Recombinant human interleukin 10 suppresses gliadin dependent T cell activation in ex vivo cultured coeliac intestinal mucosa

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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A Randomized Phase II Trial of Idiotype Vaccination and Adoptive Autologous T-Cell Transfer in Multiple Myeloma patients

Blood ◽

10.1182/blood.2020008493 ◽

2021 ◽

Author(s):

Muzaffar H Qazilbash ◽

Neeraj Y Saini ◽

Cha Soung-chul ◽

Zhe Wang ◽

Edward Stadtmauer ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Phase Ii ◽

T Cell Activation ◽

Cd8 T Cells ◽

Cell Activation ◽

Phase Ii Trial ◽

Ex Vivo ◽

Vaccine Strategy ◽

Randomized Phase Ii

We hypothesized that combining adoptively transferred autologous T cells with a cancer vaccine strategy would enhance therapeutic efficacy by adding anti-myeloma idiotype-keyhole limpet hemocyanin (Id-KLH) vaccine to vaccine-specific co-stimulated T cells. In this randomized, phase II trial, eligible patients received either the control (KLH only) or Id-KLH vaccine, an auto-transplant, vaccine-specific co-stimulated T-cells expanded ex-vivo, and two booster doses of the assigned vaccine. In 36 patients (20 in KLH, 16 in Id-KLH) enrolled, no dose-limiting toxicity was seen in either arm. At last evaluation, 6 (30%) and 8 (50%) had achieved complete remission in KLH-only and Id-KLH, respectively (p=0.22) and no difference in 3-year progression-free survival was observed (59% and 56%, respectively; p=0.32). In a 594 Nanostring nCounter gene panel analyzed for immune reconstitution (IR), compared with KLH-only patients, there was a greater change in IR genes in T-cells in Id-KLH patients relative to baseline. Specifically, upregulation of genes associated with activation, induction of effector function, and generation of memory CD8+ T cells after Id-KLH, but not after KLH control vaccination, was observed. Similarly, responding patients across both arms were associated with upregulation of genes associated with T-cell activation. At baseline, all patients had greater expression of CD8+ T-cell exhaustion markers. These changes were associated with functional Id-specific immune responses in a subset of Id-KLH patients analyzed. In conclusion, in this combination immunotherapy approach, we observed a significantly more robust IR in CD4+ and CD8+ T cells in the Id-KLH arm, supporting further investigation of vaccine and adoptive immunotherapy strategies.

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Inconsistent Reversal of HIV-1 Latency Ex Vivo by Antigens of HIV-1, CMV, and Other Infectious Agents

10.21203/rs.3.rs-35386/v1 ◽

2020 ◽

Author(s):

Thomas Vollbrecht ◽

Aaron O. Angerstein ◽

Bryson Menke ◽

Nikesh M. Kumar ◽

Michelli Faria Oliveira ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd4 T Cells ◽

Cell Activation ◽

Ex Vivo ◽

Latent Reservoir ◽

Antigen Specificity ◽

Pcr Assay ◽

Hiv 1

Abstract BackgroundA reservoir of replication-competent but latent virus is the main obstacle to a cure for HIV-infection. Much of this reservoir resides in memory CD4 T cells. We hypothesized that these cells can be reactivated with antigens from HIV and other common pathogens to reverse latency. ResultsWe obtained mononuclear cells from the peripheral blood of antiretroviral-treated patients with suppressed viremia. We tested pools of peptides and proteins derived from HIV and from other pathogens including CMV for their ability to reverse latency ex vivo by activation of memory responses. We assessed activation of the CD4 T cells by measuring the up-regulation of cell-surface CD69. We assessed HIV-expression using two assays: a real-time PCR assay for virion-associated viral RNA and a droplet digital PCR assay for cell-associated, multiply spliced viral mRNA. Reversal of latency occurred in a minority of cells from some participants, but no single antigen induced HIV-expression ex vivo consistently. When reversal of latency was induced by a specific peptide pool or protein, the extent was proportionally greater than that of T cell activation. ConclusionsIn this group of patients in whom antiretroviral therapy was started during chronic infection, the latent reservoir does not appear to consistently reside in CD4 T cells of a predominant antigen-specificity. Peptide-antigens reversed HIV-latency ex vivo with modest and variable activity. When latency was reversed by specific peptides or proteins, it was proportionally greater than the extent of T cell activation, suggesting partial enrichment of the latent reservoir in cells of specific antigen-reactivity.

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Tumor masses support naive T cell infiltration, activation, and differentiation into effectors

Journal of Experimental Medicine ◽

10.1084/jem.20092454 ◽

2010 ◽

Vol 207 (8) ◽

pp. 1791-1804 ◽

Cited By ~ 134

Author(s):

Elizabeth D. Thompson ◽

Hilda L. Enriquez ◽

Yang-Xin Fu ◽

Victor H. Engelhard

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd8 T Cells ◽

Adoptive Transfer ◽

Cell Activation ◽

Ex Vivo ◽

Tumor Infiltrating Lymphocytes ◽

T Cell Infiltration

Studies of T cell responses to tumors have focused on the draining lymph node (LN) as the site of activation. We examined the tumor mass as a potential site of activation after adoptive transfer of naive tumor-specific CD8 T cells. Activated CD8 T cells were present in tumors within 24 h of adoptive transfer and proliferation of these cells was also evident 4–5 d later in mice treated with FTY720 to prevent infiltration of cells activated in LNs. To confirm that activation of these T cells occurred in the tumor and not the tumor-draining LNs, we used mice lacking LNs. Activated and proliferating tumor-infiltrating lymphocytes were evident in these mice 24 h and 4 d after naive cell transfer. T cells activated within tumors acquired effector function that was evident both ex vivo and in vivo. Both cross-presenting antigen presenting cells within the tumor and tumor cells directly presenting antigen activated these functional CD8 effectors. We conclude that tumors support the infiltration, activation, and effector differentiation of naive CD8 T cells, despite the presence of immunosuppressive mechanisms. Thus, targeting of T cell activation to tumors may present a tool in the development of cancer immunotherapy.

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GABAA receptor α4-subunit knockout enhances lung inflammation and airway reactivity in a murine asthma model

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00107.2017 ◽

2017 ◽

Vol 313 (2) ◽

pp. L406-L415 ◽

Cited By ~ 14

Author(s):

Gene T. Yocum ◽

Damian L. Turner ◽

Jennifer Danielsson ◽

Matthew B. Barajas ◽

Yi Zhang ◽

...

Keyword(s):

T Cell ◽

T Cell Activation ◽

Immune Cells ◽

Cell Activation ◽

Cell Function ◽

Immune Cell ◽

Therapeutic Potential ◽

Ex Vivo ◽

Asthma Model

Emerging evidence indicates that hypnotic anesthetics affect immune function. Many anesthetics potentiate γ-aminobutyric acid A receptor (GABAAR) activation, and these receptors are expressed on multiple subtypes of immune cells, providing a potential mechanistic link. Like immune cells, airway smooth muscle (ASM) cells also express GABAARs, particularly isoforms containing α4-subunits, and activation of these receptors leads to ASM relaxation. We sought to determine if GABAAR signaling modulates the ASM contractile and inflammatory phenotype of a murine allergic asthma model utilizing GABAAR α4-subunit global knockout (KO; Gabra40/0) mice. Wild-type (WT) and Gabra4 KO mice were sensitized with house dust mite (HDM) antigen or exposed to PBS intranasally 5 days/wk for 3 wk. Ex vivo tracheal rings from HDM-sensitized WT and Gabra4 KO mice exhibited similar magnitudes of acetylcholine-induced contractile force and isoproterenol-induced relaxation ( P = not significant; n = 4). In contrast, in vivo airway resistance (flexiVent) was significantly increased in Gabra4 KO mice ( P < 0.05, n = 8). Moreover, the Gabra4 KO mice demonstrated increased eosinophilic lung infiltration ( P < 0.05; n = 4) and increased markers of lung T-cell activation/memory (CD62L low, CD44 high; P < 0.01, n = 4). In vitro, Gabra4 KO CD4+ cells produced increased cytokines and exhibited increased proliferation after stimulation of the T-cell receptor as compared with WT CD4+ cells. These data suggest that the GABAAR α4-subunit plays a role in immune cell function during allergic lung sensitization. Thus GABAAR α4-subunit-specific agonists have the therapeutic potential to treat asthma via two mechanisms: direct ASM relaxation and inhibition of airway inflammation.

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Blockade of Costimulation Prevents Infection-Induced Immunopathology in Interleukin-10-Deficient Mice

Infection and Immunity ◽

10.1128/iai.68.5.2837-2844.2000 ◽

2000 ◽

Vol 68 (5) ◽

pp. 2837-2844 ◽

Cited By ~ 25

Author(s):

Eric N. Villegas ◽

Ulrike Wille ◽

Linden Craig ◽

Peter S. Linsley ◽

Donna M. Rennick ◽

...

Keyword(s):

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Serum Levels ◽

Cd40 Ligand ◽

Interleukin 10 ◽

Cell Mediated Immunity ◽

Ifn Γ ◽

Oxide Synthase

ABSTRACT Interleukin-10 (IL-10) is associated with inhibition of cell-mediated immunity and downregulation of the expression of costimulatory molecules required for T-cell activation. When IL-10-deficient (IL-10KO) mice are infected with Toxoplasma gondii, they succumb to a T-cell-mediated shock-like reaction characterized by the overproduction of IL-12 and gamma interferon (IFN-γ) associated with widespread necrosis of the liver. Since costimulation is critical for T-cell activation, we investigated the role of the CD28-B7 and CD40-CD40 ligand (CD40L) interactions in this infection-induced immunopathology. Our studies show that infection of mice with T. gondii resulted in increased expression of B7 and CD40 that was similar in wild-type and IL-10KO mice. In vivo blockade of the CD28-B7 or CD40-CD40L interactions following infection of IL-10KO mice with T. gondii did not affect serum levels of IFN-γ or IL-12, nor did it prevent death in these mice. However, when both pathways were blocked, the IL-10KO mice survived the acute phase of infection and had reduced serum levels of IFN-γ and alanine transaminase as well as decreased expression of inducible nitric oxide synthase in the liver and spleen. Analysis of parasite-specific recall responses from infected IL-10KO mice revealed that blockade of the CD40-CD40L interaction had minimal effects on cytokine production, whereas blockade of the CD28-B7 interaction resulted in decreased production of IFN-γ but not IL-12. Further reduction of IFN-γ production was observed when both costimulatory pathways were blocked. Together, these results demonstrate that the CD28-B7 and CD40-CD40L interactions are involved in the development of infection-induced immunopathology in the absence of IL-10.

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Flow Cytometric Evaluation of T Cell Activation Markers after Cardiopulmonary Bypass

Surgery Research and Practice ◽

10.1155/2014/801643 ◽

2014 ◽

Vol 2014 ◽

pp. 1-6

Author(s):

Maja-Theresa Dieterlen ◽

Hartmuth B. Bittner ◽

Attila Tarnok ◽

Jens Garbade ◽

Stefan Dhein ◽

...

Keyword(s):

Cardiopulmonary Bypass ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Ex Vivo ◽

Activation Markers ◽

Increased Risk ◽

Patients At Risk ◽

Inflammatory Processes ◽

Activation Pathways

Background. Cardiopulmonary bypass surgery (CPBS) is associated with an increased risk for infections or with subsequent organ dysfunction. As T cell activation is a central mechanism during inflammatory processes, we developed an assay to evaluate T cell activation pathways in patients undergoing CPBS.Methods. Blood was obtained from eleven patients undergoing CPBS preoperatively, on postoperative day (POD)-3, and on POD-7 and was stimulated with different concentrations of Concanavalin A (ConA). Cyclosporine and sirolimus, inhibiting different pathways of the T cell cycle, were added to blood ex vivo. Expression of T cell activation markers CD25 and CD95 was analyzed by flow cytometry.Results. In untreated blood, expression of CD25 and CD95 significantly increased with higher ConA concentrations(P<0.05)and decreased for all ConA concentrations for both antigens over the study time(P<0.05). Independently from the ConA concentration, inhibition of CD25 and CD95 expression was highest preoperatively for sirolimus and on POD-3 for cyclosporine. At all time points, inhibition of CD25 and CD95 expression was significantly higher after cyclosporine compared to sirolimus treatment(P<0.001).Conclusion. Our results showed that different pathways of T cell activation are impaired after CPBS. Such knowledge may offer the opportunity to identify patients at risk for postoperative complications.

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Phase II trial of a GM-CSF-producing and CD40L-expressing cell line combined with allogeneic tumor antigen as a novel vaccine for metastatic lung adenocarcinoma.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.2559 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 2559-2559

Author(s):

Ben C. Creelan ◽

Scott Antonia ◽

David Noyes ◽

Terri B. Hunter ◽

George R. Simon ◽

...

Keyword(s):

T Cell ◽

Lung Adenocarcinoma ◽

Cell Line ◽

T Cell Activation ◽

Cell Activation ◽

Mononuclear Cells ◽

Ex Vivo ◽

Clinical Activity ◽

Metastatic Lung ◽

Grade 3

2559 Background: We created a vaccine in which irradiated allogeneic lung adenocarcinoma cells are combined with a bystander K562 cell line transfected with hCD40L and hGM-CSF. By recruiting and activating dendritic cells, we hypothesized the vaccine would induce tumor regression in metastatic lung adenocarcinoma. Methods: Intradermal vaccine was given every 14 days x3, followed by monthly x3. Cyclophosphamide (300 mg/m2 IV) was administered before 1st and 4th vaccines to deplete regulatory T-cells. All-trans retinoic acid was given (150/mg/m2/day) after 1st and 4th vaccines to enhance dendritic differentiation. Peripheral blood mononuclear cells (PBMCs) were collected at baseline and after each vaccination. T-cell activation profiles were analyzed by ELISpot assay and tested by generalized Wilcoxon for correlation to survival. Results: 24 participants were accrued at a single center from 10/2006 to 6/2008, with median age 64 and median of 3 previous lines of chemotherapy prior to entry. 20 were former smokers and 4 had brain metastases. A total of 101 vaccines were administered. Common toxicities of any grade were joint pain (79%) and fatigue (75%). Significant adverse events included a grade 3 hypotension and a grade 3 acute respiratory distress. No confirmed complete or partial radiologic responses were observed. Median overall survival (OS) was 8.0 mo (95% CI 3.5 – 12.5) and median time-to-progression was 2.4 mo (95% CI 0.3 – 4.6). Presence of HLA-A2 conferred reduced risk of progression (HR 0.37, 95% CI 0.14 -0.89, p=0.02) and trend to improved OS (HR 0.59, p = 0.06). Of 14 participants with evaluable PBMCs, 5 demonstrated sustained tumor peptide-specific T-cell activation after vaccination. Ex vivo peptide immune response correlated with improved OS compared to non-responders (23 vs. 7 mo, HR 0.48, p = 0.04). Conclusions: Vaccine administration was feasible and tolerable in a heavily pretreated population of metastatic lung cancer. These data suggest the vaccine has clinical activity in the subset with peptide-induced T-cell immune responses and warrants further investigation. A randomized trial of the vaccine is currently in development.

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T-cell redirected killing and cure of pancreatic and gastric cancer xenografts by targeting Trop-2.

Journal of Clinical Oncology ◽

10.1200/jco.2015.33.3_suppl.262 ◽

2015 ◽

Vol 33 (3_suppl) ◽

pp. 262-262

Author(s):

David M. Goldenberg ◽

Edmund A. Rossi ◽

Diane L Rossi ◽

Thomas M. Cardillo ◽

Chien-Hsing Chang

Keyword(s):

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Ex Vivo ◽

Control Group ◽

Calcium Signal ◽

Target Cells ◽

Normal Tissues

262 Background: Trop-2 [also called tumor-associated calcium signal transducer 2 (TACSTD2), EGP-1 (epithelial glycoprotein-1), GA733-1, or M1S1]is a 35 kDa transmembrane glycoprotein that is overexpressed relative to normal tissues in a variety of human cancers, including pancreatic and gastric carcinomas, where increased expression correlates with poor prognosis. Trop-2 appears to be more tumor-specific than the related molecule, EpCAM (Trop-1). MT110, the EpCAM antibody x CD3 bispecific T-cell engager (BiTE), is currently undergoing a Phase I study in various solid tumors, including lung, gastric, colorectal, breast, prostate, and ovarian cancers. We produced a similar T-cell redirecting bispecific tandem scFv, E1-3, using the variable domains of hRS7 (humanized anti-Trop-2 mAb) and Okt-3 (anti-CD3 mAb). Methods: T-cell activation, cytokine induction and cytotoxicity were evaluated ex vivo using PBMCs or purified T cells with human pancreatic (Capan-1 and BxPC3) and gastric (NCI-N87) cancer cell lines as target cells. In vivo activity was assayed with NCI-N87 xenografts that were inoculated s.c. in a mixture with twice the number of human PBMCs and matrigel. Results: In the presence of target cells and PBMCs, E1-3 potently induced T-cell activation, proliferation, and dose-dependent cytokine production of IL-2 (>2 ng/mL), IL-6 (>1 ng/mL), IL-10 (>7 ng/mL), TNF-α (>1 ng/mL) and IFN-γ (>50 ng/mL). In vitro, E1-3 mediated a highly potent T-cell lysis of BxPC3 [IC50=0.09(±0.04) pM], Capan-1 [IC50=1.2(±1.1) pM] and NCI-N87 [IC50=1.2(±1.2) pM] target cells. In vivo, two 50-µg doses of E1-3 given three days apart cured all of the mice (N=8) bearing NCI-N87 xenografts (P=0.0005; Log-Rank). Tumors in the control group (PBMCs only) reached the endpoint (TV>1 cm3) with a median of 39.5 days. All mice remained tumor-free in the E1-3 group at 78 days. Conclusions: Trop-2 is an attractive target for T-cell-mediated killing of pancreatic, gastric and other epithelial cancers.

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