A novel LPS-responsive beige-like anchor protein (LRBA) mutation presents with normal cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and overactive TH17 immunity

LPS-responsive beige-like anchor protein (LRBA) belongs to the Beige and Chediak-Higashi (BEACH)-containing proteins family that is associated with intracellular vesicle trafficking and exocytosis. LRBA deficiency is an immune dysregulation disorder in which biallelic LRBA mutations cause a disease spectrum of recurrent infections combined with autoimmune manifestations such as early-onset inflammatory bowel disease, multiorgan autoimmunity and immune dysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX)–like disease in the absence or presence of antibody deficiency.1Alangari A. Alsultan A. Adly N. Massaad M.J. Kiani I.S. Aljebreen A. et al.LPS-responsive beige-like anchor (LRBA) gene mutation in a family with inflammatory bowel disease and combined immunodeficiency.J Allergy Clin Immunol. 2012; 130: 481-482Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 2Lopez-Herrera G. Tampella G. Pan-Hammarström Q. Herholz P. Trujillo-Vargas C.M. Phadwal K. et al.Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity.Am J Hum Genet. 2012; 90: 986-1001Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 3Alkhairy O.K. Abolhassani H. Rezaei N. Fang M. Andersen K.K. Chavoshzadeh Z. et al.Spectrum of phenotypes associated with mutations in LRBA.J Clin Immunol. 2016; 36: 33-45Crossref PubMed Scopus (151) Google Scholar, 4Gámez-Díaz L. August D. Stepensky P. Revel-Vilk S. Seidel M.G. Noriko M. et al.The extended phenotype of LPS-responsive beige-like anchor protein (LRBA) deficiency.J Allergy Clin Immunol. 2016; 137: 223-230Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar Although the pathophysiological mechanisms behind the disorder remain to be fully established, a defect in regulatory T (Treg) cells is thought to be pivotal because LRBA plays a critical role in cytotoxic T lymphocyte–associated protein 4 (CTLA-4) recycling and membrane shuttling.5Lo B. Zhang K. Lu W. Zheng L. Zhang Q. Kanellopoulou C. et al.AUTOIMMUNE DISEASE. Patients with LRBA deficiency show CTLA4 loss and immune dysregulation responsive to abatacept therapy.Science. 2015; 349: 436-440Crossref PubMed Scopus (451) Google Scholar, 6Charbonnier L.-M. Janssen E. Chou J. Ohsumi T.K. Keles S. Hsu J.T. et al.Regulatory T-cell deficiency and immune dysregulation, polyendocrinopathy, enteropathy, X-linked-like disorder caused by loss-of-function mutations in LRBA.J Allergy Clin Immunol. 2015; 135: 217-227Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar The latter is supported by the similarities in clinical and immunological presentation between CTLA-4 deficiencies and LRBA deficiencies as well as by the beneficial effect of CTLA-4-Ig (abatacept) treatment on autoimmune manifestations in patients with LRBA deficiency.5Lo B. Zhang K. Lu W. Zheng L. Zhang Q. Kanellopoulou C. et al.AUTOIMMUNE DISEASE. Patients with LRBA deficiency show CTLA4 loss and immune dysregulation responsive to abatacept therapy.Science. 2015; 349: 436-440Crossref PubMed Scopus (451) Google Scholar We describe a girl, born full-term to consanguineous parents from Moroccan descent (Fig 1, A), presenting with severe life-threatening enteropathy at age 3 months. Diarrhea started after Rota virus vaccination and necessitated total parenteral nutrition. A diagnosis of autoimmune enteropathy was made on the basis of the presence of serum antienterocyte antibodies and on histologic examinations of intestinal biopsies (Fig 1, B). Treatment was started with high-dose corticosteroids and tacrolimus. Because of tacrolimus-induced hepatitis, immunosuppression was switched to sirolimus. The patient required corticosteroids for 12 months and is currently still on sirolimus maintenance therapy. Total parenteral nutrition could be stopped at the age of 15 months. Exclusive amino acid formula was given till age 19 months and from then on she has been thriving on a complete enteral cow's-milk–free diet. An overview of treatment and nutrition is given in Fig E1 in this article's Online Repository at www.jacionline.org. Under immunosuppressive treatment, we saw impressive clinical improvement and normalization of microscopic intestinal structure and inflammation (Fig 1, B). She did, however, develop autoimmune anterior uveitis, requiring topical treatment with corticosteroids and tacrolimus for 3 episodes between age 12 and 18 months. Autoimmunity in other organs has not been detected so far. The girl is considered a suitable candidate for hematopoietic stem cell transplantation and is currently, 36 months old, awaiting her transplantation in good clinical condition. The patient presented normal white blood cell differentiation, lymphocyte subsets, and serum immunoglobulins. Remarkably, despite her young age, the patient presented a well-developed switched memory B-cell compartment (6.3% SmB). The patient's complete immunological workup is provided in Table E1 in this article's Online Repository at www.jacionline.org. More in-depth phenotyping of the T cells revealed reduced effector memory T-cell populations in the PBMC fraction of our patient. Similarly, both CD4+ and CD8+ terminally differentiated effector memory cells (TEMRA) were severely reduced (see Fig E5 in this article's Online Repository at www.jacionline.org). A primary immunodeficiencies (PID) gene panel covering 226 genes (see Table E2 in this article's Online Repository at www.jacionline.org) revealed a novel homozygous mutation in the LRBA gene (NM_006726.4: c.2450-3C>A), which was confirmed by Sanger sequencing. No other potential genetic causes for autoimmunity were detected (variants detected in the PID panel are summarized in Table E3 in this article's Online Repository at www.jacionline.org). This mutation was predicted to abolish the acceptor splice site of intron 20. Indeed, cDNA sequencing revealed skipping of exon 21 (c.2450-2573), which causes a frameshift resulting in a premature stop codon in exon 22 after 29 residues (Fig 1, C; see Fig E2, A, in this article's Online Repository at www.jacionline.org). Mutant LRBA mRNA expression was 3-fold lower compared with healthy controls (Fig 1, D). Mutant LRBA protein (estimated 95 kD) was undetectable by flow cytometry and Western blot on stimulated PBMC (Fig 1, E and F) also when we used an antibody recognizing LRBA N-terminal of the premature stop (Fig E2, B). The latter indicates that mutant LRBA, like in most reported cases, is unstable leading to degradation. LRBA interacts with CTLA-4 within recycling endosomes, preventing it from being sorted to lysosomes for degradation and LRBA deficiency results in decreased CTLA-4 expression.3Alkhairy O.K. Abolhassani H. Rezaei N. Fang M. Andersen K.K. Chavoshzadeh Z. et al.Spectrum of phenotypes associated with mutations in LRBA.J Clin Immunol. 2016; 36: 33-45Crossref PubMed Scopus (151) Google Scholar, 5Lo B. Zhang K. Lu W. Zheng L. Zhang Q. Kanellopoulou C. et al.AUTOIMMUNE DISEASE. Patients with LRBA deficiency show CTLA4 loss and immune dysregulation responsive to abatacept therapy.Science. 2015; 349: 436-440Crossref PubMed Scopus (451) Google Scholar In the here-described patient, however, despite complete absence of LRBA protein, T cells (Treg and conventional) were repeatedly found to express normal levels of intracellular and mobilized CTLA-4 in steady-state and upon activation (Fig 2, A). Although defective Treg-cell function has been linked to LRBA immunopathology,6Charbonnier L.-M. Janssen E. Chou J. Ohsumi T.K. Keles S. Hsu J.T. et al.Regulatory T-cell deficiency and immune dysregulation, polyendocrinopathy, enteropathy, X-linked-like disorder caused by loss-of-function mutations in LRBA.J Allergy Clin Immunol. 2015; 135: 217-227Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar Treg-cell frequency, expression of FoxP3 and CD25, and suppressive Treg-cell activity were all normal in our patient (see Fig E3 in this article's Online Repository at www.jacionline.org; Fig 2, B). Because the patient displayed normal CTLA-4 expression and Treg-cell function, we screened for other causes of autoimmunity. Elevated circulating follicular helper T cells have been linked to immunopathology in LRBA deficiency,7Alroqi F.J. Charbonnier L.-M. Baris S. Kiykim A. Chou J. Platt C.D. et al.Exaggerated follicular helper T-cell responses in patients with LRBA deficiency caused by failure of CTLA4-mediated regulation.J Allergy Clin Immunol. 2018; 141: 1050-1059.e10Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar but these were low/normal in our patient on 2 different occasions (see Fig E4 in this article's Online Repository at www.jacionline.org). Strikingly, the proportion of IL-17–producing CD4+ T cells was strongly elevated at age of onset (3 months) and gradually normalized under aforementioned immunosuppressive treatment (Fig 2, C). In parallel, IFN-γ–producing CD4+ T cells were below age-matched controls at age of onset, and gradually normalized with treatment. To explore the effect of CTLA-4-Ig treatment, we tested ex vivo cytokine production after sirolimus wash-out and in the presence of abatacept (Fig 2, D). Rapamycin was taken along as a control, because the patient responded well to sirolimus treatment and it is known to affect cytokine secretion. Indeed, our results showed that rapamycin dampened global ex vivo T-cell cytokine secretion (IL-17A, IFN-γ, and IL-10). The effect of abatacept, however, was more selective, repressing IL-17A secretion by patient cells to a level comparable with controls, but leaving IFN-γ and IL-10 secretion relatively unaffected. Normal levels of CTLA-4 despite absent LRBA protein were an unexpected finding in our patient, because insufficient CTLA-4 expression is assumed to be the main driver of autoimmunity in LRBA deficiency.5Lo B. Zhang K. Lu W. Zheng L. Zhang Q. Kanellopoulou C. et al.AUTOIMMUNE DISEASE. Patients with LRBA deficiency show CTLA4 loss and immune dysregulation responsive to abatacept therapy.Science. 2015; 349: 436-440Crossref PubMed Scopus (451) Google Scholar, 6Charbonnier L.-M. Janssen E. Chou J. Ohsumi T.K. Keles S. Hsu J.T. et al.Regulatory T-cell deficiency and immune dysregulation, polyendocrinopathy, enteropathy, X-linked-like disorder caused by loss-of-function mutations in LRBA.J Allergy Clin Immunol. 2015; 135: 217-227Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 7Alroqi F.J. Charbonnier L.-M. Baris S. Kiykim A. Chou J. Platt C.D. et al.Exaggerated follicular helper T-cell responses in patients with LRBA deficiency caused by failure of CTLA4-mediated regulation.J Allergy Clin Immunol. 2018; 141: 1050-1059.e10Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 8Hou T.Z. Verma N. Wanders J. Kennedy A. Soskic B. Janman D. et al.Identifying functional defects in patients with immune dysregulation due to LRBA and CTLA-4 mutations.Blood. 2017; 129: 1458-1468Crossref PubMed Scopus (78) Google Scholar This suggests that in this patient, other proteins (eg, BEACH family member NBEA) may take over CTLA-4 shuttling necessary for Treg-cell function. Nevertheless, the degree of CTLA-4 loss in reported LRBA-deficient patients is variable5Lo B. Zhang K. Lu W. Zheng L. Zhang Q. Kanellopoulou C. et al.AUTOIMMUNE DISEASE. Patients with LRBA deficiency show CTLA4 loss and immune dysregulation responsive to abatacept therapy.Science. 2015; 349: 436-440Crossref PubMed Scopus (451) Google Scholar and it is described to be in the normal ranges upon T-cell activation, despite severely decreased basal levels.8Hou T.Z. Verma N. Wanders J. Kennedy A. Soskic B. Janman D. et al.Identifying functional defects in patients with immune dysregulation due to LRBA and CTLA-4 mutations.Blood. 2017; 129: 1458-1468Crossref PubMed Scopus (78) Google Scholar Treg-cell frequency and function were normal in our patient, in contrast to more than 70% of LRBA-deficient patients described by Gámez-Díaz et al4Gámez-Díaz L. August D. Stepensky P. Revel-Vilk S. Seidel M.G. Noriko M. et al.The extended phenotype of LPS-responsive beige-like anchor protein (LRBA) deficiency.J Allergy Clin Immunol. 2016; 137: 223-230Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar who had reduced Treg cells. This could be sirolimus-related because this drug is known to increase Treg cells and only 1 patient of the Gámez-Díaz et al4Gámez-Díaz L. August D. Stepensky P. Revel-Vilk S. Seidel M.G. Noriko M. et al.The extended phenotype of LPS-responsive beige-like anchor protein (LRBA) deficiency.J Allergy Clin Immunol. 2016; 137: 223-230Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar cohort was on sirolimus. Intriguingly, the patient showed very high IL-17–producing T cells at disease onset, which normalized under treatment. After sirolimus wash-out, ex vivo IL-17 secretion remained elevated, suggesting that this may be a disease-intrinsic feature in our patient. To date, there have been no reports on elevated IL-17 secretion in LRBA-deficient patients. In contrast, Alroqi et al7Alroqi F.J. Charbonnier L.-M. Baris S. Kiykim A. Chou J. Platt C.D. et al.Exaggerated follicular helper T-cell responses in patients with LRBA deficiency caused by failure of CTLA4-mediated regulation.J Allergy Clin Immunol. 2018; 141: 1050-1059.e10Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar observed lower intracellular IL-17–producing Tfh cells in 3 LRBA-deficient patients. To gain more insight in the importance of CTLA-4 and TH17 overactivity in the immunopathology of LRBA deficiency, we would recommend that known and suspected LRBA-deficient patients, as part of their diagnostic workup, would be evaluated for TH17 function in addition to LRBA and CTLA-4 protein expression and Treg-cell frequency and function. Subsequent mechanistic work should focus on TH-cell differentiation and cytokine profile, in view of the substantial developmental plasticity that is known between Treg cells and TH17. The mTOR pathway should definitely be studied in this regard, given that sirolimus (inhibiting mTOR) had a beneficial therapeutic effect and is known to restore the Treg-cell/TH17 balance by promoting Treg cells and dampening TH17. Charbonnier et al6Charbonnier L.-M. Janssen E. Chou J. Ohsumi T.K. Keles S. Hsu J.T. et al.Regulatory T-cell deficiency and immune dysregulation, polyendocrinopathy, enteropathy, X-linked-like disorder caused by loss-of-function mutations in LRBA.J Allergy Clin Immunol. 2015; 135: 217-227Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar put forward a link between LRBA deficiency and mTOR signaling. However, they reported reduced mTOR signaling in LRBA-deficient patients, which is in contrast with the reduced Treg cells they observed. Nevertheless, the normalization of ex vivo IL-17 production we observed in our patient is most likely an effect of sirolimus. Azizi9Azizi G. Abolhassani H. Yazdani R. Mohammadikhajehdehi S. Parvaneh N. Negahdari B. et al.New therapeutic approach by sirolimus for enteropathy treatment in patients with LRBA deficiency.Eur Ann Allergy Clin Immunol. 2017; 49: 235-239Crossref PubMed Scopus (33) Google Scholar et al reported earlier the successful treatment of enteropathy by sirolimus in LRBA-deficient patients. Therefore, we dare to recommend sirolimus as the immunosuppressive of choice in patients with LRBA-related enteropathy. Our experiments additionally suggest that abatacept provides a valuable alternative treatment, even in the case of normal CTLA-4 levels. In conclusion, we are the first to report normal CTLA-4 expression and normal Treg-cell function in the face of overactive TH17 immunity in an LRBA-deficient patient, illustrating that loss of CTLA-4 is not a prerequisite for autoimmunity in LRBA deficiency. The importance of TH17 overactivity in this disorder should be further studied in larger patient cohorts. We thank the patient and family for participation in this study. We thank Joris van Montfrans and Victoria Bordon for their clinical advice. We thank Veronique Debacker, Nancy De Cabooter, and Khadija Guerti for excellent technical assistance. Download .docx (.04 MB) Help with docx files Online Repository Text Download .xlsx (.02 MB) Help with xlsx files Table E3 Download .pdf (.07 MB) Help with pdf files Fig E1 Download .pdf (1.04 MB) Help with pdf files Fig E2 Download .pdf (.39 MB) Help with pdf files Fig E3 Download .pdf (.03 MB) Help with pdf files Fig E4