Jul 2026· Journal of Immunology· Vol 215· 0 citations
TL;DR
The results demonstrate that the antigen dependence of Tpex/TXp depends on context, and a subset may survive in the absence of further antigen stimulation, which has important implications for maintaining immunity to chronic infections and cancer.
Abstract
Antigen experienced T cell subsets are heterogenous, and can be described based on their phenotype, function, and migration properties. We wished to better understand how antigen stimulation regulates the trafficking, survival, differentiation and developmental plasticity of T cell subsets.
We developed a new mouse model that allows for the in vivo excision of the P14 TCR on a defined fraction of antigen-specific CD8 T cells. Specifically, we engineered a novel tamoxifen-inducible TCR-knockout transgenic mouse using a rAAV6 vector encoding for the floxed P14 TCR gene. This model allows for permanent cessation of antigen stimulation on select T cells at time points of our choosing.
After acute infection with LCMV Armstrong, elimination of the TCR on established memory CD8 T cells maintained stable subset composition, but resulted in detectable downregulation of exhaustion markers, indicating that TCR sensing occurs among steady-state memory T cells. TCR deletion during chronic infections (LCMV Minnesota and LCMV Cl13 ± aCD4 depletion) affected both the population structure and phenotype. Terminal (Tex/TXt), as well as progenitor exhausted (Tpex/TXp) T cell subsets substantially declined after TCR deletion in persistently viremic infection with LCMV Cl13 + aCD4. However, the loss of Tpex/TXp after TCR deletion was moderated in settings of chronic infections without CD4 T cell depletion, in which most host viral load was more tightly controlled.
These results demonstrate that the antigen dependence of Tpex/TXp depends on context, and a subset may survive in the absence of further antigen stimulation, which has important implications for maintaining immunity to chronic infections and cancer.
Walter Benjamin Program, DFG
Lymphocyte Differentiation and Peripheral Maintenance (LYM)
In chronic infections or cancer, stimulated CD8+ T cells progress along a trajectory towards terminal exhaustion in sites of antigen abundance. However, how these cells respond when antigen recognition is lost has not been concretely elucidated.
The Masopust lab generated a UBC-CreERT2 x P14 fl/fl mouse model that allows for the inducible excision of P14 TCR to precisely answer these questions, which has been used successfully in the setting of acute and chronic infections (unpublished work). Here, we combined this mouse model with the KP-NINJA tumor model (Fitzgerald et. al., 2021) that develop autochthonous lung tumors that progress slowly and express the gp33/H-2Db epitope that is recognized by the P14 TCR.
Preliminary results showed successful infiltration and activation of these transgenic T cells in the KP-NINJA tumor microenvironment as well as accumulation within the tumor-draining lymph node following adoptive transfer. After three weeks of antigen exposure, the TCR was eliminated from 50% of transferred T cells and flow cytometry of the tumor, tumor-draining lymph node and additional tissues was performed one-week post-excision. TCR-negative T cells persisted within solid tumors and draining lymph node, although their phenotype was distinct from co-transferred TCR-positive P14 T cells. TCR-negative cells had decreased PD-1 and TOX expression in the tumor and tumor-draining lymph node and increased CXCR6 and CD127 expression within these tissues.
Ongoing studies will define tumor-specific T cell differentiation and fate after TCR excision, investigate changes to cell localization, and test functional potential. This work will add to our understanding of the development of T cell exhaustion and the plasticity of lineage commitment in cancer to better inform future anti-cancer therapeutics.
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Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Matthew B. Watowich, Jenny Krause, O. Smith et al.· Journal of Immunology· 0 citations
This emerging view frames exhaustion as a context-dependent extension of the memory program rather than its collapse, and highlights how these insights can inform new approaches to manipulate T cell fate for therapeutic benefit.
Daniel T. Utzschneider, Stephen J. Turner· Trends in immunology· 0 citations
While intestinal epithelial cells (IECs) upregulate MHCII during inflammation and can serve as non-professional antigen-presenting cells, their specific contribution to pathogen-specific CD4+ T cell immunity and immunological memory formation remains poorly defined.
We utilized Citrobacter rodentium infection (murine model of EPEC) with single-cell RNA-seq, engineered bacteria expressing model antigens in distinct compartments, and proximity-labeling approaches. Conditional IEC-specific MHCII deletion and inducible post-infection deletion strategies assessed functional requirements for CD4+ T cell responses.
C. rodentium triggered sustained MHCII expression across all IEC lineages persisting >2 months post-clearance. IEC MHCII proved essential for pathogen-specific CD4+ TRM formation–conditional knockout shifted CD4+ T cell fate from TRM to TCM populations. scRNA-seq revealed MHCII deletion induced aberrant expression of central memory genes (Tcf7, Lef1, Bcl6) in residual TRM cells, while WT TRM showed robust TCR activation signatures (Fos, Jun, Jund) and Runx3 expression. Type III secretion-delivered cytosolic antigens drove TRM via autophagy-dependent IEC processing, whereas surface antigens promoted TFH differentiation in lymphoid tissues. Proximity labeling confirmed direct, MHCII-dependent IEC-CD4+ TRM synapses, particularly with CD103+ subsets.
We establish IECs as orchestrators of mucosal memory, demonstrating that IEC-MHCII actively suppresses TCM programming while promoting TRM differentiation through sustained TCR signaling. This antigen compartmentalization paradigm redefines CD4+ fate decisions–cytoplasmic antigen targeting via epithelial presentation could selectively generate protective TRM. These mechanisms illuminate IBD pathogenesis where dysregulated epithelial antigen presentation may disrupt TRM/TCM balance, perpetuating chronic inflammation.
F30DK135345
Mucosal and Regional Immunology (MUC)
C. Wilson, Casey T. Weaver, C. Zindl· Journal of Immunology· 0 citations
The establishment of immunological memory is crucial for durable antiviral immunity and underlies effective vaccine design in enhancing memory responses. The retinoic acid receptor—related orphan receptor α(RORα), a ligand-regulated transcription factor of the nuclear receptor superfamily, has been implicated in CD8+ T cell immunity, yet its role remains poorly defined.
To investigate this, we integrated genomic approaches with both genetic and pharmacologic perturbations of RORα to examine its role in CD8+ T cell development during antiviral responses.
RORα-deficient mice infected with acute lymphocytic choriomeningitis virus (LCMV) exhibited fewer virus-specific CD8+ T cells and higher splenic viral titers at day 5 post-infection than wild-type controls. Despite reduced numbers, RORα-deficient CD8+ T cells showed enhanced cytotoxic potential, indicating that RORα modulates effector programming rather than overall functional capacity. RORα deficiency also led to increased migration and redistribution of CD8+ T cells into non-lymphoid compartments, particularly the liver and adipose tissues, and promoted the development of memory precursors (MP), peripheral memory (TPM), and tissue-resident memory (TRM) populations. Single-cell RNA-seq and ATAC-seq revealed enrichment of transcriptional and chromatin programs linked to MP and TRM gene signatures, including elevated expression of Tcf7 and Runx family members, alongside reduced accessibility at loci governing terminal effector differentiation. Pharmacologic inhibition of RORα with the RORα-selective inverse agonist, SR3335, recapitulated the knockout phenotype, favoring memory CD8+ T cell differentiation over terminal effectors.
Collectively, our data reveal distinct programming of CD8 T effector versus memory populations attributable to RORα activity. As a ligand-regulated transcription factor, RORα may be a promising target to enhance CD8+ T cell memory responses for improved vaccines and cell-based immunotherapies.
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Immune Response Regulation: Molecular Mechanisms (IRM)
Jonathan Chuck, Sean Campbell, Anna Schell et al.· Journal of Immunology· 0 citations
Following viral infection, naïve CD8+ T cells differentiate into effector and memory populations that eliminate infected cells and establish long-term immunity. Yet, the regulatory mechanisms directing the differentiation toward a memory fate remain unclear. Here, we aim to delineate the differentiation trajectories of antigen-specific human CD8+ T cells following cognate antigen stimulation, and to identify an early-memory subset with a durable memory potential.
Sequential peripheral blood samples were collected from healthy donors, vaccinated with BNT162b2 vaccine who subsequently experienced breakthrough infection, allowing longitudinal profiling of antigen-specific responses. Single-cell RNA sequencing integrated with DNA-barcoded MHC-I multimers was used to identify SARS-CoV-2 spike-specific CD8+ T cells. To further asses epigenetic regulation, parallel single-nucleus RNA and ATAC sequencing in a murine influenza A infection model was conducted.
Regulatory network and trajectory analyses revealed a distinct early-memory population, uniquely exhibiting transcription factor FOXP1, within a spike-specific effector CD8 cluster. The analogous population in the murine model exhibited increased chromatin accessibility and FOXP1 motif enrichment. Phenotypically, FOXP1+ early-memory population showed a CD45RA+ effector memory T (TEMRA)-like surface expression with a loss of CCR7 expression while uniquely expressing CD127, suggesting functional heterogeneity within the TEMRA population. Functionally, FOXP1 knockdown reduced the expression level of TCF-1 in TCR-stimulated CD8+ T cells, implicating the role of FOXP1 in maintaining T-cell stemness.
Our results identify that the CD127+CD45RA+CCR7⁻CD8+ T cell subset represents an early-memory population with the potential to generate durable memory responses, and FOXP1 is a key regulator of this population. Our findings provide mechanistic insights into transcriptional and epigenetic regulation of memory CD8+ T-cell generation.
N/A
Lymphocyte Differentiation and Peripheral Maintenance (LYM)
So-Young Kim, J. Koh, Eui-Cheol Shin· Journal of Immunology· 0 citations
Tumor-specific CD8 T cells (TST) are often dysfunctional and fail to halt cancer progression. While chronic antigen stimulation is required to maintain the dysfunctional state , how T cell dysfunction initially arises in tumors was less clear. We recently demonstrated that TST lose effector function within hours of activation in tumor-bearing hosts; epigenetic and transcriptional analysis suggested that TST failed to activate inflammation-associated TF, resulting in loss of effector function. We therefore tested whether pathogen-associated molecular patterns (PAMP) signaling during initial priming could rescue T cell dysfunction in tumor-bearing hosts.
We used a preclinical liver cancer model in which tumors express the cognate antigen for transferred TCR-transgenic CD8 T cells (TCR-TAG), while antigen-free LM was used to provide PAMP signaling. Outcomes assessed were tumor outgrowth and T cell differentiation and function, determined by flow cytometry, RNA-SEQ, and ATAC-SEQ.
TCR-TAG adoptively-transferred into mice with advanced liver tumors rapidly became dysfunctional: unable to produce effector cytokines (TNF alpha, IFN gamma) or cytotoxic molecules (granzyme B; GZMB). Remarkably, a single LM treatment rescued effector function; TCR-TAG expressed cytokines and GZMB, resulting in remarkable tumor reduction and prolonged survival. LM-induced PAMP signaling reprogrammed TST, suppressing stem/memory-associated transcription factors (TF), such as TCF1, and activating STAT-family TF. Importantly, LM treatment rescued TST function only when delivered within 24-48h after initial priming.
Our findings demonstrate that pathogen-driven inflammatory signaling during priming is required for TST cytotoxicity. However, LM/PAMP can cause toxicity, thus we are testing genome engineering strategies to activate PAMP-associated TF in TST, independent of LM/PAMP, to generate potent tumor-targeting T cells for adoptive immunotherapy.
2T32CA009592 Microenvironment Influences in Cancer Training Program
Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
M. Wolf, M. Philip· Journal of Immunology· 0 citations
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