Rasgrp1 mutation increases naïve T-cell CD44 expression and drives mTOR-dependent accumulation of Helios+ T cells and autoantibodies

Elimination of syngeneic sarcomas in rats by a subset of T lymphocytes.

Journal of Experimental Medicine ◽

10.1084/jem.152.4.823 ◽

1980 ◽

Vol 152 (4) ◽

pp. 823-841 ◽

Cited By ~ 86

Author(s):

E Fernandez-Cruz ◽

B A Woda ◽

J D Feldman

Keyword(s):

T Cells ◽

T Cell ◽

T Lymphocytes ◽

Cell Subset ◽

Suppressor Cells ◽

Effector Cells ◽

Long Duration ◽

Ia Antigens

Established subcutaneous Moloney sarcomas (MST-1) of large size and long duration were eliminated from syngeneic rats by intravenous infusion of varying numbers of specific syngeneic effector T lymphocytes. Spleen cells from BN rats in which tumor had regressed were cultured in an in vitro mixed lymphocyte tumor cell culture (MLTC) to augment cytotoxicity of effector cells. In the MLTC a T cell subset was expanded in response to MST-1 antigens and transformed into blast elements. With these changes, there was an increase in the W3/25 antigen on the T cell surface, a decrease of W3/13 antigen, and an increase in the number of T cells with Ia antigens. The subset associated with elimination of established tumors was a blast T cell W3/25+, W3/13+, as detected by monoclonal antibodies to rat T antigens. The W3/25+ subset was poorly cytotoxic in vitro for MST-1 and apparently functioned in vivo as an amplifier or helper cell in the tumor-bearing host. The W3/25- population was a melange of cells that included (W3/13+, W3/25-) T cells, null cells, Ig+ cells, and macrophages, and was associated with enhancement of tumor in vivo, suggesting the presence of suppressor cells.

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CD40 and CD80/86 signaling in cDC1s mediate effective neoantigen vaccination and generation of antigen-specific CX3CR1+ CD8+ T cells in mice

10.1101/2020.06.15.151787 ◽

2020 ◽

Cited By ~ 1

Author(s):

Takayoshi Yamauchi ◽

Toshifumi Hoki ◽

Takaaki Oba ◽

Kristopher Attwood ◽

Xuefang Cao ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Critical Role ◽

Cell Subset ◽

Antitumor Efficacy ◽

Preclinical Model ◽

Therapeutic Vaccines ◽

T Cell Subset

AbstractThe use of tumor mutation-derived neoantigen represents a promising approach for cancer vaccines. Preclinical and early-phase human clinical studies have shown the successful induction of tumor neoepitope-directed responses; however, overall clinical efficacy of neoantigen vaccines has been limited. One major obstacle of this strategy is the prevailing lack of sufficient understanding of the mechanism underlying the generation of neoantigen-specific CD8+ T cells. Here, we report a correlation between antitumor efficacy of neoantigen/toll-like receptor 3 (TLR3)/CD40 vaccination and the generation of antigen-specific CD8+ T cells expressing CX3C chemokine receptor 1 (CX3CR1) in a preclinical model. Mechanistic studies using mixed bone marrow chimeras identified that CD40 and CD80/86, but not CD70 signaling in Batf3-dependent conventional type 1 dendritic cells (cDC1s) is required for antitumor efficacy of neoantigen vaccine and generation of neoantigen-specific CX3CR1+ CD8+ T cells. Although CX3CR1+ CD8+ T cells exhibited robust in vitro effector function, depletion of this subset did not alter the antitumor efficacy of neoantigen/TLR3/CD40 agonists vaccination, suggesting that the expanded CX3CR1+ CD8+ T cell subset represents the post-differentiated in vivo effective CX3CR1-negative CD8+ T cell subset. Taken together, our results reveal a critical role of CD40 and CD80/86 signaling in cDC1s in antitumor efficacy of neoantigen-based therapeutic vaccines, and implicate the potential utility of CX3CR1 as a circulating predictive T-cell biomarker in vaccine therapy.

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Immunomodulatory Effects of Pevonedistat, a NEDD8-Activating Enzyme (NAE) Inhibitor, in Chronic Lymphocytic Leukemia (CLL)

Blood ◽

10.1182/blood-2018-99-115086 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2946-2946

Author(s):

Scott R Best ◽

Adam Kittai ◽

Taylor Rowland ◽

Nur Bruss ◽

Stephen E Spurgeon ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Research Funding ◽

Cell Function ◽

Cell Subset ◽

Continuous Treatment ◽

Cd4 T Cell ◽

Cytotoxic Function

Abstract Introduction: T cells from patients with CLL and lymphoma show highly impaired immune synapse formation, cytotoxic function, and adhesion and migration capabilities. Recent advances in immunooncology led to the emergence of therapeutic agents that permit reversal of T-cell exhaustion in cancer. However, rational development of novel combination approaches in immunotherapy requires detailed understanding of how targeted therapies influence T-cell function. We have shown that pevonedistat (TAK-924), an investigational NAE inhibitor, abrogates NFκB activation in CLL cells. Pevonedistat forms a covalent adduct with NEDD8, a ubiquitin-like modifier, thereby disrupting its interaction with NAE. This leads to reduced activity of Cullin-RING ligases (CRLs), a group of ubiquitin ligases that require modification by NEDD8 for their function. Ultimately, a decrease in CRL activity leads to reduced ubiquitination and proteasomal degradation of CRL substrates, extending the half-life of these proteins, including inhibitor of NFκB (IκB). Moreover, NFκB is critical in T-cell function. However, limited data exist on the effects of targeting neddylation on T-cell response. Here, we demonstrate that targeting neddylation in vitro preserves T-cell functionality and may lead to favorable T-cell population shifts in CLL. Methods: Peripheral blood mononuclear cells were isolated from patients with CLL (n=50), and T cells were purified using Dynabeads. Pevonedistat was obtained from Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited (Cambridge, MA). Results: In vitro T-cell receptor (TCR; CD3/CD28) stimulation induced T-cell activation and proliferation. Continuous treatment of T cells with pevonedistat led to rapid (2 hour) disruption of cullin neddylation, followed by a significant reduction in activity of NFκB and NFAT as assessed by immunoblotting and immunofluorescence. Despite this reduction, CD4 and CD8 T cells continued to respond to TCR stimulation, with relative abundance of early markers of activation (CD40L, CD69). However, we observed reduced expression of CD25 and PD-1 at 72 hours. Continuous treatment with pevonedistat led to a dose-dependent decrease in IL-2 secretion and reduced proliferation of the CD4 T-cell subset (CFSE, Ki-67) but did not induce apoptosis. Unlike CLL cells, CD4 T cells did not undergo DNA re-replication and G2/M arrest in response to pevonedistat. We further analyzed T-cell subsets following TCR stimulation. Concurrent treatment with pevonedistat led to an increase in IFNγ-secreting CD4 T cells, whereas IL-4 production decreased, suggesting a shift toward the Th1 phenotype. Furthermore, we observed a robust decrease of the iTreg population, accompanied by downregulation of FoxP3 mRNA and protein within the CD4 T-cell subset, indicating that targeting neddylation may help to reverse the immunosuppressive phenotype in CLL. To mimic the in vivo pharmacokinetics of pevonedistat, we performed drug washouts where CLL-derived T cells were exposed to 2-hour pulse treatment with 1 µM pevonedistat prior to TCR stimulation. Under these conditions, cullin neddylation and NFκB activity began to recover by 8 hours, with near complete recovery by 24 hours. Moreover, pevonedistat did not disrupt allogeneic (OCI-LY19 cells) or autologous (CD40L-stimulated CLL cells) T-cell cytotoxicity. Meanwhile, CD8 T cells continued to produce perforin and granzyme B. Conclusions: Our data suggest that pharmacologic targeting of NAE preserves T-cell cytotoxic function and may enhance anti-tumor immunity in CLL. Combined with our earlier reports that targeting NAE kills CLL cells under lymph node-mimicking conditions, these data provide a strong rationale for continued investigation of pevonedistat in CLL and lymphoid malignancies. Disclosures Spurgeon: Bristol Myers Squibb: Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Oncternal: Research Funding; Acerta: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Pharmacyclics: Consultancy, Research Funding; MEI Pharma: Consultancy. Berger:Takeda Pharmaceuticals International Co.: Employment. Danilov:Gilead Sciences: Consultancy, Research Funding; Astra Zeneca: Consultancy; Verastem: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Aptose Biosciences: Research Funding; Takeda Oncology: Research Funding; TG Therapeutics: Consultancy; Bayer Oncology: Consultancy, Research Funding.

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Cytotoxic T Lymphocyte–associated Antigen 4 (CTLA-4) Can Regulate Dendritic Cell–induced Activation and Cytotoxicity of CD8+ T Cells Independently of CD4+T Cell Help

Journal of Experimental Medicine ◽

10.1084/jem.189.7.1157 ◽

1999 ◽

Vol 189 (7) ◽

pp. 1157-1162 ◽

Cited By ~ 53

Author(s):

Kathy D. McCoy ◽

Ian F. Hermans ◽

J. Henry Fraser ◽

Graham Le Gros ◽

Franca Ronchese

Keyword(s):

T Cells ◽

T Cell ◽

T Lymphocyte ◽

Cd8 T Cells ◽

Cytotoxic T Lymphocyte ◽

Cd4 T Cell ◽

Cd4 T Cell Help ◽

T Cell Help

The mechanisms that regulate the strength and duration of CD8+ cytotoxic T cell activity determine the effectiveness of an antitumor immune response. To better understand the antitumor effects of anti-cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) antibody treatment, we analyzed the effect of CTLA-4 signaling on CD8+ T cells in vitro and in vivo. In vitro, cross-linking of CTLA-4 on purified CD8+ T cells caused decreased proliferative responses to anti-CD3 stimulation and rapid loss of activation marker expression. In vivo, blockade of CTLA-4 by neutralizing anti–CTLA-4 mAb greatly enhanced the accumulation, activation, and cytotoxic activity of CD8+ T cells induced by immunization with Ag on dendritic cells (DC). This enhanced response did not require the expression of MHC class II molecules on DC or the presence of CD4+ T cells. These results demonstrate that CTLA-4 blockade is able to directly enhance the proliferation and activation of specific CD8+ T cells, indicating its potential for tumor immunotherapy even in situations in which CD4+ T cell help is limited or absent.

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Regulatory dendritic cells program generation of interleukin-4–producing alternative memory CD4 T cells with suppressive activity

Blood ◽

10.1182/blood-2010-05-285494 ◽

2011 ◽

Vol 117 (4) ◽

pp. 1218-1227 ◽

Cited By ~ 11

Author(s):

Xiongfei Xu ◽

Zhenhong Guo ◽

Xueyu Jiang ◽

Yushi Yao ◽

Qiangguo Gao ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Cd4 T Cells ◽

Cd4 T Cell ◽

Interleukin 4 ◽

Delayed Type Hypersensitivity ◽

Regulatory Function

Abstract The heterogeneity and mechanisms for the generation of CD4 memory T (CD4 Tm) cells remain elusive. Distinct subsets of dendritic cells (DCs) have been found to regulate a distinct T-helper (Th)–cell subset differentiation by influencing cytokine cues around CD4 T cells; however, whether and how the regulatory DC subset can regulate Tm-cell differentiation remains unknown. Further, there is no ideal in vitro experimental system with which to mimic the 3 phases of the CD4 T-cell immune response (expansion, contraction, memory generation) and/or to culture CD4 Tm cells for more than a month. By analyzing CD4 T cells programmed by long-term coculture with regulatory DCs, we identified a population of long-lived CD4 T cells with a CD44hiCD62L−CCR7− effector memory phenotype and rapid, preferential secretion of the Th2 cytokines interleukin-4 (IL-4), IL-5, IL-10, and IL-13 after antigenic stimulation. These regulatory DC-programmed Tm cells suppress CD4 T-cell activation and proliferation in vitro via IL-10 and inhibit the delayed-type hypersensitivity response once infused in vivo. We also identify their natural counterpart, which is up-regulated by regulatory DC transfusion and negatively regulates the recall response in vivo. Different from interferon-γ–producing conventional Tm cells, these IL-4–producing CD4 Tm cells act as alternative Tm cells with a regulatory function, suggesting a new way of negative immune regulation by memory T cells.

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TCRζdimlymphocytes define populations of circulating effector cells that migrate to inflamed tissues

Blood ◽

10.1182/blood-2006-12-064170 ◽

2007 ◽

Vol 109 (10) ◽

pp. 4328-4335 ◽

Cited By ~ 29

Author(s):

Zhuoli Zhang ◽

Claire L. Gorman ◽

Anna-Chiara Vermi ◽

Claudia Monaco ◽

Andrew Foey ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Subset ◽

Chronic Inflammatory Disease ◽

Cytokine Gene Expression ◽

Cell Contact ◽

Cell Reactivity ◽

Migratory Activity

Abstract The T-cell receptor ζ (TCRζ) chain is a master sensor and regulator of lymphocyte responses. Loss of TCRζ expression has been documented in infectious, inflammatory, and malignant diseases, suggesting that it may serve to limit T-cell reactivity and effector responses at sites of tissue damage. These observations prompted us to explore the relationship between TCRζ expression and effector function in T cells. We report here that TCRζdim lymphocytes are enriched for antigen-experienced cells refractory to TCR-induced proliferation. Compared to their TCRζbright counterparts, TCRζdim cells share characteristics of differentiated effector T cells but use accessory pathways for transducing signals for inflammatory cytokine gene expression and cell contact-dependent pathways to activate monocytes. TCRζdim T cells accumulate in inflamed tissues in vivo and have intrinsic migratory activity in vitro. Whilst blocking leukocyte trafficking with anti-TNF therapy in vivo is associated with the accumulation of TCRζdim T cells in peripheral blood, this T-cell subset retains the capacity to migrate in vitro. Taken together, the functional properties of TCRζdim T cells make them promising cellular targets for the treatment of chronic inflammatory disease.

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CLL B Cells Develop Resistance to Ibrutinib By Reinvigorating the IL-4R - IL-4 Axis Blocked By Bruton's Tyrosine Kinase Inhibitors Including Acalabrutinib and Zanubrutinib

Blood ◽

10.1182/blood-2019-127255 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 477-477

Author(s):

Shih-Shih Chen ◽

Constantine S. Tam ◽

Alan G. Ramsay ◽

Priyadarshini Ravichandran ◽

Natalia C. Couto-Francisco ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Cell Subset ◽

Advisory Committees

Bruton's tyrosine kinases inhibitors (BTKis) represent major advances in CLL therapy. However resistance to this form of therapy is emerging, and such patients often progress more rapidly. Hence there is an important need for therapies that address resistance. Microenvironmental input like IL-4 is critical for CLL disease progression. Compared with normal B cells, CLL cells exhibit significantly higher levels of surface membrane (sm) IL-4 receptor (IL4-R) and contain increased amounts of pSTAT6, a downstream mediator of IL-4R signaling. IL-4 stimulation of CLL B cells suppresses smCXCR4 and increases smIgM, thus promotes CLL cell retention and expansion. In this study, we aimed to examine if smIL-4R expression, IL4R signaling, and IL-4-producing cells are altered in patients sensitive or resistant to BTKis. To do so, T and B cell subset changes were studied overtime in 12 acalabrutinib-treated CLL patients, 6 zanubrutinib-treated CLL patients, 30 ibrutinib-sensitive and 5 ibrutinib-resistant CLL patients, 4 of which exhibited BTK mutations. Consistent with only ibrutinib inhibiting T-cell kinase (ITK), T-cell subset analyses revealed no changes in Th1, Th2, Th17, Th9, and Th22 cells after zanubrutinib or acalabrutinib treatment. In contrast, a Th1-biased T-cell immunity was observed in patients responsive to ibrutinib. In patients progressing on ibrutinib, significantly reduced Th2 T cells were found during the resistant as well as sensitive periods. In an in vitro T-cell function assay using T cells collected before and after the treatment with each BTKi, only ibrutinib treated patients exhibited a reduced ability of T cells to support CLL B cell survival. We next studied changes in CLL B cells, including numbers of IL-4, -10 and -13 producing B cells after BTKi treatment. IL-13 producing CLL B cells were not changed. IL-10 producing CLL B cells were reduced in both ibrutinib sensitive and resistant patients, but not in zanubrutinib or acalabrutinib treated patients. Importantly, IL-4 producing CLL B cells were significantly decreased in patients treated with all 3 BTKi. Significantly reduced smIL-4R levels, impaired IL-4R signaling, decreased smIgM and increased smCXCR4 were also seen in patients treated with each BTKi. To understand the mechanism responsible for inhibition of IL-4 production in CLL cells treated with BTKis, we stimulated CLL cells through IgM, Toll-like receptor and CD40L, finding that only anti-IgM stimulation significantly increased IL-4 production and p-STAT6 induction. We then explored the function of IL-4. IL-4 enhanced CLL B cell survival in vitro and this action was blocked by all 3 BTKis. Moreover, adhesion of CLL B cells to smIL-4R expressing stromal cells was decreased by IL-4 and IL-4R neutralizing antibodies, especially in M-CLL cases. In in vivo studies transferring autologous T cells and CLL PBMCs into alymphoid mice, we found less CLL B cells in mouse spleens post ibrutinib than zanubrutinib or acalabrutinib treatment. This might be due to the suppressed Th2 cells found only in ibrutinib, while IL-4 producing B cells were reduced in all 3 BTKi treated mice. These results support the idea that IL-4 promotes CLL B cell adhesion and growth in tissues. Finally, we investigated the IL-4/IL-4R axis in ibrutinib-resistant patients. Although IL-4 producing T cells remain reduced during the sensitive and resistant phases, CLL B cell production of IL-4 and expression of and signaling through smIL-4R returned when patients developed ibrutinib-resistance. When comparing paired ibrutinib-sensitive and -resistant CLL B cells collected from 3 patients in a xenograft model that requires T cell help, we found ibrutinib-resistant CLL B cells grew in vivo with only minimal (~15%) numbers of autologous T cells compared to B cells collected from ibrutinib-sensitive phase; this suggested a reduced requirement for T-cell help for growth of ibrutinib-resistant CLL cells. In summary, we found IL-4 is a key survival factor in the CLL microenvironment that also improves leukemia cell adhesion to stromal cells expressing smIL-4R. IL-4 production and signaling can be stimulated in CLL B cells through the B-cell receptor, and are consistently blocked by BTKis. Moreover, the recovered ability of ibrutinib-resistant CLL B cells to produce and respond to IL-4 leads to disease progression, suggesting blocking the IL-4/IL-4R axis is a potential treatment for ibrutinib-resistant CLL patients. Disclosures Chen: Pharmacyclics: Research Funding; Beigene: Research Funding; Verastem: Research Funding; ArgenX: Research Funding. Tam:Abbvie, Janssen: Research Funding; Abbvie, Janssen, Beigene, Roche, Novartis: Honoraria. Ramsay:Celgene Corporation: Research Funding; Roche Glycart AG: Research Funding. Kolitz:Boeringer-Ingelheim: Research Funding; Roche: Research Funding; Astellas: Research Funding. Zhou:BeiGene: Employment. Barrientos:Genentech: Consultancy; Gilead: Consultancy; Janssen: Consultancy; Abbvie: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding. Rai:Pharmacyctics: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Cellectis: Membership on an entity's Board of Directors or advisory committees; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees.

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MHC control of CD4+ T cell subset activation.

Journal of Experimental Medicine ◽

10.1084/jem.170.6.2135 ◽

1989 ◽

Vol 170 (6) ◽

pp. 2135-2140 ◽

Cited By ~ 80

Author(s):

J S Murray ◽

J Madri ◽

J Tite ◽

S R Carding ◽

K Bottomly

Keyword(s):

T Cells ◽

T Cell ◽

Mhc Class Ii ◽

Cd4 T Cells ◽

Cell Subset ◽

Class Ii ◽

Cd4 T Cell ◽

Selective Activation ◽

Ifn Gamma

The present results demonstrate that CD4+ T cells activated in the primary in vivo response to antigen produce distinct patterns of cytokines depending upon the MHC class II haplotype of the responding mice. I-As mice were found to selectively activate IL-2/IFN-gamma-producing CD4+ T cells, whereas I-Ab mice exhibited selective activation of IL-4-producing CD4+ T cells in response to collagen IV. The effector response phenotype was found to correlate with the cytokine phenotype of CD4+ T cells activated in vivo; IL-2/IFN-gamma-producing cells giving rise to proliferative (cell-mediated) responses, IL-4-producing cells leading to secondary IgG (humoral) responses. Together the data support the notion that the outcome of a given immune response (e.g., protection vs. onset, tolerance vs. autoimmunity) may be determined in part by the type of CD4+ T cells initially activated by antigen. Moreover, the present experiments demonstrate for the first time that polymorphism in class II MHC can determine such selective activation of different cytokine-producing CD4+ T cell phenotypes.

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Ligation of Toll-Like Receptor 5 (TLR5) by Flagellin Overcomes Antigen-Specific CD4+ T Cell Tolerance Via Inhibition of IL10 and Induction of IL12 in Antigen Presenting Cells (APCs).

Blood ◽

10.1182/blood.v104.11.3443.3443 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3443-3443

Author(s):

Ildefonso Suarez ◽

FengDong Cheng ◽

Jason B. Brayer ◽

HW Wang ◽

Horna Pedro ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Inflammatory Cytokine ◽

Tolerance Induction ◽

Cd4 T Cell ◽

High Dose ◽

Cell Responses ◽

Cognate Antigen

Abstract The immune system is very effective in fighting infections but seems not to be as efficient in recognizing and destroying cancer cells. In the cancer setting, tumor antigen uptake and presentation by APCs to antigen-specific T-cells often occurs in the absence of inflammation resulting therefore in tolerance induction. It is plausible therefore that by converting APCs from a non-inflammatory to an inflammatory phenotype through ligation of TLRs we may well overcome immune tolerance and tip the balance towards productive tumor antigen-specific T-cell responses. Among all the TLR-ligands identified to date, flagellin is the only one with a strictly proteinic nature, characteristic that make it a suitable candidate for cloning and transfection into tumor cells to generate novel tumor cell based vaccines. In this study, we first evaluated whether treatment with purified flagellin could prevent tolerance induction in vivo. Naïve CD4+ T-cells (2.5x106) specific for a MHC class II-restricted epitope of influenza hemagglutinin (HA) were adoptively transferred intravenously into BALB/c mice, 24 hours after mice were given either a tolerogenic dose of HA-peptide (200 mg), or a combination of this high dose of peptide together with Flagellin (10 mg I.V.). Two weeks later animals were sacrificed and antigen-specific CD4+ T-cell responses towards the cognate antigen evaluated in vitro. As expected clonotypic T-cells isolated from animals treated with high dose peptide were fully tolerant, in sharp contrast with those isolated from flagellin treated animals that displayed normal responses in terms of cytokine production and proliferation. Surprisingly, this preservation of T-cell function following in vivo treatment with flagellin was not observed when animals were treated with high dose HA-peptide in the presence of the TLR4 ligand, LPS. To better understand the mechanism(s) by which flagellin, and not LPS, preserved the responsiveness of antigen-specific CD4+ T-cells to cognate antigen presented by APCs, we assessed the phenotypic characteristics and the cytokine profile of macrophages and DCs treated in vitro with these TLRs ligands. Although LPS-treated APCs produce higher levels of IL-12, relative to flagellin-treated APCs, the production of this pro-inflammatory cytokine was accompanied by a parallel induction of the anti-inflammatory cytokine, IL-10. Interestingly, flagellin-treated APCs produced IL-12 but were unable to produce IL-10. This effect was dependent on ligation of TLR5, since it was not observed when RAW264.7 cells -which lack TLR5- were treated with flagellin. In vivo studies further confirmed our observations since IL-10 was not detected in the serum of animals treated with flagellin, but it was present in significant amounts in LPS-treated animals. This inhibitory effect of flagellin on IL-10 production was seen even when APCs were stimulated in vitro with strong inducers of IL-10. Given the above properties of flagellin, we generated two novel approaches to be used in the formulation of tumor cell based vaccines: 1) A bystander cell line transfected with the fliC gene from Salmonella typhimurim for flagellin expression (B78H1-Flagellin) and 2) Microspheres beads coated with flagellin. Both vaccination strategies are being currently studied in the in vivo and in vitro settings

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Circulating CD3+HLA-DR+Extracellular Vesicles as a Marker for Th1/Tc1-Type Immune Responses

Journal of Immunology Research ◽

10.1155/2019/6720819 ◽

2019 ◽

Vol 2019 ◽

pp. 1-13 ◽

Cited By ~ 3

Author(s):

Ryutaro Oba ◽

Motomichi Isomura ◽

Akira Igarashi ◽

Kinya Nagata

Keyword(s):

T Cells ◽

T Cell ◽

Extracellular Vesicles ◽

Inflammatory Diseases ◽

Cell Subset ◽

Activated T Cells ◽

Ebv Infection ◽

Hla Dr

Extracellular vesicles (EVs) are known to contain unique proteins that reflect the cells of origins. Activated T cells are reported to secrete various EVs. To establish T cell subset-specific biomarkers, we performed proteomic analysis with Th1- and Th2-derived EVs and identified HLA-DR as a Th1-dominated EV membrane protein. We designed a measurement system for CD3+CD4+, CD3+CD8+, and CD3+HLA-DR+EVs to specifically determine EV subpopulations derived from CD4+, CD8+, and Th1-type T cells, respectively.In vitroanalysis showed that CD3+CD4+EVs and CD3+CD8+EVs were selectively secreted from activated CD4+and CD8+T cells, respectively, and that CD3+HLA-DR+EVs were actively secreted from not only Th1 but also activated CD8+T (probably mostly Tc1) cells. To evaluate the clinical usefulness of these EVs, we measured the serum levels in patients with inflammatory diseases, including Epstein-Barr virus (EBV,n=13) infection, atopic dermatitis (AD,n=10), rheumatoid arthritis (RA,n=20), and osteoarthritis (OA,n=20) and compared the levels with those of healthy adults (n=20). CD3+CD4+EVs were significantly higher in all of EBV infection, AD, RA, and OA while CD3+CD8+EVs were higher in EBV infection, lower in RA, and not different in AD and OA relative to the control. The levels of CD3+HLA-DR+EVs were markedly higher in EBV infection and significantly lower in AD. The results suggest that these EV subpopulations reflectin vivoactivation status of total CD4+, total CD8+, and Th1/Tc1-type T cells, respectively, and may be helpful in T cell-related clinical settings, such as cancer immunotherapy and treatment of chronic infection, autoimmune diseases, and graft-versus-host disease.

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