With over six million cases and 376,000 deaths globally, the coronavirus pandemic is unlike anything we have experienced in modern history. Yet, we are still completely in the dark about how the virus actually causes disease in humans. A group of researchers in Belgium has pooled their technology to screen patients’ immune systems with the goal of finally understanding the virus that we’re fighting.
One of them is cancer researcher Frederik De Smet at the University of Leuven, KU Leuven, in Belgium. His knowledge of different technologies, such as flow and mass cytometry, is invaluable for clinicians at the University Hospital UZ Leuven, who recruited different researchers to study the immune systems of patients infected with the virus causing Covid-19, SARS-Cov-2, at the beginning of the coronavirus outbreak in Europe.
“We were asked by clinicians from our hospital who were first in line for treating the coronavirus patients to join forces and start working together to understand the immunological aberrations in hospitalized patients,” De Smet recalled.
The COntAGIouS trial: following virus clues in the blood
In order to better treat patients with Covid-19, it is first vital to tackle some of the unknowns in the disease. For example, why do some patients with Covid-19 have to be admitted to hospital? Why do some of these patients deteriorate so badly they have to be moved to the intensive care unit? From early immunology studies in China, the researchers could see a strong clue of where to look first: in the blood.
Together with the Flanders Institute for Biotechnology, KU Leuven and UZ Leuven started the COntAGIouS trial with around 100 patients in early March 2020. The researchers behind the trial come from various backgrounds and have expertise in a number of different technologies.
“Our clinicians have a strong focus on intensive care and lung disease, my group focuses on CyTOF® technology – also known as mass cytometry – and other colleagues use regular flow cytometry, single-cell RNA sequencing, or focus on systems biology,” De Smet explained. “We actually all do different kinds of research, but we have come together to implement these different technologies and understand this very complex disease better.”
Assembling the right technology for the job
Since the start of the COntAGIouS trial, the research teams have been collecting samples from as many Covid-19 patients as possible in the intensive care unit and from Covid-19 patients on other wards. Each day, clinicians at UZ Leuven also collect over 200 clinical parameters from every patient at the intensive care unit.
Following and analyzing all of these different clinical parameters costs precious time, which is where the teams’ different technologies come into play. “Because what’s happening is quite complex, we aim to use technologies that can measure as many parameters as possible in one dose,” De Smet told me.
One of the main concepts is the multiplexing method, in which different cells, proteins, and RNA biomarkers can be studied simultaneously in samples. In the trial, the researchers are using multiplex analysis for cytokines present in the serum, as well as for single-cell RNA sequencing of samples from the lungs and blood.
The team also uses regular flow cytometry – a technology used to detect and measure cell characteristics, like cell count and cell size – to focus on certain subtypes of the immune system, such as T cells, natural killer cells (NK), or neutrophils, which can then be studied in more depth separately.
In order to analyze the whole blood sample and gain a broad insight into the different immune cell populations present there, De Smet and his colleagues are using Fluidigm’s CyTOF technology on the Helios system hosted by the KULeuven Flow Core facility, an advanced single-cell proteomics technology which allows researchers to study over 40 markers simultaneously on millions of single cells.
CyTOF: The immune system of Covid-19 patients under the looking glass
Helios allows researchers to study the whole blood samples of Covid-19 patients and observe the different immune system players and their interactions, such as monocytes, neutrophils, NK cells, T cells, and other lymphocytes.
“For our research, we are using the off-the-shelf deep immunophenotyping kit, called the Maxpar® Direct™ Immune Profiling Assay™, that is available for the Helios system,” De Smet explained. “It is easy to implement and also allows you to add your own markers to the analysis, which is what we have done.”
According to De Smet, one major advantage of mass cytometry compared to regular flow cytometry is the fact that the technology uses metal-labeled antibodies rather than fluorescence, which greatly lowers the interference between channels, so there is less ‘noise’ in the readout.
Another point to consider is the amount of data generated by these technologies. “Let’s say you collect a few hundred thousand cells where you measure up to 40 parameters for each cell,” De Smet emphasized. “You’re generating huge amounts of data. You then again have to find some structure and understand what’s going on.”
“We have combined the Maxpar Direct kit with an easy-to-use data analysis tool from Fluidigm called Maxpar Pathsetter™ that actually helps us to analyze the data very fast without the immediate need for bioinformaticians. This combination is a great strength of the technology and allowed us to move very fast.”
“Of course, in this study, we are also performing in-depth bioinformatics data analysis as we want to ensure that we don’t miss any unexpected phenotypes,” De Smet added.
A key breakthrough: Covid-19 disrupts the immune system
By studying the different clinical parameters of Covid-19 patients who are admitted to hospital, the researchers working on the COntAGIouS trial have already collected a number of interesting findings. They have been observing the differences between the local infection in the lungs and the overall blood phenotype.
Their observations have confirmed what other research groups have already described: a fall in the number of lymphocytes – white blood cells – in patients with Covid-19. But by using CyTOF and other multiplexing methods to study lymphocyte subpopulations, De Smet and his colleagues have also discovered that most of the lymphocytes, including T cells, are crashing, while the number of neutrophils – another type of white blood cell and part of the body’s innate defense against infections – is rising.
“In a viral infection, it should be the exact opposite,” De Smet explained. “You normally have an initial phase where the innate immunity should kick in and in the second phase, the adaptive immunity should take over. Eventually, you would get to a situation where the infection can be resolved, but in this case, the virus seems to have some unusual properties because the expected immune response is not happening right away.”
So while the rising number of neutrophils indicates that the body has an infection, the plummeting number of lymphocytes is counterintuitive, because lymphocyte counts usually go up in an infection.
The researchers are still trying to understand which lymphocyte subpopulations are affected more or less by the virus, and what these counts will look like in patients with severe Covid-19 infections versus patients with mild infections.
They have also discovered that infection with SARS-Cov-2 triggers a cascade of immune activity, causing the immune system to crash. “On the one hand, you have the infection in the lungs, where cells would normally respond to a viral infection and would induce an antiviral response. That seems not to be working properly. The body has problems clearing the viral infection from the lung,” De Smet explained.
“Somehow, this triggers a second process where you get increased levels of certain cytokines, which seems to affect the rest of the immune system. So there is some kind of dual program going on, where you have a local infection in the lung, but in a second phase, the immune system is unable to respond and starts a loop. It starts to crack itself.”
It is still unclear why this immune response only happens in some patients, while others are not as badly affected. What the researchers have seen is that hospitalized Covid-19 patients seem to suffer from this immune response, and those who are transferred to intensive care seem to be suffering from a more extreme form.
Future research into the virus
As the global pandemic unfolded, the world came to a standstill. No planes, no travel, self-isolation, quarantine, and in the life sciences, many researchers were pulled from their normal work to study Covid-19 instead.
Or as De Smet put it, “When I look at how research has evolved over the last months, I think it’s kind of striking that the entire world has stopped doing what they were doing and are all focusing on one topic, on one enemy, trying to understand what’s going on with this disease.”
And although the world is slowly starting to turn again with strict measures being lifted and researchers gradually returning to their labs, there is still a lot to be done and discovered. Work on a vaccine is ongoing and the different reactions of the immune system in different people still need to be understood.
“We really need to understand immunity,” De Smet emphasized. “In Belgium, for instance, lockdown measures have already been loosened for the past week. The infection rate still keeps going down. It could either be that people are not in contact with each other as much as before and they are more aware or that we are underestimating the immunity in the population and the population might be more immune than we think.”
In the next stage of the COntAGIouS trial, De Smet and his colleagues want to follow up with people who have overcome Covid-19.
“We’d like to see the recovery phase. We’ve done some preliminary assessments and see that the immune system can bounce back. You see it crashing in patients who come to the hospital and you see it recovering in patients who can go home. Why and how does the immune system recover? That’s something we don’t know yet.”
Images via Shutterstock.com, E. Resko, and Fluidigm