Foxp3⁺ regulatory T cells (Tregs) play essential roles in maintaining the immune balance. Although the majority of Tregs are formed in the thymus, increasing evidence suggests that induced Tregs (iTregs) may be generated in the periphery from naive cells. However, unlike in the murine system, significant controversy exists regarding the suppressive capacity of these iTregs in humans, especially those generated in vitro in the presence of TGF-β. Although it is well known that IL-10 is an important mediator of Treg suppression, the action of IL-10 on Tregs themselves is less well characterized. In this article, we show that the presence of IL-10, in addition to TGF-β, leads to increased expansion of Foxp3⁺ iTregs with enhanced CTLA-4 expression and suppressive capability, comparable to that of natural Tregs. This process is dependent on IL-10R–mediated STAT3 signaling, as supported by the lack of an IL-10 effect in patients with IL-10R deficiency and dominant-negative STAT3 mutation. Additionally, IL-10–induced inhibition of Akt phosphorylation and subsequent preservation of Foxo1 function are critical. These results highlight a previously unrecognized function of IL-10 in human iTreg generation, with potential therapeutic implications for the treatment of immune diseases, such as autoimmunity and allergy.

Rationale: Genetic variation influences susceptibility to clinical tuberculosis (TB). Activation of the P2X7 receptor on human macrophages induces killing of mycobacteria. We have identified polymorphisms in the P2X7 gene that markedly reduce this killing. Objective: To determine if polymorphisms in P2X7 are associated with increased risk of TB, the prevalence of four polymorphisms was assessed in individuals from Southeast Asia, where the majority of patients with TB in our study originate. The association of these polymorphisms with clinical TB was subsequently investigated in two separate case-control cohorts and the function of P2X7 was assessed in subjects from one cohort. Methods: Genotyping of P2X7 polymorphisms was performed from subjects in a nested case-control study of a longitudinal refugee cohort and a separate case-control study. The functional capacity of P2X7 was investigated by measuring ATP-mediated mycobacterial killing and apoptosis. Results: Only the 1513A-C polymorphism was present in Southeast Asians and the allele was associated with reduced killing of Mycobacterium tuberculosis. The 1513C allele was strongly associated with extrapulmonary, but not pulmonary, TB in the first (odds ratio, 3.8; 95% confidence interval, 1.6–9.0) and second cohorts (odds ratio, 3.7; 95% confidence interval, 1.7–8.0). ATP-mediated killing of mycobacteria was ablated in macrophages from subjects homozygous for the 1513C allele and significantly impaired in macrophages from heterozygous subjects. There was strong correlation between the degree of mycobacterial killing and ATP-induced apoptosis. Conclusions: The 1513C allele increases susceptibility to extrapulmonary TB, and this defect is associated with the reduction in the capacity of macrophages to kill M. tuberculosis.

The P2X ₇ receptor is an ATP‐gated cation channel expressed in immune cells and plays a role in proinflammatory cytokine release from monocytes and macrophages. This study investigated the coinheritance of 12 functionally relevant single nucleotide polymorphisms (SNPs) in the human P2X ₇ gene ( P2RX7 ), and the functional effect of each singly and in combination was assessed by measurements of ATP‐induced currents and ethidium ⁺ uptake. Genotyping of 3430 Caucasian subjects identified 4 common haplotypes in addition to the common (wild‐type) P2X ₇ ‐1. Two haplotypes (denoted P2X ₇ ‐2 and P2X ₇ ‐4) contained various combinations of gain‐of‐function SNPs. P2X ₇ ‐4 was identified uniquely by the Gln‐460 to Arg polymorphism (rs2230912). When expressed in HEK‐293 cells, recombinant P2X ₇ ‐2, and P2X ₇ ‐4 haplotypes displayed a 3‐fold and 5‐fold increase, respectively, in receptor function compared to the wildtype P2X ₇ ‐1. Both P2X ₇ haplotypes contained the Ala‐348>Thr polymorphism (rs1718119), and this mutation was critical for the gain‐of‐function effect. Peripheral blood monocytes and erythrocytes from subjects homozygous for gain‐of‐function P2X ₇ haplotypes exhibited increased ATP‐induced ethidium ⁺ uptake and ⁸⁶ Rb ⁺ efflux, respectively, and this correlated with increased IL‐1β secretion from LPS‐primed monocytes. Inheritance of these P2X ₇ haplotypes predisposing to increased proinflammatory cytokine secretion may be important in genetic association studies of inflammatory, infectious, and psychiatric disorders.—Stokes, L., Fuller, S. J., Sluyter, R., Skarratt, K. K., Gu, B. J., Wiley, J. S. Two haplotypes of the P2X ₇ receptor containing the Ala‐348 to Thr polymorphism exhibit a gain‐of‐function effect and enhanced interleukin‐1β secretion. FASEB J . 24, 2916–2927 (2010). www.fasebj.org

The P2X₇ receptor is a ligand-gated cation channel that is highly expressed on mononuclear leukocytes and that mediates ATP-induced apoptosis and killing of intracellular pathogens. There is a wide variation in P2X₇ receptor function between subjects, explained in part by four loss-of-function polymorphisms (R307Q, E496A, I568N, and a 5′-intronic splice site polymorphism), as well as rare mutations. In this study, we report the allele frequencies of 11 non-synonymous P2X₇ polymorphisms and describe a fifth loss-of-function polymorphism in the gene (1096C → G), which changes Thr³⁵⁷ to Ser (T357S) with an allele frequency of 0.08 in the Caucasian population. P2X₇ function was measured by ATP-induced ethidium⁺ influx into peripheral blood lymphocytes and monocytes and, when compared with wild-type subjects, was reduced to 10–65% in heterozygotes, 1–18% in homozygotes, and 0–10% in compound heterozygotes carrying T357S and a second loss-of-function polymorphism. Overexpression of the T357S mutant P2X₇ in either HEK-293 cells or Xenopus oocytes gave P2X₇ function of ∼50% that of wild-type constructs. Differentiation of monocytes to macrophages, which also up-regulates P2X₇, restored P2X₇ function to near normal in cells heterozygous for T357S and to a value 50–65% of wild-type in cells homozygous for T357S or compound heterozygous for T357S/E496A. However, macrophages from subjects that are compound heterozygous for either T357S/R307Q or T357S/stop codon had near-to-absent P2X₇ function. These functional deficits induced by T357S were paralleled by impaired ATP-induced apoptosis and mycobacteria killing in macrophages from these subjects. Lymphocytes, monocytes, and macrophages from subjects homozygous for T357S or compound heterozygous for T357S and a second loss-of-function allele have reduced or absent P2X₇ receptor function.

The P2X₇ receptor is a ligand-gated channel that is highly expressed on mononuclear cells of the immune system and that mediates ATP-induced apoptosis. Wide variations in the function of the P2X receptor have been observed, explained in part by ⁷loss-of-function polymorphisms that change Glu⁴⁹⁶ to Ala (E496A) and Ile⁵⁶⁸ to Asn (I568N). In this study, a third polymorphism, which substitutes an uncharged glutamine for the highly positively charged Arg³⁰⁷ (R307Q), has been found in heterozygous dosage in 12 of 420 subjects studied. P2X₇ function was measured by ATP-induced fluxes of Rb⁺, Ba²⁺, and ethidium⁺ into peripheral blood monocytes or various lymphocyte subsets and was either absent or markedly decreased. Transfection experiments showed that P2X₇ carrying the R307Q mutation lacked either channel or pore function despite robust protein synthesis and surface expression of the receptor. The monoclonal antibody (clone L4) that binds to the extracellular domain of wild type P2X₇ and blocks P2X₇ function failed to bind to the R307Q mutant receptor. Differentiation of monocytes to macrophages up-regulated P2X₇ function in cells heterozygous for the R307Q to a value 10–40% of that for wild type macrophages. However, macrophages from a subject who was double heterozygous for R307Q/I568N remained totally non-functional for P2X₇, and lymphocytes from the same subject also lacked ATP-stimulated phospholipase D activity. These data identify a third loss-of-function polymorphism affecting the human P2X₇ receptor, and since the affected Arg³⁰⁷ is homologous to those amino acids essential for ATP binding to P2X₁ and P2X₂, it is likely that this polymorphism abolishes the binding of ATP to the extracellular domain of P2X₇.