Flt3 agonist enhances immunogenicity of arenavirus vector-based simian immunodeficiency virus vaccine in macaques.

Arenaviral vaccine vectors encoding simian immunodeficiency virus (SIV) immunogens are capable of inducing efficacious humoral and cellular immune responses in nonhuman primates. Several studies have evaluated the use of immune modulators to further enhance vaccine-induced T-cell responses. The hematopoietic growth factor Flt3L drives the expansion of various bone marrow progenitor populations, and administration of Flt3L was shown to promote expansion of dendritic cell populations in spleen and blood, which are targets of arenaviral vectors. Therefore, we evaluated the potential of Flt3 signaling to enhance the immunogenicity of arenaviral vaccines encoding SIV immunogens (SIVSME543 Gag, Env, and Pol) in rhesus macaques, with a rhesus-specific engineered Flt3L-Fc fusion protein. In healthy animals, administration of Flt3L-Fc led to a 10- to 100-fold increase in type 1 dendritic cells 7 days after dosing, with no antidrug antibody (ADA) generation after repeated dosing. We observed that administration of Flt3L-Fc fusion protein 7 days before arenaviral vaccine increased the frequency and activation of innate immune cells and enhanced T-cell activation with no treatment-related adverse events. Flt3L-Fc administration induced early innate immune activation, leading to a significant enhancement in magnitude, breadth, and polyfunctionality of vaccine-induced T-cell responses. The Flt3L-Fc enhancement in vaccine immunogenicity was comparable to a combination with αCTLA-4 and supports the use of safe and effective variants of Flt3L to augment therapeutic vaccine-induced T-cell responses.IMPORTANCEInduction of a robust human immunodeficiency virus (HIV)-specific CD4+ and CD8+ T-cell response through therapeutic vaccination is considered essential for HIV cure. Arenaviral vaccine vectors encoding simian immunodeficiency virus (SIV) immunogens have demonstrated strong immunogenicity and efficacy in nonhuman primates. Here, we demonstrate that the immunogenicity of arenaviral vectors encoding SIV immunogens can be enhanced by administration of Flt3L-Fc fusion protein 7 days before vaccination. Flt3L-Fc-mediated increase in dendritic cells led to robust improvements in vaccine-induced T- and B-cell responses compared with vaccine alone, and Flt3L-Fc dosing was not associated with any treatment-related adverse events. Importantly, immune modulation by either Flt3L-Fc or αCTLA-4 led to comparable enhancement in vaccine response. These results indicate that the addition of Flt3L-Fc fusion protein before vaccine administration can significantly enhance vaccine immunogenicity. Thus, safe and effective Flt3L variants could be utilized as part of a combination therapy for HIV cure.

Dendritic cells play no significant role in the laser-induced choroidal neovascularization model.

Age-related macular degeneration (AMD) is genetically associated with complement. Dendritic cells (DCs) play key roles during innate and adaptive immunity, and express complement components and their receptors. We investigated ocular DC heterogeneity and the role of DCs in the laser-induced choroidal neovascularization (CNV) model. In order to determine the function of DCs, we used two models of DC deficiency: the Flt3-/- and Flt3l-/- mouse. We identified three types of ocular DCs: plasmacytoid DC, classical DC-1, and classical DC-2. At steady-state, classical DCs were found in the iris and choroid but were not detectable in the retina. Plasmacytoid DCs existed at very low levels in iris, choroid, and retina. After laser injury, the number of each DC subset was up-regulated in the choroid and retina. In Flt3-/- mice, we found reduced numbers of classical DCs at steady-state, but each DC subset equally increased after laser injury between wildtype and Flt3-/- mice. In Flt3l-/- mice, each DC subsets was severely reduced after laser injury. Neither Flt3-/- or Flt3l-/- mice demonstrated reduced CNV area compared to wildtype mice. DCs do not play any significant role during the laser-induced CNV model of neovascular AMD.

Regulatory Dendritic Cells Induced by Bendamustine Are Associated With Enhanced Flt3 Expression and Alloreactive T-Cell Death.

The growth factor Flt3 ligand (Flt3L) is central to dendritic cell (DC) homeostasis and development, controlling survival and expansion by binding to Flt3 receptor tyrosine kinase on the surface of DCs. In the context of hematopoietic cell transplantation, Flt3L has been found to suppress graft-versus-host disease (GvHD), specifically via host DCs. We previously reported that the pre-transplant conditioning regimen consisting of bendamustine (BEN) and total body irradiation (TBI) results in significantly reduced GvHD compared to cyclophosphamide (CY)+TBI. Pre-transplant BEN+TBI conditioning was also associated with greater Flt3 expression among host DCs and an accumulation of pre-cDC1s. Here, we demonstrate that exposure to BEN increases Flt3 expression on both murine bone marrow-derived DCs (BMDCs) and human monocyte-derived DCs (moDCs). BEN favors development of murine plasmacytoid DCs, pre-cDC1s, and cDC2s. While humans do not have an identifiable equivalent to murine pre-cDC1s, exposure to BEN resulted in decreased plasmacytoid DCs and increased cDC2s. BEN exposure and heightened Flt3 signaling are associated with a distinct regulatory phenotype, with increased PD-L1 expression and decreased ICOS-L expression. BMDCs exposed to BEN exhibit diminished pro-inflammatory cytokine response to LPS and induce robust proliferation of alloreactive T-cells. These proliferative alloreactive T-cells expressed greater levels of PD-1 and underwent increased programmed cell death as the concentration of BEN exposure increased. Alloreactive CD4+ T-cell death may be attributable to pre-cDC1s and provides a potential mechanism by which BEN+TBI conditioning limits GvHD and yields T-cells tolerant to host antigen.

One size does not fit all: navigating the multi-dimensional space to optimize T-cell engaging protein therapeutics.

T-cell engaging biologics is a class of novel and promising immune-oncology compounds that leverage the immune system to eradicate cancer. Here, we compared and contrasted a bispecific diabody-Fc format, which displays a relatively short antigen-binding arm distance, with our bispecific IgG platform. By generating diverse panels of antigen-expressing cells where B cell maturation antigen is either tethered to the cell membrane or located to the juxtamembrane region and masked by elongated structural spacer units, we presented a systematic approach to investigate the role of antigen epitope location and molecular formats in immunological synapse formation and cytotoxicity. We demonstrated that diabody-Fc is more potent for antigen epitopes located in the membrane distal region, while bispecific IgG is more efficient for membrane-proximal epitopes. Additionally, we explored other parameters, including receptor density, antigen-binding affinity, and kinetics. Our results show that molecular format and antigen epitope location, which jointly determine the intermembrane distance between target cells and T cells, allow decoupling of cytotoxicity and cytokine release, while antigen-binding affinities appear to be positively correlated with both readouts. Our work offers new insight that could potentially lead to a wider therapeutic window for T-cell engaging biologics in general.

FLT3 Ligand Is Dispensable for the Final Stage of Type 1 Conventional Dendritic Cell Differentiation.

Conventional dendritic cells (cDCs) are comprised of two major subsets, type 1 cDC (cDC1) and type 2 cDC (cDC2). As each cDC subset differentially influences the nature of immune responses, we sought factors that would allow the manipulation of their relative abundance. Notably, cDC1 are less abundant than cDC2 in both lymphoid and nonlymphoid organs. We demonstrate that this bias is already apparent in bone marrow precommitted precursors. However, comparison of five common inbred strains revealed a disparity in precursor-product relationship, in which mice with fewer precursors to cDC1 had more cDC1. This disparity associated with contrasting variations in CD135 (FLT3) expression on cDC subsets. Hence, we characterized the response to FLT3 ligand during cDC1 and cDC2 lineage differentiation and find that although FLT3 ligand is required throughout cDC2 differentiation, it is surprisingly dispensable during late-stage cDC1 differentiation. Overall, we find that tight regulation of FLT3 ligand levels throughout cDC differentiation dictates the cDC1 to cDC2 ratio in lymphoid organs.

Allogeneic FLT3 CAR T Cells with an Off-Switch Exhibit Potent Activity against AML and Can Be Depleted to Expedite Bone Marrow Recovery.

Patients with relapsed or refractory acute myeloid leukemia (AML) have a dismal prognosis and limited treatment options. Chimeric antigen receptor (CAR) T cells have achieved unprecedented clinical responses in patients with B cell leukemias and lymphomas and could prove highly efficacious in AML. However, a significant number of patients with AML may not receive treatment with an autologous product due to manufacturing failures associated with low lymphocyte counts or rapid disease progression while the therapeutic is being produced. We report the preclinical evaluation of an off-the-shelf CAR T cell therapy targeting Fms-related tyrosine kinase 3 (FLT3) for the treatment of AML. Single-chain variable fragments (scFvs) targeting various epitopes in the extracellular region of FLT3 were inserted into CAR constructs and tested for their ability to redirect T cell specificity and effector function to FLT3+ AML cells. A lead CAR, exhibiting minimal tonic signaling and robust activity in vitro and in vivo, was selected and then modified to incorporate a rituximab-responsive off-switch in cis. We found that allogeneic FLT3 CAR T cells, generated from healthy-donor T cells, eliminate primary AML blasts but are also active against mouse and human hematopoietic stem and progenitor cells, indicating risk of myelotoxicity. By employing a surrogate CAR with affinity to murine FLT3, we show that rituximab-mediated depletion of FLT3 CAR T cells after AML eradication enables bone marrow recovery without compromising leukemia remission. These results support clinical investigation of allogeneic FLT3 CAR T cells in AML and other FLT3+ hematologic malignancies.

Route of Vaccine Administration Influences the Impact of Fms-Like Tyrosine Kinase 3 Ligand (Flt3L) on Chlamydial-Specific Protective Immune Responses.

We tested the hypothesis that the impact of the Fms-like tyrosine kinase 3-ligand (Flt3L; FL) on recombinant Vibrio cholerae ghost (rVCG) vaccine-induced chlamydial immunity is influenced by route of vaccine delivery. Female C57BL/6J mice were immunized rectally (IR) or intramuscularly (IM) with rVCG co-expressing the Chlamydia trachomatis PmpD and PorB proteins (rVCG- PmpD/PorB) with and without FL or glycoprotein D of HSV-2 (rVCG-gD2) as antigen control. Vaccine evaluation was based on measurement of T cell proliferation, Th1/Th2 cytokine, and humoral responses at systemic and mucosal compartments, and protection against intravaginal challenge infection. Results revealed that high levels of CD4+ T cell-mediated and humoral immune responses, were elicited in mice as a function of both IR and IM immunization. Unexpectedly, co-administration of vaccine with FL enhanced specific Th1-type cytokine levels and T cell proliferative responses following IR but not IM immunization. While administration of vaccine with FL enhanced the specific mucosal and systemic IgA antibody responses following both immunization routes, IgG2c responses were not enhanced following IR delivery. The vaccine-induced immune effectors protected mice against live heterologous C. muridarum infection irrespective of route of vaccine administration, with the regimen incorporating FL having a protective advantage. Further evaluation showed that protection afforded by the FL adjuvanted vaccine was facilitated by CD4+ T cells, as indicated by reduction in the intensity and duration of genital chlamydial shedding by naïve mice following adoptive transfer of immune CD4+ T cells. Taken together, the results indicate that comparable protective immunity, which is enhanced by co-delivery with FL, is elicited in the female genital tract against Chlamydia infection after mucosal and systemic administration, highlighting the ability of FL to function as an effective immunostimulator at both mucosal and systemic sites. The differential modulation of humoral and cellular immune responses, and protective immunity afforded by the FL adjuvanted vaccine following IR administration indicates that the immunomodulatory impact of FL on chlamydial-specific immunity is influenced by the route of vaccine administration. Thus, targeting of VCG-based vaccines to antigen presenting cells by co-delivery with FL is a feasible immunization approach for inducing effective chlamydial immunity in the female genital tract.

Classical dendritic cells regulate acute lung inflammation and injury in mice with lipopolysaccharide­induced acute respiratory distress syndrome.

Classical dendritic cells (cDCs) are involved in the pathogenesis of inflammatory lung diseases; however, their contributions in acute respiratory distress syndrome (ARDS), which is pathophysiologically inflammatory, remain unknown. The present study aimed to explore the regulatory effects of pulmonary cDCs on acute lung inflammation and injury in lipopolysaccharide (LPS)­induced ARDS. Fms­like tyrosine kinase 3­ligand (FLT3L) and lestaurtinib, a specific activator and an inhibitor of FLT3 signaling respectively, were used separately for the pretreatment of C57BL/6 mice for 5 consecutive days. ARDS was induced by intratracheal injection of LPS, and mice were sacrificed 6 and 24 h later. Flow cytometry was used to measure the aggregation and maturation of pulmonary cDCs. The ratio of lung wet weight to body weight (LWW/BW) and histopathological analyses were assessed to evaluate lung edema and lung injury. Tumor necrosis factor­α and interleukin (IL)­6 levels were measured by ELISA to evaluate acute lung inflammation. The levels of interferon­Î³, IL­1ß, IL­4 and IL­10, and the expression of the transcription factors T­box­expressed­in­T­cells (T­bet) and GATA binding protein 3, were quantified by ELISA, RT­qPCR and western blotting to evaluate the balance of the Th1/Th2 response. Myeloperoxidase (MPO) activity was measured to evaluate neutrophil infiltration. The results demonstrated that the aggregation and maturation of pulmonary cDCs reached a peak at 6 h after LPS challenge, followed by a significant decrease at 24 h. FLT3L pretreatment further stimulated the aggregation and maturation of pulmonary cDCs, resulting in elevated lung MPO activity and increased T­bet expression, which in turn led to aggravated LWW/BW, acute lung inflammation and injury. However, lestaurtinib pretreatment inhibited the aggregation and maturation of pulmonary cDCs, decreased lung MPO activity and T­bet expression, and eventually improved LWW/BW, acute lung inflammation and injury. The present results suggested that pulmonary cDCs regulated acute lung inflammation and injury in LPS­induced ARDS through the modulation of neutrophil infiltration and balance of the Th1/Th2 response.

Alternatively activated macrophage-derived secretome stimulates ovarian cancer spheroid spreading through a JAK2/STAT3 pathway.

High-grade serous ovarian cancer (HGSOC) metastasizes when tumor spheroids detach from the primary tumor and re-attach throughout the peritoneal cavity. Once the cancer cells have implanted in these new sites, the development of metastatic lesions is dependent on the disaggregation of cancer cells from the spheroids and subsequent expansion across the collagenous extracellular matrix (ECM). As HGSOC progresses an increase in alternatively activated macrophages (AAMs) in the surrounding ascites fluid has been observed and AAMs have been shown to enhance tumor invasion and growth in a wide range of cancers. We hypothesized that soluble factors from AAMs in the peritoneal microenvironment promote the disaggregation of ovarian cancer spheroids across the underlying ECM. We determined that co-culture with AAMs significantly increased HGSOC spheroid spreading across a collagen matrix. Multivariate modeling identified AAM-derived factors that correlated with enhanced spread of HGSOC spheroids and experimental validation showed that each individual cell line responded to a distinct AAM-derived factor (FLT3L, leptin, or HB-EGF). Despite this ligand-level heterogeneity, we determined that the AAM-derived factors utilized a common signaling pathway to induce spheroid spreading: JAK2/STAT3 activation followed by MMP-9 mediated spreading. Furthermore, immunostaining demonstrated that FLT3, LEPR, EGFR, and pSTAT3 were upregulated in metastases in HGSOC patients, with substantial patient-to-patient heterogeneity. These results suggest that inhibiting individual soluble factors will not inhibit AAM-induced effects across a broad group of patients; instead, the downstream JAK2/STAT3/MMP-9 pathway should be examined as potential therapeutic targets to slow metastasis in ovarian cancer.

Genetically engineered CAR NK cells display selective cytotoxicity against FLT3-positive B-ALL and inhibit in vivo leukemia growth.

Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells represent a promising effector cell type for adoptive cancer immunotherapy. Both, genetically modified donor-derived NK cells as well as continuously expanding NK-92 cells are currently under clinical development. To enhance their therapeutic utility for the treatment of pre-B-cell acute lymphoblastic leukemia (B-ALL), we engineered NK-92 cells by lentiviral gene transfer to express a FMS-like tyrosine kinase 3 (FLT3)-specific CAR that contains a composite CD28-CD3ζ signaling domain. FLT3 has primarily been described as a therapeutic target for acute myeloid leukemia, but overexpression of FLT3 has also been reported in B-ALL. Exposure of FLT3-positive targets to CAR NK-92 cells resulted in conjugate formation between NK and leukemia cells, NK-cell degranulation and selective cytotoxicity toward established B-ALL cell lines and primary blasts that were resistant to parental NK-92. In a SEM B-ALL xenograft model in NOD-SCID IL2R γnull mice, treatment with CAR NK-92 but not parental NK-92 cells markedly inhibited disease progression, demonstrating high antileukemic activity in vivo. As FLT3 is known to be also expressed on precursor cells, we assessed the feasibility of incorporating an inducible caspase-9 (iCasp9) suicide switch to enhance safety of our approach. Upon addition of the chemical dimerizer AP20187 to NK-92 cells coexpressing the FLT3-specific CAR and iCasp9, rapid iCasp9 activation was observed, precluding further CAR-mediated cytotoxicity. Our data demonstrate that B-ALL can be effectively targeted by FLT3-specific CAR NK cells which may complement CD19-directed immunotherapies, particularly in cases of inherent or acquired resistance to the latter.

FLT3, a prognostic biomarker for acute myeloid leukemia (AML): Quantitative monitoring with a simple anti-FLT3 interaction and flow cytometric method.

BACKGROUND: Overexpression of fms-like tyrosine kinase 3 (FLT3) protein in leukemia is highly related to poor prognosis and reduced survival rate in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients. Simple but efficient quantification of FLT3 protein levels on the leukemic cell surface using flow cytometry had been developed for rapid determination of FLT3 on intact cell surface. METHODS: Quantitation protocol for FLT3 biomarker in clinical samples was developed and validated. Cell model selection for calibration curve construction was identified and evaluated. Selected antibody concentrations, cell density, and incubation time were evaluated for most appropriate conditions. Comparison of the developed FLT3 determination protocol with the conventional Western blot analysis was performed. RESULTS: EoL-1 cell line was selected for using as positive control cells. Calibration curve (20%-120% of FLT3 positive cells) and quality control (QC) levels were constructed and evaluated. The results demonstrated good linearity (r2  > 0.99). The intra- and inter-day precision and accuracy, expressed as the coefficient of variation (%CV) and % recovery, were <20% and fell in 80%-120% in all cases. When compared with Western blotting results, FLT3 protein expression levels in leukemia patient's bone marrow samples were demonstrated in the same trend. CONCLUSIONS: The effective, reliable, rapid, and economical analytical technique using the developed flow cytometric method was demonstrated for FLT3 protein determination on leukemic cell surface. This method provided a practical analysis of FLT-3 biomarker levels which is valuable for physician decision in acute leukemia treatment.

Midostaurin reduces Regulatory T cells markers in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy in which the only curative approach is allogeneic stem cell transplant (Allo-HSCT). The recognition and elimination of leukemic clones by donor T-cells contribute significantly to Allo-HSCT success. FLT3-ITD, a common mutation in AML, is associated with poor prognosis. Recently, midostaurin became the first FDA approved FLT3-inhibitor for pre-transplant patients with FLT3-ITD in combination with standard therapy. In addition to their multikinase activity which may affect T-cell signaling, FLT3-inhibitors induce apoptosis of malignant cells which may also enhance antigen presentation to activate T-cells. Considering the increased clinical use of these inhibitors in patients with AML, and the limited clinical benefit derived from their use as single agents, understanding how FLT3-inhibitors affect T cell population and function is needed to improve their clinical benefit. We examined the effect of four different FLT3 inhibitors (midostaurin, sorafenib, tandutinib, and quizartenib) on T cell populations in peripheral blood mononuclear cells (PBMC) obtained from healthy donors and from patients with AML. Midostaurin exhibited a significant decrease in CD4 + CD25 + FOXP3+ T cell population and FOXP3 mRNA expression in healthy and AML PBMCs. Similarly, samples collected from patients with AML treated with midostaurin showed a reduction in Tregs markers. Interferon-γ(IFN-γ), tumor necrosis factor-α(TNF-α), and IL-10 levels were also reduced following midostaurin treatment. Considering the FDA approval of midostaurin for use in patients with AML in the pre-transplant setting, our finding will have important clinical implication as it provides the rationale for functional investigation of the use of midostaurin in post-transplant patients.

The Transcription Factors PU.1 and IRF4 Determine Dendritic Cell-Specific Expression of RALDH2.

RALDH2 expressed in dendritic cells (DCs) plays a critical role in the development of regulatory T cells in mesenteric lymph nodes. Despite the importance of RALDH2 in intestinal immunity, little is known about the mechanism of DC-specific expression of RALDH2. In the current study, we focused on the hematopoietic cell-specific transcription factors PU.1 and IRF4 as the determinants of Aldh1a2 gene expression. The mRNA level of Aldh1a2, and subsequently the enzyme activity, were decreased by knockdown of PU.1 and IRF4 in bone marrow-derived DCs (BMDCs) of BALB/c mice. Chromatin immunoprecipitation assays showed that PU.1 and IRF4 bound to the Aldh1a2 gene ∼2 kb upstream from the transcription start site in BMDCs. A reporter assay and an EMSA revealed that the Aldh1a2 promoter was synergistically transactivated by a heterodimer composed with PU.1 and IRF4 via the EICE motif at -1961/-1952 of the gene. The effect of small interfering RNAs for Spi1 and Irf4 and specific binding of PU.1 and IRF4 on the Aldh1a2 gene were also observed in DCs freshly isolated from spleen and mesenteric lymph nodes, respectively. GM-CSF stimulation upregulated the Aldh1a2 transcription in Flt3 ligand-generated BMDCs, in which the IRF4 expression and the PU.1 recruitment to the Aldh1a2 promoter were enhanced. We conclude that PU.1 and IRF4 are transactivators of the Aldh1a2 gene in vitro and ex vivo.

Hyperleukocytosis is associated with distinct genetic alterations and is an independent poor-risk factor in de novo acute myeloid leukemia patients.

OBJECTIVES: Acute myeloid leukemia (AML) with hyperleukocytosis (HL) is intuitively thought as a unique group with dismal prognosis. However, comprehensive studies regarding the genetic landscape and clinical outcome in this group of patients are limited. METHODS: A total of 693 newly diagnosed de novo non-M3 AML patients were consecutively enrolled. We compared relevant mutations in 20 genes between AML patients with or without HL and exposed their prognostic implications. RESULTS: Hyperleukocytosis, defined as initial white blood cell counts above 50 000/µL, occurred in 28.9% of AML patients. HL patients had higher incidences of FLT3-ITD, NPM1, DNMT3A, CEBPA, and TET2 mutations. Multivariate analysis demonstrated that HL was an independent poor prognostic factor for overall survival and disease-free survival in total patients, those with intermediate-risk cytogenetics and normal karyotype irrespective of genetic alterations. Intriguingly, HL predicted poor survival in CEBPA double mutated, NPM1 + /FLT3-ITD- and NPM1-/FLT3-ITD- patients. Further, HL patients who received allogeneic hematopoietic stem cell transplantation (allo-HSCT) in first complete remission (CR) had a significantly longer overall survival and disease-free survival than those without allo-HSCT. CONCLUSIONS: Hyperleukocytosis is an independent poor prognostic factor irrespective of cytogenetics and mutation status. Allo-HSCT in first CR seems to ameliorate the poor prognostic impact of HL.

CAR T-cells targeting FLT3 have potent activity against FLT3-ITD+ AML and act synergistically with the FLT3-inhibitor crenolanib.

FMS-like tyrosine kinase 3 (FLT3) is a transmembrane protein expressed on normal hematopoietic stem and progenitor cells (HSC) and retained on malignant blasts in acute myeloid leukemia (AML). We engineered CD8+ and CD4+ T-cells expressing a FLT3-specific chimeric antigen receptor (CAR) and demonstrate they confer potent reactivity against AML cell lines and primary AML blasts that express either wild-type FLT3 or FLT3 with internal tandem duplication (FLT3-ITD). We also show that treatment with the FLT3-inhibitor crenolanib leads to increased surface expression of FLT3 specifically on FLT3-ITD+ AML cells and consecutively, enhanced recognition by FLT3-CAR T-cells in vitro and in vivo. As anticipated, we found that FLT3-CAR T-cells recognize normal HSCs in vitro and in vivo, and disrupt normal hematopoiesis in colony-formation assays, suggesting that adoptive therapy with FLT3-CAR T-cells will require subsequent CAR T-cell depletion and allogeneic HSC transplantation to reconstitute the hematopoietic system. Collectively, our data establish FLT3 as a novel CAR target in AML with particular relevance in high-risk FLT3-ITD+ AML. Further, our data provide the first proof-of-concept that CAR T-cell immunotherapy and small molecule inhibition can be used synergistically, as exemplified by our data showing superior antileukemia efficacy of FLT3-CAR T-cells in combination with crenolanib.

Flexible fate commitment of E2-2high common DC progenitors implies tuning in tissue microenvironments.

The basic helix-loop-helix transcription factor E2-2 is essential for the development of plasmacytoid dendritic cells (pDCs) but not conventional DCs (cDCs). Here, we generated E2-2 reporter mice and demonstrated that an E2-2high fraction among common DC progenitors, which are a major source of pDCs and cDCs in the steady state, strictly gave rise to pDCs in the presence of Flt3 (Fms-like tyrosine kinase receptor-3) ligand ex vivo or in the secondary lymphoid organs when transferred in vivo. However, in the small intestine, some of these E2-2high progenitors differentiated into cDCs that produced retinoic acid. This transdifferentiation was driven by signaling via the common ß receptor, a receptor for the cytokines IL-3, IL-5 and GM-CSF, which are abundant in the gut. In the presence of GM-CSF and Flt3 ligand, E2-2high-progenitor-derived cDCs consistently induced Foxp3+ Treg cells ex vivo. Our findings reveal the commitment and flexibility of E2-2high progenitor differentiation and imply that pertinent tuning machinery is present in the gut microenvironment.

CD103+ CD11b- salivary gland dendritic cells have antigen cross-presenting capacity.

Dendritic cells (DCs) in lymphoid and non-lymphoid tissues are professional antigen-presenting cells that are essential for effective immunity and tolerance. However, the presence and characteristics of DCs in steady-state salivary glands (SGs) currently remain unknown. We herein identified CD64- CD11c+ classical DCs (cDCs) as well as CD64+ macrophages among CD45+ MHC class II+ antigen-presenting cells in steady-state murine SGs. SG cDCs were divided into CD103+ CD11b- and CD103- CD11b+ cDCs. CD103+ CD11b- cDCs expressed XCR1, CLEC9A, and interferon regulatory factor 8, whereas CD103- CD11b+ cDCs strongly expressed CD172a. Both cDC subsets in SGs markedly expanded in response to the Flt3 ligand (Flt3L), were replenished by bone marrow-derived precursors, and differentiated from common DC precursors, but not monocytes. Furthermore, ovalbumin-pulsed SG CD103+ CD11b- cDCs induced the proliferation of naïve ovalbumin-specific CD8+ T cells and production of interferon-γ from proliferating T cells. SG CD103+ CD11b- cDCs expanded by Flt3L in vivo exhibited the same properties. These results indicate that bona fide cDCs reside in steady-state murine SGs and cDCs with the CD103+ CD11b- phenotype possess antigen cross-presenting capacity. Moreover, Flt3L enhances protective immunity by expanding cDCs. Taken together, SG cDCs might play an important role in maintaining immune homeostasis in the tissues.

Immunophenotyping with CD135 and CD117 predicts the FLT3, IL-7R and TLX3 gene mutations in childhood T-cell acute leukemia.

With the combination of immunophenotyping and molecular tests, it is still a challenge to identify the characteristics of T cell acute lymphoblastic leukemia (T-ALL) associated with distinct outcomes. This study tests the possible correlation of cellular expression of CD135 and CD117 with somatic gene mutations in T-ALL. One hundred sixty-two samples were tested, including 143 at diagnosis, 15 from T-lymphoblastic lymphoma at relapse, and four relapse samples from sequential follow-up of T-ALL. CD135 and CD117 monoclonal antibodies were included in the T-ALL panel of flow cytometry. The percentage of cells positivity and the median fluorescence intensity were correlated with gene mutational status. STIL-TAL1, TLX3, FLT3 and IL7R mutations were tested using standard techniques. STIL-TAL1 was found in 24.8%, TLX3 in 12%, IL7R in 10% and FLT3-ITD in 5% of cases. FLT3 and IL7R mutations were mutually exclusive, as were FLT3-ITD and STIL-TAL1. Associations of CD135(high) (p<0.01), CD117(intermediate/high) (p=0.02) and FLT3-ITD, CD117(low) with IL7R(mutated) (p<0.01) and CD135(high) with TLX3(pos) were observed. We conclude that the addition of CD135 and CD117 to the diagnosis can predict molecular aberrations in T-ALL settings, mainly segregating patients with FLT3-ITD, who would benefit from treatment with inhibitors of tyrosine.