The Immune System in Down Syndrome: Autoimmunity and Severe Infections

Summary

Over 200,000 individuals in the US alone live with Down Syndrome (DS), the most common genetic disorder associated with intellectual disability. DS has a constellation of features across the body, including dysregulation of the immune system. Individuals with DS have both a higher frequency of autoimmunity and more severe infections than the general population, highlighting the importance of understanding the immune system in this population. Individuals with Down Syndrome present with dysregulation of both the innate and adaptive immune systems. Elevated cytokine levels, increased type I and type II IFN signaling, a shift towards memory phenotypes in T cells, and a decrease in the size of the B cell compartment are observed in individuals with DS, which contribute to both autoinflammation and severe infections. Herein we discuss the current knowledge of the immune system in individuals with Down Syndrome as well as ideas of necessary further investigations to decipher the mechanisms by which trisomy 21 leads to immune dysregulation, with the ultimate goal of identifying clinical targets to improve treatment.

Introduction

Down Syndrome (DS), otherwise known as trisomy 21, occurs in about 1 in 700 live births,¹ and about 200,000 individuals are living with DS in the US alone.² Worldwide, it is estimated that there are over five million individuals with DS. DS is the most common genetic disorder associated with intellectual disability. It presents as a constellation of features affecting multiple organ systems, including musculoskeletal and neurodevelopmental disorders, cardiac defects, and respiratory disorders.^2,3 In addition, individuals with DS display substantial immune dysregulation, with both a high frequency of autoimmunity and severe infections. Individuals with DS have higher rates of type 1 diabetes, juvenile idiopathic arthritis, celiac disease, alopecia areata, and thyroid disorders.^2,4 DS also confers a higher risk of severe infections resulting in hospitalization and mortality, despite lower rates of infectious diseases such as gastrointestinal (GI) tract infections and respiratory infections other than pneumonia.⁵ The higher risk of autoimmunity and the increased severity of infections in DS may seem paradoxical, but inflammation and susceptibility to severe infection need not be on different sides of the same coin. In this review, we discuss published findings for the innate and adaptive immune systems in DS, their relationship to autoimmunity and infection, and suggest future directions for improving our understanding of the immune system in DS.

Innate Immune System

Granulocytes

Few studies have focused on the frequency of granulocytes (neutrophils, eosinophils, and basophils) in DS. In one study on 158 neonates with DS, 80% presented with neutrophilia, with neutrophil counts of up to about three times the typical upper limit of 5,400 neutrophils/mm³ at 120 hours of age.^6,7 Another single study reported significantly lower absolute numbers of granulocytes in children aged 0 to 12 years with DS compared to children without DS. However, the granulocyte populations were not stratified in this study, so the decrease may not be consistent across neutrophils, eosinophils, and basophils as separate populations.⁸ Children and young adults with DS have significantly lower than normal mean eosinophil counts.⁹ Once individuals with DS reach adulthood, their neutrophil and eosinophil populations appear to reach levels similar to those in healthy controls.¹⁰ However, further studies are required in neonates, children and adults with DS, with stratification of neutrophils, eosinophils, and basophils, to improve our understanding of the impact of DS on these populations over the lifespan of the affected individual.

There is a consensus that the cellular chemotaxis and bactericidal capacities of neutrophils are impaired in individuals with DS. However, most papers report no difference in percent phagocytosis or phagocytic index between individuals with DS and healthy controls.^11–16 In children with DS, neutrophil intracellular calcium concentration is elevated, both at baseline and in response to stimulation. Elevated intracellular calcium concentration at baseline is also typically observed in older individuals. The high levels in children with DS may therefore potentially reflect the accelerated aging reported in this condition.¹⁷ In adults with DS, granulocytes (neutrophils and eosinophils) have higher levels of both spontaneous and anti-Fas antibody-induced apoptosis, demonstrating shorter granulocyte survival.¹⁸ All of these findings suggest that neutrophil function is altered in DS. However, there is a clear lack of studies on granulocyte development and detailed molecular function in individuals with DS, over the lifespan of individuals and in the presence of concomitantly occurring conditions. Such studies are required to determine the differences between individuals of different ages and clinical phenotypes.

Monocytes

Monocyte function and frequency have been investigated in both children and adults. Two groups have reported decreased frequency of CD14⁺ CD16⁻ classical monocytes and increased frequency of CD14^dim/- CD16⁺ non-classical monocytes in children and adults with DS.^8,19 Non-classical monocyte levels have also been reported to be upregulated in patients with SLE and to positively correlate with anti-dsDNA antibody levels, suggesting an association between these cells and autoimmunity.²⁰ However, a smaller study reported no difference in either of these monocyte populations in adults with DS,¹⁰ highlighting the need for studies of larger numbers of individuals with DS, to shed light on possible differences in monocyte populations, particularly with respect to age and clinical outcomes.

Monocytes from individuals with DS have significantly impaired phagocytosis and chemotaxis functions, but their bactericidal capacity is unaffected.^11,12,21 They express significantly higher relative levels of the cell surface interferon (IFN) receptors IFNAR2 and IFNGR2 than control cells, and their relative basal total STAT1 and pSTAT1 levels are also higher, as are their relative pSTAT1 levels after stimulation with IFN-α or IFN-γ. Downstream IFN-stimulated gene (ISG) levels are also upregulated in DS monocytes relative to healthy controls (HCs).²² As discussed in detail below, IFN signaling likely plays a substantial role in the clinical immune outcomes of DS, and monocytes may indeed be the key players.

Dendritic cells

Dendritic cell subpopulations appear to differ with age; however, the results in adults with DS have not converged upon a single narrative. Children with DS have significantly lower frequencies and absolute counts of conventional myeloid dendritic cells (cDCs) than children without DS, whereas plasmacytoid dendritic cell (pDC) levels do not differ between children with and without DS.⁸ In adults, both cDC and pDC frequencies have been reported to be higher in individuals with DS.¹⁰ However, another group reported no change in these populations in adults with DS, but an upregulation of CD1c⁺ cDCs.¹⁹ Further studies are required, ideally with large cohorts, to determine how dendritic cell populations differ between ages and clinical outcomes in DS.

NK cells

An early study on CD16⁺ CD56⁺ cytotoxic NK cells in DS reported no significant difference in the frequency of this population between children with and without DS, with significantly lower absolute numbers in children with DS. Contrastingly, higher frequencies and numbers of these NK cells in adults with DS were reported.²³ More recent studies have yielded similar results, but with some differences observed in childhood. In 41 children with DS aged 0 to 12 years, the frequency of CD3⁻ CD16⁺ CD56⁺ NK cells was found not to differ significantly from that in healthy controls, except for children with DS under the age of two years, who had a significantly higher percentage of NK cells. The absolute numbers of NK cells were lower than in controls, but not significantly.⁸ Another study of 96 children with DS reported significantly lower absolute numbers of NK cells, defined as CD3⁻ CD56⁺ and/or CD16⁺ cells, up to the age of 10 years, with no difference between children with and without DS after the age of 10 years.²⁴ In adults with DS, one study reported no difference in CD3⁻ CD56⁺ CD16⁺ NK cells,¹⁰ whereas another reported significantly higher frequencies of CD16⁺ CD56⁺ NK cells, consistent with earlier reports.¹⁹ However, this increase in frequency was apparent only in a large cohort (89 individuals in total, 38 with DS) and was not significant in the initially investigated cohort of 18 individuals with DS, suggesting that the change in the cytotoxic NK population in adults with DS is subtle and may only be detectable in large cohorts. In addition, Waugh et al. (2019) showed that the expression of CD16, CD38, CD8, and CD11c, markers associated with NK cytotoxicity and activation, was upregulated on these cytotoxic NK cells.¹⁹ Ultimately, the NK cell populations of individuals with DS appear to vary with age and the differences between adults with and without DS may be subtle, making it necessary to investigate large cohorts to capture any variability, particularly across the spectrum of comorbid conditions.

Complement System

The complement system is dysregulated in DS and may change significantly with age. One study reported a significant downregulation of multiple complement proteins, including C1R, C1QA, C3, and C6, in the circulating proteome of children and young adults with DS.²⁵ However, a recent study reported significantly higher levels of complement proteins (C1q, C3, and C9), regulators (clusterin, factor H, C1 inhibitor, and FHR4), and activation products (TCC and iC3b) in the plasma of adults with DS than in controls.²⁶ Veteleanu et al. (2023) suggested that this discrepancy might be related to age or chronic inflammation. C1q levels have been shown to increase with age²⁷ and individuals with DS have been shown to display accelerated epigenetic aging.²⁸ C1q production is stimulated by inflammatory cytokines, such as IL-6 and IFN-γ,²⁹ and C1q levels are elevated in chronic infection states.³⁰ The chronic inflammation seen in individuals with DS, as discussed below, may also affect C1q levels. In addition, complement factor H (CFH) levels are significantly decreased in the liver, brain, and spleen tissues of adults with DS, whereas miRNA-155, which is expressed on chromosome 21 and binds to CFH, is significantly upregulated in these tissues. TNFα-stressed human neuronal-glial cell cultures, which reproduce the upregulation of miRNA-155 and downregulation of CFH seen in DS, have normal levels of CFH following treatment with anti-miRNA-155, which suggests that the increased levels of miRNA-155 in DS likely contribute to the decreased levels of CFH.³¹ The DS cohort in this study was small (n=5), but the findings nevertheless suggest that complement levels differ between the tissues and plasma of adults with DS, with variable effects on each system. Further investigations of the complement system at different ages and in different organ systems are required to determine the full impact of trisomy 21 on the complement system.

Interferons Type I, II, and III, and IL-10

In addition to the alterations to innate immune cell populations and the complement system, DS is also associated with genetic and molecular changes to the innate immune system. Human chromosome 21 (HSA21) carries four of the six IFN receptor genes: IFNAR2, IL10RB, IFNAR1 and IFNGR2 (IL10RB associates with IFNLR1 but is also a subunit of IL10R).³² Many studies have shown that the triplication of these four receptor genes results in an increase in receptor expression, with the upregulation of mRNA levels for some or all of these IFNAR receptors in the fibroblasts, monocytes, EBV-B cells, and T cells of individuals with DS.^33–35 Relative levels of total STAT1 and pSTAT1 are constitutively higher in monocytes from individuals with DS compared to HCs, at a level intermediate between those of HC and STAT1 gain of function (GOF) monocytes.²² Several groups have shown that various cell types from individuals with DS exhibit elevated levels of pSTAT1 and pSTAT2 following IFN stimulation.^19,22,34 Downstream IFN-stimulated gene (ISG) levels are also upregulated in monocytes from individuals with DS relative to HCs, although less so than in the monocytes of STAT1 GOF patients and with more variation between samples, reflecting the heterogeneity observed in DS.^22,36 Analysis of the proteome of individuals with DS has also revealed a significant enrichment in interferon-related proteins.²⁵ The clinical phenotypes of individuals with DS overlap with those of patients with STAT1 GOF or STAT3 GOF variants, as discussed below, further implicating this pathway in the immune manifestations of DS.^37,38

Autoinflammation

Autoinflammation in DS is well documented but it remains unclear whether the perturbations to innate immunity cause this inflammation or vice versa. Nevertheless, we have come to the conclusion that DS can be considered a cytokinopathy, as we have shown that individuals with DS live in a perpetual state of soluble immune dysregulation. In a cytokine array assessing the levels of 29 cytokines in the plasma of individuals with DS (n = 21) and HCs, the individuals with DS clustered into three categories. The most severely affected group presented with elevated levels for 22 of the 29 soluble immune markers assessed (n = 7). The moderated affected group had high levels of a subset of cytokines (n = 9), and the least affected group clustered with HCs (n = 5). This dysregulation was stable over time, with cytokine levels remaining steady in a given individual with DS even for samples collected months apart. These findings suggest that most individuals with DS display a dysregulation of the levels of at least a subset of cytokines. There was a significant association between the cytokine groups and immune dysregulation, assessed via the number of autoimmune manifestations or infectious episodes seen in each individual. Levels of acute phase proteins, such as IL-6, IL-1α, and TNFß, are significantly higher in individuals with DS than in the general population, as are those of Th2 cytokines IL-4 and IL-13.¹⁰ Other groups have also reported soluble immune dysregulation in individuals with DS. Sullivan et al. (2017) reported significant upregulation of IL-6, IL-22, MCP-1 (CCL2), TNF-α, and VEGF-A, and noted that the upregulation of these proteins is also seen in individuals with various chronic inflammatory diseases.²⁵ A meta-analysis of studies on individuals with DS also revealed significantly higher levels of TNF-α, IL-Iß, IFN-y, and neopterin in these individuals.³⁹ Ultimately, most individuals with DS appear to live in a state of chronic soluble immune dysregulation, because of or contributing to an autoinflammatory state.

The triplication of IFN receptor genes in DS, combined with downstream effects on IFN signaling, likely also contributes to the autoimmunity observed in individuals with DS. Likewise, the clinical presentation of DS in some ways resembles that of various type I interferonopathies, such as those observed in STAT1 GOF heterozygous mutation patients and STAT3 GOF patients. All three of these groups display a propensity for both autoimmunity and infection. An autoimmune phenotype of some kind, such as thyroid disease or other endocrine diseases, is found in 37% of patients heterozygous for STAT1 GOF mutations, and the vast majority also present with chronic mucocutaneous candidiasis and bacterial infections.³⁷ STAT3 GOF patients can present with both immune dysregulation, in the form of alopecia and early-onset diabetes, and fungal, bacterial, and respiratory infections.³⁸ These both mirror the dual phenotype of autoimmunity and severe infections seen in DS and suggest that IFN and other STAT1- and STAT3-activating cytokine pathways may contribute to the clinical outcomes of DS.

Infection

Many groups have documented the greater severity of viral infections in DS for various viruses, including severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), respiratory syncytial virus (RSV), and influenza virus (IAV) H1N1.^40–42 Based on the relationship between type I IFNs (IFN-I) and viral defenses, our group demonstrated how increased IFN-I receptor expression may contribute to increased viral severity in DS through excessive negative regulation of the IFN-I pathway.³⁴ USP18, an IFN-I-induced gene, is a crucial negative regulator of the IFN-I pathway that restricts subsequent IFN-I signaling.⁴³ It does so by binding IFNAR2 and preventing JAK1 association and, thus, downstream signaling, to prevent overt inflammation. Despite their initial hyper response to IFN-I, hTERT fibroblasts from individuals with DS become hyporesponsive to IFN-I signaling after a priming event, with decreased pSTAT1 and pSTAT2 and increased USP18 levels relative to healthy controls, suggesting an overt negative regulation of IFN-I signaling in DS. DS hTERT fibroblasts primed with IFN-I present similar levels of infection with and without additional IFN-I stimulation. This situation contrasts with that reported for HC hTERT fibroblasts primed with IFN-I, in which additional IFN-I stimulation decreases the infection rate by about 15%. Ultimately, this damping down of innate immunity to viral infection in DS through higher levels of USP18 expression and hyporesponsiveness to secondary IFN-I signaling may help to explain the greater severity of viral infection in individuals with DS.³⁴

Summary

The innate immune system is dysregulated in individuals with DS. Certain cell populations, such as non-classical monocytes and cytotoxic NK cells, soluble factors, and the type I IFN pathway are affected. This dysregulation, particularly that of the type I IFN pathway, likely contributes to both the autoinflammation and increased infection severity observed in individuals with DS. However, gaps in our knowledge of the role of the innate immune system in DS remain, particularly if age and clinical phenotype are taken into account. How might the immune system change with age and the presence or absence of autoimmunity? What are the pathogenic roles of each of the cytokines found to be present at higher levels in DS? What is the combined effect of all these cytokines? Further studies are essential to provide us with a full understanding of the effects of DS on the immune system, together with the sources of heterogeneity and their impact. This research is crucial for the identification of possible treatment targets and their applications in DS.

Adaptive Immune System

T cells

T cells have been studied extensively in both children and adults with DS. Unlike in individuals with DS, in children without DS, there is a large expansion of T cells in early childhood in response to continuous antigen exposure. In a study of 96 children with DS, absolute numbers of T lymphocytes were significantly lower than those in controls for all age groups up to the age of 17 years; the most severe deficiencies were observed early in childhood, with a gradual trend towards typical levels by adulthood.²⁴ Children with DS have a significantly lower thymic weight accompanied by accelerated thymic maturation, indicated by increased representation of the medullary area in the thymus at an early age.⁴⁴ Furthermore, the thymus of children with DS under the age of two years contains a significantly lower proportion of mature T cells with high levels of TCRαβ or CD3 expression relative to that in control children.⁴⁵

There is also an imbalance of T cell subpopulations in the peripheral blood of children and young adults with DS. Mean levels of cell-surface CD3 and TCRαβ expression are normal in the peripheral blood of individuals with DS, but these individuals have lower absolute numbers of TCRαβ⁺ cells and CD3⁺ cells and a lower proportion of TCRαβ⁺ cells than controls. Children and young adults with DS also have a significantly higher proportion of TCRγδ⁺ cells in the peripheral blood.⁹ TCR excision circle (TREC) levels do not differ between the thymi of children with DS and controls, which suggests that thymic T cell production is similar in both groups, whereas TREC levels and the percentage of TREC⁺ cells are significantly lower in the periphery, suggesting potentially lower levels of thymic emigration in individuals with DS.^44,46–49 This was also noted by Bloemers et al. (2011); they showed significantly decreased PTK-7⁺ naive CD4⁺ cells, representing recent thymic emigrants, in individuals with DS, with no difference in apoptotic naive CD4⁺ and CD8⁺ T cells, no difference in recently activated CD8⁺ cells, and no difference in Ki-67⁺ naive CD4⁺ or CD8⁺ T cells, all of which suggests a decreased thymic output in DS rather than defective peripheral T cell maintenance.⁴⁸ Infants with DS display significantly lower T-cell clonal expansion rates, with a trend towards a more diverse TCR gamma repertoire.⁴⁶

It is widely reported that both children and adults with DS have significantly decreased naive CD4⁺ and CD8⁺ compartments compared to controls.^{8–10,19,22,44,47,48,50–52} They also have higher levels of memory T cells, including central memory and effector memory, although significance varies across reports.^{8,10,19,22,44,47,50–52} The majority of studies have shown Th1 and Tfh cells to be present at similar or higher levels in individuals with DS than in controls, whereas Th2 and Th17 cells are present at similar or lower levels.^{10,22,50,52,53} Several studies have also reported increased frequencies of proinflammatory Th17/1 cells.^10,50,52 In one study of 28 individuals with DS, T cell maturation markers were dysregulated in both naive CD4⁺ effector T cells and naive CD8⁺ T cells. Naive CD4⁺ effector T cells displayed significantly lower levels of CD38 and ICOS expression, whereas naive CD8⁺ T cells displayed significantly higher levels of expression for HLA-DR and KLRG1, which are upregulated when T cells are activated. Both naive CD4⁺ effector T cells and naive CD8⁺ T cells display an upregulation of CXCR3, TIGIT, and CD39, and a downregulation of CD62L, these changes again being associated with T-cell maturation and activation. All these findings suggest that naive T cells are present in a “hyperactivated” state in DS.⁵⁰ This may partly explain the imbalance in naive and memory compartments in DS, with naive T cells primed to differentiate into memory cells. In addition, CD8⁺ T cells produce significantly elevated levels of cytokines, such as TNF-α, IFN-y, GM-CSF, and IL-10, consistent with the elevated levels of cytokines observed in the plasma of individuals with DS, as discussed above.⁵²

Reports on the levels and function of regulatory T cells in DS vary. Most groups have reported a significant increase in regulatory T cell frequency in both children and adults.^{44,50,52–54} However, one study found no significant alteration to regulatory T cell levels in patients with both DS and autoimmune disorders.⁵⁴ This finding was supported by two other studies in which the majority of patients studied had at least one autoimmune manifestation and did not present with any significant modification to the frequency of regulatory T cells.^10,51 Thus, clinical phenotypes, such as autoimmune disease, may affect the frequency of regulatory T cells in individuals with DS, but larger studies are required for any firm conclusion to be drawn on this point. The functionality of regulatory T cells in DS also remains unclear. Some studies have reported reduced suppressive function,^44,54 whereas others have reported unaltered suppressive function accompanied by an increase in resistance to suppression in conventional CD4⁺ and CD8⁺ T cells from individuals with DS.⁵² One notable limitation in comparing studies on Tregs in DS is a lack of consistent markers for Treg identification in humans. As discussed below, a better understanding of the role of regulatory T cells in DS might provide a clearer picture of the contributions of the various immune cell populations to the autoimmunity seen in DS.

B cells

As seen in T cells, the early increase in absolute numbers of B cells seen in children from the general population is not seen in children with DS. However, contrary to what has been observed for T cells, this deficit of B cells does not improve with age; 88% of children with DS have B cell counts below the 10th percentile for children without DS,²⁴ and this deficit of total B cell numbers continues into adulthood. Results vary between studies, but most have reported significant decreases in transitional, naive mature, and memory B cells (MBCs), including class-switched memory B cells. There have been mixed reports for IgM memory B cells, with some papers reporting significantly lower levels of IgM MBCs, whereas others have reported only a trend towards lower levels of IgM MBCs.^{10,19,50,51,55–58} Furthermore, some groups have reported high levels of plasma cells and plasmablasts in individuals with DS.^10,19,50,56 One interesting population upregulated in individuals with DS is CD11c⁺ B cells; these cells are also known as atypical B cells and are often identified by some combination of CD11c, T-bet, and low expression of CD21.^10,19,50 This cell population is expanded in systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and other autoimmune disorders, providing a possible link with the autoimmunity seen in DS.⁵⁹ The roles of switched memory B cells in DS and infection, and of CD11c⁺ B cells in autoimmunity, are considered in more detail below.

Serum IgG and IgA levels in individuals with DS are predominantly reported to be normal, whereas IgM levels are mostly reported to be significantly decreased.^51,55,57 Some differences have been observed between IgG subclasses: IgG1 and IgG3 levels are typically high, whereas IgG2 and IgG4 levels are typically low.^58,60–62 Interestingly, the secretory rates of salivary IgA are significantly decreased in older children and adults with DS, which may be related to the increased periodontal disease and GI tract and respiratory infections observed in individuals with DS. However, given the very low salivary secretion rates in DS, these rates, rather than a deficiency of IgA production, likely contribute to this outcome.^63,64

Some deficits in germinal center formation in the tonsils of children with DS have been reported, while other groups report typical germinal centers in the tonsils. Farroni et al. (2018) reported that tonsils from children with DS had similar numbers of germinal centers, but significantly smaller germinal centers, and BCL-6+ GC B cells were significantly reduced compared to control tonsils.⁶⁵ However, Verstegen et al. (2014) reported no difference in the germinal centers of tonsils from children with DS and healthy controls, with secondary lymph follicles containing B-cell blasts, follicular dendritic cells, and helper T cells.⁵⁶ Age differences may play a role in the variable results, although more studies should be done to evaluate germinal centers in individuals with DS, across secondary lymphoid structures.

Naïve B cells from the tonsils of children with DS display a typical upregulation of AID gene expression after stimulation with anti-IgM and IL-21 plus anti-CD40 or CpG. Treatment with anti-CD40 results in normal levels of IRF4 and XBP1 expression, whereas PRDM1 expression is lower than in controls. CpG treatment results in decreased expression of PRDM1, IRF4, and XBP1 compared to controls.⁵⁶ However, PBMCs from children with DS and control children cultured in the presence of CpG had similar rates of proliferation and differentiation into plasma cells. CD27⁺ IgM⁺ B cells from children with DS had a significantly higher proliferation index compared to controls and contained significantly higher frequencies of plasma cells after CpG treatment. Switched memory CD27⁺ IgM⁻ B cells from children with DS had a similar proliferation index compared to control cells but contained significantly higher frequencies of plasma cells after CpG treatment.⁵⁵ Thus, even though naïve B cells from individuals with DS do not respond to anti-CD40 or CpG in exactly the same way as naïve B cells from individuals without DS, rates of proliferation and plasma cell differentiation appear to be similar or even higher in individuals with DS, with variation between B cell subsets.

B cells from individuals with DS also display a dysregulation of various markers and cell subpopulations, overlapping with that observed in various autoimmune conditions. As mentioned above, individuals with DS have increased levels of CD11c⁺ B cells, a feature not only of DS but also of RA, SLE, and other autoimmune disorders. Lambert et al. (2022) also showed that non-plasmablast B cells from individuals with DS had lower levels of CXCR5 and CD25 expression, as seen in SLE, and high levels of PD-1, as seen in RA. Switched memory B cells from individuals with DS have high levels of CXCR3 expression, which may indicate a greater potential for trafficking to tissues,⁶⁶ and low levels of CCR6, both of which are also observed in SLE and RA.⁵⁰ Ultimately, most B cell subsets seem to be present at lower levels in individuals with DS than in controls, with increased populations of plasmablasts and CD11c⁺ B cells in individuals with DS. Serum IgM, IgG2, and IgG4 levels are low, and IgG1 and IgG3 levels are high in individuals with DS. In addition, B cells from individuals with DS express markers common to other autoimmune conditions, including low levels of CXCR5 and high levels of CXCR3.

Autoimmunity

Multiple dimensions of the adaptive immune system in DS are likely linked to the increased rates of autoimmunity, including IFN signaling in adaptive immune cell populations, AIRE expression, levels of CD11c⁺ B cells, and autoantibody production. In an evaluation of the circulating proteome of individuals with DS, Sullivan et al. (2017) observed significant downregulation of FCRL3, an Fc receptor-like glycoprotein, ZAP70, which is involved in thymocyte development and the regulation of mature T cells, and ERAP2, which is involved in preparing epitopes for MHC I presentation. The dysregulation of these proteins has various impacts on the immune system. Mutations of FCRL3 are associated with SLE and RA, and mutations of ZAP70 are also associated with autoimmune diseases. Sullivan et al. also found that B2M was upregulated, whereas LILRB1/LILRB2 were downregulated. B2M is an interferon-stimulated gene and B2M is a subunit of the MHC class I complex, whereas LILRB1/LILRB2 negatively regulate MHC class I signaling. The observed combination of changes in these protein levels may, therefore, be related to increased antigen presentation and MHC class I signaling. Ultimately, individuals with DS display a dysregulation of the proteome that may be involved in the higher incidence of autoimmunity in these individuals.²⁵

Individuals with DS have an increased prevalence of thyroid disorders.⁴ Several groups have investigated the expression of AIRE in DS. As AIRE controls the expression of peripheral tissue antigens on medullary thymic epithelial cells, its dysregulation could affect the central tolerance of T cells.⁶⁷ AIRE is located on chromosome 21, suggesting increased expression levels in DS, but AIRE levels have only been shown to be increased at young ages. At the ages of 0 – 6 months, AIRE expression is significantly higher in individuals with DS than in the general population of the same age, with a significantly larger number of AIRE⁺ cells/mm² in the thymic medulla.⁶⁸ However, other groups have reported a significantly smaller number of AIRE⁺ cells/mm² in the thymic medulla and significantly lower levels of AIRE expression in the thymus of individuals with DS, in a population of patients aged 2 months to 12 years.^69,70 As demonstrated by Marcovecchio et al. (2019), the number of AIRE⁺ cells in the thymus likely increases at a young age but starts to decrease around one to two years old.⁴⁴ The mechanisms underlying this switch and its impact on central tolerance remain unknown.

T cells may contribute to the autoimmunity seen in DS in a number of ways. As discussed above, there is some clinical overlap between STAT3 GOF patients and individuals with DS. Patients with STAT3 GOF mutations display lymphoproliferation, susceptibility to infections, and various autoimmune disorders, including alopecia and early-onset diabetes.³⁸ Our group detected baseline pSTAT3 in naive, activated, and central memory CD4⁺ T cells in individuals with DS, potentially indicating active cytokine signaling. This baseline phosphorylation of STAT3 in CD4⁺ T cells is Jak-dependent and is predominantly mediated via elevated IL-6 levels in DS.¹⁰ Additionally, as mentioned above, individuals with DS may present a dysregulation of regulatory T cells or have CD4⁺ effector T cells that are resistant to Treg suppression. This may increase the propensity of autoreactive T cells to function and proliferate, contributing to autoimmunity. Further investigations of the contribution of these cell populations to the autoimmune phenotype seen in DS are necessary.

Several groups have reported high levels of CD11c⁺ B cells in individuals with DS.^10,19,50 CD11c⁺ B cells fall within the larger group of atypical B cells, which are defined on the basis of combinations of CD11c and T-bet expression and low levels of CD21 expression.⁵⁹ They are thought to differentiate outside of germinal centers and are predominantly described as CD27⁻ IgD⁻ (double-negative) or CD27⁻ IgD⁺ (activated naive) cells.¹⁰ These atypical B cells have been reported in systemic lupus erythematosus, common variable immunodeficiency, individuals with STAT3 GOF mutations, and rheumatoid arthritis, suggesting that they are linked to autoimmunity.^71–74 Our group has shown that levels of CD11c⁺ B cells are elevated in both children and adults with DS, with levels in adults with DS similar to those seen in SLE. In both the double-negative and activated naive compartments, T-bet, FAS (CD95), and CD86 levels are upregulated, with lower levels of CD21, CXCR5 and CCR7 expression. The frequency of CD11c⁺ B cells positively correlated with the number of autoimmune manifestations.¹⁰

In addition to phenotyping, our group sequenced the CDR3 of DS and control CD11c⁺ B cells. We found that up to 7% of BCRs were expanded in CD11c⁺ B cells, with a clonality similar to that of memory B cells, although no differences in clonality were observed between control and DS BCRs. The length of the CDR3 in CD11c⁺ B cells is similar to that in naive B cells and significantly greater than that in memory B cells, with no differences between controls and individuals with DS. CD11c⁺ B cells from individuals with DS have significantly fewer non-reference nucleotides in the V genes, which may indicate a lack of somatic hypermutation, although full BCR sequencing is required for definitive conclusions to be drawn. In addition, the rate of IGHV4–34 gene use in the CD11c⁺ B cell population is significantly higher in individuals with DS than in controls, and DS cells display greater surface 9G4 antibody expression. There are also higher circulating levels of 9G4 in the plasma of individuals with DS. The IGHV4–34 gene is associated with autoreactivity and 9G4 antibodies contribute to autoimmunity in SLE, again linking these CD11c⁺ B cells to autoimmunity in DS.¹⁰

Individuals with DS also have high levels of autoantibodies with an extensive overlap with those observed in patients with IPEX. Our group compared the autoreactivity of plasma from individuals with DS with that of plasma from healthy controls, three IPEX patients, and one APS-1 patient using the CDI HuProt protein microarray, which tests over 21,000 conformationally intact human proteins. Principal component analysis grouped DS away from HCs. Interestingly, the DS plasma samples were grouped with the IPEX plasma samples, away from the APS-1 plasma sample. In DS, there were 365 differentially abundant autoantigens compared to HCs. In total, 829 differentially abundant autoantigens were identified in IPEX, and 228 of these overlapped with DS. By contrast, there was an overlap of only two differentially abundant autoantigens between DS and APS-1 (of the 257 identified in APS-1). Autoantigens enriched in DS are expressed across organ systems and sites of autoimmunity in DS, including the pancreas, the gastrointestinal tract, the central nervous system, and the immune system.¹⁰ Another study reported that children with DS have a significantly higher frequency of autoantibodies for rheumatoid factor (28% of children with DS were reactive, versus 6.7% of controls).⁷⁵ Ultimately, it appears that individuals with DS have a sizable autoantibody repertoire that overlaps extensively with that of IPEX patients, helping to explain the autoimmunity seen in DS and possibly implicating Treg associated mechanisms in this autoimmune landscape.

Infection

Increased severity of infectious disease in individuals with DS is well documented. In children with DS, the duration of hospitalization for common respiratory conditions has been shown to be two to three times greater than that in patients without DS.⁷⁶ Pneumonia accounts for up to 40% of deaths in individuals with DS,⁷⁷ highlighting the higher risk of severe infections and mortality than in individuals without DS. However, infection rates are not necessarily higher in individuals with DS; in a retrospective study assessing the prevalence of infection in 6078 individuals with DS over a period of 28 years, individuals with DS were found to have a lower prevalence of respiratory infections other than pneumonia and a lower prevalence of GI infections than individuals without DS, but a higher prevalence of pneumonia and skin infections.⁵ As mentioned above, several studies have reported low levels of class-switched memory B cells in DS.^{51,55–57,78} Class-switched memory B cells are important for the generation of a diverse range of Ig isotype antibodies, switching from IgM to IgE, IgG, or IgA. Among other roles, switched memory B cells are a result of robust vaccination responses, and as such, decreased class switched memory B cells may indicate a less effective vaccine response in DS, increasing susceptibility to severe infection.

Vaccination responses in children with DS vary, with some differences between vaccine targets. Children with DS have significantly fewer memory B cells producing influenza-specific IgG after primary vaccination than their siblings. However, seroconversion, seroprotection, and the geometric mean titer (GMT) for children with DS meet the thresholds for immunogenicity.⁵⁷ Another study suggested that the influenza vaccine was immunogenic in children with DS, although over 50% of individuals had pre-vaccine titers for influenza A and B.⁷⁸ Children with DS have a good response to the inactivated hepatitis A virus (HAV) vaccine, with no significant difference in seroconversion rates and GMT between individuals with DS and controls (all of whom were screened for prior exposure to HAV and excluded if found to have HAV antibodies).⁷⁹ Most studies also report an adequate response to the conjugated and polysaccharide pneumococcal vaccine in individuals with DS. Children and adults with DS have lower antibody levels than controls after administration of the unconjugated polysaccharide vaccine, but both studies revealed an increase in the levels of anti-pneumococcal antibodies post vaccination.^80,81 In addition, vaccination schedules including both the conjugated and polysaccharide vaccine result in anti-pneumococcal antibody responses within the normal range.⁸²

Conversely, several studies have suggested that individuals with DS have ineffective responses to vaccines or that their antibody levels wane over time. In children with DS who received the hepatitis B vaccine during infancy, antibody levels against hepatitis B are significantly decreased compared to children without DS, although 31.9% of children with DS over the age of 10 still have adequate antibody titers for protection.⁸³ In adults with DS, receptor binding domain IgG antibody levels after administration of the BNT162b2 COVID-19 mRNA vaccine are significantly lower than those in controls after both the first and second doses, and six months after the first dose.⁸⁴ Further studies are required to determine the possible impact of defects of the adaptive immune system of individuals with DS on infection. Given the increased severity of infection in these individuals, it is important to understand how the immune system of individuals with DS responds to immunization, to make it possible to design appropriate vaccine schedules providing good protection. A modified vaccination schedule, with more frequent doses or boosters may be a valid approach to improving the control of infectious diseases in DS.

Future Directions

Research on the immune system in individuals with Down Syndrome has been conducted from many a perspective, spanning the innate and adaptive immune systems, and the impact of DS on immune cell populations and abundance is summarized in Table 1. As represented in Figure 1, multiple elements of the immune system have been tied to elevated autoimmunity and severe infections.

Table 1.

Summary of the impact of Down Syndrome on immune cell population abundance and function.

Immune Cell Population	Effect on Population Abundance	Effect on Population Function
Innate Immune System
Granulocytes	Neutrophilia at birth Lower numbers of granulocytes ages 0 to 12 Decrease in mean eosinophil counts in childhood and young adulthood Similar levels of neutrophils and eosinophils by adulthood	Impaired neutrophil cellular chemotaxis and bactericidal capacities No change in percent phagocytosis or phagocytic index Higher levels of apoptosis
Monocytes	Some reports of decreased CD14+ CD16− classical monocytes and increased CD14dim/- CD16+ non-classical monocytes in children and adults	Impaired monocyte phagocytosis and chemotaxis function Unaffected bactericidal capacity Increased IFNAR2, IFNGR2, total STAT1, pSTAT1, and IFN-stimulated gene levels
Dendritic cells	Decrease in cDCs and similar levels of pDCs during childhood Similar – increased levels of cDCs and pDCs in adulthood Increase in CD1c+ cDCs
NK cells	No difference – decreased CD16+ CD56+ cytotoxic NK cells in children Increased frequencies of CD16+ CD56+ cytotoxic NK cells in adults	Upregulated markers of NK cytotoxicity and activation (CD16, CD38, CD8, CD11c)
Complement System	Varying reports on complement proteins, may change with age and tissue
Interferons Type I, II, III, and IL-10	Upregulation of IFNAR2, IL10RB, IFNAR1, and IFNGR2 mRNA Higher total STAT1 and pSTAT1 in monocytes Upregulated IFN-stimulated gene levels in monocytes
Adaptive Immune System
T cells	Significant deficiency in T cells early in childhood, gradual trend towards typical levels of T cells by adulthood Lower numbers of TCRαβ+ cells and CD3+ in children and young adults Decreased thymic output Significantly decreased naive CD4+ and CD8+ compartments Similar - increased memory T cells Similar - increased regulatory T cells; may differ with autoimmunity status	Present with markers indicative of a “hyperactivated” state, including CXCR3, TIGIT, and CD39 CD8+ produce significantly elevated levels of cytokines Similar - reduced suppressive function of regulatory T cells Baseline pSTAT3 in naive, activated, and central memory CD4+ T cells
B cells	Significant deficiency in total B cells, from early childhood into adulthood Significant decreased in transitional, naive mature, and memory B cells, including class-switched memory B cells Similar - increased levels of plasma cells and plasmablasts Increased CD11c+ B cells Predominantly typical levels of serum IgG and IgA Significantly decreased IgM levels High levels of autoantibodies	Similar - increased rates of proliferation and plasma cell differentiation Express markers common to other autoimmune conditions, including low CXCR5 and high CXCR3 Varying vaccination responses - some reports of accelerated antibody decline after vaccination or ineffective responses to vaccines

Figure 1.

Elements of the immune system in Down Syndrome which likely contribute to autoimmunity or severe infection. Increased CD11c+ B cells, T cell dysregulation, elevated cytokines, and autoreactive antibodies all likely play a role in the increased autoimmunity seen in DS. Triplication of 4 IFN receptors, IFNAR1, IFNAR2, IFNGR2, and IL10RB, and pathways downstream of these receptors, appear to contribute to severe infections. Decreased switched memory B cells, increased USP18 negative regulation, and a subpar vaccine response also all play a probable role in severe infections.

As discussed throughout this review, our scientific understanding of many elements of the immune system in individuals with Down Syndrome would be greatly improved by large studies across ages and clinical outcomes. In particular, stratifying individuals with DS based on the presence of autoimmune diseases or regular infections might reveal how the heterogeneity of clinical outcomes in DS is linked to the immune system. Registries such as DS-Connect are valuable tools for individuals with DS and their families to connect with researchers, which in turn can provide researchers with opportunities for access to larger groups of study participants that can allow for stratification based on outcomes such as autoimmunity or regular infections. Continuing to provide resources for registries such as these is critical for advancing scientific research to understand the immune system in DS. As research on the immune system in DS progresses, we will have a clearer understanding of how various immune elements contribute to autoimmunity, infection, or both.

The role of many elements of the immune system in DS remains to be elucidated. It remains unclear how the various upregulated cytokines act in isolation or in combination to modify clinical outcomes in Down syndrome. The role of type I IFNs, in particular, requires more detailed exploration, as they appear to be important in both autoimmunity and infection. Both T cells and B cells, including effector T cells, regulatory T cells, switched memory B cells, and CD11c+ B cells, likely play important roles in clinical outcomes; their impacts are still not fully understood. Additionally, improvements in our understanding of the way in which individuals with DS respond to vaccination might reveal ways of adjusting vaccination schedules to optimize the protection of these individuals against severe infection. Transcriptional analysis, in vitro DS immune cell assays, mouse models, and clinical research all have a role in answering these questions.

Lastly, it is widely understood that an individual’s genetic makeup can influence disease outcome, as demonstrated by studies of inborn errors of immunity and their effects. Viewing DS solely as a trisomy of chromosome 21 likely underestimates the impact of genetic variants across chromosomes in tandem with the triplication of chromosome 21. Ramachandran et al. (2015) performed a genome-wide association study to investigate common genetic variants which may contribute to Down Syndrome-associated atrioventricular septal defect (AVSD); infants with DS have a 2000-fold increased risk of AVSD. Although no common autosomal (non-chromosome 21) SNPs exceeded genome-wide significance, there were four disomic regions of interest with SNPs in close proximity to genes which contribute to heart development and function. This study only genotyped 210 individuals with DS and complete ASVD compared to 242 individuals with DS and a structurally normal heart, which may suggest that large population sizes are needed to elucidate the genomic contribution outside of chromosome 21 to health outcomes commonly found in DS.⁸⁵ This is an extremely underexplored field but studies such as these and other approaches to identifying genetic variation outside of chromosome 21 in DS may help to explain the clinical heterogeneity seen across individuals with trisomy 21, both in the immune system and beyond.

As medical care for individuals with DS has improved, and lifespans increased,³ the role of the immune system has become increasingly clear. It is crucial to continue research into the links between the immune system and clinical outcomes in DS, with the ultimate goal of developing more specific and effective treatments to reduce the severity and frequency of autoimmune diseases and infections in individuals with DS.