Figure 1
Figure 1. Construction of short and long anti-TSLPR CAR constructs and lentiviral transduction of T cells. (A) Anti-TSLPR hybridoma (3G11) supernatant binds to the surface of TSLPR-overexpressing ALL. Binding was detected using phycoerytherin-conjugated goat-anti-mouse antibody. Binding of a commercially available, directly conjugated anti-TSLPR antibody (Ab) is shown for comparison. (B) Schematic representation of anti-TSLPR chimeric antigen receptor constructs. Both constructs contain an anti-TSPLR single-chain fragment variable sequence from the 3G11 hybridoma, a CD8 transmembrane domain, a CD137 (41BB) costimulatory domain, and a CD3 ζ-signaling domain. The long version of the CAR also contains a spacer region derived from an immunoglobulin CH2CH3 domain. (C) Transduction efficiency of activated CD3/CD28 bead-expanded human T cells with lentiviral-based vectors expressing short and long anti-TSLPR constructs. The left panels show detection of CAR using Protein L. The right panels show detection using a TSLPR protein Fc construct. (D) TSLPR expression on normal pediatric tissues, representative of 48 healthy donors. Colon: Weak cytoplasmic granular staining in crypt base columnar cells and weak stromal tissue positivity can be seen. Liver: Mild-moderate diffuse cytoplasmic granular staining in liver sinusoid. Kupffer cells around the lining of liver sinusoid shows stronger granular staining compared with the surrounding parenchyma. Heart: Absent or negative staining on myocardial tissue. Thymus: Weak and diffuse granular staining over thymus tissue. Tonsil: Some, but not all, lymphoid tissue in the tonsil shows weak granular cytoplasmic staining. Spleen: Weak cytoplasmic staining over splenic cords. There is also hemosiderin-laden macrophages and lipofusin (yellow droplets). Kidney: Proximal tubules, distal tubules, and collecting ducts show mild-to-moderate cytoplasmic reaction, but the glomeruli are negative. Skin: Lower level of stratum spinosum and stratum basale (basal layer) show granular cytoplasmic staining. There is no staining of keratinized squamous cell layer and collagen tissue. (E) Expression level of TSLPR on B-ALL relative to expression of CD19 and CD22. JH331 is a patient-derived xenograft model of CRLF2-rearranged ALL with endogenous TSLPR overexpression. Shaded histograms represent isotype control.

Construction of short and long anti-TSLPR CAR constructs and lentiviral transduction of T cells. (A) Anti-TSLPR hybridoma (3G11) supernatant binds to the surface of TSLPR-overexpressing ALL. Binding was detected using phycoerytherin-conjugated goat-anti-mouse antibody. Binding of a commercially available, directly conjugated anti-TSLPR antibody (Ab) is shown for comparison. (B) Schematic representation of anti-TSLPR chimeric antigen receptor constructs. Both constructs contain an anti-TSPLR single-chain fragment variable sequence from the 3G11 hybridoma, a CD8 transmembrane domain, a CD137 (41BB) costimulatory domain, and a CD3 ζ-signaling domain. The long version of the CAR also contains a spacer region derived from an immunoglobulin CH2CH3 domain. (C) Transduction efficiency of activated CD3/CD28 bead-expanded human T cells with lentiviral-based vectors expressing short and long anti-TSLPR constructs. The left panels show detection of CAR using Protein L. The right panels show detection using a TSLPR protein Fc construct. (D) TSLPR expression on normal pediatric tissues, representative of 48 healthy donors. Colon: Weak cytoplasmic granular staining in crypt base columnar cells and weak stromal tissue positivity can be seen. Liver: Mild-moderate diffuse cytoplasmic granular staining in liver sinusoid. Kupffer cells around the lining of liver sinusoid shows stronger granular staining compared with the surrounding parenchyma. Heart: Absent or negative staining on myocardial tissue. Thymus: Weak and diffuse granular staining over thymus tissue. Tonsil: Some, but not all, lymphoid tissue in the tonsil shows weak granular cytoplasmic staining. Spleen: Weak cytoplasmic staining over splenic cords. There is also hemosiderin-laden macrophages and lipofusin (yellow droplets). Kidney: Proximal tubules, distal tubules, and collecting ducts show mild-to-moderate cytoplasmic reaction, but the glomeruli are negative. Skin: Lower level of stratum spinosum and stratum basale (basal layer) show granular cytoplasmic staining. There is no staining of keratinized squamous cell layer and collagen tissue. (E) Expression level of TSLPR on B-ALL relative to expression of CD19 and CD22. JH331 is a patient-derived xenograft model of CRLF2-rearranged ALL with endogenous TSLPR overexpression. Shaded histograms represent isotype control.

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