Flow cytometry in the study of leukocyte cell surface molecules and the validation and standardization of monoclonal antibodies – Interview with Prof. Pablo Engel, University of Barcelona.

In our continued coverage of the opinions and views of leading researchers in immunology, we are pleased to present an interview with Prof. Pablo Engel, Department of Biomedical Sciences, University of Barcelona. Prof. Engel’s lab studies leukocyte cell-surface molecules. Monoclonal antibodies are very important for such studies and Prof. Engel’s lab has several years of expertise in producing them. Prof. Engel is also the President of HCDM (Human Cell Differentiation Molecules), the chair of the nomenclature and standardization committees for CD molecules and leukocytes of IUIS (International Union of Immunological Societies) and Secretary-General of EFIS (European Federation of Immunological Societies). In this interview, Prof. Engel spoke to us about his research, how CD molecules are classified and standardised, and on his views about the current research on the immune response to COVID-19 infections.

  1. Can you tell us about your lab and your research interests?

Our lab is interested in the study of the leukocyte cell surface molecules and how they control the function of the cells of the immune system. These molecules are not only excellent markers to identify and to monitor lymphocytes subsets but also very relevant molecules that regulate immune responses. Alterations in the expression or function of these molecules are responsible for a vast array of diseases, such as immunodeficiencies or autoimmune disorders. During the last 20 years, we have been studying a group of these molecules called the SLAM family receptors. Our work, and that of our collaborators, have been key to characterize the function of these co-signaling immune receptors. More recently, our group has identified several of these molecules encoded in the genome of viruses, such as cytomegalovirus. These genes have been kidnapped by the viruses from infected host cells during evolution and utilised for evading the immune system.

The production of novel monoclonal antibodies (mAbs) has been essential for the development of my research ever since I started my Ph.D. thesis on B-cells specific cell surface molecules. During the past years, we have been working to apply these mAbs for the treatment of patients. One of the most promising applications of this approach is the therapeutic use of CAR-T cells for cancer patients. It is really exciting to see that after all these years of studying the basic biology of these molecules, our research can be translated into clinically relevant applications

  1. You are the President of the International Council of Human Cell Differentiation Molecules (HCDM), and the chair of the nomenclature and standardization committees for the CD molecules and leucocytes of the IUIS. Can you tell us how CD molecules are classified and standardised?

CD (cluster of differentiation) molecules are expressed on the cell surface of leukocytes and other cells of the immune system. CD molecules are defined by groups of mAbs that recognize the same molecule. CD nomenclature is also used to designate the molecule itself. For example, CD8 designates both the group of mAbs that specifically recognize the molecule as well as the CD8 molecule itself. The CD molecule classification and standardization are established by a series of workshops, called HLDA Workshops. These workshops started in the early 1980s to cope with the need to create a standard nomenclature of the cell surface molecules that were quickly been discovered using monoclonal antibodies. Over the years these workshops have evaluated thousands of antibodies and have been recognized as one of the most important international collaborative endeavours in the field of immunology. These workshops are based on the submission of monoclonal antibodies from academic groups and companies, that are distributed among several collaborating laboratories. These labs analyze their reactivities with large panels of primary cells, cell lines, and leukemia’s and lymphomas, using flow cytometry, followed by statistical clustering analysis of the resulting expression data. The monoclonal antibodies that cluster together are further examined for the biochemical nature and molecular mass of their target molecule by immunoprecipitation. Although cellular expression analysis remains essential, molecular biology techniques, such as the study of transfected cells or the expression silencing, have become essential for the establishment of the target identity. During the 1980s and 90s, these workshops were very exciting for the immunology community. At that time, the antibodies against leukocytes were obtained by the immunization of mice with whole cells. The randomly produced antibodies were directed against a large number of unknown molecules. The HLDA workshops determined if a group of antibodies were recognising the same molecule or CD and if this represented a known or a newly identified molecule. Soon a group of companies, including BD, started to license the hybridomas that produced these antibodies and these reagents were made available to all the scientific community. The data generated by the HLDA Workshops have led to the characterization and formal designation of more than 400 CD molecules. This, together with the parallel development of flow cytometry, represented a revolution for immunology. Today, the recent HLDA workshops are focused on the validation of monoclonal antibodies. Thus, only very well-validated antibodies will get a CD designation. The 11th HLDA workshop is currently ongoing and its main focus is the study of monoclonal antibodies against GPCR proteins, such as the chemokine receptors.

  1. What are the challenges involved in producing good quality monoclonal antibodies?

Today, the production of monoclonal antibodies from different species and formats, such as recombinant antibodies, has become very easy and straightforward. This has led to an inflation in the number of companies that sell antibodies. It is estimated that more than 300 vendors offer more than 2 million monoclonal antibodies. The major problem is that a lot of these antibodies are not rigorously validated. Many of these reagents do not perform as they should. Monoclonal antibodies that do not bind or stain properly pose huge problems for the biomedical researcher. They are responsible for the waste of time and money,  the generation of ineffective experiments, and the publication of inaccurate data in the scientific literature. I believe that this is one of the major reasons for the lack of reproducibility of a lot of published results, including those in top journals. The major reason for this is the use of antibodies that are not validated for a specific application. Another important issue is the fact that a lot of antibodies are cross-reactive with other molecules of the same family or even the presence of unpredictable cross-reactivities based on sequence analysis. In my opinion, young scientists using antibodies for their research must be properly trained in their use and validation. Another important measure would be that both academic groups in their papers and vendors on their webpages, should show the data that demonstrated their validation. Unfortunately, there is no consensus about the minimal experimental evidence needed to consider an antibody as sufficiently validated. Thus, there is an urgent need to establish standardized validation guidelines.

 

  1. What role does flow-cytometry, particularly high dimensional, play in your research?

Flow cytometry has played an essential role in my research since I started my career as an immunologist in the 80s. I have witnessed the huge progress of flow cytometry from the initial studies were we used antibodies just labeled with one fluorochrome to the current revolution that allows the simultaneous determination of more than 30 markers in a single tube. This has allowed us to get an overwhelming amount of information. The biggest challenge is the development of powerful analytical tools to interpret these data, especially in the context of monitoring the patient’s immune system. My impression is that the technical improvements in flow-cytometry, new instruments, and reagents such as fluorochromes have run faster than the development of useful bioinformatics tools. For me, one of the most amazing innovations is the use of antibodies conjugated to oligos in their use for single–cells analysis and I am eager to incorporate this technology into the studies of my lab. This system will allow us to simultaneously determine the mRNA and protein expression levels in a single experiment.

  1. With the ongoing COVID-19 crisis, several groups are studying the response of the human immune system to the SARS-CoV-2 virus. What are your views on the current research in this area? What are the biggest challenges that need to be overcome to be able to find a vaccine?

The study of the immune response of patients infected with COVID-19 has proven to be challenging with a lot of surprises. The latest results seem to indicate that the antibody responses are not very persistent, and this may represent a problem for the development of an efficient vaccine. On the other hand, it seems that vaccines should boost potent T cell protective responses. This means that we also need the development of good tools to test the response of specific memory T cells in the vaccinated individuals.

Due to our expertise in the generation of monoclonal antibodies, we are currently trying to produce antibodies that could broadly neutralize not only the infection with COVID-19 virus but also other coronaviruses with the hope that these reagents can also be used as prophylactic tools against new emerging viruses of the same family. In any case, I am quite optimistic that during the next year, we will be able to have a much-needed vaccine that will end this global health problem.

 

More information about antibody validation :

-Kalina T, Lundsten K, Engel P. Relevance of Antibody Validation for Flow Cytometry. Cytometry A. 2020;97(2):126-136. doi:10.1002/cyto.a.23895

 

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