Programmed death receptor 1 (PD-1) ligand Fc fusion proteins reduce T-cell proliferation in vitro independently of PD-1.

Programmed death receptor 1 (PD-1) is an inhibitory receptor on T cells shown to restrain T-cell proliferation. PD-1 immune checkpoint blockade has emerged as a highly promising approach in cancer treatment. Much of our understanding of the function of PD-1 is derived from in vitro T-cell activation assays. Here we set out to further investigate how T cells integrate inhibitory signals such as PD-1 in vitro using the PD-1 agonist, PD-1 ligand 1 (PD-L1) fusion protein (PD-L1.Fc), coimmobilized alongside anti-CD3 agonist monoclonal antibody (mAb) on plates to deliver PD-1 signals to wild-type and PD-1-/- CD8+ T cells. Surprisingly, we found that the PD-L1.Fc fusion protein inhibited T-cell proliferation independently of PD-1. This PD-L1.Fc inhibition was observed in the presence and absence of CD28 and interleukin-2 signaling. Binding of PD-L1.Fc was restricted to PD-1-expressing T cells and thus inhibition was not mediated by the interaction of PD-L1.Fc with CD80 or other yet unknown binding partners. Furthermore, a similar PD-1-independent reduction of T-cell proliferation was observed with plate-bound PD-L2.Fc. Hence, our results suggest that the coimmobilization of PD-1 ligand fusion proteins with anti-CD3 mAb leads to a reduction of T-cell engagement with plate-bound anti-CD3 mAb. This study demonstrates a nonspecific mechanism of T-cell inhibition when PD-L1.Fc or PD-L2.Fc fusion proteins are delivered in a plate-bound coimmobilization assay and highlights the importance of careful optimization of assay systems and reagents when interpreting their influence on T-cell proliferation.

Genome-wide CRISPR screening identifies a role for ARRDC3 in TRP53-mediated responses.

Whole-genome screens using CRISPR technologies are powerful tools to identify novel tumour suppressors as well as factors that impact responses of malignant cells to anti-cancer agents. Applying this methodology to lymphoma cells, we conducted a genome-wide screen to identify novel inhibitors of tumour expansion that are induced by the tumour suppressor TRP53. We discovered that the absence of Arrestin domain containing 3 (ARRDC3) increases the survival and long-term competitiveness of MYC-driven lymphoma cells when treated with anti-cancer agents that activate TRP53. Deleting Arrdc3 in mice caused perinatal lethality due to various developmental abnormalities, including cardiac defects. Notably, the absence of ARRDC3 markedly accelerated MYC-driven lymphoma development. Thus, ARRDC3 is a new mediator of TRP53-mediated suppression of tumour expansion, and this discovery may open new avenues to harness this process for cancer therapy.

Display of Native Antigen on cDC1 That Have Spatial Access to Both T and B Cells Underlies Efficient Humoral Vaccination.

Follicular dendritic cells and macrophages have been strongly implicated in presentation of native Ag to B cells. This property has also occasionally been attributed to conventional dendritic cells (cDC) but is generally masked by their essential role in T cell priming. cDC can be divided into two main subsets, cDC1 and cDC2, with recent evidence suggesting that cDC2 are primarily responsible for initiating B cell and T follicular helper responses. This conclusion is, however, at odds with evidence that targeting Ag to Clec9A (DNGR1), expressed by cDC1, induces strong humoral responses. In this study, we reveal that murine cDC1 interact extensively with B cells at the border of B cell follicles and, when Ag is targeted to Clec9A, can display native Ag for B cell activation. This leads to efficient induction of humoral immunity. Our findings indicate that surface display of native Ag on cDC with access to both T and B cells is key to efficient humoral vaccination.

The Isolation and Enrichment of Large Numbers of Highly Purified Mouse Spleen Dendritic Cell Populations and Their In Vitro Equivalents.

Dendritic cells (DCs) form a complex network of cells that initiate and orchestrate immune responses against a vast array of pathogenic challenges. Developmentally and functionally distinct DC subtypes differentially regulate T-cell function. Importantly it is the ability of DC to capture and process antigen, whether from pathogens, vaccines, or self-components, and present it to naive T cells that is the key to their ability to initiate an immune response. Our typical isolation procedure for DC from murine spleen was designed to efficiently extract all DC subtypes, without bias and without alteration to their in vivo phenotype, and involves a short collagenase digestion of the tissue, followed by selection for cells of light density and finally negative selection for DC. The isolation procedure can accommodate DC numbers that have been artificially increased via administration of fms-like tyrosine kinase 3 ligand (Flt3L), either directly through a series of subcutaneous injections or by seeding with an Flt3L secreting murine melanoma. Flt3L may also be added to bone marrow cultures to produce large numbers of in vitro equivalents of the spleen DC subsets. Total DC, or their subsets, may be further purified using immunofluorescent labeling and flow cytometric cell sorting. Cell sorting may be completely bypassed by separating DC subsets using a combination of fluorescent antibody labeling and anti-fluorochrome magnetic beads. Our procedure enables efficient separation of the distinct DC subsets, even in cases where mouse numbers or flow cytometric cell sorting time is limiting.

Prosurvival Bcl-2 family members reveal a distinct apoptotic identity between conventional and plasmacytoid dendritic cells.

Dendritic cells (DCs) are heterogeneous, comprising subsets with functional specializations that play distinct roles in immunity as well as immunopathology. We investigated the molecular control of cell survival of two main DC subsets: plasmacytoid DCs (pDCs) and conventional DCs (cDCs) and their dependence on individual antiapoptotic BCL-2 family members. Compared with cDCs, pDCs had higher expression of BCL-2, lower A1, and similar levels of MCL-1 and BCL-XL. Transgenic overexpression of BCL-2 increased the pDC pool size in vivo with only minor impact on cDCs. With a view to immune intervention, we tested BCL-2 inhibitors and found that ABT-199 (the BCL-2 specific inhibitor) selectively killed pDCs but not cDCs. Conversely, genetic knockdown of A1 profoundly reduced the proportion of cDCs but not pDCs. We also found that conditional ablation of MCL-1 significantly reduced the size of both DC populations in mice and impeded DC-mediated immune responses. Thus, we revealed that the two DC types have different cell survival requirements. The molecular basis of survival of different DC subsets thus advocates the antagonism of selective BCL-2 family members for treating diseases pertaining to distinct DC subsets.

Bcl-2 antagonists kill plasmacytoid dendritic cells from lupus-prone mice and dampen interferon-α production.

OBJECTIVE: Interferon-α (IFNα)-producing plasmacytoid dendritic cells (PDCs) are implicated in the pathogenesis of systemic lupus erythematosus (SLE). IFNα-related genes are highlighted among SLE susceptibility alleles and are characteristically expressed in the blood of patients with SLE, while in mouse models of lupus, PDC numbers and IFNα production are increased. This study was undertaken to investigate the effects of inhibitors that selectively target different antiapoptotic molecules on the survival of PDCs. METHODS: PDC numbers, in vitro survival, and expression of antiapoptotic molecules were evaluated in lupus-prone (NZB × NZW)F1 (NZB/NZW) mice. The impact of Bcl-2 antagonists and glucocorticoids on PDCs was evaluated in vitro and in vivo. IFNα production by NZB/NZW mice was evaluated before and after treatment with Bcl-2 antagonists. RESULTS: PDCs, but not lymphoid tissue-resident conventional DCs, largely relied on the antiapoptotic protein Bcl-2 for survival. The enlarged PDC compartment in NZB/NZW mice was associated with selectively prolonged survival and increased Bcl-2 transcription. Functionally, this resulted in enhanced production of IFNα. Bcl-2 inhibitors selectively killed mouse and human PDCs, including PDCs from SLE patients, but not conventional DCs, dampened IFNα production by PDCs, and synergized with glucocorticoids to kill activated PDCs. CONCLUSION: Enhanced PDC survival is a likely contributing factor to enhanced IFNα production by lupus PDCs. Bcl-2 antagonists potently and selectively kill PDCs and reduce IFNα production. Thus, we believe that they are attractive candidates for treating PDC-associated diseases.

Maintaining dendritic cell viability in culture.

When mouse dendritic cells (DCs) are isolated from tissues, purified and placed in a nutritive culture they die more rapidly than would be expected from their normal turnover in vivo. This can distort culture assays of DC function. We therefore tested several approaches to prolonging DC survival in culture. Of several cytokines tested granulocyte-macrophage colony stimulating factor was most effective at preserving the viability of conventional DCs (cDCs) but was ineffective for plasmacytoid DCs (pDCs). Surprisingly, Fms-like tyrosine kinase 3 ligand, crucial for DC development, produced only a marginal improvement in DC survival in culture, and interleukin-3, reported to prevent apoptosis of human pDCs, produced only a minor improvement in survival of mouse DCs. Genetic manipulation of cell death pathways was also tested, to avoid activation effects exerted by cytokine signalling. The isolation of DCs from mice overexpressing Bcl-2 was especially effective in maintaining pDC viability but gave a lesser improvement in cDC viability. DCs isolated from Bim(-/-)Noxa(-/-) mice also showed improved culture survival, but in this case with pDCs showing the least improvement.

Lymphoid tissue and plasmacytoid dendritic cells and macrophages do not share a common macrophage-dendritic cell-restricted progenitor.

The relationship between dendritic cells (DCs) and macrophages is often debated. Here we ask whether steady-state, lymphoid-tissue-resident conventional DCs (cDCs), plasmacytoid DCs (pDCs), and macrophages share a common macrophage-DC-restricted precursor (MDP). Using new clonal culture assays combined with adoptive transfer, we found that MDP fractions isolated by previous strategies are dominated by precursors of macrophages and monocytes, include some multipotent precursors of other hematopoietic lineages, but contain few precursors of resident cDCs and pDCs and no detectable common precursors restricted to these DC types and macrophages. Overall we find no evidence for a common restricted MDP leading to both macrophages and FL-dependent, resident cDCs and pDCs.

GM-CSF-responsive monocyte-derived dendritic cells are pivotal in Th17 pathogenesis.

Although multiple dendritic cell (DC) subsets have the potential to induce Th17 differentiation in vitro, the key DC that is critical in Th17 induction and Th17-mediated disease remains moot. In this study, we revealed that CCR2(+) monocyte-derived DCs (moDCs), but not conventional DCs, were critical for in vivo Th17 induction and autoimmune inflammation. Functional comparison in vitro indicated that moDCs are the most potent type of Th17-inducing DCs compared with conventional DCs and plasmacytoid DCs. Furthermore, we demonstrated that the importance of GM-CSF in Th17 induction and Th17-mediated disease is its endowment of moDCs to induce Th17 differentiation in vivo, although it has little effect on moDC numbers. Our findings identify the in vivo cellular targets that can be selectively manipulated to ameliorate Th17-mediated inflammatory diseases, as well as the mechanism of GM-CSF antagonism in such diseases.

Plasmacytoid dendritic cell development.

Interferon-producing plasmacytoid dendritic cells (pDC) are a specialized branch of the dendritic cell (DC) family, and their differentiation in mice is closely linked to that of conventional DC (cDC). Several different developmental pathways retain the potential to form pDC and are likely to contribute to the steady-state pDC population. A lymphoid pathway to DC development produces mainly pDC as a branch otherwise leading to B-cell development; such pDC may carry relics of a lymphoid past such as DJ rearrangements of immunoglobulin heavy chain (IgH) genes. The myeloid pathway to pDC and cDC is better known, but recent reassessment has revealed several substreams of development with separate DC-committed precursors. One substream has a lymphoid-like aspect, involving a precursor expressing RAG-1 and producing pDC with IgH gene rearrangements. Another more biased to cDC production produces pDC without such IgH gene rearrangements. Finally, there is the production of interferon-producing pDC-like cells that are not pDC but appear to be cDC precursors; these do not express key pDC markers such as CCR9. Initiation of the DC and then the pDC developmental program overrides any surface marker-expressed developmental bias to other myeloid or lymphoid lineages, resulting in an apparent convergent differentiation to the pDC form. A DC fate is sometimes imprinted early in development, upstream of identifiable myeloid, or lymphoid precursors. This suggests that DC, including pDC, represent a distinct hematopoietic lineage separate from conventional myeloid or lymphoid cells.

The purification of large numbers of antigen presenting dendritic cells from mouse spleen.

Dendritic cells (DC) are found at low frequency in lymphoid and non-lymphoid tissues. Different DC subsets are adept at different roles in immunity in diverse scenarios of attack by infectious agents, as well as in the maintenance of self-tolerance. A key element in the ability of DC to initiate adaptive immune responses is their capacity to capture and process antigen, whether from pathogens, vaccines or self-components, and present it to T cells. Our typical procedure for isolation of the different DC types from murine spleen involves their digestion from the tissue using collagenase, selection of cells of light density, and negative selection for DC. DC may then be separated into their functionally distinct subpopulations using immunofluorescent labeling and flow cytometric cell sorting. If the availability of mice is limiting, our protocol can cater for DC numbers boosted by the administration of fms-like tyrosine kinase 3 ligand (Flt3L), directly via subcutaneous injection or via the introduction of a Flt3L secreting melanoma cell line. Large numbers of in vitro equivalents of the spleen DC subsets may also be produced by culturing bone marrow with Flt3L. If flow cytometric sorting time is a limitation splenic DC subpopulations may instead be separated using a combination of fluorescent antibody labeling and anti-fluorochrome magnetic beads. Careful segregation of these functionally distinct subpopulations of DC will enable a thorough examination of their antigen processing and presenting capabilities.

Convergent differentiation: myeloid and lymphoid pathways to murine plasmacytoid dendritic cells.

The developmental origin of IFN-producing plasmacytoid dendritic cells (pDCs) has been uncertain. In the present study, we tracked the development of pDCs in cultures of BM precursors stimulated with Flt3 ligand. Common myeloid precursors (CMPs) produced both conventional DCs (cDCs) and pDCs via the DC-restricted common DC precursor. Common lymphoid precursors (CLPs) produced only a few cDCs with variable efficiency, but produced pDCs via a transient intermediate precursor with B-cell potential. The pDCs of both origins produced IFN-α when stimulated with CpG oligonucleotides. The pDCs of CLP origin showed evidence of past RAG1 expression and had D-J rearrangements in IgH genes. Most pDCs and all cDCs of CMP origin lacked these signs of a lymphoid past. However, in these cultures, some pDCs of CMP origin showed evidence of past RAG1 expression and had D-J IgH gene rearrangements; most of these derived from a subset of CMPs already expressing RAG1.

Antibody responses initiated by Clec9A-bearing dendritic cells in normal and Batf3(-/-) mice.

Injection of antigens coupled to antibodies against the dendritic cell (DC) surface molecule Clec9A has been shown to produce strongly enhanced antibody responses even without co-administration of adjuvants, via antigen presentation by DC on MHC class II and consequent production of follicular helper T cells. A series of mutant mice were tested to determine the DC subtypes responsible for this MHC II presentation of targeted antigen, compared to presentation of antigen on MHC I. A new clec9A null mouse was developed; these mice did not give enhanced antibody production, confirming the response was dependent on Clec9A-expressing DC. However targeting of antigen to Clec9A in batf3 null mice produced enhanced antibody responses despite the marked reduction in CD8(+) DC, the major Clec9A-expressing DC subtype. This was shown to be dependent on efficient MHC II presentation by minor Clec9A-expressing DC subtypes in the environment of the Batf3(-/-) mice, namely early cells of the CD8 DC lineage and the plasmacytoid-related CD8(+) DC subset, but not by plasmacytoid cells themselves. However in normal mice most MHC II presentation of the Clec9A-targeted antigen was by the major CD8(+) DC population, the DC also responsible for presentation on MHC I.

Targeting antigen to mouse dendritic cells via Clec9A induces potent CD4 T cell responses biased toward a follicular helper phenotype.

Three surface molecules of mouse CD8(+) dendritic cells (DCs), also found on the equivalent human DC subpopulation, were compared as targets for Ab-mediated delivery of Ags, a developing strategy for vaccination. For the production of cytotoxic T cells, DEC-205 and Clec9A, but not Clec12A, were effective targets, although only in the presence of adjuvants. For Ab production, however, Clec9A excelled as a target, even in the absence of adjuvant. Potent humoral immunity was a result of the highly specific expression of Clec9A on DCs, which allowed longer residence of targeting Abs in the bloodstream, prolonged DC Ag presentation, and extended CD4 T cell proliferation, all of which drove highly efficient development of follicular helper T cells. Because Clec9A shows a similar expression pattern on human DCs, it has particular promise as a target for vaccines of human application.

Type I interferon drives dendritic cell apoptosis via multiple BH3-only proteins following activation by PolyIC in vivo.

BACKGROUND: DC are activated by pathogen-associated molecular patterns (PAMPs), and this is pivotal for the induction of adaptive immune responses. Thereafter, the clearance of activated DC is crucial to prevent immune pathology. While PAMPs are of major interest for vaccine science due to their adjuvant potential, it is unclear whether and how PAMPs may affect DC viability. We aimed to elucidate the possible apoptotic mechanisms that control activated DC lifespan in response to PAMPs, particularly in vivo. METHODOLOGY/PRINCIPAL FINDINGS: We report that polyinosinic:polycytidylic acid (PolyIC, synthetic analogue of dsRNA) induces dramatic apoptosis of mouse splenic conventional DC (cDC) in vivo, predominantly affecting the CD8α subset, as shown by flow cytometry-based analysis of splenic DC subsets. Importantly, while Bim deficiency conferred only minor protection, cDC depletion was prevented in mice lacking Bim plus one of three other BH3-only proteins, either Puma, Noxa or Bid. Furthermore, we show that Type I Interferon (IFN) is necessary and sufficient for DC death both in vitro and in vivo, and that TLR3 and MAVS co-operate in IFNß production in vivo to induce DC death in response to PolyIC. CONCLUSIONS/SIGNIFICANCE: These results demonstrate for the first time in vivo that apoptosis restricts DC lifespan following activation by PolyIC, particularly affecting the CD8α cDC subset. Such DC apoptosis is mediated by the overlapping action of pro-apoptotic BH3-only proteins, including but not solely involving Bim, and is driven by Type I IFN. While Type I IFNs are important anti-viral factors, CD8α cDC are major cross-presenting cells and critical inducers of CTL. We discuss such paradoxical finding on DC death with PolyIC/Type I IFN. These results could contribute to understand immunosuppression associated with chronic infection, and to the optimization of DC-based therapies and the clinical use of PAMPs and Type I IFNs.

Human dendritic cell subsets from spleen and blood are similar in phenotype and function but modified by donor health status.

Mouse dendritic cells (DC) have been extensively studied in various tissues, especially spleen, and they comprise subsets with distinct developmental origins, surface phenotypes, and functions. Considerably less is known about human DC due to their rarity in blood and inaccessibility of other human tissues. The study of DC in human blood has revealed four subsets distinct in phenotype and function. In this study, we describe four equivalent DC subsets in human spleen obtained from deceased organ donors. We identify three conventional DC subsets characterized by surface expression of CD1b/c, CD141, and CD16, and one plasmacytoid DC subset characterized by CD304 expression. Human DC subsets in spleen were very similar to those in human blood with respect to surface phenotype, TLR and transcription factor expression, capacity to stimulate T cells, cytokine secretion, and cross-presentation of exogenous Ag. However, organ donor health status, in particular treatment with corticosteroid methylprednisolone and brain death, may affect DC phenotype and function. DC T cell stimulatory capacity was reduced but DC were qualitatively unchanged in methylprednisolone-treated deceased organ donor spleen compared with healthy donor blood. Overall, our findings indicate that human blood DC closely resemble human spleen DC. Furthermore, we confirm parallels between human and mouse DC subsets in phenotype and function, but also identify differences in transcription factor and TLR expression as well as functional properties. In particular, the hallmark functions of mouse CD8α(+) DC subsets, that is, IL-12p70 secretion and cross-presentation, are not confined to the equivalent human CD141(+) DC but are shared by CD1b/c(+) and CD16(+) DC subsets.

The acquisition of antigen cross-presentation function by newly formed dendritic cells.

The development of Ag-presenting functions by murine dendritic cells (DCs) of the CD8(+) DC lineage was studied using a Flt-3 ligand stimulated bone-marrow culture system. Although newly formed DCs of this lineage are capable of Ag uptake and efficient presentation to T cells on MHC class II, they initially lack the ability to cross-present exogenous Ags on MHC class I. Cross-presentation capacity is acquired as a subsequent maturation step, promoted by cytokines such as GM-CSF. The development of cross-presentation capacity by the DCs in these cultures may be monitored by the parallel development of DC surface expression of CD103. However, the expression of CD103 and cross-presentation capacity are not always linked; therefore, CD103 is not an essential part of the cross-presentation machinery. These results explain the considerable variability in CD103 expression by CD8(+) DCs as well as the findings that not all DCs of this lineage are capable of cross-presentation.

Factors determining the spontaneous activation of splenic dendritic cells in culture.

Dendritic cells (DCs) serve as a link between the innate and adaptive immune systems. The activation state of DCs is crucial in this role. However, when DCs are isolated from lymphoid tissues, purified and placed in culture they undergo 'spontaneous' activation. The basis of this was explored, using up-regulation of DC surface MHC II, CD40, CD80 and CD86 as indicators of DC activation. No evidence was found for DC damage during isolation or for microbial products causing the activation. The culture activation of spleen DCs differed from that of Langerhans cells when released from E-cadherin-mediated adhesions, since E-cadherin was not detected and activation still occurred with ß-catenin null DCs. Much of the activation could be attributed to DC-DC interactions. Although increases in surface MHC II levels occurred under all culture conditions tested, the increase in expression of CD40, CD80 and CD86 was much less under culture conditions where such interactions were minimised. DC-to-DC contact under the artificial conditions of high DC concentration in culture induced the production of soluble factors and these, in turn, induced the up-regulation of co-stimulatory molecules on the DC surface.

Mouse CD8alpha+ DCs and human BDCA3+ DCs are major producers of IFN-lambda in response to poly IC.

Polyinosinic:polycytidylic acid (poly IC), a double-stranded RNA, is an effective adjuvant in vivo. IFN-λs (also termed IL-28/29) are potent immunomodulatory and antiviral cytokines. We demonstrate that poly IC injection in vivo induces large amounts of IFN-λ, which depended on hematopoietic cells and the presence of TLR3 (Toll-like receptor 3), IRF3 (IFN regulatory factor 3), IRF7, IFN-I receptor, Fms-related tyrosine kinase 3 ligand (FL), and IRF8 but not on MyD88 (myeloid differentiation factor 88), Rig-like helicases, or lymphocytes. Upon poly IC injection in vivo, the IFN-λ production by splenocytes segregated with cells phenotypically resembling CD8α(+) conventional dendritic cells (DCs [cDCs]). In vitro experiments revealed that CD8α(+) cDCs were the major producers of IFN-λ in response to poly IC, whereas both CD8α(+) cDCs and plasmacytoid DCs produced large amounts of IFN-λ in response to HSV-1 or parapoxvirus. The nature of the stimulus and the cytokine milieu determined whether CD8α(+) cDCs produced IFN-λ or IL-12p70. Human DCs expressing BDCA3 (CD141), which is considered to be the human counterpart of murine CD8α(+) DCs, also produced large amounts of IFN-λ upon poly IC stimulation. Thus, IFN-λ production in response to poly IC is a novel function of mouse CD8α(+) cDCs and their human equivalents.