Expanding the molecular and phenotypic spectrum of CTLA‐4 insufficiency

Cytotoxic T lymphocyte antigen-4 (CTLA-4), an inhibitory receptor expressed on T-cells, plays an essential role in regulating immune responses. In healthy individuals, CTLA-4 prevents overstimulation of T-cells and facilitates regulatory T-cell (Treg) suppressive function through competitive binding of CD80/CD86 on antigen-presenting cells.1 Heterozygous loss-of-function variants in CTLA4 cause the inborn error of immunity (IEI) termed CTLA-4 insufficiency, which has a variable clinical presentation often including combined immunodeficiency, autoimmunity, and lymphoproliferation.1-3 Since CTLA-4 insufficiency was first described in 2014, over 54 pathogenic variants have been identified.1, 3 Herein, we report two novel CTLA4 variants and describe the clinical presentations in two affected families, the differing functional impacts on CTLA-4-mediated transendocytosis, and the influence of prompt access to genetic testing and targeted therapy on clinical outcomes. A two-month-old boy was referred to the immunology service after he was discovered to share a variant in CTLA4 with his father. He was born at term via spontaneous vaginal delivery following an uncomplicated pregnancy and had been otherwise healthy. His father had a phenotype consistent with CTLA-4 insufficiency, including a history of recurrent infections, diarrhea, and vitiligo since childhood; hypogammaglobulinemia, eczema, nodular lymphoid hyperplasia of the liver, and type I diabetes mellitus diagnosed at 29 years of age. Genetic sequencing revealed a variant of uncertain significance (VUS) in CTLA4 c.416A > C, p.(Tyr139Ser) (Figure 1A). Testing of paternal grandparents indicated that the variant arose de novo in the patient's father (Figure 1B). Introducing p.Y139S into an expression plasmid demonstrated roughly 50% loss of function in CTLA-4-mediated acquisition of CD80 via transendocytosis when compared to wild-type CTLA-4 (Figure 1C,D and Appendix S1). Considering the genetic diagnosis, his father was started on abatacept and receives immunoglobulin replacement. Screening bloodwork at 2 months of age revealed moderate neutropenia, but an otherwise unremarkable complete blood count (CBC), immunoglobulins, T/B/NK-cell enumeration, and T/B-cell memory panel (Table 1 and Appendix S1). His CBC was repeated at 3 months of age, which reiterated neutropenia at 0.6 × 109/L. Hematology was consulted who favored an autoimmune process. His neutropenia resolved spontaneously by 11 months of age. He has otherwise remained in good health with reassuring growth and development. He is undergoing annual evaluations for immune dysregulation including physical examinations, bloodwork, and imaging coordinated by his interdisciplinary team. A 13-year-old male patient was referred to immunology for a constellation of findings including recurrent gastrointestinal (GI) infections since early childhood, Crohn disease (CD) diagnosed after developing chronic diarrhea at age 11, asymptomatic progressive pulmonary nodules, autoimmune cytopenias, and hypogammaglobulinemia. His CD was refractory to several therapies (mesalamine, budesonide, exclusive enteral nutrition, and adalimumab). Bloodwork revealed severe neutropenia, normal T/B/NK-cell enumeration, expanded proportions of PD1+ CD4 T-cells, CD57+ and TEMRA CD8 T-cells and CD21low B-cells, an elevated soluble IL-2 receptor (sIL-2R) level, and hypogammaglobulinemia (Table 1 and Figure 2A,B). Gene panel testing identified a VUS in CTLA4 c.424G > C, p.(Gly142Arg), with parental testing indicating de novo inheritance (Figure 1A,B). Introducing p.G142R into an expression plasmid demonstrated severe impairment of CTLA-4-mediated CD80 transendocytosis (Figure 1C,D and Appendix S1). Around the time of his genetic diagnosis, he developed transient expressive aphasia and agraphia, persistent headache, and fatigue. His MRI brain revealed hyperintense CNS lesions (Figure 2C), and lumbar puncture demonstrated elevated opening pressure, CSF pleocytosis, and CSF oligoclonal bands. Biopsy of his pulmonary nodules revealed granulomatous and lymphocytic interstitial lung disease (Figure S1). After a negative workup for infection and malignancy, his CNS lesions were favored to be inflammatory. He began treatment for his CTLA-4 insufficiency-associated multiorgan immune dysregulation with 2 g/kg of IVIG, pulse methylprednisolone for 3 days followed by a slow steroid taper, and abatacept IV 20 mg/kg/dose q4weeks with marked symptom improvement. Approximately 4 months later, tapering of corticosteroids below 15 mg/day led to recurrence of GI, hematologic, and CNS abnormalities (Figure 2A,C). After adjusting abatacept dosing to 15 mg/kg IV q2weeks and adding sirolimus, prednisone was successfully discontinued. Investigations revealed normalized sIL-2R and fecal calprotectin levels and a decrease in expanded PD1+CD4 and CD57+CD8 T-cell populations (Figure 2A,B). MRI brain and CT chest showed dramatic improvement in known lesions (Figure 2C, Figure S2). He remains stable on sirolimus, abatacept, IVIG (0.5 g/kg q4weeks), and cotrimoxazole prophylaxis. Here, we report the phenotype and functional validation of two novel variants causing CTLA-4 insufficiency. Case 1 is a 22-month-old infant who underwent sequencing shortly after birth, based on a paternal history of IEI and CTLA4 VUS. Early diagnosis facilitated proactive monitoring for immune dysregulation, including autoimmune neutropenia recognized at 2 months of age. Case 2 is a 13-year-old boy who at diagnosis had severe multisystem autoimmunity. These two contrasting presentations and outcomes highlight (1) the vast phenotypic profile of CTLA-4 insufficiency, (2) the importance of considering IEIs when evaluating patients with immune dysregulation, and (3) the role of genetic testing when IEIs are suspected. In Case 2, immunomodulatory treatment targeting the specific pathophysiology of CTLA-4 insufficiency led to dramatic symptom reduction, when the isolated management of each complication, before the establishment of a unifying diagnosis, had proven ineffective. The early age of presentation in Case 1, well below the median presenting age of 11 years,4 suggests that sequencing at-risk family members may reveal earlier manifestations of CTLA-4 insufficiency that would have otherwise been unrecognized. Review of this case also raises the question of whether to initiate immunomodulatory treatment for an isolated early manifestation of CTLA-4 insufficiency. We elected to monitor without initiating pharmacologic therapy and the neutropenia resolved. Further studies on the natural history and treatment of CTLA-4 insufficiency are needed to establish clinical care guidelines for these vulnerable patients. Although the described patient in Case 2 started targeted treatment for CTLA-4 insufficiency with abatacept upon diagnosis, his CNS, hematologic and GI complications recurred with corticosteroid taper. Adding sirolimus to his abatacept regimen facilitated discontinuation of steroids and helped establish clinical remission. Abatacept, a fusion protein comprising the extracellular domain of human CTLA-4 linked to human IgG Fc, provides a means to pharmacologically replace some CTLA-4-dependent functions.4 Sirolimus, a selective mTOR inhibitor that interrupts CD28 signaling pathways, expands Treg populations and enhances their suppressive capacity, further restoring immune homeostasis.5, 6 Monitoring of sIL2R levels provided a useful marker of disease activity that informed treatment. Pathologically expanded T-cell populations such as PD1+ CD4 T-cells and CD57+ and TEMRA CD8 T-cells also improved with targeted therapy, possibly reflecting a reduction in dysregulated T follicular helper cells and senescent CD8 T-cells previously implicated in CTLA-4 insufficiency.7, 8 Functional validation of these two novel variants revealed severe impairment of CTLA-4-mediated CD80 transendocytosis by the p.G142R CTLA-4 variant, while in contrast, p.Y139S retained partial activity. The p.Y139S substitution occurs within a conserved loop that interacts with CD80/CD86; p.G142R lies within an adjacent beta sheet and converts nonpolar/neutral glycine to polar/cationic arginine.9 The greater impact on transendocytosis observed with p.G142R is therefore unclear; however, it is possible that this disrupts CTLA-4 protein stability, surface expression, and/or ligand binding. Mechanistic studies support that transendocytosis predicts CTLA-4-dependent Treg suppressive function in vitro10; however, no correlation between genotype or in vitro activity and disease penetrance or phenotype has been observed in CTLA-4 insufficiency.4 To our knowledge, we present two previously unreported variants in CTLA4 and expand the genotypic profile of CTLA-4 insufficiency. These cases highlight the broad phenotypic spectrum of CTLA-4 insufficiency, and the profound impact a molecular diagnosis can have on treatment and outcomes. This work was supported by grants from the Canadian Institutes of Health Research (PJT-178054) (C.M.B., S.E.T.), Michael Smith Health Research BC (HPI-2018-2041) (C.M.B.), the Vanderbilt-Ingram Cancer Center GI SPORE P50CA236733 (J.G.M.), and the Vanderbilt Institute for Clinical and Translational Research (VICTR), which receives support from the National Center for Advancing Translational Sciences (NCATS) Clinical Translational Science Award (CTSA) Program, Award Number 5UL1TR002243-03 (J.G.M.). Catherine M. Biggs: Conceptualization; investigation; funding acquisition; writing – review and editing; project administration; formal analysis; supervision; resources; visualization; methodology; validation; data curation. Sean Duke: Conceptualization; writing – original draft; formal analysis; visualization; writing – review and editing; software; investigation; data curation. James Maiarana: Methodology; formal analysis; software; investigation; writing – review and editing; conceptualization; visualization. Pariya Yousefi: Investigation; writing – review and editing; visualization. Elijah Burks: Investigation; visualization; formal analysis. Samantha Gerrie: Investigation; visualization; writing – review and editing. Audi Setiadi: Investigation; visualization; writing – review and editing. Kyla Hildebrand: Writing – review and editing; investigation. Elliot James: Investigation; writing – review and editing. Stuart E. Turvey: Investigation; writing – review and editing. Janet G. Markle: Investigation; conceptualization; writing – review and editing; supervision; resources; project administration; funding acquisition. Ali Amid: Investigation; writing – review and editing. Cornelius Boerkel: Investigation; writing – review and editing. Stephanie Erdle: Investigation; writing – review and editing. Orlee Guttman: Investigation; writing – review and editing. Amin Kanani: Investigation; writing – review and editing. Sally Lawrence: Investigation; writing – review and editing. Anna Lee: Writing – review and editing; visualization. Persia Pourshahnazari: Investigation; writing – review and editing. Meera Rayar: Investigation; writing – review and editing. Jacob Rozmus: Investigation; writing – review and editing. Dewi Schrader: Investigation; writing – review and editing. Mehul Sharma: Investigation; writing – review and editing; methodology; formal analysis. Kevin E. Shopsowitz: Investigation; writing – review and editing; visualization. Ryan Tan: Investigation; writing – review and editing; methodology. Fergus To: Writing – review and editing; investigation. Connie L Yang: Investigation; writing – review and editing. The authors have no conflicts of interest to disclose. BC Children's and St Paul's Hospital Members: Ali Amid1, Cornelius Boerkel6, Stephanie Erdle1, Orlee Guttman1, Amin Kanani7, Sally Lawrence1, Anna F. Lee5, Persia Pourshahnazari7, Meera Rayar1, Jacob Rozmus1, Dewi Schrader1, Mehul Sharma1, Kevin E. Shopsowitz5, Ryan Tan1, Fergus To8, Connie Yang1. Appendix S1 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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