An assay for colony-stimulating factor (CSF) production by single T lymphocytes: estimation of the frequency of cells producing granulocyte-macrophage CSF and multi-lineage CSF within a T lymphocyte clone

The frequency of cells producing hemopoietic colony-stimulating factors (CSF) in a murine T lymphocyte clone has been determined by using a simple microassay that does not require clonal expansion or the addition of accessory cells. When stimulated with concanavalin A (Con A), the clone LB3 produced both granulocyte-macrophage CSF (GM-CSF) and multi-lineage CSF (Multi-CSF), which could be detected by using the cell line FDC-P1, whose proliferation is dependent on the presence of either of these factors. Limiting dilution analysis of Con A-stimulated LB3 cells indicated a requirement for cell-cell contact for optimal production of CSF, which could be bypassed by preincubation of the cells at high density with Con A for 4 hr before dilution in the assay. Limiting dilution estimates of the frequency of CSF-producing cells among Con A-pretreated LB3 cells ranged from 20 to 50%. Direct measurement of CSF production by single Con A-pretreated cells isolated by micromanipulation revealed that 10 to 20% could secrete detectable CSF. However, when isolated Con A-pretreated two-cell and three-cell aggregates were assayed, 50 to 99% were positive, indicating that 30 to 80% of the cells in the aggregates secreted CSF. Assay of the supernatants from single cells and two-cell aggregates on both FDC-P1 cells and another cell line, 32D c13, which responds only to Multi-CSF, demonstrated that many cells produced GM-CSF only, and others varied in the relative quantities of GM-CSF and Multi-CSF produced.     

Computational challenges in the analysis of ancient DNA

High-throughput sequencing technologies have opened up a new avenue for studying extinct organisms. Here we identify and quantify biases introduced by particular characteristics of ancient DNA samples. These analyses demonstrate the importance of closely related genomic sequence for correctly identifying and classifying bona fide endogenous DNA fragments. We show that more accurate genome divergence estimates from ancient DNA sequence can be attained using at least two outgroup genomes and appropriate filtering.     

Progressive differentiation and commitment of CD8+ T cells to a poorly cytolytic CD8low phenotype in the presence of IL-4

Exposure to IL-4 during activation of naive murine CD8+ T cells leads to generation of IL-4-producing effector cells with reduced surface CD8, low perforin, granzyme B and granzyme C mRNA, and poor cytolytic function. We show in this study that maximal development of these cells depended on exposure to IL-4 for the first 5 days of activation. Although IL-4 was not required at later times, CD8 T cell clones continued to lose surface CD8 expression with prolonged culture, suggesting commitment to the CD8low phenotype. This state was reversible in early differentiation. When single CD8low cells from 4-day cultures were cultured without IL-4, 65% gave rise to clones that partly or wholly comprised CD8high cells; the proportion of reverted clones was reduced or increased when the cells were cloned in the presence of IL-4 or anti-IL-4 Ab, respectively. CD8 expression positively correlated with perforin and granzyme A, B, and C mRNA, and negatively correlated with IL-4 mRNA levels among these clones. By contrast, most CD8low cells isolated at later time points maintained their phenotype, produced IL-4, and exhibited poor cytolytic function after many weeks in the absence of exogenous IL-4. We conclude that IL-4-dependent down-regulation of CD8 is associated with progressive differentiation and commitment to yield IL-4-producing cells with little cytolytic activity. These data suggest that the CD4-CD8- cells identified in some disease states may be the product of a previously unrecognized pathway of effector differentiation from conventional CD8+ T cells.     

PatMaN: rapid alignment of short sequences to large databases

We present a tool suited for searching for many short nucleotide sequences in large databases, allowing for a predefined number of gaps and mismatches. The commandline-driven program implements a non-deterministic automata matching algorithm on a keyword tree of the search strings. Both queries with and without ambiguity codes can be searched. Search time is short for perfect matches, and retrieval time rises exponentially with the number of edits allowed. AVAILABILITY: The C++ source code for PatMaN is distributed under the GNU General Public License and has been tested on the GNU/Linux operating system. It is available from http://bioinf.eva.mpg.de/patman. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Stimulator cell requirements for allospecific T cell subsets: specialized accessory cells are required to activate helper but not cytolytic T lymphocyte precursors

Murine cortisone-resistant thymocytes were separated by staining with monoclonal anti-Lyt-2 antibody and FMF into Lyt-2- and Lyt-2+ subsets in order to analyze the nature of stimulator accessory cells required to activate each of these functionally distinct T cell subpopulations. The Lyt-2- fraction was able to proliferate but not to generate cytotoxic cells when stimulated by irradiated allogeneic spleen cells. Fractionation of the stimulator population showed that low numbers of dendritic cells and splenic macrophages, but not equivalent numbers of whole spleen cells or peritoneal macrophages, were able to stimulate the Lyt-2- population. On the other hand, the Lyt-2+ population, which showed little if any proliferation in response to irradiated spleen cells, contained all the precursors of cytolytic T lymphocytes. In contrast to the highly specific stimulator requirement of the Lyt-2- fraction, allospecific cytotoxic cells were generated from Lyt-2+ cells by any alloantigen-bearing stimulator cell provided interleukin 2 was present. This was confirmed by limiting dilution analysis: alloreactive CTL-P frequencies in spleen and thymus were not influenced by the nature of the stimulator cell. These data collectively indicate that heterogeneous Ia+ accessory cells are required to stimulate helper but not cytolytic T cell precursors.     

Coordination dynamics of trajectory formation

The present study aims to understand the neurally based coordination dynamics (multistability, loss of stability, transitions, etc.) of trajectory formation in a simple task. Six subjects produced two spatial patterns of coordination in the xy plane by alternating the abduction-adduction and flexion-extension motions of their right index finger. Each pattern was characterized by a unique temporal ratio between the x and y directions of motion: (1) a figure zero, a 1∶1 temporal pattern; and (2) a figure eight, a 2∶1 temporal pattern. The patterns were produced rhythmically and movement frequency was scaled across ten frequency plateaus, with ten cycles of motion per step. As movement frequency increased, switching from a figure eight to a figure zero was observed at critical cycling frequencies. The switch from pattern (2) to pattern (1) was identified in the spatial trajectory and power spectra of x(t) and y(t). En route to the transition, enhancement of fluctuations was observed in the Fourier amplitudes of x(t) and y(t), specifically at f ₀ (the metronome frequency) and 2f ₀ (the first harmonic off ₀). Interestingly, there was no difference in the spatial variability of the two patterns. Overall, the data demonstrate that spatial patterns of coordination can be characterized in terms of the temporal relationship between the spatial components of the trajectory itself. We discuss the experimental findings in relation to other end-point planning and multijoint control strategies, as well as the much more general problem of temporal synchronization in many interlimb and intralimb coordination tasks.