InVivoMAb anti-mouse LAG-3
Product Details
The C9B7W monoclonal antibody reacts with mouse LAG-3 also known as CD223. LAG-3 is a 70 kDa type I transmembrane glycoprotein encoded by the Lag3 gene that belongs to the immunoglobulin superfamily. LAG-3 is expressed by activated T lymphocytes, NK cells, and T regulatory cells. LAG-3ās main ligand is MHC class II which it binds to with a higher affinity than even CD4 does. Upon binding LAG-3 is thought to play similar roles as CTLA-4 and PD-1 including downregulation of TCR signaling and inhibition of CD4-dependent T cell function. LAG-3 has also been demonstrated to contribute to the suppressor function of T regulatory cells. In contrast to inhibition, LAG-3 has been shown to promotes immune responses by activating antigen-presenting cells. The C9B7W antibody has been reported to block the function of murine LAG-3 in vivo and in vitro but studies suggest that the antibody does not block binding of LAG-3 to MHC class II.Specifications
| Isotype | Rat IgG1,Ā Īŗ |
|---|---|
| Recommended Isotype Control(s) | InVivoMAb rat IgG1 isotype control, anti-horseradish peroxidase |
| Recommended Dilution Buffer | InVivoPure pH 7.0 Dilution Buffer |
| Conjugation | This product is unconjugated. Conjugation is available via our Antibody Conjugation Services. |
| Immunogen | Mouse CD223-Ig fusion protein |
| Reported Applications |
in vivo LAG-3 neutralization in vitro LAG-3 neutralization Flow cytometry Western blot |
| Formulation |
PBS, pH 7.0 Contains no stabilizers or preservatives |
| Endotoxin |
<2EU/mg (<0.002EU/Ī¼g) Determined by LAL gel clotting assay |
| Purity |
>95% Determined by SDS-PAGE |
| Sterility | 0.2 Āµm filtration |
| Production | Purified from cell culture supernatant in an animal-free facility |
| Purification | Protein G |
| RRID | AB_10949602 |
| Molecular Weight | 150 kDa |
| Storage | The antibody solution should be stored at the stock concentration at 4Ā°C. Do not freeze. |
Additional Formats
Recommended Products
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Recommended Isotype Control(s)
InVivoMAb rat IgG1 isotype control, anti-horseradish peroxidase
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Recommended Dilution Buffer
InVivoPure pH 7.0 Dilution Buffer
in vivo LAG-3 neutralization
Bauche, D., et al. (2018). "LAG3(+) Regulatory T Cells Restrain Interleukin-23-Producing CX3CR1(+) Gut-Resident Macrophages during Group 3 Innate Lymphoid Cell-Driven Colitis" Immunity 49(2): 342-352 e345. PubMed
Interleukin-22 (IL-22)-producing group 3 innate lymphoid cells (ILC3) maintains gut homeostasis but can also promote inflammatory bowel disease (IBD). The regulation of ILC3-dependent colitis remains to be elucidated. Here we show that Foxp3(+) regulatory T cells (Treg cells) prevented ILC3-mediated colitis in an IL-10-independent manner. Treg cells inhibited IL-23 and IL-1beta production from intestinal-resident CX3CR1(+) macrophages but not CD103(+) dendritic cells. Moreover, Treg cells restrained ILC3 production of IL-22 through suppression of CX3CR1(+) macrophage production of IL-23 and IL-1beta. This suppression was contact dependent and was mediated by latent activation gene-3 (LAG-3)-an immune checkpoint receptor-expressed on Treg cells. Engagement of LAG-3 on MHC class II drove profound immunosuppression of CX3CR1(+) tissue-resident macrophages. Our study reveals that the health of the intestinal mucosa is maintained by an axis driven by Treg cells communication with resident macrophages that withhold inflammatory stimuli required for ILC3 function.
in vivo LAG-3 neutralization, Flow Cytometry
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 LAG-3 neutralization
Verhagen, J. and D. C. Wraith. (2014). "Blockade of LFA-1 augments in vitro differentiation of antigen-induced Foxp3(+) Treg cells" J Immunol Methods 414: 58-64. PubMed
Adoptive transfer of antigen-specific, in vitro-induced Foxp3(+) Treg (iTreg) cells protects against autoimmune disease. To generate antigen-specific iTreg cells at high purity, however, remains a challenge. Whereas polyclonal T cell stimulation with anti-CD3 and anti-CD28 antibody yields Foxp3(+) iTreg cells at a purity of 90-95%, antigen-induced iTreg cells typically do not exceed a purity of 65-75%, even in a TCR-transgenic model. In a similar vein to thymic Treg cell selection, iTreg cell differentiation is influenced not only by antigen recognition and the availability of TGF-beta but also by co-factors including costimulation and adhesion molecules. In this study, we demonstrate that blockade of the T cell integrin Leukocyte Function-associated Antigen-1 (LFA-1) during antigen-mediated iTreg cell differentiation augments Foxp3 induction, leading to approximately 90% purity of Foxp3(+) iTreg cells. This increased efficacy not only boosts the yield of Foxp3(+) iTreg cells, it also reduces contamination with activated effector T cells, thus improving the safety of adoptive transfer immunotherapy.
in vivo LAG-3 neutralization
McGray, A. J., et al. (2014). "Immunotherapy-induced CD8+ T cells instigate immune suppression in the tumor" Mol Ther 22(1): 206-218. PubMed
Despite clear evidence of immunogenicity, cancer vaccines only provide a modest clinical benefit. To evaluate the mechanisms that limit tumor regression following vaccination, we have investigated the weak efficacy of a highly immunogenic experimental vaccine using a murine melanoma model. We discovered that the tumor adapts rapidly to the immune attack instigated by tumor-specific CD8+ T cells in the first few days following vaccination, resulting in the upregulation of a complex set of biological networks, including multiple immunosuppressive processes. This rapid adaptation acts to prevent sustained local immune attack, despite continued infiltration by increasing numbers of tumor-specific T cells. Combining vaccination with adoptive transfer of tumor-specific T cells produced complete regression of the treated tumors but did not prevent the adaptive immunosuppression. In fact, the adaptive immunosuppressive pathways were more highly induced in regressing tumors, commensurate with the enhanced level of immune attack. Examination of tumor infiltrating T-cell functionality revealed that the adaptive immunosuppression leads to a progressive loss in T-cell function, even in tumors that are regressing. These novel observations that T cells produced by therapeutic intervention can instigate a rapid adaptive immunosuppressive response within the tumor have important implications for clinical implementation of immunotherapies.
in vivo LAG-3 neutralization, in vitro LAG-3 neutralization
Durham, N. M., et al. (2014). "Lymphocyte Activation Gene 3 (LAG-3) modulates the ability of CD4 T-cells to be suppressed in vivo" PLoS One 9(11): e109080. PubMed
Lymphocyte Activation Gene ā 3 (LAG-3) is an immune checkpoint molecule that regulates both T-cell activation and homeostasis. However, the molecular mechanisms underlying LAG-3ās function are generally unknown. Using a model in which LAG-3 blockade or absence reliably augmented homeostatic proliferation in vivo, we found that IL-2 and STAT5 are critical for LAG-3 function. Similarly, LAG-3 blockade was ineffective in the absence of regulatory T-cells (Treg), suggesting an important role for LAG-3 in either the responsiveness of conventional T-cells (Tconv) to regulation, or a relative defect in the ability of LAG-3 KO regulatory T-cells (Treg) to suppress the proliferation of Tconv. In this model, LAG-3 KO Treg suppressed proliferation in a manner fairly similar to wild-type (WT) Treg, but LAG-3 KO Tconv were relatively resistant to suppression. Further studies also identified a role for LAG-3 in the induction/expansion of Treg. Finally, we found that LAG-3 blockade (or knockout) led to a relative skewing of naive CD4 T-cells toward a TH1 phenotype both in vitro and in in vivo. Together, these data suggest that LAG-3 expression on Tconv cells makes them more susceptible to Treg based suppression, and also regulates the development of a TH1 T-cell response.
in vitro LAG-3 neutralization, Flow Cytometry
Erickson, J. J., et al. (2014). "Programmed death-1 impairs secondary effector lung CD8(+) T cells during respiratory virus reinfection" J Immunol 193(10): 5108-5117. PubMed
Reinfections with respiratory viruses are common and cause significant clinical illness, yet precise mechanisms governing this susceptibility are ill defined. Lung Ag-specific CD8(+) T cells (T(CD8)) are impaired during acute viral lower respiratory infection by the inhibitory receptor programmed death-1 (PD-1). To determine whether PD-1 contributes to recurrent infection, we first established a model of reinfection by challenging B cell-deficient mice with human metapneumovirus (HMPV) several weeks after primary infection, and found that HMPV replicated to high titers in the lungs. A robust secondary effector lung TCD8 response was generated during reinfection, but these cells were more impaired and more highly expressed the inhibitory receptors PD-1, LAG-3, and 2B4 than primary T(CD8). In vitro blockade demonstrated that PD-1 was the dominant inhibitory receptor early after reinfection. In vivo therapeutic PD-1 blockade during HMPV reinfection restored lung T(CD8) effector functions (i.e., degranulation and cytokine production) and enhanced viral clearance. PD-1 also limited the protective efficacy of HMPV epitope-specific peptide vaccination and impaired lung T(CD8) during heterotypic influenza virus challenge infection. Our results indicate that PD-1 signaling may contribute to respiratory virus reinfection and evasion of vaccine-elicited immune responses. These results have important implications for the design of effective vaccines against respiratory viruses.
in vivo LAG-3 neutralization, Flow Cytometry
Goding, S. R., et al. (2013). "Restoring immune function of tumor-specific CD4+ T cells during recurrence of melanoma" J Immunol 190(9): 4899-4909. PubMed
Recurrent solid malignancies are often refractory to standard therapies. Although adoptive T cell transfer may benefit select individuals, the majority of patients succumb to their disease. To address this important clinical dilemma, we developed a mouse melanoma model in which initial regression of advanced disease was followed by tumor recurrence. During recurrence, Foxp3(+) tumor-specific CD4(+) T cells became PD-1(+) and represented >60% of the tumor-specific CD4(+) T cells in the host. Concomitantly, tumor-specific CD4(+) T effector cells showed traits of chronic exhaustion, as evidenced by their high expression of the PD-1, TIM-3, 2B4, TIGIT, and LAG-3 inhibitory molecules. Although blockade of the PD-1/PD-L1 pathway with anti-PD-L1 Abs or depletion of tumor-specific regulatory T cells (Tregs) alone failed to reverse tumor recurrence, the combination of PD-L1 blockade with tumor-specific Treg depletion effectively mediated disease regression. Furthermore, blockade with a combination of anti-PD-L1 and anti-LAG-3 Abs overcame the requirement to deplete tumor-specific Tregs. In contrast, successful treatment of primary melanoma with adoptive cell therapy required only Treg depletion or Ab therapy, underscoring the differences in the characteristics of treatment between primary and relapsing cancer. These data highlight the need for preclinical development of combined immunotherapy approaches specifically targeting recurrent disease.
- Cancer Research,
- Immunology and Microbiology
Development of PVTX-405 as a potent and highly selective molecular glue degrader of IKZF2 for cancer immunotherapy.
In Nature Communications on 1 May 2025 by Chen, Z., Dhruv, H., et al.
IKZF2 (Helios) is a transcription factor that is selectively expressed by Tregs and is essential for preserving the function and stability of Tregs in the tumor microenvironment (TME), where it suppresses the anti-tumor immune response. Targeted IKZF2 degradation by small molecules represents a promising strategy for the development of a new class of cancer immunotherapy. Herein, we describe the discovery of PVTX-405, a potent, effective, highly selective, and orally efficacious IKZF2 molecular glue degrader. PVTX-405 degrades IKZF2 (DC50ā=ā0.7ānM and Dmaxā=ā91%) while sparing other CRBN neo-substrates. Degradation of IKZF2 by PVTX-405 increases production of inflammatory cytokine IL-2 and reduces the suppressive activity of Tregs, leading to an increase in Teff cell proliferation. Once-daily oral administration of PVTX-405 as single agent significantly delays the growth of MC38 tumors in a syngeneic tumor model using humanized CRBN mice. PVTX-405 in combination with anti-PD1 or anti-LAG3 significantly increases animal survival compared to anti-PD1 or anti-LAG3 alone. Together, these results demonstrate that PVTX-405 is a promising IKZF2 degrader for clinical development for the treatment of human cancers. Ā© 2025. The Author(s).
- FC/FACS,
- In Vivo,
- Mus musculus (House mouse),
- Cancer Research
Resistance to anti-LAG-3 plus anti-PD-1 therapy in head and neck cancer is mediated by Sox9+ tumor cells interaction with Fpr1+ neutrophils.
In Nature Communications on 28 April 2025 by Wang, X., Cheng, M., et al.
Relatlimab and nivolumab combination therapy shows significant efficacy in treating various types of cancer. Current research on the molecular mechanisms of this treatment is abundant, but in-depth investigations into post-treatment resistance remain notably lacking. In this study, we identify significant enrichment of SRY (sex determining region Y)-box 9 (Sox9)+ tumor cells in resistant samples using single cell RNA sequencing (scRNAseq) in a head and neck squamous cell carcinoma (HNSCC) mouse model. In addition, Sox9 directly regulates the expression of annexin A1 (Anxa1), mediating apoptosis of formyl peptide receptor 1 (Fpr1)+ neutrophils through the Anxa1-Fpr1 axis, which promotes mitochondrial fission, inhibits mitophagy by downregulating BCL2/adenovirus E1B interacting protein 3 (Bnip3) expression and ultimately prevents the accumulation of neutrophils in tumor tissues. The reduction of Fpr1+ neutrophils impairs the infiltration and tumor cell-killing ability of cytotoxic Cd8 T and Ī³Ī“T cells within the tumor microenvironment, thereby leading to the development of resistance to the combination therapy. We further validate these findings using various transgenic mouse models. Overall, this study comprehensively explains the mechanisms underlying resistance to the anti-LAG-3 plus anti-PD-1 combination therapy and identifies potential therapeutic targets to overcome this resistance. Ā© 2025. The Author(s).
- Cancer Research
Inhibitors of oncogenic Kras specifically prime CTLA4 blockade to transcriptionally reprogram Tregs and overcome resistance to suppress pancreas cancer
Preprint on BioRxiv : the Preprint Server for Biology on 4 March 2025 by Mahadevan, K. K., Maldonado, A. S., et al.
Lack of sustained response to oncogenic Kras (Kras*) inhibition in preclinical models and patients with pancreatic ductal adenocarcinoma (PDAC) emphasizes the need to identify impactful synergistic combination therapies to achieve robust clinical benefit. Kras* targeting results in an influx of global T cell infiltrates including Tregs, effector CD8 + T cells and exhausted CD8 + T cells expressing several immune checkpoint molecules in PDAC. Here, we probe whether the T cell influx induced by diverse Kras* inhibitors open a therapeutic window to target the adaptive immune response in PDAC. We show a specific synergy of anti-CTLA4 immune checkpoint blockade with Kras* targeting primed by Kras G12D allele specific inhibitor, MRTX1133 and multi-selective pan-RAS inhibitor, RMC-6236, both currently in clinical testing phase. In contrast, attempted therapeutic combination following Kras* targeting with multiple checkpoint inhibitors, including anti-PD1, anti-Tim3, anti-Lag3, anti-Vista and anti-4-1BB agonist antibody failed due to compensatory mechanisms mediated by other checkpoints on exhausted CD8 + T cells. Anti-CTLA4 therapy in Kras* targeted PDAC transcriptionally reprograms effector T regs to a naĆÆve phenotype, reverses CD8 + T cell exhaustion and is associated with recruitment of tertiary lymphoid structures (TLS) containing interferon (IFN)-stimulated/ activated B cells and germinal center B cells to enable immunotherapy efficacy and overcome resistance with long-term survival. Single cell ATAC sequencing analysis revealed that transcriptional reprogramming of Tregs is epigenetically regulated by downregulation of AP-1 family of transcription factors including Fos, Fos-b, Jun-b, Jun-d in the IL-35 promoter region. This study reveals an actionable vulnerability in the adaptive immune response in Kras* targeted PDAC with important clinical implications. Graphical abstract
- Cancer Research,
- Immunology and Microbiology
Coordinated macrophage and T cell interactions mediate response to checkpoint blockade in colorectal cancer
Preprint on BioRxiv : the Preprint Server for Biology on 17 February 2025 by Mestrallet, G., Brown, M., et al.
Summary Mismatch repair deficiency (MMRd), either due to inherited or somatic mutation, is prevalent in colorectal cancer (CRC) and other cancers. While anti-PD-1 therapy is utilized in both local and advanced disease, up to 50% of MMRd CRC fail to respond. Using animal and human models of MMRd, we determined that interactions between MHC+ C1Q+ CXCL9+ macrophages and TCF+ BHLHE40+ PRF1+ T cell subsets are associated with control of MMRd tumor growth, during anti-PD-1 treatment. In contrast, resistance is associated with upregulation of TIM3, LAG3, TIGIT, and PD-1 expression on T cells, and infiltration of the tumor with immunosuppressive TREM2+ macrophages and monocytes. By combining anti-PD-1 with anti-LAG3/CTLA4/TREM2, up to 100% tumor eradication was achieved in MMRd CRC and remarkably, in >70% in MMRp CRC. This study identifies key T cell and macrophage subsets mediating the efficacy of immunotherapy in overcoming immune escape in both MMRd and MMRp CRC settings. Abstract Figure Highlights Anti-PD-1 therapy leads to the accumulation and colocalization of MHCI/II+ C1Q+ CXCL9+ macrophages and DCs with TCF+ CCL5+ T cells that have high TCR diversity. Resistance to anti-PD-1 therapy involves multiple T cell checkpoints, TREM2+ macrophages, IL1B+ TREM1+ monocytes and neutrophils, and IFITM+ tumor cells. Simultaneous blockade of PD-1, LAG3, CTLA-4 and TREM2 dramatically prevents progression of both MMRd and MMRp tumors. Combination therapy completely eliminates tumors by leveraging MHC+ macrophage, CD4+ and CD8+ T cell interactions, facilitating durable anti-tumor effects.
- In Vivo,
- Mus musculus (House mouse)
Therapeutic potential of co-signaling receptor modulation in hepatitis B.
In Cell on 25 July 2024 by Andreata, F., Laura, C., et al.
Reversing CD8+ TĀ cell dysfunction is crucial in treating chronic hepatitis B virus (HBV) infection, yet specific molecular targets remain unclear. Our study analyzed co-signaling receptors during hepatocellular priming and traced the trajectory and fate of dysfunctional HBV-specific CD8+ TĀ cells. Early on, these cells upregulate PD-1, CTLA-4, LAG-3, OX40, 4-1BB, and ICOS. While blocking co-inhibitory receptors had minimal effect, activating 4-1BB and OX40 converted them into antiviral effectors. Prolonged stimulation led to a self-renewing, long-lived, heterogeneous population with a unique transcriptional profile. This includes dysfunctional progenitor/stem-like (TSL) cells and two distinct dysfunctional tissue-resident memory (TRM) populations. While 4-1BB expression is ubiquitously maintained, OX40 expression is limited to TSL. In chronic settings, only 4-1BB stimulation conferred antiviral activity. In HBeAg+ chronic patients, 4-1BB activation showed the highest potential to rejuvenate dysfunctional CD8+ TĀ cells. Targeting all dysfunctional TĀ cells, rather than only stem-like precursors, holds promise for treating chronic HBV infection. Copyright Ā© 2024 The Authors. Published by Elsevier Inc. All rights reserved.
- Mus musculus (House mouse),
- Cancer Research,
- Immunology and Microbiology
The diversity of inhibitory receptor co-expression patterns of exhausted CD8+ T cells in oropharyngeal carcinoma.
In IScience on 17 May 2024 by Rao, Y., Qiu, K., et al.
Exhausted CD8+ TĀ cells (Texs) are characterized by the expression of various inhibitory receptors (IRs), whereas the functional attributes of these co-expressed IRs remain limited. Here, we systematically characterized the diversity of IR co-expression patterns in Texs from both human oropharyngeal squamous cell carcinoma (OPSCC) tissues and syngeneic OPSCC model. Nearly 60% of the Texs population co-expressed two or more IRs, and the number of co-expressed IRs was positively associated with superior exhaustion and cytotoxicity phenotypes. In OPSCC patients, programmed cell death-1 (PD-1) blockade significantly enhanced PDCD1-based co-expression with other IR genes, whereas dual blockades of PD-1 and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) significantly upregulated CTLA4-based co-expression with other IR genes. Collectively, our findings demonstrate that highly diverse IR co-expression is a leading feature of Texs and represents their functional states, which might provide essential clues for the rational selection of immune checkpoint inhibitors in treating OPSCC. Ā© 2024 The Authors.
- Mus musculus (House mouse),
- Immunology and Microbiology
Combining toll-like receptor agonists with immune checkpoint blockade affects antitumor vaccine efficacy.
In Journal for Immunotherapy of Cancer on 3 May 2024 by Jeon, D., Hill, E., et al.
PubMed
T cell checkpoint receptors are expressed when T cells are activated, and modulation of the expression or signaling of these receptors can alter the function of T cells and their antitumor efficacy. We previously found that T cells activated with cognate antigen had increases in the expression of PD-1, and this was attenuated in the presence of multiple toll-like receptor (TLR) agonists, notably TLR3 plus TLR9. In the current report, we sought to investigate whether combining TLR agonists with immune checkpoint blockade can further augment vaccine-mediated T cell antitumor immunity in murine tumor models. TLR agonists (TLR3 plus TLR9) and immune checkpoint inhibitors (antibodies targeting PD-1, CTLA-4, LAG-3, TIM-3 or VISTA) were combined and delivered with vaccines or vaccine-activated CD8+T cells to E.G7-OVA or MyC-CaP tumor-bearing mice. Tumors were assessed for growth and then collected and analyzed by flow cytometry. Immunization of E.G7-OVA tumor-bearing mice with SIINFEKL peptide vaccine, coadministered with TLR agonists and Ī±CTLA-4, demonstrated greater antitumor efficacy than immunization with TLR agonists or Ī±CTLA-4 alone. Conversely, the antitumor efficacy was abrogated when vaccine and TLR agonists were combined with Ī±PD-1. TLR agonists suppressed PD-1 expression on regulatory T cells (Tregs) and activated this population. Depletion of Tregs in tumor-bearing mice led to greater antitumor efficacy of this combination therapy, even in the presence of Ī±PD-1. Combining vaccination with TLR agonists and Ī±CTLA-4 or Ī±LAG-3 showed greater antitumor than with combinations with Ī±TIM-3 or Ī±VISTA. The combination of TLR agonists and Ī±CTLA-4 or Ī±LAG-3 can further improve the efficacy of a cancer vaccine, an effect not observed using Ī±PD-1 due to activation of Tregs when Ī±PD-1 was combined with TLR3 and TLR9 agonists. These data suggest that optimal combinations of TLR agonists and immune checkpoint blockade may improve the efficacy of human anticancer vaccines. Ā© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.
- Cancer Research,
- Immunology and Microbiology
Targeting TIGIT for cancer immunotherapy: recent advances and future directions.
In Biomarker Research on 16 January 2024 by Zhang, P., Liu, X., et al.
As a newly identified checkpoint, T cell immunoreceptor with immunoglobulin and tyrosine-based inhibitory motif (ITIM) domain (TIGIT) is highly expressed on CD4+ T cells, CD8+ T cells, natural killer (NK) cells, regulatory T cells (Tregs), and tumor-infiltrating lymphocytes (TILs). TIGIT has been associated with NK cell exhaustion in vivo and in individuals with various cancers. It not only modulates NK cell survival but also mediates T cell exhaustion. As the primary ligand of TIGIT in humans, CD155 may be the main target for immunotherapy due to its interaction with TIGIT. It has been found that the anti-programmed cell death protein 1 (PD-1) treatment response in cancer immunotherapy is correlated with CD155 but not TIGIT. Anti-TIGIT alone and in combination with anti-PD-1 agents have been tested for cancer immunotherapy. Although two clinical studies on advanced lung cancer had positive results, the TIGIT-targeted antibody, tiragolumab, recently failed in two new trials. In this review, we highlight the current developments on TIGIT for cancer immunotherapy and discuss the characteristics and functions of TIGIT. Ā© 2024. The Author(s).
- Mus musculus (House mouse),
- Cancer Research,
- Immunology and Microbiology
Noninvasive Monitoring of Immunotherapy in Lung Cancer by Lymphocyte Activation Gene 3 PET Imaging of Tumor-Infiltrating Lymphocytes.
In Journal of Nuclear Medicine : Official Publication, Society of Nuclear Medicine on 2 January 2024 by Quan, Z., Han, Z., et al.
PubMed
Although immunotherapy has revolutionized the entire cancer treatment landscape, small fractions of patients respond to immunotherapy. Early identification of responders may improve patient management during immunotherapy. In this study, we evaluated a PET approach for monitoring immunotherapy in lung cancer by imaging the upregulation of lymphocyte activation gene 3 (LAG-3)-expressing (LAG-3+) tumor-infiltrating lymphocytes (TILs). Methods: We synthesized a LAG-3-targeted molecular imaging probe, [68Ga]Ga-NOTA-C25 and performed a series of inĀ vitro and inĀ vivo assays to test its specificity. Next, [68Ga]Ga-NOTA-C25 PET was used to monitor immunotherapy in murine lung cancer-bearing mice and in humanized mouse models for assessing clinical translational potential, with confirmation by immunostaining and flow cytometry analysis. Results: [68Ga]Ga-NOTA-C25 PET could noninvasively detect intertumoral differences in LAG-3+ TIL levels in different tumor models. Importantly, in Lewis lung carcinoma tumor models treated with an agonist of a stimulator of interferon genes, [68Ga]Ga-NOTA-C25 PET also detected an immunophenotyping transition of the tumor from "cold" to "hot" before changes in tumor size. Meanwhile, animals carrying "hot" tumor showed more significant tumor inhibition and longer survival than those carrying "cold" tumor. [68Ga]Ga-NOTA-C25 PET also showed markedly higher tumor uptake in immune system-humanized mice carrying human non-small cell lung cancer than immunodeficient models. Conclusion: [68Ga]Ga-NOTA-C25 PET could be used to noninvasively monitor the early response to immunotherapy by imaging LAG-3+ TILs in lung cancer. [68Ga]Ga-NOTA-C25 PET also exhibited excellent translational potential, with great significance for the precise management of lung cancer patients receiving immunotherapy. Ā© 2024 by the Society of Nuclear Medicine and Molecular Imaging.
- Mus musculus (House mouse),
- Immunology and Microbiology
Intestinal microbiota-specific Th17 cells possess regulatory properties and suppress effector TĀ cells via c-MAF and IL-10.
In Immunity on 12 December 2023 by Brockmann, L., Tran, A., et al.
Commensal microbes induce cytokine-producing effector tissue-resident CD4+ TĀ cells, but the function of these TĀ cells in mucosal homeostasis is not well understood. Here, we report that commensal-specific intestinal Th17 cells possess an anti-inflammatory phenotype marked by expression of interleukin (IL)-10 and co-inhibitory receptors. The anti-inflammatory phenotype of gut-resident commensal-specific Th17 cells was driven by the transcription factor c-MAF. IL-10-producing commensal-specific Th17 cells were heterogeneous and derived from a TCF1+ gut-resident progenitor Th17 cell population. Th17 cells acquired IL-10 expression and anti-inflammatory phenotype in the small-intestinal lamina propria. IL-10 production by CD4+ TĀ cells and IL-10 signaling in intestinal macrophages drove IL-10 expression by commensal-specific Th17 cells. Intestinal commensal-specific Th17 cells possessed immunoregulatory functions and curbed effector TĀ cell activity inĀ vitro and inĀ vivo in an IL-10-dependent and c-MAF-dependent manner. Our results suggest that tissue-resident commensal-specific Th17 cells perform regulatory functions in mucosal homeostasis. Copyright Ā© 2023 Elsevier Inc. All rights reserved.
- Cancer Research,
- Immunology and Microbiology
Differential requirements for CD4+ T cells in the efficacy of the anti-PD-1+LAG-3 and anti-PD-1+CTLA-4 combinations in melanoma flank and brain metastasis models.
In Journal for Immunotherapy of Cancer on 6 December 2023 by Phadke, M. S., Li, J., et al.
PubMed
Although the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combinations are effective in advanced melanoma, it remains unclear whether their mechanisms of action overlap. We used single cell (sc) RNA-seq, flow cytometry and IHC analysis of responding SM1, D4M-UV2 and B16 melanoma flank tumors and SM1 brain metastases to explore the mechanism of action of the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combination. CD4+ andāCD8+ Tācell depletion, tetramer binding assays and ELISPOT assays were used to demonstrate the unique role of CD4+Tācell help in the antitumor effects of the anti-PD-1+LAG-3 combination. The anti-PD-1+CTLA-4 combination was associated with the infiltration of FOXP3+regulatoryāCD4+ cells (Tregs), fewer activated CD4+T cells and the accumulation of a subset of IFNĪ³ secreting cytotoxic CD8+T cells, whereas the anti-PD-1+LAG-3 combination led to the accumulation of CD4+T helper cells that expressed CXCR4, TNFSF8, IL21R and a subset of CD8+T cells with reduced expression of cytotoxic markers. T cell depletion studies showed a requirement for CD4+T cells for the anti-PD-1+LAG-3 combination, but not the PD-1-CTLA-4 combination at both flank and brain tumor sites. In anti-PD-1+LAG-3 treated tumors, CD4+Tācell depletion was associated with fewer activated (CD69+) CD8+T cells and impaired IFNĪ³ release but, conversely, increased numbers of activated CD8+T cells and IFNĪ³ release in anti-PD-1+CTLA-4 treated tumors. Together these studies suggest that these two clinically relevant immune checkpoint inhibitor (ICI) combinations have differential effects on CD4+Tācell polarization, which in turn, impacted cytotoxic CD8+Tācell function. Further insights into the mechanisms of action/resistance of these clinically-relevant ICI combinations will allow therapy to be further personalized. Ā© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.
- Mus musculus (House mouse),
- Immunology and Microbiology
Kruppel-like factor 2+ CD4 TĀ cells avert microbiota-induced intestinal inflammation.
In Cell Reports on 28 November 2023 by Shao, T. Y., Jiang, T. T., et al.
Intestinal colonization by antigenically foreign microbes necessitates expanded peripheral immune tolerance. Here we show commensal microbiota prime expansion of CD4 TĀ cells unified by the Kruppel-like factor 2 (KLF2) transcriptional regulator and an essential role for KLF2+ CD4 cells in averting microbiota-driven intestinal inflammation. CD4 cells with commensal specificity in secondary lymphoid organs and intestinal tissues are enriched for KLF2 expression, and distinct from FOXP3+ regulatory TĀ cells or other differentiation lineages. Mice with conditional KLF2 deficiency in TĀ cells develop spontaneous rectal prolapse and intestinal inflammation, phenotypes overturned by eliminating microbiota or reconstituting with donor KLF2+ cells. Activated KLF2+ cells selectively produce IL-10, and eliminating IL-10 overrides their suppressive function inĀ vitro and protection against intestinal inflammation inĀ vivo. Together with reduced KLF2+ CD4 cell accumulation in Crohn's disease, a necessity for the KLF2+ subpopulation of T regulatory type 1 (Tr1) cells in sustaining commensal tolerance is demonstrated. Copyright Ā© 2023 The Author(s). Published by Elsevier Inc. All rights reserved.
- Mus musculus (House mouse),
- Immunology and Microbiology,
- Stem Cells and Developmental Biology
Reciprocal transmission of activating and inhibitory signals and cell fate in regenerating TĀ cells.
In Cell Reports on 31 October 2023 by Wang, P. H., Washburn, R. S., et al.
PubMed
The ability of activated progenitor TĀ cells to self-renew while producing differentiated effector cell descendants may underlie immunological memory and persistent responses to ongoing infection. The nature of stem-like TĀ cells responding to cancer and during treatment with immunotherapy is not clear. The subcellular organization of dividing progenitor CD8+ TĀ cells from mice challenged with syngeneic tumors is examined here. Three-dimensional microscopy reveals an activating hub composed of polarized CD3, CD28, and phosphatidylinositol 3-kinase (PI3K) activity at the putative immunological synapse with an inhibitory hub composed of polarized PD-1 and CD73 at the opposite pole of mitotic blasts. Progenitor TĀ cells from untreated and inhibitory checkpoint blockade-treated mice yield a differentiated TCF1- daughter cell, which inherits the PI3K activation hub, alongside a discordantly fated, self-renewing TCF1+ sister cell. Dynamic organization of opposite activating and inhibitory signaling poles in mitotic lymphocytes may account for the enigmatic durability of specific immunity. Copyright Ā© 2023 The Author(s). Published by Elsevier Inc. All rights reserved.
- Mus musculus (House mouse),
- Cancer Research,
- Cell Biology,
- Immunology and Microbiology
Autophagy inhibition in pancreatic cancer cells synergizes with immunotherapy via DC activation due to increased antigenicity and adjuvanticity
Preprint on Research Square on 18 October 2023 by Nakata, K., Oyama, K., et al.
The effect of immune checkpoint inhibitors is extremely limited in patients with pancreatic ductal adenocarcinoma (PDAC), due to the immunosuppressive tumor microenvironment (TME). Dendritic cell (DC) paucity and dysfunction are major elements contributing to this immunosuppressive TME. Autophagy, a self-degradation process, has been proposed as a therapeutic target for PDAC; however, the effect of autophagy inhibition in cancer cells on the immunosuppressive TME, and particularly DCs, remains unclear. Here, we discovered that autophagy inhibition in cancer cells induced DC activation via the intracellular accumulation of tumor antigens and increased tumor adjuvanticity. Single-cell RNA-sequencing revealed that autophagy inhibition in cancer cells also induced CD8+ T cell exhaustion, characterized by high LAG3 expression. Moreover, a triplet therapy, comprising chloroquine, Flt3L, and an anti-LAG3 antibody, markedly reduced tumor growth in an orthotopic syngeneic PDAC mouse model. Thus, this therapeutic combination may be a promising candidate for treating PDAC by overcoming the immunosuppressive TME.
- Cancer Research
Defining the mechanisms of action and resistance to the anti-PD-1+LAG-3 and anti-PD-1+CTLA-4 combinations in melanoma flank and brain models
Preprint on BioRxiv : the Preprint Server for Biology on 16 April 2023 by Phadke, M. S., Li, J., et al.
Background Although the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combinations are effective in advanced melanoma it remains unclear whether their mechanisms of action and resistance overlap. Methods We used single cell (sc) RNA-seq, flow cytometry and IHC analysis of responding SM1 and B16 melanoma flank tumors and SM1 brain metastases to explore the mechanism of action of the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combination. CD4+ and CD8+ T cell depletion and ELISPOT assays were used to demonstrate the unique role of CD4+ T cell help in the anti-tumor effects of the anti-PD-1+LAG-3 combination. Tetramer assays confirmed the loss of CD8+ tumor-reactive T cells in brain tumors resistant to the anti-PD-1+LAG-3 combination. Results The anti-PD-1+CTLA-4 combination was associated with the infiltration of FOXP3+ regulatory CD4+ cells (Tregs), fewer activated CD4+ T cells and the accumulation of a subset of IFNĪ³ secreting cytotoxic CD8+ T cells, whereas the anti-PD-1+LAG-3 combination led to the accumulation of CD4+ T helper cells that expressed CXCR4, TNFSF8, IL21R and a subset of CD8+ T cells with reduced expression of cytotoxic markers. T cell depletion studies showed a requirement for CD4+ T cells for the anti-PD-1+LAG-3 combination, but not the PD-1-CTLA-4 combination at both flank and brain tumor sites. In anti-PD-1+LAG-3 treated tumors, CD4+ T cell depletion was associated with fewer activated (CD69+) CD8+ T cells, impaired IFNĪ³ release and increased numbers of myeloid-derived suppressor cells (MDSCs) but, conversely, increased numbers of activated CD8+ T cells and IFNĪ³ release in anti-PD-1+CTLA-4 treated tumors. Analysis of relapsing melanoma brain metastases from anti-PD-1+LAG-3 treated mice showed an increased accumulation of MDSCs and a loss of gp100+ tumor reactive CD8+ T cells. An analysis of the inferred cell-cell interactions from the scRNA-seq data suggested the MDSCs interacted with multiple subsets of T cells in a bi-directional manner. Conclusions Together these studies suggest that these two clinically relevant ICI combinations have differential effects upon CD4+ T cell polarization, which in turn, impacted cytotoxic CD8+ T cell function. Further insights into the mechanisms of action/resistance of these clinically-relevant ICI combinations will allow therapy to be further personalized.
A Pilot Study to Develop Paraneoplastic Cerebellar Degeneration Mouse Model.
In Cerebellum (London, England) on 2 February 2023 by Faure, F., Yshii, L., et al.
Modeling paraneoplastic neurological diseases to understand the immune mechanisms leading to neuronal death is a major challenge given the rarity and terminal access of patients' autopsies. Here, we present a pilot study aiming at modeling paraneoplastic cerebellar degeneration with Yo autoantibodies (Yo-PCD). Female mice were implanted with an ovarian carcinoma cell line expressing CDR2 and CDR2L, the known antigens recognized by anti-Yo antibodies. To boost the immune response, we also immunized the mice by injecting antigens with diverse adjuvants and immune checkpoint inhibitors. Ataxia and gait instability were assessed in treated mice as well as autoantibody levels, Purkinje cell density, and immune infiltration in the cerebellum. We observed the production of anti-Yo antibodies in the CSF and serum of all immunized mice. Brain immunoreaction varied depending on the site of implantation of the tumor, with subcutaneous administration leading to a massive infiltration of immune cells in the meningeal spaces, choroid plexus, and cerebellar parenchyma. However, we did not observe massive Purkinje cell death nor any motor impairments in any of the experimental groups. Self-sustained neuro-inflammation might require a longer time to build up in our model. Unusual tumor antigen presentation and/or intrinsic, species-specific factors required for pro-inflammatory engagement in the brain may also constitute strong limitations to achieve massive recruitment of antigen-specific T-cells and killing of antigen-expressing neurons in this mouse model. Ā© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.
- Cancer Research
An Engineered IFNĪ³-Antibody Fusion Protein with Improved Tumor-Homing Properties.
In Pharmaceutics on 22 January 2023 by Di Nitto, C., Gilardoni, E., et al.
PubMed
Interferon-gamma (IFNĪ³) is one of the central cytokines produced by the innate and adaptive immune systems. IFNĪ³ directly favors tumor growth control by enhancing the immunogenicity of tumor cells, induces IP-10 secretion facilitating (CXCR3+) immune cell infiltration, and can prime macrophages to an M1-like phenotype inducing proinflammatory cytokine release. We had previously reported that the targeted delivery of IFNĪ³ to neoplastic lesions may be limited by the trapping of IFNĪ³-based products by cognate receptors found in different organs. Here we describe a novel fusion protein consisting of the L19 antibody, specific to the alternatively spliced extra-domain B of fibronectin (EDB), fused to a variant of IFNĪ³ with reduced affinity to its cognate receptor. The product (named L19-IFNĪ³ KRG) selectively localized to tumors in mice, showed favorable pharmacokinetic profiles in monkeys and regained biological activity upon antigen binding. The fusion protein was investigated in two murine models of cancer, both as monotherapy and in combination with therapeutic modalities which are frequently used for cancer therapy. L19-IFNĪ³ KRG induced tumor growth retardation and increased the intratumoral concentration of T cells and NK cells in combination with anti-PD-1.
- Mus musculus (House mouse),
- Cancer Research,
- Immunology and Microbiology
Targeting T cell checkpoints 41BB and LAG3 and myeloid cell CXCR1/CXCR2 results in antitumor immunity and durable response in pancreatic cancer.
In Nature Cancer on 1 January 2023 by Gulhati, P., Schalck, A., et al.
PubMed
Pancreatic ductal adenocarcinoma (PDAC) is considered non-immunogenic, with trials showing its recalcitrance to PD1 and CTLA4 immune checkpoint therapies (ICTs). Here, we sought to systematically characterize the mechanisms underlying de novo ICT resistance and to identify effective therapeutic options for PDAC. We report that agonist 41BB and antagonist LAG3 ICT alone and in combination, increased survival and antitumor immunity, characterized by modulating T cell subsets with antitumor activity, increased T cell clonality and diversification, decreased immunosuppressive myeloid cells and increased antigen presentation/decreased immunosuppressive capability of myeloid cells. Translational analyses confirmed the expression of 41BB and LAG3 in human PDAC. Since single and dual ICTs were not curative, T cell-activating ICTs were combined with a CXCR1/2 inhibitor targeting immunosuppressive myeloid cells. Triple therapy resulted in durable complete responses. Given similar profiles in human PDAC and the availability of these agents for clinical testing, our findings provide a testable hypothesis for this lethal disease. Ā© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.
- Cancer Research,
- Immunology and Microbiology
CD11b+DIP2A+LAG3+ cells facilitate immune dysfunction in colorectal cancer.
In American Journal of Cancer Research on 8 December 2021 by Kudo-Saito, C., Ogiwara, Y., et al.
PubMed
Colorectal cancer (CRC) is one of the most common malignant tumors worldwide, and tumor metastasis is the leading cause of death. Targeting immune inhibitory checkpoint inhibitory pathways has attracted great attention, since the therapeutic efficacy induced by the specific blocking antibodies has been demonstrated even in metastatic CRC patients. However, the clinical outcome is low in many cases, and thus more effective treatments are needed in the clinical settings. A SPARC family member follistatin-like 1 (FSTL1) is known as a key driver of tumor metastasis in various types of cancer. However, the immunological roles of the FSTL1 in the CRC pathogenesis remain to be elucidated. In this study, we investigated the molecular mechanisms underlying the refractory FSTL1+ CRC using murine and human FSTL1-transduced CRC cells. Also, based on the results, we evaluated anti-tumor efficacy induced by agents targeting the identified molecules using murine CRC metastasis models, and validated the clinical relevancy of the basic findings using tumor tissues and peripheral blood obtained from CRC patients. FSTL1 transduction conferred EMT-like properties, such as low proliferative (dormant) and high invasive abilities, on tumor cells. When the transfectants were subcutaneously implanted in mice, CD11b+DIP2A+LAG3+ cells were abundantly expanded locally and systemically in the mice. Simultaneously, apoptotic T cells increased and were lastly excluded from the tumor tissues, allowing tumor aggravation leading to resistance to anti-PD1/PDL1 treatment. Blocking FSTL1 and LAG3, however, significantly suppressed the apoptosis induction, and successfully induced anti-tumor immune responses in the CRC metastasis models. Both treatments synergized in providing better prognosis of the mice. FSTL1 was significantly upregulated in tumor tissues and peripheral blood of CRC patients, and the CD11b+DIP2A+LAG3+ cells were significantly expanded in the PBMCs as compared to those of healthy donors. The expansion level was significantly correlated with decrease of potent Ki67+GZMB+ CTLs. These results suggest that the FSTL1-induced CD11b+DIP2A+LAG3+ cells are a key driver of immune dysfunction in CRC. Targeting the FSTL1-LAG3 axis may be a promising strategy for treating metastatic CRC, and anti-FSTL1/LAG3 combination regimen may be practically useful in the clinical settings. AJCR Copyright Ā© 2021.
- Immunology and Microbiology
An engineered oncolytic vaccinia virus encoding a single-chain variable fragment against TIGIT induces effective antitumor immunity and synergizes with PD-1 or LAG-3 blockade.
In Journal for Immunotherapy of Cancer on 1 December 2021 by Zuo, S., Wei, M., et al.
PubMed
In addition to directly lysing tumors, oncolytic viruses also induce antitumor immunity by recruiting and activating immune cells in the local tumor microenvironment. However, the activation of the immune cells induced by oncolytic viruses is always accompanied by high-level expression of immune checkpoints in these cells, which may reduce the efficacy of the oncolytic viruses. The aim of this study is to arm the oncolytic vaccinia virus (VV) with immune checkpoint blockade to enhance its antitumor efficacy. Through homologous recombination with the parental VV, an engineered VV-scFv-TIGIT was produced, which encodes a single-chain variable fragment (scFv) targeting T-cell immunoglobulin and ITIM domain (TIGIT). The antitumor efficacy of the VV-scFv-TIGIT was explored in several subcutaneous and ascites tumor models. The antitumor efficacy of VV-scFv-TIGIT combined with programmed cell death 1 (PD-1) or lymphocyte-activation gene 3 (LAG-3) blockade was also investigated. The VV-scFv-TIGIT effectively replicated in tumor cells and lysed them, and prompt the infected tumor cells to secret the functional scFv-TIGIT. Compared with control VV, intratumoral injection of VV-scFv-TIGIT in several mouse subcutaneous tumor models showed superior antitumor efficacy, accompanied by more T cell infiltration and a higher degree of CD8+ T cells activation. Intraperitoneal injection of VV-scFv-TIGIT in a mouse model of malignant ascites also significantly improved T cell infiltration and CD8+ T cell activation, resulting in more than 90% of the tumor-bearing mice being cured. Furthermore, the antitumor immune response induced by VV-scFv-TIGIT was dependent on CD8+ T cells which mediated a long-term immunological memory and a systemic antitumor immunity against the same tumor. Finally, the additional combination of PD-1 or LAG-3 blockade further enhanced the antitumor efficacy of VV-scFv-TIGIT, increasing the complete response rate of tumor-bearing mice. Oncolytic virotherapy using engineered VV-scFv-TIGIT was an effective strategy for cancer immunotherapy. Administration of VV-scFv-TIGIT caused a profound reshaping of the suppressive tumor microenvironment from 'cold' to 'hot' status. VV-scFv-TIGIT also synergized with PD-1 or LAG-3 blockade to achieve a complete response to tumors with poor response to VV or immune checkpoint blockade monotherapy. Ā© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.
