A reply to "TCR+/BCR+ dual-expressing cells and their associated public BCR clonotype are not enriched in type 1 diabetes"

Abstract

We have recently identified a novel lymphocyte that is a dual expresser (DE) of TCRαβ and BCR. DEs in T1D patients are predominated by a public BCR clonotype (clone-x) that encodes a potent autoantigen that cross-activates insulin-reactive T cells. Betts and colleagues were able to detect DEs but alleged to not detect high DE frequency, clone-x, or similar clones in T1D patients. Unfortunately, the authors did not follow our methods and when they did, their flow cytometric data at two sites were conflicting. Moreover, contrary to their claim, we identified clones similar to clone-x in their data along with clones bearing the core motif (DTAMVYYFDYW). Additionally, their report of no increased usage of clone-x VH/DH genes by bulk B cells confirms rather than challenges our results. Finally, the authors failed to provide data verifying purity of their sorted DEs, making it difficult to draw reliable conclusion of their repertoire analysis. This Matters Arising Response paper addresses the Japp et al. (2021) Matters Arising paper, published concurrently in Cell.

Keywords: DE cells; TCR; X cells; autoimmunity; dual expresser cells; insulin-mimotope; public BCR clonotype; type 1 diabetes; x-autoantigen.

Figure 1

A: Amplification and sequencing of clone-x using method of Wilson and colleagues-(i) Cloning and sequencing of x-clonotype from sorted single DE cells. The cloning of x-clonotype was performed using the following primers; RT-PCR: (5’ L-VH 4/6); and Cloning PCR: (Forward: 5’ AgeI VH4 and Reverse 3’ SalI JH 1/2/4/5) and method as described (Smith et al., 2009). Successful cloning was confirmed by sequencing of amplified PCR product at JHU-GRCF sequencing facility (data not shown). Highlighted in blue are the sites of the forward and reverse primers used in the cloning PCR. Middle. The IMG/v-quest analysis showing the x-clonotype is comprised of the IGH-V4–38-02, IGH-D05–18*01 and IGHJ04*02 gene segments. CDR3 sequence is highlighted in green. Bottom panel. Nucleotide and amino acid sequences of CDR3 of clone-x. Figure 1B: The core motif with the identical nucleotide sequence of clone-x is present in 10 subjects of Betts at al. Top panel. Alignment of nucleotide sequences of two clones from Betts and colleagues that have the same identical nucleotide sequence (63/63) including N2 junctional region as clone-x. Bottom panel. Nucleotide sequence alignment of eight clones from eight subjects shows the exact nucleotide sequences as the core motif of clone-x except for a single silent nucleotide mismatch. Highlighted thymine (T) nucleotide constituted the N2 junctional region as determined by IMGT/v-quest in all sequences. Figure 1C: Purity of DEs depends on the gating strategy of CD5+CD19+ cells. Two gating strategies of CD5+CD19+ cells were used to check and compare purity of DE cells. PBMCs were stained with CD19, CD5, TCRab and IgD specific antibodies and sorted after exclusion of doublets, dead cells and using of CD5 and CD19 to divide live singlets into CD5+CD19+, B_con and T_con cells. Top panel. Gating on total CD5+CD19+ yields low purity of DEs. Total CD5+CD19+ are gated (left plot) and analyzed for TCR and IgD (middle plot) followed by sorting of TCRab+IgD+ cells and checking their purity (19%) by re-running sorted cells and analysis of the coexpression of TCRab+ and IgD. Bottom panel. Gating on CD5^loCD19+ yields the highest purity: Sample is processed as in the top panel except the gating was restricted to CD5^loCD19+ cells (left panel). Gating on total CD5+CD19+ yielded a purity of 82%.