Udo Holtick

Adult Human Fibroblasts Are Potent Immunoregulatory Cells and Functionally Equivalent to Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (MSC) have potent immunosuppressive properties and have been advocated for therapeutic use in humans. The nature of their suppressive capacity is poorly understood but is said to be a primitive stem cell function. Demonstration that adult stromal cells such as fibroblasts (Fb) can modulate T cells would have important implications for immunoregulation and cellular therapy. In this report, we show that dermal Fb inhibit allogeneic T cell activation by autologously derived cutaneous APCs and other stimulators. Fb mediate suppression through soluble factors, but this is critically dependent on IFN-γ from activated T cells. IFN-γ induces IDO in Fb, and accelerated tryptophan metabolism is at least partly responsible for suppression of T cell proliferation. T cell suppression is reversible, and transient exposure to Fb during activation reprograms T cells, increasing IL-4 and IL-10 secretion upon restimulation. Increased Th2 polarization by stromal cells is associated with amelioration of pathological changes in a human model of graft-vs-host disease. Dermal Fb are highly clonogenic in vitro, suggesting that Fb-mediated immunosuppression is not due to outgrowth of rare MSC, although dermal Fb remain difficult to distinguish from MSC by phenotype or transdifferentiation capacity. These results suggest that immunosuppression is a general property of stromal cells and that dermal Fb may provide an alternative and accessible source of cellular therapy.

Regulatory T-Cell Suppression of CD8+ T-Cell-Mediated Graft-Versus-Host Reaction Requires Their Presence During Priming

Background. Despite the promising therapeutic potential of regulatory T cells (Treg) in animal studies of graft-versus-host disease (GVHD), little is known about their effect on human GVHD. Whether Treg are capable of ameliorating GVHD tissue damage has never been demonstrated in humans. It is also unknown whether Treg modulation of GVH histopathologic damage relies on their presence during effector T-cell priming, or whether allogeneic Treg are safe to use clinically. Methods. To address these questions, we used an in vitro human skin explant GVHD model, which mimics the physiopathology of GVHD. First, “donor”-derived CD8+ T cells were stimulated with human leukocyte antigen-unmatched “recipient” dendritic cells (priming phase), then primed “donor” CD8+ T cells were co-cultured with “recipient” skin to induce GVH tissue damage (effector phase). “Donor”-derived Treg were added at the priming or effector phase of the GVH response. Histopathologic changes in the skin were evaluated using a clinically validated GVHD scoring system. Results. “Donor”-derived Treg significantly reduced the severity of GVH histopathologic damage when present during T-cell priming. In contrast, Treg failed to prevent GVH tissue damage when added to the skin co-culture (effector phase), concurrently with primed T cells. Importantly, “donor” Treg alone did not induce GVH tissue damage. Delayed Treg addition led to reduced and impaired Treg suppression of CD8+ T-cell activation and their cytolytic function. Conclusion. “Donor”-derived Treg effectively suppress CD8+ T-cell-mediated GVH tissue damage but are critically required during priming of effector T cells. “Donor”-derived Treg seem to be safe and do not induce GVH histopathologic damage.

Impact of Psoralen/uva-treatment on Survival, Activation, and Immunostimulatory Capacity of Monocyte-derived Dendritic Cells

Background. Extracorporeal Photopheresis (ECP) has been shown to be an effective treatment of graft-versus-host disease, solid organ graft rejection, and other T-cell-mediated diseases. The mechanisms of action of ECP include lymphocyte apoptosis, cytokine modulation, and the induction of regulatory T cells. It has been suggested that dendritic cells (DCs) are more resistant to ECP-induced apoptosis and might be directly modulated by ECP. We tested this hypothesis using in vitro Psoralen/UVA (PUVA) treatment as an in vitro model of ECP. Methods. Monocyte-derived DCs (mo-DCs) were treated with 8-methoxypsoralen /UVA and analyzed for surface molecule expression, apoptosis markers, endocytosis, and migratory and immunostimulatory capacity. Mo-DC phenotype and cytokine secretion was tested after CD40L stimulation. Naive T cells stimulated with PUVA-treated mo-DCs were tested for Th1/Th2 cytokine secretion and associated chemokine receptor patterns. Results. DCs underwent apoptosis after in vitro PUVA and in vivo ECP. In vitro, the induction of apoptosis was preceded by partial maturation of immature mo-DCs. PUVA-treated immature mo-DCs also exhibited enhanced migratory and immunostimulatory capacity. However, mo-DCs stimulation through CD40 ligation was abrogated and interleukin (IL)-12 secretion was abolished 24 hr after PUVA treatment. PUVA-treated mo-DCs skewed naive T cells toward a Th2 response as defined by increased IL-4, IL-10, and IL-13 and decreased interferon-γ levels, and the expression of the Th2-associated chemokine receptors CCR4 and CCR10. The observed Th2 shift was partially reversed by exogenous IL-12. Conclusion. These data suggest that direct modulation of DC function as well as apoptosis contribute to the immunoregulatory effects of ECP.

Overcoming tumor-mediated immunosuppression

Mechanisms of tumor-mediated immunosuppression have been described for several solid and hematological tumors. Tumors inhibit immune responses by attraction of immunosuppressive lymphocytic populations, secretion of immunosuppressive cytokines or expression of surface molecules, which inhibit immune responses by induction of anergy or apoptosis in tumor-infiltrating lymphocytes. This tumor-mediated immunosuppression represents a major obstacle to many immunotherapeutic or conventional therapeutic approaches. In this review we discuss how tumor-mediated immunosuppression interferes with different immunotherapeutic approaches and then give an overview of strategies to overcome it. Particular emphasis is placed on agents or approaches already transferred into clinical settings. Finally the success of immune checkpoint inhibitors targeting CTLA-4 or the PD-1 pathway highlights the enormous therapeutic potential of an effective overcoming of tumor-mediated immunosuppression.

OCTET-CY: a phase II study to investigate the efficacy of post-transplant cyclophosphamide as sole graft-versus-host prophylaxis after allogeneic peripheral blood stem cell transplantation.

Post‐transplant cyclophosphamide is increasingly used as graft‐versus‐host disease (GvHD) prophylaxis in the setting of bone marrow transplantation. No data have been published on the use of single‐agent GvHD prophylaxis with post‐transplant cyclophosphamide in the setting of peripheral blood stem cell transplantation (PBSCT).

Deregulation of immunoglobulin gene transcription in the Hodgkin–Reed Sternberg cell line L1236

Hodgkin–Reed Sternberg (H‐RS) cells harbour clonal immunoglobulin gene (Ig) rearrangements in almost all cases of classical Hodgkins disease but lack Ig gene expression. In the H‐RS cell line L1236, a somatic mutation of the Ig heavy‐chain gene promoter octamer motif has been described as a putative reason for absence of Ig gene expression. We addressed transcriptional activity of this mutated promoter by performing reporter gene studies and gel retardation assays. The results showed that the mutation outside the coding region of the rearranged Ig gene in L1236 cells leads to downregulation of Ig gene expression in H‐RS cells.

Targeting Tumor-Infiltrating B Cells in Cutaneous T-Cell Lymphoma

Introduction The tumor microenvironment and infiltrating immune cells are important for cancer biology and progression which has been exploited therapeutically in recent years. Whereas infiltrating T cells, macrophages, or natural-killer (NK) cells have been extensively analyzed, recent studies also suggest a role for B cells in cancer biology. Primary cutaneous T-cell lymphomas (CTCL) represent a heterogeneous group of extranodal non-Hodgkin lymphomas of which the most common subtype is mycosis fungoides (MF). At early stages ( IIA; European Organisation for Research and Treatment of Cancer [EORTC]/International Society for Cutaneous Lymphomas [ISCL]), MF usually runs an indolent course with almost normal life expectancy. However, advanced-stage MF ( IIB), the folliculotropic MF subtype (FMF) and also Sézary syndrome (SS) are more aggressive (median survival, 13-48 months). Preclinical data suggest that chronic inflammation promotes CTCL progression with a critical role for macrophages and mast cells. Occasionally, B-cell infiltrations have been reported in CTCL and although a well-established CTCL model is lacking, B-cell deficient mice exhibited significant regressions of EL4-T-cell lymphoma grafts. Therefore, we analyzed diagnostic skin biopsies of 33 consecutively treated CTCL patients for infiltrating B cells (CD20/ CD79a) and reviewed respective clinical data from 1979 to 2011 (Table 1). Psoriasis and eczema tissues served as controls. Staging followed ISCL/EORTC criteria. Lymphoma-tissues containing 50 B-cells/mm of lymphoma-infiltrate were assigned as B-cell positive. The study was approved by the institutional review board (No. 08-144). Immunohistochemistry revealed remarkable differences of CD20 B-cell infiltrates between CTCL subtypes (Fig 1A) and additional flow-cytometric analysis in available frozen samples supported the B-cell nature of CD20 cells (Fig 3A). Eighteen out of 33 CTCL patients (MF [n 25], FMF [n 5], SS [n 3]) had significantly increased median B-cell numbers within the lymphoma infiltrate, whereas controls (psoriasis [n 5], eczema [n 5]) were B-cell negative (Fig 1B and Table 1). Remarkably, all FMF and SS cases showed significantly increased median B-cell numbers compared with classic MF. However, in classic MF 40% of the cases were B-cell positive. Given the fact that FMF and SS represent more aggressive subtypes, we asked whether B-cell infiltrations correspond with the clinical stage and behavior. Comparison of B-cell infiltrates in early ( IIB) versus advanced ( IIB) stages revealed a significant correlation of B-cell positivity with advanced stages. This correlation not only applied to the entire CTCL cohort but also to classic MF cases solely (Fig 1C). Moreover, clinical data analyses revealed a significant correlation of B-cell infiltrates with progression-free survival (PFS). As shown in Figure 1D, patients with B-cell positive lymphoma had a significantly shortened median PFS, ie, 50 months (entire cohort) and 126 months (classic MF only). In contrast, median PFS in B-cell negative cases was not reached at the end of observation (360 months).

Persistent CMV infection after allogeneic hematopoietic stem cell transplantation in a CMV-seronegative donor-to-positive recipient constellation: Development of multidrug resistance in the absence of anti-viral cellular immunity

We describe a case of persistent cytomegalovirus (CMV) infection after allogeneic hematopoietic stem cell transplantation (allo-HSCT) with discordant and high-risk (D-/R+) constellation of CMV serostatus. Despite the use of different and innovative antiviral strategies, viral replication could not be suppressed successfully promoting a protracted CMV colitis associated with severe gastrointestinal graft-versus-host disease (GI GVHD). We illustrate that the development of multidrug viral resistance, the failure to mount a CMV-specific cellular immune response, as confirmed by QuantiFERON(®)-CMV (Qiagen) assay, and the refractory GVHD requiring prolonged immunosuppression were the main factors contributing to persistent viral replication and the fulminant unfavorable course.

Inhibition of Protein Geranylgeranylation Specifically Interferes with CD40-Dependent B Cell Activation, Resulting in a Reduced Capacity To Induce T Cell Immunity

Ab-independent effector functions of B cells, such as Ag presentation and cytokine production, have been shown to play an important role in a variety of immune-mediated conditions such as autoimmune diseases, transplant rejection, and graft-versus-host disease. Most current immunosuppressive treatments target T cells, are relatively unspecific, and result in profound immunosuppression that places patients at an increased risk of developing severe infections and cancer. Therapeutic strategies, which interfere with B cell activation, could therefore be a useful addition to the current immunosuppressive armamentarium. Using a transcriptomic approach, we identified upregulation of genes that belong to the mevalonate pathway as a key molecular event following CD40-mediated activation of B cells. Inhibition of 3-hydroxy-3-methylglutaryl CoA reductase, the rate-limiting enzyme of the mevalonate pathway, by lipophilic statins such as simvastatin and atorvastatin resulted in a specific inhibition of B cell activation via CD40 and impaired their ability to act as stimulatory APCs for allospecific T cells. Mechanistically, the inhibitory effect resulted from the inhibition of protein geranylgeranylation subsequent to the depletion of mevalonate, the metabolic precursor for geranylgeranyl. Thus, inhibition of geranylgeranylation either directly through geranylgeranyl transferase inhibitors or indirectly through statins represents a promising therapeutic approach for the treatment of diseases in which Ag presentation by B cells plays a role.

In vitro PUVA treatment preferentially induces apoptosis in alloactivated T cells.

tified to have double-positive disease as a cause of ESRD. Six of these patients received a renal transplant between 1999 and 2011. Detailed follow-up data is available for these six patients, representing 406 patient-months of observation. The median follow-up was 66.5 months. This patient cohort included four women and two men. There were 4 whites, 1 Native American, and 1 Asian of Middle Eastern decent. All patients received dialysis before transplantation, with a median duration on dialysis of 24 months. The median age at transplant was 63 years, with 50% patients receiving live-donor renal transplantation. At the time of initial diagnosis, five of the six patients were c-ANCA/PR3 positive, one patient was p-ANCA/MPO positive, and all were antiGBM positive. At the time of transplant, all patients were anti-GBM antibody negative; one patient was ANCA positive and remaining were ANCA negative. Four of the six patients received antibody induction therapy with antithymocyte globulin. None of the patients experienced delayed graft function. All patients were maintained on maintenance immunosuppression with mycophenolate mofetil, prednisone, and tacrolimus. There were no episodes of allograft rejection. There were no episodes of vasculitis relapse after transplantation clinically. Four of the six patients were tested routinely for ANCA and anti-GBM antibody after transplantation. The anti-GBM antibody remained negative in all four patients at 1 year after transplantation. One patient who was ANCA positive at the time of transplant remained ANCA positive after transplantation and the remaining three were ANCA negative after transplantation. One patient developed BK viremia, and there was one patient with BK viruria after transplantation. The median serum creatinine level was 1.0 mg/dL at 12 months, 0.9 mg/dL at 24 months, and 1.05 mg/dL at last follow-up. Table 1 summarizes posttransplantation outcomes for all patients. The prevalence of double-positive disease is 5% to 15% in patients with AAV (1Y4) and 20% to 40% in patients with anti-GBM disease (2, 5). The relationship between the ANCA and anti-GBM antibody in these patients is not known, but at least in some patients, ANCA has been shown to precede development of anti-GBM antibody (6). Clinically, some of these patients have multisystem presentation similar to vasculitis. The renal prognosis of these patients is poor. Our report shows favorable prognosis of our six patients that have undergone renal transplantation. The two major types of AAV, granulomatosis with polyangiitis and microscopic polyangiitis are the commonest cause of rapidly progressive renal failure. Renal transplantation for ESRD caused by AAV is safe with very low vasculitis relapse rates after transplantation with the currently used modern immunosuppressive agents (7). AntiGBM antibody disease may progress to ESRD, requiring transplantation. Recurrent anti-GBM antibody disease in the renal allograft is rare and is believed to be because of delay in transplantation until the disease has been quiescent and the antibody has been undetectable for 12 months (8Y10). Our six patients with doublepositive disease showed immediate graft function after transplantation. All were treated with mycophenolate mofetil, prednisone, and tacrolimus with four of six receiving antibody induction therapy. There were no episodes of allograft rejection and no episodes of vasculitis relapse. Two patients developed BK virus replication, one in urine and one in plasma. There was no graft loss at a median followup of 66.5 months. We conclude that renal transplantation is a safe and effective for patients with double-positive disease. The modern immunosuppressive drugs are effective in preventing vasculitis relapses.