Prenatal Cadmium Exposure Alters Proliferation in Mouse CD4+ T Cells via LncRNA Snhg7

ObjectivePrenatal cadmium (Cd) exposure leads to immunotoxic phenotypes in the offspring affecting coding and non-coding genes. Recent studies have shown that long non-coding RNAs (lncRNAs) are integral to T cell regulation. Here, we investigated the role of long non-coding RNA small nucleolar RNA host gene 7 (lncSnhg7) in T cell proliferation.MethodsRNA sequencing was used to analyze the expression of lncRNAs in splenic CD4+ T cells with and without CD3/CD28 stimulation. Next, T cells isolated from offspring exposed to control or Cd water throughout mating and gestation were analyzed with and without stimulation with anti-CD3/CD28 beads. Quantitative qPCR and western blotting were used to detect RNA and protein levels of specific genes. Overexpression of a miR-34a mimic was achieved using nucleofection. Apoptosis was measured using flow cytometry and luminescence assays. Flow cytometry was also used to measure T cell proliferation in culture. Finally, lncSnhg7 was knocked down in splenic CD4+ T cells with lentivirus to assess its effect on proliferation.ResultsWe identified 23 lncRNAs that were differentially expressed in stimulated versus unstimulated T cells, including lncSnhg7. LncSnhg7 and a downstream protein, GALNT7, are upregulated in T cells from offspring exposed to Cd during gestation. Overexpression of miR-34a, a regulator of lncSnhg7 and GALNT7, suppresses GALNT7 protein levels in primary T cells, but not in a mouse T lymphocyte cell line. The T cells isolated from Cd-exposed offspring exhibit increased proliferation after activation in vitro, but Treg suppression and CD4+ T cell apoptosis are not affected by prenatal Cd exposure. Knockdown on lncSnhg7 inhibits proliferation of CD4+ T cells.ConclusionPrenatal Cd exposure alters the expression of lncRNAs during T cell activation. The induction of lncSnhg7 is enhanced in splenic T cells from Cd offspring resulting in the upregulation of GALNT7 protein and increased proliferation following activation. miR-34a overexpression decreased GALNT7 expression and knockdown of lncSnhg7 inhibited proliferation suggesting that the lncSnhg7/miR-34a/GALNT7 is an important pathway in primary CD4+ T cells. These data highlight the need to understand the consequences of environmental exposures on lncRNA functions in non-cancerous cells as well as the effects in utero.

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Prenatal cadmium exposure alters proliferation in mouse CD4+ T cells via LncRNA Snhg7

10.1101/2021.06.04.446938 ◽

2021 ◽

Author(s):

Jamie L McCall ◽

Melinda E Varney ◽

Sebastian A. Dziadowicz ◽

Casey Hall ◽

Kathryn E Blethen ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd4 T Cells ◽

Cell Activation ◽

T Cell Proliferation ◽

Protein Levels ◽

Cd Exposure

Objective: Prenatal cadmium (Cd) exposure leads to immunotoxic phenotypes in the offspring affecting coding and non-coding genes. Recent studies have shown that long non-coding RNAs (lncRNAs) are integral to T cell regulation. Here, we investigated the role of long non-coding RNA small nucleolar RNA hostgene 7 (lncSnhg7) in T cell proliferation. Methods: RNA sequencing was used to analyze the expression of lncRNAs in splenic CD4+ T cells with and without CD3/CD28 stimulation. Next, T cells isolated from offspring exposed to control or Cd water throughout mating and gestation were analyzed with and without stimulation with anti-CD3/CD28 beads. Quantitative qPCR and western blotting were used to detect RNA and protein levels of specific genes. Overexpression of a miR-34a mimic was achieved using nucleofection. Apoptosis was measured using flow cytometry and luminescence assays. Flow cytometry was also used to measure T cell proliferation in culture. Results: We identified 23 lncRNAs that were differentially expressed in stimulated versus unstimulated T cells, including lncSnhg7. LncSnhg7 and a downstream protein, GALNT7, are upregulated in T cells from offspring exposed to Cd during gestation. Overexpression of miR-34a, a regulator of lncSnhg7 and GALNT7, suppresses GALNT7 protein levels in primary T cells, but not in a mouse T lymphocyte cell line. The T cells isolated from Cd-exposed offspring exhibit increased proliferation after activation in vitro, but Treg suppression and CD4+ T cell apoptosis are not affected by prenatal Cd exposure. Conclusion: Prenatal Cd exposure alters the expression of lncRNAs during T cell activation. The induction of lncSnhg7 is enhanced in splenic T cells from Cd offspring resulting in the upregulation of GALNT7 protein and increased proliferation following activation. miR-34a overexpression decreased GALNT7 expression suggesting that the lncSnhg7/miR-34a/GALNT7 is an important pathway in primary CD4+ T cells. These data highlight the need to understand the consequences of environmental exposures on lncRNA functions in non-cancerous cells as well as the effects in utero.

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Siplizumab, an Anti-CD2 Monoclonal Antibody, Induces a Unique Set of Immune Modulatory Effects Compared to Alemtuzumab and Rabbit Anti-Thymocyte Globulin In Vitro

Frontiers in Immunology ◽

10.3389/fimmu.2020.592553 ◽

2020 ◽

Vol 11 ◽

Author(s):

Christian Binder ◽

Felix Sellberg ◽

Filip Cvetkovski ◽

Erik Berglund ◽

David Berglund

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Mixed Lymphocyte Reaction ◽

T Cell Proliferation ◽

Allogeneic Mixed Lymphocyte Reaction ◽

Dependent Cell

Antibodies are commonly used in organ transplant induction therapy and to treat autoimmune disorders. The effects of some biologics on the human immune system remain incompletely characterized and a deeper understanding of their mechanisms of action may provide useful insights for their clinical application. The goal of this study was to contrast the mechanistic properties of siplizumab with Alemtuzumab and rabbit Anti-Thymocyte Globulin (rATG). Mechanistic assay systems investigating antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell phagocytosis and complement-dependent cytotoxicity were used to characterize siplizumab. Further, functional effects of siplizumab, Alemtuzumab, and rATG were investigated in allogeneic mixed lymphocyte reaction. Changes in T cell activation, T cell proliferation and frequency of naïve T cells, memory T cells and regulatory T cells induced by siplizumab, Alemtuzumab and rATG in allogeneic mixed lymphocyte reaction were assessed via flow cytometry. Siplizumab depleted T cells, decreased T cell activation, inhibited T cell proliferation and enriched naïve and bona fide regulatory T cells. Neither Alemtuzumab nor rATG induced the same combination of functional effects. The results presented in this study should be used for further in vitro and in vivo investigations that guide the clinical use of immune modulatory biologics.

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Allergen-Induced C5a/C5aR1 Axis Activation in Pulmonary CD11b+ cDCs Promotes Pulmonary Tolerance through Downregulation of CD40

Cells ◽

10.3390/cells9020300 ◽

2020 ◽

Vol 9 (2) ◽

pp. 300 ◽

Cited By ~ 4

Author(s):

Konstantina Antoniou ◽

Fanny Ender ◽

Tillman Vollbrandt ◽

Yves Laumonnier ◽

Franziska Rathmann ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd4 T Cells ◽

Cell Activation ◽

T Cell Proliferation ◽

C5a Receptor ◽

The Impact

Activation of the C5/C5a/C5a receptor 1 (C5aR1) axis during allergen sensitization protects from maladaptive T cell activation. To explore the underlying regulatory mechanisms, we analyzed the impact of C5aR1 activation on pulmonary CD11b+ conventional dendritic cells (cDCs) in the context of house-dust-mite (HDM) exposure. BALB/c mice were intratracheally immunized with an HDM/ovalbumin (OVA) mixture. After 24 h, we detected two CD11b+ cDC populations that could be distinguished on the basis of C5aR1 expression. C5aR1− but not C5aR1+ cDCs strongly induced T cell proliferation of OVA-reactive transgenic CD4+ T cells after re-exposure to antigen in vitro. C5aR1− cDCs expressed higher levels of MHC-II and CD40 than their C5aR1+ counterparts, which correlated directly with a higher frequency of interactions with cognate CD4+ T cells. Priming of OVA-specific T cells by C5aR1+ cDCs could be markedly increased by in vitro blockade of C5aR1 and this was associated with increased CD40 expression. Simultaneous blockade of C5aR1 and CD40L on C5aR1+ cDCs decreased T cell proliferation. Finally, pulsing with OVA-induced C5 production and its cleavage into C5a by both populations of CD11b+ cDCs. Thus, we propose a model in which allergen-induced autocrine C5a generation and subsequent C5aR1 activation in pulmonary CD11b+ cDCs promotes tolerance towards aeroallergens through downregulation of CD40.

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Demonstration of a Direct Immunomodulatory Role for Vitamin D in Human T Cells Using Flow Cytometry

Blood ◽

10.1182/blood.v112.11.2577.2577 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2577-2577

Author(s):

Richard W. Joseph ◽

Tae Kon Kim ◽

Lisa St. John ◽

Jahan Khalili ◽

Uday R Popat ◽

...

Keyword(s):

Vitamin D ◽

Flow Cytometry ◽

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

T Cell Proliferation ◽

Activated T Cells ◽

Activation Markers

Abstract Clinical and epidemiological studies have demonstrated an increasingly stronger link between Vitamin D deficiency and a broad array of illnesses characterized by inflammation, including autoimmune diseases, coronary artery disease, and cancers. Vitamin D is a steroid hormone that exerts the majority of its biologic effects via the binding of the intracellular Vitamin D receptor (VDR). While upregulation of VDR has been demonstrated in activated bulk T cells using traditional approaches (e.g., western blotting), such assays cannot precisely define VDR distribution and kinetics. To overcome these limitations, we developed what we believe to be the first flow cytometric assay to quantify VDR expression at a single-cell level. We used a primary antibody against VDR (mouse monoclonal IgG2a to human VDR) in permeabilized T cells followed by a labeled secondary antibody. We detected a positive cell population using flow cytometry that was sharply increased following activation, consistent with upregulation of VDR confirmed by immunoblotting of sorted cells. We then applied this validated assay to define the kinetics of VDR upregulation in activated T cells. We stimulated PBMC with PMA:Ionomycin (P:I) for varying intervals and assessed intracellular VDR using flow cytometry. VDR is significantly upregulated by 15 min after stimulation, reaches a plateau after 6 hr, and may remain elevated for up to 7 d. We compared VDR to classical early and late T cell activation markers (CD69 and CD25, respectively), and we found that VDR was upregulated as consistently as (but even earlier than) CD69, and that VDR and CD25 were both consistently upregulated at later intervals (p<0.0001). To examine the association between VDR expression and proliferation, we stimulated CFSE-labeled T cells with OKT3 (2mg/ml) for 5 d and found that proliferating T cells expressed a significantly higher level of VDR than resting T cells, which maintained baseline VDR expression (p<0.0001). To assess the association between T cell cytokine production and VDR expression, we stimulated T cells with (P:I) for 6 hr in the presence of brefeldin A, and we confirmed that all cytokine-producing cells (TNFα, IL-2, IFNγ) were contained within the VDR-high population. We then assessed whether physiologic concentrations of Vitamin D could inhibit T cell proliferation in vitro. We stimulated CFSE-labeled PBMC with either OKT3 or irradiated allogeneic dendritic cells (DC) in the presence or absence of physiologic concentrations of calcitriol (50 nm) for 5 to 7 d. The presence of calcitriol during OKT3 stimulation resulted in significantly reduced cell division (p=0.004, n=5). Using a previously validated phenotype to demarcate activated alloreactive CD4+ T cells (CD4hiCD38+), we demonstrated that physiologic calcitriol supplementation decreased alloreactive activation following 7 d stimulation with allogeneic DC (p=0.0003, n=10). In conclusion, VDR is a consistent and specific early and late marker of T cell activation, suggesting a direct role for the Vitamin D axis in immunoregulation. Furthermore, physiological concentrations of Vitamin D can inhibit T cell proliferation induced by polyclonal stimuli, including allogeneic DC. These data provide confirmation for a direct immunoregulatory role for Vitamin D and suggest that further mechanistic and clinical studies may yield novel therapeutic strategies for inflammatory conditions, including graft-versus-host disease.

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ATG5 Dependent Autophagy Uncouples T Cell Functions and Modulates Experimental Graft-Versus-Host Disease

Blood ◽

10.1182/blood.v126.23.149.149 ◽

2015 ◽

Vol 126 (23) ◽

pp. 149-149

Author(s):

Katherine Oravecz-Wilson ◽

Corinne Rossi ◽

Chen Liu ◽

Tomomi Toubai ◽

Hiroya Tamaki ◽

...

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

T Cell Proliferation ◽

Apoptotic Proteins

Abstract ATG5 is a key protein that regulates autophagy, a vital cellular process whose role in various immune cells is poorly understood. A recent report showed that the deficiency of autophagy gene Atg16l1 in host DCs increased graft-vs-host disease (GVHD). Nevertheless, the direct role of autophagy in regulating T cell alloreactivity after bone marrow transplant (BMT) is unknown. In order to investigate the role of autophagy in T cells, we first analyzed the changes in autophagosome marker LC3 upon WT T cell activation. TCR stimulation with anti-CD3/CD28, increased cytosolic LC3-I and its membrane-bound LC3-II form. Interestingly, we found that the upregulation of LC3 was predominant in dividing cells, which lead us to hypothesize that autophagy is essential for T cell proliferation. Therefore we next explored if the deficiency in autophagy impaired T cell proliferation utilizing B6 T cell-specific ATG5 knockout (ATG5 KO T cell) mouse and hydroxychloroquine (CQ, a known inhibitor of autophagy). As hypothesized, when compared with WT controls, both CQ treated WT T cells and the ATG5 KO T cells, in vitro, demonstrated a significant decrease in proliferation as demonstrated by 3H-thymidine incorporation and CFSE staining (p<0.0001) and were associated with a failure to upregulate LC3. These effects were observed after anti-CD3/CD28 TCR stimulation as well as following allogenic stimulation in a mixed lymphocyte reaction (MLR) with bone marrow-derived dendritic cells (DCs) from BALB/c mice. The reduction in T cell proliferation was accompanied by a significant increase in apoptosis (p<0.0001). However it was not associated with a decrease in T-helper (TH) signature cytokines (IFNγ, IL-2, IL-17, IL-4) suggesting no impact on T cell differentiation. Furthermore ATG5 deficiency also did not alter T cell activation as determined by upregulation of NFAT and ZAP70. Thus lack of autophagy lead to the decrease survival of T cell along with decreased proliferation after TCR stimulation but did not affect TH differentiation and T cell activation. Given the in vitro observations, we hypothesized that ATG5 KO T cells would also induce less GVHD following allogenic bone marrow transplantation (BMT). Utilizing clinically relevant MHC-mismatched B6 → BALB/c BMT model, we lethally irradiated (800cGy) WT-BALB/c mice and transplanted 5x106 T cell-depleted bone marrow from WT-B6 mice along with 0.5x106 splenic T cells purified from ATG5 KO or WT- B6 mice. WT-BALB/c TCD BM and T cells were used for syngeneic controls. Consistent with in vitro results, ATG5 KO T cells showed decreased proliferation in vivo but showed no difference in Th1/Th17 differentiation. Allogenic animals transplanted with ATG5 KO T cells also showed a significantly improved survival (p=0.001) and reduced GVHD severity (p=0.03). Phenotypic analyses prior to BMT showed that ATG5 KO T cells show decreased CD62L and an increased expression of CD44. Because naïve (CD62L+CD44-) T cells are critical for GVHD, we next explored if the observed improvement in GVHD could be due to a decreased population of these naïve T cells in the transplant inoculum of ATG5 KO animals. Therefore, using the same BMT model and design, we transplanted WT-BALB/c mice 0.5x106 isolated splenic CD62L+ T cells only from either ATG5 KO or WT-B6 animals and observed that GVHD mortality was reduced in the allo-recipients of ATG5 KO T cells compared with WT T cells (p=0.005). To determine the potential molecular mechanism, we next hypothesized that upon activation ATG KO T cells may show alterations in pro and anti-apoptotic proteins. We observed that ATG5 KO failed to increase Bcl-2 level and showed a decrease in Bcl-XL level upon TCR stimulation compared to WT T cells. In contrast to the relative lack of anti-apoptotic proteins, they displayed similar levels of the pro-apoptotic proteins BIM, Bak and Bax. These results suggest that an imbalance between pro- and anti-apoptotic factors is likely a cause for the reduced T cell expansion by ATG5 KO T cells. Our results collectively demonstrate that inhibition of autophagy decreases T cell expansion but not its differentiation in vitro and in vivo. Furthermore contrary to the aggravation of GVHD when autophagy is targeted in host DCs, it mitigated GVHD when targeted in donor T cells. Thus the net impact of manipulating autophagy after allogeneic BMT on GVHD is dependent on which immune cell subsets are being targeted. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

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The role of extracellular vesicles and PD-L1 in glioblastoma-mediated immunosuppressive monocyte induction

Neuro-Oncology ◽

10.1093/neuonc/noaa029 ◽

2020 ◽

Vol 22 (7) ◽

pp. 967-978 ◽

Cited By ~ 4

Author(s):

Benjamin T Himes ◽

Timothy E Peterson ◽

Tristan de Mooij ◽

Luz M Cumba Garcia ◽

Mi-Yeon Jung ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Extracellular Vesicles ◽

T Cell Activation ◽

Cell Activation ◽

T Cell Proliferation ◽

Cell Inhibition

Abstract Background Immunosuppression in glioblastoma (GBM) is an obstacle to effective immunotherapy. GBM-derived immunosuppressive monocytes are central to this. Programmed cell death ligand 1 (PD-L1) is an immune checkpoint molecule, expressed by GBM cells and GBM extracellular vesicles (EVs). We sought to determine the role of EV-associated PD-L1 in the formation of immunosuppressive monocytes. Methods Monocytes collected from healthy donors were conditioned with GBM-derived EVs to induce the formation of immunosuppressive monocytes, which were quantified via flow cytometry. Donor-matched T cells were subsequently co-cultured with EV-conditioned monocytes in order to assess effects on T-cell proliferation. PD-L1 constitutive overexpression or short hairpin RNA–mediated knockdown was used to determined the role of altered PD-L1 expression. Results GBM EVs interact with both T cells and monocytes but do not directly inhibit T-cell activation. However, GBM EVs induce immunosuppressive monocytes, including myeloid-derived suppressor cells (MDSCs) and nonclassical monocytes (NCMs). MDSCs and NCMs inhibit T-cell proliferation in vitro and are found within GBM in situ. EV PD-L1 expression induces NCMs but not MDSCs, and does not affect EV-conditioned monocytes T-cell inhibition. Conclusion These findings indicate that GBM EV-mediated immunosuppression occurs through induction of immunosuppressive monocytes rather than direct T-cell inhibition and that, while PD-L1 expression is important for the induction of specific immunosuppressive monocyte populations, immunosuppressive signaling mechanisms through EVs are complex and not limited to PD-L1.

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Anti-CD6 Monoclonal Antibody Itolizumab Efficiently Inhibits T Cell Proliferation after in Vitro TCR Stimulation in the Setting of Acute Graft Versus Host Disease

Blood ◽

10.1182/blood-2019-125721 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4517-4517 ◽

Cited By ~ 1

Author(s):

Benedetta Rambaldi ◽

Carol Reynolds ◽

Sharmila Chamling Rai ◽

Takeru Asano ◽

Yohei Arihara ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Research Funding ◽

Cell Activation ◽

T Cell Proliferation

CD6 is a co-stimulatory receptor expressed on T cells that binds activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen presenting cells and various epithelial and endothelial tissues. The CD6-ALCAM pathway plays an integral role in modulating T cell activation, proliferation and trafficking and is central to inflammation. Early studies by Soiffer et al. demonstrated that ex vivo depletion of CD6+ donor cells prior to hematopoietic cell transplantation (HCT) decreased the incidence of acute graft versus host disease (aGVHD), highlighting the importance of CD6+ cells in GVHD pathogenesis. Itolizumab, a humanized anti-CD6 monoclonal antibody, has been shown to modulate T cell activation and proliferation. The aim of this study was to characterize: (1) expression of CD6 and ALCAM, and (2) activity of itolizumab on T cell responses in peripheral blood from HCT patients pre- and post-aGvHD. We analyzed immune reconstitution in 31 adult patients who underwent HLA matched donor HCT for hematological malignancies. Patients received peripheral blood stem cell grafts and GVHD prophylaxis with tacrolimus and methotrexate. Twelve of 31 patients developed aGVHD at a median of 58 days, range 27-208, after HCT and systemic treatment was started in 83% of these cases. aGVHD grade severity was 25%, 58.3% and 16.7% of grade I, II and IV, respectively. Patient samples were collected at 1, 2 and 3 months after HCT and analyzed using multi-color flow cytometry. Nine healthy donors (HD) were analyzed as controls. Suppressive activity of itolizumab was tested using peripheral blood mononuclear cells (PBMC) obtained from HD and patients before (preGVHD) and after (postGVHD) aGvHD onset (within 30 days). PBMC were stimulated with antiCD3/CD2/CD28 coated beads in the presence of itolizumab or isotype control (cetuximab) for 72 hours. T cell proliferation was measured by CFSE dilution, while T cell activation and maturation was measured by expression of CD25 and CD45RO, respectively. For statistical analysis, non-parametric unpaired (Mann-Whitney) or paired (Wilcoxon matched-pairs signed rank) test were used. CD6+ T cells reconstituted early after transplant, accounting for 95% of positive CD3 T cells, range 57-100 at 1 month. Similar to HD PBMC, in the first 3 months after HCT, CD4 Tcon had the highest CD6 expression, while CD4 Treg had a lower CD6 expression compared to both CD4 Tcon and CD8 T cells (Fig 1A and 1B). To characterize the expression of CD6 on different T cell subsets, we used a t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm and visualized the data using a viSNE map (Fig 1C). Within the Tcon compartment, there were no differences in expression of CD6 between HD and patients at all 3 time points. Within CD4 Treg and CD8 T cells, CD6 expression was reduced in naïve CD8 T cells and CM Treg after transplant compared to HD. In HD, ALCAM expression was detected in 35% of CD14+ monocytes, 23% of CD19+ B cells, 20% of myeloid (CD11c+ CD123-) DCs and 97% of plasmacytoid (CD11c-CD123+) DCs. After HCT, expression of ALCAM in DC compartments was similar to HD. In functional studies, itolizumab inhibited CD4 and CD8 T cell proliferation in preGVHD samples, similar to HD controls. This effect was less prominent in samples collected from patients who had developed GVHD and were already receiving immunosuppressive medications, potentially confounding the ability to assess the effect of itolizumab in this assay (Fig 2A). Similar results were observed for CD25 (Fig 2B) and CD45RO (Fig 2C) expression pre- and post-aGVHD. Finally, itolizumab did not increase rates of cell death in samples from HCT patients as assessed by Annexin V expression, suggesting that itolizumab-mediated T cell inhibition was not due to increased T cell apoptosis. There was a slight increase in Annexin V expression in HD vs isotype control (21%, range 10-43 vs 15%, range 11-31, p= 0.0273). In conclusion, we demonstrate for the first time that CD6+ T cells reconstitute rapidly in peripheral blood after HCT and that CD6 expression is highest in Tcon while lowest in Treg (Tcon>CD8>Treg). Itolizumab efficiently inhibits T cell proliferation and activation after in vitro TCR stimulation of PBMC from aGvHD patients, thus representing a potential therapeutic for treating aGvHD. A phase I/II study using itolizumab as first line treatment in combination with steroids for patients with aGVHD is currently ongoing (NCT03763318). Disclosures Rambaldi: Equillium: Research Funding. Koreth:Amgen: Consultancy; Cugene: Consultancy; Equillium: Consultancy. Cutler:Pharmacyclics: Consultancy; Omeros: Consultancy; Kadmon: Consultancy; BiolineRx: Other: DSMB; Cellect: Other: DSMB; Kalytera: Other: DSMB; ElsaLys: Consultancy; Genentech: Consultancy; BMS: Consultancy; Jazz: Consultancy; Incyte: Consultancy; Fate Therapeutics: Consultancy. Nikiforow:Kite/Gilead: Honoraria; Novartis: Honoraria; NKarta: Honoraria. Ho:Omeros Corporation: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy. Soiffer:Jazz: Consultancy; Gilead, Mana therapeutic, Cugene, Jazz: Consultancy; Juno, kiadis: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Cugene: Consultancy; Mana therapeutic: Consultancy; Kiadis: Other: supervisory board. Ampudia:Equillium: Employment. Ng:Equillium: Employment, Equity Ownership. Connelly:Equillium: Employment, Equity Ownership. Ritz:Equillium: Research Funding; Merck: Research Funding; Kite Pharma: Research Funding; Aleta Biotherapeutics: Consultancy; Celgene: Consultancy; Avrobio: Consultancy; LifeVault Bio: Consultancy; TScan Therapeutics: Consultancy; Talaris Therapeutics: Consultancy; Draper Labs: Consultancy.

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ICOS-LICOS interaction is critically involved in TGN1412-mediated T-cell activation

Blood ◽

10.1182/blood-2011-12-401083 ◽

2012 ◽

Vol 119 (26) ◽

pp. 6268-6277 ◽

Cited By ~ 16

Author(s):

Sabrina Weissmüller ◽

Linda Y. Semmler ◽

Ulrich Kalinke ◽

Stefan Christians ◽

Jan Müller-Berghaus ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

Endothelial Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Umbilical Vein ◽

T Cell Proliferation ◽

Cell Contact

TGN1412, a superagonistic CD28-specific antibody, was shown to require Fc-cross-linking or immobilization as a prerequisite to mediate T-cell proliferation and cytokine release in vitro. We used primary human umbilical vein endothelial cells (HUVECs) to study their ability to induce activation of TGN1412-treated T cells. We confirmed that peripheral primary human T cells do not show activation upon stimulation with soluble TGN1412 alone. Nevertheless, cocultivation of TGN1412-treated T cells with HUVECs induced T-cell activation that was further enhanced using cytokine prestimulated HUVECs. Unexpectedly, Fc-FcγR interaction was dispensable for endothelial cell–mediated proliferation of TGN1412-treated T cells. Transwell-culture assays showed that TGN1412-treated T cells need direct cell-to-cell contact to HUVECs to induce proliferation. We found that costimulatory ICOS-LICOS interaction between T cells and endothelial cells is critically involved in TGN1412-mediated effects. Blocking LICOS reduced TGN1412-mediated T-cell proliferation significantly, whereas recombinant LICOS fully conferred TGN1412-mediated T-cell proliferation. Of note, cytokine stimulation enhanced LICOS expression on HUVECs and ICOS-LICOS interaction up-regulated ICOS expression on TGN1412-treated T cells. Hence, we provide a model of positive feedback conferred by ICOS-LICOS interaction between TGN1412-treated T cells and endothelial cells.

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Costimulation by B7 Modulates Specificity of Cytotoxic T Lymphocytes: A Missing Link That Explains Some Bystander T Cell Activation

Journal of Experimental Medicine ◽

10.1084/jem.186.10.1787 ◽

1997 ◽

Vol 186 (10) ◽

pp. 1787-1791 ◽

Cited By ~ 10

Author(s):

Pan Zheng ◽

Yang Liu

Keyword(s):

Cell Proliferation ◽

T Cells ◽

Influenza Virus ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

T Cell Proliferation ◽

Target Cells ◽

Cell Proliferation And Differentiation ◽

Proliferation And Differentiation

It has been proposed that some bystander T cell activation may in fact be due to T cell antigen receptor (TCR) cross-reactivity that is too low to be detected by the effector cytotoxic T lymphocyte (CTL). However, this hypothesis is not supported by direct evidence since no TCR ligand is known to induce T cell proliferation and differentiation without being recognized by the effector CTL. Here we report that transgenic T cells expressing a T cell receptor to influenza virus A/NT/68 nucleoprotein (NP) 366-374:Db complexes clonally expand and become effector CTLs in response to homologous peptides from either A/PR8/34 (H1N1), A/AA/60 (H2N2), or A/NT/68 (H3N2). However, the effector T cells induced by each of the three peptides kill target cells pulsed with NP peptides from the H3N2 and H2N2 viruses, but not from the H1N1 virus. Thus, NP366–374 from influenza virus H1N1 is the first TCR ligand that can induce T cell proliferation and differentiation without being recognized by CTLs. Since induction of T cell proliferation was mediated by antigen-presenting cells that express costimulatory molecules such as B7, we investigated if cytolysis of H1N1 NP peptide–pulsed targets can be restored by expressing B7-1 on the target cells. Our results revealed that this is the case. These data demonstrated that costimulatory molecule B7 modulates antigen specificity of CTLs, and provides a missing link that explains some of the bystander T cell activation.

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Activation Outcomes Induced in Naïve CD8 T-Cells by Macrophages Primed via “Phagocytic” and Nonphagocytic Pathways

Molecular Biology of the Cell ◽

10.1091/mbc.e07-07-0650 ◽

2008 ◽

Vol 19 (2) ◽

pp. 701-710 ◽

Cited By ~ 10

Author(s):

Isabel María Olazabal ◽

Noa Beatriz Martín-Cofreces ◽

María Mittelbrunn ◽

Gloria Martínez del Hoyo ◽

Balbino Alarcón ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd8 T Cells ◽

Cell Activation ◽

T Cell Proliferation ◽

Microtubule Organizing Center ◽

Antigen Uptake ◽

Soluble Peptide

The array of phagocytic receptors expressed by macrophages make them very efficient at pathogen clearance, and the phagocytic process links innate with adaptive immunity. Primary macrophages modulate antigen cross-presentation and T-cell activation. We assessed ex vivo the putative role of different phagocytic receptors in immune synapse formation with CD8 naïve T-cells from OT-I transgenic mice and compared this with the administration of antigen as a soluble peptide. Macrophages that have phagocytosed antigen induce T-cell microtubule-organizing center and F-actin cytoskeleton relocalization to the contact site, as well as the recruitment of proximal T-cell receptor signals such as activated Vav1 and PKCθ. At the same doses of loaded antigen (1 μM), “phagocytic” macrophages were more efficient than peptide-antigen–loaded macrophages at forming productive immune synapses with T-cells, as indicated by active T-cell TCR/CD3 conformation, LAT phosphorylation, IL-2 production, and T-cell proliferation. Similar T-cell proliferation efficiency was obtained when low doses of soluble peptide (3–30 nM) were loaded on macrophages. These results suggest that the pathway used for antigen uptake may modulate the antigen density presented on MHC-I, resulting in different signals induced in naïve CD8 T-cells, leading either to CD8 T-cell activation or anergy.

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