Double-positive Population Research Articles

Abstract 6606: Develop dual-targeted CAR-T cells to achieve RCC cures

Abstract Clear cell renal cell carcinoma (ccRCC) is the major type of RCC and is among the 10 most common cancers in both men and women. ccRCC is characterized by the loss of chromosome 3p, where the von-Hippel-Lindau (VHL) gene is found. Its gene product, pVHL suppresses the hypoxia inducible factor (HIF) transcription factors and the deletion of VHL leads to HIF hyperactivation, upregulating the expression of many downstream genes involved in angiogenesis, metabolism, and cell-cycle regulation. Two gene products, carbonic anhydrase IX (CAIX) and CD70 which we are pursuing as therapeutic targets are downstream genes of the HIF-1α pathway and thus commonly overexpressed in ccRCC. Despite the development of checkpoint blockade inhibitors (CBIs) for the treatment of this disease, curative therapies are rare. Chimeric Antigen Receptor (CAR)-T cells are a new type of “living drug”, in which T cells are engineered to express a single chain variable antibody fragment (scFv) linked to an intracellular signaling motif that includes CD3ζ (z) activation domain (first generation), with CD28 or 41BB (second generation), or both (third generation) costimulatory domains. CAR-T cells have proven to be a powerful, clinically translatable immunotherapy for hematologic malignancies. However, these results have not been translatable to solid tumors due to inefficient homing of CAR-T cells, the suppressive tumor microenvironment (TME), and on-target off-tumor toxicities resulting from the sharing of CAR-T target epitopes on healthy tissues. To translate CAR-T cell therapy to ccRCC, we developed dual-targeted CAR-T cells against both CAIX and CD70, in which dual CAR can target double positive (CAIX+ CD70+) and single positive (CAIX+ CD70-, CAIX- CD70+) tumor cell populations to address this heterogeneity. By IHC staining, we demonstrated that these two target antigens are overexpressed in ccRCC and represent circa 90% of the tumor cell population regardless of disease stages. Here we build on our previous findings and demonstrate that 41BB CAR and CD4/CD8 CAR-T cell mixtures exhibit the superior efficacy and persistence in our ccRCC orthotopic NSG-SGM3 mouse model compared to other CAR constructs and CD8 alone. A panel of dual-targeted CARs have been encoded in our 41BB CAR construct and assessed in vitro and in vivo by using Celigo imaging cytometry assay and humanized ccRCC orthotopic NSG-SGM3 mouse model respectively. Dual-targeted anti-CAIX/CD70 CD4/8 CAR-T cells outperformed singly-targeted CAR-T cells with elevated efficacy. In summary, anti-CAIX/CD70 CAR-T cells hold a great promise to achieve ccRCC cures. Citation Format: Yufei Wang, Marion Grimaud, Alicia Buck, Atef Fayed, Matthew Chang, Rebecca Jennings, Maura Sticco-Ivins, Miriam Ficial, Leo L. Chan, Sabina Signoretti, Quan Zhu, Wayne A. Marasco. Develop dual-targeted CAR-T cells to achieve RCC cures [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6606.

Self-Assembling 3D Spheroid Cultures of Human Neonatal Keratinocytes Have Enhanced Regenerative Properties

Relative to conventional two-dimensional (2-D) culture, three-dimensional (3-D) suspension culture of epithelial cells more closely mimics the in vivo cell microenvironment regarding cell architecture, cell to matrix interaction, and osmosis exchange. However, primary normal human keratinocytes (NHKc) rapidly undergo terminal differentiation and detachment-induced cell death (anoikis) upon disconnection from the basement membrane, thus greatly constraining their use in 3-D suspension culture models. Here, we examined the 3-D anchorage-free growth potential of NHKc isolated from neonatal skin explants of 59 different individuals. We found that 40% of all isolates naturally self-assembled into multicellular spheroids within 24 h in anchorage-free culture, while 60% did not. Placing a single spheroid back into 2-D monolayer culture yielded proliferating cells that expressed elevated levels of nuclear P63 and basal cytokeratin 14. These cells also displayed prolonged keratinocyte renewal and a gene expression profile corresponding to cellular heterogeneity, quiescence, and de-differentiation. Notably, spheroid-derived (SD) NHKc were enriched for a P63/K14 double-positive population that formed holoclonal colonies and reassembled into multicellular spheroids during 3-D suspension subculture. This study reveals marked phenotypic differences in neonatal keratinocyte suspension cultures isolated from different individuals. We present here a model system that can be readily employed to study epithelial cell behavior, along with a variety of dermatological diseases.

Concomitant decrease of double-positive lymphocyte population CD4CD8αα and Faecalibacterium prausnitzii in patients with colorectal cancer.

Changes in the composition of the gut microbiota in patients with colorectal cancer (CRC) compatible with a contribution of the gut microbiota in carcinogenesis have been reported. In particular, a decrease Faecalibacterium prausnitzii has been identified. A CD4CD8αα, double-positive lymphocyte population (DP8α) has recently been demonstrated in the human colon and blood with regulatory functions and specificity for F. prausnitzii. Here, we aimed to detect dysbiosis in the fecal microbiome of patients with CRC by metagenomic analysis, and to look for changes in the levels of DP8α circulating T cells specific for F. prausnitzii in these patients. Patients with CRC and control subjects were prospectively included. None had received antibiotics in the previous month or any anti-tumor treatment. A stool sample was collected for each participant, and analyzed by shotgun sequencing. The DP8α T cell population was identified and quantified on fresh whole blood by flow cytometry with anti-CD45, anti-CD3, anti-CD4 and anti-CD8α co-labeling. Twenty-one patients with CRC and 20 controls subjects were included. We found that mean relative abundance of five species was significantly decreased in CRC patients compared with controls, including F. prausnitzii, Barnesiella intestinihominis, Alistipes finegoldii, Bacteroides eggerthii and Eubacterium siraeum. We also found that the DP8α T cell population was significantly decreased in the blood of CRC patients compared with controls. In our work, we showed that a reduced abundance of F. prausnitzii in CRC patients was associated to a significant decrease in the circulating DP8α Treg population, suggesting a potential involvement of reduced activity of DP8α T cells in colonic carcinogenesis. These findings open new diagnostic and therapeutic strategies for CRC.

CD14+CD16+ monocytes are involved in daratumumab‐mediated myeloma cells killing and in anti‐CD47 therapeutic strategy

A deep elucidation of the mechanisms of action of anti-CD38 monoclonal antibodies (mAbs), such as daratumumab (DARA), is required to identify patients with multiple myeloma (MM) who are more responsive to this treatment. In the present study, an autologous ex vivo approach was established, focussing on the role of the monocytes in the anti CD38-mediated killing of MM cells. In bone marrow (BM) samples from 29 patients with MM, we found that the ratio between monocytes (CD14+ ) and MM cells (CD138+ ) influences the response to DARA. Further, the exposure of the BM samples to DARA is followed by the formation of a CD138+ CD14+ double-positive (DP) population, that quantitatively correlates with the anti-MM cells killing. These effects were dependent on the presence of a CD14+ CD16+ monocyte subset and on high CD16 expression levels. Lastly, the addition of a mAb neutralising the CD47/signal-regulatory protein α (SIRPα) axis was able to increase the killing mediated by DARA. The effects were observed only in coincidence with high CD14+ :CD138+ ratio, with a significant presence of the DP population and were correlated with CD16 expression. In conclusion, the present study underlines the critical role of the CD16+ monocytes in DARA anti-MM killing effects and gives a rationale to test the combination of an anti-CD47 mAb with anti-CD38 mAbs.

The transcriptional repressor Bcl6 promotes pre-TCR-induced thymocyte differentiation and attenuates Notch1 activation.

Pre-T-cell receptor (TCR) signal transduction is required for developing thymocytes to differentiate from CD4-CD8- double-negative (DN) cell to CD4+CD8+ double-positive (DP) cell. Notch signalling is required for T-cell fate specification and must be maintained throughout β-selection, but inappropriate Notch activation in DN4 and DP cells is oncogenic. Here, we show that pre-TCR signalling leads to increased expression of the transcriptional repressor Bcl6 and that Bcl6 is required for differentiation to DP. Conditional deletion of Bcl6 from thymocytes reduced pre-TCR-induced differentiation to DP cells, disrupted expansion and enrichment of intracellular TCRβ+ cells within the DN population and increased DN4 cell death. Deletion also increased Notch1 activation and Notch-mediated transcription in the DP population. Thus, Bcl6 is required in thymocyte development for efficient differentiation from DN3 to DP and to attenuate Notch1 activation in DP cells. Given the importance of inappropriate NOTCH1 signalling in T-cell acute lymphoblastic leukaemia (T-ALL), and the involvement of BCL6 in other types of leukaemia, this study is important to our understanding of T-ALL.

Perivascular macrophages in the neonatal macaque brain undergo massive necroptosis after simian immunodeficiency virus infection.

We previously showed that rhesus macaques neonatally infected with simian immunodeficiency virus (SIV) do not develop SIV encephalitis (SIVE) and maintain low brain viral loads despite having similar plasma viral loads compared to SIV-infected adults. We hypothesize that differences in myeloid cell populations that are the known target of SIV and HIV in the brain contribute to the lack of neonatal susceptibility to lentivirus-induced encephalitis. Using immunohistochemistry and immunofluorescence microscopy, we examined the frontal cortices from uninfected and SIV-infected infant and adult macaques (n=8/ea) as well as adults with SIVE (n=4) to determine differences in myeloid cell populations. The number of CD206+ brain perivascular macrophages (PVMs) was significantly greater in uninfected infants than in uninfected adults and was markedly lower in SIV-infected infants while microglia numbers were unchanged across groups. CD206+ PVMs, which proliferate after infection in SIV-infected adults, did not undergo proliferation in infants. While virtually all CD206+ cells in adults are also CD163+, infants have a distinct CD206 single-positive population in addition to the double-positive population commonly seen in adults. Notably, we found that more than 60% of these unique CD206+CD163- PVMs in SIV-infected infants were positive for cleaved caspase-3, an indicator of apoptosis, and that nearly 100% of this subset were concomitantly positive for the necroptosis marker receptor-interacting protein kinase-3 (RIP3). These findings show that distinct subpopulations of PVMs found in infants undergo programmed cell death instead of proliferation following SIV infection, which may lead to the absence of PVM-dependent SIVE and the limited size of the virus reservoir in the infant brain.

The Location of CD4+FoxP3+ Cells with Regard to CD25 and TNFRSF25 Receptor Signals Matters: Different Requirements for GI and Peripheral Tregs

Regulatory T cells (Tregs) are critical to maintaining immune homeostasis and generating tolerance in the gastrointestinal (GI) tract. GI complications play a prominent role following allogeneic HSCT (aHSCT) and we are interested in manipulating GI Tregs to regulate GVHD. Previously our lab has shown that using a two-pathway strategy of stimulating the TNFRSF25 and CD25 receptors with a TL1A-Ig fusion protein (FP) and IL-2low dose, Treg frequency and numbers in the lymphohematopoietic (LHC) compartment can be markedly increased (Wolf, BBMT 2017; Copsel, BBMT 2018). As a consequence of microbes and food antigens, Treg populations in the GI have a relative highly diverse composition compared to the lymphohematopoietic Treg compartment. This includes, but is not limited to, the presence of a stable and suppressive FoxP3+RORyt+ Treg population. Additionally, the GI tract contains various populations of innate lymphoid cells (ILCs) that interact with Tregs. ILCs express CD25 as well as TNFRSF25, and respond individually to IL-2 and TL1A administration respectively (Danny, JCI 2017; Verneris, Blood 2013). Based on the extensive differences between the lymphohematopoietic and GI Treg compartments, we evaluated how our two-pathway strategy may be differentially affecting the levels and activation status of Tregs in the GI tract. To address this question, we initially utilized B6-Nur77GFP mice, where GFP expression occurs with activation of the Nur77 promoter. However, since Nur77 is only produced following TcR engagement and not inflammatory signals, the strength of TcR stimulation in all T cell populations can be monitored by GFP levels. We first examined Tregs from 2-pathway treated B6-Nur77GFP mice and subsequently generated B6-Nur77GFPFoxP3RFP mice to readily assess the TcR signaling status of Tregs in different compartments. Mice were systemically administered FP, IL-2, or FP + IL-2 over a 1 wk period. The frequency of Tregs (FoxP3+CD4+) / Tcon (CD4+FoxP3-) in the lamina propria (LP) of the large intestine (LI) reached levels >60%. (1A). This elevation of Treg / Tcon frequency included FoxP3+RORyt- Tregs as well as FoxP3+RORyt+ double positive Treg populations. In contrast to our previous findings in the LHC, treatment with TL1A-Ig FP alone elevated levels to the same extent as the combination of FP + IL-2 (1A). Importantly, IL-2 treatment alone - as reported in the LHC - again had only a modest effect on elevating the frequency of Tregs in the LI/LP (1A). These observations suggest that the activation status of Tregs may differ based on the compartmental location. To asses activation status, we evaluated 2-pathway treated B6-Nur77GFP mice. Tregs in the LN/spleen had elevated frequency of GFP+ Tregs and higher GFP MFI than untreated mice. This elevated TcR stimulation was present in peripheral Tregs - but not CD8 - T cells (1B). Without exogenous stimulation, Tregs exhibited higher baseline TcR activation levels vs. Tcon cells (1B). The frequency of GFP+ Tregs and the GFP MFI was clearly highest (>45%) in the SI and LI LP vs. LN/spleen (<35%) (1C). In an independent experiment, we verified these findings and learned that only conjunctival Tregs demonstrated similarly increased frequency of GFP+ Tregs with elevated GFP MFI (1D). We also found that TL1A-Ig + IL-2 treatment in vivo increases the relative frequency of both innate ILC2 and ILC3 lymphoid populations (2A). The combination also increased the frequency of ILC2 cells greater than either reagent alone in the GI tract (2B). In total, our results objectively validate selective Treg vs. Tcon targeting via TNFRSF25. These findings also demonstrate that basal Treg activation status differs depending on the compartment. Notably, Tregs in mucosal vs. LHC tissue expressed higher TcR activation levels. Such Tregs have the potential for co-stimulation via TNFRSF25. Moreover, since IL-2 is required to maintain Tregs under both basal and activated conditions, our findings suggest a local source of IL-2 is present to maintain GI Tregs. ILC3 cells are recently reported to generate IL-2 (Zhou L, Nature 2019). We hypothesize that TNFRSF25 stimulation in ILC3 cells results in a local source of IL-2 which can account for our finding that TL1A-Ig alone vs. TL1A-Ig + IL-2 stimulation results in equivalent levels of GI Tregs. Ongoing experiments are examining how GI GVHD in aHSCT recipients are affected by TNFRSF25 +/- CD25 Treg/ILC stimulation. Disclosures Levy: Heat Biologics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pelican Therapeutics: Consultancy, Research Funding.

Incidence of androgen receptor and androgen receptor variant 7 coexpression in prostate cancer.

Prostate cancer (PRCA) is an androgen-driven disease, where androgens act through the androgen receptor (AR) to induce proliferation and survival of tumor cells. Recently, AR splice variant 7 (ARv7) has been implicated in advanced stages of PRCA and clinical recurrence. With the widespread use of AR-targeted therapies, there has been a rising interest in the expression of full-length AR and ARv7 in PRCA progression and how these receptors, both independently and together, contribute to adverse clinicopathologic outcomes. Despite a multitude of studies measuring the expression levels of AR and ARv7 in PRCA progression, the results have been inconsistent and sometimes contradictory due to technical and analytical discrepancies. To circumvent these inconsistencies, we used an automated multiplexed immunostaining platform for full-length AR and ARv7 in human PRCA samples and objectively quantified expression changes with machine learning-based software. With this technology, we can assess receptor prevalence both independently, and coexpressed, within specific tissue and cellular compartments. Full-length AR and ARv7 expression increased in epithelial nuclei of metastatic samples compared to benign. Interestingly, a population of cells with undetectable AR persisted through all stages of PRCA progression. Coexpression analyses showed an increase of the double-positive (AR+ /ARv7+ ) population in metastases compared to benign, and an increase of the double-negative population in PRCA samples compared to benign. Importantly, analysis of clinicopathologic outcomes associated with AR/ARv7 coexpression showed a significant decrease in the double-positive population with higher Gleason score (GS), as well as in samples with recurrence in under 5 years. Conversely, the double-negative population was significantly increased in samples with higher GS and in samples with recurrence in under 5 years. Changes in AR and ARv7 coexpression may have prognostic value in PRCA progression and recurrence. A better understanding of the prevalence and clinicopathologic outcomes associated with changes in these receptors' coexpression may provide a foundation for improved diagnosis and therapy for men with PRCA.

Stable Adult Hippocampal Neurogenesis in Cannabinoid Receptor CB2 Deficient Mice

The G-protein coupled cannabinoid receptor 2 (CB2) has been implicated in the regulation of adult neurogenesis in the hippocampus. The contribution of CB2 towards basal levels of proliferation and the number of neural progenitors in the subgranular zone (SGZ) of the dentate gyrus, however, remain unclear. We stained hippocampal brain sections of 16- to 17-week-old wildtype and CB2-deficient mice, for neural progenitor and immature neuron markers doublecortin (DCX) and calretinin (CR) and for the proliferation marker Ki67 and quantified the number of positive cells in the SGZ. The quantification revealed that CB2 deficiency neither altered overall cell proliferation nor the size of the DCX+ or DCX and CR double-positive populations in the SGZ compared to control animals. The results indicate that CB2 might not contribute to basal levels of adult neurogenesis in four-month-old healthy mice. CB2 signaling might be more relevant in conditions where adult neurogenesis is dynamically regulated, such as neuroinflammation.

PD-1 Blockade During Post-partum Involution Reactivates the Anti-tumor Response and Reduces Lymphatic Vessel Density.

Post-partum breast cancer patients, or breast cancer patients diagnosed within 10 years of last childbirth, are ~3–5 times more likely to develop metastasis in comparison to non-post-partum, or nulliparous, patients. Additionally, post-partum patients have increased tumor-associated lymphatic vessels and LN involvement, including when controlled for size of the primary tumor. In pre-clinical, immune-competent, mouse mammary tumor models of post-partum breast cancer (PPBC), tumor growth and lymphogenous tumor cell spread occur more rapidly in post-partum hosts. Here we report on PD-L1 expression by lymphatic endothelial cells and CD11b+ cells in the microenvironment of post-partum tumors, which is accompanied by an increase in PD-1 expression by T cells. Additionally, we observed increases in PD-L1 and PD-1 in whole mammary tissues during post-partum mammary gland involution; a known driver of post-partum tumor growth, invasion, and metastasis in pre-clinical models. Importantly, implantation of murine mammary tumor cells during post-partum mammary gland involution elicits a CD8+ T cell population that expresses both the co-inhibitory receptors PD-1 and Lag-3. However, upon anti-PD-1 treatment, during post-partum mammary gland involution, the involution-initiated promotional effects on tumor growth are reversed and the PD-1, Lag-3 double positive population disappears. Consequently, we observed an expansion of poly-functional CD8+ T cells that produced both IFNγ and TNFα. Finally, lymphatic vessel frequency decreased significantly following anti-PD-1 suggesting that anti-PD-1/PD-L1 targeted therapies may have efficacy in reducing tumor growth and dissemination in post-partum breast cancer patients.

Biomarker Discovery in Pre-Type 1 Diabetes; Th40 Cells as a Predictive Risk Factor.

The incidence of type 1 diabetes (T1D) is increasing worldwide. The quest to understand T1D etiology and how to predict diabetes is ongoing; and, in many ways, those goals intertwine. Although genetic components associate with T1D, not all individuals with T1D have those components, and T1D does not develop in all subjects with those components. More robust methods for prediction of T1D are needed. We investigated if high CD4+CD40+ T-cell (Th40) levels can be used as a biomarker. Th40 levels were assessed along with other parameters in blood collected from prediabetic subjects in TrialNet. In prediabetic subjects stratified according to Th40 cell level, patterns paralleled those seen between control subjects and those with T1D. Cytokine patterns were significantly different between those with high Th-40 levels (Th40-high) and those with low levels, and a CD4/CD8 double-positive population was more represented in Th40-high groups. Subjects experiencing impaired glucose tolerance had a significantly higher Th40 level than did control subjects. HLA DR4/DR4 and DQ8/DQ8 were more likely found among Th40-high subjects. Interestingly, HLA DR4/DR4 subjects were significantly older compared with all other subjects, suggesting that this haplotype, together with a high Th40 level, may represent someone in whom T1D will develop after age 30 years, which is reported for 42% of T1D cases. Considering the differences found in relation to prediabetic Th40 cell level, it may be possible to devise methods that more accurately predict who will proceed toward diabetes and, possibly, indicate prediabetic stage.

Application of a Bioactive/Bioresorbable Three-Dimensional Porous Uncalcined and Unsintered Hydroxyapatite/Poly-D/L-lactide Composite with Human Mesenchymal Stem Cells for Bone Regeneration in Maxillofacial Surgery: A Pilot Animal Study.

A novel three-dimensional (3D) porous uncalcined and unsintered hydroxyapatite/poly-d/l-lactide (3D-HA/PDLLA) composite demonstrated superior biocompatibility, osteoconductivity, biodegradability, and plasticity, thereby enabling complex maxillofacial defect reconstruction. Mesenchymal stem cells (MSCs)—a type of adult stem cell—have a multipotent ability to differentiate into chondrocytes, adipocytes, and osteocytes. In a previous study, we found that CD90 (Thy-1, cluster of differentiation 90) and CD271 (low-affinity nerve growth factor receptor) double-positive cell populations from human bone marrow had high proliferative ability and differentiation capacity in vitro. In the present study, we investigated the utility of bone regeneration therapy using implantation of 3D-HA/PDLLA loaded with human MSCs (hMSCs) in mandibular critical defect rats. Microcomputed tomography (Micro-CT) indicated that implantation of a 3D-HA/PDLLA-hMSC composite scaffold improved the ability to achieve bone regeneration compared with 3D-HA/PDLLA alone. Compared to the sufficient blood supply in the mandibular defection superior side, a lack of blood supply in the inferior side caused delayed healing. The use of Villanueva Goldner staining (VG staining) revealed the gradual progression of the nucleated cells and new bone from the scaffold border into the central pores, indicating that 3D-HA/PDLLA loaded with hMSCs had good osteoconductivity and an adequate blood supply. These results further demonstrated that the 3D-HA/PDLLA-hMSC composite scaffold was an effective bone regenerative method for maxillofacial boney defect reconstruction.

CD14+CD16+ Monocyte Binding to Myeloma Cells Is Required for Daratumumab Dependent Killing in Multiple Myeloma Patients

Recently, the introduction of anti-CD38 monoclonal antibody, Daratumumab (DARA), in multiple myeloma (MM) therapy has improved the response rate of relapsed MM patients. However only a fraction of the DARA-treated patients respond, thus further studies on DARA mechanisms of action are needed. Because the antibody dependent cellular phagocytosis (ADCP) mediated by monocyte, is one of the mechanisms through DARA exerts its anti-MM activity, an ex-vivo approach was established in order to investigate which mechanisms or patient's immunological characteristics could influence DARA-mediated killing of MM cells.Bone marrow mononuclear cells (BM-MNCs) obtained from 25 MM patients (12 newly diagnosed and 13 relapsed MM) were analyzed at time 0 (T0) by flow cytometry. We checked the % of plasma cells (PCs) (CD138+ cells), % of total monocytes (CD14+ cells) and their two subsets (CD14+CD16- or CD14+CD16+ cells), the ratio between % of CD14+ and % of CD138+ cells (CD14+:CD138+ ratio) and the median fluorescence intensity (MFI) of CD38 and of the immuno-check points CD47 on PCs and CD172a (SIRPα) on monocytes. Subsequently, BM-MNCs were treated with control IgG (10µg/ml), DARA (10µg/ml) and the F(Ab)2 portion of DARA (DARA F(Ab)2) (10µg/ml) for 48 hours and then the % variation of surviving 7AAD- CD138+ cell, the modification of the % of monocyte or subsets analyzed and the % of PCs that are attached to monocyte (identified as CD138+ cells also positive to CD14) were evaluated by flow cytometry.Firstly, we found that DARA significantly exploited its anti-MM activity (median % variation of surviving CD138+ cells: 69.05%, p=0.0007) compared to IgG control while DARA F(Ab)2 did not have any killing effect (median % variation of surviving CD138+ cells: 97.33%) compared to the control.Secondly, we detected that the % of total of monocytes and their subsets (CD14+CD16+ or CD14+CD16-) composition were not modulated by DARA or DARA F(Ab)2 treatment compared to IgG. Indeed, only in presence of DARA, we observed that a double positive population of CD138+CD14+ cell significantly increased compared to IgG control (mean %: 15.76 vs 3.14, p=<0.0001) and to DARA F(Ab)2 condition (mean %:15,76 vs 1,48, p=<0.004). Moreover, we found that monocytes involved in the double positive population CD138+CD14+ were almost completely CD14+CD16+ monocytes. Indeed, we have divided the patient' samples in high responder (HR) and low responder (LR) based on the median % of survival cell after DARA treatment (HR: <69.05% and LR:>69.05% of surviving PCs). Interestingly, in the HR group compared to LR group were significantly increased the % of double positive CD138+CD14+ (median % HR:21.59 vs LR: 7.41, p=0.035), the % of CD138+ cells bonded to CD14+CD16+ (median % HR:13.28 vs LR: 3.93, p=0.048) and the CD14+:CD138+ ratio measured at T0 (median HR:0.63 vs LR: 0.32, p=0.0158). Moreover, in 5 relapsed MM patient, we have correlated the ex-vivo response to DARA with the type of in vivo response after DARA single-agent treatment.In addition in our cohort of patients, we observed that the % of surviving CD138+ cells after DARA treatment negatively correlate with the CD14+:CD138+ ratio at T0 (r:-0.628, p=0.0023), with the % of CD138+CD14+ population (r:-0.602, p=0.0039) and with the % of CD138+CD14+CD16+ population (r:- 0.657, p=0.0238) but did not correlate with the MFI of CD38 expression on PCs and the % of CD14+CD16+ at T0.Finally, to go further inside the mechanism involved in DARA response, we have explored the role of the inhibitory axis CD47-CD172a in the ADCP DARA-induced in 5 ex-vivo samples from our cohort of MM patients. We found that the MFI of CD47 strongly positively correlated with the % of surviving CD138+ cells (r: 0.9897; p=0.010) after DARA treatment; on the other hand, we have not reported any correlation with the MFI of CD172a on monocytes. The results of these analyses are continuously updating increasing the number of samples tested.In conclusion, these data highlight that monocyte binding to PCs, in particular those CD14+CD16+, plays a central role in DARA killing effect independently of % subset composition in the pre-treatment samples, suggesting that there are mechanisms that regulate the effectiveness of the monocyte-based killing effect on malignant PCs, as the immune-suppressive axis CD47-CD172a, giving the rationale design to identify new strategies to increase the efficiency of DARA-based therapeutic regimen. DisclosuresMalavasi:Takeda Pharmaceutical Co: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceutica: Membership on an entity's Board of Directors or advisory committees, Research Funding. Aversa:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Basilea: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Giuliani:Takeda Pharmaceutical Co: Research Funding; Celgene Italy: Other: Avisory Board, Research Funding; Janssen Pharmaceutica: Other: Avisory Board, Research Funding.

Knockout of both miR-15/16 loci induces acute myeloid leukemia

MicroRNAs (miRNAs) have been extensively reported to be associated with hematological malignancies. The loss of miR-15a/16-1 at chromosome 13q14 is a hallmark of most of human chronic lymphocytic leukemia (CLL). Deletion of murine miR-15a/16-1 and miR-15b/16-2 has been demonstrated to promote B cell malignancies. Here, we evaluate the biological role of miR-15/16 clusters, crossbreeding miR-15a/16-1 and miR-15b/16-2 knockout mice. Unexpectedly, the complete deletion of both clusters promoted myeloproliferative disorders in the majority of the mice by the age of 5 months with a penetrance of 70%. These mice showed a significant enlargement of spleen and abnormal swelling of lymph nodes. Flow cytometry characterization demonstrated an expanded CD11b/Gr-1 double-positive myeloid population both in spleen and in bone marrow. The transplantation of splenocytes harvested from double-KO mice into wild-type recipient mice resulted in the development of myeloproliferative disorders, as observed in the donors. In vivo, miR-15/16 cluster deletion up-regulated the expression of Cyclin D1, Cyclin D2, and Bcl-2. Taken together, our findings identify a driver oncogenic role for miR-15/16 cluster deletion in different leukocytic cell lineages.

Abstract B060: Molecular characterization of cancer stem cells from patient-derived xenografts of advanced prostate cancer

Abstract Introduction: Androgen-deprivation therapy is the standard treatment for prostate cancer. Despite the initial response, a fraction of cases manifest progression to castration-resistant prostate cancer. The paradigm of PCa recurrence is the existence of androgen-independent cancer stem cells, which differentiate into androgen-dependent cancer cells that reinitiate tumor growth. The phenotypic properties of cancer stem cells versus highly proliferative progenitor cells/facultative stem cells remain to be further characterized in terms of androgen receptor signaling, quiescence/proliferation, tumorigenicity, and therapy resistance. Methods: To address the molecular basis of CSC switch from androgen-dependency to independency we have employed two patient-derived xenograft models (LAPC-9 and BM-18) that have different androgen sensitivity properties. We have performed microarray and proteomic analysis of BM-18 tumor tissues prior to and following castration as well as androgen replacement. To characterize the cancer stem cells in these models we have isolated different subpopulations based on combination of selected markers and assessed the transcriptomic changes, proteomic profile, and in vivo self-renewal. Results: Castration induces a rapid tumor volume decrease in the BM-18 and a stabilization of tumor burden in the LAPC-9, reflecting different androgen-dependent cell states. Proteomic analysis of bulk BM-18 tumors (intact, 14 days post castration, 50 days post androgen replacement) indicates an enrichment of stem cell markers upon castration; CD44, NKX3.1, and ALDH1 isoforms. Microarray analysis has confirmed upregulation of CD44 and ALDH1A1 at castrated state, and downregulation of expression upon androgen replacement at early time points (24 hours). We have isolated CD44+/-ALDHhigh/low subpopulation by flow cytometry and analyzed their transcriptome and proteomic changes. Castration in the BM-18 model induces an increase in the subpopulations of CD44+/ALDHlow (from 0.44% to 2.3%) and CD44+/ALDHhigh (from 0.06% to 0.38%). However, the same subpopulations are decreased in the LAPC-9 model upon castration, while only the CD44-/ALDHhigh subset is enriched (from 3.5% to 7.1%). Conclusions: Different androgen-independent cancer stem cell subpopulations may be distinguished by the ALDH activity status in combination with CD44. The androgen-independent cells in the BM-18 androgen-dependent model are reflected by enrichment of CD44 expression, as well as a rare double positive population. In the androgen-independent LAPC-9 model, CD44 expression is contrastingly decreased, potentially reflecting androgen-dependent CD44+ cells, and ALDH activity increased in CD44- cells. Ongoing analysis may elucidate the molecular mechanisms controlling the different cancer stem cell fates and their androgen sensitivity. Cancer stem cell potential of the different cell populations will have to be elucidated by transplantation experiments. Citation Format: Sofia Karkampouna, Marta de Menna, Markus Germann, Jöel Grosjean, Peter C. Gray, George N. Thalmann, Marianna Kruithof-de Julio. Molecular characterization of cancer stem cells from patient-derived xenografts of advanced prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B060.

MP54-02 DEVELOPMENT OF INTRAVESICAL HUMAN γΔT CELL BASED CHEMO-IMMUNOTHERAPY AGAINST URINARY BLADDER CANCER CELLS IN AN ORTHOTOPIC XENOGRAFT MODEL

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I1 Apr 2018MP54-02 DEVELOPMENT OF INTRAVESICAL HUMAN γΔT CELL BASED CHEMO-IMMUNOTHERAPY AGAINST URINARY BLADDER CANCER CELLS IN AN ORTHOTOPIC XENOGRAFT MODEL Teruki Shimizu, Mako Tomogane, Masatsugu Miyashita, Osamu Ukimura, and Eishi Ashihara Teruki ShimizuTeruki Shimizu More articles by this author , Mako TomoganeMako Tomogane More articles by this author , Masatsugu MiyashitaMasatsugu Miyashita More articles by this author , Osamu UkimuraOsamu Ukimura More articles by this author , and Eishi AshiharaEishi Ashihara More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1693AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Intravesical BCG treatment continues to play a central role in the treatment of non-muscle invasive urinary bladder cancer (UBC). There is currently no gold standard for salvage intravesical therapy after BCG treatment failure. To develop the substitute for BCG treatment failure, we focused on γδT cell immunotherapy. γδT cells exert potent cytotoxicity towards various cancer cells and can recognize isopentenyl pyrophosphate (IPP) through the TCRγδ receptor, and zoredronic acid (ZOL) pretreatment is widely used to induce IPP accumulation in cancer cells. In this study, we hilighted two issues; combined use of anticancer agents along with ZOL and a bladder instillation approach. The aims are to improve recognition of cancer cells by γδT cells and to increase E/T (Effector/Target) ratio. METHODS We examined the efficacy of ex vivo-expanded γδT cell immunotherapy. Flow cytometric analysis of CFSE/propidium iodide (PI) staining was used in in vitro cytotoxicity assays.After co-culture for 4 h, CFSE/PI double positive population was measured as apoptotic cancer cells. In in vivo experiment, the efficacy of ex vivo-expanded γδT cell immunotherapy was examined in an orthotopic xenograft model using In Vivo Imaging System (IVIS). RESULTS Ex vivo-expanded γδT cells showed potent cytotoxicity against UBC cells in in vitro assays. Combination treatment with standard anticancer agents showed that low dose gemcitabine pretreatment significantly enhanced the cytotoxicity of γδT cells by upregulating the expression of MICA and MICB (MICA/B), which are tumor-associated antigens recognized by γδT cells. These effects were abrogated by small interfering RNA-mediated knockdown of MICA/B in UBC cells. IVIS analysis revealed the potent cytotoxicity of weekly intravesical administration of γδT cells (Figure. 1), and weekly gemcitabine pretreatment enhanced the cytotoxicity of γδT cells in vivo. CONCLUSIONS Low dose gemcitabine pretreatment significantly enhanced the cytotoxicity of γδT cells by upregulating the expression MICA/B in in vitro. Intravesical γδT cell immunotherapy exerted potent cytotoxicity against UBC and intravesical γδT cell immunotherapy in combination with low dose gemcitabine may be a promising strategy in UBC. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e712 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Teruki Shimizu More articles by this author Mako Tomogane More articles by this author Masatsugu Miyashita More articles by this author Osamu Ukimura More articles by this author Eishi Ashihara More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Thymic stromal lymphopoietin drives the development of IL-13+ Th2 cells

T helper 2 (Th2) cells are pivotal in the development of allergy. Allergen exposure primes IL-4+ Th2 cells in lymph node, but production of effector cytokines including IL-5 and IL-13 is thought to require additional signals from antigen and the environment. Here we report that a substantial proportion of naive CD4+ T cells in spleen and lymph node express receptors for the epithelium-derived inflammatory cytokine thymic stromal lymphopoietin (TSLP). Culture of naive CD4+ T cells in anti-(a)CD3, aCD28, and TSLP-supplemented Th2 conditions enabled the development of a unique population of IL-13-single positive (IL-13-SP) CD4+ T cells; TSLP and Th2 conditions were both required for their development. Sorting experiments revealed that IL-13-SP Th2 cells originated from IL-4-negative precursors and coexpressed transcripts for the Th2 cytokines IL-5 and IL-9. In vivo, high TSLP levels acted directly on CD4+ T cells to induce the development of IL-13-SP and IL-4+IL-13+ double-positive populations in lymph node. These cells were phenotypically similar to Th2 effector cells and were CXCR5lowPD1low and expressed low levels of Bcl6 and Il21 transcripts and high levels of Gata3, Il3, and Il5 Our findings suggest a role of TSLP in directly promoting Th2 cell effector function and support the notion of TSLP as a key driver of Th2 inflammation.

Human lung branching morphogenesis is orchestrated by the spatiotemporal distribution of ACTA2, SOX2, and SOX9.

Lung morphogenesis relies on a number of important processes, including proximal-distal patterning, cell proliferation, migration and differentiation, as well as epithelial-mesenchymal interactions. In mouse lung development, SOX2+ cells are localized in the proximal epithelium, whereas SOX9+ cells are present in the distal epithelium. We show that, in human lung, expression of these transcription factors differs, in that during the pseudoglandular stage distal epithelial progenitors at the tips coexpress SOX2 and SOX9. This double-positive population was no longer present by the canalicular stages of development. As in mouse, the human proximal epithelial progenitors express solely SOX2 and are surrounded by smooth muscle cells (SMCs) both in the proximal airways and at the epithelial clefts. Upon Ras-related C3 botulinum toxin substrate 1 inhibition, we noted decreased branching, as well as increased SMC differentiation, attenuated peristalsis, and a reduction in the distal double-positive SOX2/SOX9 progenitor cell population. Thus, the presence of SOX2/SOX9 double-positive progenitor cells in the distal epithelium during the pseudoglandular stage of human lung development appears to be critical to proximal-distal patterning and lung branching. Moreover, SMCs promote a SOX2 proximal phenotype and seem to suppress the SOX9+ population.

P173 The ART(EFACT) of multicolor flow cytometry. A case of mysterious CD3/CD19 double positive lymphocytes in the flow cytometry crossmatch (FCXM) assay

Aim Over the past 3 months we have been noticing an unusual population of CD3/CD19 double positive (DP) lymphocytes in some flowcytometry crossmatches (FCXM) performed in our HLA lab. Initially, the appearance of these unusual DP cells seemed random. However, upon careful review it was noted that the DP cells were seen only in reactions with the positive control sera and only when two recently trained techs were involved in setting up FCXM. Who was to blame? Methods FCXM were performed in 96-well trays according to the Halifax protocol. In house negative control (NC) and positive control (PC) sera were used in each FCXM. T/B cells were identified using mouse anti-CD3 PerCP(BD) and anti-CD19-PE (BD) mAbs respectively. Positive FCXM reactions were identified using polyclonal goat anti-human IgG-FITC (Jackson). FCXM were acquired on BD Canto II and analysed using Diva software. Results Fig. 1A illustrates a representative dot plot showing the unusual CD3/CD19 DPcell population observed with the PC sera in FCXM set up by one of the two recently trained technologists. This DP population appeared to be a result of CD3 + T cells shifting up on the PE (FL2) scale and is not seen with the NC sera (Fig. 1B) or any patient sera in the same FCXM. Repeat FCXM performed with identical reagents (including the PC) by a senior technologist did not reveal the DP cells. While direct observation did not identify any protocol deviations by the two junior technologists, it was noted that both individuals used red sharpies to mark the rims of the PC wells (blue for NC) in crossmatch trays (Fig. 1C). Further experiments showed that marking tray rims with red sharpies caused the appearance of the DP cells. Similar effect was seen with pink or purple sharpies but not when black, blue or green sharpies were used (Fig. 1D). This was attributed to the presence of Rhodamine B (fluorescence emission spectrum similar to PE) in the ink of red, pink and purple sharpies. Conclusions This report HIGHLIGHTS that even the best intentions can create strange and unexpected artefacts in FCXM. Download : Download high-res image (303KB) Download : Download full-size image

Abstract 339: Hematopoietic-derived Progenitor Cells Identified in Mouse Heart Valves are also Present in Human Mitral Valves

Tissue engineered heart valve constructs (TEHVCs) are thought to be the solution for the increasing needs of heart valve replacements. Using decellularized xenograft valve matrices reseeded with autologous cells seems to fulfill the demands for durability, lack of coagulation and immune reactions, in the same time allowing growth and adaptability. The presently used cell sources are not viable as the TEHVCs frequently exhibit durability issues due to inefficient seeding and subsequent calcification. The natural valve interstitial cells are very heterogeneous, their role is to maintain the extracellular matrix which is responsible for bearing the mechanical and hemodynamic load. We have previously identified, in murine heart valves, a hematopoietic bone marrow-derived progenitor cell population. These progenitor cells are continuously migrating into the heart valves and appear to participate in the normal homeostasis. The heart valve progenitor cells identified in mice seems a good candidate for TEHVCs, but first, their existence must be validated in human heart valves. We used commercially available, paraffin-embedded, human mitral valve sections from 4 different individuals to immunohistochemically identify the hematopoietic-derived progenitor cells. Combination of anti-CD45 (pan hematopoietic marker) and anti-CD133 (progenitor marker) antibodies revealed a double-positive cell population in the proximal region (toward the annulus), atrial side of the mitral leaflets - exactly where we have found the similar cells in the murine system. By further phenotypic experiments, we have excluded the presence of B- and T-lymphocytes, granulocytes and macrophages. In contrast to the mouse findings, the human CD45 + /CD133 + cells are also expressing CD34 which further emphasizes their hematopoietic origin (in mouse the CD34 is not a hematopoietic marker). By co-labeling with CD31 (Pecam-1) markers, we found that the CD45 + /CD133 + cells are never part of the endocardium, although they can be observed closely underneath. In conclusion, we have identified the CD45 + /CD133 + cells, previously identified in mice as progenitor cells, in human mitral valves. Functional characterization of these cells and their use in TEHVCs is the focus of our future work.