Exploring NK cell receptor dynamics in paediatric leukaemias: implications for immunotherapy and prognosis.

Objectives: Immunotherapies targeting natural killer (NK) cell receptors have shown promise against leukaemia. Unfortunately, cancer immunosuppressive mechanisms that alter NK cell phenotype prevent such approaches from being successful. The study utilises advanced cytometry to examine how cancer immunosuppressive pathways affect NK cell phenotypic changes in clinical samples. Methods: In this study, we conducted a high-dimensional examination of the cell surface expression of 16 NK cell receptors in paediatric patients with acute myeloid leukaemia and acute lymphoblastic leukaemia, as well as in samples of non-age matched adult peripheral blood (APB) and umbilical cord blood (UCB). An unsupervised analysis was carried out in order to identify NK cell populations present in paediatric leukaemias. Results: We observed that leukaemia NK cells clustered together with UCB NK cells and expressed relatively higher levels of the NKG2A receptor compared to APB NK cells. In addition, CD56dimCD16+CD57- NK cells lacking NKG2A expression were mainly absent in paediatric leukaemia patients. However, CD56br NK cell populations expressing high levels of NKG2A were highly represented in paediatric leukaemia patients. NKG2A expression on leukaemia NK cells was found to be positively correlated with the expression of its ligand, suggesting that the NKG2A-HLA-E interaction may play a role in modifying NK cell responses to leukaemia cells. Conclusion: We provide an in-depth analysis of NK cell populations in paediatric leukaemia patients. These results support the development of immunotherapies targeting immunosuppressive receptors, such as NKG2A, to enhance innate immunity against paediatric leukaemia.

Characterization of Human Engraftment and Hemophagocytic Lymphohistiocytosis in NSG-SGM3 Neonate Mice Engrafted with Purified CD34+ Hematopoietic Stem Cells.

Immunodeficient mice bearing human immune systems, or "humanized" chimeric mice, are widely used in basic research, along with the preclinical stages of drug development. Nonobese diabetic-severe combined immunodeficiency (NOD-SCID) IL2Rγnull (NSG) mice expressing human stem cell factor, granulocyte-macrophage colony stimulating factor, and interleukin-3 (NSG-SGM3) support robust development of human myeloid cells and T cells but have reduced longevity due to the development of fatal hemophagocytic lymphohistiocytosis (HLH). Here, we describe an optimized protocol for development of human immune chimerism in NSG-SGM3 mice. We demonstrate that efficient human CD45+ reconstitution can be achieved and HLH delayed by engraftment of neonatal NSG-SGM3 with low numbers of human umbilical cord-derived CD34+ hematopoietic stem cells in the absence of preconditioning irradiation.

Justice implications of health and food security policies for Indigenous peoples facing COVID-19: a qualitative study and policy analysis in Peru.

The spread of COVID-19 in Peru resulted in the declaration of a national health emergency, in which Indigenous peoples were identified as being particularly vulnerable due to their pre-existing poor health indicators and disadvantaged social conditions. The aim of this paper is to examine how the Peruvian government responded to the health and food needs of the Shawi and Ashaninka Indigenous peoples of Peru during the first 18 months of the pandemic (March 2020-August 2021). This study uses both official policy documents and real-world experiences to evaluate policy responses in terms of their immediate impact and their longer-term sustainability and contribution to the improvement of health, well-being and justice for Indigenous communities. Four health and food security responses were evaluated: the Amazon Health Plan and Indigenous Command; food aid; cash aid; and COVID-19 vaccination. We employed the Multidimensional Injustice Framework to analyse the justice implications of the design and implementation of responses. Data collection included 71 interviews with government officials (n = 7), Indigenous leaders (n = 31) and community members (n = 33). The results show how national and regional governments released policies to address the health and food needs of Indigenous peoples directly or indirectly, as part of a broader focus on vulnerable people. However, justice implications were not sufficiently addressed in the design or implementation of the responses. On the distributive dimension, Indigenous communities were prioritized to receive health goods and services, nevertheless, the distribution had shortcomings that impeded their collection and Indigenous food systems and livelihoods were largely overlooked. On the procedural dimension, Indigenous representatives were included to provide culturally sensitive feedback on health interventions, but without funding, and furthermore, the community members had only passive participation. This paper points out the importance of considering and addressing justice implications for more effective and fairer health and food policy responses to current and future health crises.

High dimensional analysis reveals distinct NK cell subsets but conserved response to stimulation in umbilical cord blood and adult peripheral blood.

Growing interest surrounds adoptive cellular therapies utilizing Natural Killer (NK) cells, which can be obtained from various sources, including umbilical cord blood (UCB) and adult peripheral blood (APB). Understanding NK cell receptor expression and diversity in such cellular sources will guide future therapeutic designs. We used a 20-color flow cytometry panel to compare unstimulated and cytokine-activated UCB and APB NK cells. Our analysis showed that UCB NK cells express slightly higher levels of the immune checkpoints PD-1, TIGIT, and CD96 compared to their APB counterparts. Unsupervised hierarchical clustering and dimensionality reduction analyses revealed enrichment in CD56neg as well as mature NKp46neg and CD56+ CD16+ NK cell populations in UCB whereas CD57+ terminally differentiated NK cells with variable expression of KIRs and CD16 were found in APB. These populations were conserved following stimulation with IL-12, IL-15, and IL-18. Cytokine stimulation was associated with the downregulation of TIGIT and CD16 on multiple NK cell subsets in UCB and APB. Among UCB CD16- NK cell populations, TIGIT+ NK cells produced more IFN-γ than their TIGIT- counterparts. Our data demonstrate higher immune checkpoint expression on UCB NK cells compared to APB. However, the expression of TIGIT immune checkpoint is not indicative of NK cell exhaustion.

Policy responses to COVID-19 in Sri Lanka and the consideration of Indigenous Peoples.

COVID-19 has had uneven impacts on health and well-being, with Indigenous communities in the Global South facing some of the highest risks. Focusing on the experience of Sri Lanka, this study identifies key policy responses to COVID-19, documents how they evolved over two years of the pandemic, and examines if and how government responses have addressed issues pertaining to Indigenous Peoples. Drawing upon an analysis of policy documents (n = 110) and interviews with policymakers (n = 20), we characterize seven key policy responses implemented by the Sri Lankan government: i) testing for and identifying COVID-19; ii) quarantine procedures; iii) provisional clinical treatments; iv) handling other diseases during COVID-19; v) movement; vi) guidelines to be adhered to by the general public; and vii) health and vaccination. The nature of these responses changed as the pandemic progressed. There is no evidence that policy development or implementation incorporated the voices and needs of Indigenous Peoples.

Indigenous peoples and the COVID-19 pandemic: a systematic scoping review.

Past influenza pandemics including the Spanish flu and H1N1 have disproportionately affected Indigenous Peoples. We conducted a systematic scoping review to provide an overview of the state of understanding of the experience of Indigenous peoples during the first 18 months of the COVID-19 pandemic, in doing so we capture the state of knowledge available to governments and decision makers for addressing the needs of Indigenous peoples in these early months of the pandemic. We addressed three questions: (a) How is COVID-19 impacting the health and livelihoods of Indigenous peoples, (b) What system level challenges are Indigenous peoples experiencing, (c) How are Indigenous peoples responding? We searched Web of Science, Scopus, and PubMed databases and UN organization websites for publications about Indigenous peoples and COVID-19. Results were analyzed using descriptive statistics and content analysis. A total of 153 publications were included: 140 peer-reviewed articles and 13 from UN organizations. Editorial/commentaries were the most (43%) frequent type of publication. Analysis identified Indigenous peoples from 19 different countries, although 56% of publications were centered upon those in Brazil, United States, and Canada. The majority (90%) of articles focused upon the general adult population, few (<2%) used a gender lens. A small number of articles documented COVID-19 testing (0.04%), incidence (18%), or mortality (16%). Five themes of system level challenges affecting exposure and livelihoods evolved: ecological, poverty, communication, education and health care services. Responses were formal and informal strategies from governments, Indigenous organizations and communities. A lack of ethnically disaggregated health data and a gender lens are constraining our knowledge, which is clustered around a limited number of Indigenous peoples in mostly high-income countries. Many Indigenous peoples have autonomously implemented their own coping strategies while government responses have been largely reactive and inadequate. To 'build back better' we must address these knowledge gaps.

Interactions between climate and COVID-19.

In this Personal View, we explain the ways that climatic risks affect the transmission, perception, response, and lived experience of COVID-19. First, temperature, wind, and humidity influence the transmission of COVID-19 in ways not fully understood, although non-climatic factors appear more important than climatic factors in explaining disease transmission. Second, climatic extremes coinciding with COVID-19 have affected disease exposure, increased susceptibility of people to COVID-19, compromised emergency responses, and reduced health system resilience to multiple stresses. Third, long-term climate change and prepandemic vulnerabilities have increased COVID-19 risk for some populations (eg, marginalised communities). The ways climate and COVID-19 interact vary considerably between and within populations and regions, and are affected by dynamic and complex interactions with underlying socioeconomic, political, demographic, and cultural conditions. These conditions can lead to vulnerability, resilience, transformation, or collapse of health systems, communities, and livelihoods throughout varying timescales. It is important that COVID-19 response and recovery measures consider climatic risks, particularly in locations that are susceptible to climate extremes, through integrated planning that includes public health, disaster preparedness, emergency management, sustainable development, and humanitarian response.

Evaluación de la variabilidad de la práctica médica en la enfermedad isquémica coronaria / Evaluation of the variability of medical practice in coronary ischemic disease

RESUMEN Objetivo Determinar la variabilidad de práctica médica del uso de angioplastia y revascularización para tratamiento de la enfermedad isquémica coronaria en aseguradoras de salud. Materiales y Métodos Estudio descriptivo retrospectivo de tipo ecológico mixto del registro de tasa de uso de procedimientos cardiovasculares de angioplastia y revascularización empleados en el tratamiento de la enfermedad isquémica coronaria. Se realizó un análisis de regresión y un análisis de varianza (ANOVA) con los reportes de varias aseguradoras en un periodo de cinco años. Resultados La edad media de procedimientos fue 64,9 años, desviación estándar de 11,3. El procedimiento más usado fue angioplastia (75,6%) y se realizaron más procedimientos en hombres 2:1. Se encontraron diferencias estadísticamente significativas (p<0,05) en cada grupo etario para ambos procedimientos y variaciones en al menos dos aseguradoras. El análisis univariado por sexo encontró lo siguiente: variaciones de tasas de uso en angioplastia: pacientes de 40 a 49 años (p=0,017) y de 50 a 59 años (p=0,036); variaciones de tasas de uso de revascularización: pacientes de 30 a 39 años (p=0,036); de 40 a 49 años (p=0,013); de 50 a 59 años (p=0,002) y de 60 a 69 años (p<0,001). Conclusiones En las aseguradoras hay variaciones en la tasa de uso para procedimientos cardiovasculares (en todos los grupos etarios observados) después de los 30 años (también en tasa de uso por sexo). Se infirieron causas como el factor protector hormonal y terapias de reemplazo hormonal sin descartar otras causas de variación injustificadas como educación en salud a mujeres sobre detección de la enfermedad isquémica coronaria, factores culturales, sociales y económicos que limitan el uso de tecnologías.

ABSTRACT Objective To determine the variability of the medical practice of the use of angioplasty and revascularization for the treatment of Coronary Ischemic Disease in health insurers. Materials and Methods Retrospective descriptive study of mixed ecological type of the registry of the rate of use of cardiovascular procedures, angioplasty and revascularization, used in the treatment of coronary ischemic disease. Regression analysis and analysis of variance (ANOVA) were performed on five years of reports from various insurers. Results The mean age of procedures was 64.9 years, standard deviation of 11.3. The most widely used procedure was angioplasty (75.6%), and more procedures were performed in 2:1 men. Statistically significant differences (p<0.05) were found in each age group for both procedures, and variations in at least two insurers. Univariate analysis by sex, variations in angioplasty use rates 40 to 49 years (p=0.017) and 50 to 59 years (p=0.036) and revascularization 30 to 39 years (p=0.036), 40 to 49 years (p=0.013), 50 to 59 years (p=0.002) 60 to 69 years (p<0.001). Conclusions There are variations in the rate of use for cardiovascular procedures in all age groups observed after 30 years in insurers, also in rate of use by sex, causes such as the hormonal protective factor and hormone replacement therapies are inferred without ruling out other unjustified causes of modification such as health education for women on detection of coronary ischemic disease, cultural, social and economic factors that limit the use of technologies.

Elucidating the Motif for CpG Oligonucleotide Binding to the Dendritic Cell Receptor DEC-205 Leads to Improved Adjuvants for Liver-Resident Memory.

DEC-205 is a cell-surface receptor that transports bound ligands into the endocytic pathway for degradation or release within lysosomal endosomes. This receptor has been reported to bind a number of ligands, including keratin, and some classes of CpG oligodeoxynucleotides (ODN). In this study, we explore in detail the requirements for binding ODNs, revealing that DEC-205 efficiently binds single-stranded, phosphorothioated ODN of ≥14 bases, with preference for the DNA base thymidine, but with no requirement for a CpG motif. DEC-205 fails to bind double-stranded phosphodiester ODN, and thus does not bind the natural type of DNA found in mammals. The ODN binding preferences of DEC-205 result in strong binding of B class ODN, moderate binding to C class ODN, minimal binding to P class ODN, and no binding to A class ODN. Consistent with DEC-205 binding capacity, induction of serum IL-12p70 or activation of B cells by each class of ODN correlated with DEC-205 dependence in mice. Thus, the greater the DEC-205 binding capacity, the greater the dependence on DEC-205 for optimal responses. Finally, by covalently linking a B class ODN that efficiently binds DEC-205, to a P class ODN that shows poor binding, we improved DEC-205 binding and increased adjuvancy of the hybrid ODN. The hybrid ODN efficiently enhanced induction of effector CD8 T cells in a DEC-205-dependent manner. Furthermore, the hybrid ODN induced robust memory responses, and was particularly effective at promoting the development of liver tissue-resident memory T cells.

Cytokines as a marker of central nervous system autoantibody associated epilepsy.

OBJECTIVE: Autoantibodies to central nervous system (CNS) antigens are increasingly identified in patients with epilepsy. Alterations in cytokines and chemokines have also been demonstrated in epilepsy, but this has not been explored in subjects with autoantibodies. If antibody positive and antibody negative subjects show a difference in immune activation, as measured by cytokine levels, this could improve diagnostic and therapeutic approaches, and provide insights into the underlying pathophysiology. We aimed to evaluate serum and CSF cytokines and chemokines in patients with and without autoantibody positivity to identify any differences between the two groups. METHODS: We studied participants who had undergone serum and CSF testing for CNS autoantibodies, as part of their clinical evaluation. Cases were classified as antibody positive or antibody negative for comparison. Stored CSF and sera were analysed for cytokine and chemokine concentrations. RESULTS: 25 participants underwent testing. 8 were antibody positive, 17 were antibody negative. Significant elevations in the mean concentration of IL-13 and RANTES in CSF were found in the antibody positive cases and significant elevation of CSF VEGF was found in the antibody negative cases. Significant elevations in the mean concentrations of serum TNFß, INFγ, bNGF, IL-8, and IL-12 were seen in the antibody negative group, and there was poor correlation between the majority of serum and CSF concentrations. SIGNIFICANCE: Measurement of cytokines and chemokines such as IL-13 and RANTES could be useful in diagnosis of autoimmune associated epilepsy. Such markers might also guide targeted immunotherapy to improve seizure control and provide insights into the underlying pathophysiology of epilepsy associated with CNS autoantibodies.

Validation of an instrument to measure adherence to orthodontic treatment provided in dental clinics of dentistry schools / Validación de un instrumento para medir la adherencia al tratamiento de ortodoncia en servicios odontológicos de facultades de odontología

Abstract Introduction: Adherence to treatment is associated with the quality of health care given to the patient, especially in institutions with a high workload, such as dentistry schools. In these places, treatments are long and high adherence is required for them to be successful. Objective: To validate, by means of an exploratory factor analysis (EFA) and a confirmatory factor analysis (CFA), an instrument for measuring adherence to orthodontic treatment in dental clinics of dentistry schools, where a large number of patients are treated. Materials and methods: Quantitative study in which an instrument was validated by performing an EFA and a CFA. A 27-item questionnaire (adapted from the original 37-item instrument) was administered to 601 patients treated at a dentistry school of a Colombian university in two different periods: during the second semester of 2018 (n=202) and during the first semester of 2019 (n=399). The EFA and the CFA were performed using the SPSS and the LISREL software, respectively. Results: Factor analysis established that the instrument has six factors and 25 questions suitable for collecting information on adherence to treatment by obtaining the following adjustment values: x2 S B=420.09 with d.f.=260 and p<0.05; x2 S B divided by the degrees of freedom index (X2 SB/d.f.) = 1.62; CFI=0.99; RFI=0.98; NNFI= 0.99; RMSEA=0.039 (90%CI 0.032-0.046); and SRMR= 0.057. Conclusions: Based on the results obtained after performing the EFA and the CFA, it is possible to conclude that the instrument is valid and highly reliable to measure orthodontic treatment in this context. Consequently, its use in similar institutions will allow determining the levels of adherence in these patients accurately, and thus, when necessary, develop and implement actions that encourage greater engagement from this population to orthodontic treatment to obtain better outcomes.

Resumen Introducción. La adherencia al tratamiento está relacionada con la calidad de la atención en salud dada al paciente, especialmente en instituciones con un alto volumen de trabajo, como las facultades de odontología, donde los tratamientos son prolongados y se requiere de una alta adherencia para que estos sean exitosos. Objetivo. Validar, mediante un análisis factorial exploratorio (AFE) y un análisis factorial confirmatorio (AFC), un instrumento para medir la adherencia al tratamiento ortodóntico en clínicas odontológicas de facultades de odontología en las que se atiende un alto número de pacientes. Materiales y métodos. Estudio cuantitativo en el que se validó un instrumento mediante un AFE y un AFC. Se aplicó un cuestionario de 27 preguntas (adaptado del instrumento original de 37 preguntas) a 601 pacientes atendidos en una facultad de odontología de una universidad colombiana en dos periodos diferentes: el segundo semestre de 2018 y el primer semestre de 2019. El AFE y el AFC se realizaron mediante los programas SPSS y LISREL, respectivamente. Resultados. El análisis factorial permitió establecer un instrumento con 6 factores y 25 preguntas adecuado para recolectar información sobre la adherencia al tratamiento al obtener los siguientes valores de ajuste: B=420.09 con gl=260 y p<0.05; B/gl = 1.62; CFI=0.99; RFI=0.98; NNFI=0.99; RMSEA=0.039 (IC90%: 0.032-0.046), y SRMR=0.057. Conclusiones. El AFE y el AFC permitieron establecer un instrumento válido y con una alta con-fiabilidad para medir la adherencia al tratamiento ortodóntico en este escenario, por lo que su uso en instituciones similares permitirá determinar de manera confiable los niveles de adherencia en estos pacientes, y, de esta forma, cuando sea necesario, desarrollar e implementar acciones que fomenten un mayor compromiso de esta población con los tratamientos de ortodoncia para obtener mejores desenlaces.

Human CD141+ dendritic cells (cDC1) are impaired in patients with advanced melanoma but can be targeted to enhance anti-PD-1 in a humanized mouse model.

BACKGROUND: The conventional type 1 dendritic cell subset (cDC1) is indispensable for tumor immune responses and the efficacy of immune checkpoint inhibitor (ICI) therapies in animal models but little is known about the role of the human CD141+ DC cDC1 equivalent in patients with melanoma. METHODS: We developed a flow cytometry assay to quantify and characterize human blood DC subsets in healthy donors and patients with stage 3 and stage 4 metastatic melanoma. To examine whether harnessing CD141+ DCs could improve responses to ICIs in human melanoma, we developed a humanized mouse model by engrafting immunodeficient NSG-SGM3 mice with human CD34+ hematopoietic stem cells (HSCs) from umbilical cord blood followed by transplantation of a human melanoma cell line and treatment with anti-programmed cell death protein-1 (anti-PD-1). RESULTS: Blood CD141+ DC numbers were significantly reduced in patients with stage 4 melanoma compared with healthy controls. Moreover, CD141+ DCs in patients with melanoma were selectively impaired in their ability to upregulate CD83 expression after stimulation with toll-like receptor 3 (TLR3) and TLR7/8 agonists ex vivo. Although DC numbers did not correlate with responses to anti-PD-1 and/or anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) ICIs, their numbers and capacity to upregulate CD83 declined further during treatment in non-responding patients. Treatment with anti-PD-1 was ineffective at controlling tumor growth in humanized mice but efficacy was enhanced by indirectly expanding and activating DCs in vivo with fms-like tyrosine kinase-3 ligand (Flt3L) and a TLR3 agonist. Moreover, intratumoral injections of CD141+ DCs resulted in reduced tumor growth when combined with anti-PD-1 treatment. CONCLUSIONS: These data illustrate quantitative and qualitative impairments in circulating CD141+ DCs in patients with advanced melanoma and that increasing CD141+ DC number and function is an attractive strategy to enhance immunogenicity and response rates to ICIs.

Human CLEC9A antibodies deliver NY-ESO-1 antigen to CD141+ dendritic cells to activate naïve and memory NY-ESO-1-specific CD8+ T cells.

BACKGROUND: Dendritic cells (DCs) are crucial for the efficacy of cancer vaccines, but current vaccines do not harness the key cDC1 subtype required for effective CD8+ T-cell-mediated tumor immune responses. Vaccine immunogenicity could be enhanced by specific delivery of immunogenic tumor antigens to CD141+ DCs, the human cDC1 equivalent. CD141+ DCs exclusively express the C-type-lectin-like receptor CLEC9A, which is important for the regulation of CD8+ T cell responses. This study developed a new vaccine that harnesses a human anti-CLEC9A antibody to specifically deliver the immunogenic tumor antigen, NY-ESO-1 (New York esophageal squamous cell carcinoma 1), to human CD141+ DCs. The ability of the CLEC9A-NY-ESO-1 antibody to activate NY-ESO-1-specific naïve and memory CD8+ T cells was examined and compared with a vaccine comprised of a human DEC-205-NY-ESO-1 antibody that targets all human DCs. METHODS: Human anti-CLEC9A, anti-DEC-205 and isotype control IgG4 antibodies were genetically fused to NY-ESO-1 polypeptide. Cross-presentation to NY-ESO-1-epitope-specific CD8+ T cells and reactivity of T cell responses in patients with melanoma were assessed by interferon γ (IFNγ) production following incubation of CD141+ DCs and patient peripheral blood mononuclear cells with targeting antibodies. Humanized mice containing human DC subsets and a repertoire of naïve NY-ESO-1-specific CD8+ T cells were used to investigate naïve T cell priming. T cell effector function was measured by expression of IFNγ, MIP-1ß, tumor necrosis factor and CD107a and by lysis of target tumor cells. RESULTS: CLEC9A-NY-ESO-1 antibodies (Abs) were effective at mediating delivery and cross-presentation of multiple NY-ESO-1 epitopes by CD141+ DCs for activation of NY-ESO-1-specific CD8+ T cells. When benchmarked to NY-ESO-1 conjugated to an untargeted control antibody or to anti-human DEC-205, CLEC9A-NY-ESO-1 was superior at ex vivo reactivation of NY-ESO-1-specific T cell responses in patients with melanoma. Moreover, CLEC9A-NY-ESO-1 induced priming of naïve NY-ESO-1-specific CD8+ T cells with polyclonal effector function and potent tumor killing capacity in vitro. CONCLUSIONS: These data advocate human CLEC9A-NY-ESO-1 Ab as an attractive strategy for specific targeting of CD141+ DCs to enhance tumor immunogenicity in NY-ESO-1-expressing malignancies.

Human CLEC9A antibodies deliver Wilms' tumor 1 (WT1) antigen to CD141+ dendritic cells to activate naïve and memory WT1-specific CD8+ T cells.

OBJECTIVES: Vaccines that prime Wilms' tumor 1 (WT1)-specific CD8+ T cells are attractive cancer immunotherapies. However, immunogenicity and clinical response rates may be enhanced by delivering WT1 to CD141+ dendritic cells (DCs). The C-type lectin-like receptor CLEC9A is expressed exclusively by CD141+ DCs and regulates CD8+ T-cell responses. We developed a new vaccine comprising a human anti-CLEC9A antibody fused to WT1 and investigated its capacity to target human CD141+ DCs and activate naïve and memory WT1-specific CD8+ T cells. METHODS: WT1 was genetically fused to antibodies specific for human CLEC9A, DEC-205 or ß-galactosidase (untargeted control). Activation of WT1-specific CD8+ T-cell lines following cross-presentation by CD141+ DCs was quantified by IFNγ ELISPOT. Humanised mice reconstituted with human immune cell subsets, including a repertoire of naïve WT1-specific CD8+ T cells, were used to investigate naïve WT1-specific CD8+ T-cell priming. RESULTS: The CLEC9A-WT1 vaccine promoted cross-presentation of WT1 epitopes to CD8+ T cells and mediated priming of naïve CD8+ T cells more effectively than the DEC-205-WT1 and untargeted control-WT1 vaccines. CONCLUSIONS: Delivery of WT1 to CD141+ DCs via CLEC9A stimulates CD8+ T cells more potently than either untargeted delivery or widespread delivery to all Ag-presenting cells via DEC-205, suggesting that cross-presentation by CD141+ DCs is sufficient for effective CD8+ T-cell priming in humans. The CLEC9A-WT1 vaccine is a promising candidate immunotherapy for malignancies that express WT1.

Activation of human CD141+ and CD1c+ dendritic cells in vivo with combined TLR3 and TLR7/8 ligation.

Mice reconstituted with human hematopoietic stem cells are valuable models to study aspects of the human immune system in vivo. We describe a humanized mouse model (hu mice) in which fully functional human CD141+ and CD1c+ myeloid and CD123+ plasmacytoid dendritic cells (DC) develop from human cord blood CD34+ cells in immunodeficient mice. CD141+ DC are the human equivalents of murine CD8+ /CD103+ DC which are essential for the induction of tumor-inhibitory cytotoxic T lymphocyte responses, making them attractive targets to exploit for the development of new cancer immunotherapies. We used CD34+ -engrafted NSG-A2 mice to investigate activation of DC subsets by synthetic dsRNA or ssRNA analogs polyinosinic-polycytidylic acid/poly I:C and Resiquimod/R848, agonists for TLR3 and TLR8, respectively, both of which are expressed by CD141+ DC. Injection of hu mice with these agonists resulted in upregulation of costimulatory molecules CD80, CD83 and CD86 by CD141+ and CD1c+ DC alike, and their combination further enhanced expression of these molecules by both subsets. When combined, poly I:C and R848 enhanced serum levels of key cytokines associated with cross-presentation and the induction of cytotoxic T lymphocyte responses including IFN-α, IFN-ß, IL-12 and CXCL10. These data advocate a combination of poly I:C and R848 TLR agonists as means of activating human DC for immunotherapy.

Human CD141+ Dendritic Cell and CD1c+ Dendritic Cell Undergo Concordant Early Genetic Programming after Activation in Humanized Mice In Vivo.

Human immune cell subsets develop in immunodeficient mice following reconstitution with human CD34+ hematopoietic stem cells. These "humanized" mice are useful models to study human immunology and human-tropic infections, autoimmunity, and cancer. However, some human immune cell subsets are unable to fully develop or acquire full functional capacity due to a lack of cross-reactivity of many growth factors and cytokines between species. Conventional dendritic cells (cDCs) in mice are categorized into cDC1, which mediate T helper (Th)1 and CD8+ T cell responses, and cDC2, which mediate Th2 and Th17 responses. The likely human equivalents are CD141+ DC and CD1c+ DC subsets for mouse cDC1 and cDC2, respectively, but the extent of any interspecies differences is poorly characterized. Here, we exploit the fact that human CD141+ DC and CD1c+ DC develop in humanized mice, to further explore their equivalency in vivo. Global transcriptome analysis of CD141+ DC and CD1c+ DC isolated from humanized mice demonstrated that they closely resemble those in human blood. Activation of DC subsets in vivo, with the TLR3 ligand poly I:C, and the TLR7/8 ligand R848 revealed that a core panel of genes consistent with DC maturation status were upregulated by both subsets. R848 specifically upregulated genes associated with Th17 responses by CD1c+ DC, while poly I:C upregulated IFN-λ genes specifically by CD141+ DC. MYCL expression, known to be essential for CD8+ T cell priming by mouse DC, was specifically induced in CD141+ DC after activation. Concomitantly, CD141+ DC were superior to CD1c+ DC in their ability to prime naïve antigen-specific CD8+ T cells. Thus, CD141+ DC and CD1c+ DC share a similar activation profiles in vivo but also have induce unique signatures that support specialized roles in CD8+ T cell priming and Th17 responses, respectively. In combination, these data demonstrate that humanized mice provide an attractive and tractable model to study human DC in vitro and in vivo.

Human Blood CD1c+ Dendritic Cells Promote Th1 and Th17 Effector Function in Memory CD4+ T Cells.

Dendritic cells (DC) initiate the differentiation of CD4+ helper T cells into effector cells including Th1 and Th17 responses that play an important role in inflammation and autoimmune disease pathogenesis. In mice, Th1 and Th17 responses are regulated by different conventional (c) DC subsets, with cDC1 being the main producers of IL-12p70 and inducers of Th1 responses, while cDC2 produce IL-23 to promote Th17 responses. The role that human DC subsets play in memory CD4+ T cell activation is not known. This study investigated production of Th1 promoting cytokine IL-12p70, and Th17 promoting cytokines, IL-1ß, IL-6, and IL-23, by human blood monocytes, CD1c+ DC, CD141+ DC, and plasmacytoid DC and examined their ability to induce Th1 and Th17 responses in memory CD4+ T cells. Human CD1c+ DC produced IL-12p70, IL-1ß, IL-6, and IL-23 in response to R848 combined with LPS or poly I:C. CD141+ DC were also capable of producing IL-12p70 and IL-23 but were not as proficient as CD1c+ DC. Activated CD1c+ DC were endowed with the capacity to promote both Th1 and Th17 effector function in memory CD4+ T cells, characterized by high production of interferon-γ, IL-17A, IL-17F, IL-21, and IL-22. These findings support a role for CD1c+ DC in autoimmune inflammation where Th1/Th17 responses play an important role in disease pathogenesis.

Targeting CLEC9A delivers antigen to human CD141+ DC for CD4+ and CD8+T cell recognition.

DC-based vaccines that initiate T cell responses are well tolerated and have demonstrated efficacy for tumor immunotherapy, with the potential to be combined with other therapies. Targeting vaccine antigens (Ag) directly to the DCs in vivo is more effective than cell-based therapies in mouse models and is therefore a promising strategy to translate to humans. The human CD141+ DCs are considered the most clinically relevant for initiating CD8+ T cell responses critical for killing tumors or infected cells, and they specifically express the C-type lectin-like receptor CLEC9A that facilitates presentation of Ag by these DCs. We have therefore developed a human chimeric Ab that specifically targets CLEC9A on CD141+ DCs in vitro and in vivo. These human chimeric Abs are highly effective at delivering Ag to DCs for recognition by both CD4+ and CD8+ T cells. Given the importance of these cellular responses for antitumor or antiviral immunity, and the superior specificity of anti-CLEC9A Abs for this DC subset, this approach warrants further development for vaccines.