Microbiome and Metabolome driven differentiation of TGF-beta producing Tregs leads to Senescence and HIV latency

Current therapeutic interventions to eradicate latent HIV ("reservoir") and restore immune function in ART-treated HIV infection have yet to show efficacy. To explore mechanisms of HIV persistence, we apply an integrated systems biology approach and identify a distinct group of individuals with poor CD4 T-cell reconstitution (Immunologic non-responders, "INRs") and high frequencies of cells with inducible HIV. Contrary to the prevailing notion that immune activation drives HIV persistence and immune dysfunction, peripheral blood leukocytes from these subjects have enhanced expression of a network of genes regulated by cellular senescence driving transcription factors (TFs) FOXO3, SMAD2 and IRF3. In these subjects, increased frequencies of regulatory T cells and expression of the TGF-β signaling cascade are complimented by the downregulation of cell cycle, metabolic and pro-inflammatory pathways. Lactobacillaceae family and metabolites (members of the butyrate family - i.e. α-ketobutyrate) were correlated with Treg frequencies in "Senescent-INRs" ex vivo, triggered the differentiation of TGF-β producing Tregs and promoted HIV latency establishment in vitro. These cascades, downstream of PD-1/TGF-β prevent memory T cell differentiation and are associated with an increase in frequencies of cells with inducible HIV ex vivo. Our findings identify cellular senescence responses that can be targeted by PD-1 or TGF-β specific interventions that have shown safety and efficacy in cancer, and may prove to be crucial for HIV eradication.

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TGF-β associated senescence and impaired metabolism in central memory CD4 T cells promotes HIV persistence

10.21203/rs.3.rs-923015/v1 ◽

2021 ◽

Author(s):

Rafick Sekaly ◽

Khader Ghneim ◽

Ashish Sharma ◽

Susan Ribeiro ◽

Slim Fourati ◽

...

Keyword(s):

T Cells ◽

Cellular Senescence ◽

Ex Vivo ◽

Distinct Group ◽

Central Memory ◽

Therapeutic Interventions ◽

Blood Leukocytes ◽

Hiv Persistence ◽

Latent Hiv

Abstract Current therapeutic interventions to eradicate latent HIV (“reservoir”) and restore immune function in ART-treated HIV infection have yet to show efficacy. To explore mechanisms of HIV persistence, we apply an integrated systems biology approach and identify a distinct group of individuals with poor CD4 T-cell reconstitution (Immunologic non-responders, “INRs”) and high frequencies of cells with inducible HIV. Contrary to the prevailing notion that immune activation drives HIV persistence and immune dysfunction, peripheral blood leukocytes from these subjects have enhanced expression of a network of genes regulated by cellular senescence. In these subjects, increased frequencies of regulatory T cells and expression of the TGF-β signaling cascade are concomitant with the downregulation of cell cycle and metabolism in CD4 central memory T (TCM) cells. These cascades, downstream of TGF-β, lead to the accumulation of PD-1 expressing CD4 TCM and are associated with an increase in frequencies of cells with inducible HIV ex vivo. In vitro validation confirmed that this cellular profile was driven by a β-hydroxybutyrates/bile acid rich metabolic milieu and resulted in TGF-β associated latency establishment. Our findings identify targets for PD-1 or TGF-β specific interventions that can overcome cellular senescence; these therapeutic approaches have shown safety and efficacy in cancer, and may prove to be crucial for HIV eradication.

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Differentiation into an Effector Memory Phenotype Potentiates HIV-1 Latency Reversal in CD4+ T Cells

Journal of Virology ◽

10.1128/jvi.00969-19 ◽

2019 ◽

Vol 93 (24) ◽

Cited By ~ 17

Author(s):

Deanna A. Kulpa ◽

Aarthi Talla ◽

Jessica H. Brehm ◽

Susan Pereira Ribeiro ◽

Sally Yuan ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Ex Vivo ◽

T Cell Subsets ◽

Effector Memory ◽

Cell Surface Markers ◽

Cell Cycle Entry ◽

Latent Hiv ◽

Hiv 1

ABSTRACT During antiretroviral therapy (ART), human immunodeficiency virus type 1 (HIV-1) persists as a latent reservoir in CD4+ T cell subsets in central memory (TCM), transitional memory (TTM), and effector memory (TEM) CD4+ T cells. We have identified differences in mechanisms underlying latency and responses to latency-reversing agents (LRAs) in ex vivo CD4+ memory T cells from virally suppressed HIV-infected individuals and in an in vitro primary cell model of HIV-1 latency. Our ex vivo and in vitro results demonstrate the association of transcriptional pathways of T cell differentiation, acquisition of effector function, and cell cycle entry in response to LRAs. Analyses of memory cell subsets showed that effector memory pathways and cell surface markers of activation and proliferation in the TEM subset are predictive of higher frequencies of cells carrying an inducible reservoir. Transcriptional profiling also demonstrated that the epigenetic machinery (known to control latency and reactivation) in the TEM subset is associated with frequencies of cells with HIV-integrated DNA and inducible HIV multispliced RNA. TCM cells were triggered to differentiate into TEM cells when they were exposed to LRAs, and this increase of TEM subset frequencies upon LRA stimulation was positively associated with higher numbers of p24+ cells. Together, these data highlight differences in underlying biological latency control in different memory CD4+ T cell subsets which harbor latent HIV in vivo and support a role for differentiation into a TEM phenotype in facilitating latency reversal. IMPORTANCE By performing phenotypic analysis of latency reversal in CD4+ T cells from virally suppressed individuals, we identify the TEM subset as the largest contributor to the inducible HIV reservoir. Differential responses of memory CD4+ T cell subsets to latency-reversing agents (LRAs) demonstrate that HIV gene expression is associated with heightened expression of transcriptional pathways associated with differentiation, acquisition of effector function, and cell cycle entry. In vitro modeling of the latent HIV reservoir in memory CD4+ T cell subsets identify LRAs that reverse latency with ranges of efficiency and specificity. We found that therapeutic induction of latency reversal is associated with upregulation of identical sets of TEM-associated genes and cell surface markers shown to be associated with latency reversal in our ex vivo and in vitro models. Together, these data support the idea that the effector memory phenotype supports HIV latency reversal in CD4+ T cells.

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764 Expansion with IL-15 increases cytotoxicity of Vγ9Vδ2 T cells and is associated with higher levels of cytotoxic molecules and T-bet

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0764 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A812-A812

Author(s):

Pia Aehnlich ◽

Per Thor Straten ◽

Ana Micaela Carnaz Simoes ◽

Signe Skadborg ◽

Gitte Olofsson

Keyword(s):

T Cells ◽

T Cell ◽

Cell Expansion ◽

Ex Vivo ◽

Adoptive Cell Therapy ◽

Hematological Cancers ◽

Cytotoxic Molecules ◽

Vγ9vδ2 T Cells ◽

T Cell Expansion

BackgroundAdoptive cell therapy (ACT) is an approved treatment option for certain hematological cancers and has also shown success for some solid cancers. Still, benefit and eligibility do not extend to all patients. ACT with Vγ9Vδ2 T cells is a promising approach to overcome this hurdle.MethodsIn this study, we explored the effect of different cytokine conditions on the expansion of Vγ9Vδ2 T cells in vitro.ResultsWe could show that Vγ9Vδ2 T cell expansion is feasible with two different cytokine conditions: (a) 1000U/ml interleukin (IL)-2 and (b) 100U/ml IL-2+100U/ml IL-15. We did not observe differences in expansion rate or Vγ9Vδ2 T cell purity between the conditions; however, IL-2/IL-15-expanded Vγ9Vδ2 T cells displayed enhanced cytotoxicity against tumor cells, also in hypoxia. While this increase in killing capacity was not reflected in phenotype, we demonstrated that IL-2/IL-15-expanded Vγ9Vδ2 T cells harbor increased amounts of perforin, granzyme B and granulysin in a resting state and release more upon activation. IL-2/IL-15-expanded Vγ9Vδ2 T cells also showed higher levels of transcription factor T-bet, which could indicate that T-bet and cytotoxic molecule levels confer the increased cytotoxicity.ConclusionsThese results advocate the inclusion of IL-15 into ex vivo Vγ9Vδ2 T cell expansion protocols in future clinical studies.

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Dynamic Imaging of IEL-IEC Co-Cultures Allows for Quantification of CD103-Dependent T Cell Migration

International Journal of Molecular Sciences ◽

10.3390/ijms22105148 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5148

Author(s):

Karin Enderle ◽

Martin Dinkel ◽

Eva-Maria Spath ◽

Benjamin Schmid ◽

Sebastian Zundler ◽

...

Keyword(s):

T Cell ◽

Cross Talk ◽

Intestinal Inflammation ◽

Ex Vivo ◽

New Technology ◽

Surface Expression ◽

Intraepithelial Lymphocytes ◽

Dynamic Imaging ◽

The Body ◽

Therapeutic Interventions

Intraepithelial lymphocytes (IEL) are widely distributed within the small intestinal epithelial cell (IEC) layer and represent one of the largest T cell pools of the body. While implicated in the pathogenesis of intestinal inflammation, detailed insight especially into the cellular cross-talk between IELs and IECs is largely missing in part due to lacking methodologies to monitor this interaction. To overcome this shortcoming, we employed and validated a murine IEL-IEC (organoids) ex vivo co-culture model system. Using livecell imaging we established a protocol to visualize and quantify the spatio-temporal migratory behavior of IELs within organoids over time. Applying this methodology, we found that IELs lacking CD103 (i.e., integrin alpha E, ITGAE) surface expression usually functioning as a retention receptor for IELs through binding to E-cadherin (CD324) expressing IECs displayed aberrant mobility and migration patterns. Specifically, CD103 deficiency affected the ability of IELs to migrate and reduced their speed during crawling within organoids. In summary, we report a new technology to monitor and quantitatively assess especially migratory characteristics of IELs communicating with IEC ex vivo. This approach is hence readily applicable to study the effects of targeted therapeutic interventions on IEL-IEC cross-talk.

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Targeting human Acyl-CoA:cholesterol acyltransferase as a dual viral and T cell metabolic checkpoint

Nature Communications ◽

10.1038/s41467-021-22967-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Nathalie M. Schmidt ◽

Peter A. C. Wing ◽

Mariana O. Diniz ◽

Laura J. Pallett ◽

Leo Swadling ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Human Liver ◽

Lipid Droplets ◽

Ex Vivo ◽

Functional Cure ◽

Carcinogenic Activity ◽

Attractive Therapeutic Target ◽

Tcr Signalling

AbstractDetermining divergent metabolic requirements of T cells, and the viruses and tumours they fail to combat, could provide new therapeutic checkpoints. Inhibition of acyl-CoA:cholesterol acyltransferase (ACAT) has direct anti-carcinogenic activity. Here, we show that ACAT inhibition has antiviral activity against hepatitis B (HBV), as well as boosting protective anti-HBV and anti-hepatocellular carcinoma (HCC) T cells. ACAT inhibition reduces CD8+ T cell neutral lipid droplets and promotes lipid microdomains, enhancing TCR signalling and TCR-independent bioenergetics. Dysfunctional HBV- and HCC-specific T cells are rescued by ACAT inhibitors directly ex vivo from human liver and tumour tissue respectively, including tissue-resident responses. ACAT inhibition enhances in vitro responsiveness of HBV-specific CD8+ T cells to PD-1 blockade and increases the functional avidity of TCR-gene-modified T cells. Finally, ACAT regulates HBV particle genesis in vitro, with inhibitors reducing both virions and subviral particles. Thus, ACAT inhibition provides a paradigm of a metabolic checkpoint able to constrain tumours and viruses but rescue exhausted T cells, rendering it an attractive therapeutic target for the functional cure of HBV and HBV-related HCC.

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CD14 Expressing Precursors Give Rise to Highly Functional Conventional Dendritic Cells for Use as Dendritic Cell Vaccine

Cancers ◽

10.3390/cancers13153818 ◽

2021 ◽

Vol 13 (15) ◽

pp. 3818

Author(s):

Maud Plantinga ◽

Denise A. M. H. van den Beemt ◽

Ester Dünnebach ◽

Stefan Nierkens

Keyword(s):

Dendritic Cells ◽

T Cell ◽

Cord Blood ◽

T Cell Activation ◽

Cell Activation ◽

Ex Vivo ◽

Dendritic Cell Vaccine ◽

Cell Vaccine ◽

Activated T Cells

Induction of long-lasting immunity by dendritic cells (DCs) makes them attractive candidates for anti-tumor vaccination. Although DC vaccinations are generally considered safe, clinical responses remain inconsistent in clinical trials. This initiated studies to identify subsets of DCs with superior capabilities to induce effective and memory anti-tumor responses. The use of primary DCs has been suggested to overcome the functional limitations of ex vivo monocyte-derived DCs (moDC). The ontogeny of primary DCs has recently been revised by the introduction of DC3, which phenotypically resembles conventional (c)DC2 as well as moDC. Previously, we developed a protocol to generate cDC2s from cord blood (CB)-derived stem cells via a CD115-expressing precursor. Here, we performed index sorting and single-cell RNA-sequencing to define the heterogeneity of in vitro developed DC precursors and identified CD14+CD115+ expressing cells that develop into CD1c++DCs and the remainder cells brought about CD123+DCs, as well as assessed their potency. The maturation status and T-cell activation potential were assessed using flow cytometry. CD123+DCs were specifically prone to take up antigens but only modestly activated T-cells. In contrast, CD1c++ are highly mature and specialized in both naïve as well as antigen-experienced T-cell activation. These findings show in vitro functional diversity between cord blood stem cell-derived CD123+DC and CD1c++DCs and may advance the efficiency of DC-based vaccines.

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IMMU-31. QUANTITATIVE EVALUATION OF ROUTES OF ADMINISTRATION OF IMMUNOTHERAPEUTIC T CELLS TO ORTHOTOPIC GLIOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa215.461 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii111-ii111

Author(s):

Lan Hoang-Minh ◽

Angelie Rivera-Rodriguez ◽

Fernanda Pohl-Guimarães ◽

Seth Currlin ◽

Christina Von Roemeling ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Growth ◽

Ex Vivo ◽

Adoptive T Cell Therapy ◽

Whole Body ◽

T Cell Therapy ◽

Clinical Responses

Abstract SIGNIFICANCE Adoptive T cell therapy (ACT) has emerged as the most effective treatment against advanced malignant melanoma, eliciting remarkable objective clinical responses in up to 75% of patients with refractory metastatic disease, including within the central nervous system. Immunologic surrogate endpoints correlating with treatment outcome have been identified in these patients, with clinical responses being dependent on the migration of transferred T cells to sites of tumor growth. OBJECTIVE We investigated the biodistribution of intravenously or intraventricularly administered T cells in a murine model of glioblastoma at whole body, organ, and cellular levels. METHODS gp100-specific T cells were isolated from the spleens of pmel DsRed transgenic C57BL/6 mice and injected intravenously or intraventricularly, after in vitro expansion and activation, in murine KR158B-Luc-gp100 glioma-bearing mice. To determine transferred T cell spatial distribution, the brain, lymph nodes, heart, lungs, spleen, liver, and kidneys of mice were processed for 3D imaging using light-sheet and multiphoton imaging. ACT T cell quantification in various organs was performed ex vivo using flow cytometry, 2D optical imaging (IVIS), and magnetic particle imaging (MPI) after ferucarbotran nanoparticle transfection of T cells. T cell biodistribution was also assessed in vivo using MPI. RESULTS Following T cell intravenous injection, the spleen, liver, and lungs accounted for more than 90% of transferred T cells; the proportion of DsRed T cells in the brains was found to be very low, hovering below 1%. In contrast, most ACT T cells persisted in the tumor-bearing brains following intraventricular injections. ACT T cells mostly concentrated at the periphery of tumor masses and in proximity to blood vessels. CONCLUSIONS The success of ACT immunotherapy for brain tumors requires optimization of delivery route, dosing regimen, and enhancement of tumor-specific lymphocyte trafficking and effector functions to achieve maximal penetration and persistence at sites of invasive tumor growth.

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Native CGRP Neuropeptide and Its Stable Analogue SAX, But Not CGRP Peptide Fragments, Inhibit Mucosal HIV-1 Transmission

Frontiers in Immunology ◽

10.3389/fimmu.2021.785072 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jammy Mariotton ◽

Anette Sams ◽

Emmanuel Cohen ◽

Alexis Sennepin ◽

Gabriel Siracusano ◽

...

Keyword(s):

T Cell ◽

Ex Vivo ◽

Receptor Activation ◽

Humanized Mice ◽

Peptide Fragments ◽

Cgrp Receptor ◽

Trans Infection ◽

Anti Hiv ◽

Hiv 1

BackgroundThe vasodilator neuropeptide calcitonin gene-related peptide (CGRP) plays both detrimental and protective roles in different pathologies. CGRP is also an essential component of the neuro-immune dialogue between nociceptors and mucosal immune cells. We previously discovered that CGRP is endowed with anti-viral activity and strongly inhibits human immunodeficiency virus type 1 (HIV-1) infection, by suppressing Langerhans cells (LCs)-mediated HIV-1 trans-infection in-vitro and mucosal HIV-1 transmission ex-vivo. This inhibition is mediated via activation of the CGRP receptor non-canonical NFκB/STAT4 signaling pathway that induces a variety of cooperative mechanisms. These include CGRP-mediated increase in the expression of the LC-specific pathogen recognition C-type lectin langerin and decrease in LC-T-cell conjugates formation. The clinical utility of CGRP and modalities of CGRP receptor activation, for inhibition of mucosal HIV-1 transmission, remain elusive.MethodsWe tested the capacity of CGRP to inhibit HIV-1 infection in-vivo in humanized mice. We further compared the anti-HIV-1 activities of full-length native CGRP, its metabolically stable analogue SAX, and several CGRP peptide fragments containing its binding C-terminal and activating N-terminal regions. These agonists were evaluated for their capacity to inhibit LCs-mediated HIV-1 trans-infection in-vitro and mucosal HIV-1 transmission in human mucosal tissues ex-vivo.ResultsA single CGRP intravaginal topical treatment of humanized mice, followed by HIV-1 vaginal challenge, transiently restricts the increase in HIV-1 plasma viral loads but maintains long-lasting higher CD4+ T-cell counts. Similarly to CGRP, SAX inhibits LCs-mediated HIV-1 trans-infection in-vitro, but with lower potency. This inhibition is mediated via CGRP receptor activation, leading to increased expression of both langerin and STAT4 in LCs. In contrast, several N-terminal and N+C-terminal bivalent CGRP peptide fragments fail to increase langerin and STAT4, and accordingly lack anti-HIV-1 activities. Finally, like CGRP, treatment of human inner foreskin tissue explants with SAX, followed by polarized inoculation with cell-associated HIV-1, completely blocks formation of LC-T-cell conjugates and HIV-1 infection of T-cells.ConclusionOur results show that CGRP receptor activation by full-length CGRP or SAX is required for efficient inhibition of LCs-mediated mucosal HIV-1 transmission. These findings suggest that formulations containing CGRP, SAX and/or their optimized agonists/analogues could be harnessed for HIV-1 prevention.

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Augmented expansion of Treg cells from healthy and autoimmune subjects via adult progenitor cell co-culture

10.1101/2020.12.03.410316 ◽

2020 ◽

Author(s):

JL Reading ◽

VD Roobrouck ◽

CM Hull ◽

PD Becker ◽

J Beyens ◽

...

Keyword(s):

T Cell ◽

Treg Cell ◽

Regulatory T Cell ◽

Cellular Therapy ◽

Ex Vivo ◽

Fold Increase ◽

Adoptive Cellular Therapy ◽

T Cell Therapy

AbstractRecent clinical experience has demonstrated that adoptive regulatory T cell therapy is a safe and feasible strategy to suppress immunopathology via induction of host tolerance to allo- and autoantigens. However, clinical trials continue to be compromised due to an inability to manufacture a sufficient Treg cell dose. Multipotent adult progenitor cells (MAPCⓇ) promote regulatory T cell differentiation in vitro, suggesting they may be repurposed to enhance ex vivo expansion of Tregs for adoptive cellular therapy. Here, we use a GMP compatible Treg expansion platform to demonstrate that MAPC cell-co-cultured Tregs (MulTreg) exhibit a log-fold increase in yield across two independent cohorts, reducing time to target dose by an average of 30%. Enhanced expansion is linked with a distinct Treg cell-intrinsic transcriptional program, characterized by diminished levels of core exhaustion (BATF, ID2, PRDM1, LAYN, DUSP1), and quiescence (TOB1, TSC22D3) related genes, coupled to elevated expression of cell-cycle and proliferation loci (MKI67, CDK1, AURKA, AURKB). In addition, MulTreg display a unique gut homing (CCR7lo β7hi) phenotype and importantly, are more readily expanded from patients with autoimmune disease compared to matched Treg lines, suggesting clinical utility in gut and/or Th1-driven pathology associated with autoimmunity or transplantation. Relative to expanded Tregs, MulTreg retain equivalent and robust purity, FoxP3 TSDR demethylation, nominal effector cytokine production and potent suppression of Th1-driven antigen specific and polyclonal responses in vitro and xeno graft vs host disease (xGvHD) in vivo. These data support the use of MAPC cell co-culture in adoptive Treg therapy platforms as a means to rescue expansion failure and reduce the time required to manufacture a stable, potently suppressive product.

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Biogenesis of P-TEFb in CD4+ T cells to reverse HIV latency is mediated by protein kinase C (PKC)-independent signaling pathways

10.1101/2021.04.26.441433 ◽

2021 ◽

Author(s):

Uri Mbonye ◽

Konstantin Leskov ◽

Meenakshi Shukla ◽

Saba Valadkhan ◽

Jonathan Karn

Keyword(s):

T Cells ◽

Protein Kinase C ◽

T Cell ◽

Protein Kinase ◽

Signaling Pathways ◽

Cd4 T Cells ◽

Hiv Latency ◽

Kinase C ◽

Latent Hiv ◽

Memory Cd4 T Cells

The switch between HIV latency and productive transcription is regulated by an auto-feedback mechanism initiated by the viral trans-activator Tat, which functions to recruit the host transcription elongation factor P-TEFb to proviral HIV. A heterodimeric complex of CDK9 and one of three cyclin T subunits, P-TEFb is expressed at vanishingly low levels in resting memory CD4 + T cells and cellular mechanisms controlling its availability are central to regulation of the emergence of HIV from latency. Using a well-characterized primary T-cell model of HIV latency alongside healthy donor memory CD4 + T cells, we characterized specific T-cell receptor (TCR) signaling pathways that regulate the generation of transcriptionally active P-TEFb, defined as the coordinate expression of cyclin T1 and phospho-Ser175 CDK9. Protein kinase C (PKC) agonists, such as ingenol and prostratin, stimulated active P-TEFb expression and reactivated latent HIV with minimal cytotoxicity, even in the absence of intracellular calcium mobilization with an ionophore. Unexpectedly, inhibition-based experiments demonstrated that PKC agonists and TCR-mobilized diacylglycerol signal through MAP kinases ERK1/2 rather than through PKC to effect the reactivation of both P-TEFb and latent HIV. Single-cell and bulk RNA-seq analyses revealed that of the four known isoforms of the Ras guanine nucleotide exchange factor RasGRP, RasGRP1 is by far the predominantly expressed diacylglycerol-dependent isoform in CD4 + T cells. RasGRP1 should therefore mediate the activation of ERK1/2 via Ras-Raf signaling upon TCR co-stimulation or PKC agonist challenge. Combined inhibition of the PI3K-mTORC2-AKT-mTORC1 pathway and the ERK1/2 activator MEK prior to TCR co-stimulation abrogated active P-TEFb expression and substantially suppressed latent HIV reactivation. Therefore, contrary to prevailing models, the coordinate reactivation of P-TEFb and latent HIV in primary T cells following either TCR co-stimulation or PKC agonist challenge is independent of PKC but rather involves two complementary signaling arms of the TCR cascade, namely, RasGRP1-Ras-Raf-MEK-ERK1/2 and PI3K-mTORC2-AKT-mTORC1.

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