transcript signature and a treatment specific 320-transcript signature significantly diminished after 2 weeks of treatment in all cohorts, and continued to diminish until 6 months. The transcriptional response to treatment could be individually measured in each patient.Significant changes in the transcriptional signatures measured by blood tests were readily detectable just 2 weeks after treatment initiation. These findings suggest that blood transcriptional signatures could be used as early surrogate biomarkers of successful treatment response.

Staphylococcus aureus has emerged as a significant pathogen causing severe invasive disease in otherwise healthy people. Despite considerable advances in understanding the epidemiology, resistance mechanisms, and virulence factors produced by the bacteria, there is limited knowledge of the in vivo host immune response to acute, invasive S. aureus infections. Herein, we report that peripheral blood mononuclear cells from patients with severe S. aureus infections demonstrate a distinctive and robust gene expression profile which is validated in a distinct group of patients and on a different microarray platform. Application of a systems-wide modular analysis framework reveals significant over-expression of innate immunity genes and under-expression of genes related to adaptive immunity. Simultaneous flow cytometry analyses demonstrated marked alterations in immune cell numbers, with decreased central memory CD4 and CD8 T cells and increased numbers of monocytes. CD14+ monocyte numbers significantly correlated with the gene expression levels of genes related to the innate immune response. These results demonstrate the value of applying a systems biology approach that reveals the significant alterations in the components of circulating blood lymphocytes and monocytes in invasive S. aureus infections.