TCR transgenic clone selection guided by immune receptor analysis and single cell RNA expression of polyclonal responders

Reviewer #1 (Public Review):

Summary:

Debeuf et al. introduce a new, fast method for the selection of suitable T cell clones to generate TCR transgenic mice, a method claimed to outperform traditional hybridoma-based approaches. Clone selection is based on the assessment of the expansion and phenotype of cells specific for a known epitope following immune stimulation. The analysis is facilitated by a new software tool for TCR repertoire and function analysis termed DALI. This work also introduces a potentially invaluable TCR transgenic mouse line specific for SARS-CoV-2.

Strengths:

The newly introduced method proved successful in the quick generation of a TCR transgenic mouse line. Clone selection is based on more comprehensive phenotypical information than traditional methods, providing the opportunity for a more rational T cell clone selection.

The study provides a software tool for TCR repertoire analysis and its linkage with function.

The findings entail general practical implications in the preclinical study of a potentially very broad range of infectious diseases or vaccination.

A novel SARS-CoV-2 spike-specific TCR transgenic mouse line was generated.

Weaknesses:

The authors attempt to compare their novel method with a more conventional approach to developing TCR transgenic mice. In this reviewer's opinion, this comparison appears imperfect in several ways:

• Work presenting the "traditional" method was inadequate to justify the selection of a suitable clone. It is therefore not surprising that it yielded negative results. More evidence would have been necessary to select clone 47 for further development of the TCR transgenic line, especially considering the significant time and investment required to create such a line.

• The comparison is somewhat unfair, because the methods start at different points: while the traditional method was attempted using a pool of peptides whose immunogenicity does not appear to have been established, the new method starts by utilising tetramers to select T cells specific for a well-established epitope.

• Given the costs and time involved, only a single clone could be tested for either method, intrinsically making a proper comparison unfeasible. Even for their new method, the authors' ability to demonstrate that the selected clone is ideal is limited unless they made different clones with varying profiles to show that a particular profile was superior to others.

In my view, there was no absolute need to compare this method with existing ones, as the proposed method holds intrinsic value.

While having more data to decide on clone selection is certainly beneficial, given the additional cost, it remains unclear whether knowing the expression profiles of different proteins in Figure 2 aids in selecting a candidate. Is a cell expressing more CD69 preferable to a cell expressing less of this marker? Would either have been effective? Are there any transcriptional differences between clonotype 1 and 2 (red colour in Figure 2G) that justify selecting clone 1, or was the decision to select the latter merely based on their different frequency? If all major clones (i.e. by clonotype count) present similar expression profiles, would it have been necessary to know much more about their expression profiles? Would TCR sequencing and an enumeration of clones have sufficed, and been a more cost-effective approach?

Lastly, it appears that several of the experiments presented were conducted only once. This information should have been explicitly stated in the figure legends.

Reviewer #2 (Public Review):

Summary:

The authors seek to use single-cell sequencing approaches to identify TCRs specific for the SARS CoV2 spike protein, select a candidate TCR for cloning, and use it to construct a TCR transgenic mouse. The argument is that this process is less cumbersome than the classical approach, which involves the identification of antigen-reactive T cells in vitro and the construction of T cell hybridomas prior to TCR cloning. TCRs identified by single-cell sequencing that are already paired to transcriptomic data would more rapidly identify TCRs that are likely to contribute to a functional response. The authors successfully identify TCRs that have expanded in response to SARS CoV2 spike protein immunization, bind to MHC tetramers, and express genes associated with functional response. They then select a TCR for cloning and construction of a transgenic mouse in order to test the response of resulting T cells in vivo following immunization with spike protein of coronavirus infection.

Strengths:

(1) The study provides proof of principle for the identification and characterization of TCRs based on single-cell sequencing data.

(2) The authors employ a recently developed software tool (DALI) that assists in linking transcriptomic data to individual clones.

(3) The authors successfully generate a TCR transgenic animal derived from the most promising T cell clone (CORSET8) using the TCR sequencing approach.

(4) The authors provide initial evidence that CORSET8 T cells undergo activation and proliferation in vivo in response to immunization or infection.

(5) Procedures are well-described and readily reproducible.

Weaknesses:

(1) The purpose of presenting a failed attempt to generate TCR transgenic mice using a traditional TCR hybridoma method is unclear. The reasons for the failure are uncertain, and the inclusion of this data does not really provide information on the likely success rate of the hybridoma vs single cell approach for TCR identification, as only a single example is provided for either.

(2) There is little information provided regarding the functional differentiation of the CORSET8 T cells following challenge in vivo, including expression of molecules associated with effector function, cytokine production, killing activity, and formation of memory. The study would be strengthened by some evidence that CORSET8 T cells are successfully recapitulating the functional features of the endogenous immune response (beyond simply proliferating and expressing CD44). This information is important to evaluate whether the presented sequencing-based identification and selection of TCRs is likely to result in T-cell responses that replicate the criteria for selecting the TCR in the first place.

(3) While I find the argument reasonable that the approach presented here has a lot of likely advantages over traditional approaches for generating TCR transgenic animals, the use of TCR sequencing data to identify TCRs for study in a variety of areas, including cancer immunotherapy and autoimmunity, is in broad use. While much of this work opts for alternative methods of TCR expression in primary T cells (i.e. CRISPR or retroviral approaches), the process of generating a TCR transgenic mouse from a cloned TCR is not in itself novel. It would be helpful if the authors could provide a more extensive discussion explaining the novelty of their approach for TCR identification in comparison to other more modern approaches, rather than only hybridoma generation.