infection and inflammation
Name: Professor Florian Kern
Academic position: Foundation Chair in Immunology
Research focus:
Background
My interest in peripheral blood T-cells developed when I was involved in the immune monitoring of transplant recipients and patients with severe bacterial sepsis at the Institute for Medical Immunology at the Charité in Berlin.
As part of the monitoring scheme we compiled profiles of peripheral blood lymphocytes by dividing them according to lineage and surface marker expression. This 'immunophenotyping' was done by flow-cytometry, initially with only three fluorescence parameters, reflecting the state of the art in the early/mid 1990s. As a service to the clinicians we interpreted these profiles and reported back to the wards.
At the time I felt that probably the biggest restriction in so doing was the fact that we had no means of establishing the antigen-specificity of the lymphocytes, mostly T-cell subsets, that we were looking at. Interpreting changes could have been so much more meaningful if we had known in response to what antigen(s) these changes took place.
Determining the antigen specificity of the cells we analysed appeared to be absolutely key to understanding these immune profiles better. Around 1996/1997, methods for detecting T-cells specific for selected antigens were pioneered in two different ways. First, the tetramer technology was established by John Altman’s lab in Atlanta, and, second, Louis Picker’s lab in Dallas reported a method exploiting intracellular cytokine staining for the detection and enumeration of T-cells that were specifically activated by the antigens chosen for stimulation.
The tetramer technology detects T-cells that recognise specific peptides in the context of recombinant multimeric MHC complexes, and thus is limited to known combinations of peptides and presenting MHC-molecules. Even today, these are mostly class-I MHC-molecules and as a result of this, it is mostly CD8 T-cells that can be detected. The intracellular cytokine staining approach at the time was based on stimulation with whole antigens or antigen lysates, such as a lysate of Cytomegalovirus-infected human fibroblasts.
Stimulating T-cells in such a way requires that the antigens are presented by so called antigen presenting cells that have to be able to uptake the antigens, digest them and present them in an MHC-context. Nature has provided for whole antigens that are internalised by these cells to be associated with class-II MHC molecules for the most part, which means that mostly CD4 helper T-cells are activated. These stunning new technologies were able to detect very select CD8 T-cells, or CD4 T-cells in an unselected fashion.
Past discoveries
Since we needed a way of assigning antigen-specificity to the T-cells we were analysing in daily routine work, both CD4 and CD8 T-cells had to be addressed. While using complete proteins and pathogen lysates for stimulation would have provided us with the means of probing our patient samples for CD4 T-cell responses to Cytomegalovirus for example, we had no means of probing for CD8 T-cell responses in a similar way, other than using all available tetramer reagents at the same time. This would have covered only a minute fraction of possible targets but would have been prohibitively expensive.
In order to solve this problem we tested if peptides (such as the ones used in tetramers) could be used for stimulation instead of whole antigens (Kern et al., Nature Medicine, 1998). We found that both CD4 and CD8 T-cells could be very efficiently stimulated with peptides with no disadvantage for CD4 T-cells compared to the use of complete antigens. This discovery was extended by the finding that peptides combined into peptide pools spanning complete proteins could be used to replace proteins for stimulation with the added advantage that CD4 and CD8 T-cell responses to protein antigens could be analysed simultaneously (Kern et al., Eur J Immunol, 2000).
This method formed the basis of a multitude of monitoring studies carried out over the last five years and the first study ever to fully analyse the extent of CD4 and CD8 T-cell responses to a complete viral proteome (Sylwester et al., J Exp Med, 2005). By introducing short-term peptide-based stimulation to flow-cytometry we have laid the groundwork for our current research projects.
Current projects
We are now using a wide array of antigen-specific approaches to analyse in depth the T-cell (and more recently) B-cell responses to a number of different pathogens, mainly M. tuberculosis, Cytomegalovirus, and Parvovirus B19.
Apart from looking at T-cell numbers or frequencies, we are very interested in the phenotypes of the antigen-specific T-cells. Whereas antigen-specific immunology has made enormous progress over the last ten years, we have still not reached the point where we can interpret an 'immunophenotyping' profile in a satisfactory way on a fully antigen-specific level. However, we can now look at antigen-specific T-cells and attempt to decipher the blueprint underlying their functional profiles (cytokine production) and surface molecule expression. This information can be used to 'classify' T-cells into compartments along the way from non-antigen experienced (naïve) to very antigen-experienced (terminally differentiated).
By using such information to make a clinical diagnosis we have created a new paradigm in infectious diseases immunology, stating that the characteristics of an antigen-specific T-cell will be able to aid us in the diagnosis of infectious (and potentially other) diseases or disease complications. We recently reported how the expression of a co-stimulatory surface molecule, CD27, on CD4 T-cells responding to tuberculin can be used in the diagnosis of active culture and/or smear positive tuberculosis (Streitz et al., 2007).
Current projects include the development of new diagnostic assays for active tuberculosis, the analysis of T-cell mediated immune responses in the elderly and the deterioration of the immune system in old age, T-cell monitoring in transplant patients, the analysis of the antigen-specificity of T-cells invading the myocardium in PVB10 associated inflammatory myocarditis, the ability of Cytomegalovirus specific T-cells to kill CMV-infected tumour (neuroblastoma) cells, and the recognition of linear peptide epitopes by B-cells in tuberculosis, PVB19 infection and Dengue virus infection.
The research team
At BSMS:
Dr. Stephan Fuhrmann, Postdoctoral fellow
Ms. Marion Kaspari, PhD student
Ms. Paola Lanuti, PhD student
At Charite Berlin
Mr. Mathias Streitz (PhD student)
Ms. Raskit Lachmann (PhD student)
Collaborations
Prof. Helen Smith, BSMS
Prof. Tony Frew, BSMS
Dr. Melanie Newport, BSMS
Dr. Martin Llewelyn, BSMS
Dr. Simon Barry, Portsmouth
Prof. Hans-Dieter Volk, Berlin
Prof. Petra Reinke, Berlin
Dr. Rudolf Volkmer, Berlin
Dr. Constanze Schönemann, Charité, Berlin
Dr. Holger Wenschuh, JPT Peptide Technologies, Berlin/Germany
Dr. Mike Schutkowski, JPT Peptide Technologies, Berlin/Germany
Dr. Ali Quassem, Lungenklinik Lostau, Lostau/Germany
Prof. Gerd Liebetrau, Lungenklinik Lostau, Lostau/Germany