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.

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.

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.

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.

Differential uptake and cross-presentation of soluble and necrotic cell antigen by human DC subsets.

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.