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Summary Myeloid cells, including dendritic cells (DCs), granulocytes, monocytes, monocyte-derived cells and macrophages, are important players in the immune response, but their identification is not as clear as lymphocytes, especially in tissues. This protocol details the step-by-step procedure for the analysis of myeloid populations in various mouse tissues by flow cytometry. For complete details on the use and execution of this protocol, please refer to Liu et al. (2019)., Graphical Abstract, Highlights • Preparation of single-cell suspension from mouse tissues • Analysis of tissue-resident macrophages in mouse tissues • Gating strategy for myeloid cells in mouse organs, Myeloid cells, including dendritic cells (DCs), granulocytes, monocytes, monocyte-derived cells and macrophages, are important players in the immune response, but their identification is not as clear as lymphocytes, especially in tissues. This protocol details the step-by-step procedure for the analysis of myeloid populations in various mouse tissues by flow cytometry.

SUMMARYMost tissue-resident macrophage (RTM) populations are seeded by waves of embryonic hematopoiesis and are self-maintained independently of a bone-marrow contribution during adulthood. A proportion of RTMs, however, is constantly replaced by blood monocytes and their functions compared to embryonic RTM remains unclear. The kinetics and extent of the contribution of circulating monocytes to RTM replacement during homeostasis, inflammation and disease is highly debated. Here, we identified Ms4a3 as a specific marker expressed by granulocyte-monocyte progenitors (GMPs) and subsequently generated Ms4a3TdT reporter and Ms4a3Cre-RosaTdT fate mapper models to follow monocytes and their progenies. Our Ms4a3Cre-RosaTdT model traced efficiently blood monocytes (97%) and granulocytes (100%), but no lymphocytes or tissue dendritic cells. Using this model, we precisely quantified the contribution of monocytes to the RTM pool during homeostasis and inflammation. The unambiguous identification of monocyte-derived cells will permit future studies of their function under any condition.

During gestation the developing human fetus is exposed to a diverse range of potentially immune-stimulatory molecules including semi-allogeneic antigens from maternal cells, substances from ingested amniotic fluid, food antigens, and microbes. Yet the capacity of the fetal immune system, including antigen-presenting cells, to detect and respond to such stimuli remains unclear. In particular, dendritic cells, which are crucial for effective immunity and tolerance, remain poorly characterized in the developing fetus. Here we show that subsets of antigen-presenting cells can be identified in fetal tissues and are related to adult populations of antigen-presenting cells. Similar to adult dendritic cells, fetal dendritic cells migrate to lymph nodes and respond to toll-like receptor ligation; however, they differ markedly in their response to allogeneic antigens, strongly promoting regulatory T-cell induction and inhibiting T-cell tumour-necrosis factor-α production through arginase-2 activity. Our results reveal a previously unappreciated role of dendritic cells within the developing fetus and indicate that they mediate homeostatic immune-suppressive responses during gestation.

Selection of antigens for therapeutic vaccination against chronic viral infections is complicated by pathogen genetic variations. We tested whether antigens present during persistent viral infections could provide a personalized antigenic reservoir for therapeutic T cell expansion in humans. We focused our study on the HBV surface antigen (HBsAg), which is present in microgram quantities in the serum of chronic HBV patients. We demonstrated by quantitative fluorescent microscopy that, out of 6 professional APC populations in the circulation, only CD14 monocytes (MNs) retained an HBsAg depot. Using TCR-redirected CD8 + T cells specific for MHC-I–restricted HBV epitopes, we showed that, despite being constantly exposed to antigen, ex vivo– isolated APCs did not constitutively activate HBV-specific CD8 + T cells. However, differentiation of HBsAg + CD14 MNs from chronic patients to MN-derived DCs (moDCs) induced cross-presentation of the intracellular reservoir of viral antigen. We exploited this mechanism to cross-present circulating viral antigen and showed that moDCs from chronically infected patients stimulated expansion of autologous HBV-specific T cells. Thus, these data demonstrate that circulating viral antigen produced during chronic infection can serve as a personalized antigenic reservoir to activate virus-specific T cells.

Summary Dendritic cell (DC)-mediated cross-presentation of exogenous antigens acquired in the periphery is critical for the initiation of CD8+ T cell responses. Several DC subsets are described in human tissues but migratory cross-presenting DCs have not been isolated, despite their potential importance in immunity to pathogens, vaccines, and tumors and tolerance to self. Here, we identified a CD141hi DC present in human interstitial dermis, liver, and lung that was distinct from the majority of CD1c+ and CD14+ tissue DCs and superior at cross-presenting soluble antigens. Cutaneous CD141hi DCs were closely related to blood CD141+ DCs, and migratory counterparts were found among skin-draining lymph node DCs. Comparative transcriptomic analysis with mouse showed tissue DC subsets to be conserved between species and permitted close alignment of human and mouse DC subsets. These studies inform the rational design of targeted immunotherapies and facilitate translation of mouse functional DC biology to the human setting., Graphical Abstract Highlights ► Human tissues contain CD1c+ DCs, CD14+ DCs, and a CD141hi cross-presenting DC subset ► CD141hi DCs migrate to draining lymph nodes and probably arise from blood CD141+ DCs ► Human tissue CD141hi DCs are homologous to mouse CD103+ or CD8+ DCs ► Human tissue CD1c+ DCs are homologous to mouse CD4+ DCs

Cancer vaccines aim to induce antitumor CTL responses, which require cross-presentation of tumor Ag to CTLs by dendritic cells (DCs). Adjuvants that facilitate cross-presentation of vaccine Ag are therefore key for inducing antitumor immunity. We previously reported that human DCs could not efficiently cross-present the full-length cancer/testis Ag NY-ESO-1 to CTL unless formulated as either an immune complex (NY-ESO-1/IC) or with ISCOMATRIX adjuvant. We now demonstrate that NY-ESO-1/ICs induce cross-presentation of HLA-A2- and HLA-Cw3-restricted epitopes via a proteasome-dependent pathway. In contrast, cross-presentation of NY-ESO-1/ISCOMATRIX vaccine was proteasome independent and required the cytosolic protease tripeptidyl peptidase II. Trafficking studies revealed that uptake of ICs and ISCOMATRIX vaccine by DCs occurred via endocytosis with delivery to lysosomes. Interestingly, ICs were retained in lysosomes, whereas ISCOMATRIX adjuvant induced rapid Ag translocation into the cytosol. Ag translocation was dependent on endosomal acidification and IL-4-driven differentiation of monocytes into DCs. This study demonstrates that Ag formulation determines Ag processing and supports a role for tripeptidyl peptidase II in cross-presentation of CTL epitopes restricted to diverse HLA alleles.

Plasmacytoid dendritic cells (PDCs) play powerful regulatory roles in innate and adaptive immune responses and are a major source of type I interferon (IFN) following viral infection. During inflammation and mechanical stress, cells release nucleotides into the extracellular space where they act as signaling molecules via G protein–coupled P2Y receptors. We have previously reported on the regulation of myeloid dendritic cell (DC) function by nucleotides. Here, we report that human PDCs express several subtypes of P2Y receptors and mobilize intracellular calcium in response to nucleotide exposure. As a functional consequence, PDCs acquire a mature phenotype that is further enhanced in the context of CD40 ligation. Strikingly, nucleotides strongly inhibit IFN-α secretion induced by influenza virus or CpG-A. This effect is most pronounced for the uridine nucleotides UDP and UTP and the sugar nucleotide UDP-glucose, ligands of P2Y6, P2Y4, and P2Y14, respectively. Nucleotide-induced inhibition of IFN-α production is blocked by suramin, a P2Y receptor antagonist. Pharmacological data point toward a role of protein kinase C in the negative regulation of type I IFN. Manipulating PDC function with P2Y receptor agonists may offer novel therapeutic strategies for autoimmune diseases or cancer.

The interleukin-12 (IL-12) cytokine family plays important roles in the orchestration of innate and adaptive immunity by dendritic cells (DCs). The regulation of IL-12 expression has been thoroughly studied, but little is known about factors governing the expression of IL-23 and IL-27, 2 novel IL-12 family members acting on memory and naive T cells, respectively. We report that the expression of these cytokines by DCs was critically dependent on the mode of activation. DC activation by CD40L predominantly induced IL-12. Ligands of the Toll-like receptor (TLR) 3 and TLR4 induced IL-12 and IL-27, whereas exposure to intact Escherichia coli resulted in high expression of IL-12, IL-27, and IL-23. The nucleotide adenosine triphosphate (ATP) has been shown to inhibit IL-12 production by P2 receptors. We found that ATP also inhibited IL-27 expression but enhanced IL-23 expression. Interestingly, the reciprocal regulation of IL-12/IL-27 and IL-23 by ATP was mediated by 2 distinct P2 receptors and was also induced by prostaglandin E2 by cyclic adenosine monophosphate (cAMP)–elevating EP2/EP4 receptors. As a consequence, DCs were selectively impaired in their ability to induce interferon-γ (IFN-γ) in naive T cells but continued to promote IFN-γ and IL-17 production in memory T cells. These studies identify P2 receptors as promising targets for the design of novel strategies to manipulate specific stages of T-cell responses and to treat IL-12– and IL-23–mediated disorders.

Plasmacytoid dendritic cells (PDCs) are potent regulators of immune function and the major source of type I interferon (IFN) following viral infection. PDCs are found at sites of inflammation in allergic reactions, autoimmune disorders, and cancer, but the mechanisms leading to the recruitment of PDCs to these sites remain elusive. During inflammation, adenosine is released and functions as a signaling molecule via adenosine receptors. This study analyzes adenosine receptor expression and function in human PDCs. Adenosine was found to be a potent chemotactic stimulus for immature PDCs via an A1 receptor–mediated mechanism. The migratory response toward adenosine was comparable to that seen with CXCL12 (stromal-derived factor-1α [SDF-1α), the most potent chemotactic stimulus identified thus far for immature PDCs. Upon maturation, PDCs down-regulate the A1 receptor, resulting in a loss of migratory function. In contrast, mature PDCs up-regulate the A2a receptor, which is positively coupled to adenylyl cyclase and has been implicated in the down-regulation of DC cytokine-producing capacity. We show that in mature PDCs adenosine reduces interleukin-6 (IL-6), IL-12, and IFN-α production in response to CpG oligodeoxynucleotides (ODN). These findings indicate that adenosine may play a dual role in PDC-mediated immunity by initially recruiting immature PDCs to sites of inflammation and by subsequently limiting the extent of the inflammatory response induced by mature PDCs by inhibiting their cytokine-producing capacity.

Dengue is a growing global concern with 390 million people infected each year. Dengue virus (DENV) is transmitted by mosquitoes, thus host cells in the skin are the first point of contact with the virus. Human skin contains several populations of antigen-presenting cells which could drive the immune response to DENV in vivo: epidermal Langerhans cells (LCs), three populations of dermal dendritic cells (DCs), and macrophages. Using samples of normal human skin we detected productive infection of CD14+ and CD1c+ DCs, LCs and dermal macrophages, which was independent of DC-SIGN expression. LCs produced the highest viral titers and were less sensitive to IFN-β. Nanostring gene expression data showed significant up-regulation of IFN-β, STAT-1 and CCL5 upon viral exposure in susceptible DC populations. In mice infected intra-dermally with DENV we detected parallel populations of infected DCs originating from the dermis and migrating to the skin-draining lymph nodes. Therefore dermal DCs may simultaneously facilitate systemic spread of DENV and initiate the adaptive anti-viral immune response., Author Summary Dengue virus (DENV) is transmitted by mosquitoes with skin as point of entry for the virus. Here, we investigated DENV infection in primary human skin cells and their initial immune response. Using skin from normal human donors for infection with DENV in vitro we identified antigen-presenting cells (APCs) as main targets of DENV. Further analysis showed that only distinct subsets of dendritic cells (DCs) and macrophages were infected and efficiently produced viral progeny. Langerhans cells were most susceptible to infection despite lacking DC-SIGN, a previously described DENV receptor. Infection of the other DC subsets and macrophages was also independent of DC-SIGN expression. Genes of the interferon pathway and CCL5, a chemokine attracting immune cells to sites of inflammation, were highly up-regulated in the infected DC subsets. Using a mouse infection model, we showed that murine dermal DCs were also susceptible to DENV and migrated to draining lymph nodes. At the same time infiltrating monocytes differentiated into monocyte-derived cells at the site of infection and became an additional target for DENV in vivo. These data demonstrate that DENV differentially infects and activates primary human skin APCs and that infected cell types individually contribute to inflammation and the adaptive response.

Summary Mouse and human dendritic cells (DCs) are composed of functionally specialized subsets, but precise interspecies correlation is currently incomplete. Here, we showed that murine lung and gut lamina propria CD11b+ DC populations were comprised of two subsets: FLT3- and IRF4-dependent CD24+CD64− DCs and contaminating CSF-1R-dependent CD24−CD64+ macrophages. Functionally, loss of CD24+CD11b+ DCs abrogated CD4+ T cell-mediated interleukin-17 (IL-17) production in steady state and after Aspergillus fumigatus challenge. Human CD1c+ DCs, the equivalent of murine CD24+CD11b+ DCs, also expressed IRF4, secreted IL-23, and promoted T helper 17 cell responses. Our data revealed heterogeneity in the mouse CD11b+ DC compartment and identifed mucosal tissues IRF4-expressing DCs specialized in instructing IL-17 responses in both mouse and human. The demonstration of mouse and human DC subsets specialized in driving IL-17 responses highlights the conservation of key immune functions across species and will facilitate the translation of mouse in vivo findings to advance DC-based clinical therapies., Highlights • Mucosal CD11b+ DCs consist of CD24+CD64− DCs and CD24−CD64+ macrophages • Mucosal CD24+CD11b+ DCs are IRF4-dependent • IRF4-dependent CD24+CD11b+ DCs secrete IL-23α and control mucosal IL-17 responses • Human CD1c+CD11b+ DCs are functional homologs of murine CD24+CD11b+ DCs

The ability of dendritic cells (DCs) to cross-present protein tumor antigens to cytotoxic T lymphocytes (CTLs) underpins the success of therapeutic cancer vaccines. We studied cross-presentation of the cancer/testis antigen, NY-ESO-1, and the melanoma differentiation antigen, Melan-A by human DC subsets. Monocyte-derived DCs (MoDCs) efficiently cross-presented human leukocyte associated (HLA)–A2-restricted epitopes from either a formulated NY-ESO-1/ISCOMATRIX vaccine or when either antigen was mixed with ISCOMATRIX adjuvant. HLA-A2 epitope generation required endosomal acidification and was proteasome-independent for NY-ESO-1 and proteasome-dependent for Melan-A. Both MoDCs and CD1c+ blood DCs cross-presented NY-ESO-1–specific HLA-A2157-165–, HLA-B760-72–, and HLA-Cw392-100–restricted epitopes when formulated as an NY-ESO-1/ISCOMATRIX vaccine, but this was limited when NY-ESO-1 and ISCOMATRIX adjuvant were added separately to the DC cultures. Finally, cross-presentation of NY-ESO-1157-165/HLA-A2, NY-ESO-160-72/HLA-B7, and NY-ESO-192-100/HLA-Cw3 epitopes was proteasome-dependent when formulated as immune complexes (ICs) but only proteasome-dependent for NY-ESO-160-72/HLA-B7–restricted cross-presentation facilitated by ISCOMATRIX adjuvant. We demonstrate, for the first time, proteasome-dependent and independent cross-presentation of HLA-A–, B–, and C–restricted epitopes within the same full-length tumor antigen by human DCs. Our findings identify important differences in the capacities of human DC subsets to cross-present clinically relevant, full-length tumor antigens and how vaccine formulation impacts CTL responses in vivo.

Activin-A is a transforming growth factor-beta (TGF-beta) superfamily member that plays a pivotal role in many developmental and reproductive processes. It is also involved in neuroprotection, apoptosis of tumor and some immune cells, wound healing, and cancer. Its role as an immune-regulating protein has not previously been described. Here we demonstrate for the first time that activin-A has potent autocrine effects on the capacity of human dendritic cells (DCs) to stimulate immune responses. Human monocyte-derived DCs (MoDCs) and the CD1c(+) and CD123(+) peripheral blood DC populations express both activin-A and the type I and II activin receptors. Furthermore, MoDCs and CD1c(+) myeloid DCs rapidly secrete high levels of activin-A after exposure to bacteria, specific toll-like receptor (TLR) ligands, or CD40 ligand (CD40L). Blocking autocrine activin-A signaling in DCs using its antagonist, follistatin, enhanced DC cytokine (IL-6, IL-10, IL-12p70, and tumor necrosis factor-alpha [TNF-alpha]) and chemokine (IL-8, IP-10, RANTES, and MCP-1) production during CD40L stimulation, but not TLR-4 ligation. Moreover, antagonizing DC-derived activin-A resulted in significantly enhanced expansion of viral antigen-specific effector CD8(+) T cells. These findings establish an immune-regulatory role for activin-A in DCs, highlighting the potential of antagonizing activin-A signaling in vivo to enhance vaccine immunogenicity.

Summary Dendritic cells (DCs), monocytes, and macrophages are leukocytes with critical roles in immunity and tolerance. The DC network is evolutionarily conserved; the homologs of human tissue CD141hiXCR1+CLEC9A+ DCs and CD1c+ DCs are murine CD103+ DCs and CD64−CD11b+ DCs. In addition, human tissues also contain CD14+ cells, currently designated as DCs, with an as-yet unknown murine counterpart. Here we have demonstrated that human dermal CD14+ cells are a tissue-resident population of monocyte-derived macrophages with a short half-life of, Graphical Abstract, Highlights • Human dermal CD14+ cells are a transient population of macrophages • Dermal CD14+ cells are derived from circulating blood monocytes • Human CD14+ cells are homologous to murine CD11b+CD64+ monocyte-derived macrophages • Human and mouse mononuclear phagocyte network organization is conserved, It is unclear whether human dermal CD14+ cells are bona fide dendritic cells. Haniffa and colleagues demonstrate that dermal CD14+ cells are monocyte-derived macrophages and complete the human and mouse tissue dendritic cell network alignment.

Dendritic cells (DCs) are being evaluated for cancer immunotherapy due to their unique ability to induce tumor-directed T-cell responses. Here we report that the type of human DC, the mode of activation, and the strategy for delivery of antigen are 3 critical factors for efficient stimulation of tumor-specific CD8+ and CD4+ T cells. Only CD1c+ blood DCs and monocyte-derived DCs (MoDCs) were capable of presenting epitopes of the full-length tumor antigen NY-ESO-1 on both major histocompatibility complex (MHC) class I (cross-presentation) and MHC II, whereas plasmacytoid DCs were limited to MHC II presentation. Cross-presentation was inefficient for soluble protein, but highly efficient for antigen-antibody immune complexes (NY-ESO-1/IC) and for protein formulated with ISCOMATRIX adjuvant (NY-ESO-1/IMX). DC activation with CD40L further enhanced cross-presentation efficiency. The mode of antigen delivery was found to be a determining factor for cytosolic proteolysis by DCs. Immune complexes (ICs) targeted a slow, proteasome-dependent cross-presentation pathway, whereas ISCOMATRIX (IMX) targeted a fast, proteasome-independent pathway. Both cross-presentation pathways resulted in a long-lived, T-cell stimulatory capacity, which was maintained for several days longer than for DCs pulsed with peptide. This may provide DCs with ample opportunities for sensitizing tumor-specific T cells against a broad array of tumor antigen epitopes in lymph nodes.

Dendritic cells (DCs) are a family of leukocytes that initiate T- and B-cell immunity against pathogens. Migration of antigen-loaded DCs from sites of infection into draining lymphoid tissues is fundamental to the priming of T-cell immune responses. In humans, the major peripheral blood DC (PBDC) types, CD1c+ DCs and interleukin 3 receptor–positive (IL-3R+) plasmacytoid DCs, are significantly expanded in vivo with the use of Flt3 ligand (FL). DC-like cells can also be generated from monocyte precursors (MoDCs). A detailed comparison of the functional potential of these types of DCs (in an autologous setting) has yet to be reported. Here, we compared the functional capacity of FL-expanded CD1c+ PBDCs with autologous MoDCs in response to 3 different classes of stimuli: (1) proinflammatory mediators, (2) soluble CD40 ligand trimer (CD40L), and (3) intact bacteria (Escherichia coli). Significant differences in functional capacities were found with respect to changes in phenotype, migratory capacity, cytokine secretion, and T-cell stimulation. MoDCs required specific stimuli for the expression of functions. They responded vigorously to CD40L or E coli, expressing cytokines known to regulate interferon-γ (IFN-γ) in T cells (IL-12p70, IL-18, and IL-23), but required prostaglandin E2 (PGE2) during stimulation to migrate to chemokines. In contrast, PBDCs matured in response to minimal stimulation, rapidly acquired migratory function in the absence of PGE2-containing stimuli, and were low cytokine producers. Interestingly, both types of DCs were equivalent with respect to stimulation of allogeneic T-cell proliferation and presentation of peptides to cytotoxic T lymphocyte (CTL) lines. These distinct differences are of particular importance when considering the choice of DC types for clinical applications.

Dendritic cells (DCs) are specialized antigen-presenting cells residing in tissues, from which they take up antigen. Activated DCs migrate through chemokine gradients from sites of inflammation to lymph nodes to stimulate T cells. At sites of inflammation, nucleotides, such as adenosine triphosphate (ATP), are released by activated or dying cells and can function as signaling molecules through P2 receptors (P2Rs). We investigated P2R expression in different DC populations and the effect of nucleotides on chemokine-directed migration. Exposure of monocyte-derived DCs (MoDCs) and CD1a+ dermal DCs to gradients of ATP inhibited their migratory capacity in a dose-dependent manner. Studies using P2R agonists and antagonists implicated signaling through the P2Y11R. On maturation, MoDCs down-regulated P2Y11R expression and were less sensitive to ATP-mediated inhibition of migration. In contrast, ATP did not inhibit the migration of CD1c+ peripheral blood (PB) DCs or interleukin-3 receptor-positive (IL-3R+) plasmacytoid DCs. Although all 4 DC populations expressed mRNA for P2Y11R, calcium-flux studies showed that blood DC types were unresponsive to P2Y11R agonists. In conclusion, DCs use distinct subtypes of P2R. The formation of ATP gradients at sites of inflammation may transiently inhibit the migration of local DCs, thus prolonging the time of antigen encounter. P2R inhibition may represent a new strategy to improve the migration of antigen-loaded DCs from the vaccination site to lymph nodes.

Adoptive transfer (AT) of autologous T cells genetically-redirected against tumor antigens has considerable potential as cancer immunotherapy [Kershaw, Nat Rev Immunol. 2005]. However, the in vivo persistence of AT T cells is critical for tumor control and requires the development (in vitro or in vivo) of a memory T cell subset. We investigated the generation of memory T cell subsets in a novel chimeric T cell receptor-expressing T cell product prior to, and after exposure to cognate antigen. Gene-modified T cells (LeY-T) express a chimeric receptor comprising a single chain variable fragment (scFv) specific for Lewis Y (LeY) antigen coupled to the intracellular signaling domains of CD3 zeta and CD28, capable of inducing T cell effector granule release and target killing [Westwood PNAS 2005]. To produce LeY-T cells, PBMC from healthy donors (n=20) or multiple myeloma patients (n=2) were cultured with anti-OKT3 (30ng/ml) and IL-2 (600IU/ml) for three days, followed by two rounds of transduction with retroviral supernatant. Subsequently, T cells were expanded in high dose IL-2 (600IU/ml) from day 5 onwards. T cells were harvested for this study on culture days 10–12, CD8+ and CD4+ T cells expressed the chimeric protein (50–60)%. LeY CD8+ T cell subsets were assessed as naïve (N), central memory (CM), effector memory (EM) or effector (E) based on three features:- phenotype (CD45RA, CCR7, CD28, CD27 and perforin); homeostatic cytokine (IL-15/IL-7) proliferation; response to Lewis antigen contact including cell proliferation and cytokine secretion. We repeatedly observed that CD8+ LeY-T cells analyzed directly from the initial expansion culture demonstrate an effector memory (EM) phenotype (CD45RA−/CCR7−/CD28+/perforinhi and variable CD27 expression) (Figure 1A). Furthermore in vitro expanded LeY CD8 T cells express IL- 15R beta (CD122) and the common gamma chain (CD132), they proliferate in response to IL-15 (86% cell division, division index 1.82), but less with IL-7 (30% cell division, division index 0.56). Baseline expanded CD8+ LeY-T cells respond to the presence of LeY antigen by proliferating and secreting IFN-gamma (4–8% of CD8 T cells) but not IL-2. Importantly, no IFN-gamma secretion was seen in control T cells transduced with empty vector (Figure 1B, OVCAR cells). Furthermore, no IFN-gamma was secreted by the control or the CD8+ LeY-T cells in response to the Lewis antigen negative cell line (Figure 1C, HCT116 cells). To explore the memory component further, we examined the functional status of the CD8+ LeY-T cells seven and 30 days following a 48-hour exposure to LeY antigen (OVCAR cells), and compared this to CD8+ LeY-T cell functional status at baseline. Thus, direct from transduction, expansion culture LeY CD8+ T cells were largely EM phenotype (95%) a small population of cells (1–5)% had a CM phenotype (CD45RA−/CCR7+/CD28+/perforinlo). In contrast, seven days after Lewis antigen contact the EM cells had decreased to (76–88)% and CM increased to (10–21)%; this distribution was retained up to day 30 post-antigen exposure. In addition, seven days after Lewis antigen exposure, CD8+ LeY-T cells retain the capacity to proliferate in response to Lewis antigen and to secrete IFN-gamma, at no stage do these cells secrete IL-2. In conclusion, the CD8+ LeY-T cells produced by in vitro transduction and expansion culture have an EM functional status direct from in vitro culture indicating that they are an appropriate starting population for in vivo adoptive transfer. After exposure to LeY expressed on tumor cell lines in vitro, CD8+ LeY T cells show further polarization to either EM or CM cells. These results suggest that the LeY-chimeric T cells have the potential to form long-term memory populations in vivo after adoptive transfer. Figure 1. LeY T cells have an effector memory phenotype and respond to Lewis antigen expressing cell lines by secreting IFN-gamma. Following the transduction culture, the CD8+ LeY-T cells (A) expressed high levels of perforin and an EM phenotype. In (B), LeY T or empty vector control T cells were co-cultured with tumour cells overnight and intracellular cytokine secretion assay performed. The LeY CD8+ T cells responded to Lewis antigen expressing OVCAR cells by secreting IFN-gamma, whereas no response was observed with the negative cell line HCT-116. Figure 1. LeY T cells have an effector memory phenotype and respond to Lewis antigen expressing cell lines by secreting IFN-gamma. Following the transduction culture, the CD8+ LeY-T cells (A) expressed high levels of perforin and an EM phenotype. In (B), LeY T or empty vector control T cells were co-cultured with tumour cells overnight and intracellular cytokine secretion assay performed. The LeY CD8+ T cells responded to Lewis antigen expressing OVCAR cells by secreting IFN-gamma, whereas no response was observed with the negative cell line HCT-116.

The proteasome inhibitor Bortezomib (Bz) is a potent inducer of plasma cell apoptosis via suppression of NFkB activity. Bz is also associated with the dose limiting induction of thrombocytopenia as well as inhibition of dendritic cell (DC) function, which may limit the induction of autologous T cell responses to myeloma antigens in vivo. We hypothesized that the dose, and therefore the adverse effects of Bz, may be reduced by the addition of the agonistic anti-TRAIL-R1 antibody Mapatumumab (Mp). A dose of Bz that did not result in detectable apoptosis was determined for each of the cultured human plasma cell lines (RPMI8226, U266, LP-1, NCI-H929, OPM-2 and JJN3) via titration experiments in which each cell line was cultured for 24–48 hrs in Bz concentrations from 0.1–10nM. Cells were stained with annexin V-FITC and the viability dye 7-AAD and analyzed on a BD LSRII flow cytometer. Plasma cell surface expression of TRAIL-R-1 and -2 were also assessed and correlated with sensitivity to Mp induced apoptosis. Bz monotherapy at 10nM produced 85–98% apoptosis in all plasma cell lines. In contrast 4/6 cell lines were sensitive to Mp alone, RPMI8226 at 0.06ug/ml, U266 and OPM-2 at 1ug/ml and LP-1 at 10ug/ml. When non-apoptosis inducing Bz doses were used in combination with titrated doses of Mp (from 0.01 to 50ug/ml) apoptosis of RPMI8226, U266 and OPM-2 was enhanced by 32%, 30% and 15% respectively compared to Mp alone (p= Figure 1. Combination bortezomib and Mapatumamab therapy induce synergistic death of the human myeloma cell line RPMI8226. A dose response study for (A) bortezomib (B) Mapatumamab was performed on RPMI8226 cells by treating the cells with titrated doses of the monotherapy for 48 hours in triplicate wells. Subsequently RPMI8226 cells were treated with combination bortezomib and Mapatumamab therapy (C). Bortezomib dose (1nM) did not induce apoptosis above untreated controls, Mp was added at doses from 0.03 to 0.15ug/ml. A statistical comparison was made between apoptosis with Mp monotherapy versus Bz plus Mp using student t-test, * represents data points where combination therapy is statistically different to Mp alone, p