Fig. 1

The multi-tetramer approach is sensitive enough to detect antigen-specific CD4+ T cells in healthy controls. a Representative flow plots depicting the gating strategy for CD4+ T cells reactive to influenza (left) and citrullinated CILP/FGB peptides (right). b Frequency of antigen-specific CD4+ T cells is shown for seven healthy controls (different symbols and shades of grey for each buffy coat). Plotted are tetramer-positive cells per million CD4+ T cells from all fourteen experiments (one technical replicate per healthy control) for influenza, citrullinated CILP/FGB and citrullinated α-enolase. Cut-off for positivity is one tetramer-positive cell per million CD4+ T cells, marked with a dotted line. c + d Characterisation of antigen-specific CD4+ T cells by differentiation status, determined by simultaneous or singular expression of CD45RA and CCR7 according to Sallusto et al [22] in naïve (Tnaïve), central memory (Tcm, coloured in red), effector memory (Tem, coloured in salmon) and CD45RA+ effector memory (Temra) T cells. We plotted the proportion of influenza- and citrulline-specific T cells among the four different phenotypes in (c) box plots showing the mean distribution and (d) scatter plots showing the detailed proportion and distribution of influenza- (left, open symbols) and citrulline-specific (right, closed symbols) T cells among the different phenotypes