Matthias Wölfl

Priming CD8+ T cells with dendritic cells matured using TLR4 and TLR7/8 ligands together enhances generation of CD8+ T cells retaining CD28

TLRs expressed on dendritic cells (DCs) differentially activate DCs when activated alone or in combination, inducing distinct cytokines and costimulatory molecules that influence T-cell responses. Defining the requirements of DCs to program T cells during priming to become memory rather than effector cells could enhance vaccine development. We used an in vitro system to assess the influence of DC maturation signals on priming naive human CD8+ T cells. Maturation of DCs with lipopolysaccharide (LPS; TLR4) concurrently with R848 (TLR7/8) induced a heterogeneous population of DCs that produced high levels of IL12 p70. Compared with DCs matured with LPS or R848 alone, the DC population matured with both adjuvants primed CD8+ T-cell responses containing an increased proportion of antigen-specific T cells retaining CD28 expression. Priming with a homogenous subpopulation of LPS/R848-matured DCs that were CD83(Hi)/CD80+/CD86+ reduced this CD28+ subpopulation and induced T cells with an effector cytokine signature, whereas priming with the less mature subpopulations of DCs resulted in minimal T-cell expansion. These results suggest that TLR4 and TLR7/8 signals together induce DCs with fully mature and less mature phenotypes that are both required to more efficiently prime CD8+ T cells with qualities associated with memory T cells.

Primed tumor-reactive multifunctional CD62L+ human CD8+T-cells for immunotherapy

T cell-mediated immunotherapy against malignancies has been shown to be effective for certain types of cancer. However, ex vivo expansion of tumor-reactive T cells has been hindered by the low precursor frequency of such cells, often requiring multiple rounds of stimulation, resulting in full differentiation, loss of homing receptors and potential exhaustion of the expanded T cells. Here, we show that when using highly purified naïve CD8+ T cells, a single stimulation with peptide-pulsed, IFNγ/LPS-matured dendritic cells in combination with the sequential use of IL-21, IL-7 and IL-15 is sufficient for extensive expansion of antigen-specific T cells. Short-term expanded T cells were tumor-reactive, multifunctional and retained a central-memory-like phenotype (CD62L+, CCR7+, CD28+). The procedure is highly reproducible and robust as demonstrated for different healthy donors and for cancer patients. Such short-term tumor-antigen-primed, multifunctional T cells may therefore serve as a platform to target different malignancies accessible to immunotherapy.

The TGF‐β‐inducible miR‐23a cluster attenuates IFN‐γ levels and antigen‐specific cytotoxicity in human CD8+ T cells

Cytokine secretion and degranulation represent key components of CD8+ T‐cell cytotoxicity. While transcriptional blockade of IFN‐γ and inhibition of degranulation by TGF‐β are well established, we wondered whether TGF‐β could also induce immune‐regulatory miRNAs in human CD8+ T cells. We used miRNA microarrays and high‐throughput sequencing in combination with qRT‐PCR and found that TGF‐β promotes expression of the miR‐23a cluster in human CD8+ T cells. Likewise, TGF‐β up‐regulated expression of the cluster in CD8+ T cells from wild‐type mice, but not in cells from mice with tissue‐specific expression of a dominant‐negative TGF‐β type II receptor. Reporter gene assays including site mutations confirmed that miR‐23a specifically targets the 3′UTR of CD107a/LAMP1 mRNA, whereas the further miRNAs expressed in this cluster—namely, miR‐27a and ‐24—target the 3′UTR of IFN‐γ mRNA. Upon modulation of the miR‐23a cluster by the respective miRNA antagomirs and mimics, we observed significant changes in IFN‐γ expression, but only slight effects on CD107a/LAMP1 expression. Still, overexpression of the cluster attenuated the cytotoxic activity of antigen‐specific CD8+ T cells. These functional data thus reveal that the miR‐23a cluster not only is induced by TGF‐β, but also exerts a suppressive effect on CD8+ T‐cell effector functions, even in the absence of TGF‐β signaling.

TH1 predominance is associated with improved survival in pediatric medulloblastoma patients

Medulloblastoma, a primitive neuro-ectodermal tumor that arises in the posterior fossa, is the most common malignant brain tumor occurring in childhood. Even though 60–70% of children with medulloblastoma will be cured with intensive multimodal therapy, including surgery, radiotherapy, and chemotherapy, a significant proportion of surviving patients may suffer from long-term treatment-related sequelae. Therapeutic success is limited especially in younger children by radiotherapy-induced neurocognitive longterm deficits. In order to avoid or delay craniospinal radiotherapy, high-dose chemotherapy followed by autologous stem cell transplantation (HSCT) has become an established treatment modality. Data on the host immunologic environment in medulloblastoma patients are rare, notably data on cytokine expression and immune reconstitution in patients with medulloblastoma undergoing HSCT are lacking. In this present study, we therefore decided to prospectively assess immune function following 24 consecutive autologous HSCT in 17 children with medulloblastoma treated according to the German-Austrian-Swiss HIT-2000-protocol. TH1 predominance was found to be the most important factor for probability of survival. Already before HSCT, survivors showed higher IFNγ levels in sera as well as higher numbers of IFNγ-positive T-cells. After transplantation, this effect was even more pronounced. Patients with higher numbers of IFNγ- and TNFα-positive T-cells had a more favorable outcome at all analyzed time points. In addition, patients in complete remission (CR) before transplantation, known to have a better prognosis a priori, showed higher expression of IFNγ in T-cells. Taken together, this is the first report to demonstrate that high expression of IFNγ and TNFα in T-cells of medulloblastoma patients in the early post-transplant period correlates with a better prognosis. Our data point toward a potentially important influence of TH1-cytokine expression before and after transplantation on the survival of pediatric medulloblastoma patients.

Src-kinase inhibitors sensitize human cells of myeloid origin to Toll-like-receptor-induced interleukin 12 synthesis.

Src-kinase inhibitors hold great potential as targeted therapy against malignant cells. However, such inhibitors may also affect nonmalignant cells and cause pronounced off-target effects. We investigated the role of the dual kinase inhibitor dasatinib on human myeloid cells. Dasatinib is clinically used for the treatment of bcr/abl⁺ leukemias because it blocks the mutated tyrosine kinase abl. To understand its effect on the development of antigen-specific T-cell responses, we assessed antigen-specific priming of human, naïve T cells. In surprising contrast to the direct inhibition of T-cell activation by dasatinib, pretreatment of maturing dendritic cells (DCs) with dasatinib strongly enhanced their stimulatory activity. This effect strictly depended on the activating DC stimulus and led to enhanced interleukin 12 (IL-12) production and T-cell responses of higher functional avidity. Src-kinase inhibitors, and not conventional tyrosine kinase inhibitors, increased IL-12 production in several cell types of myeloid origin, such as monocytes and classical or nonclassical DCs. Interestingly, only human cells, but not mouse or macaques DCs, were affected. These data highlight the potential immunostimulatory capacity of a group of novel drugs, src-kinase inhibitors, thereby opening new opportunities for chemoimmunotherapy. These data also provide evidence for a regulatory role of src kinases in the activation of myeloid cells.

Successful treatment of an infant with CDA type II by intrauterine transfusions and postnatal stem cell transplantation

Congenital dyserythropoietic anemias are rare hematological disorders leading to ineffective erythropoiesis with chronic anemia, complicated by iron overload. Here we present a remarkable clinical course of an infant with CDA type II who first presented as a severe fetal hydrops, requiring serial intrauterine red cell transfusions. While postnatal transfusion dependency persisted, the patient was successfully transplanted with a myeloablative conditioning regimen and peripheral blood stem cells of a matched donor. We believe that allogeneic HSCT is a reasonable therapeutic approach for patients with very severe CDA, even if only a matched unrelated donor is available. Pediatr Blood Cancer 2014;61:743–745.

Pre‐differentiated human committed T‐lymphoid progenitors promote peripheral T‐cell re‐constitution after stem cell transplantation in immunodeficient mice

T‐cell re‐constitution after allogeneic stem cell transplantation (alloSCT) is often dampened by the slow differentiation of human peripheral blood CD34+ (huCD34+) hematopoietic stem cells (HSCs) into mature T cells. This process may be accelerated by the co‐transfer of in vitro‐pre‐differentiated committed T/NK‐lymphoid progenitors (CTLPs). Here, we analysed the developmental potential of huCD34+ HSCs compared with CTLPs from a third‐party donor in a murine NOD‐scid IL2Rγnull model of humanised chimeric haematopoiesis. CTLPs (CD34+lin−CD45RA+CD7+) could be generated in vitro within 10 days upon co‐culture of huCD34+ or cord blood CD34+ (CB‐CD34) HSCs on murine OP9/N‐DLL‐1 stroma cells but not in a novel 3‐D cell‐culture matrix with DLL‐1low human stroma cells. In both in vitro systems, huCD34+ and CB‐CD34+ HSCs did not give rise to mature T cells. Upon transfer into 6‐wk‐old immune‐deficient mice, CTLPs alone did not engraft. However, transplantation of CTLPs together with huCD34+ HSCs resulted in rapid T‐cell engraftment in spleen, bone marrow and thymus at day 28. Strikingly, at this early time point mature T cells originated exclusively from CTLPs, whereas descendants of huCD34+ HSCs still expressed a T‐cell‐precursor phenotype (CD7+CD5+CD1a+/−). This strategy to enhance early T‐cell re‐constitution with ex vivo‐pre‐differentiated T‐lymphoid progenitors could bridge the gap until full T‐cell recovery in severely immunocompromised patients after allogeneic stem cell transplantation.

Fulminant skin GvHD with a cytokine pattern resemblant of cytokine release syndrome successfully treated with multimodal immunosuppression including tocilizumab

Acute Graft‐versus‐Host‐Disease (GvHD) is a potentially life‐threatening complication after allogeneic stem cell transplantation. If not treated early and adequately, the complex immunological mechanisms may lead to a self‐perpetuating cycle of alloreactivity, which is then associated with a high mortality. Here we assessed the cytokine profile on a daily basis in a patient with grade 4 skin GvHD, demonstrating a signature resembling cytokine‐release‐syndrome. After multimodal immunosuppressive intervention, including treatment with the IL6 receptor‐blocking antibody tocilizumab, the severe clinical symptoms unexpectedly resolved within 48hr. Pediatr Blood Cancer

Lower TGFß serum levels and higher frequency of IFNγ-producing T cells during early immune reconstitution in surviving children after allogeneic stem cell transplantation†

Allogeneic hematopoietic stem cell transplantation (SCT) is increasingly used as a salvage therapy for patients with high‐risk malignancies as well as life‐threatening non‐malignant diseases. However, only limited data about the association between outcome and functional parameters of recovering lymphocytes are available so far.

High-grade glioma associated immunosuppression does not prevent immune responses induced by therapeutic vaccines in combination with Treg depletion

High-grade gliomas (HGG) exert systemic immunosuppression, which is of particular importance as immunotherapeutic strategies such as therapeutic vaccines are increasingly used to treat HGGs. In a first cohort of 61 HGG patients we evaluated a panel of 30 hematological and 34 plasma biomarkers. Then, we investigated in a second cohort of 11 relapsed HGG patients receiving immunomodulation with metronomic cyclophosphamide upfront to a DC-based vaccine whether immune abnormalities persisted and whether they hampered induction of IFNγ+ T-cell responses. HGG patients from the first cohort showed increased numbers of leukocytes, neutrophils and MDSCs and in parallel reduced numbers of CD4+/CD8+u2009T-cells, plasmacytoid and conventional DC2s. MDSCs and T-cell alterations were more profound in WHO IV° glioma patients. Moreover, levels of MDSCs and epidermal growth factor were negatively associated with survival. Serum levels of IL-2, IL-4, IL-5 and IL-10 were altered in HGG patients, however, without any impact on clinical outcome. In the immunotherapy cohort, 6-month overall survival was 100%. Metronomic cyclophosphamide led to >u200940% reduction of regulatory T cells (Treg). In parallel to Treg-depletion, MDSCs and DC subsets became indistinguishable from healthy controls, whereas T-lymphopenia persisted. Despite low T-cells, IFNγ-responses could be induced in 9/10 analyzed cases. Importantly, frequency of CD8+VLA-4+ T-cells with CNS-homing properties, but not of CD4+u2009VLA-4+u2009T-cells, increased during vaccination. Our study identifies several features of systemic immunosuppression in HGGs. Metronomic cyclophosphamide in combination with an active immunization alleviates the latter and the combined treatment allows induction of a high rate of anti-glioma immune responses.