Biology subjects

Straub, A.

Publications and source records attributed to Straub, A..

5 recordsLinked to original sources

bioRxiv2026

Germline-encoded V(D)J gene usage does not impose strict constraints on the epitope-specificity of T cell receptors

The theoretical diversity of T cell receptors (TCRs), generated through V(D)J recombination, is enormous, yet the diversity of TCRs capable of recognizing the same epitope remains unknown. Defining this TCR solution space is essential for uncovering basic principles that govern TCR specificity. Using single-cell RNA and TCR sequencing, we generated ultra-deep (more than 4000 unique TCRs per epitope) epitope-specific TCR libraries derived from 560 immunized C57BL/6 mice, identifying over 27,000 unique epitope-reactive TCRs across three distinct CD8+ T cell epitopes presented by two major histocompatibility complex (MHC) class I alleles. Saturation analyses indicated that the solution space for all studied epitopes comprises many tens of thousands of unique TCRs. Despite highly skewed and peptide-dependent VJ-usage patterns, nearly the entire set of functional germline V/ and J/ segments was detected at least once within each epitope-specific repertoire. Therefore, diversity of epitope-specific TCRs is not limited by distinct germline combinations but rather can emerge from a near-to-complete combinatorial space of - and -chain, V and J segments paired with compatible CDR3 sequences.

immunology↗

bioRxiv2024

Delivery of novel replicating vectors to Synechococcus sp. PCC 7002 via natural transformation of plasmid multimers

In most cyanobacteria, genetic engineering efforts currently rely upon chromosomal integration; a time-consuming process due to their polyploid nature. To enhance strain construction, here we develop and characterize two novel replicating plasmids for use in Synechococcus sp. PCC 7002. Following an initial screen of plasmids comprising seven different origins of replication, two were found capable of replication: one based on the WVO1 broad host range plasmid and the other a shuttle vector derived from pCB2.4 from Synechocystis sp. PCC 6803. These were then used to construct a set of new replicating plasmids, which were shown to be both co-transformable and stably maintained in PCC 7002 at copy numbers between 0.6-1.4 and 7-16, respectively. Lastly, we demonstrate the importance of using multimeric plasmids during natural transformation of PCC 7002, with higher order multimers providing a 30-fold increase in transformation efficiency relative to monomeric plasmids. Useful considerations and methods for enhancing multimer content in plasmid samples are also presented.

synthetic biology↗

bioRxiv2024

A mass cytometry approach to track the evolution of T cell responses during infection and immunotherapy by paired T cell receptor repertoire and T cell differentiation state analysis

T cell receptor (TCR) recognition followed by clonal expansion is a fundamental feature of adaptive immune responses. Here, we developed a mass cytometric (CyTOF) approach combining antibodies specific for different TCR V- and V{beta}-chains with antibodies against T cell activation and differentiation proteins to identify antigen-specific expansions of T cell subsets and assess aspects of cellular function. This strategy allowed for the identification of expansions of specific V{beta} and V chain expressing CD8+ and CD4+ T cells with varying differentiation states in response to Listeria monocytogenes, tumors, and respiratory influenza infection. Expanded V{beta} chain expressing T cells could be directly linked to the recognition of specific antigens from Listeria, tumor cells, or influenza. In the setting of influenza infection, we showed that the common therapeutic approaches of intramuscular vaccination or convalescent serum transfer altered the clonal diversity and differentiation state of responding T cells. Thus, we present a new method to monitor broad changes in TCR specificity paired with T cell differentiation during adaptive immune responses.

immunology↗

bioRxiv2023

Predicting T Cell Receptor Functionality against Mutant Epitopes

Cancer cells or pathogens can escape recognition by T cell receptors (TCRs) through mutations of immunogenic epitopes. TCR cross-reactivity, i.e., recognition of multiple epitopes with sequence similarities, can be a factor to counteract such mutational escape. However, cross-reactivity of cell-based immunotherapies may also cause severe side effects when self-antigens are targeted. Therefore, the ability to predict the effect of mutations in the epitope sequence on T cell functionality in silico would greatly benefit the safety and effectiveness of newly-developed immunotherapies and vaccines. We here present "Predicting T cell Epitope-specific Activation against Mutant versions" (P-TEAM), a Random Forest-based model which predicts the effect of point mutations of an epitope on T cell functionality. We first trained and tested P-TEAM on a comprehensive dataset of 36 unique murine TCRs in response to systematic single-amino acid mutations of their target epitope (representing 5.472 unique TCR-epitope interactions). The model was able to classify T cell reactivities, corresponding to in vivo recruitment of T cells, and quantitatively predict T cell functionalities for unobserved single-point mutated altered peptide ligands (APLs), or even unseen TCRs, with consistently high performance. Further, we present an active learning framework to guide experimental design for assessing TCR functionality against novel epitopes, minimizing primary data acquisition costs. Finally, we applied P-TEAM to a novel dataset of 7 human TCRs reactive to the tumor neoantigen VPSVWRSSL. We observed a similarly robust performance for these human TCRs as for the murine TCRs recognizing SIINFEKL, thus providing evidence that our approach is applicable to therapeutically relevant TCRs as well as across species. Overall, P-TEAM provides an effective computational tool to study T cell responses against mutated epitopes.

bioinformatics↗

bioRxiv2019

Salicylic acid-driven association of LENRV and NIMIN1/NIMIN2 binding domain regions in the C-terminus of tobacco NPR1 transduces SAR signal

O_LINONEXPRESSOR OF PATHOGENESIS-RELATED (PR) GENES1 (NPR1) is the central regulator of salicylic acid (SA)-induced PR-1 gene expression and systemic acquired resistance (SAR). The mechanism how SA is transduced through NPR1 is discussed controversially. Previously, we showed that Arabidopsis and tobacco (Nt) NPR1 contain two domains in their C-terminal thirds with relevance to SA signaling. SA sensitivity of NPR1 relies on the arginine residue in the LENRV motif, and SA-induced NIM1-INTERACTING (NIMIN, N) proteins bind to a highly conserved sequence termed N1/N2 binding domain (BD). C_LIO_LIWe demonstrate that LENRV and N1/N2BD regions of tobacco NPR1, separated from each other, interact in yeast, in vitro, in plant and in animal cells. Physical association of LENRV and N1/N2BD parts is enhanced considerably by SA and functional analogs, but not by a non-functional analog. Furthermore, physical association requires R431 and is most effective with intact LENRV and N1/N2BD interfaces. C_LIO_LIAssociation of separated LENRV and N1/N2BD parts by SA reconstitutes a functional NtNPR1 C-terminus, displaying transcription activity and able to interact with TGA transcription factors at two distinct sites. C_LIO_LITobacco NIMIN proteins can assemble LENRV and N1/N2BD parts into ternary complexes suggesting that NIMINs shape the NPR1 C-terminus to modulate SA signaling. C_LI

plant biology↗