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Purpose: Death-associated protein kinase 1 (DAPK1), a tumor suppressor, is a rate-limiting effector in an endoplasmic reticulum (ER) stress-dependent apoptotic pathway. Its expression is epigenetically suppressed in several tumors. A mechanistic basis for epigenetic/transcriptional repression of DAPK1 was investigated in certain forms of acute myeloid leukemia (AML) with poor prognosis, which lacked ER stress-induced apoptosis.Experimental Design: Heterogeneous primary AMLs were screened to identify a subgroup with Flt3ITD in which repression of DAPK1, among NF-κB–and c-Jun–responsive genes, was studied. RNA interference knockdown studies were carried out in an Flt3ITD+ cell line, MV-4-11, to establish genetic epistasis in the pathway Flt3ITD–TAK1–DAPK1 repression, and chromatin immunoprecipitations were carried out to identify proximate effector proteins, including TAK1-activated p52NF-κB, at the DAPK1 locus.Results: AMLs characterized by normal karyotype with Flt3ITD were found to have 10- to 100-fold lower DAPK1 transcripts normalized to the expression of c-Jun, a transcriptional activator of DAPK1, as compared with a heterogeneous cytogenetic category. In addition, Meis1, a c-Jun-responsive adverse AML prognostic gene signature was measured as control. These Flt3ITD+ AMLs overexpress relB, a transcriptional repressor, which forms active heterodimers with p52NF-κB. Chromatin immunoprecipitation assays identified p52NF-κB binding to the DAPK1 promoter together with histone deacetylase 2 (HDAC2) and HDAC6 in the Flt3ITD+ human AML cell line MV-4-11. Knockdown of p52NF-κB or its upstream regulator, NF-κB–inducing kinase (NIK), de-repressed DAPK1. DAPK1-repressed primary Flt3ITD+ AMLs had selective nuclear activation of p52NF-κB.Conclusions: Flt3ITD promotes a noncanonical pathway via TAK1 and p52NF-κB to suppress DAPK1 in association with HDACs, which explains DAPK1 repression in Flt3ITD+ AML. Clin Cancer Res; 18(2); 360–9. ©2011 AACR.

Supplemental figure legend

S1: Distribution of CD166 mRNA expression across 30 multiple myeloma and 22 breast cancer cell lines were analyzed; S2: The percentage of CD166+ cells within H929 cells from BM-homed cells were examined flow cytometrically; S3: Data are representative of 2 separate experiments (mean{plus minus}SEM, N = 6mice/group/experiment, each assayed individually); S5: Data represent 3 separate experiments done in triplicates for each group and are expressed as mean{plus minus} SEM, *P

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Supplemental table 1

Multiple myeloma is incurable once osteolytic lesions have seeded at skeletal sites, but factors mediating this deadly pathogenic advance remain poorly understood. Here, we report evidence of a major role for the cell adhesion molecule CD166, which we discovered to be highly expressed in multiple myeloma cell lines and primary bone marrow cells from patients. CD166+ multiple myeloma cells homed more efficiently than CD166− cells to the bone marrow of engrafted immunodeficient NSG mice. CD166 silencing in multiple myeloma cells enabled longer survival, a smaller tumor burden, and less osteolytic lesions, as compared with mice bearing control cells. CD166 deficiency in multiple myeloma cell lines or CD138+ bone marrow cells from multiple myeloma patients compromised their ability to induce bone resorption in an ex vivo organ culture system. Furthermore, CD166 deficiency in multiple myeloma cells also reduced the formation of osteolytic disease in vivo after intratibial engraftment. Mechanistic investigation revealed that CD166 expression in multiple myeloma cells inhibited osteoblastogenesis of bone marrow–derived osteoblast progenitors by suppressing Runx2 gene expression. Conversely, CD166 expression in multiple myeloma cells promoted osteoclastogenesis by activating TRAF6-dependent signaling pathways in osteoclast progenitors. Overall, our results define CD166 as a pivotal director in multiple myeloma cell homing to the bone marrow and multiple myeloma progression, rationalizing its further study as a candidate therapeutic target for multiple myeloma treatment. Cancer Res; 76(23); 6901–10. ©2016 AACR.

Supplementary Tables 1-3 from Gene Expression Profiling Identifies BAX-δ as a Novel Tumor Antigen in Acute Lymphoblastic Leukemia

Heterogeneity of endothelial cell (EC) populations reflects their diverse functions in maintaining tissue’s homeostasis. However, their phenotypic, molecular, and functional properties are not entirely mapped. We use the Tie2-CreERT2;Rosa26-tdTomato reporter mouse to trace, profile, and cultivate primary ECs from different organs. As paradigm platform, we use this strategy to study bone marrow endothelial cells (BMECs). Single-cell mRNA sequencing of primary BMECs reveals that their diversity and native molecular signatures is transitorily preserved in an ex vivo culture that conserves key cell-to-cell microenvironment interactions. Macrophages sustain BMEC cellular diversity and expansion and preserve sinusoidal-like BMECs ex vivo. Endomucin expression discriminates BMECs in populations exhibiting mutually exclusive properties and distinct sinusoidal/arterial and tip/stalk signatures. In contrast to arterial-like, sinusoidal-like BMECs are short-lived, form 2D-networks, contribute to in vivo angiogenesis, and support hematopoietic stem/progenitor cells in vitro. This platform can be extended to other organs’ ECs to decode mechanistic information and explore therapeutics.

The COVID-19 pandemic has triggered the first widespread vaccination campaign against a coronavirus. Most vaccinated subjects are naïve to SARS-CoV-2, however almost all have previously encountered other coronaviruses (CoVs) and the role of this immunity in shaping the vaccine response remains uncharacterized. Here we use longitudinal samples and highly-multiplexed serology to identify mRNA-1273 vaccine-induced antibody responses against a range of CoV Spike epitopes and in both phylogenetically conserved and non-conserved regions. Whereas reactivity to SARS-CoV-2 epitopes showed a delayed but progressive increase following vaccination, we observed distinct kinetics for the endemic CoV homologs at two conserved sites in Spike S2: these became detectable sooner, and decayed at later timepoints. Using homolog-specific depletion and alanine-substitution experiments, we show that these distinctly-evolving specificities result from cross-reactive antibodies as they mature against rare, polymorphic residues within these epitopes. Our results reveal mechanisms for the formation of antibodies with broad reactivity against CoVs.

SARS-CoV-2 (CoV-2) viral infection results in COVID-19 disease, which has caused significant morbidity and mortality worldwide. A vaccine is crucial to curtail the spread of SARS-CoV-2, while therapeutics will be required to treat ongoing and reemerging infections of SARS-CoV-2 and COVID-19 disease. There are currently no commercially available effective anti-viral therapies for COVID-19 urging the development of novel modalities. Here, we describe a molecular therapy specifically targeted to neutralize SARS-CoV-2, which consists of extracellular vesicles (EVs) containing a novel fusion tetraspanin protein, CD63, embedded with an anti-CoV-2 nanobody. These anti-CoV-2 enriched EVs binds SARS-CoV-2 spike protein at the receptor-binding domain (RBD) site and can functionally neutralize SARS-CoV-2. This work demonstrates an innovative EV targeting platform that can be employed to target and inhibit the early stages of SARS-CoV-2 infection., Graphical Abstract, Described here is a modular extracellular vesicle (EV) platform that can redirect EVs to target virus envelope proteins and the ability of these virus targeted EVs to broadly neutralize SARS-COV-2 variants of concern.

T cells engineered to express HIV-specific chimeric antigen receptors (CARs) represent a promising strategy to clear HIV-infected cells, but to date have not achieved clinical benefits. A likely hurdle is the limited T cell activation and persistence when HIV antigenemia is low, particularly during antiretroviral therapy (ART). To overcome this issue, we propose to use a cytomegalovirus (CMV) vaccine to stimulate CMV-specific T cells that express CARs directed against the HIV-1 envelope protein gp120. In this study, we use a GMP-compliant platform to engineer CMV-specific T cells to express a second-generation CAR derived from the N6 broadly neutralizing antibody, one of the broadest anti-gp120 neutralizing antibodies. These CMV-HIV CAR T cells exhibit dual effector functions upon

Acute lymphoblastic leukemia (ALL) is the most common malignancy in pediatric patients. About 10–15% of pediatric ALL belong to T-cell ALL (T-ALL), which is characterized by aggressive expansion of immature T-lymphoblasts and is categorized as high-risk leukemia. Leukemia initiating cells represent a reservoir that is responsible for the initiation and propagation of leukemia. Its perinatal origin has been suggested in some childhood acute B-lymphoblastic and myeloblastic leukemias. Therefore, we hypothesized that child T-ALL initiating cells also exist during the perinatal period. In this study, T-ALL potential of the hematopoietic precursors was found in the para-aortic splanchnopleura (P-Sp) region, but not in the extraembryonic yolk sac (YS) of the mouse embryo at embryonic day 9.5. We overexpressed the Notch intracellular domain (NICD) in the P-Sp and YS cells and transplanted them into lethally irradiated mice. NICD-overexpressing P-Sp cells rapidly developed T-ALL while YS cells failed to display leukemia propagation despite successful NICD induction. These results suggest a possible role of fetal-derived T-cell precursors as leukemia-initiating cells.

Medulloblastoma (MB) is a malignant childhood brain tumor which at molecular level is classified into at least four major subtypes: WNT, SHH, group C and group D differing in response to treatment. Previous studies have associated changes in expression levels and activation of certain HOX genes with MB development. In the present study, we investigate the role of HOX genes in two attributes acquired by tumor cells: migration and proliferation potential, as well as, in vivo tumorigenic potential. We analyzed UW402, UW473, DAOY and ONS-76 human pediatric MB cell lines and cerebellum primary cultures. Two-color microarray-based gene expression analysis was used to identify differentially expressed HOX genes. Among the various HOX genes significantly overexpressed in DAOY and ONS-76 cell lines compared to UW402 and UW473 cell lines, HOXA10 and HOXB4 were selected for further analysis. The expression levels of these HOX genes were validated by real-time PCR. A mouse model was used to study the effect of the HOXA10 and HOXB4 genes on the in vivo tumorigenic potential and the in vitro proliferative and migration potential of MB cell lines. Our results show that the inhibition of HOXA10 in DAOY cell line led to increased in vitro cell migration while in vitro cell proliferation or in vivo tumorigenic potential were unaffected. We also observed that induced expression of HOXB4 in the UW473 cell line significantly reduced in vitro cell proliferation and migration capability of UW473 cells with no effect on the in vivo tumorigenicity. This suggests that HOXA10 plays a role in migration events and the HOXB4 gene is involved in proliferation and migration processes of medulloblastoma cells, however, it appears that these genes are not essential for the tumorigenic process of these cells.

The increasing characterization of childhood acute lymphoblastic leukemia (ALL) has led to the identification of multiple molecular targets but has yet to translate into more effective targeted therapies, particularly for high-risk, relapsed T-cell ALL. Searching for master regulators controlling multiple signaling pathways in T-ALL, we investigated the multifunctional protein redox factor-1 (Ref-1/APE1), which acts as a signaling “node” by exerting redox regulatory control of transcription factors important in leukemia. Leukemia patients' transcriptome databases showed increased expression in T-ALL of Ref-1 and other genes of the Ref-1/SET interactome. Validation studies demonstrated that Ref-1 is expressed in high-risk leukemia T cells, including in patient biopsies. Ref-1 redox function is active in leukemia T cells, regulating the Ref-1 target NF-κB, and inhibited by the redox-selective Ref-1 inhibitor E3330. Ref-1 expression is not regulated by Notch signaling, but is upregulated by glucocorticoid treatment. E3330 disrupted Ref-1 redox activity in functional studies and resulted in marked inhibition of leukemia cell viability, including T-ALL lines representing different genotypes and risk groups. Potent leukemia cell inhibition was seen in primary cells from ALL patients, relapsed and glucocorticoid-resistant T-ALL cells, and cells from a murine model of Notch-induced leukemia. Ref-1 redox inhibition triggered leukemia cell apoptosis and downregulation of survival genes regulated by Ref-1 targets. For the first time, this work identifies Ref-1 as a novel molecular effector in T-ALL and demonstrates that Ref-1 redox inhibition results in potent inhibition of leukemia T cells, including relapsed T-ALL. These data also support E3330 as a specific Ref-1 small-molecule inhibitor for leukemia. Mol Cancer Ther; 16(7); 1401–11. ©2017 AACR.

Acute Lymphoblastic Leukemia (ALL) is the most common pediatric cancer. Despite marked clinical successes in the treatment of childhood T-ALL, leukemia relapse, refractory disease and induction failure (≈ 30% of patients) remain significant clinical problems, often life-threatening. Thus, new therapeutic strategies are needed. Activating Notch mutations have been identified in over 50% of the cases, placing Notch signaling as a central player in T-ALL. Notch regulates the differentiation, proliferation and survival of both, normal and malignant T-cells, however its therapeutic targeting has failed. Moreover, little is known about its downstream mediators and the crosstalk with other microenvironmental cues, like the IL-7 signaling pathway. IL-7 is an essential cytokine for normal lymphoid development, and its continuous signal can lead to T-cell neoplasms. Our laboratory has focused on study the crosstalk between these two signaling pathways in T-ALL. Here we show that primary T-ALL cells carrying Notch activating mutations require IL-7 for a robust proliferation and are still responsive to ligand-dependent Notch stimulation. We found that Notch activation hypersensitize T-ALL cells to IL-7 by mediating direct transcription of IL-7Rα and thus, up-regulating its surface expression. Notch blockade significantly inhibited IL-7-mediated proliferation of T-ALL whereas IL-7 stimulation greatly potentiated Notch signaling. Investigation the downstream mechanisms common to both pathways lead us to the identification of SKP2. We discovered that SKP2, a key cell cycle regulator for cell cycle entry associated with poor prognosis in cancers, is a downstream target of Notch playing an important role in T-ALL. We demonstrated that genetic ablation or pharmacological blockade of Skp2, significantly delayed T-ALL progression and increased survival in in vivo T-ALL models. SKP2 mediates mitogenic responses to various cytokines, and we showed that is upregulated following IL-7 stimulation in human T-ALL cells, suggesting that Notch and IL-7 signaling cooperate and converge in SKP2. In conclusion, our studies indicated a cooperative effect between an oncogenic event (Notch signaling) and microenvironmental cues (IL-7 signaling) for leukemia maintenance and progression, and identify the Notch/IL-7/SKP2 circuitry as a critical network and potential therapeutic target in T-ALL.

We have successfully established a clinical platform for CD19 chimeric antigen receptor (CAR) T cells and evaluated its safety and efficacy in a series of pilot clinical trials following autologous hematopoietic cell transplantation (HCT) for treatment of high-risk non-Hodgkin lymphoma (NHL) and acute lymphoblastic leukemia (ALL). However, the full potential of this therapy has been limited by the lack of engraftment and persistence of CAR T cells in patients, most likely due to the inadequate antigen drive to stimulate robust expansion and long-term persistence of the infused CAR T cells. Additionally, CD19+ NHL seems less responsive to current CAR T cell technology than does CD19+ ALL that might be explained by lower engraftment and persistence of CAR T cells in NHL. To improve the efficacy and durability of CAR T cells in these disease settings, we sought to examine a novel approach based on properties of cytomegalovirus (CMV)-specific T cells. Specifically, we select CMV pp65-specific T cells for ex vivo modification with a CAR targeting CD19. After in vitro expansion, CMV-CD19 bi-specific T cells will be infused into the patient. A second round of expansion will be done in vivo, using a CMV vaccine (Triplex), developed and clinically evaluated multiple clinical sites including City of Hope. Triplex is a multi-antigen recombinant modified vaccinia Ankara (MVA) virus vaccine expressing pp65, IE1 and IE2, that has proven safe and immunogenic in Phase I trials in healthy volunteers and transplant patients and was highly tolerable and efficacious in a completed Phase II vaccine trial in allogeneic HCT recipients. We hypothesis that shorter ex vivo expansion time will prevent CAR T cell exhaustion that results in better in vivo expansion, especially after Triplex vaccination. Thus far, we have performed six large-scale manufacturing process development (PD) runs of CMV-CD19 CAR T cells using T cells from healthy donors. Briefly, peripheral blood mononuclear cells (PBMCs) were collected and processed in the CliniMACS Prodigy® system, in which PBMCs were first stimulated with a GMP-grade PepTivator® overlapping CMV pp65 peptide pool, then enriched for CMV-responsive IFNɣ+ T cells using the IFNγ Catchmatrix reagent (Miltenyi Biotec Inc.). CMV-responsive IFNɣ+ T cells were next transduced with a lentiviral vector encoding EGFRt/CAR, and finally expanded for approximately 15 days in vitro. As summarized in Table 1, we consistently recovered 4.8%+/-1.4 ×106 CMV specific T cells with 78.3%±2.9 purity from 1×109 PBMC input, which is the maximum input number suggested by the CCS program associated with the Prodigy system (Miltenyi Biotec Inc.). During the early stage PD runs, we noticed that red blood cell contamination in the PBMC layer after ficoll separation could negatively impact the yield of CMV-specific T cells. Thus, we optimized the separation process by performing a second ficoll whenever a red buffy coat was observed, and increased the yield of CMV-specific T cells from 2.8 ×106 to 9.25×106 (Table 1, HD 187.2 PBMCs underwent 2 subsequent ficolls). Phenotypic analysis demonstrated that freshly isolated CMV-specific T cells consisted of four different memory subsets (TEMRA, Tscm, Tcm and effector memory T cells). After in vitro expansion for 15 days, CMV-CAR bi-specific T cell expansion was significantly improved from 60×106 cells in the early stage runs to 200×106 in the late stage optimized runs (Figure 1). The CMV-CD19CAR T cells expressed CD62L, though not exhaustion markers such as PD1. Bispecific T cells exhibited bi-functionality upon stimulation with CD19+ tumor cells or CMVpp65 antigen, as indicated by secretion of IFNγ. Interestingly, we detected stronger effector function against CD19+ tumor cells from CMV-CD19CAR T cells compared to conventional CD19CAR T cells that were derived from the same donor (Figure 2). To validate our manufacturing process, we are currently conducting IND-enabling studies using patient-derived PBMC as the starting population and will initiate our first clinical trial in early 2020 for patients with intermediate/advanced-grade B cell NHL post lymphodepletion or autologous/allogeneic HCT followed by Triplex vaccination 28 days after T cell infusion for in vivo expansion of bi-specific CAR T cells. The primary objectives of these two trials are to examine safety and persistence/expansion of CMV-CD19CAR T cell before and after Triplex vaccine boost. Disclosures Nakamura: Merck: Membership on an entity's Board of Directors or advisory committees; Celgene: Other: support for an academic seminar in a university in Japan; Alexion: Other: support to a lecture at a Japan Society of Transfusion/Cellular Therapy meeting ; Kirin Kyowa: Other: support for an academic seminar in a university in Japan.

There is a need to individualize assays for tumor molecular phenotyping, given variations in the differentiation status of tumor and normal tissues in different patients. To address this, we performed single-cell genomics of breast tumors and adjacent normal cells propagated for a short duration under growth conditions that enable epithelial reprogramming. Cells analyzed were either unselected for a specific subpopulation or phenotypically defined as undifferentiated and highly clonogenic ALDH+/CD49f+/EpCAM+ luminal progenitors, which express both basal cell and luminal cell–enriched genes. We analyzed 420 tumor cells and 284 adjacent normal cells for expression of 93 genes that included a PAM50-intrinsic subtype classifier and stemness-related genes. ALDH+/CD49f+/EpCAM+ tumor and normal cells clustered differently compared with unselected tumor and normal cells. PAM50 gene-set analyses of ALDH+/CD49f+/EpCAM+ populations efficiently identified major and minor clones of tumor cells, with the major clone resembling clinical parameters of the tumor. Similarly, a stemness-associated gene set identified clones with divergent stemness pathway activation within the same tumor. This refined expression profiling technique distinguished genes truly deregulated in cancer from genes that identify cellular precursors of tumors. Collectively, the assays presented here enable more precise identification of cancer-deregulated genes, allow for early identification of therapeutically targetable tumor cell subpopulations, and ultimately provide a refinement of precision therapeutics for cancer treatment. Cancer Res; 77(10); 2759–69. ©2017 AACR.

Circulating endothelial microparticles (EMPs) are emerging as biomarkers of chronic obstructive pulmonary disease (COPD) in individuals exposed to cigarette smoke (CS), but their mechanism of release and function remain unknown. We assessed biochemical and functional characteristics of EMPs and circulating microparticles (cMPs) released by CS. CS exposure was sufficient to increase microparticle levels in plasma of humans and mice and in supernatants of primary human lung microvascular endothelial cells. CS-released EMPs contained predominantly exosomes that were significantly enriched in let-7d, miR-191; miR-126; and miR125a, microRNAs that reciprocally decreased intracellular in CS-exposed endothelium. CS-released EMPs and cMPs were ceramide-rich and required the ceramide-synthesis enzyme acid sphingomyelinase (aSMase) for their release, an enzyme which was found to exhibit significantly higher activity in plasma of COPD patients or of CS-exposed mice. The ex vivo or in vivo engulfment of EMPs or cMPs by peripheral blood monocytes-derived macrophages was associated with significant inhibition of efferocytosis. Our results indicate that CS, via aSMase, releases circulating EMPs with distinct microRNA cargo and that EMPs affect the clearance of apoptotic cells by specialized macrophages. These targetable effects may be important in the pathogenesis of diseases linked to endothelial injury and inflammation in smokers.

Multiple myeloma is incurable once osteolytic lesions have seeded at skeletal sites, but factors mediating this deadly pathogenic advance remain poorly understood. Here, we report evidence of a major role for the cell adhesion molecule CD166, which we discovered to be highly expressed in multiple myeloma cell lines and primary bone marrow cells from patients. CD166+ multiple myeloma cells homed more efficiently than CD166− cells to the bone marrow of engrafted immunodeficient NSG mice. CD166 silencing in multiple myeloma cells enabled longer survival, a smaller tumor burden, and less osteolytic lesions, as compared with mice bearing control cells. CD166 deficiency in multiple myeloma cell lines or CD138+ bone marrow cells from multiple myeloma patients compromised their ability to induce bone resorption in an ex vivo organ culture system. Furthermore, CD166 deficiency in multiple myeloma cells also reduced the formation of osteolytic disease in vivo after intratibial engraftment. Mechanistic investigation revealed that CD166 expression in multiple myeloma cells inhibited osteoblastogenesis of bone marrow–derived osteoblast progenitors by suppressing Runx2 gene expression. Conversely, CD166 expression in multiple myeloma cells promoted osteoclastogenesis by activating TRAF6-dependent signaling pathways in osteoclast progenitors. Overall, our results define CD166 as a pivotal director in multiple myeloma cell homing to the bone marrow and multiple myeloma progression, rationalizing its further study as a candidate therapeutic target for multiple myeloma treatment. Cancer Res; 76(23); 6901–10. ©2016 AACR.

The extra-embryonic yolk sac (YS) is the first hematopoietic site in the mouse embryo and is thought to generate only primitive erythroid and myeloerythroid progenitor cells before definitive HSC emergence within the embryo on E10.5. Here, we have shown the existence of T cell–restricted progenitors in the E9.5 YS that directly engraft in recipient immunodeficient mice. T-cell progenitors were also produced in vitro from both YS and para-aortic splanchnopleura hemogenic endothelial cells, and these T-cell progenitors repopulated the thymus and differentiated into mature T-cell subsets in vivo on transplantation. Our data confirm that the YS produces T-lineage–restricted progenitors that are available to colonize the thymus and provide new insight into the YS as a definitive hematopoietic site in the mouse embryo.

Acute lymphoblastic leukemia (ALL) is the most frequentchildhood malignancy. Although it is well established that geneticabnormalities are critical for the development of ALL, the mechan-ismsthatconferaselectiveadvantagetotheleukemiacellsintheirsiteof origin, the bone marrow (BM), are largely unknown. The inter-actionsofALLblastswithBMstromalcellshaveapositiveimpactonleukemia cell survival and resistance to chemotherapy. ALL stimu-lates BM stromal cells, which reciprocally promote leukemia cellsurvival [1–7]. In addition, it is conceivable that leukemic cellspromote a microenvironmental change in the BM so as to inhibitnormal hematopoiesis.The interaction between ALL and BM stromal cells occurs inspecific cell niches, characterized by the expression of moleculessuchasE-selectinandstromal-cell-derivedfactor1(SDF-1)[8].BMniches are critical in sustaining ALL cell survival [9]. Close associ-ation of ALL to bone marrow stromal cells is important for ALLsurvivalinvitro.Adhesionisinpartmediatedbyb1integrins(VLA4and VLA5) and CD44, expressed on leukemia cells, and the fibro-nectinandvascularcelladhesionmolecule-1(VCAM-1),expressedonthestromallayer[2–4].InteractionwiththestromalVCAM-1celladhesion molecule renders VLA-4þleukemic cell lines resistant toAra-CandVP-16(etoposide)[10].BMStromalcellsproduceseveralgrowth factors [11,12]; some may provide a microenvironmentsuitable for leukemogenesis and leukemic progression. SDF-1,Interleukin-3 (IL-3), IL-7, stem cell factor (SCF), and FMS-liketyrosine kinase 3 ligand (Flt3-L) alone or in different combinationswere shown to promote the survival and or proliferation of B-cellprecursor ALL cells cultured onto BM stromal layers, although thedegreeofstimulationisheterogeneous betweenpatientsamplesandbetween growth factors [13–16]. Levels of basic fibroblast growthfactor (bFGF), hepatocytegrowth factor (HGF), and tumor necrosisfactor-a(TNF-a)aresignificantlyhigherinperipheralbloodplasmasamples frompatients withALL comparedwithhealthy controls, inassociation with increased angiogenesis [17]. The leukemic BMplasma contains increased levels of bFGF, and vascular endothelialfactor (VEGF) levels are increased in some patients [7]. Theabnormally high levels of these growth factors are able to promoteangiogenesis [7], and in some cases may directly benefit ALL cells.Forinstance,VEGFproducedbystromalcellsstimulatesBcl-2phos-phorylation and resistance of ALL cell lines to different chemo-therapeutic agents [18].

Despite marked clinical successes in the treatment of childhood T-ALL, leukemia relapse, refractory disease and induction failure (around 30% of patients) remain significant clinical problems. New insights in the molecular alterations of ALL have revealed new molecular targets, such as activating mutations of Notch1 (N1), identified in more than 50% of T-ALL patients. However, disruption of N1 signaling by gamma-secretase inhibitors failed to fulfill its clinical promise. Furthermore, little is known about N1 downstream mediators that can be potential therapeutic targets in T-ALL.

Purpose of review In the postnatal life, hematopoietic stem cell (HSC) niches are specialized microenvironments in the bone marrow that are essential for the maintenance and function of HSCs. The purpose of this review is to discuss the concept of HSC niche in light of recent studies that broaden its complexity and better define its molecular regulation. Also, we will discuss recent studies addressing the impact of leukemia development on HSC regulation and normal hematopoiesis, while discussing the potential regulation of leukemia-initiating cells by bone marrow niches. Recent findings Recent studies have identified new cellular and molecular components of the HSC niche and highlighted reciprocal interactions between the hematopoietic cells and their niches. These studies indicate that the HSC niche is not constituted by a single cell type but rather should be considered as a multicellular functional unit. Finally, advances have been made that provide promising insights into the the instructive role of the bone marrow microenvironment in hematological malignancies. Summary Increasing insights into the cell-cell cross talk between the hematopoietic system and its microenvironment in the bone marrow, and in particular in the interplay of HSCs with their niche(s), should provide new tools for combinatorial therapies in bone marrow failure and bone marrow cancers.

Mutations in the phosphatase and tensin homolog (PTEN) gene leading to PTEN protein deletion and subsequent activation of the PI3K/Akt signaling pathway are common in cancer. Here we show that PTEN inactivation in human T cell acute lymphoblastic leukemia (T-ALL) cells is not always synonymous with PTEN gene lesions and diminished protein expression. Samples taken from patients with T-ALL at the time of diagnosis very frequently showed constitutive hyperactivation of the PI3K/Akt pathway. In contrast to immortalized cell lines, most primary T-ALL cells did not harbor PTEN gene alterations, displayed normal PTEN mRNA levels, and expressed higher PTEN protein levels than normal T cell precursors. However, PTEN overexpression was associated with decreased PTEN lipid phosphatase activity, resulting from casein kinase 2 (CK2) overexpression and hyperactivation. In addition, T-ALL cells had constitutively high levels of ROS, which can also downmodulate PTEN activity. Accordingly, both CK2 inhibitors and ROS scavengers restored PTEN activity and impaired PI3K/Akt signaling in T-ALL cells. Strikingly, inhibition of PI3K and/or CK2 promoted T-ALL cell death without affecting normal T cell precursors. Overall, our data indicate that T-ALL cells inactivate PTEN mostly in a nondeletional, posttranslational manner. Pharmacological manipulation of these mechanisms may open new avenues for T-ALL treatment.

Background— Activated macrophages contribute to the pathogenesis of inflammatory diseases such as atherosclerosis. Although Notch signaling participates in various aspects of immunity, its role in macrophage activation remains undetermined. Methods and Results— To explore the role of Notch signaling in inflammation, we examined the expression and activity of Notch pathway components in human primary macrophages in vitro and in atherosclerotic plaques. Macrophages in culture express various Notch pathway components including all 4 receptors (Notch1 to Notch4). Notch3 selectively increased during macrophage differentiation; however, silencing by RNA interference demonstrated that all receptors are functional. The ligand Delta-like 4 (Dll4) increased in macrophages exposed to proinflammatory stimuli such as lipopolysaccharide, interleukin-1β, or minimally-modified low-density lipoprotein in a Toll-like receptor 4– and nuclear factor-κB–dependent fashion. Soluble Dll4 bound to human macrophages. Coincubation of macrophages with cells that expressed Dll4 triggered Notch proteolysis and activation; increased the transcription of proinflammatory genes such as inducible nitric oxide synthase, pentraxin 3 and Id1; resulted in activation of mitogen-activated protein kinase, Akt, and nuclear factor-κB pathways; and increased the expression of Dll4 in macrophages. Notch3 knockdown during macrophage differentiation decreased the transcription of genes that promote inflammation, such as inducible nitric oxide synthase, pentraxin 3, Id1, and scavenger receptor-A. These in vitro findings correlate with results of quantitative immunohistochemistry, which demonstrated the presence of Dll4 and other Notch components within macrophages in atherosclerotic plaques. Conclusion— Dll4-triggered Notch signaling may mediate inflammatory responses in macrophages and promote inflammation.

Extensive endothelial cell proliferation and marked neovascularization are the most pronounced microenvironmental changes consistently observed in the bone marrow (BM) of patients with acute lymphoblastic leukemia (ALL). It is not known whether ALL cells induce this phenotype and whether they receive critical signals from the tumor-associated BM endothelium. Here, we show that leukemia cells actively stimulate BM endothelium, promote de novo angiogenesis, and induce neovascularization in the leukemic BM. Soluble factors, present in the leukemic BM microenvironment, promote the proliferation, migration, and morphogenesis of BM endothelial cells, which are critical processes in tumor angiogenesis. We also show in vitro that leukemia cells display directional motion towards assembled BM endothelium and following adherence exhibit cell polarization, pseudopodia, and ultrastructural features that suggest the existence of leukemia-endothelium cross-talk. Finally, we show that BM endothelium promotes leukemia cell survival through a mechanism mediated through the anti-apoptotic molecule bcl-2. These studies indicate that ALL cells actively recruit BM endothelium and mediate the leukemia-associated neovascularization observed in ALL. Therefore, disruption of interactions between leukemia cells and BM endothelium may constitute a valid therapeutic strategy.

The malignant transformation and expansion of tumor cells involve both cell-autonomous mechanisms and microenvironment signals that regulate viability, nutrient utilization, metabolic activity and cell growth. In T-cell acute lymphoblastic leukemia (T-ALL), the co-culture of leukemic cells with stroma or the addition of particular cytokines prevents ex vivo spontaneous apoptosis. Interleukin-7 (IL-7), a cytokine produced by thymic and bone marrow stroma, increases the viability and proliferation of T-ALL cells. IL-7 induces the activation of Jak/STAT, MEK/Erk and PI3K/Akt signaling pathways in T-ALL cells. PI3K/Akt is the dominant pathway that mediates the effects of IL-7 on T-ALL. PI3K signaling is required for the induction of Bcl-2, the down-regulation of p27(kip1) and cell cycle progression. PI3K signaling is also required for the expression of the glucose transporter Glut1, uptake of glucose, activation of the metabolic machinery, increase in cell size, and maintenance of mitochondrial integrity. These observations suggest that substrates of molecular pathways activated by microenvironmental factors represent attractive molecular targets for the regulation of the viability and proliferation of T-ALL cells and provide the means for the development of novel treatment strategies.

Objectives We and others have shown that B cell precursor acute lymphoblastic leukemia cells (ALL) stimulated with CD40 ligand become efficient antigen-presenting cells (APC) capable of expanding autologous, tumor-specific T cells from patients. Translation of these preclinical findings to a novel treatment strategy required four separate issues to be determined: (1) if a CD40-ALL vaccine could be generated for clinical use; (2) whether clinical translation could be achieved; (3) whether the vaccination was safe; and (4) whether a window of time could be identified that would optimize the efficacy of vaccination. Patients and methods Nine patients with relapsed/refractory ALL were enrolled in a phase I trial of vaccination with autologous CD40-ALL. Immunologic reconstitution was measured in a separate cohort of 23 patients with newly diagnosed ALL. Results We successfully prepared autologous vaccines for all nine patients in the phase I trial. CD40-ALL were potent APC, capable of stimulating allogeneic and peptide-specific T cells in vitro. Two patients were vaccinated without adverse events. Five patients died or progressed before vaccination, suggesting that rapid disease progression limits vaccination in patients with relapse disease, thus limiting clinical translation. We therefore sought to identify a window of time for vaccination during which this approach might be feasible. To achieve this end, we evaluated immunological reconstitution in newly diagnosed patients with ALL patients. Despite recovery of myelopoiesis, most patients had profound defects in T, B, and natural killer (NK) cell numbers that failed to recover at any point during therapy. Conclusion Autologous tumor vaccination at a time of ALL relapse is not feasible. Alternative strategies for immunotherapy of ALL may require ex vivo generation of antigen specific T cells and adoptive therapy.

Improving vaccine delivery to human APCs is a way to increase the CTL response to vaccines. We report the use of a novel pH-triggered microparticle that exploits the ability of APCs to cross-present MHC I-restricted Ags that have been engulfed in the low pH environment of the phagosome. A model MHC class I-restricted peptide Ag from the influenza A matrix protein was encapsulated in spray-dried microparticles composed of dipalmitoylphosphatidylcholine and the pH-sensitive polymethacrylate Eudragit E100. Release of the peptide from the particle was triggered by a drop in pH to the acidity normally found in the phagosome. The particles were efficiently phagocytosed by human monocytes and dendritic cells with minimal cellular toxicity and no functional impairment. Encapsulation of the peptide in the microparticles resulted in efficient presentation of the peptide to CD8+ T cells by human dendritic cells in vitro, and was superior to unencapsulated peptide or peptide encapsulated in an analogous pH-insensitive particle. Vaccination of human HLA-A*0201 transgenic mice with peptide encapsulated in pH-triggering microparticles resulted in priming of CTL responses. These microparticles can be modified to coencapsulate a range of adjuvants along with the Ag of interest. Encapsulation of MHC I epitopes in pH-triggered microparticles increases Ag presentation and may improve CD8+ T cell priming to peptide vaccines against viruses and cancer.

Follicular lymphomas (FLs) localize in lymphoid tissues and recapitulate the structure of normal secondary follicles. The chemokine/chemokine receptor pair CXCL13/CXCR5 is required for the architectural organization of B cells within lymphoid follicles. In this study, we showed that CXCL13 was secreted by FL cells. FL cells expressed CXCR5 and migrated in response to CXCL13. Furthermore, we observed a synergistic effect between CXCL13 and CXCL12 (SDF-1), a chemokine produced by stromal cells in lymphoid tissues. The production of CXCL13 by FL cells and CXCL12 by stromal cells probably directs and participates in the accumulation of FL cells within specific anatomic sites.

Discovery of inter-individual functional variations in gene regulatory elements and the observation that tumor and normal tissues of the same organ are in different differentiation states necessitate rethinking of gene expression based subclassification/characterization of tumors. To address this issue, we performed single cell genomics of breast tumors and adjacent-normal cells propagated using epithelial reprogramming growth conditions for a short duration. Epithelial cells analyzed were either unselected for specific subpopulation or phenotypically defined undifferentiated ALDH+/CD49f+/EpCAM+ luminal progenitors present in the normal breast, which express both basal and luminal-enriched genes. Expression of 93 genes that included PAM50 intrinsic subtype classifier and stemness-related genes was analyzed in 420 tumor and 284 adjacent-normal cells. ALDH+/CD49f+/EpCAM+ tumor and normal cells clustered differently compared to unselected tumor and normal cells. PAM50 genes-set analyses of ALDH+/CD49f+/EpCAM+ populations efficiently identified major and minor clones of tumor cells with the major clone resembling clinical parameters of the tumor. Similarly, stemness-associated gene set identified clones with divergent stemness pathway activation within the same patient sample. This refined technique distinguished genes truly deregulated in cancer from genes that identify cellular precursors of tumors. Collectively, assays presented here may enable precise identification of genes deregulated in cancer, early identification of therapeutically-targetable minor population of tumor cells, and eventually to refinement of precision therapeutics. Citation Format: Harikrishna Nakshatri, Manjushree Anjanappa, Angelo Cardoso, Lijun Cheng, Safa Mohamad, Andrea Gunawan, Susan Rice, Yan Dong, Lang Li, Edward Srour. Refining breast cancer characterization through single-cell analysis of ex vivo reprogrammed tumor and adjacent normal cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1753. doi:10.1158/1538-7445.AM2017-1753

Sendo considerada uma das alternativas energeticas mais promissoras na busca de solucoes para reverter o quadro atual de mudancas climaticas, a energia eolica tem ganhado destaque no cenario mundial por ser uma fonte de energia renovavel e limpa quando comparada com outros tipos de geracao. O conhecimento do regime dos ventos e um dos elementos mais importantes na avaliacao.do potencial eolico de uma regiao, contudo a instalacao de torres de medicao em quantidade e disposicao adequada para estes estudos ainda apresentam um custo financeiro e logistico elevado, desta forma, o trabalho objetivou especializar a velocidade do vento no estado de Minas Gerais a partir de dados coletado por estacoes do Instituto Nacional de Meteorologia INMET. Foram empregadas 73 estacoes meteorologicas distribuidas geograficamente pelo estado e nos estados que o circundam. Os mapas de velocidade media do vento para as alturas de 50, 75 e 100 metros foram obtidos a partir de um interpolador geoestatistico chamado krigagem universal, trabalhado no Sistema de Informacoes Geograficas (SIG), ArcGIS®. Os mapas produzidos demonstram que na regiao noroeste e norte do estado, proximo aos estados do Espirito Santo e Bahia, valores iguais e superiores a velocidade de 2,5 m/s (velocidade adotada como minima para a geracao de energia) foram observados para todos os meses avaliados, demonstrando ser esta uma regiao potencial, onde estudos mais locais devem ser realizados para a confirmacao deste potencial para geracao de energia eolica. A B S T R A C T Considered one of the most promising energy alternatives in the search for solutions to reverse the current climate change picture, wind energy has gained prominence in the world scenario as a renewable and clean source of energy when compared to other types of power generation. Knowledge of the wind regime is one of the most important elements in the evaluation of the wind potential of a region. However, the installation of measurement towers in quantity and adequate layout for these studies still represents a high financial and logistical cost. This work aimed to model the wind speed in the state of Minas Gerais, Brazil from data collected by INMET National Meteorological Institute, using data from 73 meteorological stations distributed geographically by the state and in the states that surround it. The mean wind speed maps for the heights of 50, 75 and 100 meters were generated with a geostatistical interpolator called universal kriging, available on the Geographic Information System (GIS) software, ArcGIS®. The maps produced showed that in the northwest and north of the state, close to the states of Espirito Santo and Bahia. Values equal to and greater than 2.5 m/s (speed adopted as minimum for power generation) were observed for all the months evaluated, demonstrating that this is a potential region where more local studies must be carried out to confirm this potential for wind energy generation. Keywords: Wind Speed; Geographical Information System; State of Minas Gerais; kriging.

We previously showed that immature CD166(+) osteoblasts (OB) promote hematopoietic stem cell (HSC) function. Here, we demonstrate that CD166 is a functional HSC marker that identifies both murine and human long-term repopulating cells. Both murine LSKCD48(-)CD166(+)CD150(+) and LSKCD48(-)CD166(+)CD150(+)CD9(+) cells, as well as human Lin(-)CD34(+)CD38(-)CD49f(+)CD166(+) cells sustained significantly higher levels of chimerism in primary and secondary recipients than CD166(-) cells. CD166(-/-) knockout (KO) LSK cells engrafted poorly in wild-type (WT) recipients and KO bone marrow cells failed to radioprotect lethally irradiated WT recipients. CD166(-/-) hosts supported short-term, but not long-term WT HSC engraftment, confirming that loss of CD166 is detrimental to the competence of the hematopoietic niche. CD166(-/-) mice were significantly more sensitive to hematopoietic stress. Marrow-homed transplanted WT hematopoietic cells lodged closer to the recipient endosteum than CD166(-/-) cells, suggesting that HSC-OB homophilic CD166 interactions are critical for HSC engraftment. STAT3 has 3 binding sites on the CD166 promoter and STAT3 inhibition reduced CD166 expression, suggesting that both CD166 and STAT3 may be functionally coupled and involved in HSC competence. These studies illustrate the significance of CD166 in the identification and engraftment of HSC and in HSC-niche interactions, and suggest that CD166 expression can be modulated to enhance HSC function.

The localization and establishment of follicular lymphoma (FL) cells in distinct anatomic sites probably involves chemokine and adhesion receptors on the neoplastic cells and appropriate chemokines and adhesion receptor ligands in the microenvironment. Several chemokines play an important role in normal B-cell trafficking and differentiation. Monocyte chemoattractant protein-1 (MCP-1) is a C-C chemokine that induces chemotaxis of a variety of lymphoid cells through its receptor CCR2. CCR2 is also expressed on B cells, and MCP-1 induces chemotaxis of normal B cells. In this report, we investigated expression and function of CCR2 on FL cells. We found FL cells as well as the t(14; 18)+ B-cell lymphoma line H2 expressed CCR2. MCP-1 potentiated SDF-1-induced chemotaxis of FL cells and H2 cells, but MCP-1 alone did not induce chemotaxis. The specificity of the effects of MCP-1 and SDF-1 was demonstrated by antibody blocking studies. Because FL cells are generally associated with follicular dendritic cells (FDCs), FDCs may be an important source of chemokines. We found that cultured FDCs produced MCP-1, and this production was enhanced by tumour necrosis factor. These data implicate MCP-1 in the migration and localization of FL cells.

HER-2/neu is a tumor-associated antigen overexpressed in a large variety of human tumors. Eight HER-2/neu peptides displaying HLA-A*0201 anchoring motifs were selected and tested for their binding affinity to HLA-A*0201 and their capacity to elicit cytotoxic T lymphocyte (CTL) responses in both HLA-A*0201 transgenic mice and in HLA-A*0201(+) healthy donors. Two high-affinity (p5 and p48) and one intermediate-affinity (p1023) peptides triggered CTL responses in both transgenic mice and humans, comparable to those observed for the well-known HER2/neu dominant peptide p369. CTL induced in transgenic mice lysed HLA-A*0201(+) RMA cells infected with recombinant HER-2/neu but not cells infected with wild-type vaccinia virus. Human CTL lysed HLA-A*0201(+) HER-2/neu(+) tumor cells of different origins (breast, colon, lung and renal cancer) irrespective of the expression levels of HER-2/neu. Importantly, primed CTL specific for these epitopes were detected in freshly isolated tumor-infiltrating lymphocytes from three renal cell carcinoma patients. Therefore, the HER-2/neu peptides p5, p48 and p1023 may be good candidates for immunotherapy of a broad spectrum of tumors, including renal cell carcinoma.

In normal T-cell development interleukin-7 (IL-7) functions as an antiapoptotic factor by regulating bcl-2 expression in immature thymocytes and mature T cells. Similar to what occurs in normal immature thymocytes, prevention of spontaneous apoptosis by IL-7 in precursor T-cell acute lymphoblastic leukemia (T-ALL) cells correlates with up-regulation of bcl-2. IL-7 is also implicated in leukemogenesis because IL-7 transgenic mice develop lymphoid malignancies, suggesting that IL-7 may regulate the generation and expansion of malignant cells. This study shows that in the presence of IL-7, T-ALL cells not only up-regulated bcl-2 expression and escaped apoptosis but also progressed in the cell cycle, resulting in sequential induction of cyclin D2 and cyclin A. Down-regulation of p27kip1 was mandatory for IL-7–mediated cell cycle progression and temporally coincided with activation of cyclin-dependent kinase (cdk)4 and cdk2 and hyperphosphorylation of Rb. Strikingly, forced expression of p27kip1 in T-ALL cells not only prevented cell cycle progression but also reversed IL-7–mediated up-regulation of bcl-2 and promotion of viability. These results show for the first time that a causative link between IL-7–mediated proliferation and p27kip1 down-regulation exists in malignant T cells. Moreover, these results suggest that p27kip1 may function as a tumor suppressor gene not only because it is a negative regulator of cell cycle progression but also because it is associated with induction of apoptosis of primary malignant cells.

Cell vaccines engineered to express immunomodulators have shown feasibility in eliminating leukemia in murine models. Vectors for efficient gene delivery to primary human leukemia cells are required to translate this approach to clinical trials. In this study, second-generation lentiviral vectors derived from human immunodeficiency virus 1 were evaluated, with the cytomegalovirus (CMV) promoter driving expression of granulocyte-macrophage–colony-stimulating factor (GM-CSF) and CD80 in separate vectors or in a bicistronic vector. The vectors were pseudotyped with vesicular stomatitis virus G glycoprotein and concentrated to high titers (108-109 infective particles/mL). Human acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and chronic myeloid leukemia cell lines transduced with the monocistronic pHR-CD80 vector or the bicistronic pHR-GM/CD vector became 75% to 95% CD80 positive (CD80+). More important, transduction of primary human ALL and AML blasts with high-titer lentiviral vectors was consistently successful (40%-95% CD80+). The average amount of GM-CSF secretion by the leukemia cell lines transduced with the pHR-GM-CSF monocistronic vector was 2182.9 pg/106 cells per 24 hours. Secretion was markedly lower with the bicistronic pHR-GM/CD vector (average, 225.7 pg/106 cells per 24 hours). Lower amounts of CMV-driven messenger RNA were detected with the bicistronic vector, which may account for its poor expression of GM-CSF. Primary ALL cells transduced to express CD80 stimulated T-cell proliferation in an autologous mixed lymphocyte reaction. This stimulation was specifically blocked with monoclonal antibodies reactive against CD80 or by recombinant cytotoxic T-lymphocyte antigen 4–immunoglobulin fusion protein. These results show the feasibility of efficiently transducing primary leukemia cells with lentiviral vectors to express immunomodulators to elicit antileukemic immune responses.

Genetic alterations of the signaling cascade of transforming growth factor-beta (TGF-beta) are often associated with neoplastic transformation of primitive cells. This demonstrates the key role for this pleiotropic factor in the control of quiescence and cell proliferation in vivo. In the high proliferative potential-quiescent cell (HPP-Q) in vitro assay, the use of TGF-beta1 blocking antibodies (anti-TGF-beta1) allows the detection within two to three weeks of primitive hematopoietic cells called HPP-Q, which otherwise would not grow. However, the possibility of triggering cell proliferation by blocking the cell-surface TGF-beta receptors has not been investigated until now. We have tested here the efficiency of a blocking antibody against TGF-betaRII (anti-TGF-betaRII) on CD34(+)CD38(-) hematopoietic cells, a subpopulation enriched in primitive stem/progenitor cells, and compared its effect with that of anti-TGF-beta1. About twice as many HPP colony-forming cells were detected in the presence of anti-TGF-beta1 or anti-TGF-betaRII, compared to the control (p < 0.02). Moreover, anti-TGF-betaRII was as efficient as anti-TGF-beta1 for activating multipotent HPP-granulocyte erythroid macrophage megakaryocyte and HPP-Mix, bipotent HPP-granulocyte-macrophage (GM) and unipotent HPP-G, HPP-M and HPP-BFU-E. We therefore propose the use of anti-TGF-betaRII to release primitive cells from quiescence in the HPP-Q assay. This strategy could be extended to nonhematopoietic tissues, as TGF-beta1 may be a pleiotropic regulator of somatic stem cell quiescence.

We have previously shown that leukemia-specific cytotoxic T cells (CTL) can be generated from the bone marrow of most patients with B-cell precursor acute leukemias. If these antileukemia CTL are to be used for adoptive immunotherapy, they must have the capability to circulate, migrate through endothelium, home to the bone marrow, and, most importantly, lyse the leukemic cells in a leukemia-permissive bone marrow microenvironment. We demonstrate here that such antileukemia T-cell lines are overwhelmingly CD8+ and exhibit an activated phenotype. Using a transendothelial chemotaxis assay with human endothelial cells, we observed that these T cells can be recruited and transmigrate through vascular and bone marrow endothelium and that these transmigrated cells preserve their capacity to lyse leukemic cells. Additionally, these antileukemia T-cell lines are capable of adhering to autologous stromal cell layers. Finally, autologous antileukemia CTL specifically lyse leukemic cells even in the presence of autologous marrow stroma. Importantly, these antileukemia T-cell lines do not lyse autologous stromal cells. Thus, the capacity to generate anti–leukemia-specific T-cell lines coupled with the present findings that such cells can migrate, adhere, and function in the presence of the marrow microenvironment enable the development of clinical studies of adoptive transfer of antileukemia CTL for the treatment of ALL.

The identification of all CD28/CTLA-4 counterreceptors is critical to our understanding of this pivotal pathway of T cell activation. Clouding our understanding has been the reported discrepancies in expression and function of the B7-1 (CD80) molecule based upon the use of the BB1 vs other anti-B7-1 mAbs. To resolve this issue, we have cloned a BB1-binding molecule from the BB1+B7-1− NALM-6 pre-B cell line. Here, we demonstrate that this BB1-binding molecule is identical to the cell surface form of CD74 (MHC class II-associated invariant chain). CD74-transfected cells bound the BB1 mAb but not other anti-CD80 mAbs, CD28-Ig, or CTLA4Ig. Absorption and blocking experiments confirmed the reactivity of BB1 mAb with CD74. A region of weak homology was identified between CD74 and the region of B7-1 encoding the BB1 epitope. Therefore, the BB1 mAb binds to a protein distinct from B7-1, and this epitope is also present on the B7-1 protein. Many of the puzzling observations in the literature concerning the expression of human B7-1 are resolved by an understanding that BB1 staining is the summation of CD74 plus B7-1 expression. This observation requires the field to reconsider studies using BB1 mAb in the analysis of CD80 expression and function.

Although highly responsive, advanced stage follicular lymphoma (FL) is not curable with conventional treatment. This relative resistance is thought to be due to the t(14;18) that results in the constitutive overexpression of the death-inhibiting protein bcl-2. However, the observation that FL cells are sensitive to treatment in vivo and prone to apoptosis on in vitro culture questions whether bcl-2 alone is responsible for the pathogenesis and clinical behavior of this disease. Therefore, multiple genes are likely to be involved in both the lymphomagenesis and the clinical course of FL. We examined whether expression of other bcl-2 family genes might also be operative. Here, we show that FL cells display a different pattern of expression of bcl-2 family proteins from normal germinal center (GC) B cells that are thought to be their normal counterpart. FL cells express the death-suppressor proteins bcl-2, bcl-xL, and mcl-1; whereas GC B cells express bcl-xL and mcl-1 but also the proapoptotic proteins bax-α and bad. Although maintaining constitutive levels of bcl-2 and mcl-1, FL cells are not protected from apoptosis when cultured in vitro. Their propensity to undergo apoptosis is temporally associated with downregulation of bcl-xL. More importantly, activation of FL cells via CD40 not only prevents downregulation but increases the level of bcl-xL expression and results in promotion of survival. These results support the hypothesis that the overexpression of bcl-2 is not the only antiapoptotic mechanism responsible for the pathogenesis of FL. Survival of FL cells is determined by a number of death-inhibiting proteins, among which bcl-xL appears to have the most critical role. Moreover, these findings are consistent with the hypothesis that, although FL cells are malignant, they respond to microenvironmental signals such as CD40L that appear to contribute to their survival through the upregulation of death-inhibiting proteins.

Although highly responsive, advanced stage follicular lymphoma (FL) is not curable with conventional treatment. This relative resistance is thought to be due to the t(14;18) that results in the constitutive overexpression of the death-inhibiting protein bcl-2. However, the observation that FL cells are sensitive to treatment in vivo and prone to apoptosis on in vitro culture questions whether bcl-2 alone is responsible for the pathogenesis and clinical behavior of this disease. Therefore, multiple genes are likely to be involved in both the lymphomagenesis and the clinical course of FL. We examined whether expression of other bcl-2 family genes might also be operative. Here, we show that FL cells display a different pattern of expression of bcl-2 family proteins from normal germinal center (GC) B cells that are thought to be their normal counterpart. FL cells express the death-suppressor proteins bcl-2, bcl-xL, and mcl-1; whereas GC B cells express bcl-xL and mcl-1 but also the proapoptotic proteins bax-α and bad. Although maintaining constitutive levels of bcl-2 and mcl-1, FL cells are not protected from apoptosis when cultured in vitro. Their propensity to undergo apoptosis is temporally associated with downregulation of bcl-xL. More importantly, activation of FL cells via CD40 not only prevents downregulation but increases the level of bcl-xL expression and results in promotion of survival. These results support the hypothesis that the overexpression of bcl-2 is not the only antiapoptotic mechanism responsible for the pathogenesis of FL. Survival of FL cells is determined by a number of death-inhibiting proteins, among which bcl-xL appears to have the most critical role. Moreover, these findings are consistent with the hypothesis that, although FL cells are malignant, they respond to microenvironmental signals such as CD40L that appear to contribute to their survival through the upregulation of death-inhibiting proteins.

In contrast to other neoplasms, antigen-specific autologous cytolytic T cells have not been detected in patients with human pre-B–cell leukemias. The absence of efficient B7 family (B7-1/CD80; B7-2/CD86) -mediated costimulation has been shown to be a major defect in tumor cells' capacity to function as antigen-presenting cells. We show here the generation of autologous anti–pre-B–cell leukemia-specific cytolytic T-cell lines from the marrows of 10 of 15 patients with pre-B–cell malignancies. T-cell costimulation via CD28 is an absolute requirement for the generation of these autologous cytolytic T cells (CTL). Although costimulation could be delivered by either bystander B7 transfectants or professional antigen-presenting cells (indirect costimulation), optimal priming and CTL expansion required that the costimulatory signal was expressed by the tumor cell (direct costimulation). These anti–pre-B–cell leukemia-specific CTL lysed both unstimulated and CD40-stimulated tumor cells from each patient studied but did not lyse either K562 or CD40-stimulated allogeneic B cells. Cytolysis was mediated by the induction of tumor cell apoptosis by CD8+ T cells via the perforin-granzyme pathway. Although we were able to generate anti–leukemia-specific CTL from the bone marrow, we were unable to generate such CTL from the peripheral blood of these patients. These studies show that antigen-specific CTL can be generated from the bone marrow of patients with pre-B–cell leukemias and these findings should facilitate the design of adoptive T-cell–mediated immunotherapy trials for the treatment of patients with B-cell precursor malignancies.

SummaryThe microRNA miR-155 has been implicated in regulating inflammatory responses and tumorigenesis, but its precise role in linking inflammation and cancer has remained elusive. Here, we identify a connection between miR-155 and Notch signaling in this context. Loss of Notch signaling in the bone marrow (BM) niche alters hematopoietic homeostasis and leads to lethal myeloproliferative-like disease. Mechanistically, Notch signaling represses miR-155 expression by promoting binding of RBPJ to the miR-155 promoter. Loss of Notch/RBPJ signaling upregulates miR-155 in BM endothelial cells, leading to miR-155-mediated targeting of the nuclear factor κB (NF-κB) inhibitor κB-Ras1, NF-κB activation, and increased proinflammatory cytokine production. Deletion of miR-155 in the stroma of RBPJ−/− mice prevented the development of myeloproliferative-like disease and cytokine induction. Analysis of BM from patients carrying myeloproliferative neoplasia also revealed elevated expression of miR-155. Thus, the Notch/miR-155/κB-Ras1/NF-κB axis regulates the inflammatory state of the BM niche and affects the development of myeloproliferative disorders.

For efficient entry into target cells, primary macrophage-tropic and laboratory-adapted human immunodeficiency viruses type 1 (HIV-1) require particular chemokine receptors, CCR-5 and CXCR-4, respectively, as well as the primary receptor CD4 (refs 1-6). Here we show that a complex of gp120, the exterior envelope glycoprotein, of macrophage-tropic primary HIV-1 and soluble CD4 interacts specifically with CCR-5 and inhibits the binding of the natural CCR-5 ligands, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta (refs 7, 8). The apparent affinity of the interaction between gp120 and CCR-5 was dramatically lower in the absence of soluble CD4. Additionally, in the absence of gp120, an interaction between a two-domain CD4 fragment and CCR-5 was observed. A gp120 fragment retaining the CD4-binding site and overlapping epitopes was able to interact with CCR-5 only if the V3 loop, which can specify HIV-1 tropism and chemokine receptor choice, was also present on the molecule. Neutralizing antibodies directed against either CD4-induced or V3 epitopes on gp120 blocked the interaction of gp12O-CD4 complexes with CCR-5. These results suggest that HIV-1 attachment to CD4 creates a high-affinity binding site for CCR-5, leading to membrane fusion and virus entry.