Fig 2: Schematic strategy of CDR3 substitution by overlap extension polymerase chain reaction (PCR). Based on the library of TCRαβ and TCRγδ established by the described cloning platform, the strategy for CDR3 substitution using multiplex single cell PCR products and linker DNA is shown. After the sequence analysis of single-cell PCR (shown in Figure 1a), the target pairs of T-cell receptors (TCRs) are chosen from the respective second round paired PCR plates, which include TCRα/γm and TCRβ/δn (m represents a particular TRAV or TRGV subfamily; n represents a particular TRBV or TRDV subfamily). Beforehand, we generated a library of linker DNA by gBlock synthesis (Integrated DNA Technologies) (Figure 5). The linker DNA consists of TRAC/TRGC-2A-TRBVn/TRDVn (n represents the TRB/DV subfamily) sequence. Using the single cell PCR products of α/γ and β/δ chains of the desired clonotypes and the relevant linker gBlock DNA, we carried out an overlap PCR with TRAm/GVm internal sense primer and TRB/DC internal antisense primer. The PCR products were visualized on an agarose gel, and subsequently purified to use as “mega-primer” for cloning into the existing construct from our TCR cloning library (pMIC-TCRα/γm-TCR β/δn) with the same TRGV and TRDV family usage but different CDR3s by overlap extension PCR. The timeline of the whole process is on the left.

Fig 3: Peripheral blood LDH kinetics, CSF cell-free tumor DNA (ctDNA) findings, and ctDNA validation.a, Elevated Lactate Dehydrogenase (LDH) levels approximately 7–14 days following GD2-CAR T cell infusions (either IV or ICV) in N = 4 patients. Filled dot=IV, open dot=ICV. b, Cell-free tumor DNA (ctDNA) from patient CSF was evaluated using digital droplet PCR for the H3K27M mutation in the H3F3A gene. N=at least 4 technical replicates represented as Log2 of mutations per mL CSF ± SEM. Each point equals one technical replicate at the indicated timepoint; Filled dot=IV, open dot=ICV. DIPG-2 and Spinal DMG-1 demonstrated increasing cfDNA levels directly following GD2-CAR T cell ICV infusion (p < 0.0001 calculated by t-test). All available CSF samples from patients with adequate DNA extraction are represented here. DIPG-1 and DIPG-3 exhibited below the limit of detection levels and are not shown (limit of detection = 1 mutation per mL). c, Workflow for cell-free tumor DNA (ctDNA) assay. Cell-free DNA (cfDNA) was extracted from 1–4ml of CSF and subjected to 7 cycles pre-amplification followed by 40 cycles of digital droplet PCR (ddPCR). Schematic created with BioRender.com. d, Representative bioanalyzer traces of patients demonstrate adequate cfDNA quantities in Spinal DMG-1 and DIPG-2 without detectable cfDNA in DIPG-1 and DIPG-3 (i.e. there was not enough total cell-free DNA to run the assay). e, Validation of ddPCR assay through serial dilution of mutant H3K27M gBlock (IDT) against a fixed background of wild type H3K27M gBlock demonstrates linear mutant H3K27M detection. Sample concentrations (copies/ul) and dilution factors are plotted at Log10 scale. The QuantaSoft Pro Software calculates the starting concentration of each target DNA molecule by modeling as a Poisson distribution; the formula used for Poisson modeling is: Copies per droplet = -ln(1-p) where p = fraction of positive droplets. f, Inclusion of negative and positive controls for each ddPCR assay was performed alongside patient samples. Pre-amplified water served as a negative control for every run. Conditioned medium from H3K27M-mutated DIPG cell cultures was used as a positive control. Serial dilutions of H3K27M-mutated DIPG cell culture medium demonstrate a reproducible variant allele frequency (VAF) of the positive control. Source data

Fig 4: Singleplex and multiplex qPCR efficiencies. Assay optimization to compare the efficiencies of single vs. multiplex PCR assays using gBlock gene targets for (A) Necator americanus, (B) Ancylostoma ceylanicum, and (C) Ancylostoma duodenale fragments (IDT® Technologies) as standard curve controls. Ct = cycle threshold. This figure appears in color at www.ajtmh.org.

Fig 5: Schematic overview of the CAR construct and its subdomains the pHR’-derived lentiviral transfer plasmid. A second-generation human CAR was used, incorporating an Igκ leader sequence, a cytomegalovirus (CMV) promoter, a CS1-specific or non-targeting control VHH, a hinge region derived from human IgG4, a transmembrane and intracellular co-stimulatory domain derived from human CD28 and a human CD3ζ-derived T-cell activation domain. Relevant (unique) restriction sites are indicated above. Below the construct, the part that was ordered as a dsDNA molecule (gBlock) from Integrated DNA Technologies (IDT) is highlighted. CMV, cytomegalovirus; TM, transmembrane; hu, human; co-stim., co-stimulatory.