Reduced Fluorescence versus Forward Scatter Time-of-Flight and Increased Peak versus Integral Fluorescence Ratios Indicate Receptor Clustering in Flow Cytometry.

Clustering of surface receptors is often required to initiate signal transduction, receptor internalization, and cellular activation. To study the kinetics of clustering, we developed an economic high-throughput method using flow cytometry. The quantification of receptor clustering by flow cytometry is based on the following two observations: first, the fluorescence signal length (FL time-of-flight [ToF]) decreases relative to the forward scatter signal length (FSc-ToF), and second, the peak FL (FL-peak) increases relative to the integral FL (FL-integral) upon clustering of FL-labeled surface receptors. Receptor macroclustering can therefore be quantified using the ratios FL-ToF/FSc-ToF (method ToF) or FL-peak/FL-integral (method Peak). We have used these methods to analyze clustering of two immune receptors known to undergo different conformational and oligomeric states: the BCR and the complement receptor 3 (CR3), on murine splenocytes, purified B cells, and human neutrophils. Engagement of both the BCR and CR3, on immortalized as well as primary murine B cells and human neutrophil, respectively, resulted in decreased FL-ToF/FSc-ToF and increased FL-peak/FL-integral ratios. Manipulation of the actin-myosin cytoskeleton altered BCR clustering which could be measured using the established parameters. To confirm clustering of CR3 on neutrophils, we applied imaging flow cytometry. Because receptor engagement is as a biological process dependent on cell viability, energy metabolism, and temperature, receptor clustering can only be quantified by gating on viable cells under physiological conditions. In summary, with this novel method, receptor clustering on nonadherent cells can easily be monitored by high-throughput conventional flow cytometry.

Identification in CLL of circulating intraclonal subgroups with varying B-cell receptor expression and function.

Chronic lymphocytic leukemia (CLL) is a tumor of circulating B cells, variably stimulated and anergized following exposure to antigen in lymphoid tissues. Down-modulation of surface IgM (sIgM) occurs, but expression and signal capacity can recover in vitro and apparently in vivo during recirculation. We have now dissected individual circulating clones of CLL cases according to sIgM expression level by differential binding to bead-bound anti-IgM. Four clear subgroups (SG1-4) with increasing sIgM were identified in 37/37 cases. Engagement of sIgM induced phosphorylation of PLCγ2 and ERK1/2 at levels ranging from very low in SG1 to high in SG4. Phosphorylation was suppressed by the BTK inhibitor ibrutinib. Expression of CXCR4 also increased from SG1 to SG4, but markers of previous activation and proliferation were dominant in SG1. Incubation of whole CLL populations in vitro led to striking increases in CXCR4 expression as well as recovery of sIgM. Clonal analysis reveals dynamic SGs following presumed antigen stimulation in tissues. SG4 represents a fully recovered, potentially dangerous population equipped to migrate to tissue and receive a proliferative stimulus. SG1 likely represents a postmitotic unresponsive "resting" population. The effect of ibrutinib on the small SG4 population may be the critical factor in therapeutic success.

CD161 expression characterizes a subpopulation of human regulatory T cells that produces IL-17 in a STAT3-dependent manner.

Treg cells are critical for the prevention of autoimmune diseases and are thus prime candidates for cell-based clinical therapy. However, human Treg cells are "plastic", and are able to produce IL-17 under inflammatory conditions. Here, we identify and characterize the human Treg subpopulation that can be induced to produce IL-17 and identify its mechanisms. We confirm that a subpopulation of human Treg cells produces IL-17 in vitro when activated in the presence of IL-1ß, but not IL-6. "IL-17 potential" is restricted to population III (CD4(+) CD25(hi) CD127(lo) CD45RA(-) ) Treg cells expressing the natural killer cell marker CD161. We show that these cells are functionally as suppressive and have similar phenotypic/molecular characteristics to other subpopulations of Treg cells and retain their suppressive function following IL-17 induction. Importantly, we find that IL-17 production is STAT3 dependent, with Treg cells from patients with STAT3 mutations unable to make IL-17. Finally, we show that CD161(+) population III Treg cells accumulate in inflamed joints of patients with inflammatory arthritis and are the predominant IL-17-producing Treg-cell population at these sites. As IL-17 production from this Treg-cell subpopulation is not accompanied by a loss of regulatory function, in the context of cell therapy, exclusion of these cells from the cell product may not be necessary.

Comparison of regulatory T cells in hemodialysis patients and healthy controls: implications for cell therapy in transplantation.

BACKGROUND AND OBJECTIVES: Cell-based therapy with natural (CD4(+)CD25(hi)CD127(lo)) regulatory T cells to induce transplant tolerance is now technically feasible. However, regulatory T cells from hemodialysis patients awaiting transplantation may be functionally/numerically defective. Human regulatory T cells are also heterogeneous, and some are able to convert to proinflammatory Th17 cells. This study addresses the suitability of regulatory T cells from hemodialysis patients for cell-based therapy in preparation for the first clinical trials in renal transplant recipients (the ONE Study). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Healthy controls and age- and sex-matched hemodialysis patients without recent illness/autoimmune disease on established, complication-free hemodialysis for a minimum of 6 months were recruited. Circulating regulatory T cells were studied by flow cytometry to compare the regulatory T cell subpopulations. Regulatory T cells from members of each group were compared for suppressive function and plasticity (IL-17-producing capacity) before and after in vitro expansion with and without Rapamycin, using standard assays. RESULTS: Both groups had similar total regulatory T cells and subpopulations I and III. In each subpopulation, regulatory T cells expressed similar levels of the function-associated markers CD27, CD39, HLA-DR, and FOXP3. Hemodialysis regulatory T cells were less suppressive, expanded poorly compared with healthy control regulatory T cells, and produced IL-17 in the absence of Rapamycin. However, Rapamycin efficiently expanded hemodialysis regulatory T cells to a functional and stable cell product. CONCLUSIONS: Rapamycin-based expansion protocols should enable clinical trials of cell-based immunotherapy for the induction of tolerance to renal allografts using hemodialysis regulatory T cells.