The ability of cancer cells to ensure T-cell exclusion from the tumor microenvironment is a significant mechanism of resistance to anti-PD-1/PD-L1 therapy. Evidence indicates crucial roles of ...Batf3-dependent conventional type-1 dendritic cells (cDC1s) for inducing antitumor T-cell immunity; however, strategies to maximize cDC1 engagement remain elusive. Here, using multiple orthotopic tumor mouse models resistant to anti-PD-L1-therapy, we are testing the hypothesis that in situ induction and activation of tumor-residing cDC1s overcomes poor T-cell infiltration. In situ immunomodulation with Flt3L, radiotherapy, and TLR3/CD40 stimulation induces an influx of stem-like Tcf1
Slamf6
CD8
T cells, triggers regression not only of primary, but also untreated distant tumors, and renders tumors responsive to anti-PD-L1 therapy. Furthermore, serial in situ immunomodulation (ISIM) reshapes repertoires of intratumoral T cells, overcomes acquired resistance to anti-PD-L1 therapy, and establishes tumor-specific immunological memory. These findings provide new insights into cDC1 biology as a critical determinant to overcome mechanisms of intratumoral T-cell exclusion.
Given the limited efficacy of clinical approaches that rely on ex vivo generated dendritic cells (DCs), it is imperative to design strategies that harness specialized DC subsets in situ. This ...requires delineating the expression of surface markers by DC subsets among individuals and tissues. Here, we performed a multiparametric phenotypic characterization and unbiased analysis of human DC subsets in blood, tonsil, spleen, and skin. We uncovered previously unreported phenotypic heterogeneity of human cDC2s among individuals, including variable expression of functional receptors such as CD172a. We found marked differences in DC subsets localized in blood and lymphoid tissues versus skin, and a striking absence of the newly discovered Axl+ DCs in the skin. Finally, we evaluated the capacity of anti-receptor monoclonal antibodies to deliver vaccine components to skin DC subsets. These results offer a promising path for developing DC subset-specific immunotherapies that cannot be provided by transcriptomic analysis alone.
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•CyTOF reveals interindividual heterogeneity among DC subsets, especially cDC2s•Human skin harbors cDCs with a unique phenotype and lacks Axl+ DCs•Axl+ DCs display phenotypic and functional diversity, and pDCs exhibit plasticity•Receptor profiling identifies targets for antigen delivery to skin DC subsets
Dendritic cells (DCs) are potent initiators of immune responses; however, human DC subsets have yet to be successfully harnessed for immunotherapies. By combining CyTOF and unbiased analysis, Alcántara-Hernández et al. profile the heterogeneity of human DC subsets among individuals and tissues, providing comprehensive insights for the development of DC-based therapeutics.
Two subsets of conventional dendritic cells (cDCs) with distinct cell surface markers and functions exist in mouse and human. The two subsets of cDCs are specialized antigen-presenting cells that ...initiate T cell immunity and tolerance. In the mouse, a migratory cDC precursor (pre-CDC) originates from defined progenitors in the bone marrow (BM). Small numbers of short-lived pre-CDCs travel through the blood and replace cDCs in the peripheral organs, maintaining homeostasis of the highly dynamic cDC pool. However, the identity and distribution of the immediate precursor to human cDCs has not been defined. Using a tissue culture system that supports the development of human DCs, we identify a migratory precursor (hpre-CDC) that exists in human cord blood, BM, blood, and peripheral lymphoid organs. hpre-CDCs differ from premonocytes that are restricted to the BM. In contrast to earlier progenitors with greater developmental potential, the hpre-CDC is restricted to producing CD1c(+) and CD141(+) Clec9a(+) cDCs. Studies in human volunteers demonstrate that hpre-CDCs are a dynamic population that increases in response to levels of circulating Flt3L.
PD-1 checkpoint blockade has revolutionized the field of cancer immunotherapy, yet the frequency of responding patients is limited by inadequate T-cell priming secondary to a paucity of activatory ...dendritic cells (DC). DC signals can be bypassed by CD27 agonists, and we therefore investigated if the effectiveness of anti-PD-1/L1 could be improved by combining with agonist anti-CD27 monoclonal antibodies (mAb).
The efficacy of PD-1/L1 blockade or agonist anti-CD27 mAb was compared with a dual-therapy approach in multiple tumor models. Global transcriptional profiling and flow cytometry analysis were used to delineate mechanisms underpinning the observed synergy.
PD-1/PD-L1 blockade and agonist anti-CD27 mAb synergize for increased CD8
T-cell expansion and effector function, exemplified by enhanced IFNγ, TNFα, granzyme B, and T-bet. Transcriptome analysis of CD8
T cells revealed that combination therapy triggered a convergent program largely driven by IL2 and Myc. However, division of labor was also apparent such that anti-PD-1/L1 activates a cytotoxicity-gene expression program whereas anti-CD27 preferentially augments proliferation. In tumor models, either dependent on endogenous CD8
T cells or adoptive transfer of transgenic T cells, anti-CD27 mAb synergized with PD-1/L1 blockade for antitumor immunity. Finally, we show that a clinically relevant anti-human CD27 mAb, varlilumab, similarly synergizes with PD-L1 blockade for protection against lymphoma in human-CD27 transgenic mice.
Our findings suggest that suboptimal T-cell invigoration in cancer patients undergoing treatment with PD-1 checkpoint blockers will be improved by dual PD-1 blockade and CD27 agonism and provide mechanistic insight into how these approaches cooperate for CD8
T-cell activation.
.
Background
Mast cells (MC) are powerful inflammatory immune sentinel cells that drive numerous allergic, inflammatory, and pruritic disorders when activated. MC‐targeted therapies are approved in ...several disorders, yet many patients have limited benefit suggesting the need for approaches that more broadly inhibit MC activity. MCs require the KIT receptor and its ligand stem cell factor (SCF) for differentiation, maturation, and survival. Here we describe CDX‐0159, an anti‐KIT monoclonal antibody that potently suppresses MCs in human healthy volunteers.
Methods
CDX‐0159‐mediated KIT inhibition was tested in vitro using KIT‐expressing immortalized cells and primary human mast cells. CDX‐0159 safety and pharmacokinetics were evaluated in a 13‐week good laboratory practice (GLP)‐compliant cynomolgus macaque study. A single ascending dose (0.3, 1, 3, and 9 mg/kg), double‐blinded placebo‐controlled phase 1a human healthy volunteer study (n = 32) was conducted to evaluate the safety, pharmacokinetics, and pharmacodynamics of CDX‐0159.
Results
CDX‐0159 inhibits SCF‐dependent KIT activation in vitro. Fc modifications in CDX‐0159 led to elimination of effector function and reduced serum clearance. In cynomolgus macaques, multiple high doses were safely administered without a significant impact on hematology, a potential concern for KIT inhibitors. A single dose of CDX‐0159 in healthy human subjects was generally well tolerated and demonstrated long antibody exposure. Importantly, CDX‐0159 led to dose‐dependent, profound suppression of plasma tryptase, a MC‐specific protease associated with tissue MC burden, indicative of systemic MC suppression or ablation.
Conclusion
CDX‐0159 administration leads to systemic mast cell ablation and may represent a safe and novel approach to treat mast cell‐driven disorders.
This study presents the preclinical characterization, safety, pharmacokinetic and pharmacodynamic activity in a placebo‐controlled phase 1a healthy volunteer study of CDX‐0159, a specific and potent anti‐KIT inhibitory monoclonal antibody. CDX‐0159 inhibits SCF‐dependent KIT and mast cell activation. In a dose‐dependent manner, CDX‐0159 induces suppression of plasma tryptase – a marker of mast cell burden – showing a potential as a therapeutic strategy in mast cell‐driven disorders.Abbreviations: CDX‐0159, anti‐KIT inhibitory monoclonal antibody; FcR, Fc receptor; KIT, KIT proto‐oncogene, receptor tyrosine kinase; MRGPRX2, mas‐related G protein‐coupled receptor‐X2; SCF, stem cell factor
Human BDCA3(+) dendritic cells (DCs), the proposed equivalent to mouse CD8α(+) DCs, are widely thought to cross present antigens on MHC class I (MHCI) molecules more efficiently than other DC ...populations. If true, it is unclear whether this reflects specialization for cross presentation or a generally enhanced ability to present antigens on MHCI. We compared presentation by BDCA3(+) DCs with BDCA1(+) DCs using a quantitative approach whereby antigens were targeted to distinct intracellular compartments by receptor-mediated internalization. As expected, BDCA3(+) DCs were superior at cross presentation of antigens delivered to late endosomes and lysosomes by uptake of anti-DEC205 antibody conjugated to antigen. This difference may reflect a greater efficiency of antigen escape from BDCA3(+) DC lysosomes. In contrast, if antigens were delivered to early endosomes through CD40 or CD11c, BDCA1(+) DCs were as efficient at cross presentation as BDCA3(+) DCs. Because BDCA3(+) DCs and BDCA1(+) DCs were also equivalent at presenting peptides and endogenously synthesized antigens, BDCA3(+) DCs are not likely to possess mechanisms for cross presentation that are specific to this subset. Thus, multiple DC populations may be comparably effective at presenting exogenous antigens to CD8(+) T cells as long as the antigen is delivered to early endocytic compartments.
Dendritic cells (DCs) can capture extracellular antigens and load resultant peptides on to MHC class I molecules, a process termed cross presentation. The mechanisms of cross presentation remain ...incompletely understood, particularly in primary human DCs. One unknown is the extent to which antigen delivery to distinct endocytic compartments determines cross presentation efficiency, possibly by influencing antigen egress to the cytosol. We addressed the problem directly and quantitatively by comparing the cross presentation of identical antigens conjugated with antibodies against different DC receptors that are targeted to early or late endosomes at distinct efficiencies. In human BDCA1+ and monocyte-derived DCs, CD40 and mannose receptor targeted antibody conjugates to early endosomes, whereas DEC205 targeted antigen primarily to late compartments. Surprisingly, the receptor least efficient at internalization, CD40, was the most efficient at cross presentation. This did not reflect DC activation by CD40, but rather its relatively poor uptake or intra-endosomal degradation compared with mannose receptor or DEC205. Thus, although both early and late endosomes appear to support cross presentation in human DCs, internalization efficiency, especially to late compartments, may be a negative predictor of activity when selecting receptors for vaccine development.
Knockout (KO) mouse models play critical roles in elucidating biological processes behind disease-associated or disease-resistant traits. As a presumed consequence of gene KO, mice display certain ...phenotypes. Based on insight into the molecular role of said gene in a biological process, it is inferred that the particular biological process causally underlies the trait. This approach has been crucial towards understanding the basis of pathological and/or advantageous traits associated with
KO mice.
KO mice suffer from severe, early-onset retinal degeneration. MERTK, expressed in retinal pigment epithelia, is a receptor tyrosine kinase with a critical role in phagocytosis of apoptotic cells or cellular debris. Therefore, early-onset, severe retinal degeneration was described to be a direct consequence of failed MERTK-mediated phagocytosis of photoreceptor outer segments by retinal pigment epithelia. Here, we report that the loss of
alone is not sufficient for retinal degeneration. The widely used
KO mouse carries multiple coincidental changes in its genome that affect the expression of a number of genes, including the
paralog
. Retinal degeneration manifests only when the function of
is concomitantly lost. Furthermore,
KO mice display improved anti-tumor immunity. MERTK is expressed in macrophages. Therefore, enhanced anti-tumor immunity was inferred to result from the failure of macrophages to dispose of cancer cell corpses, resulting in a pro-inflammatory tumor microenvironment. The resistance against two syngeneic mouse tumor models observed in
KO mice is not, however, phenocopied by the loss of
alone. Neither
nor macrophage phagocytosis by alternate genetic redundancy accounts for the absence of anti-tumor immunity. Collectively, our results indicate that context-dependent epistasis of independent modifier alleles determines
KO traits.
Despite its prominent role as a C-type lectin (CTL) pattern recognition receptor, mannose receptor (MR, CD206)-specific signaling molecules and pathways are unknown. The MR is highly expressed ...on human macrophages, regulating endocytosis, phagocytosis, and immune responses and mediating Mycobacterium tuberculosis (M.tb) phagocytosis by human macrophages, thereby limiting phagosome-lysosome (P-L) fusion. We identified human MR-associated proteins using phosphorylated and non-phosphorylated MR cytoplasmic tail peptides. We found that MR binds FcRγ-chain, which is required for MR plasma membrane localization and M.tb cell association. Additionally, we discovered that MR-mediated M.tb association triggers immediate MR tyrosine residue phosphorylation and Grb2 recruitment, activating the Rac/Pak/Cdc-42 signaling cascade important for M.tb uptake. MR activation subsequently recruits SHP-1 to the M.tb-containing phagosome, where its activity limits PI(3)P generation at the phagosome and M.tb P-L fusion and promotes M.tb growth. In sum, we identify human MR signaling pathways that temporally regulate phagocytosis and P-L fusion during M.tb infection.
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•MR interaction with FcRγ-chain is essential for MR macrophage surface exposure•MR activation mediates Grb2 recruitment to initiate phagocytosis signaling•MR-dependent SHP-1 recruitment limits PI3K activity and phagosome-lysosome fusion
The human mannose receptor (MR) mediates macrophage phagocytosis and immune regulation. MR-specific signaling remains a major gap in the field. Rajaram et al. identify the importance of FcRγ-chain and Grb2 during MR-mediated phagocytosis and subsequent MR-dependent recruitment of SHP-1 to the M.tb phagosome, thereby limiting PI(3)P generation and phagosome-lysosome fusion.
Cyclophosphamide plus fludarabine (C/F) are currently used to improve the expansion and effectiveness of adoptive cell therapy (ACT). However, these chemotherapeutics cause pan-leukopenia and adverse ...events, suggesting that safer and more effective conditioning treatments are needed to improve ACT outcomes. Previously, we reported that varlilumab, a CD27-targeting antibody, mediates
T
reg
-preferential T cell depletion, CD8-T cell dominant costimulation, and systemic immune activation in hCD27 transgenic mice and cancer patients. We reasoned that the activities induced by varlilumab may provide an effective conditioning regimen for ACT. Varlilumab pretreatment of h
CD27
+
/
+
m
CD27
− /−
mice resulted in prominent proliferation of transferred T cells isolated from wild-type mice. These studies uncovered a critical role for CD27 signaling for the expansion of transferred T cells, as transfer of T cells from CD27 deficient mice or treatment with a CD70 blocking antibody greatly reduced their proliferation. In this model, varlilumab depletes endogenous h
CD27
+
/
+
T cells and blocks their subsequent access to CD70, allowing for more CD70 costimulation available to the m
CD27
+
/
+
transferred T cells. CD27-targeted depletion led to a greater expansion of transferred T cells compared to C/F conditioning and resulted in longer median survival and more cures than C/F conditioning in the E.G7 tumor model receiving OT-I cell therapy. We propose that translation of this work could be achieved through engineering of T cells for ACT to abrogate varlilumab binding but preserve CD70 ligation. Thus, varlilumab could be an option to chemotherapy as a conditioning regimen for ACT.
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