Esther van Rijssen

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.

Crosstalk between keratinocytes and T cells in a 3D microenvironment: a model to study inflammatory skin diseases

The interaction between keratinocytes and immune cells plays a major role in the development of inflammatory skin diseases like psoriasis and atopic dermatitis. Pharmacological intervention to inhibit T cell-derived proinflammatory mediators is an effective therapy in the treatment of psoriasis. Here, we present a model to study the interaction between keratinocytes and T cells in a three-dimensional (3D) microenvironment, based on human skin equivalents populated with CD4+ T cells. T cell migration into the dermis initiated keratinocyte activation within 2 days, with hallmarks of a psoriasiform inflammation after 4 days. Expression of epidermal psoriasis marker genes was upregulated, and proinflammatory cytokines and chemokines were highly expressed. Disturbed epidermal differentiation was shown by downregulated filaggrin expression and involucrin expression in the spinous layer. These effects were mediated via soluble factors produced by the T cells. The psoriasiform inflammation was also observed using T helper type 1 (Th1)- and Th17-polarized CD4+ T cells. We validated our model by treatment with anti-inflammatory drugs that reduced the expression of proinflammatory cytokines and chemokines and suppressed the psoriasiform inflammation. We propose that our T cell-driven inflammatory skin equivalent model has potential to study the pathogenesis of inflammatory skin diseases and may serve as a preclinical screening tool for anti-inflammatory drugs.

Immunological monitoring of renal transplant recipients to predict acute allograft rejection following the discontinuation of tacrolimus.

Background Transplant patients would benefit from reduction of immunosuppression providing that graft rejection is prevented. We have evaluated a number of immunological markers in blood of patients in whom tacrolimus was withdrawn after renal transplantation. The alloreactive precursor frequency of CD4+ and CD8+ T cells, the frequency of T cell subsets and the functional capacity of CD4+CD25+FoxP3+ regulatory T cells (Treg) were analyzed before transplantation and before tacrolimus reduction. In a case-control design, the results were compared between patients with (n = 15) and without (n = 28) acute rejection after tacrolimus withdrawal. Principal Findings Prior to tacrolimus reduction, the ratio between memory CD8+ T cells and Treg was higher in rejectors compared to non-rejectors. Rejectors also had a higher ratio between memory CD4+ T cells and Treg, and ratios <20 were only observed in non-rejectors. Between the time of transplantation and the start of tacrolimus withdrawal, an increase in naive T cell frequencies and a reciprocal decrease of effector T cell percentages was observed in rejectors. The proportion of Treg within the CD4+ T cells decreased after transplantation, but anti-donor regulatory capacity of Treg remained unaltered in rejectors and non-rejectors. Conclusions Immunological monitoring revealed an association between acute rejection following the withdrawal of tacrolimus and 1) the ratio of memory T cells and Treg prior to the start of tacrolimus reduction, and 2) changes in the distribution of naive, effector and memory T cells over time. Combination of these two biomarkers allowed highly specific identification of patients in whom immunosuppression could be safely reduced.

CTLA-4 Engagement and Regulatory CD4+CD25+ T Cells Independently Control CD8+-Mediated Responses under Costimulation Blockade

Blockade of costimulatory signals is a promising therapeutic target to prevent allograft rejection. In this study, we sought to characterize to what extent CTLA-4 engagement contributes to the development of transplantation tolerance under the cover of CD40/CD40L and CD28/CD86 blockade. In vitro, we found that inhibition of the primary alloresponse and induction of alloantigen hyporesponsiveness by costimulation blockade was abrogated by anti-CTLA-4 mAb. In addition, regulatory CD4+CD25+ T cells (TREG) were confirmed to play a critical role in the induction of hyporesponsiveness by anti-CD40L and anti-CD86 mAb. Our data indicated that CTLA-4 engagement is not required for activation or suppressor function of TREG. Instead, in the absence of either CTLA-4 signaling or TREG, CD8+ T cell division was enhanced, whereas the inhibition of CD4+ T cell division by costimulation blockade remained largely unaffected. In vivo, the administration of additional anti-CTLA-4 mAb abrogated anti-CD40L- and anti-CD86 mAb-induced cardiac allograft survival. Correspondingly, rejection was accompanied by enhanced allograft infiltration of CD8+ cells. We conclude that CTLA-4 signaling and TREG independently cooperate in the inhibition of CD8+ T cell expansion under costimulation blockade.

Targeting PKC in Human T Cells Using Sotrastaurin (AEB071) Preserves Regulatory T Cells and Prevents IL-17 Production

Regulatory T-cells (Treg) are crucial for immune homeostasis and prevention of immune pathology. Yet, Treg may lose Foxp3 and start secreting IL-17, dependent on environmental cues. Our previous data revealed that Treg from severe psoriasis patients are particularly prone to such conversion. The question of how to maintain Treg stability in the context of inflammation awaits immediate resolution. The pan-protein kinase C (PKC) inhibitor sotrastaurin has shown efficacy in clinical trials of psoriasis. Here, we show that sotrastaurin inhibited effector T-cell responses, whereas the regulatory response was enhanced. Sotrastaurin prevented TCR/CD28-induced T-cell activation and pro-inflammatory cytokine production, but preserved a stable Treg phenotype as evidenced by maintenance of suppressive capacity, high Foxp3 and CD25 expression, and lack of IL-17A and IFNγ production. Moreover, in both circulating and dermal psoriatic Treg, prone to rapid induction of IL-17, sotrastaurin enhanced Foxp3 expression and prevented IL-17A and IFNγ production even when stimulated in the presence of the helper T 17-enhancing cytokines IL-1β or IL-23. Thus, pharmacological inhibition of PKC may serve as a powerful tool to concurrently inhibit effector T cells and to facilitate Treg, thereby showing therapeutic potential for the treatment of psoriasis.

Single CD28 stimulation induces stable and polyclonal expansion of human regulatory T cells

CD4+FOXP3+ Treg are essential for immune tolerance. Phase-1 clinical trials of Treg-therapy to treat graft-versus-host-disease reported safety and potential therapeutic efficacy. Treg-based trials have started in organ-transplant patients. However, efficient ex vivo expansion of a stable Treg population remains a challenge and exploring novel ways for Treg expansion is a pre-requisite for successful immunotherapy. Based on the recent finding that CD28-signaling is crucial for survival and proliferation of mouse Treg, we studied single-CD28 stimulation of human Treg, without T cell receptor stimulation. Single-CD28 stimulation of human Treg in the presence of recombinant human IL-2(rhIL-2), as compared to CD3/CD28/rhIL-2 stimulation, led to higher expression levels of FOXP3. Although the single-CD28 expanded Treg population was equally suppressive to CD3/CD28 expanded Treg, pro-inflammatory cytokine (IL-17A/IFNγ) production was strongly inhibited, indicating that single-CD28 stimulation promotes Treg stability. As single-CD28 stimulation led to limited expansion rates, we examined a CD28-superagonist antibody and demonstrate a significant increased Treg expansion that was more efficient than standard anti-CD3/CD28-bead stimulation. CD28-superagonist stimulation drove both naïve and memory Treg proliferation. CD28-superagonist induction of stable Treg appeared both PI3K and mTOR dependent. Regarding efficient and stable expansion of Treg for adoptive Treg-based immunotherapy, application of CD28-superagonist stimulation is of interest.

DNA Methyltransferase Inhibition Promotes Th1 Polarization in Human CD4(+)CD25(high) FOXP3(+) Regulatory T Cells but Does Not Affect Their Suppressive Capacity

Regulatory T cells (Treg) can show plasticity whereby FOXP3 expression, the master transcription factor for Treg suppressor function, is lost and proinflammatory cytokines are produced. Optimal FOXP3 expression strongly depends on hypomethylation of the FOXP3 gene. 5-Azacytidine (Aza) and its derivative 5-aza-2′-deoxycytidine (DAC) are DNA methyltransferase inhibitors (DNMTi) that are therapeutically used in hematological malignancies, which might be an attractive strategy to promote Treg stability. Previous in vitro research primarily focused on Treg induction by DAC from naïve conventional CD4+ T cells (Tconv). Here, we examined the in vitro effect of DAC on the stability and function of FACS-sorted human naturally occurring CD4+CD25high FOXP3+ Treg. We found that in vitro activation of Treg in the presence of DAC led to a significant inhibition of Treg proliferation, but not of Tconv. Although Treg activation in the presence of DAC led to increased IFNγ expression and induction of a Thelper-1 phenotype, the Treg maintained their suppressive capacity. DAC also induced a trend towards increased IL-10 expression. In vivo studies in patients with hematological malignancies that were treated with 5-azacytidine (Vidaza) supported the in vitro findings. In conclusion, despite its potential to increase IFNγ expression, DAC does preserve the suppressor phenotype of naturally occurring Treg.