Tresp Cells Research Articles

Signaling through TLR7 enhances the immunosuppressive activity of murine CD4+CD25+ T regulatory cells

Although signaling through certain TLRs is known to modulate the function of T lymphocytes, the effect of TLR7 stimulation on CD4(+)CD25(+) T(reg) cell activity has not yet been elucidated. In this study, we show that mouse CD4(+)CD25(+) T(reg) cells express TLR7 mRNA and protein. We therefore used the TLR7 agonists imiquimod, gardiquimod, and single-stranded poly(U) to show that TLR7 stimulation enhanced the ability of murine T(reg) cells to suppress anti-CD3/anti-CD28 mAb-coated bead-stimulated proliferation of syngeneic CD4(+)CD25(-) T(resp) cells. In contrast, imiquimod failed to enhance the suppressor function of T(reg) cells from mice deficient in the MyD88 adaptor protein involved in TLR7 and other TLR signal transduction. Imiquimod increased murine T(reg) cell-mediated suppression of T(resp) cell proliferation induced by anti-TCRbeta mAb in the presence of syngeneic BMDCs, and T(reg) cells from gardiquimod-treated mice exhibited enhanced in vitro suppressor function. Moreover, levels of T(resp) cell-secreted IL-2 and IFN-gamma were reduced further in the presence of T(reg) cells plus imiquimod in comparison with T(reg) cells alone. In addition, imiquimod treatment increased CD25 expression by T(reg) cells and caused exogenous IL-2 to enhance T(reg) cell suppressor function. Furthermore, combined treatment with imiquimod and IL-2 increased Foxp3 expression by T(reg) cells. Collectively, these findings suggest that TLR7 signaling enhanced the suppressor function of T(reg) cells by sensitizing T(reg) cells to IL-2-induced activation. We speculate that TLR7-stimulated enhancement of T(reg) cell suppressor function may modulate host T cell responses against ssRNA viruses.

Mast Cells Down-Regulate CD4+CD25+ T Regulatory Cell Suppressor Function via Histamine H1 Receptor Interaction

Mast cells promote both innate and acquired immune responses, but little is known about the effect of mast cells on T regulatory (T(reg)) cell function. In this study, we show for the first time that the capacity of murine CD4(+)CD25(+) T(reg) cells to suppress in vitro proliferation by CD4(+)CD25(-) T responder (T(resp)) cells in response to anti-CD3/anti-CD28 mAb-coated beads was reduced in the presence of syngeneic bone marrow-derived mast cells (BMMC) activated by FcepsilonR cross-linking. Activated BMMC culture supernatants or exogenous histamine also inhibited T(reg) cell suppressor function while the histamine H1 receptor-specific antagonist loratadine, but not the H2 receptor-specific antagonist famotidine, restored T(reg) cell suppressor function in the presence of activated BMMC or activated BMMC culture supernatants. Moreover, treatment of T(reg) cells with loratadine, but not famotidine, rescued T(reg) cell suppressor function in the presence of exogenous histamine. In addition, the H1 receptor-specific agonist 2-pyridylethylamine dihydrochloride inhibited T(reg) cell suppressor function to an extent that was comparable to histamine, whereas the H2 receptor-specific agonist amthamine dihydrobromide was without effect. Both T(reg) cells and T(resp) cells expressed H1 receptors. Exposure to histamine caused T(reg) cells to express lower levels of CD25 and the T(reg) cell-specific transcription factor Foxp3. Taken together, these data indicate that BMMC-elaborated histamine inhibited T(reg) cell suppressor function by signaling through the H1 receptor. We suggest that histamine released as a result of mast cell activation by microbial products might cause a transient decrease in T(reg) cell suppressor function, thereby enhancing the development of protective immunity.

Defective Tregs expression of the NTPDase CD39 in lupus (89.24)

Abstract The role of aberrations in the regulatory T cells (Tregs) pool for the development of systemic lupus erythamatosus (lupus) remains uncertain. Treg-mediated generation of adenosine, dependent on the ectonucleotidase CD39, is an important mechanism for suppression of T-cell responses. We studied 15 SLE patients along with 6 rheumatoid arthritis and 24 healthy controls. Levels of Treg phenotypic markers, including CD39 expression, were studied by flow cytometric analysis. Varying numbers of sorted Tregs cells were co-cultured with responder T (Tresp) cells, with proliferation assessed by 3H-thymidine incorporation. The proportion of Foxp3+ CD25+high CD127-/low Tregs was similar between lupus and control populations. CD39-expressing Tregs comprised 37 ± 13% of the Treg population in healthy controls and 36 ± 21% in lupus subjects using nonsteroidal immunosuppressants to control active disease, but was nearly absent in 5 of 6 lupus subjects with minimally active disease. In lupus subjects with the CD39 defect, Treg-mediated suppression was significantly reduced and, unlike in healthy control subjects, adenosine receptor antagonism could not reverse the observed suppression. These results suggest that functional defects in Tregs, rather than reduced Treg numbers, may be important for the loss of peripheral tolerance in lupus. Supported by NIH RO1-AR39501,WFUSM Venture Funds.

Only MHC-Identical Donor CD4+CD25+ Regulatory T Cells Convey Full Protection from Lethal Graft-Versus-Host Disease

Natural CD4+CD25+ regulatory T cells (Treg) contribute to tolerance induction after transplantation. We previously showed that the adoptive transfer of donor-derived Treg cells prevents lethal graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT) in murine disease models. In contrast, host-type Treg cells failed to protect when co-transplanted under identical conditions. We now examined whether MHC compatibility between Treg cells and conventional CD25−CD4+ and CD8+ T cells (Tconv) is required for the suppression of alloresponses, or whether elimination of host-type Treg by allo-aggressive donor Tconv cells occurred. To address this issue, mixed lymphocyte reactions were performed in which CFSE-labelled responder T cells (Tresp), Treg cells and antigen presenting cells (APC) were systematically varied with regard to their MHC haplotype. When BALB/c (H-2d) Tresp cells were stimulated with mixed BALB/c and C57BL/6 (H-2b) APC, cultures contained 26.0 ± 3.1% and 86.2 ± 2.2% proliferating CD4+ and CD8+ T cells, respectively, on 6 d. In the presence of syngeneic BALB/c Treg cells, proliferation was decreased to 9.1 ± 4.7% and 25.1 ± 4.9% for CD4+ and CD8+ Tresp cells, respectively. In contrast, in cultures with allogeneic C57BL/6 Treg cells, proliferation remained at 22.1 ± 1.8% for CD4+ and 89.6 ± 0.4% for CD8+ Tresp cells. Comparable results were obtained with C57BL/6 Tresp cells after stimulation with F1 (C57BL/6 × BALB/c; H-2b/d) or 3rd party (DBA/1; H-2q) APC. Lack of suppression in co-cultures of MHC-mismatched Tresp and Treg cells was not caused by an early elimination of allogeneic Treg cells, as those were still detectable after 6 d of allostimulation. In corresponding in vivo studies, CB6F1 or DBA/1 recipients were protected from lethal GVHD only when Tconv and Treg cells were derived from MHC-identical donors, but not when they were from two MHC-disparate strains. Transplantation of 1 × 106 C57BL/6 Tconv cells resulted in 100% lethality of CB6F1 recipients by d56. When co-transplanted with 1 × 106 C57BL/6 Treg cells, all recipients survived for 100d, whereas only 40% survived after co-transfer of the same number of BALB/c Treg (n = 15; p = 0.004). Similarly, when 1 × 106 BALB/c Tconv cells were transplanted into CB6F1 recipients, all animals died from GVHD by d46. In contrast, all recipients of BALB/c Tconv and Treg cells (ratio1:1) survived for 100d, but only 10% of recipient mice survived after co-transfer of C57BL/6 Treg (n = 10; p < 0.001). Similar results were obtained after BALB/c and C57BL/6 T cell transfer into DBA/1 (3rd party) recipients. In conclusion, these data indicate that MHC-identity between Tconv and Treg cells is required for maximum suppression of an alloresponse and that Treg cells isolated from a 3rd party donor might not be suited for the prevention of GVHD after allogeneic BMT.

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

CD4+,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. CD4+,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. Comparable 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. This 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.

Only Naive CD45RA+CD4+CD25high T Cells from Human Peripheral Blood Give Rise to Homogeneous Regulatory T Cell Lines.

The adoptive transfer of donor CD4+CD25high regulatory T (Treg) cells has been suggested for the prevention of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (SCT). In preparation of such trials we previously described protocols for the efficient in vitro expansion of human Treg cells (Hoffmann et al. 2004, Blood 104:895). Strong costimulation provided by immobilized anti-CD3 and anti-CD28 antibodies together with high-dose IL-2 resulted in a more than 3-log polyclonal expansion of Treg cells with strong suppressive activity. In contrast to CD4+CD25− T cells, the majority of the CD4+CD25high Treg cells maintained expression of the lymph node homing receptors CD62L and CCR7 during in vitro culture.Detailed examination of sorted subpopulations from Treg cell lines now revealed that only CD62L+CCR7+ cells combined all phenotypic and functional characteristics of natural Treg cells, such as FOXP3 expression, lack of cytokine secretion and potent suppression of responder T (Tresp) cell proliferation. To elucidate the origin of this cell population, we initiated cultures from CD45RA+ naive as well as CD45RA− effector/memory-type CD4+CD25high Treg cells. The CD45RA+ population initially contained 95 ± 2.5% CD62L+CCR7+ cells and maintained this phenotype with still over 90% CD62L+CCR7+ cells after 2 weeks and appox.70% after 3 weeks in culture (n=10). In contrast, CD62L and CCR7 expression in CD45RA− Treg cells was less stable and decreased from 58 ± 8% CD62L+CCR7+ cells after isolation to 32 ± 20% after culture for 3 weeks. Similar differences were observed with respect to cytokine production, as determined by intracellular staining: Whereas less than 5% (n=8) of expanded CD45RA+ Treg cells expressed IL-2 and/or IFN-γ, almost 40% of expanded CD45RA− Treg cells produced one or both of these pro-inflammatory cytokines upon stimulation with PMA/ionomycin. Interestingly, expanded CD45RA− Treg cells also contained a defined subpopulation of IL-10-producing cells (6.9 ± 5.6%; n=8), which was absent in expanded CD45RA+ Treg cells. Both subpopulations showed suppressive activity after polyclonal restimulation, however, suppression of Tresp cell proliferation by expanded CD45RA+ Treg cells was much more profound with only 13.6 ± 4% proliferating cells as compared to 35.4 ± 7.7% in the presence of expanded CD45RA− Treg cells and 79.6 ± 18.7% in the absence of Treg cells, as measured in a CFSE-dilution assay at a 1:4 ratio of Treg and Tresp cells (n=7). Most importantly, when determined on a single cell level by intracellular staining, 93.6 ± 1 % (n=3) of expanded CD45RA+ Treg cells still expressed FOXP3 after 3 weeks in culture, whereas only 10.8 ± 6.8% of expanded CD45RA− Treg cells remained FOXP3+. No re-expression could be induced in FOXP3− expanded CD45RA− Treg cells by short-term restimulation via CD3/CD28, whereas expanded FOXP3+ CD45RA+ Treg cells transiently upregulated FOXP3, with peak expression levels as early as 24h after stimulation and return to baseline levels by 48 to 72h. Based on these unexpected findings that only naive CD45RA+, but not memory-type CD45RA− CD4+CD25high T cells give rise to homogeneous Treg cell lines, we suggest that isolation and expansion of CD45RA+ CD4+CD25high T cells is the best strategy for adoptive Treg cell therapies.

Human CD4+CD25+ regulatory T cells

In this report, we review studies of human CD4+CD25+ regulatory T cells (T-reg). Although lagging a few years behind the discovery of these cells in the mouse, the equivalent population of CD4+CD25+ regulatory T cells has also been isolated from human peripheral blood, thymus, lymph nodes and cord blood. In general, the characteristics of this T cell subset are strikingly similar between mouse and man. In the recent explosion of research reports on human CD4+CD25+ cells, although the majority of the characteristics ascribed to these cells appear to be consistent, contrasting results have been found primarily in regards to potential involvement of TGFβ and production of IL-10. One explanation for this variability may reside in the fact that markedly different techniques are used to isolate human CD4+CD25+ T-reg cells and thus may result in the comparison of T-reg populations that differ in cellular composition and/or activation state. Another potential explanation for differences in human T-reg function may rest on the extreme variability of the culture conditions and TCR stimuli that have been used to test the functional properties of these cells in vitro. The strength of the TCR signal provided to the culture greatly affects the functional outcome of the co-culture and can result in the difference between suppression and full activation. Surprisingly, it appears that stronger stimulation has a greater and more rapid effect on the T-resp cell than on the T-reg cell as it causes T-resp cells to quickly become resistant to suppression. Thus, the details of in vitro culture conditions may at least partially account for disparate findings in regard to the functional characterization of human CD4+CD25+ cells. Here we review the evidence regarding the identification of human CD4+CD25+ regulatory T cells and their possible mechanism(s) of function.