Biology subjects

Reinherz, E. L.

Publications and source records attributed to Reinherz, E. L..

7 recordsLinked to original sources

bioRxiv2025

Cancers modulate p53 truncal neoantigen display to evade T cell detection

Summary paragraphTP53 mutations are early truncal events across cancers1,2. These are perceived to encode tumour-specific neoantigens representing prime cytotoxic T lymphocyte (CTL) targets3,4. However, studies systematically examining the physical cell surface display of p53 peptides bound to major histocompatibility complex molecules (pMHC), their relative antigenicity, and resultant immunogenicity have yet to be conducted. Here, we develop an epitope discovery platform using p53-reconstituted lung cancer cells as well as various tumour cells as pMHC sources. Combining data-independent acquisition mass spectrometry (MS), nanoscale chromatography, and peptide detection based on probabilistic measure and three-dimensional ion visualization techniques allows attomole sensitivity identification of pMHCs. This approach excluded [~]97% of algorithm-based virtual p53 immunopeptidomes, highlighting that only a few p53 pMHCs can be presented by common human MHC (human leukocyte antigen, [HLA]) alleles. Strikingly, surface expressed neoantigens are restricted to the corresponding set of such limited self-p53 peptide arrays and unaffected by enhancing p53 proteasomal turnover. Further curtailment of MS-validated, high affinity p53 neoepitopes that are structurally deviant from self-pMHC occurs in established tumours due to immune selection against the antigen presenting MHC allele or by a novel mechanism involving p53 neoepitope destruction by endoplasmic reticulum aminopeptidase 1 (ERAP1). In contrast, given the extremely weak MHC affinity and resultant short-lived cell surface pMHC expression, the common p53 neoepitope R175H/HLA-A*02:01 escapes immune selection despite CTL with high quality T-cell receptors. Rigorous tumour-protective immunoediting makes effective truncal neoepitope targeting a challenge, requiring attentive MS analysis and functional vetting to focus protective cytolytic responses.

immunology↗

bioRxiv2025

Atomistic TCR-ligand interactions instruct memory T-cell differentiation

Memory CD8 T cells provide durable protection against recurrent infection and cancer, but how distinct memory fates are specified remains unclear. Here, we define the functional landscape of 242 murine CD8 TCR{beta} clonotypes specific for influenza NP366-374/H-2D ligand (pMHC) by integrating single-cell transcriptomics and TCR sequencing with force-dependent biophysics, structural analysis, and in vivo investigation of memory development. Fate essentially tracks with TCR mechanotransduction: TCM-associated clonotypes preferentially engage pMHC through TCR{beta}, whereas TEM-associated clonotypes preferentially engage through TCR. Clonotypes with balanced, bipolar signaling generate both memory subsets, optimize expansion, and display broad heterosubtypic crossreactivity but with exhaustion susceptibility. In contrast, diverse TCM clonotypes are largely clonal singlets with considerable mutant-epitope recognition in aggregate and stemness preservation, revealing complementary strategies for immunodefense coverage. Atomistically, subtle pMHC contact differences and load transmission through the TCR holoreceptor appear to tune phosphorylation, memory differentiation, and functional durability, features applicable to adoptive T-cell immunotherapies.

immunology↗

bioRxiv2025

Heterologous immunization modulates B-cell epitope competition between helper peptides and the MPER segment in MPER/liposome vaccines

Subdominant B-cell immune responses to conserved epitopes are major obstacles in eliciting broadly neutralizing antibodies (bnAbs) against HIV-1 through natural infection or vaccination. Although the sequence conserved membrane proximal external domain (MPER) of HIV-1 gp41 is partially occluded on the virion surface, epitope-focused immunogens could mitigate access limitations. Here, we found that a MPER/liposome vaccine delivered with single CD4 T cell helper epitope results in a post-priming response hierarchy, eliciting low affinity MPER-specific B cells. Heterologous boosting, however, promotes MPER-specific B cell clonal expansion and enhances plasma antibody functionality. This improvement is associated with increased B cell affinity for MPER and reduced competition from B cells targeting the helper epitope. While helper peptide co-delivery increases affinity of serum antibodies, the outcome of subsequent MPER antibody responses is shaped by the priming antigen. Our results offer insights into heterologous immunization strategies to potentiate subdominant B cell responses against frequently mutating viruses. Significance StatementA key challenge in vaccination against mutable viruses like HIV-1 is the immune systems focus on highly variable regions. The conserved MPER segment of gp41 elicits weak antibody responses due to poor B cell accessibility. This study evaluated a liposome-based MPER vaccine strategy. Initial priming generated low-affinity MPER-specific memory B cells, influenced by strong B cell affinity for a dominant T cell helper peptide. However, heterologous boosting overcomes subdominant MPER responses by increasing B cell affinity for booster immunogens while also reducing competition from other helper peptides. Co-delivery of helper peptides enhanced antibody affinity, though the priming antigen was critical in shaping responses. These findings suggest heterologous immunization is a promising strategy to enhance subdominant B cell responses.

immunology↗

bioRxiv2024

Load-based divergence in the dynamic allostery of two TCRs recognizing the same pMHC

Increasing evidence suggests that mechanical load on the {beta} T cell receptor (TCR) is crucial for recognizing the antigenic peptide-loaded major histocompatibility complex (pMHC) molecule. Our recent all-atom molecular dynamics (MD) simulations revealed that the inter-domain motion of the TCR is responsible for the load-induced catch bond behavior of the TCR-pMHC complex and peptide discrimination. To further examine the generality of the mechanism, we perform all-atom MD simulations of the B7 TCR under different conditions for comparison with our previous simulations of the A6 TCR. The two TCRs recognize the same pMHC and have similar interfaces with pMHC in crystal structures. We find that the B7 TCR-pMHC interface stabilizes under [~]15-pN load using a conserved dynamic allostery mechanism that involves the asymmetric motion of the TCR chassis. However, despite forming comparable contacts with pMHC as A6 in the crystal structure, B7 has fewer high-occupancy contacts with pMHC and exhibits higher mechanical compliance during the simulation. These results indicate that the dynamic allostery common to the TCR{beta} chassis can amplify slight differences in interfacial contacts into distinctive mechanical responses and nuanced biological outcomes.

biophysics↗

bioRxiv2023

Inadequate structural constraint on Fab approach rather than paratope elicitation limits HIV-1 MPER vaccine utility

Broadly neutralizing antibodies (bnAbs) against HIV-1 target conserved epitopes, thereby inhibiting viral entry. Yet surprisingly, those recognizing linear epitopes in the HIV-1 gp41 membrane proximal external region (MPER) are elicited neither by peptide nor protein scaffold vaccines. Here, we observe that while Abs generated by MPER/liposome vaccines may exhibit human bnAb-like paratopes, B-cell programming without constraints imposed by the gp160 ectodomain selects Abs unable to access the MPER within its native "crawlspace". During natural infection, the flexible hinge of IgG3 partially mitigates steric occlusion of less pliable IgG1 subclass Abs with identical MPER specificity, until affinity maturation refines entry mechanisms. The IgG3 subclass maintains B-cell competitiveness, exploiting bivalent ligation resulting from greater intramolecular Fab arm length, offsetting weak antibody affinity. These findings suggest future immunization strategies.

immunology↗

bioRxiv2022

Dynamic HIV-1 spike motion creates vulnerability for its membrane-bound tripod to antibody attack

Vaccines targeting HIV-1s gp160 spike protein are stymied by high viral mutation rates and structural chicanery. gp160s membrane-proximal external region (MPER) is the target of naturally arising broadly neutralizing antibodies (bnAbs), yet MPER-based vaccines fail to generate bnAbs. Here, nanodisc-embedded spike protein was investigated by cryo-electron microscopy and molecular-dynamics simulations, revealing spontaneous ectodomain tilting that creates vulnerability for HIV-1. While each MPER protomer radiates centrally towards the three-fold axis contributing to a membrane-associated tripod structure that is occluded in the upright spike, tilting provides access to the opposing MPER. Structures of spike proteins with bound 4E10 bnAb Fabs reveal that the antibody binds exposed MPER, thereby altering MPER dynamics, modifying average ectodomain tilt, and imposing strain on the viral membrane and the spikes transmembrane segments, resulting in the abrogation of membrane fusion and informing future vaccine development.

biophysics↗

bioRxiv2022

Pre-T cell receptor Self-MHC Sampling Restricts Thymocyte Dedifferentiation

Summary paragraphProgramming T lymphocytes to distinguish self from non-self is a vital, multi-step process arising in the thymus1-4. Signalling through the pre-T cell receptor (preTCR), a CD3-associated heterodimer comprising an invariant pT chain and a clone-specific {beta} chain, constitutes a critical early checkpoint in thymocyte development within the {beta} T-cell lineage5, 6. Recent work demonstrates that preTCRs arrayed on double negative (DN) thymocytes, like {beta} TCRs appearing on double positive (DP) thymocytes, ligate peptides bound to MHC molecules (pMHC) on thymic stroma but via a different molecular docking strategy7-10. Here we show the consequences of those distinctive interactions for thymocyte progression, using synchronized fetal thymic progenitor cultures differing in the presence or absence of pMHC on support stroma, determining single cell transcriptomes at key thymocyte developmental transitions. Although MHC negative stroma fosters {beta} T lymphocyte differentiation, the absence of pMHC-preTCR interplay leads to deviant thymocyte transcriptional programming associated with de-differentiation. Highly proliferative DN and DP subsets with antecedent characteristics of T cell lymphoblastic and myeloid malignancies emerge. Thus, at least in vitro, beyond fostering {beta} chain repertoire broadening for subsequent {beta} TCR utilization, preTCR-pMHC interaction limits cellular plasticity to facilitate normal thymocyte differentiation and proliferation that, if absent, introduces significant developmental vulnerabilities.

immunology↗