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Allo-HSCT with CCR5 Delta 32/Delta 32 donor cells is the only curative HIV-1 intervention. We investigated the impact of allo-HSCT on the viral reservoir in PBMCs and post-mortem tissue in two patients. IciS-05 and IciS-11 both received a CCR5 Delta 32/Delta 32 allo-HSCT. Before allo-HSCT, ultrasensitive HIV-1 RNA quantification; HIV-1-DNA quantification; co-receptor tropism analysis; deep-sequencing and viral characterization in PBMCs and bone marrow; and post-allo-HSCT, ultrasensitive RNA and HIV-1-DNA quantification were performed. Proviral quantification, deep sequencing, and viral characterization were done in post-mortem tissue samples. Both patients harbored subtype B CCR5-tropic HIV-1 as determined genotypically and functionally by virus culture. Pre-allo-HSCT, HIV-1-DNA could be detected in both patients in bone marrow, PBMCs, and T-cell subsets. Chimerism correlated with detectable HIV-1-DNA LTR copies in cells and tissues. Post-mortem analysis of IciS-05 revealed proviral DNA in all tissue biopsies, but not in PBMCs. In patient IciS-11, who was transplanted twice, no HIV-1-DNA could be detected in PBMCs at the time of death, whereas HIV-1-DNA was detectable in the lymph node. In conclusion, shortly after CCR5 Delta 32/Delta 32, allo-HSCT HIV-1-DNA became undetectable in PBMCs. However, HIV-1-DNA variants identical to those present before transplantation persisted in post-mortem-obtained tissues, indicating that these tissues play an important role as viral reservoirs.

Despite the application of allogeneic stem cell transplantation (SCT) since 1957 we have not been able to eradicate Graft versus Host Disease (GVHD) as a significant contributor of treatment related mortality (TRM) and morbidity. In the last years, much progress has been made in unravelling the complex pathophysiology of GVHD. These insights have led to new therapies and will hopefully further contribute to complete eradication of GVHD. Unfortunately we are not there yet and as hematologists we still have to counsel our patients about the complex risks involving allogeneic SCT. In this thesis we conducted two clinical trials and analyzed data, clinical parameters as well as soluble and cellular markers, to provide insight into the pathophysiology of GVHD. Our clinical study with Mesenchymal Stromal Cells (MSC) in steroid refractory acute GVHD patients showed a complete response rate of 50% which associates with superior overall survival. Patients treated with MSC derived from very young bone marrow donors also show improved overall survival compared to patients treated with older bone marrow donors. In patients with sclerotic chronic GVHD we tested the new combination of rituximab and nilotinib that showed promising results with response rates of 71% including 2 patients achieving a complete response. Therapeutic drug monitoring of nilotinib concentrations could possibly further enhance this effect. The combination strategy of B-cell depletion and tyrosine kinase inhibition has already been adopted in current clinical practice.

The increased understanding of the human immune system and its ability to detect and eradicate tumor cells has led to a promising wave of novel therapeutics which have the potency to become the new standard of care. This thesis aims to contribute to the development of novel cell therapy products and the GMP manufacturing thereof.

An allogeneic hematopoietic stem cell transplantation is the only curative treatment option for children suffering from chemo-refractory leukemia or inborn errors such as metabolic diseases. During this therapy, the diseased bone marrow cells of the patient are replaced by stem cells from a healthy donor. After infusion of the donor stem cells, these cells find their way into the bone marrow of the patient, where they engraft and start differentiating into new immune cells. This essential process is called immune reconstitution. Since the first successful transplantation in 1968, the treatment of patients with this therapy has drastically improved over the last years. Despite of this, complications in this risky procedure are still common, causing 30-50% of patients to die after transplantation. This is why it is important to better understand what causes these complications, and how to better predict them. During my research, I found that children with a delayed immune reconstitution after their transplantation had much more complications and lower chance of survival. In particular a fast and adequate reconstitution of T-cells, the so-called soldiers of the immune system, is essential for lowering complications such as virus infections, graft-versus-host disease, and leukemia relapse. My research also describes different factors that affect the speed of T-cell reconstitution. These novel findings enable improvement of the treatment of patients who undergo a stem cell transplantation, which are expected to drastically enhance the survival chances of future transplantation recipients.

Adenoviruses (HAdV) can cause serious life threatening infections, especially in pediatric patients after allogeneic stem cell transplantation (SCT). To improve the morbidity and mortality rate of HAdV infected patients after SCT three issues have been explored: which patients are at risk for HAdV infection, what is the best treatment for HAdV infected patients and when do we start treatment. We found that in pediatric patients HAdV-DNA positivity in nasopharyngeal aspirate before SCT is a very important risk factor for developing invasive HAdV infection after SCT. As determined with multiplex immuno-assay we also found that children with a localized HAdV infection have a different cytokine profile compared with patients with an invasive HAdV infection after SCT. Cytokine signatures can also be helpful to differentiate invasive HAdV infection from other SCT related complications as Graft versus Host Disease (GvHD) and Epstein-barr virus infection. Immune profiling could possibly help to identify patients at high risk in an earlier stage. Because no effective medication for HAdV infection exists and HAdV-specific T-cells are critically involved in virus elimination, an alternative treatment option is the adoptive transfer of HAdV-specific T-cells from the graft donor into the recipient. One of the potential risks of using HAdV-specific T-cells for adoptive therapy is the risk for GvHD. The use of a high purity of HAdV-specific CD4+ T-cells would strongly reduce this risk. By using a computer algorithm designed to predict pan HLA-DR binding T-cell epitopes we detected six 15-mer epitopes from HAdV. These epitopes induce a proliferative T-cell response in the majority of healthy controls. By culturing peripheral blood mononuclear cells (PBMCs) of healthy donors with identified HAdV epitopes it is possible to generate CD4+ HAdV-specific T-cells. With artificial antigen presenting cells, composed of liposomes harboring a HAdV-peptide and MHC-class II complex, these HAdV-specific T-cells could be isolated. Isolated HAdV CD4+ specific T-cells are fully functional, produce pro-inflammatory cytokines, express the cytotoxins perforin and granzyme B, kill HAdV-infected cells effectively and do not show alloreactivity. Because umbilical cord blood transplants (UCB) emerge as an alternative stem cell source for SCT, and immune reconstitution is delayed in patients receiving UCB, higher infection rates with HAdV are expected to emerge. In case of HAdV infection after UCB transplantation adoptive therapy with HAdV-specific T-cells derived from cord blood seems also a promising treatment option. However, the induction of HAdV-specific T-cells from UCB requires the priming and expansion of naive T-cells rather than expansion of pre-existing virus-specific memory T-cells. By stimulating cord blood mononuclear cells with 15-mer peptides we showed that HAdV-specific T-cells could be generated. The HAdV-specific T-cells produce pro-inflammatory cytokines, perforin and granzyme B, all necessary for viral elimination. The generation of HAdV-specific T-cells in response to HAdV peptides seems an essential step in developing tailor-made therapy for HAdV infection for recipients of SCT transplants. In the near future probably high-risk patients may be treated prophylactically with adoptive transfer of HAdV-specific T-cells, possibly in association with antiviral medication. These new approaches hopefully will lead to reduction in incidence and mortality rate associated with HAdV infection after SCT.