Biology Reference
In-Depth Information
system. In the second, system-focused section, the
immune system is discussed as a whole, how it is studied
using high-throughput approaches. These approaches
usually do not consider cellular context, which can make
results difficult to interpret. The last, multi-scale focused
section explores integrative models that encode informa-
tion immune system biology at multiple levels (e.g., genes,
proteins, cells, cell populations, processes etc.). The aim is
to identify emergent phenomena and knowledge gaps. It
should be noted, however, that the specification of the
models is an enormous task and only small segments of the
immune system can be studied at present. The premise for
pursuing a systems immunology approach is that devel-
oping a quantitative understanding of how the many
components of the immune system interact would yield
a true understanding of the immune system as well as
generate predictions about the immune response of a given
individual, outcomes expected to be of high clinical
relevance.
Reconstructing Cellular Networks
Immune cells are a useful model for developing network
reconstruction methodologies because they are highly
accessible and can therefore serve as a general model for
eukaryotic mammalian cells. The pathways or networks
related to innate and adaptive immunity are ultimately
involved in the determination of successful immune
responses as well as in disease. One methodology for
eukaryotic network reconstruction was originally developed
in yeast
[14]
and includes four steps: (1) global profiling of
gene expression, for instance using microarrays; (2)
computational inference to identify regulators and their
putative target genes based on co-expression; (3) repeated
measures with perturbations to obtain increasingly accurate
interactions; and (4) selected validation of predicted regu-
latory interactions (see also Chapter 4). With the develop-
ment of novel technologies, molecular resources for
perturbation as well as progress in automation, the same
approach can now be carried out in mammals, yet still
requires herculean efforts and extensive validation.
Regulatory network reconstruction has been applied to
several different hematopoietic-derived immune cell types,
including B-cell transcriptional and post-transcriptional
regulatory networks
[9
CELL-FOCUSED SYSTEMS IMMUNOLOGY
Immunologists use proteins that are expressed on the cell
surface (cell-surface markers) to quantify and isolate
heterogeneous cell populations (such as those obtained
from peripheral blood) via flow cytometry. As cells of the
immune system are highly specialized, much of the work in
immunology has been to understand the specific immune
processes mediated by each cell, and how they relate to
overall immune protection. Cells can be studied in culture,
and can be harvested then manipulated and reintroduced
into the organism. Beyond research as to their role in
immunity, their high accessibility and ease of manipulation
have made immune cells a top choice for systems biologists
interested in eukaryotic cellular
11]
, as well as CD4
þ
T cells
signaling pathways that were derived from single-cell
intracellular measurements of phosphorylated protein
abundance obtained from thousands of cells via flow
cytometry
[7]
. This impressive proof-of-concept can now be
expanded significantly using the recently developed mass
cytometry approach (
Box 25.1
,
Figure 25.2
). In the innate
system, systems biology techniques have been employed to
map out the regulatory circuitry (transcriptional and
signaling) inmacrophage and dendritic cells
[5,8,12,13]
. For
these, the focus has been on mapping the circuitry down-
stream of the Toll-like receptor (TLR) family, a class of
pattern recognition receptors each capable of recognizing
e
regulatory networks
[5,7
e
13]
.
BOX 25.1 Exploring cells in high dimension
the use of mass cytometry to quantify cell identity and function
Cells are the quanta of the immune system and their identity
and function can be understood by the degree to which they
express proteins on the cell surface or intracellularly. Tradi-
tionally, the workhorse tool of immunology has been the flow
cytometer, which optically measures the return fluorescence
from cells stained with fluorophore-labeled antibodies bound
to cell-surface and intracellular proteins. A difficulty for using
multiple fluorophores simultaneously in flow cytometry is that
the fluorescence emission spectrum spills over from its char-
acteristic wavelength to interfere with the reading from other
channel. One can use 10
e
Cytometry by time-of-flight (CyTOF) is a recently introduced
technology that measures the abundance of metal isotope labels
on antibodies and other tags (such as peptide-MHC tetramers for
labeling specific T cells) on single cells using mass spectroscopy.
The advantage of this approach is that it allows many more
molecules to be used in combination to assay a single sample
(blood or single-cell suspension of tissues) thanwith fluorescence
label-based cytometry
[1
4]
. This allows much more informa-
tion to be obtained from each cell. An illustration of this is that
whereas 10 labels gives approximately 1000 possible combina-
tions, 30 give over 1 000 000 (using the formula
e
2
n-1
, where
15 different labels before the over-
lapping emissions spectra become too complex to accurately
separate them.
e
x
¼
x
isthenumberofcombinationsand
is the number of labels).
Thus there is much greater resolving power with the number of
labels possible with this new technology.
n
