A Novel Case of IFNAR1 Deficiency Identified a Common Canonical Splice Site Variant in DOCK8 in Western Polynesia: The Importance of Validating Variants of Unknown Significance in Under-Represented Ancestries.

Advanced genomic technologies such as whole exome or whole genome sequencing have improved diagnoses and disease outcomes for individuals with genetic diseases. Yet, variants of unknown significance (VUS) require rigorous validation to establish disease causality or modification, or to exclude them from further analysis. Here, we describe a young individual of Polynesian ancestry who in the first 13 mo of life presented with SARS-CoV-2 pneumonia, severe enterovirus meningitis and adenovirus gastroenteritis, and severe adverse reaction to MMR vaccination. Genomic analysis identified a previously reported pathogenic homozygous variant in IFNAR1 (c.1156G > T, p.Glu386* LOF), which is common in Western Polynesia. Moreover, a new and putatively deleterious canonical splice site variant in DOCK8 was also found in homozygosity (c.3234 + 2T > C). This DOCK8 variant is common in Polynesians and other under-represented ancestries in large genomic databases. Despite in silico bioinformatic predictions, extensive in vitro and ex vivo analysis revealed the DOCK8 variant likely be neutral. Thus, our study reports a novel case of IFNAR1 deficiency, but also highlights the importance of functional validation of VUS, including those predicted to be deleterious, and the pressing need to expand our knowledge of the genomic architecture and landscape of under-represented populations and ancestries.

Low-level Plasmodium falciparum blood-stage infection causes dendritic cell apoptosis and dysfunction in healthy volunteers.

BACKGROUND: Dendritic cells (DCs) are highly specialized antigen-presenting cells that are crucial for initiation of immune responses. During naturally acquired malaria, DC number and function is reduced. METHODS: The timing of, parasitemia threshold of, and contribution of apoptosis to DC loss were prospectively evaluated in 10 men after experimental challenge with approximately 1800 Plasmodium falciparum-parasitized red blood cells (pRBCs) and after drug cure initiated at a parasite level of ≥ 1000 parasites/mL. RESULTS: The nadir levels of total, myeloid, and plasmacytoid DCs occurred 8 days after infection. DC loss was partially attributable to apoptosis, which was first detected on day 5 (median parasite level, 238 parasites/mL) and maximal at day 7. Remaining DCs exhibited a reduced ability to uptake particulate antigen. DC numbers recovered approximately 60 hours after antimalarial drug administration. There was no loss of DC number or function before or after drug cure in 5 men inoculated with <180 pRBCs and treated on day 6, when their parasite level was approximately 200 parasites/mL. CONCLUSIONS: Plasmodium causes DC loss in vivo, which is at least partially explained by apoptosis in response to blood-stage parasites. In primary infection, loss of DC number and function occurs early during the prepatent period and before or with onset of clinical symptoms. These findings may explain in part the inadequate development of immunity to blood-stage malaria infection.

Inborn Errors of Immunity in Children With Invasive Pneumococcal Disease: A Multicenter Prospective Study.

BACKGROUND: In settings with universal conjugate pneumococcal vaccination, invasive pneumococcal disease (IPD) can be a marker of an underlying inborn error of immunity. The aim of this study was to determine the prevalence and characterize the types of immunodeficiencies in children presenting with IPD. METHODS: Multicenter prospective audit following the introduction of routinely recommended immunological screening in children presenting with IPD. The minimum immunological evaluation comprised a full blood examination and film, serum immunoglobulins (IgG, IgA and IgM), complement levels and function. Included participants were children in whom Streptococcus pneumoniae was isolated from a normally sterile site (cerebrospinal fluid, pleura, peritoneum and synovium). If isolated from blood, features of sepsis needed to be present. Children with predisposing factors for IPD (nephrotic syndrome, anatomical defect or malignancy) were excluded. RESULTS: Overall, there were 379 episodes of IPD of which 313 (83%) were eligible for inclusion and 143/313 (46%) had an immunologic evaluation. Of these, 17/143 (12%) were diagnosed with a clinically significant abnormality: hypogammaglobulinemia (n = 4), IgA deficiency (n = 3), common variable immunodeficiency (n = 2), asplenia (n = 2), specific antibody deficiency (n = 2), incontinentia pigmenti with immunologic dysfunction (n = 1), alternative complement deficiency (n = 1), complement factor H deficiency (n = 1) and congenital disorder of glycosylation (n = 1). The number needed to investigate to identify 1 child presenting with IPD with an immunologic abnormality was 7 for children under 2 years and 9 for those 2 years old and over. CONCLUSIONS: This study supports the routine immune evaluation of children presenting with IPD of any age, with consideration of referral to a pediatric immunologist.

Immune-mediated mechanisms of parasite tissue sequestration during experimental cerebral malaria.

Cerebral malaria is a severe complication of malaria. Sequestration of parasitized RBCs in brain microvasculature is associated with disease pathogenesis, but our understanding of this process is incomplete. In this study, we examined parasite tissue sequestration in an experimental model of cerebral malaria (ECM). We show that a rapid increase in parasite biomass is strongly associated with the induction of ECM, mediated by IFN-gamma and lymphotoxin alpha, whereas TNF and IL-10 limit this process. Crucially, we discovered that host CD4(+) and CD8(+) T cells promote parasite accumulation in vital organs, including the brain. Modulation of CD4(+) T cell responses by helminth coinfection amplified CD4(+) T cell-mediated parasite sequestration, whereas vaccination could generate CD4(+) T cells that reduced parasite biomass and prevented ECM. These findings provide novel insights into immune-mediated mechanisms of ECM pathogenesis and highlight the potential of T cells to both prevent and promote infectious diseases.

Acquired Immunodeficiency from Maternal Chemotherapy and Severe Primary Pneumocystis Infection in an Infant.

Pneumocystis jirovecii is recognized as an opportunistic pathogen in immunosuppressed patients. We report a case of severe Pneumocystis pneumonia (PCP) in an infant with acquired combined immunodeficiency secondary to maternal chemotherapy exposure during the second and third trimesters of pregnancy. The infant required cardiorespiratory support with veno-venous extracorporeal membrane oxygenation (VV-ECMO) for severe respiratory failure. This case highlights the potential for severe acquired immunodeficiency in this patient cohort and further postnatal surveillance is highly recommended.

Human T cell recognition of the blood stage antigen Plasmodium hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT) in acute malaria.

BACKGROUND: The Plasmodium purine salvage enzyme, hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT) can protect mice against Plasmodium yoelii pRBC challenge in a T cell-dependent manner and has, therefore, been proposed as a novel vaccine candidate. It is not known whether natural exposure to Plasmodium falciparum stimulates HGXPRT T cell reactivity in humans. METHODS: PBMC and plasma collected from malaria-exposed Indonesians during infection and 7-28 days after anti-malarial therapy, were assessed for HGXPRT recognition using CFSE proliferation, IFNgamma ELISPOT assay and ELISA. RESULTS: HGXPRT-specific T cell proliferation was found in 44% of patients during acute infection; in 80% of responders both CD4+ and CD8+ T cell subsets proliferated. Antigen-specific T cell proliferation was largely lost within 28 days of parasite clearance. HGXPRT-specific IFN-gamma production was more frequent 28 days after treatment than during acute infection. HGXPRT-specific plasma IgG was undetectable even in individuals exposed to malaria for at least two years. CONCLUSION: The prevalence of acute proliferative and convalescent IFNgamma responses to HGXPRT demonstrates cellular immunogenicity in humans. Further studies to determine minimal HGXPRT epitopes, the specificity of responses for Plasmodia and associations with protection are required. Frequent and robust T cell proliferation, high sequence conservation among Plasmodium species and absent IgG responses distinguish HGXPRT from other malaria antigens.

Anti-NMDA-receptor Encephalitis in an Adolescent With HIV Infection and Review of the Literature.

Anti-N-Methyl-D-Aspartate-receptor encephalitis is the most common antibody-mediated autoimmune encephalopathy. A HIV-infected African boy presented with subacute psychosis as manifestation of anti-N-Methyl-D-Aspartate-receptor encephalitis. Intravenous immunoglobulin and corticosteroids induced complete remission. Although this is the first pediatric case described, 5 adult cases have been reported. The role of HIV in the etiology of this autoimmune condition requires further exploration.

The key role of CD40 ligand in overcoming tumor-induced dendritic cell dysfunction.

Overcoming dendritic cell (DC) dysfunction is a prerequisite for successful active immunotherapy against breast cancer. CD40 ligand (CD40L), a key molecule in the interface between T-lymphocytes and DCs, seems to be instrumental in achieving that goal. Commenting on our data that CD40L protects circulating DCs from apoptosis induced by breast tumor products, Lenahan and Avigan highlighted the potential of CD40L for immunotherapy. We expand on that argument by pointing to additional findings that CD40L not only rescues genuine DCs but also functionally improves populations of immature antigen-presenting cells that fill the DC compartment in patients with breast cancer.

Spontaneous apoptosis of blood dendritic cells in patients with breast cancer.

INTRODUCTION: Dendritic cells (DCs) are key antigen-presenting cells that play an essential role in initiating and directing cellular and humoral immunity, including anti-tumor responses. Due to their critical role in cancer, induction of DC apoptosis may be one of the central mechanisms used by tumors to evade immune recognition. METHODS: Spontaneous apoptosis of blood DCs (lineage negative HLA-DR positive cells) was assessed in peripheral blood mononuclear cells (PBMCs) using Annexin-V and TUNEL assays immediately after blood collection. The role of tumor products was assessed by culturing cells with supernatants derived from breast cancer cell lines (TDSN) or PBMCs (PBMC-SN, as a control). The capacity of DC stimulation to prevent apoptosis was assessed by incubating DC with inflammatory cytokines, poly I:C, IL-12 or CD40 ligand (CD40L) prior to culture with TDSN. Apoptosis was determined by flow cytometry and microscopy, and Bcl-2 expression determined by intracellular staining. RESULTS: In this study we document the presence of a significantly higher proportion of apoptotic (Annexin-V+ and TUNEL+) blood DCs in patients with early stage breast cancer (stage I to II; n = 13) compared to healthy volunteers (n = 15). We examined the role of tumor products in this phenomenon and show that supernatants derived from breast cancer lines induce apoptosis of blood DCs in PBMC cultures. Aiming to identify factors that protect blood DC from apoptosis, we compared a range of clinically available maturation stimuli, including inflammatory cytokines (tumor necrosis factor-alpha, IL-1beta, IL-6 and prostaglandin (PG)E2 as a cytokine cocktail), synthetic double-stranded RNA (poly I:C) and soluble CD40 ligand. Although inflammatory cytokines and poly I:C induced robust phenotypic maturation, they failed to protect blood DCs from apoptosis. In contrast, CD40 stimulation induced strong antigen uptake, secretion of IL-12 and protected blood DCs from apoptosis through sustained expression of Bcl-2. Exogenous IL-12 provided similar Bcl-2 mediated protection, suggesting that CD40L effect is mediated, at least in part, through IL-12 secretion. CONCLUSION: Cumulatively, our results demonstrate spontaneous apoptosis of blood DCs in patients with breast cancer and confirm that ex vivo conditioning of blood DCs can protect them from tumor-induced apoptosis.

HLA-DR+ immature cells exhibit reduced antigen-presenting cell function but respond to CD40 stimulation.

Dendritic cells (DC) have been implicated in the defective function of the immune system during cancer progression. We have demonstrated that patients with cancer have fewer myeloid (CD11c+) and plasmacytoid (CD123(hi)) DC and a concurrent accumulation of CD11c(-)CD123- immature cells expressing HLA-DR (DR(+)IC). Notably, DR(+)IC from cancer patients have a reduced capacity to stimulate allogeneic T-cells. DR(+)IC are also present in healthy donors, albeit in smaller numbers. In this study, we assessed whether DR(+)IC could have an impact on the immune response by comparing their function with DC counterparts. For this purpose, DR(+)IC and DC were purified and tested in the presentation of antigens through major histocompatibility complex (MHC) II and MHC-I molecules. DR(+)IC were less efficient than DC at presenting antigens to T-cells. DR(+)IC induced a limited activation of T-cells, eliciting poor T-helper (Th) 1 and preferentially inducing Th2-biased responses. Importantly, despite DR(+)IC's poor responsiveness to inflammatory factors, in samples from healthy volunteers and breast cancer patients, CD40 ligation induced phenotypic maturation and interleukin 12 secretion, in turn generating more efficient T-cell responses. These data underscore the importance of inefficient antigen presentation as a mechanism for tumor evasion and suggest an approach to improve the efficacy of DC-based immunotherapy for cancer.

A population of HLA-DR+ immature cells accumulates in the blood dendritic cell compartment of patients with different types of cancer.

Dendritic cell (DC) defects are an important component of immunosuppression in cancer. Here, we assessed whether cancer could affect circulating DC populations and its correlation with tumor progression. The blood DC compartment was evaluated in 136 patients with breast cancer, prostate cancer, and malignant glioma. Phenotypic, quantitative, and functional analyses were performed at various stages of disease. Patients had significantly fewer circulating myeloid (CD11c+) and plasmacytoid (CD123+) DC, and a concurrent accumulation of CD11c(-)CD123(-) immature cells that expressed high levels of HLA-DR+ immature cells (DR(+)IC). Although DR(+)IC exhibited a limited expression of markers ascribed to mature hematopoietic lineages, expression of HLA-DR, CD40, and CD86 suggested a role as antigen-presenting cells. Nevertheless, DR(+)IC had reduced capacity to capture antigens and elicited poor proliferation and interferon-gamma secretion by T-lymphocytes. Importantly, increased numbers of DR(+)IC correlated with disease status. Patients with metastatic breast cancer showed a larger number of DR(+)IC in the circulation than patients with local/nodal disease. Similarly, in patients with fully resected glioma, the proportion of DR(+)IC in the blood increased when evaluation indicated tumor recurrence. Reduction of blood DC correlating with accumulation of a population of immature cells with poor immunologic function may be associated with increased immunodeficiency observed in cancer.

Single step enrichment of blood dendritic cells by positive immunoselection.

Dendritic cells (DC) for cancer immunotherapy protocols are generated most commonly by in vitro differentiation of monocytes with exogenous cytokines (Mo-DC). However, Mo-DC differ in their molecular phenotype and function from blood DC (BDC). Clinical isolation of BDC has been limited to the use of density gradients, which result in low yields of variable purity. We have developed a DC enrichment platform, which uses the CMRF-44 (IgM) or CMRF-56 (IgG) monoclonal antibodies (mAb) to select BDC that express these antigens after a short overnight incubation. After culture of peripheral blood mononuclear cells (PBMC) in autologous/AB serum, biotinylated CMRF-44 was used to select DC in a single step immuno-magnetic bead procedure; this produced populations containing up to 99% CMRF-44(+) cells, including up to 67% CMRF-44(+) CD14(-) CD19(-) DC, from an initial starting population of approximately 0.5%. We observed consistent differences in the purities obtained from individual donors with a mean of 54% CMRF-44(+) cells (range 19-99%). Similar results were obtained using biotinylated CMRF-56 mAb, an antibody identifying a comparable population in cultured PBMC. We recovered an average of 54% and 66% of the available BDC in separations performed with the CMRF-44 and CMRF-56 mAb, respectively. The reproducibility of the procedure and the ability to perform it in a closed sterile system makes it suitable for clinical use. Larger scale preparations starting from apheresis derived PBMC will produce sufficient BDC for immunotherapy protocols. The purified BDC elicited strong allogeneic mixed leukocyte reactions and HLA classes II- and I-restricted antigen-specific primary immune responses.

Apoptosis and dysfunction of blood dendritic cells in patients with falciparum and vivax malaria.

Malaria causes significant morbidity worldwide and a vaccine is urgently required. Plasmodium infection causes considerable immune dysregulation, and elicitation of vaccine immunity remains challenging. Given the central role of dendritic cells (DCs) in initiating immunity, understanding their biology during malaria will improve vaccination outcomes. Circulating DCs are particularly important, as they shape immune responses in vivo and reflect the functional status of other subpopulations. We performed cross-sectional and longitudinal assessments of the frequency, phenotype, and function of circulating DC in 67 Papuan adults during acute uncomplicated P. falciparum, P. vivax, and convalescent P. falciparum infections. We demonstrate that malaria patients display a significant reduction in circulating DC numbers and the concurrent accumulation of immature cells. Such alteration is associated with marked levels of spontaneous apoptosis and impairment in the ability of DC to mature, capture, and present antigens to T cells. Interestingly, sustained levels of plasma IL-10 were observed in patients with acute infection and were implicated in the induction of DC apoptosis. DC apoptosis was reversed upon IL-10 blockade, and DC function recovered when IL-10 levels returned to baseline by convalescence. Our data provide key information on the mechanisms behind DC suppression during malaria and will assist in developing strategies to better harness DC's immunotherapeutic potential.

Low doses of killed parasite in CpG elicit vigorous CD4+ T cell responses against blood-stage malaria in mice.

Development of a vaccine that targets blood-stage malaria parasites is imperative if we are to sustainably reduce the morbidity and mortality caused by this infection. Such a vaccine should elicit long-lasting immune responses against conserved determinants in the parasite population. Most blood-stage vaccines, however, induce protective antibodies against surface antigens, which tend to be polymorphic. Cell-mediated responses, on the other hand, offer the theoretical advantage of targeting internal antigens that are more likely to be conserved. Nonetheless, few of the current blood-stage vaccine candidates are able to harness vigorous T cell immunity. Here, we present what we believe to be a novel blood-stage whole-organism vaccine that, by combining low doses of killed parasite with CpG-oligodeoxynucleotide (CpG-ODN) adjuvant, was able to elicit strong and cross-reactive T cell responses in mice. Our data demonstrate that immunization of mice with 1,000 killed parasites in CpG-ODN engendered durable and cross-strain protection by inducing a vigorous response that was dependent on CD4+ T cells, IFN-gamma, and nitric oxide. If applicable to humans, this approach should facilitate the generation of robust, cross-reactive T cell responses against malaria as well as antigen availability for vaccine manufacture.

Malaria vaccines: the case for a whole-organism approach.

BACKGROUND: Malaria is a significant health problem causing morbidity and mortality worldwide. Vaccine development has been an imperative for decades. However, the intricacy of the parasite's lifecycle coupled with the lack of evidence for robust infection-induced immunity has made vaccine development exceptionally difficult. OBJECTIVE: To review some of the key advances in the field and discuss potential ways forward for a whole-organism vaccine. METHODS: The authors searched PubMed using the words 'malaria and vaccine'. We searched for manuscripts detailing antigen characterisation and vaccine strategies with emphasis on subunit versus whole-parasite approaches. Abstracts were selected and relevant articles are discussed. The searches were not restricted by language or date. CONCLUSIONS: The early cloning of malaria antigens has fuelled rapid development of subunit vaccines. However, the disappointing results of clinical trials have resulted in reappraisal of current strategies. Whole-parasite approaches have re-emerged as an alternative strategy. Immunization using radiation or genetically attenuated sporozoites has been shown to result in sterile immunity and immunization with blood-stage parasites curtailed by antimalarials has demonstrated delayed parasitemia in rodent models as well as in human malaria.

Dendritic cell biology during malaria.

Malaria is an infectious disease that causes serious morbidity and mortality worldwide. The disease is associated with a variety of clinical syndromes ranging from asymptomatic to lethal infections involving anaemia, organ failure, pulmonary and cerebral disease. The molecular and cellular factors responsible for the differences in disease severity are poorly understood but parasite-specific immune responses are thought to play a critical role in pathogenesis. Dendritic cells have an essential role in linking innate and adaptive immune responses and here we review their role in the context of malaria.

Dendritic cell immunotherapy for breast cancer.

Novel adjuvant therapies are urgently needed to complement the existing treatment options for breast cancer. The advent of the use of dendritic cells (DCs) for cancer immunotherapy provides a unique opportunity to overcome the relative non-immunogenic property of breast tumours and address the underlying immunodeficiency. To date, the success of this approach has been limited, possibly due to the targeting of specific tumour antigens that rapidly mutate and, thus, become undetectable to the immune system. A more efficient approach would include preparations encompassing multiple antigens, such as those provided by loading of whole tumour cells or tumour RNA. It is proposed that targeting mammary stem cells responsible for resistance to chemo/immunotherapy, through the expression of a broad array of wild-type and mutated tumour antigens in the context of DCs, will become a mainstay for immunotherapy of breast cancer.