Sci Adv. 2025 Sep 26;11(39):eadw6755. doi: 10.1126/sciadv.adw6755. Epub 2025 Sep 26.

ABSTRACT

Glycans regulate cellular function, yet how aging affects the glycocalyx remains unclear. Here, we investigate changes in immune cell glycocalyx with age and find that α2,6-linked sialic acid, a glycan epitope associated with inhibitory signaling, is down-regulated in T cells from old animals. This reduction is tightly correlated with age-associated accumulation of effector T cells, which have little to no α2,6-linked sialic acid. To understand how α2,6-linked sialic acid affects T cell physiology, we generated a mouse model with T cell-specific deletion of sialyltransferase gene St6gal1. The lack of α2,6-linked sialic acid leads to reduced responsiveness in naïve T cells, leading to impaired T cell responses against Listeria monocytogenes infection and tumor growth. PD-1 pathway blockade partially restores St6gal1-deficient T cells' ability to control tumor growth. These findings suggest that α2,6-linked sialic acid is critical for maintaining long-term T cell responsiveness, and its loss may contribute to decreased T cell function with age.

PMID:41004592 | DOI:10.1126/sciadv.adw6755

bioRxiv [Preprint]. 2025 Sep 3:2025.08.29.673118. doi: 10.1101/2025.08.29.673118.

ABSTRACT

Regulatory T cells (Tregs) are critical guardians of immune homeostasis that must operate in diverse and often inflammatory conditions. However, the mechanisms that Tregs use to maintain their stability and function, especially in response to the stresses of distinct microenvironments, remain incompletely understood. Previous work identified the repressive chromatin modification histone 3 lysine 27 trimethylation (H3K27me3) as a rheostat for Treg function. Here, we find that loss of H3K27me3 in Tregs activates the tumor suppressor p53. Stabilization of p53 using the MDM2 inhibitor Nutlin-3 protected Tregs from losing their master transcription factor Foxp3 in vitro when cultured with the Th17 cytokines IL-6 and IL-1β, while p53 deficiency rendered Tregs more prone to Foxp3 loss. Treg-specific p53 deficiency resulted in accumulation of cells that had lost Foxp3 expression ("ex-Tregs") and reduction of suppressive markers on Tregs specifically in the colon. Additionally, these mice exhibited inflammation in the colon at homeostasis and increased severity of induced colitis. These results demonstrate a specific role for p53 in the maintenance of Treg stability in Th17-polarizing environments and present a possible target for improving Treg-based immunotherapies for diseases defined by intestinal inflammation, such as inflammatory bowel disease (IBD).

PMID:40950180 | PMC:PMC12424803 | DOI:10.1101/2025.08.29.673118

Infect Immun. 2025 Sep 5:e0034325. doi: 10.1128/iai.00343-25. Online ahead of print.

ABSTRACT

Listeria monocytogenes is a facultative intracellular pathogen that has garnered attention as a potential cancer therapeutic due to its ability to induce robust cell-mediated immunity. To ensure safe clinical administration, deletion of certain genes, such as actA, has been used to attenuate L. monocytogenes-based vaccine strains while preserving immunogenicity. Here we explored the potential inclusion of a purA gene deletion to enhance the development of L. monocytogenes-based immunotherapy. The purA gene encodes adenylosuccinate synthetase, which catalyzes the conversion of inosine monophosphate to adenosine monophosphate (AMP), a critical step in the de novo biosynthesis of purines. Since nucleotide biosynthesis is critical for the survival and pathogenesis of many bacterial pathogens, we examined the requirements of L. monocytogenes AMP synthesis in tissue culture and animal infection models. The purA mutants were able to escape from phagosomes of bone marrow-derived macrophages but were highly defective for subsequent growth in the host cell cytosol. In contrast to wild-type bacteria, the mutants did not grow in human serum or sheep blood. In intravenously infected mice, purA mutants were highly attenuated, similar to actA mutants, but displayed distinct growth kinetics during the course of infection. Remarkably, the purA mutants exhibited different localization patterns across splenic immune cells and elicited a more potent CD8+ T-cell response compared to actA mutants. These results underscore the essentiality of AMP biosynthesis for L. monocytogenes pathogenesis and provide new avenues for developing safe L. monocytogenes-based vaccines and therapeutics.

PMID:40910949 | DOI:10.1128/iai.00343-25

bioRxiv [Preprint]. 2025 Jul 11:2025.07.06.663397. doi: 10.1101/2025.07.06.663397.

ABSTRACT

A burgeoning approach to treat cancer is the pharmacological induction of ferroptotic cell death of tumor cells. However, the impact of disrupting anti-ferroptotic pathways in the broader tumor microenvironment (TME), such as in immune cells, is still undefined and may complicate treatments. Here, we show that Ferroptosis Suppressor Protein 1 (FSP1 /Aifm2 ) is critically required for regulatory T cell (Treg) resistance to ferroptosis and their immunosuppressive function within the TME. Compared to other canonical ferroptosis regulators such as GPX4, GCH1, and NRF2, only FSP1 was induced upon T cell activation. Deletion of Aifm2 in all T cells, or Tregs specifically, enhanced tumor control by selectively disrupting Treg immunosuppression within tumors without inciting autoimmune pathology in mice. As opposed to deletion of Gpx4 in all T cells, T cell deletion of Aifm2 did not impair antigen-specific CD8 + T cell responses. These results reveal a unique opportunity for targeting a regulator of ferroptosis that can not only directly target cancer cells, but also simultaneously enhance anti-cancer immune responses without inciting autoimmunity.

PMID:40672318 | PMC:PMC12265646 | DOI:10.1101/2025.07.06.663397

bioRxiv [Preprint]. 2025 Jun 28:2025.06.26.661417. doi: 10.1101/2025.06.26.661417.

ABSTRACT

Cancer cells frequently lose MHC I to evade CD8 + T cell recognition. While Natural Killer (NK) cells are poised to target MHC I-deficient cancer cells, MHC I loss alone is often insufficient to unleash fully effective NK cell responses. Here we show that selective intra-tumoral (IT) ablation of regulatory T (Treg) cells elicited potent antitumor NK cell responses that controlled MHC I-deficient and even MHC I + cancers. Tregs controlled the activation, maturation, and anti-tumor cytotoxic activity of NK cells within the tumor microenvironment. Mechanistically, IT Tregs prevented the cDC2-dependent induction of IL-2 production by CD4 + Tconv cells that was necessary for NK cell activation. Systemically administered antibodies that selectively depleted IT Tregs similarly empowered NK- dependent tumor control. These findings expand the breadth of Treg-mediated cancer immunosuppression to encompass antitumor NK cells and suggest that targeting Tregs in tumors can control CD8 + T cell-resistant cancers.

ONE SENTENCE SUMMARY: IT Treg ablation drives NK cell tumor control via CD4 + Tconv-derived IL-2, eliminating MHC I+/MHC I- cancers without systemic toxicity.

PMID:40667047 | PMC:PMC12262373 | DOI:10.1101/2025.06.26.661417

bioRxiv [Preprint]. 2025 Jun 30:2025.06.26.661786. doi: 10.1101/2025.06.26.661786.

ABSTRACT

A fundamental principle of immune responses is that innate immunity promotes adaptive immunity, but in the context of cancer immunity, the importance of innate receptor signaling is poorly defined. To study how Toll-like receptor (TLR) signaling contributes to cancer immunity, we used an attenuated strain of Listeria monocytogenes (Lm) that inhibits tumor growth in mice. We found that Lm stimulation of TLR2, but not TLR5 or TLR9, was essential for tumor control. As expected, TLR2 supported the priming of Lm-specific T cells in lymphoid tissues. However, TLR2 signaling in innate immune cells within tumors also destabilized Foxp3 expression in regulatory T cells (Tregs), a function that was not mediated by other TLRs. Reduced Treg function promoted tumor antigen cross-presentation by DC1s to enhance the functionality of recalled tumor-specific CD8+ T cells directly within tumors. These findings reveal a unique capacity for TLR2 signaling to diminish immunosuppression and promote cancer immunity.

PMID:40631163 | PMC:PMC12236835 | DOI:10.1101/2025.06.26.661786

Immunity. 2025 Jun 3:S1074-7613(25)00194-3. doi: 10.1016/j.immuni.2025.05.001. Online ahead of print.

ABSTRACT

Bacteria engineered to express tumor antigens as cancer vaccines have produced mixed results. Here, we used an attenuated strain of Listeria monocytogenes (ΔactA, Lm) that lacks tumor antigens to examine the immune response to Lm itself in tumor-bearing mice following intravenous (i.v.), intratumoral (i.t.), or combined i.v. + i.t. Lm delivery. Unexpectedly, i.t. Lm alone recruited neutrophils to tumors, which became immunosuppressive, provided an intracellular reservoir for long-term persistence of Lm in tumors, and promoted tumor growth. In contrast, prior i.v. Lm administration generated anti-Lm cytotoxic CD8+ T cells that infiltrated tumors upon i.t. Lm delivery. These Lm-specific CD8+ T cells control tumors by inducing cancer cell apoptosis, limiting cancer cell proliferation, and enhancing tumor antigen cross-presentation to tumor-specific T cells. Thus, an anti-Lm CD8+ T cell response against Lm-colonized tumors can control cancer, offering a paradigm for cancer immunotherapy that leverages systemic CD8+ T cell immunity targeting i.t. bacteria.

PMID:40480220 | DOI:10.1016/j.immuni.2025.05.001

bioRxiv [Preprint]. 2025 Jan 28:2024.02.15.580555. doi: 10.1101/2024.02.15.580555.

ABSTRACT

Bacteria engineered to express tumor antigens as a cancer vaccine have yielded mixed results. Here, we utilized an attenuated strain of Listeria monocytogenes ( ΔactA, Lm ) that does not express tumor antigen to explore the immunological response to Listeria itself in the context of intravenous (IV), intratumoral (IT), or a combination of IV+IT administration into tumor-bearing mice. Unexpectedly, we found that Lm persisted in tumors of immune competent mice, regardless of the administration route. While IT Lm alone led to the recruitment of immunosuppressive immune cells that promoted tumor growth, IV Lm followed by IT Lm controlled tumor growth. IV Lm vaccination generated a pool of anti- Lm cytotoxic CD8 T cells that killed Lm -infected non-tumor cells to control tumor growth both indirectly, by limiting cancer cell proliferation, and directly, by enhancing tumor-specific T cell responses. Our findings reveal a differential impact of IT Lm administration on tumor progression that depends on the presence of anti- Lm CD8 T cells, which alone are sufficient to promote therapeutic efficacy.

PMID:39975413 | PMC:PMC11838217 | DOI:10.1101/2024.02.15.580555

Cell Rep. 2024 Sep 24;43(9):114724. doi: 10.1016/j.celrep.2024.114724. Epub 2024 Sep 11.

ABSTRACT

The immunosuppressive function of regulatory T (Treg) cells is essential for maintaining immune homeostasis. Enhancer of zeste homolog 2 (EZH2), a histone H3 lysine 27 (H3K27) methyltransferase, plays a key role in maintaining Treg cell function upon CD28 co-stimulation, and Ezh2 deletion in Treg cells causes autoimmunity. Here, we assess whether increasing H3K27me3 levels, by using an Ezh2Y641F gain-of-function mutation, will improve Treg cell function. We find that Treg cells expressing Ezh2Y641F display an effector Treg phenotype, are poised for improved homing to organ tissues, and can accelerate remission from autoimmunity. The H3K27me3 landscape and transcriptome of naive Ezh2Y641F Treg cells exhibit a redistribution of H3K27me3 modifications that recapitulates the gene expression profile of activated Ezh2WT Treg cells after CD28 co-stimulation. Altogether, increased H3K27me3 levels promote the differentiation of effector Treg cells that can better suppress autoimmunity.

PMID:39264807 | DOI:10.1016/j.celrep.2024.114724

Nat Immunol. 2024 Aug 27. doi: 10.1038/s41590-024-01937-3. Online ahead of print.

ABSTRACT

T cell antigen receptor (TCR) recognition followed by clonal expansion is a fundamental feature of adaptive immune responses. Here, we present a mass cytometric (CyTOF) approach to track T cell responses by combining antibodies for specific TCR Vα and Vβ chains with antibodies against T cell activation and differentiation proteins in mice. This strategy identifies expansions of CD8+ and CD4+ T cells expressing specific Vβ and Vα chains with varying differentiation states in response to Listeria monocytogenes, tumors and respiratory influenza infection. Expanded T cell populations expressing Vβ chains could be directly linked to the recognition of specific antigens from Listeria, tumor cells or influenza. In the setting of influenza infection, we found that common therapeutic approaches of intramuscular vaccination or convalescent serum transfer altered the TCR diversity and differentiation state of responding T cells. Thus, we present a method to monitor broad changes in TCR use paired with T cell phenotyping during adaptive immune responses.

PMID:39191945 | DOI:10.1038/s41590-024-01937-3

Cancer Cell. 2024 Jun 10;42(6):941-942. doi: 10.1016/j.ccell.2024.05.016.

ABSTRACT

Checkpoint blockade immunotherapies, such as anti-programmed death-1 (PD-1), unleash anti-tumor CD8+ T cell responses but may also induce immunosuppressive regulatory T cells (Tregs). In this issue of Cancer Cell, Geels et al. uncover that anti-PD-1 leads to Treg expansion via interleukin-2 (IL-2)-producing CD8+ T cells. Combining anti-PD-1 with anti-ICOSL interrupts this crosstalk, thereby enhancing tumor control.

PMID:38861931 | DOI:10.1016/j.ccell.2024.05.016

bioRxiv [Preprint]. 2024 Apr 10:2024.04.05.588284. doi: 10.1101/2024.04.05.588284.

ABSTRACT

The immunosuppressive function of regulatory T (Treg) cells is essential for maintaining immune homeostasis. Enhancer of zeste homolog 2 (EZH2), a histone H3 lysine 27 (H3K27) methyltransferase, plays a key role in maintaining Treg cell function upon CD28 co-stimulation, and Ezh2 deletion in Treg cells causes autoimmunity. Here we assessed whether increased EZH2 activity in Treg cells would improve Treg cell function. Using an Ezh2 gain-of-function mutation, Ezh2 Y641F , we found that Treg cells expressing Ezh2 Y641F displayed an increased effector Treg phenotype and were poised for improved homing to organ tissues. Expression of Ezh2 Y641F in Treg cells led to more rapid remission from autoimmunity. H3K27me3 profiling and transcriptomic analysis revealed a redistribution of H3K27me3, which prompted a gene expression profile in naïve Ezh2 Y641F Treg cells that recapitulated aspects of CD28-activated Ezh2 WT Treg cells. Altogether, increased EZH2 activity promotes the differentiation of effector Treg cells that can better suppress autoimmunity.

HIGHLIGHTS: EZH2 function promotes effector differentiation of Treg cells.EZH2 function promotes Treg cell migration to organ tissues.EZH2 function in Treg cells improves remission from autoimmunity.EZH2 function poises naïve Treg cells to adopt a CD28-activated phenotype.

PMID:38645261 | PMC:PMC11030251 | DOI:10.1101/2024.04.05.588284

bioRxiv. 2024 Jan 12:2024.01.11.575237. doi: 10.1101/2024.01.11.575237. Preprint.

ABSTRACT

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 Vα- and Vβ-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 Vβ and Vα chain expressing CD8+ and CD4+ T cells with varying differentiation states in response to Listeria monocytogenes, tumors, and respiratory influenza infection. Expanded Vβ 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.

PMID:38260336 | PMC:PMC10802618 | DOI:10.1101/2024.01.11.575237

Cancer Immunol Res. 2023 Nov 15:OF1. doi: 10.1158/2326-6066.CIR-23-0857. Online ahead of print.

ABSTRACT

Regulatory T cells (Treg) can suppress antitumor immune responses, and their presence in tumors is associated with worse prognoses in most cancers. Strategies to neutralize Treg-mediated suppression in tumors without immune-related adverse events, however, are challenging due to the essential role of Tregs in maintaining immune homeostasis. In this issue, Pinioti and colleagues identify fucosylation as a critical regulator of Treg function in tumors that can be targeted therapeutically without impacting immune homeostasis. See related article by Pinioti et al., p. 1611 (3) .

PMID:37966514 | DOI:10.1158/2326-6066.CIR-23-0857

Immunity. 2023 Jun 27:S1074-7613(23)00260-1. doi: 10.1016/j.immuni.2023.06.003. Online ahead of print.

ABSTRACT

Infiltration of regulatory T (Treg) cells, an immunosuppressive population of CD4+ T cells, into solid cancers represents a barrier to cancer immunotherapy. Chemokine receptors are critical for Treg cell recruitment and cell-cell interactions in inflamed tissues, including cancer, and thus are an ideal therapeutic target. Here, we show in multiple cancer models that CXCR3+ Treg cells were increased in tumors compared with lymphoid tissues, exhibited an activated phenotype, and interacted preferentially with CXCL9-producing BATF3+ dendritic cells (DCs). Genetic ablation of CXCR3 in Treg cells disrupted DC1-Treg cell interactions and concomitantly increased DC-CD8+ T cell interactions. Mechanistically, CXCR3 ablation in Treg cells increased tumor antigen-specific cross-presentation by DC1s, increasing CD8+ T cell priming and reactivation in tumors. This ultimately impaired tumor progression, especially in combination with anti-PD-1 checkpoint blockade immunotherapy. Overall, CXCR3 is shown to be a critical chemokine receptor for Treg cell accumulation and immune suppression in tumors.

PMID:37392735 | DOI:10.1016/j.immuni.2023.06.003

Blood. 2022 Feb 28:blood.2021014557. doi: 10.1182/blood.2021014557. Online ahead of print.

ABSTRACT

Despite advances in the field, chronic graft-vs-host-disease (cGVHD) remains a leading cause of morbidity and mortality following allogenic hematopoietic stem cell transplant. As treatment options remain limited, we tested efficacy of anti-cancer, chromatin modifying enzyme inhibitors in a clinically relevant murine model of cGVHD with bronchiolitis obliterans (BO). We observed that the novel Enhancer of Zeste Homolog 2 (EZH2) inhibitor JQ5, and the BET-bromodomain inhibitor JQ1 each improved pulmonary function, impaired the germinal center (GC) reaction, a prerequisite in cGVHD/BO pathogenesis, and JQ5 reduced EZH2-mediated H3K27me3 in donor T cells. Using conditional EZH2 knockout donor cells we demonstrated that EZH2 is obligatory for the initiation of cGVHD/BO. In a sclerodermatous cGVHD model, JQ5 reduced the severity of cutaneous lesions. To determine how the two drugs could lead to the same physiological improvements while targeting unique epigenetic processes we analyzed the transcriptomes of splenic GCB cells (GCBs) from transplanted mice treated with either drug. Multiple inflammatory and signaling pathways enriched in cGVHD/BO GCBs were reduced by each drug. GCBs from JQ5 but not JQ1 treated mice were enriched for pro-proliferative pathways also seen in GCBs from BM-Only transplanted mice, likely reflecting their underlying biology in the unperturbed state. In conjunction with in vivo data these insights lead us to conclude that epigenetic targeting of the GC is a viable clinical approach for the treatment of cGVHD, and that the EZH2 inhibitor JQ5 and the BET-bromodomain inhibitor JQ1 demonstrated clinical potential for EZH2i and BETi in patients with cGVHD/BO.

PMID:35226736 | DOI:10.1182/blood.2021014557

Nat Cancer. 2021 Oct;2(10):996-997. doi: 10.1038/s43018-021-00270-0.

NO ABSTRACT

PMID:35121885 | DOI:10.1038/s43018-021-00270-0

MHC-II neoantigens shape tumour immunity and response to immunotherapy.

Nature. 2019 Oct 23;:

Authors: Alspach E, Lussier DM, Miceli AP, Kizhvatov I, DuPage M, Luoma AM, Meng W, Lichti CF, Esaulova E, Vomund AN, Runci D, Ward JP, Gubin MM, Medrano RFV, Arthur CD, White JM, Sheehan KCF, Chen A, Wucherpfennig KW, Jacks T, Unanue ER, Artyomov MN, Schreiber RD

Abstract
The ability of the immune system to eliminate and shape the immunogenicity of tumours defines the process of cancer immunoediting1. Immunotherapies such as those that target immune checkpoint molecules can be used to augment immune-mediated elimination of tumours and have resulted in durable responses in patients with cancer that did not respond to previous treatments. However, only a subset of patients benefit from immunotherapy and more knowledge about what is required for successful treatment is needed2-4. Although the role of tumour neoantigen-specific CD8+ T cells in tumour rejection is well established5-9, the roles of other subsets of T cells have received less attention. Here we show that spontaneous and immunotherapy-induced anti-tumour responses require the activity of both tumour-antigen-specific CD8+ and CD4+ T cells, even in tumours that do not express major histocompatibility complex (MHC) class II molecules. In addition, the expression of MHC class II-restricted antigens by tumour cells is required at the site of successful rejection, indicating that activation of CD4+ T cells must also occur in the tumour microenvironment. These findings suggest that MHC class II-restricted neoantigens have a key function in the anti-tumour response that is nonoverlapping with that of MHC class I-restricted neoantigens and therefore needs to be considered when identifying patients who will most benefit from immunotherapy.

PMID: 31645760 [PubMed - as supplied by publisher]

Enhanced adaptive immune responses in lung adenocarcinoma through natural killer cell stimulation.

Proc Natl Acad Sci U S A. 2019 Aug 13;:

Authors: Schmidt L, Eskiocak B, Kohn R, Dang C, Joshi NS, DuPage M, Lee DY, Jacks T

Abstract
Natural killer (NK) cells inhibit tumor development in mouse models and their presence in tumors correlates with patient survival. However, tumor-associated NK cells become dysfunctional; thus, stimulation of NK cells in cancer is emerging as an attractive immunotherapeutic strategy. In a mouse model of lung adenocarcinoma, NK cells localized to tumor stroma with immature phenotypes and low functional capacity. To test their responsiveness within established disease, we engineered a system for inducible expression of activating ligands in tumors. After stimulation, NK cells localized inside tumors, with increased cytokine production capacity. Strikingly, T cells were also recruited to tumors in an NK cell-dependent manner, and exhibited higher functionality. In neoantigen-expressing tumors, NK cell stimulation enhanced the number and function of tumor-specific T cells and, in long-term settings, reduced tumor growth. Thus, even in established disease NK cells can be activated to contribute to antitumor immunity, supporting their potential as an important target in cancer immunotherapy.

PMID: 31409707 [PubMed - as supplied by publisher]

Treg programming and therapeutic reprogramming in cancer.

Immunology. 2019 Jul;157(3):198-209

Authors: Moreno Ayala MA, Li Z, DuPage M

Abstract
Overcoming the immunosuppressive tumour microenvironment is the major challenge impeding cancer immunotherapy today. Regulatory T-cells (Tregs) are prevalent in nearly all cancers and, as immunosuppressive regulators of immune responses, they are the principal opponents of cancer immunotherapy. However, disabling Tregs systemically causes severe autoimmune toxicity, hastening the need for more selective methods to target intratumoural Tregs. In this review, we discuss a burgeoning new modality to specifically target tumour-infiltrating Tregs (TI-Tregs) by reprogramming their functionality from immunosuppressive to immune stimulatory within tumours. As the basis for therapeutic selectivity of TI-Tregs, we will focus on the defining features of Tregs within cancer: their highly activated state controlled by the engagement of key surface receptors, their distinct metabolic programme, and their unique transcriptional programme. By identifying proteins and pathways that distinguish TI-Tregs from other Tregs in the body, as well as from the beneficial antitumour effector T-cells within tumours, we highlight mechanisms to selectively reprogramme TI-Tregs for the treatment of cancer.

PMID: 30866047 [PubMed - in process]