Gillian Dao Nyesiga

This study investigated the tolerance induction and disruption of immune tolerance as strategies for immune modulation. For induction of immune tolerance, novel monocyte-derived tolerogenic dendritic cells (ItolDCs) were generated, and their ability to modulate the immune system was assessed using in vitro assays in hemophilia A patients who had developed neutralizing antibodies against their factor VIII replacement therapy. The cells were characterized, their functionality was assessed, and their feasibility as a safe cell therapy was further evaluated using both in vitro and in vivo studies for the induction of immune tolerance against factor VIII. For research on disruption of immune tolerance, meningioma, the most common brain tumor, was studied. To map the immune cell composition in meningiomas, a protocol was optimized for shorter enzymatic digestion, which breaks down the tissue into single-cell suspensions of viable immune cells. Since CD8+ T cells are vital in tumor suppression, further studies were conducted to explore their characteristics and identify possible targetable processes for immunotherapy. To investigate both induction and disruption of immune tolerance, various techniques were employed, including flow cytometry, immunohistochemistry, and functional-cell-based assays. Our investigation demonstrated that ItolDCs are a feasible and safe option for cell therapy aimed at inducing immune tolerance. Thus, factor VIII-loaded ItolDCs are ready for clinical evaluation to reduce inhibitor levels in patients with hemophilia A. Several tolerance-associated markers (PD-1, TIM-3, TIGIT, and LAG-3) were identified in CD8+ T cells in meningioma. These findings highlight how tumor cells may evade immune defenses and suggest potential immunotherapeutic targets, including immune checkpoint inhibitors. Taken together, various approaches may be employed for immune modulations to either induce or disrupt immune tolerance.

Background: Current challenges in meningioma treatment, including post-surgical complications and cognitive impairments, highlight the need for new treatment alternatives. Immunological interventions have shown promise. However, there is a knowledge gap in characterizing infiltrating immune cells in meningioma and their interplay. Further studies on immune cells in single-cell suspensions from digested meningioma tissues could identify targetable mechanisms for non-surgical treatment options with fewer side effects. This study aimed to optimize a protocol for faster digestion of meningioma tissues into viable single-cell suspensions and to identify infiltrating immune cell populations. Methods: We modified a commercial kit intended for whole skin dissociation to digest resected meningioma tissues into viable single-cell suspensions. Tumor-infiltrating immune cell populations were characterized using flow cytometry. Results: Flow cytometry analyses revealed that the digested tissue was composed of viable immune cells, including predominantly CD14⁺ macrophages and CD3⁺ T cells, with minor populations of CD56⁺ NK cells and CD19⁺ B cells. In both of the two patient samples tested, half of the tumor-associated macrophages were TIM-3⁺, with a small proportion co-expressing CD83. Women were more likely to have a lower proportion of immune cells, B cells, and NK cells. Female patients with a high proportion of immune cells had a higher proportion of macrophages. Conclusion: We successfully optimized a protocol for generating single-cell suspensions with viable immune cells from meningioma tissues, revealing infiltrating antigen-presenting cells with an immunosuppressive phenotype, and lymphocytes. This short protocol allows advanced analyses of tumor-infiltrating cells using techniques such as single-cell RNA sequencing and flow cytometry, which require live, dissociated cells.

Dendritic cells (DCs) are mediators between innate and adaptive immunity and vital in initiating and modulating antigen-specific immune responses. The most important site for induction of tolerance is the gut mucosa, where TGF-β, retinoic acid, and aryl hydrocarbon receptors collaborate in DCs to induce a tolerogenic phenotype. To mimic this, a novel combination of compounds – the synthetic aryl hydrocarbon receptor (AhR) agonist IGN-512 together with TGF-β and retinoic acid – was developed to create a platform technology for induction of tolerogenic DCs intended for treatment of several conditions caused by unwanted immune activation. These in vitro-generated cells, designated ItolDCs, are phenotypically characterized by their low expression of co-stimulatory and activating molecules along with high expression of tolerance-associated markers such as ILT3, CD103, and LAP, and a weak pro-inflammatory cytokine profile. When co-cultured with T cells and/or B cells, ItolDC-cultures contain higher frequencies of CD25+Foxp3+ regulatory T cells (Tregs), CD49b+LAG3+ ‘type 1 regulatory (Tr1) T cells, and IL-10-producing B cells and are less T cell stimulatory compared to cultures with matured DCs. Factor VIII (FVIII) and tetanus toxoid (TT) were used as model antigens to study ItolDC antigen-loading. ItolDCs can take up FVIII, process, and present FVIII peptides on HLA-DR. By loading both ItolDCs and mDCs with TT, antigen-specific T cell proliferation was observed. Cryo-preserved ItolDCs showed a stable tolerogenic phenotype that was maintained after stimulation with LPS, CD40L, or a pro-inflammatory cocktail. Moreover, exposure to other immune cells did not negatively impact ItolDCs’ expression of tolerogenic markers. In summary, a novel protocol was developed supporting the generation of a stable population of human DCs in vitro that exhibited a tolerogenic phenotype with an ability to increase proportions of induced regulatory T and B cells in mixed cultures. This protocol has the potential to constitute the base of a tolDC platform for inducing antigen-specific tolerance in disorders caused by undesired antigen-specific immune cell activation.