Intrinsic Defects in B Cell Development and Differentiation, T Cell Exhaustion and Altered Unconventional T Cell Generation Characterize Human Adenosine Deaminase Type 2 Deficiency

In 2014, biallelic loss-of-function mutations in ADA2 (encoding adenosine deaminase type 2 [ADA2]) were described as the cause of a novel rare inborn error of immunity [1, 2]. The clinical phenotype of human ADA2 deficiency includes not only recurrent fevers and vasculitis (ranging from livedo racemosa to polyarteritis nodosa and lacunar stroke) but also immunodeficiency and cytopenia, either due to autoimmunity or bone marrow (BM) failure and hematological malignancy [3‐5]. The pathophysiology of deficiency of ADA2 (DADA2) is far from resolved. Disrupted interaction between the vascular endothelium and monocytes is believed to be central to at least the vascular phenotype of the disease, with a skewing of macrophages to a pro-inflammatory M1 phenotype [1]. For patients with a predominant vasculitis phenotype, the mainstay of treatment is TNF inhibition [6]. However, cytopenias are mostly refractory to treatment with TNF inhibitors, growth factors and immunosuppressives [7, 8]. Hematopoietic stem cell transplantation results in excellent survival and cures ADA2-deficient patients from all disease manifestations, demonstrating the hematopoietic-intrinsic etiology of this condition [9, 10]. In terms of immunodeficiency, up to 30% of DADA2 patients have panhypogammaglobulinemia (IgG, IgM, IgA) [3] and can present with a common variable immunodeficiency-like phenotype, including recurrent upper and lower respiratory tract infections, bronchiectasis and associated gastrointestinal involvement. However, some patients also exhibit a phenotype mimicking auto-immune lymphoproliferative syndrome (ALPS) [11‐13]. Moreover, DADA2 patients can have particular susceptibility to herpes virus infections—cytomegalovirus (CMV), Epstein Barr virus (EBV), herpes simplex virus 1, human herpes virus 6 (HHV6)—as well as HPV and Molluscum contagiosum [12, 14].

While features of vasculitis in DADA2 are well described, mechanisms underlying immunodeficiency have been less well explored. Initial descriptions noted B cell lymphopenia, progressive hypogammaglobulinemia occasionally mimicking agammaglobulinemia, increased numbers of naïve and CD21^lo B cells, but reductions in switched memory B cells and plasmablasts [1, 15, 16]. In vitro findings of decreased T-dependent Ig secretion and increased B cell apoptosis inferred that a B cell–intrinsic defect underlies humoral immunodeficiency in DADA2 [1, 16]. However, while circulating T follicular helper (cTfh) cells—the CD4⁺ T cell subset that guides B cell differentiation [17]—have been reported to be present in normal or increased proportions in DADA2 patients, ADA2-deficient CD4⁺ T cells apparently exhibit features of impaired help for B cell differentiation such as reduced IL-21 production and CD40L expression in vitro [16]. Thus, functionally impaired cTfh cells may also contribute to the B cell defect in DADA2. Further cellular aberrations in DADA2 include increased proportions of CD4⁻CD8⁻ αß⁺ TCR⁺ cells [1, 13, 18, 19] consistent with an ALPS-like phenotype as well as reductions in frequencies of central and effector memory CD4⁺ and CD8⁺ T cell subsets [1, 13, 16]. In order to further delineate peripheral immune defects in DADA2, we conducted an in-depth clinical, immunophenotyping and functional study in 10 patients with bi-allelic pathogenic ADA2 variants from 6 unrelated families.

In this report, we provide extensive immunophenotyping and in vitro functional analyses of leukocytes from 10 patients with DADA2. We confirm the defect in terminal B cell differentiation in ADA2 deficiency [1, 2, 16, 52], evidenced by lower proportions of the total as well as IgG- and IgA-expressing memory B cells in these patients. Furthermore, the phenotype of ADA2-deficient B cell subsets suggested these cells were less mature than corresponding B cells in healthy donors. Moreover, the peripheral B cell defect correlated with a block in B cell development in the BM at the pre–B cell stage, as well as an intrinsic failure of DADA2 B cells to differentiate into IgA-producing B cells. As the proportions of CD27⁺ memory B cells increase in the peripheral blood of healthy individuals with age, it could be argued that the memory B cell deficiency in DADA2 reflects the younger age of some of the patients in our cohort (< 10 years old, Table 1). However, this is unlikely because frequencies and numbers of memory B cell in healthy individuals reach adult levels by 2–4 years of age [24, 53, 54]. Indeed, all but 1 patient (P7) had a paucity of memory B cells, including P8, P9 and P10 who were > 40 years old, indicating age is not a determinant of impaired B cell differentiation in DADA2. Collectively, these data demonstrating an intrinsic maturation B cell defect due to ADA2-deficiency may explain the hypogammaglobulinemia and B cell lymphopenia characteristic of DADA2 patients. Moreover, there was no improvement in hypogammaglobulinemia despite treatment with etanercept (Table 1, data not shown) [12]. This may reflect the requirement for TNF in establishing and maintaining the anatomical structure of B-cell follicles in mice [55], and/or the impact of TNF blockade on memory B cell maintenance in secondary lymphoid tissues [56].

Extended lymphocyte immunophenotyping and functional analysis also revealed several anomalies in DADA2 patients. First, we found a significantly reduced proportion of Treg cells. This has also been noted previously in some [13], but not all [16] studies. This is interesting as patients with DADA2 experience multiple auto-immune features including cytopenia and auto-antibody production [3]. Moreover, the frequencies of naïve CD4⁺ and CD8⁺ T cells were increased while T_CM and T_EM cells were reduced, pointing to a T cell differentiation defect. Second, ADA2-deficient CD4⁺ and CD8⁺ memory T cells exhibited a dysfunctional phenotype—elevated expression of PD-1, CD95, CX3CR1 and CD57; diminished expression of CD28—that has been associated with poor proliferation, terminal differentiation and exhaustion/senescence [57]. Third, ADA2-deficient memory CD8⁺ T cells exhibited diminished survival and granzyme production in vitro. Fourth, DADA2 patients were also found to have significantly lower proportions of MAIT, NKT, Vγδ2⁺ T cells and mature CD56^dimCD57⁻ NK cells, but increased proportions of immature CD56^bright NK cells. Interestingly, Vγδ2 T cells are the most abundant γδT cells in peripheral blood of healthy individuals and display increased cytotoxicity against multiple viral and tumoral antigens [58, 59]. Indeed, an inverse correlation between proportions of Vδ2⁺ T cells and EBV reactivation has been observed following HSCT [60, 61].

Taken together, it is likely that the senescent/exhausted phenotype and impaired production of cytolytic molecules by ADA2-deficient CD8⁺ T cells, coupled with decreased frequencies of Vγδ2⁺ T cells and an accumulation of less mature NK cells, contribute to refractory/recurrent viral infections in ADA2-deficient patients [62‐64]. From a mechanistic perspective, ADA2 deficiency may lead to increased activation of adenosine 2A receptors, reducing T cell activation, in addition to reduced TCR signaling and lytic activity of cytototoxic lymphocytes [65]. These findings are reminiscent of our previous analysis of the CD8⁺ T cell compartment in individuals with pathogenic bi-allelic inactivating variants in DOCK8 [40, 66, 67] or gain-of-function variants in PIK3CD [39] who are also susceptible to recurrent infections with herpes viruses and have an abundance of dysfunctional exhausted-type CD8⁺ T cells. The defect in ADA2-deficient cytotoxic lymphocytes may also explain why some DADA2 patients present with hemophagocytic lymphohistiocytosis (unpublished observation) or CD3⁺CD8⁺ large granular lymphocytes [68, 69].

Interestingly, DC phenotyping showed significantly increased proportions of CD16⁺CD1c^lo/− DCs. These DCs or non-classical like monocytes have potent T cell stimulatory capacity and produce proinflammatory cytokines upon TLR stimulation by TNF-α, IL6, IL12. Thus, these cells may play a role in maintaining the inflammatory state observed in DADA2. In addition, reduced proportions of mDCs may be associated with the overall defect in T and B cell immune responses observed in vivo.

DADA2 patients exhibited significantly reduced proportions of classical monocytes and increased proportions of intermediated and non-classical monocytes. Moreover, CD169/SIGLEC1 expression on each monocyte subset in DADA2 patients was significantly elevated compared to healthy controls. The increased non-classical monocytes in DADA2 may account in part for the observed inflammation in these patients. Indeed, nonclassical monocytes are considered proinflammatory and a prominent source of TNF-α compared to classical monocytes [70, 71]. Increased CD169/SIGLEC1 expression is in line with reported evidence of upregulated type I IFN signaling in DADA2. The concomitant increase in proportions of intermediate monocytes is interesting, as increases in both intermediate and nonclassical monocyte subsets have been observed in patients with lupus and sepsis, which are also characterized by type 1 IFN signatures [72]. Intriguingly, several patients with DADA2 were initially diagnosed with lupus based on clinical features and serum anti-dsDNA auto-antibodies [1, 11]. Interestingly, increased expression of SIGLEC-1 on myeloid cells has been implicated in viral dissemination in models of HSV1 and CMV infections, among other viruses [73]. Further study of the effect of increased SIGLEC-1 expression on the observed viral infections in DADA2 may shed light on the intriguing coalescence of the type I IFN signature and susceptibility to viral infection. Finally, increased SIGLEC-1 expression on monocytes from heterozygous carriers is consistent with the clinical observation that some carriers of pathogenic ADA2 mutations exhibit signs and symptoms compatible with DADA2, although the carriers tested here were reportedly asymptomatic. This biological parallel of clinical phenotype in carriers is also evident from the investigation of ADA2 deficient neutrophils [74].

In summary, we have identified a complex immunophenotype in DADA2 with impaired differentiation of CD4⁺ and CD8⁺ T cells and B cells, in addition to an exhausted/senescent CD8⁺ T cell phenotype. Unconventional T cells are diminished whereas pro-inflammatory monocytes and CD56^bright immature NK cells are increased (Fig. 9). These findings suggest a delicate balance between type I and type II IFN as well as TNF-α-driven hyperinflammation next to a predisposition to severe viral infection and diminished antibody responses. Our findings confirm and extend a recently published report [75, 76]. Although these are associations, they invite a large prospective survey on the immunophenotype of ADA2-deficient patients to further our understanding of the variable immunodeficiency manifestations. Moreover, as DADA2 may underlie humoral immunodeficiency, patients with an immunophenotype in line with these finding could be selected to have ADA2 enzyme activity tested [12]. Our study is unique as several patients were not on any treatment. Also, the median age of the patients was young, which may lead us to believe that there is less environmental influence on the findings, in terms of longer ongoing inflammation and multiple treatments. Ultimately, better knowledge may aid in designing targeted therapy to prevent viral infections in these patients with excessive inflammation as the overarching phenotype.