Optimized whole-blood assay for measurement of ZAP-70 protein expression.

Chronic lymphocytic leukemia (CLL) is characterized by a clonal expansion of small lymphocytes commonly expressing cell surface markers (CD5 and CD19) that are consistent with a population of B lymphocytes. This unit describes a technique to measure ZAP-70 protein expression in whole-blood specimens from CLL samples. The protocols presented include an optimized fixation/permeabilization technique that allows labeling of cell surface markers and intracellular ZAP-70 protein with significantly improved signal-to-noise ratio, an optimized combination of antibodies-fluorophores to maximize ZAP-70 expression levels, standardized methodology for instrument setup, including compensation, to improve inter- and intra-laboratory reproducibility, and a method to index ZAP-70 protein expression levels to internal positive and negative cell populations.

An optimized whole blood method for flow cytometric measurement of ZAP-70 protein expression in chronic lymphocytic leukemia.

Background: ZAP-70 protein expression has been proposed as a marker for immunoglobulin heavy chain mutational status, which some studies have correlated with disease course in B-cell chronic lymphocytic leukemia (CLL). Studies published to date measuring levels of expression of ZAP-70 intracellular protein using flow cytometry have demonstrated poor performance, as defined by the difference in signal in known positive and negative lymphocyte populations.

Methods: A recently published method (Chow S, Hedley DW, Grom P, Magari R, Jacobberger JW, Shankey TV, Cytometry A 2005;67:4-17) to measure intracellular phospho-epitopes was optimized using a design of experiments (DOE) approach to provide the best separation of ZAP-70 expression in positive T- or NK-cells as compared to negative B-cells in peripheral blood samples.

CD4 regulatory T cells prevent lethal autoimmunity in IL-2Rbeta-deficient mice. Implications for the nonredundant function of IL-2.

Lethal autoimmunity associated with IL-2Rbeta-deficient mice is prevented after thymic transgenic expression of wild-type IL-2Rbeta in IL-2Rbeta(-/-) mice (Tg -/- mice). Here, we show that CD4(+)CD25(+) regulatory T cells were not readily detected in IL-2Rbeta(-/-) mice, but the production of functional CD4(+)CD25(+) T cells was reconstituted in Tg -/- mice. Adoptive transfer of normal CD4(+)CD25(+) T cells into neonatal IL-2Rbeta-deficient mice prevented this lethal autoimmune syndrome.

Broad programming by IL-2 receptor signaling for extended growth to multiple cytokines and functional maturation of antigen-activated T cells.

Coincident production of IL-2 and induction of high-affinity IL-2R upon TCR engagement has precluded a clear distinction for the biological outcome of signaling through TCR/costimulatory molecules vs the IL-2R. Using a novel transgenic mouse on the IL-2Rbeta(-/-) genetic background, this study has separated the relative outcome of signaling through the TCR and IL-2R. We show that stimulation through the TCR and CD28 or CD40 ligand directly leads to T cell activation and several rounds of proliferation in an IL-2-independent fashion.

Control of T cell development in vivo by subdomains within the IL-7 receptor alpha-chain cytoplasmic tail.

IL-7/IL-7R signaling functions in both growth and differentiation during T cell development. In this study, we examined the extent these activities were controlled by signaling associated with distinct IL-7R alpha cytoplasmic domains by transgenic expression of wild-type or cytoplasmic deletion mutants of IL-7R alpha in the thymi of IL-7R alpha(-/-) mice. We show an essential requirement for the tyrosine-containing carboxyl-terminal T domain in restoring thymic cellularity, pro-/pre-T cell progression, and survival.