Phenotypic analysis of human peripheral blood regulatory T cells (CD4⁺FOXP3⁺CD127^lo/–) ex vivo and after in vitro restimulation with malaria antigens

Abstract

Regulatory T cells (Treg) play crucial roles in regulating autoimmune responses and immunity to tumors and infectious diseases. However, numerous subpopulations of Treg are now being described and the utility of various Treg markers is being reassessed. Here we report the results of a detailed phenotypic comparison of two supposedly regulatory human T-cell populations, namely CD4 1 FOXP31 T cells and CD4 (CCR), CD95 and Bcl-2, suggestive of distinct migration characteristics and susceptibility to apoptosis. Further, we propose that CD25 expression should be regarded as an activation marker rather than as a defining marker of Treg. Lastly, CD4 1 FOXP3 1 T cells activated in vitro with malaria antigen expressed the highest levels of CCR4 and CD95, and the lowest levels of CCR7, indicating that they are most likely generated from effector memory cells during an immune response and rapidly succumb to apoptosis at the end of the response. [7]. A plethora of different regulatory mechanisms has been identified (reviewed by [8]). With such a broad range of effects and mechanisms of action, it seems plausible that different subsets of natural Treg may exist. Indeed, it is increasingly recognised that human Treg are far more heterogenous than murine Treg [9], and that even CD4 [37]. Functionally distinct Treg subpopulations have also been identified on the basis of forkhead box P3 (FOXP3) expression intensity and CD45RA expression [10].We have previously reported that in areas of seasonal malaria transmission, Treg may expand alongside effector memory (EM) T cells when malaria exposure is high, and that both populations may contract once exposure decreases [38]; this contraction may involve apoptotic pathways. Treg have been shown to be highly susceptible to spontaneous cell death or cytokine-deprivation induced death [39]. As for conventional T cells, it has been shown that the majority of CD4 T cells with regard to the expression of CCR and apoptotic molecules and suggest that CD25, rather than being a marker of Treg per se, is a marker of activated Treg. Given our interest in Treg function during malaria infection [38,[44][45][46][47], we further aimed to study these markers in relation to Treg activation status in vitro, using Plasmodium falciparum schizont extract (PfSE). Importantly, we show that -irrespective of the culture conditions -the phenotype of FOXP3 1 T cells is modified when they are kept in in vitro culture, affecting both expression of CCR and molecules involved in apoptosis, indicating a need for caution when comparing data from in vitro and ex vivo studies. Results CD25 -a Treg-identifying marker or a marker of Treg activation?Whole blood from 28 adult Gambian donors was stained ex vivo and viable lymphocytes were gated for CD4 and CD25 expression.The double negative (CD4 -CD25 -) population was used to define the cut-off for CD4 and CD25 expression (Fig. 1A) (Fig. 2D) and expressing significantly higher levels of CD95 than the CD25 -or CD25 lo populations (Fig. 2E). However, importantly, both the l...

Regulatory T cells in malaria – friend or foe?

“…In line with this, we saw that T reg expressed high levels of CD95 (Fas) and low levels of the anti-apoptotic molecule Bcl-2 ex vivo [28], and CD95 expression on T reg increases in response to PfSE stimulation in vitro [34], suggesting that malaria-induced T reg are indeed prone to apoptosis.…”

CD4+CD25+Foxp3+Regulatory T Cells, Dendritic Cells, and Circulating Cytokines in Uncomplicated Malaria: Do Different Parasite Species Elicit Similar Host Responses?

“…Most studies have examined P. falciparum infections acquired under conditions of very intense transmission that prevail in Africa (3,13,27,48,49,50), and although experimental malaria models have revealed various patterns of cell activation and cytokine production according to the infecting parasite species and load (29), it remains unclear how humans regulate their immune responses when exposed to different malaria species and various transmission levels. Changes in peripheral populations of Treg cells (4,22) and DCs (22) during acute P. vivax malaria were recently characterized in Brazil and Thailand, but no data are available for P. falciparum infections in comparable settings of low endemicity.…”

Neutralization of Malaria GlycosylphosphatidylinositolIn Vitroby Serum IgG from Malaria-Exposed Individuals

“…The most obvious interpretation of these data, although it is perhaps unsurprising, is that PfSE contains other proinflammatory parasite molecules. Thus, effective antidisease immunity will probably be composed of strong neutralizing antibody responses to multiple parasite molecules as well as appropriately timed regulatory responses that moderate proinflammatory cytokine production by macrophages and other cell populations (17,26). Other parasite-derived molecules involved in the generation of proinflammatory immune responses during P. falciparum infection may include protein-DNA complexes (48) and hemozoin (11,41), which has been shown to activate the nacht domain-, leucine-rich repeat-, and Pyd-containing protein 3 (NALP3) inflammasome (41), trigger TLR9 signaling, and be an endogenous adjuvant in malaria-induced vaccination (47).…”