Myeloid-derived Cell Populations Research Articles

Multicolor flow cytometry-based immunophenotyping for preclinical characterization of nanotechnology-based formulations: an insight into structure activity relationship and nanoparticle biocompatibility profiles.

Immunophenotyping, which is the identification of immune cell subsets based on antigen expression, is an integral technique used to determine changes of cell composition and activation in various disease states or as a response to different stimuli. As nanoparticles are increasingly utilized for diagnostic and therapeutic applications, it is important to develop methodology that allows for the evaluation of their immunological impact. Therefore, the development of techniques such as immunophenotyping are desirable. Currently, the most common technique used to perform immunophenotyping is multicolor flow cytometry. We developed two distinct multicolor flow cytometry immunophenotyping panels which allow for the evaluation of the effects of nanoparticles on the composition and activation status of treated human peripheral blood mononuclear cells. These two panels assess the presence of various lymphoid and myeloid-derived cell populations as well as aspects of their activation statuses-including proliferation, adhesion, co-stimulation/presentation, and early activation-after treatment with controls or nanoparticles. To conduct assay performance qualification and determine the applicability of this method to preclinical characterization of nanoparticles, we used clinical-grade nanoformulations (AmBisome, Doxil and Feraheme) and research-grade PAMAM dendrimers of different sizes (G3, G4 and G5) and surface functionalities (amine-, carboxy- and hydroxy-). We found that formulations possessing intrinsic fluorescent properties (e.g., Doxil and AmBisome) interfere with accurate immunophenotyping; such interference may be partially overcome by dilution. In the absence of interference (e.g., in the case of dendrimers), nanoparticle size and surface functionalities determine their effects on the cells with large amine-terminated dendrimers being the most reactive.

TLR4 dependent generation of a novel bone marrow-derived inflammatory monocyte population required for elicitation of skin contact hypersensitivity responses

Abstract Contact hypersensitivity (CHS) is a CD8 T cell mediated response to hapten skin sensitization and challenge. Hapten-primed CD8 cell infiltration into the skin challenge site during elicitation of CHS requires prior infiltration and activation of a novel population of Gr-1 +CXCR2 +cells expressing FasL and perforin. The goal of these studies was to further characterize this novel population of myeloid-derived cells and to determine mechanisms generating the cells in the bone marrow during elicitation of CHS. The myeloid cells were purified from the bone marrow of hapten sensitized mice 2 hrs after hapten challenge by depletion of CD3 +, B220 +, NK1 +, F4/80 +, class II MHC +and Ly6G +cells. Flow cytometry analyses indicate >90% of the remaining bone marrow cells were CD11c +, Ly6C +, CCR2 +, expressing FasL +and perforin. This cell population was at low numbers in the bone marrow of naïve mice but increased to peak numbers 2 hrs. after hapten challenge of sensitized WT and Rag −/−mice and T-cell depleted WT mice. Generation of the myeloid cells was absent in hapten sensitized and challenged TLR4-deficient mice and was inhibited by treatment with anti-TNFa mAb at the time of hapten challenge. Whereas generation of neutrophils is inhibited by anti-G-CSF antibody, this treatment further increased numbers of these myeloid cells in the bone marrow following hapten challenge. These results indicate that generation of bone marrow-derived Ly6C +CXCR2 +cells expressing FasL and perforin in response to inflammation requires TNFa and TLR4 signaling that occurs independently of T cell activation.

CD68+ CD163+ M2-like Macrophages and Myeloid-Derived Suppressor Cells in Primary Immune Thrombocytopenia

BackgroundClinical investigations for the pathogenesis of immune thrombocytopenia (ITP) have focused on the aberrations related to adaptive immunity including platelet autoantibodies, T-cell clonality, and T helper differentiation. Recently, impaired myeloid-derived suppressor cells (MDSCs) have been implicated to participate in the pathogenesis of ITP, which could also act as a prognostic marker for treatment responses. However, another myeloid-derived cell population signified as M2 macrophages, has not been investigated properly in ITP patients. M2 macrophages, predominantly characterized as CD68+ (pan macrophages) and CD163+ (M2 specific), could promote immune modulation and tissue remodeling. Interestingly, a bidirectional cross-talk has been revealed between M2 macrophages known as “tumor associated macrophages” and MDSCs via high level of IL-10 in tumor microenvironments, which could result in facilitating tumor progression. These immunemodulation properties of MDSCs and M2 macrophages are posited to be severe disadvantages in tumor-bearing patients, while they might be beneficial in autoimmunity. In the present study, we intended to determine the features of circulating M2-like macrophages, to examine its relationship with MDSCs, and to explore their prognostic values in ITP.MethodsPBMC were isolated from the freshly collected peripheral blood of 10 healthy controls and 28 primary ITP patients. The circulating M2-like macrophages were defined as CD68+CD163+ (intracellular staining), and the phenotypic features of the circulating MDSCs were CD11b+CD33+HLA-DR- (surface staining). Stained cells were tested by FACS Aria II flow cytometer.ResultsThere are 17 patients in CR, and 11 patients in NR group. The population of circulating MDSCs expanded in newly diagnosed patients with ITP (p < 0.001), especially among the CR group (p < 0.001), but no significant difference was found in circulating M2-like macrophages. Positive linear correlation was verified between percentages of circulating M2-like macrophages and MDSCs (r= 0.15, p= 0.045). The same correlation was also determined in the CR group (r= 0.30, p= 0.021) while not in the NR group (r= 0.11, p= 0.313). Compared with those before the treatment, the percentages of both M2-like macrophages (p = 0.039) and MDSCs (p = 0.036) increased significantly after the treatment among patients in CR group, however, decreased number of MDSCs was found in NR group patients (p = 0.035).ConclusionsThe present investigation indicated critical roles of both circulating M2-like macrophages and MDSCs in the pathogenesis of ITP. The positive correlation between them might be related to some bidirectional interaction or partially due to their similar background patterns during differentiation. Further studies are required to demonstrate the cytokine profile related to the expansion of both MDSCs and M2 macrophages population in ITP patients. DisclosuresNo relevant conflicts of interest to declare.

Water extract from Euglena gracilis prevents lung carcinoma growth in mice by attenuation of the myeloid-derived cell population

The partially purified water extract from Euglena gracilis (EWE) was evaluated for its antitumor and immunomodulatory effects in cell cultures and in a mouse orthotopic lung carcinoma allograft model. In two-dimensional cell culture, the EWE treatment inhibited cell growth of both murine Lewis lung carcinoma (LLC) and human lung carcinoma cells (A549 and H1299) in a dose- and time-dependent manner. In contrast, the growth of mouse bone marrow cells (BMCs), but not mouse splenocytes (SPLs), was stimulated by the treatment with EWE. In three-dimensional spheroid culture, spheroid growth of LLC cells was significantly attenuated by EWE treatment. In a mouse LLC orthotopic allograft model, pretreatment with EWE (150–200 mg/kg/day, via drinking water) three weeks prior to the LLC cell inoculation, but not post-treatment after LLC cell inoculation, significantly attenuated the growth of LLC tumors in immunocompetent syngeneic mouse lung. This tumor growth attenuation coincided with a significant decrease in the population of myeloid-derived cells, primarily neutrophils. Flow cytometric analysis revealed that the EWE treatment significantly attenuated growth of granulocytic myeloid-derived suppressor cells (gMDSC) in BMCs and that this decrease was due to induction of gMDSC-specific apoptosis and differentiation of monocytic MDSCs (mMDSC) to macrophages. The present study provides evidence that EWE pretreatment inhibits lung carcinoma growth mainly by stimulating host antitumor immunity through attenuation of growth of gMDSCs and decreasing the number of peripheral granulocytes. This study suggests that the partially purified extract derived from Euglena gracilis contains significant bioactive materials that prevent lung carcinoma growth.

Distinguishing human peripheral blood CD16+ myeloid cells based on phenotypic characteristics.

Myeloid lineage cells present in human peripheral blood include dendritic cells (DC) and monocytes. The DC are identified phenotypically as HLA-DR+ cells that lack major cell surface lineage markers for T cells (CD3), B cells (CD19, CD20), NK cells (CD56), red blood cells (CD235a), hematopoietic stem cells (CD34), and Mo that express CD14. Both DC and Mo can be phenotypically divided into subsets. DC are divided into plasmacytoid DC, which are CD11c- , CD304+ , CD85g+ , and myeloid DC that are CD11c+ . The CD11c+ DC are readily classified as CD1c+ DC and CD141+ DC. Monocytes are broadly divided into the CD14+ CD16- (classical) and CD14dim CD16+ subsets (nonclassical). A population of myeloid-derived cells that have DC characteristics, that is, HLA-DR+ and lacking lineage markers including CD14, but express CD16 are generally clustered with CD14dim CD16+ monocytes. We used high-dimensional clustering analyses of fluorescence and mass cytometry data, to delineate CD14+ monocytes, CD14dim CD16+ monocytes (CD16+ Mo), and CD14- CD16+ DC (CD16+ DC). We sought to identify the functional and kinetic relationship of CD16+ DC to CD16+ Mo. We demonstrate that differentiation of CD16+ DC and CD16+ Mo during activation with IFNγ in vitro and as a result of an allo-hematopoietic cell transplant (HCT) in vivo resulted in distinct populations. Recovery of blood CD16+ DC in both auto- and allo-(HCT) patients after myeloablative conditioning showed similar reconstitution and activation kinetics to CD16+ Mo. Finally, we show that expression of the cell surface markers CD300c, CCR5, and CLEC5a can distinguish the cell populations phenotypically paving the way for functional differentiation as new reagents become available.

Rapid plasticity of intact axons following a lesion to the visual pathways during early brain development is triggered by microglial activation

Lesions in the central nervous system (CNS) can often induce structural reorganization within intact circuits of the brain. Several studies show advances in the understanding of mechanisms of brain plasticity and the role of the immune system activation. Microglia, a myeloid derived cell population colonizes the CNS during early phases of embryonic development. In the present study, we evaluated the role of microglial activation in the sprouting of intact axons following lesions of the visual pathways. We evaluated the temporal course of microglial activation in the superior colliculus following a contralateral monocular enucleation (ME) and the possible involvement of microglial cells in the plastic reorganization of the intact, uncrossed, retinotectal pathway from the remaining eye. Lister Hooded rats were enucleated at PND 10 and submitted to systemic treatment with inhibitors of microglial activation: cyclosporine A and minocycline. The use of neuroanatomical tracers allowed us to evaluate the time course of structural axonal plasticity. Immunofluorescence and western blot techniques were used to observe the expression of microglial marker, Iba-1 and the morphology of microglial cells. Following a ME, Iba-1 immunoreactivity showed a progressive increase of microglial activation in the contralateral SC at 24 h, peaking at 72 h after the lesion. Treatment with inhibitors of microglial activation blocked both the structural plasticity of intact uncrossed retinotectal axons and microglial activation as seen by the decrease of Iba-1 immunoreactivity. The local blockade of TNF-α with a neutralizing antibody was also able to block axonal plasticity of the intact eye following a ME. The data support the hypothesis that microglial activation is a necessary step for the regulation of neuroplasticity induced by lesions during early brain development.

Ferritin H Deficiency in Myeloid Compartments Dysregulates Host Energy Metabolism and Increases Susceptibility to Mycobacterium tuberculosis Infection.

Iron is an essential factor for the growth and virulence of Mycobacterium tuberculosis (Mtb). However, little is known about the mechanisms by which the host controls iron availability during infection. Since ferritin heavy chain (FtH) is a major intracellular source of reserve iron in the host, we hypothesized that the lack of FtH would cause dysregulated iron homeostasis to exacerbate TB disease. Therefore, we used knockout mice lacking FtH in myeloid-derived cell populations to study Mtb disease progression. We found that FtH plays a critical role in protecting mice against Mtb, as evidenced by increased organ burden, extrapulmonary dissemination, and decreased survival in Fth−/− mice. Flow cytometry analysis showed that reduced levels of FtH contribute to an excessive inflammatory response to exacerbate disease. Extracellular flux analysis showed that FtH is essential for maintaining bioenergetic homeostasis through oxidative phosphorylation. In support of these findings, RNAseq and mass spectrometry analyses demonstrated an essential role for FtH in mitochondrial function and maintenance of central intermediary metabolism in vivo. Further, we show that FtH deficiency leads to iron dysregulation through the hepcidin–ferroportin axis during infection. To assess the clinical significance of our animal studies, we performed a clinicopathological analysis of iron distribution within human TB lung tissue and showed that Mtb severely disrupts iron homeostasis in distinct microanatomic locations of the human lung. We identified hemorrhage as a major source of metabolically inert iron deposition. Importantly, we observed increased iron levels in human TB lung tissue compared to healthy tissue. Overall, these findings advance our understanding of the link between iron-dependent energy metabolism and immunity and provide new insight into iron distribution within the spectrum of human pulmonary TB. These metabolic mechanisms could serve as the foundation for novel host-directed strategies.

The histamine H4 receptor modulates the differentiation process of human monocyte-derived M1 macrophages and the release of CCL4/MIP-1β from fully differentiated M1 macrophages.

Histamine is an important mediator of biological functions and present in high amounts in inflammatory skin lesions which are characterised by a marked infiltration of myeloid derived cell populations. The aim of the study was to investigate the expression and function of histamine receptors, with a focus on the histamine H4 receptor (H4R) in detail during the differentiation process from monocytes to macrophages and on fully differentiated M1 macrophages. Quantitative PCR, ELISA technique, and flow cytometry were applied to analyze expression levels of histamine receptors, of CXCL10, CCL4, CCL3, or IL-23 and of the macrophage differentiation marker CD68, respectively. We demonstrated that monocytes and fully differentiated M1 macrophages express H1R-, H2R-, and H4RmRNA which were differentially regulated during the differentiation process and in IFN-Ƴ and LPS classically activated M1 macrophages. The H3RmRNA was not expressed. During in vitro differentiation from monocytes to macrophages, the H4R agonist ST-1006 modified the M1 phenotype by up-regulating the macrophage differentiation marker CD68, by down-regulating the production of CXCL10, and by changing the morphology. In fully differentiated M1 macrophages, histamine or ST-1006 decreased the IFN-Ƴ- and LPS-induced CCL4 mRNA expression and protein production, whereas CCL3 or IL-23 production was not regulated via H4R. We describe novel immunomodulatory functions of the H4R during the differentiation process of human monocyte-derived macrophages and in fully differentiated M1 macrophages. The down-regulation of Th1-related chemokines during the differentiation process or in classically activated macrophages via H4R may contribute to decreased migration of immune cells to the site of inflammation. This may have implications for the treatment of allergic diseases with H4R ligands regulating the dysbalance of Th2/Th1 polarizations in these disorders.

Distinct alterations of CD68+CD163+ M2-like macrophages and myeloid-derived suppressor cells in newly diagnosed primary immune thrombocytopenia with or without CR after high-dose dexamethasone treatment

BackgroundAlthough impaired myeloid-derived suppressor cells (MDSCs) recently have been studied in immune thrombocytopenia (ITP), another myeloid-derived cell population signified as M2 macrophages has not been investigated properly in ITP patients. In the present study, we intended to determine the features of circulating M2-like macrophages, to examine its relationship with MDSCs, and to explore their prognostic values in ITP.MethodsPeripheral blood mononuclear cells from healthy controls and primary ITP patients were isolated to test the circulating M2-like macrophages and MDSCs. The circulating M2-like macrophage population defined as CD68+CD163+ and circulating MDSC population as CD11b+CD33+HLA-DR− were determined by flow cytometry. Plasma inflammatory cytokines were measured by multiplex ELISA.ResultsThe percentages of MDSCs were found to be expanded in newly diagnosed patients of ITP, especially among those of the complete response (CR) group (p < 0.0001). Positive linear correlation was verified between percentages of M2-like macrophages and MDSCs. The same correlation was also determined in the CR group. After treatment, the percentages of M2-like macrophages and MDSCs were both increased significantly in CR group, while those patients among the PR + NR group manifested a significant numeric decrease of MDSCs but only a moderate decrease in M2-like macrophages. MIP-1α/CCL3 was negatively correlated with M2-like macrophages while MCP-1 possessed a positive correlation with M2-like macrophages, eotaxin-1/CCL11 was negatively correlated with MDSCs and interleukin-1β (IL-1β) was found to be negatively correlated with both M2-like macrophages and MDSCs.ConclusionsThe present findings indicated critical roles of both circulating M2-like macrophages and MDSCs in ITP. The positive correlation between them might be related to inflammatory factors-mediated bidirectional interactions or partially due to their similar background patterns during differentiation. MIP-1α/CCL3, MCP-1, eotaxin-1/CCL11 and IL-1β might play a critical role in the expansion of both M2 macrophages and MDSCs population in ITP patients, which deserves further investigation.

Hapten-induced generation of a novel myeloid-derived FasL+ perforin+cell population in the bone marrow is induced by TNFa

Abstract Contact hypersensitivity (CHS) is a CD8 T cell mediated response to hapten skin sensitization and challenge. Hapten-primed CD8 cell infiltration into the skin challenge site during elicitation of CHS requires prior infiltration and activation of a population of Gr-1+CXCR2+ cells expressing FasL and perforin. The goal of these studies was to further characterize this novel population of myeloid-derived cells and to determine mechanisms generating the cells in the bone marrow during elicitation of CHS. The myeloid cells were purified from the bone marrow of hapten sensitized mice 3 hrs after happen challenge by depletion of CD3+, B220+, NK1+, F4/80+, class II MHC+ and Ly6G+ cells. Flow cytometry analyses indicate &amp;gt;90% of the remaining bone marrow cells were CD11c+, Ly6C+, CCR2+, CX3CR1+ and FasL+ and express perforin and granzyme B. This cell population was at low numbers in the bone marrow of naïve mice but increased to peak numbers at 2 and 6 hrs. after hapten challenge of sensitized WT and Rag−/− mice and T-cell depleted WT mice. Whereas generation of neutrophils is inhibited by anti-G-CSF antibody, this treatment further increased numbers of these myeloid cells in the bone marrow following hapten challenge. Generation of the myeloid cells was absent in hapten sensitized and challenged TLR4- and IL-1 receptor-deficient mice and was inhibited by treatment with anti-TNFa mAb at the time of hapten challenge. These results indicate that generation of bone marrow-derived Ly6C+CXCR2+ cells expressing FasL and perforin/granzyme B in response to inflammation requires TNFa, IL-1 and TLR4 signaling but occurs independently of T cell activation.

Finding the evolutionary precursor of vertebrate hematopoietic lineage: Functional and molecular characterization of B. schlosseri immune system.

Abstract We are characterizing the immune system and cell populations of the tunicate model Botryllus schlosseri on functional and molecular levels. The chordate B. schlosseri belongs to a group that is considered the closest living invertebrate relative of vertebrates. Using FACS analysis we can isolate 12 populations of B. schlosseri cells by size, granularity and auto fluorescence. In order to increase the number of isolated cell populations, we used Cytof to scan large numbers of antibodies. Antibodies that differentially bind to B. schlosseri cells were validated by flow cytometry. Serum against the Botryllus Histocompatibility Factor, and lectins and fluorescent reagents activated by enzymatic activity were also used to differentiate live B. schlosseri cell types. Using these markers we isolated 34 cell populations and created RNA libraries for deep sequencing. Additionally, we developed immunological assays for cytotoxicity, phagocytosis and stem cell tracking. Using gene expression analysis we found hematopoietic stem cells and progenitors; in transplantation assays we showed their capability to induce chimerism of pigmentation and differentiation to other cell types, as well as localization to the stem cell niches. We characterized 3 different phagocytic cell types including a previously undescribed myeloid derived cell population. We describe the B. schlosseri cytotoxic cell population that originates as a large granular lymphocyte-like cell type that become morula cells upon allogeneic challenge activation. Altogether, it seems that the common ancestor of tunicates and vertebrates had a true hematopoietic myeloid lineage, while the cytotoxic cells are a convergent evolutionary system.

Abstract C107: Preclinical response of murine syngeneic tumor models to immune checkpoint inhibitor antibodies

Abstract Tumor cell viability and proliferation is regulated by the balance between tumor self-survival and host elimination mechanisms. There are multiple mechanisms whereby tumors successfully evade host immune cell surveillance, permitting continued growth and proliferation. Immunotherapies are aimed at bolstering the host immune system. Delivery of these agents has been shown to actively eliminate disease in a number of patients to date. We evaluated several syngeneic mouse tumor lines for anti-tumor activity with anti-CTLA-4 and/or anti-PD-L1 antibodies, as well as the immunological changes occurring following treatment with these checkpoint inhibitors. The effectiveness of these inhibitors was assessed in the B16.F10 (melanoma), RIF-1 (sarcoma), 4T1-luc (mammary) and CT26 (colon) murine tumor models. Cells or tumor fragments (RIF-1) were implanted SC in the axilla. The anti-CTLA-4 9D9 antibody clone from Bio X Cell (West Lebanon, NH) was employed for responsiveness in each model. All treatments were initiated 3 days post implantation, prior to the appearance of established tumor, at inoculums known to have 100% take rates. The CT26 model was highly responsive with all animals showing tumor regressions and 70% tumor-free survivors at study end. The RIF-1 model was moderately responsive with several mice showing long term progression-free survival and a 56% increase in lifespan vs. the isotype control group. Both the B16.F10 and the 4T1-luc models were non-responsive. Tumor responsiveness to the anti-PD-L1 10F.9G2 antibody clone (Bio X Cell) was also compared in the RIF-1 and CT26 models. In contrast to the anti-CTLA-4 antibody studies, RIF-1 was found to be non-responsive and CT26 was moderately responsive to the anti-PD-L1 antibody. Evaluating immunological endpoints by flow cytometry is critical when assessing the effectiveness of immune checkpoint inhibitors. To this end, the immune profiles of both responsive (CT26) and non-responsive (4T1 and B16.F10) models were examined in the absence or presence of antibody treatments. Initial studies were focused on analysis of T cells (CD4 and CD8) as well as T regulatory cells (Tregs; CD4/CD25/FoxP3) and myeloid derived suppressive cells (MDSC; CD11b/Gr1). The 4T1 model was found to be highly MDSC driven and would be suitable to evaluate agents that target myeloid-derived cell populations. While this model was not very responsive to anti-CTLA-4 treatment, a trend toward decreased MDSCs following treatment was observed with anti-CTLA-4 and not the isotype control antibody. In contrast, the CT26 model was more highly mixed and contained populations of both Tregs and MDSCs. Treatment with anti-CTLA-4 increased the CD8+ T cell population and decreased the Treg cell population with minimal effect on the MSDC population. However, in this same model, anti-PD-L1 treatment increased CD8+ T cells and decreased both Tregs and MDSCs. A more complete mechanistic understanding of how the immune system responds to checkpoint inhibitors permits identification of appropriate models for monotherapy, as well as the rationale for designing drug combination studies. Data enhancing understanding of the above, along with tumor profiling on additional syngeneic models and more flow cytometry multiplexing, is expected from further ongoing research. Citation Format: Maryland Franklin, Matt Thayer, Chris Elders, Dan Saims, Scott Wise. Preclinical response of murine syngeneic tumor models to immune checkpoint inhibitor antibodies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C107.

Collapse of the Tumor Stroma is Triggered by IL-12 Induction of Fas

Engineering CD8⁺ T cells to deliver interleukin 12 (IL-12) to the tumor site can lead to striking improvements in the ability of adoptively transferred T cells to induce the regression of established murine cancers. We have recently shown that IL-12 triggers an acute inflammatory environment that reverses dysfunctional antigen presentation by myeloid-derived cells within tumors and leads to an increase in the infiltration of adoptively transferred antigen-specific CD8⁺ T cells. Here, we find that local delivery of IL-12 increased the expression of Fas within tumor-infiltrating macrophages, dendritic cells, and myeloid-derived suppressor cells (MDSC), and that these changes were abrogated in mice deficient in IL-12-receptor signaling. Importantly, upregulation of Fas in host mice played a critical role in the proliferation and antitumor activity of adoptively transferred IL-12-modified CD8⁺ T cells. We also observed higher percentages of myeloid-derived cell populations within tumors in Fas-deficient mice, indicating that tumor stromal destruction was dependent on the Fas death receptor. Taken together, these results describe the likely requirement for costimulatory reverse signaling through Fasl on T cells that successfully infiltrate tumors, a mechanism triggered by the induction of Fas expression on myeloid-derived cells by IL-12 and the subsequent collapse of the tumor stroma.

IL-12 induced Fas expression in myeloid-derived cells within tumors delivers a co-stimulatory signal for adoptively transferred CD8+ T cells (74.10)

Abstract Engineering CD8+ T cells to deliver IL-12 to the tumor site can lead to striking improvements in the ability of adoptively transferred T cells to induce the regression of established murine cancers. We have recently shown that IL-12 triggers an acute- inflammatory environment that reverses dysfunctional antigen presentation by myeloid- derived cells within tumors and leads to an increase in the infiltration of adoptively transferred antigen-specific CD8+ T cells. Here, we find that local delivery of IL-12 increased the expression of Fas within tumor-infiltrating macrophages, dendritic cells, and myeloid-derived suppressor cells and that these changes were abrogated in mice deficient in IL-12 receptor signaling. Importantly, up- regulation of Fas in host mice played a critical role in the proliferation and anti-tumor activity of adoptively transferred IL-12 engineered CD8+ T cells. We also observed higher percentages of myeloid-derived cell populations within tumors in Fas deficient mice, indicating the resistance for stromal collapse in the absence of signaling through the death receptor Fas. Taken together, these results describe the requirement for co-stimulatory reverse signaling through Fasl on T cells that successfully infiltrate tumors, a mechanism likely triggered by the induction of Fas expression on myeloid-derived cells by IL-12 and the subsequent collapse of the tumor stroma.

IL-12 triggers a programmatic change in dysfunctional myeloid-derived cells within mouse tumors

Solid tumors are complex masses with a local microenvironment, or stroma, that supports tumor growth and progression. Among the diverse tumor-supporting stromal cells is a heterogeneous population of myeloid-derived cells. These cells are alternatively activated and contribute to the immunosuppressive environment of the tumor; overcoming their immunosuppressive effects may improve the efficacy of cancer immunotherapies. We recently found that engineering tumor-specific CD8(+) T cells to secrete the inflammatory cytokine IL-12 improved their therapeutic efficacy in the B16 mouse model of established melanoma. Here, we report the mechanism underlying this finding. Surprisingly, direct binding of IL-12 to receptors on lymphocytes or NK cells was not required. Instead, IL-12 sensitized bone marrow-derived tumor stromal cells, including CD11b(+)F4/80(hi) macrophages, CD11b(+)MHCII(hi)CD11c(hi) dendritic cells, and CD11b(+)Gr-1(hi) myeloid-derived suppressor cells, causing them to enhance the effects of adoptively transferred CD8(+) T cells. This reprogramming of myeloid-derived cells occurred partly through IFN-γ. Surprisingly, direct presentation of antigen to the transferred CD8(+) T cells by tumor was not necessary; however, MHCI expression on host cells was essential for IL-12-mediated antitumor enhancements. These results are consistent with a model in which IL-12 enhances the ability of CD8(+) T cells to collapse large vascularized tumors by triggering programmatic changes in otherwise suppressive antigen-presenting cells within tumors and support the use of IL-12 as part of immunotherapy for the treatment of solid tumors.