Notch ligands potentiate IL-7-driven proliferation and survival of human thymocyte precursors.

Notch and IL-7 are both well-characterized factors involved in T-cell development. In contrast to the mouse model, their precise requirements in the differentiation and/or proliferation of various stages of human thymic development have not been fully explored. Here, we demonstrate that IL-7 alone is sufficient to induce the differentiation of ex vivo purified CD34(+) triple negative (TN) surface (s) CD3(-) CD4(-)CD8(-) (CD3(-)CD4(-)CD8(-)), CD4 immature single positive (ISP) (sCD3(-)CD4(+)CD8(-)) and double positive (DP) (sCD3(-)CD4(+)CD8(+)) human thymic precursors to mature DP expressing sCD3 (sCD3(+)CD4(+)CD8(+)). We show that activation of Notch signaling by its ligands Delta-1 or Delta-4 potentiates IL-7-driven proliferation and survival of CD34(+) TN and to a lesser extent of CD4(+) ISP precursors. This effect of Notch is related to a sustained induction of IL-7 receptor alpha chain expression on thymocytes through a decreased methylation of its gene promoter. Thus, we show here that proliferation and differentiation of T-cell precursors are differentially modulated by IL-7 depending on the presence or absence of external signals. These results may have important implications for the clinical use of this cytokine as a strategy aimed at improving immune restoration.

Short exposure to Notch ligand Delta-4 is sufficient to induce T-cell differentiation program and to increase the T cell potential of primary human CD34+ cells.

OBJECTIVE: The Notch pathway plays a key role in cell fate choices and in T-cell development. The goal of our study was to evaluate whether a short in vitro stimulation of the Notch pathway may alter human progenitor cell behavior. METHODS: CD34+ cord blood progenitors were exposed for 4 days to either immobilized Notch ligand Delta-4 or in control conditions. Phenotypic and molecular changes induced by the short stimulation were assessed at day 4. Next, long-term alteration of the fate of these progenitors was assessed in culture conditions suitable for B (coculture with MS5 stromal cells) and T (FTOC and OP9 stromal cells expressing Delta-4 systems) cell differentiation. RESULTS: Notch activation was sufficient to trigger immunophenotypic and molecular changes consistent with early T-cell lineage differentiation. Delta-4 induced, in 4 days, CD7+cytCD3epsilon+ cells. This paralleled at the gene-transcription level with de novo expression of several T cell-related transcription factors and TCRgamma rearrangement, while B cell transcripts were simultaneous silenced. As compared to non-Delta-4 primed cells, these early changes translated to long-term alteration of the potential of cells. Delta-4 priming led to an acceleration of T-cell development, including a completion of the TCR rearrangement, when cells were cultured in systems suitable for T-cell development while B-cell development was inhibited. CONCLUSION: A transient Notch activation is sufficient to promote T-cell differentiation from cord blood CD34+ cells. This system may be a useful tool for the amplification and the quantification of the T potential of CD34+ cells in various disease conditions.

HIV-specific regulatory T cells are associated with higher CD4 cell counts in primary infection.

OBJECTIVE: Expansion of regulatory T (Treg) cells has been described in chronically HIV-infected individuals. We investigated whether HIV-suppressive Treg could be detected during primary HIV infection (PHI). METHODS: Seventeen patients diagnosed early after PHI (median: 13 days; 1-55) were studied. Median CD4 cell count was 480 cells/microl (33-1306) and plasma HIV RNA levels ranged between 3.3 and 5.7 log10 copies/ml. Suppressive capacity of blood purified CD4CD25 was evaluated in a coculture assay. Fox-p3, IL-2 and IL-10 were quantified by reverse transcriptase (RT)-PCR and intracellular staining of ex vivo and activated CD4+CD25 T cells. RESULTS: The frequency of CD4CD127CD25 T cells among CD4 T cells was lower in patients with PHI compared with chronic patients (n = 19). They exhibited a phenotype of memory T cells and expressed constitutively FoxP3. Similar to chronic patients, Treg from patients with PHI inhibited the proliferation of purified tuberculin (PPD) and HIV p24 activated CD4CD25 T cells. CD4CD25 T cells from patients with PHI responded specifically to p24 stimulation by expressing IL-10. In untreated patients with PHI, the frequency as well as HIV-specific activity of Treg decreased during a 24-month follow-up. A positive correlation between percentages of Treg and both CD4 cell counts and the magnitude of p24-specific suppressive activity at diagnosis of PHI was found. CONCLUSION: Our data showed that HIV drives Treg, as PHI and these cells persist throughout the course of the infection. A correlation between the frequency of Treg and CD4 T-cell counts suggest that these cells may impact on the immune activation set point at PHI diagnosis.

Human immunodeficiency virus-driven expansion of CD4+CD25+ regulatory T cells, which suppress HIV-specific CD4 T-cell responses in HIV-infected patients.

The present study demonstrates that CD4(+)CD25(+) T cells, expanded in peripheral blood of HIV-infected patients receiving highly active antiretroviral therapy (HAART), exhibit phenotypic, molecular, and functional characteristics of regulatory T cells. The majority of peripheral CD4(+)CD25(+) T cells from HIV-infected patients expressed a memory phenotype. They were found to constitutively express transcription factor forkhead box P3 (Foxp3) messengers. CD4(+)CD25(+) T cells weakly proliferated to immobilized anti-CD3 monoclonal antibody (mAb) and addition of soluble anti-CD28 mAb significantly increased proliferation. In contrast to CD4(+)CD25(-) T cells, CD4(+)CD25(+) T cells from HIV-infected patients did not proliferate in response to recall antigens and to p24 protein. The proliferative capacity of CD4 T cells to tuberculin, cytomegalovirus (CMV), and p24 significantly increased following depletion of CD4(+)CD25(+) T cells. Furthermore, addition of increasing numbers of CD4(+)CD25(+) T cells resulted in a dose-dependent inhibition of CD4(+)CD25(-) T-cell proliferation to tuberculin and p24. CD4(+)CD25(+) T cells responded specifically to p24 antigen stimulation by expressing transforming growth factor beta (TGF-beta) and interleukin 10 (IL-10), thus indicating the presence of p24-specific CD4(+) T cells among the CD4(+)CD25(+) T-cell subset. Suppressive activity was not dependent on the secretion of TGF-beta or IL-10. Taken together, our results suggest that persistence of HIV antigens might trigger the expansion of CD4(+)CD25(+) regulatory T cells, which might induce a tolerance to HIV in vivo.