No abstract

The CD31 ؉ subset of human naive CD4 ؉ T cells is thought to contain the population of cells that have recently emigrated from the thymus, while their CD31 ؊ counterparts have been proposed to originate from CD31 ؉ cells after homeostatic cell division. Naive T-cell maintenance is known to involve homeostatic cytokines such as interleukin-7 (IL-7). It remains to be investigated what role this cytokine has in the homeostasis of naive CD4 ؉ T-cell subsets defined by CD31 expression. We provide evidence that IL-7 exerts a preferential proliferative effect on CD31 ؉ naive CD4 ؉ T cells from adult peripheral blood compared with the CD31 ؊ subset. IL-7-driven proliferation did not result in loss of CD31 expression, suggesting that CD31 ؉ naive CD4 ؉ T cells can undergo cytokine-driven homeostatic proliferation while preserving CD31. Furthermore, IL-7 sustained or increased CD31 expression even in nonproliferating cells. Both proliferation and CD31 maintenance were dependent on the activation of phosphoinositide 3-kinase (PI3K) signaling. Taken together, our data suggest that during adulthood CD31 ؉ naive CD4 ؉ T cells are maintained by IL-7 and that IL-7-based therapies may exert a preferential effect on this population. (Blood. 2009;113: 2999-3007) IntroductionHuman naive CD4 ϩ T cells have recently been shown to contain 2 subpopulations distinguished by the expression of CD31 (platelet endothelial cell adhesion molecule-1, PECAM-1). The CD31 ϩ subset is thought to incorporate the population of cells recently emigrated from the thymus, whereas the CD31 Ϫ subset has been proposed to derive from CD31 ϩ after homeostatic cell division. 1 During T-cell development in the thymus, rearrangement of the T-cell receptor (TCR) genes generates stable episomal DNA excision circles (TRECs) that are progressively diluted with cell division. 2-4 Accordingly, CD31 ϩ naive CD4 ϩ T cells have higher TREC content compared with the CD31 Ϫ naive subset. 1 Moreover, the progressive age-associated decline in naive CD4 ϩ T cells is mainly due to a reduction in the CD31 ϩ naive subset while the CD31 Ϫ subset persists, 5,6 further supporting the contribution of thymic output to the maintenance of CD31 ϩ cells. However, the decrease in TREC levels observed during aging is disproportionally greater compared with the decline in CD31 ϩ naive T cells, implicating other mechanisms, in addition to thymic output, in the persistence of these cells into old age. 4 Cytokine-driven expansion has been proposed to significantly contribute to a low level of homeostatic proliferation that maintains naive T-cell numbers. 7 Besides its established importance in thymopoiesis, interleukin-7 (IL-7) is considered to play a key role in naive T-cell survival and proliferation in the periphery. 2,7 In vitro studies of human naive CD4 ϩ T cells cultured in the presence of IL-7 revealed, alongside with its antiapoptotic properties, an ability to induce proliferative responses without a switch to a memory phenotype. 8 IL-7 seems to exert a preferential effect on umbilic...

© F e r r a t a S t o r t i F o u n d a t i o npermeabilized in ice-cold PERM buffer III, washed in staining buffer and stained with the following antibodies: CD79a-APC; Akt-Alexa Fluor 488, PTEN-PE, phospho-Akt (S473)-Alexa Fluor 488, phospho-Akt (T308)-PE, and phospho-STAT5 (Y694)-Alexa Fluor 488. Samples were analyzed on a FACSAria or LSRFortessa using the gating strategy indicated in the Online Supplementary Appendix (Online Supplementary Figure S1). Endogenous PTEN in vitro lipid phosphatase assayPTEN phosphatase activity was measured in vitro as previously described. 13Endogenous CK2 in vitro kinase assay CK2 activity was measured in vitro as previously described. 15 Treatment with signaling inhibitorsCells were cultured in control medium or in the presence of CX-4945 or LY294002 for the indicated time points and used for protein and viability analysis. ImmunoblottingCell lysates were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and immunoblotted with the antibodies against actin, phospho-PTEN (S380), PTEN, CK2α and CK2α'. Analysis of cell viability and apoptosisCell viability was determined by double-staining with APC or FITC-conjugated Annexin V and propidium iodide (PI) and flow cytometry analysis, as previously described. 16 Statistical analysisDifferences between populations were calculated using unpaired two-tailed Students's t-test or Mann-Whitney test, as appropriate. Correlations were analyzed using the Pearson correlation coefficient. P<0.05 was considered significant. Results JAK/STAT and PI3K/Akt pathways are hyperactivated in adult B-ALL cellsHyperactivation of signaling pathways involved in promotion of proliferation and survival is commonly associated with cancer progression. Previous studies have shown that one of these signaling cascades, the JAK/STAT pathway, is constitutively activated in B-ALL patients displaying the BCR-ABL fusion (also known as Philadelphia chromosome (Ph)-positive cases) or CRFL2 overexpression in combination or not with activating mutations in JAK1 and JAK2. 3,17 Using phospho-specific flow cytometry and a gating strategy that enabled us to focus on blast cells (Online Supplementary Figure S1) to compare primary bone marrow cells from healthy donors with B-ALL blasts collected from leukemia patients at diagnosis (Table 1), we con-PI3K/Akt pathway activation in adult ALL haematologica | 2014; 99(6) 1063 TdT+, cCD79a+,CD19+, TdT+,CD10+, cCD79a+,CD19+, TdT+,cyIgM+, © F e r r a t a S t o r t i F o u n d a t i o n firmed that adult leukemia cases displayed constitutive hyperactivation of the JAK/STAT pathway ( Figure 1A; Online Supplementary Figures S1 and S2). Moreover, we found a discrete subgroup of samples that presented very high levels of STAT5 phosphorylation. In line with the knowledge that BCR-ABL drives STAT5 activation, 17,18 this group was enriched in Ph-positive cases ( Figure 1A, red labels).In contrast to JAK/STAT pathway, the activation status of PI3K/Akt signaling pathway and its potential role in adult ALL remain less well...

IgAdef, B-cell defects were mainly restricted to surface membrane (sm)IgA 1 PCs and MBCs, with 2 clear subgroups showing strongly decreased numbers of smIgA 1 PCs with mild versus severe smIgA 1 MBC defects and higher frequencies of nonrespiratory tract infections, autoimmunity, and affected family members. Patients with IgG subclass deficiency with IgA deficiency and those with CVID showed defects in both smIgA 1 and smIgG 1 MBCs and PCs. Reduced numbers of switched PCs were systematically found in patients with CVID (absent in 98%), with 6 different defective MBC (and clinical) profiles: (1) profound decrease in MBC numbers; (2) defective CD27 1 MBCs with almost normal IgG 3 1 MBCs; (3) absence of switched MBCs; and (4) presence of both unswitched and switched MBCs without and; (5) with IgG 2 1 MBCs; and (6) with IgA 1 1 MBCs. Conclusion: Distinct PAD defective B-cell patterns were identified that are associated with unique clinical profiles. (J

Naïve FoxP3-expressing regulatory T-cells (Tregs) are essential to control immune responses via continuous replenishment of the activated-Treg pool with thymus-committed suppressor cells. The mechanisms underlying naïve-Treg maintenance throughout life in face of the age-associated thymic involution remain unclear. We found that in adults thymectomized early in infancy the naïve-Treg pool is remarkably well preserved, in contrast to conventional naïve CD4 T-cells. Naïve-Tregs featured high levels of cycling and pro-survival markers, even in healthy individuals, and contrasted with other circulating naïve/memory CD4 T-cell subsets in terms of their strong γc-cytokine-dependent signaling, particularly in response to IL-7. Accordingly, ex-vivo stimulation of naïve-Tregs with IL-7 induced robust cytokine-dependent signaling, Bcl-2 expression, and phosphatidylinositol 3-kinase (PI3K)-dependent proliferation, whilst preserving naïve phenotype and suppressive capacity. Altogether, our data strongly implicate IL-7 in the thymus-independent long-term survival of functional naïve-Tregs, and highlight the potential of targeting the IL-7 pathway to modulate Tregs in different clinical settings.