Pair socks from a pile: reconstructing TCRβ and TCRα pairs through sequencing

T cell receptor (TCR) β and α chains are generated in the thymus (some in the gut) via randomly rearranged V, J, D, C segments (unlike TCRβ chains, TCRα chains lack D segments). After rearrangement, some TCRβ and TCRα chains form productive pairs, we call TCRs. Note, not every TCRβ and TCRα chains can form productive pair. Afterwards, T cells expressing surface TCR undergo positive and negative selection and enter blood circulation to start patrolling the body.

Advances in personalized medicine created a niche to study TCR specificity in clinical setting. This is especially true for cancer therapy where adoptive transfer of ex vivo expanded tumor antigen specific T cells show protection against various lymphoid malignancies.

Ordinarily TCR specificity are elucidated by tetramer or hybridoma technologies, followed, if necessary, by TCR sequencing. These two, separate steps ensure selective expansion of T cell clones of interests. However, these methods are (1) technically difficult to accomplish (few scientists have "good" hands for in vitro experiments), (2) time consuming and (3) limited in scale (10-100 specificity). 

Another method is a direct TCRβ and TCRα massive-parallel sequencing. In this regard, sequencing technologies developed by Adaptive biotechnologies from Seattle has been cited by many high profile academic papers. 

However, TCR sequencing (typically only TCRβ sequencing) per se cannot provide an answer about TCR specificity, i.e. which TCRβ chain pairs with what TCRα chain. The knowledge of genetic structure of both chains in TCR, however, would allow reconstruction of 3D structure of TCR and it's specificity determination with algorithms that work similar to MHC+peptide algorithms.

In a recent article from Science Translational Medicine, Adaptive's team tried, in collaboration with the scientists from the local Cancer Center, to develop a method that would allow such TCRβ and TCRα chain pairing determination.

The methods itself is based on simple idea, if I understood it correctly. It requires some statistical analysis but principle works the following way: 

1. T cells are collected and distributed among 96 wells. Number of T cells per well can very based on statistical analysis. 

2. In 96 well plate, each well contains an unique small oligo DNA barcode that will be amplified along side with T cells derived cDNA. 

3. T cell derived cDNA is amplified with primers specific for TCR V and C regions and then sequenced.

Afterwards, if every time a particular Vβ and Vα genes are detected together in the same randomly barcoded wells, it is assumed that those two are pairs. This is in principle. Since there are potentially hundreds of of thousands unique TCR pairs, validation of experimental design is critical. In my view, strict validation of this type of readout would require actual determination of pairing using tetramer catch or hybridoma based expansion of T cells and then specific sequencing of recovered clones. 

Validation step the authors suggested is not entirely clear from paper description and may not be sufficiently robust to be accept as a gold standard for pairing. Another weakness is amplification step since it is not clear whether every V and C primer could amplify the target region with the same optimal rate.

David Usharauli

6 antigen-specific engineered T cells per µL of blood is needed to protect against multiple myeloma

CAR-T cells express B cell receptors on T cell body. They can target cells expressing proteins. This imposes some limitations on CAR-T cells. Tumors may not express mutated surface proteins readily distinguishable from normal variants. Such differences, however, may be visible at the peptide level. This therapeutic gap could be filled with TCR-engineered T cells targeting tumor-associated MHC+peptide combinations with high affinity and avidity.

New paper in Nature Medicine has provided some early results based on such protocols with NY-ESO-1-specific TCR engineered T cells in multiple myeloma patients. The senior author on this study is Carl June from Upenn whose work on engineered T cells reinvigorated tumor immunotherapy field in recent years.   

Initially, the authors showed that myeloma antigen-specific autologous TCR engineered T cells could persist up to 1 year or more after infusion in patients (though quantification method used here could be unreliable).

Next, the authors showed that lentiviral vector-transduced T cells could migrate and then eliminate myeloma cells from the bone marrow. However, the authors observed that in one patient TCR engineered T cells failed to control myeloma cells once it escaped bone marrow to other tissues. This is concerning. 

In addition, TCR affinity or its expression level in TCR engineered T cells goes dramatically down within 1 year of infusion. This is concerning too.

Finally, the authors claimed that no patients relapsed who had more than 6 antigen-specific T cells per µL of blood. However, close examination of the data also showed that some patients were progression free even though they had less 6 antigen-specific T cells per µL of blood. This implies that T cell number per se  is not a decisive factor here.

In summary, this study showed that close to 70% of patients had a complete or near complete response after TCR-engineered T cell infusion, with minimal associated safety issues.

David Usharauli