Biology subjects

Lam, A. J.

Publications and source records attributed to Lam, A. J..

6 recordsLinked to original sources

bioRxiv2026

Cell Cycle Sensing Shapes Human T Cell Fate and Exhaustion Programs

Cell cycle (CC) dynamics are reflected in diverse T cell processes such as TCR activation, expansion, contraction, differentiation, senescence, anergy, and exhaustion; linking CC behaviors to functional and dysfunctional T cell states in development and disease. Progression through CC checkpoints is also tightly linked to cell fate decisions across development. Yet, much remains unknown about the connection between CC sensing and T cell differentiation programs. To disentangle the relationship across T cell state, time-since-activation, receptor signaling, division, and CC, we leverage high-throughput single-cell mass cytometry for parallel measurement of these diverse biological states. By modulating CC progression and receptor signaling with inhibitors as well as tonic signaling Chimeric Antigen Receptor (CAR) models of T cell exhaustion, we reveal that earlier G1/S CC programs crosstalk with receptor signaling to control T cell fate, and that exhaustion programs are downstream to aberrant, S-G2 phase CC arrest signatures in tonic CAR signaling in vitro, in situ, and in vivo across human cancers in association with CD8 T-lymphocyte dysfunction.

immunology↗

bioRxiv2024

Fourth generation CAR Tregs with PDCD1-driven IL-10 have enhanced suppressive function

The potency of regulatory T cell (Treg) therapy has been transformed through use of chimeric antigen receptors (CAR). However, to date, CAR Treg therapy has not achieved long-lasting tolerance in mouse models, suggesting that additional engineering is required to unlock the full potential of these cells. We previously found that human Tregs produce minimal amounts of IL-10 and have a limited capacity to control innate immunity in comparison to type I regulatory (Tr1) cells. Seeking to create "hybrid" CAR Tregs that were engineered with Tr1-like properties, we examined whether the PDCD1 locus could be exploited to endow Tregs with the ability to secrete high levels of IL-10 in a CAR-regulated manner. CRISPR-mediated PD1-deletion increased the activation potential of CAR Tregs without compromising in vivo stability. Knock-in of IL10 under control of the PD1 promoter facilitated CAR-mediated secretion of IL-10 in large quantities, and improved CAR Treg function, as determined by significant inhibition of dendritic cell antigen presentation and enhanced suppression of alloantigen- and islet autoantigen-specific T cells. Overall, CRISPR-mediated engineering to simultaneously remove an inhibitory signal and enhance suppressive mechanisms is a new approach to enhance the therapeutic potency of CAR Tregs.

immunology↗

bioRxiv2022

PTEN is required for human Treg suppression of costimulation

Regulatory T cell (Treg) therapy is under clinical investigation for the treatment of transplant rejection, autoimmune disease, and graft-versus-host disease. With the advent of genome editing, attention has turned to reinforcing Treg function for therapeutic benefit. A hallmark of Tregs is dampened activation of PI3K-AKT signalling, of which PTEN is a major negative regulator. Loss-of-function studies of PTEN, however, have not conclusively shown a requirement for PTEN in upholding Treg function and stability. Using CRISPR-based genome editing in human Tregs, we show that PTEN ablation does not cause a global defect in Treg function and stability; rather, it selectively blocks their ability to suppress antigen-presenting cells. PTEN-KO Tregs exhibit elevated glycolytic activity, upregulate FOXP3, maintain a Treg phenotype, and have no discernable defects in lineage stability. Functionally, PTEN is dispensable for human Treg-mediated inhibition of T cell activity in vitro and in vivo but is required for suppression of costimulatory molecule expression by antigen-presenting cells. These data are the first to define a role for a signalling pathway in controlling a subset of human Treg activity. Moreover, they point to the functional necessity of PTEN-regulated PI3K-AKT activity for optimal human Treg function.

immunology↗

bioRxiv2021

Helios is a marker, not a driver, of human Treg stability

Regulatory T cell (Treg) therapy holds promise as a potentially curative approach to establish immune tolerance in transplantation and autoimmune disease. An outstanding question is whether therapeutic Tregs have the potential to transdifferentiate into effector T cells and thus exacerbate rather than suppress immune responses. In mice, the transcription factor Helios is thought to promote Tregs lineage stability in a range of inflammatory contexts. In humans, the role of Helios in Tregs is less clear, in part due to the inability to enrich and study subsets of Helios-positive versus Helios-negative Tregs. Using an in vitro expansion system, we found that loss of high Helios expression and emergence of an intermediate Helios (Heliosmid)-expressing population correlated with Treg destabilization. We then used CRISPR/Cas9 to genetically ablate Helios expression in human naive or memory Tregs and found that Helios-knockout and unedited Tregs were equivalent in their suppressive function and stability in inflammation. Thus, high Helios expression is a marker, but not a driver, of human Treg stability in vitro. These data highlight the importance of monitoring Helios expression in therapeutic Treg manufacturing and provide new insight into the biological function of this transcription factor in human T cells.

immunology↗

bioRxiv2021

Optimized CRISPR-mediated gene knock-in reveals FOXP3-independent control of human Treg identity

Treg cell therapy is a promising curative approach for a variety of immune-mediated conditions. CRISPR-based genome editing allows precise insertion of transgenes through homology-directed repair, but use in human Tregs has been limited. We report an optimized protocol for CRISPR-mediated gene knock-in in human Tregs with high-yield expansion. To establish a benchmark of human Treg dysfunction, we targeted the master transcription factor FOXP3 in naive and memory Tregs. Although FOXP3-knockout Tregs upregulated cytokine expression, effects on suppressive capacity manifested slowly and primarily in memory Tregs. Moreover, FOXP3-knockout Tregs retained their characteristic phenotype and had few changes in their DNA methylation landscape, with FOXP3 maintaining methylation at regions enriched for AP-1 binding sites. Thus, while FOXP3 is important for human Treg development, it has a limited role in maintaining mature Treg identity. Optimized gene knock-in with human Tregs will enable mechanistic studies and the development of tailored, next-generation Treg cell therapies.

immunology↗

bioRxiv2020

A Highly Conserved 310-Helix Within the Kinesin Motor Domain is Critical for Kinesin Function and Human Health.

KIF1A, a kinesin-3 family member, plays critical roles as a long-distance cargo-transporter within neurons. Over 100 known KIF1A mutations in humans result in KIF1A Associated Neurological Disease (KAND), developmental and degenerative neurological conditions for which there is no cure. A de novo missense mutation, P305L, was recently identified in several children diagnosed with KAND, but the underlying molecular basis for the disease phenotype is unknown. Interestingly, this residue is highly conserved in kinesin-family proteins, and together with adjacent conserved residues also implicated in KAND, forms an unusual 310-helical element immediately C-terminal to loop-12 (L12, also known as the K-loop in KIF1A) in the conserved kinesin motor core. In KIF1A, the disordered K-loop contains a highly charged insertion of lysines that is thought to endow the motor with a high microtubule-association rate. Here, we characterize the molecular defects of the P305L mutation in KIF1A using genetic, biochemical, and single-molecule approaches. We find the mutation negatively impacts the velocity, run-length, and force generation of the motor. However, a much more dramatic effect is observed on the microtubule-association rate of the motor, revealing that the presence of an intact K-loop is not sufficient for its function. We hypothesize that an elusive K-loop conformation, mediated by formation of a highly conserved adjacent 310-helix that is modulated via P305, is critically important for the kinesin-microtubule interaction. Importantly, we find that the function of this proline is conserved in the canonical kinesin, KIF5, revealing a fundamental principle of the kinesin motor mechanism.

biophysics↗