Heart transplant rejection is a common occurrence, affecting 24% of patients within a year of the procedure. Acute cellular rejection is behind most cases, occurring when antigens on the allograft trigger a response from cytotoxic T cells. Less clear is the mechanism behind antibody-mediated rejection, which involves donor-specific antibodies binding to target antigens and activating the immune system.
We interviewed Dr. Patrick Bruneval, Head of Pathology at the Georges Pompidou European Hospital in Paris to learn more about his research in cardiac allograft rejection and how he and his collaborators incorporate multiplex immunofluorescence methods to better understand the cardiac rejection process.
Patrick, along with Dr. Marion Rabant, Associate Professor, Pathology at the Necker-Enfants Malades Hospital, will also speak during an upcoming webinar on Jun 22. They will demonstrate a novel in situ detection strategy, involving multiplex immunofluorescence, to better characterize the inflammatory burden during kidney and cardiac allograft rejection. Register here.
How did you develop your interest in transplantation pathology?
The cardiac transplantation program started in 1984 in my institution while I was young pathologist in charge of cardiovascular pathology. Cardiac transplant pathology was an opportunity to extend the scope of cardiovascular pathology at the interface of transplant immunology and patient management. I received training for the diagnosis of cardiac rejection on endomyocardial biopsies based only on H&E in an era when only cellular rejection was recognized. New fascinating developments came in my late twenties with the recognition of a new form of rejection, antibody-mediated rejection (AMR), with progress in immunology and immunopathology to detect AMR, and with the input of molecular pathology to characterize the molecular signatures of rejection.
What methods are researchers currently using to study cardiac rejection? What are the gaps in those methods?
Current knowledge on cardiac rejection comes from combination of sensitive techniques in immunology to detect antibodies, molecular techniques to analyze the transcripts from cardiac biopsies and skilled pathology using criteria defined by international working formulations and using immunohistochemistry. After years of unrestrained progress in these techniques working one by one, it is time to realize that the techniques should be used in an integrative approach and closely linked to clinical need.
The most demonstrative gap between the interpretations of techniques in my opinion is that between molecular analysis and histopathology, with both working on tissues obtained from cardiac biopsies. But the molecular biologists are working on a dedicated frozen biopsy bite not assessed by histology, and the pathologists are doing the job on other biopsy bites using less sensitive tools. It is now time to combine these two techniques as companion techniques to improve accuracy of rejection diagnosis and to work on the same tissue bites, performing sensitive histopathology and new molecular techniques feasible on paraffin embedded tissues.
Image acquisition and analysis with Vectra and InForm allowed us to obtain reliable quantitative data from a large volume of images and characterize the cellular profiles of immune cells.
Tell us about your work. How does multiplex immunofluorescence (mIF) broaden our understanding of the cardiac alloimmune response?
In a recent study (Am J Transplant 2015;15:526-34), we determined on endomyocardial biopsies the quantity of immune cells (CD3-T-lymphocytes, CD68-macrophages, CD20-B-lymphocytes, CD138-plasma cells) recruited in antibody-mediated rejection as compared to non-rejection biopsies. The immune cells were quantified according to their location, intra-or extravascular, as determined by plain microscopic observation. This study was conducted by using single labeling with immunoperoxidase technique, on as many paraffin-sections as necessary for all the cell markers. Multiplex immunofluorescence was the mandatory next step to analyze cell subsets engaged in cardiac rejection, allowing first, a more precise intra/extravascular location by using CD34, an endothelial cell marker, to identify the microvessels.
Second, the immune cells tested were detected and quantified on a unique paraffin section, allowing us to observe and analyze the actual distribution of cells while maintaining the spatial distribution of cells. Thus, we obtained a more precise and integrated quantification of immune cells and determined their compartmentalization in both cellular and antibody-mediated rejections.
Dr. Bruneval’s team used inForm image analysis software, along with the Vectra imaging system to study the cardiac transplant rejection process
Why did you choose the Vectra/Opal technology for your study?
Given that molecular techniques detect transcripts from several cell types which are used to characterize molecular profiles of rejection type (cellular or humoral rejection) or a profile of no-rejection, it is mandatory to link these molecular profile to cells visible in a cardiac biopsy under the microscope. This can be done under the condition that cells can be detected by a sensitive labeling technique, allowing the characterization of several cell types at a time on the same tissue section. Opal technology met the need in our experiments to perform labeling of T-lymphocytes, macrophages, NK cells and endothelial cells. Image acquisition and analysis with Vectra and the InForm program, respectively, allowed us to obtain reliable quantitative data from a large volume of images and to characterize the cellular profiles of immune cells depending on rejection status. We were thus able to link our immune cell profiles to the transcript profiles obtained in our laboratory.
Interested in learning more about multiplex immunofluorescence for allograft transplantation research? View this on-demand webinar to hear Dr. Patrick Bruneval and Dr. Marion Rabant discuss how multiplex immunofluorescence can advance our understanding of rejection processes.
