High Efficiency Isolation of Alloantigen-Reactive Foxp3+ Natural Regulatory T Cells According to Specific Activation Marker Signatures

Abstract

1915 Transplant tolerance induction and avoidance of exposure to destructive immunosuppressive drugs are still major objectives in transplantation medicine. Many preclinical animal models have proven the adoptive transfer of polyclonal CD4+CD25+Foxp3+ regulatory T (Treg) cells to be an important and effective tool for the prevention of graft rejection or graft-versus-host-disease. However, polyclonal Treg may also modulate immunity to foreign or tumor antigens. It is unclear yet, whether and how their application might lead to an unwanted general immunosuppression. Therefore the selective transfer of alloantigen-reactive Treg represents a very attractive therapeutic option. However, so far this strategy has been hampered so far by the lack of appropriate marker to assess and isolate antigen-reactive Treg cells with high efficiency. In order to get access to alloantigen-reactive Treg, we established cytometric methodologies allowing a clear dissection between (allo)antigen-specific Treg and Teff based on expression of 4-1BB and the lack of expression of CD40L. After allogeneic stimulation CD4+ T cells expressing 4-1BB but lacking CD40L expression were highly enriched in Foxp3+ T cells. Further molecular analysis of the Foxp3 gene locus revealed that only 4-1BB+CD40L− T cells were characterised by a completely demethylated TSDR (Treg specific demethylated region). 4-1BB+CD40L− T cells highly expressed the transcription factor HELIOS recently demonstrated to specify thymic-derived Treg. Alloantigen-reactive Treg isolated according to 4-1BB and CD40L were superior with respect to their alloantigen-specific in vitro suppression as compared their polyclonal Treg counterparts. Finally 4-1BB+CD40L− alloantigen-reactive Treg could be easily expanded in vitro up to 300 fold during 3 weeks culture, maintaining alloantigen-specific suppression. Our results offer the possibility to improve current approaches for adoptive cell-therapy with alloantigen-specific Treg to achieve transplantation tolerance aiming at a specific inhibition of pathology. Disclosures: Scheffold: Miltenyi Biotec GmbH: Employment.

Concordance of B and T cell responses to SARS-CoV-2 infection, irrespective of symptoms suggestive of COVID-19

“…Although IgG Spike antibody levels decline over time after natural infection, individuals were assessed on two occasions; categorisation in this study was based on contemporaneous collection of PBMCs and serology. Our laboratory methods included externally validated and published antibody testing methodology (13); and we parsed T helper and T regulatory cell responses using previously published methodologies (15,22). However, our sample size is small (in part due to travel restrictions) and predominantly female (reflecting the nature of the TwinsUK cohort).…”

“…Samples were acquired on a LSRFortessa Flow Analyser (BD Biosciences) and analysed using the software FlowJo (TreeStar Inc., version 10.7.2). As previously described, we defined activated conventional helper T cells based on the expression of CD69 and CD40L (CD154) and activated regulatory T cells based on upregulation of 4-1BB (CD137) and GARP in CD40L negative cells (15)(16)(17).…”