Genomic Spectrum and Phenotypic Heterogeneity of Human IL-21 Receptor Deficiency

Abstract

Biallelic inactivating mutations in IL21R causes a combined immunodeficiency that is often complicated by cryptosporidium infections. While eight IL-21R-deficient patients have been reported previously, the natural course, immune characteristics of disease, and response to hematopoietic stem cell transplantation (HSCT) remain to be comprehensively examined. In our study, we have collected clinical histories of 13 patients with IL-21R deficiency from eight families across seven centers worldwide, including five novel patients identified by exome or NGS panel sequencing. Eight unique mutations in IL21R were identified in these patients, including two novel mutations. Median age at disease onset was 2.5 years (0.5-7 years). The main clinical manifestations were recurrent bacterial (84.6%), fungal (46.2%), and viral (38.5%) infections; cryptosporidiosis-associated cholangitis (46.2%); and asthma (23.1%). Inflammatory skin diseases (15.3%) and recurrent anaphylaxis (7.9%) constitute novel phenotypes of this combined immunodeficiency. Most patients exhibited hypogammaglobulinemia and reduced proportions of memory B cells, circulating T follicular helper cells, MAIT cells and terminally differentiated NK cells. However, IgE levels were elevated in 50% of IL-21R-deficient patients. Overall survival following HSCT (6 patients, mean follow-up 1.8 year) was 33.3%, with pre-existing organ damage constituting a negative prognostic factor. Mortality of non-transplanted patients (n = 7) was 57.1%. Our detailed analysis of the largest cohort of IL-21R-deficient patients to date provides in-depth clinical, immunological and immunophenotypic features of these patients, thereby establishing critical non-redundant functions of IL-21/IL-21R signaling in lymphocyte differentiation, humoral immunity and host defense against infection, and mechanisms of disease pathogenesis due to IL-21R deficiency. Outcome following HSCT depends on prior chronic infections and organ damage, which should thus be considered as early as possible following molecular diagnosis.

Keywords: B cell differentiation; IL-21/IL-21R signaling; STAT3; T follicular helper cells.

Fig. 1

IL-21R domain structure and patient mutations. Domain structure and positions of mutations identified in different patients are indicated. Newly identified patients are marked in red

Fig. 2

Pathogenic variants mapped onto the crystal structure of the IL-21/IL-21R complex. IL-21R (orange cartoon) contains two external fibronectin domains, the N-terminal of which (upper) has three internal disulphides (yellow sticks), one involves residue C81 (exploded view). IL-21 (blue cartoon and transparent surface) binds the hinge between the two receptor fibronectin domains. A complex N-linked carbohydrate (gray sticks, extending from N73) bridges the two fibronectin domains and has been postulated to stabilize IL-21R [37, 38]. Residues W138, L158, D179, and R201 (orange sticks) are positioned within the C-terminal fibronectin domain proximal to the extracellular membrane. The side chains of W138 and L158 are buried within the hydrophobic interior of the domain. D179 sits on the exterior surface proximal to, but not directly interfacing, IL-21 while R201 is on an exterior surface distal to IL-21 binding. The side chain of R201 is positioned adjacent to the side chain of W214 (within the WSXWS motif) which is also glycolysylated (dark gray sticks) and may contribute to stability of the N73-projected carbohydrate bridge

Fig. 3

Clinical features of IL-21R-deficient patients. a Pedigree of the family of P1, P2, and P3. b, c Cutaneous involvement in P4 (face and arm). d H&E stain of a skin biopsy of P4 showing lymphohistiocytic inflammation. Predominant infiltration of CD3+ cells of the superficial dermis invading underlying subcutaneous tissue

Fig. 4

Laboratory features of IL-21R-deficient patients. a Proportions of lymphocyte subsets in IL-21R-deficient patients. One timepoint is displayed per patient. Gray areas indicate healthy donor control ranges [61]. b Laboratory tests of IL-21R-deficient patients. WBC white blood cell count, ALC absolute lymphocyte count

Fig. 5

IL21R mutations impact Tfh, Th17, MAIT cell formation and NK cell differentiation. PBMC from healthy controls or IL-21R-deficient (n = 5–10) patients were labeled with mAbs against CD3, CD4, CD8, CD56, CD161, TCR Vβ11, TCR Vα7-2, TCR Vα24, CCR7, and CD45RA. a–f Proportions of a total (CD3⁺) T cells, b αβ T cells, c γδ T cells, d NKT cells, e MAIT cells, and f CD4⁺ and CD8⁺ T cells. g–l Within CD4⁺ T cells, frequencies of g naïve, T_CM, or T_EM cells; h Th17 (CXCR3⁻CCR6⁺), CXCR3⁺CCR6⁺, Th1 (CXCR3⁺CCR6⁻), or CXCR3⁻CCR6⁺; i regulatory T cells; j, k cTfh cells, as well as subsets of l Th17, CXCR3⁺CCR6⁺, Th1, or CXCR3⁻CCR6⁺, cTfh cells. m, n Proportions of m naïve, T_CM, T_EM, or T_EMRA CD8⁺ T cells, and n percentages of these subsets expressing CD57. o, p Proportions of o total NK cells within lymphocytes, and of p CD56^hi, CD56^loCD57⁻, and CD56^loCD57⁺ NK cell subsets. For all graphs, values represent mean ± SEM, with individual symbols corresponding to one healthy donor or IL-21R-deficient patient. Statistics performed using t tests with Mann-Whitney tests. *p < 0.05, **p < 0.01. FACS data for some of the patients has been published previously [–27, 33] but is included here to provide an overview of all available patients

Fig. 6

Impaired generation of memory B cells and induction of class switching due to IL-21R deficiency. PBMC from healthy controls or IL-21R-deficient (n = 5–9) patients were labeled with mAbs against CD19, CD20, CD10, CD27, IgG, IgA, CD123, CD11c, CD141, CD16, and CD1c. a–d Proportions of a total B cells within lymphocytes; b transitional, naïve, and memory B cells within the total B cell population; c naive B cells with a IgM⁻IgD⁺ or IgM^loIgD^hi phenotype; and d memory B cells expressing IgG or IgA. e–g Proportions of e DC within all leukocytes, f plasmacytoid (CD123⁺CD11c⁻) and myeloid cells (CD11c⁺CD123⁺) within the total DC population, and g mDC subsets [cDC1 (CD141⁺), CD16⁺ DC (CD16⁺), cDC2 (CD1c⁺)]. For all graphs, values represent mean ± SEM, with individual symbols corresponding to one healthy donor or IL-21R-deficient patient. Statistics performed using t tests with Mann-Whitney tests. *P < 0.05, **** P < 0.0001

Fig. 7

Functional defects in B and T cells due to inactivating biallelic IL21R variants. a–d Memory (a, c) or naïve (b, d) CD4⁺ T cells were sort-purified from healthy donors (HD, n = 22–24), or individuals with biallelic variants in IL21R (IL21R, n = 6) or DN variants in STAT3 (STAT3 DN, n = 6–9), and then cultured for 5 days in vitro under non-polarizing Th0 conditions (a, c), Th17 conditions (b) or Tfh-polarizing conditions (d). Secretion of the indicated Th17 (a, b; IL-17A, IL-17F, IL-22) or Th2 (c; IL-4, IL-5, IL-13) cytokines, or expression of the Tfh-cytokine IL-21 (d) was then determined. e, f Naive B cells were sort-purified from healthy donors (HD, n = 35), or individuals with biallelic variants in IL21R (IL21R, n = 6) or STAT3 DN variants (n = 10), and then cultured for 7 days in vitro with CD40L and IL-21. Secretion of IgM (e) or IgG and IgA (f) was then determined. NB: the data depicted in these graphs has been presented in previous publications [26, 33, 34], but is reproduced here to enable comparison between flow cytometric and functional defects, as well as clinical features, resulting from IL-21R deficiency and STAT3 DN mutations