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Primary antibody deficiency Primary antibody deficiencies (PAD) encompass various disorders characterized by reduced antibody production and are a common form of primary immunodeficiency. They include specific polysaccharide antibody deficiency (SPAD), immunoglobulin G subclass deficiency (IgSD), common variable immunodeficiency (CVID), and agammaglobulinemia (XLA). B cell dysfunction is a key feature of PAD, leading to recurrent respiratory and gastrointestinal infections due to compromised mucosal protection. Although preventive measures such as vaccination, prophylactic antibiotics, and immunoglobulin replacement therapy (IRT) have been effective in reducing infectious complications, some sequelae, including bronchiectasis, hearing loss, and malnutrition, still occur in PAD patients. Regular pulmonary screening is recommended, and further research is needed to optimize the use of IRT and prophylactic antibiotics in specific types of PAD and identify high-risk patients for bronchiectasis. Non-infectious complications contribute significantly to the morbidity and mortality in CVID patients, despite receiving appropriate IRT. These complications affect multiple organ systems and include enteropathy, granulomatous lymphocytic interstitial lung disease (GLILD), and secondary hemophagocytic lymphohistiocytosis (HLH). Enteropathy, characterized by gastrointestinal inflammation, affects a significant proportion of CVID patients but lacks effective treatment options. GLILD, observed in a subset of CVID patients, leads to respiratory symptoms, and treatment guidelines are currently insufficient. HLH, a life-threatening immune dysregulation syndrome, can be triggered by severe infections or non-infectious complications in CVID patients, necessitating early intervention, although its diagnosis remains challenging due to non-specific criteria and delays in obtaining the necessary criteria. Genetic studies have revealed that the genetic landscape of PAD is diverse, with over 40 genes identified as causative or associated with PAD using next-generation sequencing. These genes are involved in critical immunological signaling pathways and B cell development, highlighting the intricate interactions required for proper B cell maturation and immune function. However, monogenic causes are only identified in a small percentage of cases, underscoring the complex nature of PAD. T cell dysfunction has also been implicated in PAD, extending beyond the B cell abnormalities typically associated with the condition. Specific monogenetic PAD-like diseases, as well as CVID patients without monogenetic disease experiencing non-infectious complications, demonstrate T cell dysregulation. Increased effector memory T cells and elevated expression of IFN-γ response genes have been observed in CVID patients with noninfectious complications. Additionally, dysregulated T cells and elevated IFN-γ production contribute to the activation of monocytes, leading to the production of B cell activating factor of the TNF family (BAFF). BAFF has been linked to the pathogenesis of non-infectious complications, particularly in GLILD patients. To address the knowledge gaps in PAD diagnosis and management, this thesis aims to improve our understanding of the genetic landscape, identify optimal strategies for preventing infectious complications, and develop treatment approaches for non-infectious complications. The thesis highlights the need for larger cohort studies and randomized clinical trials to establish evidence-based recommendations. Additionally, identifying predictive risk factors for specific complications, such as bronchiectasis and progressive GLILD, is crucial for effective screening and treatment decisions. Given the role of T cell functionality in non-infectious complications, the thesis also focuses on exploring methods to normalize T cell function through clinical and preclinical assays. Overall, the research conducted in this thesis strives to advance our knowledge of PAD, enhance our understanding of T cell dysfunction, and identify strategies to improve the management of infectious and non-infectious complications associated with PAD.
Part 1: Diagnosis and pathophysiology of PAD
The first part of this thesis aims to further unravel the hampered crosstalk between B and T cells in PAD by studying the functional T and B cell profiles of patients with a known monogenetic disease and patients with newly discovered genes potentially associated with PAD. In chapter 2, we report on a new dominant activating variant in the RAC2 gene, RAC2-E62K, that leads to constitutively active RAC2. RAC2 plays an important role in actin polymerization in immune cells.9 Remodeling of the actin skeleton is vital for important immune cell functions like cell division, phagocytosis and cell migration.10 Previously, dominant negative mutations in RAC2 have been shown to mainly cause phagocyte disorders.11 Here, we and others use patient material, cellular models and a mouse model to show that the E62K variant causes constitutively active RAC2. Constitutively active RAC2 not only leads to a phagocyte disorder, but also to T cell and, potentially, B cell dysfunction. In chapter 3, we report on new dominant negative mutations in the DNA binding domain of c-Myb, a transcription factor involved in T and B cell development.13,14 This report discusses two patients who exhibited combined immunodeficiency-like symptoms and bone marrow failure. Both carried dominant negative mutations that potentially limit the DNA binding capacity of c-Myb, preventing transcription of important target genes and leading to T and B cell dysfunction. This study showed defective CD8+ cell signaling in one affected patient potentially caused by his specific c-Myb variant. This report provides the first evidence that c-Myb might potentially be a candidate gene for PAD. In chapter 4, we compare the peripheral immune phenotype of primary immunoregulatory disorders (PIRD) subtypes associated with PAD-like disease and explore the intestinal immune phenotype. Peripheral T cell subsets were found to distinguish between PIRD subgroups, while intestinal T cells showed signs of limited infiltration, activation, and maturation. The study confirmed some previously observed differences in PIRD, but also showed conflicting results compared to previous studies, highlighting the variable immune phenotype within PIRD subgroups. Our findings show that peripheral T cells potentially play a vital role in PIRD and that their activation status, exhaustion status and homing status can potentially be used to distinguish between PIRD subtypes. Our findings also suggest that the intestinal T cell compartment may not be as important in intestinal pathology in PIRD as previously believed, suggesting the need for studying other intestinal immune subsets. In chapter 5, we explore an in vitro T cell assay for predicting individual treatment responses in common variable immunodeficiency with complications (CVIDc). The results show that T cell proliferation induced by aCD3 stimulation can be inhibited by immunosuppressants, and the effectiveness of inhibition differs among CVID subgroups. Intracellular cytokine production, however, was not a reliable predictor of clinical response. Our results suggest that combined T cell subset proliferation could moderately predict clinical response.
Part 2: Management of PAD
The second part of this thesis aims to give new insights in the prevention of infectious complications and the management of non-infectious complications in PAD. To this aim we used retrospective, prospective and randomized clinical studies to further elucidate risk factors associated with a poor outcome of infectious and non-infectious-complications and compare effectivity of treatment regimens that prevent infectious and non-infectiouscomplications. In chapter 6, we study potential risk factors for airway disease and airway disease related bronchiectasis in patients with PAD that are treated with IRT. Here we find that airway disease and airway disease related bronchiectasis are stable in most patients during a follow-up of ten years. This study also shows that patients with XLA, patients with recurrent infections and patients with pre-existent airway disease are more at risk for progression of airway disease. Prophylactic antibiotics were potentially given more frequently to patients with airway disease progression, but did not manage to halt the progression. In chapter 7, we compare the efficacy and safety of prophylactic antibiotics and immunoglobulin replacement therapy (IRT) in patients with IgSD and SPAD. Both treatment regimens have previously been reported to be effective in these milder forms of PAD.26 In this study these treatment regimens were equally effective. Moreover, patients treated with prophylactic antibiotics suffered less adverse events and prophylactic antibiotics are easier to administer and less costly. Based on these findings, we recommend prophylactic antibiotics as first-line treatment for these milder forms of PAD. Importantly, we also found that a subgroup of patients still suffered from recurrent infections, despite treatment with prophylactic antibiotics. We found that IRT could reduce the infectious burden in these patients. We could not identify other clinical characteristics that could distinguish patients with milder forms of PAD that did not respond to prophylactic antibiotics. In chapter 8, we elaborate on clinical signs, symptoms, and the natural course of granulomatous-lymphocytic interstitial lung disease (GLILD). The study confirms that GLILD is associated with an increased female/male ratio, splenomegaly, lymphadenopathy, autoimmune cytopenia, hepatopathy, enteropathy, reduced switched memory B cells, increased CD21low B cells, and reduced CD4+ naive cells. Without treatment, the majority of GLILD patients experience progression. A poor clinical outcome, defined as severely impaired pulmonary function tests, pulmonary hypertension, or oxygen dependency, was observed in 47% of patients. Hepatopathy, high ILD scores on baseline CT, and high NK cell counts can potentially serve as clinical markers to identify patients at risk for a poor clinical outcome, guiding early treatment initiation.
In chapter 9 we provide a systematic review of the existing evidence on treatment options for GLILD. The majority of evidence consists of case reports and small cohort studies, with a lack of well-controlled studies with quantitative outcome measures. However, studies have reported qualitative or quantitative outcomes for treatment with corticosteroids, rituximab monotherapy, and rituximab combined with azathioprine or mycophenolate mofetil. Rituximab, both as monotherapy and in combination, showed qualitative improvements in pulmonary function tests, radiological findings, and clinical symptoms in 75% to 95% of cases. Combination therapy appeared to be more effective, with more frequent reporting in the literature. Corticosteroid therapy showed limited efficacy (27%), possibly due to reporting bias and the assumption of its effectiveness as first-line treatment. In chapter 10 we focus on the efficacy and safety of corticosteroids as a first-line treatment for GLILD. This study shows that corticosteroids can induce a long-lasting remission in 42% of patients, and side effects and infections were primarily associated with prolonged exposure to corticosteroids during maintenance therapy. Short-term treatment, consisting of an induction phase and tapering phase, was equally effective in maintaining remission as long-term therapy with maintenance therapy. Given the ability of short-term corticosteroid treatment to maintain long-lasting remission with minimal serious side effects, the study proposes it as the first-line treatment. Combination therapy with rituximab, although more effective, has less known safety data and is considered a good candidate for second-line treatment when corticosteroids fail. In chapter 11, we used a retrospective analysis of a discovery and a validation cohort, to assess the diagnostic value of the HLH-2004 criteria for early identification of the HLH syndrome. By utilizing statistical methods such as hierarchical cluster analysis and principal component analysis, this study identified a parameter set that could exclude HLH syndrome and a minimal parameter set consisting of major and minor criteria for positive identification. The minimal parameter set, including two major criteria (hemophagocytosis and splenomegaly) and three minor criteria (cytopenia, increased ferritin, and increased triglycerides/low fibrinogen), predicted HLH with high sensitivity (95%) and specificity (94%). Implementing this approach could improve patient outcomes by enabling rapid diagnosis and treatment of HLH syndrome.
In this thesis, we present important clinical recommendations for the diagnosis, monitoring, and treatment of primary antibody deficiencies (PAD). Based on our findings and recent research in the field, we propose several key recommendations. Firstly, we recommend that all PAD patients should be offered next-generation sequencing (NGS) based panel diagnostics to improve the diagnostic yield. While NGS has its limitations, a genetic diagnosis can have a significant clinical impact, leading to changes in management for a majority of patients. To facilitate the identification of relevant genetic variants affecting multiple individuals, we propose the development of an automated international platform that connects clinicians and researchers. This platform would allow for the efficient sharing and detection of variants of unknown significance and (likely) pathogenic variants, accelerating research on new monogenetic causes of PAD.
Secondly, we emphasize the heterogeneity of PAD and the need for individualized treatment approaches. To aid clinicians in selecting optimal treatments, we suggest the use of in vitro assays such as targeted proteomics and advanced immunophenotyping. These assays can help us identify potential responders to new immunomodulators, allowing for personalized treatment options. Additionally, they can be used to confirm the efficacy of these immunomodulators, enabling their compassionate use in PAD patients. Thirdly, we highlight the importance of intensive monitoring and proactive measures for patients at risk of recurrent infections and infectious complications. This may include considering prophylactic antibiotics and higher target trough levels of immunoglobulin replacement therapy (IRT). Patients with preexistent bronchiectasis and ongoing infections, despite adequate trough levels, may benefit from further raising the target trough levels. Furthermore, we believe that efforts should be made to reduce the diagnostic delay of PAD, including the development of predictive algorithms that can identify high-risk patients in first-line care. In our research, we find that X-linked agammaglobulinemia (XLA) patients are particularly susceptible to airway disease. We propose exploring alternative treatment options such as hematopoietic stem cell transplantation (HSCT) and gene editing for XLA patients at risk of severe airway disease. Finally, we advocate for standardized treatment approaches for granulomatous-lymphocytic interstitial lung disease (GLILD) to reduce its associated morbidity and mortality. Early treatment initiation is crucial for patients at risk of disease progression, and individualized treatment options can be considered when standard approaches fail. Future perspectives and final conclusion While providing these clinical recommendations, we acknowledge that there are still many unanswered research questions in the field of PAD. We emphasize the need for collaborative efforts, including multi-center, international collaborations involving experts in various disciplines. These collaborations would facilitate observational studies and randomized clinical trials, which are essential for obtaining higher degrees of evidence for therapeutic interventions in PAD. In conclusion, this thesis sheds light on the pathophysiological mechanisms of PAD and proposes recommendations to improve diagnosis, monitoring, and treatment. By implementing genetic diagnostics, utilizing in vitro assays for personalized treatment, and ensuring intensive monitoring and proactive measures, we can enhance the care provided to PAD patients. However, further research and collaborative efforts are necessary to address remaining knowledge gaps and advance our understanding and management of this complex disease.