Immunodeficiency and EBV-induced lymphoproliferation caused by 4-1BB deficiency

Abstract

Background: The tumor TNF receptor family member 4-1BB (CD137) is encoded by TNFRSF9 and expressed on activated T cells. 4-1BB provides a costimulatory signal that enhances CD8⁺ T-cell survival, cytotoxicity, and mitochondrial activity, thereby promoting immunity against viruses and tumors. The ligand for 4-1BB is expressed on antigen-presenting cells and EBV-transformed B cells.

Objective: We investigated the genetic basis of recurrent sinopulmonary infections, persistent EBV viremia, and EBV-induced lymphoproliferation in 2 unrelated patients.

Methods: Whole-exome sequencing, immunoblotting, immunophenotyping, and in vitro assays of lymphocyte and mitochondrial function were performed.

Results: The 2 patients shared a homozygous G109S missense mutation in 4-1BB that abolished protein expression and ligand binding. The patients' CD8⁺ T cells had reduced proliferation, impaired expression of IFN-γ and perforin, and diminished cytotoxicity against allogeneic and HLA-matched EBV-B cells. Mitochondrial biogenesis, membrane potential, and function were significantly reduced in the patients' activated T cells. An inhibitory antibody against 4-1BB recapitulated the patients' defective CD8⁺ T-cell activation and cytotoxicity against EBV-infected B cells in vitro.

Conclusion: This novel immunodeficiency demonstrates the critical role of 4-1BB costimulation in host immunity against EBV infection.

Keywords: 4-1BB; CD137; EBV; Hodgkin lymphoma; immunodeficiency.

Figure 1.. EBV viremia EBV driven lymphoproliferation and impaired IFN-γ secretion in the patients.

A. Circulating EBV viral load in P1. B. Immunohistochemistry of cervical LN from P1 showing a cluster of CD20⁺ B cells (left) and positive EBV (EBER) in situ hybridization (right) C. IFN-γ and IL-2 levels in supernatants of PHA Stimulated PBMCs from P1 and controls (n=3) in two independent experiments. D. Circulating EBV viral load P2. E. H&E stain of cervical LN from P2 showing a diffuse growth pattern (upper left), and immunohistochemistry showing negative CD20 staining (upper right), positive CD38 staining (lower left) and positive EBV (EBER) in situ hybridization (lower right) consistent with a DLBCL of plasmablastic subtype. F. IFN-γ and IL-2 levels in supernatants of PHA Stimulated PBMCs from P2 and controls (n=3) in two independent experiments. Results represent the mean, columns and bars represent mean + SEM. *** p<0.001, ns= not significant.

Figure 2.. The 4-1BB^G109S mutation and its impact on 4-1BB expression.

A. Sanger sequencing around the missense TNFRSF9 mutation (c.325G>A: p.Gly109Ser) in a reference control and patients. The mutated nucleotide is indicated in red and by an asterisk. B. Evolutionary conservation of the S109 a.a. in 4-1BB. Protein sequence orthologous to human 4-1BB was aligned in six non-human vertebrate species, all of which shared the p.S109 amino acid. C. Linear schematic of 4-1BB showing its domains and the location of the G109S mutation (red) in the extracellular domain. The 4-1BB fragment crystallized in complex with 4-1BB (PDB:6PCR) is indicated D. Ribbon diagram of the 4-1BB extracellular in complex 4-1BBL. The 4-1B trimer (blue) is shown bound to its trimeric ligand 4-1BBL (green). The red dot corresponds to the location of the mutated G109 residue. The insets illustrate the potential of the mutated S109 residue to form hydrogen bonds with neighboring polar residues. E. Immunoblot analysis of PHA stimulated PBMCs from both patients and controls using a polyclonal antibody directed against the intracellular domain of 4-1BB. Actin was used as loading control. F. FACS analysis of 4-1BB expression (left), 4-1BBL binding (center) and CD25 expression(right) in PHA stimulated CD8⁺ T cells from the patients and a control. Similar results were obtained from two independent experiments in E and F.

Figure 3.. The 4-1BB^G109S mutation Impairs the generation of allo- and EBV-specific CD8⁺ T cells.

A. Experimental protocol for the generation and testing of cytotoxic CD8⁺ T cells against EBV-B cells. B. CD8⁺ T cell numbers on days 0, 14 and day 21 post-stimulation with allogeneic EBV-B cells of PBMCs from patients (n=2) controls (n=4) and controls with addition of anti-4-1BB blocking antibody BBK-2 (n=2). C. Representative FACS analysis (left) quantitative analysis (center) and MFI (right) of CD8⁺IFN-γ⁺ and CD8⁺Perforin⁺ cells on Day 14 post stimulation of PBMCs stimulated with allogeneic EBV-B cells as described in B. D. Cytotoxic activity of CD8⁺ T cells against the stimulatory allogeneic EBV B cells. CD8⁺ T cells were purified on Day 21 from cultures of stimulated PBMCs from patients (n=2) controls (n=4). The effect of anti-4-1BB blocking antibody BBK-2 was examined in 2 of the 4 controls (n=2). E-G. CD8⁺ T cell numbers (F) numbers of CD8⁺IFN-γ⁺ and CD8⁺Perforin⁺ cells (H) and cytotoxicity of CD8⁺ T cells (G) on day 21 post-stimulation of PBMCs from P1 with HLA-matched EBV-B cells and of normal PBMCs stimulated with autologous EBV-B cells without or with addition of anti-4-1BB blocking antibody BBK-2 (n=4 and n=2, respectively). We were able to study P1 CD8⁺ T cell cytotoxicity against HLA-matched EBV-B cell targets once, because the patient underwent HSCT. Symbols and bars in B, D, E and G and columns and bars in C and F represent mean and SEM. *= p<0.05, ** p<0.01, *** p<0.001, ns= not significant.

Figure 4.. Defective mitochondrial biogenesis and function in the patients’ activated CD8⁺ T cells.

A. Schematic of factors determining the oxygen consumption rate (OCR). Complexes I – IV comprise the electron transport chain. The stepwise addition of reagents used during the extracellular flux analysis (1 – 3) are depicted in red. B. Extracellular flux analysis of T cells purified from PHA-stimulated PBMCs for 3 days from P2 (data pooled from two independent experiments) and controls without or with 4-1BB blocking antibody BBK-2 (n=2). C, D. Representative FACS analysis (left) and quantitative analysis (MFI, right) of mitochondrial mass measured using MitoTracker Green (MTG) (C) and of mitochondrial membrane potential measured using MitoTracker Red CMXRos (D) of CD8⁺ cells in PHA stimulated cultures of PBMCs from patients (n=2) and controls (n=3). Columns and bars represent mean + SEM. ** p<0.01, ns= not significant.