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Study Using Mass Cytometry Correlates Diversity of Innate Immune Cells to Increased Infection Risk


NEW YORK (GenomeWeb News) – Researchers from Stanford University have found via a mass cytometry-based study that high diversity of the innate immune system is correlated with increased susceptibility to infection.

Detailed in a paper published this week in Science Translational Medicine, the study used Fluidigm's CyTOF mass cytometer to explore the diversity of innate natural killer (NK) cell populations and its relationship to response to HIV-1 infection both in vitro and in clinical subjects.

Their findings, which Stanford researcher and senior author Catherine Blish characterized as "extremely surprising," indicate that high levels of NK cell diversity is linked to an increased risk of HIV-1 infection.

NK cells are key components of the body's innate immune system, which serves as a sort of first line of response, quickly attacking infection while also releasing proteins that can help induce the body's adaptive response.

The response of NK cells to infection is determined by the combination of receptors on their cell surface. The cells bind to targets on infected or compromised cells, which they then kill. There are around 30 different such receptors, Blish said, noting that the different combinations of these receptors account for the diversity of NK cell populations.

Prior to their recent findings, Blish said that she and her colleagues assumed that the more diverse a subject's NK cell population, the more effective it would be at protecting against infection, the thinking being that high diversity would mean a wide repertoire of NK cell populations, each specialized for target specific infections.

In fact, they found "the precise opposite in that exposure causes specialization, but that that [specialization] may not be good," Blish said, noting that after a certain point this specialization appears to exhaust the NK repertoire, making it less effective in mounting a response to new infections. As the authors wrote, NK anti-viral response led to increased diversity "resulting in terminal differentiation and cytokine production at the cost of cell division and degranulation."

While unexpected, the discovery does "make sense in terms of thinking about the yin and yang of your adaptive and innate immune response," Blish added, observing that from an evolutionary perspective, swapping a speedy response for eventual NK cell exhaustion might seem a reasonable trade.

"If you think about the timeline, evolutionarily, the pressure is on anything that happens before reproductive age, and so this specialization and differentiation as it piles up to, say, age 20, is probably ok, and you are probably willing to have the trade-off," she said.

Blish and her colleagues arrived at their findings by investigating samples from the Mama Salama Study, which looked at 1,304 Kenyan women, tracking who did and did not acquire HIV-1 over a nine to 12 month follow-up period. They examined samples from 25 patients, 12 of whom were infected with HIV-1, and developed a measure of NK cell diversity by using Boolean gating to define for each surface marker the cell populations in which it was present.

In this way they were able to calculate the frequency of more than 11,000 single-cell marker combinations. With this information, they generated an index measuring the diversity of these combinations in different individuals, finding that for each 100-point increase in diversity on this index, a patient's risk of HIV-1 infection went up 2.5-fold.

The CyTOF was "absolutely critical" to measuring NK cell diversity given the high number of parameters involved. The instrument combines capabilities of flow cytometry and atomic mass spectrometry, which allows it to measure large numbers of proteins in single cells with high throughput. Atomic mass spectrometry detects proteins using antibodies linked to stable isotopes of elements, which can then be read with high resolution via time-of-flight mass spectrometry.

In the STM study, Blish and her colleagues measured 41 analytes per cell on the CyTOF.

By way of comparison, she noted, even the most advanced flow cytometry systems typically top out at around 20 analytes, "so the CyTOF really was critical for us to look at all the receptors at once."

Beyond that, Blish said, accurately identifying NK cells for analysis required measurement of a variety of markers aside from the cell surface proteins that contribute to diversity.

"NK cells also are defined more by what they are not than what they are," she said. "There's no single receptor that will allow you to identify [a cell] as an NK cell or not. So we had to use a lot of lineage markers to exclude T-cells, B-cells, monocytes, etc., in order to identify the NK cells."

Additionally, the multiplexing capacity of the CyTOF allowed the researchers to include in their analysis a variety of functional markers like cytokines.

Looking ahead, Blish said the researchers hope next to confirm the findings in additional cohorts and see if the correlation between NK diversity and infection risk holds true for other viruses.

She added that down the road the findings could have clinical implications. For instance, measures of NK diversity could be used to identify patients at highest risk of infection when choosing who to give HIV-1 vaccines.

Additionally, Blish and her colleagues "are trying to figure out how to turn back the clock and return NK cells to a more naïve, flexible state," she said. "We know that NK cells are extremely responsive to cytokines and that that can change their repertoire, so that's one path forward."

Manipulation of NK cells has uses outside infectious disease, Blish said. In particular, "right now there is a lot of work on using NK cells for cancer therapy," she noted. She added that interest appeared to be growing in using the CyTOF for NK analysis, as several outside groups have recently contacted her lab to discuss the technology.