Successful Regulatory T Cell-Based Therapy Relies on Inhibition of T Cell Effector Function and Enrichment of FOXP3+ Cells in a Humanized Mouse Model of Skin Inflammation.

BACKGROUND: Recent clinical trials using regulatory T cells (Treg) support the therapeutic potential of Treg-based therapy in transplantation and autoinflammatory diseases. Despite these clinical successes, the effect of Treg on inflamed tissues, as well as their impact on immune effector function in vivo, is poorly understood. Therefore, we here evaluated the effect of human Treg injection on cutaneous inflammatory processes in vivo using a humanized mouse model of human skin inflammation (huPBL-SCID-huSkin). METHODS: SCID beige mice were transplanted with human skin followed by intraperitoneal (IP) injection of 20-40 × 106 allogeneic human PBMCs. This typically results in human skin inflammation as indicated by epidermal thickening (hyperkeratosis) and changes in dermal inflammatory markers such as the antimicrobial peptide hBD2 and epidermal barrier cytokeratins K10 and K16, as well as T cell infiltration in the dermis. Ex vivo-expanded human Treg were infused intraperitoneally. Human cutaneous inflammation and systemic immune responses were analysed by immunohistochemistry and flow cytometry. RESULTS: We confirmed that human Treg injection inhibits skin inflammation and the influx of effector T cells. As a novel finding, we demonstrate that human Treg injection led to a reduction of IL-17-secreting cells while promoting a relative increase in immunosuppressive FOXP3+ Treg in the human skin, indicating active immune regulation in controlling the local proinflammatory response. Consistent with the local control (skin), systemically (splenocytes), we observed that Treg injection led to lower frequencies of IFNγ and IL-17A-expressing human T cells, while a trend towards enrichment of FOXP3+ Treg was observed. CONCLUSION: Taken together, we demonstrate that inhibition of skin inflammation by Treg infusion, next to a reduction of infiltrating effector T cells, is mediated by restoring both the local and systemic balance between cytokine-producing effector T cells and immunoregulatory T cells. This work furthers our understanding of Treg-based immunotherapy.

Anti-B cell therapy with rituximab as induction therapy in renal transplantation.

Traditionally, antirejection therapy in organ transplantation has mainly been directed at T cells. During recent years, the role of B cells in acute rejection has attracted more attention. In the Radboud University Medical Center (Nijmegen, The Netherlands) we performed a randomized, placebo controlled study to assess the efficacy and safety of rituximab as induction therapy after renal transplantation. In parallel we investigated the effects of rituximab on the numbers and function of B and T cells. An overview of the results, which have largely been published in peer reviewed papers, is presented below.

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.

Balance of Treg vs. T-helper cells in the transition from symptomless to lesional psoriatic skin.

BACKGROUND: In the pathogenesis of psoriasis, proinflammatory T cells are strongly involved in the inflammatory process, where regulatory T-cell (Treg) function is impaired. OBJECTIVES: As effective Treg function is associated with a numerical balance between Treg and effector T cells, we wondered whether Treg/T-helper cell ratios may be associated with certain stages of the inflammatory process. We opted for the margin zone model as a dynamic approach. METHODS: From nine patients with chronic plaque psoriasis, 3-mm punch biopsies were obtained from the centre and margin of the lesion, perilesional skin and distant uninvolved skin. Skin biopsies of 10 healthy volunteers were included as a control. Samples were analysed using immunohistochemistry and immunofluorescence. RESULTS: In the transition from symptomless to lesional skin, a significant increase of CD3+, CD4+ and Foxp3+ cells was found. In seven of nine patients the ratio of Treg (Foxp3+) vs. CD4+ T cells was higher in the distant uninvolved skin than in the perilesional and lesional skin. Interestingly, the Foxp3/CD4 ratio in the distant uninvolved skin was even higher than in the skin of healthy controls. Notably, we found that most of the interleukin (IL)-17 expression was not related to CD4+ cells, but to mast cells. CONCLUSIONS: The relatively high Foxp3/CD4 ratio in symptomless skin of patients with psoriasis suggests an active immune controlling mechanism distant from the psoriatic plaque. In the margin and centre of the plaque the ratio appears skewed towards effector cells associated with inflammation. IL-17, an important driver of the psoriatic process, is mostly related to mast cells, and only sporadically to T cells.

In vitro effects of rituximab on the proliferation, activation and differentiation of human B cells.

Rituximab is a chimeric anti-CD20 monoclonal antibody (mAb) used in B-cell malignancies, various autoimmune disorders and organ transplantation. Although administration of a single dose of rituximab results in full B-cell depletion in peripheral blood, there remains a residual B-cell population in secondary lymphoid organs. These nondepleted B cells might be altered by exposure to rituximab with subsequent immunomodulatory effects. Therefore, we analyzed in vitro the effects of rituximab on proliferation, activation and differentiation of CD19(+) B cells by means of carboxyfluorescein succinimidyl ester (CFSE)-based multiparameter flow cytometry. Rituximab inhibited the proliferation of CD27(-) naïve, but not of CD27(+) memory B cells. Interestingly, upon stimulation with anti-CD40 mAb and interleukin-21 in the presence of rituximab there was an enrichment of B cells that underwent only one or two cell divisions and displayed an activated naïve phenotype (CD27(-)IgD(+)CD38(-/+)). The potency of prestimulated B cells to induce T-cell proliferation was increased by exposure of the B cells to rituximab. Of note, after stimulation with rituximab-treated B cells, proliferated T cells displayed a more Th2-like phenotype. Overall, these results demonstrate that rituximab can affect human B-cell phenotype and function, resulting in an altered outcome of B-T cell interaction.

Mycophenolic acid-mediated suppression of human CD4+ T cells: more than mere guanine nucleotide deprivation.

Mycophenolic acid is the active ingredient of the immunosuppressant mycophenolate mofetil that is widely used in transplantation medicine and autoimmunity. Mycophenolic acid inhibits inosine monophosphate dehydrogenase, an enzyme involved in biosynthesis of guanine nucleotides required for lymphocyte clonal expansion. Here, we present novel insights into the mechanisms underlying mycophenolic acid-mediated suppression of human CD4+ T cells. Upon CD3/CD28 stimulation, mycophenolic acid inhibited T cell IL-17, IFN-γ and TNF-α production but not IL-2 production. Phenotypic analysis showed that drug treatment enhanced the expression of negative co-stimulators PD-1, CTLA-4 and the transcription factor FoxP3 and decreased the expression of positive co-stimulators CD27 and CD28, whereas CD25 was unaffected. Mycophenolic acid-treated cells were anergic, but not suppressive, and at the same time proved hyperblastoid with high metabolic activity. Moreover, a reduced Akt/mTOR and STAT5 signaling was observed. Interestingly, the co-stimulatory molecule CD70 was uniquely and dose-dependently upregulated on mycophenolic acid-treated T cells and found to be directly linked to target enzyme inhibition. CD70 on mycophenolic acid-treated cells proved functional: an anti-CD70 agonist was found to restore both STAT5 and Akt/mTOR signaling and may thereby prevent apoptosis and promote survival. These novel insights may contribute to optimization of protocols for MPA-based immunosuppressive regimens.

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

The immune modulating capacity of vitamin D(3) is well-recognized. Ultra-violet (UV) exposure determines production of vitamin D(3) in 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 D(3) , the effect of 1,25(OH)(2) D(3) on peripheral blood mononuclear cells (PBMC) cytokine response was first determined in vitro. 1,25(OH)(2) D(3) 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)D(3) and 1,25(OH)(2) D(3) were higher during summer (P<0·05) and a down-regulation of TLR-4-mediated IL-1ß, IL-6, TNF-α, interferon (IFN)-γ and IL-10 production in summer was observed compared to winter (P<0·05). 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 D(3) status through the four seasons of the year can lead to alteration in the innate immune responses. Elevated vitamin D(3) level in vivo is associated with down-regulation of cytokine response through diminished surface expression of pattern recognition receptors.

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 (T(REG)) 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 T(REG) 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 T(REG) 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(+) T(REG) in vivo. As peripheral tolerance induction is a prerequisite for successful treatment outcome after allogeneic SCT, these findings are of potential clinical relevance.

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 alpha-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.