InVivoMAb anti-mouse CD28
Product Details
The 37.51 monoclonal antibody reacts with mouse CD28, a 45 kDa costimulatory receptor and a member of the Ig superfamily. CD28 is expressed by thymocytes, most peripheral T cells, and NK cells. CD28 is a receptor for CD80 (B7-1) and CD86 (B7-2). Signaling through CD28 augments IL-2 and IL-2 receptor expression as well as cytotoxicity of CD3-activated T cells. The 37.51 antibody has been shown to stimulate the proliferation and cytokine production by activated T and NK cells and provide a costimulatory signal for CTL induction.Specifications
| Isotype | Syrian Hamster IgG2 |
|---|---|
| Recommended Isotype Control(s) | InVivoMAb polyclonal Syrian hamster IgG |
| Recommended Dilution Buffer | InVivoPure pH 6.0T Dilution Buffer |
| Conjugation | This product is unconjugated. Conjugation is available via our Antibody Conjugation Services. |
| Immunogen | C57BL/6 mouse T cell lymphoma EL-4 cells |
| Reported Applications |
in vitro T cell stimulation/activation in vivo CD28 blockade |
| Formulation |
PBS, pH 6.0 0.01% Tween Contains no stabilizers or preservatives |
| Endotoxin |
<2EU/mg (<0.002EU/μg) Determined by LAL gel clotting assay |
| Purity |
>95% Determined by SDS-PAGE |
| Sterility | 0.2 µm filtration |
| Production | Purified from cell culture supernatant in an animal-free facility |
| Purification | Protein G |
| RRID | AB_1107624 |
| Molecular Weight | 150 kDa |
| Storage | The antibody solution should be stored at the stock concentration at 4°C. Do not freeze. |
Recommended Products
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Recommended Isotype Control(s)
InVivoMAb polyclonal Syrian hamster IgG
-
Recommended Dilution Buffer
InVivoPure pH 6.0T Dilution Buffer
in vitro T cell stimulation/activation
Lacher, S. M., et al. (2018). "NF-kappaB inducing kinase (NIK) is an essential post-transcriptional regulator of T-cell activation affecting F-actin dynamics and TCR signaling" J Autoimmun 94: 110-121. PubMed
NF-kappaB inducing kinase (NIK) is the key protein of the non-canonical NF-kappaB pathway and is important for the development of lymph nodes and other secondary immune organs. We elucidated the specific role of NIK in T cells using T-cell specific NIK-deficient (NIK(DeltaT)) mice. Despite showing normal development of lymphoid organs, NIK(DeltaT) mice were resistant to induction of CNS autoimmunity. T cells from NIK(DeltaT) mice were deficient in late priming, failed to up-regulate T-bet and to transmigrate into the CNS. Proteomic analysis of activated NIK(-/-) T cells showed de-regulated expression of proteins involved in the formation of the immunological synapse: in particular, proteins involved in cytoskeleton dynamics. In line with this we found that NIK-deficient T cells were hampered in phosphorylation of Zap70, LAT, AKT, ERK1/2 and PLCgamma upon TCR engagement. Hence, our data disclose a hitherto unknown function of NIK in T-cell priming and differentiation.
in vitro T cell stimulation/activation
Wendland, K., et al. (2018). "Retinoic Acid Signaling in Thymic Epithelial Cells Regulates Thymopoiesis" J Immunol 201(2): 524-532. PubMed
Despite the essential role of thymic epithelial cells (TEC) in T cell development, the signals regulating TEC differentiation and homeostasis remain incompletely understood. In this study, we show a key in vivo role for the vitamin A metabolite, retinoic acid (RA), in TEC homeostasis. In the absence of RA signaling in TEC, cortical TEC (cTEC) and CD80(lo)MHC class II(lo) medullary TEC displayed subset-specific alterations in gene expression, which in cTEC included genes involved in epithelial proliferation, development, and differentiation. Mice whose TEC were unable to respond to RA showed increased cTEC proliferation, an accumulation of stem cell Ag-1(hi) cTEC, and, in early life, a decrease in medullary TEC numbers. These alterations resulted in reduced thymic cellularity in early life, a reduction in CD4 single-positive and CD8 single-positive numbers in both young and adult mice, and enhanced peripheral CD8(+) T cell survival upon TCR stimulation. Collectively, our results identify RA as a regulator of TEC homeostasis that is essential for TEC function and normal thymopoiesis.
in vitro T cell stimulation/activation
Ron-Harel, N., et al. (2016). "Mitochondrial Biogenesis and Proteome Remodeling Promote One-Carbon Metabolism for T Cell Activation" Cell Metab 24(1): 104-117. PubMed
Naive T cell stimulation activates anabolic metabolism to fuel the transition from quiescence to growth and proliferation. Here we show that naive CD4(+) T cell activation induces a unique program of mitochondrial biogenesis and remodeling. Using mass spectrometry, we quantified protein dynamics during T cell activation. We identified substantial remodeling of the mitochondrial proteome over the first 24 hr of T cell activation to generate mitochondria with a distinct metabolic signature, with one-carbon metabolism as the most induced pathway. Salvage pathways and mitochondrial one-carbon metabolism, fed by serine, contribute to purine and thymidine synthesis to enable T cell proliferation and survival. Genetic inhibition of the mitochondrial serine catabolic enzyme SHMT2 impaired T cell survival in culture and antigen-specific T cell abundance in vivo. Thus, during T cell activation, mitochondrial proteome remodeling generates specialized mitochondria with enhanced one-carbon metabolism that is critical for T cell activation and survival.
in vitro T cell stimulation/activation
Gu, A. D., et al. (2015). "A critical role for transcription factor Smad4 in T cell function that is independent of transforming growth factor beta receptor signaling" Immunity 42(1): 68-79. PubMed
Transforming growth factor-beta (TGF-beta) suppresses T cell function to maintain self-tolerance and to promote tumor immune evasion. Yet how Smad4, a transcription factor component of TGF-beta signaling, regulates T cell function remains unclear. Here we have demonstrated an essential role for Smad4 in promoting T cell function during autoimmunity and anti-tumor immunity. Smad4 deletion rescued the lethal autoimmunity resulting from transforming growth factor-beta receptor (TGF-betaR) deletion and compromised T-cell-mediated tumor rejection. Although Smad4 was dispensable for T cell generation, homeostasis, and effector function, it was essential for T cell proliferation after activation in vitro and in vivo. The transcription factor Myc was identified to mediate Smad4-controlled T cell proliferation. This study thus reveals a requirement of Smad4 for T-cell-mediated autoimmunity and tumor rejection, which is beyond the current paradigm. It highlights a TGF-betaR-independent role for Smad4 in promoting T cell function, autoimmunity, and anti-tumor immunity.
in vitro T cell stimulation/activation
Choi, Y. S., et al. (2015). "LEF-1 and TCF-1 orchestrate TFH differentiation by regulating differentiation circuits upstream of the transcriptional repressor Bcl6" Nat Immunol 16(9): 980-990. PubMed
Follicular helper T cells (TFH cells) are specialized effector CD4(+) T cells that help B cells develop germinal centers (GCs) and memory. However, the transcription factors that regulate the differentiation of TFH cells remain incompletely understood. Here we report that selective loss of Lef1 or Tcf7 (which encode the transcription factor LEF-1 or TCF-1, respectively) resulted in TFH cell defects, while deletion of both Lef1 and Tcf7 severely impaired the differentiation of TFH cells and the formation of GCs. Forced expression of LEF-1 enhanced TFH differentiation. LEF-1 and TCF-1 coordinated such differentiation by two general mechanisms. First, they established the responsiveness of naive CD4(+) T cells to TFH cell signals. Second, they promoted early TFH differentiation via the multipronged approach of sustaining expression of the cytokine receptors IL-6Ralpha and gp130, enhancing expression of the costimulatory receptor ICOS and promoting expression of the transcriptional repressor Bcl6.
in vitro T cell stimulation/activation
Xu, H., et al. (2015). "Regulation of bifurcating B cell trajectories by mutual antagonism between transcription factors IRF4 and IRF8" Nat Immunol . PubMed
Upon recognition of antigen, B cells undertake a bifurcated response in which some cells rapidly differentiate into plasmablasts while others undergo affinity maturation in germinal centers (GCs). Here we identified a double-negative feedback loop between the transcription factors IRF4 and IRF8 that regulated the initial developmental bifurcation of activated B cells as well as the GC response. IRF8 dampened signaling via the B cell antigen receptor (BCR), facilitated antigen-specific interaction with helper T cells, and promoted antibody affinity maturation while antagonizing IRF4-driven differentiation of plasmablasts. Genomic analysis revealed concentration-dependent actions of IRF4 and IRF8 in regulating distinct gene-expression programs. Stochastic modeling suggested that the double-negative feedback was sufficient to initiate bifurcation of the B cell developmental trajectories.
in vivo CD28 blockade
Rouhani, S. J., et al. (2015). "Roles of lymphatic endothelial cells expressing peripheral tissue antigens in CD4 T-cell tolerance induction" Nat Commun 6: 6771. PubMed
Lymphatic endothelial cells (LECs) directly express peripheral tissue antigens and induce CD8 T-cell deletional tolerance. LECs express MHC-II molecules, suggesting they might also tolerize CD4 T cells. We demonstrate that when beta-galactosidase (beta-gal) is expressed in LECs, beta-gal-specific CD8 T cells undergo deletion via the PD-1/PD-L1 and LAG-3/MHC-II pathways. In contrast, LECs do not present endogenous beta-gal in the context of MHC-II molecules to beta-gal-specific CD4 T cells. Lack of presentation is independent of antigen localization, as membrane-bound haemagglutinin and I-Ealpha are also not presented by MHC-II molecules. LECs express invariant chain and cathepsin L, but not H2-M, suggesting that they cannot load endogenous antigenic peptides onto MHC-II molecules. Importantly, LECs transfer beta-gal to dendritic cells, which subsequently present it to induce CD4 T-cell anergy. Therefore, LECs serve as an antigen reservoir for CD4 T-cell tolerance, and MHC-II molecules on LECs are used to induce CD8 T-cell tolerance via LAG-3.
in vitro T cell stimulation/activation
Rabenstein, H., et al. (2014). "Differential kinetics of antigen dependency of CD4+ and CD8+ T cells" J Immunol 192(8): 3507-3517. PubMed
Ag recognition via the TCR is necessary for the expansion of specific T cells that then contribute to adaptive immunity as effector and memory cells. Because CD4+ and CD8+ T cells differ in terms of their priming APCs and MHC ligands we compared their requirements of Ag persistence during their expansion phase side by side. Proliferation and effector differentiation of TCR transgenic and polyclonal mouse T cells were thus analyzed after transient and continuous TCR signals. Following equally strong stimulation, CD4+ T cell proliferation depended on prolonged Ag presence, whereas CD8+ T cells were able to divide and differentiate into effector cells despite discontinued Ag presentation. CD4+ T cell proliferation was neither affected by Th lineage or memory differentiation nor blocked by coinhibitory signals or missing inflammatory stimuli. Continued CD8+ T cell proliferation was truly independent of self-peptide/MHC-derived signals. The subset divergence was also illustrated by surprisingly broad transcriptional differences supporting a stronger propensity of CD8+ T cells to programmed expansion. These T cell data indicate an intrinsic difference between CD4+ and CD8+ T cells regarding the processing of TCR signals for proliferation. We also found that the presentation of a MHC class II-restricted peptide is more efficiently prolonged by dendritic cell activation in vivo than a class I bound one. In summary, our data demonstrate that CD4+ T cells require continuous stimulation for clonal expansion, whereas CD8+ T cells can divide following a much shorter TCR signal.
in vitro T cell stimulation/activation
Xiao, N., et al. (2014). "The E3 ubiquitin ligase Itch is required for the differentiation of follicular helper T cells" Nat Immunol 15(7): 657-666. PubMed
Follicular helper T cells (T(FH) cells) are responsible for effective B cell-mediated immunity, and Bcl-6 is a central factor for the differentiation of T(FH) cells. However, the molecular mechanisms that regulate the induction of T(FH) cells remain unclear. Here we found that the E3 ubiquitin ligase Itch was essential for the differentiation of T(FH) cells, germinal center responses and immunoglobulin G (IgG) responses to acute viral infection. Itch acted intrinsically in CD4(+) T cells at early stages of T(FH) cell development. Itch seemed to act upstream of Bcl-6 expression, as Bcl-6 expression was substantially impaired in Itch(-/-) cells, and the differentiation of Itch(-/-) T cells into T(FH) cells was restored by enforced expression of Bcl-6. Itch associated with the transcription factor Foxo1 and promoted its ubiquitination and degradation. The defective T(FH) differentiation of Itch(-/-) T cells was rectified by deletion of Foxo1. Thus, our results indicate that Itch acts as an essential positive regulator in the differentiation of T(FH) cells.
in vitro T cell stimulation/activation
Tang, W., et al. (2014). "The oncoprotein and transcriptional regulator Bcl-3 governs plasticity and pathogenicity of autoimmune T cells" Immunity 41(4): 555-566. PubMed
Bcl-3 is an atypical member of the IkappaB family that modulates transcription in the nucleus via association with p50 (NF-kappaB1) or p52 (NF-kappaB2) homodimers. Despite evidence attesting to the overall physiologic importance of Bcl-3, little is known about its cell-specific functions or mechanisms. Here we demonstrate a T-cell-intrinsic function of Bcl-3 in autoimmunity. Bcl-3-deficient T cells failed to induce disease in T cell transfer-induced colitis and experimental autoimmune encephalomyelitis. The protection against disease correlated with a decrease in Th1 cells that produced the cytokines IFN-gamma and GM-CSF and an increase in Th17 cells. Although differentiation into Th1 cells was not impaired in the absence of Bcl-3, differentiated Th1 cells converted to less-pathogenic Th17-like cells, in part via mechanisms involving expression of the RORgammat transcription factor. Thus, Bcl-3 constrained Th1 cell plasticity and promoted pathogenicity by blocking conversion to Th17-like cells, revealing a unique type of regulation that shapes adaptive immunity.
in vitro T cell stimulation/activation
Choi, Y. S., et al. (2013). "Bcl6 expressing follicular helper CD4 T cells are fate committed early and have the capacity to form memory" J Immunol 190(8): 4014-4026. PubMed
Follicular helper CD4 T (Tfh) cells are a distinct type of differentiated CD4 T cells uniquely specialized for B cell help. In this study, we examined Tfh cell fate commitment, including distinguishing features of Tfh versus Th1 proliferation and survival. Using cell transfer approaches at early time points after an acute viral infection, we demonstrate that early Tfh cells and Th1 cells are already strongly cell fate committed by day 3. Nevertheless, Tfh cell proliferation was tightly regulated in a TCR-dependent manner. The Tfh cells still depend on extrinsic cell fate cues from B cells in their physiological in vivo environment. Unexpectedly, we found that Tfh cells share a number of phenotypic parallels with memory precursor CD8 T cells, including selective upregulation of IL-7Ralpha and a collection of coregulated genes. As a consequence, the early Tfh cells can progress to robustly form memory cells. These data support the hypothesis that CD4 and CD8 T cells share core aspects of a memory cell precursor gene expression program involving Bcl6, and a strong relationship exists between Tfh cells and memory CD4 T cell development.
in vivo CD28 blockade
Eberlein, J., et al. (2012). "Multiple layers of CD80/86-dependent costimulatory activity regulate primary, memory, and secondary lymphocytic choriomeningitis virus-specific T cell immunity" J Virol 86(4): 1955-1970. PubMed
The lymphocytic choriomeningitis virus (LCMV) system constitutes one of the most widely used models for the study of infectious disease and the regulation of virus-specific T cell immunity. However, with respect to the activity of costimulatory and associated regulatory pathways, LCMV-specific T cell responses have long been regarded as relatively independent and thus distinct from the regulation of T cell immunity directed against many other viral pathogens. Here, we have reevaluated the contribution of CD28-CD80/86 costimulation in the LCMV system by use of CD80/86-deficient mice, and our results demonstrate that a disruption of CD28-CD80/86 signaling compromises the magnitude, phenotype, and/or functionality of LCMV-specific CD8(+) and/or CD4(+) T cell populations in all stages of the T cell response. Notably, a profound inhibition of secondary T cell immunity in LCMV-immune CD80/86-deficient mice emerged as a composite of both defective memory T cell development and a specific requirement for CD80 but not CD86 in the recall response, while a related experimental scenario of CD28-dependent yet CD80/86-independent secondary CD8(+) T cell immunity suggests the existence of a CD28 ligand other than CD80/86. Furthermore, we provide evidence that regulatory T cells (T(REG)s), the homeostasis of which is altered in CD80/86(-/-) mice, contribute to restrained LCMV-specific CD8(+) T cell responses in the presence of CD80/86. Our observations can therefore provide a more coherent perspective on CD28-CD80/86 costimulation in antiviral T cell immunity that positions the LCMV system within a shared context of multiple defects that virus-specific T cells acquire in the absence of CD28-CD80/86 costimulation.
in vitro T cell stimulation/activation
Angkasekwinai, P., et al. (2010). "Regulation of IL-9 expression by IL-25 signaling" Nat Immunol 11(3): 250-256. PubMed
The physiological regulation of the expression of interleukin (IL)-9, a cytokine traditionally regarded as being T(H)2 associated, remains unclear. Here, we show that IL-9-expressing T cells generated in vitro in the presence of transforming growth factor-beta and IL-4 express high levels of mRNA for IL-17 receptor B (IL-17RB), the receptor for IL-25. Treatment of these cells with IL-25 enhances IL-9 expression in vitro. Moreover, transgenic and retroviral overexpression of IL-17RB in T cells results in IL-25-induced IL-9 production that is IL-4 independent. In vivo, the IL-25-IL-17RB pathway regulates IL-9 expression in allergic airway inflammation. Thus, IL-25 is a newly identified regulator of IL-9 expression.
- Biochemistry and Molecular biology,
- Cancer Research,
- Cell Biology
Dual Ribosome Profiling reveals metabolic limitations of cancer and stromal cells in the tumor microenvironment.
In Nature Communications on 19 May 2025 by Aviles-Huerta, D., Del Pizzo, R., et al.
The tumor microenvironment (TME) influences cancer cell metabolism and survival. However, how immune and stromal cells respond to metabolic stress in vivo, and how nutrient limitations affect therapy, remains poorly understood. Here, we introduce Dual Ribosome Profiling (DualRP) to simultaneously monitor translation and ribosome stalling in multiple tumor cell populations. DualRP reveals that cancer-fibroblast interactions trigger an inflammatory program that reduces amino acid shortages during glucose starvation. In immunocompetent mice, we show that serine and glycine are essential for optimal T cell function and that their deficiency impairs T cell fitness. Importantly, immune checkpoint blockade therapy imposes amino acid restrictions specifically in T cells, demonstrating that therapies create distinct metabolic demands across TME cell types. By mapping codon-resolved ribosome stalling in a cell‑type‑specific manner, DualRP uncovers metabolic crosstalk that shapes translational programs. DualRP thus offers a powerful, innovative approach for dissecting tumor cell metabolic interplay and guiding combined metabolic-immunotherapeutic strategies. © 2025. The Author(s).
- Mus musculus (House mouse),
- Cancer Research,
- Immunology and Microbiology
SPP1 + macrophages cause exhaustion of tumor-specific T cells in liver metastases.
In Nature Communications on 7 May 2025 by Trehan, R., Huang, P., et al.
Functional tumor-specific CD8+ T cells are essential for effective anti-tumor immune response and immune checkpoint inhibitor therapy. Here we show that, compared to other organ sites, primary, metastatic liver tumors in murine models contain a higher number of tumor-specific CD8+ T cells which are also dysfunctional. High-dimensional, multi-omic analysis of patient samples reveals a higher frequency of exhausted tumor-reactive CD8+ T cells and enriched interactions between these cells and SPP1+ macrophages in profibrotic, alpha-SMA rich regions specifically in the liver. Differential pseudotime trajectory inference analysis reveals that extrahepatic signaling promotes an intermediate cell (IC) population in the liver, characterized by co-expression of VISG4, CSF1R, CD163, TGF-βR, IL-6R, and SPP1. Analysis of premetastatic adenocarcinoma patient samples reveals enrichment of this population may predict liver metastasis. These findings suggest a mechanism by which extrahepatic tumors drive liver metastasis by promoting an IC population that inhibits tumor-reactive CD8+ T cell function. © 2025. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.
- Immunology and Microbiology
Alpha-1 Antitrypsin Overexpressing Mesenchymal Stem/Stromal Cells Reverses Type 1 Diabetes via Promoting Treg Function and CD8+T cell exhaustion
Preprint on BioRxiv : the Preprint Server for Biology on 21 April 2025 by Wei, H., Gou, W., et al.
ABSTRACT Mesenchymal stem/stromal cell (MSC) therapy holds great promise as both a therapeutic option and as a biofactory, as cells produce therapeutic proteins to augment their efficacy in disease treatment. This study investigates the therapeutic effects and the mechanistic insights of alpha-1 antitrypsin overexpressing MSCs (AAT-MSCs) in diabetes prevention and treatment. A single infusion of AAT-MSCs not only delayed diabetes onset but reversed new-onset type 1 diabetes (T1D) in the nonobese diabetic (NOD) mice. Using single-cell RNA sequencing, flow cytometry, and functional analyses, we characterized the impact of AAT-MSCs on immune cells, particularly CD4 + and CD8 + T cells, in pancreatic lymph nodes (PLNs) and islets of NOD mice. AAT-MSCs enhanced the immunosuppressive function and the communication of regulatory T cells (Tregs) with other immune cells while reducing the numbers of T helper 1 (Th1) cells and CD8 + cytotoxic T cells. In vitro experiments further confirmed the capacity of AAT-MSCs to promote the proliferation of Tregs, which consequently fostered an exhausted phenotype in CD8 + T cells, thereby facilitating β cell survival and potentially aiding in diabetes remission. Thus, our findings underscore the significant protective effects of AAT-MSCs, delineate their novel mechanistic insight on recipient immune cells, and provide evidence for the clinical application of AAT-MSCs in treating T1D.
- Cancer Research,
- Immunology and Microbiology
A Spatially Distributed Microneedle System for Bioorthogonal T Cell-Guided Cancer Therapy.
In Advanced Science (Weinheim, Baden-Wurttemberg, Germany) on 1 April 2025 by Li, L., Wang, F., et al.
Chimeric antigen receptor (CAR)-T cell therapy represents a promising strategy for cancer treatment. However, the diversity of solid tumor antigens and the poor infiltration of CAR-T cells significantly hinder the efficacy of CAR-T therapies against tumors. Here, a spatially distributed microneedle system (SDMNS) is developed that leverages bioorthogonal reactions to activate and guide endogenous T cells to tumors for effective destruction. The SDMNS consists of two dissolving microneedles, each loaded with complementary bioorthogonal groups and applied separately to lymph nodes and tumor sites. One microneedle loaded with two dibenzocyclooctyne (DBCO)-modified antibodies activates T cells and labels them with bioorthogonal groups in lymph nodes. The other microneedle, containing N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz) for glycometabolic labeling of tumor cells, and the T cell chemotactic factor IP10, is applied directly to the tumor site. The in vivo studies demonstrate that SDMNS effectively directs the migration and infiltration of endogenous activated T cells into the tumors. Through a bioorthogonal click reaction, DBCO-modified T cells conjugate with azide (N3)-modified tumor cells, eliciting robust antitumor immune responses and durable immune memory. The SDMNS offers a novel strategy to overcomes tumor heterogeneity by facilitating the directed migration of endogenous T cells. © 2025 The Author(s). Advanced Science published by Wiley‐VCH GmbH.
- Cancer Research,
- Immunology and Microbiology
VDAC2 loss elicits tumour destruction and inflammation for cancer therapy.
In Nature on 1 April 2025 by Yuan, S., Sun, R., et al.
Tumour cells often evade immune pressure exerted by CD8+ T cells or immunotherapies through mechanisms that are largely unclear1,2. Here, using complementary in vivo and in vitro CRISPR-Cas9 genetic screens to target metabolic factors, we established voltage-dependent anion channel 2 (VDAC2) as an immune signal-dependent checkpoint that curtails interferon-γ (IFNγ)-mediated tumour destruction and inflammatory reprogramming of the tumour microenvironment. Targeting VDAC2 in tumour cells enabled IFNγ-induced cell death and cGAS-STING activation, and markedly improved anti-tumour effects and immunotherapeutic responses. Using a genome-scale genetic interaction screen, we identified BAK as the mediator of VDAC2-deficiency-induced effects. Mechanistically, IFNγ stimulation increased BIM, BID and BAK expression, with VDAC2 deficiency eliciting uncontrolled IFNγ-induced BAK activation and mitochondrial damage. Consequently, mitochondrial DNA was aberrantly released into the cytosol and triggered robust activation of cGAS-STING signalling and type I IFN response. Importantly, co-deletion of STING signalling components dampened the therapeutic effects of VDAC2 depletion in tumour cells, suggesting that targeting VDAC2 integrates CD8+ T cell- and IFNγ-mediated adaptive immunity with a tumour-intrinsic innate immune-like response. Together, our findings reveal VDAC2 as a dual-action target to overcome tumour immune evasion and establish the importance of coordinately destructing and inflaming tumours to enable efficacious cancer immunotherapy. © 2025. The Author(s).
- Cell Biology,
- Immunology and Microbiology
TMEM41B is an endoplasmic reticulum Ca2+ release channel maintaining naive T cell quiescence and responsiveness.
In Cell Discovery on 4 March 2025 by Ma, Y., Wang, Y., et al.
In mammalian cells, endoplasmic reticulum (ER) passively releases Ca2+ under steady state, but channels involved remain elusive. Here, we report that TMEM41B, an ER-resident membrane protein critical for autophagy, lipid metabolism, and viral infection, functions as an ER Ca2+ release channel. Biochemically, purified recombinant TMEM41B forms a concentration-dependent Ca2+ channel in single-channel electrophysiology assays. Cellularly, TMEM41B deficiency causes ER Ca2+ overload, while overexpression of TMEM41B depletes ER Ca2+. Immunologically, ER Ca2+ overload leads to upregulation of IL-2 and IL-7 receptors in naive T cells, which in turn increases basal signaling of JAK-STAT, AKT-mTOR, and MAPK pathways. This dysregulation drives TMEM41B-deficient naive T cells into a metabolically activated yet immunologically naive state. ER Ca2+ overload also downregulates CD5, lowering the activation threshold of TMEM41B-deficient T cells and leading to heightened T cell responses during infections. In summary, we identify TMEM41B as a concentration-dependent ER Ca2+ release channel, revealing an unexpected role of ER Ca2+ in naive T cell quiescence and responsiveness. © 2025. The Author(s).
Single-Cell Landscape of Bronchoalveolar Lavage Fluid Identifies Specific Neutrophils during Septic Immunosuppression.
In Advanced Science (Weinheim, Baden-Wurttemberg, Germany) on 1 March 2025 by Shen, R., Jiang, Y., et al.
Sepsis-induced immunosuppression is related to increased susceptibility to secondary infections and death. Lung is the most vulnerable target organ in sepsis, but the understanding of the pulmonary immunosuppression state is still limited. Here, single-cell RNA sequencing of bronchoalveolar lavage fluid (BALF) is performed to map the landscape of immune cells, revealing a neutrophil-driven immunosuppressive program in the lungs of patients with immunosuppressive sepsis. Although immunosuppressive genes are upregulated in different immune cells, only neutrophils dramatically increase in the BALF of patients in immunosuppressive phase of sepsis. Five neutrophil subpopulations in BALF are identified, among which CXCR2+ and CD274 (PD-L1 coding gene)+IL1RN+ neutrophil subpopulations increased significantly during septic immunosuppression. Interestingly, a developmental trajectory from CXCR2+ to CD274+IL1RN+ neutrophil subpopulation is disclosed. Moreover, the therapeutic effect of CXCR2 blockade is observed on the survival of septic mice, along with a decreased number of PD-L1+ neutrophils. Taken together, the CXCR2+ neutrophil subpopulation is discovered as a contributor to immunosuppression in sepsis and identified it as a potential therapeutic target in sepsis treatment. © 2025 The Author(s). Advanced Science published by Wiley‐VCH GmbH.
- Cancer Research
CAR T cells, CAR NK cells, and CAR macrophages exhibit distinct traits in glioma models but are similarly enhanced when combined with cytokines.
In Cell Reports Medicine on 18 February 2025 by Look, T., Sankowski, R., et al.
Chimeric antigen receptor (CAR) T cell therapy is a promising immunotherapy against cancer. Although there is a growing interest in other cell types, a comparison of CAR immune effector cells in challenging solid tumor contexts is lacking. Here, we compare mouse and human NKG2D-CAR-expressing T cells, natural killer (NK) cells, and macrophages against glioblastoma, the most aggressive primary brain tumor. In vitro we show that T cell cancer killing is CAR dependent, whereas intrinsic cytotoxicity overrules CAR dependence for NK cells, and CAR macrophages reduce glioma cells in co-culture assays. In orthotopic immunocompetent glioma mouse models, systemically administered CAR T cells demonstrate superior accumulation in the tumor, and each immune cell type induces distinct changes in the tumor microenvironment. An otherwise low therapeutic efficacy is significantly enhanced by co-expression of pro-inflammatory cytokines in all CAR immune effector cells, underscoring the necessity for multifaceted cell engineering strategies to overcome the immunosuppressive solid tumor microenvironment. Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.
- FC/FACS,
- Mus musculus (House mouse),
- Immunology and Microbiology,
- Pharmacology
Cannabinoid receptor 2 selective agonist ameliorates adjuvant-induced arthritis by modulating the balance between Treg and Th17 cells.
In Frontiers in Pharmacology on 17 February 2025 by Tian, N., Yang, C., et al.
Adjuvant-induced arthritis (AIA) serves as a classic model for rheumatoid arthritis (RA), typified by inflammatory cell infiltration and joint damage. This study explores the therapeutic efficacy of HU-308, a CB2 receptor-specific agonist, on inflammation and immune balance in AIA. AIA was induced in mice by CFA injection. AIA mice were treated with HU-308 or vehicle, and effects on paw swelling, spleen index, histopathology, and immune cell profiles were evaluated. Flow cytometry, in vitro differentiation assays, and Western blot analysis were performed to examine Th17 and Treg cells, as well as signaling pathways involved in their differentiation. HU-308 reduced paw swelling, lowered spleen index, and preserved joint integrity in AIA mice, mitigating inflammatory cell infiltration and bone erosion. Flow cytometry revealed that HU-308 restored the Th17/Treg imbalance in AIA, decreasing Th17 cell frequency and enhancing Treg cell infiltration. In vitro assays confirmed HU-308s role in promoting Treg differentiation and inhibiting Th17 polarization. Western blot analysis indicated that HU-308 modulated immune balance through the JAK/STAT5 and TGF-β/SMAD signaling pathways, increasing Foxp3 and TGF-β expression. HU-308 demonstrates significant anti-inflammatory effects in AIA by restoring Th17/Treg balance and reducing joint damage. The findings indicate that HU-308 holds potential as an immunomodulatory agent for RA, providing valuable insights into CB2-mediated therapeutic strategies for autoimmune diseases. Copyright © 2025 Tian, Yang, Du, Chen, Li, Li and Dai.
- Biochemistry and Molecular biology,
- Cancer Research,
- Cell Biology
Dual Ribosome Profiling reveals metabolic limitations of cancer and stromal cells in the tumor microenvironment
Preprint on BioRxiv : the Preprint Server for Biology on 8 January 2025 by Aviles-Huerta, D., Rossella, D. P., et al.
Cancer cells, immune cells, and stromal cells within the tumor microenvironment (TME) collaboratively influence disease progression and therapeutic responses. The nutrient-limited conditions of the TME, particularly the scarcity of glucose, amino acids, and lipids, challenge cancer cell survival 1–4 . However, the metabolic constraints faced by immune and stromal cells in comparison to cancer cells, and how these limitations affect therapeutic outcomes, remain poorly understood. Here, we introduce Dual Ribosome Profiling (DualRP), a method that allows for simultaneous analysis of translation and identification of ribosome stalling, revealing amino acid shortages in different cell types within tumors. Using DualRP, we uncover that interactions between cancer cells and fibroblasts trigger an inflammatory response, mitigating amino acid limitations during glucose starvation. In immunocompetent mouse models, we observe that immune checkpoint blockade therapy induces serine and glycine restrictions specifically in T cells, but not in cancer cells. We further demonstrate that these amino acids are essential for optimal T cell function both in vitro and in vivo , highlighting their critical role in effective immunotherapy. Our findings show that therapeutic interventions create distinct metabolic demands across different tumor cell types, with nutrient availability significantly influencing the success of immunotherapy. DualRP’s ability to explore cell type-specific metabolic vulnerabilities offers a promising tool for advancing our understanding of tumor biology and improving therapeutic strategies.
- Mus musculus (House mouse)
Potent antitumor activity of a designed interleukin-21 mimic
Preprint on BioRxiv : the Preprint Server for Biology on 7 December 2024 by Chun, J., Lim, B. S., et al.
Long-standing goals of cancer immunotherapy are to activate cytotoxic antitumor T cells across a broad range of affinities while dampening suppressive regulatory T (Treg) cell responses, but current approaches achieve these goals with limited success. Here, we report a de novo IL-21 mimic, 21h10, designed to have augmented stability and high signaling potency in both humans and mice. In multiple animal models and in ex vivo human melanoma patient derived organotypic tumor spheroids (PDOTS), 21h10 showed robust antitumor activity. 21h10 generates significantly prolonged STAT signaling in vivo compared with native IL-21, and has considerably stronger anti-tumor activity. Toxicities associated with systemic administration of 21h10 could be mitigated by TNFα blockade without compromising antitumor efficacy. In the tumor microenvironment, 21h10 induced highly cytotoxic antitumor T cells from clonotypes with a range of affinities for endogenous tumor antigens, robustly expanding low-affinity cytotoxic T cells and driving high expression of interferon-
- Genetics
Genetic evidence against involvement of TRPC proteins in SOCE, ROCE, and CRAC channel function.
In Proceedings of the National Academy of Sciences of the United States of America on 3 December 2024 by Susperreguy, S., Yamashita, M., et al.
Using genetically engineered mice and cell lines derived from genetically engineered mice we show that depletion of ER delimited Ca2+ stores activates heteromeric Ca2+ entry (SOCE) channels formed obligatorily, but not exclusively by Orai1 molecules. Comparison of Orai-dependent Ca2+ entries revealed Orai1 to be dominant when compared to Orai2 and Orai3. Unexpectedly, we found that store-depletion-activated Ca2+ entry does not depend obligatorily on functionally intact TRPC molecules, as SOCE monitored with the Fura2 Ca2+ reporter dye is unaffected in cells in which all seven TRPC coding genes have been structurally and functionally inactivated. Unexpectedly as well, we found that TRPC-independent Gq-coupled receptor-operated Ca2+ entry (ROCE) also depends on Orai1. Biophysical measurements of Ca2+ release activated Ca2+ currents (Icrac) are likewise unaffected by ablation of all seven TRPC genes. We refer to mice and cells carrying the seven-fold disruption of TRPC genes as TRPC heptaKO mice and cells. TRPC heptaKO mice are fertile allowing the creation of a new homozygous inbred strain.
- Mus musculus (House mouse),
- Immunology and Microbiology
Targeted protein degradation via CAR endocytosis of antigen in T cells
Preprint on BioRxiv : the Preprint Server for Biology on 21 November 2024 by Wang, Y., Yin, N., et al.
Targeted protein degradation (TPD) relies on molecules engaging host protein degradation machinery. Here, we developed a novel TPD platform based on antigen endocytosis and degradation by chimeric antigen receptor (CAR) T cells. T cells expressing a CAR with TNFR1 as the antigen-binding domain (TNFR1T) were able to bind, endocytose, and degrade TNF in vitro. To enhance in vivo expansion and persistence of TNFR1T cells, BCOR and ZC3H12A were depleted, generating TNFR1T IF cells. In a human TNF transgenic mouse model of rheumatoid arthritis, a single infusion of TNFR1T IF cells sustainably reduced serum hTNF to near wild-type levels, leading to long-term disease remission. This approach extends CAR T cell targets from cells to extracellular proteins, enabling long-term degradation of inflammatory cytokines and durable remission in chronic inflammatory diseases.
- Biochemistry and Molecular biology,
- Cell Biology,
- Immunology and Microbiology
Neurotrophic factor Neuritin modulates T cell electrical and metabolic state for the balance of tolerance and immunity.
In eLife on 20 November 2024 by Yu, H., Nishio, H., et al.
The adaptive T cell response is accompanied by continuous rewiring of the T cell's electric and metabolic state. Ion channels and nutrient transporters integrate bioelectric and biochemical signals from the environment, setting cellular electric and metabolic states. Divergent electric and metabolic states contribute to T cell immunity or tolerance. Here, we report in mice that neuritin (Nrn1) contributes to tolerance development by modulating regulatory and effector T cell function. Nrn1 expression in regulatory T cells promotes its expansion and suppression function, while expression in the T effector cell dampens its inflammatory response. Nrn1 deficiency in mice causes dysregulation of ion channel and nutrient transporter expression in Treg and effector T cells, resulting in divergent metabolic outcomes and impacting autoimmune disease progression and recovery. These findings identify a novel immune function of the neurotrophic factor Nrn1 in regulating the T cell metabolic state in a cell context-dependent manner and modulating the outcome of an immune response. © 2024, Yu et al.
- Immunology and Microbiology
HIF1α-regulated glycolysis promotes activation-induced cell death and IFN-γ induction in hypoxic T cells.
In Nature Communications on 30 October 2024 by Shen, H., Ojo, O. A., et al.
Hypoxia is a common feature in various pathophysiological contexts, including tumor microenvironment, and IFN-γ is instrumental for anti-tumor immunity. HIF1α has long been known as a primary regulator of cellular adaptive responses to hypoxia, but its role in IFN-γ induction in hypoxic T cells is unknown. Here, we show that the HIF1α-glycolysis axis controls IFN-γ induction in both human and mouse T cells, activated under hypoxia. Specific deletion of HIF1α in T cells (Hif1α-/-) and glycolytic inhibition suppresses IFN-γ induction. Conversely, HIF1α stabilization by hypoxia and VHL deletion in T cells (Vhl-/-) increases IFN-γ production. Hypoxic Hif1α-/- T cells are less able to kill tumor cells in vitro, and tumor-bearing Hif1α-/- mice are not responsive to immune checkpoint blockade (ICB) therapy in vivo. Mechanistically, loss of HIF1α greatly diminishes glycolytic activity in hypoxic T cells, resulting in depleted intracellular acetyl-CoA and attenuated activation-induced cell death (AICD). Restoration of intracellular acetyl-CoA by acetate supplementation re-engages AICD, rescuing IFN-γ production in hypoxic Hif1α-/- T cells and re-sensitizing Hif1α-/- tumor-bearing mice to ICB. In summary, we identify HIF1α-regulated glycolysis as a key metabolic control of IFN-γ production in hypoxic T cells and ICB response. © 2024. The Author(s).
- Cancer Research,
- Immunology and Microbiology
Non-classical action of Ku70 promotes Treg suppressive function through a FOXP3-dependent mechanism in lung adenocarcinoma.
In The Journal of Clinical Investigation on 24 October 2024 by Huang, Q., Tian, N., et al.
Ku70, a DNA repair protein, binds to the damaged DNA ends and orchestrates the recruitment of other proteins to facilitate repair of DNA double-strand breaks. Besides its essential role in DNA repair, several studies have highlighted non-classical functions of Ku70 in cellular processes. However, its function in immune homeostasis and anti-tumor immunity remains unknown. Here, we discovered a marked association between elevated Ku70 expression and unfavorable prognosis in lung adenocarcinoma, focusing specifically on increased Ku70 levels in tumor-infiltrated Treg cells. Using a lung-colonizing tumor model of in mice with Treg-specific Ku70 deficiency, we demonstrated that deletion of Ku70 in Treg cells led to a stronger anti-tumor response and slower tumor growth due to impaired immune-suppressive capacity of Treg cells. Furthermore, we confirmed that Ku70 played a critical role in sustaining the suppressive function of human Treg cells. We found that Ku70 bound to FOXP3 and occupied FOXP3-bound genomic sites to support its transcriptional activities. These findings not only unveil a non-homologous end joining (NHEJ)-independent role of Ku70 crucial for Treg suppressive function, but also underscore the potential of targeting Ku70 as an effective strategy in cancer therapy, aiming to both restrain cancer cells and enhance pulmonary anti-tumor immunity.
- Immunology and Microbiology
Progranulin protects against Clostridioides difficile infection by enhancing IL-22 production.
In Gut Microbes on 1 October 2024 by Huang, J., Liu, B., et al.
Enhanced mortality, relapse rates, and increased prevalence of Clostridioides difficile infection (CDI) emphasize the need for better therapies and management approaches. Modulating host immune response to ameliorate CDI-associated immunopathology may provide new advantages to currently inadequate antibiotic therapies. Here, we identified progranulin (PGRN) as an important immune target upregulated in response to CDI. PGRN-deficient mice displayed dramatically higher mortality and aggravated epithelial barrier disruption compared with wild type (WT) mice after CDI despite equivalent levels of bacterial burden or toxin in the large intestine. Mechanistically, PGRN protection was mediated by IL-22 production from CD4+ T helper cells, as demonstrated by a decrease in colonic IL-22-producing CD4+ T helper cells in the intestine of PGRN-deficient mice upon CDI and a boost of IL-22-producing CD4+ T helper cells activated by PGRN ex vivo. Clinical evidence suggests that CDI patients had significantly higher serum levels of PGRN compared with healthy controls, which was significantly and positively correlated with IL-22. Our findings thus indicate a critical role for PGRN-promoted CD4+ T cell IL-22 production in shaping gut immunity and reestablishing the intestinal barrier during CDI. As an alternative to pathogen-targeted therapy, this study may provide a new host-directed therapeutic strategy to attenuate severe, refractory CDI.
- Mus musculus (House mouse),
- Immunology and Microbiology
Development of an antigen-based approach to noninvasively image CAR T cells in real time and as a predictive tool.
In Science Advances on 20 September 2024 by Fröse, J., Rowley, J. E., et al.
CAR T cell therapy has revolutionized the treatment of a spectrum of blood-related malignancies. However, treatment responses vary among cancer types and patients. Accurate monitoring of CAR T cell dynamics is crucial for understanding and evaluating treatment efficacy. Positron emission tomography (PET) offers a comprehensive view of CAR T cell homing, especially in critical organs such as lymphoid structures and bone marrow. This information will help assess treatment response and predict relapse risk. Current PET imaging methods for CAR T require genetic modifications, limiting clinical use. To overcome this, we developed an antigen-based imaging approach enabling whole-body CAR T cell imaging. The probe detects CAR T cells in vivo without affecting their function. In an immunocompetent B cell leukemia model, CAR-PET signal in the spleen predicted early mortality risk. The antigen-based CAR-PET approach allows assessment of CAR T therapy responses without altering established clinical protocols. It seamlessly integrates with FDA-approved and future CAR T cell generations, facilitating broader clinical application.
- Immunology and Microbiology
The CXCL10/CXCR3 axis regulates Th1 cell differentiation and migration in experimental autoimmune prostatitis through the PI3K/AKT pathway.
In Andrology on 1 September 2024 by Yue, S. Y., Niu, D., et al.
To investigate the mechanism of the CXCL10/CXCR3 axis regulating Th1 cell differentiation and migration through the PI3K/AKT pathway in chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). Experimental autoimmune prostatitis (EAP) model, a well-described and validated animal model of CP/CPPS, was used in our study. After treatment with CXCL10, the severity of EAP and Th1 cell proportion were respectively measured by HE stains, immunohistochemistry, and flow cytometry. Then, the protein expression of the PI3K/AKT pathway in CXCL10/CXCR3-regulated Th1 cell differentiation and migration was evaluated by western blotting. Additionally, by the CXCR3 antagonist AMG487 and the PI3K inhibitor LY294002 applications, the effects of CXCL10/CXCR3 through PI3K/AKT pathway on the Th1 cell differentiation and migration were further assessed. The EAP model was successfully built. CXCL10 increased the proportion of Th1 cells in EAP mice, accompanied by upregulation of the PI3K/AKT pathway. Additionally, the PI3K/AKT pathway was found to be involved in CXCL10/CXCR3 axis-mediated Th1 cell differentiation and migration. Our investigations indicate that the CXCL10/CXCR3 axis regulates Th1 cell differentiation and migration in EAP through the PI3K/AKT pathway, which provides a new perspective on the immunological mechanisms of CP/CPPS. © 2023 American Society of Andrology and European Academy of Andrology.
- Cancer Research,
- Endocrinology and Physiology,
- Immunology and Microbiology
Tumor-associated macrophage-derived itaconic acid contributes to nasopharyngeal carcinoma progression by promoting immune escape via TET2.
In Cell Communication and Signaling : CCS on 27 August 2024 by Zhang, X., Qian, S., et al.
Nasopharyngeal carcinoma (NPC) is a malignant tumor of epithelial origin in head and neck with high incidence rate in South China, Southeast Asia and North Africa. The intervention of tumor-associated macrophages (Mφs) (TAMs)-mediated immunosuppression is a potential therapeutic strategy against tumor metastasis, but the exact mechanisms of TAM-mediated immunosuppression in nasopharyngeal carcinoma are unclear. Furthermore, how TAM affects the occurrence and development of nasopharyngeal carcinoma through metabolism is rarely involved. In this work, we revealed that NPC cells promoted M2-type Mφ polarization and elevated itaconic acid (ITA) release. Also, TAMs facilitated NPC cell proliferation, migration, and invasion through immune response gene 1 (IRG1)-catalyzed ITA production. Then, IRG1-mediated ITA production in TAMs repressed the killing of CD8+ T cells, induced M2-type polarization of TAMs, and reduced the phagocytosis of TAMs. Moreover, we demonstrated ITA played a tumor immunosuppressive role by binding and dampening ten-eleven translocation-2 (TET2) expression. Finally, we proved that ITA promotes NPC growth by facilitating immune escape in CD34+ hematopoietic stem cell humanized mice. In Conclusion, TAM-derived ITA facilitated NPC progression by enhancing immune escape through targeting TET2, highlighting that interfering with the metabolic pathway of ITA may be a potential strategy for NPC treatment. © 2024. The Author(s).
