Primary Human CD4 Research Articles

DNA hypomethylating agents increase activation and cytolytic activity of CD8+ T cells

We demonstrate that DNA hypomethylating agent (HMA) treatment can directly modulate the anti-tumor response and effector function of CD8+ Tcells. Invivo HMA treatment promotes CD8+ Tcell tumor infiltration and suppresses tumor growth via CD8+ Tcell-dependent activity. Exvivo, HMAs enhance primary human CD8+ Tcell activation markers, effector cytokine production, and anti-tumor cytolytic activity. Epigenomic and transcriptomic profiling shows that HMAs vastly regulate Tcell activation-related transcriptional networks, culminating with over-activation of NFATc1 short isoforms. Mechanistically, demethylation of an intragenic CpG island immediately downstream to the 3' UTR of the short isoform was associated with antisense transcription and alternative polyadenylation of NFATc1 short isoforms. High-dimensional single-cell mass cytometry analyses reveal a selective effect of HMAs on a subset of human CD8+ Tcell subpopulations, increasing both the number and abundance of a granzyme Bhigh, perforinhigh effector subpopulation. Overall, our findings support the use of HMAs as a therapeutic strategy to boost anti-tumor immune response.

Integrin Crosstalk Modulates Stiffness-independent Motility in CD4+ T Lymphocytes

CD4+ T lymphocytes interact with various cell types and tissue substrates in response to inflammation.Mechanotransduction in T lymphocytes is frequently studied at immunological synapses (IS) during T cell activation, after engagement with T cell receptors (TCR) and costimulatory receptors (i.e. CD3/CD28) and LFA-1 integrins. Recently, in vitro and in vivo studies suggest T cells can migrate upstream (i.e. in the opposite direction of shear flow) on surfaces expressing ICAM-1 through interaction with LFA-1 integrins. LFA-1 likely interacts as a mechanosensor, and thus we hypothesized that substrate mechanics might affect the ability of LFA-1 to support upstream migration of T cells under flow. To test the hypothesis, we measured motility of activated primary human CD4+ T lymphocytes on polyacrylamide gels with pre-determined stiffnesses, functionalized with protein A/G and ICAM-1, VCAM-1, or 1:1 mixture of ICAM-1/VCAM-1 Fc chimeras. We found that CD4+ T cells exhibit stiffness-dependent motility on ICAM-1 surfaces, with a maximum random motility appearing at an elasticity of about 60 kPa. in contrast, motility on VCAM-1 or 1:1 mixture of VCAM-1/ICAM-1 mixed surfaces was independent of elasticity. This mechanosensitivity on ICAM-1 surfaces is overcome when VLA-4 is ligated with soluble VCAM-1. Lastly, we observed that CD4+ T cells can migrate upstream under flow on hydrogels functionalized with ICAM-1, independent of substrate stiffness. In this study, we show that CD4+ T cells can sense stiffness with integrins alone under static conditions and that integrin crosstalk can activate an elasticity-independent motility. interestingly, CD4+ T lymphocytes migrated upstream under flow on ICAM-1 substrates regardless of the substrate stiffness.

Single-Molecule, Super-Resolution, and Functional Analysis of G Protein-Coupled Receptor Behavior Within the T Cell Immunological Synapse.

A central process in immunity is the activation of T cells through interaction of T cell receptors (TCRs) with agonistic peptide-major histocompatibility complexes (pMHC) on the surface of antigen presenting cells (APCs). TCR-pMHC binding triggers the formation of an extensive contact between the two cells termed the immunological synapse, which acts as a platform for integration of multiple signals determining cellular outcomes, including those from multiple co-stimulatory/inhibitory receptors. Contributors to this include a number of chemokine receptors, notably CXC-chemokine receptor 4 (CXCR4), and other members of the G protein-coupled receptor (GPCR) family. Although best characterized as mediators of ligand-dependent chemotaxis, some chemokine receptors are also recruited to the synapse and contribute to signaling in the absence of ligation. How these and other GPCRs integrate within the dynamic structure of the synapse is unknown, as is how their normally migratory Gαi-coupled signaling is terminated upon recruitment. Here, we report the spatiotemporal organization of several GPCRs, focusing on CXCR4, and the G protein Gαi2 within the synapse of primary human CD4+ T cells on supported lipid bilayers, using standard- and super-resolution fluorescence microscopy. We find that CXCR4 undergoes orchestrated phases of reorganization, culminating in recruitment to the TCR-enriched center. This appears to be dependent on CXCR4 ubiquitination, and does not involve stable interactions with TCR microclusters, as viewed at the nanoscale. Disruption of this process by mutation impairs CXCR4 contributions to cellular activation. Gαi2 undergoes active exclusion from the synapse, partitioning from centrally-accumulated CXCR4. Using a CRISPR-Cas9 knockout screen, we identify several diverse GPCRs with contributions to T cell activation, most significantly the sphingosine-1-phosphate receptor S1PR1, and the oxysterol receptor GPR183. These, and other GPCRs, undergo organization similar to CXCR4; including initial exclusion, centripetal transport, and lack of receptor-TCR interactions. These constitute the first observations of GPCR dynamics within the synapse, and give insights into how these receptors may contribute to T cell activation. The observation of broad GPCR contributions to T cell activation also opens the possibility that modulating GPCR expression in response to cell status or environment may directly regulate responsiveness to pMHC.

MRNA Transfection of T-Lymphocytes by Electroporation.

Electroporation enables the transfection of different cell types including microbial, plant, and animal cells with charged molecules, such asnuclear acids or proteins. During electroporation, an electrical field is applied to the cells leading to a transient permeabilization of the cell membrane allowing exogenous molecules to enter the cells. Here we report the electroporation of human primary CD4+ -T cells with in-vitro transcribed mRNA to facilitate gene editing (knockout) of the CC-chemokine receptor 5 (CCR5), the coreceptor of the human immunodeficiency virus 1 (HIV1) predominantly used during primary infection. Using such strategy of transient expression of a CCR5-specific Transcription-activator-like-effector nuclease (TALEN), we aim toprotect helper T cells from de novo HIV infection.

The Proteomic Landscape of Resting and Activated CD4+ T Cells Reveal Insights into Cell Differentiation and Function.

CD4+ T cells (T helper cells) are cytokine-producing adaptive immune cells that activate or regulate the responses of various immune cells. The activation and functional status of CD4+ T cells is important for adequate responses to pathogen infections but has also been associated with auto-immune disorders and survival in several cancers. In the current study, we carried out a label-free high-resolution FTMS-based proteomic profiling of resting and T cell receptor-activated (72 h) primary human CD4+ T cells from peripheral blood of healthy donors as well as SUP-T1 cells. We identified 5237 proteins, of which significant alterations in the levels of 1119 proteins were observed between resting and activated CD4+ T cells. In addition to identifying several known T-cell activation-related processes altered expression of several stimulatory/inhibitory immune checkpoint markers between resting and activated CD4+ T cells were observed. Network analysis further revealed several known and novel regulatory hubs of CD4+ T cell activation, including IFNG, IRF1, FOXP3, AURKA, and RIOK2. Comparison of primary CD4+ T cell proteomic profiles with human lymphoblastic cell lines revealed a substantial overlap, while comparison with mouse CD+ T cell data suggested interspecies proteomic differences. The current dataset will serve as a valuable resource to the scientific community to compare and analyze the CD4+ proteome.

Upregulated microRNA-132 in T helper 17 cells activates hepatic stellate cells to promote hepatocellular carcinoma cell migration in vitro.

MicroRNAs play an important role in the modulation of the immune system. T helper 17 (Th17) cells are involved in the modulation of the tumour microenvironment. However, the function of miRNA in Th17 cells in the tumour microenvironment is unclear. In this study, we analysed miR-132 expression in Th17 cells and assessed the function of miR-132 on Th17 cell differentiation. In addition, the effect of miR-132 on Th17 cells in the tumour microenvironment, especially hepatic stellate cells (HSCs), was confirmed. CD4+ IL-17∓cells were isolated from hepatocellular carcinoma (HCC) tumour tissues. The expression of miR-132 was higher in CD4+ IL-17+cells than in CD4+ IL-17- cells. Human primary CD4+ T cells were used for Th17 cell differentiation. Compared with primary CD4+ T cells, Th17 cells expressed high levels of miR-132. During Th17 cell differentiation, a miR-132 mimic and inhibition were applied. After treatment with the miR-132 mimic, the differentiation of Th17 cells accelerated, showing a a higher percentage of Th17 cells and the expression and secretion of IL-17 and IL-22. Smad nuclear interacting protein 1 (SNIP1), as one of the targets of miR-132, decreased during Th17 cell differentiation-related Th17 differentiation and IL-17 expression. The conditioned medium of miR-132-overexpressing Th17 cells could increase the activation of the HSCs, which strongly promoted HCC cell migration and epithelial-mesenchymal transition (EMT). In summary, miR-132 positively regulates Th17 cell differentiation and improves the function of Th17 on HSCs for their tumour-promoting effects.

Activated human NK and CD8+ T cells express both TNF-related apoptosis-inducing ligand (TRAIL) and TRAIL receptors but are resistant to TRAIL-mediated cytotoxicity

AbstractThe expression of tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) and TRAIL receptors was investigated in resting and cytokine-activated purified primary human natural killer (NK) and CD8+ T cells. Resting NK and CD8+ T cells expressed the mRNA for all TRAIL receptors, but TRAIL-R4 was the only receptor clearly detectable on the surface of both cell types. NK cells were activated by interleukin 2 (IL-2) or IL-15, whereas CD8+ T cells were activated by phytohemagglutinin (PHA) + IL-2 followed by IL-2 alone for up to 10 days. On activation, both cell types rapidly expressed TRAIL-R2 and TRAIL-R3, whose expression peaked at day 10 of culture. TRAIL-R1, however, was never expressed at any time point examined, whereas the expression of TRAIL-R4, which showed a progressive increase in CD8+ T cells, remained constant in NK cells. Notwithstanding the expression of TRAIL-R2, recombinant TRAIL did not show any cytotoxic activity on either NK or CD8+ T cells. Both resting and activated NK and CD8+ T cells were found to express high levels of the 2 isoforms of c-FLIP (cellular Fas-associated death domain protein [FADD]–like IL-1–converting enzyme [FLICE]–inhibitory protein). Small interference RNA-mediated inhibition of c-FLIP expression in NK cells abrogated their resistance to the apoptotic effect of soluble TRAIL. Thus, once activated the major cytotoxic effector cells are potentially sensitive to TRAIL but are physiologically protected from its apoptotic action by intracellular level of c-FLIP.

Interorganelle Tethering to Endocytic Organelles Determines Directional Cytokine Transport in CD4+ T Cells

Delivery of vesicles to their desired destinations plays a central role in maintaining proper cell functionality. In certain scenarios, depending on loaded cargos, the vesicles have spatially distinct destinations. For example, in T cells, some cytokines (e.g., IL-2) are polarized to the T cell-target cell interface, whereas the other cytokines are delivered multidirectionally (e.g., TNF-α). In this study, we show that in primary human CD4+ T cells, both TNF-α+ and IL-2+ vesicles can tether with endocytic organelles (lysosomes/late endosomes) by forming membrane contact sites. Tethered cytokine-containing vesicle (CytV)-endocytic organelle pairs are released sequentially. Only endocytic organelle-tethered CytVs are preferentially transported to their desired destination. Mathematical models suggest that endocytic organelle tethering could regulate the direction of cytokine transport by selectively attaching different microtubule motor proteins (such as kinesin and dynein) to the corresponding CytVs. These findings establish the previously unknown interorganelle tethering to endocytic organelles as a universal solution for directional cytokine transport in CD4+ T cells. Modulating tethering to endocytic organelles can, therefore, coordinately control directionally distinct cytokine transport.

Human Orthogonal IL-2/IL-2Rβ As a Tunable Approach to Enhance CD19-Speific CAR-T Cell Antitumor Activity

Immunotherapy using T cells engineered with a chimeric antigen receptors (CAR) can produce deep and durable control of B-cell malignancies that is associated with the engraftment and persistence of the CAR T cells following adoptive transfer. Interleukin-2 (IL-2) is a central T cell cytokine that promotes T cell proliferation and effector function with a potential to enhance CAR T cell immunotherapy; however, the use of recombinant IL-2 to support T cell immunotherapy is limited by the significant toxicity associated with IL-2 infusion. Previous work has demonstrated the feasibility of engineering murine IL-2 and it's receptor to create an orthogonal (ortho) cytokine-receptor pair capable of delivering IL-2's signal without the associated toxicity (Sockolosky JT et al. Science 2018). In order to translate this novel approach to humans, we engineered a human orthogonal IL-2 (ortho-hIL-2) and human orthogonal IL-2Rβ (ortho-hIL-2Rβ) pair based upon similar mutations to those required in the murine system. We show that hoIL-2 induces JAK-STAT signaling and supports proliferation of the IL-2 dependent cutaneous T-cell lymphoma (CTCL) line, SeAx, and primary human CD8+ primary T cells when hoIL-2Rb is expressed in these cells. HoIL-2 at concentrations 1000-fold higher than wild-type IL-2 failed to induce signaling in the absence of the orthogonal receptor illustrating the high selectivity of ortho-hIL-2 for the ortho-hIL-2Rβ. Using a CD19-specific, 4-1BB-costimulated CAR T cell (CART19), we show that ortho-hIL-2 induced a dose-dependent increase in ortho-hIL-2Rβ CAR-T cell expansion and serum cytokines in vivo by as much as 1000-fold at 2 weeks following adoptive transfer into NSG mice bearing CD19+ Nalm6 leukemia xenografts and daily ortho-hIL-2 treatment. We further demonstrate that hoIL-2 can rescue the anti-leukemic effect of an otherwise suboptimal CAR T cell dose. In addition to augmenting initial CAR T cell engraftment and efficacy, we also show that ortho-hIL-2 administration initiated at the time of leukemic relapse following CAR T cell therapy can rescue an otherwise failed anti-leukemic response. In aggregate, these data demonstrate the ability of the human orthogonal IL-2 cytokine-receptor system to support T cell expansion in vivo and the potential of combining this approach with CAR T cell immunotherapy to augment the anti-tumor efficacy of engineered T cells. Disclosures Sockolosky: Synthekine Therapeutics: Current equity holder in private company. Garcia:Synthekine: Current equity holder in private company, Other: founder. Milone:Novartis: Patents & Royalties.

Gene Transfer in Adeno-Associated Virus Seropositive Rhesus Macaques Following Rapamycin Treatment and Subcutaneous Delivery of AAV6, but Not Retargeted AAV6 Vectors.

Adeno-associated virus (AAV) vectors such as AAV6, which shows tropism for primary human CD4+ T cells in vitro, are being explored for delivery of anti-HIV therapeutic modalities in vivo. However, pre-existing immunity and sequestration in nontarget organs can significantly hinder their performance. To overcome these challenges, we investigated whether immunosuppression would allow gene delivery by AAV6 or targeted AAV6 derivatives in seropositive rhesus macaques. Animals were immune suppressed with rapamycin before intravenous (IV) or subcutaneous (SC) delivery of AAV, and we monitored vector biodistribution, gene transfer, and safety. Macaques received phosphate-buffered saline, AAV6 alone, or an equal dose of AAV6 and an AAV6-55.2 vector retargeted to CD4 through a direct ankyrin repeat protein (DARPin). AAV6 and AAV6-55.2 vector genomes were found in peripheral blood mononuclear cells and most organs up to 28 days postadministration, with the highest levels seen in liver, spleen, lymph nodes (LNs), and muscle, suggesting that retargeting did not prevent vector sequestration. Despite vector genome detection, gene expression from AAV6-55.2 was not detected in any tissue. SC injection of AAV6 facilitated efficient gene expression in muscle adjacent to the injection site, plus low-level gene expression in spleen, LNs, and liver, whereas gene expression following IV injection of AAV6 was predominantly seen in the spleen. AAV vectors were well tolerated, although elevated liver enzymes were detected in three of four AAV-treated animals 14 days after rapamycin withdrawal. One SC-injected animal had muscle inflammation proximal to the injection site, plus detectable T cell responses against transgene and AAV6 capsid at study finish. Overall, our data suggest that rapamycin treatment may offer a possible strategy to express anti-HIV therapeutics such as broadly neutralizing antibodies from muscle. This study provides important safety and efficacy data that will aid study design for future anti-HIV gene therapies.

Identification of a Distal Locus Enhancer Element That Controls Cell Type-Specific TNF and LTA Gene Expression in Human T Cells.

The human TNF/LT locus genes TNF, LTA, and LTB are expressed in a cell type-specific manner. In this study, we show that a highly conserved NFAT binding site within the distal noncoding element hHS-8 coordinately controls TNF and LTA gene expression in human T cells. Upon activation of primary human CD4+ T cells, hHS-8 and the TNF and LTA promoters display increased H3K27 acetylation and nuclease sensitivity and coordinate induction of TNF, LTA, and hHS-8 enhancer RNA transcription occurs. Functional analyses using CRISPR/dead(d)Cas9 targeting of the hHS-8-NFAT site in the human T cell line CEM demonstrate significant reduction of TNF and LTA mRNA synthesis and of RNA polymerase II recruitment to their promoters. These studies elucidate how a distal element regulates the inducible cell type-specific gene expression program of the human TNF/LT locus and provide an approach for modulation of TNF and LTA transcription in human disease using CRISPR/dCas9.

Broadly active zinc finger protein-guided transcriptional activation of HIV-1

Human immunodeficiency virus type 1 (HIV-1) causes a persistent viral infection resulting in the demise of immune regulatory cells. Clearance of HIV-1 infection results in integration of proviral DNA into the genome of host cells, which provides a means for evasion and long-term persistence. A therapeutic compound that specifically targets and sustainably activates a latent HIV-1 provirus could be transformative and is the goal for the “shock-and-kill” approach to a functional cure for HIV-1. Substantial progress has been made toward the development of recombinant proteins that target specific genomic loci for gene activation, repression, or inactivation by directed mutations. However, most of these modalities are too large or too complex for efficient therapeutic application. We describe here the development and testing of a novel recombinant zinc finger protein transactivator, ZFP-362-VPR, which specifically and potently enhances proviral HIV-1 transcription both in established latency models and activity across different viral clades. Additionally, ZFP-362-VPR-activated HIV-1 reporter gene expression in a well-established primary human CD4+ T cell latency model and off-target pathways were determined by transcriptome analyses. This study provides clear proof of concept for the application of a novel, therapeutically relevant, protein transactivator to purge cellular reservoirs of HIV-1.

Canonical Wnts Mediate CD8+ T Cell Noncytolytic Anti-HIV-1 Activity and Correlate with HIV-1 Clinical Status.

CD8+ T cells do not rely solely on cytotoxic functions for significant HIV control. Moreover, the noncytotoxic CD8+ T cell antiviral response is a primary mediator of natural HIV control such as that seen in HIV elite controllers and long-term nonprogressors that does not require combined antiretroviral therapy. In this study, we investigated the biological factors contributing to the noncytotoxic control of HIV replication mediated by primary human CD8+ T cells. We report that canonical Wnt signaling inhibits HIV transcription in an MHC-independent, noncytotoxic manner and that mediators of this pathway correlate with HIV controller clinical status. We show that CD8+ T cells express all 19 Wnts and CD8+ T cell-conditioned medium (CM) induced canonical Wnt signaling in infected recipient cells while simultaneously inhibiting HIV transcription. Antagonizing canonical Wnt activity in CD8+ T cell CM resulted in increased HIV transcription in infected cells. Further, Wnt2b expression was upregulated in HIV controllers versus viremic patients, and in vitro depletion of Wnt2b and/or Wnt9b from CD8+ CM reversed HIV inhibitory activity. Finally, plasma concentration of Dkk-1, an antagonist of canonical Wnt signaling, was higher in viremic patients with lower CD4 counts. This study demonstrates that canonical Wnt signaling inhibits HIV and significantly correlates with HIV controller status.

Persistent STAT5 activation reprograms the epigenetic landscape in CD4+ T cells to drive polyfunctionality and antitumor immunity.

The presence of polyfunctional CD4+ T cells is often associated with favorable antitumor immunity. We report here that persistent activation of signal transducer and activator of transcription 5 (STAT5) in tumor-specific CD4+ T cells drives the development of polyfunctional T cells. We showed that ectopic expression of a constitutively active form of murine STAT5A (CASTAT5) enabled tumor-specific CD4+ T cells to undergo robust expansion, infiltrate tumors vigorously, and elicit antitumor CD8+ T cell responses in a CD4+ T cell adoptive transfer model system. Integrated epigenomic and transcriptomic analysis revealed that CASTAT5 induced genome-wide chromatin remodeling in CD4+ T cells and established a distinct epigenetic and transcriptional landscape. Single-cell RNA sequencing analysis further identified a subset of CASTAT5-transduced CD4+ T cells with a molecular signature indicative of progenitor polyfunctional T cells. The therapeutic significance of CASTAT5 came from our finding that adoptive transfer of T cells engineered to coexpress CD19-targeting chimeric antigen receptor (CAR) and CASTAT5 gave rise to polyfunctional CD4+ CAR T cells in a mouse B cell lymphoma model. The optimal therapeutic outcome was obtained when both CD4+ and CD8+ CAR T cells were transduced with CASTAT5, indicating that CASTAT5 facilitates productive CD4 help to CD8+ T cells. Furthermore, we provide evidence that CASTAT5 is functional in primary human CD4+ T cells, underscoring its potential clinical relevance. Our results implicate STAT5 as a valid candidate for T cell engineering to generate polyfunctional, exhaustion-resistant, and tumor-tropic antitumor CD4+ T cells to potentiate adoptive T cell therapy for cancer.

Human CD8+ T Cells Exhibit a Shared Antigen Threshold for Different Effector Responses.

T cells recognizing cognate pMHC Ags become activated to elicit a myriad of cellular responses, such as target cell killing and the secretion of different cytokines, that collectively contribute to adaptive immunity. These effector responses have been hypothesized to exhibit different Ag dose and affinity thresholds, suggesting that pathogen-specific information may be encoded within the nature of the Ag. In this study, using systematic experiments in a reductionist system, in which primary human CD8+ T cell blasts are stimulated by recombinant peptides presented on MHC Ag alone, we show that different inflammatory cytokines have comparable Ag dose thresholds across a 25,000-fold variation in affinity. Although costimulation by CD28, CD2, and CD27 increased cytokine production in this system, the Ag threshold remained comparable across different cytokines. When using primary human memory CD8+ T cells responding to autologous APCs, equivalent thresholds were also observed for different cytokines and killing. These findings imply a simple phenotypic model of TCR signaling in which multiple T cell responses share a common rate-limiting threshold and a conceptually simple model of CD8+ T cell Ag recognition, in which Ag dose and affinity do not provide any additional response-specific information.

IL-10 provides cardioprotection in diabetic myocardial infarction via upregulation of Heme clearance pathways.

Diabetes is a risk factor for myocardial infarction, and outcomes after myocardial infarction are worse among diabetics compared with nondiabetics. Diabetes is associated with impaired Heme clearance. Here, we determined whether heme toxicity and impaired heme clearance contribute to diabetic myocardial infarction injury and assessed IL-10 as a therapeutic agent for diabetic myocardial infarction. Plasma-free hemoglobin was significantly elevated in diabetic mice compared with nondiabetic mice after myocardial infarction. Infarct size had strong correlation to the level of plasma-free hemoglobin. Hemoglobin and reactive iron deposition within the infarct zone were also demonstrated in diabetic MI. IL-10 significantly reduced infarct size and improved cardiac function in diabetic mice. Moreover, IL-10 improved capillary density, reduced apoptosis, and decreased inflammation in the border zone of the infarcted hearts, findings that were partially inhibited by Tin protoporphyrin (a heme oxygenase-1 inhibitor). IL-10 upregulated CD163, the hemoglobin:haptoglobin scavenger receptor, and heme oxygenase-1 in THP-1–derived and primary human CD14+ macrophages. IL-10 significantly protected against ischemic injury when HL-1 cardiomyocytes were cotreated with hemoglobin. Together, our findings indicate that IL-10 is cardioprotective in diabetic myocardial infarction via upregulation of heme clearance pathways. These findings implicate heme clearance as a potentially novel therapeutic direction for diabetic myocardial infarction.

CARD8 inflammasome activation triggers pyroptosis in human T cells

Inflammasomes execute a unique type of cell death known as pyroptosis. Mostly characterized in myeloid cells, caspase‐1 activation downstream of an inflammasome sensor results in the cleavage and activation of gasdermin D (GSDMD), which then forms a lytic pore in the plasma membrane. Recently, CARD8 was identified as a novel inflammasome sensor that triggers pyroptosis in myeloid leukemia cells upon inhibition of dipeptidyl‐peptidases (DPP). Here, we show that blocking DPPs using Val‐boroPro triggers a lytic form of cell death in primary human CD4 and CD8 T cells, while other prototypical inflammasome stimuli were not active. This cell death displays morphological and biochemical hallmarks of pyroptosis. By genetically dissecting candidate components in primary T cells, we identify this response to be dependent on the CARD8‐caspase‐1‐GSDMD axis. Moreover, DPP9 constitutes the relevant DPP restraining CARD8 activation. Interestingly, this CARD8‐induced pyroptosis pathway can only be engaged in resting, but not in activated T cells. Altogether, these results broaden the relevance of inflammasome signaling and associated pyroptotic cell death to T cells, central players of the adaptive immune system.

Trehalose Inhibits Human Immunodeficiency Virus Type 1 Infection in Primary Human Macrophages and CD4+ T Lymphocytes through Two Distinct Mechanisms.

Autophagy is a highly conserved recycling pathway that promotes cell survival during periods of stress. We previously reported that induction of autophagy through the inhibition of the mechanistic target of rapamycin (MTOR) inhibits HIV replication in human macrophages and CD4+ T lymphocytes (T cells). However, the inhibition of MTOR has modulatory effects beyond autophagy that might affect viral replication. Here, we examined the effect on HIV replication of trehalose, a nontoxic, nonreducing disaccharide that induces autophagy through an MTOR-independent mechanism. Treatment of HIV-infected macrophages and T cells with trehalose inhibited infection in a dose-dependent manner. Uninfected and HIV-infected macrophages and T cells treated with trehalose exhibited increased markers of autophagy, including LC3B lipidation with further accumulation following bafilomycin A1 treatment, and increased levels of LAMP1, LAMP2, and RAB7 proteins required for lysosomal biogenesis and fusion. Moreover, the inhibition of HIV by trehalose was significantly reduced by knockdown of ATG5 Additionally, trehalose downregulated the expression of C-C motif chemokine receptor 5 (CCR5) in T cells and CD4 in both T cells and macrophages, which reduced HIV entry in these cells. Our data demonstrate that the naturally occurring sugar trehalose at doses safely achieved in humans inhibits HIV through two mechanisms: (i) decreased entry through the downregulation of CCR5 in T cells and decreased CD4 expression in both T cells and macrophages and (ii) degradation of intracellular HIV through the induction of MTOR-independent autophagy. These findings demonstrate that cellular mechanisms can be modulated to inhibit HIV entry and intracellular replication using a naturally occurring, nontoxic sugar.IMPORTANCE Induction of autophagy through inhibition of MTOR has been shown to inhibit HIV replication. However, inhibition of the mechanistic target of rapamycin (MTOR) has cellular effects that may alter HIV infection through other mechanisms. Here, we examined the HIV-inhibitory effects of the MTOR-independent inducer of autophagy, trehalose. Of note, we identified that in addition to the inhibition of the intracellular replication of HIV by autophagy, trehalose decreased viral entry in human primary macrophages and CD4+ T cells through the downregulation of C-C motif chemokine receptor 5 (CCR5) in T cells and CD4 in both T cells and macrophages. Thus, we showed that trehalose uniquely inhibits HIV replication through inhibition of viral entry and intracellular degradation in the two most important target cells for HIV infection.

Metformin and 2-Deoxyglucose Collaboratively Suppress Human CD4+ T Cell Effector Functions and Activation-Induced Metabolic Reprogramming.

Metabolic reprogramming plays a central role in T cell activation and differentiation, and the inhibition of key metabolic pathways in activated T cells represents a logical approach for the development of new therapeutic agents for treating autoimmune diseases. The widely prescribed antidiabetic drug metformin and the glycolytic inhibitor 2-deoxyglucose (2-DG) have been used to study the inhibition of oxidative phosphorylation and glycolysis, respectively, in murine immune cells. Published studies have demonstrated that combination treatment with metformin and 2-DG was efficacious in dampening mouse T cell activation-induced effector processes, relative to treatments with either metformin or 2-DG alone. In this study, we report that metformin + 2-DG treatment more potently suppressed IFN-γ production and cell proliferation in activated primary human CD4+ T cells than either metformin or 2-DG treatment alone. The effects of metformin + 2-DG on human T cells were accompanied by significant remodeling of activation-induced metabolic transcriptional programs, in part because of suppression of key transcriptional regulators MYC and HIF-1A. Accordingly, metformin + 2-DG treatment significantly suppressed MYC-dependent metabolic genes and processes, but this effect was found to be independent of mTORC1 signaling. These findings reveal significant insights into the effects of metabolic inhibition by metformin + 2-DG treatment on primary human T cells and provide a basis for future work aimed at developing new combination therapy regimens that target multiple pathways within the metabolic networks of activated human T cells.

CCL21-loaded 3D hydrogels for T cell expansion and differentiation

Recent achievements in the field of immunotherapy, such as the development of engineered T cells used in adoptive cell therapy, are introducing more efficient strategies to combat cancer. Nevertheless, there are still many limitations. For example, these T cells are challenging to manufacture, manipulate, and control. Specifically, there are limitations in producing the large amounts of therapeutic T cells needed for these therapies in a short period of time and in an economically viable manner. In this study, three-dimensional (3D) poly(ethylene) glycol (PEG) hydrogels covalently combined with low molecular weight heparin are engineered to resemble the lymph nodes, where T cells reproduce. In these hydrogels, PEG provides the needed structural and mechanical properties, whereas heparin is used as an anchor for the cytokine CCL21, which is present in the lymph nodes, and can affect cell migration and proliferation. The 3D structure of the hydrogel in combination with its loading capacity result in an increased primary human CD4+ T cell proliferation compared to the state-of-the-art expansion systems consisting of artificial antigen presenting cells. Thus, we present a new tool for adoptive cell therapy to help achieving the large numbers of cells required for therapy of selected phenotypes targeted against cancer cells, by mimicking the lymph nodes.