**3. Epigenetics in the Immune System**

A growing body of evidence suggests that epigenetic mechanisms, including DNA methylation, play a key role in hematopoiesis, contributing to the differentiation of the hematopoietic stem cell (HSC) into different subsets of immune cells, namely towards the lymphoid and myeloid lineages. Indeed, each cell subset exhibits a unique methylation profile, with remarkable differences between the cells of the myeloid and lymphoid lineages [24].

DNA methylation is increased with lymphoid differentiation but reduced in myeloid differentiation [25] (Figure 1B). Interestingly, in humans, the inactivation of DNA demethylating enzymes TET has been associated with several myeloid malignancies, as myeloproliferation replaces cell differentiation [26].

During each step of their development, B cells undergo methylation changes in up to one third of all their genome CpGs. In the early phases of differentiation in the bone marrow, these changes are considered lineage-determining. Non-CpGs demethylation occurs upon B-cell commitment in pre-B2 cells, while CpG methylation changes in effector genes are detected in all other stages of B-cell maturation and activation after B-cell receptor stimulation by antigen binding in the spleen [27].

DNA methylation and histone acetylation are also involved in V(D)J recombination, a process that causes changes in chromatin structure and allows recombination steps through the activity of RAG1/2 enzyme, which recognizes specific signal sequences [28].

As for the T-cell compartment, when the lineage choice of T cells occurs, DNA methylation of the *Cd4* locus is required for its repression in CD8+ cells and its expression in CD4+ cells, as demonstrated in mouse models [29]. In the thymus, DNMT1 interaction with FOXP3 (Forkhead Box P3) transcription factor induces Tregs development. Tregs are a heterogeneous population of CD4-positive T cells characterized by a high expression of CD25 and a low expression of CD127 [30]. After T cell activation, active DNA demethylation is essential for interleukin-2 (IL2) synthesis and for lineage polarization into T helper-1 (Th1), Th2, and Th17 [31,32].

DNA methylation plays a critical role in CD4+ T-cell differentiation: DNMT1 loss leads to decreased peripheral T-cell proliferation and the increased expression of cytokines such as IL-2, IL-3, IL-4 and IFNγ, in activated CD4+ (and CD8+) T cells, suggesting a repressive function of DNMT1 towards cytokine production. Under TH2 polarizing conditions, DNMT1 dissociates from the IL4 locus, enabling the demethylation of the locus and the increased expression of IL-4 [33].

The shift to a memory-like phenotype induced in NK cells by some viral infections may also rely on changes in the methylome profiling of promoters of cytokines, including IL13, IL5, and IFN, which become demethylated, as observed in T-cell activation [33,34].

The role of DNA methylation machinery has also been described in the mononuclearphagocyte system during monocyte differentiation into macrophages and their polarization to a "M1" state or an anti-inflammatory "M2" phenotype, as well as in keeping the neutrophil phenotype fully differentiated [35].

Extensive mRNA expression profiling has widely demonstrated how hematopoiesis and cell lineage commitment are also accompanied and orchestrated by changes in mRNA signatures [36]. For instance, relevant steps in both T- and B-cell lymphopoiesis rely on gene regulation by specific sets of miRNA [37]. Notably, hematopoiesis also undergoes regulation by lncRNAs that stimulate the proliferation and differentiation of erythroid progenitors by targeting GATA1, TAL1 and KLF1, as well as granulocyte differentiation, thanks to HOTAIRM1, that acts as a regulator of cell cycle [38–40].

#### **4. Epigenetic Alterations in Inborn Errors of Immunity**

Since the proper establishment of DNA methylation patterns is necessary for the differentiation of cells of the immune system, the impairment of DNA methylation machinery

results in immune dysfunction and diseases. Historically known as primary immunodeficiencies, Mendelian disorders of the immune system are now referred to as Inborn Errors of Immunity (IEI), a more precise and wider definition that takes into account the traditionally known feature of increased susceptibility to infections along with remarkable immune dysregulation and/or hyperinflammation [41,42]. More than 400 genes have been included in the most recent classification of by the International Union of Immunological Sciences [43,44]. In the following sections, we review the potential involvement of epigenetic alterations in the pathogenesis of some inborn errors of immunity, whose features are summarized in Table 1.

**Table 1.** Representative gene defects causing epigenetic changes and immunological alterations within defined syndromes.


#### *4.1. Inborn Errors of Humoral Immunity*

Common Variable Immunodeficiency (CVID) is a heterogeneous group of disorders characterized by hypogammaglobulinemia and impaired response to vaccinations. CVID is characterized by marked genetic and phenotypic heterogeneity and monogenic variants have been identified in no more than 10% of patients [45]. Thus, the majority of CVID patients lack a monogenic basis and a polygenic origin may be assumed in most cases. Since a genetic diagnosis of CVID can be achieved only in a small percentage of patients [46], epigenetic alterations, such as DNA methylation and histone modifications, may be theoretically envisioned as potential mechanisms implicated in genetically undefined cases, as a few studies, described below, seem to suggest.

In the early stages of B cell differentiation, during the transition from pro-B to pre-B cells, an alteration in DNA methylation occurs, especially in intragenic and intronic regions [47] closely associated with transcription factor sites related to B cell development, such as *EBF1*, *E2F*, and *PAX5* [48]. Tallmadge et al. analyzed the transcriptome sequencing of horses affected by CVID, revealing a significant down-regulation in *PAX5* expression. The suspicion of an epigenetic mechanism responsible for this down-regulation was confirmed by the analysis of the epigenomic profile, which revealed a hypermethylation of the *PAX5* enhancer in the bone marrow of CVID-affected horses [49].

However, the most important alterations in DNA methylation are observed in the transition from naïve B cells to germinal center memory and plasma cells. B-cell differentiation is associated with a gradual DNA demethylation [27], with a similar grade of DNA methylation in memory and plasma cells, although these two cell lines have different transcriptional profiles [50]. A study on CVID-discordant monozygotic twins revealed an increase in the DNA methylation of critical B lymphocyte genes, such as *PIK3CD*, *BCL2L1*, *RPS6KB2*, *TCF3* and *KCNN4* in the affected sibling, as compared to the healthy sibling. This hypermethylation, observed in both unswitched- and switched-memory B cells, led to a down-regulation of those genes and, consequently, to B cell dysfunction [51].

In another study, the DNA methylome of CVID patients was compared with that of healthy donors, underpinning the hypothesis that altered demethylation during B cell differentiation may contribute to the pathogenesis of CVID, with a reduction in memory B cells paralleling the degree of demethylation impairment [51].

Immunodeficiency with centromeric instability and facial anomalies syndrome (ICF) is a rare disease caused by biallelic mutations in DNA methyltransferases, characterized by instability of the pericentromeric heterochromatin of chromosomes 1, 9 and 16, peculiar facial anomalies and immune deficiency. The latter may have a variable degree of severity, ranging from complete agammaglobulinemia to decreased levels of single classes of immunoglobulins, lymphopenia, T-cell proliferative response [52–54] and, rarely, autoimmunity [52]. Recurrent respiratory and gastrointestinal infections are typical features.

ICFs are classified according to genetic defects in ICF1, ICF2, ICF3 and ICF4, due to mutations in the *DNMT3B*, *ZBTB24*, *CDCA7* and *HELLS* genes, respectively [55–57].

As for ICF1, most patients harbor mutations in the catalytic domain of DNMT3B and show hypomethylation of DNA at determined noncoding repetitive sequences and genes located in inactive heterochromatin, causing chromatin decondensation and chromosomal instability [58]. It has been hypothesized that dysregulated DNA methylation underlies an abnormal maturation of B cells and the generation of immunologic memory [59]. Indeed, lymphoblastoid B cell lines from patients with ICF1 show an impaired expression of the genes involved in critical processes such as lymphocyte signaling, maturation and migration. When compared with controls, almost half of these genes appear to be upregulated. Additionally, the finding of an increased histone trimethylation at lysine-4, H3K4me3 supported this hypothesis [59].

*ZBTB24* is another regulator of hematopoietic development and, being highly expressed in naïve B cells, has a paramount role in B-cell differentiation [60]. In ICF patients with *ZBTB24* mutations, a normal number of total B lymphocytes, as well as naïve and

unswitched-memory B cells, has been described, associated with a decrease in switchedmemory B cells [53].

Finally, the *HELLS* gene, causing ICF4, encodes a lymphoid-specific, ATP-dependent, chromatin-remodeling enzyme, which forms a complex with CDC7A protein, whose gene defect underlies ICF3. Together, they activate chromatin-remodeling activity and, presumably, as in mouse models, exert epigenetic control over B cell development [61].

Kabuki syndrome (KS), a rare, multisystemic genetic syndrome associated with an immune disorder, has an estimated prevalence of 1:30,000–1:40,000 individuals. It is characterized by typical facial features, mild-to-moderate developmental delay, cardiac, skeletal and/or renal malformations and immunological abnormalities [62]. Children with KS might share some immune system abnormalities overlapping with CVID, such as hypogammaglobulinemia, increased susceptibility to upper and lower respiratory tract infections and a higher risk of lymphoproliferation [63]. Autoimmune manifestations have also been reported, the most common being autoimmune thrombocytopenia, with or without hemolytic anemia, followed by thyroiditis, celiac disease and vitiligo [64,65]. Seventy percent of KS cases are caused by mutations in the histone methyltransferase *KMT2D* [66], whereas the remaining cases are due to mutations in the histone demethylase *KDM6A* [67]. Both genes contribute to gene expression during embryogenesis. In particular, KMT2D is a lysine H3K4 mono-methyltransferase belonging to the SET domain containing 1/Mixed-Lineage Leukemia (SET1/MLL) protein family, whereas KDM6A acts on H3K27 methylated lysine to remove a repressive mark [68]. Antibody deficiency, as well as a reduction in B cells, total-memory B cells and class-switched-memory B cells, have been detected in KS patients [69].

The immune defects described in KS patients may depend on a loss of H3K4 methylation occurring at crucial transcription factors, dysregulating T and B lymphocyte differentiation. KMT2D loss-of-function might also cause a direct alteration of the antibody maturation, reducing the efficiency of class-switch recombination, while autoimmunity may derive from B-cell tolerance breakage or defective Treg generation [70].

#### *4.2. Inborn Errors of Adaptive Immunity*

The 22q11.2 Deletion Syndrome (22q11.2 DS) is the most common chromosomal microdeletion disorder. It is characterized by a wide phenotypic spectrum and includes multi-organ defects with congenital heart disease, immunodeficiency, hypoparathyroidism, genitourinary problems, palatal abnormalities, developmental delay and psychiatric symptoms [71]. So far, no single gene has been identified to explain all the features of 22q11.2DS and epigenetic mechanisms have been proposed to explain the clinical variability [72]. The phenotype of 22q11.2 DS could be the sum of the haploinsufficiency of 22q11.2 genes, as well as histone and DNA methylation defects [73]. *TBX1* (T-box 1) is the main candidate gene to explain the disease manifestations and it is involved in chromatin accessibility and transcriptional regulation [74]. *TBX1* was found to co-localize with three H3K4 methyltransferases in ChIP–Western blot analyses of co-immunoprecipitation experiments. In mouse models, *Tbx1* haploinsufficiency is associated with a global reduction in H3K4me1 histone monomethylation levels, causing the differential expression of some protein-coding genes [75]. A genome-wide DNA methylation analysis conducted on 22q11.2DS patients by Rooney et al. [73] led to the identification of 160 differentially methylated CpG probes, retained for the epigenetic signature of the syndrome. Moreover, the DNA methylation profile described was different in patients carrying typical deletions as compared to patients with atypical distal deletions. Identifying the target genes and functional consequences of the histone and DNA methylation alteration in 22q11.2 DS will help to better understand the pathogenesis of the syndrome.

The dysregulation of miRNAs and lncRNAs due to microdeletion may also partially account for the heterogeneity of the immunological and clinical phenotypes of the syndrome.

Moreover, a reduced function of miR1857, among others, may contribute to a decreased expression of Bruton's tyrosine kinase (Btk) and marginal-zone B1 protein (Mzb1), thus explaining a subsequent reduction in memory B cells [72].

Schimke immuno-osseous dysplasia (OMIM 242900) is an autosomal recessive disorder, due to mutations in *SMARCAL1* gene encoding SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A like 1, a chromatin-remodeling enzyme. The function of SMARCAL1 is to regulate transcription through chromatin remodeling [76]. The clinical phenotype includes: dysmorphic features, short stature with skeletal abnormalities, such as spondyloepiphyseal dysplasia and exaggerated lumbar lordosis; and arteriopathy. Impaired kidney function and immune deficiency consisting in recurrent bacterial, viral, or fungal infections have also been reported [77]. Laboratory tests show lymphopenia, absent mitogen-induced proliferation response, reduced CD8 and CD3/CD4 T cells [78]. Loss-of-function mutations in *SMARCAL1* may lead to genome instability, since the enzyme recognizes transitions from single- to double-stranded DNA.
