Orientation-dependent CD45 inhibition with viral and engineered ligands

Abstract

CD45 is a cell surface phosphatase that shapes the T cell receptor signaling threshold but does not have a known ligand. A family of adenovirus proteins, including E3/49K, exploits CD45 to evade immunity by binding to the extracellular domain of CD45, resulting in the suppression of T cell signaling. We determined the cryo-EM structure of this complex and found that the E3/49K protein is composed of three immunoglobulin domains assembled as "beads on a string" that compel CD45 into a closely abutted dimer by cross-linking the CD45 D3 domain, leading to steric inhibition of its intracellular phosphatase activity. Inspired by the E3/49K mechanism, we engineered CD45 surrogate ligands that can fine-tune T cell activation by dimerizing CD45 into different orientations and proximities. The adenovirus E3/49K protein has taught us that, despite a lack of a known ligand, CD45 activity can be modulated by extracellular dimerizing ligands that perturb its phosphatase activity and alter T cell responses.

Fig. 1.. Biochemical characterization and cryoEM structure of CD45:E3/49K complex.

(A) SPR plot of CD45 and E3/49K strain D19a. (B) Example CD69 expression upon pretreatment with E3/49K then stimulated with OKT3 in Jurkat T cells (left) and quantification from replicates (right). (C) Superdex 200 size-exclusion chromatography CD45:E3/49K complex. (D) CryoEM density map of CD45 dimer (red and purple) bound by a single E3/49K (green) resolved to 3.8-Å resolution.

Fig. 2.. Structural basis for CD45 engagement by viral E3/49K.

(A) Ribbon diagram of the 2:1 CD45:E3/49K complex. Dashed boxes indicate magnified interface views in later panels. (B) Binding footprint of E3/49K R1 (left) and -R2 (right) to the front of CD45 D3. (C) Binding footprint of E3/49K-R3 to the back of CD45-D3. (D) Interface 1 view of CD45-D3 and E3/49K R1. (E) Interface 2 view of CD45-D3 and E3/49K R2. (F) Interface view of CD45-D3 and linker between E3/49K R1-R2. (G) Interface 3 view of CD45-D3 and E3/49K R3. (H) Amino acid sequence alignment of E3/49K R1, linker and R2 between different D strains of adenovirus using Jalview (2.11.0). Highlighted residues are conserved (black), H-bond contacts (yellow) or vdW contacts (light blue). Arrows and helices indicate secondary structures. (I) SPR plot of CD45 and E3/49K strain D30. (J) CD69 expression upon incubating with E3/49K strain D30 compared with D19a, then stimulating with OKT3.

Fig. 3.. R1R2 domains of E3/49K and its VHH mimics are sufficient to downregulate T cells.

(A) (top) Cartoon representation of E3/49K construct and its R1-R3 domains. (bottom) CD69 expression upon incubating with different combinations of linked E3/49K domains, then stimulating with OKT3. (B) Same constructs tested in PBMC CD4+ T cells and (C) CD8+ T cells. (A-C), Data are mean ± s.d. from n = 3 replicates from different donors. Statistical significance is determined by one-way ANOVA with Fisher’s LSD multiple comparison test, (ns > 0.05; *P < 0.05; **P < 0.01). (D) (left) Cartoon representation of R1-R2 dimerizer and its VHH mimics binding to different domains of CD45 and inducing different conformations. (right) Table detailing targeted CD45 domains by each tested VHH mimic. (E) CD69 expression upon pretreatment with different VHH mimics then stimulated with OKT3 in Jurkat T cells. Data are mean ± s.d. from n = 3 replicates. Statistical significance is determined by one-way ANOVA with Fisher’s LSD multiple comparison test (ns > 0.05; *P < 0.05; **P < 0.01, ***P < 0.001; ****P < 0.0001).

Fig. 4.. CD45 dimerization reduces T cell signaling through Lck and Zap70.

(A) Detection of specific phosphorylation in Lck and Zap70 in Jurkat cells by protein immunoblot after various treatments. Compound 211 and NQ301 are purchased from different sources with identical chemical formula. Numbers below panels denote quantification of band intensity normalized as: (*) Lck pY394/Lck ratio, (**) Lck pY505/Lck ratio, (***) pZap70/Zap70 ratio. (B) Schematic depiction of CD45-mediated T cell signaling before (left) and after (right) binding of E3/49K binding.

Fig. 5.. CD45 distancing tunes T cell signaling.

(A) Cartoon representation of E3/49K constructs with extended flexible or rigid linkers. (B) PBMCs were pretreated with E3/49K with different types of linkers then stimulated with OKT3 and assayed for CD69 expression in CD4+ (left) and CD8+ (right) T cells. Data are mean ± s.d. from n = 5 replicates from different donors. Statistical significance is determined by a one-way ANOVA with Fisher’s LSD multiple comparison test (ns > 0.05; *P < 0.05; **P < 0.01, ***P < 0.001; ****P < 0.0001).