Inherited human RelB deficiency impairs innate and adaptive immunity to infection

doi:10.1073/pnas.2321794121

Case Reports

. 2024 Sep 10;121(37):e2321794121.

doi: 10.1073/pnas.2321794121. Epub 2024 Sep 4.

Inherited human RelB deficiency impairs innate and adaptive immunity to infection

Tom Le Voyer^#^{1

2

3}, Majistor Raj Luxman Maglorius Renkilaraj^#^{1

2}, Kunihiko Moriya^#^{1

2}, Malena Pérez Lorenzo^#^{1

2}, Tina Nguyen^#^{4

5}, Liwei Gao^#^{1

2}, Tamar Rubin^#⁶, Axel Cederholm^#⁷, Masato Ogishi^#⁸, Carlos A Arango-Franco^{1

2

9}, Vivien Béziat^{1

2

8}, Romain Lévy^{1

2

8}, Mélanie Migaud^{1

2}, Franck Rapaport⁸, Yuval Itan¹⁰, Elissa K Deenick^{4

5}, Irene Cortese¹¹, Andrea Lisco¹², Kaan Boztug^{13

14

15}, Laurent Abel^{1

2

8}, Stéphanie Boisson-Dupuis^{1

2

8}, Bertrand Boisson^{1

2

8}, Patrick Frosk¹⁶, Cindy S Ma^{4

5}, Nils Landegren⁷, Fatih Celmeli¹⁷, Jean-Laurent Casanova^#^{1

2

8

18

19}, Stuart G Tangye^#^{4

5}, Anne Puel^#^{1

2

8}

Affiliations

¹ Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM UMR 1163, Paris 75015, France.
² Imagine Institute, Paris Cité University, Paris 75015, France.
³ Clinical Immunology Department, Assistance Publique Hôpitaux de Paris, Saint-Louis Hospital, Paris 75010, France.
⁴ Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia.
⁵ School of Clinical Medicine, Faculty of Medicine and Health, University of New South Wales Sydney, Sydney, NSW 2052, Australia.
⁶ Division of Pediatric Clinical Immunology and Allergy, Department of Pediatrics and Child Health, University of Manitoba, Winnipeg, MB R3A 1S1, Canada.
⁷ Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala SE-751 05, Sweden.
⁸ St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY 10065.
⁹ Group of Inborn Errors of Immunity, Department of Microbiology and Parasitology, School of Medicine, University of Antioquia, Medellín 050010, Colombia.
¹⁰ Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029.
¹¹ Experimental Immunotherapeutics Unit, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD 20892.
¹² Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892.
¹³ St. Anna Children's Cancer Research Institute, Vienna 1090, Austria.
¹⁴ Medical University of Vienna, Department of Pediatrics and Adolescent Medicine, Vienna 1090, Austria.
¹⁵ CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna 1090, Austria.
¹⁶ Department of Biochemistry and Medical Genetics, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB R3E 0W2, Canada.
¹⁷ Department of Allergy and Immunology, University of Medical Science, Antalya Education and Research Hospital, Antalya 07100, Türkiye.
¹⁸ Pediatric Hematology-Immunology Unit, Necker Hospital for Sick Children, Paris 75015, France.
¹⁹ HHMI, New York, NY 10065.

^# Contributed equally.

PMID: 39231201
PMCID: PMC11406260
DOI: 10.1073/pnas.2321794121

Case Reports

Inherited human RelB deficiency impairs innate and adaptive immunity to infection

Tom Le Voyer et al. Proc Natl Acad Sci U S A. 2024.

. 2024 Sep 10;121(37):e2321794121.

doi: 10.1073/pnas.2321794121. Epub 2024 Sep 4.

Authors

Affiliations

¹ Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM UMR 1163, Paris 75015, France.
² Imagine Institute, Paris Cité University, Paris 75015, France.
³ Clinical Immunology Department, Assistance Publique Hôpitaux de Paris, Saint-Louis Hospital, Paris 75010, France.
⁴ Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia.
⁵ School of Clinical Medicine, Faculty of Medicine and Health, University of New South Wales Sydney, Sydney, NSW 2052, Australia.
⁶ Division of Pediatric Clinical Immunology and Allergy, Department of Pediatrics and Child Health, University of Manitoba, Winnipeg, MB R3A 1S1, Canada.
⁷ Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala SE-751 05, Sweden.
⁸ St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY 10065.
⁹ Group of Inborn Errors of Immunity, Department of Microbiology and Parasitology, School of Medicine, University of Antioquia, Medellín 050010, Colombia.
¹⁰ Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029.
¹¹ Experimental Immunotherapeutics Unit, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD 20892.
¹² Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892.
¹³ St. Anna Children's Cancer Research Institute, Vienna 1090, Austria.
¹⁴ Medical University of Vienna, Department of Pediatrics and Adolescent Medicine, Vienna 1090, Austria.
¹⁵ CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna 1090, Austria.
¹⁶ Department of Biochemistry and Medical Genetics, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB R3E 0W2, Canada.
¹⁷ Department of Allergy and Immunology, University of Medical Science, Antalya Education and Research Hospital, Antalya 07100, Türkiye.
¹⁸ Pediatric Hematology-Immunology Unit, Necker Hospital for Sick Children, Paris 75015, France.
¹⁹ HHMI, New York, NY 10065.

^# Contributed equally.

PMID: 39231201
PMCID: PMC11406260
DOI: 10.1073/pnas.2321794121

Abstract

We report two unrelated adults with homozygous (P1) or compound heterozygous (P2) private loss-of-function variants of V-Rel Reticuloendotheliosis Viral Oncogene Homolog B (RELB). The resulting deficiency of functional RelB impairs the induction of NFKB2 mRNA and NF-κB2 (p100/p52) protein by lymphotoxin in the fibroblasts of the patients. These defects are rescued by transduction with wild-type RELB complementary DNA (cDNA). By contrast, the response of RelB-deficient fibroblasts to Tumor Necrosis Factor (TNF) or IL-1β via the canonical NF-κB pathway remains intact. P1 and P2 have low proportions of naïve CD4⁺ and CD8⁺ T cells and of memory B cells. Moreover, their naïve B cells cannot differentiate into immunoglobulin G (IgG)- or immunoglobulin A (IgA)-secreting cells in response to CD40L/IL-21, and the development of IL-17A/F-producing T cells is strongly impaired in vitro. Finally, the patients produce neutralizing autoantibodies against type I interferons (IFNs), even after hematopoietic stem cell transplantation, attesting to a persistent dysfunction of thymic epithelial cells in T cell selection and central tolerance to some autoantigens. Thus, inherited human RelB deficiency disrupts the alternative NF-κB pathway, underlying a T- and B cell immunodeficiency, which, together with neutralizing autoantibodies against type I IFNs, confers a predisposition to viral, bacterial, and fungal infections.

Keywords: NF-κB pathway; RelB deficiency; autoantibodies; immunodeficiency; type I IFNs.

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Conflict of interest statement

Competing interests statement:Tim Niehues was a co-author on publications with the following co-authors within the last 4 y: K.B., C.S.M., and S.G.T. (https://doi.org/10.1182/blood.2020006738) and K.B. (https://doi.org/10.1016/j.jaci.2019.11.051; https://doi.org/10.1002/eji.202048713). Andrew R. Gennery was a co-author with S.G.T. (https://doi.org/10.1016/j.jaci.2020.09.010; https://doi.org/10.1016/j.jaci.2022.09.002) within the last 4 y.

Figures

**Fig. 1.**
AR RelB deficiency in two unrelated patients. (A) Pedigrees of the two unrelated families. The familial segregation of the variants identified in P1 and P2 is depicted. The double lines connecting P1’s parents indicate consanguinity. An arrow indicates the probands. Solid symbols indicate disease status. “E?” indicates individuals of unknown genotype. (B) The CADD score and MAF of the variants of P1 and P2 (shown in red), the two previously reported disease-causing variants (shown in blue) (7, 23, 24), and the two homozygous missense variants found in the gnomAD 2.1 database (shown in black). All variants from P1, P2, and the four previously reported patients are private. The variants are plotted according to their CADD scores (y-axis) and MAF (x-axis). The black horizontal dotted line indicates the MSC. (C) Schematic diagram of the RelB protein. The regions corresponding to functionally significant domains are shown in dark gray (LZ: leucine zipper domain; RHD: Rel homology domain; TAD: transactivation domain). The variants of P1 and P2 are shown in red; the two previously reported disease-causing variants are indicated in blue, and the variants reported in the homozygous state in gnomAD appear in black. (D) Immunoblot analysis of RelB expression on total protein extracts from non-transfected HEK 293 T (NT) cells, or HEK 293 T cells transfected with an empty pCMV6 plasmid (EV), or pCMV6 plasmids containing the WT, P1’s variant (c.C212dup, p.Q72Tfs*152, or p.Q72fs), P2’s variant (c.433G>A/c.1091C>T, p.E145K/p.P364L), the previously reported disease-causing variants (c.1191C>A, p.Y397*, and c.400_c.401insAGC/p.Q135dup), or the two missense (c.1249G>A, p.D417N, and c.1459G>T, p.D487Y) variants of *RELB* present in the homozygous state in gnomAD. RelB was detected with a mAb specific for the C- or N-terminal region or with an antibody against the DDK tag. Actin was used as a loading control. The results shown are representative of three independent experiments. (E) Luciferase activity of HEK 293 T cells after 48 h of transfection with an NF-κB reporter plasmid together with an empty pCMV6 vector (EV), or a pCMV6 vector encoding wild-type (WT), P1’s variant (c.C212dup/p.Q72Tfs*152), P2’s variant (c.433G>A/p.E145K and c.1091C>T/p.P364L), the previously reported disease-causing (c.1191 C>A/p.Y397* and c.400_c.401insAGC/p.Q135dup) variants, and the two missense (c.1249 G > A/p.D417N; c.1459 G>T/p.D487Y) RelB variants present in the homozygous state in gnomAD, with or without the pCMV6-NFKB2-DDK plasmid encoding p100. Results were normalized against *Renilla* luciferase activity. Results were then normalized against the level of transcriptional activity for the WT p52/RelB dimer. The mean ± SD of one representative experiment (performed with a biological duplicate) is shown.

**Fig. 2.**
Impaired *RELB* expression and impaired activation of the alternative NF-κB pathway in the fibroblasts of P1 and P2. (A) RNAs extracted from SV40-immortalized fibroblasts (SV40-F) from healthy controls (C), P1, and P2 were subjected to RT-qPCR for total *RELB*. Data are expressed as 2-^Δ^C^t relative to the mean expression of controls, after normalization against *GUS* (endogenous control) expression (ΔCt). (B and C) RNA extracted from primary fibroblasts from three different healthy subjects (C), P1, P2, and a patient with AR complete NIK deficiency stimulated with lymphotoxin α1β2 (Lt) for 24 h and subjected to RT-qPCR for *NFKB2* (B) or *VCAM1* (C). (D) Immunoblot analysis of phosphorylated p100 (P-p100), NF-κB2 (p100/p52), RelB, NF-κB1 (p105/p50), and RelA levels in primary fibroblasts from healthy controls (C1 and C2), P1, P2, and a NIK-deficient patient (NIK^−/−) (8). Actin immunoblotting was used as a loading control. The results shown are representative of two independent experiments. (E) Graph showing the p100/p52 ratio in primary fibroblasts from healthy controls (C1 and C2), P1, P2, and a NIK-deficient patient (NIK^−/−), based on data from two independent experiments. (F) Primary fibroblasts from controls (C1 and C2), the two RelB-deficient patients (P1 and P2, in red), a patient with a p52^LOF/IκBδ^GOF variant (6) and a NIK-deficient patient (NIK^−/−) (8), were left unstimulated or were stimulated with TWEAK for 48 h and stained with a rabbit anti-C-terminal RelB or a mouse anti-N-terminal p100 mAb. Nuclei were stained with DAPI.

**Fig. 3.**
Rescue of defective non-canonical NF-κB signaling in P1 fibroblasts. (A) SV40-F from a healthy control (C) and P1 were stably transduced with an empty vector (EV) or the WT *RELB* cDNA. SV40-F from the healthy control and P1 were also transduced with the Q72Tfs, E145K, P364L, or Y397* mutant *RELB* cDNA. The RNA extracted from these cells was subjected to RT-qPCR for total *RELB*. Data are displayed as 2^−ΔCt relative to EV-transduced cells, after normalization against *GUSB* (endogenous control) expression (ΔCt). The result of one representative experiment is shown. (B) SV40-F from a healthy control (*Left*) or from P1 (*Right*) were stably transduced with an EV, the WT or a mutant (Q72fs, E145K, P364L, or Y397*) *RELB* cDNA. The cells were either left unstimulated (NS, gray bars for the control or red bars for P1) or were stimulated for 24 h with Lt (black bars). The RNA extracted from these cells was subjected to RT-qPCR for total *NFKB2*. Data are displayed as 2^−ΔCt values relative to unstimulated EV-transduced cells, after normalization against *GUSB* (endogenous control) expression (ΔCt). The result of one independent experiment is shown.

**Fig. 4.**
Normal activation of the canonical NF-κB pathway in fibroblasts from P1 and P2. (A) Immunoblot analysis of the cytoplasmic and nuclear fractions of SV40-F from healthy controls (C1 and C2), P1, P2, or SV40-F deficient for NEMO (NEMO^Y/−) left unstimulated or stimulated with TNF for 4 h, and assessed for the levels of NF-κB1 (p105/p50), RelA (p65), RelB, and NF-κB2 (p100). The (*) symbol indicates non-specific bands. Tubulin and β-actin were used as loading controls. (B) SV40-F from controls (C1 and C2), the two RelB-deficient patients (P1 and P2, in red), and NEMO-deficient SV40-F (NEMO^Y/−) were stimulated with TNF for 4 h, and stained with a mouse anti-C-terminal RelA or a mouse anti-N-terminal p105 antibody. Nuclei were labeled with DAPI.

**Fig. 5.**
Auto-Abs against type I IFNs in P1 and P2. (A) Auto-Abs neutralizing IFN-α2, IFN-ω, or IFN-β at concentrations of 10 ng/mL or 100 pg/mL were assessed in an ISRE luciferase assay. The gray area corresponds to the normal ISRE activity obtained with non-neutralizing plasma from healthy controls. (B) Protein microarray showing the distribution of auto-Ab reactivity against 20,000 human proteins in plasma samples from patients with AR RelB deficiency (n = 2, P1 and P2, aged 19 and 32 y, respectively). Data are expressed as the fold-change relative to 22 plasma samples from healthy donors. Red dots represent type I IFNs and yellow dots represent type III IFNs. Data for HuProt and neutralization assay experiments are presented as the mean for at least two technical replicates. (C) Venn diagram showing the enriched autoantigen profile (log₂-fold change relative to healthy controls >1.5) of patients with APS-1 (n = 14), p52^LOF/IκBδ^GOF variants (n = 12), or AR RelB deficiency (n = 2). Type I IFNs are indicated in bold. (D) Proportion of shared (by ≥2 patients) and private reactive autoantigens in the group of patients with APS-1, a p52^LOF/IκBδ^GOF variant, or AR RelB deficiency. (E) Reactive autoantigens common to the two patients with AR RelB deficiency included in the analysis. Red dots indicate type I IFNs.

**Fig. 6.**
RelB deficiency compromises T cell development and functions. Immunophenotyping of PBMCs from 4-13 healthy controls (C, black dots), P1 (red dots), and P2 (blue dots). (A–C) Frequency of naïve (CD45RA⁺CCR7⁺), central memory (CM, CD45RA⁻CCR7⁺), effector memory (EM, CD45RA^+/−CCR7⁻), and terminally differentiated effector memory (TEMRA, CDR45RA⁻CCR7⁻) cells among the CD4⁺ (A) and CD8⁺ (B) T cells of healthy subjects (C, n = 4), P1 before HSCT, and P2. (C) Frequency of T_reg (CD3⁺CD4⁺CD25^hiFoxP3⁺) cells in the CD4⁺ T cell compartment in controls (n = 4), P1 before HSCT, and P2. (D) Frequencies of T_h subsets among memory CD4⁺ T cells from controls (n = 5), P1 before HSCT, and P2. Subsets were defined as follows: T_h1 (CXCR5⁻CXCR3⁺CCR4⁻CCR6⁻), T_h1* (CXCR5⁻CXCR3⁺CCR4⁻CCR6⁺), T_h2 (CXCR5⁻CXCR3⁻CCR4⁺CCR6⁻), and T_h17 (CXCR5⁻CXCR3⁻CCR4⁺CCR6⁺). Dotted horizontal lines represent the median value in each distribution. (E) Percentages of cells expressing IL-17A, IL-17F, IL-22, and IFN-γ ex vivo, as determined by flow cytometry, among memory CD4⁺ T cells from PBMCs activated by incubation with PMA and ionomycin for 8 h. (F) IL-17A and IL-22 production assessed by specific ELISA on whole-blood supernatants stimulated by incubation for 24 h with PMA and ionomycin. The two experiments were conducted in parallel for healthy subjects (n = 10), the RelB-deficient patient (P1), and patients with heterozygous gain-of-function *STAT1* mutations (n = 6). (G) Cytokine production, measured by ELISA, for IL-17A and IL-17F after 4 d of culture in T_h0 or T_h17 conditions, for cells from nine different healthy donors, and from P1 before (pre-) and after (post-) HSCT.

**Fig. 7.**
RelB deficiency impairs peripheral B cell development and function in P1. (A and B) Immunophenotyping of PBMCs from healthy donors (C, n = 10, filled black circles), and P1 before (pre, filled red circles) and after (post, red circles) HSCT. Subsets were defined by labeling with mAbs against CD20, CD10, CD27, IgG, IgA, and IgM. The proportions of B (CD20⁺) cells within the lymphocyte gate (A), and of transitional (IgM⁺IgD^+/−CD10⁺CD27⁻), naïve (IgM⁺IgD⁺CD27⁻), and memory (CD27⁺) cells within the B cell compartment (B) are shown. (C) Proportions of IgM⁺, IgG⁺, and IgA⁺ cells within the memory B cell population, as determined by flow cytometry. (D–F). Naïve B cells from six healthy subjects, and P1 before (pre) and after (post) HSCT were sorted and incubated with CD40L alone or together with CpG, BCR ligand, or IL-21 for 7 d. The secretion of IgM (D), IgG (E), and IgA (F) into the supernatant was assessed by Ig heavy chain–specific ELISA. The limit of detection corresponds to 1.

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References

1. Hayden M. S., Ghosh S., NF-κB in immunobiology. Cell Res. 21, 223–244 (2011). - PMC - PubMed
1. Sun S. C., The non-canonical NF-κB pathway in immunity and inflammation. Nat. Rev. Immunol. 17, 545–558 (2017). - PMC - PubMed
1. Shih V. F.-S., Tsui R., Caldwell A., Hoffmann A., A single NFκB system for both canonical and non-canonical signaling. Cell Res. 21, 86–102 (2011). - PMC - PubMed
1. Fusco A. J., et al. , The NF-κB subunit RelB controls p100 processing by competing with the kinases NIK and IKK1 for binding to p100. Sci. Signal. 9, ra96 (2016). - PubMed
1. Millet P., McCall C., Yoza B., RelB: An outlier in leukocyte biology. J. Leukoc. Biol. 94, 941–951 (2013). - PMC - PubMed

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[1] Hayden M. S., Ghosh S., NF-κB in immunobiology. Cell Res. 21, 223–244 (2011). - PMC - PubMed

[2] Hayden M. S., Ghosh S., NF-κB in immunobiology. Cell Res. 21, 223–244 (2011). - PMC - PubMed

[3] Sun S. C., The non-canonical NF-κB pathway in immunity and inflammation. Nat. Rev. Immunol. 17, 545–558 (2017). - PMC - PubMed

[4] Sun S. C., The non-canonical NF-κB pathway in immunity and inflammation. Nat. Rev. Immunol. 17, 545–558 (2017). - PMC - PubMed

[5] Shih V. F.-S., Tsui R., Caldwell A., Hoffmann A., A single NFκB system for both canonical and non-canonical signaling. Cell Res. 21, 86–102 (2011). - PMC - PubMed

[6] Shih V. F.-S., Tsui R., Caldwell A., Hoffmann A., A single NFκB system for both canonical and non-canonical signaling. Cell Res. 21, 86–102 (2011). - PMC - PubMed

[7] Fusco A. J., et al. , The NF-κB subunit RelB controls p100 processing by competing with the kinases NIK and IKK1 for binding to p100. Sci. Signal. 9, ra96 (2016). - PubMed

[8] Fusco A. J., et al. , The NF-κB subunit RelB controls p100 processing by competing with the kinases NIK and IKK1 for binding to p100. Sci. Signal. 9, ra96 (2016). - PubMed

[9] Millet P., McCall C., Yoza B., RelB: An outlier in leukocyte biology. J. Leukoc. Biol. 94, 941–951 (2013). - PMC - PubMed

[10] Millet P., McCall C., Yoza B., RelB: An outlier in leukocyte biology. J. Leukoc. Biol. 94, 941–951 (2013). - PMC - PubMed

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Inherited human RelB deficiency impairs innate and adaptive immunity to infection

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Inherited human RelB deficiency impairs innate and adaptive immunity to infection

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