The dysregulation of self-tolerance in the immune system leads to the generation of autoimmune diseases (AID), but the precise pathogenesis of various AID remains unclear. Autologous haematopoietic stem cell transplant (AHSCT) is an effective therapeutic modality for selected patients with aggressive forms of AID such as severe Systemic Sclerosis  and is also capable of suppressing CNS inflammatory activity and inducing sustained disease control in patients with aggressive Multiple Sclerosis. Therefore, polychromatic flow cytometry during AHSCT provides a unique opportunity to investigate immune deregulation in humans. 

   We designed four complex flow cytometry panels with 9 to 14 colours including  differentiation markers, chemokine receptors and trafficking markers, and tested  them using PBMC from 6 normal adult controls. These panels successfully define numerous characterised subpopulations in PBMC including pro-inflammatory disease-causing subsets such as CCR6⁺CD161^high mucosal associated invariant T (MAIT) cells, as well as suppressive disease-stabilising subsets such as Tregs, combined with cell surface integrins that direct trafficking to differing tissue compartments. The panels have begun to be applied to longitudinal cryopreserved PBMC samples from MS patients undergoing AHSCT. Results will be analysed cross-sectionally between patients and healthy controls, and longitudinally within successfully treated patients.

   The subpopulations detectable by these panels will allow for the characterisation of changes associated with immune reconstitution following AHSCT in autoimmune disease patients. Overall, this study will help to understand the mechanisms underlying AHSCT clinical effectiveness and lead to further improvements of this therapy.