Periodic Reporting for period 1 - Remin-T (Dissecting the molecular basis of immunological memory in human T cells)
Reporting period: 2023-09-01 to 2025-08-31
T cells are essential for immunity to pathogens and malignancies. While the initial activation of a naive T cell – that has never encountered its target antigen (such as a pathogen- or tumour-derived protein) – is slow (multiple days), experienced or 'memory' T cells launch powerful ‘recall’ responses that are both faster (hours) and greater in magnitude. These memory T cells provide long-lasting immunological protection against previously encountered harmful agents – forming the basis of the current vaccination strategies. However, the formation of memory T cells is not without risk. Aberrant memory T cells targeting harmless (self)antigens can cause autoimmunity (worldwide prevalence of ~4%) or allergy (most prevalent type of chronic disease in Europe affecting 10-40% of EU citizens). In addition, in patients with cancer – who account for an estimated global death toll of 10 million per year – T cells often fail to adopt functional memory phenotypes and instead enter a state of relative dysfunction. This leads to failed anti-tumor immunity and partly explains the modest clinical success of current cancer immunotherapies.
Unraveling the molecular basis of memory in T cells is key to developing improved vaccination strategies and T cell–based immunotherapies. Evidence is emerging that the rapid recall ability of memory T cells may rely on dynamic changes at the chromatin or epigenome level. However, we lack a mechanistic understanding of how memory T cells durably ‘remember’ gene regulatory processes essential for high-magnitude protective immune responses. Most importantly, the molecular determinants that coordinate the maintenance of transcriptional memory in T cells remain unknown.
This HE-MSCA-PF project employs an innovative epigenomics approach to uncover the molecular circuitry underlying immunological memory in primary human T cells. Specifically, this project addresses the following key objectives:
• Objective 1: Multidimensional epigenomics of naive and memory T cells during activation
• Objective 2: Computational data integration to identify mechanisms and drivers of memory recall
• Objective 3: Functional validation of candidate genes, gene regulatory elements and/or biological processes underlying T cell memory recall
Unraveling the molecular basis of memory in T cells is key to developing improved vaccination strategies and T cell–based immunotherapies. Evidence is emerging that the rapid recall ability of memory T cells may rely on dynamic changes at the chromatin or epigenome level. However, we lack a mechanistic understanding of how memory T cells durably ‘remember’ gene regulatory processes essential for high-magnitude protective immune responses. Most importantly, the molecular determinants that coordinate the maintenance of transcriptional memory in T cells remain unknown.
This HE-MSCA-PF project employs an innovative epigenomics approach to uncover the molecular circuitry underlying immunological memory in primary human T cells. Specifically, this project addresses the following key objectives:
• Objective 1: Multidimensional epigenomics of naive and memory T cells during activation
• Objective 2: Computational data integration to identify mechanisms and drivers of memory recall
• Objective 3: Functional validation of candidate genes, gene regulatory elements and/or biological processes underlying T cell memory recall
Technical approach:
To model immunological memory in T cells, CD8+ naive and effector memory T cells were isolated from the peripheral blood of healthy human volunteers. Cells were cultured for 24h in the presence or absence of beads coated with antibodies against CD3/CD28 to mimic natural activation via the T cell receptor and coreceptor. From these CD8+ T cell populations, we profiled the transcriptome using RNA-sequencing (RNA-Seq; n = 3 or 4 independent biological replicates), chromatin accessibility by Assay for Transposase-Accessible Chromatin using sequencing (ATAC-Seq; n = 4 independent biological replicates) and 3D chromatin architecture by chromosome conformation capture coupled to high-throughput sequencing (Hi-C; n = 4 independent biological replicates). We used a combined single cell RNA-Seq/ATAC-Seq protocol (10X Genomics Multiome technology; n = 3 independent biological replicates) to jointly capture transcriptional/epigenomic heterogeneity and to dissect the crosstalk between transcriptome and epigenome dynamics in individual CD8+ T cells. CRISPR/Cas9-mediated gene perturbation followed by downstream analyses using flow cytometry, RNA-Seq and ATAC-Seq was performed for functional validation, aiming to gain mechanistic insight into the molecular underpinnings that maintain epigenetic priming in memory CD8+ T cells.
Outcomes:
Using this multidimensional epigenomics approach, we identified regions of transcription-permissive accessible chromatin specifically enriched in memory CD8+ T cells compared to naive CD8+ T cells, often residing near genes associated with memory recall. Joint gene expression and epigenome profiling of CD8+ T cells at single cell resolution using 10X Multiome technology showed that memory CD8+ T cells exhibit precisely regulated accessible chromatin dynamics underlying unique transcriptional signatures, and revealed that chromatin priming mechanisms are concentrated in a specific subset of effector memory CD8+ T cells. We pinpointed sets of accessible chromatin sites specifically overrepresented in memory CD8+ T cell already at baseline (i.e. prior to activation), which are enriched for 3D regulatory interactions and specific transcription factor binding sites. Compared to naive CD8+ T cells, memory CD8+ T cells selectively upregulated three key members of these transcription factor families – already at the resting state. Combined CRISPR/Cas9-mediated knock-out of this trio of transcription factors in memory CD8+ T cells caused a loss of epigenetic priming, culminating in an impaired capacity to produce inflammatory molecules essential for CD8+ T cell-mediated immunity. Together, our findings show that memory CD8+ T cells maintain a transcription-permissive chromatin landscape, which is imprinted by cooperating transcription factors to enable the rapid reactivation of inflammatory genes crucial for protective immune responses.
To model immunological memory in T cells, CD8+ naive and effector memory T cells were isolated from the peripheral blood of healthy human volunteers. Cells were cultured for 24h in the presence or absence of beads coated with antibodies against CD3/CD28 to mimic natural activation via the T cell receptor and coreceptor. From these CD8+ T cell populations, we profiled the transcriptome using RNA-sequencing (RNA-Seq; n = 3 or 4 independent biological replicates), chromatin accessibility by Assay for Transposase-Accessible Chromatin using sequencing (ATAC-Seq; n = 4 independent biological replicates) and 3D chromatin architecture by chromosome conformation capture coupled to high-throughput sequencing (Hi-C; n = 4 independent biological replicates). We used a combined single cell RNA-Seq/ATAC-Seq protocol (10X Genomics Multiome technology; n = 3 independent biological replicates) to jointly capture transcriptional/epigenomic heterogeneity and to dissect the crosstalk between transcriptome and epigenome dynamics in individual CD8+ T cells. CRISPR/Cas9-mediated gene perturbation followed by downstream analyses using flow cytometry, RNA-Seq and ATAC-Seq was performed for functional validation, aiming to gain mechanistic insight into the molecular underpinnings that maintain epigenetic priming in memory CD8+ T cells.
Outcomes:
Using this multidimensional epigenomics approach, we identified regions of transcription-permissive accessible chromatin specifically enriched in memory CD8+ T cells compared to naive CD8+ T cells, often residing near genes associated with memory recall. Joint gene expression and epigenome profiling of CD8+ T cells at single cell resolution using 10X Multiome technology showed that memory CD8+ T cells exhibit precisely regulated accessible chromatin dynamics underlying unique transcriptional signatures, and revealed that chromatin priming mechanisms are concentrated in a specific subset of effector memory CD8+ T cells. We pinpointed sets of accessible chromatin sites specifically overrepresented in memory CD8+ T cell already at baseline (i.e. prior to activation), which are enriched for 3D regulatory interactions and specific transcription factor binding sites. Compared to naive CD8+ T cells, memory CD8+ T cells selectively upregulated three key members of these transcription factor families – already at the resting state. Combined CRISPR/Cas9-mediated knock-out of this trio of transcription factors in memory CD8+ T cells caused a loss of epigenetic priming, culminating in an impaired capacity to produce inflammatory molecules essential for CD8+ T cell-mediated immunity. Together, our findings show that memory CD8+ T cells maintain a transcription-permissive chromatin landscape, which is imprinted by cooperating transcription factors to enable the rapid reactivation of inflammatory genes crucial for protective immune responses.
This project has produced a first-in-class, multidimensional epigenomic atlas of human CD8+ T cell activation and memory recall at both the bulk (cell population-wide) and single cell levels. For the first time, we integrate the potentially instructive information encoded by 3D genome topology, (2) putative single cell heterogeneity and (3) the dynamics of the transcriptome-epigenome crosstalk in individual memory CD8+ T cells. Combined, my findings show that the functional capacity of memory T cells ultimately resides within their epigenome, which is instructed by dedicated transcription factors and primes genes essential for protective immune responses. These findings underscore the transformative potential of the multi-dimensional epigenomics strategy employed in this study to unravel fundamental immunological mechanisms.
More specifically, this project has produced the following key outcomes:
• A unique technological platform that can be used to molecularly dissect other populations of (immune) cells in (patho)physiological processes. This includes improved genome-wide approaches for interrogating transcriptome and epigenome dynamics at both the bulk (cell population-wide) and single-cell levels, compatible with low input cell numbers (as few as 10K). In addition, we have optimized CRISPR/Cas9-based assays for targeted (epi)genome perturbation in primary human immune cells.
• First-in-class high-resolution epigenomics datasets of primary human naive and memory CD8+ T cells, which will serve as important resources for follow-up research on lymphocyte biology in health and disease.
• Integrated analysis of primary human naive and memory CD8+ T cells in both resting and activated states, exposing the molecular circuits linked to T cell activation and memory recall.
• Multimodal single cell analysis of primary human naive and memory CD8+ T cells, revealing the transcriptome-epigenome crosstalk dynamics in individual cells.
• Mechanistic insights into how dedicated transcription factors cooperate to imprint epigenetic priming for memory recall in human T cells.
Collectively, these results provide a strategic foundation for (1) the rational design of follow up studies aimed at elucidating how aberrant epigenetic rewiring underpins T cell dysfunction in patients with cancer or immune-related disease; and (2) the exploration of strategies to modulate epigenetic programs in T cells to benefit human health, thereby paving the way for improved vaccines and T cell-based immunotherapies.
More specifically, this project has produced the following key outcomes:
• A unique technological platform that can be used to molecularly dissect other populations of (immune) cells in (patho)physiological processes. This includes improved genome-wide approaches for interrogating transcriptome and epigenome dynamics at both the bulk (cell population-wide) and single-cell levels, compatible with low input cell numbers (as few as 10K). In addition, we have optimized CRISPR/Cas9-based assays for targeted (epi)genome perturbation in primary human immune cells.
• First-in-class high-resolution epigenomics datasets of primary human naive and memory CD8+ T cells, which will serve as important resources for follow-up research on lymphocyte biology in health and disease.
• Integrated analysis of primary human naive and memory CD8+ T cells in both resting and activated states, exposing the molecular circuits linked to T cell activation and memory recall.
• Multimodal single cell analysis of primary human naive and memory CD8+ T cells, revealing the transcriptome-epigenome crosstalk dynamics in individual cells.
• Mechanistic insights into how dedicated transcription factors cooperate to imprint epigenetic priming for memory recall in human T cells.
Collectively, these results provide a strategic foundation for (1) the rational design of follow up studies aimed at elucidating how aberrant epigenetic rewiring underpins T cell dysfunction in patients with cancer or immune-related disease; and (2) the exploration of strategies to modulate epigenetic programs in T cells to benefit human health, thereby paving the way for improved vaccines and T cell-based immunotherapies.