William G. Telford

Donor-derived CD19-targeted T cells cause regression of malignancy persisting after allogeneic hematopoietic stem cell transplantation

New treatments are needed for B-cell malignancies persisting after allogeneic hematopoietic stem cell transplantation (alloHSCT). We conducted a clinical trial of allogeneic T cells genetically modified to express a chimeric antigen receptor (CAR) targeting the B-cell antigen CD19. T cells for genetic modification were obtained from each patients alloHSCT donor. All patients had malignancy that persisted after alloHSCT and standard donor lymphocyte infusions (DLIs). Patients did not receive chemotherapy prior to the CAR T-cell infusions and were not lymphocyte depleted at the time of the infusions. The 10 treated patients received a single infusion of allogeneic anti-CD19-CAR T cells. Three patients had regressions of their malignancies. One patient with chronic lymphocytic leukemia (CLL) obtained an ongoing complete remission after treatment with allogeneic anti-CD19-CAR T cells, another CLL patient had tumor lysis syndrome as his leukemia dramatically regressed, and a patient with mantle cell lymphoma obtained an ongoing partial remission. None of the 10 patients developed graft-versus-host disease (GVHD). Toxicities included transient hypotension and fever. We detected cells containing the anti-CD19-CAR gene in the blood of 8 of 10 patients. These results show for the first time that donor-derived allogeneic anti-CD19-CAR T cells can cause regression of B-cell malignancies resistant to standard DLIs without causing GVHD.

Quantitative Comparison of Long-wavelength Alexa Fluor Dyes to Cy Dyes: Fluorescence of the Dyes and Their Bioconjugates

Amine-reactive N-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins. These conjugates were compared with spectrally similar protein conjugates of the Cy3, Cy5, Cy5.5, Cy7, DY-630, DY-635, DY-680, and Atto 565 dyes. As N-hydroxysuccinimidyl ester dyes, the Alexa Fluor 555 dye was similar to the Cy3 dye, and the Alexa Fluor 647 dye was similar to the Cy5 dye with respect to absorption maxima, emission maxima, Stokes shifts, and extinction coefficients. However, both Alexa Fluor dyes were significantly more resistant to photobleaching than were their Cy dye counterparts. Absorption spectra of protein conjugates prepared from these dyes showed prominent blue-shifted shoulder peaks for conjugates of the Cy dyes but only minor shoulder peaks for conjugates of the Alexa Fluor dyes. The anomalous peaks, previously observed for protein conjugates of the Cy5 dye, are presumably due to the formation of dye aggregates. Absorption of light by the dye aggregates does not result in fluorescence, thereby diminishing the fluorescence of the conjugates. The Alexa Fluor 555 and the Alexa Fluor 647 dyes in protein conjugates exhibited significantly less of this self-quenching, and therefore the protein conjugates of Alexa Fluor dyes were significantly more fluorescent than those of the Cy dyes, especially at high degrees of labeling. The results from our flow cytometry, immunocytochemistry, and immunohistochemistry experiments demonstrate that protein-conjugated, long-wavelength Alexa Fluor dyes have advantages compared to the Cy dyes and other long-wavelength dyes in typical fluorescence-based cell labeling applications.

Dynamics and mechanisms of quantum dot nanoparticle cellular uptake

BackgroundThe rapid growth of the nanotechnology industry and the wide application of various nanomaterials have raised concerns over their impact on the environment and human health. Yet little is known about the mechanism of cellular uptake and cytotoxicity of nanoparticles. An array of nanomaterials has recently been introduced into cancer research promising for remarkable improvements in diagnosis and treatment of the disease. Among them, quantum dots (QDs) distinguish themselves in offering many intrinsic photophysical properties that are desirable for targeted imaging and drug delivery.ResultsWe explored the kinetics and mechanism of cellular uptake of QDs with different surface coatings in two human mammary cells. Using fluorescence microscopy and laser scanning cytometry (LSC), we found that both MCF-7 and MCF-10A cells internalized large amount of QD655-COOH, but the percentage of endocytosing cells is slightly higher in MCF-7 cell line than in MCF-10A cell line. Live cell fluorescent imaging showed that QD cellular uptake increases with time over 40 h of incubation. Staining cells with dyes specific to various intracellular organelles indicated that QDs were localized in lysosomes. Transmission electron microscopy (TEM) images suggested a potential pathway for QD cellular uptake mechanism involving three major stages: endocytosis, sequestration in early endosomes, and translocation to later endosomes or lysosomes. No cytotoxicity was observed in cells incubated with 0.8 nM of QDs for a period of 72 h.ConclusionsThe findings presented here provide information on the mechanism of QD endocytosis that could be exploited to reduce non-specific targeting, thereby improving specific targeting of QDs in cancer diagnosis and treatment applications. These findings are also important in understanding the cytotoxicity of nanomaterials and in emphasizing the importance of strict environmental control of nanoparticles.

Side population analysis using a violet-excited cell-permeable DNA binding dye.

Hoechst 33342 side population (SP) analysis is a common method for identifying stem cells in mammalian hematopoietic and nonhematopoietic tissues. Although widely employed for stem cell analysis, this method requires an ultraviolet (UV) laser to excite Hoechst 33342. Flow cytometers equipped with UV sources are not common because of the cost of both the laser and optics that can transmit light UV light. Violet laser sources are inexpensive and are now common fixtures on flow cytometers, but have been previously shown to provide insufficient Hoechst dye excitation for consistent resolution of SP cells. One solution to this problem is to identify additional fluorescent substrates with the same pump specificity as Hoechst 33342, but with better violet excitation characteristics. DyeCycle Violet reagent has emission characteristics similar to those of Hoechst 33342, but with a longer wavelength excitation maxima (369 nm). When this dye is loaded into hematopoietic cells, a sharply resolved side population was also observed, similar in appearance to that seen with Hoechst 33342. Unlike Hoechst SP, DCV SP was similar in appearance with both violet and UV excitation. DCV SP could be inhibited fumitremorgin C, and showed the same membrane pump specificity as Hoechst 33342. Simultaneous immunophenotyping with stem cell markers in mouse bone marrow demonstrated that DCV SP was restricted to the stem cell lineage− Sca‐1+ c‐kit+ cells population, as is Hoechst SP. Pending confirmation by functional analysis of DCV SP cells, these results suggest that DCV efflux identified approximately the same stem cell population as did Hoechst 33342 efflux. Substituting DCV for Hoechst 33342 in the SP technique may, therefore, allow side population analysis on flow cytometers with violet lasers.

HOX decoy peptide enhances the ex vivo expansion of human umbilical cord blood CD34+ hematopoietic stem cells/hematopoietic progenitor cells.

The isolation and characterization of living human epithelial stem cells is difficult because distinguishing cell surface markers have not been identified with certainty. Side population keratinocytes (SP‐KCs) that efflux Hoechst 33342 fluorescent dye, analogous to bone marrow‐derived side population (SP) hematopoietic stem cells, have been identified in human skin, but their potential to function as keratinocyte stem cells (KSCs) in vivo is not known. On the other hand, human keratinocyte populations that express elevated levels of β1 and α6 integrins and are distinct from SP‐KCs, which express low levels of integrins, may be enriched for KSCs based on reported results of in vitro cell culture assays. When in vitro assays were used to measure total cell output of human SP‐KCs and integrin‐bright keratinocytes, we could not document their superior long‐term proliferative activity versus unfractionated keratinocytes. To further assess the KSC characteristics in SP‐KCs and integrin‐bright keratinocytes, we used an in vivo competitive repopulation assay in which bioengineered human epidermis containing competing keratinocyte populations with different human major histocompatibility (MHC) class I antigens were grafted onto immunocompromised mice, and the intrinsic MHC class I antigens are used to quantify expansion of competing populations. In these in vivo studies, human SP‐KCs showed little competitive expansion in vivo and were not enriched for KSCs. In contrast, keratinocytes expressing elevated levels of α6 integrin and low levels of CD71 (α6‐bright/CD71‐dim) expanded over 200‐fold during the 33‐week in vivo study. These results definitively demonstrate that human α6‐bright/CD71‐dim keratinocytes are enriched with KSCs, whereas SP‐KCs are not.

Regulatory T Cells and Human Myeloid Dendritic Cells Promote Tolerance via Programmed Death Ligand-1

Human regulatory T cells inhibit graft-versus-host disease that can occur after tissue transplantation, in part through expression of programmed death ligand 1 and modulation of antigen-presenting cells.

Accompanying protein alterations in malignant cells with a microtubule-polymerizing drug-resistance phenotype and a primary resistance mechanism

Microtubules (MTs) are cytoskeletal components whose structural integrity is mandatory for the execution of many basic cell functions. Utilizing parental and drug-resistant ovarian carcinoma cell lines that have acquired point mutations in beta-tubulin and p53, we studied the level of expression and modification of proteins involved in apoptosis and MT integrity. Extending previous results, we demonstrated phosphorylation of pro-survival Bcl-x(L) in an epothilone-A resistant cell line, correlating it with drug sensitivity to tubulin-active compounds. Furthermore, Mcl-1 protein turned over more rapidly following exposure to tubulin-modifying agents, the stability of Mcl-1 protein paralleling the drug sensitivity profile of the paclitaxel or epothilone-A resistant cell lines. The observed decreases in Mcl-1 were not a consequence of G(2)M arrest, as determined by flow cytometry analysis, which showed prominent levels of Mcl-1 in the absence of any drug treatment in populations enriched in mitotic cells. We also observed that a paclitaxel-resistant cell line expressed Bax at a much lower level than the sensitive parental line [A2780(1A9)], consistent with its mutant p53 status. MT-associated protein-4 (MAP4), whose phosphorylation during specific phases of the cell cycle reduces its MT-polymerizing and -stabilizing capabilities, was phosphorylated in response to drug challenge without a change in expression. Phosphorylation of MAP4 correlated with sensitivity to tubulin-binding drugs and with a dissociation from MTs. We propose that the tubulin mutations, which result in a compromised paclitaxel:tubulin or epothilone:tubulin interaction and paclitaxel or epothilone resistance, indirectly inhibit downstream events that lead to cell death, and this, in turn, may contribute to the drug-resistance phenotype

Semiconductor nanocrystal conjugates, FISH and pH

To the editor: Properties of high luminescence, reduced photobleaching and single-wavelength excitation make quantum dots (QDs) ideal for bioimaging1. Fluorescence in situ hybridization (FISH) applications have been limited for lack of reagents, stable surface treatments2 and optimization. We previously demonstrated QD-FISH detection of DNA probes with (CdSe)/ZnSstreptavidin conjugates3. Here we show pH effects on fluorescence of QD-detected hybridization signals in FISH experiments that are not evident in solution experiments. FISH (Fig. 1a) revealed major hybridization sites irrespective of the type of conjugate (organic or nanocrystal). We were able to detect the centromeres of other human chromosomes with organic fluorophores, but not with quantum dots (Fig. 1a). We investigated the effects of pH on fluorescence with biotinylated total human genomic DNA probes in FISH experiments. At each pH, cell manipulations were identical before the final incubation with fluorophore-streptavidin conjugates. Fluorescence intensities of chromosome band 1q12 (ref. 4) and total chromosomal signal were plotted (Fig. 1b). QD fluorophore intensities varied with pH in two different imaging systems (Fig. 1b,c). QD signals were optimal at ~pH 6–7, and diminished at pH 4 and pH 8 (Fig. 1b, manually quantified FISH). Standard deviations at pH 6 and pH 7 overlapped (Fig. 1b). Laser scanning cytometry (LSC) experiments confirmed this finding (5,000–10,000 cells at each pH; Fig. 1c). LSC measurements included the number of signals in each cell and the integrated density of all QD signals (Fig. 1c). These data confirmed that borate buffers at pH 6 and 7 gave superior fluorescence intensity. Quantification showed fluorescence intensity was greater at pH 6 than pH 7 by twofold (Fig. 1c). Previously, we3 noted qualitative distribution differences of fluorescence sites between fluorescein isothiocyanate (FITC) and QD detection systems. Here we quantitatively show that spot number at pH 6 was greater than at lower or higher pH (Fig. 1c). Rescanning FISH/LSC experiments demonstrated that QD fluorescence was more stable (98% after five scans) than Alexa Fluor 488 (75% after five scans), extending this analysis to high-throughput imaging (Supplementary Fig. 1 online). Singlecell FISH with dynamic changes in pH via microfluidics using a third imaging system also confirmed this (Supplementary Data and Supplementary Fig. 2 online). Thus, fluorescence intensity of streptavidin-QD conjugates used to detect FISH hybridization probes varied with pH of the final incubation buffer. Control solution experiments with QD conjugates in the presence or absence of DNA showed no pH dependence (Supplementary Data). It is unclear why fluorescence intensity of the QD-streptavidin probe in the FISH format exhibited pH dependence. pH control, however, may allow optimization of QD fluorophores in clinically useful formats such as FISH and other hybridization-based assays. Additional information is available in Supplementary Methods online.

miR-155 augments CD8+ T-cell antitumor activity in lymphoreplete hosts by enhancing responsiveness to homeostatic γc cytokines

Significance We describe here a strategy based on microRNA therapeutics to augment the efficacy of T-cell–based therapies without the requirement of toxic maneuvers such as lymphodepletion preconditioning and the administration of high doses of exogenous cytokines. These findings can lead to the development of safer and more effective T-cell–based therapies for the treatment of patients with advanced cancer. Lymphodepleting regimens are used before adoptive immunotherapy to augment the antitumor efficacy of transferred T cells by removing endogenous homeostatic “cytokine sinks.” These conditioning modalities, however, are often associated with severe toxicities. We found that microRNA-155 (miR-155) enabled tumor-specific CD8+ T cells to mediate profound antitumor responses in lymphoreplete hosts that were not potentiated by immune-ablation. miR-155 enhanced T-cell responsiveness to limited amounts of homeostatic γc cytokines, resulting in delayed cellular contraction and sustained cytokine production. miR-155 restrained the expression of the inositol 5-phosphatase Ship1, an inhibitor of the serine-threonine protein kinase Akt, and multiple negative regulators of signal transducer and activator of transcription 5 (Stat5), including suppressor of cytokine signaling 1 (Socs1) and the protein tyrosine phosphatase Ptpn2. Expression of constitutively active Stat5a recapitulated the survival advantages conferred by miR-155, whereas constitutive Akt activation promoted sustained effector functions. Our results indicate that overexpression of miR-155 in tumor-specific T cells can be used to increase the effectiveness of adoptive immunotherapies in a cell-intrinsic manner without the need for life-threatening, lymphodepleting maneuvers.

“Rainbow” Reporters for Multispectral Marking and Lineage Analysis of Hematopoietic Stem Cells

Hematologic diseases potentially benefiting from gene‐based therapies involving hematopoietic stem cells (HSCs) include hereditary hemoglobinopathies, immunodeficiency syndromes, and congenital bleeding disorders such as hemophilia A, as well as acquired diseases like AIDS. Successful treatment of these blood diseases with gene‐modified HSCs requires high efficiency gene delivery to the target cell population and persistence of transgene expression following differentiation. We review flow cytometric procedures that permit simultaneous, noninvasive measurements of transgene expression and phenotypic discrimination of hematopoietic cell subsets. Central to this approach has been the recent development of a spectrum of blue, cyan, and yellowish‐green fluorescent reporters based on the jellyfish Aequorea victoria green fluorescent protein and the discovery of a red fluorescent protein in Discosoma coral. This methodology should facilitate the optimization of oncoretroviral and lentiviral vectorology and HSC transduction protocols for the ultimate purpose of HSC‐directed gene therapy.