Rational Design of T Cell Receptors with Enhanced Sensitivity for Antigen

Enhancing the affinity of therapeutic T cell receptors (TCR) without altering their specificity is a significant challenge for adoptive immunotherapy. Current efforts have primarily relied on empirical approaches. Here, we used structural analyses to identify a glycine-serine variation in the TCR that modulates antigen sensitivity. A G at position 107 within the CDR3β stalk is encoded within a single mouse and human TCR, TRBV13-2 and TRBV12-5 respectively. Most TCR bear a S107. The S hydroxymethyl side chain intercalates into the core of the CDR3β loop, stabilizing it. G107 TRBV possess a gap in their CDR3β where this S hydroxymethyl moiety would fit. We predicted based on modeling and molecular dynamics simulations that a G107S substitution would increase CDR3β stability and thereby augment receptor sensitivity. Experimentally, a G107S replacement led to an ∼10–1000 fold enhanced antigen sensitivity in 3 of 4 TRBV13-2+ TCR tested. Analysis of fine specificity indicated a preserved binding orientation. These results support the feasibility of developing high affinity antigen specific TCR for therapeutic purposes through the identification and manipulation of critical framework residues. They further indicate that amino acid variations within TRBV not directly involved in ligand contact can program TCR sensitivity, and suggest a role for CDR3 stability in this programming

Identification of errors introduced during high throughput sequencing of the T cell receptor repertoire

<p>Abstract</p> <p>Background</p> <p>Recent advances in massively parallel sequencing have increased the depth at which T cell receptor (TCR) repertoires can be probed by >3log10, allowing for saturation sequencing of immune repertoires. The resolution of this sequencing is dependent on its accuracy, and direct assessments of the errors formed during high throughput repertoire analyses are limited.</p> <p>Results</p> <p>We analyzed 3 monoclonal TCR from TCR transgenic, Rag^-/-mice using Illumina^®sequencing. A total of 27 sequencing reactions were performed for each TCR using a trifurcating design in which samples were divided into 3 at significant processing junctures. More than 20 million complementarity determining region (CDR) 3 sequences were analyzed. Filtering for lower quality sequences diminished but did not eliminate sequence errors, which occurred within 1-6% of sequences. Erroneous sequences were pre-dominantly of correct length and contained single nucleotide substitutions. Rates of specific substitutions varied dramatically in a position-dependent manner. Four substitutions, all purine-pyrimidine transversions, predominated. Solid phase amplification and sequencing rather than liquid sample amplification and preparation appeared to be the primary sources of error. Analysis of polyclonal repertoires demonstrated the impact of error accumulation on data parameters.</p> <p>Conclusions</p> <p>Caution is needed in interpreting repertoire data due to potential contamination with mis-sequence reads. However, a high association of errors with phred score, high relatedness of erroneous sequences with the parental sequence, dominance of specific nt substitutions, and skewed ratio of forward to reverse reads among erroneous sequences indicate approaches to filter erroneous sequences from repertoire data sets.</p

Chronic TLR stimulation controls NLRP3 inflammasome activation through IL-10 mediated regulation of NLRP3 expression and caspase-8 activation

While the molecular mechanisms promoting activation of the Nod-like Receptor (NLR) family member NLRP3 inflammasome are beginning to be defined, little is known about the mechanisms that regulate the NLRP3 inflammasome. Acute (up to 4 hours) LPS stimulation, followed by ATP is frequently used to activate the NLRP3 inflammasome in macrophages. Interestingly, we observed that the ability of LPS to license NLRP3 is transient, as prolonged (12 to 24 hours) LPS exposure was a relatively ineffective priming stimulus. This suggests that relative to acute LPS, chronic LPS exposure triggers regulatory mechanisms to dampen NLRP3 activation. Transfer of culture supernatants from macrophages stimulated with LPS for 24 hours dramatically reduced ATP- and nigericin-induced NLRP3 inflammasome activation in naive macrophages. We further identified IL-10 as the secreted inflammasome-tolerizing factor that acts in an autocrine manner to control activation of the NLRP3 inflammasome. Finally, we demonstrated that IL-10 dampens NLRP3 expression to control NLRP3 inflammasome activation and subsequent caspase-8 activation. In conclusion, we have uncovered a mechanism by which chronic, but not acute, LPS exposure induces IL-10 to dampen NLRP3 inflammasome activation to avoid overt inflammation

Identification of errors introduced during high throughput sequencing of the T cell receptor repertoire

<p>Abstract</p> <p>Background</p> <p>Recent advances in massively parallel sequencing have increased the depth at which T cell receptor (TCR) repertoires can be probed by >3log10, allowing for saturation sequencing of immune repertoires. The resolution of this sequencing is dependent on its accuracy, and direct assessments of the errors formed during high throughput repertoire analyses are limited.</p> <p>Results</p> <p>We analyzed 3 monoclonal TCR from TCR transgenic, Rag^-/-mice using Illumina^®sequencing. A total of 27 sequencing reactions were performed for each TCR using a trifurcating design in which samples were divided into 3 at significant processing junctures. More than 20 million complementarity determining region (CDR) 3 sequences were analyzed. Filtering for lower quality sequences diminished but did not eliminate sequence errors, which occurred within 1-6% of sequences. Erroneous sequences were pre-dominantly of correct length and contained single nucleotide substitutions. Rates of specific substitutions varied dramatically in a position-dependent manner. Four substitutions, all purine-pyrimidine transversions, predominated. Solid phase amplification and sequencing rather than liquid sample amplification and preparation appeared to be the primary sources of error. Analysis of polyclonal repertoires demonstrated the impact of error accumulation on data parameters.</p> <p>Conclusions</p> <p>Caution is needed in interpreting repertoire data due to potential contamination with mis-sequence reads. However, a high association of errors with phred score, high relatedness of erroneous sequences with the parental sequence, dominance of specific nt substitutions, and skewed ratio of forward to reverse reads among erroneous sequences indicate approaches to filter erroneous sequences from repertoire data sets.</p