Hans J. P. M. Koenen

Human CD25highFoxp3pos regulatory T cells differentiate into IL-17-producing cells.

The effector T-cell lineage shows great plasticity. Th17 cells are acknowledged to be instrumental in the response against microbial infection, but are also associated with autoimmune inflammatory processes. Here, we report that human regulatory T cells (CD4(pos)CD25(high)Foxp3(pos)CD127(neg)CD27(pos)) can differentiate into IL-17-producing cells, when stimulated by allogeneic antigen-presenting cells, especially monocytes, in the presence of rhIL-2/rhIL-15. These regulatory T cell (Treg)-derived IL-17-producing cells showed high expression of the Th17-related transcription factor RORgammat and were positively identified by CCR6 expression. This differentiation process was enhanced by exogenous IL-1beta, IL-23, and IL-21, whereas IL-6 or TGFbeta did not affect the emergence of IL-17-producing cells. The addition of IL-1 receptor antagonist (IL-1Ra), but not anti-IL-23 antibody, reduced IL-17-producing cell numbers. When an histone deacetylase (HDAC) inhibitor trichostatin A (TSA) was evaluated, we found a profound negative effect on the emergence of IL-17-producing cells from Tregs, implying that Treg differentiation into IL-17-producing cells depends on histone/protein deacetylase activity. Thus, the data suggest that epigenetic modification underlies the phenomenon of Treg plasticity here described.

Rapamycin, not cyclosporine, permits thymic generation and peripheral preservation of CD4 + CD25 + FoxP3 + T cells

Graft-versus-host-disease (GVHD) is the most common cause of poor outcome after allogeneic stem cell transplantation (SCT). Of late, exploitation of FOXP3+ regulatory T-cell (TREG) function is emerging as a promising strategy in suppression of GVHD, while preserving graft-versus-leukemia (GVL). Cyclosporine and rapamycin reduce the expansion of effector T cells by blocking interleukin (IL)-2, but signaling by IL-2 is pivotal for TREG homeostasis. The resolution of GVHD is critically dependent on thymus-dependent reconstitution of the immunoregulatory system. Thus, there has been concern about the impact of blocking IL-2 signaling by immunosuppressive agents on TREG homeostasis. Here we demonstrate in a mouse model that in contrast to rapamycin, cyclosporine compromises not only the thymic generation of CD4+CD25+FoxP3+ T cells but also their homeostatic behavior in peripheral immune compartments. Treatment with cyclosporine resulted in a sharp reduction of peripheral CD25+FoxP3+ T cells in all immune compartments studied. Prolonged rapamycin treatment allowed for thymic generation of CD4+FoxP3+ T cells, whereas treatment with cyclosporine led to a reduced generation of these cells. In conclusion, cyclosporine and rapamycin differentially affect homeostasis of CD4+FoxP3+ TREG in vivo. As peripheral tolerance induction is a prerequisite for successful treatment outcome after allogeneic SCT, these findings are of potential clinical relevance.

Regulation of cytokine responses by seasonality of vitamin D status in healthy individuals

The immune modulating capacity of vitamin D3 is well‐recognized. Ultra‐violet (UV) exposure determines production of vitamin D3in vivo and varies through the course of the year, especially in temperate regions. However, it is not known whether the human innate immune response differs due to seasonality. To validate the seasonal effects of vitamin D3, the effect of 1,25(OH)2D3 on peripheral blood mononuclear cells (PBMC) cytokine response was first determined in vitro. 1,25(OH)2D3 decreased interleukin (IL)‐6 and tumour necrosis factor (TNF)‐α release by PBMC stimulated with tripalmitoyl‐S‐glycerylcysteine (Pam3Cys) or lipopolysaccharide (LPS). Subsequently, ex‐vivo stimulation studies were performed in 15 healthy volunteers through the course of the four seasons of the year. PBMC were isolated and stimulated with Toll‐like receptor (TLR)‐2 and TLR‐4 ligands Pam3Cys and LPS, respectively. Circulating concentrations of 25(OH)D3 and 1,25(OH)2D3 were higher during summer (Pu2003<u20030·05) and a down‐regulation of TLR‐4‐mediated IL‐1β, IL‐6, TNF‐α, interferon (IFN)‐γ and IL‐10 production in summer was observed compared to winter (Pu2003<u20030·05). The variation in cytokine response upon TLR‐2 (Pam3Cys) stimulation was moderate throughout the four seasons. The repressed cytokine production during the summer months could be explained partly by the reduced cell‐membrane expression of TLRs. Physiological variation in vitamin D3 status through the four seasons of the year can lead to alteration in the innate immune responses. Elevated vitamin D3 level in vivo is associated with down‐regulation of cytokine response through diminished surface expression of pattern recognition receptors.

CD27/CFSE-based ex vivo selection of highly suppressive alloantigen-specific human regulatory T cells.

Naturally occurring CD4+CD25+ regulatory T cells (Treg) are crucial in immunoregulation and have great therapeutic potential for immunotherapy in the prevention of transplant rejection, allergy, and autoimmune diseases. The efficacy of Treg-based immunotherapy critically depends on the Ag specificity of the regulatory T cells. Moreover, the use of Ag-specific Treg as opposed to polyclonal expanded Treg will reduce the total number of Treg necessary for therapy. Hence, it is crucial to develop ex vivo selection procedures that allow selection and expansion of highly potent, Ag-specific Treg. In this study we describe an ex vivo CFSE cell sorter-based isolation method for human alloantigen-specific Treg. To this end, freshly isolated CD4+CD25+ Treg were labeled with CFSE and stimulated with (target) alloantigen and IL-2 plus IL-15 in short-term cultures. The alloantigen-reactive dividing Treg were characterized by low CFSE content and could be subdivided by virtue of CD27 expression. CD27/CFSE cell sorter-based selection of CD27+ and CD27− cells resulted in two highly suppressive Ag-specific Treg subsets. Each subset suppressed naive and Ag-experienced memory T cells, and importantly, CD27+ Treg also suppressed ongoing T cell responses. Summarizing, the described procedure enables induction, expansion, and especially selection of highly suppressive, Ag-specific Treg subsets, which are crucial in Ag-specific, Treg-based immunotherapy.

Following Anti‐CD25 Treatment, A Functional CD4+CD25+ Regulatory T‐Cell Pool Is Present in Renal Transplant Recipients

Daclizumab, a humanized antibody directed against the α‐chain of the interleukin‐2 receptor (CD25), has shown efficacy in the prevention of acute rejection following organ transplantation. However, anti‐CD25 therapy can be expected to affect not only alloreactive effector T cells, but also CD4+CD25+ regulatory T (Treg) cells that are shown to play an important role in the induction of transplantation tolerance. Therefore, the size and function of the Treg pool in human renal allograft recipients after single‐dose daclizumab administration was investigated in this study. Approximately 8 weeks after administration, daclizumab was cleared from the circulation and the Treg population then present appeared not different from that observed before transplantation. Functional analysis revealed that the Treg possessed a normal capacity to suppress mixed lymphocyte reactions in vitro. These data indicate that after daclizumab therapy a Treg population, normal in number and function, is present in the peripheral blood of renal transplant recipients.

In Vivo Induction of Cutaneous Inflammation Results in the Accumulation of Extracellular Trap-Forming Neutrophils Expressing RORγt and IL-17

Clinical trials successfully using antibodies targeting IL-17 in psoriasis support the importance of IL-17 in the pathophysiology of this disease. However, there is a debate concerning the source and dynamics of IL-17 production in inflamed skin. Here we characterized IL-17-producing immune cells over time, using two established in vivo models of human skin inflammation that share many histological features with psoriasis, i.e., leukotriene B4 application and tape-stripping. Both treatments revealed a clear influx of neutrophils and T cells. Staining for IL-17 revealed that the majority of IL-17 was expressed by neutrophils and mast cells, in both models. Neutrophils, but not mast cells, coexpressed the IL-17-associated transcription factor RORγt and were able to form extracellular traps. While the presence of mast cells remained steady during the skin inflammatory process, the presence of neutrophils was clearly dynamic in time. Therefore, it is attractive to hypothesize that IL-17+/RORγt+ neutrophils contribute to human skin inflammation in vivo and possibly to the pathogenesis of skin diseases such as psoriasis. Surprisingly, T cells represented a minority of the IL-17-expressing cell population. These observations challenge the classical opinion that IL-17 is predominantly associated with T cells in skin inflammation.

A single dose of rituximab does not deplete B cells in secondary lymphoid organs but alters phenotype and function

A single dose of the anti‐CD20 monoclonal antibody rituximab induces a nearly complete B cell depletion in peripheral blood, but not in secondary lymphoid organs. Modulation of this remaining B cell population due to rituximab treatment may contribute to the therapeutic effects of rituximab. To assess the in vivo effects of rituximab we used lymph nodes (LNs) collected during renal transplant surgery in patients who had received rituximab 4 weeks earlier in preparation for an ABO‐incompatible transplantation. Rituximab treatment resulted in a lower percentage of naïve (IgD+CD27−) and a higher percentage of switched memory (IgD−CD27+) B cells. Remarkably, transitional (CD24++CD38++) B cells were virtually lacking in the LNs of rituximab‐treated patients. Moreover, LN‐derived B cells from rituximab‐treated patients produced different amounts of various Ig‐subclasses after anti‐CD40/IL‐21 stimulation ex vivo. Finally, after stimulation of allogeneic T cells with LN‐derived B cells from rituximab‐treated patients, the proliferated T cells showed a decreased production of IL‐17. In conclusion, after treatment with rituximab there remains a B cell population with different functional capacities. Consequently, the effect of rituximab on the immune response will not only be determined by the extent of B cell depletion, but also by the functional properties of the remaining B cells.

Translating the role of vitamin D3 in infectious diseases

Vitamin D3 affects both the innate as well as adaptive immune responses. Epidemiological studies have established that vitamin D3 deficiency plays an important role in tuberculosis (TB) and viral influenza prevalence as well as susceptibility to active disease in TB. Vitamin D3 status has been associated with the clinical course of HIV infection and drug interaction with anti-retroviral therapy. This article reviews the immunomodulatory capacity of vitamin D3 and examines the impact of vitamin D3 supplementation as a preventive or therapeutic intervention with the intent to uncover its potential therapeutic application in infectious diseases and to identify novel areas for future research. We present a review of randomized, controlled clinical studies conducted in humans which included assessment of the immune function or clinical outcome as study end points. Current data support vitamin D3 supplementation as risk-modifying intervention in tuberculosis and viral respiratory tract infection, but the optimal dosage regimen remains to be determined. However, to date the knowledge on its role in fungal infection and sepsis is limited although a potential benefit could be harnessed from its ability to curtail the unrestrained pro-inflammatory response and therefore prevent excessive collateral tissue damage.

Monitoring regulatory T cells in clinical samples: consensus on an essential marker set and gating strategy for regulatory T cell analysis by flow cytometry

Regulatory T cell (Treg)-mediated immunosuppression is considered a major obstacle for successful cancer immunotherapy. The association between clinical outcome and Tregs is being studied extensively in clinical trials, but unfortunately, no consensus has been reached about (a) the markers and (b) the gating strategy required to define human Tregs in this context, making it difficult to draw final conclusions. Therefore, we have organized an international workshop on the detection and functional testing of Tregs with leading experts in the field, and 40 participants discussing different analyses and the importance of different markers and context in which Tregs were analyzed. This resulted in a rationally composed ranking list of “Treg markers”. Subsequently, the proposed Treg markers were tested to get insight into the overlap/differences between the most frequently used Treg definitions and their utility for Treg detection in various human tissues. Here, we conclude that the CD3, CD4, CD25, CD127, and FoxP3 markers are the minimally required markers to define human Treg cells. Staining for Ki67 and CD45RA showed to provide additional information on the activation status of Tregs. The use of markers was validated in a series of PBMC from healthy donors and cancer patients, as well as in tumor-draining lymph nodes and freshly isolated tumors. In conclusion, we propose an essential marker set comprising antibodies to CD3, CD4, CD25, CD127, Foxp3, Ki67, and CD45RA and a corresponding robust gating strategy for the context-dependent analysis of Tregs by flow cytometry.

Rituximab as induction therapy after renal transplantation: a randomized, double-blind, placebo-controlled study of efficacy and safety

We evaluated the efficacy and safety of rituximab as induction therapy in renal transplant patients. In a double‐blind, placebo‐controlled study, 280 adult renal transplant patients were randomized between a single dose of rituximab (375u2009mg/m2) or placebo during transplant surgery. Patients were stratified according to panel‐reactive antibody (PRA) value and rank number of transplantation. Maintenance immunosuppression consisted of tacrolimus, mycophenolate mofetil and steroids. The primary endpoint was the incidence of biopsy proven acute rejection (BPAR) within 6 months after transplantation. The incidence of BPAR was comparable between rituximab‐treated (23/138, 16.7%) and placebo‐treated patients (30/142, 21.2%, pu2009=u20090.25). Immunologically high‐risk patients (PRA >6% or re‐transplant) not receiving rituximab had a significantly higher incidence of rejection (13/34, 38.2%) compared to other treatment groups (rituximab‐treated immunologically high‐risk patients, and rituximab‐ or placebo‐treated immunologically low‐risk (PRAu2009≤u20096% or first transplant) patients (17.9%, 16.4% and 15.7%, pu2009=u20090.004). Neutropenia (<1.5u2009×u2009109/L) occurred more frequently in rituximab‐treated patients (24.3% vs. 2.2%, pu2009<u20090.001). After 24 months, the cumulative incidence of infections and malignancies was comparable. A single dose of rituximab as induction therapy did not reduce the overall incidence of BPAR, but might be beneficial in immunologically high‐risk patients. Treatment with rituximab was safe.