Inherited human RelB deficiency impairs innate and adaptive immunity to infection

Significance We report two unrelated patients with combined T- and B-cell immunodeficiency and autoantibodies neutralizing type I interferons (IFNs) who developed recurrent viral, bacterial, and fungal diseases. The patients carried biallelic rare loss-of-function variants of V-Rel Reticuloendotheliosis Viral Oncogene Homolog B (RELB). Their thymic stromal cells, T cells, and B cells were defective in vivo, ex vivo, and in vitro. Human RelB is essential for T- and B-cell immunity to various pathogens and for thymic stromal cell-driven tolerance to type I IFNs.


Case report for P1
A 13-year-old female patient was referred to the pediatric immunology clinic for recurrent sinopulmonary infections beginning at the age of six years.The patient was born at full term to consanguineous parents, after a normal pregnancy.The delivery was uneventful.There was no family history of recurrent infections or immunological disorders.The patient had received all regular vaccinations, including those based on live vaccines (BCG and polio), without complications.At six years of age, she was diagnosed with oral candidiasis, otitis and recurrent respiratory tract infections leading to repeated hospital admissions.The patient had had multiple episodes of bronchopneumonia, resulting in bronchiectasis.At the age of 10 years, hypogammaglobulinemia was detected and the patient received short-term intravenous immunoglobulin treatment.During follow-up, at the age of 12 years, the patient presented with Cryptococcus neoformans meningitis complicated by cranial nerve palsy resulting in a loss of vision (26).The infectious phenotype of the patient, including chronic sinusitis, chronic otitis, oral candidiasis, and recurrent skin infections, was associated with a failure to thrive (weight and height below the third percentile for age) and mild splenomegaly.A marked macular eruption with hyperpigmentation was observed on the lower extremities.
Laboratory test results were as follows: hemoglobin, 11 g/dL; platelets, 158000/mm 3 ; WBC, 9500/mm 3 and an absolute lymphocyte count of 3000 cells/mm 3 .Mantoux tests for TB and serological tests for HIV were negative.Immunological testing revealed normal T-, B-and NK-lymphocyte counts, although the levels of recent thymic emigrants and TREC were below the lower limit of the normal range for age, and chest CT scans showed a thymus smaller than that in an age-matched healthy subject (Table S4; Fig. S3A-B).P1 had no pathologically enlarged lymph nodes during or independently of infectious disease.Serum Ig isotypes were evaluated before IVIg replacement therapy, at the age of 13 years: IgM levels were high, but IgG and IgA levels were extremely low (Table S4).It was not possible to assess levels of antigen-specific antibodies whilst the patient was on IVIg replacement therapy.The patient had no autoimmune manifestations.She was negative for anti-dsDNA, ANA, RF, anti-cardiolipin, anti-M2, ANCA, anti-HTG, anti-TPO, anti-GAD, anti-insulin, and anti-islet antibodies (Table S4).The patient was diagnosed with CID.
At the age of 14 years, she underwent HSCT with peripheral blood stem cells donated by a fully HLAmatched sibling, with a low-intensity conditioning regimen.The patient is currently in good health, with donor myeloid and T-cell chimerism levels > 90%, without GVHD.In particular, she has not presented any of the infections observed before transplantation, or any other severe infections.She suffered no serious complications from SARS-CoV-2 infection.No thymic hyperplasia was observed.The patient has been off IVIg replacement therapy since the age of 17 years.She has also been able to mount specific antibody responses to hepatitis B, rubella, and mumps vaccination.She has remained negative for anti-dsDNA, ANA, RF, anti-cardiolipin, anti-M2, ANCA, anti-HTG, anti-TPO, anti-GAD, anti-insulin, and anti-islet antibodies.
However, auto-Abs against IFN-α2 and IFN-ω were detected five years after HSCT (100 pg/mL).The patient's IgG levels have normalized, and her IgM levels have decreased towards the normal range.No neutralizing auto-Abs against type I IFNs were detected in the plasma samples from the P1's parents (I.1 and I.2).No material from P1's siblings (II.2 and II.3) was available for testing.

Case report for P2
We recruited a second patient, a 33-year-old man of Irish, Scottish, French, and German descent.His mother had had chronic lymphocytic leukemia, neither the patient's non-consanguineous parents nor his brother presented any other unusual infectious, autoimmune or autoinflammatory manifestations.At the age of three months, P2 presented with bilateral chorioretinitis of unknown cause.He suffered from one episode of varicella pneumonia requiring mechanical ventilation at the age of three years.Since infancy, P2 has had several episodes of bacterial peritonitis, pneumonia, otitis media, sinusitis, and oral and esophageal candidiasis.The patient reported a lower frequency of episodes of sinusitis and pneumonitis whilst on IVIg replacement therapy.Between the ages of five and six years, he suffered one episode of osteomyelitis.He reported having "sparse body hair" when assessed for delayed puberty during adolescence.A biopsy of warts on the hand and feet of P2 at the age of about 12 years revealed epidermodysplasia verruciformis.
HPV typing revealed the presence of HPV 23.The warts progressed to the skin of the groin, anterior neck, trunk, arms, and temple areas.The lesions were refractory to multiple treatments and required debridement surgery.The patient developed an early-onset malabsorption syndrome associated with chronic asymmetric lymphedema of both legs from birth onwards and had suffered from diarrhea and failure to thrive since infancy.Gastrointestinal (GI) endoscopy and biopsies provided no evidence of lymphangiectasia.Alphaone antitrypsin clearance studies did not detect protein-losing enteropathy in this patient, so the etiology of his early GI issues remains unclear.The patient also developed cirrhosis with cholestatic liver disease and portal hypertension.He had primary hypothyroidism, high TSH levels, and low gonadotropin and testosterone levels, but no detectable anti-TPO or anti-thyroglobulin auto-Abs.At the age of 22 years, P2 was diagnosed with EBV-negative diffuse large B-cell lymphoma (DLBCL) with meningeal and diffuse gastrointestinal involvement.Short-term treatment with IV dexamethasone was administered for DLBCLrelated intracranial hypertension.The patient went into remission after chemotherapy with rituximabcyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) and intrathecal cytarabine (Ara-C).
At the age of 31 years, he was treated for JC virus encephalitis.He developed alopecia, which worsened after chemotherapy, and suffered from chronic lymphopenia.A comprehensive immunological analysis at the age of 17 years showed severe lymphopenia with low CD4 + T-cell counts.The lymphocyte responses of this patient to pokeweed mitogen, phytohemagglutinin, concanavalin A, and Candida antigen ranged from normal to 20 to 30% the levels in normal healthy subjects, with low levels of proliferation in response to VZV.TCR Vβ repertoire analysis showed an overrepresentation of TCR Vβ21.3, with parallel decreases in the levels of the other TCR Vβ families.P2 had low levels of CD56 + natural killer cells, associated with a profound decrease in the function of these cells (in chromium release assays).P2 had 56 B cells/µL of blood at the age of 22 years, before rituximab infusion.B cells remained almost undetectable after the rituximab infusions, with fewer than 5 circulating CD19 + cells/mm 3 at the age of 32 years.The patient responded poorly to immunization with several vaccines (poor response to mumps and varicella, despite prior vaccination against measles mumps and rubella, and despite having had chicken pox and shingles; anti-tetanus titers were 0.29 IU/mL and anti-diphtheria and anti-mumps antibody titers were below the limit of detection).P2 had hypogammaglobulinemia requiring IVIg replacement therapy from the age of 18 years.
On clinical laboratory screening, P2 tested positive for anti-dsDNA autoantibodies, but negative for ANA and RF (Table S5).IgM levels were normal to high, IgA levels were low to normal and no circulating IgE antibodies were detected.P2 received trimethoprim/sulfamethoxazole prophylaxis continuously from the age of 22 years.He died from PML at the age of 34 years.

Cell purification and culture of the patients' cells
Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density centrifugation (Amersham Pharmacia Biotech, Sweden) from whole blood.Primary human fibroblasts obtained from skin biopsy specimens, and SV40 fibroblasts obtained after the immortalization of primary fibroblasts with SV-40T antigen (SV40-immortalized fibroblasts or SV40-F) were cultured in DMEM (Gibco BRL, Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL, Invitrogen, USA).All cells were grown at 37°C, under an atmosphere containing 5% CO2.

Generation of EBV-B cells with RELB variants
PBMCs were isolated by Ficoll-Hypaque density centrifugation (Amersham-Pharmacia-Biotech) from whole-blood samples obtained from healthy volunteers or patients.EBV-immortalized lymphoblastoid cell lines were cultured in RPMI medium supplemented with 10% FBS.

Full-length RT-PCR for RELB and quantitative PCR analysis
Total RNA was extracted (Qiagen, France) and used as a template for cDNA synthesis with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems).The cDNA for the full-length transcript was amplified with the following primers: forward primer: GTGCATGCTTCGGTCTGGGC, reverse primer: CTACGTGGCTTCAGGCCCC. Real-time quantitative PCR (qPCR) was performed with the Biosystems Assays-on-Demand probes/primers for TaqMan for RELB (Hs00232399_m1, Thermo Fisher Scientific), NFKB2 (Hs00174517_m1, Thermo Fisher Scientific), IL6 (Hs00985639_m1, Thermo Fisher Scientific) or CSF2 (Hs00929873, Thermo Fisher Scientific).GAPDH (00402869, Thermo Fisher Scientific) was used for normalization.For RT-qPCR on primary leukocyte and myeloid subsets, cells from freshly isolated PBMCs were negatively (NK, naïve and memory CD4 + and CD8 + T cells) or positively (CD19 + , CD14 + , pDC, mDC) sorted by magnetic separation (Miltenyi Biotec).The results are expressed according to the 2− ΔΔCt method, as described by the manufacturer.
Antibodies against GAPDH (FL-335, Santa Cruz Biotechnology), tubulin (B-7, Santa Cruz Biotechnology) and laminin A/C (H-110, Santa Cruz Biotechnology) were used as loading controls.The appropriate HRPconjugated secondary antibodies were incubated with the membrane for the detection of antibody binding with the ChemiDoc MP system (Bio-Rad).Quantification was performed with Image Labo software (Biorad).

Lentiviral transduction of SV40 fibroblasts
Retroviral vectors expressing the WT RELB, a variant from one of our patients, c.212 dup, the previously reported RELB mutant, c.1191 C>A, or one of the two missense gnomAD variants, c.1249 G>A or c.1459 G>T, were generated by inserting the RELB sequence from the corresponding pCM6-RELB-DDK plasmid into pTRIP-SFFV-ΔNGFR-2A.The plasmids were used to transfect HEK293T cells along with pCMV-VSV-G (Addgene Plasmid #8454), psPAX2 (Addgene Plasmid #12260) and pHXB2 (NIH-AIDS Reagent Program, 1069), with Opti-MEM (Gibco) and X-tremeGENE 9 (Roche), according to the manufacturers' instructions.Six hours after transfection, the medium was replaced, and 24 hours later, the supernatants were collected, filtered through 25 mm Acrodisc Syringe Filters with a 0.2 µm-mesh Supor Membrane (Pall Corporation), and subjected to precipitation with the Retro-X concentrator (Clontech), according to the manufacturer's instructions.One million SV40 fibroblasts were mixed with lentivirus-containing supernatant in a total volume of 2 mL.After 24 h of incubation, 200 μL of FCS was added and the cells were incubated for a further four days.Transduced cells were purified by positive MACS with an anti-NGFR-biotin antibody and an anti-biotin antibody conjugated with magnetic beads (Miltenyi Biotec), according to the manufacturer's protocol.The levels of NFKB2 and RELB expression were assessed by RT-qPCR.

Luciferase neutralization assay for auto-Abs against type I IFNs
The blocking activity of anti-IFN-α2 and anti-IFN-ω auto-Abs was determined with a reporter luciferase assay.Briefly, HEK293T cells were transfected with a plasmid containing the firefly luciferase gene under the control of the human ISRE promoter in the pGL4.45backbone, and a plasmid constitutively expressing Renilla luciferase for normalization (pRL-SV40).Cells were transfected in the presence of the X-tremeGene 9 transfection reagent (Sigma Aldrich, Cat#6365779001) for 24 hours.Cells in Dulbecco's modified Eagle medium (DMEM, Thermo Fisher Scientific) supplemented with 2% fetal calf serum (FCS) and 10% healthy donor or patient serum/plasma were either left unstimulated or were stimulated with  or IFN-ω (Merck, Cat#SRP3061) at 10 ng/mL or 100 pg/mL, or with  at 10 ng/mL, or 1 ng/mL, or with one of the 13 IFN-α subtypes for 16 hours at 37°C.Each sample was tested once for each cytokine and dose, in at least two independent experiments.Finally, cells were lysed for 20 minutes at room temperature and luciferase levels were measured with the Dual-Luciferase® Reporter 1000 assay system (Promega, Cat#E1980), according to the manufacturer's protocol.Luminescence intensity was measured with a VICTOR X Multilabel Plate Reader (PerkinElmer Life Sciences, USA).Relative luciferase activity (RLA) was calculated by normalizing firefly luciferase activity against Renilla luciferase activity, and then normalizing against nonstimulated conditions.Samples were considered to be neutralizing if the luciferase activity signal, normalized against non-stimulated conditions, was below 5.

Protein microarray
Protein microarrays (HuProt, CDI Laboratories) were performed as previously described ( 6).Protein microarrays were incubated for 90 minutes in 5 mL of a blocking solution consisting of 2% bovine serum albumin and 0.05% Tween-20 in PBS.The arrays were then immersed overnight in 5 mL blocking solution per array with serum from either a blood donor or a patient at a 1:2,000 dilution.Each array then underwent five five-minute wash cycles with 5 mL PBS-T (PBS + 0.05% Tween-20).Alexa Fluor 647 goat anti-human IgG (Thermo Fisher Scientific, A-21445, RRID:AB_2535862) and Dylight 550 goat anti-GST (Columbia Biosciences, D9-1310) were diluted in blocking solution (at dilutions of 1:2,000 and 1:10,000, respectively).
Each array was immersed in 5 mL of this mixture for 90 minutes.Five washes were then performed as described above.The incubations and washes were carried out on an orbital shaker, with aluminum foil used to block out light during steps involving fluorescent antibodies.Ultimately, each array was thoroughly rinsed three times with deionized water and centrifuged for approximately 30 seconds for drying purposes.
Later the same day, an Innoscan 1100AL Fluorescence scanner (Innopsys) in tandem with Mapix v.9.1.0was used to scan the arrays.The resulting images underwent analysis using the Jan 18-22 Huprot v4.0 Genepix Array List file, with either GenePix Pro v.5.1.0.19 or GenePix Pro 7. A normalization procedure was applied to address variations in signal intensity across experiments.The dataset also included data from additional healthy donors from independent protein array experiments.Signal intensities were derived from the scanned image by subtracting the local background.IgG-reactive proteins were identified as proteins with a fluorescence intensity log2[fold change] ≥ 1.5.
Data were then analyzed with Flow-Jo 10.1r5 software.
Subsets were manually gated with FlowJo software and further analyzed in R.
The cells were incubated with 40 ng/mL PMA and 10 -5 M ionomycin.Supernatants were collected after 24 h of incubation, for ELISA (R&D Systems).

In vitro proliferation and activation of B cells
Naïve B cells were sorted from peripheral blood.They were cultured in the presence of CD40L (200 ng/mL; R&D Systems) alone or with CpG 2006 (1 µg/mL; Sigma) ± F(ab')2 fragments of goat anti-human IgM (Jackson Laboratories), BAFF (PeproTech) or IL-21 (50 ng/mL; Peprotech).Their proliferation rate (with CD40L ± IL-21) was assessed by CFSE dilution.After five days, the induction of surface-cell activation markers (HLA-DR, CD80, CD86, CD95, IL-21R) was evaluated (with CD40L ± CpG ± BCR or BAFF or IL-21).After seven days, the production of IgG, IgA and IgM was assessed by Ig heavy chain-specific ELISA on the culture supernatant.Viability was assessed by FACS after Zombie dye fixation (Biolegend).