The expansion of the CD8+CD28− Treg population in both the PB or SF of RA(MTX) patients was similar to the reported increase in CD4+ Tregs in RA patients [9]. We confirmed the findings from a previous report [8] that CD8+CD28− Treg numbers correlate with age. Indeed, the expansion may simply highlight the accelerated immune ageing in RA patients resulting in terminally differentiated T cells lacking CD28 expression [10]. In the synovial fluid this growth may be accelerated further

by the local cytokine milieu, where high local concentrations of IL-7 and IL-15 promote CD8+CD28− growth [11], while high TNF-α concentrations abrogate Small molecule library CD28 transcription [12]. The inability of ex-vivo RA(MTX) CD8+CD28− Treg to suppress activation of autologous responder cells raised three questions: (i) what is the mechanism of action of this particular Treg; (ii) are RA(MTX) CD8+CD28− capable of suppressing healthy allogeneic responder cells; and (iii) would the addition of TNFi in vitro or in vivo restore their

function? TW cultures established that HC and RA(TNFi) CD8+CD28− Treg, in contrast to CD4+CD25+ Treg [13], required little or no direct responder cell contact, suggesting that soluble mediators were the dominant mode of action. IL-10 is a critical mediator for CD8+ Tregs [14]. IL-10 was detected at significantly higher levels in Arachidonate 15-lipoxygenase RA(MTX) compared with HC CD8+CD28− Treg cultures, therefore we hypothesized that this may be due partially to defective uptake and signalling AG-014699 concentration by IL-10 in the RA(MTX) cells. Indeed, we show evidence that IL-10R is not up-regulated to the same extent by activated RA(MTX) as it is on HC T cells. This may be exacerbated by the

concomitant low expression of ICOS CD8+CD28− Treg in RA(MTX), which stabilizes IL-10R [15]. In contrast, IL-10 levels were reduced compared with HC in anti-CD3 antibody stimulated RA(TNFi) CD3+CD8+CD28−Treg cultures. An explanation for this finding may be the counter-regulation between IL-10 and TNF-α. IL-10 production requires the initial presence of TNF-α but IL-10 regulates the stability of TNF-α mRNA [16]. Inconsistent inhibition of suppression, using neutralizing anti-IL-10, may be due to IL-10 gene polmorphisms that relate to high/low IL-10 production [17]. Less variable results may be obtained by blocking the IL-10 receptor. In addition to IL-10, it has been reported that TGF-β is critical for both CD4+ and CD8+ Treg suppressor function; we show that blocking TGF-β in vitro reduces suppression of responder PBMC proliferation by CD8+ CD28− Treg. Further analysis of this mechanism will be explored in future studies. In addition, all activated CD8+CD28− Treg cultures produced high levels of IFN-γ similar to that produced by CD4+CD28− T cells [18].

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