Growth Factor Fms-like Tyrosine Kinase Research Articles

Phenotypic and functional differences of dendritic cells in tumor.

Dendritic cells (DCs) are a unique class of immune cells vital to the immune system, functioning as antigen-presenting cells that play a key role in launching both cellular and humoral immune responses. They are crucial in preventing infectious diseases and regulating tumor growth. DCs can be categorized based on various criteria such as phenotype, function, and tissue location, resulting in several subgroups. Generally, DCs are divided into two primary groups: plasmacytoid DCs (pDCs) and conventional DCs (cDCs), which are further classified into Type I classical DCs (cDC1) and Type II classical DCs (cDC2). cDC1 cells are distinguishable by specific gene programs and associated markers, while cDC2 cells display more diversity. Moreover, there is an ongoing debate surrounding a recently identified subgroup called DC3, and whether it can be considered a distinct cell type in the maturation process of DCs remains uncertain. Most of these DC subgroups rely on the growth factor Fms-like tyrosine kinase 3 ligand (FLT3L) for differentiation from a common DC precursor (CDP), guided by various cytokines. Although the general classification of DC subgroups is similar in both humans and mice, numerous phenotypic and functional variations exist within each subgroup. Therefore, comprehending these differences between DC subgroups in humans and mice holds the potential to significantly advance relevant research.

Leveraging STING, Batf3 Dendritic Cells, CXCR3 Ligands, and Other Components Related to Innate Immunity to Induce A "Hot" Tumor Microenvironment That Is Responsive to Immunotherapy.

Simple SummaryThe conceptualization of a “hot” tumor microenvironment has been characterized by mainly T cell infiltration, whereas, “cold” or “immune-deserted” tumors were seen as lacking T cells. However, the presence of antigen-presenting myeloid cells is equally important. In particular, Batf3-lineage DCs are highly efficient in priming effector T cells. Additionally, they can recruit T cells with CXCR3 ligands and re-activate dysfunctional T cells with co-stimulatory molecules in the tumor microenvironment.In a T-cell-inflamed phenotype, tumor eradication works best and is potentiated by immunotherapy such as checkpoint blockade. However, a majority of patients die despite receiving immunotherapy. One reason is insufficient T cell priming and infiltration in the tumor. Nature provides us with innate immune mechanisms in T-cell-inflamed tumors that we can adopt for more personalized immunotherapy strategies. Tumor sensing through innate signaling pathways and efficient antigen-presenting possess a significant role in bridging innate and adaptive immunity and generating a T-cell-inflamed tumor. One approach to strengthen these innate immune mechanisms is to deliver innate immune factors such as STING or activated DCs into the tumor microenvironment, in particular in patients resistant to checkpoint blockade. The low number of DCs in the tumor bed could potentially be increased with the growth factor FMS-like tyrosine kinase 3 ligand (Flt3L). CD103+ DCs are integral for three phases of anti-tumor immunity: priming, recruiting, and re-invigoration of effector T cells. Re-activation of dysfunctional T cells is achieved via co-stimulatory molecules such as the 4-1BB ligand. The presence of myeloid-cell-derived CXCL9 and CXCL10 in the tumor microenvironment can predict response to immunotherapy. We outline recent preclinical and clinical approaches to deliver these crucial components bridging innate and adaptive immunity into the tumor microenvironment.

FLT3 ligand (CDX-301) and stereotactic radiotherapy for advanced non-small cell lung cancer.

9618 Background: In a murine non-small cell lung cancer (NSCLC) model, we demonstrated synergy between localized radiotherapy and the dendritic cell growth factor fms-like tyrosine kinase 3 (FLT3) ligand. We now present results from a phase II study testing this combination in patients with advanced and treatment-refractory NSCLC. Methods: Advanced NSCLC patients with multifocal active disease after at least one line of systemic therapy and ECOG performance status 0-2 received 5 daily subcutaneous injections of CDX-301 (75 µg/kg) concurrent with stereotactic body radiotherapy (SBRT, 30-54 Gy in 1-5 fractions based on target size and location) directed at a single site of disease. Additional “cycles” of SBRT and CDX-301 could be administered at least four months after the initial study treatment, at the discretion of the treating physicians. The primary endpoint was progression-free survival four months after treatment initiation (PFS4), with a hypothesis that the PFS4 rate would exceed 40%. Secondary endpoints included overall survival (OS) duration, responses on PET (PERCIST criteria) and CT (RECIST criteria), and dose-limiting toxicities (grade ≥3 adverse events within 30 days). Lesions targeted with SBRT were excluded from response assessments. The intended sample size was 29 subjects. Blood samples were obtained for flow cytometry and other analyses of immune activation. Results: Twenty-nine subjects received study therapy between October 2016 and January 2020. Subjects received a median of 3 lines (range: 1-5) of systemic therapy prior to study enrollment, including immune checkpoint inhibitors targeting the PD-1/PD-L1 axis in 26 subjects (90%). At the time of this analysis, the actuarial PFS4 rate is 60%, which exceeds our pre-specified efficacy objective. With a median follow-up duration for living patients of 12 months, the actuarial 12-month OS rate is 55%. Partial response of lesions not targeted with SBRT (“abscopal effect”) was observed in 9 subjects (31%) using PET criteria and in 4 subjects (14%) using CT criteria. Seven subjects (24%) received a second course of SBRT and CDX-301 after initial study therapy. No dose-limiting toxicities have been observed. Only six subjects (21%) have received additional chemotherapy or immunotherapy after study treatment. Conclusions: The combination of CDX-301 and SBRT is well-tolerated and has activity as systemic therapy for advanced NSCLC. Additional studies to maximize the efficacy of this in situ vaccination approach with the addition of an agonist anti-CD40 antibody (CDX-1140) are planned. Clinical trial information: NCT02839265 .

Adoptive cellular therapy with T cells expressing the dendritic cell growth factor Flt3L drives epitope spreading and antitumor immunity.

Adoptive cell therapies using genetically engineered T cell receptor or chimeric antigen receptor T cells are emerging forms of immunotherapy that redirect T cells to specifically target cancer. However, tumor antigen heterogeneity remains a key challenge limiting their efficacy against solid cancers. Here, we engineered T cells to secrete the dendritic cell (DC) growth factor Fms-like tyrosine kinase 3 ligand (Flt3L). Flt3L-secreting T cells expanded intratumoral conventional type 1 DCs and substantially increased host DC and T cell activation when combined with immune agonists poly (I:C) and anti-4-1BB. Importantly, combination therapy led to enhanced inhibition of tumor growth and the induction of epitope spreading towards antigens beyond those recognized by adoptively transferred T cells in solid tumor models of T cell receptor and chimeric antigen receptor T cell therapy. Our data suggest that augmenting endogenous DCs is a promising strategy to overcome the clinical problem of antigen-negative tumor escape following adoptive cell therapy.

Abrogation of tolerance with Flt3L requires CD154 expression (98.9)

Abstract Soluble proteins are poorly immunogenic, and will often lead to Ag-specific tolerance. Similarly, Ag associated with apoptotic cells will frequently induce tolerance. These types of Ag are not very “dangerous”, so that recruitment and stimulation of professional APC is minimal. Treatment with the hematopoietic growth factor Fms-like tyrosine kinase 3 ligand (Flt3L) results in tremendous DC expansion, and previous studies have shown that Flt3L treatment can prevent the establishment of tolerance. Yet, the exact mechanism by which Flt3L treatment enhances immune responses remains undefined. I.V. injection of apoptotic cells or soluble Ag into wt mice induces tolerance, but this tolerance is abrogated by treatment with Flt3L. Interestingly, similar abrogation of tolerance was not observed in CD154-/- mice, suggesting a role for CD40-CD40L interaction. Further analysis of Flt3L-treated mice found reduced expansion of CD11c+ cells in CD154-/- mice versus B6 mice. This reduction in CD11c+ cells correlated with reduced T proliferation, as determined by CFSE dilution, in Flt3L-treated CD154-/- mice compared to B6 mice. Phenotypic analysis of the CD11c+ DC from Flt3L-treated B6 and CD154-/- mice showed reduced costimulatory molecule expression, especially CD40, in the CD154-/- CD11c+ DC. Together, these data suggest that maximal DC mobilization by Flt3L requires CD40-CD154 binding, which primes the DC for stimulating immunity even against Ag that normally induce tolerance.

Characterization of an Immediate Splenic Precursor of CD8+ Dendritic Cells Capable of Inducing Antiviral T Cell Responses

Mouse spleens contain three major dendritic cell (DC) populations: plasmacytoid DC, conventional CD8(+)CD24(+) DC (CD8(+) DC), and conventional CD8(-)CD24(-) DC (CD8(-) DC). We have previously shown that CD8(+) DC are the major cross-presenting subtype in vivo and are the main inducers of antiviral cytotoxic T lymphocyte responses. Here we show that after depletion of CD8(+) DC, the only DC capable of viral Ag presentation was a small subset that expresses CD24 but not CD8. This CD8(-)CD24(+) DC population is greatly expanded in mice treated with the DC growth factor FMS-like tyrosine kinase 3 ligand. The CD8(-)CD24(+) DC represent an immediate precursor of CD8(+) DC, as demonstrated by their expression pattern of characteristic markers of CD8(+) DC, their capacity to cross-present in vitro, and their conversion into CD8(+) DC upon adoptive transfer into recipient mice. Accordingly, the lifespan of transferred CD8(-)CD24(+) DC in vivo was greatly enhanced as compared with terminally differentiated CD8(+) DC. Moreover, in a vaccination protocol, CD8(-)CD24(+) DC induced stronger T cell responses and accelerated viral clearance of HSV-1 compared with CD8(+) DC. Our results demonstrate that the ability to cross-present first appears in an immediate precursor population of CD8(+) DC that does not yet express CD8. The enhanced capacity of CD8(-)CD24(+) DC to induce immune responses upon adoptive transfer makes them an attractive novel tool for DC-based immunotherapies.

Disease-modifying capability of murine Flt3-ligand DCs in experimental autoimmune encephalomyelitis

Dendritic cells (DCs) bridge the innate and adaptive immune response, are uniquely capable of priming naïve T cells, and play a critical role in the initiation and regulation of autoimmune and immune-mediated disease. At present, in vivo expansion of DC populations is accomplished primarily through the administration of the recombinant human growth factor fms-like tyrosine kinase 3 ligand (hFL), and in vitro DCs are generated using cytokine cocktails containing GM-CSF +/- IL-4. Although hFL has traditionally been used in mice, differences in amino acid sequence and biological activity exist between murine FL (mFL) and hFL, and resultant DC populations differ in phenotype and immunoregulatory functional capabilities. This study developed and characterized mFL-generated DCs and determined the therapeutic capability of mFL DCs in the autoimmune disease experimental autoimmune encephalomyelitis (EAE). Our findings demonstrate that mFL and hFL expand splenic DCs equally in vivo but that mFL-expanded, splenic DCs more closely resemble normal, resting, splenic DCs. In addition, a novel method for generating mFL-derived bone marrow-derived DCs (BM-DCs) was developed, and comparison of mFL with hFL BM-DCs found mFL BM-DCs to be less mature (i.e., lower MHC Class II, CD80, and CD86) than hFL BM-DCs. These immature mFL DCs up-regulated costimulatory molecules in response to maturation stimuli LPS and TNF-alpha. Mature mFL BM-DCs were immunogenic and exacerbated the clinical disease course of EAE.

Effects of DNA- and Mycobacterium bovis BCG-Based Delivery of the Flt3 Ligand on Protective Immunity to Mycobacterium tuberculosis

The control of intracellular pathogens such as Mycobacterium tuberculosis is dependent on the activation and maintenance of pathogen-reactive T cells. Dendritic cells (DCs) are the major antigen-presenting cells initiating antimycobacterial T-cell responses in vivo. To investigate if immunization strategies that aim to optimize DC function can improve protective immunity against virulent mycobacterial infection, we exploited the ability of the hematopoietic growth factor Fms-like tyrosine kinase 3 ligand (Flt3L) to expand the number of DCs in vivo. A DNA fusion of the genes encoding murine Flt3L and M. tuberculosis antigen 85B stimulated enhanced gamma interferon (IFN-gamma) release by T cells and provided better protection against virulent M. tuberculosis than DNA encoding the single components. Vaccination of mice with a recombinant Mycobacterium bovis BCG strain secreting Flt3L (BCG:Flt3L) led to early expansion of DCs compared to immunization with BCG alone, and this effect was associated with increased stimulation of BCG-reactive IFN-gamma-secreting T cells. BCG and BCG:Flt3L provided similar protective efficacies against low-dose aerosol M. tuberculosis; however, immunization of immunodeficient mice revealed that BCG:Flt3L was markedly less virulent than conventional BCG. These results demonstrate the potential of in vivo targeting of DCs to improve antimycobacterial vaccine efficacy.

Spleen but not tumor infiltration by dendritic and T cells is increased by intravenous adenovirus-Flt3 ligand injection

Dendritic cell (DC) expansion is regulated by the hematopoietic growth factor fms-like tyrosine kinase 3 ligand (Flt3L). DCs are critical to the control of tumor growth and metastasis, and there is a positive correlation between intratumoral DC infiltration and clinical outcome. In this report, we first demonstrate that single intravenous (i.v.) injections of adenovirus (Adv)-Flt3L significantly increased splenic dendritic, B, T and natural killer (NK) cell numbers in both normal and mammary tumor-bearing mice. In contrast, the numbers of DCs and T cells infiltrating the tumors were not increased. Consistent with the minimal effect on immune cell infiltration, i.v. Adv-Flt3L injections had no therapeutic activity against orthotopic mammary tumors. In addition, we noted tumor and Adv-Flt3L expansion of Gr1(+)CD11b(+) immature myeloid suppressor cells (IMSCs), which may inhibit the therapeutic efficacy of Adv-Flt3L-expanded DCs.

Recruitment and expansion of dendritic cells in vivo potentiate the immunogenicity of plasmid DNA vaccines.

DCs are critical for priming adaptive immune responses to foreign antigens. However, the utility of harnessing these cells in vivo to optimize the immunogenicity of vaccines has not been fully explored. Here we investigate a novel vaccine approach that involves delivering synergistic signals that both recruit and expand DC populations at the site of antigen production. Intramuscular injection of an unadjuvanted HIV-1 envelope (env) DNA vaccine recruited few DCs to the injection site and elicited low-frequency, env-specific immune responses in mice. Coadministration of plasmids encoding the chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) and the DC-specific growth factor fms-like tyrosine kinase 3 ligand with the DNA vaccine resulted in the recruitment, expansion, and activation of large numbers of DCs at the site of inoculation. Consistent with these findings, coadministration of these plasmid cytokines also markedly augmented DNA vaccine---elicited cellular and humoral immune responses and increased protective efficacy against challenge with recombinant vaccinia virus. These data suggest that the availability of mature DCs at the site of inoculation is a critical rate-limiting factor for DNA vaccine immunogenicity. Synergistic recruitment and expansion of DCs in vivo may prove a practical strategy for overcoming this limitation and potentiating immune responses to vaccines as well as other immunotherapeutic strategies.

Costimulation blockade promotes the apoptotic death of graft-infiltrating T cells and prolongs survival of hepatic allografts from FLT3L-treated donors.

Mouse liver grafts are accepted across major histocompatibility complex (MHC) barriers and induce donor-specific tolerance without immunosuppressive therapy. By contrast, hepatic allografts from donors treated with the hematopoietic growth factor fms-like tyrosine kinase 3 ligand (FL), which dramatically increases hepatic interstitial dendritic cells (DC), are rejected acutely (median survival time 5 days). This switch from tolerance to rejection is associated with a marked reduction in apoptotic activity of graft-infiltrating T cells. We hypothesized that T-cell costimulation, provided by markedly enhanced numbers of donor antigen presenting cells (APCs), might inhibit apoptosis, promote expansion of T helper 1 cells and play a key role in acute liver rejection. C3H (H2k) recipients of orthotopic liver grafts from FL-treated B10 (H2b) donors were given cytotoxic T-lymphocyte antigen 4: immunoglobulin (CTLA4Ig), a chimeric fusion protein that blocks the B7-CD28 costimulatory pathway, or control human immunoglobulin (200 microg) on the day of transplantation (day 0). Livers and spleens were removed on day 4. Cryostat sections were stained for interleukin (IL)-12 or by terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL). Expression of mRNA encoding interferon (IFN)-gamma and IL-10 was determined by RNase protection assay. Suspensions of graft-infiltrating cells (GICs) and spleen cells were analyzed for apoptotic (TUNEL+) T-cell subsets by flow cytometry. CTL activity of GICs and circulating alloantibody levels were determined by cytotoxicity assays. Survival of liver grafts from FL donors was markedly prolonged by CTLA4Ig administration. This effect was associated with reductions in IFN-gamma and IL-10 gene transcripts within the GIC population, and with decreases in donor-specific CTL and NK cell activities and circulating anti-donor alloantibody levels. At the same time, there were marked increases in TUNEL+ CD4+ and especially CD8+ cells, both within the grafts and in the spleens of CTLA4Ig-treated mice. Signaling via the B7-CD28 pathway appears to play a key role in the switch from tolerance to rejection that is precipitated by markedly enhanced numbers of donor DCs. Inhibition of acute liver allograft rejection by CTLA4Ig, linked to restoration of apoptotic activity of graft-infiltrating T cells, further suggests that deletion of these cells may be critical for promotion of long-term allograft survival.