PD-1 immune checkpoint blockade against inhibitory receptors such as receptor programmed cell death-1 (PD-1), has revolutionized cancer treatment. Effective immune reactivity against tumour antigens ...requires the infiltration and activation of tumour-infiltrating T-cells (TILs). In this context, ligation of the antigen-receptor complex (TCR) in combination with the co-receptor CD28 activates the intracellular mediator AKT (or PKB, protein kinase B) and its downstream targets. PD-1 inhibits the activation of AKT/PKB. Given this, we assessed whether the direct activation of AKT might be effective in activating the immune system to limit the growth of tumors that are resistant to PD-1 checkpoint blockade. We found that the small molecule activator of AKT (SC79) limited growth of a B16 tumor and an EMT-6 syngeneic breast tumor model that are poorly responsive to PD-1 immunotherapy. In the case of B16 tumors, direct AKT activation induced (i) a reduction of suppressor regulatory (Treg) TILs and (ii) an increase in effector CD8+ TILs. SC79 in vivo therapy caused a major increase in the numbers of CD4+ and CD8+ TILs to express interferon-γ (IFN-γ). This effect on IFN-γ expression distinguished responsive from non-responsive anti-tumor responses and could be recapitulated ex vivo with human T-cells. In CD4+FoxP3+Treg TILs, AKT induced IFN-γ expression was accompanied by a loss of suppressor activity, the conversation to CD4
helper Th1-like TILs and a marked reduction in phospho-SHP2. In CD8+ TILs, we observed an increase in the phospho-activation of PLC-γ. Further, the genetic deletion of the transcription factor T-bet (Tbx21) blocked the increased IFN-γ expression on all subsets while ablating the therapeutic benefits of SC79 on tumor growth. Our study shows that AKT activation therapy acts to induce IFN-γ on CD4 and CD8 TILs that is accompanied by the intra-tumoral conversation of suppressive Tregs into CD4
Th1-like T-cells and augmented CD8 responses.
Objective
To study the involvement of Treg cells expressing tumor necrosis factor receptor type II (TNFRII) in exerting control of inflammation in experimental models and in the response to anti‐TNF ...treatments in patients with rheumatoid arthritis (RA) or spondyloarthritis (SpA).
Methods
The role of TNFRII in Treg cells was explored using a multilevel translational approach. Treg cell stability was evaluated by analyzing the methylation status of the Foxp3 locus using bisulfite sequencing. Two models of inflammation (imiquimod‐induced skin inflammation and delayed‐type hypersensitivity arthritis DTHA) were induced in TNFRII−/− mice, with or without transfer of purified CD4+CD25+ cells from wild‐type (WT) mice. In patients with RA and those with SpA, the evolution of the TNFRII+ Treg cell population before and after targeted treatment was monitored.
Results
Foxp3 gene methylation in Treg cells was greater in TNFRII−/− mice than in WT mice (50% versus 36.7%). In cultured Treg cells, TNF enhanced the expression, maintenance, and proliferation of Foxp3 through TNFRII signaling. Imiquimod‐induced skin inflammation and DTHA were aggravated in TNFRII−/− mice (P < 0.05 for mice with skin inflammation and P < 0.0001 for mice with ankle swelling during DTHA compared to WT mice). Adoptive transfer of WT mouse Treg cells into TNFRII−/− mice prevented aggravation of arthritis. In patients with RA receiving anti‐TNF treatments, but not those receiving tocilizumab, the frequency of TNFRII+ Treg cells was increased at 3 months of treatment compared to baseline (mean ± SEM 65.2 ± 3.1% versus 49.1 ± 5.5%; P < 0.01). In contrast, in anti‐TNF–treated patients with SpA, the frequency of TNFRII+ Treg cells was not modified.
Conclusion
TNFRII expression identifies a subset of Treg cells that are characterized by stable expression of Foxp3 via gene hypomethylation, and adoptive transfer of TNFRII‐expressing Treg cells ameliorates inflammation in experimental models. Expansion and activation of TNFRII+ Treg cells may be one of the mechanisms by which anti‐TNF agents control inflammation in RA, but not in SpA.
IL-33 is strongly involved in several inflammatory and autoimmune disorders with both pro- and anti-inflammatory properties. However, its contribution to chronic autoimmune inflammation, such as ...rheumatoid arthritis, is ill defined and probably requires tight regulation. In this study, we aimed at deciphering the complex role of IL-33 in a model of rheumatoid arthritis, namely, collagen-induced arthritis (CIA). We report that repeated injections of IL-33 during induction (early) and during development (late) of CIA strongly suppressed clinical and histological signs of arthritis. In contrast, a late IL-33 injection had no effect. The cellular mechanism involved in protection was related to an enhanced type 2 immune response, including the expansion of eosinophils, Th2 cells, and type 2 innate lymphoid cells, associated with an increase in type 2 cytokine levels in the serum of IL-33-treated mice. Moreover, our work strongly highlights the interplay between IL-33 and regulatory T cells (Tregs), demonstrated by the dramatic in vivo increase in Treg frequencies after IL-33 treatment of CIA. More importantly, Tregs from IL-33-treated mice displayed enhanced capacities to suppress IFN-γ production by effector T cells, suggesting that IL-33 not only favors Treg proliferation but also enhances their immunosuppressive properties. In concordance with these observations, we found that IL-33 induced the emergence of a CD39(high) Treg population in a ST2L-dependent manner. Our findings reveal a powerful anti-inflammatory mechanism by which IL-33 administration inhibits arthritis development.
Melanoma is the deadliest form of skin cancer. Although targeted therapies and immunotherapies have revolutionized the treatment of metastatic melanoma, most patients are not cured. Therapy ...resistance remains a significant clinical challenge. Melanoma comprises phenotypically distinct subpopulations of cells, exhibiting distinct gene signatures leading to tumor heterogeneity and favoring therapeutic resistance. Cellular plasticity in melanoma is referred to as phenotype switching. Regardless of their genomic classification, melanomas switch from a proliferative and differentiated phenotype to an invasive, dedifferentiated and often therapy-resistant state. In this review we discuss potential mechanisms underpinning melanoma phenotype switching, how this cellular plasticity contributes to resistance to both targeted therapies and immunotherapies. Finally, we highlight novel strategies to target plasticity and their potential clinical impact in melanoma.
COVID-19, caused by SARS-CoV-2, can lead to a severe inflammatory disease characterized by significant lymphopenia. However, the underlying cause for the depletion of T-cells in COVID-19 patients ...remains incompletely understood. In this study, we assessed the presence of different T-cell subsets in the progression of COVID-19 from mild to severe disease, with a focus on TCF1 expressing progenitor T-cells that are needed to replenish peripheral T-cells during infection. Our results showed a preferential decline in TCF1+ progenitor CD4 and CD8+ T-cells with disease severity. This decline was seen in various TCF1+ subsets including naive, memory and effector-memory cells, and surprisingly, was accompanied by a loss in cell division as seen by a marked decline in Ki67 expression. In addition, TCF1+ T-cells showed a reduction in pro-survival regulator, BcL2, and the appearance of a new population of TCF1 negative caspase-3 expressing cells in peripheral blood from patients with severe disease. The decline in TCF1+ T-cells was also seen in a subgroup of severe patients with vitamin D deficiency. Lastly, we found that sera from severe patients inhibited TCF1 transcription ex vivo which was attenuated by a blocking antibody against the cytokine, interleukin-12 (IL12). Collectively, our findings underscore the potential significance of TCF1+ progenitor T-cells in accounting for the loss of immunity in severe COVID-19 and outline an array of markers that could be used to identify disease progression.
Immunotherapy using checkpoint blockade (ICB) with antibodies such as anti-PD-1 has revolutionised the treatment of many cancers. Despite its use to treat COVID-19 patients and autoimmune diseases ...such as systemic lupus erythematosus and rheumatoid arthritis, the effect of hydroxychloroquine (HCQ) on cancer immunotherapy has not been examined. In this study, remarkably, we find that HCQ alone, or in combination with azithromycin (AZ), at doses used to treat patients, decreased the therapeutic benefit of anti-PD-1 in cancer immunotherapy. No deleterious effect was seen on untreated tumors. Mechanistically, HCQ and HCQ/AZ inhibited PD-L1 expression on tumor cells, while specifically targeting the anti-PD-1 induced increase in progenitor CD8+CD44+PD-1+TCF1+ tumor infiltrating T cells (TILs) and the generation of CD8+CD44+PD-1+ effectors. Surprisingly, it also impaired the appearance of a subset of terminally exhausted CD8+ TILs. No effect was seen on the presence of CD4+ T cells, FoxP3+ regulatory T cells (Tregs), thymic subsets, B cells, antibody production, myeloid cells, or the vasculature of mice. This study indicates for the first time that HCQ and HCQ/AZ negatively impact the ability of anti-PD-1 checkpoint blockade to promote tumor rejection.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Flow cytometry is an essential tool for studying the tumor-immune microenvironment. It allows us to quickly quantify and identify multiple cell types in a heterogeneous sample. This chapter provides ...an overview of the flow cytometry instrumentation and a discussion of the appropriate considerations and steps in building a reproducible flow cytometry staining panel. We present an updated lymphoid tissue and solid tumor-infiltrating leucocyte flow cytometry staining protocol and an example of flow cytometry data analysis.
Abstract
Objective:
Phosphorylated Eukaryotic translation initiation factor 4E (p-eIF4E) is a critical regulator of protein synthesis and is phosphorylated by MNK1/2 to promote the translation of a ...mRNA subset. We have shown that blocking the eIF4E phosphorylation increased the anti-tumor immune response, especially the CD8 T cell activation. However, the role of p-eIF4E in CD4 T cell subsets, and in particular regulatory T cells (Tregs) is still unknown. The aim of this study was to explore the impact that the absence of p-eIF4E could have on the Treg activity as well as in an inflammatory context.
Methods:
To investigate the role of p-eIF4E on Tregs activity, we used genetically modified mice expressing a non-phosphorylatable form of eIF4E (KI mice). First, we analyzed Treg activity in WT and KI mice and the same mice subjected to dextran sulfate sodium (DSS)-induced colitis. We also analyzed the colonic immune cell infiltration using spectral flow cytometry and CODEX technology.
Results:
Using our mouse models, we observed that the p-eIF4E lack led to a decrease in Treg stability as well as a decrease in their ability to control the helper T cell (Th) proliferation. In the colitis context, we observed an increase in the disease severity in KI compared to WT mice characterized by an increase in colonic immune infiltration. Moreover, our mesenteric lymph node and colon immunophenotyping revealed a significant decrease in Treg, and an increase in Th expressing IFNγ in KI compared to WT mice. Finally, we observed a decrease in KI Treg ability to migrate to the lymph nodes.
Conclusion:
These results demonstrate for the first time the preponderant role of p-eIF4E in the control of Treg stability and Treg migration but also in their ability to regulate inflammation.
Abstract
Immune checkpoint inhibitors (ICIs) are associated with high rates of toxic side effects known as immune related adverse events such as inflammatory arthritis (ir-RA). ir-RA is empirically ...treated with drugs for rheumatoid arthritis such as methotrexate (MTX) and hydroxychloroquine which was recently shown to reduce the anti-tumor benefit of ICIs in mouse models of cancer. We have set out to determine the safety of MTX use in combination with ICIs, its effect on the anti-tumoral immune response and its efficacy in treating ir-RA in a novel arthritis-B16 PD-L1 tumor mouse model. We observed that MTX did not diminish the anti-tumor benefit of ICIs in our melanoma model and significantly altered the intra-tumoral CD8 +T cell population by promoting the formation of central memory cells and diminishing PD1 expression. We have identified that MTX acts on neuropilin-1 (NRP1), a receptor identified as a CD8 +T cell exhaustion marker promoting terminal exhaustion and limiting memory cell formation in tumors. We have shown that MTX treatment in vitro reduces the expression of NRP1 on stimulated CD8 +T cells. We have also advanced in the development of a novel arthritis-tumor model showing that MTX can control and diminish inflammation in the delayed type hypersensitivity arthritis (DTHA) and reduced the frequency of Th1 and Th17 cells in the popliteal lymph nodes of mice, with Th17 cells known to have a crucial role in ir-RA. Together these results show that MTX is safe for use in combination with ICIs in our tumor model and is effective in our DTHA model that mimics the immune profile of ir-RA. With the development of our combined arthritis-tumor model we will be able to assess MTX’s efficacy in controlling arthritis in ICIs treated tumor bearing mice.
La polyarthrite rhumatoïde (PR) est une maladie inflammatoire chronique d’étiologie inconnue. L’inflammation présente dans cette pathologie est fortement dépendante de la cytokine pro-inflammatoire ...qu’est le TNFα. Cette molécule possède deux récepteurs : le TNFR1 et le TNFR2. Le TNFR1 est un récepteur exprimé à la surface de toutes les cellules. L’activation de sa voie de signalisation déclenche la mort cellulaire et elle est souvent associée à des phénomènes inflammatoires. Le TNFR2, quant à lui, est exprimé à la surface des cellules immunitaires, des cellules endothéliales et des cellules neuronales. L’activation de la signalisation du TNFR2 conduit à la survie et à la prolifération cellulaire. Le TNFR2 est de plus, associé à des mécanismes anti-inflammatoires. Les lymphocytes T régulateurs (Treg), cellules clé dans le contrôle de la réponse immunitaire, sont caractérisés par l’expression du facteur de transcription Forkhead box P3 (FoxP3) et sont défectueux chez des patients atteints de PR. Ces cellules expriment les deux récepteurs du TNFα et sont capables d’inhiber l’action des cellules inflammatoires et particulièrement des T effecteurs par différents mécanismes d’immunosuppression. Les Treg exprimant le TNFR2 représentent la population la plus immunosuppressive actuellement recensée. L’objectif de notre travail a été de mieux comprendre le rôle des Treg exprimant le TNFR2 dans le contrôle de l’inflammation dépendante du TNFα. Tout d’abord, nous avons montré que la signalisation TNFα-TNFR2 sur les Treg augmentait le maintien de l’expression de FoxP3 ainsi que la prolifération de ces cellules. L’expression du TNFR2 est en outre liée à une stabilité accrue de ces cellules. Ces résultats peuvent expliquer le rôle important que pourraient jouer les Treg TNFR2+ dans le contrôle de l’inflammation dépendante du TNFα. Afin de confirmer cette hypothèse, nous avons démontré, dans deux modèles expérimentaux d’inflammation dépendants du TNFα (arthrite et psoriasis), que les Treg TNFR2+ jouaient un rôle prépondérant dans le contrôle de l’inflammation. Enfin, des expériences effectuées chez des patients atteints de PR ont mis en évidence que les traitements anti-TNFα conduisaient à une augmentation de la fréquence des Treg TNFR2+ circulants chez des patients répondeurs. En démontrant le rôle prépondérant des Treg TNFR2+ dans la résolution de l’inflammation, ce travail ouvre la voie vers l’élaboration de thérapies ciblant le système TNFα/TNFR plus spécifiques pour le traitement de la PR et d’autres pathologies dépendantes du TNF.
Rheumatoid arthritis (RA) is a chronic inflammatory disease with unknown etiology. In this pathology, inflammation is mainly dependent on the pro-inflammatory cytokine TNFα. This molecule acts through two receptors: TNFR1 and TNFR2. TNFR1 is expressed on almost all cell types. Activation of this pathway mainly leads to cell death and is often associated with pro-inflammatory response. In contrast, TNFR2 is expressed on immune, epithelial and neuronal cells. Activation of TNFR2 signaling triggers cellular survival and cell proliferation. Furthermore, TNFR2 pathway is associated with anti-inflammatory mechanisms. Regulatory T cells (Treg) play a pivotal role in the control of inflammation and are defective in RA. They are characterized by the expression of transcriptional factor Forkhead box P3 (FoxP3). Tregs express both TNFα receptors and are able to inhibit inflammatory cells, specifically effector T cells using various immunosuppressive mechanisms. Treg expressing TNFR2 have been identified as the most suppressive Treg population. The aim of this study was to elucidate the role of TNFR2+ Tregs in TNFα mediated - inflammation by. Firstly, we have shown that TNFα-TNFR2 signaling on Tregs increased their proliferation and helped to maintain FoxP3 expression. Moreover, TNFR2 expression was associated with increased Treg stability. These results could explain the potential role of TNFR2+ Tregs in control of TNFα mediated - inflammation. To confirm this hypothesis, we demonstrated, in two models of inflammation mediated by TNFα (arthritis and psoriasis), that TNFR2+ Tregs play a major role in the control of inflammation. Finally, our experiments in RA patients highlighted that anti-TNFα treatments increased circulating TNFR2+ Treg frequency in responder RA patients. By demonstrating the major role of TNFR2+ Tregs in resolution of inflammation, our work paves the way for therapies targeting more specifically TNFα/TNFR system to cure RA and others TNFα - mediated pathologies.
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