Overexpression of c-Met and CD44v6 receptors contributes to autocrine TGF-ß1 signaling in interstitial lung disease.

The hepatocyte growth factor (HGF) and the HGF receptor Met pathway are important in the pathogenesis of interstitial lung disease (ILD). Alternatively spliced isoforms of CD44 containing variable exon 6 (CD44v6) and its ligand hyaluronan (HA) alter cellular function in response to interaction between CD44v6 and HGF. TGF-ß1 is the crucial cytokine that induces fibrotic action in ILD fibroblasts (ILDFbs). We have identified an autocrine TGF-ß1 signaling that up-regulates both Met and CD44v6 mRNA and protein expression. Western blot analysis, flow cytometry, and immunostaining revealed that CD44v6 and Met colocalize in fibroblasts and in tissue sections from ILD patients and in lungs of bleomycin-treated mice. Interestingly, cell proliferation induced by TGF-ß1 is mediated through Met and CD44v6. Further, cell proliferation mediated by TGF-ß1/CD44v6 is ERK-dependent. In contrast, action of Met on ILDFb proliferation does not require ERK but does require p38(MAPK). ILDFbs were sorted into CD44v6(+)/Met(+) and CD44v6(-)/Met(+) subpopulations. HGF inhibited TGF-ß1-stimulated collagen-1 and α-smooth muscle cell actin expression in both of these subpopulations by interfering with TGF-ß1 signaling. HGF alone markedly stimulated CD44v6 expression, which in turn regulated collagen-1 synthesis. Our data with primary lung fibroblast cultures with respect to collagen-1, CD44v6, and Met expressions were supported by immunostaining of lung sections from bleomycin-treated mice and from ILD patients. These results define the relationships between CD44v6, Met, and autocrine TGF-ß1 signaling and the potential modulating influence of HGF on TGF-ß1-induced CD44v6-dependent fibroblast function in ILD fibrosis.

Phenotypic complexity of T regulatory subsets in patients with B-chronic lymphocytic leukemia.

Increased numbers of T regulatory (T(reg)) cells are found in B-chronic lymphocytic leukemia, but the nature and function of these T(regs) remains unclear. Detailed characterization of the T(regs) in chronic lymphocytic leukemia has not been performed and the degree of heterogeneity of among these cells has not been studied to date. Using 15-color flow cytometry we show that T(reg) cells, defined using CD4, CD25, and forkhead box P3 (FOXP3), can be divided into multiple complex subsets based on markers used for naïve, memory, and effector delineation as well as markers of T(reg) activation. Furthermore FOXP3(+) cells can be identified among CD4(+)CD25(-) as well as CD8(+)CD4(-) populations in increased proportions in patients with chronic lymphocytic leukemia compared with healthy donors. Significantly different frequencies of naïve and effector T(regs) populations are found in healthy donor controls compared with donors with chronic lymphocytic leukemia. A population of CCR7(+)CD39(+) T(regs) was significantly associated with chronic lymphocytic leukemia. This population demonstrated slightly reduced suppressive activity compared with total T(regs) or T(regs) of healthy donors. These data suggest that FOXP3-expressing cells, particularly in patients with chronic lymphocytic leukemia are much more complex for T(reg) sub-populations and transitions than previously reported. These findings demonstrate the complexity of regulation of T-cell responses in chronic lymphocytic leukemia and illustrate the use of high-dimensional analysis of cellular phenotypes in facilitating understanding of the intricacies of cellular immune responses and their dysregulation in cancer.

High dimensional flow cytometry for comprehensive leukocyte immunophenotyping (CLIP) in translational research.

New paradigms in translational research are focused on deep understanding of all aspects of the human immune system in response to diseases or perturbations such as vaccination or therapy. To obtain this knowledge, coordinated, comprehensive assessments by genomics, proteomics, and cytomics are necessary. One component of this assessment is comprehensive leukocyte immunophenotyping (CLIP) that not only provides a deep and broad description of the entire immune system at any given moment, but also encompasses all leukocyte lineages, including activation states, functional markers, and signaling molecules. As envisioned, a CLIP panel could study nearly 400 antigens utilizing 17-parameter flow cytometry. The CLIP panel is structured in a manner that tubes are grouped by lineage and, within lineage each of the tubes, while having some redundant markers, characterize distinct populations. To date, a preliminary 10 tube CLIP panel has been developed with the following 17 parameter tubes: T(reg), T(h17), T(h1/2), B(general), B(naive/memory), B(intracellular), NK1, NK2, myeloid/monocyte, and dendritic cells (DC). Together these tubes have the potential to identify over 28,000 subsets of leukocytes. The feasibility of developing these tubes has been demonstrated, as well as their utility in describing complex alterations of the immune system in the context of disease and vaccination. The plethora of data accrued in the preliminary CLIP panel highlights the need for novel data analysis and reduction strategies, while at the same time illustrates the power of CLIP.