Clinically applicable CD34+-derived blood dendritic cell subsets exhibit key subset-specific features and potently boost anti-tumor T and NK cell responses.

Allogeneic stem cell transplantation (alloSCT), following induction chemotherapy, can be curative for hemato-oncology patients due to powerful graft-versus-tumor immunity. However, disease recurrence remains the major cause of treatment failure, emphasizing the need for potent adjuvant immunotherapy. In this regard, dendritic cell (DC) vaccination is highly attractive, as DCs are the key orchestrators of innate and adaptive immunity. Natural DC subsets are postulated to be more powerful compared with monocyte-derived DCs, due to their unique functional properties and cross-talk capacity. Yet, obtaining sufficient numbers of natural DCs, particularly type 1 conventional DCs (cDC1s), is challenging due to low frequencies in human blood. We developed a clinically applicable culture protocol using donor-derived G-CSF mobilized CD34+ hematopoietic progenitor cells (HPCs) for simultaneous generation of high numbers of cDC1s, cDC2s and plasmacytoid DCs (pDCs). Transcriptomic analyses demonstrated that these ex vivo-generated DCs highly resemble their in vivo blood counterparts. In more detail, we demonstrated that the CD141+CLEG9A+ cDC1 subset exhibited key features of in vivo cDC1s, reflected by high expression of co-stimulatory molecules and release of IL-12p70 and TNF-α. Furthermore, cDC1s efficiently primed alloreactive T cells, potently cross-presented long-peptides and boosted expansion of minor histocompatibility antigen-experienced T cells. Moreover, they strongly enhanced NK cell activation, degranulation and anti-leukemic reactivity. Together, we developed a robust culture protocol to generate highly functional blood DC subsets for in vivo application as tailored adjuvant immunotherapy to boost innate and adaptive anti-tumor immunity in alloSCT patients.

CLEC12A-Mediated Antigen Uptake and Cross-Presentation by Human Dendritic Cell Subsets Efficiently Boost Tumor-Reactive T Cell Responses.

Potent immunotherapies are urgently needed to boost antitumor immunity and control disease in cancer patients. As dendritic cells (DCs) are the most powerful APCs, they are an attractive means to reinvigorate T cell responses. An appealing strategy to use the effective Ag processing and presentation machinery, T cell stimulation and cross-talk capacity of natural DC subsets is in vivo tumor Ag delivery. In this context, endocytic C-type lectin receptors are attractive targeting molecules. In this study, we investigated whether CLEC12A efficiently delivers tumor Ags into human DC subsets, facilitating effective induction of CD4(+) and CD8(+) T cell responses. We confirmed that CLEC12A is selectively expressed by myeloid cells, including the myeloid DC subset (mDCs) and the plasmacytoid DC subset (pDCs). Moreover, we demonstrated that these DC subsets efficiently internalize CLEC12A, whereupon it quickly translocates to the early endosomes and subsequently routes to the lysosomes. Notably, CLEC12A Ab targeting did not negatively affect DC maturation or function. Furthermore, CLEC12A-mediated delivery of keyhole limpet hemocyanin resulted in enhanced proliferation and cytokine secretion by keyhole limpet hemocyanin-experienced CD4(+) T cells. Most importantly, CLEC12A-targeted delivery of HA-1 long peptide resulted in efficient Ag cross-presentation by mDCs and pDCs, leading to strong ex vivo activation of HA-1-specific CD8(+) T cells of patients after allogeneic stem cell transplantation. Collectively, these data indicate that CLEC12A is an effective new candidate with great potential for in vivo Ag delivery into mDCs and pDCs, thereby using the specialized functions and cross-talk capacity of these DC subsets to boost tumor-reactive T cell immunity in cancer patients.

Positional identification of RT1-B (HLA-DQ) as susceptibility locus for autoimmune arthritis.

Rheumatoid arthritis (RA) is associated with amino acid variants in multiple MHC molecules. The association to MHC class II (MHC-II) has been studied in several animal models of RA. In most cases these models depend on T cells restricted to a single immunodominant peptide of the immunizing Ag, which does not resemble the autoreactive T cells in RA. An exception is pristane-induced arthritis (PIA) in the rat where polyclonal T cells induce chronic arthritis after being primed against endogenous Ags. In this study, we used a mixed genetic and functional approach to show that RT1-Ba and RT1-Bb (RT1-B locus), the rat orthologs of HLA-DQA and HLA-DQB, determine the onset and severity of PIA. We isolated a 0.2-Mb interval within the MHC-II locus of three MHC-congenic strains, of which two were protected from severe PIA. Comparison of sequence and expression variation, as well as in vivo blocking of RT1-B and RT1-D (HLA-DR), showed that arthritis in these strains is regulated by coding polymorphisms in the RT1-B genes. Motif prediction based on MHC-II eluted peptides and structural homology modeling suggested that variants in the RT1-B P1 pocket, which likely affect the editing capacity by RT1-DM, are important for the development of PIA.

Natural polymorphisms in Tap2 influence negative selection and CD4∶CD8 lineage commitment in the rat.

Genetic variation in the major histocompatibility complex (MHC) affects CD4∶CD8 lineage commitment and MHC expression. However, the contribution of specific genes in this gene-dense region has not yet been resolved. Nor has it been established whether the same genes regulate MHC expression and T cell selection. Here, we assessed the impact of natural genetic variation on MHC expression and CD4∶CD8 lineage commitment using two genetic models in the rat. First, we mapped Quantitative Trait Loci (QTLs) associated with variation in MHC class I and II protein expression and the CD4∶CD8 T cell ratio in outbred Heterogeneous Stock rats. We identified 10 QTLs across the genome and found that QTLs for the individual traits colocalized within a region spanning the MHC. To identify the genes underlying these overlapping QTLs, we generated a large panel of MHC-recombinant congenic strains, and refined the QTLs to two adjacent intervals of ∼0.25 Mb in the MHC-I and II regions, respectively. An interaction between these intervals affected MHC class I expression as well as negative selection and lineage commitment of CD8 single-positive (SP) thymocytes. We mapped this effect to the transporter associated with antigen processing 2 (Tap2) in the MHC-II region and the classical MHC class I gene(s) (RT1-A) in the MHC-I region. This interaction was revealed by a recombination between RT1-A and Tap2, which occurred in 0.2% of the rats. Variants of Tap2 have previously been shown to influence the antigenicity of MHC class I molecules by altering the MHC class I ligandome. Our results show that a restricted peptide repertoire on MHC class I molecules leads to reduced negative selection of CD8SP cells. To our knowledge, this is the first study showing how a recombination between natural alleles of genes in the MHC influences lineage commitment of T cells.

PD-L1 microSPECT/CT Imaging for Longitudinal Monitoring of PD-L1 Expression in Syngeneic and Humanized Mouse Models for Cancer.

Antibodies that block the interaction between programmed death ligand 1 (PD-L1) and PD-1 have shown impressive responses in subgroups of patients with cancer. PD-L1 expression in tumors seems to be a prerequisite for treatment response. However, PD-L1 is heterogeneously expressed within tumor lesions and may change upon disease progression and treatment. Imaging of PD-L1 could aid in patient selection. Previously, we showed the feasibility to image PD-L1+ tumors in immunodeficient mice. However, PD-L1 is also expressed on immune cell subsets. Therefore, the aim of this study was to assess the potential of PD-L1 micro single-photon emission tomography/computed tomography (microSPECT/CT) using radiolabeled PD-L1 antibodies to (i) measure PD-L1 expression in two immunocompetent tumor models (syngeneic mice and humanized mice harboring PD-L1 expressing immune cells) and (ii) monitor therapy-induced changes in tumor PD-L1 expression. We showed that radiolabeled PD-L1 antibodies accumulated preferentially in PD-L1+ tumors, despite considerable uptake in certain normal lymphoid tissues (spleen and lymph nodes) and nonlymphoid tissues (duodenum and brown fat). PD-L1 microSPECT/CT imaging could also distinguish between high and low PD-L1-expressing tumors. The presence of PD-L1+ immune cells did not compromise tumor uptake of the human PD-L1 antibodies in humanized mice, and we demonstrated that radiotherapy-induced upregulation of PD-L1 expression in murine tumors could be monitored with microSPECT/CT imaging. Together, these data demonstrate that PD-L1 microSPECT/CT is a sensitive technique to detect variations in tumor PD-L1 expression, and in the future, this technique may enable patient selection for PD-1/PD-L1-targeted therapy.

MCLA-117, a CLEC12AxCD3 bispecific antibody targeting a leukaemic stem cell antigen, induces T cell-mediated AML blast lysis.

Objective: We report the characterization of MCLA-117, a novel T cell-redirecting antibody for acute myeloid leukaemia (AML) treatment targeting CD3 on T cells and CLEC12A on leukaemic cells. In AML, CLEC12A is expressed on blasts and leukaemic stem cells. Methods: The functional capacity of MCLA-117 to redirect resting T cells to eradicate CLEC12APOS tumor cells was studied using human samples, including primary AML samples. Results: Within the normal hematopoietic compartment, MCLA-117 binds to cells expressing CD3 and CLEC12A but not to early myeloid progenitors or hematopoietic stem cells. MCLA-117 induces T cell activation (EC50 = 44 ng/mL), T cell proliferation, mild pro-inflammatory cytokine release, and redirects T cells to lyse CLEC12APOS target cells (EC50 = 68 ng/mL). MCLA-117-induced targeting of normal CD34POS cells co-cultured with T cells spares erythrocyte and megakaryocyte differentiation as well as preserves mono-myelocytic lineage development. In primary AML patient samples with autologous T cells, MCLA-117 robustly induced AML blast killing (23-98%) at low effector-to-target ratios (1:3-1:97). Conclusion: These findings demonstrate that MCLA-117 efficiently redirects T cells to kill tumour cells while sparing the potential of the bone marrow to develop the full hematological compartment and support further clinical evaluation as a potentially potent treatment option for AML.

Hematopoietic stem cell-derived myeloid and plasmacytoid DC-based vaccines are highly potent inducers of tumor-reactive T cell and NK cell responses ex vivo.

Because of the potent graft-versus-tumor (GVT) effect, allogeneic stem cell transplantation (alloSCT) can be a curative therapy for hematological malignancies. However, relapse remains the most frequent cause of treatment failure, illustrating the necessity for development of adjuvant post-transplant therapies to boost GVT immunity. Dendritic cell (DC) vaccination is a promising strategy in this respect, in particular, where distinct biologic functions of naturally occurring DC subsets, i.e. myeloid DCs (mDCs) and plasmacytoid DCs (pDCs), are harnessed. However, it is challenging to obtain high enough numbers of primary DC subsets from blood for immunotherapy due to their low frequencies. Therefore, we present here an ex vivo GMP-compliant cell culture protocol for generating different DC subsets from CD34+ hematopoietic stem and progenitor cells (HSPCs) of alloSCT donor origin. High numbers of BDCA1+ mDCs and pDCs could be generated, sufficient for multiple vaccination cycles. These HSPC-derived DC subsets were highly potent in inducing antitumor immune responses in vitro. Notably, HSPC-derived BDCA1+ mDCs were superior in eliciting T cell responses. They efficiently primed naïve T cells and robustly expanded patient-derived minor histocompatibility antigen (MiHA)-specific T cells. Though the HSPC-pDCs also efficiently induced T cell responses, they exhibited superior capacity in activating NK cells. pDC-primed NK cells highly upregulated TRAIL and possessed strong cytolytic capacity against tumor cells. Collectively, these findings indicate that HSPC-derived DC vaccines, comprising both mDCs and pDCs, may possess superior potential to boost antitumor immunity post alloSCT, due to their exceptional T cell and NK cell stimulatory capacity.

Association of MicroRNA-618 Expression With Altered Frequency and Activation of Plasmacytoid Dendritic Cells in Patients With Systemic Sclerosis.

OBJECTIVE: Plasmacytoid dendritic cells (PDCs) are a critical source of type I interferons (IFNs) that can contribute to the onset and maintenance of autoimmunity. Molecular mechanisms leading to PDC dysregulation and a persistent type I IFN signature are largely unexplored, especially in patients with systemic sclerosis (SSc), a disease in which PDCs infiltrate fibrotic skin lesions and produce higher levels of IFNα than those in healthy controls. This study was undertaken to investigate potential microRNA (miRNA)-mediated epigenetic mechanisms underlying PDC dysregulation and type I IFN production in SSc. METHODS: We performed miRNA expression profiling and validation in highly purified PDCs obtained from the peripheral blood of 3 independent cohorts of healthy controls and SSc patients. Possible functions of miRNA-618 (miR-618) on PDC biology were identified by overexpression in healthy PDCs. RESULTS: Expression of miR-618 was up-regulated in PDCs from SSc patients, including those with early disease who did not present with skin fibrosis. IFN regulatory factor 8, a crucial transcription factor for PDC development and activation, was identified as a target of miR-618. Overexpression of miR-618 reduced the development of PDCs from CD34+ cells in vitro and enhanced their ability to secrete IFNα, mimicking the PDC phenotype observed in SSc patients. CONCLUSION: Up-regulation of miR-618 suppresses the development of PDCs and increases their ability to secrete IFNα, potentially contributing to the type I IFN signature observed in SSc patients. Considering the importance of PDCs in the pathogenesis of SSc and other diseases characterized by a type I IFN signature, miR-618 potentially represents an important epigenetic target to regulate immune system homeostasis in these conditions.

The Aryl Hydrocarbon Receptor Antagonist StemRegenin1 Improves In Vitro Generation of Highly Functional Natural Killer Cells from CD34(+) Hematopoietic Stem and Progenitor Cells.

Early natural killer (NK)-cell repopulation after allogeneic stem cell transplantation (allo-SCT) has been associated with reduced relapse rates without an increased risk of graft-versus-host disease, indicating that donor NK cells have specific antileukemic activity. Therefore, adoptive transfer of donor NK cells is an attractive strategy to reduce relapse rates after allo-SCT. Since NK cells of donor origin will not be rejected, multiple NK-cell infusions could be administered in this setting. However, isolation of high numbers of functional NK cells from transplant donors is challenging. Hence, we developed a cytokine-based ex vivo culture protocol to generate high numbers of functional NK cells from granulocyte colony-stimulating factor (G-CSF)-mobilized CD34(+) hematopoietic stem and progenitor cells (HSPCs). In this study, we demonstrate that addition of aryl hydrocarbon receptor antagonist StemRegenin1 (SR1) to our culture protocol potently enhances expansion of CD34(+) HSPCs and induces expression of NK-cell-associated transcription factors promoting NK-cell differentiation. As a result, high numbers of NK cells with an active phenotype can be generated using this culture protocol. These SR1-generated NK cells exert efficient cytolytic activity and interferon-γ production toward acute myeloid leukemia and multiple myeloma cells. Importantly, we observed that NK-cell proliferation and function are not inhibited by cyclosporin A, an immunosuppressive drug often used after allo-SCT. These findings demonstrate that SR1 can be exploited to generate high numbers of functional NK cells from G-CSF-mobilized CD34(+) HSPCs, providing great promise for effective NK-cell-based immunotherapy after allo-SCT.

The aryl hydrocarbon receptor antagonist StemRegenin 1 promotes human plasmacytoid and myeloid dendritic cell development from CD34+ hematopoietic progenitor cells.

The superiority of dendritic cells (DCs) as antigen-presenting cells has been exploited in numerous clinical trials, where generally monocyte-derived DCs (Mo-DCs) are injected to induce immunity in patients with cancer or infectious diseases. Despite promising expansion of antigen-specific T cells, the clinical responses following vaccination have been limited, indicating that further improvements of DC vaccine potency are necessary. Pre-clinical studies suggest that vaccination with combination of primary DC subsets, such as myeloid and plasmacytoid blood DCs (mDCs and pDCs, respectively), may result in stronger clinical responses. However, it is a challenge to obtain high enough numbers of primary DCs for immunotherapy, since their frequency in blood is very low. We therefore explored the possibility to generate them from hematopoietic progenitor cells (HPCs). Here, we show that by inhibiting the aryl hydrocarbon receptor with its antagonist StemRegenin 1 (SR1), clinical-scale numbers of functional BDCA2(+)BDCA4(+) pDCs, BDCA1(+) mDCs, and BDCA3(+)DNGR1(+) mDCs can be efficiently generated from human CD34(+) HPCs. The ex vivo-generated DCs were phenotypically and functionally comparable to peripheral blood DCs. They secreted high levels of pro-inflammatory cytokines such as interferon (IFN)-α, interleukin (IL)-12, and tumor necrosis factor (TNF)-α and upregulated co-stimulatory molecules and maturation markers following stimulation with Toll-like receptor (TLR) ligands. Further, they induced potent allogeneic T-cell responses and activated antigen-experienced T cells. These findings demonstrate that SR1 can be exploited to generate high numbers of functional pDCs and mDCs from CD34(+) HPCs, providing an alternative option to Mo-DCs for immunotherapy of patients with cancer or infections.

Ex vivo generation of interstitial and Langerhans cell-like dendritic cell subset-based vaccines for hematological malignancies.

Autologous, patient-specific, monocyte-derived dendritic cell (MoDC) vaccines have been successfully applied in the clinical studies so far. However, the routine application of this strategy has been hampered by the difficulties in generating sufficient numbers of DC and the poor DC vaccine quality because of pathology or prior treatment received by the patients. The immunotherapeutic potential of other subsets of DC has not been thoroughly investigated because of their rarity in tissues and difficulties associated with their ex vivo generation. The high expansion and differentiation potential of CD34 hematopoietic progenitor cells (HPC), isolated from umbilical cord blood (UCB), into different DC subsets make them an attractive alternative DC source for cancer immunotherapy. Therefore, the aim of this study was to generate a large number of different DC subsets from CD34 HPC and evaluate their functionality in comparison with MoDC. Our culture protocol generated a clinically relevant number of mature CD1a myeloid DC and CD207 Langerhans cells (LC)-like DC subsets from CD34 HPC with >95% purity. Both DC subsets exhibited a cytokine profile that favors cytotoxic T-cell responses. Furthermore, UCB-DC and UCB-LC demonstrated superior induction of proliferation of both allogeneic as well as viral antigen-specific CD8 T cells, both in vitro and in vivo. Additional studies revealed that UCC-DC and UCB-LC can efficiently expand minor histocompatibility antigen (MiHA) HA-1-specific cytotoxic T cells in the peripheral blood of leukemia patients and prime MiHA HA-1-specific and HA-2-specific cytotoxic T cells in vitro. These preclinical findings support the pharmaceutical development of the described culture protocol for clinical evaluation.