The immune system affects a wide variety of disease states and areas of study—ranging from vaccine development to leukemia, infectious disease to arthritis, pediatrics to psychiatry, immunotherapy to autoimmunity. The quest for understanding (and intervention) similarly runs the gamut of techniques and instrumentation from cell-mediated cytotoxicity assays to mass spectrometry. Many of these are becoming more intertwined, with fields and technologies not only borrowing from one another but overlapping and forming synergies. Witness, for example, immunotherapy combining principles from immunology and cell therapy, or the Luminex platform taking the concept of ELISA to flow cytometry. And let’s not forget all the techniques taking advantage of single-cell technologies and next-generation sequencing (NGS).
Individual labs looking at specific questions will almost certainly have their own sets of techniques. But a few overall trends seem to be emerging. No doubt these are not unique to immunology. But then again, immunological questions are not unique to any single discipline, either.
Multidimensionality
Think about immunology, and you typically think of analyzing (often sorting) up to many millions of individual white blood cells on the basis of their size and granularity along with fluorescently labeled markers—in other words, flow cytometry. It’s possible to simultaneously phenotype immune cells by flow cytometry on the basis of the presence and specificity of their antigen receptor, for example, the expression of the CD4 and CD8 antigens, co-stimulatory molecules and a host of other cell-surface makers. Staining of intracellular antigens (following permeabilization) has become commonplace, and secreted proteins such as cytokines can even be captured and visualized at the same time.
“There is a trend toward higher-dimensional assays, so that there can be better phenotypic resolution of the cells,” remarks Eline Luning Prak, scientific director of the human immunology core in the Perelman School of Medicine at the University of Pennsylvania. This enables, among other things, better tracking of minimal residual disease following treatment of leukemias and lymphomas as well as the ability to look for clonal evolution, if the phenotype of the malignancy changes. Similarly, it allows researchers to follow maturational schemes and relationships of even relatively rare lineages.
“Up to even 15 colors … flow is the best thing out there,” says Holden Maecker, director of the Human Immune Monitoring Center (HIMC) at the Stanford University School of Medicine.
Yet “the chance to basically do a lot more probes in parallel, and do them with little or no spillover between channels—you’ve got close to 50 channels to work with—is exciting to us,” he says of the Fluidigm Helios system, a CyTOF mass cytometer. The CyTOF instrument is like a flow cytometer, except that instead of using an optical detector to distinguish fluorescently conjugated antibodies by the wavelengths they emit, the CyTOF uses an atomic mass spectrometer to differentiate isotopic metal-conjugated antibodies.
“It allows us to build panels where we basically throw every T-cell marker that we can think of in there and see what falls out. Or do rather broad lineage markers and then look at signaling in every one of those cell types. It really expands the possibilities of what you can do in a single tube and intersects all the data,” Maecker explains.
Of course, nothing is perfect. A CyTOF instrument is more expensive than a flow, and it’s slower—about 500 events per second compared with five to 10,000 events per second on a good fluorescence analyzer. “That makes a real difference when you’re talking about rare events, or if you want to run 500 samples,” Maecker says.
Information overload
Having the ability to analyze so many parameters often means the technology may outstrip our ability to analyze the data, comments Prak. Researchers are using approaches such as probability binning, SPADE (spanning-tree progression analysis of density-normalized events) and probability state modeling to analyze multicolor flow cytometry and CyTOF data.
“It’s not just looking at bivariate plots—you can really analyze the interrelationships between multiple different markers on the same cell with some of these other approaches,” Prak notes. “You can’t keep track of it in your head—you need ways of looking at the data in aggregate. You can create a model in however many dimensions your dataset has and then systematically compare different samples from the same patient over time or compare patients with one particular disease type to patients with another disease type, and you can do it in a quantitative and rigorous way by letting the computer figure out what the differences are in your samples rather than just eyeballing it.”
Get the message?
Flow cytometry can be used to query not just protein but RNA, as well. Using eBioscience’s PrimeFlow™ RNA assays, permeabilized cells are incubated with complementary oligonucleotide probes. These are then detected, and the signal is amplified, by sequential hybridization of labeled branch DNA to “build up this fluorophore Christmas tree that can be excited by a laser on a conventional cytometer,” explains Dara Grantham Wright, senior vice president/general manager of eBioscience, a business unit of Affymetrix. “We can do up to three RNAs and up to five-plus protein[s] simultaneously.”
EMD Millipore’s SmartFlare™ Technology accomplishes similar aims using gold nanoparticles conjugated to oligonucleotides duplexed with quenched fluorophores, according to the company’s website. The probes enter live cells by endocytosis. After they bind to their target RNA, the reporter “flares” are released and unquenched, allowing them to be detected.
“We’re seeing more of a convergence of traditional immunological techniques with molecular analysis techniques,” Wright says. “People at CYTO [the 30th Congress of the International Society for Advancement of Cytometry, held in June] were using the term ‘molecular cytometry,’ integrating these workflows [such] that you can enrich single cells and do genomic analyses on them.”
RNA in situ hybridization [ISH] on tissue samples can also be performed with products such as Affymetrix’ ViewRNA® assay, which is available in both manual and automated formats. The technique allows researchers to examine transcripts for many targets, which can enable characterization of tumor infiltrating lymphocytes (TILs), to detect the RNA coding for secreted proteins such as cytokines, to examine non-coding RNAs, and to look for transcripts of proteins for which antibodies are difficult to make, notes Affymetrix’ VP, R&D Clinical Applications Eric Fung.
An alternative RNA ISH technology is offered by Advanced Cell Diagnostics and ts RNAscope®. The platform uses advance proprietary probe design to amplify target-specific signals within intract cells.
Seq and ye shall find RNA
RNA from single cells is also being queried by NGS. “We call it targeted RNA-Seq. Basically we sort single T cells, either using tetramers as a probe—so we have antigen-specific T cells—or, if we have a biopsy specimen from a tumor sample, we pull all the T cells from that sample, because we assume they’re there for a reason and so they’re probably interesting,” explains Maecker. “Then we do a cDNA-synthesis step and an amplification with primers for about 70 or so genes that are of interest in T-cell immunology, including the T-cell receptor (TCR). So we can get the TCR sequences of each cell, and we can get expression levels of a bunch of other genes.”
By looking at the TCR sequences, “we can see clonal history in a way, and we can also look at the phenotypes of those clones,” Maecker adds. “For the first time, we have a connection between the specificity and the phenotype, which you otherwise don’t get: You were either doing sequencing, or you were doing phenotyping.”
Using well-defined sequence barcodes (as tags), which allows cDNA from hundreds of wells to be combined into a single, deep-sequencing run, makes single-cell targeted RNA-Seq far more focused, and far less expensive, than whole RNA-Seq.
Of course, NGS can also be used to look at the genome. For example, “There’s repertoire profiling: high-throughput sequencing of B-cell receptor and TCR rearrangements that is a major area on the research side, and also on the translational side,” says Prak. “It’s not quite yet in the clinics, but soon will be, I imagine, for things like minimal residual disease detection, and eventually perhaps for autoimmunity and of analysis of drug therapies.”
Tying this all together
The only thing that really ties immunology together is the focus on the immune system. We try to understand the workings of innate or specific immunity for its own sake, as well as when it causes problems—as it does in lymphoma, autoimmunity or transplant rejection, for example. And we use the immune system and its products (such as antibodies) for research and therapeutic intervention. From the latest and greatest developments emerging from cutting-edge research labs to the tried-and-true methods performed by med techs on a routine basis, there’s no shortage of ways to look at, and to use, the immune system.
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