Multiparameter flow cytometry: advances in high resolution analysis.

Over the past 40 years, flow cytometry has emerged as a leading, application-rich technology that supports high-resolution characterization of individual cells which function in complex cellular networks such as the immune system. This brief overview highlights advances in multiparameter flow cytometric technologies and reagent applications for characterization and functional analysis of cells modulating the immune network. These advances significantly support high-throughput and high-content analyses and enable an integrated understanding of the cellular and molecular interactions that underlie complex biological systems.

Single and coexpression of CXCR4 and CXCR5 identifies CD4 T helper cells in distinct lymph node niches during influenza virus infection.

Influenza virus infection results in strong, mainly T-dependent, extrafollicular and germinal center B cell responses, which provide lifelong humoral immunity against the homotypic virus strain. Follicular T helper cells (T(FH)) are key regulators of humoral immunity. Questions remain regarding the presence, identity, and function of T(FH) subsets regulating early extrafollicular and later germinal center B cell responses. This study demonstrates that ICOS but not CXCR5 marks T cells with B helper activity induced by influenza virus infection and identifies germinal center T cells (T(GC)) as lymph node-resident CD4(+) ICOS(+) CXCR4(+) CXCR5(+) PSGL-1(lo) PD-1(hi) cells. The CXCR4 expression intensity further distinguished their germinal center light and dark zone locations. This population emerged strongly in regional lymph nodes and with kinetics similar to those of germinal center B cells and were the only T(FH) subsets missing in influenza virus-infected, germinal center-deficient SAP(-/-) mice, mice which were shown previously to lack protective memory responses after a secondary influenza virus challenge, thus indicting the nonredundant functions of CXCR4- and CXCR5-coexpressing CD4 helper cells in antiviral B cell immunity. CXCR4-single-positive T cells, present in B cell-mediated autoimmunity and regarded as "extrafollicular" helper T cells, were rare throughout the response, despite prominent extrafollicular B cell responses, revealing fundamental differences in autoimmune- and infection-induced T-dependent B cell responses. While all ICOS(+) subsets induced similar antibody levels in vitro, CXCR5-single-positive T cells were superior in inducing B cell proliferation. The regulation of T cell localization, marked by the single and coexpression of CXCR4 and CXCR5, might be an important determinant of T(FH) function.

Human ABCB6 localizes to both the outer mitochondrial membrane and the plasma membrane.

Expression of the ATP-binding cassette transporter ABCB6 has been associated with multiple cellular functions, including resistance to several cytotoxic agents, iron homeostasis, and porphyrin transport. To further elucidate its physiological function and/or role in drug resistance, we determined the subcellular location of ABCB6. Using three novel ABCB6-specific antibodies, Western blot analysis of cells expressing cDNA-derived or endogenous ABCB6 revealed two distinct molecular weight forms. Confocal microscopy indicates that the protein localizes to both mitochondria and the plasma membrane. Differential centrifugation revealed that the lower molecular weight form predominantly resides in the mitochondria, while the larger protein form is more abundant in the plasma membrane. Preliminary studies indicate that ABCB6 is functionally relevant in the plasma membrane, where its expression prevents the accumulation of specific porphyrins in the cell. Digitonin solubilization of mitochondria demonstrated that ABCB6 is present in the outer mitochondrial membrane, while back-titration assays with the ABCB6-specific antibodies reveal that the nucleotide binding domain of ABCB6 is cytoplasmic. These studies are the first to demonstrate that ABCB6 exists in two molecular weight forms, is localized to both the outer mitochondrial membrane and the plasma membrane, and plays a functional role in the plasma membrane.

Differential effects of CD28 costimulation upon cytokine production by CD4+ and CD8+ T cells.

The impact of CD28 ligation upon CD4+ and CD8+ T lymphocyte proliferation and cytokine production was assessed. Although costimulation increased the proliferative response of both T cell subsets, cytokine production was most markedly increased in the CD4+ subset, as evidenced by a 40-fold increase in interleukin-2 (IL-2), a 14-fold increase in interleukin-3 (IL-3) and 5-fold increases in interferon gamma and GM-colony-stimulating factor (CSF) production. The CD8+ T cell response to CD28 ligation was less marked, maxima being a 5-fold increase in IL-2 production and 2-fold increases in IL-3 and GM-CSF production. Resolution of CD4+ and CD8+ T cells into their CD44lo (naïve) and CD44hi (memory/effector) subsets revealed that naive CD4+ T cells were the most CD28-responsive subsets. CD28-mediated costimulation promotes distinct differentiation programs in CD4+ versus CD8+ T cells.

Age-related changes in mature CD4+ T cells: cell cycle analysis.

T cell proliferative responses decrease with age, but the mechanisms responsible are unknown. We examined the impact of age on memory and naive CD4(+) T cell entry and progression through the cell cycle using acridine orange to identify cell cycle stage. For both subsets, fewer stimulated cells from old donors were able to enter and progress through the first cell cycle, with an increased number of cells arrested in G(0) and fewer cells in post G(0) phases. The number of dead cells as assessed by sub-G(0) DNA was also significantly greater in the old group. CD4(+) T cells from old mice also exhibited a significant reduction in clonal history as assessed by CFSE staining. This was associated with a significant decline in cyclin D2 mRNA and protein. We propose that decreases in cyclin D2 are at least partially responsible for the proliferative decline found in aged CD4(+) T cells.