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Next-generation humanised mouse models and single-cell RNA sequencing (scRNAseq) approaches enable in-depth studies into human immune cell biology. Here we used NSG-SGM3 mice engrafted with human umbilical cord haematopoietic stem cells to investigate how human immune cells respond to and/or are changed by traumatic spinal cord injury (SCI). We hypothesised that the use of such mice could help advance our understanding of spinal cord injury-induced immune depression syndrome (SCI-IDS), and also how human leukocytes change as they migrate from the circulation into the lesion site. Our scRNAseq experiments, supplemented by flow cytometry, demonstrate the existence of up to 11 human immune cell (sub-) types and/or states across the blood and injured spinal cord (7 days post-SCI) of humanised NSG-SGM3 mice. Further comparisons of human immune cell transcriptomes between naïve, sham-operated and SCI mice identified a total of 579 differentially expressed genes, 190 of which were 'SCI-specific' (that is, genes regulated only in response to SCI but not sham surgery). Gene ontology analysis showed a prominent downregulation of immune cell function under SCI conditions, including for T cell receptor signalling and antigen presentation, confirming the presence of SCI-IDS and the transcriptional signature of human leukocytes in association with this phenomenon. We also highlight the activating influence of the local spinal cord lesion microenvironment by comparing the transcriptomes of circulating versus infiltrated human immune cells; those isolated from the lesion site were enriched for genes relating to both immune cell activity and function (e.g., oxidative phosphorylation, T cell proliferation and antigen presentation). We lastly applied an integrated bioinformatics approach to determine where immune responses in humanised NSG-SGM3 mice appear congruent to the native responses of human SCI patients, and where they diverge. Collectively, our study provides a valuable resource and methodological framework for the use of these mice in translational research., (© 2024. The Author(s).)

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) IL2Rγ null (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., Competing Interests: Conflict of Interest Disclosure The authors do not have any conflicts of interest to declare in relation to this work., (Copyright © 2023 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Hypoglycemia in type 1 diabetes associates with changes in the pancreatic islet α cells, where the receptor for advanced glycation end products (RAGE) is highly expressed. This study compared islet RAGE expression in donors without diabetes, those at risk of, and those with type 1 diabetes. Laser-dissected islets were subject to RNA bioinformatics and adjacent pancreatic tissue were assessed by confocal microscopy. We found that islets from type 1 diabetes donors had differential expression of the RAGE gene (AGER) and its correlated genes, based on glucagon expression. Random forest machine learning revealed that AGER was the most important predictor for islet glucagon levels. Conversely, a generalized linear model identified that glucagon expression could be predicted by expression of RAGE signaling molecules, its ligands and enzymes that create or clear RAGE ligands. Confocal imaging co-localized RAGE, its ligands and signaling molecules to the α cells. Half of the type 1 diabetes cohort comprised of adolescents and a patient with history of hypoglycemia-all showed an inverse relationship between glucagon and RAGE. These data confirm an association between glucagon and islet RAGE, its ligands and signaling pathways in type 1 diabetes, which warrants functional investigation into a role for RAGE in hypoglycemia., (© 2023. Springer Nature Limited.)

Antiviral CD8 + T cell immunity depends on the integration of various contextual cues, but how antigen-presenting cells (APCs) consolidate these signals for decoding by T cells remains unclear. Here, we describe gradual interferon-α/interferon-β (IFNα/β)-induced transcriptional adaptations that endow APCs with the capacity to rapidly activate the transcriptional regulators p65, IRF1 and FOS after CD4 + T cell-mediated CD40 stimulation. While these responses operate through broadly used signaling components, they induce a unique set of co-stimulatory molecules and soluble mediators that cannot be elicited by IFNα/β or CD40 alone. These responses are critical for the acquisition of antiviral CD8 + T cell effector function, and their activity in APCs from individuals infected with severe acute respiratory syndrome coronavirus 2 correlates with milder disease. These observations uncover a sequential integration process whereby APCs rely on CD4 + T cells to select the innate circuits that guide antiviral CD8 + T cell responses., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Poor maternal diet during pregnancy is a risk factor for severe lower respiratory infections (sLRIs) in the offspring, but the underlying mechanisms remain elusive. Here, we demonstrate that in mice a maternal low-fiber diet (LFD) led to enhanced LRI severity in infants because of delayed plasmacytoid dendritic cell (pDC) recruitment and perturbation of regulatory T cell expansion in the lungs. LFD altered the composition of the maternal milk microbiome and assembling infant gut microbiome. These microbial changes reduced the secretion of the DC growth factor Flt3L by neonatal intestinal epithelial cells and impaired downstream pDC hematopoiesis. Therapy with a propionate-producing bacteria isolated from the milk of high-fiber diet-fed mothers, or supplementation with propionate, conferred protection against sLRI by restoring gut Flt3L expression and pDC hematopoiesis. Our findings identify a microbiome-dependent Flt3L axis in the gut that promotes pDC hematopoiesis in early life and confers disease resistance against sLRIs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Dendritic cell (DC) vaccines are a safe and effective means of inducing tumor immune responses, however, a better understanding of DC biology is required in order to realize their full potential. Recent advances in DC biology have identified a crucial role for cDC1 in tumor immune responses, making this DC subset an attractive vaccine target. Human cDC1 exclusively express the C-type-lectin-like receptor, CLEC9A (DNGR-1) that plays an important role in cross-presentation, the process by which effective CD8 + T cell responses are generated. CLEC9A antibodies deliver antigen specifically to cDC1 for the induction of humoral, CD4 + and CD8 + T cell responses and are therefore promising candidates to develop as vaccines for infectious diseases and cancer. The development of human CLEC9A antibodies now facilitates their application as vaccines for cancer immunotherapy. Here we discuss the recent advances in CLEC9A targeting antibodies as vaccines for cancer and their translation to the clinic.

Type 1 diabetes is an autoimmune disease with no cure, where clinical translation of promising therapeutics has been hampered by the reproducibility crisis. Here, short-term administration of an antagonist to the receptor for advanced glycation end products (sRAGE) protected against murine diabetes at two independent research centers. Treatment with sRAGE increased regulatory T cells (Tregs) within the islets, pancreatic lymph nodes, and spleen, increasing islet insulin expression and function. Diabetes protection was abrogated by Treg depletion and shown to be dependent on antagonizing RAGE with use of knockout mice. Human Tregs treated with a RAGE ligand downregulated genes for suppression, migration, and Treg homeostasis (FOXP3, IL7R, TIGIT, JAK1, STAT3, STAT5b, CCR4). Loss of suppressive function was reversed by sRAGE, where Tregs increased proliferation and suppressed conventional T-cell division, confirming that sRAGE expands functional human Tregs. These results highlight sRAGE as an attractive treatment to prevent diabetes, showing efficacy and reproducibility at multiple research centers and in human T cells., (© 2022 by the American Diabetes Association.)

Introduction: The use of dendritic cell (DC)-based cancer vaccines over three decades has shown them to be a safe therapeutic approach against a range of hematological and solid malignancies. However, underwhelming and inconsistent results from clinical trials have seen them move in and out of favor. The limitations of ex vivo generated monocyte-derived DC (MoDC) in these therapies provide a pointed explanation for the varying and somewhat disappointing clinical outcomes. The identification of a specialized role for the rare conventional type 1 dendritic cell (cDC1) subset in orchestrating tumor immunity via the initiation of CD8 +  T cell responses has led to a new concept of targeting cDC1 as a therapeutic option to address the unmet need of enhancing the immune response in cancer patients., Areas Covered: This article reviews several current challenges and key opportunities associated with the development and use of next generation cDC1 cancer vaccines., Expert Opinion: Manipulation of cDC1 quantity and quality holds enormous potential to improve tumor immunogenicity, as already demonstrated in preclinical models. New technologies are rapidly advancing the understanding of cDC1 in human cancer patients and facilitating the generation of these extremely rare cells in vitro , providing feasible new approaches toward clinical translation.

Stimulator of Interferon Genes (STING) is a cytosolic sensor of cyclic dinucleotides (CDNs). The activation of dendritic cells (DC) via the STING pathway, and their subsequent production of type I interferon (IFN) is considered central to eradicating tumours in mouse models. However, this contribution of STING in preclinical murine studies has not translated into positive outcomes of STING agonists in phase I & II clinical trials. We therefore questioned whether a difference in human DC responses could be critical to the lack of STING agonist efficacy in human settings. This study sought to directly compare mouse and human plasmacytoid DCs and conventional DC subset responses upon STING activation. We found all mouse and human DC subsets were potently activated by STING stimulation. As expected, Type I IFNs were produced by both mouse and human plasmacytoid DCs. However, mouse and human plasmacytoid and conventional DCs all produced type III IFNs (i.e., IFN-λs) in response to STING activation. Of particular interest, all human DCs produced large amounts of IFN-λ1, not expressed in the mouse genome. Furthermore, we also found differential cell death responses upon STING activation, observing rapid ablation of mouse, but not human, plasmacytoid DCs. STING-induced cell death in murine plasmacytoid DCs occurred in a cell-intrinsic manner and involved intrinsic apoptosis. These data highlight discordance between STING IFN and cell death responses in mouse and human DCs and caution against extrapolating STING-mediated events in mouse models to equivalent human outcomes., Competing Interests: HH is an employee of Bavarian Nordic GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pang, Daraj, Balka, De Nardo, Macri, Hochrein, Masterman, Tan, Shoppee, Magill, Jahan, Bafit, Zhan, Kile, Lawlor, Radford, Wright and O’Keeffe.)

Objectives: DROSHA and DICER have central roles in the biogenesis of microRNAs (miRNAs). However, we previously showed that in the murine system, DROSHA has an alternate function where it directly recognises and cleaves protein-coding messenger (m)RNAs and this is critical for safeguarding the pluripotency of haematopoietic stem cells (HSCs). Maintenance of murine HSC function is dependent on DROSHA-mediated cleavage of two mRNAs, Myl9 and Todr1 . The goal of this study is to determine whether this pathway is conserved in human HSCs., Methods: DROSHA and DICER were knocked down in human cord blood CD34 + HSCs with short hairpin RNAs. The function of HSCs was analysed in vitro and in humanised mice. Analysis of mRNA cleavage was performed by capture of 5' phosphorylated RNAs., Results: Consistent with murine HSCs, DROSHA knockdown impaired the differentiation of human HSCs in vitro and engraftment into humanised mice, whereas DICER knockdown had no impact. DROSHA cleaves the MYL9 mRNA in human HSCs and DROSHA deficiency resulted in the accumulation of the mRNA. However, ectopic expression of MYL9 did not impair human HSC function. We were unable to identify a human homolog of Todr1., Conclusion: A miRNA-independent function of DROSHA is critical for the function of human HSCs. DROSHA directly recognises and degrades mRNAs in humans HSCs. However, unlike in murine HSCs, the degradation of the MYL9 mRNA alone is not critical for human HSC function. Therefore, DROSHA must be inhibiting other targets and/or has another miRNA-independent function that is essential for safeguarding the pluripotency of human HSCs., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

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., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Half of the mortality in diabetes is seen in individuals <50 years of age and commonly predicted by the early onset of diabetic kidney disease (DKD). In type 1 diabetes, increased urinary albumin-to-creatinine ratio (uACR) during adolescence defines this risk, but the pathological factors responsible remain unknown. We postulated that early in diabetes, glucose variations contribute to kidney injury molecule-1 (KIM-1) release from circulating T cells, elevating uACR and DKD risk. DKD risk was assigned in youth with type 1 diabetes ( n = 100; 20.0 ± 2.8 years; males/females, 54:46; HbA 1c 66.1 [12.3] mmol/mol; diabetes duration 10.7 ± 5.2 years; and BMI 24.5 [5.3] kg/m 2 ) and 10-year historical uACR, HbA 1c , and random blood glucose concentrations collected retrospectively. Glucose fluctuations in the absence of diabetes were also compared with streptozotocin diabetes in apolipoprotein E -/- mice. Kidney biopsies were used to examine infiltration of KIM-1-expressing T cells in DKD and compared with other chronic kidney disease. Individuals at high risk for DKD had persistent elevations in uACR defined by area under the curve (AUC; uACR AUC0-10yrs , 29.7 ± 8.8 vs. 4.5 ± 0.5; P < 0.01 vs. low risk) and early kidney dysfunction, including ∼8.3 mL/min/1.73 m 2 higher estimated glomerular filtration rates (modified Schwartz equation; P adj < 0.031 vs. low risk) and plasma KIM-1 concentrations (∼15% higher vs. low risk; P < 0.034). High-risk individuals had greater glycemic variability and increased peripheral blood T-cell KIM-1 expression, particularly on CD8 + T cells. These findings were confirmed in a murine model of glycemic variability both in the presence and absence of diabetes. KIM-1 + T cells were also infiltrating kidney biopsies from individuals with DKD. Healthy primary human proximal tubule epithelial cells exposed to plasma from high-risk youth with diabetes showed elevated collagen IV and sodium-glucose cotransporter 2 expression, alleviated with KIM-1 blockade. Taken together, these studies suggest that glycemic variations confer risk for DKD in diabetes via increased CD8 + T-cell production of KIM-1., (© 2021 by the American Diabetes Association.)

The endothelial adhesion protein E-selectin/CD62E is not required for leukocyte homing, unlike closely related family member P-selectin/CD62P. As transmigration through the endothelium is one of the first steps in generating a local immune response, we hypothesized that E-selectin may play additional roles in the early stages of immune activation. We found contact with E-selectin, but not P-selectin or vascular cell adhesion molecule 1 (CD106), induced phosphorylation of protein kinase B (AKT) and nuclear factor-κB in mouse bone marrow-derived macrophages (BMDMs) in vitro. This occurred within 15 min of E-selectin contact and was dependent on phosphatidylinositol-3 kinase activity. Binding to E-selectin activated downstream proteins including mammalian target of rapamycin, p70 ribosomal protein S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1. Functionally, adhesion to E-selectin induced upregulation of CD86 expression and CCL2 secretion. We next asked whether contact with E-selectin impacts further BMDM stimulation. We found enhanced secretion of both interleukin (IL)-10 and CCL2, but not tumor necrosis factor or IL-6 in response to lipopolysaccharide (LPS) stimulation after adhesion to E-selectin. Importantly, adhesion to E-selectin did not polarize BMDMs to one type of response but enhanced both arginase activity and nitric oxide production following IL-4 or LPS stimulation, respectively. In cultured human monocytes, adhesion to E-selectin similarly induced phosphorylation of AKT. Finally, when E-selectin was blocked in vivo in mice, thioglycollate-elicited macrophages showed reduced CD86 expression, validating our in vitro studies. Our results imply functions for E-selectin beyond homing and suggest that E-selectin plays an early role in priming and amplifying innate immune responses., (© 2021 Australian and New Zealand Society for Immunology Inc.)

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., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The dendritic cell receptor Clec9A facilitates processing of dead cell-derived antigens for cross-presentation and the induction of effective CD8 + T cell immune responses. Here, we show that this process is regulated by E3 ubiquitin ligase RNF41 and define a new ubiquitin-mediated mechanism for regulation of Clec9A, reflecting the unique properties of Clec9A as a receptor specialized for delivery of antigens for cross-presentation. We reveal RNF41 is a negative regulator of Clec9A and the cross-presentation of dead cell-derived antigens by mouse dendritic cells. Intriguingly, RNF41 regulates the downstream fate of Clec9A by directly binding and ubiquitinating the extracellular domains of Clec9A. At steady-state, RNF41 ubiquitination of Clec9A facilitates interactions with ER-associated proteins and degradation machinery to control Clec9A levels. However, Clec9A interactions are altered following dead cell uptake to favor antigen presentation. These findings provide important insights into antigen cross-presentation and have implications for development of approaches to modulate immune responses., Competing Interests: KT, PT, HP, RS, NM, RL, AP, EG, CH, AF, JD, PC, LW, JM, GR, KR, IC, MO, JV, MW, MB, WH, KS, AP, NN, JZ, ML No competing interests declared, (© 2020, Tullett et al.)

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-1β, 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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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 β-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., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Dendritic cells are a specialized subset of hematopoietic cells essential for mounting immunity against tumors and infectious disease as well as inducing tolerance for maintenance of homeostasis. DCs are equipped with number of immunoregulatory or stimulatory molecules that interact with other leukocytes to modulate their functions. Recent advances in DC biology identified a specific role for the conventional dendritic cell type 1 (cDC1) in eliciting cytotoxic CD8+ T cells essential for clearance of tumors and infected cells. The critical role of this subset in eliciting immune responses or inducing tolerance has largely been defined in mice whereas the biology of human cDC1 is poorly characterized owing to their extremely low frequency in tissues. A detailed characterization of the functions of many immunoregulatory and stimulatory molecules expressed by human cDC1 is critical for understanding their biology to exploit this subset for designing novel therapeutic modalities against cancer, infectious disease and autoimmune disorders., (© 2020 Elsevier Inc. All rights reserved.)

Many adenocarcinomas, including colorectal cancer (CRC), overexpress the MUC13 cell surface mucin, but the functional significance and mechanisms are unknown. Here, we report the roles of MUC13 in colonic tumorigenesis and tumor progression. High-MUC13 expression is associated with poor survival in two independent patient cohorts. In a comprehensive series of in vivo experiments, we identified a critical role for MUC13 in the development of this malignancy, by promoting survival and proliferation of tumor-initiating cells and driving an immunosuppressive environment that protects tumors from checkpoint inhibitor immunotherapy. In Muc13-deficient mice, fewer tumors are generated after exposure to carcinogens and inflammation, they have markedly reduced β-catenin signaling, have more tumor-infiltrating CD103 + dendritic cells and CD8 + T lymphocytes, fewer myeloid-derived suppressor cells, and are rendered sensitive to checkpoint inhibitor immunotherapy (anti-PD-L1). Mechanistically, we show that MUC13 protects β-catenin from degradation, by interacting with GSK-3β, which increases β-catenin nuclear translocation and promotes its signaling, thereby driving cancer initiation, progression, invasion, and immune suppression. Therefore, MUC13 is a potential marker of poor prognosis in colorectal cancer, and inhibiting MUC13 may be useful in the treatment of colitis-associated cancer and sensitizing tumors to immunotherapy.

Leukemias are clonal proliferative disorders arising from immature leukocytes in the bone marrow. While the advent of targeted therapies has improved survival in certain subtypes, relapse after initial therapy is a major problem. Dendritic cell (DC) vaccination has the potential to induce tumor-specific T cells providing long-lasting, anti-tumor immunity. This approach has demonstrated safety but limited clinical success until recently, as DC vaccination faces several barriers in both solid and hematological malignancies. Importantly, vaccine-mediated stimulation of protective immune responses is hindered by the aberrant production of immunosuppressive factors by cancer cells which impede both DC and T cell function. Leukemias present the additional challenge of severely disrupted hematopoiesis owing to both cytogenic defects in hematopoietic progenitors and an abnormal hematopoietic stem cell niche in the bone marrow; these factors accentuate systemic immunosuppression and DC malfunction. Despite these obstacles, several recent clinical trials have caused great excitement by extending survival in Acute Myeloid Leukemia (AML) patients through DC vaccination. Here, we review the phenotype and functional capacity of DCs in leukemia and approaches to harness DCs in leukemia patients. We describe the recent clinical successes in AML and detail the multiple new strategies that might enhance prognosis in AML and other leukemias.

Current HLA-typing methods are typically designed to provide exquisitely-detailed identification of multiple HLA-alleles to satisfy the requirements for organ and bone marrow transplantation or genetic studies. Many human immunological studies, on the other hand, focus around only a small number of HLA alleles that are abundant or of relevance to specific diseases. Consequently, for such studies, many HLA typing approaches are not cost-effective and are potentially complicated, slow and not easily performed in-house. Work-flow would be streamlined by a simple, inexpensive and rapid typing method able to be performed in-house. We outline a straightforward approach that provides appropriate data for much immunological research. In a predominantly Caucasian population, flow cytometry using anti-HLA-A2, -B8 and -B7 antibodies consistently and accurately screened for samples carrying the highly-abundant HLA class I alleles HLA-A*02:01, -B*08:01 and -B*07:02 that form the focus of immunological studies. Next, we describe a straightforward and simple strategy for design and use of allele-specific PCR primers to identify, at high-resolution, alleles of interest. When combined with a simple gDNA extraction technique this provides reliable, simple and inexpensive in-house HLA typing demonstrated here for highly-abundant HLA class I alleles., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cancer immunotherapy harnesses the ability of the immune system to recognize and eliminate cancer. The potent ability of dendritic cells (DCs) to initiate and regulate adaptive immune responses underpins the successful generation of anti-tumor immune responses. DCs are a heterogeneous leukocyte population comprised of distinct subsets that drive specific types of immune responses. Understanding how DCs induce tumor immune responses and the mechanisms adopted by tumors to evade DC surveillance is essential to render immunotherapies more effective. This review discusses current knowledge of the roles played by different DC subsets in human cancer and how these might be manipulated as new immunotherapeutics to improve CD8 + T cell-mediated immune responses, with a particular focus on the conventional type 1 DCs (cDC1)., (© 2019 Elsevier Inc. All rights reserved.)

Since the 1997 discovery that the first identified human homolog of Drosophila Toll could activate the innate immune system, the innate arm of immunity has rapidly taken on a new light as an important player in the recognition of pathogens and damaged self. The recognition of danger by dendritic cells (DC) is a crucial step in activating the adaptive immune system. Different DC express varied subsets of pattern recognition receptors (PRR), enabling both overlap and exclusivity in the recognition of danger signals by DC. PRR-mediated DC maturation and activation can be measured by changes in the surface expression of costimulatory as well as coinhibitory molecules, changes in size and shape of the DC and by their production of different cytokines.

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-β, 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., (© 2018 Australasian Society for Immunology Inc.)

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 .

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-1β, 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-1β, 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.

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.

Cross-presentation is the mechanism by which exogenous Ag is processed for recognition by CD8(+) T cells. Murine CD8α(+) DCs are specialized at cross-presenting soluble and cellular Ag, but in humans this process is poorly characterized. In this study, we examined uptake and cross-presentation of soluble and cellular Ag by human blood CD141(+) DCs, the human equivalent of mouse CD8α(+) DCs, and compared them with human monocyte-derived DCs (MoDCs) and blood CD1c(+) DC subsets. MoDCs were superior in their capacity to internalize and cross-present soluble protein whereas CD141(+) DCs were more efficient at ingesting and cross-presenting cellular Ag. Whilst cross-presentation by CD1c(+) DCs and CD141(+) DCs was dependent on the proteasome, and hence cytosolic translocation, cross-presentation by MoDCs was not. Inhibition of endosomal acidification enhanced cross-presentation by CD1c(+) DCs and MoDCs but not by CD141(+) DCs. These data demonstrate that CD1c(+) DCs, CD141(+) DCs, and MoDCs are capable of cross-presentation; however, they do so via different mechanisms. Moreover, they demonstrate that human CD141(+) DCs, like their murine CD8α(+) DC counterparts, are specialized at cross-presenting cellular Ag, most likely mediated by an enhanced capacity to ingest cellular Ag combined with subtle changes in lysosomal pH during Ag processing and use of the cytosolic pathway., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Central to adaptive immunity is the interaction between the αβ T cell receptor (TCR) and peptide presented by the major histocompatibility complex (MHC) molecule. Presumably reflecting TCR-MHC bias and T cell signaling constraints, the TCR universally adopts a canonical polarity atop the MHC. We report the structures of two TCRs, derived from human induced T regulatory (iT(reg)) cells, complexed to an MHC class II molecule presenting a proinsulin-derived peptide. The ternary complexes revealed a 180° polarity reversal compared to all other TCR-peptide-MHC complex structures. Namely, the iT(reg) TCR α-chain and β-chain are overlaid with the α-chain and β-chain of MHC class II, respectively. Nevertheless, this TCR interaction elicited a peptide-reactive, MHC-restricted T cell signal. Thus TCRs are not 'hardwired' to interact with MHC molecules in a stereotypic manner to elicit a T cell signal, a finding that fundamentally challenges our understanding of TCR recognition.

The method of choice for the development of new vaccines is to target distinct dendritic cell subsets with antigen in vivo and to harness their function in situ to enhance cell-mediated immunity or induce tolerance to specific antigens. The innate functions of dendritic cells themselves may also be targeted by inhibitors or activators that would target a specific function such as interferon production, potentially important in autoimmune disease and chronic viral infections. Importantly targeting dendritic cells requires detailed knowledge of both the surface phenotype and function of each dendritic cell subset, including how they may respond to different types of vaccine adjuvants, their ability to produce soluble mediators and to process and present antigens and induce priming of naïve T cells. This review summarizes our knowledge of the functional attributes of the human dendritic cell subsets in the steady state and upon activation and their roles in human disease.

Preclinical studies have suggested that purified populations of CD1c (BDCA-1) blood-derived dendritic cells (BDC) loaded with tumor-specific peptides may be a feasible option for prostate cancer immunotherapy. We performed an open-label dose-finding Phase I study to evaluate the safe use of CD1c BDC in patients with advanced metastatic hormone refractory prostate cancer. HLA-A*0201-positive patients with advanced metastatic prostate cancer were recruited and consented. The vaccine was manufactured by pulsing autologous CD1c BDC, prepared by magnetic bead immunoselection from apheresed peripheral blood mononuclear cells, with a cocktail of HLA-A*0201-restricted peptides (prostate-specific antigen, prostate acid phosphatase, prostate specific membrane antigen, and control influenza peptide) and keyhole limpet hemocyanin. The vaccine was administered intradermally or intravenously and peripheral blood was taken at predetermined intervals for clinical and immunologic monitoring. The vaccine was manufactured with a median purity of 82% CD1c BDC and administered successfully to 12 patients. Each patient received between 1 and 5 × 10 fresh CD1c BDC on day 0, followed by cryopreserved product in the same dose on days 28 and 56. The vaccine was well tolerated in all patients, with the most frequent adverse events being grade 1-2 fever, pain, or injection-site reactions. Vaccination with CD1c BDC is therefore feasible, safe, and well tolerated in patients with advanced-stage metastatic prostate cancer.

Antigen-presenting cells survey their environment and present captured antigens bound to major histocompatibility complex (MHC) molecules. Formation of MHC-antigen complexes occurs in specialized compartments where multiple protein trafficking routes, still incompletely understood, converge. Autophagy is a route that enables the presentation of cytosolic antigen by MHC class II molecules. Some reports also implicate autophagy in the presentation of extracellular, endocytosed antigen by MHC class I molecules, a pathway termed "cross-presentation." The role of autophagy in cross-presentation is controversial. This may be due to studies using different types of antigen presenting cells for which the use of autophagy is not well defined. Here we report that active use of autophagy is evident only in DC subtypes specialized in cross-presentation. However, the contribution of autophagy to cross-presentation varied depending on the form of antigen: it was negligible in the case of cell-associated antigen or antigen delivered via receptor-mediated endocytosis, but more prominent when the antigen was a soluble protein. These findings highlight the differential use of autophagy and its machinery by primary cells equipped with specific immune function, and prompt careful reassessment of the participation of this endocytic pathway in antigen cross-presentation.

Dendritic cells (DC) play an essential role in the induction and regulation of immune responses, including the generation of cytotoxic T lymphocytes (CTL) for the eradication of cancers. DC-based cancer vaccines are well tolerated with few side effects and can generate anti-tumour immune responses, but overall they have been of limited benefit. Recent studies have demonstrated that CD141(+) DC play an important role in anti-tumour responses. These are now attractive targets for the development of vaccines that directly target DC in vivo. An understanding of the functional specialisations of DC subsets, strategies for the delivery of tumour Ag to DC and for enhancing immune responses, point to promising new avenues for the design of more effective DC-based cancer vaccines., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

We established a humanized mouse model incorporating FLT3-ligand (FLT3-L) administration after hematopoietic cell reconstitution to investigate expansion, phenotype, and function of human dendritic cells (DC). FLT3-L increased numbers of human CD141(+) DC, CD1c(+) DC, and, to a lesser extent, plasmacytoid DC (pDC) in the blood, spleen, and bone marrow of humanized mice. CD1c(+) DC and CD141(+) DC subsets were expanded to a similar degree in blood and spleen, with a bias toward expansion of the CD1c(+) DC subset in the bone marrow. Importantly, the human DC subsets generated after FLT3-L treatment of humanized mice are phenotypically and functionally similar to their human blood counterparts. CD141(+) DC in humanized mice express C-type lectin-like receptor 9A, XCR1, CADM1, and TLR3 but lack TLR4 and TLR9. They are major producers of IFN-λ in response to polyinosinic-polycytidylic acid but are similar to CD1c(+) DC in their capacity to produce IL-12p70. Although all DC subsets in humanized mice are efficient at presenting peptide to CD8(+) T cells, CD141(+) DC are superior in their capacity to cross-present protein Ag to CD8(+) T cells following activation with polyinosinic-polycytidylic acid. CD141(+) DC can be targeted in vivo following injection of Abs against human DEC-205 or C-type lectin-like receptor 9A. This model provides a feasible and practical approach to dissect the function of human CD141(+) and CD1c(+) DC and evaluate adjuvants and DC-targeting strategies in vivo.

Dendritic cells (DC) play a pivotal role in the induction and regulation of immune responses, including the induction of cytotoxic T lymphocytes (CTL) responses. These are essential for the eradication of cancers and pathogens including HIV and malaria, for which there are currently no effective vaccines. New developments in our understanding of DC biology have identified the key DC subset responsible for CTL induction, which is now an attractive candidate to target for vaccination. These DC are characterized by expression of novel markers Clec9A and XCR1, and a specialized capacity to cross-present antigen (Ag) from tumors and pathogens that do not directly infect DC. New generation DC vaccines that specifically target the cross-presenting DC in vivo have already demonstrated potential in preclinical animal models but the challenge remains to translate these findings into clinically efficacous vaccines in man. This has been greatly facilitated by the recent identification of the equivalent Clec9A(+) XCR1(+) cross-presenting DC in human lymphoid tissues and peripheral tissues that are key sites for vaccination administration. These findings combined with further studies on DC subset biology have important implications for the design of new CTL-mediated vaccines.

The distribution and function of the C-type lectin Mincle has not previously been investigated in human cells, although mouse models have demonstrated a non-redundant role for Mincle in the host response to fungal infections. This study identified an unusual pattern of reciprocal expression of Mincle on peripheral blood monocytes or neutrophils isolated from the same donor. Expression on monocytes was inversely correlated with phagocytosis and yeast killing, but was necessary for the induction of inflammatory cytokines in response to ex vivo Candida challenge. In contrast, Mincle expression on neutrophils was associated with phagocytic and candidacidal potential of those cells. Candida challenge upregulated Mincle expression but only in Mincle+ cells. These data highlight species-specific differences between the regulation of Mincle expression in mouse and man. Reciprocal expression of Mincle modified the candidacidal potential of monocytes or neutrophils, suggesting it may also polarize the type of host response to fungal infection.

Human blood myeloid DCs can be subdivided into CD1c (BDCA-1)(+) and CD141 (BDCA-3)(+) subsets that display unique gene expression profiles, suggesting specialized functions. CD1c(+) DCs express TLR4 while CD141(+) DCs do not, thus predicting that these two subsets have differential capacities to respond to Escherichia coli. We isolated highly purified CD1c(+) and CD141(+) DCs and compared them to in vitro generated monocyte-derived DCs (MoDCs) following stimulation with whole E. coli. As expected, MoDCs produced high levels of the proinflammatory cytokines TNF, IL-6, and IL-12, were potent inducers of Th1 responses, and processed E. coli-derived Ag. In contrast, CD1c(+) DCs produced only low levels of TNF, IL-6, and IL-12 and instead produced high levels of the anti-inflammatory cytokine IL-10 and regulatory molecules IDO and soluble CD25. Moreover, E. coli-activated CD1c(+) DCs suppressed T-cell proliferation in an IL-10-dependent manner. Contrary to their mouse CD8(+) DC counterparts, human CD141(+) DCs did not phagocytose or process E. coli-derived Ag and failed to secrete cytokines in response to E. coli. These data demonstrate substantial differences in the nature of the response of human blood DC subsets to E. coli., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Immunotherapy is a promising new treatment for patients with advanced prostate and ovarian cancer, but its application is limited by the lack of suitable target antigens that are recognized by CD8+ cytotoxic T lymphocytes (CTL). Human kallikrein 4 (KLK4) is a member of the kallikrein family of serine proteases that is significantly overexpressed in malignant versus healthy prostate and ovarian tissue, making it an attractive target for immunotherapy. We identified a naturally processed, HLA-A*0201-restricted peptide epitope within the signal sequence region of KLK4 that induced CTL responses in vitro in most healthy donors and prostate cancer patients tested. These CTL lysed HLA-A*0201+ KLK4 + cell lines and KLK4 mRNA-transfected monocyte-derived dendritic cells. CTL specific for the HLA-A*0201-restricted KLK4 peptide were more readily expanded to a higher frequency in vitro compared to the known HLA-A*0201-restricted epitopes from prostate cancer antigens; prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA) and prostatic acid phosphatase (PAP). These data demonstrate that KLK4 is an immunogenic molecule capable of inducing CTL responses and identify it as an attractive target for prostate and ovarian cancer immunotherapy.

Acute lymphocytic leukemia (ALL) patients who relapse after transplantation have few therapeutic options. An immunotherapeutic approach that enhances the graft versus leukemia effect may improve their survival. We postulate that cytotoxic T lymphocytes (CTLs) generated from total RNA loaded cord blood CD34+-derived dendritic cells can control the kinetics of leukemic growth in a nonobese diabetic/severe combined immunodeficient (NOD-SCID) mouse model of human ALL. CD34+-derived dendritic cells electroporated with total RNA from an ALL xenograft generate antileukemic CTL with specificity for the ALL xenograft while sparing autologous cord blood mononuclear cells. The CD3+ T-cell compartment of the CTL was dominated by CD4+ T cells, although CD8+ T cells accounted for an average of 30% of the CD3+ T cells present. Expansion of both CD4+ and CD8+ memory and terminal effector memory subsets from predominantly naive cells was evident. Natural killer (NK) cells accounted for an average of 13% of the final antitumor lymphoid cells produced. Blocking experiments confirmed that the CD8+ T-cell compartment was responsible for the antileukemic activity of the polyclonal CTL pool. Administration of antileukemic CTL to NOD-SCID mice bearing ALL xenograft cells was able to delay, but not prevent the growth of ALL in vivo. Coadministration of antigen-loaded antigen-presenting cells did not further improve upon the delay in ALL engraftment kinetics observed with CTL alone. The efficacy of adoptively transferred polyclonal CTL can be improved with coadministration of recombinant human interleukin-2. However, in NOD-SCID mice, the efficacy of these adoptively transferred cells is masked by interleukin-2 stimulation of murine NK cells, which facilitate killing of ALL cells. Our data highlights the role for NK cells in antileukemic responses posttransplant. Collectively, our results support the notion that ALL-specific adoptive immunotherapy could be used clinically and provide an alternative strategy for preventing and treating disease relapse posttransplant and that the success of this therapy is likely to be maximized if given in the setting of minimal residual disease.

The characterization of human dendritic cell (DC) subsets is essential for the design of new vaccines. We report the first detailed functional analysis of the human CD141+ DC subset. CD141+ DCs are found in human lymph nodes, bone marrow, tonsil, and blood, and the latter proved to be the best source of highly purified cells for functional analysis. They are characterized by high expression of toll-like receptor 3, production of IL-12p70 and IFN-beta, and superior capacity to induce T helper 1 cell responses, when compared with the more commonly studied CD1c+ DC subset. Polyinosine-polycytidylic acid (poly I:C)-activated CD141+ DCs have a superior capacity to cross-present soluble protein antigen (Ag) to CD8+ cytotoxic T lymphocytes than poly I:C-activated CD1c+ DCs. Importantly, CD141+ DCs, but not CD1c+ DCs, were endowed with the capacity to cross-present viral Ag after their uptake of necrotic virus-infected cells. These findings establish the CD141+ DC subset as an important functionally distinct human DC subtype with characteristics similar to those of the mouse CD8alpha+ DC subset. The data demonstrate a role for CD141+ DCs in the induction of cytotoxic T lymphocyte responses and suggest that they may be the most relevant targets for vaccination against cancers, viruses, and other pathogens.

Human blood dendritic cells (DCs) are a rare, heterogeneous cell population that comprise approximately 1% of circulating peripheral blood mononuclear cells (PBMCs). Their isolation has been confounded by their scarcity and lack of distinguishing markers and their characterisation perplexed by the recent discovery of phenotypic and functionally distinct subsets. Human blood DCs are broadly defined as leukocytes that are HLA-DR positive and lack expression of markers specific for T cell, B cell, NK cell, monocyte and granulocyte lineages. They can be subdivided into the CD11c(-) (CD123(+)CD303(+)CD304(+)) plasmacytoid DC and CD11c(+) myeloid DC, which can be further subdivided into three subsets based on differential expression of CD1c, CD141 and CD16. DC can be isolated from peripheral blood by using an initial density gradient centrifugation step to enrich for mononuclear cells followed by immunomagnetic depletion of cells expressing markers specific for leukocyte lineages and undesired DC subsets. Subsequent flow cytometry-based cell sorting allows the isolation of highly pure individual DC subsets that can then be used for functional studies.