T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral ...infection and cancers. Under some conditions, TIM-3 expression has also been shown to be stimulatory. Considering that TIM-3, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), is being targeted for cancer immunotherapy, it is important to identify the circumstances under which TIM-3 can inhibit and activate T-cell responses. Here we show that TIM-3 is co-expressed and forms a heterodimer with carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition. Biochemical, biophysical and X-ray crystallography studies show that the membrane-distal immunoglobulin-variable (IgV)-like amino-terminal domain of each is crucial to these interactions. The presence of CEACAM1 endows TIM-3 with inhibitory function. CEACAM1 facilitates the maturation and cell surface expression of TIM-3 by forming a heterodimeric interaction in cis through the highly related membrane-distal N-terminal domains of each molecule. CEACAM1 and TIM-3 also bind in trans through their N-terminal domains. Both cis and trans interactions between CEACAM1 and TIM-3 determine the tolerance-inducing function of TIM-3. In a mouse adoptive transfer colitis model, CEACAM1-deficient T cells are hyper-inflammatory with reduced cell surface expression of TIM-3 and regulatory cytokines, and this is restored by T-cell-specific CEACAM1 expression. During chronic viral infection and in a tumour environment, CEACAM1 and TIM-3 mark exhausted T cells. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses with improved elimination of tumours in mouse colorectal cancer models. Thus, CEACAM1 serves as a heterophilic ligand for TIM-3 that is required for its ability to mediate T-cell inhibition, and this interaction has a crucial role in regulating autoimmunity and anti-tumour immunity.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Objective
Eosinophils are hallmarks in allergic type 2 inflammation and are known to release cytotoxic granule proteins that contribute to inflammation. Eosinophils develop in the bone marrow from ...hematopoietic stem cells and once mature, have a limited lifespan in culture, making them difficult to study
ex vivo
. IL-33 has increasingly been shown as a key regulator of type 2 inflammation
via
signaling through its receptor, ST2. The present study was conducted to detail a method of eosinophil differentiation from hematopoietic stem cells and determine the response to IL-33.
Methods
CD34+ and CD14+ cells were isolated from donor apheresis cones and differentiated into eosinophils or macrophage controls, respectively. Morphologic, transcriptional and protein analyses were performed to validate this method of eosinophil differentiation. The effect of IL-33 on differentiated eosinophils was assessed using qPCR, immunofluorescence, and multiplex cytokine array.
Results
CD34 differentiated eosinophils appear morphologically similar by H&E and express eosinophil peroxidase (EPX) protein as well as the conventional eosinophil transcripts
EPX
,
CLC
, and
MBP
. In addition, differentiated eosinophils expressed both isoforms of the IL-33 receptor,
ST2L
and s
ST2
throughout the differentiation process. Transcript levels of both IL-33 receptors were up-regulated by treatment with IL-33 at earlier timepoints in the differentiation. These cells also expressed
IL-4
and
IL-13
mRNA which were up-regulated by IL-33 as well. Notably,
IL-13
expression was significantly higher with IL-33 treatment compared to media control at every timepoint measured. IL-33 significantly increased cellular secretion of IL-13 protein at most timepoints throughout differentiation. IL-8, LIF, CCL1, CCL5, CCL7, and CCL8 were also significantly secreted after IL-33 stimulation.
Conclusions
Our findings suggest that CD34 differentiated eosinophils are morphologically and phenotypically similar to peripheral eosinophils. The release of specific cytokines in direct response to IL-33 may contribute to the pathogenesis of type 2 inflammation and facilitates new avenues for studying eosinophils as effector cells
in vitro
.
Recombinant protein therapeutics often suffer from short circulating half-life and poor stability, necessitating multiple injections and resulting in limited shelf-life. Conjugation to polyethylene ...glycol chains (PEG) extends the circulatory half-life of many proteins, but the methods for attachment often lack specificity, resulting in loss of biological activity. Using four-helix bundle cytokines as an example, we present a general platform that uses sortase-mediated transpeptidation to facilitate site-specific attachment of PEG to extend cytokine half-life with full retention of biological activity. Covalently joining the N and C termini of proteins to obtain circular polypeptides, again executed using sortase, increases thermal stability. We combined both PEGylation and circularization by exploiting two distinct sortase enzymes and the use of a molecular suture that allows both site-specific PEGylation and covalent closure. The method developed is general, uses a set of easily accessible reagents, and should be applicable to a wide variety of proteins, provided that their termini are not involved in receptor binding or function.
Coordinated regulation of genes is key to determining cell fate. Although this is best understood for master regulator transcription factors, posttranscriptional regulation of mRNA stability and ...nuclear export can be equally effective at altering gene expression. Indeed, the heterogeneity of RNA-binding proteins and miRNAs suggests a deep complexity to posttranscriptional regulatory processes. In this issue, Wu and colleagues report a new mechanism for TGFβ-mediated immune suppression via regulation of mRNA-binding proteins in CD8
T cells.
.
The therapeutic potential of recombinant cytokines has been limited by the severe side effects of systemic administration. We describe a strategy to reduce the dose-limiting toxicities of monomeric ...cytokines by designing two components that require colocalization for activity and that can be independently targeted to restrict activity to cells expressing two surface markers. We demonstrate the approach with a previously designed mimetic of cytokines interleukin-2 and interleukin-15-Neoleukin-2/15 (Neo-2/15)-both for trans-activating immune cells surrounding targeted tumor cells and for cis-activating directly targeted immune cells. In trans-activation mode, tumor antigen targeting of the two components enhanced antitumor activity and attenuated toxicity compared with systemic treatment in syngeneic mouse melanoma models. In cis-activation mode, immune cell targeting of the two components selectively expanded CD8
T cells in a syngeneic mouse melanoma model and promoted chimeric antigen receptor T cell activation in a lymphoma xenograft model, enhancing antitumor efficacy in both cases.
Carpenter and colleagues analyze organ donors to find that pancreatic intraepithelial neoplasia (PanIN), the precursor lesions of pancreatic ductal adenocarcinoma, are highly prevalent in the average ...healthy adult starting from a young age. Why these precursor lesions do not progress to cancer in most people is a mystery. See related article by Carpenter et al., p. 1324 (1).
During eukaryotic development, the induction of a lineage-specific transcription factor typically drives differentiation of multipotent progenitor cells, while repressing that of alternative ...lineages. This process is often mediated by some extracellular signaling molecules, such as cytokines that can bind to cell surface receptors, leading to activation and/or repression of transcription factors. We explored the early differentiation of naive CD4 T helper (Th) cells into Th1 versus Th2 states by counting single transcripts and quantifying immunofluorescence in individual cells. Contrary to mutually exclusive expression of antagonistic transcription factors, we observed their ubiquitous co-expression in individual cells at high levels that are distinct from basal-level co-expression during lineage priming. We observed that cytokines are expressed only in a small subpopulation of cells, independent from the expression of transcription factors in these single cells. This cell-to-cell variation in the cytokine expression during the early phase of T helper cell differentiation is significantly larger than in the fully differentiated state. Upon inhibition of cytokine signaling, we observed the classic mutual exclusion of antagonistic transcription factors, thus revealing a weak intracellular network otherwise overruled by the strong signals that emanate from extracellular cytokines. These results suggest that during the early differentiation process CD4 T cells acquire a mixed Th1/Th2 state, instructed by extracellular cytokines. The interplay between extracellular and intracellular signaling components unveiled in Th1/Th2 differentiation may be a common strategy for mammalian cells to buffer against noisy cytokine expression.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
CDK4/6 inhibitors are approved to treat breast cancer and are in trials for other malignancies. We examined CDK4/6 inhibition in mouse and human CD8
T cells during early stages of activation. Mice ...receiving tumor-specific CD8
T cells treated with CDK4/6 inhibitors displayed increased T-cell persistence and immunologic memory. CDK4/6 inhibition upregulated MXD4, a negative regulator of MYC, in both mouse and human CD8
T cells. Silencing of
in mouse CD8
T cells demonstrated the importance of this axis for memory formation. We used single-cell transcriptional profiling and T-cell receptor clonotype tracking to evaluate recently activated human CD8
T cells in patients with breast cancer before and during treatment with either palbociclib or abemaciclib. CDK4/6 inhibitor therapy in humans increases the frequency of CD8
memory precursors and downregulates their expression of MYC target genes, suggesting that CDK4/6 inhibitors in patients with cancer may augment long-term protective immunity. SIGNIFICANCE: CDK4/6 inhibition skews newly activated CD8
T cells toward a memory phenotype in mice and humans with breast cancer. CDK4/6 inhibitors may have broad utility outside breast cancer, particularly in the neoadjuvant setting to augment CD8
T-cell priming to tumor antigens prior to dosing with checkpoint blockade.
.
Standard genetic approaches allow the production of protein composites by fusion of polypeptides in head-to-tail fashion. Some applications would benefit from constructions that are genetically ...impossible, such as the site-specific linkage of proteins via their N or C termini, when a remaining free terminus is required for biological activity. We developed a method for the production of N-to-N and C-to-C dimers, with full retention of the biological activity of both fusion partners and without inflicting chemical damage on the proteins to be joined. We use sortase A to install on the N or C terminus of proteins of interest the requisite modifications to execute a strain-promoted copper-free cycloaddition and show that the ensuing ligation proceeds efficiently. Applied here to protein-protein fusions, the method reported can be extended to connecting proteins with any entity of interest.
Transforming growth factor-b (TGFb) plays pleiotropic roles in pancreatic cancer, including promoting metastasis, attenuating CD8 T-cell activation, and enhancing myofibroblast differentiation and ...deposition of extracellular matrix. However, single-agent TGFb inhibition has shown limited efficacy against pancreatic cancer in mice or humans.
We evaluated the TGFβ-blocking antibody NIS793 in combination with gemcitabine/nanoparticle (albumin-bound)-paclitaxel or FOLFIRINOX (folinic acid FOL, 5-fluorouracil F, irinotecan IRI and oxaliplatin OX) in orthotopic pancreatic cancer models. Single-cell RNA sequencing and immunofluorescence were used to evaluate changes in tumor cell state and the tumor microenvironment.
Blockade of TGFβ with chemotherapy reduced tumor burden in poorly immunogenic pancreatic cancer, without affecting the metastatic rate of cancer cells. Efficacy of combination therapy was not dependent on CD8 T cells, because response to TGFβ blockade was preserved in CD8-depleted or recombination activating gene 2 (RAG2−/−) mice. TGFβ blockade decreased total α-smooth muscle actin–positive fibroblasts but had minimal effect on fibroblast heterogeneity. Bulk RNA sequencing on tumor cells sorted ex vivo revealed that tumor cells treated with TGFβ blockade adopted a classical lineage consistent with enhanced chemosensitivity, and immunofluorescence for cleaved caspase 3 confirmed that TGFβ blockade increased chemotherapy-induced cell death in vivo.
TGFβ regulates pancreatic cancer cell plasticity between classical and basal cell states. TGFβ blockade in orthotropic models of pancreatic cancer enhances sensitivity to chemotherapy by promoting a classical malignant cell state. This study provides scientific rationale for evaluation of NIS793 with FOLFIRINOX or gemcitabine/nanoparticle (albumin-bound) paclitaxel chemotherapy backbone in the clinical setting and supports the concept of manipulating cancer cell plasticity to increase the efficacy of combination therapy regimens.
Blockade of the cytokine transforming growth factor-β enhances pancreatic tumor cell chemotherapy sensitivity by promoting a basal-like to classical cell state transition. Transforming growth factor-β blockade and combination chemotherapies are currently in clinical trials.
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