Towards a T cell subset that can control Mycobacterium tuberculosis infection
Led by Dr Munyaradzi Musvosvi (University of Cape Town, South Africa)
Project Aims
It is clear that T cells play a critical role in the control of Mycobacterium tuberculosis (M.tb) infection, however, it is not known to what degree certain T cell attributes contribute to the outcome of M.tb infection. Recently, we observed that M.tb-infected persons who controlled the bacterium and did not progress to active disease had much higher abundance of a type of T cell population bearing a particular antigen-receptor compared to persons who progressed to active disease. We propose that, if targeted by vaccination, this T cell subset may afford protective immunity against TB. However, a more complete understanding of this T cell subset is needed to determine its function, the bacterial components that it targets and to what extent it can be targeted by vaccination. To achieve this, we will develop and optimise new research tools that identify and isolate this T cell clone. In this project we will exploit the conserved nature of the antigen-receptor sequence expressed by this T cell clone to develop molecular probes, monoclonal antibodies, and TCR tetramers to study this T cell clone and identify the cognate antigen(s) that it recognises. The tools we will develop as part of this project will be critical to better understand the nature of this T cell clone and determine to what extent this clone can be targeted by vaccines.
Project Outcomes
The project aimed to replicate a finding that a public TCR was enriched in IGRA+ persons who did not progress to disease during a two year follow up (i.e. controllers) compared to IGRA+ persons who developed disease during follow up (i.e. proprogressors). We also observed that healthy IGRA+ persons had higher frequencies of this public TCR compared to persons with active TB. We termed this CDR3a sequence as kiif.tb. To achieve our objective of replicating our initial findings, we employed a digital PCR (dPCR) approach targeting the kiif.tb CDR3a sequence. The dPCR approach was used because we reasoned that it would be a cost-effective alternative to bulk TCR sequencing. Initially, in a subset of samples that had previously been analysed using the bulk TCR sequencing, we observed a modest correlation between the dPCR and the bulk TCR sequencing results. However, when we extended our analysis to an independent group of healthy IGRA+ persons and persons and persons with active TB, we were unable to replicate the findings observed in the initial cohort. It appeared as if both groups had similar frequencies of kiif.tb expressing T cells measured by dPCR.
The question arises whether the dPCR approach was the most suitable approach to measure kiif.tb. We are exploring alternative methodologies to measure and characterise T cells expressing this public TCR. This includes the PrimeFlow assay, an in-situ RNA hybridisation approach compatible with flow cytometry. Our custom PrimeFlow assay targets the public CDR3a sequence expressed by the clone of interest. Another aspect of this project was to test whether monoclonal antibodies could be generated against the CDR3a sequence of this public TCR. Mice were immunized with the CDR3a sequences of the public TCR, and we are now testing whether any of the monoclonal antibodies bind to a Jurkat cell line expressing the kiif.tb TCR. Overall, this project involved a various investigative approaches to measure the frequencies and phenotype of kiif.tb expressing T cells. These efforts will furthering our understanding of the role of kiif.tb expressing T cells in tuberculosis.
