Charles H. Pletcher

CD25 Blockade Depletes and Selectively Reprograms Regulatory T Cells in Concert with Immunotherapy in Cancer Patients

CD25 monoclonal antibody therapy rapidly and durably depletes Tregs in cancer patients through a mechanism consistent with reprogramming. Rehabilitating Tregs In A Clockwork Orange, a young criminal undergoes a controversial rehabilitation therapy that reprograms him to become sick at even the thought of violence. Indeed, rehabilitation, although carried out with much less extreme measures, is a critical strategy for reforming many types of bad apples. Rech et al. now extend this concept to the delinquent regulatory T cells (Tregs) that suppress antitumor immune responses. Tregs perform a critical role in healthy individuals—they prevent immune cells from the attacks on their host that causes autoimmunity. However, in the tumor microenvironment, Tregs are insidious—inhibiting immune responses against the tumor itself. Rech et al. hypothesized that daclizumab, a Food and Drug Administration–approved antibody to CD25, which is expressed on Tregs, could deplete Tregs and restore the antitumor immune response. They found in vitro that Tregs treated with daclizumab lost their suppressive function and secreted interferon-γ, which is consistent with reprogramming to effector T cells. They then treated patients with daclizumab in conjunction with an experimental vaccine for metastatic breast cancer. These patients had fewer Tregs and increased levels of vaccine antigen-specific effector T cell responses. Despite the loss of Tregs, these patients did not develop autoimmunity. Although these are still early studies, they provide hope that daclizumab may help to restore antitumor immunity without inducing autoimmunity. Regulatory T cells (Tregs) are key mediators of immune tolerance and feature prominently in cancer. Depletion of CD25+ FoxP3+ Tregs in vivo may promote T cell cancer immunosurveillance, but no strategy to do so in humans while preserving immunity and preventing autoimmunity has been validated. We evaluated the Food and Drug Administration–approved CD25-blocking monoclonal antibody daclizumab with regard to human Treg survival and function. In vitro, daclizumab did not mediate antibody-dependent or complement-mediated cytotoxicity but rather resulted in the down-regulation of FoxP3 selectively among CD25high CD45RAneg Tregs. Moreover, daclizumab-treated CD45RAneg Tregs lost suppressive function and regained the ability to produce interferon-γ, consistent with reprogramming. To understand the impact of daclizumab on Tregs in vivo, we performed a clinical trial of daclizumab in combination with an experimental cancer vaccine in patients with metastatic breast cancer. Daclizumab administration led to a marked and prolonged decrease in Tregs in patients. Robust CD8 and CD4 T cell priming and boosting to all vaccine antigens were observed in the absence of autoimmunity. We conclude that CD25 blockade depletes and selectively reprograms Tregs in concert with active immune therapy in cancer patients. These results suggest a mechanism to target cancer-associated Tregs while avoiding autoimmunity.

A plastid segregation defect in the protozoan parasite Toxoplasma gondii.

Apicomplexan parasites—including the causative agents of malaria (Plasmodium sp.) and toxoplasmosis (Toxoplasma gondii)—harbor a secondary endosymbiotic plastid, acquired by lateral genetic transfer from a eukaryotic alga. The apicoplast has attracted considerable attention, both as an evolutionary novelty and as a potential target for chemotherapy. We report a recombinant fusion (between a nuclear‐encoded apicoplast protein, the green fluorescent protein and a rhoptry protein) that targets to the apicoplast but grossly alters its morphology, preventing organellar segregation during parasite division. Apicoplast‐deficient parasites replicate normally in the first infectious cycle and can be isolated by fluorescence‐activated cell sorting, but die in the subsequent host cell, confirming the ‘delayed death’ phenotype previously described pharmacologically, and validating the apicoplast as essential for parasite viability.

CD34 + , kit + , rhodamine123 low phenotype identifies a marrow cell population highly enriched for human hematopoietic stem cells

We hypothesized that human hematopoietic cells displaying a CD34+, kit+, rhodamine123low phenotype would be highly enriched for cells with stem-like properties. To test this hypothesis, we employed fluorescence activated cell sorting (FACS) to isolate cells with this phenotype from normal light density marrow mononuclear cells (MNC). CD34+, kit+, rhodamine123low cells comprised from 0.05–0.01% of the total MNC population. They were small, had scant cytoplasm, and contained nuclei with dense, hyperchromatic chromatin and inconspicuous nucleoli. Additional immunophenotyping revealed that these cells were CD33−, CD38−, CD20−, and glycophorin A−. When plated in semisolid cultures containing optimal concentrations of IL-3, GM-CSF, KL, EPO, IL-6, and IL-1 these cells did not form colonies. However, when cultured over irradiated stromal cells, cobblestone areas were observed to form after 3 weeks, and harvested cells were able to initiate long-term cultures. To further demonstrate that these cells were indeed stem like, we also tested their ability to engraft and mature in immunocompromised (SCID) mice. Irradiated (400 cGy) SCID mice were transplanted with 2 × 103 candidate stem cells which were then injected with recombinant human growth factors every other day. Two months post-transplant the animals were sacrificed. PCR and FACS analysis of marrow and spleen cell samples revealed the presence of cells expressing human CD45 consistent with engraftment of human stem cells and the establishment of murine–human chimerism. Moreover, MNC isolated from transplanted mice formed unambiguously human BFU-E, CFU-GM and B cell colonies when stimulated with the appropriate growth factors. Accordingly, we have identified a relatively rapid and simple mechanism for isolating primitive human hematopoietic cells with stem cell-like properties. We anticipate that this strategy will be useful for experimental and therapeutic applications that require human stem cells in quantity.

Recombinant human thrombopoietin (TPO) stimulates erythropoiesis by inhibiting erythroid progenitor cell apoptosis

Thrombopoietin (TPO) has been reported to stimulate erythropoiesis, but the stimulatory mechanism has not been defined. To address this issue, we performed serum‐free cell‐culture experiments with recombinant human TPO and purified human progenitor cells. We found that TPO alone was able to stimulate the megakaryocyte colony formation in serum‐free cultures, but erythroid colonies were never observed. Only in the presence of EPO (erythropoietin) +IL‐3 was TPO able to stimulate a small increase (∼25%) in erythroid colony formation. Accordingly, we hypothesized that TPO might have an effect on erythroid progenitor cell viability, rather than a direct stimulatory effect. To test this idea, CD34+ cells were cultured for 7 d in serum‐free methylcellulose in the presence or absence of TPO, after which time KL+ EPO was added to the cultures. Cells which were pre‐cultured for 7 d in the presence of TPO gave rise to approximately 6 times as many burst forming unit‐erythroid (BFU‐E) colonies as cells which were pre‐cultured in the absence of TPO. Further, when primitive CD34+, Kit+ MNC were cultured for 3–7 d under serum‐free conditions in the presence or absence of TPO, significantly fewer cells cultured in the presence of TPO displayed apoptotic changes when compared to cells cultured in the absence of TPO. Taken together, these results suggest that TPO has little direct stimulatory effect on erythroid progenitor cells, but might indirectly enhance erythropoiesis by preventing very early erythroid progenitor cells from undergoing apoptotic cell death.

Effect of basic (FGF-2) and acidic (FGF-1) fibroblast growth factors on early haemopoietic cell development

Basic fibroblast growth factor (FGF‐2) and acidic fibroblast growth factor (FGF‐1) are mitogens for a variety of cell types. Many reports suggest that haemopoietic cells are among these. Nevertheless, when we examined the effect of recombinant human FGF‐1 or 2 on normal human marrow cell proliferation in vitro, only minimal stimulatory activity could be detected. In this regard, the addition of either growth factor to cultures of ancillary cell depleted marrow mononuclear cells (MNC), or to highly enriched CD34+ MNC, failed to enhance haemopoietic colony number and induced only a slight increase in colony size. Perturbation of FGF receptor (FGF‐R) expression on CD34+ MNC with antisense (AS) oligodeoxynucleotides (ODN) was also without apparent effect on cell growth. Neither could we demonstrate any effect of FGF‐1 or 2 on survival of early progenitor cells in serum‐free culture. To explain these findings, we examined progenitor cells for expression of the FGF‐R at the mRNA and protein level using RT‐PCR and flow cytometry. Primitive CD34+/KIT+ MNC had no detectable FGF‐R (FGF‐R1, 2, 3 or 4) mRNA or protein expression. In fact, direct immunofluorescence labelling of MNC for CD34 antigen and FGF‐R1 demonstrated that expression of these markers was mutually exclusive in the populations examined. FGF‐R1 expression was detected on subpopulations of MNC and on cells derived from day‐6 CFU‐GM and BFU‐E colonies. Accordingly, FGF‐R1 is either absent, or present at very low levels, on primitive haemopoietic cells. This fact, combined with our in vitro culture data, suggest that receptors are unlikely to play a significant role in the development of these early cells. Nevertheless, the development of mature cells may be influenced by the FGFs since the FGF‐Rs are expressed on more mature cells.

Entry of Human Immunodeficiency Virus‐1 Into Glial Cells Proceeds via an Alternate, Efficient Pathway

Although the CD4 molecule is the cellular receptor for human immunodeficiency virus‐1 (HIV‐1) in cells of the lymphocyte/monocyte lineage, a number of investigators have also been able to infect cells, including several of central nervous system (CNS) origin, that do not express CD4 protein or mRNA. These infections are generally nonpermissive. To ascertain whether the nonpermissive nature of infection in glial cells is due to an inefficient entry pathway, we prepared a permanently transfected U373‐MG cell line expressing the CD4 molecule and demonstrated that HIV‐1 still replicates at a low level. Furthermore, a virus uptake assay indicated that HIV‐1 enters glial cells effectively, even in the absence of CD4. These results demonstrate that HIV‐1 entry is efficient and that the restrictive nature of the infection in glial cells is due to postentry mechanisms. In addition, these findings support the existence of an alternate, efficient, entry pathway in some glial cells.

Novel approach for simultaneous evaluation of cell phenotype, apoptosis, and cell cycle using multiparameter flow cytometry

Apoptosis is a vital process for organism development and, when disrupted, can lead to abnormalities including cancer and autoimmune diseases. We demonstrate a novel multicolor flow cytometry approach for quantifying apoptosis and cell cycle information of phenotypically distinct populations, using less than 2 x 10(5) cells per sample. We used incorporation of Cy5-dUTP into DNA strand breaks by the terminal dUTP nucleotide end labeling (TUNEL) method to determine apoptosis, while cell cycle information was assessed with an ultraviolet DNA binding dye, DAPI. To simultaneously determine surface phenotype, we used paraformaldehyde fixation and a gentle permeabilization protocol combined with FITC- and PE-labeled surface antibodies. Using these fluorochromes, and three-laser instrumentation, we quantified apoptosis and cell cycle phase in lymphocyte subpopulations from heterogeneous human and murine cell sources, subjected to various culture conditions. Further, we used this method to detect divergent rates of apoptosis in a human, heterogeneous lymphocyte tumor population, demonstrating a potential application for clinical and/or research settings. Thus, we describe a six-parameter, four-color flow cytometry approach for evaluating apoptosis and cell cycle with dual surface labels. This method may also be useful as a generalized scheme to assess simultaneously two intracellular targets in a mixed cell population.

International Society for the Advancement of Cytometry Cell Sorter Biosafety Standards

Flow cytometric cell sorting of biological specimens has become prevalent in basic and clinical research laboratories. These specimens may contain known or unknown infectious agents, necessitating precautions to protect instrument operators and the environment from biohazards arising from the use of sorters. To this end the International Society of Analytical Cytology (ISAC) was proactive in establishing biosafety guidelines in 1997 (Schmid et al., Cytometry 1997;28:99–117) and subsequently published revised biosafety standards for cell sorting of unfixed samples in 2007 (Schmid et al., Cytometry Part A J Int Soc Anal Cytol 2007;71A:414–437). Since their publication, these documents have become recognized worldwide as the standard of practice and safety precautions for laboratories performing cell sorting experiments. However, the field of cytometry has progressed since 2007, and the document requires an update. The new Standards provides guidance: (1) for laboratory design for cell sorter laboratories; (2) for the creation of laboratory or instrument specific Standard Operating Procedures (SOP); and (3) on procedures for the safe operation of cell sorters, including personal protective equipment (PPE) and validation of aerosol containment. Published

Analysis of Tumor Thiol Concentrations: Comparison of Flow Cytometric With Chemical and Biochemical Techniques

The importance of glutathione (GSH) in contributing to cancer therapy resistance is well established. Various advantages may accrue from the ability to determine the distribution of GSH content in individual tumor cells disaggregated from solid tumors using flow cytometric techniques compared with biochemical or chemical measurements of the average GSH level in bulk tissue samples. The flow cytometric technique requires a thiol-reactive fluorescent adduct which is stable and which can differentiate cellular GSH from protein thiols. Thiol-reactive compounds specific for GSH require facilitated conjugation by endogenous cellular enzymes, but such compounds have not been found to accurately monitor GSH in human cells. Compounds which react generally with all thiols require a GSH-depleted calibration control to assess GSH vs. non-GSH components of fluorescent-adduct formation. Our report addresses this question, and compares three different thiol assays in several cell lines. We have found a simple way to control for non-GSH adduct formation. This involves a selective permeabilization process to release low molecular weight adducts (dominated by GSH and cysteine). In application to the desired goal of assessing the distribution of GSH in cells disaggregated from tumors, we have identified a problem with cell-line-specific thiol loss during the tumor cell disaggregation process.

An integrated flow cytometry-based platform for isolation and molecular characterization of circulating tumor single cells and clusters

Comprehensive molecular analysis of rare circulating tumor cells (CTCs) and cell clusters is often hampered by low throughput and purity, as well as cell loss. To address this, we developed a fully integrated platform for flow cytometry-based isolation of CTCs and clusters from blood that can be combined with whole transcriptome analysis or targeted RNA transcript quantification. Downstream molecular signature can be linked to cell phenotype through index sorting. This newly developed platform utilizes in-line magnetic particle-based leukocyte depletion, and acoustic cell focusing and washing to achieve >98% reduction of blood cells and non-cellular debris, along with >1.5 log-fold enrichment of spiked tumor cells. We could also detect 1 spiked-in tumor cell in 1 million WBCs in 4/7 replicates. Importantly, the use of a large 200μm nozzle and low sheath pressure (3.5 psi) minimized shear forces, thereby maintaining cell viability and integrity while allowing for simultaneous recovery of single cells and clusters from blood. As proof of principle, we isolated and transcriptionally characterized 63 single CTCs from a genetically engineered pancreatic cancer mouse model (n = 12 mice) and, using index sorting, were able to identify distinct epithelial and mesenchymal sub-populations based on linked single cell protein and gene expression.