Thaddeus C. George

Mucosal CD8α+ DC, with a plasmacytoid phenotype, induce differentiation and support function of T cells with regulatory properties

Repetitive stimulation of naïve T cells by immature splenic dendritic cells (DC) can result in the differentiation of T‐cell lines with regulatory properties. In the present study we identified a population of DC in the mucosae that exhibit the plasmacytoid phenotype, secrete interferon‐α (IFN‐α) following stimulation with oligodeoxynucleotides containing certain cytosine‐phosphate‐guanosine (CpG) motifs and can differentiate naïve T cells into cells that exhibit regulatory properties. Although these DC appear to be present in both spleen and mesenteric lymph nodes (MLN), only CpG‐matured DC from the MLN (but not the spleen) were able to differentiate naïve T cells into T regulatory 1‐like cells with regulatory properties. The activity of these DC failed to sustain robust T‐cell proliferation and thereby enhanced the suppressive efficacy of CD4+ CD25+ T regulatory cells. These DC are the major CD8α+ DC population in the Peyers patches (PP). Given their significant presence in mucosal tissue, we propose that these DC may provide a mechanistic basis for the homeostatic regulation in the gut by eliciting regulatory cell suppressor function and poorly supporting T helper cell proliferation at a site of high antigenic stimulation like the intestine.

Distinguishing modes of cell death using the ImageStream multispectral imaging flow cytometer.

Here we demonstrate the ability of the ImageStream® 100 Multispectral Imaging Cytometer to discriminate between live, necrotic, and early and late apoptotic cells, using unique combinations of photometric and morphometric features.

High antigen dose and activated dendritic cells enable Th cells to escape regulatory T cell-mediated suppression in vitro.

CD4+CD25+ regulatory T cells (Tregs) are critical for peripheral tolerance and prevention of autoimmunity. In vitro coculture studies have revealed that increased costimulation breaks Treg‐mediated suppression in response to anti‐CD3 or antigen. However, it was unclear whether loss of suppression arose from inactivation of Tregs or whether increased stimulationcaused Th cells to escape suppression. We have investigated conditions that allow or override Treg‐mediated suppression using DO11.10 TCR‐transgenic T cells and chicken ovalbumin peptide 323–339‐pulsed antigen‐presenting cells. Treg suppression of Th proliferation is broken with potent stimulation, using activated spleen cells and high antigen dose, but is intact at low antigen dose. Costimulation with CD80 and CD86 expressed on activated dendritic cells was essential for Th cell escape from suppression at a high antigen dose. Potently stimulated Tregs were functional since they reducedlevels of IL‐2, IFN‐γ, IL‐4 and Th CD25 expression in cocultures. Furthermore, Tregs responding to high antigen dose and activated splenocytes retained the ability to suppress proliferation, but only of Th cells responding to a sub‐optimal dose of independent antigen. Together, our results demonstrate that under conditions of strong antigen‐specific stimulation, Tregs remain functional, but Th cells escape Treg‐mediated suppression.

Interferon regulatory factor 7-mediated responses are defective in cord blood plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDC) are specialized in massive production of type I interferons (IFN) upon viral infections. Activation of IFN regulatory factor (IRF)‐7 is critically required for the synthesis of type I IFN in pDC. IRF‐7 is highly expressed by resting pDC and translocates into the nucleus to initiate type I IFN transcription. In a previous work, we observed an impaired IFN‐α production in enriched cord blood pDC following a TLR9 stimulation using CpG oligonucleotides. Herein, we show that highly purified pDC from cord blood exhibit a profound defect in their capacity to produce IFN‐α/β in response to TLR9 as well as to TLR7 ligation or human CMV or HSV‐1 exposure. Microarray experiments indicate that expression of the majority of type I IFN subtypes induced by a TLR7 agonist is reduced in cord blood pDC. We next demonstrated a reduced nuclear translocation of IRF‐7 in cord blood pDC following CpG and HSV stimulation as compared to adult pDC. We conclude that impaired IRF‐7 translocation in cord blood pDC is associated with defective expression of type I IFN genes. Our data provide a molecular understanding for the decreased ability of cord blood pDC to produce type I IFN upon viral stimulation.

Numbers matter: quantitative and dynamic analysis of the formation of an immunological synapse using imaging flow cytometry

Activation of T lymphocytes by antigen-presenting cells (APC) results in the formation of an immunological synapse. Following contact with the target cell, key signaling and adhesion molecules polarize within minutes to hours to the T cell-APC interface. Multispectral imaging flow cytometry, a new technology which combines flow cytometry with imaging, was used to visualize and quantify the recruitment of the CD3epsilon and Lck signaling molecules during the evolution of an immune synapse. Using this technology, thousands of T cell/macrophage conjugates could be analyzed for each experimental time point. Following Ca++ triggered T cell activation, the dynamics of Lck and CD3epsilon recruitment to the synapse, analyzed by two independent methods, were comparable. However, CD3epsilon exhibited longer residence times (>8 min) at the synapse than Lck.

High Throughput, Parallel Imaging and Biomarker Quantification of Human Spermatozoa by ImageStream Flow Cytometry

Spermatid specific thioredoxin-3 protein (SPTRX-3) accumulates in the superfluous cytoplasm of defective human spermatozoa. Novel ImageStream technology combining flow cytometry with cell imaging was used for parallel quantification and visualization of SPTRX-3 protein in defective spermatozoa of five men from infertile couples. The majority of the SPTRX-3 containing cells were overwhelmingly spermatozoa with a variety of morphological defects, detectable in the ImageStream recorded images. Quantitative parameters of relative SPTRX-3 induced fluorescence measured by ImageStream correlated closely with conventional flow cytometric measurements of the same sample set and reflected the results of clinical semen evaluation. Image Stream quantification of SPTRX-3 combines and surpasses the informative value of both conventional flow cytometry and light microscopic semen evaluation. The observed patterns of the retention of SPTRX-3 in the sperm samples from infertility patients support the view that SPTRX3 is a biomarker of male infertility.

Visualization and quantification of cytotoxicity mediated by antibodies using imaging flow cytometry

Conventional approaches for the detection of antibody dependent cell-mediated cytotoxicity (ADCC) activity rely on quantification of the release of traceable compounds from target cells or flow cytometry analysis of population-wide phenomena. We report a new method for the direct imaging and quantification of ADCC of cancer cells. The proposed method using imaging flow cytometry combines the statistical power of flow cytometry with the analytical advantages of cell imaging, providing a novel and more comprehensive perspective of effector/target cell interactions during ADCC events. With this method we can quantify and show in detail the morphological changes in target and effector cells, their apoptotic index, the physical interaction between effector and target cells, and a directional transfer of cytosolic contents from effector to target cells. As a model system we used the therapeutic anti-CD20 antibody rituximab to target CFSE labeled Ramos human Burkitts lymphoma cells, to CMTPX-labeled human monocytic U-937 effector cells. We expect that similar studies using different effector and target cell populations may contribute to the pre-clinical evaluation of therapeutic antibodies and help to identify mechanisms that could be beneficial in the immunotherapy of cancer.