Ram Kumar Venigalla

Reduced CD4+,CD25− T cell sensitivity to the suppressive function of CD4+,CD25high,CD127−/low regulatory T cells in patients with active systemic lupus erythematosus

OBJECTIVEnCD4+,CD25high regulatory T (Treg) cells play a crucial role in the maintenance of self tolerance and prevention of organ-specific autoimmunity. The presence of many in vivo-preactivated CD4+,CD25++ T cells in patients with systemic lupus erythematosus (SLE) poses a difficulty in discriminating CD25++ activated T cells from CD25high Treg cells. To overcome this problem, we analyzed the phenotype and function of CD4+,CD25high,CD127(-/low) natural Treg (nTreg) cells isolated from the peripheral blood of patients with SLE.nnnMETHODSnCD4+,CD25high,CD127(-/low) nTreg cells and CD4+,CD25- responder T (Tresp) cells from patients with SLE and normal donors were separated by fluorescence-activated cell sorting. Cell proliferation was quantified by 3H-thymidine incorporation, and immunophenotyping of the cells was done using FACScan.nnnRESULTSnComparable percentages of CD4+,CD25high,FoxP3+ T cells were observed in patients with SLE and normal donors. Proliferation of SLE nTreg cells sorted into the subset CD4+,CD25high,CD127(-/low) was significantly decreased compared with that of SLE nTreg cells sorted into the subset CD4+,CD25high (mean +/- SEM 2,223 +/- 351 counts per minute versus 9,104 +/- 1,720 cpm, respectively), while in normal donors, these values were 802 +/- 177 cpm and 2,028 +/- 548 cpm, respectively, confirming that effector cell contamination was reduced. Notably, the suppressive activity of nTreg cells was intact in all groups. However, CD4+,CD25- Tresp cells isolated from patients with active SLE were significantly less sensitive than those from patients with inactive SLE to the suppressive function of autologous or normal donor CD4+,CD25high,CD127(-/low) nTreg cells. Furthermore, a significant inverse correlation was observed between the extent of T cell regulation in suppressor assays and the level of lupus disease activity.nnnCONCLUSIONnThis study is the first to show that, in human SLE, impaired sensitivity of Tresp cells to the suppressive effects of a comparably functional, highly purified nTreg cell population leads to a defective suppression of T cell proliferation in active SLE. Studies aiming to define the mechanisms leading to Tresp cell resistance might help in the development of highly specific, alternative immunotherapeutic tools for the control of systemic autoimmune diseases such as SLE.

Induction of CD4 + T-cell anergy and apoptosis by activated human B cells

B cells are well-known mediators of humoral immunity and serve as costimulators in the generation of T cell-mediated responses. In several mouse models, however, it was observed that B cells can also down-regulate immune reactions, suggesting a dual role for B cells. Due to this discrepancy and so far limited data, we directly tested the effects of primary human B cells on activated CD4(+) T helper cells in vitro. We found that under optimal costimulation large, activated CD25(+) B cells but not small CD25(-) B cells induced temporary T-cell anergy, determined by cell division arrest and down-regulation of cytokine production. In addition, large CD25(+) B cells directly induced CD95-independent apoptosis in a subpopulation of activated T cells. Suppression required direct B-T-cell contact and was not transferable from T to T cell, excluding potential involvement of regulatory T cells. Moreover, inhibitory effects involved an IL-2-dependent mechanism, since decreasing concentrations of IL-2 led to a shift from inhibitory toward costimulatory effects triggered by B cells. We conclude that activated CD25(+) B cells are able to costimulate or down-regulate T-cell responses, depending on activation status and environmental conditions that might also influence their pathophysiological impact.

Impaired suppressive capacity of activation-induced regulatory B cells in systemic lupus erythematosus.

B cells with immunoregulatory properties (Breg cells) have been described in mice, but their role in the control of human immune responses is not well defined. We recently identified a human population of activated FSChigh B cells that exhibited regulatory activity toward T helper cells. The aim of the present study was to test such induced Breg (iBreg) cells in patients with autoimmune disease.

Identification of a human Th1-like IFNγ-secreting Treg subtype deriving from effector T cells

Characteristics and function of effector T-cells with regulatory properties (induced Treg, iTreg) in humans are ill defined. Here we report that a proportion of activated, initially CD4(+)CD25(-)CD127(+) effector T-cells from human peripheral blood can convert into T-cells with regulatory activity while concomitantly secreting IFNγ. Upon short-term culture in vitro these cells expressed a panel of common Treg markers, including FOXP3, CD25, GITR, HLA-DR and CTLA-4 in parallel with the Th1-specific transcription factor T-bet. Despite their own IFNγ secretion they effectively suppressed IFNγ secretion in effector T cells in parallel with inhibition of their proliferation. Highly purified IFNγ(+)iTreg shared many functional properties with nTreg: Their suppressive activity was antigen-independent, contact-mediated and cytokine-independent. Of note, in contrast to nTreg an inhibitor of TGF-β1 signalling promoted the proliferation of IFNγ(+)iTreg, without abrogating their suppressive function. In addition in vivo in tonsils of patients with chronic tonsillitis an IFNγ-secreting subpopulation of the CD4(+)CD25(-)CD127(+)CD45RA(-) memory T helper cell population was detected, which exhibited regulatory properties as well. Our results support the existence of Th1-like adaptive Tregs in humans that express a robust regulatory phenotype, comparable to nTreg and at the same time share characteristics of Th1 cells. According to our in vitro data IFNγ(+)iTreg can emerge from activated effector T cells and downregulate Th1-mediated immune responses, supporting the hypothesis of effector T cell plasticity as a means for proper initiation and self regulation of inflammatory processes. This report characterizes a new subpopulation of human adaptive regulatory T-cells that derive from effector Th-cells and concomitantly express Th1-specific T-bet and IFNγ with Foxp3.

Analysis of FOXP3+ regulatory T cell subpopulations in peripheral blood and tissue of patients with systemic lupus erythematosus

Regulatory T cells (Tregs) are critical mediators of immune tolerance, yet their involvement in the autoimmune disease systemic lupus erythematosus (SLE) is incompletely understood. We analyzed CD4+ T cell subpopulations with Treg-related phenotypes and their association with disease activity in peripheral blood (PB) and tissues of patients with SLE. In detail, we quantified subpopulations regarding CD25, FOXP3, CD62L, CCR6, CD27, CD45RA, and CD45RO expression in PB from 31 patients with SLE divided into two disease activity groups and 32 healthy controls using flow cytometry. CD4+ and FOXP3+ T cells in skin and kidney biopsies of patients with SLE were quantified by immunohistochemistry. CD4+CD25+/++FOXP3+ and CD4+CD25+CD45RA−/CD45RO+ T cell frequencies were significantly higher in PB from patients with active compared to inactive SLE. The fraction of CD4+CD25++FOXP3+ Tregs and CD4+CD25+CD45RA+/CD45RO− naïve Tregs was not significantly different between these groups. CD4+CD25++ Tregs from active SLE patients comprised significantly less CD27+ cells and more CCR6+ cells compared to patients with inactive SLE. The percentage of CD4+FOXP3+ T cells among inflammatory infiltrates in skin and kidney biopsies of SLE patients was not different from other inflammatory skin/kidney diseases. In conclusion, although CD4+FOXP3+ T cell frequencies in the inflamed tissues of SLE patients were comparable to other inflammatory diseases, distinct T cell subpopulations appeared misbalanced in PB of patients with active SLE. Here, cells phenotypically resembling activated T cells, but not Tregs, were increased compared to patients with inactive SLE. Within Tregs of patients with active SLE, markers related to Treg function and homing were altered.